3.-r1b
BY
WARREN GARY HILL M.Sc.
Thesis Submitted For The Degree Of Doctor of PhilosoPhY
tn The University of Adelaide (Faculty of Medicine)
Lysosomal Diseases Research Unit Department of Chemical PathologY Women's and Children's HosPital South Australia
and
Department of Paediatrics FacultY of Medicine Women's and Children's HosPital South Australia
July 1995 ERRATUM
The following corrections to this thesis should be noted
On page 6 line l4;'lumenal' should read 'luminal' \ On page 9 figure l. l; the abbreviation CFTR was used but not defined. It should have been defined as Cystic Fibrosis Transmembrane Conductance Regulator On page 2l line 20; 'cAMP and phosphorylation activatable' should read 'oAMP- and phosphorylation-activatable' On page 2l line 24;'was lymphocytes' should read 'was the lymphocyte' Onpage32 lines I 8 &. 2l ;' forskolin stimulated' should read'forskolin-stimulated' On page 34 line2l,page36line22 and page 38 lines 2 &3; glcNAc and galNAc should read GlcNAc and GalNAc Onpage 44 line I l; the fullstop should be at the end of the previous line On page 69 line 18; IMDM unà OlvfEIVf should have been listed in the abbreviations as Iscove's Modified Dulbeccos Medium and Dulbecco's Modified Eagles Medium On page 70 line l' G418 should have been listed in the abbreviations as Geneticin On page 154 line 22;'(F508's' should read '(F508 cell lines' On page 159 line 16; UNC should be listed in the abbreviations as Universþ of North Carolina and the reference Snouwaert et al., (L992) added after'mouse' On page 187 line l8; a fullstop is missing Onpage224 Conclusions B, number l; the sentence should read 'Differences in [35S].[3H] ratios appear to be due to differences in sulphate-utilisation by CFPAC rather than glucosamine-utilisation by PANC' Onpage228 line 8;the equation [SOa2-]i: [SO42-]c + i.248lll2 + [Cl-]o should be expressed as [SO42-]i : [SO42-]c + 2.248/(ll2 + [Cl-]o) On page 256 line 6; 'cysteine sulphate' should read 'cysteine sulphur' There was a man who sat each day tooking out through a narrow vertical opening where a single board had been removed from a tall wooden fence' the Each day a witd ass of the desert passed outside the fence and across narrow openinglirst the nose, then the head, the forelegs, the long brown back, the hindlegs and tastty the tait. One day, the man leaped to his feet with "lt the light of discovery in his eyes and he shouted for all who could hear him: is obvious! The nose causes the tail!"
Frank Herbert 'Heretics of Dune' DEDICATION
This thesis is dedicated to the memory of Nicky May
1 972-1 988 LIST OF ABBREVIATIONS v¡¡
THESIS ABSTRACT ix
STATEMENT xi ACKNOWLEDGEMENTS xiii
CHAPTER ON E . INTRO DUCTION
1.1 CYSTIC FIBROSIS 1
1.2 PATHOPHYSIOLOGY OF CYSTIC FIBROSIS...... 2 1.2.1 MECONIUM ILEUS: 3
1 .2.2 PANCREATIC INSUFFICIENCY:'.....".'
1.2.3 INFERTILITY:
1 .2.4 RESPIRATORY INVOLVEMENT:...'..."'
1.3 ROLE OF CHLORIDE CHANNELS lN EPITHELIA ...... """' 6
1.3.1 CHLORIDE TRANSPORT lN AIRWAYS:.. .7 I.3.2 CHLORIDE TRANSPORT lN PANCREAS:"""""" """"""""""' 8 I.3.3 CHLORIDE TRANSPORT lN SWEAT DUCT:"""" """"""""""10
1.4 THE ION TRANSPORT DEFECT IN CYSTIC FIBROSIS...... 10
1.4.1 SELECTIVE ADVANTAGE FOR CF CARRIERS:. 12
1.5 THE CYSTIC FIBROSIS GENE ..."....13
1.6 THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR)
1.6.1 STRUCTURAL SIMlLARITY WITH ABC TRANSPORTERS: """"""
1.6.2 ION CHANNEL OR TRANSPORTER?
1.6.3 TRANSPORTER FUNCTIONS:'......
1.6.4 REGULATION OF CFTR:
1.6.5 LOCALISATION OF CFTR:
1.6.6 MOLECULAR BASIS FOR CFTR DYSFUNCTION:"""""'
1 .6.7 G ENOTYPE/PHENOTYPE CORRELATION :'...... "
1.7 OTHER FUNCTIONS OF CFTR
1 .7.1 STIMULUS/RESPONSE COUPLING:...... 1.8.1 MUCUS AND MUCIN: ...... 34
1 .8.2 GLYCOSAMINOGLYCANS (GAGS): 35
1.8.2.1 Structure and function of GAGs: ...... 35
1 .8.2.2 Biosynthesis of GAGs q
1.9 SULPHATE BIOCHEMISTRY...... 41
1.10 GLYCOCONJUGATE SYNTHETIC DEFECTS IN CF...... 46 .I.10.1 ALTERED GLYCOSYLATION IN CF: 46
1.10.2 ALTERED SULPHATION IN CF: ß
1.10.3 POTENTIAL ROLE OF SULPHATE lN CF: ...... 49
1.1 1 POTENTIAL PATHOPHYSIOLOGICAL CONSEQUENCES OF GLYCOCONJUGATE DEFECTS.. .52 1.11.1 PULMONARY INFECTION IN CYSTIC FIBROSIS: .52
1.11.2 WHY THE CF LUNG? ...... 53
1.12 RATIONALE FOR THIS STUDY...... 57
1.13 ArMS.. 59
CHAPTER TWO . MATERIALS AND METHODS
2.1 MATERIALS/REAGENTS...... 63
2.1.1 CELL LINES USED IN THESE STUDIES: 63
2.1 .2 RADIOCHEMICALS: ...... 64
2.1 .3 ENZYMES/ANTIBODIES:...... 64
2.1.4 CHROMATOGRAPHIC MEDIA: ...... 65
2.1.5 CELL CULTURE MATERIALS:...... 65
2.1 .6 MISCELLANEOUS MATERIALS ...... 66
2.1.7 CHEMICALS:.,...... 67 ...... 69
2.2.1 CELL CULTURE:. 69
2.2.2 CRYOPRESERVATION OF CELL LINES: 70 2.2.3 REVIVAL OF CRYOPRESERVED CELLS: 70 iii
2.2.4IMMUNOFLUORESCENT STAINING OF CELLS: ..'....'...... '....71
2.2.5 MEASUREMENT OF CHLORIDE EFFLUX: ."'.'...'.' 71
2.2.6 SUBCELLULAR FRACTIONATION: 72
2.2.7 ø-HEXOSAMI N IDASE ASSAY:....'.. 73 2.2.8 ACIDIFICATION ASSAY: ...... 74
2.2.9 SCINTILI-ATION COUNTING: ...... 7A
2.2.10 METABOLIC RADIOLABELLING OF ADHERENT CELLS 74
2.2.1 1 M ETABOLIC RADI OLABELLI NG OF LYM PHOBI-ASTS:...... 75
2.2.12 ALKALI EXTRACTION OF GAGS: ...'..'....'...... 76
2.2.13 GAG ASSAY: ...... 77
2.2.1 4 SEPHADEX GsO CHROMATOG RAPHY: ...... 77
2.2.1 5 ION-EXCHANG E CHROMATOGRAPHY: "...... 78
2.2.16 HYALURONIDASE DIG ESTION ...... 79
2.2.17 NITROUS ACID CLEAVAGE OF HEPARAN SULPHATE:....' ...... 79
2.2.18 CHONDROITINASE ABC DIGESTION:....', 80
2.2.19 DESALTING OF CS/DS DISACCHARIDES 80
2.2.20 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) OF CS/DS DISACCHARIDES: .81 2.2.21GENE TRANSFECTION OF CELL LINES: ".'....'. .82 (CPC) ...... 84 2.2.22 CETYLPYR I DI N IUM CHLORI DE PRECI PITATION OF GAGS:
2.2.23 PROTEIN ASSAY: u
2.2.24 STATI STI CAL ANALYSES : ... "'...... ".... " 85
CHA R3.SULP HATION BY YSTIC FI BROSIS AND OL PANCREATIC CELLS
3.1 TNTRODUCTION.... .89 .90
3.2. 1 GENERAL CHARACTERISATION: .... 90 90 3.2. 1 .1 Anti-Q¡tokeratin Staining: .....
3.2.1.2 Acidification of Lysosomes: .. ..91
3.2.1.3 Chloride TransPort: ..92
3.2.2 ANALYSIS OF GLYCOCONJUGATES ..98 IV
3.2.2.1 Metabolic Radiolabellin g, An ion-Exchange Chromatog raphy and Peak Assignment:...... 98 3.2.2.2 Analysis of Peak I on Sepharose CL4B ...106
3.2.2.3 ['uS]:['H] Ratios in Glycoconjugates Synthesised by CFPAC and PANC: ...... 106 3.2.2.4 ["S]:['H] Ratios in Chondroitin/Dermatan Sulphate Synthesised by CFPAC and P 109
3.2.2.5 Demonstration of Heparan Sulphate in Peak lll: ..... 118
3.3 DrSCUSSrON...... 125
CHAPTER 4. SULPHATION OF GLYCOSAMINOGLYCANS BY CYSTIC FIBROSIS LYMPHOBLASTS
4.1 INTRODUCTION 135
4.2 EXPERIMENTAL AIMS ...... 1 38
4.2.1 CELL LINES: ...... 1 38
4.2.2 SPECIFIC METHODS ...... 1 39 4.3 RESULTS...... 140 4.3.1 CHARACTERISATION OF GLYCOCONJUGATE SYNTHESIS BY LYMPHOBLASTS: ...... 140 4.3.2 ['-S]:['H] RATIOS lN GAGS PRODUCED BY CF AND CONTROL LYMPHOBLASTS: ...... 141 4.3.3 INFLUENCE OF CELL CYCLE SYNCHRONISATION ON LYMPHOBLASTS: ...... 146 4.4 DrSCUSSrON......
CHAPTER 5 . SULPHATION OF GLYCOSAMINOGLYCANS BY THE
CFTRGÊ) MOUSE
5.1 tNTRODUCT|ON... 159 5..I.1 EXPERIMENTAL DESIGN 162
5.2 RESULTS f63
5.2.1 T'S]SULPHATE INCORPORATION INTO MURINE GAGS: 163 5.2.2 T'S]SULPHATE INCORPORATION INTO MURINE HEPARAN SULPHATE: ...... 166 5.2.3 ALTERATIONS IN CS/DS:HS RATIOS IN ORGANS FROM NORMAL AND CFTR(-/-) MICE: 172 5.2.4 ANALYSIS OF CS/DS DISACCHARIDES BY HPLC:" """"""'172
5.2.5 SPECIFIC ACTIVITY OF T'S]SULPHATE IN THE 45 DISACCHARIDE OF CS/DS: """"""176 5.3 DrSCUSSlON...... 181 5.3.1 CONCLUDING REMARKS: ...... 189
R PA
CELLS AND THE EF FECT OF EXPRESSI ON ON
SULPHATION
6.1 TNTRODUCTION....
6.2 RESULTS: 4...... 6.2.1 GENERATION OF THE RECOMBINANT CFTR-EXPRESSING CELL LINE 'TR20': """"""'198
6.2.2 [..S]:[.H] RATIOS OF GAGS lN LONG TERM LABELLING STUDIES:...... 198
6.3 DISCUSSION: A...... """""""205 6.3.1 CONCLUSIONS: 4...... 211
6.4 RESULTS: 8...... 6.4.1 SHORT TERM LABELLING: INFLUENCE OF CLo AND p-D- XYLOSIDE:212 6.4.1.1 Experimental Design: ...... 212
6.4.1.2 Results:...... 214
6.5.1 CONCLUSIONS: 8..' 224
6.6 RESULTS: C...... """"'226 6.6.1 SULPHATE UPTAKE AND POOL SIZE IN PANCREATIC CELLS:"..''""..226 6.6.1.1 Experimental Design: """"""226 6.6.1'2 Results:'.... """""227
6.6.2 CYSTEINE AS A SOURCE OF SULPHATE IN PANCREATIC CELLS: "..'2U 6.6.2.1 Experimental Design ...234
6.6.2.2 Results: ...236 ...238
6.7.1 CONCLUSIONS: C 249 6.8 RESULTS: D ...... 250
6.8.1 TNFLUENCE OF ALTERED SULPHATE POOLS AND p-D-XYLOSIDE ON ["S]:['H] RATIOS lN CS/DS:.. 250 6.8.1.1 Experimental Design: 250 6.8.1.2 Results: .250
258 6.9.1 CONCLUSIONS: D.... 259 6.10 OVERVIEW: A MODEL FOR THE EFFECT OF CFTR ON SULPHATE UTILISATION 260
CHAPTER SEVEN . REFLECTION
7.1 SULPHATION IN CYSTIC FIBROSIS .271
7.2 PHILOSOPHICAL CONSIDERATIONS .277
REFERENCES 283 VI
ABBREVIATIONS
ABC ATP binding cassette APS adenosine-5'-phosPhosulPhate
ATP adenosine-5'{riPhosPhate bp base pair cAMP cyclic adenosine-5'-monophosphate
CBAVD congenital bilateral absence of the vas deferens
CF cystic fibrosis
CFTR cystic f ibrosis transmem brane cond uctance reg ulator
CSi DS chondroitin sulphate/dermatan sulphate
CV coetficient of variation
EBV epstein bar virus
ECM extracellular matrix
ER endoplasmic reticulum
FBS loetal bovine serum
GAG glycosaminoglycan galNAc N acetylgalactosamine
glcNAc N acetylglucosamine
HPLC high performance liquid chromatography
HS heparan sulphate
mRNA messenger RNA
NBF nucleotide binding fold
PAPS 3'-phosphoadenosine-5'-phosphosulphate
PCR polymerase chain reaction
PI pancreatic i nsuff iciencY
PKA protein kinase A
PS pancreatic sufficiencY
RESP rapidly exchangeable sulphate pool
SESP slowly exchangeable sulphate pool
V-ATPase vacuolar-ATPase vlil
(0s) 2-acetamido-2-deoxy-3-O-(B-D-gluco-4-enepyranosyluronic ^dios acid)-D-galactose
(4s) 2-acetamido-2-d eoxy-3-O-(F- D-g I uco-4-enepyranosyluron ic acid)- ^d¡4s 4-O-sulfo- D-galactose
(6s) 2-acetamido-2-deoxy-3-O-(F- D-g I uco-4-enepyranosyl u ronic acid)- ^di6s 6-O-sulfo-D-g alactose
(2s) 2-acetam ido-2-deoxy-3-O-(2-O-su lfo-B- D-g I uco-4- ^dizs enepyranosyluronic acid)-D-galactose
Adi-di4,2s (diB) 2 -acetam ido-2-deoxy-3-O- (2-O-su lfo-p-D-g I uco-4- enepyranosyluronic acid)-4-O-sullo-D-galactose
(diD) 2-acetam ido-2-deoxy-3-O- (2-O-su lfo-p-D-g I uco-4- ^di-d¡6,2s enepyranosyluronic acid)-6-O-sulfo-D-galactose
Adi-di4,6s (d¡E) 2-acetam id o-2-d eoxy-3-O- (F- D-g I uco-4-enepyranosylu ronic acid)-
4, 6-d i-O-sulfo- D-g alactose. tx
Cystic fibrosis (CF) is a lethal inherited disease. MorbidiV relates to the production of thick mucus in the ainruays, which predisposes the lung to chronic infection. The presence of glycoprotein abnormalities in the mucus of CF patients has not been well defined. The purpose of this study was to establ¡sh whether altered sulphation in CF could be demonstrated in different experimental systems, by focusing on glycosaminoglycans (GAGs). The second aim was to establish the molecular basis for such a phenomenon.
Results Obtained: were 1. [ruS]:[.H] ratios of GAGs synthesised by two CF lymphoblast lines ^FsoB/AFso, (p<0'05) higher compared to two controls and two CF lines of undefined genotype after radiolabelling with l"S]sulphate and ['H]glucosamine. This difference was more significant for chondroitin/dermatan sulphate (CS/DS) (p<0.002) than heparan sulphate (HS) (not different). The structure of CSiDS synthesised by cell lines (examined by HPLC) was identical. However ["S]:['H] ratios within the 4-sulphated
(4S) disaccharide of ÂF*r/AFso, CS/DS was higher, implying differences in the specific
activity of precursor pools for GAG synthesis. Z. CF mice injected with [.'S]sulphate exhibited higher incorporation into GAGs of liver (except and pancreas than normal controls (p<0.05). All other CF organs examined (not nasal mucosa which had less (p<0.05)) had higher l*SJsulphate incorporation significant). Nasal septum cartilage exhibited lower ["S]sulphate into keratan sulphate (p<0.05). 3. lleum and gall bladder of CF mice contained less CS/DS as a proportion of total GAGs, than normals (P<0.05)' 4. CS/DS in ileum and liver of CF mice was oversulphated þ<0'02 and p<0'05)' 5. Specific activity of sulphate whhin the 45 disaccharide was higher in CF mice, implicating a difference in intracellular specilic activity' 6. A CF pancreatic cell line (CFPAC), exhibited higher ["S]:['H] ratios in GAGs than a control (PANC). Gene-corrected CFPAC cell lines (PLJ6, TR20, PLJ4.7) exhibited normal¡sation (to varying degrees) of ["S]:['H] ratios. Differences in ["S]:['H] ratíos in CS/DS was attributable to ditferent specific activities within disaccharide species, not to levels of sulphation. 7. Higher ["S]:['H] ratios were due to altered kinetics of dual intracellular sulphate pools.
ln CF cells the proportion of sulphate derived from the rapidly exchangeable pool was
greater than in controls. ['uS]:['H] ratios in PLJ6 (not the others) reverted to control levels, indicating CFTR expression had corrected sulphate utilisation pathways.
8. CFPAC cells incorporated 7.4 times as much l"S]cysteine into GAG sulphate as PANC. PLJ6 was corrected, implying that CFTR influenced this pathway also.
These findings add to our understanding of sulphate utilisation pathways in epithelial cells, provide compelling evidence of the involvement of CFTR in sulphate metabolism rn vivo, and warn that therapíes based on gene replacement need to be aware of secondary metabolic conseq uences.
I owe an enormous debt of gratitude to Dr Greg Harper and Prolessor John Hopwood for their roles in the initiation, smooth progression and completion of this PhD project. To Greg, thanks mate. You've been both friend and mentor. At times your enthusiasm left me breathless. You even knew when I needed to start developing some scientific independence and left me to get on with it. To John Hopwood, many thanks for the resources, the training, the direction and your support of a project which was initially slow to make headway.
A great deal of credit for the breadth of this project and the amount of ground that was covered must go to Tina Rozaklis for her excellent technical assistance and unstinting dedication to the CF project. Much of the tissue culture, column chromatography and immunofluorescence (not to mention Western blotting) as well as transport experiments were performed by Tina. I thank her and realise it can't always have been easy working with an obsessive PhD student.
I am indebted to the Nicky May Cystic Fibrosis Research Foundation and to Ken May in particular. The Foundation provided not only extended scholarship suppoñ, but a
refrigerator for the lab and financial assistance for an overseas study trip.
Thanks to the Australian Cystic Fibrosis Research Trust who provided grant support to our studies, and especially to the firemen who ran around Australia fund raising for the ACFRT appeal, which we were fortunate to benefit from'
Many thanks must go to the people in other lnstitutions and other pafts of the world who
assisted this project. Dr Jeremy Turnbull for training in strong anion-exchange HPLC and lor providing disaccharide standards, Dr Brandon Wainwright for providing, and Dr Richard Gregory for permission to Lise, a CFTR-gene containing plasmid. Professor XIV
Richard Boucher for the opportunity to perform an experíment on the CF mouse, and Drs Barbara Grubb and Pi-Wan Cheng for assisting me with those experiments. To Dr Ada Elgavish for looking after me while in Birmingham, Alabama and Professor Ray Frizzell for providing the CFTR expressing CFPAC clones.
On a personal level, thanks Mum and Dad, for tireless emotional and financial support. I promise, my days as a penniless student are almost over.
Without intelligent (opinionated), sociable (church-going) and passionate (argumentative) friends my sanity would not have prevailed. So thanks Doug, Maria, Erik, Sonia, Maftin,
Sven, Michael, Jill, John and Ed.
And finally, thanks to my beautiful wife, Angela. You know what you have done. Words do not suffice.
1.1 CYSTIC FIBROSIS
Cystic fibrosis (CF) is an autosomal, recessively inherited disease which has a tragic impact on families and the community by virtue of its lethality and high prevalence. lt occurs with high frequency in Caucasian populations with about 1:2500 live births affected (Boat et a1.,1 989) and as such is the most common fatal genetic disease of that population. Asian and African-American communities suffer a much lower incidence ol
CF; 1:90000 and 1:12-17000 respectively (di Sant'Agnese and Davis, 1976). lt has been hypothesised that CF may provide a selective advantage for heterozygotes in European populations, in orderto explain its high prevalence (Rodman and Zamudio, 1991). The nature of such an advantage is still somewhat speculative, but there is evidence of a protective effect against cholera, for CF carriers (see section 1.4'1).
CF is primarily a disease of exocrine organ systems with airways, intestine, pancreas,
reproductive tract and sweat gland exhibiting significant involvement. Characteristically the disease manifests itself by the production of thick viscous mucus, particularly in the tracheobronchialtree and pancreas, although nearly every mucus secreting epithelium in
the body is affected (Gerken and Gupta, 1993). Historically the condition was termed
'cystic fibrosis of the pancreas' to reflect the loss ol pancreatic function (Anderson, 1938)
and then later'mucoviscidosis' in recognition of the more ubiquitous nature and exocrine
involvement of the disease (Farber, 1945). lt wasn't until 1983 that the true nature of the
biochemical abnormality was revealed by the work of Knowles et al. 0983) who showed
that chloride ion permeability in respiratory epithelium was defective and that of Quinton 2
(1983) who pinpointed a similar defect in the sweat duct. lt was the sweat gland abnormality which formed the lirst diagnostic basis lor CF both in ancient times and today (di Sant'Agnese and Powell, 1962). The lack of a normal chloride permeability in the sweat gland results in a secretion which is excessively salty due to an inability of the cells within the duct to resorb chloride ions from isotonic sweat secreted into the coil
(euinton and Reddy, 1989). This was readily identifiable in the newborn infant simply by
predict licking the skin. lT the baby tasted 'salty' the midwife of yesteryear was able to the child's imminent death, a few years hence, of failure to thrive and pulmonary complications (Taussig, 1984). The pilocarpine iontophoresis test based on the measurement of NaCl in sweat (Gibson and Cooke, 1959) is used today in modern paediatric hospital pathology laboratories as the 'gold standard' diagnostic indicator of cF (stewart, et a1.,1995).
1.2 PATHOPHYSIOLOGY OF CYSTIC FI ROSIS'
CF is a disease of uncertain outcome for those affected. lt is characterised by a high degree of clinical variability ranging from extremely mild organ involvement where a diagnosis may be made in adulthood (Robinson and Richardson, 1991); to extremely
palliative severe, with death occurring before adolescence. With improvements in care, (Collins, the life expectancy for someone born with CF has steadily increased 1992).
One study based on English and Welsh patient data predicted the median age of survival
(1989) in ,Many of the clinical descriptions ol the disease in this section are taken from Boat et al' Metabotic Basis of tnherited Disease 6th Ed. Other sources are referenced in the main text' 3 lor someone born with CF in 1990 to be 40 years (Elborn et al., 1991), double that ol someone born with cF twenty years ago'
Clinical heterogeneity results from the complex interplay of genetic and environmental factors. lt is possible that there are, as yet unknown, genetic loci in CF which contribute to a patients phenotype (Kiesewetter ef a/., 1993; Dean and Santis, 1994)' A numberof studies have attempted to establish correlations between genotype and phenotype with limited success (see section 1.6.7).
The discovery of a basic ion transport defect in affected CF tissues provided at least a partial explanation for the major pathophysiological consequence of the disease, namely thick dehydrated mucous secretions. Affected epithelial surfaces Jose not only the ability to transport chloride ion, but equally importantly, fluid (Wine, 1991). The regulated secretion and absorption of fluids by the body is a lundamental aspect of numerous metabolic processes, from thermoregulation to the utilisation of ingested food, by secretion ol digestive enzymes. The mechanisms by which regulated ion and fluid movements occur will be discussed in later sections. Suffice to say that the ramilications of an inability to control water and ion movements would be predicted to be severe' This
prediction is borne out in CF.
1.2.1 MECONIUM ILEUS:
Meconium ileus is a blockage of the intestine in the neonate. lt occurs in approximately
10% of CF infants and is due to the presence ol desiccated muco-fecal material or
meconium, which creates an obstruction in the intestinal tract which can only be
corrected surgically. The presence of meconium ileus at birth is virtually diagnostic for 4
CF as its occurrence in the absence of CF is exceedingly rare. The condition appears to predispose the individualto a four lold higher risk of liver disease later in life (Colombo et a1.,1994).
1.2.2 PANCREATIC INSUFFICIENCY:
The pancreas is one of the major sites of pathology in CF, secondary only to the airways (Pl) rn the impact that dysfunction has on the patient. Pancreatic insufficiency was a term coined to describe those individuals with fat malabsorption to due a deficiency of
'fatty digestive enzymes. One of the consequences is steatorrhoea or stools'- The infant who is not detected with CF before, or shortly after birth, inevitably fails to thrive as a result of nutritional deficiency. This deficit affects 85-90% of all CF sufferers
(Widdicombe and Wine, 1991; Kristidis et al., 1992) and appears to be due to mucus
plugging of ducts and subsequent acinar atrophy. Histological examination of the CF
pancreas at postmortem reveal fibrosis, inspissation of secretions and occasionally
pancreatic epithelial lined cysts and calcification. Among those individuals who exhibit (Couper sufficiency (PS) at a very young age, a small subset will convert to Pl et al',
1 992).
1.2.3 INFERTILITY:
and lnfertility of males is an almost universal feature, with the vas deferens, epididymis
a result of seminal vesicles, atrophied (Collins, 1992). Pathogenesis probably occurs as
is slightly early genital tract obstruction by inspissated secretions. ln women fertility
and failure higher at 1gyo, compared with 2-37o in males. However menstrual irregularity
to ovulate are common, due to poor nutrition and chronic lung infection' 1.2.4 RESPIRATORY INVOLVEMENT:
The lile+hreatening consequences of CF derive mainly from the eflect the disease has on the airways. There is chronic pulmonary obstruction and inlection from birth, both ol which relate to the production of thick, abnormally viscous, mucus (Geddes and Shiner,
1g8g). As lung disease progresses there is increasing evidence of bronchitis, bronchiolitis, and submucosal gland hypertrophy. There is usually extensive bronchiectasis later in life. The formation of nasal polyps and oedema of the nasal mucosa are common in CF. lt is the predisposition of the CF lung and airways to infection by pathogenic Pseudomonas and Staphylococcalspecies which results in the gradual destruction of lung tissue due to inflammatory processes. Once infection is established it is virtually impossible to eradicate.
The hypersecretion of mucus by these tissues appears to be the initiating pathogenic event and the lack of mucociliary clearance a contributing factor to infection. This does not appear to be the complete explanation however. Although the mucus is assumed to be dehydrated as a logical consequence of the ion-transport defect there are many studies which point to chemical differences between CF and normal bronchial secretions
(see sections 1.10, 1.10.1,1.10.2). These secondary consequences may play an important role in the pathophysiology of the disease ffhiru et al., 1990; King et a/., 1990;
Frates et a1.,1983; Chace et a1.,1983; Carnoy et a1.,1993). 1.3 ROLE OF CHLORIDE C NELS IN E THELIA
It is interesting that despite the gross organ pathology which characterises CF, many clinicians and researchers today, tend to regard the disease primarily as one of defective fluid absorption and secretion (Quinton, 1989). All cells require mechanisms for moving solutes across their plasma membranes in order to regulate their internal environment. ln epithelia these mechanisms are highly developed, rellecting the specialised role that the epithelium plays as an interactive membrane which separates, in simple terms, the outside world from the inside. Because it performs many roles, not least of which is the formation and maintenance of a protective mucus blanket, it has developed highly specialised mechanisms for regulating its external and internal milieu. The coordinated secretion of water, ions and mucus in response to a range of agonists is central to its lunction (Liedtke, 1989; Wine, 1991).
Epithelial cells are polarised, i.e. they perform different functions on their apical (or lumenal) surface than on their basolateral (serosal). ln terms of fluid movement we can think ol two processes occurring; 1) secretion and 2) absorption. Epithelial cells are organised in such a way that they are able to precisely regulate both processes depending on their physiological status and as a result have the ability to pedorm vectorial solute flow. An important component in the control of fluid movement is chloride ion movement. Chloride fluxes occur through channels embedded in the plasma
(1989) membrane. The number of known channels is growing rapidly. Landry et a/. isolated three unique chloride channels from bovine kidney and trachea using traditional 7 approaches such as alfinity chromatography whilst new molecular techniques such as expression cloning have given researchers tools for discovering ion-channels without
(Paulmichl having to isolate and reconstitute functional forms ol the proteins et a1.,1992;
Ho ef a/., 1993). Biophysical methods, revealing characteristics such as single channel conductance, gating properties and regulatory features, allow discrimination between types of chloride channels. So far there have been seven different chloride channel
(Paulmichl protein-families described, of which one is the chloride channel involved in CF et al., 1gg3). The way in which different epithel¡a regulate their ion lluxes is highly complex and usually involves the coordinated movements of Na., K', H*, Cl and HCO; by a combination of co-transporters, counter-transporters and passive diffusion, under the influence of numerous effectors. An in-depth look at these is beyond the scope of this introduction; the interested reader is referred to (Quinton, 1989; Liedtke, 1989; Field,
1993; Boucher,1994).
1.3.1 CHLORIDE TRANSPORT IN AIRWAYS:
Transepithelial secretion of NaCl in the airways is accomplished by the coordinated action of a number of pathways. Efflux of chloride at the apical membrane is primarily regulated by p-adrenergic agonists which activate either the oAMP second messenger cascade (Boucher and Larsen, 1988; Hwang et al., 1989; Verbeek et al., 1990) or
et al., release of intracellular calcium (Clancy et a|.,1990; Clarke et a\.,1992b; Yamaya
pathways 1gg3; Haas and McBrayer, 1994). Chloride channels activated by the two are
(see distinct, with the cAMp-mediated channel defective in CF Fig. 1.1). To maintain
apparent chloride efflux at the apical membrane requires the concomitant activation and
upregulation of the electroneutral NaYK'/2Cl co-transporter on the basolateral
membrane. This allows replenishment of intracellular chloride (Haas et al., 1993). Co- I transport ol ions across the basolateral membrane is driven by the inwardly directed chemical gradient for K' and Na' (see Fig. 1,2B.)
1.3.2 CHLORIDE TRANSPORT ¡N PANCREAS:
pancreatic acinar cells, which secrete digestive enzymes, appear to be quite different from duct cells in the regulatory mechanisms they employ to control electrolyte transport.
The main role of duct cells is to secrete the bicarbonate rich fluid necessary for
(Liedtke, neutralising the acidic chyme which enters the duodenum from the stomach
1 989).
The secretion of pancreat¡c juice which contains high concentrat¡ons of bicarbonate,
takes place via a bicarbonate/chloride anion exchanger in the plasma membrane of duct
cells, and is stimulated by secretin and vasoactive intestinal peptide. These peptide
hormones act through the cAMP second messenger cascade. Stimulation by secretin
can result in the production of a large volume of fluid with bicarbonate concentrations
reaching 140-160 mEq/l (Hootman and Deondaza, 1993). However in CF the volume of
fluid is reduced to approximately 40o/o ol normal, and this diminution correlates with
reduced bicarbonate and chloride secretion. lt has been proposed that lack of ion
movement is the driving force behind reduced fluid secretion and the pancreatic defect in
CF (Kopelman ef a/., 1988; Durie, 1989). The movement of potassium across the
basolateral membrane also appears to be important to duct secretion'
by ln contrast with the duct cell, acinar cells secrete a chloride rich fluid upon stimulation
calcium acetylcholine and cholecystokinin. These elfectors act by promoting intracellular
(Liedtke, release via the phosphatidylinositol/phospholipase C pathway 1989)' lt has ßecgptor
J Oe" Activated J cAMP Adenylde Protein Kinase A Cyclaee :: ATP cl- ATP
J tr Gplc J PLC 0 ct- J g Activated +lPs + c^2*l + J 'c"2*-caM - CaM Kinase DAG
FIGURE 1.1 Controt Of Plasma Membrane Chloride Channels by Parallel cAMP and Calcium in an ldealised Airway Epithelial Cell. pLC, phospholipase C; Gs and GpLc, stimulatory and PlC-specific guanine nucleot¡de binding proteins respect¡vely; PlP2, phosphatidylinositol 4,5-bisphosphate; DAG, diacylglycerol; caM, calmodulin (copied lrom wagner et al. (1992)). 10 been suggested that chloride channels present in pancreatic zymogen granule membranes may be inserted into the plasma membrane and play a role in exocytosis
(Delisle and Hopfer, 1986)
1.3.3 CHLORIDE TRANSPORT IN SWEAT DUCT:
The principle mechanism by which humans dissipate heat is through evaporative cooling.
To accomplish this and not lose excessive quantities of electrolytes, we have evolved a specialised structure in the form of the eccrine sweat gland. The gland is composed of a secretory coil at the base and a duct structure which leads to the surface ol the skin
Fluid secretion in the coil is essentially isotonic, but as it flows through the duct, chloride and sodium are reabsorbed so that the final composition of the sweat is hypotonic
(Quinton and Reddy, 1989). The general principles of salt absorption in epithelia can be seen in Figure 1.2A. ln CF, ductal chloride conductance is absent which results in excessively salty sweat. The cells lining the duct are unresponsive to p-adrenergic agonists as well as calcium mobilising agents in terms of their ability to activate chloride conductance pathways (Liedtke, 1 989).
1.4 THE lON TRANSPORT DEFECT IN CYSTIC FIBROSIS
It was the series of landmark studies by Knowles et a/. (1983), Quinton (1983) and Sato and Sato (1984) all of which demonstrated defective chloride permeability in ep¡thelia, which provided for the {irst time, a unifying hypothesis for the cause of CF. Testing of that hypothesis pinpointed a failure ol CF epithelia to secrete chloride ion in response to
B-adrenergic stimulation. Specifically it was found that increases in intracellular cAMP 11
A B
ATP d Na' g Na+ 3Na + 2K
Na+ ct ADP + K + K ct Na' 2ct
+ Na
Salt Absorpt¡on Salt Secretion
FIGURE 1.2 General Model of Transepithelial lon Transport Pathways in Salt Absorbing ß) or Secreting (B) Tissue. A general feature of both cell types is the presence of a 3Na/2K ATPase in the basolateral membranes which maintains the h¡gh potass¡um/low sodium concentration of the cell (Copied from Bijman (1989)). 1 caused an increased chloride permeabil¡ty ¡n normal but not in CF epithel¡a. The abnormality was localised to the apical membrane of ep¡thelial cells (Widdicombe et al.,
1985; Cotton et al., 1987) and it was established that cAMP levels in response to hormone stimulation were elevated appropriately in CF epithelia (Frizzell et al., 1986;
Welsh and Liedtke, 1986). Hence it did not appear that the abnormality was due to a defect in receptor signalling. Further studies demonstrated that the second messenger cascade involved cAMP-dependent protein kinase and protein kinase C both of which were capable of phosphorylating and opening CF chloride channels in excised
membrane patches from normal airway epithelia, but not from CF epithelia (Li et al.,
1988; Hwang et a1.,1989; Li et a1.,1989). These studies and others pointed strongly to the likelihood that CF was a disease caused either by the lack of a chloride channel or a
molecule which regulated a chloride channel in epithelial cells. Confirmation required
isolation of the gene.
1.4.1 SELECTIVE ADVANTAGE FOR CF CARRIERS:
ln cholera, the normal secretory mechanisms of the gut epithelium are disrupted and the consequences are a massive secretory diarrhoea. This causes the loss of not only fluid from the body but salts as well, with potentially fatal consequences. CF heterozygosity
appears to confer a selective advantage against challenge by the microorganism and the toxin responsible for cholera. Baxter et al. (988) have shown that intestinal tissue from
CF heterozygotes has a decreased responsiveness to cholera toxin. Also, Gabriel et al.
(1994) have shown a direct correlation between the number ol CF alleles and fluid
accumulation in response to cholera toxin by an animal model lor CF. CF homozygous
mice secreted no lluid when challenged with cholera toxin whilst heterozygotes secreted
500/o that ol normals. Thus there is mounting evidence that CF carriers historìcally, would 1 3 have had a survival benefit from possession of one CF allele. This may account for the high proportion of the gene in European populations
1.5 THE CYSTIC FIBROSIS ENE
The CF gene was cloned in 1989 and reported in three landmark papers (Kerem et al., j9B9; Riordan et a1.,1989; Rommens et al.,1989). The anticipation and hope embodied in this achievement was seen in the image of the young boy on the cover of that issue of
Science (overleaO. This was the first disease gene to have been identified strictly by
,reverse genetics' or what is now known as 'positional cloning'. Collins defined positional cloning as 'the strategy whereby a gene is identified by its map position without making
any assumptions about its function' (Collins, 1991). This process became possible once
(Wainwright the gene had been localised to a small enough region on chromosome 7 ef
a/., 1g8S). The definition offered by Collins was particularly apt in the case of CF
because, despite the indications of ion transport abnormalities in affected tissues, the
nature of the gene product was unknown.
The CF gene encoded a 6.5 kb mRNA which was expressed primarily in tissues which
glands, were affected in cF. The 6.5 kb transcripts were detected in lung, colon, sweat
placenta, liver, pancreas and in nasal polyps. The best evidence that it actually was the
gene for CF came from the finding that transcripts from 70o/o ol CF chromosomes
position encoded a 3 bp deletion resulting in the loss of a phenylalanine residue at 508
ol the protein (Kerem et a:.,1 989; Riordan et al.,1 989). This mutation, termed AF,o,, was 14 not found on any non-CF chromosome. The gene spanned 250 kb ol genomic DNA, contained 27 exons and encoded a protein of 1480 amino acids with a predicted
molecular weight ol 168138 daltons. lt was predicted to contain two membrane spanning
domains of six membrane spanning segments each, two cytoplasmic consensus
nucleotide binding folds capable of binding ATP, and a highly charged cytoplasmic
domain in the middle of the polypeptide called the R or regulatory domain (see Fig. 1.3
A). Riordan et al. (1989) named this protein the 'cystic fibrosis transmembrane
conductance regulator'. The major mutation, ÂF.or, was located in the first of the two
highly conserued nucleotide binding folds (NBF's).
1.6 THE CYSTIC FIBROSIS TRANSMEMBRA NE
CONDUCTANCE REGULATOR (CFTR)
1.6.1 STRUCTURAL S¡MILARITY WITH ABC TRANSPORTERS:
The ATP-binding motifs and the two-by-six transmembrane spanning domains of CFTR
were recognised as bearing remarkable similarity to the mammalian multidrug resistance
P-glycoprotein (which has been shown to mediate resistance to a wide variety of
cytotoxic drugs in human tumours (Nielsen and Skovsgaard, 1992)), as well as to other
membrane bound proteins with transporter functions. The R-domain however was
unique and bore little similarity to any other known protein (Dulhanty and Riordan, 1994).
Nonetheless CFTR became the newest member ol the ABC (ATP-Binding Cassette)
superfamily ol transporlers. Also known as the traffic ATPases, they are characterised
by a highly conserued ATP binding motif and otten utilise the energy of ATP hydrolysis,
15
A.
R Domain
NBFl NtsF2
cAMP
TM TM
NBFl P
P P
FIGURE 1.3 Domain Structure (A) and Regulation (B) of CFTR. 1. ln the absence of R domain phosphorylation' the channel is closed. 2. After cAMP poìsed stimulates pKA to phosphorylate a number of serines in the R domain, the channel is to bind ATp. 3. ATp is bound. 4. Cleavage results in a conformalional change which opens (Adapted the chloride channel. The channel then decays back to a closed state (2). from Collins (1992)). 16 to pump substrate across membranes aga¡nst a concentration gradient. Over fifty ABC transporters have been identified (lor a comprehensive review see (Higgins, 1992)).
Other members ol this lamily included the STE6 a-mating factor transporter of yeast
(Raymond et al., 1992), pfMDRl, (the Plasmodium falciparum equivalent of P- glycoprotein, and the molecule whose presence is associated with chloroquine-resistant malaria (Foote et al., 1990)), a host of bacterial periplasmic transporters, and the transporters associated with antigen processing and peptide transport in T cells, TAPI and TAP2 (Shepherd et a1.,1993). This family of proteins is thus represented throughout the evolutionary spectrum from yeast to man. They are often, but not always, associated with a transporter function, and although each ABC transporter is relatively specific for its substrate, the variety of molecules transported is enormous. Transponers lor amino acids, Sugars, ions peptides and even proteins have all been characterised.
1.6.2 ION CHANNEL OR TRANSPORTER?
Because of the structural similarity, there was intense speculation as to whether CFTR was a transporter, instead of an ion channel or a regulator of an ion channel as the electrophysiological data suggested. With the discovery of the gene, workers were able to use molecular techniques to transduce cells and express the protein. Rich ef a/.
(1990) were able to demonstrate correction of the chloride transport defect in cultured CF airway epithelial cells and were also able to show that expression of CFTR carrying the
ÂFro, mutation did not correct the defect. Gregory et al. (1990) in an accompanying
paper expressed CFTR in HeLa cells and demonstrated that it was a membrane bound glycoprotein that could be phosphorylated in vitro by cAMP-dependent protein kinase'
Drumm et al. (990) demonstrated correction of the chloride transport defect in a CF
pancreatic cell line derived from a pancreatic adenocarcinoma (CFPAC-1) and the linear 17 nature of the current-voltage relationship in whole cell patch clamp experiments. The hypothesis that cFTR was itself a chloride channel, received a lurther boost when
Anderson et at. (991c) demonstrated the appearance of characteristic CFTR chloride currents in HeLa, CHO and NIH 3T3 libroblasts, cells which d¡d not have cAMP-
proof, dependent currents prior to retroviral-transfection. This did not constitute but an alternative explanation would have had to assume cryptic cAMP-dependent chloride channels which only became activated upon expression of CFTR. Confirmation was obtained from the work of Kartner et at. (991) who showed the presence of CFTR-like
(derived Fall conductances upon transfection in Sf9 insect cells from ovarian tissue of the armyworm) which normally had no ion conductances at all. They established the channel had a low conductance of 5-10 pS and confirmed the linear current-voltage relationship obserued by others. Strong evidence that CFTR was itself a chloride
(991b) basic channel came with the demonstration by Anderson et at. that mutation of
selectivity' residues within the transmembrane spanning domains altered CFTR's anion
Final confirmation was provided by Bear et at. (ggÐ who purified CFTR and then reconstituted the protein into proteoliposomes. chloride channel biophysical
CFTR previously. characteristics were consistent with all of the observations made about
Thus, CFTR, with many of the features of a transpoder was in fact an ion channel'
1.6.3 TRANSPORTERFUNCTIONS:
chloride channel, Despite the clear evidence that CFTR was a phosphorylation regulated to look for the strong similarity it bore to other ABC transporters prompted investigators
(1992) hypothesised that additional transport related Tunctions. Riordan and Chang have
the protein the transport capability of CFTR, although real, has been modified such that
R domain transpons its own R domain into and out of the channel pore. As such, the 18 could be considered a tethered substrate involved in its own 'abortive'transport. Despite the appealing simplicity of this model, CFTR has been shown to participate in the transport of molecules other than chloride ions. These include neutral amino acids
(Rotoli et al., 1994) and ATP (Cantiello et al., 1994; Reisin et al., 1994). The homologous ABC transporter, P-glycoprotein, has recently been shown to lunction as an
ATP channel also (Abraham et al., 1993).
A number of observations regarding the interaction of ATP with CFTR and with airway epithelia make this potential transport activity of great interest. They are:
1) extracellular ATP and UTP increased chloride secretion in the airways of patients
with CF, and in nasal epithelial cells in culture (Knowles et al., 1991; Clarke and
Boucher,1992),
2) external ATP activated CFTR by a cAMP-independent mechanism, even in cells that
were depleted of intracellular ATP (Cantiello et a1.,1994),
3) intracellutar ATP at high levels (5 mM) activated CFTR via a non-hydrolytic
interaction, leading to the proposal that CFTR may be 'sensing'the energy status of
the cell and coupling it to the energy demands of ion transport (Quinton and Reddy,
1992; Bell and Quinton, 1993).
The physiological consequences of these properties are unknown but it would appear that the energy status of the cell plays a role in controlling chloride secretion, through multiple pathways. High intracellular ATP concentration leading to CFTR channel opening, concomitant ATP elflux and then act¡vation of further chloride channels via extracellular and possibly paracrine interaclions. CFTR has also been shown to regulate 1
(Gabriel the activity of another channel - the 'outwardly rectifying chloride channel' et al.,
1gg3). The nature of this regulatory interaction has not been elucidated but could conceivably occur by mechanisms similar to those outlined.
