ASSOCIATION OF A NONSENSE MUTATION AT THE CODON FOR GLU 54 IN THE GM2A WITH AB VARIANT CiMZGANGLIOSIDOSIS: CHARACTERIZING THE INTRONf EXON JUNCTIONS OF THE GENE

by Biao Chen

Thesis submitted in conformity with the requirements For the degree of Master of Science Department of Laboratory Medicine & Pathobiology University of Toronto National Library Biblïatheque natiar\ale l*l OfCamda du Canada Acquisitions and Acquisitions et Bibliographie Senrices services bibliographiques 395 Weüington Sireet 395. rue Wellington Ottawa ON K1A ON4 -ON K1AW canada canade

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THESIS ABSTRACT...... ~...... v ACKNOWLEDGMENT...... vi .. LIST OF FIGURES ...... vit LIST OF TABLES ...... ix AE3BREVIATIONS...... -x PUBLICATIONS AND PRESENTATIONS ...... xi

CHAPTER 1 GENERAL INTRODUCTION

1 -1 HISTORICAL INTRODUCTION ...... -2 1.2 ...... 5 1 .2.1 Structure and nomenclature ...... 5 1.2.2 Synthesis and degradation ...... 5 1.2.3 Functions ...... 6 .. 1.2.4 Gm gangliosrde ...... 6 1.3 AND THE ACTIVATOR ...... 8 1.3.1 Stmcture and properties ...... -3 1-3 -2 Biosynthesis, processing and transport ...... 9 1.4 THE INTERACTION BETWEEN THE ACTNATOR, Gm AND A ...... -14 1.4.1 The substrates of hexosaminidase A ...... 14 1.4.2 Role of the Activator in Gw hydrolysis by hexosaminidase A ...... 15 1.4.3 Binding of the Activator with gangliosides ...... -17 1.4.4 The binding domains in the Activator ...... 18 1 -5 STRUCTURE OF HEXOSAMINfDASE AND THE ACTIVATOR ...... 18 1 S.1 Hexosaminidase genes ...... -18 1S.2 The GM2Activator gene ...... 19 1.6 OTHER LYSOSOMAL ACTIVATOR PROTELNS...... -21 1.7 GM2 GANGLIOSIDOSES AND MUTATIONS IN THER RELATED GENES ...... 22 1.7.1 Classification of Gm gangliosidoses ...... 22 1.7.2 Clinical phenotypes...... 22 1.7 -3 Mutations associated with Tay-Sachs disease and Sandhoff disease ...... 25

1.7.4 Mutations associated with the AB variant form of Cim ...... ,...... 25 1.8 MOUSE Gm ACTIVATOR AND MOUSE MODELS OF Gm GANGLIOSIDOSES

...... ,...... 29 .. 1.8.1 The Gm activator protein in mice ...... 29 1.8.2 Mouse mode1 of Gm gangliosidoses ...... -29 1 -9THESIS OBJECTIVES...... 30 1.10 REFERENCES ...... -31

CHAPTER II CHARACTERIZATION OF THE EXON/ INTRON JUNCTIONS OF THE GM2A GENE

2.1 INTRODUCTION...... -42 2.2 MATERIALS AND METHODS ...... 45 2.2.1 Isolation of genomic DNA ...... 45 2.2.2 Long PCR to ampli@ intron 1 and intron 2 of the GM2A gene ...... 45 2.2.3 Restriction analysis of intron 1 and intron 2 ...... -48 2.2.4 Subcloning of both ends of introns 1 and 2 into the pBluescnpt vector ...... 48 2.2.5 Nucleotide sequencing fkom both ends of intron 1 and 2 ...... 48 2.2.6 Use of PCR to ampli@ ail of the exons and exon/ intron junctions of the GM2A gene ...... -49 2.2.7 Direct sequencing...... 5 1 2.3 RESULTS ...... 51 2.4 DISCUSSION ...... 55 2.5 ACKNOWLEDGMENT ...... 62 2.6 REFERENCES ...... -63 CHAPTER III ASSOCIATION OF A NONSENSE MUTATION AT THE CODON FOR GLU54 IN THE GMZA GENE WITH ACUTE AB VARIANT Cm GANCZIOSlDOSIS

3.1 INTRODUCTION...... 67

3.2 MATERIALS AND METHODS ...... , ...... 68 3.2.1 Patient information...... 68 3.2.2 Ce11 lines and leukocyte sample ...... -69 3.2.3 Western blot analysis...... 69 3.2.4 Poly A' RNA isolation and Northem blot analysis...... -70 3.2.5 Total RNA isolation and RT-PCR ...... 71 3.2.6 Cloning and sequencing of the normal and patient Activator cDNA ...... 72 3.2.7 PCR amplification of genomic DNA fragments containing nucleotide 160 ...... 72 3.2.8 Direct sequencing, ...... 73 3.2.9 Determination of the length of intron 1 & 2 of the GM2A gene fiom the patient's genomic DNA by long PCR ...... 73 3.2.10 Detection of the Activator rnRNA lacking exon 2 in normal genomic DNA ...... 73 3.3 RESULTS ...... 74 3.4 DISCUSSION...... 87 3.5 ACKNO WLEDGEMENT ...... 93 3.6 REFERENCES...... -94

CHAPTER IV FUTURE WORK

4.1 Expression of the exon 2-lacking activator in E coli ...... 98 4.2 Testing the Iipid binding bction of the exon 2-lacking activator ...... 99 4.3 EstabIishing a pennanently exon 2-lacking cDNA transfected CHO cell line ...... -99 4.4 Determination of the levels of exon 2-lacking mRNA in variant tissue ...... 100 4.5 REFERENCES ...... 101 ASSOCIATION OF A NONSENSE MUTATION AT CODON FOR GLU 54 IN THE GM2A GENE WITH AB VARIANT C;M2 GANGLIOSIDOSIS: CHARACTERIZING THE INTRON/ EXON SUNCTIONS OF THE GENE by

Biao Chen

Department of Laboratory Medicine & Pathobiology University of Toronto 1999

THESIS ABSTRACT

The AB variant form of Gm gangliosidosis is an inherited lysosorna1 storage disease caused by mutations in the GM2A gene. In this study, the introd exon junction of GMZA gene was characterized, and a PCR procedure was developed to quickly analyze the GM2A gene for mutations. Meanwhile, a new AB variznt patient was detected and characterized. The patient was found to be deficient in both Gm activator protein and niRNA. RT-PCR and sequencing detected some normal size cDNA (containing a single nonsense mutation in exon 2 (Glu54STOP)) along with a lower-level of a smaller cDNA species (an infiame deletion of exon 2 (AE2)). Further experiments excluded the possibility that AE2 specie was a product of abnonnal splicing from a second mutant allele. Finally, through restriction digestion and nested amplification of RT-PCR product, the normal sample was also found to contain AE2 species mRNA. Therefore, a

Glu54STOP nonsense mutation and a naturally occwring transcript oPAE2 were identified. 1 would like to extend my sincere gratitude to my supervisor, Dr. Don Mahuran, for his constant support and guidance. 1 wish to thank Dr. John Callahan for his critical comments on the manuscripts of rny thesis. 1 also like to thank the other members of my advisory cornmittee, Dr.

Paul Thorner and Dr. Janet Forstner, for their participation in evaluating my work.

1 highly appreciate al1 colleagues in Dr. Mahuran and Dr. Callahan's laboratories for their friendship and assistance. 1 would like to thank Sunqu Zhang, Brigitte Rigat, Roderick Tse,

Yongmin Hou, Scott Bukovac, Huinan Deng and Natasha Smilljanic-Georgijev for their helpful advice and discussion. 1 specially thank Amy Leung for her great technical assistance and Mana

Chow for her kindly help. 1 would also like to acknowledge Rick Bugshow, Irené Warren and

Marie-Anne Skomorowski for making an enjoyable place to work.

1 would like to appreciate Dr. Raymond Tellier for his critical advisors in long PCR technique. 1 would like to thank Dr. Chi-Chung Hui for his continued encouragement and support and Dr. Qi Ding for her valuable experimental suggestion. 1 also like to thank Dr. Joe Clarke for offering clinical data and Dr. Paula Strasberg for her technical advisors.

