Molecular and kinetic analysis of human glyoxylate/hydroxypyruvate
reductase
David Peter Cregeen
A thesis submitted for the degree of Doctor of
Philosophy
University College London ProQuest Number: 10010104
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This thesis describes the elucidation of the genetic basis of primary hyperoxaluria type 2 (PH2), an autosomal recessive disease characterised by recurrent calcium oxalate kidney stone formation leading to renal failure and, in some cases to an early death. The enzyme deficient in this condition, glyoxylate / hydroxypyruvate reductase (GRHPR), has been partially purified and characterised from human liver and is distinct from lactate dehydrogenase which also has hydroxypyruvate reductase activity. GRHPR utilises hydroxypyruvate and glyoxylate as substrates with a preference for hydroxypyruvate with NADPH as preferred cofactor. Chromatofocused partially purified human liver proteins show no GR activity associated with LDH, using NADPH as cofactor.
Although sharing low protein sequence similarity to the GR/HPR enzymes of plants and bacteria, a human cDNA encoding GRHPR has been identified from an EST database. Purified recombinant GRHPR has similar kinetic properties to the partially purified hver protein, with a preference for hydroxypyruvate as substrate and NADPH as cofector at pH7.0. Antibodies raised against this recombinant enzyme identify a single protein in human liver of approximately
38kDa which is absent in patients with PH2. The protein demonstrates charge heterogeneity on chromatofocusing and isoelectric focusing, although at a different pi range unless subunits are chemically crosslinked as a dimer (the preferred enzyme conformation). Northern blot analysis of GRHPR mRNA shows its primary location to be the liver but with RNA present in many other tissues. This ubiquitous expression is not reflected by distribution of immunoreactive protein which was essentially restricted to the liver. DNA jfrom patients with PH2 has been screened for mutations in the GRHPR gene by direct sequencing of cDNA where available, or SSCP analysis and sequencing of genomic DNA. 6 mutations and a single polymorphism have been identified including a Ibp deletion in codon 35, found in 33% of all PH2 alleles. Abbreviations
AGT alaninerglyoxylate aminotransferase bp base pair cDNA conq)lementary DNA
DNA deoxyribonucleic acid
EDTA Ethyienediaminetetra-acetic acid
HPR hydroxypyruvate reductase mRNA messenger RNA
NAD+ p-Nicotinamide adenine dinucleotide
NADH p-Nicotinamide adenine dinucelotide, reduced form
NADP+ p-Nicotinamide adenine dinucleotide phosphate
NADPH p-Nicotinamide adenine dinucleotide phosphate, reduced form
PHI primary hyperoxaluria type 1
PH2 primary hyperoxaluria type 2
RNA ribonucleic acid
SDS Sodium dodecyl sulphate
Tris Tris[hydroxymethyl]aminomethane Contents
Chapter Index
Title...... 1
Abstract...... 2
Abbreviations...... 4
Contents...... 5
Chapter Index ...... 2
List of Figures...... 9
List of Tables...... 13
Acknowledgements...... 14
Chapter 1: Introduction...... 15
1.1 Oxalate in biological systems ...... 15
1.1.1 Calcium oxalate stones...... 20 1.1.2 Dietary sources of oxalate in humans...... 20 1.1.3 Intestinal oxalate transport...... 21 1.1.4 Renal oxalate transport...... 22 1.1.5 Metabolic precursors of oxalate...... 23 1.1.6 Glyoxylate cycle in animals ...... 30 1.1.7 Glycolate dehydrogenase...... 33 1.1.8 Hepatic serine metabolism ...... 33 1.1.9 Tissue specificity of metabolic pathways...... 35 1.1.10 Intracellular transport of metabolites...... 35
1.2 Hyperoxaluria ...... 36
1.2.1 Enteric hyperoxaluria...... 36 1.2.2 Primary hyperoxaluria...... 37 1.2.3 Primary hyperoxaluria type 1 ...... 38 1.2.4 Primary hyperoxaluria type 2 ...... 40 1.2.5 Atypical primary hyperoxaluria ...... 42 1.2.6 Treatment of primary hyperoxaluria...... 42
1.3 Hydroxypyruvate reductase / glyoxylate reductase ...... 45
1.3.1 GR/HPR in plants...... 45 1.3.2 GR/HPR in bacteria...... 46 1.3.3 GR/HPR in algae...... 47 1.3.4 GR/HPR in mammals...... 47
1.4 Aims of the project ...... 49
Chapter 2: General materials and methods...... 50
2.1 Bacterial culture reagents...... 50 2.2 Bacterial culture protocol...... 50 2.3 Plasmid DNA isolation from E.coli...... 51 2.4 PCR reagents...... 51 2.5 PCR protocol...... 52 2.6 Restriction digestion...... 52 2.7 Extracting DNA bands from agarose gels...... 53 2.8 DNA quantitation...... 53 2.9 Sequencing...... 53 2.10 SDS-PAGE for protein samples...... 53 2.11 Immunoblotting...... 54 2.12 Determination of tissue protein concentration by the Lowry procedure...... 55 2.13 Spectrophotometric determination of protein concentration...... 56 2.14 Coomassie blue staining of precast polyacrylamide gels...... 56 2.15 Silver staining of precast polyacrylamide gels...... 57 2.16 Assays for GR and HPR activity...... 57 2.17 Isoelectric focusing...... 58 2.18 Chromatofocusing...... 58 2.19 Preparation of human tissue sonicates...... 59 2.20 Dephosphorylation of vector 5' ends...... 59 2.21 Ligation of insert DNA into plasmid vector...... 60 2.22 Transformation of plasmid DNA into E.coli...... 61 2.23 Induction of GRHPR expression in BL21 cells...... 62 2.24 Preparation of bacteria for enzyme assays...... 63 2.25 Northern blot...... 63 2.26 DNA/protein analysis packages...... 63
Chapter 3: Purification of GRHPR from human liver and enzyme kinetic studies...... 64
3.1 Introduction...... 64
3.2 Methods ...... 66
3.2.1 Partial purification of GRHPR from human liver...... 66 3.2.2 Purification of GRHPR for protein sequencing...... 67 3.2.3 Two substrate kinetics...... 68 3.2.4 LDH measurement...... 69
3.3 Results...... 70
3.3.1 Purification of GRHPR for protein sequencing...... 71 3.3.2 Two substrate kinetics...... 75
3.4 Discussion...... 76
6 Chapter 4: Identification, expression and characterisation of human
GRHPR cDNA and GRHPR protein ...... 80
4.1 Introduction...... 80
4.2 Methods ...... 82
4.2.1 Nickel affinity chromatography...... 82 4.2.2 Western blots...... 82 4.2.3 Isoelectric focusing...... 82 4.2.4 Chemical crosslinking of pure GRHPR...... 83 4.2.5 Enzyme kinetic studies...... 83
4.3 Results...... 84
4.3.1 Identification of putative GRHPR cDNA clone...... 84 4.3.2 Expression ofcDNA...... 89 4.3.3 Induction of GRHPR expression in BL21 cells...... 90 4.3.4 Purification of recombinant human GRHPR...... 92 4.3.5 Antibody production...... 93 4.3.6 Antibody characterisation...... 94 4.3.7 Tissue distribution...... 95 4.3.8 Tissue distribution of GRHPR protein...... 97 4.3.9 Western blots of PH2 liver sonicates...... 98 4.3.10 GRHPR chemical crosslinking...... 98 4.3.11 Charge heterogeneity of GRHPR...... 99 4.3.12 DTT treatment...... 102 4.3.13 Chemical dénaturation of GRHPR...... 103 4.3.14 Enzyme kinetics...... 104
4.4 Discussion...... 110
Chapter 5: Molecular Genetics of PH2 ...... 123
5.1 Introduction...... 123
5.2 Methods ...... 124
5.2.1 RNA isolation...... 124 5.2.2 Genomic DNA isolation from leucocytes...... 124 5.2.3 Reverse transcriptase PCR (RT-PCR)...... 125 5.2.4 Polyacrylamide gel electrophoresis (PAGE) for DNA samples...... 125 5.2.5 Single strand conformation polymorphism analysis...... 126
5.3 Results...... 127
5.3.1 Identification of sequence variants...... 127 5.3.2 Deletion of 28bp in exon 1...... 127 5.3.3 G deletion in codon 35...... 129 5.3.4 Expression of a truncated form of the GRHPR protein...... 131 5.3.5 GTAA deletion at 5’ splice donor site of intron 4...... 133 5.3.6 Mutation screening by SSCP analysis...... 135 5.3.7 Exon 4 ...... 135 5.3.8 Exon 6 ...... 137 5.3.9 Exon 7 ...... 140 5.3.10 Exon 9...... 141 5.3.11 Summary...... 144
5.4 Discussion...... 145
Concluding Remarks...... 155
Appendices...... 158
Appendix 1 - PCR primers and amplification conditions ...... 158
Appendix 2 - Kinetics on partially purified GRHPR from human liver 159
Appendix 3 - GRHPR cDNA, EST and protein sequences ...... 161
Appendix 4 - Calibration curve for isoelectric focusing ...... 163
Appendix 5 - EST sequences matching GRHPR with 28bp deletion 164
Appendix 6 - Summary of PH2 patient diagnosis, HPR/GR activity and
GRHPR mutations...... 165b
References...... 166
Publications arising from this work...... 192 List of Figures
Chapter 1 - Introduction
Figure L I Oxalate transport and utilisation in Oxalobacter formigenes 18 Figure 1.2 Oxalate metabolism in bacteria...... 19 Figure 1.3 Compounds involved in glyoxylate and oxalate metabolism 23 Figure 1.4 Suggested pathways leading to oxalate production...... 24 Figure 1.5 Summary o f the pathways linking serine entry into gluconeogenesis and glyoxylate metabolism ...... 29 Figure 1.6 The glyoxylate cycle ...... 31 Figure 1.7 The tricarboxylic acid (TCA) cycle ...... 32 Figure 1.8 Metabolic pathway relevant to PHI and PH2 ...... 38
Chapter 3 - Purification of GRHPR from human liver and enzyme kinetic
studies
Figure 3.1 CM-Sephadex ion exchange chromatography profile o fsemi-purified human liver GRHPR ...... 70 Figure 3.2 Fractionation o f human GRHPR by chromatofocusingfrom pH 7.2 to p H 4 .0 ...... 71 Figure 3.3 Elution profile of human liver GRHPR from 2 ’5 ’ADP sepharose... 72 Figure 3.4 Elution profile o f GRHPR from Sephadex G-200...... 73 Figure 3.5 Coomassie blue stained SDS-PAGE illustrating samples from various points in the GRHPR protein purification procedure ...... 74
Chapter 4 - identification, expression and characterisation of human GRHPR cDNA and GRHPR protein Figure 4.1 Full cDNA and predicted protein sequences o f human GRHPR 85 Figure 4.2 Human GRHPR protein sequence ...... 86 Figure 4.3 Mulitiple protein sequence alignment o f HPR/GR ...... 87 Figure 4.4 Cloning and expression strategy for the putative human GRHPR cDNA...... 88 Figure 4.5 HPR and GR activity in BL21 cells transfected with pTrcHisB- GRHPR...... 90 Figure 4.6 E.Coli BL21 proteins during induction o f recombinant protein expression ...... 91 Figure 4 .7 Purification on nickel affinity column o f recombinant GRHPR enzyme...... 92 Figure 4.8 Dot blot of primary antibody dilutions against various pure recombinant GRHPR concentrations to determine optimal titre 94 Figure 4.9 Multiple tissue northern blot hybridised with full length GRHPR cDNA...... 95 Figure 4.10 Quantitation o f GRHPR content o f human by comparison with known amounts o f pure recombinant GRHPR ...... 96 Figure 4.11 Tissue distribution o f GRHPR immimoreactivity ...... 97 Figure 4.12 GRHPR and AGT immunoreactivity on a western blot containing human liver protein from PH2 patients and controls...... 98 Figure 4.13 Chemical crosslinking o f pure recombinant GRHPR with BS3 99 Figure 4.14 HPR and GR activity o f recombinant human GRHPR separated on chromatofocusing ...... 100 Figure 4.15 Coomassie blue^tainedAmpholine^^PAGplate (pHrange 3.5-9.5) isoelectric focusing gels...... 101 Figure 4.16 Investigation o f charge heterogeneity in GRHPR ...... 102 Figure 4.17 Single substrate kinetics o f pure recombinant human GRHPR.... 105 Figure 4.18 Spontaneous dehydrogenation o f NADPH and NADH with increasing glyoxylate concentration ...... 108 Figure 4.19 Effect of hydroxypyruvate on reaction rate o f recombinant GRHPR ...... 109 Figure 4.20 Active site ofD-GDHfrom Hmethylovorum ...... I l l
10 Chapter 5 - Molecular Genetics of PH2 Figure 5.1 GRHPR exon 1 & 2 cDNA sequences obtained from RT-PCR with primers EHPRl andEHPR2 ...... 127 Figure 5.2 Protein sequence comparison between proteins derived from the normal (undeleted) form o f GRHPR and the sequence with a 28bp deletion at the 3 ’ end o f exon 1 ...... 127 Figure 5.3 Silver stained PAGE gel containing PCR products amplified from cDNA of various tissues with HPRA and HPR5 primers...... 128 Figure 5.4 Codon 34/35 G deletion in a PH2 patient compared to wild-type sequence ...... 129 Figure 5.5 PCR with BsmF and BsmR primers produces a 152 bp product which digests to 91 and 6Ibp fragments in the wild-type sequence or remains undigested in the presence o f the Ibp G deletion in codon 34/35... 130 Figure 5.6 Western blot and GRHPR activity o f bacterial homogenates expressing the EHPR3/EHPR2 PCR product in pTrcHisB expression vector...... 132 Figure 5.7 GTAA/AAGTdeletion at the 5 ’ splice donor site in intron 4 sequenced from genomic DNA using primers EX4F1 and EX4R1 ...... 133 Figure 5.8 Demonstration o f the effect o f the GTAA deletion on GRHPR splicing ...... 134 Figure 5.9 Exon 4 SSCP ...... 135 Figure 5.10 Sequence analysis o f GRHPR exon 4 ...... 136 Figure 5.11 Exon 6 SSCP...... 137 Figure 5.12 Sequence analysis o f GRHPR exon 6 (G494A) ...... 138 Figure 5.13 Sequence analysis o f GRHPR exon 6 (A579G) ...... 139 Figure 5.15 Sequence analysis o f GRHPR exon 7 ...... 141 Figure 5.16 Exon 9 SSCP ...... 142 Figure 5.\1 Sequence analysis o f GRHPR exon 9...... 143 Figure 5.18 Slipped mispairing model o f Ibp deletion in GRHPR codon 34/35 ...... 147 Figure 5.19 Putative conformation o f a short palindromic sequence in GRHPR exon 2 around the codon 34/35 region G deletion ...... 148 Figure 5.20 Slipped-mispairing model for exon 4/intron 4 4bp deletion 149
11 Figure 5.21 Modified slipped-mispairing model for exon 4/intron 4 4bp deletion ...... 150 Figure 5.22 Sequence surrounding GRHPR exon 7 CT deletion ...... 151
12 List of Tables
Chapter 1 - Introduction Table 1.1 Substrate specificity o f bacterial GR/HPR enzymes ...... 46
Chapter 3 - Purification of GRHPR from human liver and enzyme kinetic studies Table 3.1 Kinetic analysis o f peaks A and B from chromatofocusing ...... 75
Chapter 4 - Identification, expression and characterisation of human GRHPR cDNA and GRHPR protein Table 4.1 Schedule o f immunisation of rabbits with pure recombinant GRHPR. 93 Table 4.2 Kinetics for GR, HPR and D-GDH activities ofpurified recombinant GRHPR with various substrate-cofactor combinations...... 104 Table 4.3 Putative phosphorylation sites within the human GRHPR protein sequence ...... 116
Chapter 5 - Molecular Genetics of PH2 Table 5.1 Summary o f DNA changes found in PH2 patient cohort studied 144
13 Acknowledgements
Firstly, I would like to thank my supervisor Dr. Gill Rumsby for her advice,
encouragement, enthusiasm, patience and willingness to check and recheck
papers and this thesis. Thanks also go to my second-supervisor Cohn Samuell for
taking such an interest in my work.
Many thanks to everyone I have worked with in the lab over the last few years
for their help and the relaxed working atmosphere. In particular I would like to
thank Tim Weir for help with most aspects of my lab work and for being a
constant source of usefiil informatioa Also Emma Williams for all the
interesting discussion and brainstorming sessions we have had.
I would also like to express my gratitude to everyone in the department of
Chemical Pathology at UCLH for letting me loose in their labs and for so many
good nights out. Also thanks to the department of (Immunology and) Molecular
Pathology.
Finally I would like to thank the Sir Jules Thom Charitable Trust and the
Oxalosis and Hyperoxaluria Foundation whose funding enabled this work to be carried out and my thesis to be completed.
14 Chapter 1 : Introduction Primary hyperoxaluria type 2 (PH2) is a rare disease of endogenous oxalate
overproduction characterised by recurrent calcium oxalate renal stones leading to
impaired renal function. PH2 patients show decreased glyoxylate reductase (GR) and
hydroxypyruvate reductase (HPR) enzyme activities, however it is unclear if these are
activities of a single protein such as that found in plants and bacteria. This thesis aims
to characterise the human enzyme/s with GR and HPR activity and identify the
molecular basis of PH2. In order to understand the role of this enzyme in oxalate
metabolism, a wider view needs to be taken.
1 .1 Oxalate in biological systems
Oxalate is the anionic form of oxalic acid, found in many plants, microbes and
animals as its crystalline calcium salt. Calcium oxalate is thought to serve many
roles in nature including provision of an intracellular calcium pool, structural and
defensive roles in plants and a microbial carbon source. In animals oxalate seems
to serve no useful purpose and is thought to be an end product of normal
metabolism. The insolubility of the calcium salt at physiological pH in higher
mammals can be regarded as a pathological nuisance and calcium oxalate is a
major constituent in the majority of renal stones in man.
Oxalate, and more specifically calcium oxalate, is found to accumulate in
significant quantities in most higher plants and is produced from many different
sources (reviewed in [1]). Glycolate derived from photosynthesis and
photorespiration is the major precursor of oxalate, first being converted
enzymatically to glyoxylate in the peroxisome. Photorespiration is the uptake of
O2 and the formation of CO 2 in light which results from glycolate synthesis in
15 the chloroplast and glycolate metabolism in the peroxisomes and mitochondria
[2]. The relative amount of oxalate formed from glyoxylate depends on nitrogen
status; when nitrogen is abundant most glyoxylate is transammated to glycine
hence keeping oxalate production relatively low [2], Ascorbic acid has been
shown to be catabolicaUy converted to glycolate and glyoxylate in the dark [3]
and in some systems oxaloacetate and formate are also thought to be precursors
of oxalate [1], Soluble oxalate accumulation may function to maintain an ion or
pH balance in some cells [4]. Calcium oxalate crystallisation occurs in plants
mainly in specialised cells known as crystal idioblasts [4] and various
suggestions for this accumulation have been put forward [1,4]. Accumulation in
some plants such as oxalis, spinach and rhubarb may produce a bitter taste and
discourage animal foraging [5] or the crystals may simply be a way of removing
excess unusable oxalate from the plant system [4]. It has also been suggested that
calcium oxalate may function as a calcium store in the plant [6] or provide
structural strength [4].
Many species of oxalate-degrading bacteria have been isolated to date including
Methylobacterium extorquens, Bacillus oxalophilus, Pseudomonas oxalaticus
and Oxalobacter formigenes (reviewed in [7]). Much attention has been fr>cussed
on O.formigenes due to its presence in human faeces [8] however this may not be the only oxalate degrading bacterial species present therein. O.formigenes is an
anaerobic organism found in the rumen of herbivores as well as the gastrointestinal tracts of non-ruminant herbivores and humans and also aquatic sediments. The O.formigenes strain was originally isolated from the rumen of sheep [9] and cattle [10, 11] which were shown to cope with increased oxalate
16 containing diets by an increase in the number of oxalate degrading rumenal microbes. The 21 strains of O.formigenes are gram negative and utilise oxalate as their sole energy-yielding substrate [12]. Oxalate is transported into the cell via an oxalate/formate antiporter [13, 14] followed by activation to oxalyl-coenzyme
A by acy 1-coenzyme A transferase [15] (EC 4.1.1.8) (see Figure 1.1 for reaction scheme). Oxalyl-coenzyme A is then decarboxylated to form formyl-coenzyme
A by oxalyl-coenzyme A decarboxylase (EC 4.1.1.8) [16]. O.formigenes produces formate as the end product of oxalate metaboHsm [12] and a proton gradient is established by the consumption of H ^ by the decarboxylase reaction
[13]. The proton gradient is utilised by an indirect proton pun^ to generate ATP
[13]. This is in contrast to other oxalate degrading species such as M. extorquens
[17], and P.oxalaticus [18, 19] which generate energy directly from the oxidation of formate. It is also thought that the metaboHc pathways utilising oxalate differ between O.formigenes and other oxalate degrading bacteria mentioned. Figure
1.2 shows that in O.formigenes [7] and P.oxalaticus [18, 20] glyoxylate is converted to glycerate via tartronic semialdehyde and not via hydroxypyruvate as seen in M extorquens [17].
17 interiorexterior XJ
CO oxalate 7T oxalate Formyl-CoA OxfT oxalate:fonnate CoA Oxalyl-CoA antiporter transferase decarboxylase
formate formate Oxalyl-CoA
ADP+Pi
3H+ F„R ATPase 3H+
ATP
Figure L I oxalate transport and utilisation in Oxalobacter formigenes (adapted from [7])
18 oxalate i Oxalyl-CoA I - ORk r — ►NAD(P)^ glyoxylate S G l\^
Tartronic semialdehyde hydroxypyruvate
HPR — ►NAD* glycerate
f " ^ A D P 3-phosphoglycerate A I A biosynthesis
O.formigenes M.extorquens B.oxalophilus P.oxalaticus
Figure 1.2 Oxalate metabolism in bacteria. OR, Oxalyl-CoA reductase (glyoxylate dehydrogenase EC 1.2.1.17) GC, Glyoxylate carboligase (EC 4.1.1.47) TSR, Tartronic semialdehyde reductase (EC 1.1.1.60) SGT, serine:glyoxylate transaminase (EC 2.6.1.45) HPR, Hydroxypyruvate reductase (EC 1.1.1.29) GK, Glycerate kinase (EC 2.7.1.31) (adaptedfrom [7P
Oxalobacter formigenes colonises the human gut and it is believed that the bacteria can scavenge some dietary oxalate to decrease the amount available for absorption in the colon [21]. In the process it produces a transepithelial gradient to enable colonic oxalate secretory pathways to operate [21]. Children start to be colonised by the bacteria at approximately one year of age with nearly 100% colonised by the age of 3 to 10 years [22]. However, up to 25% lose colonisation during adolescence or adulthood which may be due to antibiotic use.
19 1.1.1 Calcium oxalate stones
Kidney stones are common in industrialised countries affecting up to 10% of the
population [23], more frequently in men than women and with a high recurrence
rate. Up to 80% of stones contain calcium salts [24] the majority of which are
calcium oxalate. Calcium oxalate is found as monohydrate or dihydrate crystals,
the monohydrate being indicative of primary hyperoxaluria. Crystallisation is
influenced by many factors such as hypercalciuria, hyperoxaluria,
hyperuricosuria, low urine volume, urine pH, hypercitraturia or absence of
crystal inhibitors [25]. The solubility of calcium oxalate is very low, with only
T.lmg soluble in 1 litre of water at body temperature compared with 285g of
potassium oxalate, 37g sodium oxalate and 0.4g magnesium oxalate [5],
therefore presence or absence of these factors may influence crystal formation.
As the molar ratio of calcium to oxalate is high [26] changes in urinary oxalate
will have a greater effect on calcium oxalate saturation than urinary calcium.
1.1.2 Dietary sources of oxalate in humans
A normal diet contains 70-93Omg/day of oxalate but in countries with a large
amount of vegetables in the diet intake may be up to 2000mg/day [27]. High
amounts of oxalate are found in spinach, rhubarb and chocolate which contain
750mg, 860mg and 117mg per lOOg of material respectively [28], however there
is considerable variation in oxalate content of plants by season, species, variety,
age, maturity, soil conditions and part of plant [28]. It has recently been shown
that capillary electrophoresis and ion chromatography may be a more sensitive
method of measuring the oxalate content of food [29] than the enzymatic method
[28] commonly used.
20 The amount of oxalate available for gastrointestinal absorption depends on the
amount present as insoluble salts in food [30] and is also inversely related to the
intralumenal calcium concentration [31]. It bas been estimated that only 10 to
15% of the oxalate excreted in the urine of bumans comes from the diet in
normal subjects [31-34], the remaining gastrointestinal oxalate being bound by
intralumenal calcium in the gastrointestinal tract and excreted in the &eces. A
recent report suggests this figure may vary in individuals and may account for
between 10 and 72% of oxalate excreted [35], measured as a decrease in urinary
oxalate excretion after dietary restriction. However, as oxalate content of the diet
was not regulated or measured prior to restriction it is unclear whether the
decrease in urinary oxalate concentration actually corresponds to the percentage
dietary contribution, or merely that urinary oxalate can be reduced by dietary
restriction.
1.1.3 Intestinal oxalate transport
Although only a small percentage of oxalate excreted in the urine comes from
dietary sources, oxalate transport across the gastrointestinal tract remains an
important influence on the amount of oxalate reaching the kidney. In the intact
gastrointestinal tract the stomach may be an important site for oxalate absorption
[36] as well as the proximal small bowel [37, 38]. Oxalate can passively difiuse
across all portions of the normal intestine [39] which may be the predominant
method of transport when intraluminal oxalate concentrations are high. Oxalate
exchange for anions has been shown in the brush border membrane of the rabbit
ileum, where hydroxyl and chloride ions can be exchanged for oxalate [40,41].
21 In addition oxalate has been shown to inhibit the transport of sulphate across the
basolateral membrane of rabbit ileal enterocytes, probably due to competition
with sulphate for the proposed sulphate-bicarbonate exchange system [42].
Oxalate transport may also be affected by the oxalate concentration gradient
across the colon. Rats with chronic renal failure have been shown to secrete
oxalate into the colon [43] (as opposed to the normal net absorption) which may
work in concert with renal excretion under these circumstances. As previously
mentioned, it has also been suggested that the oxalate degrading bacterium
O.formigenes ; helps to maintain the oxalate gradient in the normal
colon [21].
1 1.4 Renal oxalate transport
Oxalate is freely filtered at the glomerulus, however this does not account for the
amount of oxalate present in the urine, consistent with a net renal tubular
secretory mechanism. However both net absorptive [44-46] and secretory [47,
48] transport mechanisms have been identified in the renal tubule. The reason for
this discrepancy is unclear although it has been shown that absorption/secretion
can vary from day to day [47]. The transport of oxalate across the basolateral
(facing the blood supply) and apical (feeing the tubular fluid) membranes of
epithelial cells is mediated by anion-exchange mechanisms. Oxalate is a
substrate for the sulphate (bicarbonate) transport system at the basolateral
membrane [49-52] which is coupled to the chloride/bicarbonate exchanger [53].
At the apical membrane electroneutral secretion of oxalate occurs in exchange
for Na^ dependent resorption of sulphate [54]. Several other oxalate transporters
22 have been identified in isolated membrane vesicles fi"om rat and rabbit (reviewed
in [55], [54]).
1/ 1,5 Metabolic precursors of oxalate
H H H I I I H 0-C = 0 H -C = 0 H-C-NH, H-C-OH H-C-OH I I I I I H O-C=0 H 0-C = 0 H 0 -C = 0 H 0 -C = 0 H -C = 0 Oxalate Glyoxylate Glycine Glycolate Glycolaldéhyde
CHj CH3 CHgOH CH-OH OHjOH I H-C-NH, 0=0 H-C-NH. 0 = 0 H-O-OH I I I I I H O-C=0 H O -C = 0 H O -C = 0 H 0 -0 = 0 H 0 - 0 = 0 Alanine Pyruvate Serine Hydroxypyruvate Glycerate
Figure 1.3 Compounds involved in glyoxylate and oxalate metabolism
Several metabolic precursors of oxalate have been described in the literature,
including glycine, ethylene glycol, xylitol and finctose/sorbitol, and the amino
acids phenylalanine, tyrosine and tryptophan. The structure of some of the
relevant compounds in this pathway are illustrated in Figure 1.3. Potential
oxalate precursors have either been radiolabelled and shown to produce
radiolabelled oxalate (usually in rat liver), or have been shown to produce
oxalate (and hyperoxaluria) when ingested or applied in excessive amounts.
23 These potential routes feeding into oxalate production are described in more detail below and summarised in Figure 1.4.
hydroxypyruvate O-xylulose xylitol
HPD xylulose-1 -phosphate fructose-1 -phosphate glycolaldéhyde ADH ethylene glycol ALDH*
glycolate
GCDH, GR PhefT rp/Tyr glycine -(? glyoxylateglyoxylate — diet DAO GGT glycine LDH
oxalate ^ ascorbate cytosolperoxisome
Figure 1.4 Suggested pathways leading to oxalate production. GR, glyoxylate reductase (EC 1.1.1.26) LDH, lactate dehydrogenase (EC 1.1.1.27) GCDH, glycolate dehydrogenase GGT, glutamate:glyoxylate aminotransferase (EC 2.6.1.4) DAO, D-amino acid oxidase (EC 1.4.3.3) ADH, alcohol dehdrogenase (EC 1.1.1.1) ALDH, aldehyde dehydrogenase (EC 1.2.1.21) HPD, hydroxypyruvate decarboxylase (EC 4.1.1.40) * indicates enzyme fourui in both cytosol and mitochondria.
Hyperoxaluria is observed in some cases of transurethral resection of prostate
(TURP) following glycine irrigation [56, 57]. This hyperoxaluria is also associated with elevated glycolate excretion and is suggested to result from the conversion of glycine to glyoxylate by peroxisomal D-amino acid oxidase.
However kinetic studies of this reaction in rats show that at physiological concentrations of glycine this reaction is unlikely to occuJ in vivo [58].
24 Ethylene glycol intoxication has been shown to cause metabolic acidosis, renal
failure and tissue oxalosis [59]. Ethylene glycol itself has a low toxicity but may
be converted to an oxalate precursor, glycolaldéhyde, by alcohol dehydrogenase.
Only 0.2-0.5% of the ethylene glycol which is ingested will be converted to
oxalate [60], however this is sufficient to deposit calcium oxalate crystals in
virtually all tissues. Current treatment for ethylene glycol intoxication involves
ethanol/fomepizole administration to compete with ethylene glycol for alcohol
dehydrogenase [61, 62].
Post operative hyperoxaluria and calcium oxalate deposition in the renal tubules
can be associated with methoxyfluorane anaesthesia. Two pathways have been
proposed for the oxalate synthesis, via dichloroacetate or difluorohydroxy-acetic
acid [63].
Xylitol is a 5 carbon sugar alcohol which naturally occurs as an intermediate in
the glucuronate-xylulose pathway and was considered a useful form of parenteral
nutrition as an alternative to glucose. However at a high infusion rate adverse
reactions including renal and liver dysfunction, metabolic acidosis and tissue
oxalosis were shown to occur [64, 65]. Xylitol was subsequently shown to be a precursor of glycolate and glyoxylate using radioactive precursors [60].
Normally 98% of xylitol is converted to D-xylulose then D-xylulose-5- phosphate [60], however, in situations with increased levels of xylitol, the
alternative pathway may become more important in which D-xylulose is converted to xylulose-1-phosphate by fructokinase which is further converted to glycolaldéhyde and fed into glyoxylate, glycolate and oxalate metaboUsm.
25 Fructose and sorbitol can also be used for parenteral nutrition in glucose
intolerant patients. Along with hyperphosphataemia, hyperuricaemia and
impaired liver fimction, infusions of fructose or sorbitol can produce tissue
oxalosis in some patients although the metabolic pathway remains unclear [66].
It may be that the route involves the oxidation of glyceraldehyde to
hydroxypyruvate followed by decarboxylation to glycolaldéhyde.
Phenylalanine, tyrosine and tryptophan have also been identified as precursors of
oxalate [67]. It has been suggested that phenylalanine and tyrosine are converted
to oxalate via glycolate and glycolate dehydrogenase and that tryptophan is
metabolised via indolepyruvate and glyoxylate [68]. However, there is no
evidence to suggest hyperoxaluria is associated with conditions such as
phenylketonuria or tyrosinaemia which would be expected if these pathways
exist.
A futher potential precursor to glyoxylate described in rat liver is a-keto-y-
hydroxyglutarate, produced in the catabolism of hydroxyproline [69], a
component of collagen.
Ascorbate has been suggested to account for up to 40% of oxalate found in the
urine [68]. However, early experiments involving high dose vitamin C ingestion
failed to account for the spontaneous non-enzymatic conversion of ascorbate to
oxalate in non-acidified urine. Recent studies have shown that when urine is
correctly acidified large doses of ascorbate do not lead to anything more than a minimal increase in urinary oxalate level [70, 71].
26 The most likely major precursor of oxalate in vivo is glyoxylate (with an
uncertain role of glycolate as a direct precursor). This organic acid is produced as
an intermediate of a number of metabohc pathways. As discussed above it seems
unlikely that glycine is a direct precursor of glyoxylate therefore the most
important precursor appears to be glycolate. Glycolate is converted to glyoxylate
by the Kver specific peroxisomal enzyme glycolate oxidase (GO) [72, 73] (also
referred to as L-2-hydroxyacid oxidase A (HAG-A)) [74] yielding hydrogen
peroxide as a byproduct which is removed by catalase.
As glyoxylate is hydrated in aqueous solution, it is a suitable substrate for
oxidation to oxalate by GO [75], LDH [76-79] or xanthine oxidase (XOD) [80].
It seems unlikely that XOD has any significant role in vivo in glyoxylate
metabolism however as the specific inhibitor allopurinol has little effect on urinary oxalate excretion [81]. The relative importance of GO and LDH in glyoxylate oxidation has been subject to much discussion. Glyoxylate is a poorer substrate for GO than glycolate [82] but as good a substrate for LDH as lactate
[77], however glyoxylate is less abundant in the cytosol than lactate [82].
Contradictory observations make it difficult to definitely say which enzyme is more important, not least because most studies have either involved in vitro observations on rats or observations with isolated perfused rat Hver. In liver perfusion studies the redox state may not allow LDH to proceed at its normal rate
[83] and substrates which are normally produced in the peroxisome (such as glyoxylate) are directly introduced into the cytosol. Inhibitors of GO, such as phenyllactate and n-heptanoate, completely inhibit glyoxylate to oxalate
27 oxidation in isolated perfused rat liver [84]. This implies a major role for the
enzyme in glyoxylate metabohsm in the rat, however this may not be the case in
humans where other enzymes involved in glyoxylate metabolism such as
alanine:glyoxylate aminotransferase are located in different subcellular
organelles. Similarly, both phenyllactate and the LDH inhibitor NAD-pyruvate
adduct have been shown to inhibit glyoxylate/glycolate oxidation in isolated rat
hepatocytes [83] which would implicate both the enzymes. However, LDH
deficient patients do not have a noticeably lower urinary oxalate concentration
than controls [82] and the enzyme is present in the cytosol away from glyoxylate
production in the peroxisome [82]. One suggestion is that both GO and LDH act
cooperatively to oxidise glyoxylate, GO catalysing peroxisomal glyoxylate
oxidation when concentrations are low and LDH removing excess glyoxylate
leaking out of the peroxisomes in situations where it accumulates such as PHI
[85]. Another suggestion is that GO, LDH and XOD each have their own specific
function and oxalate production is an unwanted by-product resulting from a lack
of specificity of the enzymes [75].