1.6.4 REGULATION OF CFTR:
Rote Of The R Domain-fhe observation that elevations in intracellular cAMP activated
CFTR, pointed to a role for cAMP-dependent protein kinase (PKA) in the regulation of the channel. The R domain was a likely target for this enzyme due to the presence of numerous consensus phosphorylation sites. This speculation was confirmed when it was shown that the catalytic subunit of PKA could activate CFTR in cell free patches ol membrane (Berger et al., 1991). There were however, ten predicted PKA consensus phosphorylation sites within the R domain, and attempts to ascertain which were phosphorylated rn vivo and what role individual sites played in the overall regulatory mechanism produced conflicting results. Cheng et al. (1991) showed that four serine residues were phosphorylated in vivo and that it required mutation of all four sites to
(1993) abolish chloride channel opening. ln contrast with these results, Chang ef a/. demonstrated that all ten PKA sites could be mutated and the channel still activated by pKA. lt was thus apparent that channel opening in response to phosphorylation ol serine residues contained some degeneracy which suggested complex regulatory
mechanisms. The ability of pKA to open the channel after removal of all ten serines also
hinted at the presence of other cryptic phosphorylation sites. Deletion of the R domain
resulted in a constitutively open channel (Rich et al., 1 991 ) while replacement of serines
with aspartates (conferring altered charge properties) generated open chloride channels
in the absence of cAMp (Rich ef a/., 1993). These observations led to the idea of the R
pore domain as a 'ball' plugging the ion pore of the channel but lorced out of the by 20 electrostatic repulsion upon phosphorylation, thus releasing the block and allowing ion passage (see Fig. 1.38 lor general features of CFTR regulation). A number of studies also demonstrated phosphorylation and channel activation by protein kinase C, thereby implicating convergent kinase regulatory pathways in the overall functioning of CFTR
(Berger et a1.,1993; Dechecchi et a1.,1993).
Rote Of The Nucleotide Binding Domains-The presence of two NBF's suggested ATP could play an important role in regulating the activity of CFTR. Elucidation of the precise role of these structural motifs was important because they were the site of a number of naturally occurring, disease causing mutations, including ÂF*' (cutting et a/'' 1990)' An understanding of their role might point the way to therapeutic interventions. Anderson ef a/. (1991a) demonstrated that once the R domain was phosphorylated, hydrolysable ATP was absolutely required for channel activation. An interesting observation was that only
NBFI was required, with mutations in NBF2 not prohibiting channel opening. This pointed to functional non-equivalence for the two NBF's. ATP was subsequently shown to interact directly with the NBF's and indeed it could be shown that could bind ATP ^FsoB equally as well as wild type NBFl (Hartman et al., 1992) suggesting AF,or's ability to hydrolyse ATP may be impaired. ln contrast G551D (the second most common CF mutation) in NBFI and G1349D in the analogous position of NBF2, showed reduced binding of ATp (Logan et al.,1gg4). This pointed to a multiplicity ol functional mutations in the CF gene. R domain phosphorylation was found not to be a prerequisite for ATP binding (Travis et a;.,1993). Studies ol the roles ol NBFl and NBF2 in which the highly conserued 'Walker motifs' were mutated, confirmed that the two domains were functionally distinct. Mutations in NBF1 caused CFTR to have a decreased sensitivity to 2 1 activation whilst mutat¡ng NBF2 had the opposite effect with an increase in sensitivity to activation (Smit er a/., 19g3). Gunderson and Kopito (1994) found that ADP could inhibit channel opening by directly compet¡ng with ATP and that pyrophosphate in the presence ol ATp caused CFTR to 'lock' into a prolonged open burst state. These observations have been clarified recently by Hwang et al. (1994) who suggest that incremental phosphorylation may differentially regulate interactions between nucleotides and NBF's.
Their findings argue that ATP hydrolysis at one NBF controls channel opening and ATP hydrolysis at the other regulates channel closing.
1.6.5 LOCALISATION OF CFTR:
Since Riordan et at. (98g) first identified CFTR mRNA in a number of exocrine tissues implicated in the pathology of CF, many studies have validated the concept of CFTR being localised to the apical membrane of epithelial surfaces throughout the body. (Marino These include the gastrointestinal tract (Strong ef al., 1994), pancreas et al., (Cohn 1gg1), lung (although levels are low), kidney (Crawford et al., 1991), liver et al.,
1gg3), airway epithelium (Puchelle et al., 1992; Jacquot et al., 1993; Gaillard et al.,
1gg4), gall bladder and reproductive tissues (Tizzano et al., 1993). There has however been considerable debate about the presence of CFTR in non-epithelial cells and tissues. Lin and Gruenstein (1987) found defective cAMP stimulated chloride channels in CF fibroblasts and concluded that the CF defect was expressed in this cell type.
in Likewise Bear (1ggg) found cAMp and phosphorylation activatable chloride channels
normal fibroblasts. Other investigators however, were unable to find any difference in chloride conductances between CF and control fibroblasts (Vasseur et al'' 1992;
Squassoni eta;.,1990; Mastrocola and Rugolo, 1990; Dho and Foskett, 1993). Another
cell type scrutinised for the presence of CFTR was lymphocytes. ln this case the 22 evidence looked clearer with a number ol reports confirming the existence of cAMP- dependent chloride channels in normal lymphocytes, but absent in CF lymphocytes
(Chen et a1.,1989; Krauss et al., 1992a; McDonald et al., 1992). However the presence
of CFTR in lymphocytes could be cell-cycle dependent (Bubien et al., 1990; Krauss el
at., i992b) which may explain why others were unable to demonstrate CAMP stimulated
chloride transport (Hagiwara et a\.,1989). These studies on fibroblasts and lymphocytes
involved searching for evidence of lunctional CFTR-like chloride channels. Yoshimura ef
a/. (1991) approached the problem differently and were able to demonstrate CFTR
mRNA in lymphocytes and fibroblasts by quantitative PCR, at levels higher than is
explicable by 'illegitimate' transcription. They hypothesised that low levels of
transcription (even at less than one copy of mRNAJcell) could still be physiologically
relevant.
1.6.6 MOLECULAR BASIS FOR CFTR DYSFUNCTION:
Over 400 mutations in the CF gene have been described (Dean and Santis, 1994) and
the consequences of these on CFTR biosynthesis and function are myriad. They
depend on the type of mutation and its physical location within the protein. Welsh and
Smith (1ggg) have proposed a classification scheme which categorises CF mutations
into four classes.
Class I mutations result in defective protein production due to nonsense, frameshift, or
splice mutations. These would be expected to produce unstable mRNA and no
detectable protein. 23
Class ll mutations are due to defective protein processing and are those in which the cellular quality control mechanisms detect and degrade CFTR protein before it is traflicked to the plasma membrane. Usually this will occur as a result of incorrect folding upon translocation of newly synthesised polypeptide into the lumen of the endoplasmic reticulum (ER). The most common CF mutation ÅF,0,, belongs to this class. A number ol studies have demonstrated biosynthetic arrest of AF'.TCFTR in the ER, incomplete glycosylation and a failure to traflic to the plasma membrane (Cheng et al., 1990;
Dalemans et al.,1gg2; Denning et a\.,1992b). lnterestingly, this mutat¡on is temperature sensitive. When the temperature was dropped from 37"C to less than 30'C, ^Fs.BCFTR (Denning was able to escape the biosynthetic block, and traffic to the plasma membrane
(993) when et al.,1g92a). This property was utilised by Lukacs et al. to show that even
was fully glycosylated and present at the plasma membrane, where it retains ^F..'CFTR (Dalemans 1991), it some chloride channel activity in response to elevated cAMP et al., has a greatly reduced half life (< 4 hours compared to > 24 hours for wild type CFTR).
This finding dampened expectations that it might be possible to remove or circumvent the biosynthetic arrest of ÂF..,CFTR, thereby ameliorating pathology' Ì
These result Mutations which affect the NBF's constitute most of the class lll category.
and trafficked in defective regulation of CFTR, which has been synthesised by the cell
properly, or normally to the plasma membrane. These mutants may not bind ATP some of these alternatively binding may be unaffected, but hydrolysis compromised'
wild type. (Welsh mutants have little or no activity whilst others are reduced compared to
and smith, 1993). CFTR is also regulated by phosphorylation, however the mulliple 24 redundancy which appears to be built into the R domain may explain the relative paucity of mutations which appear in this parl of the protein.
Class lV mutants manifest defective conductance as a result ol alterations to the
membrane spanning regions which constitute the pore. Sheppard et al. (1993)
expressed three such CFTR mutants in heterologous cells (R117H, R334W, R347P) and
characterised their regulation and channel kinetics. All three were processed correctly
and generated chloride currents upon cAMP stimulation. Those currents were
macroscopically normal, however the amount of current was reduced. Not surprisingly,
these mutations were correlated with mild CF disease and pancreatic sufficiency.
1.6.7 GENOTYPE/PHENOTYPECORRELATION:
The extensive clinical heterogeneity exhibited in CF has inspired attempts to correlate
genetic information with clinical phenotype. Variables such as severity of pulmonary
disease, pancreatic status, age of diagnosis, sweat chloride concentration, age at which
colonisation with certain microorganisms occurs, nasal polyps, meconium ileus, diabetes,
rectal prolapse, cirrhosis and gall bladder disease, have all been examined for
associations with common mutations. The comparisons have been somewhat hampered
by the high number of rare mutations which are often lound in combination with ÀF*'.
Nonetheless, attempted correlations have been almost uniformly unsuccessful, with the
only clinical correlate being pancreatic function status. PS or Pl are strongly correlated
with certain mutant alleles (Kristidis et a|.,1992). ÂFro, homozygosity strongly correlated
with Pl as did ÀF*, when paired with a number ol other'severe' CF alleles. PS however
could be conlerred by inheriting one or two 'mild' CF alleles. Thus 'mild' alleles appears
to dominate over 'severe'. Early reports suggested that patients with PS had better pulmonary function than those with Pl (Gaskin et al., 198Ð but this does not appear to have been borne out. A recent survey of 399 patients of defined genotype (all compound heterozygotes with AFrr) were compared with age and sex matched AF'' homozygotes. There was no correlation between pancreatic status and lung disease or its progression (The Cystic Fibrosis Genotype-Phenotype Consortium, 1993)' There was wide variability of lung function irrespective of genotype. These findings strongly implied that there are environmental or other genetic factors influencing the severity of lung disease in CF. Kiesewetter et al. (1993) have shown that the phenotypic expression of one CF allele (R117H) varies depending on the length of the polypyrimidine tract in an
¡ntron splice acceptor site. Hence the genetic context that a mutation exists within, can
group modify phenotypic expression. This may be well illustrated by the example of a of infertile men with a condition known as congenital bilateral absence of the vas deferens
(CBAVD). Whilst normal in all other respects this condition has been shown to be associated with mutations in the CFTR gene (Bienvenu et a/., 1993). lt has thus been argued by some that CBAVD represents a particularly mild form of CF *ìtf' onty genital involvement, whilst others who have failed to find two CF alleles in some CBAVD patients argue that ¡t is a separate clinical entity to cF but one which paradoxically
the involves the gene for CFTR (Osborne et a1.,1993; Culard et al., 1994). On top of genetic context it is undoubtedly true that environmental factors such as exposure to pathogens, pollution, and passive smoke inhalation, also contribute to the onset and progression of pulmonary symptoms in CF (Rosenstein, 1994; Sm¡h et al',1994)' 26
1.7 OTHER FUNCT IONS OF CFTR
How does a mutation in the CF gene result in the gross pathophysiological consequences observed? Most would agree the pathology is a direct result of the abnormal mucus secretions which lead to plugging of glands, tissue atrophy and the infection processes which characterise the respiratory tract. Despite the passage of more than five years since the discovery of the gene and intensive research elforts by many groups, the link between a defective chloride channel and the production of abnormal mucus, which in some way appears to facilitate infection, eludes us. The following sections attempt to review what we know about CFTR in terms of its secondary effects
1.7.1 STIMULUS/RESPONSE COUPLING:
A link between chloride transport and mucin secretion was established by Lloyd Mills et
at. (992) who showed that B-adrenergic stimulation ol mucus secretion could be inhibited by an antibody to CFTR. Huflejt et at. (1994) showed that forskolin stimulation of T84, cells resulted in stimulation of exocytosis of l"S]-labelled glycosaminoglycans, conlirming that secretory processes were linked with the CAMP second messenger cascade (see section 1,7.3). Rogers et at. (1993) showed that baseline secretion of mucus from bronchial segments of both CF and non-CF subjects were similar but that secretion rates in CF explants exposed to p-adrenergic agonists were impaired. The authors postulated a defect in autonomic control ol bronchial secretion in CF upon receptor mediated stimulation. Studies performed in CFTR expression systems have
.A chloride secreting colonic epithelial cell line known to express high levels of CFTR' also been informative and seem to confirm the link between CFTR and mucin secretion.
Kuver et at. (gg4) transduced CFTR into dog gall bladder epithelial cells and showed that a live lold increase in CFTR expression was correlated with a four fold increase in mucin secretion. Engelhardt et al. (994) expressed CFTR in the skin of Xenopus laevis and demonstrated intimate coupling between cAMP mediated chloride secretion and mucous cell degranulation in this experimental system. ln contrast to these observations it appears that the car.-dependent pathway for mucus secretion remains intact in cF nasal and tracheobronchial tissue (Lethem et al., 1993). The mechanistic basis for
CFTR's influence on secretion is still unknown but it has been hypothesised that CFTR may reside on the membranes of secretory granules and contribute to exocytic events from this location (McPherson and Dormer, 1994). The theories of Verdugo et al'
(19g7a, b) are of interest in this context. They have hypothesised that high density mucus packaging within secretory vesicles is achieved by Ca" shielding of intragranular charge. Upon fusion with the membrane there is massive and explosive post-exocytic swelling of mucins as the Ca" charge shielding is removed. This has been termed a
'Jack in the Box' effect. One of their postulates for a means of removing the charge shielding involves an anion channel present in the granule membrane which becomes activated upon membrane fusion events'
1.7.2 INTRAVESICULAR PH:
The maintenance of compartmentally discrete acidic microenvironments is a critical
pH an metabolic requirement for all cells. Precise regulation ol intraorganellar is
Vacuolar ¡mportant feature of the biosynthetic, degradative and endocytic pathways'
action of organelles are able to generate a more acidic internal environment by the
vacuolar.ATPases(V.ATPases)whichtranslocatepfotonSacrossthevacuole 28 membrane, utilising the energy of ATP hydrolysis in doing so. V-ATPases are present in large numbers in the organelles ol eukaryotic cells and can be found in synaptic vesicles, chromaffin granules, clathrin coated vesicles, the Golgi complex, lysosomes and secretory granules (Nelson, 1991). They function as proton-specific electrogenic pumps and do not counter-transport other cations. An important corollary of their electrogeniciÇ is the requirement to dissipate the membrane potential which accumulates from lumenal proton concentration. This is achieved primarily by chloride ion channels which provide a counter-ion conductance (Cuppoletti ef a1.,1987; Al-Awqati et a1.,1992;Zen et al',1992;
Myers and Forgac, 1993).
The functional role that differentially acidified compartments play in normal cells is best
(see described for two diametrically opposed processes i.e. endocytosis and secretion
Fig. 1.4).
Endocytosr's-Upon receptor mediated uptake from extracellular sources, ligands are internalised in clathrin coated vesicles. These vesicles rapidly uncoat and fuse with early endosomes; the slightly acidified interiors of which, cause ligand-receptor uncoupling
(Mellman, 1992). The divorced receptor is then free to recycle back to the plasma membrane. yamashiro et a/. (1984) demonstrated that'sorting' of internalised ligands depended on differently acidified compartments. Transferrin, destined for recycling, trafficked to mildly acidified vesicles (pH 6.4), whilst cr.-macroglobulin, destined for degradation, was routed to more acidic vesicles (pH 5.a). Macromolecules to be
degraded, ultimately reached their intracellular destination in the highly acidic lysosomal
companment þH 4.5-5.0), which contains high concentrations of acid hydrolases. 16ã|rì Extracellular Fluid
@t x @ I / X t ,/, iiütâii:i:::: \
i::+g :1: c- ::.\:l::: ::::: \ ,.,: Ill::ìi ATP ::: ::::::::'':::' 2H \ ADP + Pi \ \ \ x x I ¡El a ,ë a $,r \ I
i:¡ i]:
::::: ::::
@ @
@ aÐ r@ @ GD
FIGUR E 1.4 The Acidified Vacuolar Network: RecepÌor-ligand complexes are internalised removed from the coated via clathrin-coated P its and vesicles. Alter clathrin has been (compartment vesicles, they fuse and mature into an acidic comparlmenl called CURL lor plasma uncoupling of recePtor and lig and). From here many receptors are recycled to the membrane in recYcling vesicl es. Ligands most often are moved to the lysosome for where degradation. Clathrin coated vesicles are also formed at the trans-Golgi network fiGN) they carry lysosomal enzYmes bou nd to the mannose-6-phosphate receptor. After uncoatlng, the Golgi and VESIC es luse with CURL, mannose-6-P hosphate receptors shuttle back to (1993)) lysoso mal enzymes are de ivered to the lysosome (Adapted from Myers and Forgac Symbo ls: receptorf , tigand ,clathrin x ,V-ATPase o ,chloridechannel O, lysosomal enzyme ?, mannose- O-phosphate receptor J, PM-plasma membrane, N-nucleus Secretion-lertaining to the secretory pathway, a well characterised example of the role ol pH in controlling vesicular transport and molecular recycling is the mannose-6-
phosphate receptor mechanism for trafficking of lysosomal enzymes to the lysosome
(Griffiths and Simons, 1986). ln the absence of an added mannose-6-phosphate
residue, proteins are secreted from the cell via the default pathway. The addition of
mannose-6-phosphate to newly synthesised glycoprotein in the ER rescues it from the secretory pathway and presages its ultimate destination - the lysosome. The mannose-
$-phosphate receptor which is responsible for binding and shepherding the enzyme, recycles between the Golgi apparatus and endosomes. Mechanistically it achieves this by discharging its ligand at the much lower pH typically found in late endosomes, whilst receptors which return to the plasma membrane dissociate at the higher pH found in mildly acidic vesicles (Mellman, 1992). pH asymmetry thus provides valuable intracellular signposting for complex molecular trafficking pathways
Chloride channels play an integral role in the generation and maintenance of pH gradients in different membrane structures (Wada et al., 1992; Van Dyke and Belcher,
1994) including the trans-Golgi network (Brightman et al., 1992). Second messengers appear able to regulate the internal pH of some vesicles by directly controlling chloride channel activity (Barasch et al., 1988; Zen et al., 1992). However not all membrane structures have the same requirement for chloride channels. Rat liver lysosomes have a lower pH than endosomes but a reduced rather than increased permeability to chloride. lnstead they are more permeable to potassium and phosphate ions (Van Dyke, 1993).
Some acidified organelles have no requirement at all for chloride ion conductance e.g. phagosomes from murine macrophages (Lukacs et al., 1991). 31
The possibility that CFTR was not solely localised to the plasma membrane, but might be
present and functional within intracellular membrane structures, lirst received
experimental support from Barasch ef a/. (1991). They showed that a CF ainrvay
epithelial cell line when compared with a normal cell line was defective in acidification of
its trans-Golgi network, endosomes and prelysosomes. Lysosomes appeared to be
unaflected; a result later supported by Van Dyke et al. (99Ð. These observations lent
themselves to the intriguing idea that the CFTR chloride channel might normally be
present in subcellular vesicles. Thus CFTR dysfunction might compromise normal
problems acidification mechanisms and potentially cause biosynthetic and trafficking or
pathologies. lndeed, Barasch et at. $991) were able to demonstrate post{ranslational
processing abnormalities. These included decreased sialylation of proteins and lipids by
protein the CF cell line. They speculated that abnormal intravesicular pH might influence
glycosylation and sulphation (by virlue of glycosyl- and sulphotransferase enzymes no
longer operating at their pH optimum), the rheological properlies of secretions, bacterial
pathogen binding to secretions and inappropriate lysosomal enzyme secretion' Bae and
Verkman (Bae and Verkman, 1990) have shown the existence of a PKA-activatable
chloride conductance in endocytic vesicles from rabbit proximal tubule. Also, direct
evidence has now been obtained that functional CFTR is present in clathrin-coated
(Lukacs Biwersi and vesicles (Bradbury et al., 1994) and in endosomes et al., 1992;
(1992) that CFTR Verkman, 1994; Webster et a1.,1994). However, Lukacs et al. clalm
does not influence endosomal pH, and suggest that factors other than counter-ion
permeability are the major determinants. Another study also disputed the acidification 32 hypothesis. Using CFPAC-1 and CFTR corrected CFPAC-1 cells, Dunn et al. (1994) found that CFTR had no elfect on the pH regulation ol endosomes.
More evidence is obviously required to implicate CFTR in a physiological role within intracellular organelles, and to show that its absence/dyslunction in CF leads to defectivd acidification. A role for CFTR in regulating intracellular pH still remains however, an enticing hypothesis to explain the plethora of observations in CF which don't appear explicable by a simple defect in plasma membrane chloride conductance.
1.7.3 ENDOCYTIC TRAFFICKING:
Bradbury and colleagues have postulated that CFTR plays a role in the endocytic removal and exocytic insertion of membrane (and membrane components) in epithelial cells. lnitial work from their laboratory showed that endocytosis was inhibited by stimulation of the cAMP second messenger cascade in T84 cells. As the number of chloride channels in the plasma membrane represents a dynamic equilibrium mediated by continuous exocytic insertion and endocytic retrieval, they postulated that a cAMP mediated decrease in endocytosis may enhance plasma membrane chloride permeability
(Bradbury et a1.,1992b). The potential role of CFTR in this process was explored using
CFPAC-1 and CFPAC-1 cells retrovirally transfected wilh the gene for CFTR. A number 2 of clonal transfected cell lines exhibited forskolin stimulated inhibition of horse radish peroxidase (a fluid phase marker) endocytosis, but there was no effect on CFPAC-1 or mock-transfected cells. Likewise, the CFTR-corrected cell lines could be shown to have elevated rates of exocytosis upon forskolin stimulation, an effect not observed with
CFPAC-1 (see Fig. 1.5). Thus both arms of the membrane recycling machinery
(endoc¡ic retrieval and exocytic insertion) appeared to be affected in CF cells and were CFTR e
- {-/
ct- ct- HzO HzO
FIGURE 1.5 Regulation of Membrane Recycling by CFTR' Wh"" tnggered by cAMP, CFTR allows Cl- and water to leave the cell, but also causes endocytic vesicles in the cell to fuse with the plasma membrane (Copied from Barinaga (1ee2)). 34 corrected by the presence ol wild type oFTR (Bradbury et al., 1992a). Prince et al.
(1994) demonstrated that CFTR exists within a rapidly recycling pool of endosomes at the plasma membrane. They also found that endocytic retrieval of CFTR was inhibited under conditions of cAMP stimulation. Further support for this hypothesis came from the finding that CFTR was present in clathrin coated vesicles (Bradbury et al., 1994) and from the observations of Fuller et at. (1994) that cAMP-stimulated (but not Caà- stimulated) chloride secretion in T84 cells, is dependent on an intact microtubule network. This was consistent with, but does not prove, that exocytic insertion of chloride channels may be a consequence of second messenger stimulation. Two lurther studies were unable to f¡nd any effect of CFTR on membrane recycling (Dunn et al., 1994;
Santos and Reenstra, 1994). As with so many of the phenomena observed in CF, this hypothesis will require further study before its validity is established.
1.8 G LYCOCONJUGATE BIOCH EM ISTRY
1.8.1 MUCUS AND MUCIN:
Mucins are the predominant molecular component of mucus and confer upon it many of the special properties which allow it to perform protective and lubricative functions on exposed epithelial surfaces. They are extremely high molecular weight glycoconjugates with 60-80% of their mass due to carbohydrate. The carbohydrate exists as hundreds of oligosaccharide side chains (possessing from one to twenty sugar residues) O-linked to serine or threonine residues within the polypept¡de core of the mucin. The carbohydrate side chains contain N-acetylglucosamine (glcNAc), N-acetylgalactosamine (galNAc), 35 galactose, sialic acid and fucose. ln addition the galactose and glcNAc residues may be
(Rose, Gerken and sulphated (for reviews of mucin structure and function see 1992;
Gupta, lggg)). Mucins exhibit extensive heterogeneity both between and within particular tissue sources. This makes structural analysis extremely diflicult. Humans with normal respiratory systems secrete only small quantities of mucus. Hence, most ol
people the work on human mucin chemistry has been on material obtained from suflering hypersecretory pathological conditions such as chronic bronchitis and asthma. Caution
processes may have needs to be taken in the interpretat¡on of such data as inflammatory altered the physico-chemical properties and even the chemistry of these complex
problems molecules (Rose et al., 1987; Kim, 1991). Not only are there in obtaining
of terminology in the unmodified normal mucin to work with, but there is some confusion
literature, with different investigators applying differing standards of stringency in
Mucus a assigning the label 'mucin' to purified fractions from whole mucus' is
glycoproteins, glycolipids, viscoelastic gel containing, in addition to mucin; salts, proteins,
glycosaminoglycans and proteoglycans (Rose, 1992). ln the inflamed airways, DNA can
and be a major component due to infihration and subsequent breakdown of neutrophils
other leukocytes.
1.8.2 GLYCOSAMINOGLYCANS (GAGS):
1.8.2.1 Structure and function of GAGs:
proteoglycans are a ubiquitous class of structurally diverse glycoproteins which can be
which can be found in virtually all tissues. They are characterised by a protein core to
chains attached as few as one, or as many as hundreds, of unbranched carbohydrate
their role in known as GAGs (see Fig. 1.6). Proteoglycans are most widely known for extracellular matrix (ECM) and particularly tissues such as cartilage where they are present in very high concentrations. Although proteoglycans have been identilied in invertebrates such as the sea urchin and Drosophila, and are present throughout the body, the only structural feature they have in common is the presence ol GAGs. The protein core may be anywhere from 10 to 400 000 kDa in size. lt is generally accepted that proteoglycans derive their unique properties from GAGs and it is the nature of these polysaccharides which confers some exceptional properties on them e.g. the high resistivity to compression found in cartilage (Maroudas , 1972;1975; 1976). Biophysically this resistance results from osmotic pressure generated by localisation of the proteoglycans within the cartilage. This imbibes the tissue with water and micro-ions and resists the expulsion of water out of the tissue upon compression (Comper and Laurent,
1 978)
GAGs are linear, polyanionic, carbohydrate chains usually found attached to proteins through serine residues. Except for keratan sulphate and hyaluronan there is invariably a tetrasaccharide linkage region with the sequence, serine-xylose-galactose-galactose- glucuronic acid. GAGs are composed of two monosaccharides strung together in alternating fashion and hence can be considered chains of repeating disaccharide motifs. The chains are of variable length depending on the tissue in which they are synthesised. There are five classes of GAG which derive from three basic disaccharide units. The disaccharide unit in all cases has a hexuronic acid residue in conjunction with an N-acetyl hexosamine. Heparan sulphate (HS) and hyaluronan are both synthesised from glucuronic acid and glcNAc although the glycosidic linkages ol the two chains are 37
cH2oo- coo- cuú|
coo- heparan sulphale
oo- HNAc ldoA?S G/c/VSdS GlcNAc
coo- c+l2otl -GlcA 9o coo- sulphate
H},IAC HNAe ldoA2S Galî,lAc4S GlcA GalNAc4S
cH2oH coo- cH2oH - coo-
HNAc Ffl$AC GleA- GalNAc4S GlcA Ga¡tlAc4S
- coo-
Ftl.!Ac Gail\lAc65 GlcA
coo- cF.l2oH
HNAc HNAc GlaA GfcNAc GlcA GleNAc
curo@- cn.o@-- cnro@- QHzOF{
ratan sulphate
HNAc HNAÈ
Gal6S G/ciV,4c6S Gal G/c/VAc6S
-
FIGURE 1.6 Structures of the glycosaminoglycans heparan sulphate, dermatan sulphate, chondroitin sulphate, hyaluronan and keratan sulphate. (Copied from Freeman ('1991)). 38
diflerent'. Chondroitin and dermatan sulphate (CS and DS) both derive from a parent
disaccharide consisting of glucuronic acid and galNAC whilst keratan sulphate is composed ol galactose and glcNAc'. Despite the simplícity of the basic structure, each
ol these species (with the exception of hyaluronan) is capable of being subjected to
lurther chemical modification, which generates a high degree of heterogeneity and hence
f unctional versatility.
HS can be modified in five different ways. The glucuronate can be epimerised to
iduronate, N-deacetylation and N-sulphation can occur on the glucosamine, and further
sulphate esters may be added atC2 of iduronate and C3 and/or C6 of glucosamine. The
addition ol up to three sulphate esters per disaccharide results in a molecule which is
highly negatively charged. ln fact the bulk of sulphate acquired by a cell is utilised in the
synthesis of GAGs (Humphries et a1.,1988)
The distinction between CS and DS arises as a consequence of the epimerisation of
glucuronic to iduronic acid. Chains with this modification are called dermatan sulphate whereas those with no iduronic acid are called chondroitin sulphate. Both are able to be
sulphated at C2 of the uronic residue and C4 or C6 of the amino sugar. Disaccharides
may be disulphated in various combinations or even trisulphated. There is no N-
sulphation in these GAGs and as a result they have less heterogeneiÇ than HS.
.Heparan sulphate has the repeating structure lD-glucuronic acid p(1+4) D-N-acetylglucosamine u(1--+4)1" and hyaluronan the structure [D-glucuronic acid Þ(1-+3) D-N-acetylglucosamine þ(1+4)J" olD-glucuronic acid p(1 -+3) D-N-acetylgalactosamine p(1 -+4)1" '[D-galactose Þ(1 -+4) D-N-acetylglucosamine þ('l -+3)J" 39
glucosamine and/or Keratan sulphate may be modified only by sulphation at C6 of the
others and c6 of galactose. This GAG does not have the broad tissue distribution of the
the cornea it appears to be a specialised polymer found only in avascular tissues such as
for and cartilage (scott, 1gg4). lt has been hypothesised to be a functional substitute cs where oxygen is in shoft suPPlY.
As a result of these modifications and the extensive heterogeneity that ensues, there is
The study considerable potential lor coding of biological information in these molecules.
merely of GAGs in recent years, has revealed that a group, which was once considered as scaffolding in the ECM, can interact specifically with a variety of effector molecules
two of the and in doing so have a role to play in numerous cellular processes. Perhaps most well known examples are the anticoagulant activity of heparin resulting lrom
fibroblast growth specific interactions with antithrombin lll, and the binding of HS to basic
ln the factor; a prerequisite for subsequent binding of the growth factor to its receptor.
which former case, a sulphated pentasaccharide of unique structure was identified
(Lindahl ln the contained a 3-O-sulphate group on one of the disaccharides et a1.,1984)'
HS containing latter, Turnbull and colleagues identified a 7-disaccharide fragment from
groups as its dominant structural feature a high number of ldoA(2-OSO.)a1,4GlcNSO.
(Turnbulletal.,1992).ThiswasararesequenceinHsandtheywereabletoshowthat
growth factor binding' both the 2-O-sulphate and N-sulphate groups were necessary for
proteoglycans are also known to play many other roles in cellular physiology, including
playing role in cellto cell regulating the morphology, proliferation and migration of cells; a
of the adhesion and matrix assembly, as well as sequestration and indeed, augmentation
efTects is activity of growth factors. At this stage the molecular basis for many of these largely undefined, but lurther research in the field is sure to illuminate complex structure- function relationships in which sulphated carbohydrates mediate important developmental processes. More comprehensive reviews of proteoglycan structure and function may be found in (Roden, 1980) and (Fransson, 1987))
1.8.2.2 Biosynthesis of GAGs:
The biosynthesis of GAG chains within the Golgi occurs by the initial transfer of xylose from UDP-xylose to serine residues in the already synthesised core protein. Formation of a protein/polysaccharide linkage region then occurs by the sequential addition of two galactose and one glucuronic acid residue from the respective UDP-sugar. Synthesis of the GAG chain proper occurs by alternating addition of uronic acid and hexosamine residues. Concomitant with chain elongation there is movement of the nascent proteoglycan through the Golgi. Sulphation of chondroitin chains at C4 and C6 of galNAc residues appears to take place during chain polymerisation in the same medial or trans Golgi site (SilberI et a1.,1993). ln HS this series of highly coordinated modifications include N-deacetylation which is closely linked to N-sulphation of glcNAc units. lt now appears that one enzyme is responsible for both of these activities (Pettersson et al.,
1991 ; Zheng et al., 1993). Further modifications to the chain include epimerisation at C5 of glucuronic acid to iduronic acid and O-sulphation at C2 of glucosamine units. lt has been proposed that unlike CS, sulphation of HS chains is predominantly a post- polymerisation event (Gallagher et al., 1992). Polymer formation and modification are rapid processes and involve up to sixteen different enzymatic reactions (Uhlin-Hansen and Yanagishita, 1993). GAG synthesis therefore, results from the concerted action of a highly organised membrane associated multi-enzyme system. There remains however no detailed information on how the different enzymes are organised, in which 41 subcompartments of the ER/Golgi complex they reside and how they recognise core proteins in order to initiate appropriate GAG synthesis. one study however has lound
protein that the enzymes responsible for synthesising HS chains are specific for HS core and did not initiate GAG lormation on DS core protein indicating a high level of recognition and speciliciry (Uhlin-Hansen and Yanagishita, 1993).
1.9 SULP TE BI HEMIS Y
Sulphate is required by cells for a diverse array of sulphoconjugation reactions. lt is
proteins, necessary for the normal biosynthesis and post{ranslational modification of glycoproteins, mucins, GAGs and proteoglycans, with the latter two providing the major substrate for sulphate in most cells (Humphries et al., 1988). lt is an impodant constituent of the detoxification pathway by which the liver and other tissues,
sulphate can sulphoconjugate xenobiotics priorto excretion (Schwaz, 1982). ln addition
(Mulder lt be found conjugated to steroids, biogenic amines and bile acids etal-,1982).
proteins has appears to play many different roles. Sulphation of tyrosine residues within
(Pittman been shown to be necessary for the normal catalytic activity of some enzymes
(Hinkle al-, 1992)' Sulphation of et al., 1992) and for normal secretion of others et
properties mucins and proteoglycans is essential for their myriad physical and functional
postulated as a (see sections 1.8.1 and 1.8.2). For some molecules sulphation has been
amines; mechanism to ¡nit¡ally inactivate and then facilitate excretion e.g. the biogenic
(Wong, However' sulphation adrenaline, noradrenaline, dopamine and serotonin 1982)'
properties may also allow sequestration of these compounds. The physical and chemical 42
of dopamine are known to dilfer considerably from its sulphated counterpart. The
sulphate conjugated form is more resistant to degradation and appears able to cross
membranes more easily. lt may therefore represent a transport and storage form of free
dopamine (Buu ef al., 1982). Sulphates of steroids have also been seen as 'storage'
forms. There is some experimental evidence that they may also be intermediates in the
conversion of steroids and that tissues may be able to sulphate and de-sulphate (through
the action of sulphatases) steroids as they are required (Mulder, 1982). Brooks et a/.
(1982) have shown that oestrogen sulphotransferase plays a key role in the regulation of
the oestrous cycle. Sulphation of bile salts changes their physicochemical properties
making them more polar. ln healthy individuals bile salt sulphation appears to occur to a
minor extent, but in clinical conditions with cholestasis, renal excretion of bile salt
sulphates is markedly increased (Loof, 1982). This may be a protective mechanism, as
some bile salts are toxic.
The predominant source of intracellular sulphate for conjugation reactions has been, and remains, the subject of some uncertainty. ln the 1940's and 1950's two schools of thought developed as some believed that inorganic sulphate obtained from extracellular sources constituted the majority of sulphate accessed by a cell, whilst others felt that catabolism of the sulphur containing amino acids (cysteine and methionine) provided most of the cells metabolic requirement (Curtis, 1982). lt is likely that the answer depends on which tissue is being examined, as well as the relative availability of sulphate and cysteine from different sources. Sulphate liberated by the lysosomal degradation ol GAGs may also participate in fuñher sulphation reactions although at least one study found that most of the sulphate released in enzyme-supplemented 43
Maroteaux-Lamy libroblasts (a genetic disease in which the enzyme, N-
storage of acetylgalactosamine-4-sulphatase is delicient, causing subsequent lysosomal
(Harper DS) d¡d not appear to be reutilised by the cells, but was lost to the medium et al.,
1gg3). A number of studies have tested the abili$ of different cell types in culture to
utilise cysteine as a source of sulphate for GAGs and proteoglycans. Chinese hamster
ovary cells appear not to requrre exogenous sulphate in order to synthesise lully (Esko sulphated GAGs when given l,uSlcysteine as a source of sulphur et a1.,1986) and
(Templeton this ability is shared by cultured rat glomeruli, mesangial cells and Wang,
the 1992), and bovine aortic smooth muscle cells (Humphries et al',1988)' lnterestingly'
for addition of sulphate to smooth muscle cells did not inhibit the utilisation of cysteine
sulphation, indicating a preferred pathway. ln contrast to these results cartilage, lung (lmai fibroblasts (Humphrie s et al., 1988) and rat ovarian granulosa cells et al., 1994)
showed limited ability to utilise cysteine (as judged by the production of undersulphated
(Humphries GAGs) whilst endothelial cells were completely unable to use this substrate
high et a1.,1988). ln an intriguing'whole body'experiment, it was shown that a single
A second dose of phenol given to rats, resulted in rapid depletion of sulphate in serum. as the first dose of phenol given one hour later was sulphated to the same extent
stores to allow indicating that sulphate had been rapidly mobilised from unknown tissue
normal metabolic clearance of the xenobiotic (schwaz, 1982).
of cells There are a number of different sulphate transporters in the plasma membrane
These which localise to different tissues and have different functional characteristics.
include a SOo"/Cl- antiporter found in many tissues including lung fibroblasts,
hepatocytes, erythrocytes and tracheal epithelium; a SOo"/HCO." antiporter in 44
hepatocytes; a SO4¿/SO¿¿ exchange system in erythrocytes and hepatocytes and a SOoa
/Na' symporter in hepatocytes, ileal ep¡thelium and kidney cortex (Schwaz, 1982; Lucke
et al., 1981; Lucke et al., 1979; Elgavish and Meezan, 1989). The first two have a
number of features in common with the well characterised erythrocyte band 3 anion
exchanger. This is a major integral membrane protein of red blood cells which facilitates
the movements of various ions into and out of the cell. Some of the features it shares
with non-erythroid anion exchangers are; extracellular chloride is a competitive inhibitor
of sulphate uptake and stimulates sulphate efflux, anion exchange is inhibitable by 4,4'-
diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), and low extracellular pH stimulates
sulphate uptake (Elgavish et a1.,1988). The carrier also displays a high affinity for HCO;
Anion exchange catalysed by band 3 consists of a tightly coupled, electrically silent,
one for one exchange. Although the kinetics of exchange are complex it appears that
exchange occurs by a 'ping pong' mechanism in which anions take turns traversing the
membrane. An intracellular anion such as chloride binds to an inward facing
configuration of the protein, is transported to the exterior by conformational alteration in
the protein, is released and then an extracellular anion such as sulphate may bind and
be transported to the interior (Jennings, 1989). Whilst most cells appear to have the
ability to access sulphate from the extracellular milieu by means of specific plasma
membrane transporters, the late ol sulphate once released into the cytoplasm is unclear
Mohapatra et al. (1993) have shown that inorganic sulphate in respiratory epithelial cells
resides in tvvo discrete compartments. They were unable to define how sulphate was compartmentalised, but showed at normal external chloride concentrations, only 107o of the intracellular sulphate was readily exchangeable with the external medium. The remaining 90% was sequestered in some way and only exchanged slowly with newly taken up sulphate. This pool may represent a storage mechanism, or contain sulphate
predestined for biosynthesis, and therefore in some way inaccessible to the plasma
membrane anion transporters.
Sulphate in the cytoplasm becomes a substrate for the enzyme ATP sulphurylase which
converts ATP to adenosine-5'-phosphosulphate (APS) by the following reaction,
SO+2 + ATP APS + PPi
ATp sulphurylase is unusual in that the reaction equilibrium lies far to the left. This is
due to ApS being a potent product inhibitor of the enzyme. Formation of the universal
(PAPS), activated sulphate donor, S'-phospoadenosine-5'-phosphosulphate is achieved
by conversion of ApS to pApS by APS kinase (Schmidt et al., 1982). The overall
production of pApS in vivois therefore promoted by the hydrolysis of pyrophosphate and
the favourable ApS kinase reaction. PAPS which is the product of this two step
known activation sequence serves as the sulphate donor for the biosynthesis of all
macromolecular sulphate esters (schmidt et a1.,1982). ln order to become available for
into lumen of the biosynthesis, pAps must then be translocated from the cytoplasm the
(Mandon Once in Golgi apparatus by the action of a specific transporter et al-, 1994).
which the Golgi interior, PAPS becomes a substrate for numerous sulphotransferases
transfer to accomplish the post-translational modifications. lt appears that sulphate
glycoprotein and proteoglycans is not restricted to one part of the Golgì apparatus'
in Uhlin-Hansen and yanagishita (Uhlin-Hansen and Yanagishita, 1993) demonstrated
occurred in rat ovarian granulosa cells that sulphation and synthesis of heparan sulphate 46
the ER/proximal part ol the Golgi, whilst dermatan sulphate was completely synthesised
in the trans-Golgi network. Thus, sulphotransferases and presumably PAPS transporters
may be found throughout the Golgi.
1.10 GLYCOCON JUGATE SYNTHETIC DE FECTS IN CF
The literature on CF priorto 1983 (and the elucidation of the biochemical abnormality),
presented a confusing picture to researchers. There were a welter of observations which
investigators pursued as they attempted to define causes for the underlying
pathophysiology of the disease (Matalon and Dorfman, 1968; Bowman et al., 1973).
Many of these related to the nature of the mucus produced by CF patients. This
secretion was thick, tenacious and purulent. What made it so different from normal
exocrine exudates? W¡th the finding that CFTR was a chloride channel, some believed
that the peculiar propert¡es of CF secretions could be explained purely in terms of water
content and relative dehydration. However, many studies found evidence of chemical
differences in the carbohydrate moieties of high molecular weight mucus glycoproteinso.
1.10.1 ALTERED GLYCOSYLATION IN CF:
Glycoprotein abnormalities are a feature of CF glycoconjugates. The abnormality is
manifest in the extent to which glycoconjugates are post{ranslationally modified by the
addition of fucose, sialic acid and sulphate. The latter will be discussed in more detail in
the following sect¡on. Fucosylation has been shown to be increased in the membrane
uFor a review ol earlier work in this area, see Boat ef a/. (1989). 47
(Clamp glycoproteins of CF fibroblasts (Wang et al., 1990) as well as meconium and
Gough, 1g7g) and intestinal mucus from CF pat¡ents (Wesley et al., 1983; Thiru et al.,
(Boat l ggo), but does not seem to be increased in mucus from the cF respiratory tract et
a/., l ggg). CF salivary mucins were shown to have increased fucose and sialic acid
compared with normal controls (Carnoy et a1.,1993). lnterestingly, most studies of CF
glycoconjugates have observed decreases in sialic acid content. Possibly there are
(1991) tissue specific abnormalities resulting from CFTR dysfunction. Barasch ef a/.
lound that an immortalised CF airway epithelial cell line displayed a generalised under-
(Saiman sialylation of its glycoproteins and glycolipids. Saiman and Prince and Prince,
lggg) demonstrated lower amounts of asialo-GM1 glycolipid on the surface of primary (1994) CF epithelial cells compared with normals and Dosanjh et al. showed that
expression of AFsosCFTR in heterologous cells resulted in the expression of
undersialylated membrane associated glycoconjugates. Untransfected and CFTR
transfected mouse mammary C127 epithelial cells in this study had unaltered levels of
sialylation. These recent studies are of great interest because they utilise cells in culture
that rather than recovered mucus or tissue explants. Because of this it can be assumed
infection or the observed alterations to glycosylation are not secondary consequences of
sialic acid inflammatory processes. lt has been hypothesised that fucose, sulphate and
due to the compete for sites on mucins during post-translational processing and that
at perturbation present in cF, there is a preponderance of fucose and sulphate addition
(994) however the expense of sialic acid (Al-Awqati et al., 1992). Lo-Guidice et al.