Most of al1 1 would like to thank my wife, Meili, for her understanding, encouragement and love, and my son, Richard, for making my life joyous 2nd worthwhile. Chapter 1

Fig. 1-1. Stnicture of G, ganglioside. Fig. 1-2. Lysosomal degradation pathway for gangiioside GM,. Fig. 1-3. Disulfide bonds in the Activator protein Fig. 1-4. Mode1 for lysosomal protein targeting to Fig- 1-5. A mode1 of the two known functions of the Activator Fig. 1-6. The known GM2A gene structure in 1996 Fig. 1-7. The variants of G, gangliosidosis Fig. 1-8. Mutations and polymorphisms in the GM2A gene

Chapter 2

Fig. 2- 1. Determination of the length of intron 1 and intron 2 of the GM2A gene Fig. 2-2. Digestion of intron 1 and intron 2 with BarnHI and Ssd Fig. 2-3. PCR amplification of al1 4 exons and digestion with unique restriction endonucleases Fig. 2-4. Restriction map of the GM2A gene Fig. 2-5. Nucleotide sequences of GM2A exons and their flanking intronic sequences

Chapter 3

Fig. 3- 1. Western blot analysis Fig. 3-2. Northem blot analysis Fig. 3-3. Reverse transcription and PCR analysis Fig. 3-4. Nucleotide sequence of cDNA fiom hvo normal individuals Fig. 3-5. Nucleotide sequence of the larger cDNA fiom the patient Fig. 3-6. Nucleotide sequence of the smaller cDNA fiom the patient Fig. 3-7. (A) Digestion of exon 2 flanking region with MdV (B) The digestion diagram of exon 2 flanking region with WV

vii Fig. 3-8, Nucleotide sequence of a PCR amplifïed region of the GM2A gene containhg nucleotide 175 fiom three normal individuals Fig. 3-9. Direct sequencing of PCR products fkom the patient's genomic DNA Fig 3- i 0. Digestion of intron 1 of the GM2A gene obtained by PCR with EcoM and BamHI Fig. 3- 1 1. (A). Digestion of wild type cDNA and the exon 2-lacking cDNA (AE2) by Hinf l. (B). Digestion diagram for fiil1 length and AE2 cDNA by Hinf 1 Fig. 3- 12. Nested PCR amplification of RT-PCR product fiom the patient and a normal individual

Chapter 4

No figures

viii LIST OF TABLES

Chapter 1

Table 1 - 1 Naturally occurring mutations in the GWAgene Table 1-2 GM2A gene polyrnorphisms

Chapter 2

Table 2-1 Pnmers used to ampli@ intron 1 and uitron 2 of GMZA gene Table 2-2 Primers used to ampli@ the exons and the exon/ intron junctions of GM2A gene

Chapter 3

No tables

Chapter 4

No tables 4-MUG 4-methylumbelliferyl PN-acetylglucosamuie 4-MUGS 4-methylumbelliferyl p-N-acetylglucosamine-6-sulfate Activator the human G, activator protein bp cDNA complementary DNA Cer CHO ceil Chinese Hamster Ovary ce11 cpm counts per minute CRM cross reacting material Da Dalton DTT dithiothreitol ER endoplasmic reticulum FCS fetal calf serurn Ga1 galactose GalNAc N-acetylgalactosamine Glc glucose GlcNAC N-acetylglucosamine Hex B-N-acetylhexosaminidase(EC 3-2.1.52) IVS intervening sequence kb kilobase pairs M6P mannose-6-phosphate MEM minimum essential media NeuNAc N-acetylneuraminic acid (sialic acid) nt nucleo tide(s) PAGE poIyacrylamide gel electrophoresis PCR polymerase chain reaction SAP sphingolipid activator protein SDS sodium dodecyl suIfate WT wild type AE2 a deletion of exon 2 Erom Activator mRNA Publications & Presentations

B. Chen, B. Rigat, C. Curry and D. J. Mahuran. Structure of the GMZA gene: Identification of an exon 2 nonsense mutation and a naturally accu-g transcript with an inframe deletion of exon 2 (1999). Arnerican Journal of Human Genetics, Volume 65: 77-87.

B. Chen, B. Rigat, J. T. R. Clarke, and D. J. Mahuran, G175A transition (Val59Ile substitution) is a novel polymorphism in human GWAgene. Presented in the Garrod Association of Canada Conference, April 1997, Toronto CHAPTER 1

GENERAL INTRODUCTION 1.1 HISTORICAL INTRODUCTION

The GMzactivator protein (the Activator) was identified and characterized as a result of the discovery of its participation in the hydrolysis of GMt ganglioside (GaiNAccS(1-4)-(NeuNAca(2-

3)) -Galp( 1-4)-Glc-ceramide) (Fig 1- 1) by ~hexosaminidaseA (Hex A) (Conzelmann and Sandhoff

1979). The interest in Hex A derived fiom the discovery that deficiency in its activity is associated with the Tay-Sachs disease (Okada and O'Brien 1969). This disease was first described late in the last century (Sachs 1887; Tay 1881) as a common hereditary disease in Jews (Sachs 1896)- Tay-

Sachs disease is the most common of three disorders caused by the intralysosomal storage of GM2 ganglicside (Kienk 1935), which are known as Gm gangliosidosis (Suzuki and Chen 1967). Gm ganglioside contains a terminal, nonreducing fblinked GalNAc residue, which can be cleaved by hexosaminidase (Hex) (Makita and Yamakawa 1963; Svennerholm 1962). Hex can be separated into two major isoenzymes, Hex A and Hex B (Robinson and Stirling 1968). The patients with

Tay-Sachs disease were found to lack the A but not the B isozyme (Okada and O'Brien 1969). It was found that whereas Hex A is composed of an acidic a subunit and a basic subunit, Hex B is composed of two P subunits (Snvastava and Beutler 1973). Thus classic Tay-Sachs disease results from defects in the unique a subunit which preclude the formation of only Hex A. Other studies demonstrated that some non-Jewish patients presumed to have Tay-Sachs disease were missing both

Hex A and Hex B or, even more paradoxically, had normal levels of both isoenzymes (Sandhoff

1969). The lack of both Hex A and Hex B was referred as O variant of GM~gangliosidosis or

Sandhoff disease. It results fiom a deficiency of the $ subunit, precluding the formation of either isoenzyrne. The existence of normal Hex A and Hex B levels in patients with Gm Gangliosidosis, referred to as the AB variant form, suggested other r'actor(s), in addition to Hex A, participated in the hydrolysis of ClhlZ ganglioside. Site of Hex A cleavage i oligosaccharide ceramide

O - C CCH r?c A, I II wcn GalNAc Gal Glc Cm11 - CWOH t cm CHOH 1 IL2 O. cn2 - If CH2 stearic acid -I L - 1 II CH2 I; NeuNAc

d2 1 CH2

Fig. 1-1. Structure of Gw ganglioside. The cleavage site of Gm hy Hex A is indicated. Gal, galactose; Glc, glucose; NeuNAc, N-acetylneuraminic acid (sialic acid); GalNAc, N- acetylgalactosamine. Through the study on the patients with AB variant Gm gangliosidosis, Conzelmann and

Sandhoff suggested that a "stimulating factor", which can stimulate Hex A to hydrolyze Gm, was deficient (Conzelmann and Sandhoff 1978). Li and colleagues also found that "a heat stable factor" obtained fiom a crude Hex fraction fiom human Iiver could stimulate the hydrolysis of Gm by Hex

A, but not Hex B (Li et al. 1973). This "heat stable factor" was identified as a protein, the

Activator, without any inherent activity towards ganglioside (Hechtman 1977). The

Activator was purified and further characterized as a small (Mr=22,000), acidic (p14.8) monomer

(Conzelmann and Sandhoff 1979). Further study indicated that the Activator did not activate Hex

A, but functioned as a transport protein by solubilizïng a single molecule of ganglioside Gm fkom the lysosomal membrane and presenting it to Hex A for hydrolysis (Conzelmann et al. 1982).

In the 1990s, the Activator was Mercharacterized at the molecular level. The Activator is encoded by GM2A gene on 5 Weng et al. 1993). Its deduced sequence includes 193 amino acids with the N-terminal23 amino acids as a signal peptide, the following 8 amino acids as a propeptide and the remaining 162 amino acids as the mature fom that contains one site for N- linked gl ycosy iation. The complete localization of the Activator's four disulfide bonds has been reported (Schütte et al. 1998) and a mouse mode1 for the AB variant fonn of Gm gangliosidosis has been established (Liu et al. 1997). Four mutations in the GM2A gene have been reported to be responsible for deficiencies of the Activator protein, causing the AB variant form of GM2 gangliosidosis (Schepers et al. 1996; Schroder et al. 1991; Xie et al. L992b). Recently, other functions of the Activator have been identified. In addition to its fùnction as a cofactor for the hydrolysis of GM2by Hex A, the Activator has been shown to bind, solubilize and transport a broad spectrum of Iipid molecules (reviewed in (Mahuran 1998)). Furthemore. the Activator's intracellular transport is only partially dependent on the Mannose-6-phosphate receptor (MPR) .