In humans glyoxylate is transaminated to glycine by hepatic peroxisomal
alanineiglyoxylate aminotransferase (AGT) concomitantly converting alanine to pyruvate [86] (Figure 1.5). Glyoxylate can also be transaminated to glycine by
glutamate:glyoxylate aminotransferase (GGT) with the concomitant conversion of glutamate to 2-oxoglutarate [87]. GGT activity is mainly found in the cytosol but at substantially lower levels than AGT [87]. In the rat, AGT is located in both the peroxisome and the mitochondria of the hver and also other tissues. In addition, a second AGT enzyme not found in humans is located in the
28 mitochondria [88]. These findings make the use of rat liver as a model for human
liver glyoxylate metabolism less than adequate. Similarly, other species may have different subcellular distributions of enzymes and varying enzyme activities which should not be disregarded.
gluconeogenesis cytosol
3-phospho-serine<4-3-phospho-hydroxypyruvate<^ 3-phospho-D-glycerate PSA PGDH D-GK PSP D-glycerate
D-GDH^ |HPR senne ^ hydroxypyruvate hydroxypyruvate HPD*
glycolaldéhyde
SHMT pyruvate alanine ALDH* glycolate ^ ^ glycolate
-/?Wglycine m glyoxylate ^ ^ glyoxylate GO LDH
peroxisome oxalate < ►oxalate
Figure 1.5 Summary o f the pathways linking serine entry into gluconeogenesis and glyoxylate metabolism. GR, glyoxylate reductase (EC 1.1.1,26) HPR, hydroxypyruvate reductase (D-GDH, D-glycerate dehydrogenase) (EC 1.1.1.29) LDH, lactate dehydrogenase (EC 1.1.1.27) GO, glycolate oxidase (hydroxy-acid oxidase A) (EC 1.1.3.15) AGT, alanine:glyoxylate aminotransferase / serine .pyruvate aminotransferase (SPT) (EC 2.6.1.44) PSP, phosphoserine phosphatase (EC 3.1.3.3) PSA, phosphoserine transaminase (EC2.6.1.52) PGDH, phosphoglycerate dehydrogenase (EC 1.1.1.95) D-GK, glycerate kinase (EC2.7.1.31) SHMT, serine hydroxymethyltransferase (EC 2.1.2.1) ALDH, aldehyde dehydrogenase (EC 1.2.1.21) HPD, hydroxypyruvate decarboxylase (EC 4.1.1.40) * indicates enzyme found in both cytosol and mitochondria. Dotted lines indicate minor or putative pathways in normal metabolism.
It has also been shown that glyoxylate may be decarboxylated in the presence of
2-oxoglutarate to form 2-hydroxy-3-oxo-adipate or 2-oxo-3-hydroxyadipate by
29 the action of 2-oxoglutarate:glyoxylate carboligase [89, 90]. The fimction of this
reaction at present remains unclear.
1.1.6 Glyoxylate cycle in animals
The glyoxylate cycle for plants {Figure 7.6) is a modification of the tricarboxylic
acid (TCA) cycle {Figure 1.7) where a prominent intermediate is glyoxylate and
from which most higher plants, certain micro-organisms and algae are able to
synthesise all required carbohydrates from any substrate that is a precursor of
acetyl Co A. Until recently it was thought that animals did not have a functional
glyoxylate cycle until isocitrate lyase and malate synthase, two glyoxylate cycle
specific enzymes, were identified in the liver peroxisomes of alloxan-treated rats
[91] and also in the peroxisomes of human liver [92]. It has been suggested that
the glyoxylate cycle may function in extreme conditions as a way of bypassing
isocitrate dehydrogenase and a-ketoglutarate dehydrogenase to convert carbons
derived from fetty acid oxidation (in the mitochondria and peroxisome) into
glucose [92].
30 NAD+ malate NADH H+
oxaloacetate
Acetyl-CoA H2OCoA-SH
CoA-SH AcetylCoA HgO Glyoxylate cycle citrate
glyoxylate isocitrate
succinate i TCA cycle
Figure 1.6 The glyoxylate cycle. 5, malate dehydrogenase (EC 1.1.1.37) 6, citrate synthase (EC 4.1.3.7) 7, aconitase (EC 4.2.1.3) 9, isocitrate lyase (EC 4.1.3.1) 10, malate synthase (EC 4.1.3.2)
31 NAD+ malate NADH H+ HgQ
fumarate oxaloacetate FADH2 \ ✓Acetyl-CoA HgO
FAD VCoA-SH succinate TCA cycle citrate
GTP CoA-SH
Pi GDB succinyl-CoA isocitrate
NAD+ NADH H+ CO; a-ketoglutarate NADH H+ CO. NAD+ CoA-SH
Figure 1.7 The tricarboxylic acid (TCA) cycle. 1, a-ketoglutarate dehydrogenase (EC 1.2.4.2) 2, succinyl-CoA synthetase (EC 6.2.1.4) 3, succinate dehydrogenase (EC 1.3.99.1) 4, fumarase (EC 4.2.1.2) 5, malate dehydrogenase (EC 1.1.1.37) 6, citrate synthase (EC 4.1.3.7) 7, aconitase (EC 4.2.1.3) 8, isocitrate dehydrogenase (EC 1.1.1.41)
The two major precursors of glyoxylate are glycolate and glycolaldéhyde, the
latter probably being derived from hydroxypyruvate. If a pathway from hydroxypyruvate via glycolate and glyoxylate to oxalate exists it may function as
a route whereby carbohydrates are able to be converted to oxalate [93, 94]. The conversion of hydroxypyruvate to glycolaldéhyde in the hver is catalysed by hydroxypyruvate decarboxylase [95], an enzyme present in the rat mitochondria and cytosol and also in various other tissues [96, 97]. It is thought the glycolaldéhyde is then oxidised to glycolate by aldehyde dehydrogenase [88] an enzyme present in the cytosol and mitochondria [98]. The relevance of this
32 hydroxypyruvate to oxalate pathway in vivo is unclear where HPR would also be
present and able to convert hydroxypyruvate to D-glycerate, but if it exists it may
be of significance when HPR is lacking.
1.1.7 Glycolate dehydrogenase
GO converts glycolate to oxalate via glyoxylate [72, 73], however there is some
dispute as to the importance or even existence of a glycolate dehydrogenase in
the liver (reviewed by [88]) which would convert glycolate directly to oxalate.
Glycolate dehydrogenase activity without glyoxylate as an intermediate has been
demonstrated in metabohc studies on rat and human liver [99, 100] in which no
detectable glyoxylate was observed upon labelling and no glyoxylate was
captured by Tris buffer [100]. However, other studies, also on rat and human
liver fractions have failed to detect this activity at all [82] and the conversion of
an alcohohc group to a carboxyhc group in one step would not seem
energetically feasible [58]. If glycolate dehydrogenase activity does exist, there
would be 2 enzymes, GO and glycolate dehydrogenase, able to convert glycolate
to oxalate.
1.1.8 Hepatic serine metabolism
In mammalian Hver there are three major pathways involved in serine
metabohsm (reviewed in [101]). The first is initiated by the action of serine
dehydratase (SDH) which converts serine to pyruvate. Secondly serine may be
converted to hydroxypyruvate by the action of serine:pyruvate/alanine:glyoxylate
aminotransferase (SPT/AGT), the hydroxypyruvate then being fiinnehed into
gluconeogenesis via 3-phosphoglycerate, summarised m Figure 1.5.
33 Lastly, serine can be interconverted to glycine by serine
hydroxymethyltransferase (SHMT) either in the cytosol (cSHMT) or
mitochondria (mSHMT) [102], the glycine subsequently metabolised by the
glycine cleavage enzyme system (GCS). Rat has been shown to metabolise serine
in the liver quite differently to rabbit, humans and dogs with a major flux through
SDH shown both in vitro and in vivo [101,102]. SPT/AGT can be
induced/enhanced by glucagon administration but still remains only 10% of the
flux through SDH at quasi-physiological conditions in vitro (ImM serine,
0.25mM pyruvate) or 10-20% in vivo [101]. In rabbit, human and dog liver the
major flux appears to be through SPT/AGT in vitro and in vivo and this remains
a substantial contribution whether the SPT/AGT is peroxisomal (rabbit and
human) or mitochondrial (dog) [102]. The peroxisomal location of SPT/AGT in
herbivores is thought to be a measure to cope with the large amount of glycolate
contained in an herbivorous diet [103]. Glycolate will be converted to glyoxylate
in the peroxisome and hence SPT/AGT may be necessary in herbivores to remove this glyoxylate. In carnivores where the glycolate concentration will be
lower [103] there will be less need for peroxisomal SPT/AGT, however the
function of the mitochondrial SPT/AGT remains unclear. Flux through cSHMT is fast [102], maintaining the intracellular glycine and serine concentrations.
Therefore the slow decarboxylation of the glycine pool may also be regarded as a metabolic exit for serine.
34 1.1.9 Tissue specificity of metabolic pathways
It seems likely that the main organ of interest in pathways leading to oxalate
synthesis is the liver, on which most studies have concentrated. Quantitatively
the liver is most important in oxalate synthesis from substrates such as xylitol,
glyoxylate, glycolate, glycine and serine (reviewed in [88]) and contains organ
specific enzymes such as AGT and GO. Other enzymes such as LDH and GR are
found elsewhere including the kidney although the majority of oxalate deposition
elsewhere in the body in times of excess synthesis is thought to originate from
the liver [104]. It also seems plausible that, in the absence of AGT, GR may have
to deal exclusively with glyoxylate detoxification in the kidney. However,
isolated perfused rat kidney does not seem to produce significant oxalate from a
variety of sources including glycine, serine, glyoxylate and glycolate and thus the
liver may well be the most important source of oxalate [105].
1.1.10 Intracellular transport of metabolites
Little is known about the transport of oxalate precursors within the cell between
the organelles involved in glyoxylate metabolism (peroxisome and mitochondria)
and the cytosol. This may occur via passive diffusion across the membranes as
the metabolites involved are small; by carrier-mediated transport, possibly by
transporters such as the monocarboxylate transporter (reviewed in [106]) or by a
combination of both. The distribution of enzymes and their substrates within the
subcellular organelles will determine whether in vitro reactions are also likely to
occur in vivo.
35 1 2 Hyperoxaluria
Hyperoxaluria can be defined as an oxalate concentration >0.46mmol / 24hours
It is most commonly the result of secondary causes e.g. excess dietary intake or
gastrointestinal disorders but can also be primary i.e. due to an enzyme
deficiency.
1.2.1 Enteric hyperoxaluria
Enteric hyperoxaluria results from hyperabsorption of dietary oxalate in the gut.
Hyperabsorption was first observed in patients with ileal disease or resection
[107] and has subsequently been shown in patients with malabsorption of fetty
acids, bile acids or both. The colon has been suggested as the primary site of
hyperabsorption [108], whereby fatty acids/bile acids increase the colonic
permeability with respect to oxalate [108-111]. This enhanced oxalate absorption
in the colon resulting in hyperoxaluria is associated with Crohn’s disease,
steatorrhea and jejuno-ileal bypass surgery [112-114]. The solubility theory
suggests that the fetty acids/bile acids form soaps with the intraluminal calcium,
leaving soluble oxalate to be absorbed [115, 116]. It has also been suggested that
enteric hyperoxaluria may result from enhanced secretory pathways for oxalate
in the kidney (reviewed in [55]), reduced secretory oxalate flux in the gut [41] or
absence of the gut dwelling oxalate degrading bacterium O.formigenes. Cystic
fibrosis patients are at high risk for nephrocalcino sis and calcium oxalate
urohthiasis, probably due to hyperoxaluria secondary to malabsorption [117],
and possibly exacerbated by loss of O.formigenes as a result of antibiotic therapy
[118]. In patients with jejuno-ileal bypass hyperoxaluria has been associated with
36 an absence of O. formigenes and a low rate of oxalate degradation [8]. This loss
of O.formigenes in hyperoxaluria patients may be due to diarrhoea (common in
jejunoileal bypass) or increased bile salts which may inhibit bacterial growth [7].
A lack ofO.formigenes has also been associated with the number of recurrent
kidney stone episodes in patients with a high risk for calcium oxalate urolithiasis
[119]. Studies in normally non-coIonised rat showed that animals colonised with
live O.formigenes, or a preparation of the oxalate degrading enzymes derived
from O.formigenes, had reduced oxalate excretion, absence of crystalluria and
resistance to the formation of calcium oxalate crystals in the nephron compared
to controls [8]. From these studies it seems that absence of O.formigenes from
the gut may increase the risk of hyperoxaluria and recurrent kidney stone disease
and replacement therapy may be of some use.
1.2.2 Primary hyperoxaluria
The primary hyperoxalurias are a group of rare inherited disorders characterised
by raised urinary oxalate concentrations. Two conditions, primary hyperoxaluria
type 1 (PHI) and primary hyperoxaluria type 2 (PH2), have been described in
some detail. The molecular basis of PHI has been determined, however studies
on the basis of PH2 are at a much earlier stage with the molecular basis yet to be
determined. Further conditions may also belong to this group, possibly including
primary enteric hyperabsorption [120] and atypical hyperoxaluria [121], however
these conditions are poorly documented and their genetic basis has not been
determined. The clinical phenotype of PHI and PH2 apposa to be similar,
characterised by progressive urohthiasis, nephrocalcinosis and systemic oxalosis
following renal failure. Definitive diagnosis of PHI and PH2 is currently made
37 by measurement of enzyme activity in liver biopsies [122, 123]. The relevant
metabolic pathway for PHI and PH2 is illustrated in Figure 1,8.
serine pyruvate H ,a (PLP) I AGT AGT l(PLP)
hydroxypyruv; alanine glyoxylate L GO glycolate
peroxisome
cytosol hydroxypyruvate------»L-g|ycerate glycolate
NAD(P)H NAD(P) NADH NAD GR HPR, D-GDH NAD(P)H glyoxylate NAD(P) NAD D-glycerate I LDH NADH
oxalate gluconeogenesis
Figure 1.8 Metabolic pathway relevant to PHI and PH2. AGT, alanine:glyoxylate aminotransferase PLP, pyridoxalphosphate GO, glycolate oxidase (hydroxy-acid oxidase A) LDH, lactate dehydrogenase GR, glyoxylate reductase HPR, hydroxypyruvate reductase (D-GDH, D- glycerate dehydrogenase) Dashed lines indicate minor pathways in normal (non-PH) metabolism.
1.2.3 Primary hyperoxaluria type 1
Most primary hyperoxaluria type 1 (PHI) patients present under the age of 5
with symptoms resulting from urolithiasis [124-126] such as renal colic and
gross haematuria, however onset of disease can vary from a few months of age
up to the seventh decade of life [127, 128]. Nephrocalcinosis and urolithiasis
eventually impair renal function leading to systemic oxalosis, with calcium
oxalate deposition being found in many tissues including the skeleton, heart.
38 arteries, nerves and kidney (reviewed in [124]). Deposition in the bone is
associated with bone pain, multiple fractures and osteosclerosis and in the heart
can result in heart block, myocarditis and stroke [124, 125]. Death frequently
results from conq)lications secondary to oxalate deposition such as arrhythmia
and cardiac feilure. Approximately 10 percent of patients present at an early age,
typically in the first few months of life, with renal failure as a result of
nephrocalcinosis sometimes without urolithiasis [11, 129]. These patients may
also present with severe metabolic acidosis, anorexia and anaemia and have
systemic oxalosis at the time of presentation.
Along with an elevated oxalate excretion in the urine, as patients undergo renal
failure the plasma oxalate-creatinine ratio increases by a fector of 4 to 5 relative
to normal controls and patients with renal failure of other causes [130].
Hyperglycolic aciduria may also be present but not in all cases [127]. The
incidence of PHI has been reported in several populations with a prevalence in
France of 1.05/million population and an incidence rate of 0.12 per million per
year [131]. However, this is likely to be an underestimate due to the clinical
heterogeneity of the condition [127] and lack of metabolic investigation of adults
presenting with recurrent calcium oxalate stones. Disease severity may also be
under the influence of other factors including genetic, such as other enzyme
activities involved in the same pathway, and environmental, such as gut
colonisation with oxalate degrading bacteria, diet and hydration.
PHI is a result of an absence of the liver specific peroxisomal enzyme alanineiglyoxylate aminotransferase (AGT) [132] (see Figure 1.8). AGT is a
39 pyridoxal phosphate (PLP) dependent transaminase, the gene for which (AGXT)
is located at the tip of the long arm of chromosome 2 [133]. One third of PHI
patients have a combination of a C154T polymorphism (present in 15-20% of the
normal Caucasian population) which produces a weak mitochondrial targeting
sequence [134] and a G630A mutation which facilitates mitochondrial uptake by
slowing AGT dimérisation [135]. This combination mistargets approximately
90% of the enzyme to the mitochondria. Over 20 ftirther mutations have been
identified in the AGXT gene known to cause PHI (reviewed in [136]) accounting
for approximately 40 to 50 percent of patients.
The diagnosis of PHI is made by assay of AGT activity in needle liver biopsies
by utilising alanine and glyoxylate as substrates (along with the cofector
pyridoxal-5-phosphate) and measuring the pyruvate product in a linked LDH
reaction, following the reaction spectrophotometrically by a decrease in
absorbance at 340nm upon oxidation of the cofector NADH [122].
1.2.4 Primary hyperoxaluria type 2
Primary hyperoxaluria type 2 (PH2) is generally regarded to be a rarer and less
severe disease than PHI [137-139] and is distinguished by the presence of L-
glycerate in addition to oxalate in the urine [138, 140-144]. L-glycerate is not
found in the urine of healthy subjects and hence PH2 is also referred to as L-
glyceric aciduria [140]. The condition, like PHI, is inherited in an autosomal
recessive fashion [140, 145]. PH2 was first described in 4 patients (3 of them
siblings) with recurrent calcium oxalate kidney stones, hyperoxaluria and hyper-
L-glyceric aciduria [140]. These symptoms were attributed to a deficiency of D-
40 glycerate dehydrogenase (D-GDH) activity in leucocytes. L-glycerate is produced by the lactate dehydrogenase (LDH) catalysed reduction of hydroxypyruvate, which accumulates due to the absence of hydroxypyruvate reductase activity, the reverse reaction to D-GDH [140]. A subsequent study identified glyoxylate reductase and D-GDH deficiencies in the hver of PH2 patients [145] (see Figure 1.8). D-GDH and GR activities are primarily located in the cytosol in normal subjects and were absent in the PH2 patient studied
[145], with both activities attributed to the same enzyme (which also has HPR activity) [146, 147]. The classical presentation of PH2 is urolithiasis however
PH2 patients have been described with end stage renal disease [137, 144] and nephrocalcinosis [141], a common presenting feature of PHI. The infrequent observation of nephrocalcinosis in PH2 but much higher frequency in PHI has not been explained, however a recent study showed higher urinary oxalate and lower urinary calcium, magnesium and citrate concentrations in PHI patients compared to PH2 patients [148]. Hyperoxaluria in PH2 results from the accumulation of glyoxylate in the cytosol, due to absence of GR activity, which in turn is converted to oxalate by LDH [77, 78,145,149,150]. Some studies have suggested that in PH2, hydroxypyruvate is decarboxylated to glycolaldéhyde and subsequently converted to glycolate and oxalate [151].
However this seems unlikely as no hyperglycolic aciduria is associated with PH2 and the bulk of accumulated hydroxypyruvate is converted to L-glycerate by
LDH. To date, 28 PH2 patients have been described in the literature [137,138,
140-144], however this only represents 8 to 16 distinct femilies. This is a much lower figure than for PHI but it is not clear whether this is a result of underdiagnosis, misclassification or whether the disease is actually less common.
41 Underdiagnosis may result from confusion with the much better described PHI,
or due to adults presenting with hyperoxaluria not being investigated for
metabohc causes. Generally patients seem to present with symptoms of stones in
childhood (<10 years) [138, 140-144] however at least 2 PH2 patients have
presented with first symptoms in their 20’s [140, 144].
1.2.5 Atypical primary hyperoxaluria
Several children have recently been characterised with a clinical presentation
similar to the primary hyperoxalurias, that is hyperoxaluria and urohthiasis in
several cases, and in whom secondary causes of hyperoxaluria have been ruled
out [121]. However, these children do not have raised urinary glycolate or L-
glycerate and their hepatic AGT, D-GDH and GR activities are in the normal
range. It may be that these patients have an, as yet, uncharacterised metabohc
defect leading to excess oxalate synthesis.
1.2.6 Treatment of primary hyperoxaluria
An initial approach to the treatment of primary hyperoxaluria is to attempt the
reduction of oxalate levels in the body. This may foUow a similar course to the
treatment of secondary hyperoxaluria (idiopathic calcium oxalate stone disease,
enteric and milder hyperoxaluria) including maintenance of adequate hydration,
dietary oxalate restriction and inhibition of calcium oxalate crystalhsation in the
renal tract [124]. Urinary volumes in excess of 3.0 htres [126, 152] must be
maintained in order to reduce the risk of calcium oxalate supersaturation. Dietary
oxalate can be restricted, however due to the small amount of oxalate absorbed
42 from the gut [31-34] thiS| approach is thought to be inadequate for the treatment
of PH, probably better suited to the secondary hyperoxalurias. The solubility of
oxalate in the renal tract can be increased by magnesium oxide administration
[153], magnesium oxalate having a higher solubility than calcium oxalate.
Orthophosphate [154] and citrate [155, 156] administration have also been
shown to prevent stone formation in some cases although the mechanism of
action is not clearly understood. Restriction of dietary calcium would prove
counterproductive, abolishing calcium oxalate formation and subsequent
excretion from the gut and increasing oxalate uptake from the gut.
Several pharmacological approaches to treatment of PH have been made
(reviewed in [157]). Three enzymes have been implicated in oxalate production
from glyoxylate including GO [75], LDH [76-79] and XOD [80] however
inhibitors of GO and XOD have shown little or no effect on urinary oxalate in
non-hyperoxaluric lab animals [81, 158] and the role of LDH is thought to be too
metabolically important to alter pharmacologically. Pyridoxine/vitamin B6 is
commonly used in treatment of PH and produces lowering of urinary oxalate
levels in some PHI patients [159-161]. Pyridoxal phosphate is the cofector for
AGT (and all transaminases) and is thought to either boost residual AGT activity
[162] or possibly increase the activities of other glyoxylate metabolising enzymes such as GGT.
For those patients who develop calcium oxalate stones, surgery or extracorporeal shockwave lithotripsy may be used to clear urine flow/improve renal function.
However, if renal failure develops, dialysis and/or kidney transplantation may
43 become necessary. Some success has been shown in Europe where patients have had combined liver/kidney transplantation [163], both removing the cause and result of the hyperoxaluria. However, systemic oxalosis results in calcium oxalate deposits throughout the body resolubilising upon kidney transplantation which may cause damage to the kidney.
An obvious solution to the enzyme deficiencies in PHI and PH2 would be enzyme replacement, directly correcting the basic defect. Oxalate decarboxylase may also be a useful enzyme for gene therapy, although the consequences of adding an unnatural enzyme on other metabolites and pathways is unknown and antibodies may be raised against the foreign protein. Current enzyme replacement strategy for PHI involves liver transplantation (the European experience of which has been summarised on several occasions, most recently
[163]), which although showing positive results in many patients is not without its own long-term side effects and reduced life expectancy. At the present time it is unclear whether liver transplantation would be as effective for treatment of
PH2.
44 1.3 Hydroxypyruvate reductase / glyoxylate reductase
HPR converts the 3 carbon metabolite hydroxypyruvate to D-glycerate, its
reverse reaction being referred to as D-GDH. GR converts the 2 carbon
metabolite glyoxylate to glycolate.
1.3.1 GR/HPR in plants
Three enzymes with GR and HPR activities are found in plants; NADHiHPRl,
NADPH:HPR2 and NADPHiGR [164]. NADHiHPRl is a peroxisomal enzyme
with a preference for NADH as its cofector and hydroxypyruvate as its substrate
[165]. Glyoxylate is also reduced by the enzyme but the Km is much higher than
for hydroxypyruvate [165]. NADPH:HPR2, in contrast, has a preference for
NADPH as cofactor and is able to utilise both hydroxypyruvate and glyoxylate
as substrates [166]. Cytosolic NADPHiGR, which is also found in chloroplasts,
has a preference for NADPH but is only able to utilise glyoxylate as a substrate
[167, 168]. It seems likely that the fimction of the extraperoxisomal enzymes is
to prevent the accumulation of glyoxylate which would otherwise inhibit
Rubisco in the chloroplasts hence impairing photosynthesis [169]. However the
relative contributions of NADPH:HPR2 and NADPHiGR vary widely between
plants [164]. In addition these enzymes may also fimction as scavengers of
cytosolic glyoxylate and hydroxypyruvate which are released fi*om the
peroxisome, recycling them to glycolate. Two variations of the NADHiHPR-1
peptide encoded by the same gene have been identified in pumpkin cotyledons
which differ in their carboxy terminal amino acid sequences [170], one form
containing a peroxisomal targeting sequence. The relative amounts of the
45 transcripts are influenced by light which influences the subcellular localisation of
the HPR proteins [171].
1.3.2 GR/HPR in bacteria
Bacterial GRHPR is generally referred to as HPR only and has been
characterised in several species (summarised in [172]) with considerable
variation in substrate/cofector preference. The methylotrophic bacteria
M.exorquens AMI and Hmethylovorum GM2 both have a preference for
hydroxypyruvate rather than glyoxylate as a substrate and both utilise NADH as
their preferred cofactor [172, 173] {Table 1.1).
K M (mM ) Organism Subunit Optimal Cofector Hydroxy Glyoxylate Glycerate structure pH -pyruvate Methylobacterinm Homo 4.5 NADH 0.1 1.5 2.6 extorquens AMI dimeric NADPH HyphonÜCTobium Homo 6.8 NADH 0.175 10.8 methylovorum GM2 dimeric Pseudomonas Inducible Homo 5.5-8.0 NADH 8 0.76 acidovorans dimeric NADPH Constitutive Homo 5.3 NADH 0.13 dimeric NADPH Parococcus denitrificans 5.0 NADH 0.10 NADPH
Table 1.1 Substrate specificity o f bacterial GR/HPR enzymes (preferred cofactor in bold)
In addition M.extorquens is shown to have a lower Km for both reduction
reactions con^ared to D-GDH activity. HPR has also been studied in
Pseudomonas acidovorans [174] and Parococcus denitrificans [175] although
kinetics studies are fer less comprehensive merely showing cofactor preference
46 and ability to perform the HPR or D-GDH reactions. Interestingly,
P. acidovorans has been shown to have a lower Km for glycerate than
hydroxypyruvate possibly indicating a different function to the HPR of the
methylotrophic bacteria [174]. The HPR gene has been cloned in both
methylotrophic bacteria described above and investigated extensively in
M.extorquens [176]. In addition the crystal structure of HPR from
Hmethylovorum has been elucidated [177] and gives information on the
symmetrical homodimer which HPR forms (a homodimeric conformation has
been described for all the bacterial HPR enzymes mentioned above except for
P. denitrificans where no information is available).
1.3.3 GR/HPR in algae
Two separate GRHPR enzymes have been described in the unicellular algae
Chlamydomonas reinhardtii. NADH-HPR has a preference for hydroxypyruvate
over glyoxylate (Km of O.OSmM compared to lOmM) whereas NADPH-GR has
no HPR activity (Km for hydroxypyruvate of O.lmM) [178].
1.3.4 GR/HPR in mammals
Enzymes with GR and HPR activities have been traditionally referred to as D-
GDH (reverse of HPR) in mammalian species [147, 179-182]. More recently the
lesser importance of the D-GDH reaction has been recognised and GR/HPR is
the preferred designation.
47 Comprehensive kinetic investigations of rat and bovine GRHPR enzymes have
been undertaken following protein purification from bovine liver. The results of
these studies are discussed in relation to the human GRHPR enzyme in chapters
3 and 4 of this thesis.
Kinetic analysis of HPR, GR and D-GDH activities in crude human liver samples
indicates the enzyme or enzymes involved have a lower Km for hydroxypyruvate
than for glyoxylate which is in turn lower than for D-glycerate (0.5mM, 1.25mM
and 20mM respectively) using NADPH as cofactor [123]. The tissue distribution
of GR and HPR activities has also been determined in humans with marked
differences between the distribution of activities. HPR activity was found at
similar levels in liver, kidney, fibroblast^and leucocytes[123]. GR activity, in
contrast, was highest in liver and significantly lower in all other tissues tested. In
addition to this observation it was shown that not only do PH2 patients have residual hepatic HPR with undetectable GR activity but leucocyte HPR fell
within the normal reference range [123]. These observations suggested that there are 2 enzymes with HPR activity only one of which has associated GR activity.
These findings agree with the observations in rat and ox liver where both
GRHPR and LDH have HPR activity [146, 179].
48 1.4 Aims of the project
The aim of this thesis is to purify human hepatic GRHPR and to describe the
kinetic properties of the enzyme. In addition, by identifying the gene ;
encoding the human enzyme it will be possible to C5q)ress the enzyme in vitro
and characterise the recombinant protein. Confirmation of the role of this enzyme
in PH2 will be obtained by sequence analysis of the GRHPR gene fi'om patients
withPH2.
49 Chapter 2: General materials and methods
All chemicals were of analytical grade and were purchased from BDH (Poole,
UK) unless otherwise stated.
2.1 Bacterial culture reagents
LB (Luria-Bertani) broth 43 mM sodium chloride
lOmg/ml tryptone (Difco, Detroit, USA)
5mg/ml yeast extract (Difco)
Adjusted to pH7.5 with lOM NaOH solution.
LB-agar 12mg/ml Bactoagar (Difco)
2 2 Bacterial culture protocol
Bacterial colonies were streaked on LB-agar plates containing 50pg/ml
ampicillin and 40pg/ml X-gal and were incubated overnight at 37°C. Single
white colonies were picked and inoculated into 5ml LB broth containing 50pg/ml
ampicillin and were incubated at 37°C overnight with shaking. Stock solutions of
bacteria were also made at this stage in 15% glycerol and were stored at -80°C.
IMAGE Consortium clones were received cloned into the pT7T3-pac vector
transformed into E.Coli, which was stabbed into agar plugs. These plugs were
incubated overnight at 37®C and the resulting colonies seen were streaked onto
50 LB-agar plates containing ampicillin and X-gal (as above). Single colonies were
grown up in the usual method.
2.3 Plasmid DMA isolation from E. ooli
Plasmid DNA was extracted from 3 ml of bacterial suspension using the
QIAPrep*^ Spin Mini-prep kit (QIAGEN, Crawley, UK) with DNA eluted into
50|il of lOmM Tris-HCl, pH 8.5. Mini-prep DNA was stored at -20°C until use.
2.4 PCR reagents
5x THE: 0.5M Trizma base (Sigma, Poole, UK)
0.4M orthoboric acid
5mM Ethylenediammetetra-acetic acid (EDTA)
disodium salt
lOx DNA loading buffer 2.5mg/ml bromophenolblue (Sigma)
2.5mg/ml xylene cyanol (Sigma)
250mg/ml Ficoll (Sigma)
DNA molecular weight markers
Lambda DNA/Hindlll (used on 0.8% agarose gels)
(MBI Fermentas,St.Leon-Rot, Germany)
PhiX174 D N A/H aelll (used on 2 or 0.8% agarose gels)
(Promega, Southan^ton, UK)
51 2.5 PCR protocol
DNA (5ng plasmid or 80-200ng genomic) was added to a PCR solution
containing PCR buffer (Promega)(comprising 50mM potassium chloride, lOmM
tris.HCl, pH9.0 and 0.1% Triton X-100), l-2mM magnesium chloride, |200pM
dNTP (Amersham Pharmacia Biotech, Little Chalfont, UK), 10.6pM sach primer
(Sigma Genosys), 0.25U taq polymerase (Promega) in a volume of 25pLPrimer
sequences, PCR conditions and product sizes are listed m. Appendix L lOpl PCR
product was mixed with 2pl loading dye and electrophoresed on a 0.8/2.0%
agarose gel (Life Technologies Ltd., Paisley, UK) containing 0.5pg/ml ethidium
bromide (Sigma) at 100mA, 200V for 10 to 20 mins. DNA was visualised by UV
light.
2 .6 Restriction digestion
DNA was digested with 1-10 units of restriction enzyme in the presence of an
appropriate buffer for the enzyme(s), 0.1 mg/ml bovine serum albumin (New
England Bio labs, Hitchin, Hertfordshire, UK) and water to a final volume of
lOjil. Restriction enzymes, reaction buffers and incubation times and
temperatures are listed in Appendix 2. lOp.1 of undigested and digested vector
were mixed with 2|li1 loading dye and were run on ethidium bromide stained
agarose gels.
52 2.7 Extracting DNA bands from agarose gels
Bands were extracted from agarose gel slices using the Geneclean® spin kit
(Anachem, Luton, UK) with elution in 25 pi of DNase/RNase free water.
2.8 DNA quantitation
Band intensity on agarose gels was compared to bands of similar size and known
concentration in either the lambda DNA/Hindlll or PHX174 D NA/H aelll
marker.
2.9 Sequencing
Plasmid DNA was sequenced by MWG-Biotech UK Ltd. (Milton Keynes, UK)
in both directions using M13-universal and Ml 3-reverse primers. PCR products
from genomic DNA were sequenced using Ml 3-tailed nested primers.
2.10 SDS-PA GE for protein samples
5X sangle buffer: 0.12mM Trizma^ base (Sigma)
5ml 10% SDS (Sigma)
25pM DL-dithiothreitol (Sigma)
O.lmg/ml bromophenol blue (Sigma)
Adjusted to pH to 7.5 with acetic acid
Sandies were electrophoresed on the Multiphorll unit with reagents purchased
from Amersham Pharmacia Biotech as follows. Protein samples and Rainbow™
53 coloured protein molecular weight markers in IX sample buffer were heated for
3 mins and 2 mins respectively at 95°C and immediately transferred to ice. 7pl of
protein sample or 5 pi of marker were loaded onto Cleangel 36S precast 10%
polyacrylamide gels rehydrated in SDS pH 8.0 Gel Buffer. Buffer strips were
soaked with SDS pH 8.0 anode and cathode buffers and loaded along with the
gel onto the Multiphorll electrophoresis unit cooled to 10°C and run at 200V,
70mA and 40W for 10 min followed by 600W, 100mA and 40W for
approximately 90 min . Gels were either silver stained to visualise proteins, or
blotted onto nitrocellulose transfer membrane.
2.11 Immunoblotting
Blotting buffer 200mM glycine
25mM Trizma^ base (Sigma)
in 4:1 (v/v) waterimethanol
0.45pm nitrocellulose transfer membrane (molecular weight cut-off 12 to 14
kDa, Anderman and Co. Ltd., Kingston-Upon-Thames, UK) soaked first in water
then blotting buffer was overlayed onto 2 pieces of 3MM chromatography paper
(Scientific Laboratory Supplies Ltd., Nottingham, UK) soaked in blotting buffer
on a Trans-blot^ SD semi-dry transfer apparatus (BioRad, Hemel Hempstead,
UK). Precast polyacrylamide CleanGels were soaked for 3mins in blotting
buffer and stripped fi*om their backing plastic and overlayed onto the
nitrocellulose. Two further pieces of 3MM were overlayed and the blot run at / 15V for 1 hour. Filters were immersed for 10 mins in 3% (w/v) dried skimmed
milk powder (Tesco)(milk proteins) in phosphate buffered saline (PBSA),
followed by overnight incubation at 4°C with
54 the primary antibody diluted in 3% milk proteins. Two washes in PBSA were
performed prior to incubation with the secondary antibody, also diluted in 3%
milk proteins. After two final, 10 min washes in PBSA, the blot was immersed in
alkaline phophatase Colour Development reagent (BioRad) for 20 min. Rabbit
anti-human alanineiglyoxylate aminotransferase (AGT) antibody was purchased
from Prof. C. Danpure (Department of Biology, University College London).
2.12 Determination of tissue protein concentration by the Lowry procedure
[183]
Lowry reagent A: 1.9mM anhydrous sodium carbonate
ImM sodium hydroxide
Lowry reagent B : 3.1 pM copper II sulphate pentahydrate
Dissolved in 10ml trisodium citrate (Sigma)
Lowry reagent C: 50ml reagent A mixed with 1ml reagent B.
Made immediately before use.
Lowry reagent D: Folin-Ciocalteu reagent (Fisons, Loughborough,
UK)
Diluted 1:1 with ddH20.
Bovine serum albumin (BSA) (Pierce & Warriner, Warrington, UK) solution was
used to prepare standards as follows:
Protein concentration (mg/ml) 0 0.025 0.05 0.125 0.25 0.375 0.5 ddHsO (pi) 200 195 190 175 150 125 100 BSA 1 mg/ml (pi) 0 5 10 25 50 75 100
55 Samples were diluted 1 in 30 with ddHiO, i.e. 20pl sample plus SSOpl water with
both samples and standards run in duplicate. 1ml of reagent C was added to each
tube. This was vortexed and allowed to stand for 10 mins at room temperature.
lOOpl of reagent D was added to each tube, which was then vortexed rapidly.