(of triple found lour ollgosaccharides isolated from CF mucus twenty four) were
chain, which substituted with fucose, sialic acid and sulphate on a single carbohydrate
may cast some doubt on the hypothesis' 1.10.2 ALTERED SULPHATION IN CF:
There have been reports that secretions lrom CF respíratory epithelia contain higher amounts of sulphate than is seen in normal secretions. Early work noted that CF sputum contained greater amounts of acidic glycoprotein components than sputum from people with chronic bronchitis (Lamblin et a1.,1977). One study showed a significant correlation
(p<0.002) between the amount of highly sulphated mucin and the severity of the disease in seventeen CF patients (Chace et al., 1983) thus indicating that sulphated glycoconjugates might be pathogenic determinants in CF. A later study from the same group demonstrated two fold higher sulphate content in mucins from CF patients (Chace et al., 1989). Tissue explants of CF trachae, bronchi and nasal polyp tissue slices exhibited three to six times higher secretion rates, greater quantities of acidic glycoprotein and higher sulphation in the acidic components than the equivalent tissues from normal subjects (Frates et al., 1983). The glycopeptides from CF saliva have also been shown to possess more sulphate than normal (Carnoy et a1.,1993)
All of these studies had the limitation of utilising patient samples which left them open to the criticism that they were potentially observations of secondary effects. Sputum from infected lungs may have been modified; ainruays tissue as a response to infection may synthesise a different type of mucin; fresh tissue explants could be in a state of inflammatory arousal. Strong evidence that greater sulphation was an intrinsic property of CF, came with the finding that primary CF airway epithelial cells in culture, over- sulphated newly synthesised high molecular weight glycoconjugates. Those secreted into the culture medium were two fold more highly sulphated, and those glycoconjugates associated with the cell surface, three to four fold higher. The conclusions were based 49 on [.uS]sulphate incorporation normalised against ['H]glucosamine and ['H]serine incorporation. Disease control cells (lrom patients with allergic rhinitis) were not diflerent lrom the normal controls (Cheng et a1.,1989). Ol particular interest was the finding that a testicular hyaluronidase susceptible fraction in the culture medium, was three and a half times more highly sulphated than that produced by normals. This implicated GAGs as substrates for greater sulphate addition by CF cells'
1.10.3 POTENTIAL ROLE OF SULPHATE IN CF:
The involvement of glycoconjugate sulphate esters in the adhesion mechanics of pseudomonas is not well defined. However, Chace et al. (983) showed a significant correlation between the content of highly sulphated mucin in CF secretions and the severity of pulmonary disease in CF. This observation implicated sulphated carbohydrate in some unknown way to palhology in CF. Further circumstantial, rather
of than direct evidence that sulphated glycoconjugates may be important in the aetiology infection in CF are provided by the following observations:
1) lncreased sulphation of mucous glycoconjugates enhances the binding abiliV of influenza virus (Boal et a1.,1976);
Z) The host-defensive aggregation ability of salivary mucus toward oral bacteria resides
in the acidic sulpho-mucin fraction (Piotrowski et a1.,1994);
Helicobacter 3) Certain bacteria have specific adhesins for sulphated glycoconjugates'
pytori(the organism responsible for gastroduodenal ulcer disease) and Staphylococcus
(Liang al',1992; Ascencio ef aureus have both been shown to specifically bind to HS et
to bind a/., 1993). The respiratory pathogen, Bordetetlaperfussishas also been shown
to HS present on cell surface glycolipid (Hannah et a1.,1994); 50
q The basement membrane of pulmonary epithelium exhibits a number ol
morphologically distinct domains which have been shown to be differentially sulphated
(Khosla et al., 1994). The ability ol Pseudomonas to adhere to basement membrane
components might have ramifications in CF il epithelialcells lay down an altered ECM;
5) Highly sulphated mucins have been shown to have functions other than simply
lubricative and protective. Slomiany et a/. (1993) have shown that salivary sulpho-mucin can regulate the activity of a calcium channel in buccal epithelial cells. lt is possible therefore that sulphated macromolecules may have regulatory roles on other epithelial surfaces;
6) lt has been postulated that highly sulphated glycoproteins found in the alimentary canal may have enhanced resistance to degradation by bacteria. Thus, observations of decreased sulphation of mucins in the colon and rectum have been implicated as the cause of ulcerative colitis and Crohn's disease (Raouf et al., 1992; Morita et al., 1993).
Likewise, defects in proteoglycan sulphation and secretion have been postulated to play a fundamental role in the pathogenesis of autosomal dominant polycystic kidney disease
(Carone e¡ a/., 1993);
7) lmportantly, the presence of sulphated GAGs has been associated with, and hypothesised to contribute to, hypersecretory disease conditions of the human respiratory tract. These include CF, chronic bronchitis and bronchial hypersecretion in acute quadriplegia (Bhaskar et al., 1991 ; Rahmoune et al., 1991). ln the latter condition it has been noted that approximately 20o/o ol patients develop unexplained production of tenacious bronchial mucus which is similar in quantity and physical characteristics to that seen from CF patients. The glycoprotein composition of the secretion was shown to abnormal and in one patient who subsequently died, there was evidence for the 5 1 presence of CS and hyaluronan (Bhaskar et al., 1991). An investigation into the
(CB) presence of CS in sputum from sulferers of CF and chronic bronchitis revealed that eleven ol thirteen patients with CF and one non-infected sputum sample from a patient with CB had the GAG present. CS was not seen in sputa from patients with CB who were not infected, nor was it identified in two CF samples from mildly infected donors.
They conclude that in all cases but one, the presence of CS appeared to be related to
the severity of bronchial infection (Rahmoun e et a\.,1 991 );
B) The normal metabolism of sulphate (see section 1.9) involves complex enzyme
pathway systems and regulatory control. Perturbation to the sulphate utilisation may be
a secondary consequence of some other primary defect, as seems to occur in
galactosemia (where the enzyme galactose-1-phosphate uridyltransferase is delicient).
reduced by l.uS]sulphate incorporation into glycoconjugates by dþeaSe fibroblasts is
65%. The pathophysiological consequences ol this for patients is unknown as most of
the pathology is assumed to be the result of cellular toxicity resulting from galactose-1-
phosphate accumulation (Tedesco and Miller, 1979). Galactosemia may be somewhat
analogous to CF in exhibiting secondary effects on sulphate metabolism. Diastrophic
results in dysplasia on the other hand is due to the loss of a sulphate transporter and
mutations in under-sulphation of proteoglycans (Hastbacka et al., 1994). Patients with
the gene suffer from spinal deformation and dwarfism. Primary defects in sulphate 'natural' metabolism may therefore be severe and it is possible that lew examples of
defects in this pathway are known because of a high lethality. 52
1.11 POTENTIAL PATHOPHYSIOLOGICAL CONSEQ UENCES OF
G LYCOCO N J U-GATE DE FECTS
1.11.1 PULMONARY INFECTION IN CYSTIC FIBROSIS:
The tracheobronchial tree of the human respiratory tract normally maintains a sterile
surface despite continuous challenge from inhaled microorganisms. The initial line of
host defence is ain¡rays mucus which entraps bacteria and is then rapidly translocated
upwards by ciliary beating to the throat where it is swallowed (Lamblin and Roussel,
1993). Even when there is colonisation by pathogenic bacteria, host immune defences
usually suffice to eradicate the invading organism. The CF lung, however, is in some
critical way different. For reasons which are still to be defined, the respiratory tract of
people with CF are prone to chronic colonisation, predominantly by Staphylococcus
aureus and Pseudomonas aeruginosa. At b¡rth, the lungs of babies born with CF are ostensibly normal, but almost inevitably there is progression to respiratory infection with
S. aureus, which is then often supplanted by long term colonisation with P. aeruginosa.
Even in older patients with mild lung disease who are clinically well, pathogenic microorganisms can be isolated with indications that they are sutfering from ongoing inflammation and concomitant tissue damage (Konstan et al., 1994). These pathogens appear competent to employ a myriad array of immuno-evasive strategies. ln the case of
P. aeruginosa, one of the major persistence mechanisms is conversion from non-mucoid to mucoid form. This conversion has been shown to correlate strongly with lung lunction deterioration and a worsened prognosis for the patient. The molecular basis for this conversion has been defined recently as being due to inactivation of a gene (muc A) which locks the organism into constitutive oversecretion of the exopolysaccharide alginate (also known as biofilm). This is a favourable adaptive mechanism lor the pathogen which has increased anti-phagocytic properties as a result (Martin et a\.,1993). ln addition to this transition, the organism is able to modulate the hosts immune response in a multifactorial way which employs pili, membrane proteins, protease and exotoxin release among others, to enhance its own persistence, and indeed the damage the host sustains in attempting to fight the infection (for a comprehensive review see
(Buret and Cripps, 1993)). lnterestingly, the systemic immunity of CF patients 1o the organism has been shown to remain intact, with the site of colonisation restricted to the lung. Also worth reiterating is that the non-CF respiratory tract (unless immuno- compromised) has little difficulty in dealing with and eradicating Pseudomonas. Clearly there are factors residing within the environmental milieu of the CF lung which prove conducive to colonisation, establishment of infection and ultimately the life-shortening consequences which accrue.
1.11.2 WHY THE CF LUNG?
Establishment of infection for the microorganism is predicated on first contact. What happens at this critical juncture? For the pathogen, adhesion is of paramount importance and is generally regarded as a prelude to colonisation and infection'
Adhesion is accomplished at the molecular level through the interactions of bacterial
There is surface proteins with either the mucus layer or with the epithelial cell surface' some debate about which is the more imporlant contact surface but it appears that
(Ramphal, lt mucus interactions are extremely impodant and may be paramount 1990)'
is clear from many studies that bacterial pathogens have a number of lectin-like
'adhesins' with different properlies and affinities, and that there may be a number ol 54
'docking sites'which can be utilised?. Tsang et al. (1994) investigating the binding ol P
aeruginosa with adenoid tissue in organ cullure showed that this species only
infrequently adhered to normal epithelium but adhered to areas of epithelial damage and
to basement membrane. There were also preferential associations with epithelial cil¡a
and mucus at four hours, but not earlier. Colonisation was shown to actually cause
tissue damage. ln a study of nine strains ol StaphylococcLrs, Thomas et al. (1993)
demonstrated that adhesion to mucin was accomplished via two distinct mechanisms
One of these utilised bacterial) Ca'?.-binding surface proteins and was enhanced
significantly by elevated Ca2. (it is worth noting that the Ca'. concentration of CF mucus is
higher than that of normal mucus (Potter et al., 1967)) whilst the second employed
different proteins and was unaffected by Ca'.. S. aureus strains isolated from CF
respiratory tracts were shown to bind to normal ep¡thelial cells far more avidly than
isolates from non-CF ainrrays. ln some way the CF airway either selected certain strains
or induced an adherent phenotype by virtue of CF specific factors within the lung
(Schwab et al., 1993). Reddy (Reddy, 1992) showed that P. aeruginosa strains
interacted directly with highly purified tracheobronchial mucins through a 16 kDa non-
pilus surface protein whilst others have shown that pili are also important in attachment to epithelial cells (Ramphal et a1.,1984; Doig ef a/., 1988). Haemophilus influenzae and
Streptococcus pneumoniae (pathogens also implicated in CF lung disease, albeit to a lesser extent) also adhere to mucus (Ramphal, 1990).
'A comprehensive review of the literature is not intended in this section, but selected recent studies have been highlighted to convey current thinking in the field. The question that needs to be answered with respect to CF, is whether these mechanisms are important in the predisposition to infection. What is the host context which allows and even facilitates the establishment ol lung infection. lt has been argued that colonisation in the cF respiratory tract is a consequence of dehydrated secretions
and that poor mucociliary clearance allows establishment of chronic airways inlection
(Gerken and Gupta, 1gg3). There are studies however which point to differences ol a
molecular rather than rheological nature which enhance binding of bacterial pathogens.
Saiman et at. (992) found that twice as many P. aeruginosa bound to primary CF airway
epithelial cells as to normal cells and this was due to a greater number of receptors on
the CF cell surface. From binding inhibition studies using different monosaccharides
they concluded that the receptors on the two cell types were qualitatively different'
pseudomonas supernatant containing protease and neuraminidase activity enhanced
binding to both cell types and appeared to expose asialoganglioside binding sites.
Devaraj et at. (1gg4) showed greater binding interactions between P. aeruginosa and cF
tracheobronchial mucins as opposed to normal mucins. Reports suggesting that
pseudomonas preferentially bound asialo-GM1 residues, prompted Saiman and Prince
(Saiman and prince, 1993) to quantify the numbers of these receptors on respiratory
epithelial cells. They found significantly greater numbers present on CF cells'
(a non- lnterestingly, Carnoy et at. (g93) showed that three strains of P- aeruginosa
mucoid, a non-piliated and a piliated variant) all bound more highly to CF salivary
glycopeptides, but demonstrated decreased binding to cF glycopeptides upon treatment
with neuraminidase suggesting that sialylated residues were important to binding' All ol these data paint a seemingly confused píøure of multiple substrata suitable lor
binding (i.e. basement membrane, epithelial cell surface, mucín), multiple adhesion
molecules and elfectors and apparently conllicting data on the resídues required for
binding interactions to occur. What clearly emerges however are two facts: 1) there are chemical alterations not only to CF mucus but to molecules associated with the epithelial cell surface which can act as receptors for bacteria, 2) the differences (which require greater clarification) enhance the binding ol Pseudomonas and Staphytococcus bacteria.
It is possible, that the post-translational modifications to CF secretions, and the greater affinity of CF mucus for certain microorganisms have a cause and effect relationship. 57
1.12 RATIONALE THIS STUDY
The major goal of this PhD project was to investigate one aspect of the pathophysiology of cystic fibrosis in the hope that it might aid in the understanding and treatment ol the disease. The second goal was to shed light on the over-sulphation phenomenon reported for the glycoprotein secretions of cystic fibrosis patients. The relationship between over-sulphation and the well established primary gene defect in CF has not been thoroughly investigated. Further, the potential of gene-supplementation to influence sulphate metabolism is highly relevant at this time. This study attempted to address some of the gaps in the 'sulphation story' by;
1. focusing on highly sulphated model substrates,
2. investigating sulphation of those model substrates by cystic
fibrosis tissues in vivo, and
3. performing gene-correction experiments on cF cells and
obseruing the subsequent effects on sulphation and sulphate
metabolism.
Accumulated evidence is suggestive of the reality of altered glycoconjugate sulphation in
CF, but not conclusive. lt is of concern that those reports which do exist in the literature, are disparate and lacking in follow-up studies. This may reflect the investigation of a phenomenon which manifests only in select tissues or under particular circumstances.
For example, there may be cell cycle dependence or it may affect only certain molecules.
ln addition, variables which have yet to be considered could influence its expression. 58
Nevertheless, the paucity of studies confirming a link between CF and sulphate related
abnormalities in glycoconjugate synthesis was a faclor in the decision to pursue this
question.
lf over-sulphation of glycoconjugates does occur in response to the loss of CFTR
chloride channels it raises several important issues. Firstly, how does it affect
pathophysiology and what organs are involved. lt is st¡ll not known for example why the
CF lung becomes infected. ln the face of challenge by similar pathogens to those seen
in CF lung disease, the normal lung maintains a sterile surface. Explanations which
invoke mucus stas¡s, appear intuitively unsatisfactory. Other hypersecretory illnesses
such as chronic bronchitis do not manifest the highly destructive infection cycle seen in
CF. Even the lungs of long term smokers subjected to continuous chemical insult for
many years and which gradually lose the ability to clear mucus normally, do not succumb
to infections which the body is unable to eradicate. Despite our progress in
understanding CF on many other fronts it would appear that unknown factors still
determine some of the clinical outcomes. Altered sulphate metabolism might be one of
those determinants.
Secondly, if sulphation plays a role in the pathophysiology of CF it has ramifications for therapy. Particularly those approaches which envisage the application of gene therapy.
lf CFTR is implicated in secondary metabolic consequences, then expression levels and implicitly, cellular localisation of the protein may need to be controlled more rigorously than is currently belng attempted. Finally, there is the question: why should carbohydrates be the recipient of higher amounts ol sulphate because of dysfunction in a seemingly unrelated chloride channel? cystic fibrosis represents an 'experiment of nature'. we can observe the consequences of that experiment and in doing so, gain further insights into normal epithelial cell biology.
Part of our raison d'etre as scientists is to try to understand the workings of the natural world. lt was hoped that the study of this phenomenon might yield insights into new molecular communication pathways, with potentially unforseen benefits to our understanding of normal as well as disease processes'
1.13 AIMS
1. To use GAGs as a model acceptor substrate for the demonstration of sulphation
abnormalities by cystic fibrosis cells in tissue culture'
2. To examine the composition and sulphation of GAGs by a range of cystic fibrosis
affected organs in vivo.
3. To elucidate a biochemical mechanism for abnormal sulphate metabolism in cystic
fibrosis and to gain an understanding of the linkage between CFTR and sulphate
utilisation in normal ep¡thelial cells
63
2.1 MATERIALS/REAGENTS
2.1.1 CELL LINES USED IN THESE STUDIES:
NAME/CELL TYPE SOURCE CODE CULTURE MEDIUM
PANC-1/ ATCCT cRL 1469 lscoves Modified Dulbecco's Adenocarcínoma Medium/10% (v/v) FBS3 (IMDM)
CFPAC-1/ ATCC cRL 1918 IMDM/10o/o FBS Adenocarcinoma
184t ATCC CCL2ß 1 :1 Ham's F12:Dulbecco's Modified Adenocarcinoma Eagle's Medium/1 0olo FBS
TR2O/CFTR Lysosomal Diseases IMDM/1 0% FBS/0.4m9/ml G41 I transfected CFPAC Research UnitÞ
PLJ 4.7/CFTR Professor R.Frizzelll I MDM/1 0%FBS/O.75m9/ml G41 8 transfected CFPAC
PLJ 6/CFTR Professor R.Frizzell IMDM/1 0o/oFBS/O.75m9/ml G41 8 transfected CFPAC
Lymphoblast Coriell lnstituter GMO3714 RPMI 1640/1Oo/oFBs
Lymphoblast Coriell lnstitute GM03299 RPMI 1640/1O%FBS
Lymphoblast Coriell lnstitute GMO4330 RPMI 1640/1O%FBS
Lymphoblast Coriell lnstitute GMO7227A RPMI 1640/1Oo/oFBS
Lymphoblast Coriell lnstitute GMO7904 RPMI 1640/1Oo/oFBS
hoblast Coriell lnstitute GMO4540A RPMI 1640/1Oo/oFBS
1 American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852. s Foetal bovine serum. References to FBS assume percentage concentration as (v/v). " Dept. Chemical Pathology, women's and children's Hospital, Adelaide. ' Dept. Physiology and Biophysics, University of Alabama at Birmingham. ' NIGMS Human Genetic Mutant Cell Repository, Coriell lnstitute for Medical Research,401 Haddon Ave, Camden, NJ 08103' 64
2.1.2 RADIOCHEMICALS:
Hydrogen ["Cl']chloride NEN Research Products (Wilmington, (13-15 mOi/g) U.S.A)
D-[6-'Hlg lucosamine hyd rochloride Amersham (Buckinghamshire, England) (37 Ci/mmol)
D-[6-'H]glucosamine hyd rochloride NEN Research Products (30-40 Ci/mmol)
L-["S]cysteine NEN Research Products (1075 Ci/mmol)
Sodium ["SJsulphate NEN Research Products ê550 mCi/mmol)
2.1.3 ENZYMES/ANTIBODIES:
Anti-Cytokeratin (A combination of 2 Boehringer Mannheim (Mannheim, monoclonal antibodies: AE1 /AE3) Germany)
Chondroitinase ABC (Proteus vulgaris) ICN Biomedicals (Costa Mesa, U.S.A.)
EcoRV Restriction Endonuclease Boehringer Mannheim
FITC-Conjugated Sheep Silenus (Hawthorn, Australia) Anti-Mouse lgG
Monoclonal Anti-Human CFTR Genzyme Corp. (Cambridge, U.S.A.) (C-terminus specific)
Stre ptomyces Hyaluronidase Sigma Chemical Co. (St Louis, U.S.A.) 65
2.1.4 CHROMATOGRAPHICMEDIA:
BioGel P2 Bio-Rad Laboratories (Richmond, U.S.A.)
DEAE-Sephacel Pharmacia LKB Biotechnology (Uppsala, Sweden)
Propac PA1 Strong Anion Exchange Dionex (Surrey, U.K.) Columns Ø x250 and 4 x 50 mm)
Sephadex G10 Pharmacia LKB Biotechnology
Sephadex G50 Pharmacia LKB Biotechnology
Sepharose CL-48 Sigma Chemical Co.
2.1.5 CELL CULTURE MATERIALS:
Cryotubes Nunc (Roskilde, Denmark)
Disposable 12 ml plugged pipettes costar (cambridge, u.s.A.)
Foetal bovine serum Gibco BRL (Glen Waverley, Australia)
L-glutamine Gibco BRL
Penicillin/streptomyci n antibiotic Gibco BRL solution (5000 U/ml)
Powdered culture medium Gibco BRL
Sterile phosphate buffered saline Gibco BRL
(Toongabbie, Sterile pyrogen free water Baxter Healthcare Australia)
Trypsin/versene Gibco BRL
All remaining plasticware Corning (Corning, U.S.A.) 2.1.6 MISCELLANEOUSMATERIALS:
Ampicillin Sigma Chemical Co.
Bacto-agar Dífco (Detroit, U.S.A.)
Bacto-tryptone Difco
Bacto-yeast Difco
Chondroitin sulphate A Sigma Chemical Co.
Chondroitin sulphate ICN Biomedicals disaccharide standards
Eight-well chamber slides Nunc
ELISA trays (96 well, vinyl) Costar
Eppendorf tubes Treff Lab (Switzerland)
Filters (0.2pm,45 mm diameter) Millipore
Laboratory sealing film Whatman lnternational Ltd. (Maidstone, England)
Minisart 0.2¡rm syringe filters Sartorius (Chadstone, Australia)
Optiphase Hisafe scintillation fluid Fison Chemicals (Homebush, Australia)
QuickSealtubes Beckman (Palo Alto, U.S.A.) 2.1.7 CHEMICALS:
Alt chemicals were of Analar or HPLC grade and were supplied by the following compantes.
Acridine orange Sigma Chemical Co
Acrylamide solution (LiquiGel) G rad ipore (Pyrmont, Australia)
Adenosine triphosphate (ATP) Sigma Chemical Co.
Ammonium formate Ajax Chemicals
Ammonium persulphate Bio-Rad Laboratories
Ammonium phosphate Sigma Chemical Co.
Carbonylcyanid e-m-chlorophenyl Sigma Chemical Co hydrazone (CCCP)
Cetylpyrid inium chloride (CPC) Sigma Chemical Co
1,4 Dioxan BDH Chemicals Ltd. (Poole, England)
Disodium hydrogen phosPhate BDH Chemicals Ltd
DMSO (d imethylsulphoxide) Ajax Chemicals
E DTA (ethylenediami netetraacetic Ajax Chemicals acid)
Ethanol Ajax Chemicals
Formaldehyde Ajax Chemicals
Forskolin Sigma Chemical Co.
Glucose Ajax Chemicals
Geneticin (G418) Gibco BRL
Glycine BDH Chemicals Ltd.
Hemi-calcium g luconate Sigma Chemical Co 68
Hemi-magnesium gluconate Sigma Chemical Co.
HEPES (N-2-hydroxyethylpiperazine- Sigma Chemical Co.
Hydrochloric acid Ajax Chemicals lonomycin Sigma Chemical Co
Lithium chloride BDH Chemicals Ltd
L-Mannitol Sigma Chemical Co
4-Methylumbelliferyl-2-acetamido-2- Koch-Light Ltd. (Suffolk, England) d eoxy-beta-D-g I ucopyranoside
4-Methlumbelliferyl-p-D-xyloside Sigma Chemical Co.
Methanol Ajax Chemicals
Nigericin Sigma Chemical Co.
Phosphate buffered saline (PBS) Sigma Chemical Co. tablets
Phosphoric acid Ajax Chemicals
Potassium chloride Ajax Chemicals
Potassium gluconate Sigma Chemical Co
SDS (sodium dodecyl sulphate) BDH Chemicals Ltd.
Sodium acetate Ajax Chemicals
Sodium azide Sigma Chemical Co.
Sodium bicarbonate Ajax Chemicals
Sodium borohydride Sigma Chemical Co
Sodium carbonate Ajax Chemicals
Sodium chloride Ajax Chemicals
Sodium citrate Ajax Chemicals
Sodium gluconate Sigma Chemical Co. Sodium hydroxide Ajax Chemicals
Sodium nitrite Ajax Chemicals
Stainsall (1 -ethyl-2-[3-(1 -ethylnaphthol Bio-Rad Laboratories
11,2 - dlthiazo I i n - 2 -yl i d e nd -2- methylpropenyllnaphthol[1,2-d]- thiazolium bromide
Sucrose Ajax Chemicals
Sulphuric acid Ajax Chemicals
TEMED (N,N, N', N'-tetramethyl Bio-Rad Laboratories ethylenediamine)
Tris (tris(hydroxymethyl)aminomethane Boehringer Mannheim Corp.
Triton X-100 Ajax Chemicals
Tween 20 BDH Chemicals Ltd
Valinomycin Sigma Chemical Co
2.2 METHODS
2.2.1 CELL CULTURE:
Cell culture was performed essentially as recommended by the cell repositories which provided cell lines. PANC-1 cells however, were cultured in IMDM instead of DMEM in order to carry out experiments in which metabolic activities could be compared directly with CFpAC-1 cells. The PLJ and TR20 cell lines were transduced with a genetic construct containing the CFTR gene and a selectable marker to impart neomycin resistance. ln order to keep selection pressure on the introduced genetic elements, cells 70 were grown in the presence of 0.75 mg/ml G418 continuously Q.q mg/ml lor TR20).
Seven days prior to radiolabelling of these cell lines, G418 was removed from the culture medium.
2.2.2 CRYOPRESERVATION OF CELL LINES:
Confluent 75 cm' flasks were emptied of medium and washed twice with 10 ml PBS
Trypsin-versene solution (1-2 ml) was added and flasks were incubated either at room temperature or at 37"C, for 5-15 minutes. When cells could be seen to have lifted from the surface of the plastic, clumps were broken up by gently suckìng up and down several times in a sterile, plugged, pasteur pipette. Cells were transferred to a 10 ml centrifuge tube containing 8 ml medium/107o FBS, centrifuged at 1500 rpm for 5 minutes and resuspended in 3 ml medium/SO7o FBS (per flask). To this solution, an equal volume of medium containing 30% (v/v) DMSO was added slowly with gentle mixing. Cell suspension was then aliquotted into Nunc cryotubes (1.5-1.8 ml) and the tubes slowly cooled by inserting inside polystyrene foam containers and placing in a -80"C lreezer
Approximalely 24 hours later cryotubes were transferred into liquid nitrogen for long term storage
2.2.3 REVIVAL OF CRYOPRESERVED CELLS:
Ampoules were removed from liquid nitrogen and immediately immersed and thawed in a
37'C waterbath. The contents ol the ampoule were diluted by slow dropwise addition of an equalvolume ol medium/sO% FBS. The cell suspension was then left undisturbed for
15 minutes before slow addition of another 6 ml of medium/sO% FBS. After a further 15 minutes undisturbed, the cells were centrifuged at 1500 rpm for 5 minutes and washed 71 twice with medium/10% FBS to remove DMSO. Cells were resuspended in 15 ml medium/10% FBS in a 75 cm' llask and incubated at 37"Cl5o/o CO".
2.2.4 IMMUNOFLUORESCENT STAINING OF CELLS:
Cells were grown to confluence in Nunc eight chamber slides. Medium was aspirated, the chambers washed quickly with PBS (2 x 0.5 ml) and then cells fixed in 2o/o formaldehyde/PBS for 10 minutes. Chambers were rinsed with methanol, followed by
PBS and then washed thoroughly 3x with PBS/10% FBS for 15 minutes. Primary antibody diluted 1:250 (unless othen¡vise stated) in PBS/100/o FBS at 100 plichamber was incubated with cells for 2 hours at room temperature before washing 3 x 5 minutes with
PBS/10% FBS (v/v). Detection of antibody binding was accomplished by incubation of chambers for t hour at room temperature with FITC conjugated anti-mouse lgG at 1:15 dilution in PBS/10% FBS, followed by washing as above and then application of a drop of 507o (v/v) glycerol/PBS and a cover slip. Slides were examined and photographed on a Leitz Diaplan fluorescence microscope at excitation/emission wavelengths of 450 nm and 490 nm. lncubation steps were carried out with slide covers on, and from the point where FITC conjugated secondary antibody was added, the slides were kept in the dark as much as possible. Control incubations containing no primary antibody were always performed to assess non-specific staining of cells.
2.2.5 MEASUREMENT OF CHLORIDE EFFLUX:
Confluent monolayers of cells in 35 mm diameter plastic wells were washed twice with bicarbonate-free DMEM/2O mM HEPES pH7.2 and then cells were incubated in 1 ml of the same medium containing 2.5 ¡rCi/ml f'Cll. Radiolabel came as H['uCllCl, therefore it was necessary to adjust the pH of the radiolabel containing medium fo7.2 before adding 72 to cells. Wells were incubated at 37'C lor at least two hours under ambient CO, and then washed six times with ice cold efflux buffer (300 mM L-mannitol, 1 mM KCl, 10 mM
HEPES; pH 7.Ð to remove extracellular radiolabel. Elflux at room temperature was started by the addition of 1 ml efflux buffer (containing either agonist or vehicle), and the wells were rocked gently on a platform rocker throughout the experiment. Unless othen¡rise stated the concentrations of agonist used were: forskolin, 13 pM and ionomycin, 5 pM. Concentrated stock solutions of each were prepared in DMSO and kept at -20oC. At time points, zero, one, two, three, five, seven and ten minutes, a 200
¡rl aliquot was removed from each well for scintillation counting, and immediately replaced with 200 pl of fresh buffer containing appropriate additions. After sampling of the aliquot at ten minutes, the remaining buffer was discarded the cell layer washed with ice-cold efflux buffer and 1 ml 0.2 M NaOH added to each well for ten minutes with rocking, to recover intracellular radiolabel. After recovery of cell lysate, the wells were further washed with 1 ml of water and the two samples pooled for scintillation counting
Results are expressed as the percentage of 36Cl-counts remaining intracellular at each of the time points measured
2.2.6 SUBCELLULARFRACTIONATION:
Cells from 3 x 175 cm2 confluent culture flasks were harvested by trypsinisation and then centrifuged at 1500 rpm in a bench top centrifuge. Supernatant was aspirated off and replaced by 9 ml 0.25 M sucrose (pH to 7.0 with Tris) and this procedure repeated twice more The cell pellet was resuspended in 6 ml sucrose buffer and then lysed in 6 x 1ml aliquots by drawing up into a 10 ml syringe, placing several layers of parafilm over the end of the syringe and then drawing back the plunger, creating a vacuum and releasing.
This was repeated 20 times and from this point forward samples were kept on ice as 7 much as possible. The cell 'lysate' was centriluged at 1900 rpm for 5 minutes and the supernatant removed for centrifugation at 17000 g(av) lor 15 minutes in a Beckman
L5758 Ultracentrifuge. The pellet or 'granular fraction' from this centrifugation step was resuspended in 0.5 ml sucrose buffer for immediate acidification assay. lf the granular fraction was destined for Percoll gradient lractionation, the pellet was taken up in 3 ml ol
0.2S M sucrose, 10 mM HEPES, pH 7.0 and layered onto a 36 ml solution ol 20o/o (vlv)
Percoll in the same buffer in Beckman Quickseal tubes. The tubes were centrifuged for
60 minutes at 30000 g(av) and then 1.5 ml fractions from the gradient were recovered by piercing the top and then the bottom of the tube with a 26G needle. The contents of the tube were collected as drops from the bottom, in eppendorf tubes.
2.2.7 p-HEXOSAMINIDASEASSAY:
o/o(vlv) Samples were made up to 100 pl by addition of 12.5p1 Triton X-100 and an appropriate volume of citrate-phosphate buffer (13 mM sodium citrate, 20 mM disodium hydrogen phosphate, pH 4.4). Substrate solution (200 pl citrate-phosphate buffer containing 1 .24 mM 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside) was added and the tubes incubated for 10 minutes in a 37"C waterbath before the reaction was stopped by addition of 1.5 ml glycine butfer (200 mM glycine, 125 mM sodium carbonate, 150 mM NaOH, pH 10.7). Tubes and solutions were kept on ice until the incubation commenced. Fluorescence was measured for all tubes, plus a blank and a known standard, on a Perkin Elmer LSS Luminescence Spectrometer at excitation and emission wavelengths of 366 and 446 nm respectively. 74
2.2.8 ACIDIFICATION ASSAY:
Organelles in 0.25 M sucrose/Tris/HCl or 0.25 M sucrose, 10 mM HEPES/HCI pH 7.2 were added to a 3 ml quartz cuvette containing 1 .95 ml of acidilicatíon medium (13 mM sucrose, 100 mM KCl, 500 ¡rM MgOlr, 1 ¡rM valinomycin, 2.5 ¡rM acridine orange, 12 mM
HEPES pH 7.0) made up from 0.22 ¡m filtered stock solutions. The cuvette was mounted in the cuvette holder of a Perkin Elmer LS50B fluorimeter connected to a personal computer. Fluorescence was monitored at excitation/ emission wavelengths of
492 and 540 nm using Perkin Elmer FL Data Manager sottware until a steady baseline was achieved. Acidification was initiated by the addition of freshly made ATP to 500 ¡rM and mixing. To demonstrate that decreasing fluorescence was due to acidification, the proton ionophores carbonylcyanide-m-chlorophenylhydrazone (CCCP) or nigericin were added to final concentrations of 45 ¡rM or 0.5 ¡rM respectively.
2.2.9 SCINTILLATION COUNTING:
Samples containing radioisotopes (usually less than 1 ml) were scintillation counted after addition of 4 ml of Optiphase Hisafe scintillation fluid, in either a Nuclear Chicago lsocap/3O0, or Wallac 1409 scintillation counter. Data from the Nuclear Chicago lsocap/30O is presented as cpm. Samples counted on the Wallac 1409 are presented as dpm and these were calculated based on quench curves for different isotopes programmed in by the manufacturer. All l'sS]sulphate data presented in this thesis has been corrected for decay.
2.2.10 METABOLIC RADIOLABELLING OF ADHERENT CELLS:
Cells were grown to the required degree of confluence (usually 100% unless otherwise stated) in 3.5 cm diameter plastic wells. ln one experiment 5 cm petri dishes were used 75 instead. Medium was aspirated and cell monolayers were washed with PBS and lhen 2
ml (3 ml lor 5 cm dishes) ol fresh medium supplemented with 40 ¡rOi/ml D-[6-'H(N)]-
glucosamine-HCl and 100 ¡rCi/ml Nar['uS]SO4 was added to each well. Cells were
incubated at 37"C in a 5o/o CO, atmosphere lor 24 hours, unless othenruise stated. At the
end of the radioisotope labelling period, medium was collected, the cells washed with 2
ml PBS and the two were pooled to become the 'medium fraction'. Cells and cell
associated material were harvested by addition oI 2 ml4M guanidine-HCl, 2o/o Trilon X-
100, 50 mM sodium acetate pH 6.0 (guanidine bufler). Atter 30 minutes at room
temperature with rocking, the contents of the wells were collected after scraping the
bottom with a cell scraper. These samples became 'cell layer'fractions.
2.2.11 METABOLIC RADIOLABELLING OF LYMPHOBLASTS:
Cell lines were obtained from the Coriell lnstitute for Medical Research and cell culture
was perlormed according to instructions supplied. Cells were pelleted by centrifugation
at 10OO rpm for 5 minutes in a bench top centriluge and viable cell numbers assessed by
trypan blue exclusion and counting in a Neubauer counting chamber (Weber). Dilution
factors were calculated to achieve a final cell suspension containing 0.5-0.75 x 10u
cells/ml culture medium. A radiolabel containing stock solution was made up which
contained l.rS]sulphate and l.H]glucosamine at 200 ¡rOi/ml and 80 pCi/ml respectively.
This was added at a ratio of 1:1 to a 2x cell suspension (also in culture medium) in order
to achieve the correct cell density and radiolabel concentrations of 40 ¡rOiiml
[.H]glucosamine and 100 pCi/ml l3sS]sulphate. This ensured that each cell suspension
received exposure to identical concentrations of radiolabel precursors. A standard
volume ol 2 ml/petri dish was used, unless otherwise staled. Cell densities of 0.5-0.75 x
10. cells were chosen to ensure that, lor at least part of the radiolabelling period, cells 76
were in a growth phase. From inlormation supplied by the cell repository, lymphoblasts
enter stationary phase at 1 00 cells/ml and have a doubling time of approximately 15
hours. Atter 18-24 hours incubation at 37"C in 5olo COr, (unless otherwise stated)
'medium' and 'cell-associated' material were recovered separately. Cells were centrifuged at 1000 rpm for 5 minutes and the medium decanted. Pelleted cells were washed with 2 ml PBS and centriluged again. The medium and PBS wash were pooled to become the 'medium' or'secreted'fraction. Cellular and cell-associated material e.g. extracellular matrix, were obtained by solubilising the pellet with guanidine buffer.
Samples were buffer exchanged and unincorporated radiolabel removed by chromatography on 9 ml Sephadex G50 columns (see section 2.2.14). A manipulation employed in one experiment was to expose cells to 1mM hydroxyurea lor 27 hours in order to synchronise them at the G1-S phase of the cell cycle prior to the start of labelling. ln order to maintain their cell-cycle synchronised state, hydroxyurea was present during labelling also.
2.2.12 ALKALI EXTRACTION OF GAGS:
Mouse organs that had been lyophilised subsequent to dissection and weighing, were minced thoroughly with scissors to increase surface area to volume ratio and then transferred to 20 ml glass scintillation counting vials. 6 ml of 0.5 M NaOH was added to each vial and the tissues incubated overnight at 4"C. All solutions were then neutralised with 600 ¡rl 5M HCI and 500 ¡rl 1 M Tris/HCl pH 8.0. Tissues were centrifuged at 5000 rpm for 15 minutes at4"C, and the GAG-containing supernatants recovered. Except for the small organs; gall bladder, nasal septum, nasal mucosa and trachea; all tissues were then extracted a second time in order to obtain quantitative recovery of GAGs. 2.2.13 GAG ASSAY:
GAGs were assayed by a modilication of the method of Homer et al. (1993) which is based on the spectral shift exhibited by the dye 'Stainsall' in the presence ol chondroitin, dermatan and heparan sulphate at 475 nm. Reagents were prepared as specilied in the original method, however the proportion of sample to other reagents for the 96 well microtitre tray assay were altered, allowing the assay to be performed in the more shallow Costar 96 well microtitre trays. The modifications did not result in any loss of sensitivity. Briefly; 130 pl dye reagent (0.1 mM Stainsall, 1 mM acetic acid,0.5 mM ascorbic acid, 507o (v/v) 1,4-dioxan) was added to 20 ¡rl of sample or standard in a microtitre tray. Water (50 pl) was then added and the contents of the wells mixed before measurement of absorbance al 475 nm in a Biotek lnstruments Ceres 900HDi ELISA reader. Standards were either bovine heparan sulphate or chondroitin sulphate A alone, or a 1:1 mixture of the two, when GAGs of unknown composition were assayed'
2.2.14 SEPHADEX G5O CHROMATOGRAPHY:
Rapid desalting, buffer exchange, preparation of samples for lyophilising and analysis of enzyme digestions throughout this project, were performed in 12 ml disposable plastic
Costar pipettes containing Sephadex G50. The pipettes had their tops removed
(containing cotton wool) and were plugged with wetted glass wool. Sephadex G50 slurry was poured in to the 1O ml mark thus providing a bed volume of approximately I ml when allowance was made lor the glass wool. Columns were equilibrated by washing with two column volumes of the appropriate buffer and then 1-2 ml samples applied and eluted.
For some applications 10-12 x 1 ml fractions were collected and for others a peak eluting in the void volume Uo) was collected based on the known elution characteristics of these columns. This column system was chosen as an analytical tool because of the
facility with which it allowed rapid throughput of large numbers of samples. Using a
specially designed rack it was possible to run ten columns simultaneously. The size of
the columns didn't allow complete separation of high molecular weight material which ran
in the void volume (for example in enzymatic GAG digests) from the included material. lt
was felt however that the sacrifice in our ability to gain absolutely quantitative information
on the proportions of undegraded versus degraded material was offset by our abiliÇ to
handle large numbers of samples. ln the early stages of this project a number of
experiments were performed e.g. on fibroblasts, which allowed us to screen cell culture
systems for CF related biosynthetic effects. Rapid column procedures allowed us to
focus more quickly on the experimental models of interest
2.2.15 ION.EXCHANGE CHROMATOGRAPHY:
DEAE-Sephacel chromatography of samples from metabolic labelling experiments, were
performed according to the method of Harper et al. (1987) Briefly; samples that had
been chromatographed on 9 ml Sephadex G50 columns to remove unincorporated
radiolabel and transfer macromolecules into dissociative buffer (8 M urea, 1%o Triton X-
100,0.15 M NaCl,50 mM sodium acetate/acetic acid pH 5.8); were applied to 2 ml
DEAE-Sephacel columns equilibrated in the same dissociative buffer. An unbound peak
was collected and then a 0.15-1.0 M NaCl gradient in dissociative buffer was applied
Fractions were collected, scintillation counted and appropriate peaks pooled for further
analysis. Mouse organ extracts which were predominantly GAG chains (due to B-
elimination reaction w¡th alkali - see section 2.2.12) rather than intact proteoglycan, were
anion exchanged differently in that the buffer system was simplified by removal of urea and Triton X-100. After determining that these changes did not alter the separation 79 properties of the anion exchange system, a batch procedure was devised in which the
DEAE-Sephacel and samples were equilibrated in 0.3 M NaCl, 50 mM sodium acetate/acetic acid, pH 5.8. Hyaluronan elutes under these conditions between 0.2 and
0.3 M NaCl. By utilising this altered buffer system, the only molecules to bind to the column were sulphated GAGs. A one-step elution with 2 x 5 ml aliquots of 1 M NaCl, 50 mM sodium acetate/acetic acid pH 5.8 gave quantitative recovery ol GAGs as evidenced by recovery of radiolabel. Purified GAGs were then lyophilised.
2.2.16 HYALURONIDASE DIGESTION:
Hyaluronan was digested according to the method of Harper et al. (1987). Briefly, 200 pl samples were dialysed into 50 mM sodium acetate pH 5.0 and then incubated with 5-10 units of Streptomyces hyaluronidase for 5 hours at 60"C. The products of digestion were analysed by collecting 1 ml fractions after chromatography on 9 ml Sephadex G50 columns in PBS.
2.2.17 NITROUS ACID CLEAVAGE OF HEPARAN SULPHATE:
Nitrous acid deamination of heparan sulphate al pH 1.5 was performed according to the method of Shively and Conrad (Shively and Conrad, 1976). Samples that had been buffer exchanged into 100 mM ammonium formate and lyophilised, were exposed to 5 ml nitrous acid pH 1.5 for 10 minutes at room temperature and then neutralised with 1 ml 2
M sodium carbonate. Products of the digestion were lyophilised and resuspended in 1 ml water. Chromatographic analysis was performed either on 9 ml Sephadex G50 columns or on a BioGel P2 column (dimensions 98 cm x 1.4 cm) equilibrated in 100 mM ammonium bicarbonate. Samples on BioGel P2 were eluted at 0.14 ml/min. and 70 x 2 ml lractions collected for scintillation counting. 80
Nitrous acid cleavage of heparan sulphate at pH 4 was performed by taking up
lyophilised samples in 500 pl water, adding 750 pl 28o/o (vlv) acetic acid and 750 pl 0.65
M sodium nitrite then incubating for 2 hours at room temperature. lf the pH of the
solution was not approximately 4 (assessed with pH indicator paper), it was modilied by
addition of aliquots of 3 M sulphuric acid ensuring that pH did not go below 4. After 2
hours, 1.1 ml 1M sodium carbonate was added.
2.2.18 CHONDROITINASE ABC DIGESTION:
Samples for enzyme digestion were buffer exchanged on 9 ml Sephadex G50 columns
¡nto 0.1 M ammonium formate, lyophilised and then dissolved in 200 pl 50 mM Tris-
acetate, pH 8.0. ChondroitinaseABC (0.1 unit') in 10 pl of the same bufferwere added,
and then incubated at 37"C for four hours, unless othenryise stated. Upon completion,
samples were either lrozen immediately or placed in a boiling water bath for 60 seconds.
Analysis of the products of digestion was performed on 9 ml Sephadex G50 columns
Sample volumes were made to 1ml with ammonium formate prior to chromatography and
12 x 1 ml fractions collected lor scintillation counting. Usually, fractions 3-5 containing
high molecular weight, undigested material and fractions 7-11 containing disaccharides
were pooled and lyophilised (Edwards Modulyo)
2.2.19 DESALTING OF CS/DS DISACCHARIDES:
Lyophilised disaccharides were resuspended in 250 pl 10% (v/v) ethanol and then
chromatographed on a 36 cm x 1.4 cm Sephadex G10 desalting column equilibrated in the same. Sixty 1 ml fractions were collected at a flow rate ol 0.2 ml/min and aliquots of
' Quantity of enzyme which catalyses the lormation of 1 ¡rmole of unsaturated disaccharide from chondroitin 6-sulphate per minute at 37"C, pH 8.0. 81 fractions 11-30 were routinely scintillation counted and fractions containing radioactivity
pooled and lyophilised.
2.2.20 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) OF
CS/DS DISACCHARIDES:
Priorto HPLC, disaccharides (10 pl in water) were reduced by exposure to 10 pl 100 mM
sodium borohydride in 200 mM sodium bicarbonate pH 9.0 overnight at room
temperature. Solutions were subsequently neutralised by addition of 10 pl 100mM acetic
acid. Disaccharide separation utilised a Dionex Propac PA1 strong anion exchange
column (4 x 250 mm) and guard column for the experiment involving mouse samples
(Chapter 5) and a smaller column (4 x 50 mm) was used for all other data presented.
Reduced disaccharides were loaded and eluted at 1 ml/min with a 0-750 mM NaCliHCl
(pH 3.5) gradient applied over 45 minutes on a Pharmacia LKB FPLC system (LKB LCC
2252, LKB 2150 HPLC pump, LKB 2210 2 channel recorder, Dionex Eluant Degas
Module). Fractions (0.5 or 1 ml) were collected, and detection of disaccharides was
accomplished by measurement of UV absorbance at 226 nm (LKB Uvicord Fixed
Wavelength 2158) or by scintillation counting. Buffers were filtered and degassed before
use and sparged daily with helium. The column was equilibrated in 0 mM NaCl/HCl
(pH3.5) for 10 minutes between runs. The elution position of disaccharides was
confirmed by running CS standards both on their own and spiked into sample. The
elution times were generally consistent to within + 0.1 minutes.