Besides the lysosomal form, secretory forms of the Activator are also present, and once secreted they can be re-captured fkom the extracellular fluid through a second carbohydrate-independent rnechanism (Rigat et al. 1997).

1.2 GANGLIOSIDES

1 -2.1 Structure and nomenclature

Gangliosides are a group of consisting of a hydrophobic ceramide and a hydrophilic oligosaccharide chain. The presence of one or more sialic acid residues separate these compound from glycolipids (Sandhoffet al. 1989). Gangliosides are classified based on their oligosaccharide moiety alone, i-e.regardless of variations in the lengths of the hydrocarbon chains comprising the s2hingosine and fatty acid components. Members of the ganglio-family are designed by "G". The number of sialic acid residues in a ganglioside is designated by a capital letter: A (asialo-), M (monosialo-), D (disialo-), T (trisialo-), Q (quatrosialo-), etc. The length of the neutrd sugar chain is designated by a number following the formula "5-n", where "n" is the number of neural sugars in the ganglioside.

1.2.2 S-wthesis and derrradation

The synthesis of gangliosides begins in the smooth ER where the ceramide portion is synthesized from serine and palmitoyl CoA. After the addition of an amide-linked fatty acid, the ceramide is transferred to the Golgi apparatus (Zeller and Marchase 1992)' where ceramide is glycosylated by the transfer of the individual sugar from the respective uridine-5'-diphosphate

(UDP) derivatives. Glucose first links with ceramide to forrn glucosylceramide (GlcCer) in the presence of glucosyltransferase, then galactose joins to GlcCer to forrn lactosylceramide (LacCer) catalyzed by gdactosyltransferase. The sequential addition of monosaccharide or sialic acid residues to the growing oligosaccharide chain, yielding ganglioside CiM3 and more complex gangliosides, is catalyzed by membrane-bound .

Afier their synthesis in the Golgi, gangliosides are transported to the plasma membrane by vesicular flow and anchored to the outer leaflet of the membrane by their ceramide moieties, with their ~Iigosaccharidesextending into the extracellufar space (Van Echten and Sandhoff 1993).

Gangliosides on the plasma membrane are ultimately transported to the lysosomal cornpartment for degradation after endocytosis. Degradation occurs in the through the sequential removal of the monosaccharides tiom the non-reducing terminal end of the molecule, each by a specific lysosomal glycosidase (Fig 1-2), in the reverse of their order of synthesis. In most cases, a deficiency of any one of these results in a lipid storage disease (reviewed in (Neufeld

199 1)).

1.2.3 Functions

Gangliosides anchor to the plasma membrane through their hydrophobie ceramide moiety so that their hydrophilic oligosaccharide chains extend into the extracellular space and form cell-type specific patterns on the cell surface. Gangliosides play important roles in ce11 recognition and adhesion, and in signal transduction (Zeller and Marchase 1992). They are especially abundant in neurones where they assist with synaptic transmission and neuro-protection (Thomas and Brewer i 990)-

1.2.4 G- M-> an di oside

Guz ganglioside is an intermediate in both the synthesis and degradation of Gui, which is more abundant in normal neuronal cells. GM2 ganglioside contains ceramide core and a trisaccharide (gangliotriaose) chain with one sialic acid (Fig 1-1). GM2ganglioside is almost exclusively degraded through the removal of the terminal GalNAc by Hex A to produce Gm in Gal-GalNAc-Gd-Glc-Cer I NeuNAc (G,,gangiioside)

wlioside 8-cialactosidase Ga1 GaihiAc-Gal-Glc-Cer I NeuNAc (G, ganglioside) I osaminidase A GaiNAc 1- (G, ganglioside) NeuNAc A- NeuNAc Gal-Glc-Cer (lactosylceramide)

&galactosidase Ga1 Glc-Cer (glucosylceramide)

ocerebroside &glucosidase Glc Cer + fatty acid + sphingenine

Fig. 1-2. Lysosomal degradation pathway for ganglioside GMI. The names of the hydrolysis

products are in the brackets, while the hydrolases are underlined. human, however, in mice, Gm is also slowly degraded through the removal of the other terminal residue, NeuAc, by sialidase to fom GAZ(Sandhoff et al. 1989).

1.3 HEXOSAMINIDASES AM) THE GmACTIVATOR

1 -3.1 Structure and moperties

Lysosomal p-hexosaminidase (&N-acetylhexosaminidase, EC 3.2.1.52, Hex) cleaves terminal b-linked N-acetylglucasamine (GlcNAc) and N-acetylgalactosamine (GalNAc) residues from (including GM~,GAZ and ), glycoprotein-derived oligosaccharides and glycosaminoglycans; as well as Ciom artificial substrates which contain fluorescent or chromogenic properties after hydrolysis. The family of hex isozymes results from the three possible dimeric combinations of two subunits, a and p, Le. Hex A (a$),Hex B (PB) and Hex S (aa)(Beutler 1979).

In normal human tissue, Hex A and Hex B are the two major isoenzymes. Hex S can only be detected in samples from the patients with Sandhoff disease (see GM2gangliosidoses) in which p-subunits are deficient. Hex A is thermolabile at 50°C while Hex B is thermostable at this temperature. Hex S is highly thermally labile (I~oM~et al. 1975; Sandhoff 1969).

Mature Activator contains 162 amino acid residues and a single N-linked carbohydrate moiety bound at Asn 63 (Fürst et al. 1990). It is a srnaIl (22kDa) and acidic (PI, 4.8) monomeric protein, which is heat stable up to 60°C (Conzelmann and Sandhoff 1979; Li et al. 1981). The

Activator contains 8 cysteine residues, which fonn four disulfide bonds at Cys39- 183, Cys99- 106,

Cys 1 12- 138 and Cys 125- 136 (Schütte et al. 1998) (Fig 1-3). The latter three disulfide bridges fa11 within a stretch of 39 residues Iocated in the central third of the molecule. This region also contains seven out of seventeen prolines, and may serve to keep the central part of the activator in a highly restricted conformation. This structural element may play a critical role with regard to the stability and hctionality of the Activator (Schütte et al- 1998).

The Activator is present in various body fluids and tissues such as kidney, placenta, brain, spleen, Iiver, and serum, but is highest in kidney (800 nglmg protein) and urine (600 ng/mg protein)

(Banerjee et al. 1984). It is laborious to puri@ the Activator from human tissues and the yield is also low, e.g. using one kg of human kidney as the starting material only about 1 mg of the

Activator can be isolated (Conzelmann and Sandhoff 1979). Furthemore, the purification of the

Activator often does not totally exclude contamination of other sphingdipid activator (see

Section 1.6). This problem has been solved by the production of hctional re-folded Activator from transformed bacteria. The recombinant functional Activator has been produced by three laboratories (Klima et al- 1993; Wu et al. 1994; Xie et ai. 1998). The CO-factoractivity of the unglycosylated refolded protein was found to be similar to that of the wild-type Activator isolated from the media of transfected CHO cells (Rigat er al. 1997; Smiljanic-Georgijev et al. 1997).

1-3.2 Biosynthesis. processine and trans~ort

Like other lysosomal glycoproteins, the Hex isoenzymes are synthesized and processed through a complex biosynthetic pathway which includes the rough ER and Golgi apparatus

(reviewed in (Grave1 et al. 1995)). Briefly, the a and P subunits of hexosaminidases are synthesized on ribosomes bound to the rough ER as prepropolypeptides, which are cleaved to propeptides CO-translationally. Both pro-a and pro-p chains are glycosylated at selected Asn-X-

Ser/Thr (Kornfeld and Kornfeld 1985; Komfeld 1986), fold to their near native conformation

(Pelham 1989) and fom into dimers in ER/cis Golgi network (Hurtley and Helenius 1989). The phosphorylation Fig. 1-3 Disulfide bonds in the Activator protein. Four disulfide bonds are indicated with dot lines.