Tubes were left to stand for 30 mins at room temperature and their absorbance
was read at 660nm. Absorbances of standards against protein concentration were
plotted and the standard curve produced was used to determine unknowns.
Unknowns were finally multiplied by 30 to give mg protein / ml.
2.13 Spectrophotometric determination of protein concentration
For an uncontaminated pure protein the absorbance at 280nm is approximately
equal to the protein concentration in mg/ml [184].
2.14 Coomassie blue staining of precast polyacrylamide gels
Fixing solution 6.5:2.5:1 (v/v) water:isopropanol:acetic acid
Destain #1 5.3:4:0.7 (v/v) water:methanol:acetic acid
Destain #2 8.8:0.7:0.5 (v/v) wateriacetic acidrmethanol
Staining solution 0.25mg/ml Coomassie brilliant blue R (Sigma)
In 10.6:8:1.4 (v/v) water:methanol:acetic acid
The gel was stained with rocking according to the following protocol:
1 Fix 20 mins Room tenqjerature 2 Destain#! 3 mins Room temperature 3 Coomassie blue stain 1 hour 60°C 4 Destain#! 30 mins Room temperature 5 Destain #2 overnight Room temperature
56 2.15 Silver staining of precast polyacrylamide gels
The PlusOne™ DNA silver staining kit (Amersham Pharmacia Biotech) was used
according to the manufacturers instructions.
2.16 Assays for GR and HPR activity
Assay procedure adapted from the method of Giafi and Rumsby [123]
HPR assay
0.1M potassium phosphate buffer, pH6.0 6.15ml 0. IM K2HPO4
43.85ml O.IMKH 2PO4
1.2mM NADPH tetrasodium salt (Sigma) in 0.1 M phosphate buffer, pH6.0.
50mM p-hydroxypyruvic acid lithium salt (Sigma) made up in water.
GR assay
0.1M potassium phosphate buffer, pH7.6 43.5ml 0.1M K2HPO4
6.5ml O.IM KH2PO4
1.2mM NADPH tetrasodium salt in O.IM phosphate buffer, pH7.6.
208mM glyoxylic acid monohydrate (Sigma) made up in water.
Reactions were initiated with the addition of the non-nucleotide substrate and
were followed on a Uvikon 922 spectrophotometer (Bio-Tek Kontron, Watford,
UK) for 10 min at 37°C in duplicate using 1ml cuvettes with the following
reagent volumes: Enzyme blank Sample Substrate (hydroxypyruvate/glyoxylate) 0 120pl NADPH (pH6.0/pH7.6) 500pl 500pl ddH20 450pl 330pl Supernatant 50|il 50pl
57 Converting absorbance change at 340nm into nmoles NADPH oxidised/
min /m g protein
0.6mM NADPH gives an absorbance at 340nm of 3.1 units. Hence, an
absorbance change of lU corresponds to an NADPH concentration of
190p,mol/L or 190nmol/ml.
Therefore absorbance change per min multiplied by 190pmol/L NADPH divided
by the amount of protein in the 50pl supemantant sample, gives the number of
nmoles NADPH oxidised / min/ mg protein.
2.17 Isoelectric focusing
Samples were applied to rehydrated ampholine^ PAGplate isoelectric focusing
gels, pH gradient 3.5 to 9.5 (Amersham Pharmacia Biotech), and electrophoresed
at 1500V, 50mA and 30W for approximately 1 hour at 10°C. Proteins were
visualised by staining with coomassie blue. Western blots were prepared as
previously described.
2.18 Chromatofocusing
Sandies were initially dialysed overnight at 4®C against chromatofocusing buffer
(25mM imidazole.HCl pH7.2) and were then applied to a 36 x 1.6cm
chromatography column (Amersham Pharmacia Biotech) containing PBE 94
(Amersham Pharmacia Biotech) equilibrated with chromatofocusing buffer at
flow rate of 0.5ml/minute. The sample was washed into the column with 5ml
chromatofocusing buffer. GRHPR proteins bound to the column were eluted with
58 a descending pH gradient from pH7.2 to pH4, produced by application of
Poiybufier 74 (Amersham Pharmacia Biotech) diluted 1 in 8 with ddH20 and
brought to pH4 with concentrated HCl. The flow rate used was 0.5ml/minute.
2.19 Preparation of human tissue sonicates
Human tissue was obtained from UCL Hospitals Primary Hyperoxaluria
diagnostic service and leucocytes from normal volunteers. Ethics approval for
these studies was obtained from the Joint UCLH committees on the Ethics of
Human Research.
Tissue was suspended in O.IM potassium phosphate buffer, pHS.O, 0.24M
sucrose at approximately 2% w/v. The tissue was sonicated on ice for 10 second
bursts from a Microson XL-sonicator (Heat Systems Inc., New York) with 30
second intervals between bursts until the mass was uniformly disrupted.
Unsonicated particulate material was removed by centrifuging at 15 000 x g for
10 mins at 4°C. Protein concentration was determined by the method of Lowry
(as described earlier) and samples were frozen at -80°C until used.
2.20 Dephosphorylation of vector 5' ends
The number of pmoles of linear double stranded DNA ends was determined
using the following formula:
(jig DNA / kb size of DNA) x 3.04 = pmol of ends
0.01 units of calf intestinal alkaline phosphatase (ClAP) (Promega) per pmol of
ends was incubated at 37°C for 15 min then 56°C for 15 min in a thermal cycler
along with DNA, ClAP reaction buffer (Promega) and water to a final volume of
59 lOOjLil. A further 0.01 units of ClAP per pmol of ends were then added and this
reaction incubated for a further 30 min at 37°C. To terminate the reaction 2|il of
0.5M EDTA was added and a final incubation performed at 65°C for 20 min.
DNA was then purified away fi-om other components using the Wizard™ DNA
Clean-up System (Promega) with elution in 50pl of lOmM Tris-HCl, pH 7.6,
ImM EDTA.
2.21 Ligation of insert DNA into plasmid vector
1:1 and 1:3 molar ratios of vectoriinsert were chosen such that approximately
50ng of plasmid vector DNA was used in each case. 1 unit of T4 DNA ligase
(Promega) was added to each reaction along with T4 DNA ligase buffer
(Promega) and water to a final volume of lOpl. Controls to determine incomplete
restriction enzyme digestion and dephosphorylation of vector were included (no
insert DNA, or no insert DNA and no T4 DNA ligase, respectively). Reactions
were incubated at 4°C overnight.
60 2.22 Transformation of plasmid DNA into E. coii
SOB media 20mg/ml tryptone (Difco)
5mg/ml yeast extract (Difco) 8.6mM sodium chloride
2.5mM potassium chloride (Sigma)
lOmM magnesium chloride hexahydrate
pH to 7.0 with lOM sodium hydroxide solution
SOC media As for SOB except contains
20mM D-glucose
100|il aliquots of competent cells stored at -80°C were thawed on ice. 1 pi of a
1:10 dilution of 14.2M p-mercaptoethanol in ddHiO was added to each tube and
swirled followed by incubation on ice for 10 min swirling gently every 2 min.
Approximately 20ng in 2pl of plasmid vector containing insert or 42ng of vector
alone as a control were added to the reaction and swirled. This was followed by
incubation on ice for 30 min:, a heat pulse in a 42°C water bath for 45 seconds
and a further incubation on ice for 2 min. 0.9ml SOC media was added and
incubated for 37°C for 1 hour with shaking (250rpm). A sterile spreader was
used to plate 700pl of the transformation reaction onto an LB agar plate
containing 50pg/ml ampicillin. Single colonies were inoculated into 5ml SOB
medium (containing 50pg/ml ampicillin) and cultured overnight at 37°C with
shaking.
61 2.23 Induction of GRHPR expression in BL21 cells
Maimfecturers instructions for the Xpress™ System with pTrcHIsB (Invitrogen)
were followed. 2ml SOB (containing 50ug/ml ampicillin) was innoculated with a
single colony of BL21 containing pTrcHisB vector + recombinant insert. This
culture was grown overnight at 37°C with shaking. 50ml of SOB (containing
50pg/ml ampicillin) were then inoculated with 0.2ml of this overnight culture
and grown at 37°C with shaking to an absorbance at 600nm of 0.6 (mid-log
phase). 1ml of cells were removed prior to induction and spun at 10 000 x g for 1
min in a microcentriluge. The supernatant and pellet were separated and frozen
at -20°C. Isopropyl p-D-thiogalacto-pyranoside (IPTG)(Sigma) was added to the
50ml culture to give a final concentration of ImM. The culture was then
incubated at 37°C with shaking, removing 1ml samples every hour for 5 hours.
Each 1ml sample was spun and supernatant and pellets stored at 4°C. After aU
samples were collected each pellet was resuspended in lOOpl of 20mM
potassium phosphate buffer at pH 7.0 and frozen in liquid nitrogen. The frozen
lysate was then thawed at 42°C. This freeze-thaw cycle was repeated 3 additional
times after which the sample was spun at 10 000 x g for 10 mins to pellet
insoluble protein. lOOpl of this supernatant was removed to a fresh tube
containing lOOpl of 2X SDS-PAGE sample buffer. The pellet was resuspended
in lOOpl IX SDS-PAGE sample buffer. Supernatant stored at -20°C was thawed
and SDS-PAGE sample buffer was also added to give a final concentration of
IX. 7pl of initial supernatant, final supernatant and final pellet resuspension were
run on SDS-PAGE.
62 2 24 Preparation of bacteria for enzyme assays
Single colonies of pTrcHisB and pTrcHisB plus the EHPR12 insert in BL21 cells
were grown in SOB medium (containing 50pg/ml ampicillin) as described above
to a final volume of 50ml with a final 37°C incubation post-IPTG induction.
Cells were spun down at 1500 x g for 10 mins, washed once with PBSA, pelleted
again and the pellet sonicated on ice in 10ml O.IM potassium phosphate buffer,
pH8.0 containing 240mM sucrose using three, 10 s bursts from a Microson XL
sonicator. The cell debris was pelleted by centrifugation at 10 000 x g for 10 min
at 4°C. HPR and GR activity was determined in the supernatant as described m
above.
2.25 Northern blot
Northern blots containing mRNA from a variety of tissues (MTN™ Blot,
Clontech, Palo, Alto, CA, USA) were hybridised with ^^P-dCTP-labelled GRHPR
cDNA prepared by random priming according to manufacturer instructions
(Rediprime^ Amersham Pharmacia).
2.26 DNA/protein analysis packages
ENTREZ http ://www. ncbi. nlm. nih. gov/Entrez/
BLAST http://www.ncbi.nlm.nih.gov/Blast/
CLUSTALW http:/Avww. clustalw. genome.ad.jp/
WebCutter http://wv/w.firstmarket.com/cutter/cut2.html
Translate http://ww'v. expasy. cliy'tools/dna.html
KEGG http://ww^v. genome, ad.jp/kegg/metaboiism.html
63 Chapter 3: Purification of GRHPR from human liver and enzyme
kinetic studies
3.1 Introduction
The mammalian enzyme with GR, HPR and D-GDH activities has been
historically referred to as ‘D-GDH’ although this may not reflect the true
function of the enzyme. Previous work on the human enzyme found the Km for
hydroxypyruvate to be lower than for glyoxylate, which was in turn lower than
for D-glycerate [123]. This suggests that the reductase activities may be
physiologically more important than dehydrogenase activity, a finding supported
by data on the rat [146] and bovine [179] ‘D-GDH’ enzymes. In both these cases
the enzyme was separated away from LDH, an enzyme known to utilise
hydroxypuyruvate [146] as a substrate with NADPH as cofector. In this chapter I
describe the first kinetic analysis of the human ‘D-GDH’ enzyme away from the
influence of LDH. I have designated the human enzyme ‘GR’, reflecting the
GRiNADPH activity specific to this enzyme, absence of which is thought to lead
to the accumulation of glyoxylate and its subsequent conversion to oxalate in
PH2.
This chapter also describes purification of human GR to enable protein
sequencing and ultimately gene identification. Mammalian D-GDH (GR) has
been semi-purified from rat [146,182] and bovine [147] liver and also
completely purified to homogeneity from bovine fiver [180, 181]. In these earlier
studies two different purification procedures were used to purify the enzyme, the
first using selective ammonium sulphate precipitation and calcium phosphate gel
64 treatment [146, 147], the second using ammonium sulphate precipitation in conjunction with anion- and cation-exchange chromatography [181].
65 3.2 Methods
3.2.1 Partial purification of GRHPR from human liver
Homogenisation buffer 154mM KCi
lOOmM Manganese chloride MnCl2.4H20 (Sigma)
0.5% CHAPS (3-[(3-cholamidopropyl)-
dimethylammonio]- 1-propanesulfonate) (Sigma)
Complete™ protease inhibitor tablet (Boehringer
Mannheim, Lewes, UK)
O.lmM EDTA (disodium salt)
Twelve grams of human liver in 50ml homogenisation buffer was homogenised
using an Ultra-Turrax homogeniser (Janke & Kunkel, Staufen, Germany).
Homogenisation was performed on ice using five,30 second bursts separated by
60 second intervals, to allow cooling. The resulting lysate was maintained on ice
for 1 hour followed by centrifugation at 30 000 x g in a fixed angle rotor for 30
minutes at 4°C. The supernatant was passed over glass wool to remove lipids and
further centrifuged at 1000 x g for 10 minutes at 4°C, discarding the resulting
pellet. 8.2g of ammonium sulphate was added gradually to give a final saturation
of 30%, with constant stirring on ice, and the solution was centrifiiged at 30 000
X g for 20 minutes at 4°C. Ammonium sulphate saturation in the supernatant was
increased to 60% by adding a fiirther 9.05g as described above and, following
centrifugation, the pellet was resuspended in 20ml of equilibration buffer (20mM
potassium phosphate, 2mM EDTA, pH 5.8). The solution was dialysed overnight
against 1 litre of equilibration buffer. The dialysed sample was mixed thoroughly
in a 50ml falcon tube at 4°C with 25ml of DEAE Sephadex A-50 (Sigma) in
66 equilibration buffer. This mixture was applied to a Buchner funnel and washed
several times with 10ml of the same buffer. Those fractions containing HPR and
GR activity (measured as described in Chapter 2) were pooled and applied to a
40 X 2.6cm chromatography column (Amersham Pharmacia Biotech) containing
carboxymethyl(CM)-sephadex C50 (Sigma) in equilibration buffer, at a rate of 3
ml/minute. The column was washed with 2 volumes (approximately 400ml) of
equilibration buffer. GRHPR proteins were eluted with 150mM NaCl in
equihbration buffer at a rate of 5ml/min. The HPR/GR fraction was dialysed
against chromatofocusing buffer (25mM imidazole.HCL pH7.2) overnight at
4°C and applied to a 36 x 1.6cm chromatography column containing PBE 94
equilibrated with 25mM chromatofocusing buffer at flow rate of O.Sml/minute.
The sample was washed into the column with a fiirther 5ml chromatofocusing
buffer. HPR/GR proteins bound to the column were eluted with a descending pH
gradient from pH7.2 to pH4, produced by application of Polybuffer 74 diluted 1
in 8 with water and brought to pH4 with HCl. The flow rate used was
O.Sml/minute.
3.2.2 Purification of GRHPR for protein sequencing
GRHPR proteins eluted from CM-sephadex CSG were sealed in Cellu-Sep
Regenerated Cellulose Tubular Membrane T3 (Pierce & Warriner, Warrington) and
placed in a bed of sucrose at 4°C overnight. The concentrated solution was then
dialysed for 1 hour against 1 litre of equilibration buffer, followed by a fresh litre of
equilibration buffer overnight. The resulting solution was applied to a 8.5 x 1.5cm
chromatography column (Amersham Pharmacia Biotech) containing 2’5’ ADP
sepharose 4B (Amersham Pharmacia Biotech in lOmM potassium phosphate,
67 2mM EDTA, pH 7.2, at a rate of 1 ml/minute. NADP—specific dehydrogenases
were then eluted with lOmM NADP^(Boehringer) in lOmM potassium
phosphate, 2mM EDTA pH 7.2. Fractions containing GRHPR proteins were
pooled and concentrated in sucrose (as above) and applied to a 40 x 2.6cm
chromatography column (Amersham Pharmacia Biotech) containing Sephadex
G-200 (Amersham Pharmacia Biotech) in equilibration buffer, at a rate of
3ml/min. Fractions eluted were assayed for GR and HPR activity. Following
elution from Sephadex G-200 fractions containing GR and HPR activity were
pooled and once again concentrated in sucrose and dialysed overnight against
equilibration buffer. As a measure of purity proteins were subjected to SDS-
PAGE and coomassie blue stained (as described in Chapter 2).
3.2.3 Two substrate kinetics
Prior to kinetic analysis the partially purified proteins were dialysed against
50mM potassium phosphate buffer, 2mM EDTA at either pH 6 (HPR analysis)
or pH 7.6 (GR analysis). Two-substrate kinetics were performed using the
method described by Fell [185] by varying the concentrations of substrates,
hydroxypyruvate or glyoxylate, and the cofector, NADPH at 37°C. The reaction
was followed spectrophotometrically on the Cobas Bio centrifiigal analyser
(Roche Diagnostic System, Welwyn Garden City, UK) as a decrease in
absorbance at 340nm
68 3.2.4 LDH measurement
LDH activity was measured on the Cobas Integra (Roche Diagnostic System)
using the ‘P-L’ cassette [186]. This cassette provides pyruvate and NADH as
substrate and cofactor for the enzyme in a buffer containing Tris, sodium
chloride and sodium azide. The reaction is followed spectrophotometrically by
the system as a decrease in absorbance at 340nm as NADH is oxidised.
69 3.3 Results
The elution profile of HPR activity after CM-sephadex ion-exchange
chromatography shows two major peaks of activity which are not fully resolved
by this method {Figure 3.1).
0.8
0.7
0.6
0.5
0.4
^ 0.3 0.2
0.1
100 150 200 250 Volume eluted from column (ml)
Figure 3.1 CM-sephadex ion exchange chromatography profile of semi-purified human liver GRHPR, showing HPR activity only.
Therefore, the whole double peak of HPR activity was pooled and applied to the
chromatofocusing column {Figure 3.2). Two peaks of HPR activity were seen
upon elution. The first (peak A) was not retained by the column, had no GR
activity and co-eluted with LDH. The second peak (peak B), eluted between pH
6.5 and 5.5 and had GR activity. No LDH activity was associated with this peak.
70 PolybufFer applied pH 7.2 pH 6.5 pH 5.5 1400 n I
1200
1000 -
800 - ■o
600 -
400 -
200
■ p ■ mh ## " " I------ÉKB------,------1 100 200 300 400 500 600 Volume eluted from column (ml)
Figure 3.2 Fractionation o f human GRHPR by chromatofocusing from pH 7.2 to pH 4.0. HPR activity (% ) GR activity (M )
3.3.1 Purification of GRHPR for protein sequencing
GRHPR was retained by the 2’5’ ADP sepharose column, as would be expected
for anNADP"^ specific dehydrogenase. Figure 3.3 illustrates the GR/HPR elution
profile fi*om this column. lOmM NADP^ eluted a sharp peak of HPR/GR activity
off the column in a volume of approximately 30ml. Although this column will
enable GRHPR to be separated firom enzymes which are not NADP^ specific
dehydrogenases, the wide elution profile of the GR and HPR activities indicates
that if other NADP^ specific dehydrogenases were present, such as mitochondrial
glutamate or isocitrate dehydrogenase (Genbank accession no. P00367 and
71 AAH09244 respectively), they are unlikely to be separated from GRHPR by this method.
2 0 0 0 1
1800
1600
1400
1200
1000 - 10mM NADP 800
600 -
400
200
20 40 60 80 100 volume eluted from column (ml)
Figure 3.3 Elution profile o f human liver GRHPR from 2 '5 ’ ADP sepharose, showing HPR (% ) and GR (A) activity.
Figure 3.4 illustrates the elution profile from Sephadex G-200, with a large protein peak at 50ml corresponding to the void volume of the column followed by the peak of GR/HPR activity. Care was taken to only pool fractions from the central region of the GR/HPR containing peak to avoid contamination with the initial protein peak however the GRHPR peak is particularly wide, spanning
40ml, and therefore is likely to contain other contaminating proteins.
72 400 T 0.25
350 0.2 300
250 - 0.15
200 CO
150 - 0.1
100 - 0.05
40100 120 140 volume eluted from column (ml)
Figure 3.4 Elution profile o f GRHPR from Sephadex G-200., showing HPR (% ) and GR ( ^ ) activity and absorbance at 280nm (dashed line).
Figure 3.5 illustrates liver proteins before purification, after elution from the 2’5’
ADP sepharose column and after elution from the Sephadex G-200 column. The
GRHPR-containing fractions eluted from Sephadex G-200 contain 2 main bands between 30 and 40kDa. Less intense bands can also be seen at higher molecular weights, either indicating contaminating proteins present in this fraction or multimeric forms of the GR/HPR protem. Protein sequencing of the larger protein isolated from SDS-PAGE (performed by Dr. Nick Tottie, Ludwig
Institute, UCL) identified peptides matching fructose-bisphosphate aldolase B sequence (Genbank Accession no. P05062), but no novel proteins. This result suggests that aldolase is a major contaminant of the preparation and that GRHPR has not been purified to homogeneity at this stage.
73 220kDa
130kDa
90kDa
70kDa 60kDa
40kDa
30kDa
3 2 1
Figure 3.5 Coomassie blue stained SDS-PAGE illustrating samples from various points in the GRHPR protein purification procedure. 1) pre-DEAE- sephadex A-50 2) post-2 ’5 ’ADP sepharose 3) post-Sephadex G-200 column
74 3.3.2 Two substrate kinetics
In a separate experiment kinetic analysis was performed on the HPR and
GR/HPR activity eluting as peaks A and B respectively {Figure 3,2). For
measurement of Km values for the two enzymes, substrate concentrations were
chosen such that the reaction was first order with respect to substrate. All
reactions were performed at 37®C in 50mM potassium phosphate buffer (pH 6.0
for HPR and pH 7.6 for GR), using NADPH as co&ctor. Reactions were
monitored as a decrease in absorbance at 340nm during a period of 3 to 5 min.
Km values for hydroxypyruvate, glyoxylate and NADPH are shown in Table 3.1.
Primary and secondary plots for each reaction are illustrated m Appendix 2.
Km(mM) HPR GR Substrate Hydroxypyruvate NADPH Glyoxylate NADPH Peak A 8 0.6 No activity No activity Peak B 0.1 0.2 2.3 0.1
Table 3.1 Kinetic analysis of peaks A and B from chromatofocusing
75 3.4 Discussion
Results obtained on chromatofocusing indicate that there are at least 2 enzymes
with HPR activity in human liver, both of which use NADPH as a substrate. The
bulk of HPR activity (peak A) eluted from the column before the pH gradient
was applied indicating the pi of this enzyme or enzymes is above pH 7.2. Under
these conditions we cannot exclude the possibility that there may be more than
one enzyme with HPR activity in this peak, a higher starting pH would be
necessary to retain the protein(s) and possibly resolve separate peaks. It is
unlikely that the HPR activity in peak A could be the result of column
overloading as care was taken not to exceed the capacity of the column and there
was no associated GR activity which would be expected if the HPR/GR passed
straight through. Peak B, with lower HPR activity, was retained by the
chromatofocusing column, eluting in an irregular peak between pH 5.5 and 6.5,
and also had GR activity. As PH2 liver has been shown to have a deficiency of
both HPR and GR activities [145, 182], the enzyme in peak B is more likely to
be the protein deficient in PH2. In view of the wide pi range over which peak B
eluted from the column it may also represent multiple enzymes with HPR and
GR activity.
HPR activity in rat and ox liver also appears to be present in two proteins [146,
179], one of which is known to be LDH. The finding of LDH activity in peak A
but not in peak B suggests that at least part of the HPR activity is due to this
ubiquitous enzyme. The iso form of LDH predominantly found in liver is LDH5,
a homotetramer formed of LDH-H subunits (Genbank accession no. P07195).
LDH5 does not bind to DEAE-sephadex (A50) at pH7.4 [79]. As DEAE-
76 sephadex is positively charged and LDH5 does not bind, LDH5 must also be positively charged at this pH and therefore has an isoelectric point > 7.4. Peak A on chromatofocusing has a pi > 7.2 which is therefore consistent with LDH5.
Hepatic LDH although commonly reported as having no activity with NADPH has been shown to utilise hydroxypyruvate but not glyoxylate with NADPH as cofector [146]. LDH has both GR and HPR activities with NADH as cofactor in addition to the ability to oxidise glyoxylate in the presence of NAD^ [78, 149,
150]. For this reason and to more easily differentiate GRHPR and LDH by the presence or absence of GR activity, NADPH was used as the cofector for this study.
Even after selective ammonium sulphate precipitation, anion- and cation-ion exchange chromatography, chromatofocusing, afiSnity and size exclusion chromatography GRHPR has not been purified to homogeneity. This was clearly shown by the several bands visible on SDS-PAGE of the final purified firaction, and also upon protein sequencing where no novel proteins were identified.
Interestingly, the only peptides sequenced were those matching fructose-1,6- bisphosphate aldolase B (aldolase) (Genbank accession no. P05062) which is not an NADP-dependent dehydrogenase so would not be expected to be retained by the 2’5’ ADP sepharose column. It is unclear therefore how this enzyme has been retained throughout the purification protocol. Aldolase B has carbon-carbon lyase activity and forms part of glycolysis converting fimctose-1,6-bisphosphate to glycerone phosphate and glyceraldehyde 3-phosphate. The enzyme has no reductase/dehydrogenase activity, and its absence is associated with fiiictose intolerance and hypoglycaemia (OMIM entry 229600), a quite different
77 phenotype to that seen in PH2. It is therefore clear that Aldolase B is not the
human GRHPR enzyme or deficient in PH2. The inability to identify the GRHPR
protein by sequencing implies that the GRHPR protein is present at a lower
concentration than Aldolase B in the purified fi-action. Aldolase B is tetrameric
with a subunit molecular weight of 38kDa [187] corresponding to one of the two
darkest bands observed upon SDS-PAGE post-Sephadex G-200. The GRHPR
protein purified fi-om bovine liver has a subunit molecular weight of 34kDa
which is very similar to Aldolase B and so the two proteins cannot be resolved
on SDS-PAGE, however, 2D-PAGE may separate the proteins if they have
different isoelectric points. As the 2’5’ ADP sepharose column should separate
GRHPR fi'om aldolase it may also be possible to use several passes of this
column at this stage in the protocol. However, due to a significant decrease in
GR/HPR activity throughout the purification protocol used so fer this addition
would not be feasible and may require the entire purification protocol to be
altered to obtain enough GRHPR protein for sequencing.
Kinetic analysis of the two peaks showed that the enzyme in peak B had a Km for hydroxypyruvate lower than that of peak A and therefore a higher affinity for this substrate. Peak B is therefore more likely to be of importance physiologically where concentrations of hydroxypyruvate are of the order of
5|iM [182]. The enzyme contained in peak B has a higher activity with hydroxypyruvate than glyoxylate, a finding which has also been described for the non-LDH containing HPR enzymes in both rat [146] and ox [179] liver. It should be noted, however, that all these studies were performed at different pH values. For example, the hydrogen ion concentrations used in the present study
78 allowed maximum enzyme activity to be obtained for GR and HPR but were not physiological [123]. Further kinetic studies at the pH of hver cytosol, are required to compare GR and HPR activities directly and this will be addressed with recombinant human GRHPR in Chapter 4.
Confirmation of the role of peak B in glyoxylate metabolism could come from chromatofocusing of partially purified liver proteins from a PH2 patient. If the entire retained peak (B from Figure 3.2) was absent this would indicate that all the GR/HPR activity bound by the chromatofocusing column was due to one enzyme. If some HPR or GR activity remained it would either be due to residual
GRHPR activity in the hver sample patient or other enzymes able to perform the
HPR/GR reactions. This may help to explain the wide elution profile of GRHPR but the experiment would require 12 grams of hver from a PH2 patient which was not available. The wide elution profile could also be further investigated by chromatofocusing of completely purified GRHPR, however several further purification steps would be needed in addition to those employed in this study.
Pure enzyme is essential to enable more specific kinetic studies to be undertaken, giving more information on the role of the enzyme in the cell. For this reason I have used a second approach to obtaining pure human GRHPR avoiding the need for time consuming multiple purification steps from human hver, and this is described in chapter 4.
79 Chapter 4: Identification, expression and characterisation of human
GRHPR cDNA and GRHPR protein
4.1 Introduction
Recent developments in human genetics including initial stages of the human
genome project have produced hbraries of human expressed sequence tags
(ETSs), cDNAs from a variety of sources which have been partially sequenced at
their 5’ and 3’ ends [188]. These libraries can be searched for homology to DNA
and/or proteins from other species enabling the isolation of a variety of expressed
proteins.
Enzymes with GR and HPR activities have been identified in several species of
plant [189-192], several bacteria [172, 175, 193], algae [178] and also in rat
[146], bovine [147, 179] and human [140] liver, but only in a few is the gene
actually cloned. The protein sequence of several, non-human, GRHPR enzymes
has been determined [171, 176, 177, 194, 195] and this sequence information can
now be used to identify human cDNAs with protein sequence homology to
GRHPR, without the extremely time consuming process of protein purification
and sequencing.
The availability of unlimited supphes of pure protein enables a wide variety of
information to be gathered about the enzyme, which would not previously have
been possible with crude tissue homogenates, including subunit associations,
isoelectric point (pi), kinetic data, protein structure and also quantification of
protein. Polyclonal antibody raised against the purified protein can be used for
80 detection of GRHPR on western blots thus allowing the specific detection of the
GRHPR protein in human liver sonicates.
81 4.2 Methods
4.2.1 Nickel affinity chromatography
Bacterial pellets from 500ml culture media were subjected to 4 freeze thaw
sonication cycles as previously described (Induction of GRHPR expression.
Chapter 2). The sonicate containing His-tagged protein was applied to a nickel
column (Xpress™ system, Invitrogen) according to manufacturers instructions
and eluted with imidazole (50mM - 500mM) or pH (pH6 - 4). Purified protein
was dialysed overnight in T3 dialysis membrane (Pierce and Warriner) against
400 volumes of 20mM potassium phosphate buffer, pH7.0 and was quantified by
absorbance at 280nm.
4.2.2 Western blots
Blots were produced using standard techniques with an alkaline phosphatase
conjugated second antibody as described previously (Chapter 2). Anti-Xpress™
antibody (Invitrogen) and alkaline phosphatase conjugated goat anti-mouse IgG
(Sigma) were used at manufacturers suggested dilutions.
4.2.3 Isoelectric focusing
Samples were applied to rehydrated Ampholine™ PAGplate isoelectric focusing
gels, pH gradient 3.5 to 9.5 (Amersham Pharmacia Biotech), along with marker
proteins (lEF-MIX 3.6-9.3, Sigma) and electrophoresed at 1500V, 50mA and
30W for approximately 1 hour at 10°C. Proteins were visualised by staining with
82 coomassie blue. Western blots were prepared as previously described (Chapter
2).
4.2.4 Chemical crosslinking of pure GRHPR
Recombinant GRHPR in 20mM potassium phosphate buffer, pH7.0 was
concentrated to dryness in a Microcon30 microconcentrator (Millipore, Watford,
Herts, UK) by centrifugation at 13 000 rpm for 12 mins. 500|il of reaction buffer
(20mM sodium phosphate, 0.15M sodium chloride pH 7.5) was added and the
tube again spun to dryness. The sample was finally eluted from the column in
500p,l reaction buffer and the final concentration measured by absorbance at
280nm. 7.4pg of GRHPR in 23pi reaction buffer was added to 2ul of BS3
(Bis(sulfosuccinimidyl) suberate. Pierce and Warriner) in 5mM citrate buffer, pH
5.0 giving final concentrations of 1 to lOmM. After incubation at room
temperature for 30 min, 1 pi of IM Tris.HCl pH 7.5 was added and the reaction
incubated for a fiirther 30 mins at room temperature. 8pl of the resulting solution
was electrophoresed on SDS-PAGE and western blots prepared.
4.2.5 Enzyme kinetic studies
The Km apparent (Km’) was determined for each substrate after plotting rate of
change in absorbance at 340nm against substrate concentration. Km and Vmax
were obtained from a computer-generated line of best fit to satisfy the Michaelis-
Menten equation.
83 4.3 Results
4.3.1 Identification of putative GRHPR oDNA clone
Using the tblastn tool (http ;//www. ncbi. iilm. nih. gov/BLAST/1 the peptide
sequence of HPR üom Hyphomicrobium methylovorum {Appendix 3a, Accession
no. S48189) was used to search for human ESTs which, when translated, had
peptide sequence homology. The cDNA which was identified {Appendix 3b,
Accession no. D63259) when translated shares 37% identity with the
H methylovorum HPR amino acid sequence {Appendix 3c). This cDNA was then
used to screen the EST database for clones containing additional 5’-sequence
using the Blast tool {Appendix 3d). The longest cDNA {Appendix 3e, Accession
no. AA148891) was obtained from the IMAGE Consortium [LLNL]
(http://image.llnl.gov/) (clone ID: 503200) in the vector pT7T3D-pac (IMAGE
Consortium) and sequenced in full.
The cDNA identified from the IMAGE hbrary is 1235 bp, with 41bp of 5’-
untranslated region and an open reading frame of 987 bp {Figure 4.1). The 3’-
untranslated region is 225 bp with a putative polyadenylation signal sequence
(AATAA) 14 nucleotides from the end of the cDNA.
84 5'gcttctgtactgccaggtccgggtcggcggctgcactgcgg 1 atg aga ccg gtg cga etc atg aag gtg ttc gtc acc cgc agg ata ccc gcc gag 1 M R PV R LM K V F V T RRIPA E
55 ggt agg gtc gcg etc gcc egg gcg gca gac tgt gag gtg gag cag tgg gac teg 19 G R V ALARA ADC E VE Q w DS
109 gat gag ccc ate cet gcc aag gag eta gag cga ggt gtg gcg ggggcc cac ggc 37 D EPIP A K ELE R G VAG A HG
163 ctg etc tgc etc etc tcc gac cac gtg gac aag agg ate ctg gat get gca ggg 55 L L C L LSDH VDKRILDAAG
217 gcc aat etc aaa gtc ate age acc atg tct gtg ggc ate gac cac ttg get ttg 73 A N L KVI S T MS V G IDHL A L
271 gat gaa ate aag aag cgt 999 ate cga gtt ggc tac acc cca gat gtc ctg aca 91 D E IK KR G IR V G YT P D V LT
325 gat acc acc gcc gaa etc 9 ca gtc tcc ctg eta ctt acc acc tgc cgc egg ttg 109 D TTA ELAVS LL LTTCRRL
379 ccg gag gcc ate gag gaa 9tg aag aat ggt ggc tgg acc teg tgg aag ccc etc 127 P E AI E EVKN G G w T S W K p L
433 tgg ctg tgt ggc tat gga etc acg cag age act gtc ggc ate ate gggctg ggg 145 w L c G Y G LT Q S T V GIIGLG
487 cgc ata ggc cag gcc att 9 ct egg cgt ctg aaa cca ttc ggt gtc cag aga ttt 163 R I G Q A IARR LK PF G V Q RF
541 ctg tac aca 999 cgc cag ccc agg cet gag gaa gca gca gaa ttc cag gca gag 181 L Y T G R Q PRPEE AAE F Q A E
595 ttt gtg tct acc cet gag ctg get gcc caa tct gat ttc ate gtc gtg gcc tgc 199 F V ST P E L A A Q S DFI VV A C
649 tcc tta aca cet gca acc gag gga etc tgc aac aag gac ttc ttc cag aag atg 217 SL TPA T EGL C N K D ' F F Q K M
703 aag gaa aca get gtg ttc ate aac ate age agg ggc gac gtc gta aac cag gac 235 K E TA V FINI S R GD VVN Q D
757 gac ctg tac cag gcc ttg gcc agt ggt aag att gca get get gga ctg gat gtg 253 D L Y Q AL ASGK I AA A G LDV
811 acg age cca gaa cca ctg cet aca aac cac cet etc ctg acc Ctg aag aac tgt 271 T SPEPLP T NHP LL TL KN C
865 gtg att ctg ccc cac att ggc agt gcc acc cac aga acc cgc aac acc atg tcc 289 V I LP H IG S ATH RT RNTM s
919 ttg ttg gca get aac aac ttg ctg get ggc ctg aga ggggag ccg atg cet agt 307 L L A A N NLLAGL RGE P M P S
973 gaa etc aag ctg tag 325 E L K L stop ccaaacagtagagatggagggccgggaagcaaaccgtgccctggtattgtcagacacacccaggcttgatttggatccaca ggcagagccaagggaaggtgtgattctctgaggaaagagtgattctgatatatgtacttgtcacattggtgttggacacat ttgcgccaaaagtatggtaattctattattaaataattctctgagaaaaaaaaaaaaaaaaaa
Figure 4.1 Full cDNA and predicted protein sequences o f human GRHPR now assigned GenBank ID (accession number) NM 012203. Putative polyA addition signal sequence (AATAA) is underlined
85 Analysis of the peptide sequence using the PROSITE program [196] revealed a
2-hydroxyacid dehydrogenase signature (MKETAWINISRGDVVN) starting at codon 232 {Figure 4.2),
1 mrpvrlmkvf vtrripaegr valaraadce veqwdsdepi pakelergva gahgllclls 61 dhvdkrilda aganlkvist itisvgidhlal deikkrgirv gytpdvltdt taelavslll 121 ttcrrlpeai eevknggwts wkplwlcgyg Itqstvgiig Igrigqaiar rlkpfgvqrf 181 lytgrqprpe eaaefqaefv stpelaaqsd fiwacsltp ateglcnkdf fqkmketavf 241 inisrqdwn qddlyqalas gkiaaagldv tspeplptnh plltlkncvi Iphigsathr 301 trntmsllaa nnllaglrge pmpselkl
Figure 4.2 Human GRHPR protein sequence (protein accesion number NP_036335). 2-hydroxyacid dehydrogenase signature starting at codon 232 is underlined.