Chapter 5 describes the analysis of CS/DS disaccharides extracted from murine organs.
Typically samples contained a number ol unidentified contaminants which eluted early.
Each tissue exhibited characteristic diflerences in the absorbance profile near the I injection peak. As the mice had been injected with ['uS]sulphate it was impossible to quantitate the amount of zero sulphated disaccharide in samples by scintillation counting.
Therefore, quantitation ol individual disaccharide species was accomplished by analysing
peak areas from UV absorbanc e Q26 nm) chart recordings. Peak areas were calculated
by triangulation.
2.2.21 GENE TRANSFECTION OF CELL LINES:
CFpAC cells were transfected with plasmid CMV-CFTR936C which was kindly provided
by Dr Brandon Wainwright (Centre for Molecular Biology and Biotechnology, University of
(Genzyme The eueensland) with permission from Dr Richard Gregory Corporation).
plasmid had 4.5 kb of the human oFTR gene (1 22-4620 from the Aval site to the sacl
promoter. site in human CFTR) cloned into a 5.4 kb plasmid under the cytomegalovirus
genes. It also contained selectable markers in ampicillin and neomycin resistance
Plasmid was prepared in the following way. 100 ng of plasmid DNA provided by Dr
(cells Wainwright was added to 100 ¡rl of DHS E. cotithat had been made competent
were prepared by Maria Fuller, Dept. of Chemical Pathology, Women's and Children's
shocked at Hospital, Adelaide). The bacteria were incubated on ice for 35 minutes, heat
(LB) medium 42"C lor g0 seconds and then placed back on ice. 250 ¡rl of Luwia-Bertani
(w/v)) and (bacto-tryptone 1Yo (w/v), bacto-yeast extract 0.5% (w/v), NaCl 17o was added
plates the tube incubated at 37.C for 45 minutes before bacteria were plated on LB agar
plate was (1.5% (w/v) bacto-agar in LB medium) conta¡ning 0.01% (w/v) ampicillin. The
picked off and incubated overnight at 37"C and the following morning individual colonies
medium' grown up separately lor 16 hours at 37"C with aeration in 10 ml LB/ampicillin Mini-plasmid preparations to assess transformed colonies were performed as follows.
Bacterial culture (0.5 ml) was transferred to an eppendorf tube, the tube microfuged at
12000 rpm lor 1 minute and the supernatant removed. 100 ¡rl of LiCl buffer (2.5 M LiCl,
50 mM fris,4o/o (v/v) Triton X-100, 62.5 mM EDTA, pH 8.0) was added to the pellet and the bacteria resuspended and lysed by extensive vortexing. Water-saturated phenol- chlorolorm (100 ¡rl) was added and the eppendorf vortexed lor 20 seconds then microfuged for 5 minutes. The aqueous phase (90 pl) was transferred to a new eppendorf, 60 ¡rl isopropanol added, the tube vortexed and then microfuged for 5 minutes. The supernatant was aspirated and the DNA pellet washed with 30 ¡i ol70o/o
(v/v) ethanol before microfuging once more. Ethanol was aspirated off and the pellet air dried before being dissolved in 20 ¡rl TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5).
Restriction endonuclease digests of 0.5 pg DNA with Eco RV were carried out according to conditions specified by the manufacturer. Gel electrophoresis of digested plasmid was performed in a 17o (w/v) agarose gel in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM
EDTA, pH 8,3) in Pharmacia GNA100 tanks for horizontal submerged gel electrophoresis. Samples were prepared in loading buffer (5% (v/v) glycerol, 0.1% (w/v)
SDS, 10 mM EDTA, 0.017o (w/v) bromophenol blue, pH 8.0) and were electrophoresed at 5-10 V/cm until bromophenol blue had migrated a sufficient distance to ensure adequate separation of DNA fragments. DNA was visualised under UV light after brief staining of the gel in 10 pg/ml ethidium bromide.
(Potter Gene transfection was attempted by electroporation using the method of Potter ef a/., 1g84)). Electroporation was performed on 10' cells in 0.8 ml PBS to which 22 pg oI plasmid DNA had been added. Cells pre-cooled on ice, were electro-pulsed at 0'C using 84
10 minutes on ice, cells had 10 ml of a BRL Cell-Porator set at 330 pF and 275 V. After culture llask. Medium was IMDM added and were transferred to a 75 cm2 tissue the cells were trypsinised and changed after 16 hours. 48 hours post-electroporation,
containing 0'4 mg/ml G418' Medium split into 4 x 10 cm diameter petri dishes with |MDM
were harvested by the use of glass was changed every 4_5 days and at 14 days coronies
When wells had become confluent cloning rings and replated in a 24 well culture dish' culture flasks and each line the transfected cell lines were grown up in 25 cm',
cryopreserved in liquid nitrogen'
OF GAGS: 2.2.22 CETYLPYRIDINIUM CHLORIDE (CPC) PRECIPITATION
method of Hopwood and p-eliminated GAGs were precip¡tated from solution by the the samples had 300 pg carrier Harrison (Hopwood and Harrison, 1982). Briefly,
(from stock made up in 0'0125 M Nacl' 0'01 M chondroitin sulphate A added a 3 mg/ml
Tris/HClpH7.4)andwerethendilutedl:1with0'1%(w/v)CPCin0.054Mcitratebuffer
minutes at 37"C' samples were then (pH to 4.8 with citric acid) before incubation for 30
supernatant decanted and the tubes well centrifuged at 3000 rpm for 10 minutes, the by vortexing to resuspend' drained. To pellets were added 150 pl 2 M Licl followed
by vortexing and centrilugation at 3000 Ethanol was then added (g00 pl/tube) followed
off and the pellet resuspended prior to rpm for 10 minutes. supernatant was decanted
scintillation countlng
2.2.23 PROTEIN ASSAY:
(BCA) method (smith et al'' 1985) using protein was assayed by the bicinchoninic acid
bovine serum albumin as a standard 2.2.24 STATISTICAL ANALYSES:
Unless othen¡rise stated statistics were calculated on a PC using Microsoft Excel
software.
3.1 INTRODUCTION
The pancreas is an organ which is severely affected in most patients with cystic fibrosis.
Pancreatic insufliciency manifests in 85-90% of all cases of CF and appears to be caused by duct blockage and eventual tissue atrophy as a consequence ol rheologically abnormal mucous secretions. With the establishment of a pancreatic adenocarcinoma ductal epithelial cell line from a CF patient (CFPAC-1), a valuable tool became available for the study of CF pancreatic duct physiology (Schoumacher et al., 1990).
Serendipitously, CFPAC-1 was homozygous for AFror, the dominant CF mutation and one classically defined as severe. The cells were shown to exhibit typical epithelial polarisation with the presence of apical microvilli, tight junctions and gap junctions
(Schoumacher ef a/., 1990). We chose to examine this cell line for evidence of elevated sulphation of secreted and/or cell-associated glycoconjugates. For a normal control we obtained PANC-1, a cell line established from a (non-CF) pancreatic adenocarcinoma. pANC-1 had the same tissue origin as CFPAC-f i.e. the ductal epithelium, and importantly, has been shown to possess CFTR both at the mRNA (Ward et a\.,1991) and functional protein level (Kopelman et al., 1993). PANC-1 has maintained many of the differentiated characteristics of normal ductal epithelial cells (Madden and Sarras, 1988) including a cuboidal morphology, tight junctions and normal ultrastructural features, as well as exhibiting biochemical markers of pancreatic duct such as T- glutamyltranspeptidase, carbonic anhydrase and Na',K'ATPase. lt was therefore considered a reasonable control for these experiments.
'The two cell lines will be referred to as CFPAC and PANC subsequently pancreas was due We hypothesised that, at least in part, the mucous abnormality in the
be demonstrable to over-sulphation of secreted glycoconjugates. lt was felt this should
in cell culture with experimentally by metabolic radiolabelling ol PANC and CFPAC cells
incorporation, the medium was [.rS]sulphate. To quantitate and normalise l"S)sulphate inorganic arso suppremented with lrH]grucosamine. As the majority of intracellular
(Humphries ef a/', 1988)' it sulphate for biosynthesis is channelled into GAG synthesis
likely to manifest itsell was felt that a generalised defect in sulphate metabol¡sm would be
were focused in the chemical structure of these molecules. Accordingly, our efforts towards GAG analYsis
3.2.1 GENERALCHARACTERISATION:
3.2.1.1 Anti-Cytokeratin Staining:
of the two cell lines, a ln order to confirm the epithelial origins and differentiated status
in an immunofluorescence commercially available antibody to cytokeratin was employed by epithelial protocol. cytokeratin is an intermediate filament phenotypically expressed and PANC cells. Figure 3.1 illustrates that there was strong staining of both CFPAC
cells. A B
c D
FIGURE 3.1 IMMUNOFLUORESCENT STA¡NING OF CYTOKERATIN IN PANCREATIC CELLS: Staining was performed as detailed in section 2.2.4. Slides were photographed on a Leitz Diaplan fluorescent microscope with top-mounted camera using Kodak Ektachrom 400 AsA slide film at an excitation wavelength of 450 nm and emission wavelength of
490 nm. a) PANC (10x magnification) b) PANC (25x magnification) c) CFpAC (10x magnification) d) CFPAC (25x magnification)
91
3.2.1.2 Acidification of Lysosomes:
Barasch et at. (991) published work indicating that trans-Golgi network and endosomes of CF airway cells were delectively acidified. To determine whether CF pancreatic cells might exhibit similar characteristics, subcellular fractionation by differential centrifugation was perlormed. A granular lraction containing endosomes, lysosomes and microsomes from PANC cells was tested for acidification competency in an acridine orange based dynamic fluorescence assay (F¡g. 3.2). Membranes (50 pl) in 0.25 M sucrose pH 7.0
(with minimum Tris) were added to a cuvette containing acidification medium.
Fluorescence was monitored for three minutes and a steady baseline achieved.
Subsequent addition of freshly prepared ATP, initiated acidilication by the activation of membrane bound V-ATPases (for a review of practical and theoretical principles see
(Lowe and Al-Awqati, 1988)). The immediate increase in fluorescence was an artifact due to intrinsic ATP fluorescence. Strongly acidifying vesicles could be seen as the fluorescence dropped rapidly. lt can be seen fhat the rate of fluorescence change begins to slow as proton influx is reduced - this is due to vesicles becoming lully acidified. To demonstrate that the time based reduction in fluorescence was due to
(CCCP) acidification, the proton ionophore, carbonylcyanide-m-chlorophenylhydrazone was added with a consequent rapid dissipation of ÂpH.
To investigate the acidification activities of endosomes and lysosomes individually' granular fractions were subjected to centrifugation on 20o/o (vlv) Percoll gradients. With some cell types it is possible to demonstrate clear separation of a dense lysosomal fraction from endosomes which are lighter and sediment differently on Percoll' ln our hands the results of applying this technique to pancreatic cells was disappointing. Figure 3.3 shows the activity of p-hexosaminidase assayed across Percoll gradients to which PANC and CFPAC granular fractions were applied. p-hexosaminidase is a soluble
enzyme marker normally associated with lysosomes and to lesser extent endosomes.
Neither cell line appears to have appreciable B-hexosaminidase activity associated with a
lighter, endosomal fraction whilst both exhibit significant activity at the bottom (dense)
end of the gradient. The density gradient was measured by weighing 200 pl aliquots of
each 1.5 ml fraction from the PANC run, and showed an increase in densiÇ, from the top
to the bottom of the tube.
Membranes from fractions 1 and 24 ol the CFPAC, and 6 and 23 of the PANC loaded
gradients, were assayed for their ability to acidify. There was evidence for weak
acidification of lysosomes from both cell lines and reversal of the effect by the addition of
nigericin. The rate and extent of acidification were similar (data not shown). This is
consistent with the findings of others who have postulated that CFTR plays no role in the
acidification of lysosomes (Van Dyke et al., 1992). There was no evidence for
acidification of either of the putative endosomal fractions.
3.2.1.3 Chloride TransPort:
We acquired the colonic adenocarcinoma cell line T84 which is known to express high
levels of CFTR (Yoshimura et al., 1991; Barrett, 1993) for use as a positive control to
validate our chloride efflux assay. The assay employed a pre-loading phase with
radioisotope, and then upon initiation of efflux, a sample removal/replacement strategy.
The data was corrected for this and presented as the percentage of 'uCl counts
remaining intracellular at each time point sampled. F luorescence lntensitY 520 0 ATP c
6t0
500
190
480
470
4€Ð
¡l50
¡l¡+0
¡¡{Ð
420
410
{qr0 po {{) 60 80 loo l?o f40 160 l8{¡ 200 2æ 210 60 280 300 320 340 360 3æ 400-o Iime (minutes)
FIGURE 3.2 ACIDIFICATION OF A SUBCELLULAR GRANULAR FRACTION FROM PANC CELLS:
Membrane suspension (50 rrl) in 0.25 M sucrose (pH 7.0 by addition of minimum quantity of Tris) was added to 1.95 ml acidification medium (see section 2.2.8 lor details). A steady baseline was achieved at room temperature once the cuvette was mounted in the fluorimeter and monitoring of fluorescence commenced. The FL Data Manager programme monitored and recorded fluorescence intensity every 0.1 seconds. Acidification of organelles was initiated by addition of 500 pM ATP. The pH gradient was
dissipated by the addition of 45 pM CCCP. Fluorescence intensity units on the y-axis are arbitrary. 94
It could be shown that T84 cell monolayers pre-loaded with "Cl- and exposed to 10 pM fo fncrea-secfthêir ráte of rádioisoTope efflux compãrêd to baseline efflux (Fig. 3.4a). This response was modest but reproducible and it was interesting to note that baseline efflux and forskolin stimulated efflux did not diverge until two minutes following addition of agonist containing medium. Presumably this 'lag' represented the time required to recruit or activate the enzyme systems involved in the
çAMP second messenger cascade i.e. G proteins, adenylate cyclase, protein kinase A etc. When the calcium ionophore, ionomycin was added to efflux buffer, there was rapid loss of ..Cl from the interior of the cell. This 'dumping' of Cl-through Ca'.-activatable chloride channels represented more than 50% of the intracellular radioisotope within the first 60 seconds of the experiment. From this analysis we could say that the concentrations of forskolin and ionomycin employed were effective in activating second messenger cascades responsible for the opening of Ca2.-dependent as well as cAMP- dependent chloride channels in T84 cells. When CFPAC cells were exposed to ionomycin there was a similar massive efflux of 'uCl-within the first minute (F¡9. 3.4b).
However there was no evidence for any effect of forskolin on chloride secretion. This result was expected and confirmed similar results found by others (Schoumacher et al.,
1990). The experiment demonstrated that Ca'.dependent chloride channels remained functional in CFPAC cells but that elevated intracellular cAMP could not be shown to stimulate chloride efflux. The basal rate of efflux was much lower than that observed in
T84 cells, possibly reflecting fewer ion channels on the apical surface through which hormone-independent flux could occur. PANC cells failed to respond to forskolin with an increase in chloride transport (Fig. 3.4c). This result was disappointing, and might raise questions about the sensitivity of the assay we were using and the level of expression of 95
ToP Bottom 10 1125 I
I
I
E 8-r 1 .105 o E E ! o c o l o- E 6- 1.085 o- o (so f o Ø U) (ú 4- 1.065 c p (o .E E t 3 (ú U' o a ox 2 --a 1.045
-cI o
0 1.025 0 5 10 15 20 25 fraction number
FIGURE 3.3 PERCOLL DENSITY GRADIENT CENTRIFUGATION OF PANC AND CFPAC GRANULAR FRACTIONS:
Percoll solutions (20% (v/v)) were prepared in Beckman Quickseal tubes and overlayed with 3 ml of granular fraction from PANC and CFPAC cells which had been prepared by differential centrifugation as described in section 2.2.6. The self-forming gradients were generated by centrifugation at 30 000 g for 60 min. Fractions (26 x 1.5 ml) were collected from the bottom (dense end) of the gradient and 50 ¡rl aliquots ol each fraction were assayed for B-hexosaminidase activity (details in section 2.2.7) for 10 minutes at 37"C. The formation of the gradient was assessed by weighing 200 ¡rl aliquots of each fraction. 96 FIGURE 3.4 CHLORIDE TRANSPORT IN T84, CFPAC AND PANC CELLS:
Chloride transport experiments were carried out on confluent cell monolayers that had been pre-loaded with 2.5 p9i "Cl'for 2 hours at 37"C under ambient CO.. After removal of extracellular radiolabel by washing, efflux was initiated by addition of mannitol buffer containing either vehicle or agonist (see section 2.2.5 for details). Agonist containing efllux buffer was made up on the day of the experiment from concentrated stock solutions of forskolin and ionomycin in DMSO stored at -20"C. Efllux buffer containing vehicle had 0.625To (v/v) DMSO added, the same concentration as both agonist- containing etflux buffers. Forskolin was used at a final concentration of 13 ¡rM except for T84 cells which were exposed to 1O¡rM, and ionomycin for all cells was used at 5 pM.
A 200 pl aliquot of efflux buffer was removed for scintillation counting and immediately replaced with fresh bulfer at each time point in the experiment. Following sampling of the 10 minute time point, the remaining efllux buffer was aspirated, the cells washed with ice cold mannitol bufler and the cell monolayer lysed with 0.2 M NaOH. The entire lysed cell sample was counted to ascertain counts remaining intracellular at the end of the experiment. Results are presented as the percentage of 'uOl counts remaining intracellular at each time point sampled. Error bars represent the spread of duplicate values. Where none are shown the spread was not larger than the symbol. a) T84 cells b) CFPAC cells c) PANC cells 97
100 OVehicle I Forskolin 80 I lonomycin
60 I. 40
20- a (ú 0 f õ 100 (õo .= 80 o) .g C '(5 60 E E 40 (J (o (f) 20 àe
0 100
80
60
40
20 c 0 0 1 2 3 4 5 6 7 I 9 10 time (minutes) 98
CFTR in this cell line (Ward et a1.,1 991). lonomycin could be shown to induce a chloride secretory response in PANC cells but at a much slower efflux rate than either CFPAC or
T84. The basal rate of chloride secretion was higher for PANC than for CFPAC and was similar to that seen in T84 cells. lt is worth noting that even in T84 cells, which express
CFTR naturally at higher levels than virtually any other cell line, the response to lorskolin is modest.
3.2.2 ANALYSISOFGLYCOCONJUGATES:
3.2.2.1 Metabotic Radiotabelling, Anion-Exchange Chromatography and Peak
Assignment:
Glycoconjugates produced by both cell lines over a 24 hour period were characterised at two stages of confluence - 50-60% and 1007o. This was to explore the possibility that
cellular metabolism, and consequently the nature of the glycoconjugates being
produced, might change upon cells reaching confluence. Accordingly, cells were grown
in 5 cm diameter plastic petri dishes at two different seeding densities. When the
required degree of confluence was reached, radiolabelling of cells was performed lor 24
hours as described in section 2.2.10.
Eight samples were obtained from this experiment. They were derived from the two cell
lines at two states of confluence and two sources ('medium' and 'cell layer'). Samples
were chromatographed on Sephadex G50 and then subjected to anion exchange
chromatography on DEAE-Sephacel. Figure 3.5 illustrates the profiles obtained for each
of the 'medium' or secreted samples. After collection of an unbound peak at a NaCl
concentration of 150 mM (labelled lin Fig.3.5a), a 0.15-1.0 M NaCl gradient in dissociative buffer was applied. This resulted in the elution ol two lurther peaks from the column (ll and lll in Fig. 3.5a). The first of these eluted at approximately 200 mM NaCl whilst the second, eluted at 400-500 mM NaCl. Peaks I and ll had only minor amounts of l"S]sulphate associated with them and the majority of "S-counts could be seen to have partitioned to peak lll. There was little dilference between the pre-conlluent profiles
(Figs. 3.5c, 3.5d) and the conlluent profiles (Figs. 3.5a, 3.5b) for each cell type but there were interesting differences between the type of glycoconjugates synthesised by PANC compared with CFPAC. PANC cells (pre- and post-confluence) produced larger amounts of peak ll material and as a result proportionally less ['H]glucosamine was incorporated into peak l. The distribution of ['H]glucosamine between peaks is illustrated in Figure
3.7. Peaks I and ll were subjected to enzymatic digestion by Streptomyces hyaluronidase in an effort to determine whether either contained hyaluronan. Figure 3.6 shows the profiles obtained for PANC medium (confluent cells) after incubation with enzyme or vehicle, and analysis of the digestion products on Sephadex G50. There was no conversion of high molecular weight material to lower molecular weight forms when any peak I samples were exposed to enzyme (Fig. 3.6a). However peak ll was composed entirely of hyaluronan as evidenced by a 100% shift to low molecular weight disaccharide products (Fig. 3.6b). This was also found for CFPAC samples (data not shown). This analysis confirmed the identity of peak ll as hyaluronan for both celltypes. FIGURE 3.5 DEAE.SEPHACEL ANION EXCHANGE CHROMATOGRAPHY OF
SECR ETED G LYCOCONJUGATES:
Medium lrom cells that had been radiolabelled with [3sS]sulphate and ['H]glucosamine lor 24 hours was desalted and bulfer exchanged on Sephadex G50 into dissociative buffer (section 2.2.14). The samples were then applied to DEAE-Sephacel columns equilibrated in dissociative buffer and 12 x 1ml fractions collected before bound components were eluted with a 0.15-1.0 M NaCl gradient in dissociative buffer (see section 2.2.15 for further details). Aliquots (50 ¡rl ) of collected fractions were scintillation counted. Protein assays were performed on the solubilised 'cell layers' and data corrected for total protein. Data is presented as the total cpm/mg protein in each fraction. y-Axes in panels b) and d) correspond to those of a) and c) respectively. l, ll, and lll in panel a) indicate the nomenclature used to refer to corresponding peaks from all DEAE-Sephacel runs in this chapter. a) CFPAC (Confluent cells), b) PANC (Confluent cells), c) CFPAC (Pre-confluent cells), d) PANC (Pre-confluent cells). NaCl Concentration (mM) o oo o o o ôo o (o gRoã @ Iv NI rf)o
o 3
o (f)
o c\,1
U) o r.l) o (f) -o t .cl E E - o f cf) c o __,_.t-_.,_ ___ _-1- lr)o c õ o o o z (ú to E
(Ðo
o N
o (E ll o .e T ------'l-' I o o o o o o o (oo o o o o c.l o @ (o g C.l C.¡ co \t (spuesnoqÐ utolold Ou¡utdc 1
PANC PEak lll CFPAC Peak lll 23.Ooß 26.1%
il 17.70Á
32.60Â
Peak I 44.40ß Peak I 56.3%
FIGURE 3.7 PROPORTION OF I'H]GLUCOSAMINE IN EACH PEAK AFTER DEAE-SEPHACEL CHROMATOGRAPHY
It appeared therefore, that PANC cells synthesised greater amounts of hyaluronan than
CFpAC and correspondingly, channelled a greater proportion of the l'H]glucosamine taken up from the culture medium into the synthesis ol this GAG. Peak lll material proved to be susceptible to both chondroitinase ABC digestion and nitrous ac¡d deamination which confirmed the presence of CS/DS and HS (details of these analyses will be presented in section 3.2.2.4 and 3.2.2.5). Peak I material was not directly analysed, but due to its low charge density (¡t did not bind to the column at 150 mM
NaCl) and relatively low sulphate content was likely to be comprised primarily ol glycoprotein. These peak assignments are consistent with what others have observed
using the same buffers and separation protocols Harpet et a1.,1986). Figure 3.8 shows the anion exchange profiles obtained when the four'cell layer' samples were analysed
on DEAE-sephacel. There was little diflerence between any of these but a striking 1
7 a OVehicle 6 U) lEnzyme E (ú 5 )U' o 4 .C,
E 3 o- O I 2 c) 1
0 6 ttU) C (ú 5 U)) o 4 3 E o- o 2 I CÐ 1
0 0 1 234567 89 fraction number
FIGURE 3.6 HYALURONIDASE DIGESTION OF PEAKS PURIFIED ON DEAE.SEPHACEL:
Aliquots (200 rrl) of pooled peaks off DEAE-Sephacel were dialysed for 24 hours against 50 mM sodium acetate pH 5.0 and then digested for 5 hours at 60"C with 10 U Streptomyces hyaluronidase. Controls were incubated under the same conditions but had bulfer without enzyme added. Samples were made up to 1 ml with PBS and then chromatographed on Sephadex G50 in PBS. Fractions (l ml) were collected and scintillation counted. Samples illustrated in this figure come from PANC Medium
(Confluent cells). a) Peak I b) Peak ll 1 o4 FIGURE 3.8 DEAE.SEPHACEL ANION EXCHANGE CHROMATOGRAPHY OF CELL LAYER GLYCOCONJUGATES:
Cell monolayers that had been radiolabelled with l"S]sulphate and ['H]glucosamine for 24 hours were dissolved in 4 M guanidine-HCl buffer then desalted and buffer exchanged on Sephadex G50 into dissociative buffer. The samples were applied to DEAE-Sephacel columns equilibrated in dissociative butfer and 12 x 1ml fractions collected before bound components were eluted with a 0.15-1.0 M NaCl gradient in dissociative buffer (see section 2.2.15 for details). Aliquots of collected lractions (50 pl) were scintillation counted. Protein assays were performed on aliquots of these samples and data corrected for total protein. Data is presented as the total cpm/mg protein in each fraction. a) CFPAC (Confluent cells), b) PANC (Confluent cells), c) CFPAC (Pre-confluent cells), d) PANC (Pre-confluent cells) 105
mM NaCl e3H +35S
300 000 a b I 600 i I O
o I :r 800 ; I I I 450 200 ! i
I 600
i
300 I - 400
I 00 z U' Ê) õc a 150 (ú I zæO U' 1 o f f o o -c o J Co 0 'o Ê) o c d t* õ' f o- 450 450 o) 3 E 60O:-z E o- o 3oo 300
I 400
I I
150 150 i ! 200
I
l
0 0 0 01020304050 01020304050
fraction number 106 difference from all of the 'medium' samples was the greatly reduced amounts of GAGs present i.e. peaks ll and lll. This was not ent¡rely unexpected as proteoglycans are normally exported lrom the cell. However some may become embedded in the plasma membrane andlor become associated with ECM.
3.2.2.2 Anatysis of Peak I on Sepharose CL4B ln order to assess whether either pancreatic cell line was producing high molecular weight mucin type glycoprotein, peak I material was run on a Sepharose CL4B column
(F¡g. 3.9). This gel liltration matrix has a molecular weight exclusion of several million and whilst not confirming the chemical identity as mucin, any species running at or near the void volume would be a good mucin candidate. CFPAC cells produced two peaks of high molecular weight material which may have represented mucin or mucin like glycoproteins, but PANC cells had no material eluting at these positions' This precluded a comparison of mucin structure and sulphation by these cell lines.
3.2.2.3 [ssS]:FHl Ratios in Glycoconjugates Synthesised by CFPAC and PANC:
[.'S]:[,H] ratios were calculated and tabulated for peaks I and lll in all eight samples'o
3'S (Tables 3.1 and 3.2). ln peak I the ratios of to'H incorporated were very low but there was evidence of higher levels of sulphation by the CF cell line compared with the control. This was more pronounced for cellular glycoprotein than secreted glycoprotein.
The state of confluence increased the differences between PANC and CFPAC. This occurred because sulphation levels increased as CFPAC reached confluence but also because PANC metabolites became less sulphated upon reaching confluence. For
associated with this peak 'oHyaluronan does not possess esterìlied sulphate residues. "S-counts were likely to represent'crossover'from'H in the scintillation counting channels. 7
1,200 a e3H 1,000 + 35S
800
E Vo o- 600 o I
400
200
0 1,000 k) Vo I 800
E 600 o- o 400
200
0 0 10 20 30 fraction number
FIGURE 3.9 SEPHAROSE CL-48 CHROMATOGRAPHY OF PEAK I MACROMOLECULES FROM PANC AND CFPAC MEDIUM: CFPAC and PANC medium lrom pre-conlluent cells were anion exchanged on DEAE- Sephacel and 500 pl of peak I was run on a 90 cm x 0.5 cm Sepharose CL-48 column equilibrated in PBS/0.1% (w/v) sodium azide, at a flow rate of 0.14 ml/min. 35 x 1 ml (Vo) fractions were collected and 200 ¡rl aliquots scintillation counted. The void volume ol the column was established by running blue dextran under the same conditions. a) PANC b) CFPAC 1 08
TABLE 3.1 QQMPARISOñ€F [.'Sl:f.HI RAflOS lN PEA.K I-AFTER-EEAE-SEPHAÇEt CHROMATOGRAPHY
0.073 0.054 1.4
0.092 0,046 2.0
0.081 0.036 2.3
0.102 0.032 3.2
TABLE 3.2
COMPARISON OF ["SI:['H] RATIOS lN PEAK lll AFTER DEAE-SEPHACEL CHROMATOGRAPHY
1.26 1.10
1.56
1.22 0.83
1.58 1.07 109 example, with 'medium'samples, CFPAC's ratio increased lrom 0.073 pre-conlluent to
0.092 at confluence, whilst PANC secret¡ons move from having a ratio of 0.054 to 0.046.
The reasons for these subtle differences in metabolic behaviour are unknown but may occur as a result of the CF delect in CFPAC cells. The higher sulphation by CFPAC is more pronounced for 'cell layer' associated macromolecules than lor secreted ones.
Table 3.2 shows the sulphation ratios for the GAGs of both cell lines. lt can be seen that some of the trends apparent for peak I material are reproduced, with a tendency for the
GAGs synthesised by CFPAC to be more highly sulphated than those synthesised by
PANC. The dilferences are more modest than for peak I samples ranging from 10-507o higher. The GAGs produced by both cell lines were more highly sulphated at conlluence than when pre-confluent. Although the ratio differences between cell lines in peak lll were not great, it was felt there might by a subset of molecules within peak lll which were more highly sulphated, therefore further analysis of this material was perlormed.
5.2.2.4 P'Sl:FHl Ratios in Chondroitin/Dermatan Sulphate Synthesised by CFPAC and
PANC:
Figures 3.10 and 3.11 illustrate the Sephadex G50 profiles obtained after peak lll samples were digested with chondroitinase ABC for four hours. This enzyme cleaves both CS and DS chains down to disaccharides and these can be detected as an included peak by gel filtration (indicated in Fig.3.10a). Figure 3.10 shows comparisons of 'cell
layer' samples and Figure 3.11 the 'medium' samples. All samples contained significant
amounts of CSiDS. The elution patterns were very similar for pre-confluent and confluent samples indicating that there had not been a radical shitt in the biosynthetic
activities of either CFPAC or PANC upon reaching confluence. lt can readily be seen
however (Fig. 3.10 a and b compared with Fig. 3.1 1 a and b) that the amount ol CS/DS 11 FIGURE 3.10 CHONDROITINASE ABC DIGESTION OF PEAK III MACROMOLECULES FROM CELL LAYER SAMPLES:
Chondroitinase ABC digestions were perlormed on peak lll material isolated after DEAE- Sephacel anion exchange of cell layer samples as described in section 2.2.15. The digestion products were chromatographed on Sephadex G50, 1 ml fractions collecled and 100 pl aliquots, scintillation counted. Due to relatively low numbers of radioactive counts the samples were counted for 10 minutes to attain greater counting accuracy. The peak representing the disaccharide product of digestion is indicated in a). a) PANC (Pre-confluent cells) b) PANC (Confluent cells) c) CFPAC (Pre-confluent cells) d) CFPAC (Confluent cells) 11 1
e3H +35S
70æ 8000 a b 60æ
6000 5000
4000 É. E o o 4mo o- CS/DS Disaccha¡ides o- o o 3000
2000 2000
10m
0 0 0 1 2 3 4 5 6 7 I I 1011 12 0 2S4567I9101t12 fraction number lraction number
2ffi 4000 d
2000 3000 a
1500 oE oE 2000 o- o o 1m
1000 500
o o 0 1 2 3 4 5 6 7 8 I 1011 12 0l 34567I9101112 frælion number frætion number 112 FIGURE 3.11 CHONDROIT¡NASE ABC DIGESTION OF PEAK III MACROMOLECULES FROM MEDIUM SAMPLES:
Chondroitinase ABC digestions were performed on peak lll material isolated after DEAE- Sephacel anion exchange of medium samples as described in section 2.2.15. The digestion products were chromatographed on Sephadex G50, 1 ml fractions collected and 100 pl aliquots, scintillation counted. Due to relatively low numbers of radioactive counts the samples were counted for 10 minutes to attain greater counting accuracy.
a) PANC (Pre-conlluent cells) b) PANC (Confluent cells) c) CFPAC (Pre-confluent cells) d) CFPAC (Conlluent cells) 11 3
Ogx csss
20æ 7000 a b 6000
1 500 50co
4000 Ê E o 10æ o o- o- o o 3000
2000 5æ
1 000
0 0 0 1 2 3 4 5 6 7 I I 1011 0 2 3 4 5 6 7 I I 1011 12 fraction number fraction number
5000 1 2000 d
4000 9000
3000 E E o o 6000 o- o- o o 2000
3æO 't000
0 0 0 | 2 3 4 5 6 7 I 9101'1 12 0 3 4 5 6 7 I 9 1011 12 fraction number fractìcn number 11 4 in PANC 'cell layer' is a smaller proportion ol the total than in the 'medium' suggesting that secreted proteoglycans are markedly dilferent than those destined for cell matrix deposition. The interesting linding from this analysis was the higher ["S]:['H] ratios in
CS/DS produced by CFPAC cells.
Sephadex G50 chromatography using I ml columns" allowed semi-quantitative information from the analysis. 3H-counts in fractions 2-4 were added and taken to represent the total quantity of undegraded material, whilst counts in fractions 6-12 were assumed to represent all of the disaccharide component of the reaction mixture.
Because fraction 5 included material from both peaks it was not included in the calculation',. The results of this are summarised in Table 3.3. The proportions of undegraded material did not change markedly with confluence. For PANC cells there was no change at all. lt was interesting to note however that GAGs secreted by PANC cells have twice as much CS/DS as do the GAGs of the 'cell layer'. CFPAC cells also secreted proportionally more CS/DS than was associated with cell matrix (approximately
30Yo). Of greater interest however, was the comparison ol ["S]:['H] ratios to be found in
GAGs. Table 3.4 lists the ratios obtained by summing the'H- and "S-counts of fractions
3 and 4 (undegraded) and 7-9 (CS/DS). These fractions were chosen as they had the highest number ol counts and therefore the greatest accuracy. By not including fraction
6 there was no contribution from the overlapping peak. The CS/DS molecule produced by PANC cells under all conditions appeared to be fairly uniform in terms of its sulphation. The values ranging from 1.05 to 1.31. CS/DS produced by CFPAC
"See sectio n 2.2.14 for discussion of limitations associated with this technique. ':ln Fig. 3.10c it can be seen that the radioactivity distributed slightly ditferently and hence fractions 2-S were taken aS the undegraded component and fractìon 6 was not included in the calculatlons. 115
TABLE 3.3
PROPORTIONS OF GAGS IN PEAK III
68% 32%
70o/o 30%
35o/o 65%
36% 64o/o
41Yo 59%
50% 50%
25o/o 75o/o
33o/o 67o/o 116 appeared to be more variable depending on whether the cells were confluent or pre- conlluent. Upon reaching confluence the ['uS]:['H] ratio in secreted CS/DS increases lrom 1.64 to 2.16 whilst the shift with cellular CS/DS is also dramatic, going lrom 1.52 to
2.36, a 50% increase. This may reflect biosynthetic changes which occur as the cells alter their growth cycle from one of cell division to one of confluent equilibrium.
Of most interest from the perspective of what this project hoped to ident¡fy, was the consistently higher sulphation ratio exhibited by CFPAC CS/DS compared with that produced by PANC. The relative increase when PANC was compared with CFPAC is indicated in the final column of the table and ranges lrom 260/o to 225% higher. The putative HS fraction does not exhibit the over-sulphation that CS/DS does' HS ratios of
CFPAC 'cell layer' samples when compared with those of PANC 'cell layers' are similar whilst CFPAC 'medium' material is lower than PANC's.
Although it was not considered likely, peak I material was tested for the presence of under-sulphated chondroitin. When peak I material from all samples was subjected to chondroitinase ABC digestion there was no evidence for the formation of low molecular weight products. Figure 3.12 shows the products of digestion when PANC'cell layer'
(pre-confluent) was exposed to enzyme for four hours and then run on Sephadex G50.
Macromolecular material remains undegraded and elutes in the void volume. Testing ol
peak ll material for the presence of CS/DS with this enzyme was not a possibility as chondroitinase ABC is able to utilise hyaluronan as a substrate, albeil at a much lower
rate (0.02 times that lor chondroitin sulphate C at pH 8.0)". Moreover, our
"lnformation supplied by the manufacturer 117
TABLE 3.4 ["Sl:['HI RATIOS lN GAGs
0.50 1.21
0.44 1.52 1.26
0.39 1.05
0.43 2.36 2.25
0.76 1.07
0.39 1.64 1.53
0.91 1.31
0.37 2.16 1.65 118 demonstration that peak ll was completely susceptible lo Streptomyces hyaluronidase made the possibility unlikely
3.2.2.5 Demonstration of Heparan Sulphate in Peak lll:
Each of the undegraded peaks remaining after chondroitinase ABC digestion of peak lll were subjected to nitrous acid (pH 1.5). The products of the reaction after neutralisation with 2 M NarCO, were chromatographed on Sephadex G50. This analysis enabled us to draw some qualitative rather than quantitative conclusions about the presence of HS.
This was not considered problematical as the aim was to establish whether chondroitinase ABC resistant material in peak lll contained HS. Results of this analysis are presented in Figures 3.13 and 3.14. Because the products of the reaction arc a
heterogeneous population of different molecular weight species the profiles do not
usually fall into the pattern seen previously for chondroitinase digestion with a peak at the void volume Uo) and a symmetrical, reasonably well separated included peak comprising disaccharides. lt can be seen in all eight profiles that there are considerable
amounts of 3H-labelled material appearing to the right of Vo (usually fraction 4) and large
amounts of l"S]sulphate also in the lower molecular weight region. Some of these 'uS-
counts will be liberated ["S]sulphate and some will be O-linked sulphate on
oligosaccharides of variable length. The appearance of this material in fractions 6-12
indicates the presence of HS.
An inspection of the elution profiles suggests that there are broad similarities between
the confluent and pre-confluent samples (Fig.3.13 a vs b, c vs d; Fi$.3.14 a vs b, c vs
d) and that there are differences between PANC HS and CFPAC HS in their response to
nitrous acid. lt is interesting to look atthe four profiles in Figure 3.13 in the light of data 119
10 VO +3H .- 35S I
EØ C 6 (d U, o:J -c. E o- 4 c)
2
0 0 1 2 3 4 5 6 7 I 9 101112 fraction number
FIGURE 3.12 CHONDROITINASE ABC DIGESTION OF PEAK I MACROMOLECULES: Chondroitinase ABC digestion was pedormed on peak I material isolated after DEAE- Sephacel anion exchange chromatography as described in section 2.2.15. The digestion products were chromatographed on Sephadex G50, 1 ml fractions collected and 100 ¡rl aliquots scintillation counted. lllustrated in this figure is PANC 'cell layer' from pre- confluent cells. 1 20 F|GURE3.13 NITROUS ACID (pH 1.5) ON PEAK tll MACROMOLECULES FROM MEDIUM:
Material from medium samples that remained undegraded after chondroitinase ABC digestion was subjected to nitrous acid deamination as described in section2.2.17. fhe products ol this reaction were chromatographed on Sephadex G50, 1 ml fractions collected and 100 pl aliquots scintillation counted. Due to relatively low numbers of radioactive counts the samples were counted for 10 minutes to attain greater counting accuracy a) PANC (Pre-confluent cells) b) PANC (Confluent cells) c) CFPAC (Pre-confluent cells) d) CFPAC (Confluent cells) 1 I
e3H +35S
20æ 2m a b
1 500 1 5CO
E oE o 1000 000 o o- I O a
5æ 500
0 0 o 1 2 3 4 5 6 7 I I 101112 0 1 2 3 4 5 6 7 I I 1011 12
1500 500 d
400
1000
3æ C 5 d o- o a
200 500 L
100
I I I
0 0 0 2 3 4 5 6 7I91 11 1 2 3 4 5 6 7 I I 10111
fraction number 1 F|GURE3.l4 NITROUS ACID (pH 1.5) ON PEAK lll MACROMOLECULES FROM CELL LAYER:
Material from cell layer samples that remained undegraded atter chondroitinase ABC digestion was subjected to nitrous acid deamination as described in section 2.2.17. The products of this reaction were chromatographed on Sephadex G50, 1 ml fractions collected and 100 pl aliquots scintillation counted. Due to relatively low numbers of radioactive counts the samples were counted for 10 minutes to attain greater counting accuracy a) PANC (Pre-confluent cells) b) PANC (Confluent cells) c) CFPAC (Pre-confluent cells) d) CFPAC (Conlluent cells) e3H +35S
10æ 10æ ib
I 600 8æ :
I
600 600
E E o- ô- a
400 4æ-
200 2æ
0 0
2W 800
i d
I
I
æoo I 6æ
I
I
1500 I c E o ¿m L o o. o o
1m I
2æ t
5æ I
0 o 012345ô7A9101112 't 2 3 4 5 6 7 I 910 11 12
fraction number 124 contained in Table 3.4. The ['uS]:['H] ratio in undegraded material was much lower lor
CFPAC medium than for PANC medium. This appears to be reflected in Figure 3.13 c and d, where there is a smaller amount of l"S]sulphate appearing in the included fractions (6-12) than in the equivalent PANC samples (Fig.3.13 a and b). This may mean that CFPAC cells are producing a HS for secretion that is less N-sulphated than that produced by PANC. Given that both cell lines are ductal epithelium from the pancreas it would be predicted that they would produce the same type ol GAG molecules. Such differences as we observe may reflect the effect of the CF mutation on the metabolic activity of CFPAC or alternatively the alterations may result from cellular transformation.
Although there was clear evidence of substantial amounts of HS in the samples analysed, of some concern was the relatively high number of 'H-counts which eluted at
Vo. One of two explanations for this result appear feasible. Firstly that the HS chains have been cleaved but a significant proportion of the oligosaccharides generated are of a molecular weight around l OOOO or greater (the exclusion size for dextran on this gel matrix). Alternatively there was some substance in the sample which interfered with the
reaction going to completion. Triton X-100 was present in the sample preparations
because it has a very low critical micellar concentration and is impossible to remove from
macromolecular mixtures by dialysis. The series of column chromatography steps
employed throughout these analyses would not have separated the Triton X-100 from
macromolecules. Attempts to remove the detergent by adsorption onto polar SM2 Bio-
beads (Bio-Rad) was attempted but resulted in the removal of a high percentage of 125 counts from the samples tested. This was not deemed a feasible approach. No further analytical steps were taken to resolve this question.
Another chemical cleavage method was attempted , to ascertain whether the Vo material after nitrous acid þH 1.5) might contain HS with significant quantities of the normally rare
N¡ree glucosamine residues i.e. neither acetylated nor sulphated. Nitrous acid at pH 4 cleaves the polymer at glucosamine residues. The results of this analysis were negative for all samples. Figure 3.15 shows the data for PANC and CFPAC medium (confluent cells) when exposed to the reagent and the digestion products analysed on Sephadex
G50. There was no significant shift to lower molecular weight products
3.3 DISCUSSION
Transformed pancreatic cells in tissue culture were chosen as an experimental system to explore whether sulphation differences would arise as a consequence of mutalions to
CFTR. CFPAC cells, established from the pancreatic adenocarcinoma of a ÀF'0, homozygous CF patient and PANC, our designated normal control of similar developmental and tissue origin, were partially characterised as a prelude to sulphation studies.
The evidence presented by Barasch et al. (991) which showed defective acidification in the Golgi apparatus and endosomes of CF cells was extremely relevant to the aims of this project, which was to find evidence and mechanisms for post{ranslational 126 processing abnormalities in CF. As a consequence we measured the acidifying potential of endosomes and lysosomes isolated from pancreatic cells. Lysosomes from both cell
lines acidified at a similar rate and to a similar extent in acridine orange lluorescence assays but we were unable to purify a significant endosomal population from either cell
line. We were primarily interested in the differences Barasch et al. (1991) had attributed to CF endosomes. Their approach to demonstrating these effects had utilised
intracellular partitioning of the weak base DAMP (3-Q,4-dinitro anilino)-S' amino-N-
methyldipropylamine) to acidic vesicles, and detection by electron microscopic
immunocytochemistry. lt was concluded from the preliminary experiments we had
performed with subcellular fractionation and acidification of isolated organelles, that the techniques we were using were unlikely to be sensitive enough to demonstrate what
might be subtle differences between our CF and control cell lines. Consequently this line
of experimentation was not pursued further.
The CF defect is characterised by a failure of p-adrenergic stimulation to initiate the
opening of chloride channels. lt was reasoned that at least a part¡al characterisation of
the chloride secretory behaviour of the CFPAC and PANC cell lines was required as a
pre-requisite to further studies on sulphation which were the main aim of this project.