The eight cystine residues are indicated with bold numbers. The bIank area refers to the

mature form of the Activator, and the cross-hatching areas represent signal peptide. The gray

area represents the 8 residues of the propeptide that are cleaved to form the mature Activator

protein. of selected mannose residues on the oligosaccharide chains specifically targets the enzymes to the lysosome via the mannose-6-phosphate receptor @PR) in the trans Golgi network (Mahuran 1991 ;

Sonderfeld-Fresko and Proia 1989). In the lysosome, Merproteolytic and glycosidic processing occur to form the mature enzyme. The pro-a chain is cleaved into two disulfide-linked chains of 53 kDa (a,) (Hasilik and Neufeld 1980; Mahuran and Lowden 1980) and 7-kDa (ap)(Hubbes et al.

1989). Similarly, the pro+ chain is cleaved into three chains of 30-kDa (Ba), 24-26 kDa (Pb)

(Mahuran and Gravel 1988; Mahuran et al- 1982), and 7-10 kDa (Bp) (Hubbes et al. 1989). The two (c(,c(,) and three (&&f&) polypeptide chains are held together in their respective mature subunits by disulfide bonds to form the mature Hex A and Hex B, respectively.

The Activator is also synthesized as a prepropolypeptide (193 residues, Mr=20,000) on ribosomes attached to the rough ER (reviewed in (Gravel et ai. 1995)). The signal peptide (residue

1-23) is cleaved by signal peptidase in the lumen of the ER, resulting in a pro-polypeptide of 170 residues and a Mr of 18,000. This event may be followed by the addition of an oligosaccharide chain to the asparagine 63 residue contained in the consensus sequence, Asn-X-Ser/Thr. The final conformation of the monomor is the formation of its 4 disulfide bonds (Fürst er a[. 1990; Xie et al.

1998). Depending on the composition of its oligosaccharide, the pro-polypeptide can have a Mr as determined by SDS-PAGE, of 22,000 Da (hi& mannose type), 24,000-27,000 Da (complex type) or

20,000 Da (no oligosaccharide) (Rigat et ai. 1997) (Glombitza et ai. 1997).

AAer the newly synthesized propolypeptides are properly folded, they pass out of ER/ cis

Golgi network and enter the cis Golgi where continued Golgi transport is via bulk blow. Mannose-

6-phosphate (M6P) markers may be added to its high mannose-type oligosaccharides in the cis

Golgi networkl cis Golgi in order to specifically target the propolypeptides to the lysosome.

Addition of the M6P marker also prevents high mannose oligosaccharide fiom being processed to a complex-type structure, which is typical of secretory proteins. The M6P markers are generated by 12 the sequential action of two Golgi enzymes. First, GlcNAc-phosphotransferase transfers GlcNAc-

1-phosphate £tom the nucleotide sugar uridine diphosphate-GlcNAc to select mannose residues to give rise to a phosphodiester intermediate. Then, GlcNAc-1-phosphodiester glycosidase removes the GlcNAc residue to expose the recognition signal (Komfeld and Sly 1995; Lang er al. 1984).

The importance of this process is underlined by the occurrence of 1-ce11 disease which is a severe, fatal disease caused by the deficiency of GlcNAc-phosphotransferase. FibrobIasts fiom 1-ce11 patients secrete a large percentage of their newly synthesized lysosomal proteins into the culture media because the pathway for targeting to the lysosome is defective in these cells (Burg et a/.

1985; Hasilik and von Figura 198 1).

The phosphorylation of propolypeptides specially targets them to the lysosome through their interaction with M6P receptors (Ml?R) in the trans Golgi network (TGN) (Griffiths et al. 1988).

There are two distinct MPRs, CI-MPR (cation-independent) and CD-MPR(cation-dependent). The large (270 kDa) CI-MPR is also the receptor for insulin-like growth factor II (Morgan et al. 1987).

Both type of MPRs are transmembrane proteins and can be concentrated in clathrin-coated vesicles on the TGN membrane by the adaptor proteins. The adaptor proteins include the AP-2 adaptors which are responsible for coated-pit formation at the plasma membrane and M-1 adaptor which act at the TGN membrane (Glickrnan et al. 1989; Pearse and Robinson 1990). Both MPRs function in the Golgi-endosome-lysosome pathway, but only the CI-MPR hctions at the plasma membrane and accounts for the "re-capture" activity of cells towards M6P-containing proteins with subsequent transport to the lysosome (Kornfeld 1990) (Fig 1-4).

Although the major intracellular transport pathway for most lysosomal proteins that do not contain a transmembrane domain is via a MPR, MPR-independent pathways also exist in some cells. For example, skin fibroblasts corn 1-ce11 disease patients are deficient in most soluble lysosomal enzymes, however, other cells, e-g. lymphoblasts, liver and kidney, often contain near Biosynthetic pathway I 1

RER lysosome

Golgi & cis

trans rn

O

secretory+ pathway

0w 0 Secretory proteins

lysosomal enzymes

-f mannose-6-phosphate receptor a cytosolic vesicle (clathrin coated)

Fig. 1-4. Mode1 for lysosomal protein targeting to lysosomes normal levels of the enzyme (Nolm and Sly 1989). Recently, the transport of the Activator in human fibroblasts has been studied in our laboratory (Rigat et al. 1997). In this report, we identify the MPR pathway as the major biosynthetic route for the incorporation of the Activator into the lysosomes of fibroblasts. We also demonstrate that a large percentage of the newly synthesized

Activator does not contain the M6P tag, but contains complex-type oligosaccharides and is normally secreted. The refolded Activator from bacteria with no oligosaccharide cm be endocytosed by a carbohydrate-independent mechanism. Simiiar results were also reported tiom the transport of the Activator in human epidermal keratinocytes (Glombitza et al. 1997). However, in the latter study the author estimated that only 10% of the Activator was phosphorylated and that

70% was retained intracellularly; thus, they concluded that there must be a major MPR-independent biosynthetic pathway for the Activator. These data suggest that a large fraction of the Activator synthesized in normal cells is treated as secretory rather than Iysosomely-targeted proteins and this secretory form could serve as a transport protein.

Afier the Activator enten the lysosome, the propolypeptide is processed by proteolytic and glycosidic enzymes to form 22 kDa mature protein. The activator precursor is the major form found in the culture medium, while the mature fom is detected in cells, suggesting a rapid processing compared to the low biosynthetic rate (Bwg et al. 1985).

1.4 THE INTERACTION BETWEEN THE ACTIVATOR, GMZGANGLIOSIDE AND HEXOSAMINIDASE A

1 -4.1 The substrates of hexosarninidase A

Although al1 three Hex isoenzymes cm hydrolyze substrate with terminal $-GlcNAc or P-

GalNAc residues, only Hex A can hydrolyze Gw ganglioside with the Activator in vivo in humans

(Conzelmann et al. 1982). In vitro certain detergents can replace the Activator function, however in this case Hex S as well as Hex A can hydrolyse GMz. An uncharged fluorogenic substrate, 4- 15 methylumbelliferone-GlcNAc (4MüG), is recognized by al1 Hex isozymes and does not require the

Activator. However, a related negatively charged compound, 4-methylumbelliferyl-GlcNAc-6- sulfate (4MUGS), can be cleaved efficiently by Hex A and Hex S, and only slowly by Hex B

(Bayleran 1984). This suggests a unique charged binding site in the a-subwiit.

1.4.2 Roie of the Activator in GMz- hvdrolvsis - bv hexosaminidase A

The Activator can extract Gm and several other fkom micelles or liposomes, forming stable water-soluble 1: 1 complexes, but it appears unable to penetrate the liposomal membrane (Conzelmann et al. 1982). Thus, the Activator acts primarily as a substrate- specific CO-factorof Hex A, instead of ccactivating"the enzyme (Sandhoff et al. 1989). Briefly, the

Activator solubilizes a single molecule oEGW fiom the lysosomal membrane to form a complex, and presents it to Hex A for hydrolysis. AAer the reaction, the Activator participates in another round of catalysis (Fig 1-5) (Grave1 et al. 1995).

The mechanism by which the Activator acts as a CO-factorin hydrolyzing Gm by Hex A has not been fully elucidated and some controversy still exists in the literature. Sandhoff and colleagues believe that primary function of the Activator is to remove Gm fkom its membranous environment that sterically hinders Hex A (Meier et al. 199 1). On the other hand, Li and colleagues believe that the Function of the Activator in Gm hydrolysis is more than simply solubilizing the lipid substrates.