This signature is derived from comparison of conserved regions in several other
2-hydroxyacid dehydrogenase enzymes including ‘D-GDH’ from
H methylovorum andM.extorquens.
The sequence is predicted to encode a protein of 328 amino acids with a molecular weight of 36.5kDa (http://www.expasv.ch/tools/pi-tool.htmn. The protein has sequence similarity with HPR/GR from H. methylovorum (32%),
Cucumis sativus (31%) and m.extorquens (21%) {Figure 4.3).
86 Human ------MRPVRLMKVFVTRRIPAEGRVALARAADCEVEQWDSDEPIPAKELERGVAG hypho ------MSKKKILITWPLP-EAAMARARESYDVIAHGD-DPKITIDEMIETAKS cucurbit MANRVQVEVWNPNGKYRWSTKPMPGTRWINLLIEQDCRVEICTEKKTILSVEDIVALIG methylo ------MTKKWFLDRES---LDATVREFNFPHEYKEYESTWTPEEIVERLQG human — AHGLLCLLSDHVDKRILDAAG-ANLKVISTMSVGIDHLALDEIKKRGIRVGYTPDVLT hypho — VDALLITLNEKCRKEVIDRIP-ENIKCISTYSIGFDHIDLDACKARGIKVGNAPHGVT cucurbit DKGDGVIGQLTEDWGEVLFSALSRAGCKAFSNMAVGYNNVDVNAANKYGIAVGNTPGVLT methyl o AE lAMINKVPMRADTLKQLP— DLKL lAVAATGTDWDKAAAKAQGITWNI RNYAF human DTTAELAVSLLLTTCRRLPEAIEEVKNGGWTS-- WKPLWLCGYGLTQSTVGIIGLGRIG hypho VATAEIAMLLLLGSARRAGEGEKMIRTRSWPG-- WQPLQLVGQRLDNKTLGIYGFGKIG cucurbit ETTAELAASLSLAAARRIVEADEFMRAGHYDG-- WLPNLFVGNLLKGQTVGVIGAGRIG methylo NTVPEHWGLMFALRRAIVPYANSVRRGDWNKSKQFCYFDYPIYDIAGSTLGIIGYGALG human QAIARRLKP-FGVQRFLYTGRQ------PRPEEAAEFQAEF-- VSTPELAAQS hypho QALAQRARG-FDMNVHYYDIYR------AKPEVEAKYNATYH—DSLDSLLKVS cucurbit SAYARMMVEGFKMNLIYFDLYQSTRLEKFVTAYGEFLKANGEVPVTWRRASSMDEVLREA methylo KSIAKRAEA-LGMKVLAFDVFP------QDGLVDLET------ILTQS human DFIWACSLTPATEGLCNKDFFQKMKETAVFINISRGDWNQDDLYQALASGKIAAAGLD hypho QFFSINAPSTPETRYFFNKETIEKLPQGAIWNTARGDLVKDDDVIAALKSGRLAYAGFD cucurbit DVISLHPVLDKTTFHLVNKESLKAMKKDAILINCSRGPVIDEAALVEHLKENPMFRVGLD methylo DVITLHVPLTPDTKNMIGAEQLKKMKRSAILINTARGGLVDEAALLQALKDGTIGGAGFD human VTSPEPLP— TNHPLLTLKNCVILPHIGSATHRTRNTMSLLAANNLLAGLR-----GEP- hypho VFAGEPN INEGYYDLPNTFLFPHLGSAAIEARNQMGFEALDNIDAFFA-----GKD- cucurbit VFEDEPY MKPGLADMKNAIIVPHIASASKWTREGMATLAALNVLGKIKQYPVWADPN methylo WAQEPPKDGNILCDADLPNLIVTPHVAWASKEAMQILADQLVDNVEAFVA-----GKP- ★ ★★ * * « * * « • • • * . human — MPSELKL------328 hypho — MPFKLA------322 cucurbit RVEPFLDENAPPPAASPSIVNAKALGI 381 methylo QNWEA ------314
Sequences (human:hypho) Score: 32% identity Sequences (human:cucurbit) Score: 31% identity Sequences (human:methylo) Score: 21% identity Sequences (hypho:cucurbit) Score: 25% identity Sequences (hypho:methylo) Score: 24% identity Sequences (cucurbit:methylo) Score: 24% identity
Figure 4.3 Mulitiple protein sequence alignment o f HPR/GR from: human GRHPR (Accession no. NP 036335), Hyphomicrobium methylovorum (Hypho) NADH dependent HPR/GR (Accession no. S48189), Cucumis sativus (Cucurbit) HPR (Accession no. BAA08411), Methylobacterium extorquens (Methylo) GDH/NADH dependent HPR/GR (HPR-A) (Accession no. Q59516). indicates positions which have a single, fully conserved residue, V ' indicates that one o f the following 'strong' groups is fully conserved: STANEQKNHQKNDEQQHRKMLVMILFHYFYW, indicates that one o f the following 'weaker' groups is fully conserved: CSA ATVSAG STNKSTPA SGND SNDEQKNDEQHKNEQHRKFVLIHFYM.
87 The strategy for expression of GRHPR is shown in Figure 4.4.
PCR of GRHPR cDNA with Digestion of pTrcHisB vector EHPR1/EHPR2 primers with Kpnl and Hindill restriction enzymes I Digestion with Kpnl and Dephosphorylation of 5’ Hindill restriction enzymes ends
Ligation of cDNA insert into pTrcHis 3 vector
Transformation of Epicurian Coli BL21 (DE3) with plasmid
Induction ofi expression
Enzyme kinetic studies Protein characterisation
Figure 4.4 Cloning and expression strategy for the putative human GRHPR cDNA (GenBank accession AA148891).
The fiill length cDNA clone (GenBank accession AA148891) was amplified by
PCR using oligonucleotide primers EHPRl and EHPR2 designed to introduce restriction sites for Kpnl and Hindill at the 5’- and 3’-ends, respectively (see
Appendix 1 for primer sequences and PCR conditions). Following digestion with
Kpnl and Hindill (see Appendix 2 for digestion conditions), the PCR product was ligated into the pTrcHis B expression vector (Invitrogen) which had been cut with the same enzymes and the cut ends dephosphorylated. The cDNA was inserted into the multiple cloning site of pTrcHis B downstream of the His tag and Anti-Xpress antibody epitope and in frame with the plasmid initiation codon.
The construct (pTrcHisB-GRHPR) was transfected into Epicurian co//BL21
(DE3) competent cells (Stratagene). Individual colonies were picked and cultured
88 overnight in SOB medium containing ampicillin at a final concentration of
50|Lig/ml. Following plasmid purification, restriction enzyme digestion with Kpnl
and Hindill confirmed the presence of an insert of the correct size (1093bp). To
confirm the correct cDNA sequence had been inserted into the expression vector,
and in the absence of a useful sequencing primer site in the pTrcHisB vector, the
cDNA was excised from the vector and inserted into the pCR2.1 TA cloning
vector (as described for the pTrcHisB vector) and sequenced in full.
4.3.2 Expression of cDNA
Expression of the cDNA in BL21 bacterial cells produced a protein with GR and
HPR activities of 631 ±22 and 509±17 nmol NADPH oxidised/min/mg protein
respectively (mean ± IS.D. from six analyses) compared with activities of 124±7
nmol/min/mg protein and 16±8 nmol/min/mg protein in cells transfected with
vector alone {Figure 4.5). A fusion protein of 43kDa was detected on Western
blot analysis which was not present in sonicates of cells transfected with vector
alone. As the fusion protein adds an additional 3kDa to the protein size, this
result indicates that the protein translated from the cDNA is approximately
40kDa, slightly bigger than the 36.5kDa predicted from the amino acid sequence.
89 =■ 700
a . 600
1 5 300
< 100
pTrcHisB + pTrcHisB pTrcHisB + pTrcHisB GRHPR GRHPR
Figure 4.5 HPR (shaded boxes) and GR (open boxes) activity in BL21 cells transfected with pTrcHisB-GRHPR andpTrcHisB, respectively (error bars indicate ISD).
4.3.3 Induction of GRHPR expression in BL21 cells
Samples taken before and during induction of recombinant protein expression in
BL21 cells were electrophoresed on SDS-PAGE and coomassie blue stained
{Figure 4.6a). Changes in protein concentrations post-induction were followed
using a band of similar molecular weight to GRHPR as a reference (indicated by
an arrow). The amount of soluble bacterial protein (lanes C0-C5) increases
dramatically from almost nothing pre-induction (CO) to a maximum at 1 hour
post-induction (Cl), remaining stable for up to 5 hours (C5). Coomassie blue
staining can give an indication of general protein expression but not specific
GRHPR expression. Therefore, samples were also visualised by staining with the
anti-Xpress™ antibody {Figure 4.6b). Immunoreactive protein was visible after 1
hour (Cl) and increased in intensity to a maximum level after 2 hours (C2),
90 remaining stable up to 5 hours (C5) (no signal was seen pre-induction).
Expressed GRHPR was also seen in the protein pellet (B1-B5) suggesting that the enzyme forms inclusion bodies, a common observation with recombinant protein expression. However inclusion bodies do not need to be investigated further as GRHPR was found in the soluble cell fraction. No recombinant protein was observed in the extracellular medium (lanes A0-A5), which would be expected with the expression of a cytosolic protein.
220kDa g
97.4kDa ^
66kDa g
46kDa 0
30kDa f 21.5kDa 14.3kDa t >>>>>> 00 00 00 00 00 m o oooo0 o -»• Fo c*> -tik cn o NJ c*> ^ oi o N3 05 01
f • i
o o O O O o ro 05 2 cn
Figure 4.6 E.Coli BL21 proteins during induction o f recombinant protein expression, a) Coomassie blue stained SDS-PAGE. Lanes A0-A5 Bacterial growth medium 0 to 5 hours after IPTG induction. Lanes B0-B5 Resolubilised pellet post-freeze/thaw sonication disruption of cells 0 to 5 hours after IPTG induction. Lanes C0-C5 Supernatant from freeze/thaw sonication disruption of cells 0 to 5 hours after IPTG induction. Arrow indicates band chosen to follow intensity changes, b) Western blot using anti-Xpress™ antibody. Lanes C0-C5 Supernatant from freeze/thaw sonication disruption o f cells 0 to 5 hours after IPTG induction.
91 4.3.4 Purification of recombinant human GRHPR
The bacterial sonicate containing His-tagged protein was applied to a Nickel
affinity column and eluted either by decreasing pH to 4, or by stepwise elution
with imidazole at concentrations from 50mM to 500mM. Elution with 500mM
imidazole yielded a single protein of 43kDa {Figure 4.7a) which was detected on
western blots with the anti-Xpress^'^ antibody, confirming this protein as
recombinant GRHPR {Figure 4.7b). pH elution was less effective and not
subsequently used.
Imidazole elution pH elution
Pre-nickel column
66kDa
O)0) 46kDa o I Post-nickel column 30kDa
ongin Distance from origin 1 2 3
Figure 4.7 Purification on nickel afflnity column of recombinant GRHPR enzyme, a) Densitometric scans o f silver stained SDS-PAGE gels of recombinant GRHPR samples before and after application to the nickel column and elution either with 500mM imidazole or pH4 elution buffer, b) Western blot of recombinant GRHPR detected with anti-Xpress™ antibody. Lane 1- markers proteins, lane 2 - pre-nickel column, lane 3 - post-nickel column with 500mM imidazole elution.
92 Purified GRHPR had GR activity of 21 500 nmoles NADPH oxidised / min / mg
protein, a 23-fold increase in specific activity over the pre-purification cell
sonicate (950 nmoles NADPH oxidised / min / mg protein).
4.3.5 Antibody production
3.3mg of purified recombinant GRHPR protein in 15ml 50mM potassium
phosphate buffer pH 7.0 was fi-eeze dried and used for immunisation of 2 rabbits
to produce a polyclonal anti-GRHPR antibody (Sigma Genosys) according to the
schedule illustrated in Table 4.1.
Day 0 Pre-Bleed + Antigen injection
Day 14 Antigen Injection
Day 28 Antigen Injection
Day 35 Test Bleed (7.9.99)
Day 42 Antigen Injection
Day 49 Bleed (21.9.99)
Day 56 Antigen Injection
Day 63 Bleed (5.10.99)
Day 70 Antigen Injection
Day 77 Bleed
Day 84 Antigen Injection
Day 91 Final Bleed
Table 4.1 Schedule of immunisation o f rabbits with pure recombinant GRHPR (performed by Sigma Genosys).
93 Serum samples were stored in 1ml aliquots frozen at -80®C. On thawing, sodium
azide was added to a final concentration of 0.1% and antiserum stored at 4°C.
4.3.6 Antibody characterisation
Dot blots were performed on the Bio-Dot microfiltration system (Bio-Rad,
Hemel Hempsted, UK) according to manufacturers instructions with 20pl of pure
recombinant GRHPR in water loaded into each sample well. No difference in
signal intensity was observed between 1 : 1 0 0 0 and 1 : 2 0 0 0 primary antibody
(rabbit anti-human GRHPR) dilutions at enzyme concentrations down to 6.4ng
{Figure 4.8).
Primary antibody titre
6400ng e
640ng #
128ng # or. 0. X 64ng e CD 26ng # 2 3 13ng 0. e 6.4ng #
blank
o o o o o ino
Rabbit anti-human GRHPR antibody dilution
Figure 4.8 Dot blot of primary antibody dilutions against various pure recombinant GRHPR concentrations to determine optimal titre.
94 At a 1:5000 dilution signal intensity was much lower and was therefore not
deemed suitable for low enzyme concentrations such as those found in human
liver. Hence, a 1:2000 dilution was adopted for the primary antibody. For the
secondary antibody (alkaline phosphatase conjugated goat anti-rabbit IgG,
Sigma) manufacturers instructions were followed using a dilution of 1:2000.
4.3.7 Tissue distribution
Northern blots showed a predominant RNA species at approximately 1.45 kb in
all tissues {Figure 4.9), which corresponds approximately to the size expected for
the GRHPR mRNA (Chapter 3).
4.2kb 2.9kb
# < ------1.45kb
c CO CD 3 $ E S ill III II Ia. iliCO f I It (/) 2 1— CL 8 g CD CD CD E o CO g .d Q. \_ CD CL
Figure 4.9 Multiple tissue northern blot hybridised with full length GRHPR
95 This highest intensity was seen in liver followed by kidney, heart, skeletal muscle and pancreas. Higher molecular weight species at 2.9 and 4.2 kb were also seen in liver, kidney, heart and pancreas but are of unknown significance.
Antibody raised against the recombinant GRHPR detected a protein of approximately 38kDa in human liver. The signal in bpg of liver proteins is at a similar intensity to 12ng of purified recombinant GRHPR {Figure 4.10), and thus the concentration of GRHPR in human liver was estimated to be approximately
0.2% of soluble non-nuclear liver proteins. This result is only approximate as two different methods were used to measure protein concentration, the Lowry method for liver proteins and absorbance at 280nm for pure recombinant
GRHPR.
66kDa
46kDa
30kDa
Û: Û: tr ÛC a: Ct q : g)
Figure 4.10 Quantitation of GRHPR content of human by comparison with known amounts o f pure recombinant GRHPR.
96 4.3.8 Tissue distribution of GRHPR protein
1 Opg of liver tissue gave a clearly visible, single band of GRHPR cross reacting
material (CRM) whereas only a very weak signal was obtained with the same
amount of kidney {Figure 4.1 la). Muscle and leucocyte protein had no
significant CRM when as much as 40|iig of muscle protein was present on the
blot {Figure 4.11b).
46kDa
SOkDa
0) e g CO ) 0 E o > O) O) O)O) O) C =L =L o o o o o o 46kDa SOkDa E E E o > O) O) O) =L =L 1 1 Io IO oi iO I E CO e g T— Figure 4.11 Tissue distribution of GRHPR immunoreactivity with rabbit anti human GRHPR antibody, a) various tissues b) various concentrations of muscle proteins compared to lOpg liver protein. 97 4.3.9 Western blots of PH2 liver sonicates A western blot containing liver sonicates from 5 PH2 patients and 4 controls is shown in Figure 4.12. All control samples (lanes 6-9) have both AGT (upper band) and GRHPR (lower band) cross-reacting material (CRM) while those from PH2 patients (lanes 1-5) have AGT but no GRHPR. One sample (lane 4) appeared to have several fainter bands present around 30kDa with anti-GRHPR, which may represent truncated versions of the GRHPR protein. 66kDa AGT —^ V?-' ^ 46kDa 4 “ 30kDa 1 234567 89 10 Figure 4.12 GRHPR and AGT immunoreactivity on a western blot containing 12fag o f human liver protein from PH2 patients (lanes 1-5), controls (lanes 6-9). Lane 10 protein molecular weight marker. 4.3.10 GRHPR chemical crosslinking With increasing concentration of BS3 crosslinker the intensity of the uncrosslinked recombinant GRHPR band at 43kDa decreased while the intensity of a 92kDa band increased to a maximum at lOmM BS3 {Figure 4.13). This result is consistent with dimérisation of the protein. Faint bands at sizes larger than 1 OOkDa were observed at 1 mM BS3 however these bands did not increase 98 in intensity with increased BS3 concentrations and are present at much lower levels than dimeric GRHPR. 220kDa ------ 97.4kDa ------■92kDa 66kDa ------►I 46kDa ■43kDa 30kDa I 21.5kDa 14 3kDa E E E CN m o in [BS3] mM [BS3] mM Figure 4.13 Chemical crosslinking of pure recombinant GRHPR with BS3 a) western blot with rabbit anti-human GRHPR b) coomassie blue stained 4.3.11 Charge heterogeneity of GRHPR Pure recombinant GRHPR, applied to a chromatofocusing column under the same conditions as used for human liver (Chapter 3), was completely retained on the column and eluted in three distinct peaks containing both GR and HPR activity {Figure 4.14). The two largest peaks eluted between pH 5.2 to 5.8 and the third peak at a pH below 5.0.1 have shown in chapter 3 that partially purified human liver had two peaks of HPR activity; one with pi greater than pH 7.2 was 99 not retained by the chromatofocusing column and had no associated GR, while the second peak, eluting between pH 6.5 and 5.5, had GR activity. It would therefore appear that the second (i.e. retained) peak in human liver corresponds to GRHPR. 1000 1 900 - pH6pH5.5 pH5 E 800 - c E 700 - ■D $ 600 - y X o 500 - X 0_ Q 400 - < Z S 300 - 0 1 200 - 100 - 0 100 200 300 400 volume eluted from column (ml) Figure 4.14 HPR (circles) and GR (triangles) activity o f recombinant human GRHPR separated on chromatofocusing The recombinant enzyme did not behave as a homogeneous species on this column, but appeared to have at least 3 pi’s. This result suggests that charge heterogeneity is present, a phenomenon further investigated by isoelectric focusing, treatment with chemical dénaturants and reducing agents as follows. 100 Uncrosslinked recombinant GRHPR was seen as three distinct bands upon coomassie blue staining of the polyacrylamide gel at approximately pH 7.3, 7.15 and 7.0 {Figure 4.15a). Following crosslinking the three bands focused at pH 5.6, 5.5 and 5.3 {Figure 4.15b) i.e. similar to the pattern observed on the chromatofocusing column and suggesting that the enzyme is present as a dimer on the chromatofocusing column. A representative calibration plot for these gels is given in Appendix 4. 1.2—^ ^ ^ pi 7.3 7.2—► ^___ pi 7.3 pi 7.15 ^-2----- pi 7.15 pi 7.0 — pi 7.0 6.8 6.8 —► . * 6.6 6.6 5.9 5,9 ^ ------pi 5.6 <------pi 5.5 M pi 5.3 5.1 4.6 5.1 4.6 —► ^ 3.6 —► 0 3_6 H CO CO CO CO CO (D CO CO CO CO III II m 00 00 CD 00 2 I g 2 E Q . CL E E EEE < < o in OJ o Figure 4.15 Coomassie blue stained Ampholine^^ PAGplate (pH range 3.5-9.5) isoelectric focusing gels a) 2.3 pg ofpure recombinant GRHPR b) 2.3 pg of pure recombinant GRHPR treated with various concentrations of the chemical crosslinker BS3. 101 Following lEF, western blots of human liver sonicates also showed at least 3 bands, confirming the presence of charge heterogeneity in native GRHPR {Figure 4.16a). 0)0)0) CN U ) O ) o o o o + DTT - DTT 2M 4M 6M Figure 4.16 Investigation o f charge heterogeneity in GRHPR. a) Western blot o f various amounts o f human liver protein subjected to isoelectric focusing on Ampholine PAGplate (pHrange 3.5-9.5) gels, detected with rabbit anti human GRHPR b) Coomassie blue stained stained Ampholine PAGplate (pH range 3.5-9.5) isoelectric focusing gel of 5 pg ofpure recombinant GRHPR and 5pg of pure recombinant GRHPR in 5mM DTT c) Coomassie blue stained Ampholine PAGplate (pH range 3.5-9.5) isoelectric focusing gel of 2.3pg of pure recombinant GRHPR treated with various concentrations o f guanidine. 4.3.12 DTT treatment 5pg of pure recombinant GRHPR was resuspended in 5mM dithiothreitol (DTT, Sigma) and heated at 95®C for 3 mins then cooled on ice and subjected to isoelectric focusing. Multiple bands were still observed after DTT treatment 102 {Figure 4.16b) indicating charge heterogeneity of GRHPR is not due to multiple subunit associations formed during isolation of the protein in an oxidising environment. 4.3.13 Chemical dénaturation of GRHPR 2.3pg aliquots of purified GRHPR were freeze dried, resuspended in either 7|li1 of water, 2M, 4M or 6M guanidine hydrochloride (Sigma) and subjected to isoelectric focusing. 3 or more bands were still observed on isoelectric focusing after 6M guanidine hydrochloride treatment {Figure 4.16c) indicating charge heterogeneity of GRHPR is unaffected by unfolding of the protein. In summary, charge heterogeneity in the GRHPR protein is observed in the recombinant and native forms of the enzyme and is unaffected by reduction to give monomeric GRHPR only or chemical denturation to remove secondary structure from the enzyme. The reason for this heterogeneity remains unclear. 103 4.3.14 Enzyme kinetics Table 4.2 shows apparent Km values determined by single substrate kinetics with computer generated lines of best fit. Results fi*om Hanes plots and curve fitting plots are shown side by side with data plots in Figure 4.17. Both methods gave similar results {Table 4.2) however curve fitting is likely to produce the best results as it does not require reciprocal plots necessary for earlier, non-computer based plots. In most cases the Km apparent (Km’) was determined because of substrate inhibition and other limitations as described below. Actual Km values determined with saturating concentrations of the fixed substrate (i.e. 20 x Km) were possible for glyoxylate with NADPH as cofactor and NADH with glyoxylate as substrate. Substrate cofactor Curve Fitting Hanes Plot Km (mM) Km (mM) hydrcxypyruvate NADPH hydrcxypyruvate 0.06 0.04 NADPH 0.01 0.01 NADH hydrcxypyruvate 0.43 0.41 NADH 0.37 0.38 glyoxylate NADPH glyoxylate 2.18* 1.25* NADPH 0.03 0.03 NADH glyoxylate 8.75 8.57 NADH 0.49* 0.52* D-glycerate NADP D-glycerate 2.30 2.12 NADP 0.45 0.45 D-glycerate NAD negligible activity Glycolate NADP/NAD negligible activity Table 4.2 Kinetics for GR, HPR and D-GDH activities ofpurified recombinant GRHPR with various substrate-cofactor combinations. Values are Km apparent (Km ’) in all cases except those marked * which illustrate actual Km values. 104 To determine the K,^ for hydroxypyruvate (with NADPH as cofactor) y - 77.305* + 3.0443 0.007 0.006 £ 0.005 E o 0.004 s s E < 0.003 f 0.002 0.000 -0.045-0.025 -0.005 0.015 0.035 0.005 0.01 0.015 0.02 P iydroxypyninitel mM IbydiDxypyrnvate] mM Hanes plot Curve fitting plot To determine the K,^ for NADPH (with hydroxypyruvate as substrate) y = 149.95* + 1.2586 0.007 -, 0.006 - £ 0.005 - 0.004 < 0.003 • 0.002 0.001 0.000 -0.01 -0.005 0.005 0.01 0.015 0.02 0.025 0.005 0.01 0.015 0.02 0.025 [NADPH] mM [NADPH] mM Hanes plot Curve fitting plot To determine the for glyoxylate (with NADPH as cofactor) y » 61.814X + 77.214 0.018 0.016 E 700 - ^ 0.014 I 1 0.012 a g 0.010 a < 0.008 a 5 0.006 .i' a 200 . Î “ 0.004 & 0.002 a 0.000 -4 2 0 2 4 6 8 10 12 (glyoxylate] mM bilyoxylate) mM Hanes plot Curve fitting plot To determine the for NADPH (with glyoxylate as substrate) 25 1 0.01 -, y = 74.449*+ 2.3218 0.01 - 20 - 15 - 0.01 - 10 - 0.00 _ -0.050.05 0.15 0.2 0.25 0.05 0.1 0.15 0.2 0.25 [NADPH] mM [NADPH] mM Hanes plot Curve fitting plot Figure 4.17 Single substrate kinetics o f pure recombinant human GRHPR. Dashed line in curve fitting plot indicates calculated rate and points indicate measured rate. Error bars ± 1 S. D. 105 To determine the \< ^ for hydroxypyruvate (with NADH as cofactor) 25 -, y = 28.241X+ 11.701 0020 0.018 20 0016 1 “ 014 .--t o °.012 .010 008 j:006 ° 0 004 0 002 0000 -0.45-0.25 -0.05 0.15 0.35 0.1 0.2 0.3 ]mM [h y d ro x y p y ru v a ta ] mM Hanes plot Curve fitting plot To determine the K,^ for NADH (with hydroxypyruvate as substrate) 0.0035 y = 169.91X + 65.26 S 0.0030 e B 0.0025 I 0 .0 0 2 0 a 80 g < 0.0015 0.0010 I 20 - 0.0005 0.0000 -0.5 -0.3 -0.1 0.1 0.3 [NADH] mM [NADH] mM Hanes plot Curve fitting plot To determine the for glyoxylate (with NADH as cofactor) 250-1 y = l6 .3 6 9 x + 140.3 0.025 -I I 200 - 0.02 I I 150 - Ç 0.015 & 9 ? 100 - s 0.01 Ô 50 - 0.005 B! 0 -10 -5 0 5 [gl^xylate] nM [glyoxylata] mM Hanes plot Curve fitting plot To determine the for NADH (with glyoxylate as substrate) 120 -, y = 110.79x + 58.064 0.0045 0.0040 100 I 0.0035 80 - 1 0.0030 I 0.0025 5 60 Ô 0.0020 40 = 0.0015 0.0010 0.0005 0.0000 -0.6 -0.4 - 0.2 0 0.2 0.4 0.2 [NADH]mM [NADH]mM Hanes plot Curve fitting plot Figure 4.17 (continued) Single substrate kinetics o f pure recombinant human GRHPR. Dashed line in curve fitting plot indicates calculated rate and points indicate measured rate. Error bars ± 1 S. D. 106 To determine the for D-glycerate (with NADP as cofactor) y = 275.35x + 583.92 ê 0.04 a 0.03 IS 0 . 0 2 9 0.02 - 0.01 - -0.5 0.7 0 [D-glycsrate] mM [D-glyctrate] mM Hanes plot Curve fitting plot To determine the for NADP (with D-glycerate as substrate) 9000 y = 1817.4x + 818.37 0.0007 8000 0.0006 7000 'g 0.0005 6000 5000 5 0.0004 .I..-.{ 4000 ^ 0.0003 3000 g 0.0002 2000 0.0001 - 1 0 0 ^ ,------0 1 2 2 3 [NADP] mM [NADHnM Hanes plot Curve fitting plot Figure 4.17 (continued) Single substrate kinetics o f pure recombirumt human GRHPR. Dashed line in curve fitting plot indicates calculated rate and points indicate measured rate. Error bars ±1 S. D. The enzyme showed a preference for NADPH as cofector and hydroxypyruvate as substrate {Table 4.2). Although Km values for glyoxylate and D-glycerate were similar (2.18 and 2.30mM), it should be noted that the dehydrogenase reaction was only feasible if the reaction product, hydroxypyruvate, was captured with hydrazine. Minimal activity was seen in the absence of this reagent. Computer generated lines of best fit satisfying the MichaeHs-Menten equation (curve-fitting) and the more traditional Hanes plots gave very similar results {Table 4.2, Figure 4.17). Due to the reciprocal nature of the Hanes plot which will expand error in the results, ‘curve-fitting’ is likely to give the most reliable results. 107 NADPH and NADH both underwent spontaneous, non-enzymatic dehydrogenation reactions at glyoxylate concentrations of 30mM and above precluding the use of high concentrations of glyoxylate in the reaction {Figure 4.18). 1.6 1.4 12 1 cofactor if nà-NADH 0.8 NADPH 0.6 X)(0 0.4 & c 02 I 0 0 10 20 30 40 50 glyoxylate concn. mM Figure 4.18 Spontaneous dehydrogenation o f NADPH and NADH with increasing glyoxylate concentration Hydroxypyruvate was found to inhibit the HPR reaction in the presence of NADPH. Maximum activity occurred at 0.3mM hydroxypyruvate, felling to approximately 30% of maximum at lOmM {Figure 4.19a). However, with NADH as cofector a maximum rate was seen at approximately ImM hydroxypyruvate which was not significantly different at higher hydroxypyruvate concentrations {Figure 4.19b). 108 fi) Absorbance change at 340nm per Absorbance change at 340nm minute per minute (booooooooooo oooooooooooo oooooooooooo ro -‘ O-k-kKjrOCOUAAC/lUl § oi ooo o î 1 N3 I I S 1 S3 O 4.4 Discussion The fiill length GRHPR cDNA isolated from human liver shares approximately 30% sequence similarity with HPR and GR from a number of species of plants and bacteria including Hmethylovorum, C.sativus and M.extorquens. Figure 4.3 illustrates a protein sequence alignment between human, bacterial and plant GRHPR enzymes. ‘D-GDH’ has been crystallised from Hmethylovorum and the 3D structure elucidated at 2.4À resolution [177]. Goldberg and co-workers identified the N and C termini of the enzyme as being involved in substrate binding while the central region is involved in nucleotide binding. Figure 4.20 illustrates 3 important residues involved in the active site fimction of H.methylovorum D-GDH, comprising a substrate orientating Arg residue (Arg240) and a glutamate/histidine catalytic pair (Glu269, His287). These residues are shown to be important in hydroxypyruvate binding and are invariant in all species illustrated in Figure 4.3 including human GRHPR described in this thesis (residues Arg245, Glu274 and His293). The GR reaction was not investigated structurally in H.methylovorum and hence whether additional residues are involved when bound to a 3 carbon substrate remain unclear. 110 Glu269 0 ^ 0 / / hydroxypyruvate y y H O H ^ c ^ n = ^ N = ^ = 0 H — N HÎS287 H,N O X / .Ni-Iz .NH X HOH2C .H Arg240 \ / C -OOC'^ ^OH D-glycerate Figure 4.20 Active site of D-GDH from Hmethylovorum (adaptedfrom [177p. D-GDH amino acid residues are shown in grey. Substrate and product molecules are shown in black. Antibodies to the enzyme recognise a protein of approximately 38kDa in human liver, 5kDa smaller than the GRHPR fusion protein. Crosslinking studies indicate that GRHPR forms homodimers. Faint bands were observed on western blots at sizes larger than lOOkDa which may represent over-crosslinked GRHPR in conformations which would not normally occur in nature but which are forcibly introduced by the non-physiological covalent amide bonding of lysine residues between subunits which are the product of BS3 treatment. These results show a similar pattern to those previously seen for GRHPR in bovine liver with a subunit size of 34kDa [180] which forms homodimers of 65-72kDa [180,181]. Comparisons between plant and human GR/HPR enzymes are not as easy given that plants have several GR/HPR enzymes with different subcellular locations. I l l However, the spinach ‘HPR-2’ enzyme, which appears to be most similar to human GRHPR, with a preference for NADPH, ability to utilise both hydroxypyruvate and glyoxylate as substrates and cytosohc location, is composed of two identical subunits of 38kDa [166]. Similarly peroxisomal HPR- 1, which has preferences for hydroxypyruvate and NADH as substrates, is also formed of homodimers of molecular weight 41kDa. In contrast, the cytosolic spinach NADPH-GR enzyme, which is also able to use hydroxypyruvate as a substrate albeit with a lower affinity, has a homotetrameric conformation con^osed of 33kDa subunits and therefore quite different to the human GRHPR enzyme. The bacterial GR/HPR enzymes of M.extorquens and H.methylovorum are once again composed of homodimers with 37 and 38kDa subunits respectively. It therefore appears that GRHPR enzymes from different species of plants, bacteria and mammals favour a homodimeric conformation, despite sharing low protein sequence similarity {Figure 4.3). The GRHPR gene is expressed primarily in the hver and, to a lesser degree, in a wide variety of tissues as determined by northern blot analysis. The significance of this ubiquitous expression is unclear. Higher molecular weight species at 2.9kb and 4.2kb were also seen in liver, kidney, heart and pancreas on the northern blot but are also of unknown significance. It is possible that these larger species may represent alternatively spliced transcripts, a common occurrence found in up to 38% of human ESTs [197]. However, the human EST database does not seem to include any alternative versions of the GRHPR mRNA which would result in transcripts of this length. Cramer et al. [198] have described a human cDNA which, when translated, has GR, HPR and D-GDH activity 112 although the rate of catalysis was much higher for the reductase than dehydrogenase reactions. Northern blot analysis was not performed by this group but they did describe a search of the EST database identifying the GRHPR gene in many tissues including adrenal, heart, testis, kidney, blood, liver, brain, lung, breast, ovary, colon, placenta and prostate. However, as my northern blot illustrates, the level of expression in these tissues is very variable and therefore the presence of an EST tells us little about its role in these tissues, Huang et al. [199] have also subsequently performed a northern blot of an identical gene they call D-2-Hydroxyacid Dehydrogenase. A very similar result was obtained to my blot, however no comment has been made on higher molecular weight bands also observed on this blot. Both groups reported the chromosomal location of the GRHPR gene assigned by radiation hybrid mapping [198,199], localised to the centromeric portion of chromosome 9. The genomic structure of the GRHPR gene has also recently been described [198] which spans approximately 9kb and contains 9 exons and 8 introns. Tissue distribution studies with the anti-GRHPR antibody showed similar results to those previously documented for GR enzyme activity [123] with the greatest enzyme activity and immunoreactivity in the liver, a lesser but still detectable amount in the kidney and httle activity and no immunoreactivity in muscle and leucocytes. It is not known whether reduction of glyoxylate is important in other tissues as the enzyme catalysing the subsequent step of glycolate oxidation, glycolate oxidase, is restricted to the liver [200]. 113 In all cases of PH2 in which liver was available for analysis, the disease was found to be associated with lack of GRHPR immunoreactive protein. This test has now been incorporated into the UCLH Primary Hyperoxaluria Diagnostic Service. In chapter 3 1 have described the behaviour of HPR and GR enzyme activities from semi-purified human liver upon chromatofocusing in which more than one protein with HPR activity was found. One of these peaks, which was not retained by the column i.e. pl>7.2, had HPR activity only, and we hypothesised that this was possibly due to LDH. The other, which had GR and HPR activity, eluted over a pH range of 1 unit suggesting that multiple isoforms might be present. Recombinant GRHPR was found to be fully retained on the column but also focused as multiple peaks in the same medium, suggesting that charge heterogeneity is present and therefore the multiple peaks of GR and HPR activity seen in the semi-purified human liver were in fact due to the behaviour of one protein and not isoenzymes. The chromatofocusing data were supported by lEF, which showed the same charge heterogeneity although at a higher pi (7.3, 7.15 and 7.0). Following dimérisation of the protein by chemical crosslinking, the 3 bands focused at pH 5.3, 5.5 and 5.6 which is the range seen for human liver GRHPR and recombinant GRHPR on chromatofocusing. It therefore seems likely that on chromatofocusing we observe the enzyme in its native, dimeric form whereas upon lEF, possibly due to the high voltage and hence running temperature inherent in this technique, we see the uncrosshnked enzyme in its monomeric state. 