The methodology we employed was based on 'uCl radioisotope efflux. This approach to
examining the regulation of anion flux is relatively insensitive compared to the technique
of patch clamping which can examine single, ion channel conductances as well as single
channel regulation. Chloride transpon activated by the release of intracellular Ca" was
shown to be intact in CFPAC and PANC but there were quantitative differences in the
degree ol responsiveness. CFPAC cells appeared more sensitive to stimulation by the 2500 a
2000
E I o 1 500 o- C) I I 1000 _ cr)
1
500 t-
0 1 200
1000 E o 800 ro 3 600 I (f) - 400 200
0 0 2 3 456789101112 fraction number
FIGURE 3.15 NITROUS ACID (PH 4) ON PEAK III MACROMOLECULES FROM MEDIUM: Material from medium (confluent) samples that remained undegraded after chondroitinase ABC digestion and nitrous acid deamination at pH 1.5 were subjected to nitrous acid at pH 4 as described in 2.2.17. The products of this reaction were chromatographed on Sephadex G50, 1 ml fraclions collected and 100 ¡rl aliquots scintillation counted. Due to relatively low numbers ol radioactive counts the samples were counted for 10 minutes to attain greater counting accuracy. a) PANC b) CFPAC 12 I
Ca" ionophore than PANC cells. Neither cell line could be shown to respond to forskolin even though forskolin elficacy in stimulating chloride efflux was demonstrated in T84 cells. Basal efflux of chloride was greater in PANC than in CFPAC possibly as a consequence of the CF defect. Limits to the sensitivity of the assay combined with low levels ol CFTR expression in PANC may have thwarted our attempts to visualise cAMP- dependent channel opening. Yoshimura et al. (1991) have estimated that T84 cells express 20 CFTR mRNA transcripts/cell, whereas other epithelial cell lines tested had an average o10.2 copies/cell. This 100 fold difference in transcript levels might renderthe detection of stimulated chloride efflux by our assay, unlikely. Unfortunately, at the time of these experiments, this information was unknown to us. Ward et al. (1991) tested four epithelial cell lines for the presence of CFTR mRNA using PCR of poly (A). RNA and demonstrated a 1000 fold variation in the levels of CFTR transcripts. PANC CFTR transcripts were est¡mated to be only 1-5% of that seen in T84. Therefore we concluded our inability to demonstrate cAMP-dependent chloride efflux in PANC did not invalidate its use as a normal control.
Metabolic radiolabelling experiments were carried out to ascenain whether CFPAC over- sulphated some or all of its glycoconjugates. lnitial work focused on characterising the glycoconjugates produced by each cell line. Anion exchange chromatography of
'medium'and 'cell layer'samples on DEAE-Sephacel revealed three peaks. Peak ll was demonstrated to be hyaluronan whilst peak lll contained considerable amounts of CS/DS and HS. Peak l, likely to contain glycoprotein, was analysed for ìhe presence of high molecular weight material which might have constituted a 'mucin-like' molecule. CFPAC cells synthesised two peaks ol high molecular weight material whilst PANC produced 129 none. A comparison ol mucin produced by the two cell lines was therefore not possible.
There was little dilference between the proportions ol glycoconjugates synthesised when cells were pre-confluent compared to when conlluent. PANC cells under both conditions produced almost twice as much hyaluronan as CFPAC. Each cell line produced similar quantities of GAG. 'Cell layer' fractions had l¡ttle hyaluronan or sulphated GAG associated with them. The majority was secreted into the medium'
A comparison of [3'S]:['H] ratios in peak I material revealed low levels of sulphation but
CFPAC macromolecules had 1.4-2.2 times higher ratios than PANC, possible evidence of a generalised over-sulphation by the CF cell line. These differences in the sulphation of peak I material are intriguing but difficult to define. They may represent a generalised over-sulphation of glycoprotein, presumably occurring on tyrosine residues or at glycosylation sites, or possibly a subset of molecules which CFPAC specifically sulphates to higher levels. Some or all of the difference could be due to the high molecular weight
(mucin?) species that CFPAC synthesises but that PANC does not. The ditficulties inherent in attempting to precisely define the chemical diÍferences between lowly sulphated molecules in a highly heterogeneous population were a major reason for our decision to focus on the GAGs as model substrates for sulphation.
Peak lll material containing most of the sulphate, was also higher for CFPAC samples than for PANC, but to a lesser extent. Samples ranged from 10-507o higher in ["S]:['H] ratio. An examination of GAGs within peak lll revealed that CS/DS produced by CFPAC was over-sulphated compared to the normal control by 1.3-2.3 times. CS/DS produced by conlluent cells had higher ratios than that produced by pre-confluent cells. An 130 analysis of the ["S]:['H] ratios in peak lll GAGs that had been depleted of CS/DS, revealed no evidence lor over-sulphation by CFPAC. This indicated that the over- sulphation of CS/DS by CFPAC may be specific to this class of GAG. This was unexpected and slightly perplexing, but possibly explicable by the observations of Uhlin-
Hansen and Yanagishita (Uhlin-Hansen and Yanagishita, 1993). They were able to demonstrate in rat ovarian granulosa cells that the synthesis of HS and DS occurred in different regions of the Golgi apparatus and that the entire complement o{ biosynthetic enzymes required for each molecule was compartmentally discrete. lf this were true for pancreatlc cells then a compartmentalisation of sulphate pools (for which we present experimental evidence in Chapter Six) might also occur, with the result that newly synthesised HS might have a different specific activity of l"S]sulphate from CS.
The presence of HS in peak lll was tested for, by nitrous acid. The D-glucosamine residue which occurs at every second saccharide position in HS is usually either N- acetylated (GlcNAc) or N-sulphated (GlcNSO.). Heparan sulphate chains may carry
GlcNAc containing disaccharides interspersed with GlcNSO. containing disaccharides or may exhibit blocks of disaccharides containing only GlcNAc or GlcNSO. residues (Guo and Conrad, 1989). These regions, high in N-sulphation and separated by variable length stretches of N-acetylated disaccharides, have been postulated to be functional domains which may provide a template for binding to and organising structural proteins in the ECM (Gallagher et al., 1992). Nitrous acid at pH 1.5 cleaves the HS polymer at
GlcNSO. residues but not at GlcNAc residues. ln this reaction the GlcNSO. residues are converted into 2,5 anhydro-D-mannose with the release of SOo* and Nr. Hence, where there are blocks ol high N-sulphation the product will be mainly disaccharides, but the 131 blocks that contain high numbers of GlcNAc residues are converted into a heterogenous mixture of tetra- hexa- and greater oligosaccharides. HS was demonstrated ¡n peak lll by the use of nitrous acid. There was some indication after analysis of the digestion products of a reduced degree of N-sulphation in the HS produced by CFPAC cells. This would require further analysis to confirm
ln conclusion, there appears to be evidence for a specific over-sulphation ol chondroitinidermatan sulphate by the CF cell line (mean 67%o highet, î=4) and possibly a generalised over-sulphation of glycoprotein appearing in peak I (mean 123o/o higher, n=4). This experimental system may therefore be appropriate for an examination of sulphate metabolism in CF cells and may allow a mechanistic assessment of how sulphate metabolism is perturbed.
135
4.1 INTRODUCTIO N
Because the most severely alfected tissues in patients with cystic fibrosis are those with an epithelial integument, the proposal that CFTR is expressed in cells ol non-epithelial origin has been controversial. This debate exists, in part, because ol the extremely low levels of CFTR expression claimed for many non-epithelial cell types (Yoshimura et al.,
1991). The advent of powerful techniques for amplifying low abundance mRNA species has raised the question of whether they have any physiological signilicance (Guo and
Conrad, 1989; Harris, 1992). Despite early failures using traditional techniques such as
Northern blotting (Riordan et al., 1989) there is now a growing literature to support the thesis that lymphocytes express CFÏR.
Chen et al. (1989) used patch clamping to demonstrate the presence of CAMP- dependent chloride channels in B and T lymphocyte cell lines. These channels were not activatable in CF lymphocytes. However, Hagiwara et al. (1989) were unable to duplicate the cAMP-dependency of channel opening noted by Chen ef a/. Bubien ef a/.
(1990) made the intriguing observation that the appearance of cAMP-dependent chloride currents in lymphocytes appeared to be cell cycle dependent. Using both patch clamping and SPQ (a chloride sensitive fluorophore) to assess macroscopic chloride fluxes, they demonstrated that cells which had been synchronised at the G'-S phase ol the cell cycle had chloride conductances which could be stimulated by elevated oAMP.
Correspondingly, these conductances were absenl in CF derived lymphocytes. The appearance of CFTR-like chloride conductances at G,-S, but not at the Go or S phases of cell growth were postulated to be the result of chloride channels either being expressed or becoming active, and may have explained the dilliculty some groups had in reliably demonstrating this channel activity in lymphocytes. Further published studies by the same group confirmed the original observations. By utilising antisense oligonucleotides against the first 36 nucleotides downstream of the start codon for CFTR, they eliminated cAMP-dependent chloride currents in a normal B lymphoblast cell line, thereby elfectively inducing a CF phenotype (Krauss et a1.,1992a). Cells were shown to possess mRNA for CFTR, but consistent with the low levels of expression seen by others in these types of cells (Yoshimura et a1.,1991; Ward et a1.,1991 ; Bremer et al., 1992), Krauss ef at. (992a) were unable to demonstrate the presence of CFTR protein. ln another study they showed that transfecting the gene for wild type CFTR into a CF B lymphoblast line restored the appropriate channel activity (Krauss et a\.,1992b). Although several groups were in agreement about the presence of CFTR in lymphocytes, there were interesting differences in the characterìstics they ascribed to the channel. McDonald et al. (992) demonstrated CFTR mRNA in B and T cell lines and their patch clamping studies recapitulate the findings of others with regard to activation of a CFTR-like chloride channel. However, they were unable to demonstrate any dependence of chloride eonductances on cell cycling. Another poìnt of contrast between the two groups, was the finding by Krauss et at. (992b) that not only were cAMP-dependent currents atfected in
CF lymphocytes, but Ca,.-dependent chloride fluxes also. These were restored upon transfection of the gene for wild type CFTR. McDonald et al. (992) however, found no evidence for an impairment of Ca2'-dependent chloride currents ìn CF cell lines. lt appeared therefore that whilst there was good evidence for lunctional CFTR in lymphocytes, the precise nature ol its regulation and role remain to be determined. 137
The physiological signilicance of the CF defect being expressed in lymphocytes is currently unknown, but the prospect that it may play an immunomodulatory role in the pathogenesis ol CF explains the high level of interest in the question. A number of studies have pinpointed altered immune responses in CF e.g. hypogammaglobulinemia
(Matthews et a\.,1980), and reduced helper and cytotoxic T cell function (Knutsen et al.,
1988; Lahat et al., 1989). Whether these elfects are linked to defective CFTR in lymphocytes awaits experimental evidence.
As there was sufficient reason to believe that lymphocytes possessed CFTR, it was decided they would provide a suitable experimental model with which to investigate over- sulphation in cystic fibrosis. The availability and accessibility through cell culture repositories of Epstein Barr virus (EBV) transformed lymphoblast cell lines made them a convenient cell type with which to experiment. However, it was also felt they olfered the opportun¡ty to address a number of other important questions. Firstly, are defects or alterations to sulphate metabolism a feature of cell types other than epithelial cells?
Secondly, if differences could be demonstrated, how might they contribute to an impairment of the immune system in CF? Although many of the immunoevasive properties ol Pseudomonas aeruginosa have been elucidated it remains an attractive proposition that some alteration to the functioning of the hosf immune system allows the establishment and subsequent rampant colonisation of the lungs.
ln terms of the potential impoftance of sulphation, it has been demonstrated that proteoglycans are constitutively synthesised by lymphocytes and that synthesis is 1 markedly stimulated by lymphocyte activation (Bartold et al., 1989). Thus, elevated proteoglycan synthesis (and necessarily, appropriate synthesis) might be related to cellular lunction in immune responses. Finally, using lymphoblasts allowed us to investigate the hypothesis that alterations to sulphate metabolism might be CFTR genotype-dependent i.e. sulphation might be aflected to greater or lesser degree depending on the type of mutation carried. As genotype/phenotype correlations remain the 'holy grail' ol human biochemical genetics, it was an important reason for attempting to define sulphation defects in lymphoblasts.
4.2 EXPERIM L AIMS
To study the sulphation behaviour of lymphoblasts with different genotypes and to test the hypothesis of Bubien et at. (1990) that CFTR was expressed or activated at G'-S of the cell cycle in normal lymphoblasts. lf true, then we should maximise any sulphation differences being manifested and hence our ability to detect them.
4.2.1 CELL LINES:
Six EBV transformed lymphoblast cell lines were obtained lrom the Coriell lnstitute for
Medical Research. These lines comprised two normals (GMO3714, GMO3299 - subsequently referred to as N'l and N2 respectively), two CFs of undefined genotype
(GMO7227A, GMO433O - referred to as U1 and U2 respectively), and two ^F'* homozygotes,(GMO7904, GMO454OA - DF1 and DF2 respectively). The controls and cell lines of undefined genotype (U1 and U2) were chosen because they were the same cell lines used by McDonald et a/. (1992). ln this study they demonstrated the presence 1 of CFTR mRNA (by reverse transcription PCR) and cAMP-dependent chloride fluxes in the normals, and a failure to reproduce those currents ¡n the CFs. This had the advantage of providing validation about the chloride secretory behaviour of all lour cell lines prior to our experiments on sulphation. U2 was known to carry AFro, on one allele, but the other three alleles had no information provided about them except that they were not ÂFror. ln an attempt to determine whether they carried any of the more common CF mutations, Paul Nelson of the Dept. of Chemical PathologV (Women's and Children's
Hospital, Adelaide) kindly screened them for the presence of ÂFror, alro., G..rD, Rru.X and
G*rX mutations. None were identified except for the AFuo, allele on U2 as expected
Although of undefined genotype, both CF cell lines were derived from donors who experienced severe CF. lnformation provided by the cell repository noted that donor U1 was pancreatic insufficient with elevated sweat electrolytes, whilst U2 experienced severe clinical episodes including bronchitis and pneumonia and required hospitalisation for the illness and secondary complications.
4.2.2 SPECIFIC METHODS:
Petri-dishes were set up containing an I ml cell suspension supplemented with appropriate radiolabels (see section 2.2.11 for details). After 24 hours incubation, the dishes were swirled to resuspend cells and 4 ml removed. The remaining 4 ml was incubated for a further 48 hours, before cells and medium were harvested. After butfer exchange on Sephadex G50, GAGs were purified on DEAE-Sephacel. This was achieved by elution with 1 M NaCl in dissociative buffer, after the unbound material at
0.15 M NaCl had been collected. As there was only one peak ol bound material, gradìent elution was not considered necessary. 1
CS/DS disaccharides were subjected to analysis by HPLC. This system allowed good separation of unsulphated, monosulphated and disulphated disaccharides by elution from the strong anion exchange matrix with a NaCl gradient. lt was also able to separate monosulphated disaccharides (with sulphate esters at dilferent carbon positions) although to a lesser degree. Because they eluted closely together the interpretation ol the data was confined to the proportions of unsulphated, monosulphated and disulphated disaccharides in each of the samples.
ln the cell cycle synchronisation experiment, four lymphoblast cell lines were tested (N1,
N2, DF1 and DF2). All four were synchronised at G,-S of the cell cycle as described in section 2.2.11(Krauss etal.,1992b). ln total eight samples (4cell lines synchronised,4 cell lines unsynchronised) were radiolabelled lor 24 hours. Only the 'medium' was analysed in this experiment.
4.3 RESULTS
4.3.1 CHARACTERISATION OF GLYCOCONJUGATE SYNTHESIS BY
LYMPHOBLASTS:
(N1) The profiles obtained forthe'cell-associated'and'medium'fractions of one normal
and one CF (U1) cell line are illustrated in Figure 4.1. The other cell lines gave
comparable results (not shown). Macromolecules from all samples and all cell lines
produced separated into two distinct peaks. This was in contrast to the glycoconjugates
by pancreatic cells, which partitioned inlo three peaks, and was due to the absence of 141 hyaluronan. This GAG was not synthesised in any appreciable quantity by lymphoblasts.
Like pancreatic cells there were only small amounts of the highly sulphated component we had previously identilied as GAGs, present in 'cell-associated' material. Most had been constitutively secreted during the period of radiolabelling. Chondroitinase ABC digestion revealed that 62.2 ! 6.4% (mean + S.D., n = 24) of the GAG produced by all cell lines was CS/DS
4.3.2 ["Sl:['HI RATIOS lN GAGS PRODUCED BY CF AND CONTROL
LYMPHOBLASTS:
['uS]:['H] ratios in GAGs isolated from cells and the'medium'were combined for each pair of cell lines and compared (Fig. 4.2). Each bar of the histogram represents the mean of four samples derived from two cell lines. lt was noted that the ratios were essentially unchanged at 72 hours compared with 24 hours. The second observation to be made was that there was little difference between the GAGs produced by the normals and the
CF lymphoblasts of undefined genotype, however there was a difference displayed by the lymphoblasts. When the data was analysed by two factor ANOVA with ^F.o,i^Fuo, repeated sampling (24 and 72 hour) the difference attributable to genotype was statistically significant (p<0.05). There was no significant effect of radiolabelling time on the ratios
The GAGs from all 24 samples were digested with chondroitinase ABC and the [3sS]:[3H] ratios of both susceptible and resistant material examined for evidence ol genotype consistent differences (Fig. 4.3a, and b). Two factor ANOVA (with repeat sampling) revealed that the differences in CS/DS ratios exhibited by the AF,oB/^Fso, lymphoblasts 142 FIGURE 4.1 DEAE-SEPHACEL ANION EXCHANGE CHROMATOGRAPHY
OF LYMPHOBLAST GLYCOCONJUGATES:
Cells were prepared as described in section2.2.11, resuspended at 0.5 x 10u cells/ml in
2 ml culture medium supplemented with l'uS]sulphate and ['H]glucosamine, then
radiolabelled for 18 hours. 'Cell-associated' and 'medium' samples were recovered,
buffer exchanged and subjected to chromatography on DEAE-Sephacel with elution by
NaCl gradient. Aliquots (50 pl) of 1 ml fractions were counted and the data corrected for
cellular protein.
a) N1 (Cell-associated), b) N1 (Medium),
c) U1 (Cell-associated), d) U1 (Medium). 600 600 1,000 a mM NaCl b O sl-l r35S 500 500 : 800
I
400 400 rñ I 600
I 300 300
I (, l I a 400
I 200 I a 200 þ z c 9) (ú I o U' 200 J 100 ; 100 o o I f o o .c J o 0 -t o 0 0È. l- 800 o o- c 250 d f, o, 1,OOO E a 3 il E i 5 o_ oi o j 200 600 800
150 600 400
\y 100 400 I U
l 200
I 50 200
./ I
o 0 0 0 10 20 30 40o 10 20 30 40 fraction number 144 were h¡ghly signilicant (p<0.002) whilst there was no difference between the ratios incorporated into HS by these cells. There was no inlluence ol time for either CS/DS or
HS.
CS/DS disaccharides were analysed further by HPLC. We analysed twenty three of the twenty four samples on a 4 x 50 mm Dionex Propac PA1 strong anion exchange column according to the method of Turnbull et al. (1992). Despite our inability to accurately quantitate individual disaccharide species (because the 45, 25 and 65 species eluted closely together), all samples were characterised by a high proportion of 4-sulphated disaccharides. ln fact many of the samples did not exhibit a distinct 63 peak at all with most of the radioactivity confined to the 45 disaccharide.
Quantitation of the variously sulphated disaccharides (Table 4.1a and b) revealed that for all disaccharide samples the unsulphated component was relatively minor (less than
(in 10% for 20 of 23 samples) and the monosulphated component dominated all cases greater than 74o/o). Thus there didn't appear to be an obseruable shitt towards a more highly sulphated phenotype by the AF,o, homozygous lymphoblasts. Two-factor ANOVA with repeated sampling confirmed that there were no significant differences between the mean quantities of unsulphated, monosulphated and disulphated disaccharides. There was also no evidence for a ditference between 24 and 72 hour samples. Because the composition of CS/DS chains was unchanged between cells of ditferent genotype' the question arose as to how to explain the significantly different ['uS]:['H] ratios in chondroitinase ABC digested material. The mean ['uS]:['H] ratio exhibited by ¿F'*
yet lymphoblasts was almost 40% higher than either the normal or undefined CFs and 0.7 ffi24 Hours -72 Hours 0.6
0.5
o (ú 0.4 I CY)
U)- ro 0.3 CÐ
0.2
0.1
0 normal undefined delta F/delta F
FIGURE 4.2 ["Sl:['Hl RATIOS lN GAGs SYNTHESISED BY LYMPHOBLASTS AT 24 AND 72 HOURS: GAGs isolated by DEAE-Sephacel chromatography were scintillation counted and the ratios calculated from dpm. Bars represent the mean of 'medium' and 'cell-associated' fortwo cell lines i.e. n=4. Error bars represent S.D. 46 there was no compositional difference seen after HPLC analysis. The answer to this apparent anomaly was provided by the ["S]:['H] ratio within disaccharides. Figure 4.4 shows the mean ratios for the major disaccharide peak (4S). Two{actor ANOVA (repeat sampling) revealed there was no significant difference between the means of 45 disaccharide [.'S]:['H] ratios by the three cell types. However the differences were close to being significant (p<0.06). A comparison of the means showed that the ÂF'0, lymphoblasts with a mean ratio of 0.58, were 30 7o higher than the normals at 0.44. This explained most of the difference we had obserued between chondroitinase ABC digested samples.
4.3.3 INFLUENCE OF CELL CYCLE SYNCHRONISATION ON
LYMPHOBLASTS:
Tabf es 4.2a and 4.2b detail the ["S]:['H]ratios in macromolecules secreted by the four cell lines under standard and cell synchronised conditions. Synchronisation had no effect on ["S]:['H] ratio, either on the normals or on the CFs. This applied equally to the glycoprotein peak and the GAG peak. Bracketed figures are the ratios generated by the same cell lines in a previous radiolabelling experiment. Their close agreement provided evidence that the metabolic activities we were interested in were reproducible in these cell lines. When the ['uS]:['H] ratios of GAGs produced by the CF cells were compared with controls, it appeared that they were higher by approximately 507o (comparing means of unsynchronised cells). The ["S]:['H] ratios for glycoprotein material from the AF'o' CFs also appeared higher, but were less convincing when the synchronised values were also considered. 147 s24 Hours r72 Hours 0.8 a
o 0.6 (ú I (Ð 0.4 - Ø LrO cÐ 0.2
0 normal undefined delta F/delta F 0.5 b
o.4 o (ú L- 0.3 I c)
Ø o.2 LrO CÐ
0.1
0 normal undefined delta F/delta F
FIGURE 4.3 ["SI:['Hl RATIOS lN CS/DS AND HS SYNTHESISED BY LYMPHOBLASTS OVER 24 AND 72 HOURS: GAGs were isolated by DEAE-sephacel chromatography, digested with chondroitinase ABC and the products analysed on 9 ml Sephadex G50 columns. Data is presented as the mean value obtained from 'cell-associated' and 'medium' for two cell lines i.e. n=4. Error bars indícate S.D. a) CS/DS b) HS 148 TABLE 4.1a
MEAN PROPORTIONS OF DISACCHARIDES IN'CELL.ASSOCIATED' CS/DS
7.9 88.1 4.0 6.3 85.2 8.6
8.3 85.3 6.4 5.1 83.6 11.4
3.7 90.9 5.5 4.5 85.4 10.2
TABLE 4.1b
MEAN PROPORTIONS OF DISACCHARIDES IN'MEDIUM' CS/DS
6.8 90.3 3.0 6.6 BB.1 5.4
7.8 89.1 3.2 9.4 83.B 6.8
5.7 84.4 9.9 8.7 83.6 7.8 149
0.8 ffi24 Hours -72 Hours
0.6
_9 (ú
-(Y) 0.4 (n LO CÐ
o.2
0 normal undefined delta F/delta F
FIGURE 4.4 MEAN ["SI:['HI RATIOS lN THE 45 DISACCHARIDE SYNTHESISED BY LYMPHOBLASTS: Ratios were calculated from dpm. Data is presented as the mean value obtained from 'cell-associated' and 'medium' for two cell lines i.e. n=4. Error bars indicate S.D. 150
TABLE 4.2a
["Sl:['HI RATIOS lN GLYcoPRorElN SEORETED BY OELL cycLE
SYNCHRONISED AND UNSYNCHRONISED LYMPHOBLASTS
0.019 (0.029) 0.027
0.018 (0.020) 0.020
0.027 0.027
0.024 0.021
TABLE 4.2b
['uSI:['H] RATIOS lN GAGs SECRETED BY CELL CYCLE SYNCHRONTSED
AND UNSYNCHRONISED LYMPHOBLASTS
0.94 (1.00) 0.80
1.29 (1.32) 1.38
1.64 1.71
1.85 1.80 15 1
The GAG containing fractions were pooled and subjected to chondroitinase ABC digestion. Examination of Figure 4.5 reveals that none of the cell lines altered their
['sS]:['H] ratio in either CS/DS or HS in response to synchronisation. Therefore, if CFTR was expressed by normal lymphoblasts only at G,-S of the cell cycle, it didn't appear to have any phenotypic impact on either the types of GAG synthesised or the extent of their sulphation. The aFro, homozygous cell lines both had higher ["S]:['H] ratios than the two normals, confirming earlier evidence of an apparent over-sulphation. This trend was repeated for the HS containing fractions, albeit less strongly. When a two-factor analysis of variance (ANOVA) was applied to the data, the difference between the means for the control and CF cell lines was significant for CS/DS þ<0.01) and for HS (p<0.05). There was no significant difference between synchronised and unsynchronised cells. Thus there was no evidence that cell cycle synchronisation had affected sulphation
4.4 DISCUSSION
The classification scheme proposed by Welsh and Smith (1993) grouped CF mutations into four classes (see section 1.6.6) each with potentially differing effects on the molecular dysfunction. We attempted to control for this variable by obtaining cells of known genotype. AFro, homozygotes were chosen because the deletion is the most commonly occurring mutation in CF and also the most well studied in terms of its biosynthesis and residual channel characteristics. Homozygosity would eliminate any complex phenotypic expression which might occur in compound heterozygotes. Another reason for studying the effect of this mutation was that ÂFro, is strongly associated with 1 pancreatic insufficiency and therefore severe CF. lt was hypothesised that a severe mutation was more likely to exhibit sulphation abnormalities than milder ones.
Analysis ol newly synthesised glycoconjugates on DEAE-Sephacel provided us with two pieces of information. Firstly that there was little hyaluronan synthesised by this cell type, which simplified our approach to the purification of GAGs; and secondly, that cells were secreting large amounts of GAG. The majority of GAG synthesised was CS/DS with this species accounting for approximately two-thirds of the total.
When the [.'S]:[.H] ratios in GAGs were analysed, the AFuo, homozygous cells exhibited significantly higher ratios, whilst there was no difference between the normals and CFs of undefined genotype. This data presented strong evidence for the existence of CFTR related abnormalities in lymphoblasts and for a genotype specific effect. Unfortunately the effect is modest and it is difficult to speculate on the possible role of ÂF.0, in this process without knowing the nature of the CF mutations carried by the other cells. ÀFror-
CFTR is biosynthetically arrested in the endoplasmic reticulum. Other mutations resulting in altered channel properties or unstable mRNA for example, might interact with the metabolic pathways involved in GAG synthesis in different ways or not at all. lt may be some property peculiar to the ÀFro, mutation or the class of mutation that it belongs to.
The existence of genotype specific effects on glycoconjugate synthesis has a number of implications. lt may be part of the reason that altered sulphation and other metabolic abnormalities have been difficult to reliably demonstrate. Samples derived from patients or cells with different genotypes may have served to produce conflicting results. 1 2.5 EUnsynchronised a Isynchron¡sed
2
.9 (s 1.5 I CÐ - Ø ro 1 (f)
0.5
0 N1 N2 DF1 DF2
mUnsynchronised 1.6 Isynchronised b
o 1.2 (ú
(Ð - 0.8 U) ro cf)
o.4
0 N1 N2 DF1 DF2
FIGURE 4.5 ['sSl:['HI RATIOS lN GAGs SYNTHESISED BY CONTROL AND
AF.OU HOMOZYGOUS LYMPHOBLASTS:
GAGs were isolated by DEAE-Sephacel chromatography, digested with chondroitinase
ABC and the products analysed on 9 ml Sephadex G50 columns. Data calculated from cpm. a) CS/DS b) HS 154
Certainly we have shown here that two cell lines from CF patients are not different from
two normals. lt may also provide a partial explanation for the spectrum of clinical severity
encountered in CF
Analysis of individual GAG species revealed an intriguing result, with CS/DS synthesised
by the ÂFso, c€lls apparently very different from HS. The alteration to ["S]:['H] ratios
appeared to be specific to one class of GAG. A similar observation was noted in CFPAC
cells (also homozygous for ÂFroJ, and the observations of Uhlin-Hansen and Yanagishita
(1993) may be relevant in explaining how such an effect might be manifested (see
'Discussion' Chapter 3)
HPLC was used to define precisely what type of CS/DS molecule the various
lymphoblast cell lines were synthesising and whether there were shifts in the pattern or
extent of sulphation. GAGs synthesised by haemopoietic cells have a number of
distinctive structural features. One of these is that the great majority of peripheral blood
cells (e.g. lymphocytes, granulocytes, platelets, monocytes, NK cells) synthesise simple
homopolymeric chondroitin sulphate chains which are predominantly 4-sulphated (Kolset
and Gallagher, 1990). Our findings are in agreement with this. ln fact the analysis
showed no difference in the structure of CS/DS synthesised by the ÂF,0, cells. However the differences in ["S]:['H] ratio did appear explicable upon examination of the individual
disaccharide peaks. lt is interesting to speculate on how a 45 disaccharide synthesised
by one cell can have a different ratio from that produced by another cell. They are an
identical molecular entity with one sulphate ester. The most obvious explanation for the
30% difference exhibited by the was that one of the pools from which the ^F*/s, 155 precursors were derived i.e. the sulphate pool or the glucosamine pool, may have possessed different specilic activities for the radioisotope. As the pulse radiolabelling of cell lines was carried out over a relatively long interval i.e. 24 and 72 hours and there was no significant dilference between the ratios recorded at each time point'o then the altered specific activities could not be explained on kinetic grounds . Differences in the specilic activity of equilibrated precursor pools are possibly a reflection of the size ol those pools. ln simple terms a smaller pool might have a higher specific activity for the substrate in question. This observation led us to an important realisation, namely that higher ['uS]:['H] ratios did not necessarily predict that higher amounts of sulphate were being incorporated. That conclusion relies on the assumption of equal pool sizes for the radiolabel precursors of interest.
An examination oi the effect of synchronising cells at G'-S of the cell cycle found no differences in ["S]:['H] ratios induced as a result of the manipulation. Therefore we can provide no further support to the hypothesis of Bubien et al. (1990). This experiment did however lend more weight to the observations of altered ["S]:['H] ratios being exhibited by ÂFuor/AFro, lymphoblasts, and in fact provided some evidence that HS was ditferent also (an obseruation which had not been seen in the initial experiments). The apparently conflicting nature of these results points to the need for further experiments with a greater number of cell lines in order to confirm the observations we have made. lssues which remain to be resolved are:
'.This assumption was tested by application of the Wilcoxon signed-ranks test for tvvo-related samples - a non-parametric test which doesn't require the assumption of a normal distribution. When applied to both medium and cell-associated samples there was no evidence to reject the null hypothesis lha|72 hour ratios were unchanged from 24 hour ratios. See Appendix A for working. 156
1. can the ellect of the mutation be conlirmed, ^F5o8 2. can other mutations (and for these studies, cells with fully delined genotypes
would be necessary) be shown to exhibit similar effects,
3. is HS aflected to a lesser degree than CS and what might the mechanistic
basis for this specificity be
ln conclusion, no evídence was found for an over-sulphation of macromolecules synthesised by CF lymphoblasts. There was some indication of a difference in the way
lymphoblasts utilised glycoconjugate precursors intracellularly. The effect ^F508/^F.0, however, if real, was subtle.
5.1 INTRODUCTION
Remarkable progress in cystic fibrosis research in recent years has combined with recently developed gene{argeting technologies to enable the design and development of animal models for CF (Bronson and Smithies, 1994). Three years after identification of the CF gene, the first 'CF mouse' containing targeted interruptions to its CFTR gene was developed. Known colloquially as 'knockouts' these animals have provided researchers with an invaluable tool.for developing new therapies lor CF as well as greater understanding of the pathophysiology of the disease (Alton et al., 1993; Gabriel et a1.,1993).
The first CF mouse contained an engineered chain termination codon at position 489 of the murine homologue of the CFTR gene (Koller et al., 1991). This artificial mutation
(S489X) which resulted in a mRNA truncated in exon 10, mimicked a naturally occurring human mutation known to result in severe CF. A number of other mouse models were generated shortly afterwards and each appeared to have slightly differing characteristics in terms of the pathology exhibited (Dorin et al., 1992; Oneal et al., 1993; Ratcl¡ff et al.,
1993). As the work conducted in this project was based on the'UNC mouse'further discussion is limited to the characteristics of that model.
Mice homozygous for S489X exhibited pathology which was highly reminiscent of the meconium ileus otten seen in human infants born with CF. The mice exhibited a high rate of morlality in the first weeks of life, with approximately 90% dying prior to thirty days 160
of age as a result ol severe intestinal obstruction by muco-lecal material. Animals which
died prior to weaning tended to exhibit a pattern of ileal blockage, whilst those dying atter
weaning usually had obstructions of the large intestine (Snouwaert et al., 1992)
Localisation studies using ribonuclease protection assays of CFTR mRNA in normal
mice, found high levels of CFTR expression in all regions of the gut. ln fact levels were
higher in the jejunum, ileum, caecum and colon than in any other tissue tested
lnterestingly, mice which survived the early weeks of life appeared to be quite healthy
and possessed normal longevity (personal communication, Dr Barbara Grubb)
Examination of other organ systems revealed a pattern of pathology somewhat dissimilar to CF in humans. The pancreas and liver of CF mice did not appear to be overtly
affected, but the gall bladder was often distended or ruptured. Perhaps disappointingly the respiratory tract appeared relatively normal. There were minor histological changes
apparent in the nasal sinuses with evidence of serous gland tissue atrophy. Glands in the nasal mucosa and proximal trachea showed dilation of ducts but there was no
evidence of mucus plugging, the hallmark of human disease.
Electrophysiological studies of gut and ainruays tissue from transgenic mice confirmed the
chloride secretory defect which is the defining feature of human CF. Thus, cAMP-
activated chloride currents could be demonstrated in airways and gut tissue of both
normal and heterozygous CF mice, but not in CFTRC/-) mice (Clarke et al., 1992a).
Further work revealed important differences in the ion-transport mechanisms utilised by
mice compared with humans. Nasal epithelia from CFTR(J-) mice exhibited a raised
potential difference which was consistent with elevated Na'transport and had a greater
basal equivalent short-circuit current. These features were analogous to the 1 electrophysiological signature noted with human CF patients. ln response to the Caa ionophore, ionomycin, normal murine nasal tissue did not secrete Cl- whilst CF mice exhibited a large rise in Cl- conductance (Grubb et a1.,1994b). These results suggested that CFTR(J-) mice had responded to the loss ol CFTR by upregulating Ca"-mediated pathways of chloride secretion. The authors speculated that this might provide an explanation for the lack of severe ainruays pathology in these mice. Studies of tracheal epithelium also yielded surprises, with the tissue responding quite differently from the nasal epithelium. Firstly, there was no difference in basal short-circuit current between
CFs and controls, and ionomycin stimulated large increases in the short-circuit current of both CFs and normals. Completely unexpected was the finding that forskolin, a B- adrenergic agonist which increases cAMP levels, also increased intracellulat Ca"' concentration. Murine tracheal epithelial tissue was therefore physiologically quite different from human and displayed no difference between CFTR(-i-) and normal animals. Again, the preponderance of Ca''dependent pathways in the regulation of chloride fluxes may have cushioned the severity of airways pathology in this animal model. A survey of the chloride transport capabilities of pancreatic, respiratory and intestinal tissues supported this hypothesis. There was a clear correlation between the level of Car.-regulated conductance and the protection of an organ from the elfects of disrupted CFTR (Clarke et al., 1994). lt has since been concluded that studies on the upper airways of CFTR(-/-) mice are likely to be a more informative model for assessment of gene transfer protocols because of their similarity to human airways epithelium, whilst the lower airways of mice have significant physiological differences from humans (Grubb et a1.,1994a). 1
The development ol an animal model lor CF allowed us to consider a new approach to the study ol sulphation in CF. lt was reasoned that injecting radioactive sulphate into groups ol control and CF mice might allow the metabolic labelling of tissues in vivo.
These tissues could then be harvested and analysed for levels and patterns of sulphate incorporation. The obvious advantages to this approach lay in circumventing limitations associated with studying glycoconjugates synthesised by cells in culture, or analysing the purulent and degraded components of expectorated sputum from human subjects - traditionally the only ways of addressing the problem. The other major advantage was that for the first time it would be possible to examine a large range of organs and tissues which are affected in CF and potentially identify tissue specific pathology associated with sulphation abnormalities. With the generous assistance of Professor Richard Boucher,
Dr Barbara Grubb and Dr Pi-Wan Cheng (University of Nth Carolina, Chapel Hill) a number of animals were made available for an experiment aimed at examining the effect of targeted mutagenesis of CFTR on the sulphation of GAGs by a murine model for CF.
5.1.1 EXPERIMENTALDESIGN
Four CFTRC/-) mice and five control mice were used in a blinded protocol. Nine mice
(four female, five male) whose genetic identities were unknown, were injected intraperitoneally with 2.6 mCi NaJ"SlSOo Radiolabel was purchased as 25 ¡rl carrier free ataconcentrationof 1.019Ci/ml. Sterilesaline(950rrl)wasaddedtothevial and 100p| aliquots injected into each animal. Females and males were caged separately, and then after 48 hours mice were despatched by lethal injection with sodium pentobarbital and twelve organs and tissue samples dissected from each. These were: ileum, caecum,
Colon, jejunum, spleen, liver, pancreas, gall bladder, lungs, trachea, naSal mucOSa and nasal septal cartilage. ln total there were 107 samples. Due to an oversight one ileum 1 sample was not collected during the dissection. All tissue samples were weighed and lyophilised. Analyses were performed without knowledge of which mice were CF and which were controls.
5.2 RESULTS
5.2.1 T'SISULPHATE INCORPORATION INTO MURINE GAGS: lncorporation of l"S]sulphate by CF mice and controls was compared for each of the tissues (F¡g.5.1). Unfortunatelyone recalcitrant mouse managed to avoid receiving the full dose of radiolabel at the injection stage. lnformation obtained later revealed that it belonged to the CF group. As a result, n=3 for incorporation data pertaining to CF mice.
When normalised for the amount of GAG present in each sample, there was a clear trend for most CF tissues to have higher l"S]sulphate incorporation than normal mice.
The exceptions to this trend were the lung which exhibited similar incorporation and the nasal mucosa which surprisingly showed the CF group to have markedly lower radiolabel incorporation. Also notable was the greater variability exhibited by the CF group. When tests of significance were considered for analysing the data, the higher variances exhibited by the CF mice were taken into account. Tables of critical values of the F distribution were used to test for homogeneity of variance. Data pertaining to trachea, gall bladder and nasal septum did not fulfil the criterion for homogenous variances and were therefore tested by t-test with an assumption of unequal variance . All others were analysed by standard t-test. GAGs isolated from the liver and pancreas of CF mice had 164 lleum Caecum
6 at U' E 5 Þc c (ú (d 5 at faD 4 o o E 4 r o (9 3 (5 3 o o) o, 2 f 2 f E E o- o- 1 õ 1 E
0 0 Normal's (4) CF's (3) Normal's (5) CF's (3) Colon Jejunum
7 I
t) an Eaû 'tt c C (ú C' 6 ah 5 at l J o o .c 4 ! o o 4 o 3 o ct¡ JO¡ f 2 2 E E o- CL E 1 io
0 0 Normal's (5) CF's (3) Normal's (5) CF's (3) Liver Spleen
7 5 U' 6 E Eu, c C (ú 4 (ú an 3t 5 =o J E o 3 ! 4 o o (5 3 2 o ol o, f J 2 E o- E ! o- E 1
0 0 Normal's (4) CF's (3) Normal's (5) CF's (3)
FIGURE 5.1 f'SISULPHATE INCORPORATION INTO GAGs: GAGs were extracted from murine organs as detailed in section 2.2.12. Aliquots were scintillation counted and GAGs assayed by the 'Stainsall' method. Error bars indicate standard deviation. Figures in brackets indicate the number of samples used to * calculate the data. Significance tested for by one-tailed t-test. indicates p<0.05. 165 Trachea Lung J
! C ".25C (ú (ú (h^ A^ az =Zo -c -c. (5 15 o=- 15 o (t o)1 o, 1 I' l' È E oo 05 oo 05
n Normal's (4) CF's (2 Normal's (4) CF's (3) Gall Bladder A/asa/ Mucosa 4 4
.D G ! ! c c J (ú 3 (ú ø o f l o * -c. -c. (t 2 (t 2 (t (t O) O¡ f l E E è o_ ! !
0 0 Normal's (5) CF's (3) Normal's (5) CF's (3) Nasa/ Septum Pancreas 25 *
ah ul b ! 2 C C (ú (ú ú, Ø l = o '1 5 E -c. 4 (t o (5 (, 1 o, o, f f E 2 E o_ 0 Ã
U 0 Normal's (5) CF's (3) Normal's (4) CF's (3)
FIGURE 5.1 (CONT'D) P'SISULPHATE INCORPORATION INTO GAGs: 166 significantly higher ['uSJsulphate incorporation than the controls (p<0.05). lnterestingly, the only other tissue to show a significant difference was the nasal mucosa in which the
CF group demonstrated a significantly lower l"S]sulphate incorporation (p<0.05). The gut tissues which were anticipated to be most likely to exhibit significant differences, did not do so. However the data for the ileum and the colon (the two regions of the gut implicated in lethal blockages in young CF mice) were both close to attaining significance
(p=0.06). This was suggestive of a CF related effect and indicated that further work with greater numbers of mice should be undertaken.
5.2.2 T'SISULPHATE INCORPORATION INTO MURINE
HEPARAN SULPHATE:
GAGs were subjected to chondroitinase ABC digestion and high molecular weight material which remained after digestion was subjected to nitrous acid deamination to test for the presence of HS. Figure 5.2 shows the BioGel P2 profile obtained when chondroitinase resistant material from the jejunum of a CF mouse was run on a column with, and without, nitrous acid treatment. The large arrow represents the position at which ["S]sulphate ran. Sulphate ion ran at a position inconsistent with its molecular weight and did so under all conditions testedls
It can be seen that macromolecules not exposed to nitrous acid, eluted in the void volume of the column as a sharp peak. Digested material displayed two high molecular weight peaks before the majority of sulphate eluted in a position consistent with that of
'uOonsideration of the hydrodynamic properties and interactions of anions in solution with nonpolar surfaces, is beyond the scope to this thesis. However for a discussion of the non-ideal behaviour of ions on gel sievíng matrices see (Collins, 1995). lPre-Nitrous Acid Vo ePost-Nitrous Acid 1 600 J 600
þ E o o- Ø 1f I 1200 f Ø 450 LrO o c) c Ø E 0) 'õ o. (ú o- a 800 f 300 (^) o (¡l CN =c I o- o E o- 3 400 150
0 0 10 20 30 40 50 60 70 fraction number
FIGURE 5.2 NITROUS ACID DIGESTION OF CHONDROITINASE ABC RESISTANT GAGS:
A GAG sample resistant to chondroitinase ABC and derived from the jejunum of a CF mouse was divided in two, and one half subjected to nitrous acid deamination (pH 1.5) as described in section 2.2.17. Both samples were then analysed on a BioGel P2 column equilibrated in 0.1 M ammonium bicarbonate as detailed in section 2.2'17 Fractions were collected and aliquots scintillation counted. The arrow indicates the
elution position ol l"S]sulphate. Vo = void volume of the column. free sulphate. This peak is likely to be mostly liberated N-sulphate. Elution of free l.uSJsulphate is followed by a number ol small l'uS]sulphate peaks which correspond to
O-sulphate groups on saccharides ol diminishing size. The l"S]sulphate associated with high molecular weight material is most likely O-sulphate esters on stretches of HS low in
N-sulphation and hence resistant to chemical cleavage. That peaks do not precisely coincide is due to technical reasons. Peak elution varied by one or two fractions between runs. All samples subjected to nitrous acid'u gave similar results and we concluded that with the exception of one tissue, most of the chondroitinase resistant material was HS. Cartilage contains high concentrations of the proteoglycan aggrecan, which has no HS. lt would be reasonable therefore, to assume that chondroitinase resistant material from the nasal septum was likely to be keratan sulphate rather than
HS. CS/DS in nasal septum represented 957o of the total GAG extracted (Table 5.1).