They believe that the effectiveness of the Activator in stimulating the hydrolysis of Guz is due to its ability to recognize the specific trisaccharide structure of the GLI<~epitope, GalNAcp144

(NeuAcaZJ3) Gal-, and to modify the strong hydrogen bond between GalNAc and NeuAc

Fig 1-5 A mode1 of the two known functions of the Activator. 1) its role as a substrate-specific co- factor for the hydrolysis of Gw by Hex A, and 2) its role as a glycolipid uansport protein. Fig 1-5 A mode1 of the two known Functions of the Activator. 1) its role as a substrate-specific co- factor for the hydrolysis of Gmby Hex A, and 2) its role as a glycolipid transport protein. (Wu et al. 1994). Further work is needed to dari@ the relative importance of the Activator's detergent-like function versus its interactions with the terminal GalNAc and NeuAc residue in the hydrolysis of Gm.

1.4.3 Binding of the Activator with ~aneliosides

The Activator interacts with both the hydrophilic oligosaccharide and hydrophobic ceramide moieties of gangliosides to fom Activator-ganglioside cornplex. Because complex-formation is reversible, the Activator is able to extract a ganglioside ffom one membrane and replace it in another, i e. serve as a general ganglioside transport protein. The spectrum of glycolipids that interacts with the Activator is prirnarily determined by their oligosaccharide moieties. In vitro binding studies have indicated that the terminal GalNAc and interna1 NeuAc residues of ganglioside play an important role in determining binding afinity, i-e. GW >> GMl> GDI, = Gm = GAZ(Fürst and Sandhoff 1992; Fürst er al. 1990).

The hydrophobic binding site for the ceramide portion of gangliosides may be composed of a pocket formed by amphiphilic a helices predicted from the amino acid sequence of the Activator

(Fürst er al. 1990). Recently, a fluorescence-dequenching assay specific for the hydrophobic binding pocket has been evaluated and optimized in our laboratory (Smiljanic-Georgijev et al.

1997). This assay was developed based on the investigation of endosotne/ lysosome fusion by a fluorescence dequenching method (Kuwana et al. 1993; Kuwana et al. 1995). The investigation by

Kuwana et al suggested that the Activator couid act as a transfer protein of the fluorescence lipid probe, octadecylrhodamine (R- 18), between egg phosphatidylchoIine liposomes, as well as isolated endosornes and lysosomes. In our study, we first develop a fluorescence dequenching assay that could be used to evaluate the hydrophobic binding function of the Activator. The optimal time course was detennined to be fiom the 5" to 10" minute afier the initiation of the assay. The optimal amount of the Activator and optimal pH used in this assay were found to be fiom 0.75 pg to 4 pg 18 and pH 5, respectively. Because addition of glycosphingolipid (GSL) could inhibit R-18 transport, the percentage inhibition could also be used to assess the oligosaccharide binding sites in the

Activator. Thus the fluorescence dequenching assay was used to extend the spectrum of GSL- binding affinity with the Activator, i. e. GM~(90% inhibition) >> GTI~(62%) >> GMI (25%) z Gw

(24%) > GW (17%) >> GA2(3%).

1.4.4 The binding domains in the Activator

The possible binding domains with GM~and Hex A in the Activator have been proposed from the following experiments. Three truncated fonns of the Activator, truncated at L157 plus extra residues WSCPVGSPPGTTA, or tmncated at C 183 and K185 by introducing a STOP codon, were tested for a fiinctional hydrophobic binding site. The experiments indicated that the hydrophobic binding function in each mutant protein was lost (Rigat er ai. 1997; Smiijanic-

Georgijev er ai. 1997; Xie et ai. 1998). These data suggest that the hydrophobic binding site is located in the C-terminus of the Activator. On the other hand, the shidy of naturally occuming

Cys138Arg substitution in the Activator suggested the location of its Hex A-binding domain. This mutated activator did not lack the Activator Iipid transport activity (Smiljanic-Georgijev et al.

1997), but did lack the ability to assist Hex A in hydrolysis of Gm (Xie et al. 1998). Thus Cys138 or the loop structure formed with Cysll2 is likely cnticai in foming the recognition site for Hex A, localizing this domain to the middle region of the protein.

1.5 STRUCTURE OF HEXOSAMINIDASE AND THE ACTIVATOR GENES

1 -5.1 Hexosaminidase aenes

The genes encoding the a- (HEXA) and & (HEXB) subunits have been isolated and charactenzed. The HEU gene is 35 kb long, contains 14 exons (Proia and Soravia 1987) and is mapped to chromosome lSq23-q24 (Nakai et al. 199 1). The HEYB gene is about 45 kb long and also contains 14 exons (Neote et al. 1988; Proia 1988). HEXB gene locates on chromosome 5q 13

(Biklcer et al. 1988). The promoters of both HEAX and HEXB have been identified (Neote et aL

1 988; Proia and Soravia 1987). The HEXA and HEXB genes encode propro-polypeptides of 529 and 556 residues, respectively. A cornparison of the deduced primary sequences from the both cDNAs reveals an overall60% identity. As well, both genes show a striking degree of homology in both the number and the placement of exodintron junctions. These data indicate the both genes are derived from a common ancestor (Korneluk et al. 1986; Proia 1988); thus structure-function retationships within the two subunits shoufd be conserved.

1-52The Gu7- activator gene

The Activator is encoded by GMZA gene. GMZA is a mal1 gene of at least 16 kb, and its hl 1 length cDNA has been isolated (Klima et al. 199 1; Nagarajan et al. 1992; Xie et al. 199 1). The promoter of the GMZA gene has not been characterized. The 2.5 kb cDNA is transcribed fiom four exons in the GMZA with exon 1 containing the 5' untranslated region and exon 4 containing more than 1.5 kb of 3' untranslated sequence. At the begiming of my thesis work, only intron 3 had been fùlly sequenced (Klima et al. 1991), and the sequences of intron 1, intron 2 and their exonic junctions were incomplete (Fig 1-6). A second altematively spliced mRNA product containing exons 1-3 and part of intron 3 had also been identified (Nagarajan et al. 1992). Due to the presence of a STOP codon early in the retained intron 3 sequence, the product of the alternatively spliced mRNA is a truncated fom of the Activator, missing residues 142-193 and containing an additional three residues encoded by the intron 3, Val-Ser-Thr. The GMZA gene has been mapped to chromosome 5q3 2-33, while a processed pseudogene related to the functional, G MZA P, was identified and localized to ciuomosome 3 (Heng et ai. 1993; Swallow et al. 1993; Xie et al- 1992a). Fig 1-6 The known GM2A gene structure in 1996. The larger open boxes refer to the exons, and

the black boxes refer to the 5' and 3' end untranslated region. The smaller open boxes refer

to the introns. The regions with known sequences of the gene are indicated with double

underline. 21

Three polymorphisms, ASSG, G205A and G582A, have been reported in the exons of the GM2A gene through screening of cDNA library and sequencing (Xie et a' 1991) (Table 1-2) (Fig 1-8).

1.6 OTHER LYSOSOMAL SPHINGOLLPID ACTIVATOR PROTEINS

In addition to the Activator, the lysosomal degradation of other with short h ydrophi lic groups is also dependent on small nonenzymic gl ycoproteins, tenned sphingolipid activator proteins (SAP). Four homologous SAPs, SapA, Sap-B, Sap-C and Sap-D, have been characterized. These proteins al1 arise from the processing of a single SAP precursor polypeptide, (Sandhoff et al. 1999, which is encoded by a gene on . The prosaposin has a total of 524 amino acids with five N-glycosylation sites. The four homologous domains, each encoding about 80 amino acids, result in SAP A-D (Roman and Grabowski 1989). Most of the precursor is first transported to the ce11 surface and then endocytosed into the lysosomal cornpartment, where it is processed into the four mature glycoprotein forms. The Activator is evolutionarily distinct from the other SAPs. It functions as a monomer while Sap A-D are homodimers. The Activator is encoded by a separate gene that shares no significant deduced pnmary structure homology with the others (Schroder et al. 1989).

The functions of Sap A-D differ significantly. Saposin B is known as a nonspecific activator protein and has been found to have a detergent-like activity which stimulates the hydrolyses of various glycolipids by different glycosidases (Li et al. 1988). e.g. it activates the hydrolysis of cerebroside sulfate, GMI,and globotriaosylceramide by arylsulphatase A, p- galactosidase, and a-galactosidase, respectively. Sap-C stimulates the hydrolysis of glucosy lceramide, , and by B -glucosylceramidase,

B-galactosidase, and sphingomyelinase, respectively. The clinical findings in Sap-C defciency are thought to be similar to thcse in Gaucher disease type 3 (Beutler and Grabowski 1995). In vitro,

Sap-A activates glucosylceramidase and , while Sap-D shows some stimulatory effect on the degradation of ceramide in vitro and in vivo (Azurna et al. 1994; Klein et al. 1994).