114 Charge heterogeneity can arise as a result of post-translational modifications such as glycosylation, phosphorylation, myristylation, palmitoylation and acétylation which are common events in eukaryotic cells. However these processes are unlikely in the case of GRHPR because the recombinant enzyme behaved in a similar manner and bacteria do not generally modify proteins in the same way as eukaryotes. An alternative possibility is that charge heterogeneity is caused by slight differences in the protein folding (tertiary structure) [201]. The charge differences were not affected by isolation in the presence of 5mM DTT or 6M guanidine.HCl suggesting that they were not due to variations in disulphide bonding or protein conformation and hence were due to some property of the protein sequence itself or a modification therein. Charge heterogeneity has previously been described in a number of enzymes including bovine liver arginase [202] and the ATi angiotensin II receptor subtype [203], however no reason for this heterogeneity was identified in either case. Further work should initially concentrate on phosphorylation as a cause of this charge variation. Reversible phosphorylation is used extensively in eukaryotes as a means of enzyme regulation [204, 205]. Analysis of the GRHPR protein sequence with the PROSITE program [196] revealed several putative phosphorylations sites illustrated in Figure 4.21. 115 Protein kinase C phosphorylation site Residue numbers Recognition sequence 12-14 TRR 122-124 TCR 140-142 SWK 183-185 TOR 260-262 SGK 284-286 TLK 298-300 THR Casein kinase 11 phosphorylation site Residue numbers Recognition sequence 110-113 TTAE 201-204 STPE 244-247 SRGD 271-274 TSPE Table 4.3 Putative phosphorylation sites within the human GRHPR protein sequence identified by PROSTFE. Protein kinase C and casein kinase II phosphorylate serine and threonine residues. However, whether any, or all, or these sites are actually suitable substrates for these enzymes would depend on the accessibility of the consensus sequence on the surface of the enzyme. As recombinant GRHPR expressed in bacteria retains charge heterogeneity, if this phenomenon is due to phosphorylation then this must also be possible in bacteria. Relatively little has been published on bacterial serine and threonine phosphorylation [206-209] however, recent reports suggest there may be many families of bacterial enzymes with high similarity to protein serine/threonine kinases which have yet to be described in any detail [210, 211]. Hence, phosphorylation at sites on eukaryote proteins expressed recombinantly in bacteria may be possible and cannot be 116 ruled out in this case. Phosphorylation can be investigated initially either by immunostaining with anti-phosphoserine/threonine/tyrosine antibodies (Sigma) or by gel staining reagents such as the GelCode phosphoprotein staining kit (Perbio Science UK Ltd., Tattenhall, Cheshire, UK) which stains phosphoserine and phosphothreonine containing proteins on acrylamide gels. The GRHPR also contains a putative N(asparagine)-glycosylation site at residues 242-245 (NISR). Again, this sequence motif alone does not prove whether the protein is glycosylated at this residue, which would depend on the protein’s 3 dimensional structure and whether this region is accessible on its surface. GRHPR is a cytosohc protein and is therefore unlikely to be glycosylated, especially in bacteria, which are generally not beheved to glycosylate proteins. Eukaryotes are able to glycosylate some cytosohc and nuclear proteins but this is m an 0-linked (rather than N-linked) fashion [212]. To investigate glycosylation of GRHPR further, N-linked carbohydrate chains can be removed from the protein with N-glycanase and isoelectric focusing performed to see if one or more bands remain. This procedure was performed for the angiotensin II receptor [203] however N-glycanase treatment did not alter the protein’s charge heterogeneity. The result of charge heterogeneity on protein fimction needs to be investigated further and may involve altered subunit associations and/or kinetic behaviour depending on the metabohc state of the hepatocyte (as suggested for bovine hver arginase [202]). Kinetic investigations could be made by separating peaks more fiiUy on chromatofocusing and determining the Km for hydroxypyruvate and 117 glyoxylate for each peak. To enable peaks of recombinant GRHPR to be resolved completely, a tighter pH gradient would need to be applied to the chromatofocusing column requiring the same column packing media (PBE 94, Amersham Pharmacia) and a polybuffer 74 dilution of 1:10 to give a pH gradient from 6 to 5. For kinetic studies of recombinant GRHPR it was not possible to use 2 substrate kinetics, where both substrates are varied simultaneously, because at the low substrate concentrations required the absorbance changes were too small to measure accurately. Single substrate kinetics, where one substrate concentration is varied while keeping the other constant, were therefore applied in all cases. However, it was not possible to use the ideal saturating (20 x Km) substrate concentrations in all cases due to inhibition of the HPR reaction by hydroxypyruvate concentrations above 0.3mM, spontaneous oxidation of NADPH by glyoxylate at concentrations greater than 30mM and limitation on using NADPH and NADH concentrations higher than 0.6mM and O.TmM respectively. Thus the Km’ was determined in all but two cases (glyoxylate with NADPH as cofactor and NADH with glyoxylate as substrate). Inhibition of GRHPR by hydroxypyruvate above 0.3mM is only seen with the preferred cofactor NADPH, an observation also made with the rat hver enzyme above 0.2mM hydroxypyruvate [182]. This inhibition is unlikely to be of physiological relevance as intracellular concentrations of hydroxypyruvate are of the order of0.005mM [182], however if concentrations did reach levels that would inhibit HPR hydroxypyruvate would be converted to L-glycerate by LDH. 118 It seems unlikely that the non-enzymatic dehydrogenation of NADPH and NADH at high glyoxylate concentrations plays any significant role in vivo where the amount of glyoxylate, even in PH2 patients, would be unlikely to reach this concentration. Glyoxylate concentration in the liver has been estimated as between 5nmol/g and lOnmol/g wet weight of homogenised liver tissue in rat and guinea pig respectively [213,214]. These values do not tell us the actual concentration in the cytosol, however calculations made on similar measurements for glycolate reveal its concentration in the cytosol to be 0.17mM [214]. As the amount of glyoxylate per gram of liver is less than that of glycolate [214] the concentration of glyoxylate in the cytosol is likely to be lower than 0.17mM. There are no equivalent data for human liver but one would expect the concentration of glyoxylate to be kept at low levels due to its reactive nature. In plants GRHPR enzymes play an important role in the glyoxylate cycle. Three variants of the enzyme have been described: peroxisomal NADiHPRl ; cytosolic NADPH:HPR2 and cytosolic/chloroplast NADPHiGR [164]. The human GRHPR enzyme is most like NADPH:HPR2, with a similar preference for NADPH as cofactor and utilising hydroxypyruvate and glyoxylate as substrates [166]. The function of NADPH:HPR2 may be to capture glyoxylate in the cytosol and chloroplast (reviewed in [164]) to prevent it exerting inhibitory effects on other processes such as inhibition of RUBISCO in the leaf chloroplast [169]. GR/HPR enzymes and the genes coding for them have also been described extensively in bacteria [172] where they are part of the metabolic pathway whereby oxalate is assimilated into biosynthetic pathways. 119 Mammals all appear to contain a single enzyme with HPR, GR and D-GDH activities, which can utilise either NADPH or NADH as cofactors [146, 179- 182]. This enzyme is distinct from LDH which can also reduce hydroxypyruvate and glyoxylate [146] and, as shown in Chapter 3, can be physically separated from GR (Chapter 3 and [215]). In common with other species [146, 179] and the partially purified human liver enzyme [215], recombinant human GRHPR prefers hydroxypyruvate as substrate. The reverse dehydrogenation reaction (i.e. D-glycerate dehydrogenase) is not thought to be of physiological importance [216], a behef supported by the need for a high enzyme concentration to obtain adequate reaction rates and observation that little activity was found in the absence of a hydrazine capture system. Human and rat GRHPR favour NADP(H) as cofector with both glyoxylate and hydroxypyruvate [182] in contrast to the bovine enzyme which prefers NAD(H) [179]. Both NAD(H) and NADP(H) are present at similar concentrations in the cytosol [217] but the ratio of the reduced and oxidised cofactors are different. Cytosolic NAD/NADH and NADPH/NADP ratios are high [217, 218] favouring reduction reactions for an enzyme with a preference for NADP(H). LDH is known to reduce both hydroxypyruvate and glyoxylate with NADH as cofactor, and hydroxypyruvate alone with NADPH [146, 219]. Rat liver LDH has a preference for pyruvate over hydroxypyruvate with either NADH (Km values of O.lmM and 0.4mM respectively) or NADPH (Km values of 0.67mM and ImM respectively) as cofector [146], thus metabolism of hydroxypyruvate by LDH in vivo may depend on the concentration of this metabohte. This reasoning is supported by the absence of L-glycerate, the product of LDH 120 catalysed metabolism of hydroxypyruvate, in the urine of normal subjects. By contrast, when GRHPR is defective, as seen in PH2, there is usually excessive production of L-glycerate from hydroxypyruvate presumably as a result of LDH action. The overproduction of oxalate in PH2 is believed to arise from absence of GR and the subsequent oxidation of glyoxylate by LDH, a reaction fevoured by the high NAD^/NADH ratio in liver cytosol [218]. Rabbit muscle LDH has also been shown to reduce glyoxylate to glycolate [78,149,150] with a similar Km to that for lactate [220]. From my work it becomes apparent that GRHPR would have a preference for hydroxypyruvate as a substrate over glyoxylate if both substrates were present at similar concentrations in liver cytosol. However, there is very little data available on the concentrations of hydroxypyruvate or glyoxylate in mammalian liver, the data which is available not being directly comparable (molar concentration versus moles / g wet weight of liver tissue) [182, 213]. In the normal non diseased liver the HPR activity of GRHPR is an important step in the metabolic pathway whereby serine is fed into gluconeogenesis [102]. However, as alternative pathways are able to feed serine into gluconeogensis by the action of serine dehydratase or via serine hydroxymethyltransferase (reviewed in [101]), absence of HPR in PH2 may not significantly impair this function. It seems likely that in normal non-diseased liver GR activity has a similar function to that predicted for plant HPR [169] detoxifying any cytosolic glyoxylate by conversion to glycolate. In PH2 absence of GR and HPR activity will lead to accumulation of both hydroxypyruvate and glyoxylate in the cytosol. Rabbit LDH has a lower Km for hydroxypyruvate than glyoxylate (0.73mM and 3mM 121 respectively) [78, 146] hence favouring conversion of accumulated hydroxypyruvate to L-glycerate in a situation where equal amounts of substrate are accumulating. However, this theory does not take into account other substrates of LDH which would also influence glyoxylate and hydroxypyruvate usage, the Km for pyruvate being approximately 100 fold lower than for hydroxypyruvate [78] and the Km for glyoxylate and lactate being similar [220]. Until accurate hydroxypyruvate and glyoxylate concentrations have been determined in human liver the nature of this metabolic pathway will remain unclear. The predominantly hepatic expression of GRHPR along with other glyoxylate utilising enzymes, such as AGT [221] and glycolate oxidase [74, 200], confirms the importance of the liver in glyoxylate metabolism and prevention of endogenous oxalate production. These findings need to be considered when choosing whether to ofifer kidney only or liver-kidney transplantation for the treatment of PH2. A liver specific location for GRHPR would fevour liver transplantation as a good means to replace the majority of defective enzyme. 122 Chapter 5: Molecular Genetics of PH2 5.1 Introduction The University College London Hospitals Chemical Pathology department offers a diagnostic service for PHI screening for the most common mutations and also prenatal testing based on closely associated linkage markers to the AGXT gene. Up until this point, the absence of an associated gene has meant diagnosis of PH2 has completely depended on enzyme analysis, and therefore family studies have been precluded. With the identiûcation of the human GRHPR gene (Chapter 4) the genetic basis for GRHPR deficiency in our patient cohort can be determined which will offer a valuable extension to the PH diagnostic service. It is hoped that under certain circumstances diagnosis may be made from genomic DNA isolated from blood, a far less risky procedure to the patient than liver biopsy. The approach used to identify potential mutations in RNA was reverse transcription-PCR (RT-PCR) and single-stranded conformation polymorphism analysis (SSCP). However, mRNA was not available in all cases. During the course of this work the GRHPR genomic DNA sequence was submitted into GenBank (accession number AF146689) [198] enabling intronic primers to be designed to allow amplification of genomic DNA exon by exon which could then be analysed by PCR-SSCP. Any differences observed on SSCP can be investigated further by direct sequencing ofPCR product and confirmation of any changes observed made by restriction endonuclease digestion where possible. 123 5.2 Methods Patients Leucocyte DNA was obtained from patients with PH2 diagnosed either by elevated urinary oxalate and L-glycerate (n=8) or by lack of GR activity in liver biopsies (n=7). A total of 39 samples were obtained from first degree relatives in 15 unrelated families. Ethics approval for these studies was obtained from UCLH ethics committee (reference 95/0084). 5.2.1 RNA isolation Total RNA was extracted from up to lOOmg human tissue using the RNA Isolator kit (Sigma Genosys) according to the manufacturers instructions. Tissue was homogenised in a glass homogeniser in 1ml RNA Isolator and RNA was eluted and quantitated by absorbance at 280nm. Aliquots were frozen at -80°C in 0.1% diethyl pyrocarbonate (DEPC, Sigma) treated ddH20. RNA was isolated from whole blood using the QlAamp*^ RNA blood mini kit (QIAGEN), using 1ml whole blood and eluting RNA into 30pl DEPC treated water. 5.2.2 Genomic DNA isolation from leucocytes Genomic DNA isolation was performed according to the method of Higuchi [222]. Whole blood was mixed with lysis buffer containing sucrose, tris-HCl (pH 7.5), MgCh and 1% Triton-XlOO, spun and resuspended several times to lyse blood cells and pellet cell nuclei. The resulting pellet was then treated with Proteinase K and nonionic detergents to break open the nuclei and digest away proteins. 124 5.2.3 Reverse transcriptase PCR (RT-PCR) 0.5-lug of RNA was added to an RT solution containing PCR buffer (GibcoBRL)(containing 50mM potassium chloride, 20mM tris-HCl, pH8.4), 5mM magnesium chloride (GibcoBRL), ImM dNTP (Amersham Pharmacia Biotech), Ix hexanucleotide mix (Boehringer Mannheim), 20U RNasin^ RNase inhibitor (Promega), 50U M-MLV reverse transcriptase (GibcoBRL) in a volume of 20p,l. The reaction was incubated at room temperature for 10 min followed by incubations of 42°C 15 min, 99°C 5 min and 5°C 5 min in an OmniGene thermal cycler (Hybaid, Ashford, Middlesex, UK). PCR was performed using 5ul of cDNA as template as described previously (Chapter 2). 5.2.4 Polyacrylamide gel electrophoresis (PAGE) for DMA samples DNA samples in IX bromophenolblue / xylene cyanol loading buffer plus 150ng PhiX174 DNA/LTae///molecular weight marker were applied directly to rehydrated CleanGel 48 S precast 10% polyacrylamide gels (Amersham Pharmacia Biotech). CleanGels and anode and cathode buffer strips soaked with 5X TBE were loaded onto the Multiphorll electrophoresis unit (Amersham Pharmacia Biotech) cooled to 15°C. Electrophoresis was performed at 600V, 30mA, 18W for 60 mins. DNA was visualised by silver staining using the PlusOne DNA analysis kit (Amersham Pharmacia Biotech). 125 5.2.5 Single strand confonnation polymorphism analysis Formamide SSCP loading dye Mix solutions A and B in a 7:5 ratio. Store at -20®C. Solution A 90% formamide lOmM EDTA 0.05% bromophenol blue 0.05% xylene cyanol Solution B 0.1% SDS lOmM EDTA l-2p.l of PCR product were added to 6pl of formamide SSCP loading dye, heated for 3 minutes to 98°C and chilled on ice. Samples were loaded and run as described for DNA PAGE at either 10®C or 15®C, for 60 - 90 mins according to fragment size. PAGE gels were then silver stained to visualise DNA. 126 5.3 Results 5.3.1 Identification of sequence variants 5.3.2 1. Deletion of 28bp in exon 1 Sequencing of the entire cDNA sequence from a normal subject (produced by RT-PCR with EHPRl and EHPR2 primers. Appendix 1) revealed a 28bp deletion of the 3’ end of exon 1 {Figure 5. 7). WT 1 atg aga ccg gtg ega ete atg aag gtg tte gte aee ege agg ata eee Del 1 at g aga ccg gtg ega ete atg aag gtg tte gte aee ege agg at a eee WT 49 gcc gag ggt agg gte gcg ete gee egg gcg gea gae tgt gag gtg gag Del 49 gcc gag g [ 28 bp deletion ] e tgt gag gtg gag WT 97 cag tgg gac teg gat gag eec ate eet gee aag gag eta gag ega ggt Del 69 cag tgg gac teg gat gag eee ate eet gee aag gag eta gag ega ggt WT 145 gtg gcg ggg gee eae gge etg etc tge ete ete tec gae eae gtg gae Del 117 gtg gcg ggg gee eae gge etg ete tge ete ete tec gae eae gtg gae WT 193 aag agg ate etg gat get gea Del 165 aag agg ate et g gat get gea Figure 5.1 GRHPR exon 1 & 2 cDNA sequences obtained from RT-PCR with primers EHPRl and EHPR2. WT, denotes wild-type sequence. Del, denotes sequence containing a 28bp deletion. This 28bp section of exon has the 5’ splice consensus sequence found in introns i.e. GT at the 5’ end, which may indicate a weak splice site or alternative splicing of unknown fimction. A deletion at this point results in altered protein sequence and premature protein termination at the equivalent of codon 36 {Figure 5.2). Undeleted 1 MRPVRLMKVFVTRRIPAEGRVALARAADCEVEQWDSDEPIPAKELERGVA... I I I I I I I I I I I I I I I I I I Deleted 1 MRPVRLMKVFVTRRIPAEAVRWSSGTRMSPSLPRSX Figure 5.2 Protein sequence comparison between proteins derived from the normal (undeleted) form of GRHPR and the sequence with a 28bp deletion at the 3 ’ end o f exon 1 (deleted). Both sequences are produced using the same reading frame. X indicates a stop codon. 127 The 28bp deletion was also found in several EST sequences submitted to GenBank including accession numbers BE747283 and BE897871 (from ovary and skin respectively, full sequences m Appendix 5). RT-PCR from various tissues with primers HPRA (exon 1 ) and HPR5 (exon 2)(see Appendix 1 for PCR conditions) produced 2 bands, one of the correct size for the wild-type undeleted GRHPR sequence and the other consistent with containing the 28bp deletion {Figure 5.3). N ative \ sequence r% . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Figure 5.3 Silver stained PAGE gel containing PCR products amplified from cDNA o f various tissues with HPRA and HPR5 primers. Lanes 1-2 normal liver, lanes 3-6 PH2 liver, lane 7 phiXl 74 DNA/Haelll marker, lanes 8-9 leucocyte, lanes 10-11 adrenal, lanes 12-13 chorionic villus, lanes 14-15 cultured HepG2 cells, lanes 16-17 testis, lanes 18-19 cloned GRHPR DNA, lanes 20-21 cloned DNA with 28hp deletion, lanes 22 & 23 mixture of cloned DNA samples from lanes 18 & 20 and 19 & 21 respectively. The larger band was at a higher intensity in all cases but both bands were observed in a variety of tissues {Figure 5.3). Products amplified from cloned DNA with (lanes 20, 21) and without (lanes 18, 19) the 28bp deletion were included as controls. When these PCR products were mixed and run (lanes 22 and 23) a similar pattern was observed to PCR products from cDNA, supporting the identity of the 2 bands analysed. The fact that the larger (undeleted) band is at a higher intensity may indicate the shorter product is merely the result of a weak splice site. However the function, if any, of a protein produced from this 128 sequence remains unclear. It is also not yet clear whether this mRNA is translated at all. 5.3.3 2. Gdeletion in codon 35 Amplification of GRHPR cDNA with primers EHPRl and EHPR2 yielded a full- length cDNA which on sequence analysis identified a I bp G deletion in a run of 3 G residues in codons 34 and 35 {Figure 5.4). Exon 2 codon 34/35 G deletion Gin Trp Thr Arg 33 34 35 36 r A GTGG # CT CGG Codon 34/35 G deletion Gin I Trp I Asp I Ser I 33 I 34 I 35 I 36 I C* &TGGGA CT CC G Wild type sequence Figure 5.4 Codon 34/35 G deletion in a PH2 patient compared to wild-type sequence This leads to a frameshift from codon 35 onwards resulting in premature protein termination at the equivalent of codon 44. This mutation removes a BsmFI restriction endonuclease site. Primers BsmF and BsmR (see Appendix 1 for 129 sequence and conditions) were used to amplify a 152bp fragment from genomic DNA, which after digestion with BsmFI restriction enzyme produces 2 fragments of 91 bp and 61 bp in normal sequence and remains undigested in the presence of the G deletion. Figure 5.5 illustrates the patterns obtained when the digested PCR fragments were electrophoresed on agarose gels. The mutation was found in 10/30 (33%) PH2 alleles and 0/20 non-PH2 alleles tested. Interestingly this mutation was seen only in PH2 patients of Caucasian origin (n= 8 ) and not in any of Asian origin (n= 6 ). 152bp intro n 1 GGGACTCGGATGAGC exon 2 4 a BsmFI 91 bp fragment 61 bp fragment intron 1 GGACTCGGATGAGC exon 2 A T b G deletion (codon 34/35) BsmFI site removed — + — + — + Figure 5.5 PCR with BsmF and BsmR primers produces a 152 bp product (lanes 1,3,5 with their respective digested products in lanes 2,4,6) which digests to 91 and 61 bp fragments in the wild-type sequence (lane 4) or remains undigested in the presence of the Ibp G deletion in codon 34/35 (lane 6). Heterozygotes show 3 bands o f 152, 91 and 6Ibp (lane +2). denotes presence of restriction endonuclease, - denotes absence of restriction endonuclease. 130 5.3.4 Expression of a truncated form of the GRHPR protein To investigate whether a functional GRHPR enzyme is produced from truncated GRHPR beginning at methionine 81, a PCR product produced from the primers EHPR3 and EHPR2 (see Appendix 1 for PCR conditions) was cloned into the pTrcHisB expression vector as described in Chapter 4. This construct when translated produces a recombinant version of the GRHPR protein missing the first 80 amino acids at the N terminus of the protein (the region encompassing the 28bp deletion and codon 35 mutation). Figure 5.6a illustrates that the recombinant protein product was produced in E.coh BL21 (DE3) cells and on western blotting shows immunoreactivity with the anti-GRHPR antibody. However, the protein had no functional HPR and GR activity {Figure 5.6b\ only background levels were observed, as seen with transfected vector alone. This indicates that the 80 amino acid N-terminal region is essential for GRHPR activity and that any protein product from the Ibp G deletion in codon 34/35 or the deleted version of GRHPR from Met81 would have no GRHPR activity. Background HPR and GR activity in BL21 cells is higher than in the original control in Chapter 4 {Figure 4.5) however this may reflect the more efficient homogenisation protocols used in these later methods. 131 1 2’ 3 350 1 300 66kDa 46kDa Ç 200 30kDa 21.5kDa T- CM CO >» c c c o O o o o o o m Ü o o w CN CM CM X CO CO CO Ü (X. a : ÜC EHPR32 EHPR32 EHPR32 pTrcHisB CL CL CL XX X colony 1 colony 2 colony 3 alone HI HI HJ Figure 5.6 a) Western blot o f bacterial homogenates expressing the EHPR3/EHPR2 PCR product in pTrcHisB expression vector (lanes 1,2,3); pTrcHisB vector alone, b) HPR (black) and GR (white) activity of bacterial homogenates expressing either the EHPR3/EHPR2 construct (EHPR32 colonies 1-3) or pTrcHisB vector alone. Bars denote mean ± 1 S.D. (n=3). 132 5.3.5 3. GTAA deletion at 5’ splice donor site of intron 4 Sequencing of a full-length GRHPR cDNA derived from hepatic tissue identified 2 splice variants in one individual. 15 and 74bp fragments were inserted at the same plaee in the cDNA sequence at a location which later turned out to be the exon 3/intron 4 boundary. Amplification of exon 4 and the 5’ end of intron 4 with primers EX4F1 and EX4R1 revealed a 4bp deletion at the 5’ splice donor site {Figure 5.7a). Asn SAAeAArGTftAeTeAAC...... intror^.GCAG GAAGAAiGTGAAC1^ 135...... GC!.n?.r.9.G^i AG 1 Wild type sequence Homozygous GTAA/AAGT deletion 5’ donor site ____ A----- A G G T — A G T Splice donor site consensus sequence G exon GAAGT GAAGAAGT A A G T GRHPR intron 4 splice donor site (wild-type) GAAGT GAAGAAGTG AAC Intron 4 splice site with GTAA/AAGT deletion Figure 5.7 GTAA/AAGT deletion at the 5 ' splice donor site in intron 4 sequenced from genomic DNA using primers EX4F1 and EX4R1 a) sequencing profile of the wild-type cxon4/intron4 boundary and the sequence from a PH2 patient homozygous for the GTAA/AAGT deletion ’ b)splice 5 donor sites for consensus sequence, wild-type GRHPR intron 4 site and GRHPR intron 4 with GTAA/AAGT deletion. 133 Due to the repetition of surrounding sequence at this site, it is impossible to say whether this deletion is of the last 2 bases of exon 4 and the first 2 bases of intron 4 i.e. AAGT, or the first 4 bases o f intron 4 (GTAA). Figure 5.7b illustrates how this deletion affects the conserved sequence at the 5’ splice donor site of intron 4. Upon sequencing of the whole GRHPR cDNA of one patient with the 4bp deletion (using primers EHPRl and EHPR2) 2 variant sequences were identified at the boundary of exons 4 and 5. These sequences are illustrated in Figure 5.8, comprising 2 small portions of the 5’ end of intron 4 which were retained after splicing. Pre-splicing a ... GAAGAA GTAAGTGAACGC 642bp intron TGGTGG... Exon 4 Intron 4 Exon 5 ...GAAGAA GTGAACGC 642bp intron TGGTGG... Post-splicing 0 ...GAAGAA TGGTGG.... Exon 4 Exon 5 ...GAAGAA GTGAACGCAGACCAG TGGTGG... Exon 4 Exon 5 ...GAAGAA GTGAACGCAGACCAG 59 bases of intronAG TGGTGG... Exon 4 Exon 5 Figure 5.8 Demonstration o f the effect o f the GTAA deletion on GRHPR splicing. GRHPR intron 4 pre-splicing a) wild-type sequence b) containing 4bp GTAA deletion. GRHPR splice variants observed in cDNA from patients homozygous for the deletion containing d) 15 and e) 7 4 bp o f intron 4. Normal spliced variant shown in c). Dashed lines show AAGT deletion, dotted lines show GTAA deletion. 134 This deletion was found to be homozygous in 2 brothers with PH2, but not in any other of the cohort (2/30 PH2 alleles, 6 .6 %). These short insertions both introduce termination codons immediately after the last amino acid introduced by exon 4 (also changing this residue fi'om asparagine to lysine). No immunoreactivity was seen in this patient (Chapter 4, Figure 4.12, lane 5). 5.3.6 Mutation screening by SSCP analysis 5.3.7 Exon 4 Genomic DNA from all PH2 samples was amplified using primers EX4F1 and EX4R1 (see Appendix 1). SSCP analysis of the resulting 313bp PCR product revealed a single variant (lane 2, Figure 5.15) compared to control (lane 1). Figure 5.9 Exon 4 SSCP. Run at 15"^C for55 min. Lane 1 - control, lane 2 - PH2 Upon sequencing this pattern was found to be due to homozygosity for a C295T transition resulting in the replacement of the arginine at codon 99 with a stop codon {Figure 5.10). It would be expected that protein termination at this point would abolish all enzyme activity, truncating the protein to under a third of its usual length. The liver biopsy from a patient homozygous for this mutation showed no immunoreactivity (Chapter 4, Figure 4.12, lane 1). This sequence change results in the loss of a Baw/// restriction endonuclease site. The PCR 135 fragment of 313bp on digestion with BamHI would normally be digested to 11 Ibp and 202bp (lane 2, Figure 5.10c). In the presence of the C295T change the PCR product remains undigested (lane 4, Figure 5.10c). This method confirmed results of SSCP analysis showing that only one of 15 PH2 patients was homozygous for this change. I Glv lie Arg Val iGly + — + — Intron 3 9 7 98 99 1 0 0 1101 A T CC G AC T TG G C 313bp 2 0 2 bp 1 1 1 bp Wild type sequence 1 2 3 4 5 Gly lie IStop Intron 3 97 98 99 AC:T G6GRTCT GAGTTGGC Homozygous C295T Figure 5.10 Sequence analysis of GRHPR exon 4 showing a) wild-type sequence and b) homozygosity for C295T. c) BamHI digest of PCR product from primers EX4F1 and EX4R1. Lane 1 PhiX174 DNA/Haelll molecular weight marker, Lanes 2&3 PCR product from an individual homozygous for wild- type sequence undigested (-) and digested with BamHI (+), Lanes 4&5 PCR product from an individual homozygous for C295T undigested (-) and digested with BamHI (^). 136 5.3.8 Exon 6 PCR was performed from genomic DNA using primers EX6F1 and EX6R1 (see Appendix 1). SSCP analysis of the resulting 222bp PCR product from patients and controls revealed several patterns illustrated in Figure 5.11. Figure 5.11 Exon 6 SSCP. Run at 15^C for55 min. Lanes 6, 7, 11, 14, 15, 16, 19 and 20 show banding patterns which are different to control (non-PH2) samples. Upon sequencing,the pattern in lanes 14 & 15 was shown to be the result of a G494A transition resulting in a Gly 165Asp substitution {Figure 5.12). Until this mutant has been expressed in the bacterial expression system it remains unclear what affect it has on enzyme function. This change results in the addition of an ATjfl/restriction endonuclease site which was used to screen for this mutation in other patients following amplification of genomic DNA with primers EX6F1 and EX6R1 (see Appendix 1 for PCR conditions). The 222bp fragment (lanes 10 & 11, Figure 5.12) was cut to 7Ibp and 15Ibp in DNA with the G494A change. Two patients were homozygous for the mutation (lanes 7 & 9, Figure 5.12) and their parent was heterozygous (lane 8 , Figure 5.12). Another unrelated PH2 patient was homozygous for this change (lane 4, Figure 5.12). Thus the mutation accounts for 4/30 PH2 alleles in unrelated patients, a frequency of 13%. 137 a Gly I Gin I Ala I He Intron 5 165 166 167 168 T G c T C f AG C CACCC CAT I 222bp 151bp 71bp 23456 789 10 11 T-j Wild type seq u en ce b Asp I Gin Ala I lie Intron 5 165 166 167 168 T TGCTCTAGIAC CAGGC CAT I Homozygous G494A Figure 5.12 Sequence analysis of GRHPR exon 6 showing a) wild-type sequence and h) homozygosity for G494A c) DNA fragments produced from PCR with primers EX6FI and EX6RI undigested (lanes 10 & II) or digested with Xhal (lanes 2-9) and PhiXl 74 DNA/Haelll molecular weight marker (lane I). Lanes 2,3,5 & 6 show the wild-type pattern, lane 8 shows the pattern observed when heterozygous for a G494A change, and lanes 4, 7 & 9 show the pattern observed when homozygous for a G494A mutation. Upon sequencing the variants seen in Figure 5.11, lanes 6 & 7 were shown to be the result of an A579G transition {Figure 5.13) resulting in no change of amino acid at position 193. This sequence variant introduces an Wcz/ restriction endonuclease site which was used to screen for this change in our patient cohort. Exon 6 was amplified as described above (using primers EX6F1 and EX6R1) producing a fragment 222bp in length. In subjects with wild-type sequence this fragment was digested to 181bp and 41 bp with Acil (lanes 2, 4 & 9, Figure 5.13d). The A579G change introduced an additional Acil site and the PCR 138 fragment digested to 4Ibp, 121bp and 60bp (shown homozygously in lanes 5, 6 , 10, II and heterozygously in lanes 3, 7, 8 , Figure 5.13d). All four controls tested were homozygous for the A579G change which, in combination with the PH2 patient data, may imply that G at this position is in fact the more common occurrence. As control samples (n=4) are homozygous for this sequence change it is assumed that it is a polymorphism, although to verify its frequency in the general population further screening of normal controls will be required. Ala Ala Glu Phe Gin Ala Ala Glu Phe Gin 192 193 194 195 196 192 193 194 195 196 AAGCA GCAGBATTCCAG AAGCAGC® GAATTCCAG Wild type seq uence Heterozygous A579G Ala Ala Glu Phe Gin 192 193 194 195 196 ft' AACCAGC GGAATTCCAG - ^ 222bp ^ 181 bp -4— 121bp "4-60bp "4— 41 bp 2 3 4 5 6789 10 11 1213 Homozygous A579G Figure 5.13 Sequence analysis of GRHPR exon 6 showing a) wild-type sequence, b) homozygosity for A579G and c) heterozygosity for A 579G. d) Demonstration of A579G variant in exon 6 EX6F1/EX6R1 PCR products undigested (lanes 12 & 13) or digested with Acil (lanes 2-11). PhiXl 74 DNA/Haelllmolecular weight marker is shown in lane 1. Lanes 2,4 & 9 show the wild-type pattern, lanes 3,7 & 8 show the pattern observed in heterozygotes, and lanes 5, 6, 10 & 11 show the pattern observed in homozygotes for the A579G variant/poly morphism. 139 5.3.9 Exon 1 PCR was performed from cDNA using primers GLXR5F and GLXR7R (see Appendix 1). SSCP analysis of the resulting 197bp PCR product revealed differences in 2 samples (lanes 2 & 3, Figure 5.14). 