The data for [.'S]sulphate incorporation into HS is presented in Figure 5.3. Consistent with the data obtained for total GAGS, the pancreas and the liver of CF mice displayed significantly higher incorporation of l'uS]sulphate into HS. The nasal mucosa was no longer significantly different but the trend towards lower incorporation by the CF mice was maintained. ln another interesting finding, the nasal septum, which, when total
GAGs were considered had shown little difference between CF and normal mice,
showed a significant under-incorporation of sulphate into putative keratan sulphate by
CFs.
l.Because some to the tissues we attempted to analyse in this study were very small e.g nasal mucosa, nasal septum, gall bladder and trachea, the quantities of GAG extracted were small. This difficulty was compounded by the problem of decaying l"S]sulphate. We were thereiore unable to analyse the tissues listed above by nitrous acid. 169
TABLE 5.1
PROPORTION OF CHONDROITIN SULPHATE lN GAGs
* 74.0+3.7 (n=4) 66.7t2.5 (n=4)
64.9¡8.2 (n:5) 64.514.8 (n:4)
70.4t4.6 (n=5) 71.6t6.9 (n:4)
64.418.8 (n:4) 67.2t12.8 (n:4)
67.8r9.9 (n:4) 76.1x2.6 (n:3)
77.9!4.5 (n=5) 77.1+2.3 (n:4)
97.011.1 1n=5) 97.4x0.6 (n:3)
73.2¡S.B (n=S) 7 j.1x1.Q (n=4)
S0.Sr14.3 (n=5) 57.4x12.5 (n=4)
94.113.5 (n:5) 94.9t3.6 (n=4)
90.9t4.1 (n=5) 84.5¡2.1 (n:4) *
61.3t8.2 (n=5) 64.616.8 (n:4)
* Data expressed as mean + standard deviation. indicates p<0.05 for two-tailed t-test 1 lleum Caecum 35 5 T I Eat, c 4 (ú 2.5 at f o ! 2 3 U) U) 1.5 - I O) 2 o, f = 1 E E o- o- E ! 0.5
0 0 Normal's (4) CF's (3) Normal's (4) CF's (3) Colon Jejunum 6 6
U' 5 u, 5 ! E C c (ú (ú at o 4 f 4 l o o ! E 3 q) 3 U)- r o, O¡ f 2 f 2 E E CL o ! 1 E 1
0 0 Normal's (5) CF's (3) Normal's (5) CF's (3) Liver Spleen 4 * 7
u, 6 Eo ! C C (tt 3 (ú 5 .tt o l f o o E E 4 2 U) U)- T 3 o, fO¡ = 2 E E o- oo- !
0 0 Normal's (5) CF's (3) Normal's (5) CF's (3)
FIGURE 5.3 I''SISULPHATE INCORPORATION INTO HEPARAN SULPHATE:
On the basis of nitrous acid treatment, heparan sulphate was assumed to be the material remaining after chondroitinase ABC digestion of GAGs. Aliquots of high molecular weight mater¡al excluded from Sephadex G50 columns were scintillation counted and GAGs assayed by the 'stainsall' method. Error bars indicate standard deviation. Figures in brackets indicate the number of samples used to calculate the data. Significance differences were tested for by one-tailed t-test. " indicates p<0.05. Note: Nasal septum included in this figure is likely to be keratan sulphate rather than heparan sulphate. 171 Trachea Lung 4
at, ø ! õ C 3 1s (ú (tF u) l l o o _c. 2 u)l -U) - o, o, f È 05 oE o o !
0 0 Normal's (5) CF's (3) Normal's (5) CF's (3) Gall Bladder Nasa/ Mucosa
3.5 5
3 ø Ø ! 4 ! C (úc (! th 25 .lt f f o 2 -c. 2 E U) U) 5 I 2 -(', o) f f 1 E E è o_ E 0 5
0 0 Normal's (2) CF's (2) Normal's (5) CF's (3) Nasa/ Septum Pancreas o 4 * UI 5 !U' C C (ú 3 (ú at * Ø 4 f f o o E E J 2 U) IU) I o, o, 2 l f E E o o_ o E 1
0 0 Normal's (3) CF's (2) Norma s (5) CF s (3)
FIGURE 5.3 (CONT'D) T'SISULPHATE INCORPORATION INTO HEPARAN SULPHATE: 172
The gut regions which were of considerable interest, did not show significant differences
between groups of mice. However, the ileum with p=0.065 was close to exhibiting a
statistically significant difference. Data from colon samples, this time, required analysis
by t-test assuming unequal variances. lt did not approach significance in the same way
as the total GAGs from this organ had. The jejunum in this analysis exhibited a difference between groups which also came close to being significant (p=0.055)
5.2.3 ALTERATIONS lN CS/DS:HS RATIOS lN ORGANS FROM NORMAL
AND CFTRGÊ) MICE:
The proportion of GAG susceptible to chondroitinase ABC in each organ and for each group of mice, is summarised in Table 5.1. Because one CF mouse received the incorrect amount of l"S]sulphate, analyses requiring radiolabel incorporation data were not able to take into account the tissues from this animal. Compositional data from its organs were able to be used however. The proportion of CS/DS ranged from 50% in nasal mucosa lo 97o/o in the trachea, therefore CS/DS tended to be the predominant
GAG for all of the organs examined. Unexpectedly, two organs displayed a significantly altered proportion of CS/DS between CF and normal mice. The ileum and the gall bladder of CF mice contained significantly less CS/DS and by extension, significantly more HS.
5.2.4 ANALYSIS OF CS/DS DISACCHARIDES BY HPLC:
Figure 5.4 illustrates the HPLC absorbance profiles generated by CS/DS samples derived from a CF ileum (Fig. 5.4a) and a normal ileum (Fig. 5.4b). For several tissues quantitation ol peak areas from these types of profiles was not feasible. ln general they were the tissues of smallest mass, the nasal mucosa, nasal septum and gall bladder, none of which yielded enough material lor accurate quantitation. ln addition it was found to be impractical to quantitate the disaccharides of lung tissue because of a contaminant which eluted with the unsulphated disaccharide. Data for the remaining tissues is presented in Table 5.2 with the proportions of dilferently sulphated disaccharides presented as percentages of the total. The table lists both 4-sulphated data and the total proportion of monosulphated residues to illustrate the preponderance of the 4-sulphated species produced by all murine tissues
Of the tissues able to be examined, only the ileum and the liver exhibited a significant difference between the CF group and the normal group in the sulphation of CS/DS. The ileum, which in previous analyses had hinted at significantly higher sulphate incorporation, exhibited a clear structural alteration in the composition of its CS/DS
There was a relatively high level of zero-sulphation in normal mice which in the CFs was markedly reduced (this shift can be seen in the two HPLC profiles illustrated in Figure
5.4, where the amount of 0S disaccharide compared to 45 can be seen to change). This was accompanied in the CFs by a concomitant increase in the level of 4-sulphation and other monosulphated residues. The livers of CF mice had also increased the sulphation of their CS/DS chains, but for this tissue the change was accomplished by a decrease in mono-sulphation and an increase in di-sulphation
The extent of sulphation of CS/DS in dilferent organs may be appreciated more easily il we apply an index of sulphation. This simplifies the large amounts of data contained in
Table 5.2 and allows the comparison of single numbers to reveal trends. The index consolidates the percentages of zero sulphated, monosulphated and disulphated 174 FIGURE 5.4 HPLC ABSORBANCE PROFILES OF CS/DS DISACCHARIDES:
,^ôrnô )i^^^^l^^åA^^ hrr nhnnr{rnilinaca ÂFllì r{inacfinn nl êAêc evl¡acla¡l v9 vvrrvrqrvv^^ñ^'^tn¡{ v, v from murine organs, werclreeze-dried, desalted on Sephadex G10 and then borohydride reduced overnight as described in sections 2.2.18-2.2.20. Disaccharides were subjected to strong anion-exchange HPLC and the UV absorbance at 226 nm monitored. Samples lrom the ileum of a) a CF mouse and b) a normal mouse are shown. The elution positions of disaccharide standards on their own and when spiked into sample are indicated. Elution times of standards were cons¡stent to within + 0.1 minute. For full chemical definitions of disaccharides see'Abbreviations'. 4S d¡B d¡D a ll
OS
b
0 45 Time (minutes) 176 residues by assuming that a CS chain 100% zero sulphated has an index of 0, a chain that is 100% m-nozulphated has ah indêx of 1 ãnd a chain thaf¡s 100% disulphated an index of 2. The results are presented in Table 5.3. Excepting the ileum and liver, all organs, whether normal or CF, have sulphation indices between 0.86 and 1.01. This is consistent with the high level of mono-sulphation most organs exhibit. The normal ileum has an index of 0.73 which reflects the relatively high proportion of zero-sulphation whilst the normal liver is 1.08. These tissues in CF mice have exhibited an increase in the sulphation index, and this is seen in the ratio of CF:normal. W¡th the exception of the ileum, the liver and possibly the pancreas (which had higher di-sulphation and lower mono-sulphation but differences were not significant), the CF mice have synthesised a
CS/DS molecule that is identical to their normal counterparts i.e. ratios are indistinguishable from 1 .0.
5.2.5 SPECIFIC ACTIVITY OF FSISULPHATE lN THE 45 DISACCHARIDE
OF CS/DS: l.'S]sulphate incorporation data had suggested that GAGs were being over-sulphated by
CF mice and yet the fine structural analysis of CS/DS by HPLC, in most cases, did not support this conclusion. ln an attempt to find an explanation for this apparent anomaly, an analysis of the specific activity of l"S]sulphate in the most abundant disaccharide i.e. the 45 disaccharide was performed. The amount of 45 disaccharide was quantitated by measuring the peak area from chart recordings. Total dpm in the 45 peak of each sample was then normalised against the peak area and the quantity expressed as dpm/mm'. Results of this analysis are presented in Table 5.4. TABLE 5.2
DISACCHARIDE COMPOSITION OF CS/DS SYNTHESISED IN ORGANS OF CF AND NORMAL MICE
CONTROL CF CONTROL CF CONTROL CF CONTROL CF * 33112.5 (4) 8.914.5 *(4) 49.0t11.3 71.6x7.1n 61.1113.0 84.7t5.6 5.917.0 6.3t2.9
9.013.1 (4) 10.7t2.8 (3) 75.7¡6.1 76.2x3.7 81.0t5.1 79.8x2.1 10.0t2.8 9.610.8
5.8t0.9 10.4t4.5 (4) 11.2!4.7 (4) 77.1x3.3 73.1x10.7 82.2x4.9 83.1 t4.1 7.4!1.2 \l\¡
7.1!1.7 (5) 5.912.5 (4) 77.2t4.5 82.0t4.0 85.2t2.2 87.2t3.6 7.7x1.0 7.0x1.7 * 11.2t5.3 (4) 11.913.9 (4) 58.7t9.2 49.6t3.5 70.8t14.0 51 .2t5.1 18.7t14.8 35.3t2.2
16.312.5 (5) 20.3t3.3 (4) 71.5L4.8 64.h4.0 76.4x3.7 71.6x4.0 7.3t1.9 8.1t1.8
1.2x1.8 (5) 2.2x1.5 (4) 87.5x7.0 92.6¡2.7 98.8t1.7 97.8t1.5
17.3t11.2 (3) 17.9!4.0 (4) 68.3t5.4 67.0¡2.7 79.3t10.9 73.7x5.0 3.313.9 8.4t1.6
* ** Data expressed as mean t standard deviation. indicates p<0.05, indicates p<0.02 for two-tailed t-test. Numbers in brackets are n values 178
TABLE 5.3
SULPHATION INDEX OF CS/DS IN ORGANS
OF NORMAL AND CFTR('' MICE
0.97 1.33
1.01 0.99 0.98
0.97 0.95 0.98
1.01 1.01 1.00
1.08 1.22 1.13
0.91 0.88 0.97
0.99 0.98 0.99
0.86 0.91 1.06 TABLE 5.4
SPECIFIC ACTIVITY OF T'S]SULPHATE IN THE 45 DISACCHARIDE
OF CS/DS FROM ORGANS OF NORMAL AND CFTRGÊ) MICE
52.6x7.7 (4) 102.8149.8 (3)
51.117.3 (4) 73.8t31.7 (3)
60.6119.3 (5) 81.1140.2 (3)
58.719.6 (5) 89.3136.1 (3)
57.919.0 (4) 80.8136.3 (3)
63.1t17 .4 (5) 94.6132.2 (3)
10.912.4 (5) 12.715.0 (3)
53.9t11.6 (5) 75.3t20.1 (2\
40.8!21.4 (5) 16.3110.8 (3)
56.5127.8 (5) 80.8133.1 (3)
Data expressed as mean + standard d eviation. Units are dpm/mm'. Numbers in brackets are n values 1 80
There was a clear and consistent trend for all of the organs from CF mice, except the nasal mucosa, to exhibit a higher mean specific activ¡ty ol l"S]sulphate in the 45 disaccharide. The CF data however, exhibited wide variability. The reason for the variability was that whilst two animals displayed much higher values than the normals, the third CFTR(-/-) animal consistently exhibited values within the normal range. ln contrast, the normal group displayed much lower variability (the pancreas and nasal mucosa being exceptions). The mean percentage coefficient ol variation (%CV) for all ten normaltissues was 26.5% whilst for CFs it was 43-4o/o.
Non-homogeneous variances precluded the application of standard parametric analyses to test the data for significant differences. lt is noteworthy however that the means for each group i.e. the normals and CFs are very consistent. lf we preclude data from the trachea and the nasal mucosa (which responds to CFTR disruption in its own unique way), and calculate the mean of the means for each group of animals, an interesting comparison can be made. With n=8 organs for each group, the mean and standard deviation of the normal means is 56.814.1 and for the CFs is 84.8t10.0. Thus, the specific activity of l"S]sulphate in the 45 disaccharide, is remarkably consistent for all eight organs from normal mice (%CV=7.2) and only slightly less consistent for all eight organs from CF mice (%CV=11.8). This trend represents a mean specific activity
increase of 50% in CF tissues. 181
5.3 DISCUSSION
Results showing three organs with a statistically signilicant difference in ["S]sulphate incorporation into GAGs between CF mice and normal controls were both intriguing and somewhat counter-intuitive. The liver and pancreas in normal mice have very low levels of CFTR and yet exhibited the strongest changes in GAG sulphation when CFTR was disrupted. ln contrast, the gut contains the highest levels of CFTR and greatest signs of overt pathology, but failed to record statistically significant differences in [3sS]sulphate incorporation.
ln early studies of the animal model, the pancreas exhibited low level pathology, with enlarged acini containing eosinophilic material in two out of five CF mice, whilst no lesions were found in the livers of relatively young CF mice (Snouwaert et al., 1992). ln another study, the weights of pancreata from CF mice were found to be significantly lower than normal and the activities of some enzymes were significantly reduced, pointing to pathological alterations without gross morphological changes (lp et a\.,199Ð.
It must therefore be concluded that there is not a strong correlation between signiticant alterations to l"S]sulphate incorporation into GAGs and the organs which exhibited profound pathology. Altered sulphation is a secondary consequence of dysfunctional
CFTR. Hence the response of various tissues, to what may be subtle interventions in sulphate usage, probaOly depends on the sensitivity of that tissue to correct macromolecular sulphation. Tissues sensitive to correct sulphation levels may exhibit a 18 high degree ol pathology, whilst others may have greater tolerance to such metabolic abnormalities.
Of great interest was the linding of significantly reduced l'uSlsulphate ¡ncorporation into
GAGs of the nasal mucosa. As described in the introduction to this section, the nasal mucosa shows good similar¡ty to the phys¡ology of human airways and has demonstrated electrophysiological characteristics consistent w¡th classical CF in humans (Grubb et al.,
1994b). lt is a tissue with moderate levels ol CFTR but only relatively minor pathology, usually associated with ducts. ln humans with CF, evidence of nasal pathology usually manifests in the form of nasal polyps. lt would be interesting to investigate whether
GAGs extracted from human CF polyps exhibit altered sulphation.
The finding of reduced levels of l"S]sulphate incorporation is reminiscent of the situation that exists in foetal life. Sulphation of GAGs has been shown to be critical to the normal development of an organism. ln foetal or embryonic life, under-sulphation predominates
(Bouziges et al., 1991). This is due not only to the presence of high quantities of hyaluronan, but where sulphated GAGs have been identified in fetal tissues, they generally possess fewer sulphate residues. Sulphate appears to be important to the processes of cellular differentiation and inhibition of proliferation. For example, undersulphated GAG are found associated with highly proliferative and undifferentiated cells such as exist in foetal intestinal crypts. Sulphated GAG are more often identified as being associated with differentiated cells such as villous epithelial cells (Murch, 1995).
The correlation between low sulphation and cellular expansion, and high sulphation with differentiation, appears to continue postnatally. lt is possible that, as in humans with CF 183 who develop nasal polyps (due to inappropriate growth regulation and consequent proliferation of nasaltissue) the CF mouse may manifest reduced sulphation in the nasal mucosa due to the loss of CFTR.
These speculations await further evidence. ln the absence of more information, it would be true to say that in all of the organs where the CF mice exhibit significant dilferences in l.'S]sulphate incorporation, we appear to have identified a biochemical phenomenon which doesn't result in obvious pathological consequences. This does not mean however, that the biochemical alteration we have observed is necessarily without consequence. ln human CF disease the greatest pathology occurs in the lung and ainrvays, and yet CFTR expression is known to be low in this tissue (Cravrford et al.,
199'1; Trapnell et al., 1991). Paradoxically, it is high in the kidney and yet there is little pathology associated with this organ. CFTR expression therefore, is not the sole determinant of whether an organ will exhibit pathology or to what extent that pathology manifests in gross dyslunction. As discussed in the introduction to this chapter (section
5.1), where the murine organ possesses alternative Ca'.dependent chloride channels, the etfect of CFTR disruption is minimised (Clarke et al., 1994). Thus compensating mechanisms can play a role regardless of whether CFTR expression is high or low. The manifestation of sulphation abnormalities may also depend on other (presently unknown) organ specific, biochemical determinants.
The finding of reduced sulphation of a possible keratan sulphate component with¡n cartilage was of great interest. Data showing abnormal pulmonary mechanics in CF mice appear to be best explained by abnormal compliance of the chest wall (personal communication, Prolessor Richard Boucher). This may implicate widespread cartilage abnormalities and be consistent with an under-sulphation of keratan sulphate chains.
The charge density of the predominant proteoglycan of cartilage (aggrecan), is extremely important to the tissues biomechanical properties i.e. its rigidity and deformability
(Maroudas, 1972). Alterations to the amount of sulphate incorporated might lead to compromise of these propefties. lt should be emphasised however that this data needs to be treated with caution as only small amounts ol material were available to be assayed. A number of the samples were below the detection limit of the GAG assay and therefore only three data points from the normal group and two from the CFs was available for comparison.
The analysis of material impervious to degradation by chondroitinase ABC, provided evidence that CF mice were incorporating significantly more ['uS]sulphate into HS synthesised by liver and pancreas. lt has been shown that the sulphation of HS is critical to its ability to interact with and construct basement membrane structures (Brauer et al., lggg). The consistent differences obserued with several gut components, although not significant, provided grounds for suspicion that some regions, in particular the ileum, were implicated in metabolic changes involving sulphate. This prediction was borne out by further analyses.
The ileum and the gall bladder were shown to have an alteration to the proportions of
CS/DS and HS we were able to extract from tissue (Table 5'1)' lt was reassuring to note the consistency of the data in this analysis, not only within groups but between groups for most tissues. Both organs are major sites of pathology in the animal model, and this 185 observation raises the question of whether CF mice may be exhibiting mult¡factorial alterations to their matrix biochemistry i.e. not only differences in sulphate incorporation but sh¡tts in the biosynthetic machinery to lavour the production of inappropriate GAGs.
The actual difference was not large, with the ileum reducing lromT4o/olo66.70/o and the gall bladder from 90.9% to 84.5o/o. However, these alterations may induce pathology associated with abnormal basement membrane structures or in the secretory or absorptive functioning of the tissue.
When CS/DS disaccharides were examined by HPLC, most tissues were shown to synthesise a molecule of similar structure with respect to levels of sulphation. Given that many tissues had an apparent elevation in l'sS]sulphate incorporation into GAGs and
HS, this finding was somewhat surprising. lt was anticipated that even where the differences between CF and normal mice were not significant, that altered sulphation would account for the trends observed. The data is noteworthy in fact for the consistency of results between CFs and normals for many tissues.
It is interesting to focus on the composition of these polysaccharides in the light of which
(33%) organs exhibited alterations. The normal ileum has a high level of zero-sulphation whilst in all other tissues, the proportion of zero-sulphation does not exceed 20To-
Examination of data for the liver reveals that it ¡s the only organ to have an appreciable component of its disaccharides disulphated (18.7o/o in normals). No other tissue has more than 107o disulphated. CS/DS synthesised by control mice in all other tissues examined was; less than 20% zero sulphated, 75olo or more monosulphated and 107o or less disulphated. lt is possible that the ileum, in synthesising a significant proportion ol 186 disaccharides without sulphate in normal animals, thereby provided a substrate for lurther sulphation in CFs. The jejunum for example only has 60/o ol its disaccharides unsulphated Assuming the sulphotransferase enzyme systems within the Golgi apparatus of cells in the jejunum do not readily lacilitate dual sulphate substitution/disaccharide residue (an assumption supported by our analysis showing only
To/o ol residues disulphated), then the capacity to mono-sulphate further by CF animals, is severely circumscribed by the high level (85%) which occurs normally. The liver with a high capacity for di-sulphation in control animals, appears to have upregulated this enzyme activity in the CF animals to produce CS/DS which contains signilicantly more sulphate. There is a precedent for elevated enzyme activities occurring as a result of
CFTR disruption. As described in the introduction to th¡s section, CF mice appear to have upregulated 'alternative' Cat.-dependent mechanisms in the nasal mucosa and trachea to achieve chloride transpod in the absence of CFTR channels. ln this case, upregulation acted to buffer the animal from developing airways pathology' Higher sulphation of CS/DS in the ileum and liver, may be the result of an equilibrium shift towards greater sulphate addition, combined with substrate or enzyme systems which allow it to occur
A possible explanation for higher l"S]sulphate levels in CF GAGs in the absence of
structural alterations to GAG chains was the higher specific activity in disaccharides. Of
note however, was the high variability in this parameter exhibited by the CF mice.
Survival characteristics ol the animal model suggests that there is variable disease
that severity or vai'iable impact of the gene knockout on phenotype. What factor is it
of allows 10% suruival beyond 30 days? Cystic fibrosis in humans has a wide spectrum 187 clinical severity, even in patients with identical genotype (Welsh et a1.,1995). CFTR(-/-)
mice which survive beyond the first month of life may represent animals with a milder
phenotype. lf this hypothesis is correct, then our attempts to def¡ne pathological
alterations to metabolic parameters like sulphation, may encounter high variation
between CF animals, such as has been observed.
The trachea and nasal mucosa show interesting differences in specific activity of their
CSiDS disaccharides from trends exhibited by the other tissues. lt is likely that the low
specific activities recorded for the trachea in both CF and normal mice simply reflects the
high canilage content of the tissue. GAGs are unlikely to turn over rapidly in cartilage
and therefore in the 48 hours allowed for metabolic labelling only a small proportion of
the total CS/DS may have incorporated l"S]sulphate. The nasal mucosa has high
variability which is likely to be due to accumulated errors from measuring small peaks
and counting small amounts of radioactivity. Of great interest though, is the fact that it
was the only tissue in this analysis to exhibit a trend towards lower specific activity in CF
mice. This is consistent with data presented earlier showing significantly lower
incorporation of l.,S]sulphate into CF GAGs (See Fig. 5.1). The consistency of this trend
through consecutive analyses of GAGs, HS and CS/DS is highly suggestive of a real
phenomenon
The differences in specific activity of ['uS]sulphate in a defined molecular entity such as
the 4-sulphated disaccharide of CS/DS, must rellect the specific activity of the sulphate
pool from which the sulphate was accessed. As expressed in section 4.4 where there
was an analogous situation with lymphoblasts displaying altered ["S]:['H]glucosamine 1 ratios in the 45 disaccharide, this altered specilic activity of sulphate pools may be a reflection of the pool size. However unlike the situation with lymphoblasts where cells were bathed in radioisotope-containing medium constantly, and it can reasonably be assumed that intracellular pools equilíbrate with the medium, these mice received an intraperitoneal 'pulse'of radioisotope which was then lree to be utilised or excreted. lt ¡s possible that the altered specilic activity of sulphate pools may be due to altered kinetics of uptake from the blood supply rather than an intrinsic diflerence in the pool itself. While
CFTR itself is unlikely to participate in sulphate uptake from extracellular sources, it has been shown to regulate the activity of another chloride channel in CF mice (Gabriel et al.,
1993). By extension therefore, CFTR may regulate the activities of, as yet unknown, enzymes. ll the differential uptake hypothesis is correct then it presupposes that disruption of CFTR can have opposite effects in different tissues, in order to explain the nasal mucosa data.
Whether knocking-out CFTR affects sulphate uptake at the plasma membrane or in some way alters the dynamics or size of intracellular pools as suggested by the altered specific activities observed, we have evidence also, of alterations to the structure of
CS/DS in the liver and ileum, and to the proportions of GAGs synthesised by the ileum and gall bladder. The altered specific activity of sulphate pools in many organs which express CFTR is an indication of sulphate metabolism being perturbed. This ubiquitous
phenomenon supports the contention, that the normal equilibrium of sulphate utilisation
has been shifted. When certain conditions are met this can result in higher sulphate
addition as was seen for CS/DS synthesised by ileum and liver. Although we have 1 locused on GAGs as a substrate lor sulphate addition it is possible that other relevant
macromolecules e.g. mucins may also be affected
ln discussing differences in l"S]sulphate incorporation into GAGs by CF mice, it needs to
be remembered that we are in most cases dealing with a heterogeneous population of
cell types, some ol which probably do not express CFTR (e.9. fibroblasts). Therefore the
GAGs extracted from each tissue represent an average of the metabolic output ol a
number of cell types present in diflerent proportions. The only exception to this
generalisation is cartilage, which possesses only one cell type - the chondrocyte. The
implications of this are that, in tissues where we have failed to demonstrate statistically
significant differences, there may be specific cells e.g. epithelial cells, which are
synthesising over-sulphated GAGs or mucins. This possibility needs to be borne in mind
for future studies which utilise the animal model.
5.3.1 CONCLUDING REMARKS:
These data point to a biochemical perturbation involving sulphate which affects many
organs, including, surprisingly, non-epithelial tissue such as cartilage. The exact nature
of this perturbation is unknown. The results obtained in this series of analyses provide
intriguing proof of the reality of CFTR mediated effects on sulphation rn vivo. This is the
first time that this has been achieved. The CFTR(JI mouse offers an excellent model for
addressing questions related to the basic pathophysiology of CF. Not only will it allow
the assessment of new therapies for CF but it should allow dissection of the molecular
events which entwine CFTR with so many other seemingly unrelated metabolic
consequences.
193
6.1 INTRODUCTI
Our examination of sulphation by a cystic librosis pancreatic cell line (CFPAC) had indicated that CS/DS and glycoprotein were over-sulphated compared to a control cell line (PANC)''. Clearly this observation by itself did not prove or disprove the hypothesis that mutations in the CFTR gene result in sulphation abnormalities. The differences observed may have been the result of cell line to cell line variation, or alternatively they could be a phenotyp¡c change which occurred as a result of transformation. ln order to secure more convincing evidence that the differences between CFPAC and PANC were due to the ÂFro, mutation in the CFTR gene, protocols were designed with the aim of producing a gene-corrected CFPAC cell line. The underlying assumption being, that if sulphation abnormalities were the result of dysfunctional CFTR, then gene-corrected cell lines displaying functional CFTR in the correct cellular location should in theory, return sulphate metabolism to normal. Gene augmentation has been used by others to demonstrate the acquisition of cAMP-dependent chloride fluxes in a number of dilferent cell types (Anderson et al., 1991c; Rommens et al., 1991; Kartner et al., 1991;
Cunningham et a1.,1992; Egan et al., 1992). We intended to take this approach one step further and examine whether it was possible to influence, not just the primary defect, but in addition, a secondary manifestation such as abnormal sulphation of GAGs, in
CFPAC.
"See Chapter 3. 194
Data to be presented in this chapter will show that we were successful in generating a
CFPAC derived clone (by electroporation ol plasmid DNA) which expressed CFTR at the plasma membrane. We also obtained two cell lines (a generous gift from Professor Ray
Frizzell, Dept. of Physiology and Biophysics, University of Alabama at Birmingham) which had been transduced by retroviral gene transfection and shown to express CFTR chloride channels (Drumm eta1.,1990; Cliff et al., 1992). Using thethree'daughter'cell lines, the parent cell line and the normal control, in standard metabolic labelling protocols, evidence was obtained which indicated that CFTR expression did influence the sulphation of GAGs, and in fact can, but does not always, cause reversion to the normal phenotype. These results are presented in section 6.2.2 (A: F SI:FHl Ratios of
GAGs in Long Term Labelling StudieÒ.
Many of the experiments that had been conducted in this project seemed to indicate some perturbation of precursor pools for GAG synthesis. Examination by HPLC of the
CS/DS disaccharides synthesised by CFPAC and PANC cells (data presented in this chapter) reinforced this impression. We had previously obserued alterations to the specific activity of sulphate in CS/DS disaccharides produced by CF mice rn vivo
(Chapter 5), and alterations to ["S]:['H] ratios in the 45 disaccharide of CF lymphoblasts
(Chapter 4). A similar phenomenon was now seen in the 45 disaccharide of CS/DS synthesised by CF pancreatic cells. lf CFTR was influencing sulphate metabolism, the combined data from three completely different experimental systems implicated some change to sulphate pools even where there was no evidence of over-sulphation. The cell lines now at our disposal provided an ideal experimental model with which to explore a number of aspects of sulphate metabol¡sm and to try to identity at which point CFTR 195 might be involved in the sulphation pathway. Experiments were therefore designed with the aim ol probing the molecular basis for higher ["S]:['H] ratios.
A number of studies have identified a 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid
(DIDS) sensitive SOo''iCl anion exchanger in the plasma membrane as the key mechanism by which epithelial cells procure sulphate from the extracellular environment
(Elgavish et a1.,1987), 1991). This activiÇ has been demonstrated in CFPAC and PANC cells also (Elgavish and Meezan, 1992). Because sulphate and chloride ion compete for binding to the exchanger, it is possible to influence the uptake or efflux of either, by manipulating the composition of the extracellular medium. Mohapatra et al. (1993) have shown in bronchial epithelial cells that it is possible to increase the intracellular concentration of sulphate five fold by exposing cells to chloride free media. By varying the concentration of extracellular chloride (Cl") between 0 and 140 mM it was anticipated that we would influence the intracellular concentration of sulphate in the pancreatic cell lines and hence alter the physiological pool. An initial set of experiments was conducted in which we exposed CFPAC and PANC to varying concentrations of chloride ion in a simple defined medium conta¡ning radiolabel precursors for GAG synthesis. Because the medium for these experiments was essentially just a buffered salt solution, labelling was carried out over a shorter interval than we had used previously. Our interest in the results was three-fold: would ["S]:['H] ratios be altered under conditions in which sulphate was actively driven into the cell; would the biosynthetic machinery be compromised by low chloride containing medium; would a time frame of only a few hours allow suflicient synthesis of GAGs for analysis? As an adjunct to these experiments, further work was performed in which we attempted to 'stress'the sulphation machinery in 196
CFPAC and PANC by markedly increasing the rcle ol GAG synthesis. This could be lacil¡tated by use of the artificial GAG acceptor, 4-methylumbelliferyl-9-D-xyloside" - a compound which acts to remove the rate-limiting step of core protein availability from
GAG synthesis, and in doing so stimulates cells to massively upregulate production of these molecules (Galligani et a1.,1975; Schwartz, 1977; Robinson and Robinson, 1981)
The results of these experiments are presented in section 6.4.1 (B: Short Term
Labelting: lnfluence of Cf . and B-D-xylosidd.
By manipulating cells in the ways described and assessing the impact on ["S]:['H] ratios we were attempting to define aspects of sulphate utilisation indirectly. ln essence we were simply providing cells with a small fraction of isotopically labelled sulphate in the medium and then examining its eventual fate in a metabolic end product. The ability to draw conclusions about alterations to a complex pathway with such an approach was found to be limiting. Direct measurement of total inorganic sulphate within different cell lines, was therefore deemed to be the appropriate next step in understanding the effect that loss of CFTR was having on pancreatic cells and whether gene transfection could modify such elfects. lt represented another tier of information, and could be used to confirm that alterations to medium chloride concentrations had increased the size of intracellular pools. By incorporating a small amount of radioisotope in the medium and running a brief uptake protocol we could also obtain valuable information about the ability of cells to import sulphate lrom the extracellular medium via anion exchange mechanisms. To provide lurlher information it was felt that the other arm of the sulphoconjugation pathway i.e. the catabolism of cysteine to provide intracellular
"subsequently referred to as þ-D-xyloside. 197 sulphate, should be examined. lntracellular sulphate derives not only from the extracellular milieu, but can be generated ¡n some cells via the cysteine catabolic pathway. To our knowledge, the contribution of sulphate from cysteine has never been measured in pancreatic cells. As we were interested in compiling as much information as possible about the sulphation reactions within pancreatic cells, the conversion of cysteine to sulphate for sulphoconjugation to GAGs, was examined and quantitated. The results of this series of experiments is presented in sections 6.6.1 and 6.6.2 (C: Sulphate uptake and Pool Size in Pancreatic Cells and Cysteine as a Source of Sulphate in
Pancreatic Celld.
The final series of experiments which represented the completion of our enquiry, involved an examination of the ['uS]:['H] ratios incorporated into GAGs by all five cell lines (both transfected and control), after subjecting them to a range of chloride concentrations as well as I p-D-xyloside stimulation. With the information that was available from previous experiments it was hoped we might be able to pinpoint where CFTR was interacting in the sulphation pathway, what parameters were affected, and whether addition of wild- type CFTR to CF cells was able to correct aspects of altered metabolic behaviour. The final data are presented in section 6.8.1 (D: lnfluence of Altered Sulphate Pools and B-
D-xyloside on F'Sl:FHl Ratios in CS/DÐ.
To conclude the work contained in this chapter, section 6.10, entitled'Overvievl', synthesises the findings from sections (A-D) and a model is presented to explain the influence of CFTR on sulphation pathways 1
6.2 RESU TS: A
6.2.1 GENERATION OF THE RECOMBINANT CFTR.EXPRESSING CELL
LINE'TR2O':
Attempts to create stable CFPAC cell lines incorporating plasmid CMV-CFTR936C by electroporation, were successful in generating a number of colonies which were stable in the presence of 0.4 mg/ml G418 in culture medium. A number of these clones were tested for the presence of CFTR by immunofluorescent staining of cells with a commercially available monoclonal antibody to the C-terminus ol CFTR. Testing of a limited number of the total number of G418 resistant clones revealed one which showed strong surface staining with the monoclonal antibody (Fig.6.1b). We called this cell line
TR2O. As we had recently obtained two well characterised CFTR gene transfected
CFPAC cell lines from Professor Frizzell (PLJ-CFTR-6 and PLJ-CFTR-4'7 - subsequently referred to as pLJ6 and PLJ4.7), no further effofts were made to identify CFTR positive clones from the electroporation experiment. Figures 6.1c and 6.1d reveal binding of the
CFTR antibody to PLJ6 and PLJ4.7 respectively, confirming the presence of appropriately localised CFTR protein. CFPAC exhibited low level cross-reactivity to the antibody with some cytoplasmic staining visible. There was not the high intensity surface staining characteristic of the three CFTR expressing cell lines.
6.2.2 [..S]:['Hl RATlos oF GAGS lN LONG TERM LABELLING STUDIES:
cell GAGs were isolated by DEAE-Sephacel chromatography from the 'medium' of all live lines which had been metabolically radiolabelled with ["S]sulphate and ['H]glucosamine (Fig' lor 24 hours and 72 hours. When ["S]:['H] ratios of secreted GAGs were compared A B
c D
FIGURE 6.1 IMMUNOFLUORESCENT STAINING OF CELL LINES WITH MONOCLONAL ANTIBODY SPECIFIC FOR C.TERMINUS OF CFTR: Staining was performed as detailed in section 2.2.4. Slides were photographed on a Leilz Diaplan fluorescent microscope with top-mounted camera using Kodak Ektachrom 400 ASA slide film at an excitation wavelength of 450 nm and emission wavelength ol 490 nm. Magnilication 25x. A) CFPAC b) TR2O C) PLJ6 d) PLJ4'7
1
0.6 a 0.5
.9 (ú 0.4
I
I I cr) 0.3 t
- L CD ro CÐ 0.2
0.1
0 0.7 b
0.6 .Fo (ú 0.5
(Ð 0.4 - Ø ro 0.3 CÐ 0.2
0.1
0 PANC TR2O CFPAO PLJ4.7 PLJ6
FIGURE 6.2 ["SI:['HI RATIOS lN GAGS ISOLATED FROM THE'MEDIUM' OF TRANSFECTED AND UNTRANSFECTED CELLS:
GAGs were purified on DEAE-Sephacel according to the batch ion-exchange procedure detailed in section 2.2.15. Aliquots were scintillation counted and ratios calculated from dpm. N.B. All subsequent graphs showing ratios were calculated from dpm also.
@) 24 hours radiolabelling, (b) 72 hours radiolabelling. 200
6.2), there was evidence ol an elevation in CFPAC compared to PANC. GAGs recovered from CFPAC medium had ratios 1.7 times and 2.4 times higher than PANC medium, after 24 and 72 hours respectively. Sulphation by CFTR{ransfected CFPAC cells was variable in response to the presence ol the gene product. The PLJ6 cell line had an ['uS]:['H] ratio 15% higher than CFPAC after 72 hours. PLJ4.7 however, exhibited an ["S]:['H] ratio close to that of PANC at both 24 and 72 hours and therefore an apparent correction of the CF phenotype. TR20 fell midway between the CF and control cell lines indicating parlial correction or normalisation. To investigate one explanation for the differences, we attempted to correlate the rate of production of 'H- labelled GAGs for each of the five cell lines (over 24 and 72 hours) with the [3sS]:[3H] ratio
(data not shown). A correlation coefficient (r) of -0.41 indicated the two variables were not significantly correlated (from critical values for correlation coefficients). This observation argued against a simple kinetic explanation for our data i.e. that cell lines synthesising GAG at a faster rate were as a consequence incorporating less sulphate into them. ln fact this conclusion was supported by observations made in subsequent experiments with p-D-xyloside, an accelerater of GAG synthesis. Not only did ["S]:['H] ratios not decrease with increased rates of synthesis but they were seen to increase, and it could be noted that the relationships between ["S]:['H] values between cell lines in the absence of p-D-xyloside were maintained in the presence of the compound.
When GAGs isolated from the 'medium'were digested with chondroitinase ABC it was observed that [.'S]:[.H] ratios in CS/DS disaccharides ol CFPAC cells were 1.5 times higher than those of PANC after 24 hours and 2.6 times higher after 72 hours in culture
(Fig. 6.3). The three CFTR gene-transfected cell lines behaved in a similar but not 201
1 I24Hours
,,..E T2Hours
08 ¡
o 0.6 (u I cf) (/) lr) (Ð 0.4
0.2
0 PANC TR2O CFPAC PLJ4.7 PLJ6
FIGURE 6.3 COMPARISON OF f'SI:['H] RATIOS lN CS/DS SECRETED BY TRANSFECTED AND UNTRANSFECTED CELL LINES:
Total GAGs were digested with chondroitinase ABC (details in seclion 2.2.18) and the products of digestion separated on Sephadex G50. Fractions containing disaccharides were pooled and aliquots scintillation counted. identical manner to the complete GAG lraction. The ["S]:['H] ratio ol PLJ4.7 was the same as PANC at 24 hours, although higher al 72 hours. PLJ6 and TR20 were unchanged from CFPAC at both time points
The high molecular weight material remaining after chondroitinase ABC digestion was partially degraded by nitrous acid suggesting it was HS (data not shown). Keratan sulphate, was unlikely to be produced by these cells as it is only synthesised by specialised avascular tissues such as cornea and cartilage (Scott, 1994). The sulphation ratios of HS are shown in Figure 6.4. HS synthesised by CFPAC had higher ["S]:['H] ratios than PANC at 24 and 72 hours (1.6 times and 2.2 times higher respectively). HS from PLJ6 had a reduced ["S]:['H] ratio at 24 hours but not al72 hours. The ratio in HS from TR20 cells was similar to control at 24 hours but slightly elevated at 72 hours. lnterestingly, HS from PLJ4.7 cells had an ["S]:['H] ratio half that of PANC aL 24 hours and717o of PANC, al72 hours.
Having demonstrated what appeared to be over-sulphation of GAGs by the CFPAC cells, it was important to define the structural changes that had occurred. HPLC analysis of
CS/DS disaccharides allowed the purilication and quantitation of different disaccharide species. All five cell lines produced a CS/DS molecule that was similar in terms of its sulphation (Table 6.1). None of the cell lines produced appreciable quantities of unsulphated disaccharide i.e or disulphated disaccharide e.g. Adi- ^di-0s, ^d¡-di4,2S; di6,25 or ln fact 90% or more of the disaccharides were monosulphated. ^di-di4,6s. The explanation for the higher ["S]:['H] ratios in CFPAC CS/DS could be seen in the greater specilic act¡vity of l'uS]sulphate in each CFPAC disaccharide peak (Tables 6.2 0.6 r I I24Hours
I
I I-72Hours I I I
0.5 i
I
0.4 o ot-
(Ð 0.3 (t) rr) CÐ
0.2
0.1
0 PANC TR2O CFPAC PLJ4.7 PLJ6
SECRETED BY FIGU RE 6.4 COMPARISON OF ["SI:['H] RATIOS lN HS TRANSFEoTEDANDUNTRANSFECTEDcELLLINES: products digestion Total GAG. were digested with chondroitinase ABC and the of weight undegraded separated on Sephadex G50. Fractions containing high molecular materialwere pooled and aliquots scintillation counted. TABLE 6.1
COMPOSITION OF SECRETED CS/DS PRODUCED BY TRANSFECTED AND
UNTRANSFECTED CELL LINES AFTER 24 HOURST
4.5 92.2 3.3
2.2 92.8 5.0
2.1 96.8 1.0
3.1 89.8 7.1
4.0 92.6 3.4
CS/DS disaccharides from cells which had been radiolabelled for 24 hours were
generated by chondroitinase ABC digestion of 'medium'GAGs and after Sephadex G10
chromatography and borohydride reduction were separated by strong anion exchange
HPLC as detailed in section 2.2.20. Quantitation of disaccharide species was done on
the basis of 'H dpm, with disaccharide standards monitored by in-line UV absorbance at
226 nm used to confirm peak assignment.
ì The composition of CS/DS produced by all cell lines was unchanged atler 72 hours of radiolabelling. 205
peaks and 6.3). For example, after 72 hours the ["S]:['H] ratios in the and Âdi-6S ^dl-4s, (Table are 0.40 and 0.40 respectively for PANC and 0.83, and 0.78 for CFPAC 6.3).
These values compare with pre-HFLC ratios ol 0.30 and 0'77. This analysis also showed that at 24 hours where the difference between the sulphation ratios of intact
CS/DS from CFpAC and PANC was at a minimum (i.e. 0.56 and 0.38 respectively), that
(0.87 there was almost a two fold difference in the ratios in disaccharides and 0.48).
presence The reason for the discrepancy pre-HPLC versus post-HPLC was due to the of unsulphated disaccharide and unidentified, low molecular weight, radiolabelled contaminants. Tables 6.2 and 6.3 show the [3sS]:[3H] ratios in the individual disacoharide peaks from all five cell lines. The average ratios for all peaks are also shown and have been calculated relative to PANC in order to illustrate the relationship to the normal sulphation phenotype. lt can be seen that the earlier conclusions regarding normalisation of the CF sulphation phenotype (based on chondroitinase ABC digestion and shown in Fig. 6.3) are confirmed by the ratio data obtained from the disaccharide analysis. Where CS/DS contains disulphated disaccharide, the ["S]:['H] ratio in that peak is approximately two times that of the monosulphated disaccharides.
6.3 DISCUSSION: A
A CFTR expressing cell line was successfully generated in our laboratory by
the electroporating CFpAC cells with plasmid CMV-CFTR936C. This construct contained
named gene for CFTR under control of the cytomegalovirus promoter. A clonal cell line
localisation TR20 was shown to express OFTR on the cell surface by immunofluorescent TABLE 6.2
24 HOUR [.'SI:[.HI RATIOS IN SECRETED CS/DS DISACCHARIDES PURIFIED BY HPLC
AD|-4S MeantS.D. o1\) ^Di-6S ^Di-4.6S c') 0.56 0.76 0.94 1.92 0.89t0.11 1.9x
0.56 0.75 0.78 1.50 0.76t0.02 1.6x
0.60 0.62 0.64 1.47 0.67t0.06 1.4x
0.33 0.42 0.38 0.63 0.37t0.05 0.8x
0.38 0.46 0.48 0.93 0.47t0.01 1.0x
All ratio values are calculated from ["SJdpm and ['H]dpm. Mean values are calculated from the three monosulphated pealis and 0.5x the ratio of the disulphated peak. TABLE 6.3
72 HOUR ["S]:['H] RATIOS lN SECRETED CS/DS DISACCHARIDES PURIFIED BY HPLC
ADi-4S Api-6s Mean+S.D ^pi-4.6s 0.77 0.84 0.79 1.97 0.87t0.10 2.4x
0.76 1.01 1.05 2.08 1.03t0.02 2.8x
0.72 0.87 0.95 1.79 0.91t0.04 2.5x
0.46 0.52 0.49 1.04 0.51t0.02 1.4x
0.30 0.41 0.41 0.57 0.37t0.07 1.0x 2 with a monoclonal antibody. Using the same technique it was confirmed that two further
CFTR transduced CFPAC cell lines (tne pt¡ clones which had been generated by retroviral gene transfer) also still expressed CFTR. W¡th these CFTR expressing cell lines, labelling experiments were run over 24 and 72 hours to see whether earlier observations (described in Chapter 3) could be duplicated, and whether CF cells would exhibit normalised sulphate metabolism in response to the presence of CFTR.