1.7 GM~GANGLIOSIDOSES AND MUTATIONS LN THEIR RELATED GENES

1 -7.1 Classification of GMT- aandiosidoses

Deficiency of f3-hexosaminidase isoenzymes or the Activator causes a group of autosomal recessive inherited disorders, known as GMZgangliosidosis. The major characteristic of these disorders is the excessive intralysosornal accumulation of ganglioside GM~,particularly in neuronal cells where ganglioside synthesis is greatest. There are three variants in Gm gangliosidosis (Fig I-

7), i. e. Tay-Sachs disease (B variant), Sandhoff disease (O variant) and AB variant (Sandhoff et al.

1989). Tay-Sachs disease results fiom Hex A deficiency through mutations of the HEXA gene encoding the a subunit (Hex B activity is normal or increased in these patients). Sandhoff disease is characterized by combined Hex A and Hex B deficiency caused by mutations in the HEXB gene encoding the common subunit. The AB variant is caused by mutation of the GM2A gene encoding the Activator. In this variant, Hex A and Hex B are both structurally and hnctionally normal, however, hydrolysis of ganglioside GM~is prevented by the absence of the GM~-Activator complex, the true substrate for Hex A (reviewed in (Grave1 et aL 1995)).

1.7.2 Clinka1 ~henotypes

GM2is an intermediate in the degradation of GMi(Fig 1-2). Since GM~is particularly rich in neurons, GMtgangliosidosis is principally a neurological disorder. The most pronounced cellular change is the presence of swollen neurons with massiv~accumulation of storage matenal in HEXA HEXB GM2A Gene chi 15q23-24 chr 5q13 chr 5q32-33

Poly- peptide

Gm

Deficiency Tay-Sachs Sandhoff AB variant Disease Disease Form (B variant) CO variant)

Fig. 1-7 The variants of Gm gangliosidoses lysosomes throughout the nervous system. These form characteristic inclusions, the "membranous

cytoplasmic bodies", which are lamellar structures consisting of dense concentnc membranes

(Terry and Weiss 1963).

Patients with Gw gangliosidosis display a wide spectrurn of clinka1 severity, which is Iikely

related to the amount of residual Hex A activity present (Leinekugel et uL 1992). On the basis of

t heir di fferent cl inical phenotypes, patients are generally classi fied into acute (the classical infantile

type), subacute (late infantile and juvenile type) and chronic forms (adult type) (reviewed in

[Mahuran, in press #492]). Generally, the earlier the onset of symptoms the more severe the

resulting disease. A correlation between residual Hex A activity and the severity of the resulting

disease in the patients with Gw gangliosidosis has been determined. Residual Hex A activities

found for acute, subacute, and chronic patients were O.1%, OS%, and 2-4% of normal controls,

respectively (Conzelmann et al. 1983). Al1 three forms of Gm gangliosidoses appear in Tay-Sachs

disease and Sandhoff disease, however, only the infantile form of AB-variant has been described.

The acute form of al1 three variants is the most common, and also is the clinically and biochemicaIly least variable. While affected infants generally appear completely normal at birth, they usually begin to show motor weakness in the first 3 to 5 months. An exaggerated startle response is often one of the first signs recognized by parents. More profound neurological symptoms, such as hypotonia, ataxia, and development retardation are found and develop rapidly with death normally occurring by the age of about 4 years. Cherry-red spot in retina is a common sign in this form. The subacute phenotypes usually show evidence of neurological symptoms, such as ataxia and progressive psychomotor retardation, at 2-6 years of age with death occurring between

10- 15 years of age. The chronic fonn of Gm gangliosidosis displays highly variable symptoms and clinical course even in the same family (Argov and Navon 1984; Mcinnes et al. 1992). Some of chronic patients have motor difficulties, psychosis, mental deterioration and progressive dystonia. However, other chronic patients present with abnonnalities of gait and posture between 2 and 5 years of age, and most of these patients with this condition are still living in their third or fourth decade of life (reviewed in (Grave1 et al. 1995)). Al1 Gu2 gangliosidoses exhibit an autosomal recessive pattern of inhentance, and heterozygous forms for any of the defects are completely asymptomatic.

1.7.3 Mutations associated with Tav-Sachs disease and Sandhoff disease

Mutations in genes encoding the subunits of hexosaminidase A, HEXA and HEAB, have been widely reported. At least 87 H.gene mutations and 23 HEXB gene mutations have been characterized [Mahuran, in press #492]. These mutations can be placed into a number of broad categories, i,e, partial gene deletions, mutations producing early stop codons, mutations affecting mRNA processing and missense mutations (Mahuran 1997).

1.7.4 Mutations associated with the AB variant form of GE gannliosidosis

The AB variant fom of GM~gangliosidoses is extremely rare and only four different mutations in GM2A have been described at the molecular level (Table 1-1) (Fig 1-8). Of these mutations, the Cysl38Arg (T412jC) substitution was the first to be described (Schroder et al.

199 1 ; Xie et al. 1992b) and is the most interesting one fiom a structure-function point of view (Xie et al. 1998). This mutation did not affect GMZA mRNA transcription, but it caused its encoded protein to be retained and degraded in the ER (Xie et al. 1992b). Bacterial expression and refolding studies indicate that the mutant protein retained 1.2% of the wild type's specific Hex A CO-factor activity. The presence of this srna11 amount of activity in the mutant protein coupled with a nearly normal CD spectmm strongly suggested that no major tertiary or secondary structural changes had occurred due to the mutation (Xie et al. 1998). However, the mutant protein was found Fig. 1-8 Mutations and polymorphisms in the GM2A gene. Four reported mutations are localized

with arrows: 1). AAG 262-264 deletion, 2). A410 deletion, 3). T412+C transition

(Cys l38Arg), 4). G5063C transversion (Argl69Pro). Three polymorphisms, ASSG,

G205A and G582A, are indicated. El, E2, E3 and E4 represent exonl-4. Table 1-1 Naturally Occurring Mutations in the GM2A gene

Mutation 1 Location 1 Rcsult 1 Biochemical phenotype Klinical 1 Hcritagc 1 Rcferencc Phenotypc 1) A AAG Exon 3 A Lys 88 Normal rnRNA, no maturc CRM,transport mutation,8% Acute Saudi Arabian (Schepcrs el al. 1996) 262-264 1 1 rcsidual activitv after bactcrial exoression ' 2) A A410 Normal mRNA, no maturc CRM, transport mutation, 3% (Sc hepers et ai. 1 996) 'Ion 1 ::_Cid, I rcsidual aciivity derbacterial cxpreuion consangiiinity loss of 24 Exon 3 Cys 138 3 No dctcctable CRM or function in patient cclls, COS-Act; (Sctirodcr et al, 1993; Xie precursor detecicd no signilicant function, transpoct et al. 1998; Xie et al. mutation, bacterial expression dcmonstrated the protein 1992) rctained ganglioside transport function, but rcduccd interaction with Hex A - Exon 4 Arg I69JPro Premature degradation of thc mutant GM2 activaior, Acutc (Schriidcr et al, 1993) 1' l I 1 transport mutation I

Table 1-2 GM2A gene polymorphisms

Varicty Locaiion Rcsul t Commcnts Rc fercncc

1 AS53G 1 Exon 1 1 Alal93Thr ( Located in the putative ( (Schradcr et al. 1989; Xie et al. 1 signal peptide i99i) A2053G Exon 2 Va1693Mct Found in a number of (Fürst CI al, 1990; Schroder et diffcrciit sources al, 1989; Xic et al. 199 1) A5829G Exon 4 One Stop codon 40 (Xic et RI. 199 1) 1 1 1 anothcr 1 1 1 28 to have a 14-fold reduction in its heat stability at 60°C likely due to the loss of a disulfide, Cys 1 12-

Cys 138 (Schroder et al. 199 1; Xie et al. 1992b). The fluorescence dequenching assay for R-18 binding and its inhibition by Gw (SmiIjanic-Georgijev et al, 1997) detected no differences between the wild type and mutant proteins, indicating that the Cys 1 12-Cys 138 loop is not involved in lipid transportation and Gm binding. Kinetic analysis demonstrated a -10 fold increase in the Km of

Hex A for the mutant Activator-Gm complex with Little or no change in V,,,. Therefore, the mutation specifically affects a domain in the Activator that is responsible for the recognition of the

Activatod G- complex by Hex A-

Another AB variant patient was found to be homozygous for a G506 to C (Argl69Pro) mutation (Schroder et al. 1993). BHK cells -sfected with the corresponding mutant cDNA constmct produced no detectable Activator protein. Therefore, the Argl69Pro substitution, like rnany others in HEX4 and HEXB genes, appears to result in premature degradation of the mutant

Activator in ER.