12 3 4 Figure 5.14 Exon 7 SSCP run at 15°C for 45 min. Sequence analysis showed homozygosity for a deletion of bases C608 and T609 {Figure 5.15b) resulting in a frameshifr and premature protein termination at codon 210. As this premature termination removes over 1/3 of the GRHPR protein, it would be expected to have a deleterious effect on enzyme activity. This change results in the addition of an Aval restriction endonuclease site which was used to screen other cases by amplification of genomic DNA with primers EX7F2 and GLXR7R (see Appendix 1 for PCR conditions). The 134bp fragment remained undigested with ^vo/ in the wild-type sequence but was digested to 85bp and 49bp in the presence of the 2bp deletion {Figure 5.15). This method identified two siblings who were homozygous for the deletion. 140 c - Ser 1 Thr Pro Glu 1 Leu 1 Ala 20 1 1 2 0 2 203 204 1 205 1 206 TCTCTftC CCCTCflGC T G GC T ■134bp •85bp Wild type seq u en ce Ser Thr 1 Arg Ala Gly 20 1 2 0 2 1 T G T C TAC C C G A G C T G G C T \ .L.V'j'fJ LMXk Homozygous C108 & T109 deletion Figure 5.15 Sequence analysis of GRHPR exon 7 showing a) wild-type sequence, h) homozygosity for deletion of C608 and T609. c) Aval digestion of PCR product from primers EX7F2 and GLXR7R. Lanes I & 2 PCR product from an individual homozygous for C608 & T609 deletion undigested (-) and digested with Aval (+), Lanes 3 & 4 PCR product from an individual homozygous for the wild-type sequence undigested (-) and digested with Aval (+). PhiXl 74 DNA/Haelll molecular weight marker is shown in lane 5. 5.3.10 Exon 9 PCR was performed from genomic DNA using primers EX9F2 and EX9R2 (see Appendix 1). SSCP analysis of the resulting 219bp PCR product revealed several banding patterns in both single and double stranded DNA {Figure 5.16). However, upon sequencing only one patient showed a difference to the wild-type sequence (lane 4, Figure 5.16). 141 1 2 3 4 5 6 Figure 5.16 Exon 9 SSCP. Run at 15°C for 50 min This patient was found to be heterozygous for a C904T transition resulting in an Arg302Cys substitution {Figure 5.17b). Expression studies have not yet been completed to determine whether enzyme activity is affected. C904T results in the removal of an Acil restriction endonuclease site which was used to screen other genomic DNA samples. The 219bp EX9F2-EX9R2 fragment when cut with Acil generates 2 bands of 99bp and 120bp {Figure 5.17d, lanes 2 & 4). PCR product with the C904T change remains undigested. This method identified a single patient who was heterozygous for this change {Figure 5.17d, lane 1). Figure 5.17d illustrates the inheritance of this mutation in the family of this patient in which the mother (lane 3) is also heterozygous while the father (lane 2) and the patients brother are homozygous for the C904 i.e. normal allele. The other mutation in this patient is the codon 34/35 G deletion inherited from the father {Figure 5.17c). 142 a) c) Arg Thr Arg I Asni Thr LIVI 300 301 302 303 304 1:1 1:2 AC ilG a AC C C CC AAC AC CA C904T C | | C C l l T Codon35delG N I I M n | | n 11:1 :2 Wild type seq u en ce C904T C I I T c lie Codon35delG M 1 1 N N I I N b) I Arg I Thr CysAsn I Thr 300 301 302 303 304 »C ftC ftftC CT CCBllCftC C A d) 11.1 1.1 1.2 11.2 Control (uncut) 219bp 120bp 99bp Heterozgous C904T Figure 5.17 Sequence analysis of GRHPR exon 9 showing a) wild-type sequence, h) heterozygosity for C904T. c) family pedigree for patient heterozygous for C904T mutation indicating nucleotide at base 904 and presence (M) or absence (N) of codon 34/35 Ibp G deletion d) Acil digestion of EX9F2- EX9R2 PCR product in members o f the same family and an undigested control sample showing heterozygosity for C904T in II. I and 1.2 and homozygosity for wild-type sequence in 1.1 and II. 2. 143 5.3.11 Summary Table 5.1 illustrates a summary of mutations identified in GRHPR from unrelated patients with PH2 and their corresponding allele frequency determined by mutation screening as described above. Exon DNA change Protein change Frequency Restriction site (30 unrelated affected PH2 alleles) Exon 2 Codon 34/35 del G Asp35Thr 10/30(33%) removesBsmFI termination at codon 44 Exon 4 C295T Arg99Stop 2/30 (6.6%) removesBamHI Intron 4 4bp (GTAA/AAGT) Insertion of intronic 2/30 (6.6%) none deletion in intron 4 sequence into RNA. splice donor site Asn135Lys termination at codon 136 Exon 6 A579G Aia193Ala 5/30 (17%) adds Acil Exon 6 G494A Gly165Asp 4/30 (13%) adds Xbal Exon 7 0608 & T609 del Pro203Arg 2/30 (6.6%) adds Aval termination at codon 210 Exon 9 C904T Arg302Cys 1/30 (3.3%) removesAcil Table 5.1 Summary o f DNA changes found in PH2 patient cohort studied. A summary of how the PH2 patients were diagnosed, their hepatic HPR and GR activity (if measured) and mutations identified in GRHPR is illustrated in Appendix 6. 144 5.4 Discussion In this chapter I have described several changes in the GRHPR DNA sequence from PH2 patients compared to the wild-type sequence. With one exception, the splice variant, the effect of these mutations have not been confirmed in vitro. However, the mutations track with disease in families and it is not unreasonable to expect premature stop codons and aberrant spHcing to have a substantial effect on enzyme activity. Function becomes more difficult to predict in the case of amino acid substitutions and hence expression work will be the only way to verify that the G494A and C904T mutations cause decreased GR/HPR activity. Work is currently underway in our laboratory to express these DNA changes to observe their functional significance. DNA mutations can occur by many mechanisms including errors of DNA replication and repair, and chemical or radiation induced damage. The CpG dinucleotide is a known hotspot for mutation in vertebrate genomes, having a mutation rate approximately 8.5 times that of the average dinucleotide [223], as the C nucleotide is susceptible to méthylation and deamination to thymine. Two C to T mutations were identified in our PH2 patient group, C295T and C904T. The amino acid substitution introduced by the C904T mutation results in an arginine (302) to cystine substitution, i.e. a positively charged amino acid substituted for an uncharged SH-containing amino acid. With this type of change there may be an effect on the surrounding protein, either structurally or on enzyme activity. Arg302 is conserved in Hmethylovorum and C.sativus, but not M.extorquens where an uncharged methionine residue is present at this position. 145 Hence, it is unclear whether this change will have an effect on protein function, and needs to be confirmed by expression studies. The C295T mutation introduces a premature stop codon which will result in protein termination at codon 99. However, it is unlikely this truncated protein will be produced at all due to nonsense-mediated mRNA decay, a process whereby RNA surveillance mechanisms identify mRNA containing a premature stop codon which is then rapidly degraded [224, 225]. In the unlikely event that truncated polypeptide is produced, termination in exon 4 is very unlikely to result in functional enzyme as the hydroxyacid-dehydrogenase signature in exons 7 and 8 is removed, this region obviously being an important feature of the GRHPR enzyme. Another possibility is skipping of the exon containing the premature stop codon, and subsequent usage of the correct stop codon. As RNA was not available for most patients, exon skipping cannot be verified or discounted. Two other mutations which I have identified introduce frameshifts and premature stop codons downstream of the mutations (codon 34/35 G deletion and C108/T109 del). Again, it is unlikely that these transcripts result in truncated proteins or that any protein produced would have any associated enzyme activity. Short gene deletions are a feature of several mutations seen in PH2 patients. Deletions commonly occur in regions where direct repeats, palindromes (inverted repeats), ‘symmetric elements’ (sequences with an axis of internal symmetry) or mutation ‘hotspots’ are present [226]. Figure 5.18 illustrates the region of GRHPR exon 2 where the Ibp G deletion was observed in several DNA samples. Several sequence types associated with short gene deletions are illustrated 146 including a symmetric element (underlined) and deletion hotspot consensus sequence (in green derived from: TGA/GA/GG/TA/C [226]) however, neither of these motifs overlap the base which is deleted and therefore are unlikely to be involved in the process. 5' CTGTGAGGTGGAGCAGTGGGACTCGGATGAG 3' ^ p^niiratinn fnrU 3 ' GACACTCCScCfÜGTCACCCTGAGCCTACTC 5 ' ^ Slipped mispairing ^ 5 ' ----- CTGTGAGG#GGAG#AGTGGACTCGGATGAG ------3' Excision and repair of ^ 3'— GACACTCCACCTCGTCACC„ ^ DNA synthesis misaligned base followed by replication of the altered DNA strand Q 5 ' ---- CTGTGAGG|GGAOSeAGTGGACTCGGATGAG 3 ' Wild-type and mutated 3 ' -----GACACTCCA c CTCSTCACCTGAGCCTACTC ------5' daughter duplexes 5 ' -----CTGTGAGGfGGAGGAGTGGGACTCGGATGAG------3 ' 3 ' -----GACACTCCILCCTCGTCACCCTGAGCCTACTC------5 ' Figure 5.18 Slipped mispairing model of Ibp deletion in GRHPR codon 34/35. a) Double stranded DNA containing several sequence motifs; sequence in green is a putative deletion hotspot consensus sequence, sequence in yellow is a putative palindromic sequence, underlined sequence is a symmetric element, b) Slipped mispairing o f a G residue and subsequent excision, repair and DNA synthesis using the mutant strand as template, c) Wild-type and mutant (G deleted) daughter duplexes. A palindromic sequence is illustrated in yellow which, after the sequence is rendered single-stranded by a replication fork, may form the structure indicated in Figure 5.19. It is unclear how this would facilitate removal of a single G residue however, this may be an attempt by error repair mechanisms to eliminate non-paired bases. Figure 5.18 illustrates the most likely mechanism of deletion 147 of this base by the slipped-mispairing model put forward by Kunkel [227]. A single G residue becomes misaligned in a run of 3, and this misalignment is incorporated and fixed into the sequence of the newly generated strand. In a short run of 3 G residues the first base would be more likely to be mispaired and remain that way long enough to be excised in this way as the next 2 bases would stabilise the misalignment. G - G g ' \ ® \ ^ C I \ I G C / G 5’ Figure 5.19 Putative conformation o f a short palindromic sequence in GRHPR exon 2 around the codon 34/35 region G deletion seen in 33% of PH2 patients (one o f the 3 G residues seen at the top o f the figure). Circles indicate base pairing. A similar slipped-mispairing scheme can be produced for the GTAA/AAGT deletion seen at the exon 4/intron 4 boundary {Figure 5.20) whereby the first of 2 neighbouring AAGT sequences loops out, is excised by DNA repair mechanisms and is subsequently copied into the daughter DNA strand. 148 a 5 GGAAGTGAAGAAGTAAGTGA— 3^ Replicationfork 3' CCÏ.TCÂCTTCTTCATTCACT 5 '\ AAGT-v Excision 3' b 5 ' GGAAGTGAAG&AGT 3' CCTTGACTTCTTCATTCACT 5' c 5 '-- GGAAGT GAAGAAGT GA 3 ' 3 '-- CCTTGACTTCTTCACT 5 ' 5 '-- GGAAGTGAAGAAGTAAGTGA--- 3 ' 3'-- CCTTGACTTCTTCATTCACT--- 5' Figure 5.20 Slipped-mispairing model for exon 4/intron 4 4bp deletion, a) Double stranded DNA containing 3 AAGT repeats in green, yellow and blue, b) Slipped-mispairing with an AAGT sequence looped out and excised by DNA repair mechanisms, c) Wild-type and mutant (AAGTdeleted) daughter duplexes. Interestingly, a further modified slipped-mispairing deletion mechanism proposed by Krawczak and Cooper [226] can also be applied to this situation, this time resulting in a GTAA deletion but resulting in the same mutated sequence. In this case misalignment occurs between a third AAGT repeat 5’ to the previous repeats (in green. Figure 5.21) and an interrupted combination of the other two repeats, with the middle 4 bases of the two adjacent repeats ‘looping out’. The initial looped region between the green and yellow copies of the repeat is merely a transient mispairing which lasts only long enough to template the formation of the second misalignment, the looped region within which is excised and copied into the daughter DNA strand. 149 a GGA^TGAAGAAGTAAGTGA— 3' Replication fork 3 ' — CCT5^CACTTCTTCATTGACT 5 ' GTAAx Excision Transient - remains undeleted Y 3' 5 ' — ggaagtga b 3' CCtTCACTTCTTCATTCACT 5 Q 5 ' ------GGAAGTGAAGAAGTGA 3 ' 3 ' ------CCTfCfCTTCTTCACT 5 ' 5 '------GGâÀGTGAAGAAGTAAGTGA ----3 ' 3 ' ------CCiTCACTTCTTCATTCACT----5 ' Figure 5.21 Modified slipped-mispairing model for exon 4/intron 4 4 bp deletion, a) Double stranded DNA containing 3 AAGT repeats in green, yellow and blue, b) Transient slipped-mispairing of an interrupted combination o f the yellow and blue repeats with the green TTCA sequence. Looped-out region o f the yellow and blue AAGT repeats is excised and copied into the daughter DNA strand whereas the green region is unchanged, c) Wild-type and mutant (GTAA deleted) daughter duplexes. It is unclear which hypothesis is more likely, although the original slipped mispairing model is certainly much simpler. In addition to showing this mutation in the intron 5’ splice donor region in genomic DNA, I have also sequenced 2 resulting splice variants present in the mRNA of this PH2 patient. The splice variants produce immediate protein termination at codon 136 which removes over 50% of the protein and would not be expected to give rise to functional enzyme. The patient had no hepatic GRHPR immunoreactivity (Chapter 4, Figure 4.12, lane 5). indicating that mispliced GRHPR enzyme predominates over any which may be correctly spliced. The 3’ splice acceptor sites are partially 150 conserved in the small portions of intron retained in that both end with AG residues, which is the most conserved part of the acceptor site. Due to the small length of one of the inserts it is unlikely to contain the conserved branch site sequence usually seen 20bp or more upstream of the splice acceptor site. It seems likely that these short sequences are weak splice sites and only function when the splice donor site is weakened by mutation, in this case the GTAA deletion. The mechanism by which the exon 7 CT deletion came about is more difficult to predict from the surrounding sequence. Figure 5.22 illustrates 2 repeated sequences in the region of the deletion, one of which, the CCCT repeat, contains both deleted bases. However, it is unclear how a slipped mismatch-type mechanism could produce such a deletion. 5 ' CCTGCCCTCCCTCAGTGTCTACCc: ÎAGCTGGCTGCCCAATCTG 3 ' 3 ' GGACGGGAGGGAGTCACAGATGGGGA : T CGACCGACGGGT TAGAC 5 ' Figure 5.22 Sequence surrounding GRHPR exon 7 CT deletion. Sequence in green is a CCCT direct repeat; sequence in yellow is a GCTG direct repeat; boxed sequence indicates site of CT deletion. The 2bp deletion in exon 7 (C608/T609) causes premature protein termination at codon 210 truncating the enzyme by 118 amino acids i.e. well over a third of the protein is missing. It is therefore not surprising that no GRHPR immunoreactivity was observed for a patient homozygous for this mutation (lane 2, Figure 4.12, Chapter 4). GR activity was reduced at 21 nmol / min / mg protein (reference range 49-213) [228] but surprisingly, not eompletely absent. It is unclear how any residual activity can be retained by such a large enzyme truncation removing the highly conserved hydroxyacid-dehydrogenase signature 151 region in exons 7 and 8. Interestingly, L-glycerate concentration was not elevated in the patient’s urine [228], an observation made in all PH2 patients tested to date [138, 140-144]. The brother of this patient also presented with stones, elevated urinary oxalate and detectable but not elevated urinary L- glycerate, however GR activity was not measured. This observation would be consistent with the presence of residual GRHPR enzyme using hydroxypyruvate as a substrate in preference for glyoxylate, which would remain available for conversion to oxalate by LDH. Goldberg and co-workers identified the N- and C- termini to be involved in substrate binding in Hmethylovorum D-GDH [177] which may explain reduced, but not absent, GR and HPR activity. Clearly, the presence of GR activity and the unusual phenotype of this patient in addition to high degree of protein truncation illustrates that this mutated version of GRHPR should be recombinantly expressed and the resulting protein assayed for GR and HPR activity. Two DNA changes were caused by G to A/A to G transitions (A579G & G494A). These changes may simply be the result of misincorporation of an incorrect base and subsequent failure of DNA mismatch mechanisms at this position or other chemical modifications to the base/nucleotide resulting in altered base pairing. The exon 6 A579G transition results in a synonymous substitution where alanine at codon 193 remains unchanged. The transition is in the third base position of the codon, a fourfold degenerate site where any base substitution is synonymous, a situation found at 16% of the base positions in human codons. Although a synonymous substitution will not alter the protein sequence directly, it may alter an exonic enhancer sequence (ESE). ESEs are 6-8 152 nucleotide sequences required for efficient splicing of exons and are found throughout gene sequences [229]. Mutations in these sequences can therefore alter splicing. However, as the A579G change is also found in the homozygous state in normal subjects as well as in PH2 patients where mutations in both alleles have already been found, it seems unlikely that this change resides within an ESE but is probably a polymorphism. The G494A transition results in a Glyl65Asp substitution i.e. an uncharged amino acid changed to a negatively charged amino acid. This glycine residue is conserved in all orthologous sequences illustrated in Figure 4.3 (Chapter 4), a good indication that substitution at this point will have an effect on enzyme activity. This mutation has been expressed in Cos cells [230] and shown to have no associated GR, HPR or D-GDH activity. The 28bp deletion at the end of exon 1 seen in multiple tissues by RT-PCR is of unknown significance, the putative protein product sharing 18 amino acids with the wild-type sequence followed by 17 amino acids before premature termination. It seems likely that this represents a weaker splice site than that which produces functional GRHPR enzyme. A recent study has estimated that 38% of human mRNAs contain possible alternative splice forms by comparing multiple EST sequences [197]. As mentioned previously nonsense-mediated mRNA decay is likely to occur in any transcript containing premature stop codons and therefore this mRNA is unlikely to be translated. AJtematively translation may be initiated at a later methionine residue after the frameshift produced by the 28bp deletion or the G deletion in codon 34/35 (for example 153 methionine 81). Expression of GRHPR from codon 81 onwards produced immunoreactive GRHPR but with no GR and HPR activity. Such a large 5’ end deletion would undoubtedly affect protein structure and also substrate binding residues which are suggested to be in the N and C terminal regions of bacterial D-GDH [177]. Mutation screening has proved successful in our patient cohort so far and with added information on the effect of these changes on enzyme function it may be usefiil to screen for some or all of these changes as an addition to diagnosis, especially if the common G deletion in exon 2 continues to be seen at a frequency of 33%. Known mutations in a family will also facilitate the prenatal diagnosis of subsequent children for PH2 if desired. Work is currently underway in our lab to determine linkage markers associated with the GRHPR gene to aid diagnosis in a similar way to that offered for PHI [231]. The common Ibp G deletion in codon 34/35 has also been described by Cramer et al. [198] who established the genomic structure of the GRHPR gene. Subsequent work published from their group [230] has identified 5 further DNA changes found in PH2 patients of which 3, C295T, G494A and intron 4 AAGT/GTAA deletion have also been found m our sanqjles. In addition this group described a G to A transition in intron 7 at the 3’ splice site and and a T965G transversion in exon 9. The G494A and T965G changes were expressed in mammalian Cos ceUs and showed no significant D-GDH, HPR or GR activity. No RNA studies were performed in the case of the AAGT/GTAA deletion and hence altered splicing as a result has not been demonstrated until this thesis. 154 Concluding Remarks The main aims of this thesis have been achieved; native human GRHPR has been separated from LDH and characterised; the gene encoding GRHPR has been identified and the recombinant protein expressed and characterised. Native human GRHPR has a higher affinity for hydroxypyruvate than glyoxylate with NADPH as cofactor which suggests that the HPR reaction is favoured under normal physiological conditions. The enzyme has a pi ranging from pH 5.5 to 6.5, indicating charge heterogeneity and can be physically separated from the HPR activity of LDH, which can also utilise NADPH as cofactor. As GR activity with NADPH as cofactor is the only activity specific to the GRHPR enzyme, this assay must be used as the definitive test for GRHPR deficiency, i.e. for the diagnosis of PH2. The GRHPR gene was isolated from a cDNA library by virtue of its amino acid sequence homology with the hydroxypyruvate reductase enzyme from H.methylovorum. Recombinant GRHPR was expressed in bacterial cells isolated from the soluble fraction of cell homogenates. This expression system is quick and produces large amounts of recombinant enzyme which can be easily purified on a nickel column by virtue of a His tag expressed at the N terminus of the recombinant enzyme. The pure enzyme has been used to raise a polyclonal anti human GRHPR antibody, which detects a single protein in normal human liver. Screening of PH2 patients has shown a lack of immunoreactive GRHPR in all cases tested so far, hence making GRHPR immunoreactivity a useful addition to 155 the PH diagnostic service currently offered at this centre (UCLH Chemical Pathology). In addition this antibody enables fiirther studies of the GRHPR enzyme in heterogeneous tissue preparations. Northern blots indicate that GRHPR mRNA is present in a wide variety of tissues, however this ubiquitous expression is not seen for immunoreactive GRHPR protein, which was predominantly in liver and a lesser amount in kidney. Hence, PH2 can be regarded as a liver specific disease and therefore, like PHI, liver transplantation as a means of replacing defective or absent GRHPR may be the preferred mode of treatment in severely affected cases. Crosslinking of purified recombinant GRHPR explained the observed differences between pi ranges seen upon chromatofocusing and lEF as charges resulting from monomeric or dimeric conformations of GRHPR. Charge heterogeneity of the enzyme is not due to incomplete reduction or subunit associations and remains, as yet, unexplained. Pure human GRHPR, like the semi-purified liver GRHPR enzyme, has a higher affinity for hydroxypyruvate as substrate and NADPH as cofector. Although capture systems enable the reverse (D-GDH) reaction to be observed, it is unlikely that this reaction is of any consequence physiologically and therefore the role of the enzyme appears to be that of a reductase. Further investigation of the kinetics of recombinant GRHPR including additional potential substrates and the kinetics of inhibition of GR/HPR by non-substrate/product confounds such as oxalate will be required in the friture. 156 Mutations in the GRHPR gene have been identified in PH2 patients and restriction endonuclease digests have verified the sequencing and been used to determine firequency within our patient cohort. Future investigations are needed to examine the effect of these mutations on GRHPR enzyme activity in the bacterial expression system used in this study. Genetic analysis can be used to identify additional affected family members if desired. The results presented in this thesis firmly place GRHPR as an hepatic enzyme and PH2 as a disease of the liver. Thus it would appear that the prime organ for glyoxylate metabohsm (and therefore oxalate production) is the fiver. Future direction for this research is therefore to determine whether gene therapy, targeted at the fiver, can provide a cure for these diseases. 157 Appendices Appendix 1 - PCR primers and amplification conditions Primers produced by Genosys Biotechnologies, Pampisford, UK Primer Primer sequence Product Primer Annealing Extension MgCl2 size (mM) temp. (°C) time (secs) (mM) (bp) EHPR1 5’ ATGGTACCGGGTCGGCGGCTG 1119 0.6 66 90 2 EHPR2 5' GCAAGCTTCCCTTGGCTCTGC EHPR3 5’ ATGGTACCTGTGAGGTGGAG 999 0.6 64 30 2 EHPR2 5* GCAAGCTTCCCTTGGCTCTGC BsmF 5’ TTCTCCTGAGGGCCTCCCTTT 152 0.6 62 20 2 BsmR 5' TCCAGGATCCTCTTGTCCACG EX6F1 5' GCTGTTCCGGAAATGCTGGG 222 0.6 56 60 1.5 EX6R1 5’ CAACTGGGCACAGATAGGC EX4F1 5’ GGCAGGCAGATCAAAGAGGG 313 0.6 58 30 1.5 EX4R1 5’ CTCCTAACCTCATGATCCGC EX9F2 5’ GCTGAAGGCTGCTGAACC 219 0.6 54 30 1.5 EX9R2 5’ GCACGGTTTGCTTCCCGG GLXR5F 5' AGAGCACTGTCGGCATCATC 197 0.6 60 30 1.5 GLXR7R 5’ TAAGGAGCAGGCCACGACGA EX7F2 5’ AGCAAGGGGCTGGTCTCC 134 0.3 58 30 1.5 GLXR7R 5’ TAAGGAGCAGGCCACGACGA PCR was performed as follows: 2 minute dénaturation at 94°C 10 second dénaturation at 94^C 10 second annealing at ten^erature listed above 30 cycles 30-90 second extension at 72°C depending on product length 7 minute extension at 72®C 158 Appendix 2 - Kinetics on partially purified GRHPR from human liver 2 substrate kinetics to determine Km values for hydroxypyruvate and NADPH - peak A {Figure 3.2) rhvdroxvDvruvateT*finterceDts from primaiv olott vs rhvdroxvDvruvatel y = 2.9472x + 1.4866 slope KwNADPH/V y = 2.6936x+ 1.3714 y = 2.4009X + 1.2885 y intercept (K,NADPH)(K^hydmxypymvate) / V y = 2.3219x+ 1.1949 X intercept -(K,hydroxypyruvate) y = 2.1327x+1.1358 fhvdroxvDvruvate1*fslooes from primary plot) vs rhvdroxvDvruvatel 1 -0.1 0.4 slope 1V [NADPH] m M y intercept (K^hydroxypyruvate) / V Primary plot X intercept -(K„hydroxypyruvate) y = 0.6909X + 4.056 25 r 2 y= 1.1329X+9.1646 s 10 -- 20 8 -- a a 6 -- k Î 2 £ 7 2 3 8 -9 -4 1 6 [hydroxypyruvat*] ItlM [hydroxypyruvat*] m M Secondary plots 2 substrate kinetics to determine Km values for hydroxypyruvate and NADPH - peak B {Figure 3.2) rhvdroxvDvruvate1*finterceDts from primarvplot) y " 7.905X + 0.848 vs fhvdroxvDvruvatel y = 6.287x + 0.8325 y = 5.7471X + 0.758 slope K^,NADPH/V y = 5.441X+ 0.73 y intercept (K,NADPH)(K^hydroxypyruvate) / V X intercept -(K,hydroxypyruvate) * 0.1 ■ 0.15 fhvdroxvDvruvateT*fsloDes from primary plot) A 0.2 vs fhvdroxvDvruvatel XO.25 - 0.2 0.2 n 4 0.6 slope 1 / V [NADPH] m M y intercept (K^hydroxypyruvate) / V Primary plot X intercept -(K„hydroxypyruvate) 0.2 j y = 0.6396x + 0.024 y = 3.831 2x+ 0.3903 0.18 - 0.16 - 0.14 -- 0.12 - 0.08 0.06 0.04 O.Og. -0.05 0.1 0.15 0.2 0.250.05 -0.15 0.15 1 mM 1 mM Secondary plots 159 2 substrate kinetics to determine Km values for glyoxylate and NADPH - peak B {Figure 3.2) y = 42.996x+ 1.8518 rglyoxylate1*(interœpts from primarv plot) vs y = 24.563X + 1.2224 rgtvoxvlatel y = 17.972X + 0.8179 slope K„NADPH/V y = 13.644X + 0.6801 y = 11.5 9 3 x + 0.6508 y intercept (KjNADPH)(K^lyoxylate) / V X Intercept -(Kjglyoxylate) ♦ 0.2 ■ 0 .4 rgtvoxviatertstopes from primarv plot) vs A 0.6 [glyoxylate] xO.8 slope 1 / V -0 .0 5 0.0 5 0 .1 5 0.2 5 • 1 [NADPH] mM y Intercept (Kjglyoxylate) / V Primary plot X Intercept -(K^lyoxylate) y = 0.308X + 0.3242 0 .7 j y = 3.5388x+8.2199 0.6 - 12 -- 0 .5 - 0 .4 - 6 - 0.2 - 0.1 - 0.6 - 0.1 0.40.9 -2.5 -1.5 -0.5 0.5 [glyoxylato] mM [gtyoxyiata] mM Secondary plots 160 Appendix 3 - GRHPR cDNA, EST and protein sequences a) Hyphomicrobium m ethylovorum hydroxypyruvate reductase Accession number S48189 1 mskkkilitw plpeaamara resydviahg ddpkitidem ietaksvdal 51 litlnekcrk evidripeni kcistysigf dhidldacka rgikvgnaph 101 gvtvataeia mllllgsarr agegekmirt rswpgwqplq Ivgqrldnkt 151 Igiygfgkig qalaqrargf dmnvhyydiy rakpeveaky natyhdslds 201 llkvsqffsi napstpetry ffnketiekl pqgaiwnta rgdlvkdddv 251 iaalksgrla yagfdvfage pninegyydl pntflfphlg saaieamqm 301 gfealdnida ffagkdmpfk la b) EST identified by tblastn program (at http://www.ncbi.nlm.nih.gov/BLAST/) using H.methylovorum HPR sequence Accession number D63259 1 gccaggtccg ggtcggcggc tgcactgcgg atgagaccgg tgcgactcat 51 gaaggtgttc gtcacccgca ggatacccgc cgagggtagg gtcgcgctcg 101 cccgggcggc agactgtgag gtggagcagt gggactcgga tgagcccatc 151 cctgccaagg agctagagcg aggtgtggcg ggggcccacg gcctgctctg 201 cctcctctcc gaccacgtgg acaagaggat cctggatgct gcaggggcca 251 atctcaaagt catcagcacc atgtctgtgg gcatcgacca cttggctttg 301 gatgaaatca agaagcgtgg gatccgagtt ggctacaccc cagatgtcct 351 gacagatacc accgccgaac ttgcagtctc cctgctactt accacctgcc 401 gccggttgcc g c) Result of tblastn search with H.methylovorum HPR sequence A: H.methylovorum HPR sequence (accession number S48189) B: D63259 Human placenta polyA+ mRNA 5' end Length = 411 37% identity A: 5 KILITWPLP-- EAAMARARESY DVIAHGDDPKITIDEMIETAKS VDALLITLNEKCRKE K+ +T +P A+ARA + +V D I E+ LL L++ K B: 52 KVFVTRRIPAEGRVALARAADC-EVEQWDSDEPIPAKELERGVAGAHGLLCLLSDHVDKR A: 62 VIDRIPENIKCISTYSIGFDHIDLDACKARGIKVGNAPHGVTVATAEIAMLLLLGSARR ++D N+K 1ST S+G DH+ LD K RGI+VG P +T TAE+A+ LLL + RR B:229 ILDAAGANLKVISTMSVGIDHLALDEIKKRGIRVGYTPDVLTDTTAELAVSLLLTTCRR 161 d) D63259 blast against other human ESTs to get longer 5' region Clone AA148891 (A) against D63259 (B) A:1 gcttctgtact B:1 gccaggtccgggtcggcggctgcactgcggatgagaccggtgcgactcatgaaggtgttc I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I M I I I I M I I I I I I I I I I I I I I I I A:12 gccaggtccgggtcggcggctgcactgcggatgagaccggtgcgactcatgaaggtgttc B : 61 gtcacccgcaggatacccgccgagggtagggtcgcgctcgcccgggcggcagactgtgag I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I A:72 gtcacccgca-gatacccgccga-ggtagggtcgcgctcg-ccgggcggcagactgtgag B : 121 gtggagcagtgggactcggatgagcccatccctgccaaggagctagagcg-aggtgtggc I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I A: 129 gtggagcagtgggactcggatgagcccatccctgccaaggagctagagcgaaggtgtggc B : 180 gggggcccacggcctgctctgcctcctctccgaccacgtggacaagaggatcctggatgc I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I A:189 ggggg-ccacgcctgcntctgcctcctctccgaccacgtggacaagaggatcctggatgc B : 24 0 tgcaggggccaatctc-aaagtcatcagcaccatgtctgtgggcatcgaccacttggctt I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I A:248 tgcaggggccaatctcaaaagtcatcagcaccatgtctgtgggcatcgaccacttggctt B : 299 tggatgaaatc-aagaagcgtgggatccgagttggctacacccca I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I A:308 tggatgaaatcaaagaagcgtgggatccgagttggctacacccca e) Clone AA148891 Soares_pregnant_uterus_NbHPU Homo sapiens cDNA clone 5', mRNA sequence 1 gcttctgtac tgccaggtcc gggtcggcgg ctgcactgcg gatgagaccg gtgcgactca 61 tgaaggtgtt cgtcacccgc agatacccgc cgaggtaggg tcgcgctcgc cgggcggcag 121 actgtgaggt ggagcagtgg gactcggatg agcccatccc tgccaaggag ctagagcgaa 181 ggtgtggcgg gggccacgcc tgcntctgcc tcctctccga ccacgtggac aagaggatcc 241 tggatgctgc aggggccaat ctcaaaagtc atcagcacca tgtctgtggg catcgaccac 301 ttggctttgg atgaaatcaa agaagcgtgg gatccgagtt ggctacaccc caagattgtc 361 cttgacaaga taccacccgn cgaacttcgc aagtcttccc ttgcttactt tagnaccctt 421 gcccgncggg ttgc 162 Appendix 4 - Calibration curve for isoelectric focusing 100 -1 90 - - 80 - ë 70 - -14.31X + 146.32 60 - 1 50 - E I 40- S 30 - C» 2 0 - 3 4 5 6 7 8 9 10 pi Calibration curve o f distances migrated by isoelectric focusing marker proteins against their p i values for uncrosslinked isoelectric focusing gel (Figure 4.15a) 163 Appendix 5 - EST sequences matching GRHPR with 28bp deletion BE747283 from human ovary GRHPR + del is the GRHPR cDNA sequence containing a 28bp deletion in exon 1 GRHPR + del: 20 cgggtcggcggctgcactgcggatgagaccggtgcgactcatgaaggtgttcgtcacccg 79 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I BE747283: 1 cgggtcggcggctgcactgcggatgagaccggtgcgactcatgaaggtgttcgtcacccg 60 GRHPR + del: 80 caggatacccgccgaggctgtgaggtggagcagtgggactcggatgagcccatccctgcc 139 I I II I I I I I I I I II IiI I I I I I IiI I I I I I I IiI I II I I I I I I I I I I I I I I M I I I I I I I BE747283: 61 caggatacccgccgaggctgtgaggtggagcagtgggactcggatgagcccatccctgcc 120 GRHPR + del: 140 aaggagctagagcgaggtgtggcgggggcccacggcctgctctgcctcctctccgaccac 199 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE747283: 121 aaggagctagagcgaggtgtggcgggggcccacggcctgctctgcctcctctccgaccac 180 GRHPR + del : 200 gtggacaagaggatcctggatgctgcaggggccaatctcaaagtcatcagcaccatgtct 259 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE747283: 181 gtggacaagaggatcctggatgctgcaggggccaatctcaaagtcatcagcaccatgtct 240 GRHPR + del: 260 gtgggcatcgaccacttggctttggatgaaatcaagaagcgtgggatccgagttggctac 319 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE747283: 241 gtgggcatcgaccacttggctttggatgaaatcaagaagcgtgggatccgagttggctac 300 GRHPR + del: 320 accccagatgtcctgacagataccaccgccgaactcgcagtctccctgctacttaccacc 379 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE747283: 301 