Differences observed between CFPAC and PANC raises the question of the appropriateness of PANC as a 'normal'control. As the cell line was established from the
pancreas of a person without CF and is also derived from the pancreatic duct, it may be considered a reasonable choice. ln addition, PANC cells possess both CFTR mRNA
(Ward et a1.,1991) and protein (Kopelman el a/., 1993). ln the latter study functional
CFTR was demonstrated by the presence of cAMP-dependent chloride currents as
measured by cell volume change, and the abolition of those currents by incubation of
cells with anlisense oligodeoxynucleotides to CFTR (Kopelman et a1.,1993). The level of
expression however, appears to be low and it is not known how this level compares with
normal tissue in vivo. An additional important control in this study was the genetically
matched parent cell line CFPAC. Any metabolic changes which occurred in the gene-
transfected cell lines may be assumed to be the result of gene{ransfection. The data
demonstrating a trend towards lower ["S]:['H] ratios with the gene-corrected cell lines
was evidence that CFTR was able to have an effect on sulphate utilisation. The addition
of CFTR to CFPAC cells appeared to shift (to varying degrees) sulphate metabolism
towards the assumed normal phenotype. CFPAC cells exhibited a 1.7-2.4 times higher ["S]:['H] ratio in its 'medium' GAGs than the control (Fig. 6.2) and this apparent over-sulphation could be demonstrated in both oS/DS (F¡g. 6.3) and HS (F¡g. 6.4)' A two{old elevation in ["S]:[3H] ratio for secreted glycoconjugates corresponded with previous data obtained using CF nasal epithelial cells (Boat and Cheng, 1989). ln order to ascertain whether these differences resulted from defective CFTR, we compared ['uS]:['H] ratios in GAG produced by gene- transfected CFPAC cell lines. The presence of CFTR in these cell lines resulted in variable effects on the ['uS]:['H] ratios in GAGs. PLJ4.7 had a ratio the same as the control cell line in its total GAGs (Fig. 6.2) and its CS/DS (F¡9. 6.3) whilst HS (Fig. 6.a) had a lower ratio than that of PANC, thus demonstrating an apparent correction of the
CF phenotype. PLJ-CFTR-6 cells were not different from CFPAC in the CS/DS (Fig. 6.3) they synthesised. ln contrast, HS produced by this cell line, demonstrated some correct¡on at 24 hours (Fig. 6.4). TR20 cells were interesting in that they showed no correction of their CS/DS whilst the HS they produced was close to normal.
Higher l.uS]sulphate incorporation was not a transient phenomenon, as the trends present at 24 hours were still observable after 72 hours of radiolabelling. There was a tendency for'medium'GAGs from CFPAC and related cell lines to increase their [*S]:['H] ratio betwe en 24 and 72 hours whilst PANC secretions had a lower ratio after a longer period of radiolabelling. The reason for this trend is unclear, however there were differences in the growth characteristics ol PANC and the CFPAC derived cell lines at confluence. 21 0
An interesting observation was that HS ratios for dilferent cell lines did not mimic the pattern seen lor CS/DS produced by those same cells. This implied that the altered precursor pool for HS synthesis was compartmentally discrete from that used to synthesise CS/DS. ln rat ovarian granulosa cells, HS has been shown to be synthesised in the endoplasmic reticulum/proximal region of the Golgi whilst DS is completely synthesised in the trans-Golgi network (Uhlin-Hansen and Yanagishita, 1993). Each subcellular location appears to contain the entire complement of enzymes for the synthesis of these molecules. Different regions of synthesis within the Golgi may therefore provide an explanation for the subtle pool size and hence specific activity differences observed between GAGs.
We attempted to define the 'over-sulphation' we had observed more rigorously, by analysing CS/DS disaccharides on HPLC. There was no evidence of extra sulphate incorporated into these molecules. ln fact, the composition in terms of proportions of unsulphated, monosulphated and disulphated disaccharides was similar for all five cell lines (see Table 6.1). The explanation for the ratio differences lay in difTerential
incorporation of a radiolabelled precursor. As the disaccharides are chemically defined
and contain specified numbers of sulphate esters, the only way they can possess
different [.rS]:[,H] ratios is if the pools, from which the biosynthetic precursors were
drawn, have ditferent specific activities. The question arises, how could differences in
specific activity, for a substrate like sulphate, occur between two cell lines? Our initial
reasoning was that the specific activity of a pool committed to macromolecular
biosynthesis was most likely a reflection of its size. Pulse labelling was carried out over a
sufliciently long period (72 hours) that the differences between cell lines were not simply 21 due to dilferent rates of uptake for sulphate or glucosamine from the external medium' with the subsequent analysis ol molecules synthesised lrom non-equilibrated pools. We
'sulphate were led to conclude at that point that possibly one of the manifestations ol the delect' in CF was an altered pool for glycoconjugate precursors. Unfortunately the data at this point did not allow us to define whether it was the sulphate pool or the glucosamine poolwhich was being affected.
6.3.1 CONCLUSIONS: A
1. CFTR gene transduction of a CF cell line has altered ["S]:['H] ratios incorporated into
GAGs by three clonal cell lines,
Z. There is no dilference in the actual amount of sulphate incorporated into GAGs:
therefore altered [..S]:[.H] ratios reflect an isotope related effect and must be due to
the specific activity of pools from which sulphate (or glucosamine) for synthesis is
drawn. 21 2
6.4 RESULTS: B
Because the data from the 24 and 72 hour labelling studies (Section A) pointed to some alteration in the specific activity of precursor pools for glycoconjugate synthesis, and an effect of CFTR on those pools, experiments aimed at manipulating sulphate pools and sulphate flux through the biosynthetic machinery were conducted
6.4.1 SHORT TERM LABELLING: INFLUENCE OF CL'" AND P-D-
XYLOSIDE:
6.4.1.1 Experimental Design:
BUFFERS AND SOLUTIONS:
Physiological Medium: 132 mM NaCl, 1 mM CaOlr, 1 mM MgCl', 5 mM KCl,
1 mM KH,PO.,5 mM glucose, 10 mM HEPES-Tris pH7.4
Gluconate Medium: 140 mM Na-gluconate,2 mM hemi-Mg-gluconate,
2 mM hemi-Ca-gluconate,5 mM K-gluconate,
10 mM HEPES-Tris pH 7.4
To obtain solutions containing varying amounts of chloride ion, gluconate medium had
NaCl added and the concentration of Na-gluconate was reduced accordingly.
PROTOCOL 1: INFLUENCE OF Cl'"
CFPAC and PANC cells were grown to 90-100% conlluence in 35 mm plastic petri dishes, washed twice with sterile PBS and then incubated in gluconate medium (0.75 ml) containing 0 mM, 10 mM or 140 mM NaCl. At time points 0 minutes, 90 minutes and150 minutes the medium was replaced with the same solution containing 100 pCiiml 213
each 30 minute Na,[.'S]SO o and 40 pçi/ml l.H]glucosamine for 30 minutes. At the end of
,pulse' the medium was aspirated off and the 'cell layer' washed then solubilised in guanidine buffer for subsequent analyses including protein estimation. lt was felt that over such a shon labelling period that there was likely to be greater synthesis and incorporation of GAG into ECM than secretion into the medium.
This protocol allowed us to compare the rate of incorporation of sulphate and glucosamine as well as the [,.S]:[.H] ratio of molecules synthesised after cells had been exposed for varying times, to solutions of low or normal chloride ion concentration'
Hence an assessment of whether the cellular metabolic machinery was being rapidly
ion was compromised by incubation in the artificial medium or by the lack of chloride possible and it also provided information about the effect that increasing the sulphate poolwould have on ["S]:[3H] ratios in GAGs'
PROTOCOL 2: INFLUENCE OF p-D-XYLOSIDE
twice with CFpAC and pANC cells were grown as described above and then washed physiological medium before being incubated for 30 minutes, 120 minutes or 240
at minutes in 0.75 ml physiological medium containing l"SJsulphate and l'H]glucosamine
was either the concentrations stated above. Also added at the start of the incubation B- 0.1 mM, or D-xyloside in DMSO (100 mM stock solution) to a final concentration of
'mediuml were collected vehicle. Upon completion of the incubation both 'cell layer' and
on for analysis. The purpose of this experiment was to assess the effect of B-D-xyloside
how long it would the rate of synthesis and the [3sS]:[3H] ratio in GAGs, and to determine
take for cells to synthesise enough GAG to allow meaningful analysis. 214
6.4.1.2 Results:
GAGs were isolated by anion-exchange chromatography and the ["S]:['H] ratios compared (Fig. 6.5). lnterestingly, there was no clear cut effect of external chloride concentration on ratio. CFPAC cells exhibited some variability, but in the absence of a consistent trend it was reasonable to conclude that the concentration of external chloride was not affecting ['uS]:['H] ratios. We were also able to conclude that the length of time spent under these minimal media conditions did not affect GAG ratios. CFPAC had a mean ratio that was 4.5 times higher than PANC. This was a greater difference than we had previously observed with longer labelling times. A breakdown of the data to observe the relative contributions of l'H]glucosamine (Fig. 6.6a) and l"S]sulphate (Fig. 6.6b) to the ratios, revealed that l'H]glucosamine and l"S]sulphate incorporation by CFPAC
(normalised lor cell protein) were both relatively constant over 3 hours. The significance of the low sulphate incorporation at 0 mM Cl" in the 4th and 6th 30 minute labelling periods are unknown (diamond symbols indicate the mean value for combined chloride concentrations at each time point). PANC cells exhibited declining incorporation of both isotopes with time. Thus, although the ratios lor both cell lines were unchanged with time spent in gluconate buffer, the rate of synthesis of GAGs by PANC appears to decline. This may reflect a deleterious effect of the Na-gluconate medium on cellular metabolism. Such an effect was unlikely to be the result of low Cl-", as the rate of synthesis slowed even when cells were exposed to 140 mM NaCl (F¡9.6.6). Two further observations were ol interest. Firstly CFPAC was able to obtain and utilise considerably greater amounts of l"S]sulphate, whilst PANC's incorporation of l'H]glucosamine was slightly higher than CFPAC. Secondly, there was no dependency ol incorporation by either isotope on external chloride concentration. Cells Placed ln Gluconate Medium J 30 60 90 120 150 Mn
1 t 1 I 1 i
7 I - I I0 mM NaCl I, I iæto mM Nact I f --l Ø140 mM NaCl 6 t
l-
5 --..1 t t-
I .9 F (ú 4
cf) - U) ro 3 (Ð
2
1
0 1st 30 min. 6th 30 min 4th 30 min. 4th 30 min. 1st 30 min. 6th 30 min CFPAC PANC
FTGURE 6.5 EFFECT OF TIME SPENT IN GLUCONATE MEDIUM ooNTAINING VARIABLE CHLORIDE ION CONCENTRATIONS, ON [*S]:[.HI RATIOS IN GAGS SYNTHESISED BY CFPAC AND PANC: Cells were grown and radiolabelled as per the protocol in section 6.4.1.1. 'Cell layer' GAGs were purified on DEAE-sephacel using the batch procedure described in section 2.2.15. Aliquots were scintillation counted. The schematic illustrates the labellìng strategy Black bars are the intervals between addition and removal of radiolabel 21 6 FIGURE 6.6 INCORPORATION OF INDIVIDUAL ISOTOPES INTO GAGS SYNTHESISED BY CFPAC AND PANC IN GLUCONATE MEDIUM
-€el*T*ll.ll¡¡G+ARl AALÉeHtORf Þ E{ ON-eONe EilTRA-Tl Olff*-
Cells were grown and radiolabelled as per the protocol in section 6.4.1.1. 'Cell layer'
GAGs were purified on DEAE-Sephacel using the batch procedure described in section 2.2.15 and scintillation counted. Samples were assayed for protein and the data corrected for this variable.
(a) [3H]glucosamineincorporation (b) l"S]sulphateincorporation 217
100 I0 mM NaCl a 810 mM NaCl Ø'140 mM NaCl o Mean 'õC 80 o o o) f C 60 E o co o- oL o 40 .= oE ! T arj 20
0 1st 30 min 6lh 30 m¡n. 4th 30 min 4th 30 min 1st 30 min 6th 30 min. CFPAC PANC
200 I0 mM NaCl b 810 mM NaCl ø140 mM NaCl o Mean 'õc # - go 150 o- I o) L
J l t- ! E o cf) 100 - o- oL C) .ç E Eo- U) LO 50 cf)
0 1st 30 min. ôth 30 min 4th 30 min. 4th 30 min. 'l st 30 min. 6th 30 min. CFPAC PANC 218 FIGU E 6.7 EFFECT OF p-D-XYLOSTDE ON GAe SYNTHESIS By CFPAC AND PANC:
Cells were grown and radiolabelled for varying periods of time either ín the presence of vehicle or 0.1 mM 4-methylumbelliferyl-p-D-xyloside as per the protocol in section
6.4.1.1. 'Medium'and'cell layer'samples were recovered and GAGs were purilied on DEAE-Sephacel using the batch procedure described in section 2.2.15. Aliquots were scintillation counted and the total amount of each isotope incorporated is presented.
(a) 'H-incorporation into'medium'GAGs, (b) "S-incorporat¡on into'medium'GAGs, (c) 'H-incorporation into'cell layer'GAGs, (d) "S-incorporation into'cell layer'GAGs 21 I
180 1 000 Panc Med þXyl) Med CXyl) Panc Med (+XYl) a Med 1+¡Y¡¡ 160 FPAC Med (-Xyl) EcFPAc Med GXyl) ICFPAC Med IcFPAc Med 800 140 -
Ø th 120 - E E o (U th 600 ]J, 100 - o o= .c,
E E 80 -r o- o E 400 E U) I r.l) (a 60- (Y'
40- 200
20-r
0 0 0 60 90 120 150 180 210 240 o 30 60 90 120 150 180 210 240 time of radiolabelling (minutes) time of radiolabelling (minutes)
120 1 000 OPanc cL þXyl) cL GXyl) #Panc CL (+¡Y¡¡ c CL (+¡Y¡¡ d CL cL GXyl) FPAC GXYI) CL 1 00 - CL (+ PAC 800 '
(t, ?ll 80- Þ E t C o (E v, 600 tt, oJ o 60- E E o- o- p E 400 U) I ú) (a 40-r c., :
: 200 20- E 0 0 '180 o 30 60 90 120 150 180 210 240 0 30 60 90 120 150 210 240 time of radiolabelling (minutes) time of radiolabelling (minutes) 220
PANC and CFPAC cells were radiolabelled in the presence or absence of p-D-xyloside lor up to four hours according to the protocol outlined. Figure 6.7 revealed that the synthesis of GAGs secreted into the medium as measured by l'H]glucosamine and
p-D- [,,SJsulphate incorporation (Figs. 6.7a and 6.7b) was increased by exposure to xyloside''. This effect appears to accelerate over a period of four hours with the incorporation of isotopes at 30 minutes not much different between stimulated and unstimulated cells and with the profiles of l'uS]sulphate and l'H]glucosamine closely mirroring each other. The accumulation of each of the radiolabels is higher in the 'cell layer' (Fig. 6.7c and d) than in the 'medium' (Fig. 6.7a and b) in the absence of B-D-
generally xyloside. ln the presence of B-D-xyloside, ['H]glucosamine incorporation is higher in the 'cell layer' but l"S]sulphate is higher in the 'medium'. This data supported our decision to focus on the GAGs which became associated with either cellular material or the ECM rather than those secreted into the medium during short term labelling experiments. This strategy has been used by others who noted similar effects (Silbert ef
proportion their newly a/., 1993). B-D-xyloside causes cells to redistribute a greater of synthesised GAGs into the medium than occurs normally.
(F¡9. An examination of combined [,,S]:['H] ratios for'medium'and 'cell layer' GAGs 6.8) revealed that there was little change over the 4 hour duration of the experiment, and that
higher [,'S]:[.H] ratios of GAGs synthesised in the presence of the artificial acceptor were than those synthesised in its absence (F¡g. 6.8). This applied to both PANC and CFPAC biosynthetic products. CFPAC GAGs maintained higher ratios than PANC under all conditions, consistent with our past observations.
'oOne data point is missing due to accidental loss of sample 221
6.5 DISCUSSION: B
When short 'pulse labelling' experiments were carried out over three hours in buffered salt solutions containing varying amounts ol NaCl, two phenomena were encountered.
The first was that the difference between ['uS]:['H] ratios incorporated by CFPAC and
PANC were greater than we had previously observed with the 24 hour and longer labelling protocols. This may have been due to some difference in the rate the two cell lines accessed sulphate for macromolecular conjugation and which pools were drawn upon. Alternatively, over the relatively short time of the experiment there may not have been equilibration of externally available isotope with glucosamine pools inside the cell.
Nevertheless it reinforced the impression that the two cell lines were metabolically very different and lent greater significance to the dilferences we had observed previously. A second, somewhat surprising observation, was that external chloride had no effect on
[.uS]:[.H] ratios even though we were confident that the extracellular concentration and therefore availability of sulphate would be higher at 0 mM Cl-". Elgavish and Meezan
(Elgavish and Meezan, 1992) had shown previously that both CFPAC and PANC displayed carrier-mediated sulphate transport by anion exchange and that chloride was a competitive inhibitor of sulphate uptake. This result implied that a larger sulphate pool did not result in greater sulphate incorporation. We conclude that under these conditions, sulphate availability was not a rate limiling factor in GAG synthesis.
From the data shown in Figure 6.6 it is interesting to note the greater incorporation of l,'S]sulphate by CFPAC. The diflerence is approximately three fold. PANC'S greater use of l'H]glucosamine contributes to the 4.5 lold difference in ["S]:['H] ratio between the two 222 cell lines. A three fold greater use of ["S]sulphate by CFPAC when normalised for cell protein would argue strongly that the ["S]:['H] differences in CS/DS 45 disaccharides seen in section A were due more to an alteration in the specific activity of sulphate rather than glucosamine.
The slowdown in the rate of synthesis by PANC may be due to some nutrient deficit experienced by these cells. This wasn't considered a major problem in the conduct ol further experiments because ["S]:['H] ratios remained unchanged. Although synthesis was slowing, qualitatively the GAGs remained structurally similar. Because of this effect however, further experiments were limited to two hours labelling.
The influence of p-D-xyloside on GAG synthesis was predictable in all respects except one. Because the action of this compound is to act as a GAG chain acceptor in the
Golgi, the h¡gher [.'S]:['H] ratios exhibited in its presence were unexpected. The best explanation for this effect is a kinetic one. ln producing more PAPS for elevated GAG synthesis, cells were deriving a greater proportion of the sulphate required, from new sulphate obtained from the medium. Figure 6.9 illustrates schematically the model we have proposed in order to explain higher ['uS]:['H] ratios in GAGs synthesised by cells exposed to B-D-xyloside. According to the dual compartment model of Mohapalra et al.
(1993), sulphate in airway epithelial cells resides in two pools, one of which rapidly exchanges with the medium (RESP - rapidly exchangeable sulphate pool) and one of which does not (slowly exchangeable sulphate pool - SESP). Evidence to be presented in this chapter confirms this model in pancreatic cells. Thus, under B-D-xyloside 223
e PANC (-Xyl) x PANC 1+xyl) e CFPAC (-Xy¡) * CFPAC (+Xyl)
15
o 10 (ú t- I (f) - U) ro c)
5
0 o 30 60 90 120 150 180 210 240 time of radiolabelling (minutes)
FIGURE 6.8 ["Sl:['H] RATIOS lN TOTAL GAGS SYNTHESISED BY CFPAC AND PANC rN THE PRESENCE OR ABSENCE OF p-D-XYLOSIDE:
Cells were grown and radiolabelled for varying periods of time either in the presence of vehìcle or 0.1 mM 4-methylumbelliferyl-B-D-xyloside as per the protocol in section 6.4.1.1. 'Medium' and 'cell layer' samples were recovered and GAGs were purified on DEAE-Sephacel using the batch procedure described in section 2.2.15. Aliquots were scintillation counted and the combined ["S]:['H] ratios (weighted average based on
[3H]glucosamine incorporation) presented. 224 stimulation we would speculate that more l"SJsulphate is being channelled into GAG synthesis lrom the RESP than occurs when not stimulated. This results in a 'hotter' GAG molecule. An alternative explanation might invoke an exhaustion ol the SESP due to the greater sulphate demand and then subsequent synthesis would necessarily require sulphate sourced from the RESP. This scenario is unlikely due to the fact that in Figure
6.8 it can be seen after 30 minutes that ratios under B-D-xyloside stimulation are higher and yet there is no increase in GAG production evident at this early point (Fig. 6.7a-d). lt has been shown that B-D-xyloside increases the demand for sulphate in mouse mastocytoma cells and that at low medium sulphate concentrations, chondroitin sulphate becomes undersulphated (Silberl et al., 1993). When medium sulphate levels were raised, the chondroitin sulphate molecule became fully sulphated but there was no evidence for over-sulphation. This would then argue against the explanation, that the effect of B-D-xyloside we have observed in pancreatic cells is due to higher than normal levels of sulphate being added to GAGs.
6.5.1 CONCLUSIONS: B
1. Differences in ['uS]:['H] ratios appear to be due more to differences in sulphate
utilisation by CFPAC than glucosamine by PANC,
2. Enhanced GAG synthesis elicited in the presence of the chain initiator, B-D-xyloside
also results in increased ["S]:['H] ratios. Under conditions of elevated sulphate
demand this may be due to an increased proportion of sulphate for GAG synthesis
coming from newly acquired medium sulphate. a GAGs
Golgi T SO4 PAPS ->
ct-
s04 * so¿
GAGs b
Golgi
soa) eaes )
t-
s04 * so¿
FIGURE 6.9 PROPOSED MODEL TO EXPLAIN HIGHER [..SI:[.HI RATIOS IN THE PRESENCE OF p-D-XYLOSIDE: a) Under normal conditions a certain proportion of sulphate must derive from the rapidly exchangeable sulphate pool (RESP) and a certain proportion from the slowly exchangeable sulphate pool (SESP) for the synthesis of PAPS and ultimately GAGS in the Golgi, b) When p-D-xyloside stimulates higher levels of GAG synthesis, a greater proportion *SO. (represented by thicker arrows) of the sulphate required, derives from the RESP. = sulphate isotoPe. 6.6 RESU TS: C
6.6.1 SULPHATE UPTAKE AND POOL SIZE IN PANCREATIC CELLS:
6.6.1.1 Experimental Design:
CFpAC, PANC, PLJ6, PLJ4.7 and TR20 cells were grown as described above and washed six times with ice-cold Na-gluconate buffer. At time 0 minutes, 1 ml Na- gluconate buffer containing varying chloride ion concentrations (0, 10, 20,70, and 140 mM) and supplemented with Narsoo and 6 ¡rci/ml Na,[*S]soo (final sulphate concentration, 100 pM) was added to each dish and incubated for 10 minutes at room temperature. The cell monolayers layers were washed six times with ice-cold Na- gluconate buffer, the cells lysed with 1 ml 0.01 M NaOH overnight at 4"C and then cellular contents were recovered. Each condition was performed in triplicate. Of the 1 ml recovered from each sample, 600 pl was lyophilised and sent to Amdel lndustrial
Services (a National Association of Testing Authorities [NATA] certified testing laboratory) for inorganic sulphate analysis by ion chromatography. Three samples of medium sent away with the cell extract samples for sulphate analysis returned results of
ion- g7.O t 2.3 ¡rM, thereby providing an internal control as to the accuracy of the exchange analysis. Aliquots of the remaining samples were tested for protein and scintillation counted. To confirm that none of the ["S]sulphate had been incorporated into macromolecules during the 1o minute uptake, samples from each cell line were run on Sephadex G50 columns. There was no evidence of any radioactivity eluting in the void volume of columns i.e. associated with macromolecules. All glassware used in this 227 experiment was washed ten times with distilled water and then several more times wilh highly purified Milli-Q water (Millipore Corp.) in order to remove any traces of residual sulphate which may have derived from glass washing detergent.
6.6.1.2 Results:
Total intracellular sulphate for each of the cell lines (normalised for cellular protein) is shown in Figure 6.10. PANC cells exhibited a steady increase in the concentration of intracellular sulphate with decreasing Cl". This was the anticipated response on the basis of known characteristics of other anion exchange mechanisms in epithelial cells.
CFPAC in contrast showed little or no sensitiv¡ty to extracellular chloride, suggesting some differences from the kinetics of normal anion-exchange as seen in PANC. There was some evidence to suggest that at 0 mM Cl" the internal sulphate concentration in
CFpAC was higher. Unexpectedly there was no ditference in the size of the sulphate pool between CFPAC and PANC at physiologicalchloride concentration.
The CFTR transfected cell lines showed radical departures from the parent cell line
(CFPAC) in terms ol their responsiveness to Cl" and the size of their sulphate pools. pLJ4.7 and TR20 were similar in the changes that CFTR expression had induced. They showed the same lack of responsiveness to Cl-" as CFPAC had, but the size of the sulphate pool was hugely increased at all concentrations of chloride ion. At physiological
of Cl-. the mean inorganic sulphate concentration of PLJ4.7 and TR20 was 5.2 times that
CFPAC and pANC. PLJ6 has had sensitivity of its anion exchange mechanism to 0 mM
Cl" restored to normal levels. The overall response isn't completely normalised however, in that pLJ6 cells do not display the graded response to Cl-" that PANC cells do' lf the data are viewed in a dilferent way (Fig.6.11) it can be seen how much more closely 228
PLJ6 now represents the sulphate pool size profile ol PANC than of CFPAC. Certain characteristics of the parent cell line have been maintained however e.g the 'blip' in sulphate concentration that occurs with Cl'" at 70 mM.
We attempted to fit the data from this analysis to a mathematical model for ainruay epithelial cells derived by Mohapatra et al. (1993). They were able to relate the intracellular sulphate concentration to chloride ion concentration in the medium by the following equation:
lSO.'1, = [SO,'-]" + 2.248111 2 + [Cl-]" where tSO.''1, is total intracellular sulphate concentration, [SOo"]" is a non-exchangeable intracellular sulphate compartment (which I have termed the SESP for our pancreatic studies), and [ClJ" is the extracellular chloride ion concentration. They experimentally determined the volume of ain,ray epithelial cells to be 7 pl/pg protein. lf we make the assumption that PANC and CFPAC have the same cellular volume, then the actual concentration of intracellular sulphate can be calculated, to determine whether the compartmental model they have demonstrated, applies to another epithelial cell type. By graphing the chloride ion concentration against 1/[SO.'], a linear relationship is predicted.
Figure 6.12a shows that PANC cells do indeed conïorm to the suggested model, with a correlation coefficient of r'= 0.99. However it can be seen that neither CFPAC nor any of the CFTR expressing clones lit the relationship (Fig. 6.12b). Correlation coeflicients for PLJ4.7, TR20, PLJ6 and CFPAC were r'=0.23,0.21,0.18 and 0.004 respectively.
This indicated a profound difference in either the distribution of intracellular sulphate or response to external chloride by these cell lines compared to both airway epithelial cells and normal pancreatic ep¡thelial cells. 229 3
PANC PLJ4.7 TR2O PLJ6 CFPAC 2.5
'õc
o-I 2 o o) -) o 1.5 (u 2 -c 2 _o- J U' o) C 1 (u o 2
2 0.5 2
0 0 20 140 10 70 0 20 140 10 70 0 20 140 10 70 0 20 140 10 70 0 20 140 10 70 external chloride concentration (mM)
FIGURE 6.10 TOTAL INTRACELLULAR SULPHATE CONCENTRATIONS IN
TRANSFECTED AND UNTRANSFECTED CELL LINES EXPOSED TO VARYING EXTRACELLULAR CHLORIDE CONCENTRATION: cells were exposed to gluconate medium containing 100 pM Na,soo, 6 pci/ml Na'["S]SOo and varying concentrations of NaCl (in triplicate) for 10 minutes as per the protocol described in section 6.6.1.1. After washing, the cellular contents were
recovered by lysing overnight at 4"C in 1 ml 0.01 M NaOH. 600 pl was freeze-dried and
analysed for inorganic sulphate by ion-exchange chromatography. Protein estimation on all samples was performed and the sulphate concentration data corrected for this variable. Error bars represent the standard deviation from three readings except where a '2' indicates that only two data points were used due to outliers. 230
The concentration of intracellular sulphate in PANC at 140 mM NaCl was calculated to be 0.34 mM based on a cell volume assumption oÍ 7 ¡tllpg protein. This compares favourably with the value of 0.33 mM obtained for ainruay cells, lending support to the validity of the assumption. lt also showed that PANC was able to concentrate intracellular sulphate approximately three fold over a medium sulphate concentration of
0.1 mM under conditions of normal chloride. lt is worth noting that the concentration of sulphate found in human serum is approximately 0.3 mM. Whilst pancreatic and airway cells appear to have similar sulphate content at normal chloride concentrations, at 0 mM
Cl'", PANC concentrated sulphate almost eleven fold to 3.6 mM, whilst airway epithelial cells only reached 1.8 mM.
Having established the size of total intracellular sulphate pools between cell lines it was of interest to observe the rate of sulphate uptake as measured over a single short interval (10 minutes). Figure 6.13 illustrates the uptake of ["S]sulphate by different cell lines when exposed to different Cl'". PANC cells obtain sulphate from the external medium more rapidly than any of the other cell lines at all values of Cl'". PANC took up almost four times as much as CFPAC at 140 mM Cl-. whilst at 0 mM Cl. PANC took up more than nine times as much ['uSìsulphate. All cell lines show chloride-dependent influx of sulphate, consistent with a SOo"/Cl- exchange mechanism, but CFPAC and its transduced clones take up considerably less sulphate from the surrounding medium
The inset graph shows sulphate uptake by CFPAC and related cell lines, plotted on a different scale on the opposite y-axis. lt can be seen that the difference between PANC and the other cell lines is quantitative rather than qualitative 1
3 + Panc +PLJ 6 ECFPAC 2.5 .ç o o o- 2 o o t o) J L
1 o 1.5 (õ - -c _o-
I Ø i co) 1 õ o 0.5
0 0 20 40 60 80 1 00 120 140 external chloride concentration (mM)
FIGURE 6.11 COMPARISON OF THE SIZE OF INTRACELLULAR SULPHATE POOLS IN PLJ6, CFPAC AND PANG: Cells were exposed to gluconate medium containing 100 ¡rM Na.SOo, 6 pCi/ml Nar["S]SOo and varying concentrations of NaCl (in triplicate) for 10 minutes as per the protocol described in section 6.6.1.1. After washing, the cellular contents were recovered by lysing overnight at 4"C in 1 ml 0.01 M NaOH. 600 ¡rl was freeze-dried and analysed for inorganic sulphate by ion-exchange chromatography. Protein estimation on all samples was performed and the sulphate concentration data corrected for this variable. Combining the ["SJsulphate uptake and intracellular sulphate data allowed us to calculate the intracellular specific activity ol ['uS]sulphate at diflerent Cl'". This data is presented graphically in Figure 6.14. The specific activity of the external medium (at all
Cl") is shown as the horizontal line (mean value, 1085 t 34 dpm/ng sulphate). Under no conditions do any of the cell lines achieve an equilibration of internal and external specific activity. At physiological Cl'" the specific activity in PANC cells is 35% that ol the medium while for CFPAC it was only 10.5%o. All three transduced cell lines had specific activities ol 5% or less that of the medium after 10 minutes uptake. Under conditions of reduced Cl-" specific activities increased, which was consistent with the elevated rate of sulphate uptake seen in Figure 6.13. Striking differences between the cell lines were observed. CFPAC had a lower specific activity than PANC at all chloride concentrations, whilst the three transduced cell lines had markedly lower specific activity than the parental cell line. lt was interesting to note that all cell lines except PLJ4.7 displayed sigmoidal specific activity profiles (see inset Fig.6.14 for enhanced scale on PLJ4.7,
PLJ6 and TR20) indicating a plateauing out at very low Cl'". Thus, even where the equilibrium constant favoured maximal sulphate uptake (i.e. at 0 mM Cl-.), there was a limit to the specific activity that could be achieved by cells because of the presence of the
SESP. This result provided experimenfal evidence which strongly supported the concept that sulphate was compartmentalised within RESP and SESP in pancreatic epithelial cells, and that the majority was not freely and rapidly exchangeable with the external medium
An analysis was performed to see whether the higher inorganic sulphate content of cells at 0 mM Cl'" could be explained by the l3ss]sulphate uptake data. ln other words, was 3.5 +PANC a 3 o (õ -c. 2.5 -9- J U' 2 (ú f õ 1.5 o (ú +. c 1
0.5
0 IPLJ6 +TR2O (j^PLJ4.7 ËCFPAC 5
o b (ú -c 4 _o- J U' (ú 3 f õ o (ú 2 c
1
0 0 20 40 60 80 1 00 120 140 mM chloride (external)
FIGURE 6.12 RELATIONSHIP BETWEEN INTRACELLULAR SULPHATE CONCENTRATION AND EXTERNAL CHLORIDE CONCENTRATION FOR PANCREATIC CELL LINES: The Mohapatra dual sulphate compartment model predicts that intracellular sulphate concentration is inversely proportional to the chloride concentration in the medium. This relationship holds only for PANG. See text for details of the model. 234 the increase in total inorganic sulphate at 0 mM Cl. (compared to 140 mM Cl'.), correlated to the influx in P'slsutphafe that we observed during the ten minute duration of the experiment? ln performing the calculations we have assumed that the increase in tofal sulphate was due to an increase in the size of the RESP while the size ol the SESP remained constant. Plotting the increase in inorganic sulphate (at 0 mM compared to
140 mM) againstthe increase in l"S]sulphate dpm (at 0 mM compared to 140 mM and corrected forthe fractional size olthe RESP) yielded the result in Figure 6.15. The line represents the hypothetical fit if the hypothesis is correct and the increase in total sulphate is solely accounted for by uptake from the medium into the RESP- All of the cell lines fall close to, or on the line, and therefore fit the model fairly well except for pLJ6. ln Figure 6.10 it can be seen that PLJ6 at 0 mM Cl-" increases its sulphate concentration to normal levels. However, this analysis would indicate that the additional sulphate was not sourced from the medium.
6.6.2 CYSTEINE AS A SOURCE OF SULPHATE IN PANCREATIC CELLS:
6.6.2.1 Experimental Design:
PANC, CFPAC, pLJ6 and PLJ4.7 were grown as described above, until at or near
confluence. Culture medium was aspirated off and the cells were washed twice with
gluconate buffer containing either 0 mM or 140 mM NaCl. Cells were then incubated for
(supplemented 100 pM 2 hours at g7 "C in 5olo CO., in 1 ml gluconate medium with
1 with either no NarSOo) containing 10 ¡rCi/ml l"S]cysteine (1075 Ci/mmol, 1 mCi/ml)
vehicle. chloride ion or 140 mM NaCl in the presence of either 0.1 mM B-D-xyloside or
layers Each condition was run in triplicate. After 2 hours the medium was collected, cell
washed twice with ice-cold gluconate buffer and then cells lysed with 500 ¡rl 0.05 M 235
rt3ssl in PANC +[3sS] in CFPAC +[35S] in PLJ6 +[35S] inPLJ4.7 e[35S] in TR20 2500 2500 500 ¡n PANC rn CFPAC in PLJ6 400 iî PLJA 7 rn TR20 c, cl t 'q, E -l\ o 500 300 3 2000 iì ji o- (oc o) -o = o É, 200 t CL 000 o. : ! a 'c)c I 500 100 1 500 - o I 0 0 20 ¿ß 60 80 100 120 140 o- L o) L
J I E 1 000 oo-
500
0 0 20 40 60 80 1 00 120 140 external chloride concentration (mM)
FIGURE 6.13 EFFECT OF EXTERNAL CHLORIDE ION CONCENTRATION ON ffS]SULPHATE UPTAKE BY TRANSFECTED AND NATIVE CELL LINES: Cells were exposed to gluconate medium containing 100 pM NarSOo, 6 pCi/ml Nar[*S]SOo and varying concentrations of NaCl (in triplicate) for 10 minutes as per the protocol described in section 6.6.1.1. After washing, the cellular contents were recovered by lysing overnight at 4"C in 1 ml 0.01 M NaOH. Protein estimation on all samples was performed and aliquots were scintillation counted. Data points are the
mean of three readings. The inset graph shows CFPAC, PLJ4.7, PLJ6 and TR20 plotted on the right y-axis in order to d¡splay l"S]sulphate uptake compared to PANC (for which values are plotted on the left y-axis. 236
NaOH lor t hour at room temperature. Aliquots (50 rrl) were removed for protein estimation. The concentration of NaOH was then increased to 0.5 M with the addition of
1 M NaOH to achieve p-elimination of GAG chains from 'cell layer'proteoglycans. This reaction was allowed to proceed for 16 hours at 4"C after which the solutions were neutralised by addition of 45 ¡rl concentrated HCI and 100 pl 1 M Tris/HCl, pH 7.0. GAG chains and high molecular weíght material remaining were separated from free
[,,S]cysteine by Sephadex G50 chromatography and then GAG chains were precipitated with CPC as described in section 2.2.22.
6.6.2.2 Results:
Figure 6.16 shows the level ol incorporation of l"SJsulphate into GAGs by each of the cell lines tested when l"S]cysteine is used in the medium. lt should be noted that the gluconate buffer contained 100 ¡rM sodium sulphate and therefore cysteine was not the sole source of sulphate available to cells. A number of observations can be made from this data. Firstly, it is clear that each of the cell lines has the ability to utilise this pathway for the generation of at least some sulphate for macromolecular biosynthesis. Even with sulphate in the medium this pathway was utilised. lt is interesting to note that p-D- xyloside has no inlluence on the extent to which cysteine is converted to sulphate for
GAG synthesis, an unexpected result given that the cells demand for sulphate in the face of accelerated GAG synthesis is increased.
A striking obseruation to arise from this experiment was the difference in sulphate incorporation from cysteine by CFPAC, when compared to PANC at physiological chloride ion concentration. This difference (7.4 times higher) points to another lundamental difference between the CF cell line and the normal control in terms of their 237
lPanc *PLJ 4.7 ãTR2O OPLJ 6 TCFPAC +Medium 1200 250 LPLJ 4.7
200 Opr-¡ o
1 000 c) 150 (õ -c _g
Ø= 800 50 cC') 0 E o 20 40 60 80 100 120 140 o- E 600
= 2 o (ú 400 C) 2 'õ 2 o o- ú, 200
2 0 0 20 40 60 80 1 00 120 140 external chloride concentration (mM)
FIGURE6.14 SPECIFIC ACTIVITY OF INTRACELLULAR [''S]SULPHATE IN RESPONSE TO DIFFERENT EXTERNAL CHLORIDE CONCENTRATIONS:
Combined data from the total intracellular sulphate and l3sS]sulphate uptake allowed the calculation of specific activity of sulphate within cells after 10 minutes influx in gluconate medium containing dilfering NaCl concentrations. Error bars represent the standard deviation lrom three readings except where a '2' indicates that only two data points were used due to outliers. Medium specific activity is the mean of three measurements The inset shows three cell lines which had low specific activ¡ty on an expanded scale. sulphation pathways. One way ANOVA testing of the four cell lines showed statistically signilicant diflerences for each condition. Levels of signilicance were greater for cells in
140 mM Cl'" CB-D-xyloside, p<0.01; +p-D-xyloside, p<0.001) than at 0 mM Cl-" (-B-D- xyloside, p<0.005; +B-D-xyloside, p<0.01). The finding that PLJ6 showed normalised activity in this pathway lends credibility to the conclusion that CFTR may be involved in influencing another sulphation related pathway. PLJ4.7 on the other hand shows little difference from the parent cell line despite being transfected with the gene for CFTR.
One lurlher finding of interest from this experiment was the reduced quantity of cysteine derived sulphate in GAGs when the cells were incubated in 0 mM Cl". This was most obvious for CFPAC and PLJ4.7. One way ANOVA of each cell lines response to the four conditions showed no significant effects of chloride concentration or B-D-xyloside on pANC or PLJ6 whilst CFPAC and PLJ4.7 did exhibit significant ditferences (p<0.05).
6.7 DISCUS SION: C
The possibility that sulphate availability might be affected in the CF cell line was supported by the observations of Elgavish and Meezan (Clitf et al-, 1992) who demonstrated a ten fold lower capacity of the plasma membrane sulphate anion- exchanger in CFPAC to internalise sulphate compared to PANC. This difference was correctable in their hands by retrovirus mediated CFTR gene transfection. The size of the intracellular pool lor sulphate depends on the contribution of this transport mechanism as well as the ability ol cells to utilise the sulphur-containing amino acids cysteine and methionine (Humphries et al., 1988). Hence the dynamics involved in 239
12
tt o E 10 ()I
E o PAN (E I o (g .c. -gf a Ø 6 ro (f)
(ü f õ C) 4 (ú L .ç .= o CF U' (d 2 X PLJ6 Lo C) L TR2O PLJA.7 0 o 2 4 6 I 10 12 increase in total intracellular sulphate at 0 mM Cl- (times)
FIGURE 6.15 FOLD INCREASE IN TOTAL INTRACELLULAR SULPHATE AT O MM cL." (CoMPARED To 140 mM CL.o) vS FoLD INGREASE lN r,S]SULPHATE UPTAKE
AT O MM CL." (COMPARED TO 140 MM GL-o): The line represents the theoretical relationship that exists if the increase in intracellular sulphate is completely accounted for by sulphate taken up from the medium during the 10 minute uPtake exPeriment' determ¡ning pool size are complex. Nevertheless it was possible that such a profound difference in the ability of CFPAC to take up sulphate from extracellular sources, compared with PANC, would have an effect on the total available intracellular sulphate.
This in turn has been postulated to regulate the extent to which macromolecular sulphation may occur within the Golgi (Elgavish and Meezan, 1991)'
The experiment which measured intracellular sulphate revealed some intriguing results.
Our hypothesis that CFPAC and PANC would exhibit different sulphate pool sizes and that this might form the basis for higher ["S]:['H] ratios in GAGs produced by CFPAC, was found to be incorrect. At physiological Cl" (which was the condition used for all of the earlier labelling experiments presented in this thesis) the inorganic sulphate concentration in CFPAC and PANC was very similar (F¡9.6.10). However there were other differences. PANC displayed a high level of sensitivity to extracellular chloride and at 0 mM Cl-. its internal sulphate concentration was over ten times higher than at physiological Cl". This massive increase occurs within ten minutes (the duration of the experiment), a time which was chosen in order to be certain that external sulphate would fully equilibrate across the plasma membrane. Sulphate influx has been shown to be a rapid process. Mohap atra et a/. (1993) found that intracellular sulphate in nasal epithelial cells reached a steady state within five minutes of exposure to medium. lt was felt that ten minutes would give cells ample time to equilibrate with extracellular sulphate whilst
not allowing sufficient time for newly acquired l"S]sulphate to become incorporated into
GAG. This was subsequently shown to be true, with none of the intracellular
[.uS]sulphate associated with high molecular weight glycoconjugate. 241
l0 mM Cl- ØO mM Cl- (+xyl) 1 500 @140 mM Cl- w14O mM Cl- (+xyl)
-oc Po o- 1 000 o) J E oo- U) ro cÐ 500
0 PANC CFPAC PLJ6 PLJ4.7
FIGURE 6.16 ABILITY OF PANCREATIC CELLS TO CONVERT T'SICYSTEINE INTO SULPHATE FOR INCORPORATION INTO GAGs: Cells were radiolabelled in gluconate buffer containing 10 pCl/ml l"S]cysteine and 100
¡rM NarSOo for two hours as detailed in section 6.6.2.1. GAGs associated with the 'cell layer' were recovered by p-elimination with alkali (section 2.2.12) and CPC precipitation
(section 2.2.22). Protein estimations were performed on an aliquot of the solubilised 'cell layer'. Error bars represent the standard deviation from three measurements. 242 ln their study of ainruay epithelial cells Mohapatra ef al. (1993) demonstrated one sulphate compartment (comprising 10o/o of the total); at physiological chloride concentrations; was rapidly exchangeable with the external medium via the anion- exchanger (RESP). The remaining sulphate was only slowly exchangeable and comprised the remaining 90% of sulphate within the cell (SESP). This was also the case for pancreatic cells as can be seen from the specific activity data. At normal chloride levels the proportion of sulphate freely exchangeable with the medium was about 35% for PANC cells and about 10% for CFPAC. Even when the medium contained no chloride, the proportion of sulphate which was rapidly exchangeable did not go much higher than 80o/o in PANC or 50% in CFPAC. For the transfected cell lines it was much less
The CFTR-expressing cell lines all exhibit alterations to their sulphate pools under these conditions. The presence of CFTR has caused PLJ4.7 and TR20 to increase their sulphate pool size approximately five fold compared to the parent cell line. lt should be noted that these large pools which exist at physiological chloride concentrations cannot be explained by the relatively low uptake of sulphate from the medium, but in all likelihood pre-existed when the experiment started. ln this case it represents a major pefturbation to normal sulphate dynamics or utilisation; a change which makes these cells unlike both the genetically matched parent and the normal control. There is some increase in these pools at 0 mM Cl" and as was seen in the analysis in Figure 6.15 this increase could be attributed to uptake. However, it must be surmised that a five-fold increase in sulphate content (95% or more, of which belongs to the SESP as judged by the specific activities in Fig.6.14) is the result of CFTR transfection. Considering the phenomenon occurs in two cell lines it is unlikely to be an artifact of gene translection' A reasonable hypothesis to explain the phenomenon would be that there is some block to the normal exit ol sulphate from the SESP and possibly elevated influx, with the consequence that sulphate accumulates to higher levels.