Recently, two small deletion mutations in GMîA gene have been reported; a three base deletion, AAG(262-264), resulting in the deletion of Lys88 (referred as AK88), and a single base deletion, A 410, causing a fiameshifi (referred as fsH137) with the substitution of 33 amino acids and the loss of another 24 amino acid residues (Schepers et al. 1996). Each patient was homoallelic for their respective mutations. Although the cultured fibroblasts of both patients produce normal levels of GMZA mRNA, they iacked detectable Activator protein. Pulsekhase and in vitro translation study indicated a premature degradation of each mutant Activator in the ER. 1.8 MOUSE Gw ACTIVATOR PROTEIN AND MOUSE MODELS OF Gm GANGLIOSIDOSES

1.8.1 The GE activator rote in in mice

There are proteins similar to the hurnan Activator in rats, mice, cattle and pigs (Burg et ai.

1983). The mouse Gx activator protein has been the best investigated among these species. The cDNA of mouse Gm activator was isolated and characterized in 1993 (Bellachioma et al. 1993).

The mouse activator mRNA is similar in overall structure to that of human- It has a similar length of mRNA, its coding sequence is also at the 5' end, containing a similar long untranslated region in

3' end. The cornparison of mouse and human sequences downstream from the termination codon indicates a 67% nucleotide and 68% amino acid identity. The greatest divergence between the sequences is found in the first 3 1 amino acids, which includes the signal peptide and pro-peptide sequence in hurnan activator protein, while the regions from residue 32 to the C-terminal are 75% identical. Ttius the tertiary structures of the human and mouse GiM2activator proteins are likely to be very similar. Al1 eight cysteines of the human sequence are conserved in the mouse; however, the N-glycosylation sites are not (Bellachioma et ai. 1993). The mouse Gm2a gene is mapped to a region on mouse chromosome 1 1 that is homologous with a segment of hurnan .

1.8.2 Mouse mode1 of GhA2pangliosidoses

Through the targeted disruption of the hem, , and gmîa genes, mouse models of Tay-

Sachs, Sandhoff, and the AB-variant form of Gm gangliosidosis have been established (Liu et al.

1997; Sango et ai. 1995). Although the three human disorders are very similar in their clinical phenotypes, each of the mouse models is distinct. The Sandhoff mouse exhibited the most extensive ganglioside accumulations which produced the most severe neurological disease, while

Tay-Sachs mouse appeared to be phenotypically normal (Sango et ai. 1995). The AB variant mouse was of an intermediate phenotype (Liu et 41. 1997). The possible reason for the difference between the human disorders and the mouse models is that there is an alternative degradative patbway for

GMZganglioside in mice. In the primary pathway, the Gm/activator is degraded to Gw by Hex A

(the major and almost exclusive pathway in humans). In the second normally minor pathway

specific to the mouse, Gm is degraded by sialidase to Ge and then by Hex A or, to a lesser extent,

Hex B to lactosylceramide. Therefore, the Tay-Sachs mouse accumulates iower amounts of GW,

but not Ga,and appears asymptomatic. The Sandhoff mouse accumulates both Gw and GAZdue to

blocks in both pathways and shows a severe phenotype. The AB variant mouse accumulates GM~

with a small amount of Ga. These data indicate the hexosaminidase-mediated degradation of GAZ

can proceed to some extent in the absence of the activator possibly with SAP-B. However, mouse

GM2activator is likely required for this reaction to proceed at an optimal rate (Liu et al. 1997). In summary, because the catabolic pathways for Gw in mouse and human are clearly not identical, these mouse models of GM2gangliosidosis cannot truly reflect their cornterparts in humans (Yuziuk el al. 1 998).

1.9 THESIS OBJECTIVES

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Physiol. 262:C 134 1 -C1355 CHAPTER II

CHARACTERIZATION OF THE EXONI INTRON

JUNCTIONS OF THE GM2A GENE 2.1 INTRODUCTION

nie GM~activator protein (Activator) is a substrate specific cofactor for degradation of Gm ganglioside by lysosomal ~hexosaminidaseA and is encoded by the GUZA gene on chromosome 5

(Heng et al. 1993; Xie et al. 1992). GM2A mRNA is about 2.5 kb and contains a 582 nucleotide coding region and a long 3' untranslated end sequence (Klima et al. 199 1; Nagarajan et al. 1992;

Xie et al. 1991). GM2A is a small gene of at least 16 kb whose promoter has not been charactenzed. Three exons (exon 2-4) have been identified and a fourth (exon 1) extrapolated to account for the remaining 81 bp of 5' coding sequence. Arnong these three introns (1-3)' only intron 3 has been hlly sequenced, and the sequences of intron 1 and intron 2 and their exonic junctions remain to be determined.

Sandhoff and colleagues previously screened a genomic DNA library and found that two genomic clones contained part of the GM2A gene (Klima et al. 1991). Based on data fiom these clones, they demonstrated that intron 2 was 7.3 kb in length and predicted that intron 1 contained more than 1.8 kb. They sequenced the full length of intron 3 (38 1 bp), 20 bp each of the 5' and 3 ' ends of intron 2 and 20 bp of the 3' end of intron 1. However they could not obtain the 5' end sequence of intron 1, as neither genomic clones contained this part of the sequence. In our laboratory, we also screened a hDash human genomic library and obtained a GM2A related clone that contained a 13 kb insert. This 13 kb insert produced six fragments of approximately 4.5 kb, 3 kb, 2.5 kb, 2 kb, 0.7 kb, and 0.3 kb with EcoR I digestion. The 0.7 kb fragment was found to contain part of GM2A cDNA sequence and the 2.5 kb fiagrnent to contain its 3' untranslated end.

In order to confirm that the hDash insert was an authentic fragment of the GM2A gene, the 0.7 bp fragment was subcloned into pBluescript vector and sequenced. Sequence results indicated that the

0.7 kb fragment contained the 3' end of intron 2, exon 2, intron 3 and part of exon 4. However, it was found that none of the other fragments fiom the 13 kb insert contained the complete 5' end of

GMZA including the end of its intron 1.

It is well known that specific intronic nucieotides, especially those close to exodintron junctions, are required for mRNA splicing. Mutations in these nucleotides often cause alternative

mRNA splicing which in turn results in a severe reduction or deficiency in the protein product and

an abnormal clinical phenotype (E3reathnach and Chambon 198 1; McKeown 1992; Sharp 1994).

The most critical nucleotides in the introns are those of the "invariant splice sites", Le. IVS

(intervening sequence) +lg and +2t, and IVS -2a and -lg, and branch sites (Lewin 1997). When

mRNA is splicing, the reaction with the 5' splice site involves the formation of a lariat like structure

that joins the "gt" end of the intron (5' splice site) via a 5' - 2' Iinkage to the "A" at the position of

the branch site which is close to 3 ' end of the intron, Then the 3 ' -OH end of the exon attacks the 3 '

splice site and ligates to the next exon. Krawczak analyzed 101 different cases of splice junction

mutations, and he estimated that 15% of al1 point mutations causing human genetic disease result

from a rnRNA splicing defect. Most junction mutations are caused by point mutations at splice sites

while others are caused by a mutation of intronic nucleotides 3-15 upstream fiom the intron 3' end

(Krawczak et al. 1992). More recently, branch site mutations which cause alternative rnRNA

splicing have been reported (Hara et al. 1995; Webb et al. 1996). in yeast, branch site,

UACUAAC, is conserved and lies 18-40 nucleotides upstream of the 3 ' splice site (Zhuang et al.