accccagatgtcctgacagataccaccgccgaactcgcagtctccctgctacttaccacc 360 GRHPR + del: 380 tgccgccggttgccggaggccatcgaggaagtgaagaatggtggctggacctcgtggaag 439 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE747283: 361 tgccgccggttgccggaggccatcgaggaagtgaagaatggtggctggacctcgtggaag 420 GRHPR + del: 440 cccctctggctgtgtggctatggactcacgcagagcactgtcggcatcatcgggctgggg 4 99 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE747283: 421 cccctctggctgtgtggctatggactcacgcagagcactgtcggcatcatcgggctgggg 480 GRHPR + del: 500 cgcataggccaggccattgctcggcgtctgaaaccattcggtgtccagagatttctgtac 559 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE747283: 481 cgcataggccaggccattgctcggcgtctgaaaccattcggtgtccagagatttctgtac 540 GRHPR + del: 560 acagggcgccagcccaggcctgaggaagcagcagaattccaggcagagtttgtgtctacc 619 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE747283: 541 acagggcgccagcccaggcctgaggaagcagcggaattccaggcagagtttgtgtctacc 600 GRHPR + del: 620 cctgagctggctgcccaatctgatttcatcgtcgtggcctgctccttaacacctgcaacc 679 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE747283; 601 cctgagctggctg-ccaatctgatttcatcgtcgtggcctgctccttaacacctgcaacc 659 GRHPR + del: 680 gagggactctgcaacaaggacttcttccag-aagatgaaggaaacagctgtgttcatcaa 738 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE747283: 660 gagggactctgcaacaaggacttcttccagaaagatgaaggaaacagctgtgttcatcaa 719 GRHPR + del: 739 catcagcaggggcgacgtcgtaaaccaggacgacctg-taccaggccttggccagtggta 797 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I III I I I I I I I BE747283: 720 catcagcaggggcgacgtcgtaaaccaggacgacctgttaccaggcc-tgggcagtggt- 777 164 BE897871 from human skin GRHPR + del is the GRHPR cDNA sequence containing a 28bp deletion in exon 1 GRHPR + del: 14 caggtccgggtcggcggctgcactgcggatgagaccggtgcgactcatgaaggtgttcgt 73 I I I I I I I I I Ii I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I BE897871: caggtccgggtcggcggctgcactgcggatgagaccggtgcgactcatgaaggtgttcgt 60 GRHPR + del: 74 cacccgcaggatacccgccgaggctgtgaggtggagcagtgggactcggatgagcccatc 133 I I I I I I I I I I I I I i I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE897871: 61 cacccgcaggatacccgccgaggctgtgaggtggagcagtgggactcggatgagcccatc 120 GRHPR + del: 134 cctgccaaggagctagagcgaggtgtggcgggggcccacggcctgctctgcctcctctcc 193 II I I II I I I I I I I I I I I I I I I I I I I I I I I I i I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE897871: 121 cctgccaaggagctagagcgaggtgtggcgggggcccacggcctgctctgcctcctctcc 180 GRHPR + del : 194 gaccacgtggacaagaggatcctggatgctgcaggggccaatctcaaagtcatcagcacc 253 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE897871: 181 gaccacgtggacaagaggatcctggaggctgcaggggccaatctcaaagtcatcagcacc 240 GRHPR + del: 254 atgtctgtgggcatcgaccacttggctttggatgaaatcaagaagcgtgggatccgagtt 313 I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II II I II I I I I I I I I BE897871: 241 atgtctgtgggcatcgaccacttggctttggatgaaatcaagaagcgtgggatccgagtt 300 GRHPR + del: 314 ggctacaccccagatgtcctgacagataccaccgccgaactcgcagtctccctgctactt 373 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE897871: 301 ggctacaccccagatgtcctgacagataccaccgccgaactcgcagtctccctgctactt 360 GRHPR + del: 374 accacctgccgccggttgccggaggccatcgaggaagtgaagaatggtggctggacctcg 433 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE897871: 361 accacctgccgccggttgccggaggccatcgaggaagtgaagaatggtggctggacctcg 420 GRHPR + del: 434 tggaagcccctctggctgtgtggctatggactcacgcagagcactgtcggcatcatcggg 493 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE897871: 421 tggaagcccctctggctgtgtggctatggactcacgcagagcactgtcggcatcatcggg 480 GRHPR + del: 494 ctggggcgcataggccaggccattgctcggcgtctgaaaccattcggtgtccagagattt 553 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE897871: 481 ctggggcgcataggccagg-cattgctcggcgtctgaaaccattcggtgtccagagattt 539 GRHPR + del: 554 ctgtacacagggcgccagcccaggcctgaggaagcagcagaattccaggcagagtttgtg 613 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE897871: 540 ctgtacacagggcgccagcccaggcctgaggaagcagcggaattccaggcagagtttgtg 599 GRHPR + del: 614 tctacccctgagctggctgcccaatctgatttcatcgtcgtggcctgctccttaacacct 673 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE897871: 600 tctacccctgagctggctg-ccaatctgatttcatcgtcgtgg-ctgctccttaacacct 657 GRHPR + del: 674 gcaaccgagggactctgcaacaaggacttcttccagaagatgaaggaaacag 725 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I BE897871: 658 gcaaccgagggactctgcaacaaggacttcttccagaagatgaaggaaacag 709 165 Appendix 6 - Summary of PH2 patient diagnosis, HPR/GR activity and GRHPR mutation Family Sex Diagnosis of Liver Liver Mutation in DNA/ PH2 made GR HPR GRHPR RNA by: activity activity extracted * * from: 1 Female Siblings Affected tL-glycerate Not identified Blood Female Affected tL-glycerate Not identified Blood Female Affected tL-glycerate Not identified Blood Female Affected tL-glycerate Not identified Blood Male Blood Male Father Blood 2 Male Siblings Affected 35delG nm Blood Male Affected Blood Female Affected GR activity 1 410 35delG nm LivCT 3 Male Siblings Affected tL-glycerate 35delG mm Blood Male Affected tL-glycerate 35delG mm Blood 4 Male Siblings Affected GR activity 16 185 Ex4/I4 delAAGT Blood Male Affected Ex4/I4 delAAGT Blood 5 Female Affected GR activity 2.8 35delG mm Blood 6 Female Affected tL-glycerate 35delG mm Blood Female Mother 35delG nm Blood Male Father 35delG nm Blood 7 Female Affected GR activity 0 77 Not identified Blood 8 Male Affected tL-glycerate G494A mm Blood 9 Female Affected GR activity 13 180 35delG mm Liver 10 Male Affected tL-glycerate G494A mm Blood Male Father G494A nm Blood Female Mother Affected tL-glycerate G494A mm Blood 11 Male Father Blood Male Sibling Affected tL-glycerate Not identified Blood Female Twins Affected tL-glycerate Not identified Blood Female Affected tL-glycerate Not identified Blood 12 Female Affected tL-glycerate Not identified Blood 13 Female Affected GR activity 16 143 35delG nm Blood C904T nm Male Fatha" 35delG nm Blood C904Tnn Female Mother 35delG nn Blood C904Tnm Male Sibling 35delG nn Blood C904Tnn 14 Female Affected GR activity 32 201 C295T mm Blood 15 Male Siblings Affected GR activity 21 133 Del C608/T609 Blood mm Male Affected Del C108/T109 Blood mm HPR and GR activity were measured at pH6.0 and pH7.6 respectively as described on p57. * Units for HPR and GR activity are nmoles NADPH oxidised/min/mg protein. Key: 35delG Codon 35 G deletion Ex4/I4 delAAGT Exon4/Intron 4 boundary 4bp deletion Del C608/T609 Deletion of exons 608 and 609 nm Heterozygous for the mutation in question. mm Homozygous for the mutation in question. tL-glycerate Raised L-glycerate levels 165b References 1. Homer HT, Wagner BL. Calcium oxalate formation in higher plants. In: Khan S, editor. Calcium oxalate in biological systems. Florida: CRC Press Inc; 1995. p. 53-72. 2. Tolbert NE. Metabolic pathways in peroxisomes and glyoxysomes. Ann Rev Biochem 1981; 50: 133-157. 3. Franceschi V. Oxalic acid metabolism and calcium oxalate formation in Lemna minor L. Plant, Cell and Environment 1987; 10: 397-406. 4. Franceschi V, Homer H J. Calcium oxalate crystals in plants. Bot Rev 1980; 46: 361-427. 5. Hodgkinson A, Oxalic acid in biology and medicine. 1977, London: Academic Press. 6. Kirkby EA, Pilbeam D J. Calcium as plant nutrient Plant Cell Environ 1984; 7: 397-405. 7. Allison M, Daniel S, Comick N. 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A fresh look at "prokaryotic" protein phosphorylation. JBacteriol 1996; 178(16): 4759- 4764. 209. Saier M, Wu L, Reizer J. Regulation of bacterial physiological processes by three types of protein phosphorylating systems. Trends Biochem Sci 1990; 15(10): 391-395. 188 210. Leonard C, Aravind L, Koonin E. Novel families of putative protein kinases in bacteria and Archaea: evolution of the 'eukaryotic' protein kinase superfamily. Genome Research 1998; 8: 1038-1047. 211. Torshin I. Molecular surface sequence analysis of several E.coli enzymes and implications for existence of casein kinase-2 bacterial predecessor. Frontiers in Bioscience 1999; 4: d394-407. 212. Kreppel L, Blomberg M, Hart G. Dynamic glycosylation of nuclear and cytosolic proteins. J Biol Chem 1997; 272(14): 9308-9315. 213. Funai T, Ichiyama A. High-performance hquid chromatographic determination of glyoxylate in rat Hver. J Biochem 1986; 99: 579-589. 214. Holmes RP, Hurst CH, Assimos DG, et al. Glucagon increases urinary oxalate excretion in the guinea pig. Am J Physiol (Endocrin Metab) 1995; 32: E568-E574. 215. Cregeen D, Rumsby G. Recent developments in our understanding of primary hyperoxaluria type 2. J Am Soc Nephrol 1999; 10: S348-S350. 216. Van Schaftingen E. D-glycerate kinase deficiency as a cause of D- glyceric aciduria FEBS Lett 1989; 243: 127. 217. Williamson D, Brosnan J. Concentrations of metaboHtes in animal tissues. In: Bergmeyer H editor. Methods of Enzymatic Analysis. New York: Academic Press; 1974. p. 2266-2302. 218. Williamson D, Lund P, Krebs H. The redox state of free nicotinamide- adenine dinucleotide in the cytoplasm and mitochondria of rat Hver. Biochem J 1961', 103: 514-527. 219. Anderson S, Florini J, Vestling C. Rat Hver lactate Dehydrogenase. JBiol Chem 1964; 239(9): 2991-2997. 189 220. Sawaki S, Hattori H, Yamada K. Glyoxylate dehydrogenase activity of lactate dehydrogenase. J Biochem 1967; 62(2): 263-268. 221. Kamoda N, Minatogawa Y, Nakamura M, et al. The organ distribution of human alanine-2-oxoglutarate aminotransferase and alanine-glyoxylate aminotransferase. Biochem Med 1980; 23: 25. 222. Higuchi R. Single and rapid preparation of samples for PCR. In: Erlich H editor. PCR Technology. New York: Stockton Press; 1989. p. 31-38. 223. Cooper D, Antonarakis S, Krawczak M. The nature and mechanisms of human gene mutation. In: Scriver C et al. editors. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill; 1995. p. 259-291. 224. Hentze M, Kulozik A. A perfect message: RNA surveillance and nonsense-mediated decay. Cell 1999; 96: 307-310. 225. Culbertson M. RNA surveillance: unforseen consequences for gene expression, inherited genetic disorders and cancer. Trends in Genet 1999; 15(2): 74-80. 226. Krawczak M, Cooper D. Gene deletions causing human genetic disease: mechanisms of mutagenesis and the role of the local DNA sequence environment Hum Genet 1991; 86: 425-441. 227. Kunkel T. Misalignment-mediated DNA synthesis errors. Biochemistry 1990; 29: 8003-8011. 228. Rumsby G, Sharma A, Cregeen D, et al Primary hyperoxaluria type 2 without L-glycericaciduria: is the disease under-diagnosed? Nephrol Dial Transplant 2001; 16: 1697-1699. 190 229. Liu H-X, Cartegni L, Zhang M, et al A mechanism for exon skipping caused by nonsense or missense mutations in BRCAl and other genes. Nature Genet 2001; 27: 55-58. 230. Webster K, Ferree P, Holmes R, et al Identification of missense, nonsense and deletion mutations in the GRHPR gene in patients with primary hyperoxaluria type II (PH2). Human Genetics 2000; 107(2): 176- 185. 231. von Schnakenburg C, Weir T, Rumsby G. Linkage of microsatellites to the AGXT gene on chromosome 2q37.3 and their role in prenatal diagnosis of primary hyperoxaluria type 1. Ann Hum Genet 1997; 61: 365-368. 191 J Am Soc Nephrol 10: S348-S350, 1999 Recent Developments in Our Understanding of Primary Hyperoxaluria Type 2 DAVID P. CREGEEN and GILL RUMSBY Department of Molecular Pathology, Windeyer Institute of Medical Sciences, University College London, London, United Kingdom. Abstract. Hydroxypyruvate reductase (HPR) has been partially enzyme(s) in peak A (8 mM) was 80 times greater than that of purified from human liver and can be separated into at least peak B (0.1 mM), suggesting that the HPR enzyme contained two forms by chromatofocusing; these forms therefore differ in in peak B may be more important physiologically, where the their pi values. Both forms, one with a pi of >7.2 (peak A) and hydroxypyruvate concentrations are in the micromolar range. the other with a pi between pH 6.5 and 5.5 (peak B), use The data presented provide a biochemical explanation for the NADPH as a cofactor. However, only peak B was able to previously observed differences in the tissue distribution of reduce hydroxypyruvate and glyoxylate, with a of 2.3 mM HPR and glyoxylate reductase activities in human subjects and for the latter substrate. Peak A coeluted with lactate dehydro support the claim that diagnoses of primary hyperoxaluria type genase and could represent lactate dehydrogenase (which is 2 should be made by measurement of glyoxylate reductase known to reduce hydroxypyruvate) alone or a mixture of activity in the liver. proteins with HPR activity. The for hydroxypyruvate of the Primary hyperoxaluria type 2 (PH2) is a rare autosomal reces hver. Because the human studies were performed on unfrac sive disorder characterized by hyperoxaluria and the presence tionated hver tissue, these results presumably reflect more than of urinary L-glycerate (1). Previous metabolic studies sug one protein. We have sought to characterize the different forms gested that the underlying defect could involve reduced cyto of HPR and GR in human hver according to their reaction solic hydroxypyruvate reductase (HPR) activity (1), with the kinetics, after partial purification. excess hydroxypyruvate being reduced to L-glycerate by lactate dehydrogenase (LDH) (EC 1.1.1.27). HPR also has D-glycerate dehydrogenase (D-GDH) (EC 1.1.1.29) (2) and glyoxylate Materials and Methods reductase (GR) (EC 1.1.1.26/79) (3) activities, with reduced All reagents were of analytical grade and were obtained from D-GDH activity being demonstrated in the liver (4) and leu Sigma Chemical Co. (Poole, Dorset, United Kingdom), except where kocytes (1) and reduced GR activity being shown in the liver otherwise stated. T3 dialysis tubing (molecular weight cutoff, 12,000 to 14,000) was obtained from Pierce and Warriner (Warrington, (4) of patients with PH2. It is now thought that the enzyme United Kingdom). Polybuffer 74, polybuffer exchanger 94, and all functions primarily as a reductase, rather than a dehydrogenase chromatography columns were obtained from Amersham Pharmacia (3,5), producing the gluconeogenic precursor D-glycerate from Biotech (St. Albans, United Kingdom). HPR and GR activities were hydroxypyruvate. The hyperoxaluria observed for patients with measured using a Cobas Bio centrifugal analyzer (Roche, Welwyn PH2 is probably attributable to the failure of GR to remove Garden City, United Kingdom), as described previously (6). glyoxylate from the cytosol and the subsequent oxidation of Twelve grams of human liver in 50 ml of homogenization buffer, glyoxylate to oxalate by LDH (1). Previous studies of the tissue containing 154 mM KCl, 100 mM MnClg, 0.5% 3-[(3-cholamidopro- distributions of HPR and GR activities indicated that there are pyl)dimethylammonio]propanesuLfonate, a Complete™ protease in at least two enzymes with HPR activity, only one of which has hibitor tablet (Boehringer Mannheim, Lewes, United Kingdom), and significant GR activity and is deficient in PH2 (6). The latter 0.1 mM ethylenediaminetetraacetic acid (EDTA), was homogenized enzyme seems to be predominantly concentrated in the hver, using an Ultra-Turrax homogenizer (Janke & Kunkel, Staufen, Ger many). Homogenization was performed on ice in five 30-s bursts and there are some indications that the two forms can be separated by 60-s intervals, to allow cooling. The resulting lysate was separated by chromatofocusing (7). maintained on ice for 1 h, followed by centrifugation at 30,000 X g in Kinetics of the D-GDH and GR reactions have been de a fixed-angle rotor for 30 min at 4°C. The supernatant was passed over scribed for the enzymes from bovine (8), rat (3), and human (6) glass wool to remove lipids and then centrifuged at 1000 X g for 10 min at 4°C; the resulting pellet was discarded. Ammonium sulfate was added gradually to 30% saturation, with constant stirring on ice, and Conespondence to Dr. GUI Rumsby, Department of Chemical Pathology, Windeyer the solution was centrifuged at 30,000 X g for 20 min at 4°C. BuUding, Cleveland Street, London WIP 6DB, United Kingdom. Hione: 0207-504- Ammonium sulfate saturation in the supernatant was increased to 60% 9229; Fax: 0207-504-9496; E-maU: [email protected] as described above and, after centrifugation, the pellet was resus 1046-6673/1011-0348 pended in 20 ml of equihbration buffer A (20 mM potassium phos Journal of the American Society of Nephrology phate, 2 mM EDTA, pH 5.8). The solution was dialyzed overnight Copyright © 1999 by the American Society of Nephrology against 1 L of equilibration buffer A. J Ara Soc Nephrol 10: S348-S350, 1999 HPR Isoforms S349 The dialyzed sample was thoroughly mixed with 25 ml of dieth- polybuffer ylaminoethyl-Sephadex in buffer A, in a 50-ml Falcon tube, at 4°C. applied This mixture was apphed to a Buchner funnel and washed several pH 6.5 pH 5.5 times with 10 ml of the same buffer. The fractions containing HPR pH 7.2 and GR activities were pooled and applied, at a rate of 3 ml/min, to a 1.4 40 X 2.6-cm chromatography column containing carboxymethyl- Sephadex in buffer A. The column was washed with 2 vol (approxi mately 400 ml) of buffer A. HPR/GR proteins were eluted with 150 1.2 mM NaCl in equihbration buffer, at a rate of 5 ml/min. The HPR/GR fraction was dialyzed against buffer B (25 mM imidazole • HCl, pH I o 7.2) overnight at 4°C and apphed, at a flow rate of 0.5 ml/min, to a T)- 36 X 1.6-cm chromatography column containing PBE 94 equihbrated m 1 with 25 mM imidazole • HCl, pH 7.2. The sample was washed into the column with 5 ml of buffer B. HPR/GR proteins bound to the column were eluted with a descending pH gradient from pH 7.2 to pH 4; the 0.8 pH was established by apphcation of Polybuffer 74 diluted 1/8 with water and was brought to pH 4 with HCl. The flow rate used was 0.5 ■s ml/min. 0.6 Before kinetic analysis, the partiahy purified proteins were dia lyzed against 50 mM potassium phosphate buffer with 2 mM EDTA, W) at either pH 6 (HPR analysis) or pH 7.6 (GR analysis). Two-substrate kinetics were assayed using the method described by FeU (9), by t 0.4 varying the concentrations of substrates, hydroxypyruvate or glyoxy late, and cofactor (NADPH) at 37°C. The reaction was spectropho- tometrically monitored as a decrease in absorbance at 340 nm, which 0.2 was measured using the Cobas Bio centrifugal analyzer (Roche). The I LDH assay (10) was performed on the Cobas Integra (Roche). 0 Results 0 100 200 300 400 500 600 Under the conditions used, a single peak of HPR/GR activity Volume eluted from column (ml) was observed in carboxymethyl-Sephadex ion-exchange chro Figure 1. Fractionation of human hydroxypyruvate reductase (HPR)/ matography (data not shown); this peak was applied to the glyoxylate reductase (GR) by chromatofocusing from pH 7.2 to pH chromatofocusing column. Figure 1 presents the elution profile 4.0. # , HPR activity; ■, GR activity. obtained with the chromato^cusing column. Two peaks of HPR activity were seen. lEbe first peak (peak A) was not retained by the column, had no GR activity, and coeluted with Table 1. Kinetic analysis of peaks A and B^ LDH. The second peak (peak B) eluted between pH 6.5 and 5.5 (mM) and had GR activity. No LDH activity was associated with this peak. Substrate HPR GR For measurement of values for the two enzymes, sub strate concentrations were chosen so that the reaction was Hydroxy pyruvate NADPH Glyoxylate NADPH first-order with respect to substrate. values for hydroxy pyruvate, glyoxylate, and NADPH are shown in Table 1. Peak A 8 0.6 No activity No activity Peak B 0.1 0.2 2.3 0.1 Discussion “ AU reactions were performed at 37°C in 50 mM potassium Results obtained by chromatofocusing indicate that there are phosphate buffer (pH 6.0 for HPR and pH 7.6 for GR), using It least two enzymes with HPR activity in the liver that use NADPH as a cofactor. Reactions were monitored as a decrease in S^ADPH as a substrate. The bulk of the HPR activity (peak A) absorbance at 340 nm during a period of 3 to 5 min. duted from the column before the pH gradient was applied, ndicating that the pi of this enzyme or enzymes is greater than )H 7.2. Under these conditions, we cannot exclude the possi- cusing column (eluting between pH 5.5 and 6.5) and exhibited )ility that there may be more than one enzyme with HPR less HPR activity but significant GR activity. PH2 liver has activity in this peak; a higher starting pH would be necessary been shown to have deficiencies of both HPR and GR activities 0 retain the protein(s) and possibly resolve separate peaks. It (4,6), and therefore the enzyme in peak B is more likely to be s unlikely that the HPR activity in peak A was the result of the protein that is deficient in PH2. Because peak B eluted olumn overloading, because care was taken not to exceed the from the column in a wide pi range, this peak may also apacity of the column and there was no associated GR activ- represent multiple enzymes with HPR and GR activities. ty, which would be expected if the HPR/GR passed straight HPR activity in rat and ox liver also seems to be present in irough the column. Peak B was retained by the chromatofo- two proteins (3,11), one of which is known to be LDH. The S350 Journal of the American Society of Nephrology J Am Soc Nephrol 10: S348-S350, 1999 finding of LDH activity in peak A but not in peak B suggests References that at least part of the HPR activity is attributable to this 1. Williams H, Smith LJ: L-Glyceric aciduria: A new genetic vari ubiquitous enzyme. Hepatic LDH, although commonly re ant of primary hyperoxaluria. 7 Med 278: 233-239, 1968 ported as having no activity with NADPH, has been shown to 2. Willis IE, Sallach HJ: Evidence for a mammalian D-glyceric acid utilize hydroxypyruvate, but not glyoxylate, with NADPH as a dehydrogenase. J Biol Chem 237: 910-915, 1962 cofactor (3). 3. Dawkins P, Dickens F: The oxidation of d - and L-glycerate by rat Kinetic analysis of the two peaks showed that the enzyme in liver. Biochem J 94: 353-367, 1965 4. Mistry J, Danpiure C, Chalmers R: Hepatic D-glycerate dehydro peak B had a for hydroxypyruvate lower than that of peak genase and glyoxylate reductase deficiency in primary hyperox A and thus had a higher affinity for this substrate. Peak B is aluria type 2. Biochem Soc Trans 16: 626-627, 1988 therefore more likely to be of importance physiologically, 5. Van Schaftingen E, Draye J-P, Van Hoof FV: Coenzyme spec where concentrations of hydroxypyruvate are on the order of 5 ificity of mammalian liver D-glycerate dehydrogenase. Eur J Bio jaM (5). The enzyme in peak B showed greater activity with chem 186: 355-359, 1989 hydroxypyruvate than with glyoxylate, a finding that has also 6. Giafi CF, Rumsby G: Kinetic analysis and tissue distribution of been reported for the non-LDH-containing HPR enzymes in human D-glycerate dehydrogenase/glyoxylate reductase and its both rat (3) and ox (11) liver. It should be noted, however, that relevance to the diagnosis of primary hyperoxaluria type 2. Ann all of these studies were performed at different pH values. For Clin Biochem 35: 104-109, 1998 example, the hydrogen ion concentrations used in this study 7. Giafi CF: Characterisation of human D-glycerate dehydrogenase/ allowed determination of maximal enzyme activities for GR glyoxylate reductase, PhD. thesis, London, University of London, 1998 and HPR but were not physiologic. Additional kinetic studies 8. Rosenblum lY, Antkowiak DH, Sallach HJ, Flanders LE, Fahien at pH 7.1 (the pH of liver cytosol) are required for direct LA: Purification and regulatory properties of beef liver D-glyc comparison of GR and HPR activities. In conclusion, the erate dehydrogenase. Arc/i Rioc/iem Riop/iyj 144:375-383, 1971 9. Fell D: Understanding the control of metabolism. In: Frontiers in finding of different forms of HPR provides a biochemical Metabolism 2, edited by SneU K, London, Portland Press, 1997, explanation for the previously observed differences in the pp 47-84 tissue distribution of HPR and GR activities (6) and reinforces 10. Société Française de Biologie Clinique: Recommendations for our recommendation that measurement of GR activity is the determining the catalytic concentration of lactate dehydrogenase method of choice for diagnosis of PH2 (12). in human serum at 30°C. Ann Biol Clin 40: 160-164, 1982 11. Heinz F, Bartelsen K, Lamprecht W: D-Glycerat-Dehydrogenase Acknowledgment aus Leber. Hoppe-Seyler’s Z Physiol Chem 329: 222—240, 1962 We acknowledge the Oxalosis and Hyperoxaluria Foundation for 12. Giafi CF, Rumsby G: Primary hyperoxaluria type 2: Enzymol- support of this research. ogy. J Nephrol ll[Suppl 1]: 29-31, 1998 BIOCHIMICA ET BI0PHY5ICA ACTA BBa ELSEVIER Biochimica et Biophysica Acta 1446 (1999) 383-388 www.elsevier.com/locate/bba Short sequence-paper Identification and expression of a cDNA for human hydroxypyruvate/glyoxylate reductase G. Rumsby *, D.P. Cregeen Department of Molecular Pathology, University College London, Windeyer Institute of Medical Sciences, Cleveland St, London WIP 6DB, UK Received 26 March 1999; received in revised form 4 June 1999; accepted 17 June 1999 Abstract The isolation and expression of a human liver cDNA encoding a 40-kDa protein with glyoxylate and hydroxypyruvate reductase activities is described. The cDNA (GLXR) is 1235 bp and consists of a predicted open reading frame o f987 bp with a 225-bp 3'-untranslated region. The 328-amino add protein has partial sequence similarity to hydroxypyruvate and glyoxylate reductases from a variety of plant and microbial spedes, © 1999 Elsevier Sdence B.V. All rights reserved. Two inherited disorders which cause endogenous [3] activities. It is now believed that the enzyme func overproduction of oxalate have been described in the tions primarily as a reductase rather than a dehydro literature. Both diseases lead to recurrent renal genase [3,5] producing the gluconeogenic precursor stones and, in the most severe cases, to end stage D-glycerate from hydroxypyruvate and acting as a renal failure and death. Primary hyperoxaluria type mechanism to remove the highly reactive oxalate pre 1 (PHI) is caused by decreased activity of alanine: cursor glyoxylate from the cytosol thus preventing its glyoxylate aminotransferase, an hepatic peroxisomal conversion to oxalate (Fig. 1). enzyme. Primary hyperoxaluria type 2 (PH2) is less Tissue distribution studies suggest that there is well documented but is characterised by hyperoxalu more than one enzyme with hydroxypyruvate reduc ria and the presence o f elevated L-glycerate in the tase activity in humans [6] and we have recently dem urine. Metabolic studies suggested that the underly onstrated that human liver HPR can be fractionated ing defect in PH2 could be explained by reduced by chromatofocusing into two peaks differing in their cytosolic hydroxypyruvate reductase (HPR) activity isoelectric points and substrate aflBmities [7]. Only one [1], with the excess hydroxypyruvate reduced to of these forms reduces both hydroxypyruvate and L-glycerate in a reaction catalysed by lactate dehy glyoxylate when NADPH is used as cofactor and is drogenase (LDH; EC 1.1.1.27). HPR also has presumed to be the enzyme lacking in PH2. D-glycerate dehydrogenase (o-GDH; EC 1.1.1.29) This paper offers the first full description of a gene [2] and glyoxylate reductase (GR; EC 1.1.1.26/79) encoding human HPR/GR and will provide the means to elucidate the role of this gene product in the metabolism of oxalate precursors as well as en abling the molecular diagnosis of PH2. * Corresponding author. Fax: +44-171-504-9496; The Entrez database (http://www.ncbi.nlm.nih. E-mail: [email protected] gov/htbin-post/Entrez/) was screened using the term 0167-4781/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PH: 8 0 1 6 7 -4 7 8 1 (9 9 )0 0 1 0 5 -0 384 G. Rumsby, D.P. Cregeen ! Biochimica et Biophysica Acta 1446 (1999) 383-388 peroxisome cytosol hydroxypyruvate hydroxypyruvate D-gly carat* HPR AGT alanin*pyruvate AGT glyoxylate ✓ GO^ glycolate glyoxylate GR oxalate Fig. 1. Proposed pathway of glyoxylate and hydroxypyruvate metabolism in mammalian liver. AGT, alaninerglyoxylate aminotransfer ase; HPR, hydroxypyruvate reductase; GR, glyoxylate reductase; GO, glycolate oxidase. ‘hydroxypyruvate reductase’ and a cDNA identified sion vector (Invitrogen, The Netherlands) which had with peptide sequence homology to HPR fromHy been cut with the same enzymes and the cut ends phomicrobium methylovorum. The DNA sequence of dephosphorylated. The cDNA was inserted into the this cDNA was used to screen the EST database for multiple cloning site downstream of the His tag and clones containing additional 5'-sequence and the lon Anti-Xpress antibody epitope and in frame with the gest cDNA clone obtained from the IMAGE Con plasmid initiation codon. The construct (pTrcHisB- sortium [LLNL] (web site http://bbrp.llnl.gov/bbrp/ HPR) was transfected into Epicurean coli BL21 image) [8] and sequenced in full. (DE3) competent cells (Stratagene, Cambridge, The full-length cDNA clone (IMAGE Consortium UK). Individual colonies were cultured overnight in clone ID:503200) was amplified by polymerase chain SOB medium (20 g/1 tryptone, 5 g/1 yeast extract, reaction (PCR) using oligonucleotide primers 0.5 g/1 sodium chloride, 2.5 mmol/1 potassium chlo (Genosys Biotechnologies, Pampisford, UK) de ride, 10 mmol/1 magnesium chloride pH 7.0) contain signed to introduce restriction sites for KprH and ing ampicUlin at a final concentration of 50 pg/ml. HindlW at the 5'- and 3'-ends, respectively (forward Following plasmid purification, digestion withKpnl primer, 5'-ATGGTACCGGGTCGGCGGCTG; re and Hintilll confirmed the presence of an insert of verse primer, 5 -GCAAGCTTCCCTTGGCTCT- the correct size (1093 bp). GC). PCR conditions were as follows: 5 ng DNA, Single colonies of pTrcHisB and pTrcHisB-HPR 50 mmol/1 potassium chloride, 10 mmol/1 Tris HCl, in BL21 cells were grown up overnight in 2 ml pH 9.0, 0,1% Triton X-100, 2 mmol/1 magnesium SOB medium plus ampicillin (50 pg/ml). Aliquots chloride, 200 pmol/1 dNTP, 0.6 pmol/1 each primer, (50 ml) of SOB medium containing ampicillin 0.25 U Taq polymerase (Promega, Southampton, (50 pg/ml) were inoculated with 0.2 ml of the over UK) in a volume of 25 pi. Thirty cycles of 94®C night cultures and cells grown with aeration to an 0.5 min, 66®C 0.5 min, 72*C for 1.5 min were carried absorbance at 600 nm of 0.6. Isopropylthio-P-D-gal- out. actoside (final concentration 1 mmol/1) was added Following digestion with Hpnl and Hindlll, the and the culture incubated for a further 5 h with PCR product was ligated into the pTrcHis B expres shaking. The cells were spun down (3000 rpm for G. Rumsby, D.P. Cregeen j Biochimica et Biophysica Acta 1446 (1999) 383-388 385 5'gcttctgtactgccaggtccgggtcggcggctgcactgcgg 1 atg aga ccg gtg cga etc atg aag gtg ttc gtc acc cgc agg ata ccc 1 M R PV R L MK V FVT R R I p 49 gcc gag ggt agg gtc gcg etc gcc egg gcg gca gac tgt gag gtg gag 17 AE GR VALARAAD C B VE 97 cag tgg gac teg gat gag ccc ate cct gcc aag gag eta gag cga ggt 33 Q W D S D E p IPA KE L ER G 145 gtg gcg ggg gcc cac ggc ctg etc tgc etc etc tcc gac cac gtg gac 49 V AGAH GL LC L L S D H VD 193 aag agg ate ctg gat get gca ggg gcc aat etc aaa gtc ate age acc 65 KR IL D A A 6 A NL K V I S T 241 atg tct gtg ggc ate gac cac ttg get ttg gat gaa ate aag aag cgt 81 M S VGID H LALD E I K KR 289 ggg ate cga gtt ggc tac acc cca gat gtc ctg aca gat acc acc gcc 97 G IR VGYT P DV L T DT TA 337 gaa etc gca gtc tcc ctg eta ctt acc acc tgc cgc egg ttg ccg gag 113 E L A VS LL LTTC RRL PE 385 gcc ate gag gaa gtg aag aat ggt ggc tgg acc teg tgg aag ccc etc 129 AIEB VKNGGWT S W K p L 433 tgg ctg tgt ggc tat gga etc acg cag age act gtc ggc ate ate ggg 145 W L C GY G L T Q S TV G II G 481 ctg ggg cgc ata ggc cag gcc att get egg cgt ctg aaa cca ttc ggt 161 LG RI G Q AIAR RL K PF G 529 gtc cag aga ttt ctg tac aca ggg cgc cag ccc agg cct gag gaa gca 177 V Q R F LYTGR Q p R P E E A 577 gca gaa ttc cag gca gag ttt gtg tct acc cct gag ctg get gcc caa 193 A E F Q A B F V S T p E LA A Q 625 tot gat ttc ate gtc gtg gcc tgc tcc tta aca cct gca acc gag gga 209 S D FI V V AC S L T P A T E G 673 etc tgc aac aag gac ttc ttc cag aag atg aag gaa aca get gtg ttc 225 L C NK D F F Q KMK E TA V F 721 ate aac ate age agg ggc gac gtc gta aac cag gac gac ctg tac cag 241 IN I SRGD V V N 0 DDL Y Q 769 gcc ttg gcc agt ggt aag att gca get get gga ctg gat gtg acg age 257 A L AS G KI A A A 6 L 0 V TS 817 cca gaa cca ctg cct aca aac cac cct etc ctg acc ctg aag aac tgt 273 P EPL PT N H p L LT L K NC 865 gtg att ctg ccc cac att ggc agt gcc acc cac aga acc cgc aac acc 289 V I LP H IG S A T H R T R NT 913 atg tcc ttg ttg gca get aac aac ttg ctg get ggc ctg aga ggg gag 305 MS L LA A NN LL A G L RGE 961 ccg atg cct agt gaa etc aag ctg tag ccaaacagtagagatggagggccggga 321 PM P S E L K L stop agcaaaccgtgccctggtattgtcagacacacccaggcttgatttggatccacaggcagagccaaggga aggtgtgattctctgaggaaagagtgattctgatatatgtacttgtcacattggtgttggacacatttg cgccaaaagtatggtaattctattattaaataattctctgagaaaaaaaaaaaaaaaaaa Fig. 2. cDNA and predicted protein sequence of human HPR/GR (GLXR). Putative polyA addition signal sequence (AATAA) is underlined. Sequence deposited in GenBank submission no. API 34895. 