Like CFpAC, PLJ4.7 and TR20 exhibit a lack of sensitivity to Cl'". PLJ6 in contrast has
had at least partial sensitivityto Cl" restored (Fi9.6.11). Given this pattern ol apparent
insensitivity to Cl-., it was interesting to note that l'sS]sulphate uptake is indeed stimulated
(Fig' by decreasing Cl'", albeit to a much smaller extent than for PANC 6'13)'
We would be tempted to speculate on the basis of what looks to be normal anion
exchange in these cell lines, albeit at reduced levels compared to the normal control, that
(seen the lack of conformation to the Mohapatra 'dual sulphate compartment model' in
Fig.6.12), implicates the distribution of sulphate within these cells as the altered
parameter. This appears plausible from an examination of the abnormal pool sizes
noted in pLJ4.7 and TR20 (i.e. 5 fold larger than control at all chloride concentrations) in
contrast to which there is low uptake of isotope from the surrounding medium. The
Mohapatra model relies on a number of assumptions which, whilst providing an excellent
fit for the experimental data in airway epithelial cells, may not be true for CFPAC and
CFTR-expressing CFPAC cells. For example, they use a figure of 40 mM internal
in chloride concentration (when the RESP is empty). Given the chloride secretory deficit
cFpAc, that assumption may not hold. The influx of sulphate into the RESP requires
to exit of two chloride ions, an electroneutral exchange. Pancreatic cells have the ability
pH was transport bicarbonate ion which may confound this prediction. Finally the internal 244 assumed to be 7.15. For PANC cells the internal pH has been measured as 7.28, however CFPAC has an intracellular pH of 7.83, a marked dilference (Elgavish, 1991)
Whatever the reason for the profound divergence in these cell lines from a model which
PANC appears to adhere to, it would seem that gene transfection has not restored the relationship between extracellular chloride and intracellular sulphate distribution seen in normal airway and pancreatic epithelial cells.
Our data for the uptake kinetics of CFPAC and PANC are in agreement with the work of
Elgavish and Meezan (1992) who found a ten-fold lower capacity of the SOo'-¡4,' anion exchanger in CFPAC compared with PANC at physiological chloride concentration.
However, in their study they also found that retroviral transduction with CFTR (to varying degrees depending on the clonal cell line) restored sulphate uptake (V.*) to higher levels. As is clear from Figure 6.13 none of the three CFTR transfected cell lines has had normal sulphate uptake restored under our conditions. Elgavish and Meezan (1992) found that the K. of sulphate uptake in the transduced clones had increased to greater than 4 mM. As we only exposed cell lines to 100 pM sulphate (which is at the lower end of the physiological range in serum) a definitive kinetic analysis was not performed
The behaviour of PLJ6 appeared to be contradictory. At 0 mM Cl-" the total sulphate pool was as large as in PANC. However, it took up no more ["S]sulphate from the extracellular environment than CFPAC, PLJ4.7 and TR20. The analysis shown in Figure
6.15 confirmed this and showed that PLJ6 was unlike any of the other cell lines tested. lt was anticipated that the reversion of PLJ6 to a more normal phenotype in response of the total sulphate pool to Cl'", would rellect in an elevated specilic activity. This did not 245 occur, as PLJ6 was seen to have intracellular specific activities even lower than CFPAC
(F¡g. 6.14). This apparently anomalous behaviour of PLJ6 at 0 mM Cl'. seemed explainable only if we postulated that it was able to supplement its intracellular pool with sulphate derived from the sulphur containing amino acids, cysteine and methionine.
Extracellular chloride has been shown to influence the metabolic activities of a range of different cell types. The contractile response of muscle cells is dependent on extracellular chloride Øhang et al., 1991) and may be influencing a Cl"-dependent intracellular calcium pool (Rangachari and Triggle, 1986). Reduced external chloride ion has been shown to inh¡bit the receptor-activated mobilisation of intracellular calcium in
CD3 T cells (Rosoll et a1.,1988) and to inhibit insulin release by islet cells (Tamagawa and Henquin, 1983). The authors of this last study propose that the effect of Cl-" may be due to altered intracellular pH which could have multiple pleiotropic effects. Cultured rat mesangial cells have also been shown to increase their concentration of intracellular calcium in low chloride medium with subsequent nitric oxide production via the
(Graf cyclooxygenase pathway ffsukahara et at., 1994). From these and other studies and petersen, 1978; Dilibe¡1o et a1.,1989; Kurz and Schomig, 1989) it seemed plausible that the total replacement of external chloride with gluconate in our experiment could stimulate PLJ6 cells to upregulate a pathway which generates sulphate from sulphur containing amino acids. Conversion ol methionine to cysteine and subsequent oxidation of organic sulphate via the sulphinyl pyruvate pathway is the only mechanism known in eukaryotic cells that could logically account for this observation ffempleton and Wang,
1 992). 2
Dilferent cell types have different capacities to utilise the cysteine -+ sulphate pathway, with some unable to generate inorganic sulphate for macromolecular sulphation at all e.g bovine aortic smooth muscle cells (Humphries et al., 1988). Others are able to generate some of their sulphate requirement in this way e.g. fibroblasts and chick chondrocytes, whilst Chinese hamster ovary cells (Esko et al., 1986), glomerular and rat mesangial cells (Templeton and Wang, 1992) can synthesise fully sulphated proteoglycans in the absence of exogenously added sulphate. The hypothesis was tested by the experiment described in section 6.6.2 and was found not to be supported by the results which can be seen in Figure 6.16. lf anything, the opposite was true with
PLJ6 in medium containing no chloride, able to convert cysteine to sulphate less rapidly than any of the other cell lines. At this point we are unable to explain how PLJ6 increased its sulphate pool in 0 mM chloride without invoking sources of sulphate or mechanisms previously not reported. The only other potential source of sulphate might be from the lysosomal degradation of GAGs, however this seems unlikely to be able to account for the magnitude of the increase in such a short period of time
The experiment in which we tested cysteine as a source of sulphate for macromolecular synthesis was able to rule out one explanation for the behaviour of PLJ6 at 0 mM Cl'.
However, the major reason for running the analysis was to examine the other route by which cells may obtain sulphate. To our knowledge this pathway has not been examined in pancreatic cells before. While all of the cell lines tested were able to utilise this amino acid for the generation of sulphate, and therelore must possess the relevant enzyme systems, they did so to widely varying degrees. CFPAC converted 7.4 times as much cysteine into GAG sulphate as PANC did at physiological Cl". Significantly however, 247 pLJ6 showed reversion to normal levels of incorporation whilsl, PLJ4.7 was unchanged
way. It must be concluded that CFTR is interacting with this pathway in some
The catabolism of cysteine to sulphate involves the lollowing steps: l. uptake of cysteine from the medium via amino acid transporters, ll. conversion in the cytoplasm to L-cysteinesulphinic acid by cysteine oxygenase, lll. transport into mitochondria, p-sulphinylpyruvate' lV. transamination with either o-ketoglutarate or oxaloacetate to
V. desulphination to pyruvate and sulphite, vl. conversion of sulphite to sulphate by sulphite oxidase,
Vll. transport out of mitochondria in order to become accessible to PAPS
synthesising enzymes in the cytosol (Singer, 1975)'
occur entirely ln addition to this pathway there is another pathway which is thought to
an intermediate within mitochondria and which doesn't involve cysteinesulphinic acid as
(Singer, 1g7S). At what point in this complicated catabolic sequence CFTR might be
interacting is imPossible to saY.
was not the sole source Because sulphate was included in the uptake medium, cysteine
low compared to the of sulphate lor GAG synthesis. However, 100 pM sulphate is ln these normal physiological availability of sulphate (200-3OO pM) to these cells'
responsible lor conditions it is possible the cells would upregulate the enzyme systems
in cysteine cysteine catabolism. As we saw in Figure 6.16 there was no increase
indicate they were conversion to sulphate under B-D-xyloside stimulation which may
is that there is low operating at V.* under control conditions. An alternative explanation but constitutive usage of this pathway by cells and that higher sulphate demand is unable to increase turnover.
The reduction of ['uS]sulphate incorporation by CFPAC and PLJ4.7 at 0 mM Cl-. in the l"S]cysteine experiment is most likely due to a dilution effect. At low Cl-", sulphate levels intracellularly are increased. As cysteine conversion contributes sulphate to this enlarged pool the expected result would be to see less incorporation of ['uS]sulphate. lntriguingly, PANC cells do not show this diminution at 0 mM Cl-" even though we have demonstrated that the total sulphate content of the cell increases eleven fold, while perhaps more relevantly the size of the RESP increases nine times. How is it that these cells fail to manifest some evidence of isotope dilution under this condition? lt can only be concluded that the sulphate generated from cysteine in the mitochondria does not mix with the RESP. From the rapid rate of incorporation (preliminary experiments not shown indicated high numbers of dpm were incorporated by PANC within 30 minutes) sulphate from cysteine must route either directly to the Golgi, or alternatively via the SESP. These obseruations provide fascinating insights into complicated sulphate utilisation pathways within cells. To our knowledge these have not been described before'
The absence of a dilution effect in PANC and presence of it in CFPAC also raises the question ol whether this re-routing is in some way connected to the functioning of CFTR channels. ANOVA testing ol PLJ6 revealed no statistical evidence of a dilution effect, which implies that not only has its cysteine pathway been down-regulated to control levels, but that the channelling of liberated sulphate follows ditferent pathways to
CFPAC. 6.7.1 CONCLUSIONS: C
(at 140 mM 1. No diflerence in the size of the sulphate pool between CFPAC and PANC
chloride),
Z. The RESp represented 35% of thetotal ¡n PANC, 107o in CFPAC and lessthan 5%
in PLJ6, PLJ4.7 and TR20, g. Five-fold higher sulphate pools in TR20 and PLJ4.7, 95o/o of which was in the
SEsp-this implied an accumulation and therefore possibly an exit block from the
Pool,
4. pLJ6 increased its sulphate pool to normal levels at 0 mM Cl-. but the source of
or additional sulphate could not be identified as it did not derive from the medium
cysteine,
(albeit kinetics), 5. All cell lines displayed normal SOo.-/Cl anion exchange with different
for by influx and increases in intracellular sulphate at 0 mM Cl-" could be accounted
from the medium (excePt for PLJ6),
sulphate as 6. CFPAC was able to convert 7.4 times as much cysteine into GAG
PANG-a difference which was fully corrected in PLJ6'
but not in PANC 7. Dilution of sulphate generated from cysteine in CFPAC and PLJ4'7
and PLJ6 implicated ditferent routing of sulphate between cell lines' 6.8 RESULTS:D
6.8.1 TNFLUENCE OF ALTERED SULPHATE POOLS AND p-D-XYLOSIDE
ON ["SI:['H] RATIOS lN CS/DS:
6.8.1.1 Experimental Design:
All five cell lines were grown as described above and then washed six times with ice-cold
Na-gluconate buffer. Na-gluconate buffer (0.75 ml) containing varying concentrations of chloride (0, 10, 20,70 and 140 mM), 100 ¡rM NarSOo,0'1 mM B-D-xyloside orvehicle,
and 56 pQi/ml Nar['uS]SOo and 22.4 ¡ßilml l3H]glucosamine was added to cells which were incubated at 37'C for 2 hours. After incubation the medium was recovered, the
cells washed with a furlher 0.75 ml Na-gluconate buffer and the wash combined with the
medium. The 'cell layer' was recovered by solubilisation in guanidine buffer. After
samples were buffer exchanged on Sephadex G50 into 0.1 M ammonium formate they
were lyophilised and subjected directly to chondroitinase ABC digestion after which
disaccharides were recovered and scintillation counted in the usual way.
6.8.1.2 Results:
The [.uS]:[.H] ratios of CS/DS disaccharides synthesised by CFPAC and PANC in the
p-D-xyloside presence and absence of B-D-xyloside are presented in Figure 6.17a.
stimulates higher ["S]:['H] ratios than are found in its absence for both PANC and
CFPAC, consistent with what we observed previously. This elfect appears greater for
CFPAC than for PANC, however ¡f the p-D-xyloside ratios are compared with vehicle
ratios, the average increase exhibited by CFPAC is 4.5 times higher and for PANC is 3.9 251 times higher. PANC has lower ratios than CFPAC under both conditions. Ratios do not seem to be greatly influenced by Cl'" except when chloride ion is absent. ln fact the ratios at all other chloride concentrations are lakly constant. For example, CFPAC exposed to B-D-xyloside has a significantly lower ratio at 0 mM but at all other Cl" the ratio remains fairly steady around a value of 1.6. As we know that the RESP is increased at lower chloride concentrations (see specilic activities in Fig 6.14) it can be stated that the size of the RESP has no influence on sulphation or the production of sulphated GAGs. From an assessment of the ['H]glucosamine incorporation into GAGs it can also be said that Cl'. does not affect the amount of GAG produced by any cell line
(data not shown).
The behaviour of the transduced cell lines is interesting. ln Figures 6.17b and 6.17c it can be seen that TR2O and PLJ4.7 behave in a very similar manner to CFPAC i.e. high ratios (of a comparable level to CFPAC) in the presence of B-D-xyloside and the same
'dip' in ratio at 0 mM Cl'". ln the absence of the chain initiator, ratios are very similar to the parent cell line also. For these two transduced cell lines, the presence of sulphate
pools fiveJold higher than CFPAC, results in no real difference in ["S]:['H] ratios. This is
further evidence that the size of the pools is not a key lactor in the dynamics of
["S]sulphate incorPoration.
The behaviour of PLJ6 (Fig.6.17d) is once again diflerent from its transduced
counterparts and particularly at the higher chloride concentrations of 70 and 140 mM,
[..S]:[.H] ratios seem to be comparable to the normal control. At lower Cl'" the behaviour
is more complex. ln the presence of p-D-xyloside the pattern is similar to that exhibited 252 FIGURE 6.17 f"SI:f'Hl RATIOS lN CS/DS SYNTHESISED BY CELLS UNDER VARYTNG CHLORTDE rON CONCENTRATTONS AND rN THE PRESENCE OF p- D.XYLOSIDE OR VEHICLE:
Cells were radiolabelled for two hours as per the protocol described in section 6.8.1.1.
'Cell layers'were recovered by solubilisation in guanidine buffer and after Sephadex G50 chromatography into ammonium formate buffer were lreeze-dried and then subjected directly to chondroitinase ABC digestion. Disaccharides were collected after G50 chromatography and aliquots scintillation counted. (a) CFPAC and PANC CS/DS ratios, (b) CFPAC, PANC and TR20 ratios (c) CFPAC, PANC and PLJ4.7 ratios (d) CFPAC, PANC and PLJ6 ratios
N.B. The lines for TR20, PLJ4.7 and PLJ6 (b, c, and d) are in bold to assist in comparing their behaviour to CFPAC and PANC a b
GXvl) (+xyr) rTR20 (Xyl) GXvl) (+Xyl) -1R20 2 2
i I 16 tt 161
o 12- .o 1.2 (u (E x I q? c? Ø Ø U. ro (f) 08 (Ð 0.8
o4 0.4
0 0 0 60 80 100 120 1& 0 20 & 60 80 100 120 1Æ 20 4 (mM) external chloride concentration (mM) external chloride concentration
c d
(+Xyl) OPLJ 6 GXyt) xPLJ 4.7 (+XYl) GXyD GXvl) 'tPLJ 4.7 (-Xyl) r¡PLJ 6 CXvl) 2 2
1.6 1.6
.o 1.2 .9 1.2 t! ß t I e e U) Ø r¡) r.o (r) 0.8 (o 0.8
0.4 0.4
60 80 100 1n 140 0 20 40 60 80 100 '120 140 n & (mM) external chloride concentration (mM) external chloride concentration 254
by PANC. ln the absence of p-D-xyloside, PANC ratios go from 0.3 at 0 mM Cl'" down to
0.1 at 20 mM. PLJ6 ratios go up in this areaol the graph and then suddenly revert to
normal control levels. lt appears that some mechanism switches the ratios to lower
values at the higher chloride concentrations.
When the ["S]:['H] ratios at 140 mM, 70 mM and 20 mM are averaged for each of the cell
lines and presented as a histogram (Fig. 6.18) ¡t is easier to appreciate the differences
between the live cell lines, and to observe how closely PLJ6 now resembles PANC, both
normally and under conditions of accelerated GAG synthesis. Because of the dilference
in ratios between t p-D-xyloside the data have been presented on different y-axes. Also
of note in this figure are the relatively small error bars, confirming that despite a seven
fold difference in Cl'" (20-140 mM) there is little effect on ratios
With the data obtained in this experiment for l"S]sulphate incorporation at 140 mM Cl'" it
was possible to estimate the total incorporation of sulphate into GAGs by isotope dilution
calculations. When combined with the results of the cysteine conversion experiment
described in section 6.6.2, lor which a similar calculation can be performed, it was
possible to estimate the relative contribution of each source of sulphate to total GAG
sulphation. For the medium sulphate contribution it was assumed that all of the sulphate incorporated in the two hour labelling experiment derived lrom the RESP. For the cysteine calculation it was assumed that the intracellular specilic activity of cysteine rapidly equilibrated with the specific activity in the medium and that all of the l"S]sulphate generated was incorporated into GAGs. Because the final experiment quantitated
["S]:['H] ratios in CS/DS and not total GAGs (as was done in the cysteine experiment), 255
0.6 2 IVehicle ffiXyloside 0.5 o o .9 1.5 ß) -c f o (¡) 0.4 (Jl .9 Ø (ú cù - 1 I 0.3 .1 CÐ 0) - a+ Ø õ' LO cÐ x 0.2 :S. c o (ú Ø o 0.5 d o 0.1
0 0 PANC TR2O CFPAC PLJ6 PLJ4.7
FIGURE 6.18 MEAN ["SI:['H] RATIOS lN CS/DS SYNTHESISED BY CELL LrNES UNDER CONTROL CONDITIONS AND ß-D-XYLOSIDE STIMULATTON: Data at 20 mM, 70 mM and 140 mM NaCl taken from the analysis in Figure 6.17 was averaged and presented as a histogram with error bars showing standard deviations. Data for PLJ6 contains only the values from 70 mM and 140 mM NaCl due to fluctuations which appeared to affect this cell line but not the others. Data for p-D-xyloside stimulation are plotted on the right y-axis. factors accounting for the known proportion of CS/DS and HS synthesised by PANC and
the CFPAC clones was used (see Table 3.3). Equations O, Ø and O were derived: (see
appendix B for details)
(amol/¡rg orotal sulphate incorporated into GAGs protein) by pANC = 1 1662
ØTotal sulphate incorporated into GAGs (amol/pg protein) by CFpAC = 17452
9Total cysteine sulphate incorporated into GAGs (amol/pg protein) = 0.41Bgz
where z is the FuSlsulphate dpm/pg protein.
Table 6.4 shows the results for all cell lines at 140 mM Cl". The contribution of sulphate
from cysteine is less than 0.17o under all conditions tested (this applied to cells in 0 mM
Cl'" also - data not shown). Consistent with the higher conversion of cysteine into GAG
sulphate by CFPAC cells seen in Figure 6.16, the proportion of the total sulphate coming
from cysteine was higher in these cells than in PANC (0.0857o compared with 0.036%).
This was a difference ol 2.4 times. PLJ6 cells show levels of sulphate coming from
cysteine which are close to the normal control (0.020o/o), while PLJ4.7 at 0.065yo falls
between the CF and control values. Under p-D-xyloside stimulation the expected
reduction in contribution from cysteine is seen. This correlates with the lack of increase
in cysteine conversion seen under p-D-xyloside stimulation as compared to the greater total sulphate incorporation into GAGs. This greater incorporation can clearly be seen in
Table 6.4 in the'Medium Sulphate'column. TA E 6.4
ToTAL SULPHATE INCORPORATION INTO GAGS AND THE CONTRIBUTION
FROM CYSTEINE AT 140 mM Cl'o
45 1 25 800 0.036 54 1 005 000 0.005
328 384 800 0.08s 340 1 839 600 0.018
74 364 900 0.020 49 2 000 900 0.002
508 753 750 0.067 404 2 945 500 0.014 6.9 DISCUSSION: D
Our results show that the size ol the RESP (which increases for all cell lines at lower Cl-")
has no effect on [3sS]:[3H] ratios. This argues that sulphate entry via the band 3 like anion
exchanger does not play a regulatory role in macromolecular sulphation in pancreatic cells. Mohapatra et al. (1993) arrived at the same conclusion for ainvay epithelial cells.
lntuitively this makes sense as plasma chloride concentrations would not be expected to fluctuate greatly, therefore the supply of sulphate to cells via the exchanger would be fairly constant. PLJ6 has had its ["S]:['H] ratios normalised except at low Cl-". This indicates that CFTR gene transfection hasn't completely normalised sulphate utilisation in these cells but clearly, under some conditions, it becomes difficult to distinguish from the normal control (see Fig. 6.18). For different reasons, PLJ4.7 and TR20 are also interesting. Despite the large sulphate pools they possess in comparison to CFPAC, their ["S]:['H] ratio profiles are the same as that generated by CFPAC. This argues strongly that the large SESP does not contribute quantities of sulphate to GAG synthesis propodional to its size - even under p-D-xyloside stimulation. lf this were not true then there would be a dilution of the isotope in the RESP being channelled into GAG synthesis.
The proportion of sulphate generated from cysteine was very low - less than 0.17o. This raises the question of what physiological relevance the pathway has in pancreatic cells.
The answer to this is unclear. 259
calculations which certain assumplions were required belore we could attempt the was that only provided this information. For sulphate quantitation, the requirement
we don't have direct sulphate from the RESP was utilised lor GAG synthesis' Although for most of the data evidence for this, and in lact the model we propose to account
isotopically labelled necessitates a contribution from this pool, it is certainly true that
to the medium. ln our hands GAGs can be isolated very soon after addition of radiolabel
GAGs within 30 minutes' we have shown considerable quantities of l'uS]sulphate labelled
have been overestimated, ln the event that it is incorrect, the contribution of cysteine will
from the SESP' as our calculations have not taken into account sulphate incorporated in the medium The other assumption was that there would be equilibration of cysteine
standard assumption in with ce¡urar poors. we have no evidence ror this, but it is a
a1.,1993; lmai ef al',1994)' many isotopic labelling studies (Esko et a1.,1986; Silbert et
(compared to PANC - see lf the increase in medium sulphate incorporated by CFPAC oFPAC (i'e' Table 6.4) is multiplied by the higher contribution of cysteine in This rheoreticar 3g4goo/125g00 x 0.0g5/0.036), a figure or 7.2 times is obtained. to find incorporated from difference betvveen the amount of sulphate one would expect
value ol 7 times that was cysteine by CFPAC compared to PANC' is close to the '4 6.6.2.2 (F¡g. 6.16)' actua¡y observed in the cysteine experiment described in section support to the such a conjunction of experimental and theoretical values lends
assumptions discussed above.
6.9.1 CONCLUSIONS: D
1. The size of the RESP is irrelevant to ['uS]:['H] ratios in GAGs'
2. The size of the SESP is irrelevant to ["S]:['H] ratios in GAGs' 2
3. PLJ6 produces GAGs with normal ["S]:['H] ratios at higher medium chloride
concentrations, whilst TR20 and PLJ4.7 - despite changes to overall sulphate pools
and sulphate uptake kinetics - incorporate [.uS]:[3H] ratios essent¡ally identical to
CFPAC,
4. The contribution of cysteine to the sulphation of GAGs is too low to be affecting
["S]:['H] ratios in GAGs.
6.10 OVERVIEW: A MODEL FOR THE EFFECT OF CFTR ON
SULP HATE UTILISATION
The observation that we have endeavoured to find an explanation for, is higher ["S]:['H] ratios in GAGs synthesised by a CF cell line compared with a normal control. Evidence that the phenomenon was possibly CF related came from one of three CFTR-transduced cell lines (PLJ6) which exhibited lower ["S]:['H] ratios consistent with normal values.
ln formulating a model to explain the complex metabolic behaviour we have observed in these cell lines it is informative to start by eliminating those variables which have been shown notlo affect ["S]:['H] ratios:
l. The structure of CS/DS synthesised by all cell lines is identical. Therefore the
explanation is not due to higher amounts of sulphate on GAGs, 261 ll. PANC cells take up sulphate four times faster than CFPAC at 140 mM chloride and
yet have lower [..S]:[.H] ratios; therefore the rate of sulphate uptake from the medium
is not critical,
|il. The size of the RESp. 10% in CFPAC vs 35% in PANC, but CFPAC has higher
['uS]:['H] ratios, lV. The size of the SESp. [..S]:[.H] ratios in GAGs produced by TR20 and PLJ4.7 are
the same as those synthesised by CFPAC despite five fold higher sulphate pools,
95% of which resides in the SESP,
V. The contribution of cysteine to sulphate for GAG synthesis. At less than 0.1% this
pathway is unlikely to be significant.
These conclusions facilitate the formation of a model which predicts that the difference
from different between the CF and the normal cell lines lies in the utilisation of sulphate
propoftion pools for GAG synthesis, The key feature of this model revolves around the of sulphare obtained from the RESP vs the sEsP (see Fig. 6.19).
in its It is necessary to invoke some contribution of sulphate from the SESP because absence we would see no difference in [3sS]:[3H] ratios' Medium sulphate equilibration with the RESp is rapid and if this were the only source of sulphate for GAG synthesis, the then, despite the difference in relative size between CFPAC and PANC, ["S]:['H]
kinetic ratios would be identical. Diflerences in ["S]:['H] ratios occur by the simple
RESP in CFPAC' For explanation that a grealer percentage of sulphate comes from the
that the equivalent example, if 50yo of GAG sulphate derives from RESP in PANC, but
figure in cFpAc is 75%, the net result is a three lold diflerence in ["s]:['H] ratios 262 FIGURE 6.19 PROPOSED MODEL TO EXPLAIN HOW LOSS OF CFTR
INTERFERES WITH SULPHATE METABOLISM IN CYSTIC FIBROSIS: Nsrmal-cells (P-ANC) are hypothesised to have a CFÏR-mediated rate=limiting step on the supply of sulphate from the RESP. ln CF cells (CFPAC) this limitation has been removed with resulting higher ["S]:['H] ratios. lt is likely that there is normally some communication between the two sulphate pools i.e. slow equilibration. ln PLJ6 cells the rate limit¡ng step is proposed to have been restored by CFTR expression. ln PLJ4.7 and TR20 cells, that has not occurred, however there appears to have been a redirection of sulphate from the RESP to the SESP. Because efflux from the SESP is limited, the size of that pool is increased. ['uS]:['H] ratios remained unchanged from those exhibited by the parent cell line, CFPAC. 263 GAGs
,ì Golgi 0 ooo () (f oo PANC so4 PAPS il{ll 7.
ct-
s04 * so¿ GAGs
ì t35Sl:[3H]
so4 --)
ct-
s04 * so¿
GAGs
ì
so4 -->
ct-
s04 * so¿
GAGs
o C 'oQo [35S]:[3H] so4 PAPS => PLJ4.7/TR20 -> ililn
ct-
s04 264
incorporated into GAGs. p-D-xyloside must increase further the proportion of sulphate
from the RESP - both lor CFPAC and PANC. Therefore during elevated GAG synthesis,
higher demand is placed on the RESP lor sulphate. As a consequence, [.'S]:[.H] ratios
are increased. A possible corollary ol this theorem is that the level of sulphate donation
from the SESP may be fixed and therefore not able to respond to higher demand
ln order to explain the characteristics of the three transduced cell lines it is necessary to
postulate a'brake'or perhaps more accuralely, a rate-limiting step induced by CFTR on the contribution of sulphate from the RESP. This rate-limiting mechanism normally
present in PANC results in lower ["S]:['H] ratios. When restored to PLJ6 the result is a return to normal ratios. The increase in size of the SESP and lack of effect of CFTR as seen in TR20 and PLJ4.7 is more problematical. The best explanation for this behaviour is that in some way the expression of CFTR has enhanced the transfer of sulphate from the RESP to the SESP. As the exit of sulphate from the SESP seems to occur at a fixed rate, the size of the pool must increase. Therefore, rather than imposing a limit or restriction on the high flow of sulphate from the RESP -+ Golgi, CFTR has caused a rerouting of sulphate in TR20 and PLJ4.7. The contribution of sulphate from RESP and
SESP to GAGs remains the same however with the result that ["S]:[3H] ratios are unchanged from CFPAC
These observations raise the question, 'what is the SESP?' At present we are unable to answer this question. lt exists purely as a kinetic phenomenon because we cannot say how or where the sulphate is comparlmentalised. Mohapatra et a/. speculated that it may represent a pool of sulphate generated from cysteine. As such, an intracellular 2
shown that location within mitochondria might be plausible. lndeed it has been
lransport mitochondria possess high concentrations of sulphate Munn 1974 and can
we have shown sulphate elfectively (Crompton et al., 1974),1975) ln pancreatic cells
the that the contribution of cysteine to GAG sulphation is very small. However, il
pool lrom the RESP, catabolism of cysteine to sulphate results in a sulphate separate
then our calculations may be incorrect (because we have assumed there is a rapid
generated becomes equilibration of external and internal cysteine and that the sulphate
sulphate which incorporated rather than compartmentalised) and the contribution of
present only way we have of originally derives from cysteine may be much higher. At the
exchanger. defining the sEsp is by its inaccessibility to the plasma membrane anion
it All of the data obtained appear to be consistent with the proposed model, however
affected but not requires that we believe two of the CFTR expressing cell lines were
the data corrected. what could be the molecular basis for such an effect? Unfortunately
potential of CFTR do not answer this question. However current theories regarding the
may hold the answer' to reside in intracellular membrane systems and to function there,
protein and responses may be dictated by the level of expression of the cellular ,its low abundance subsequent localisation to the correct intracellular site' oFTR is a
specific regulation chloride channel known to be under tight temporal and tissue
(Montrose-Rafizadeh et a1.,1g91; Trezise and Buchwald, 1991; Trezise ef a/-, 1992)'
in growth arrest of schiavi et al. (1gg3) have shown that over-expression of CFTR results in transgenic rabbits monkey kidney cells and a high incidence of stillborn male births
lines could result in expressing CFTR. Over or under-expression in our transfected cell
pathway we have identilied' the partial interuention or partial correction of the metabolic 266 ln the context of human disease this might exacerbate rather than improve the original defect. lt is possible that other classes of mutations e.g. resulting in altered channel characteristics rather than biosynthetic arrest in the endoplasmic reticulum as we know 1o happen with ÂF'or, might influence the pathway in different ways. More information is obvíously required on the intracellular location and functioning of CFTR before we can speculate on how sulphate metabolism might be affected by inappropriate expression ol the protein
An apparent contradiction in some of the data presented in this chapter needs to be pointed out. ln section A, the results of long term labelling experiments with these cell lines produced results in terms of ["S]:['H] GAG ratios which suggested that PLJ4.7 was the cell line exhibiting correction (i.e. most similar to PANC) and that PLJ6 had ratios comparable with CFPAC. The nature of these discrepancies is not readily understood but may reflect the fact that over long periods of exposure to l"SJsulphate there may have been a partial or full equilibration of the RESP and SESP with unpredictable consequences for ["S]:['H] ratios in GAGs. lt was noted earlier that short term labelling studies generated greater differences between CFPAC and PANC than was seen for the longer labelling experiments e.g. the differences seen in section A were of the order of two{old whilst in section B they were 4.5 fold. Given the complexity of metabolic pathways involving sulphate and glucosamine, particularly over long periods in tissue culture, it would be fair to say that the later experiments in which cells were incubated for short periods in defined medium allow us to say with greater certitude that the relationships between ratios are reflecting the eflect of CFTR expression 267
As was noted with earlier experiments involving lymphoblasts (Chapter 4), this study
The demonstrates the need for caution in interpreting radiolabel incorporation data.
is inference that higher [.'S]sulphate incorporat¡on is indicative of higher total sulphation founded on the assumption that cell lines (or other experimental systems) being
When compared have equivalent pool sizes and also access precursors in similar ways'
previous this is not so, the conclusions drawn can be incorrect. With this in mind, work claiming evidence lor over-sulphation based on increased ['uS]:['H] ratios by CF cells may need to be re-evaluated'
The model we have proposed is testable but requires an accurate chemical
A comparison determination of the total amount of sulphate in newly synthesised GAGs' of the specific activity of sulphate within GAG chains compared to the known specific
minutes) would activity of the RESP (which will be the same as the medium within a lew
the contribution ol allow quantitation of any differences. Such differences must be due to
sulphate from the sESp. Upon correcting for the relative size of the sEsP, a
pool could be made. stoichiometric assessment of the contribution of sulphate from each
lf the This would be a particularly valuable analysis for an assessment of PLJ6 cells'
of model is correct there will have been a readjustment of the proportional contribution
sulphate from the RESP down to levels exhibited by PANC.
271
7.1 LPHATIO rNc FIBR
Cystic fibrosis has historically been slow to yield its secrets. Following the demonstration by di Sant, Agnese and colleagues in 1953 ol salt loss experienced by CF children during
years before the a summer heat wave (di Sant' Agnese et al., 1 953), it was to be many biochemical defect was discovered by Knowles et al. (1981). This project did not and has not proved any more forthcoming in terms of providing quick answers' Ostensibly the aim of this study was to demonstrate over-sulphation of glycoconjugates in CF by focusing on a defined model substrate and by testing a number of experimental systems.
With two notable exceptions (the liver and the ileum of CF mice) we failed in this aim.
This was despite examining two pancreatic cell lines (one CF), six lymphoblast cell lines
(four cF) and seven individualtissues and organs (by HPLC) from CF mice.
I use the word ostensibly because in a real sense the project quickly became an
per The examination of 'sulphate-related abnormalities' rather than over-sulphation se. experimental approach taken was simple and has been often used. Supplementing culture medium with l'sS]sulphate and l'H]glucosamine allowed an assessment of the incorporation of both radiolabels and their relative ratios, into newly synthesised glycoconjugates or GAGs. lnitial results with pancreatic cells and lymphoblasts seemed
analysis of to support the contention that GAGs were being over-sulphated. However
aberration of the structure of cs/DS in both, revealed the phenomenon was due to some
the specilic activity of a precursor pool. There were higher ["S]:['H] ratios in the GAGs
produced by CF cells but no difference in the structure of those molecules. The finding 272 ol a similar phenomenon i.e. some dynamic affecting the specilic activity of sulphate pools, in two completely dilferent celltypes suggested to us that the etfecl was real. The magnitude of the eflect in lymphoblasts, admittedly was small, and cognisance is taken ol that fact. lf the effect in lymphoblasts can be conlirmed with further work, the linding of genotype-specific effects has important implications for our understanding ol CFTR function and dysfunction and possibly for genotype/phenotype correlations.
Our decision to focus on GAGs - a group of molecules with well defined chemistry, and our ability to precisely analyse the structure of those molecules, permitted several insights into the way CF cells were utilising sulphate from different intracellular pools.
Strong evidence that pancreatic cells lrom a CF patient were sourcing sulphate for biosynthesis from different pools than those used by a normal control, came from experiments with gene-corrected cell lines. One CFTR expressing CF cell line exhibited a reversion of [.'S]:['H] ratios to control values, while in two others the presence of CFTR
appeared to affect sulphation but with unintended consequences which did not normalise
behaviour. This finding may have important implications for gene therapy. Simply
providing cells with the gene for CFTR using a shotgun style approach may elicit
unintended consequences, if expression levels and targeting are not able to be
controlled. A further intriguing finding from the final series of experiments was that, in
addition to altered sulphate channelling through the RESP in CF pancreatic cells, the
ability to utilise cysteine as a source of sulphate was enhanced. Again, transfection ol
CF cells with the gene lor CFTR led in one instance, to a lull correction i.e. a reduction in
usage of the pathway to levels exhibited by the normal control. This effect of CFTR on a
cells ability to utilise an amino acìd catabolic pathway for sulphation was completely 273 unexpected. lt will be interesting to see whether this observation can be repeated in other cell types
What could be the molecular basis for a CFTR-mediated check point on sulphate utilisation from the RESp. One possibility would be an interaction with the sulphate activating enzyme, ApS kinase (see section 1.9). This enzyme catalyses a potential rate-limiting step in the formation of PAPS, because of the unfavourable thermodynamic equilibrium that exists for formation of one of its substrates, APS. Because APS is a potent product inhibitor of ATP sulphurylase, APS kinase must rapidly convert APS to pAps in order to favour the synthesis of more APS. Therefore if APS kinase activity,
(or synthesis or trafficking, is normally moderated by CFTR by something it transports,
CFTR (due such as ATp which is intimately involved in this pathway), then an absence of to a mutation such as which results in arrest and degradation in the ER) may result ^F508 in inappropriately high conversion of sulphate within the RESP to PAPS, and subsequent incorporation of sulphate from that source into GAGS. This is of course highly speculative, but might form a logical basis upon which to extend this work'
from the Can the data obtained from cells in culture, be reconciled with observations
higher levels of tissues of CF mice? Firstly, two organs from CF mice actually did exhibit sulphation. Conversely, the nasal septum and the nasal mucosa of CF mice exhibited
we did not have significant reductions to the incorporation of l"S]sulphate. Unfortunately
results of sufficient material to analyse the CS/DS disaccharides from these organs' the
which may have been very informative. A number of other tissues exhibited higher
did not translate into higher [.'S]sulphate incorporation by CF mice than controls, but this 274 total sulphate levels. Thus we were observing a similar phenomenon to that which had been noted with cells in culture. An examination ol the specific activity of ['sS]sulphate in
43 disaccharides from mice confirmed this, with the CF mice having 50% higher
['uSìsulphate per unit GAG than the normal controls. Therefore, and this point is of great significance, what had been observed in pancreatic cells and lymphoblasts was demonstrable in vivo. fhe ubiquity ol this phenomenon is compelling. lt argues strongly that the effect is real, and that the intracellular utilisation of sulphate within tissues of the
CF mouse may be altered in a similat way to the model proposed for pancreatic cells
That it manifests in such a wide range of tissues and cell types leads to speculation that it may be a universal feature of the CFTR expressing cell.
Although we conclude that there is some alteration to the dynamics of sulphate utilisation in CF, it is impossible, lrom the information as it stands, to speculate on consequences.
Others have observed alterations to sulphation in cystic fibrosis. We have demonstrated over-sulphation of CS/DS in the ileum and the liver of CF mice. This may have important pathological consequences in terms of the function of these organs. Basement membranes for example are highly specialised structures which help to determine tissue shape, stab¡l¡ty and architecture. ECM components of which they are composed, influence numerous important cellular activities, including adhesion, spreading, dilferentiation, polarisation and proliferation (Khosla et al., 1994). ln the gut, the major physiological role of GAG and mucous glycoprotein is to control the movement of albumin and ions. HS on vascular and basement membranes restricts albumin flux, whilst within the glycocalyx of enterocytes, GAGs function to sequester K" and Ca" and exclude Cl' (Murch, 1995). There are numerous disease conditions in which proteoglycan synthesis and/or sulphation is alfected and in which it is suspected these metabolic aberrations actively contribute to pathogenesis. The lollowing examples point to the potential importance of inappropriate matrix assembly and sulphation: l. ln hyaline membrane disease of the lung there is a disruption to the tight interaction
between collagen and dermatan sulphate proteoglycan and an enrichment in large
chondroitin sulphate proteoglycan (Juul et a\.,1993). ll. Mouse spleen infected with myeloproliferative sarcoma virus to induce a
myeloproliferative syndrome, increased the production of GAG five times over normal
spleen stromaltissue (Smadja Joffe ef a1.,1992)' lll. ln amyloidosis ol liver and spleen, GAGs have been identilied as tightly associated
with amyloid fibrils. specilically DS and HS have been found associated with fibrils in
different organs and different types of fibrils, supporting the view that they may be of
pathogenic significance (Nelson et al., 1991). lV. ln colorectal adenocarcinomas there is a decrease in GAG produced by neoplastic
colon, the proportions of GAGs normally present is altered and HS appeared to be
under-sulphated (Bouziges et al., 1990). Pancreatic tumour tissue has also been
shown to synthesise different proteoglycans (Fukata et a\.,1989).
V. Congenital nephrotic syndrome (CNS) is proposed to occur as a result of abnormal
glomerular permeability. One study examined glomerular basement membranes from
a deceased cNS patient and found the quantity of HS massively decreased from
59% of the total GAGs to 3%. There was also an increase observed in the level of
HS excreted in urine of these patients (Vermylen et a1.,1989).
Vl. ln human liver fibrogenesis, the pattern of GAGs in fibrotic liver matrix is altered by
several fold increases in hyaluronan, CS and DS (Meyer et a1.,1990)' 2
Vll.ln a model of renal proximal tubule tumorigenesis, cells from rabbits exhibited
alterations to the amount of proteoglycan released into culture medium, an increase
in the synthesis ol free GAG chains and a dramatic change in the composition ol
GAG chains with an increase in CS synthesis at the expense of HS (Lelongt et al.,
1992). A finding with parallels to our data in the gall bladders and ilia of CF mice.
Also of great importance, is the disparate way in which the tissues of CF mice responded to the provision of a dose of l"S]sulphate. lt informs us without doubt that there is fissue speciÍic utilisation of sulphate by CF organs i.e. each organ responds differently.
Although this might seem self-evident, it demonstrates an important principle, namely that there is no pan-cellular mechanism which results in global over- or under-sulphation.
Hence, the tissue or cell Wpe one chooses to examine for sulphation-related effects will be important, as will the molecule examined. We chose GAGs, (and in fact focused on
CS/DS) for the facility with which they could be analysed structurally. Maybe GAGs, for reasons peculiar to their mode of synthesis, Golgi localisation, or some regulatory constraint, do not, except in extreme circumstances, become over-sulphated. Other molecules may not only exhibit more consistent over-sulphation, but have greater pathophysiological relevance in CF. Suffice to say that we have pinpointed a number of interesting and complex, CFTR mediated effects. lt remains to be established, the importance (or othenryise) of those effects, and to deduce how a cAMP-dependent chloride channel interacts with the cellular mechanisms which atfect not only sulphate utilisation but also the catabolism of amino acids. This project has laid the foundation for a new appreciation of the role of CFTR and for new understandings about the subtle ways in which it may affect metabolic and catabolic pathways. The clinical relevance of 277 these lindings are unclear, however sulphate and sulphation may still prove to be of great importance in the pathophysiology of cystic librosis
7.2 PHI PHICAL SIDERATIONS
The CF story is still unfolding... slowly. The discovery of the gene heralded a new era of understanding and the promise of a cure. Such hopes have dimmed somewhat, as we struggle to comprehend some of the bewildering manifestations of the condition. The focus of our efforts must fall predominantly on CF, the disease, and not solely on CFTR, the protein. CFTR is a chloride channel regulated by exquisitely complex pathways and governed in its activity by subtle inter- and intra- molecular signals. As such it will yield to scrutiny. But, how to explain lung disease in CF; how to explain the pathological progression of a disease which in two individuals with identical genotype results in vastly different outcomes; how to explain CBAVD - CF or not CF? These remain of vital
impodance and are unlikely to be answered by studying CFTR in isolation'
Our understanding is sorely tested because some of our assumptions are simplistic. For
example, why should CFTR-genotype correlate with CF-phenotype? We have seen that
CFTR can regulate another chloride channel, that Na. transport is elevated in CF
epithelia, that CF mice suffer no lung disease, that the human lung in CF appears
incapable of preventing infection, whilst the immune system apparently remains intact'
These observations challenge our understanding, and our wish to simplify complex
genetic, biochemical and environmental interactions. Further, they point to secondary man¡festations (such as were demonstrated in this project) about which we know little and some would wish to ignore. As scientists we should not dwell overlong on the magnitude of what we try to comprehend nor attempt to deny it. A need to appreciate that magnitude is vital. ln the search for understanding we must learn to hold a little more loosely, the comforting assumptions/simplifications we hold, and at times defend with such vigour. The words of Freeman Dyson are apposite; "natures imagination is richerthan ourd'. This is the lesson we continue to learn as we confront an enigmatic disease that defies our best efforts and in doing so continues to claim young lives.
281
APPENDTX A: WILCOXON SIGNED-RANKS TEST FOR TWO-RELATED SAMPLES TO DETERMINE WHETHER ["S]:['H] RATIOS lN THE 45 DISACCHARIDE OF LYMPHOBLASTS WERE SIGNIFICANTLY DIFFERENT AT 72 HOURS THAN AT 24 HOURS:
MEDIUM SAMPLES: Tabulating differences and ranks d- 0.01 -0.02 0.04 0.06 0.08 0.16 rank= -2 3 4 5 6
Observed sum of Positive ranks = 19 Observed sum of negative ranks = 2 + T=2 pr*o.".,(T=2) = 0.188, therefore we accept the null hypothesis that the two sets of data are not different between the two sampled time points (for n=6, T must equal 0 for
Pr*.o,, -0.14 d -0.02 0.02 -0.06 0.08 -0.09 rank= -1.5 1.5 -3 4 -5 -6 Observed sum ol positive ranks = 5.5 Observed sum of negative ranks = 15.5 + T=5.5 hypothesis. pr*-,..,(T=5.S) = 0.31 ... As lor medium samples we accept the null 2 APPENDIX B We know the amount of cysteine or sulphate added to each dish (mol) and the specific activity of the medium (dpm/pg protein) and the specilic activity incorporated into GAGs (dpm/pg protein). Hence a simple proportional calculation can be performed to yield the number of moles of total sulphate incorporated from the nom¡nated source. t . ,. sp. act. in GAGs I I mol cystelne ln meolum r I L sp. act. rn medruml cysteine sulphate incorp (mol / ug protein) : protein (ug) - By rearranging the term for specific activity in medium, the protein term cancels leaving the number of dpm added to the medium, cysteine sulphate incorp. 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