386 G. Rumsby, D.P. Cregeen!Biochimica et Biophysica Acta 1446 (1999) 383-388 10 min), washed once with PBSA, pelleted again and Anti-Xpress antibody (Invitrogen) at a final dilution the pellet sonicated on ice in 1 ml 100 mmol/1 potas of 1 in 5000. Excess antisera was removed by wash sium phosphate buffer, pH 8.0 containing 240 mmol/1 ing in PBSA (2 X 10 min) and the blot incubated with sucrose using three, 10-s bursts from a Microson XL alkaline phosphatase conjugated, goat-antimouse sonicator. The cell debris was pelleted by centrifuga IgG (Sigma, Poole) for 3 h at room temperature. tion at 13000 rpm for 10 min at 4®C. HPR and GR After two, 10-min washes in PBSA, the blot was activity was determined in the supernatant as previ immersed m alkaline phosphatase Colour Develop ously described [6]. ment reagent (Bio-Rad, Welwyn Garden City) for Six ng of total ceU protein from sonicate super 25 min. natants were electrophoresed in rehydrated poly The cD N A identified from the IMAGE library is acrylamide gels (Clean gel 36S, Amersham Pharma 1235 bp, with 41 bp o f 5'-untranslated region and an cia Biotech, St Albans, UK). After blotting onto open reading frame of 987 bp (Fig. 2). The gene nitrocellulose, non-specific binding sites were blocked encoded by this cDNA has been assigned the symbol by immersion in 3% (w/v) milk proteins in PBSA, GLXR by the Nomenclature Committee of the Hu followed by incubation of the blot overnight with man Genome Project. The 3'-untranslated region is human HPR/GR — ------MRPVRLMKVFVTRRIPAEGRVALARAADCEVEQWDSDEPIPAKELERGVAG hypho HPR/GR ------MSKKKILITWPLP-E2UMCmRKESYDVIAHGD-DPKITIDEMIETAKS cucurbit HPR/GR MRKPV0IEVWNPNGKYRWSTKPMPGTRWIMLLIEQDCRVEICTEKKTIL3VEDILM.IG methylo HPR/GR ------MTKKWFLDR— E--SLDATVREEÈÏFPHEYKEYESTÎITPEEIVERLQG human HPR/GR --AHGLLCLLSDHVAKRILDAAG-ANLKVISTMSVGIDHLALDEIKKRGIRVGYTPDVLT hypho HPR/GR — VnaiilTLNEKCaiKEVIDRIP-ENlKCISTySIGPDHIDLDACKABGIKVQiAPHGVT cucurbit HPR/GR DKCDGVIGQLTEDWGEVLFSMSRAGGFSVFSNMAVGYNNVDVtlAfiNKÏGVAVGNTPGVLT methylo HPR/GR AEIAMINKVPMRADTLKQLP— DLKLHWaATGTDWDIBtftAKaQGITWNIRNYAF human HPR/GR ETTADLAVSLLLTTCRRIPEAIEEVKNGGWTSWKPLWLCG YGLTQSTVGIIGLGRIG hypho HPR/GR VftTAEiaMLLLLGSARBAGEGEKMIRTRSWPGWQPLQLVG QRLDNKTLGIYGFGKIG cucurbit HPR/GR ETTAELAASLSLAAARRIVEADEFMRAGRYDGWLPNLFVG HLLKGQTVGVIGAGRIG methylo HPR/GR NTVPEHWGLMFALRRAIVPYANS VRRGDWIKSKQFCYFDYP3YD1AGSTLGIIGYGRLG . * I * : : : : . : * * : * human HPR/GR QAIARRLKP- FGVQRFLYTGRQP------RPEEAAEFQAEF-VSTPEIAAQS hypho HPR/GR QALAQRARG-FDMNVHYYDIYRA------EŒ’EVEAKYNATYHDSLDSLLKVS cucurbit HPR/GR SAYARMMVEGFKMNLIYFDLYQSTRLEKFVTAYGEFLIQUïGEAPVTWRRaSSMDEVLREA methylo HPR/GR KS TRKMARA— PQ------D------GLVDXiET IIiTQS human HPR/GR DFIWACSLTP:!»fEGLCNKDFF{^a>fKSTAVFIN-ISRGDWMQ0DLTQALAS6KIAAA^D hypho HPR/GR QFFSINAPSTPETRYFFNKETIEKLPQeAIWNTARGDLVKDDDVIAALKSraLAYAGFD cucurbit HPR/GR DVISLHPVLDKTTFHLVNKESLKMffiKDAILINCSRGPVIDEAALVDHLRDNPMFRVGLD methylo HPR/GR DVITLHVPLTPDTKNMIGAEQLEOMKRSAILINTARGGLVDEAALLQALKDGTIGGAGFD human HPR/GR VTSPEPLP— TNHPLLTLKNCVILFHIGSATHRTRNTMSLLAaNNLLAGLRCTPMPSELK hypho HPR/GR VFAGEPN INEGYYDLPNTFLFPHLGSAAIEARNQMGFEALDNIDAFFAGKDMPFKLA cucurbit HPR/GR VFEDEPY MKPGLADMKNAIIVPHIASASKWTREGMATLAALNVLGKIKGYPVWSDPN methylo HPR/GR WAQEPPKDGNILCDADLPNLIVTPHVAWASKEAMQILADQLVDNVEAFVAGKPQNWEA * * * # * ! * # _ * * * # . . * human HPR/GR 328 hypho HPR/GR 322 cucurbit HPR/GR RVEPFLDENVSPPAASPSIVNAKALGNA 382 methylo HPR/GR 314 Fig. 3. Peptide sequence of human HPR/GR aligned with glycerate dehydrogenase (NADH-dependent HPR/GR) from Hyphomi crobium methylovorum (hypho) (GenBank P36234), Cucumis sativus (cucurbit) (Genbank P13443) andMethylobacterium extorquens (metbylo) (Genbank Q59156). G. Rumsby, D.P. Cregeen IBiochimica et Biophysica Acta 1446 (1999) 383-388 387 g 300 100 - pTicHteB-HPR pTncHtoB pTrcHIsB-HPR pTrcHItB Fig. 4. HPR (shaded boxes) and GR (open boxes) activity in BL21 cells transfected with pTrcHisB-HPR and pTrcHisB, respectively. 225 bp with a putative polyadenylation signal se pression of the cDNA in BL21 cells produced a fu quence (AATAA) 14 nucleotides from the end of sion protein of 43 kDa with the ability to reduce the cDNA. The sequence is predicted to encode a both glyoxylate and hydroxypyruvate in the presence protein of 328 amino acids with a molecular weight of NADPH as cofactor. As the plasmid transcript of 36.5 kDa (http://www.expasy.ch/tools/pi-tool. provides an additional 40 amino acids, the actual html). The protein has sequence similarity with size of the expressed HPR/GR protein is of the order HPR/GR from H. methylovorian (32%), Cucucmis of 40-kDa. The deduced protein sequence shares ap sativus (32%) and Methylobacterium extorquens proximately 30% sequence similarity with HPR and (21%) (Fig. 3). GR from a number of species of plants and bacteria Expression of the cD NA in BL21 bacterial cells including H. methylovorum, C. sativus and M. extor produced a protein with GR and HPR activities of quens (Fig. 3). There was no significant similarity 631 ±22 and 509 ± 1 7 nmol NADPH oxidised/min/ with any of the mammalian lactate dehydrogenase mg protein respectively (mean ± 1 S.D. from six anal genes. Analysis of the peptide sequence using the yses) compared with activities of 124 ±7 nmol/min/ Prosite program [9] revealed a 2-hydroxyacid dehy mg protein and 16 ±8 nmol/min/mg in cells trans drogenase signature starting at codon 232 (MKE- fected with vector alone (Fig. 4). A fusion protein TAVFINISRGDWN). of 43 kDa was detected on Western blot analysis A cDNA of similar size encoding a protein with which was not present in sonicates of cells trans d -GDH activity has been described in abstract form fected with vector alone. As the fusion protein adds [10], but no information was given as to its activity an additional 3 kDa to the protein size, this result with hydroxypyruvate or glyoxylate or its nucleotide indicates that the protein translated from the cDNA or peptide sequence and therefore no further com is approximately 40 kDa, slightly bigger than the parisons are possible. However, in view of the fact predicted 36.5 kDa predicted from the amino acid that GR, HPR and d -GDH activities are all de sequence. The same size band was detected under creased in PH2 [1,4] and therefore that one enzyme denaturing and non-denaturing conditions suggesting appears to have all three activities, it is possible that that the enzyme is present as a monomer and is ac the two cDNA species are the same. Further studies tive in this form. will elucidate the kinetics of this enzyme, physical In the present study, we have identified a full- properties and role in metabolism. The discovery of length cDNA clone from human liver by virtue of this gene also enables the molecular genetics of PH2 its homology to HPR fromH. methylovorum. Ex to be established. 388 G. Rumsby, D.P. Cregeen!Biochimica et Biophysica Acta 1446 (1999) 383-388 This project was funded by a grant from the Ox [5] E. Van Schaftingen, J.-P. Draye, F.V. Van Hoof, Eur. J. alosis and Hyperoxaluria Foundation. Biochem. 186 (1989) 355-359. [Q C.F. Giah, G. Rumsby, Ann. Clin. Biochem. 35 (1998) 104- 109. [7] D. Cregeen, G. Rumsby. J. Am. Soc. Nephrol., in press. References [8] G.G. Lennon, C. AuflFray, M. Polymeropoulos, M B. Soares, Genomics 33 (1996) 151-152. [1] H. Williams, L.J. Smith, New Engl. J. Med. 278 (1968) 233- [9] A. Bairoch, P. Bucher, K. Hofmann, Nucleic Acids Res. 25 239. (1997) 217-221. [2] J.E. Willis, H.J. SaUach, J. Biol. Chem. 237 (1962) 910-915. [10] S.D. Cramer, K. Lin, R.P. Hohnes, J. Urol. 159 (5S) (1998) [3] P. Dawkins, F. Dickens, Biochem. J. 94 (1965) 353. 661. [4] J. Mistry, C.J. Danpure, R.A. Chalmers, Biochem. Soc. Trans. 16 (1988) 626-627. BIOCHIMICA ET BIOPHYSICA ACTA BB3 ELSEVIER Biochimica et Biophysica Acta 1493 (2000) 246-248 www.elsevier.com/Iocate/bba Short sequence-paper Identification and expression of a cDNA for human glycolate oxidase E. Williams, D. Cregeen, G. Rumsby * Department of Molecular Pathology, University College London, Windeyer Institute of Medical Sciences, Cleveland St, London WIP 6DB, UK Received 15 March 2000; received in revised form 7 June 2000; accepted 8 June 2000 Absfiract The isolation and expression of a human liver cDNA encoding a 43 kDa protein with glycolate oxidase activity is described. The cDNA (HAOl) is 1128 bp and has a 1113 bp open reading frame. Northern blot analysis detects a 1.8 kb mRNA with expression restricted to the liver. The 370 amino acid protein has 89% sequence similarity to glycolate oxidase from mouse liver and 53% similarity to the spinach and Arabidopsis enzymes. The protein has glycolate oxidase activity in vitro, as measured by the reduction of 2,6-dichlorophenolindophenol in the presence of flavin mononucleotide and glycolate. The genomic sequence is contained within eight exons and encompasses approximately 57 kb of chromosome 20pl2. This paper offers the first full functional description of a gene encoding human glycolate oxidase. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Glycolate oxidase; Hydroxy acid oxidase A; Primary hyperoxaluria Human liver glycolate oxidase is a flavin mononucleo nation codon indicated that the clone was missing the 3' tide (FMN)-dependent protein which oxidises a number of end. A subsequent BLAST search identified an almost medium and short chain length L-a-hydroxy acids [1] with identical cDNA (AB024079) which included additional 3' a preference for glycolate. Oxidation of glycolate produces sequence. PCR primers (Genosys Biotechnologies Ltd, glyoxylate, which is rapidly utilised by alanine:glyoxylate Pampisford, UK) were designed based upon the 5' end aminotransferase for the peroxisomal synthesis of glycine. of the IMAGE clone and the 3' end of AB024079, incor This pathway appears to be an important step for the porating restriction sites forPstl and Hindlïl at the 5' and detoxification of glyoxylate which, if allowed to accumu 3' ends respectively (forward primer: 5 -AACTGCAGT- late, may be metabolised to oxalate. Glycolate oxidase can GAAAATGCTCCCCC, reverse primer: 5 -TCAAGCTT- also utilise glyoxylate, but the substrate affinities difler TGTGCACTGTCAGAT). between the human and rat enzymes. The rate of oxida Total RNA was extracted from human liver tissue using tion of glyoxylate relative to glycolate is reported to be the RNA Isolator Kit (Sigma Genosys Biotechnologies Ltd, 40% for the rat [2] but only 7% for the human enzyme [1]. Pampisford, UK). A full length cDNA clone was synthe The pivotal role of the enzyme in peroxisomal glyoxylate sised by reverse transcription-polymerase chain reaction production makes it of interest to workers in the hyper (RT-PCR) using the following conditions: 2 pg RNA, 50 oxaluria field, as activity of this enzyme could be expected mmol/1 potassium chloride, 20 mmol/1 Tris-HCl pH 8.4, to have impact on the amount of residual glycolate avail 5 mmol/1 magnesium chloride, 1 mmol/1 dNTP, 20 units able in the cell for oxalate production. RNasin RNase inhibitor (Promega, Southampton, UK) The Entrez database (http://www.ncbi.nlm.nih.gov/ and 50 U M-MLV reverse transcriptase in a final volume htbin-post/Entrez/) was screened using the term ‘glycolate of 20 pi. The reaction was carried out at 42®C for 15 min oxidase’ which identified a cDNA with peptide sequence followed by 99°C for 5 min. 5 pi was added to a reaction mix homology to glycolate oxidase fromArabidopsis thaliana. containing 50 mmol/1 potassium chloride, 10 mmol/1 Tris- The cDNA clone (IMAGE 84824) was obtained from the HCl, pH 9.0,0.1% Triton X-KX), 2 mmol/1 magnesium chlo I.M.A.G.E Consortium [LLNL] (http://bbrp.llnl.gov/bbrp/ ride, 200 pmol/1 dNTP, 0.6 pmol/1 each primer and 0.25 U image) [3] and sequenced in full. The absence of a termi Taq polymerase in a final volume of 25 pL Thirty cycles of 94“C 10 s, 66®C 10 s and12°C for 1.5 min were carried out. The PCR product was ligated into the pCR2.1 vector * Corresponding author. Fax: +44-20-7679-9496; (Invitrogen, The Netherlands), transfected into competent E-mail: [email protected] One Shot cells (Invitrogen, The Netherlands) and se- 0167-4781/00/$ - see front matter ® 2000 Elsevier Science B.V. All rights reserved. PH: 80167-4781(00)00161-5 E. Williams et aL ! Biochimica et Biophysica Acta 1493 (2000) 246-248 247 S ' g t g a a a 1 atg etc ccc cgg cta att tgt atc aat gat tat gaa caa cat get aaa chloride, 2.5 mmol/1 potassium chloride, 10 mmol/1 mag 1 M LPRLTCIlfDTBQHAK nesium chloride pH 7.0) containing ampicillin (50 pg/ml) 49 tea gta ctt cca aag tôt ata tat gmc tat tac agg tct ggg gca aat 17 SVLPKSITDTYSSGAH and 0.2 ml of the overnight cultures was inoculated into 50 ml aliquots of SOB medium containing ampicillin (50 pg/ 97 gat gaa gaa act ttg get gat aat att gca gca ttt tcc aga tgg aag 33 OEETLADKIAAFSRVK ml). The cells were grown with aeration until an absor 145 ctg tat cca agg atg etc cgg aat gtt get gaa aca gat ctg tcg act bance at 600 nm of 0.6 was reached, at which time iso- 49 LYPRHLRNVA8TDL3T propylthio-P-D-galactoside (final concentration 1 mmol/1) 193 tct gtt tta gga cag agg gtc agc atg cca ata tgt gtg ggg get acg 65 SVIGORVSNPICVGAT was added and the culture incubated for a further 3 h. The 241 goc atg cag cgc atg get cat gtg gac ggc gag ctt gcc act gtg aga cells were harvested by centrifugation at 3(XK) rpm for 10 81 AKQRM AEVDGELATVR min and the pellet resuspended in 25 mmol/1 potassium 289 goc tgt cag tcc ctg gga acg ggc atg atg ttg agt tcc tgg gcc acc 97 ACQSLGTGHM LSSW AT phosphate buffer pH 8, containing 1 mmol/1 EDTA and 337 tcc tea att gaa gaa gtg gcg gaa get ggt cet gag gca ctt cgt tgg 0.1 mmol/1 FMN. 113 S3IBBV A BA G PBA LRW The cells were treated with lysozyme at a concentration 385 ctg caa ctg tat atc tac aag gac cga gaa gtc acc aag aag cta gtg 129 IQ LTIY R O R B V TK B liV of 100 pg/ml on ice for 15 min. The cell suspensions were 433 cgg cag gca gag aag atg ggc tac aag gcc ata ttt gtg aca gtg gac sonicated by three 10 s bursts from a Microson XL soni- 145 RO A EK H G Y X A I FVTTD cator, followed by flash freezing in liquid nitrogen and 481 aca cet tac ctg ggc aac cgt ctg gat gat gtg cgt aac aga ttc aaa 161 tpylghrldovrbrfk thawing at 37®C. This cycle was repeated three more times, 529 ctg ccg cca caa etc agg atg aaa aat ttt gaa acc agt act tta tea following which the cell lysates were treated with RNase at 193 I>FPQ LR IiK B FETSTLS a final concentration of 5 pg/ml. The cell debris was pelleted 577 ttt tct cet gag gaa aat ttt gga gac gac agt gga ctt get gca tat 193 FS PB B B FG D D 3G LA A Y by centrifugation at 13 (XX) rpm for 10 min at 4“C, and the 625 gtg get aaa gca ata gac cca tct atc agc tgg gaa gat atc aaa tgg supernatants cleared by passage through a syringe filter. 209 7A K A IO PSISB EO X B B Glycolate oxidase activity in the supernatant was mea 673 ctg aga aga ctg aca tea ttg cca att gtt gca aag ggc att ttg aga sured by reduction of 2,6-dichlorophenolindophenol 225 B R R I.T 3L PIV A X 6IX .R (DCIP) at 600 nm in the presence of FMN with glycolate 721 ggt gat gat goc agg gag get gtt aaa cat ggc ttg aat ggg atc ttg 241 G D D A REA V BRG LN G II. as substrate, as previously described [4]. Crude extracts 769 gtg tcg aat cat ggg get cga caa etc gat ggg gtg cca gcc æt att from cells containing the pTrcHisB-GO construct had ac 257 V 8 H H G A R 0 LDGVPATI tivity of 474 ±20 nmol DCIP reduced/min/mg protein 817 gat gtt ctg cca gaa att gtg gag get gtg gaa ggg aag gtg gaa gtc 273 D V LPBITEA V EG K V BV (mean±2 S.D.) compared to 5±3 in those cells trans 865 ttc ctg gac ggg ggt gtg cgg aaa ggc act gat gtt ctg aaa get ctg fected with vector alone. 289 FLDGGFREGTDVLKAL Total cell protein from sonicate supernatants (6 pg) was 913 get ctt ggc gcc aag get gtg ttt gtg ggg aga cca atc gtt tgg ggc 305 algaxavfvgrpivwg electrophoresed in rehydrated polyacrylamide gels (Clean 961 tta get ttc cag ggg gag aaa ggt gtt caa gat gtc etc gag ata cta gel 36S, Amersham Pharmacia Biotech, St Albans, UK). 321 IAFQGBKGTQOVLEIL After blotting onto nitrocellulose, non-specific binding 1009 aag gaa gaa ttc cgg ttg gcc atg get ctg agt ggg tgc cag aat gtg 337 KEBFRLAHAL3GCQH7 sites were blocked by immersion in 3% (w/v) milk proteins 1057 aaa gtc atc gac aag aca ttg gtg agg aaa aat cet ttg goc gtt tcc in PBSA, followed by incubation of the blot overnight 351 XV I DXT1VRXBPLAV3 with Anti-Xpress antibody (Invitrogen) at a final dilution 1105 aag atc tga cagtgcaca 369 I_ stop of 1 in 5000. Excess antiserum was removed by washing twice in PBSA for 10 min and the blot incubated with Fig. I. cDNA sequence of human glycolate oxidase (GeoBank AF244134). The FMN-dependent a-hydroxyadd dehydrogenase active alkaline phosphatase conjugated, goat anti-mouse IgO site (residues 258-264) and peroxismnal targeting signal (residues 368- (Sigma Chemical Co., Poole, UK) for 3 h at room temper 370) are underlined. ature. After two 10 min washes in PBSA, the blot was immersed in alkaline phosphatase Colour Development quenœd (Fig. 1). Comparison with peptide sequences from reagent (Bio-Rad, Welwyn (jarden City, UK) for 25 mouse liver, spinach and Arabidopsis glycolate oxidases is min. A single band of 46 kDa was detected, of which shown in Fig. 2. approximately 3 kDa is contributed by the plasmid vector. Following excision of the plasmid insert withPstl and This result approximates the predicted size of 40.9 kDa HindlTL, it was cloned into the pTrcHisB expression vector (PROSITE, http://www.expasy.ch). (Invitrogen, The Netherlands) which had been cut with the Northern blots containing mRNA from a variety of same restriction enzymes and the cut ends dephosphory- tissues (Clontech MTN, Clontech, Palo Alto, CA, USA) lated. The cDNA was inserted into the multiple cloning were hybridised with the ^^P-labelled insert from IMAGE site, in frame with the plasmid initiation codon and down clone 84824. These blots showed a single species of ap stream of the His tag and Anti-Xpress antibody epitope. proximately 1.8 kb with expression restricted to the liver. The pTrcHisB-GO construct was transfected into Epicur The cDNA has been mapped to chromosome 20pl2 ean coli BL21 (DE3) competent cells (Stratagene Ltd, (www.ncbi.nlm.nih.gov/UniGene). Comparison of the full Cambridge, UK). Individual colonies of pTrcHisB and length cDNA to the genomic sequence deposited in Gen- pTrcHisB-GO were cultured overnight in 2 ml SOB me Bank (accession number AL021879) revealed eight exons, dium (20 g/1 tryptone, 5 g/1 yeast extract, 0.5 g/l sodium ranging in size from 70 to 256 bp (Fig. 3). 248 E. Williams et aL l Biochimica et Biophysica Acta 1493 (2000) 246-248 H u m a n MLPKLlCINOYSt^lAKSVLPKSIYCYTBSGANDEErrLADNIAAFSRIIKI.'yFBlfLRNVAET M ouse HLFRLVCISDYEQBVRSVLQKSVYDYYItSGANDQErrLADin;gAFSBlIKZ.YFBIILBNVAOI S p in a c h — HEITNVREYE3U;AKQKLPKMVYI>rXASGAE3X2in:LAElIRMAFSKILFKFRILIDVTNX Arabidopsis Human DX.STSVLGQRVSNPICVGATAM0BNAH9DGELATVRACQSI.GTGNMLSSHATSSIEEVAE M ouse DLSTSVLGt^VSMPICVGATAMQCMAHVDGELATVIlACQTHGTGMMLSSIlATSSIEEVAE S p in a c h DMTTTILGFKZSMPXMIAPTAMQKMAHPEGEYATABAASAAGTlHn.SSmVTSSVEEVAS Arabidopsis ------—-— ------———ATSSVBKIAS ****;*:;*. Human AGPEAUafLQLYZYXOREVTKKLVSQASXMGTKAZFVTVDTPYLrantLDDVKNKlf'RLPPQ M ouse AGPEALRHHQLYIYKDREZSRQIVKBAEKQGYKAIFVTVDTFYLranUDDVRinirKLPPQ S p in a c h TGFG-IHFFQLYVYKDBHWAOLVRBAEItAGFKAIAIfTVDTPHLGRREAOIKilRFVLPPF A r a b i d o p s is TGPG-IRFFQLYV¥KHBKWEQLVBKAEKAGFKA1AI>TVNTPBLGPKKSDIKHR£TLPPH .** . *••*** ♦** Human LPMKHFETSTI.SFSPEEIIF6D0SGLAAYVAKAZ0PSISflEDIK>UtRLTSl.PIVAKGILR Mouse LRMKNFETNDLAFSPKQIFGOIISGIAEYVAQAIDPSLSHDDITIILRRLTSLPIWKGILR S p in a c h LTLKNFEGIDLGKMDKAN OSGLSSYVAGQZDRSLSMKDVAJILQTITSLPZLVKGVIT A r a b id o p s is LTLKHFEGLDLGKHDEAH DSGLASYVAGQIDRTLSWKDIQRLQTITIIMPXLVKGVLT * •«*** * ; * .***; *** ** » «** * « **; î*,î**!.**î! Human GDDABEAVKHGLM6ILVSMHGABQLDGVPATIDVU>EIVEAVEGKVEVFL0G6VRKGTI)V M ouse GDDAKEAVKHGVDGILVSMHGARQLDGVPAYIDVLFEZVBAVBGKVEVFLDGGVRKGTDV S p in a c h AEDARLAVQHGAAGIIVSNHGARQLDyVPATIMALEEWKAAQGRIPVFLDGGVRRGTDV A r a b i d o p s is GEQARIAIQAGAAGIIVSMHGARQWTVPATISALEEWEATQGGVPVFLDGGVRBGTDV « * **.***♦****** ***** _ * *«*.* «* j ************* Human LKALALGAKAVFV(æPIVHGLAFQGEKGVQDVL2CILKEBnaAMAI.SGCQHVRVII)KTI,V M ouse UKALALGAKAVFVGRPIX«SLAFC26£KGVQ17VLEXLKEErHLAHALSGCQNVKVIOKTLV S p in a c h FKALALGAAGVFZGRPWFSIiAA£(%A6VKXVLGKMRDEFELTHALSGCRSLKEZSItSHI A r a b id o p s is FKALALGTSGXFZGRPWFALAAE(a»GVKKVLQMLROEFELTHALSGCBSISElTIU]HZ .******: •*•***•-• ** -** ♦*• ** «•••** * ******** • * • Human ware LAVSKi M ouse BKNP — LAVSKI S p in a c h AADMDGPSSRAVARL Arabidopsis VTEMDIP— RHLPRL Fig. 2. Peptide sequence erf’ human glycolate oxidase aligned with those from mouse (GenBank AAD25332) spinach (GenBank AAA34030) and Arabi dopsis (GenBank AAB80700). This is the first paper describing the expression of hu described by Jones et al., J. Biol. Chem. 275 (2000) 12590- man glycolate oxidase from cloned DNA, although the 12597. enzyme was first purified from human liver in 1979 [1,5]. The cDNA is very similar to that described for the mouse and both show the same liver specific expression [6]. The References identification and expression of human glycolate oxidase will permit further studies of its role in the metabolism of [1] D.W. Fry, K.E. Richardson, Biochim. Biophys. Acta 568 (1979) 135- oxalate precursors and the potential for deficiency of this 144. [2] Y, Ushijima, Arch, Biochem. Biophys. 155 (2) (1973) 361-367. enzyme to be an underlying cause of hyperoxaluria. [3] G.G. Lennon, C. Auffiray, M. Polymeropoulos, M.B. Scares, Ge This project was funded by a grant from the Oxalosis nomics 33 (1996) 151-152. and Hyperoxaluria Foundation. [4] L.L. Liao, K.E. Richardson, Arch. Biochem. Biophys. 154 (1973) 68- 75. [5] H. Schwam, S. Mitchelson, W.C. Randall, J.M. Sondey, R. Hirsch mann, Biochemistry 18 (1979) 2828-2833. Note added in proof [6] SA. Kohler, E. Menotti, L.C. Kuhn, J. Biol. Chem. 274 (1999) 2401- 2407. This sequence is identical to that of HAOXl recently Skb 1 1 Exon 67 8 f - f Size (bp) 137 152 256 176 92 159 70 71 Fig. 3. Genomic organisation of the human glycolate oxidase gene. Boxes represent exons, lines introns. Nephrol Dial Transplant (2001) 16: 1697-1699 Nephrology Dialysis Case Report Transplantation Primary hyperoxaluria type 2 without L-glycericaciduria: is the disease under-diagnosed? Gill Rumsby', Abhishek Shanna^ David P. Cregeen' and Laurie R. Solomon^ 'Department of Chemical Pathology, UCL Hospitals, London and "Department of Renal Medicine, Royal Preston Hospital, Sharoe Green Lane North, Preston, UK Keywords: primary hyperoxaluria type 2; L-glyceri- glyoxylate reductase protein but who did not have caciduria; glyoxylate reductase; hydroxypyruvate excess L-glycerate in the urine, reductase; oxalate Case Introduction The patient had formed renal stones since the age of four when an IVP showed renal calculi and he was The primary hyperoxalurias (PH) are inherited dis admitted for their removal from the ureter. After this, orders of endogenous oxalate overproduction. Type 1 he remained asymptomatic until aged 39 when in June (PHI) is caused by deficiency of hepatic peroxisomal 1988 he had a road traffic accident which caused alanine: glyoxylate aminotransferase (AGT) [1] and spastic quadriparesis. In Jtme 1990 he presented to his type 2 (PH2) by glyoxylate reductase (GR) deficiency general practitioner with frank haematuria. Investig [2]. The differential diagnosis formerly relied on ations showed plasma creatinine 280 pmol/1 and a the finding of hyperglycolic aciduria in PHI and midstream urine grew Escherichia coli, which was L-glycericaciduria in PH2, but it is now known that treated with antibiotics. Ultrasound scan and IVP were approximately one third of PHI patients have urine normal but he passed a small stone 3 months later and glycolate within the reference range [3] and that raised analysis showed calcium oxalate. In January 1992 a urinary glycolate may occur in patients without 24 h urine contained 1.08 mmol oxalate. Between 1992 PHI [4]. There is less information about PH2 and it and 1995 he had several more episodes of renal colic has been believed that the absence of L-glycericaciduria and suspected urinary infection, four IVPs showed excluded the diagnosis of PH2. stones in both kidneys and he passed at least five more In PH2 glyoxylate and hydroxypyruvate are stones. metabolized by lactate dehydrogenase to oxalate and He was referred to the renal out-patient depart L-glycerate respectively [5] (Figure 1). This observation ment in July 1996 at the age of 47 for investigation is consistent with deficiency of a single protein having of hypertension, renal impairment and proteinuria. both glyoxylate and hydroxypyruvate reductase Investigations included serum creatinine 273 pmol/1, (HPR) activity and this has now been demonstrated urea 15.3 mmol/1, calcium 2.44 mmol/1, creatinine by identification and expression of the gene [6,7]. The clearance 20 ml/min, urinary protein 0.49 g/24 h, urine major role of this enzyme may be the prevention of calcium 0.8 mmol/24 h, urinary oxalate 1.58 and cytosolic glyoxylate accumulation. Loss of the enzyme 1.48 mmol/24 h (0.08-0.49) in separate samples. therefore increases the amount of glyoxylate available Urinary organic acid analysis failed to demonstrate for metabolism by lactate dehydrogenase to oxalate. L-glycerate (measured by gas chromatography) and We describe a patient with hyperoxaluria, renal provisionally excluded PH2. Glycolate excretion was stones and renal failure who had PH2 diagnosed 0.08 mmol/24 h (reference range 0.1-0.33). A renal by measurement of GR and HPR activity in a liver biopsy in December 1996 showed 18 of 28 glomeruli biopsy and confirmed by lack of immuno-reactive were totally sclerosed but the remainder looked normal. There was focal tubular atrophy and mild chronic inflammation and occasional crystals in Correspondence and offprint requests to: G. Rumsby, Department of some of the tubules. He was commenced on pyridoxine Chemical Pathology, UCL Hospitals, Windeyer Building, Cleveland 50 mg once daily and calcium carbonate 1.25 g twice street, London WIP 6DB, UK. daily. 2001 European Renal Association-European Dialysis and Transplant Association 1698 G. Rumsby et al. glycine hydroxypyruvate giyoxytate ( GO glycolate peroxisome cytosol hydroxypyruvate L-glycerate glycolate LDH I NAD(P)H NAD(P) HPR NAD(P) NAD(P)H D-glycerate oxalate ^ ^ ■ glyoxylate Fig. 1. Glyoxylate metabolism in the hepatocyte. Solid bar denotes site of block in PH2 and metabolites typically associated with the disease are given in parentheses. AGT, alanine; glyoxylate aminotransferase; GR, glyoxylate reductase; HPR, hydroxypyruvate reductase; GO, glycolate oxidase; LDH, lactate dehydrogenase. He was lost to follow-up in the renal clinic until October 1999 when he presented with loin pain and advanced renal failure (urea 23 mmol/1, creatinine ^ 66kDa 780 pmol/1) three months after having extracorporeal shock wave lithotripsy for a left-sided staghom cal culus. An ultrasound scan excluded obstruction. 46K08 Suspicion remained that he had underlying metabolic cause for hyperoxaluria and he therefore had a liver biopsy in December 1999. Urine glycerate was reported 30kDa detectable but not raised. He started haemodialysis in February 2000. ■21.5kDa Liver enzyme analysis The liver biopsy was analysed for AGT, GR and HPR 4 3 2 1 activities as previously described [8,9]. AGT activity Fig. 2. Western blot analysis of human liver sonicates hybridized was within normal limits but HPR and GR activities with antibodies to AGT and GR. Lanes 1 and 2, control; lanes 3 and were both reduced at 133 nmol.min~'.mg protein"' 4, patient. (reference range 322-1002) and 21 nmol.min"'.mg protein"*.-1 (reference range 49-213) respectively. 0.73 mmol/24 h (reference range 0.1-0.46 mmol/24 h). Absence of GR-specific protein in the hver was Urine glycerate was reported as present but not demonstrated on Western blots using antibody raised elevated. Plasma urea and creatinine were 4.7 mmol/1 to expressed human GR (Figure 2). Immunoreactive and 84 pmol/1 respectively. Liver biopsy was not AGT was used as a control for sample application. undertaken. Family study Discussion One of the patient’s two brothers reported stones, which had been removed surgically when he was a This case demonstrates that PH2 may occur without baby. He had further episodes of renal colic at the ages L-glycericaciduria and reliance on this finding may of 16 and 31, when an abdominal X-ray suggested a miss the diagnosis, which requires liver biopsy. The stone at the lower end of the left ureter and he passed absence of r-glyceric aciduria may have resulted from debris in the urine. He was reviewed at age 48, when partial enzyme impairment. GR activity was not zero he was found to have a raised urinary oxalate of as in most of the liver biopsies analysed from patients Primary hyperoxaluria type 2 without L-glycericaciduria 1699 with this disease [9] [Rumsby, unpublished data] References although there was significant reduction of immuno reactive GR protein compared to control liver biopsies. 1. Danpure CJ, Jennings PR, Watts RW. Enzymological diagnosis of primary hyperoxaluria type 1 by measurement of hepatic The residual enzyme activity may be sufficient to alanine: glyoxylate aminotransferase activity. Lancet 1987; metabolize hydroxypyruvate normally while inade 1: 289-291 quate for the reduction of glyoxylate, the enzyme 2. Mistry J, Danpure CJ, Chalmers RA. Hepatic o-glycerate having a higher affinity for hydroxypyruvate than for dehydrogenase and glyoxylate reductase deficiency in primary glyoxylate [10]. hyperoxaluria type 2. Biochem Soc Trans 1988; 16: 626-627 3. Danpure CJ. Molecular and clinical heterogeneity in primary Both brothers gave a history of stones in infancy and hyperoxaluria type 1. Am J Kidney Dis 1991; 17: 366-369 probably have the same metabolic defect. The propo 4. Van Acker KJ, Eyskens FJ, Espeel MF, Wanders RJA, situs developed frequent stones and renal failure after Dekker C, Kerckaert lO, Roels F. Hyperoxaluria with hyper- a road accident caused quadriparesis. Immobilization glycoluria not due to alanine: glyoxylate aminotransferase defect: A novel type of primary hyperoxaluria. Kidney Int hypercalciuria may have been a second contributory 1996; 50: 1747-1752 factor. It is possible that his brother has a more 5. Williams H, Smith LJ. L-glyceric aciduria. A new genetic variant typical phenotype and that similar partial enzyme of primary hyperoxaluria. N Engl J Med 1968; 278: 233-239 defects will be found in other patients with moderate 6. Rumsby G, Cregeen D. Identification and expression of a cDNA hyperoxaluria and occasional stone formation. for human hydroxypyruvate/glyoxylate reductase. Biochim Biophys Acta 1999; 1446: 383-388 It is already known that PH2 may be underdia 7. Cramer SD, Ferrce PM, U n K, Milliner DS, Holmes RP. The gnosed either by misclassification as PHI or through gene encoding hydroxypyruvate reductase (GRHPR) is mutated the lack of available urinary L-glycerate assays. For in patients with primary hyperoxaluria type II. Hum Mol Genet example Milliner and colleagues [11] found that more 1999; 8: 2063-2069 examination 8. Rumsby G, Weir T, Samuell CT. A semiautomated thorough of 30 patients with PHI alanine:glyoxylate aminotransferase assay for the tissue diag identified five who had PH2. It is also particularly nosis of primary hyperoxaluria type 1. Ann Clin Biochem 1997; difficult to diagnose metabolic hyperoxaluria in cases 34: 400-404 presenting in end-stage renal failure and a true 9. Giafi CF, Rumsby G. Kinetic analysis and tissue distribution of diagnosis may be missed [12]. Our patient illustrates human D-glycerate dehydrogenase/glyoxylate reductase and its relevance to the diagnosis of primary hyperoxaluria type 2. that PH2 may also be excluded despite the standard Ann Clin Biochem 1998; 35: 104-109 investigation because some patients do not have sig10. Cregeen DP, Rumsby G. Recent developments in our under nificant L-glycericaciduria. Liver biopsy, although standing of primary hyperoxaluria type 2. J Am Soc Nephrol invasive, is currently the most reliable method of 1999; 10: S348-S350 11. Chlebeck FT, Milliner DS, Smith LH. Long-term prognosis in confirming the diagnosis of both types of PH [8,13] primary hyperoxaluria type II (L-glyceric aciduria). Am J Kidney and should be undertaken more often following Dis 1994; 23: 255-259 thorough biochemical investigation. 12. Marangella M, Petrarulo M, Cosseddu D, Vitale C, Cadario A, Portigliatti Barbos M, Gurioli L, Linari F. Detection of pri mary hyperoxaluria type 2 (L-glyceric aciduria) in patients with end stage renal failure. Nephrol Dial Transplant 1995; Acknowledgements. The developmental aspects of this work were 10: 1381-1385 funded by a grant to GR from the Sir Jules Thom Charitable 13. Giafi CF, Rumsby G. Primary Hyperoxaluria type 2: cnzymol- Trust. ogy. J Nephrol 1998; 11 [Suppl 1]: 29-31 Received for publication: 18.1.01 Accepted in revived form: 16.3.01