1989). The branch site in higher eukaryotes is not well conserved, but has a preference for purines

or pyrimidines at each position and the target "A" nucleotide which usually lies less than 100

nucleotides upstream fiom the 3'splice site (Lewin 1997). One point mutation in an intronic

nucleotide, 26 bp upstream of the 3' splice site in IVS 30 of the human FBN2 gene, which resuited

in severe congenital contractural arachnodactyly (CCA), has been reported. This point mutation

was concliided to be located at the branch site in the intronic sequences (Maslen et al. 1997). Based on the above reports, in order to completely analyze genornic DNA for disease causing mutations, it is necessary to characterize the introdexon junction sequence and at least -100 bp at each end of the intronic sequences.

Among the other two genes whose protein products are needed to degrade Gmganglioside,

HEM. and HEXB, 13 splicing disease-causing mutations have been reported. Of these 13 mutations, 7 involve one of four splice site nucleotides, "gt" at 5' end and "ag" at 3' end (reviewed in (Grave1 et al. 1995)). Thus the characterization of the introdexon junction of the GM2A gene is necessary in order to fûlly screen the gene for a disease causing mutations.

Previously our lab and others have tried to characterize the complete structure of the GM2A gene by isolating gene hgrnents fiom a genomic DNA library. However, in both cases the 5' end including intron 1 was not isolated. Screening a genomic DNA library is also a time-consuming and complicated technique. Thus, I decided to take advantage of new PCR techniques that have been used to ampli@ more than 10 kb of A phage DNA CKainz et al. 1992; Ohler and Rose 1992).

Cheng and colleagues have amplified up to 22 kb of the p-globulin gene cluster fiom human genomic DNA and up to 42 kb fiom phage h DNA (Cheng 1994), while Barnes have amplified up to 35 kb of DNA with high fidelity and high yield from A phage (Bames 1994). As the exon sequences of GM2A gene have been identified, my objective was to ampli@ the full length of intron

1 and intron 2 with exonic primers using long PCR techniques. In this study, 1 amplified both introns either of two DNA polymerases, rTth XL (Perkin Elmer) or Klan Taq-1 DNA polymerase

(Clontech). Afier subcloning both ends of each intron from the digested PCR fragments, 1 sequenced more than 500 nucleotides fiom each end of the introns. I next designed four pairs of primers based on these intronic sequences which could ampli@ al1 four exons and their flanking regions in the GM2A gene using a single set of PCR conditions. Finally, 1 deveioped a restriction map for the full length of the GMZA gene (except part of 3' untransiated region). 2.2 MATERIALS AND METHODS

2.2.1 Isolation of ~enomicDNA

Normal cultured skin fibroblast ceIl lines were obtained fkom the Tissue Culture Facility, the

Hospital for Sick Children, Toronto and the leukocytes of normal individuals were obtained from the Clinical Laboratory in Hospital for Sick Children, Toronto- Confluent fibroblast celts were harvested by scraping *th a rubber policeman. They were resuspended in 2 ml of phosphate buffered saline (PBS), centrifuged and the ce11 pellet was collected. Both fibroblast cells and

Ieukocytes were resuspended in lysis buffer (100 mM Tris-CI pH 8, 40 mM EDTA pH 8, 0.2%

SDS, and 0.6 mglm1 protease K) and were grounded, then incubated in 5S°C water bath for 10 hours. The sample was extracted with one volume of phenol/chloroform, and DNA was precipitated by adding 2 volumes of 100% ethanol. The DNA pellet was rinsed with 70% ethanol and resuspended in 10 rnM Tris-Cl pH 8, 1 mM EDTA pH 8 (TE bufler).

2.2.2 Long PCR to amdifi intron 1 and intron 2 of the GM2A gene

Two pairs of primers were designed through RightPrimer software (BioDisk, San Francisco,

California) to be used for amplification of intron 1 and intron 2 of the GMZA gene (Table 2-1). The primers for intron 1 each contained 18 nt of exonic sequence plus 10 nt to produce an Xho I site, i-e.

5'-GCGCCTCGAGGACCCACCCTTCCCGATG (#2833, upstream, - 15+3, counting from the

"A" of the first ATG as the nucleotide 1, and 5'-GCGCCTCGAGCAGGGGGACACTGGTGCT

(#2834, downstream, 228->2 1 1, Xho I sites are underlined). The primers for amptification of intron

2 each contained 32 nt of exonic sequence plus 10 nt to generate an Xho I site, i. e, 5'-

GCGCCTCGAGTI'CCTGGGATAACTGTGATGAAGGGAAGGAC !#805, upstream, 1023 46

1 3 3) and 5 '-GCGCCTCGAGGGCAGGGCTCCCCAGTAGGAATTAACATTA (#806, downstream, 376+345) (Table 2-1).

The rTrh extra Long DNA polymerase (rTth XL) was purchased fiom Perkin Elmer. intron 1 was amplified with prirners #2833 and #2834 in a GeneAmp PCR system 2400 (Perkin-Elmer). An

Ampliwax PCR Gem 100 bead (Perkin Elmer) was added to perform a "hot start9' reaction. The

PCR was performed in a 100 pl reaction volume with 0.4 pg template, 0.5 FM primers, 4 unit rTth

XL, 200 pM each dNTP, 1.1 mM M~(OAC)~(magnesium acetate). The cycling parameters used were 1 min at 94OC for denaturation, and 35 cycles each of 15 sec at 94"C, 30 sec at 60°C and 5 min at 72"C, then extra extension for 10 min at 72OC.

Intron 2 was amplified using Advantage KlenTaq Polymerase Mix (Clontech, Cat# 8417- 1), which contained Klen Taq-l DNA polymerase, TaqStart anti-Taq Antibody and minor Deep Vent

DNA polymerase, based on several reports (Tellier et ai. 1996; Barnes et al. 1994). Intron 2 was arnplified with primers #805 and #806 in Robocycler 40 (Stratagene). The PCR was performed in a

50 fl reaction volume with 0.4 - 0.6 pg templates, 0.5 primers, 1 FI Advantage KlenTaq

Polymerase Mix, 200 j.M each dNTP in a buffer of 40 mM Triche-KOH @H=9.2), 15 mM KOAc,

3.5 mM M~(OAC)~and 75 pg/ml bovine serum albumin. The cycling parameters were: denaturation at 99°C for 35 sec, annealing at 67OC for 30 sec, and elongation at 6g°C for 9 min during cycles 1-1 5, then for 1 1 min during cycles 16-25, and finally 13 min during cycles 26-35.

Ten each of the PCR products were mixed with DNA loading dye and were analyzed by eIectrophoresis in a 1% agarose gel. Table 2-1 Primers used to amplify intron 1 and intron 2 of GM2A gene

Intron Primers " 5 ' Size of PCR Size of Pol yinerase location product intron 1 GCGCCTCGAGGACCCACCCTTCCCGATG El (-15) 6.7kb 6.45kb rTth extra long GCGCCTCGAGCAGGGGGACACTGGTGCT E2 (228) ( Perkinpl Elmer) 2 GCGCCTCGAGTTCCTGGGATAACTGTGATGAAGGGAAGGACC E2(102) 6.60kb Advantagc Klcn GCGCCTCGAGGGCAGGGCTCCCCAGTAGGAATTAACATGTCA E3(376) Taq mix(C1ontech) a. The MoI site in the primers is underlined. b. Exonic location is based on the "A" of thc first ATG as nucleotide 1. 2.2.3 Restriction analvsis of intron I and intron 2

The PCR products of intron 1 and intron 2 were digested by the following endonucleases:

BamH Sst 1, EcoR i, Ba1, Kpn I, Hind 1'and Xho 1. The digestion results were analyzed in 1% agarose gel. Four BamH I digestion fragments of intron 1 were separated with QiAEX II gel extraction kit (Qiagen) and were Merdigested with the other six endonucleases. Two Sst I digestion fragments of intron 2 were also separated and were further digested with the other six endonucleases.

2.2.4 Subcloning- of both ends of introns 1 and 2 into the ~Bluescri~tvector

Restriction analysis identified two BamH I hgments of intron 1 (0.6 kb and 1.2 kb) as the

5' and 3'ends of intron 1. These two fragments were independently digested with Xho I and cleaned with QIAEX II gel extraction kit (Qiagen). One hundred ng of each fragment was ligated inro 30 ng BamH I/Xho 1 sites of pBluescnpt vector @BS, Stragagene) with T4 ligase (Borhinger,

Cat# 799 099). The ligation products were transformed into XL-Blue MRF' competent cell

(Stratagene, Cat# 200230). Through white/ blue selection in 50 pg/ml ampicillin LB plates with X-

Ga1 and IPTG, the white colonies were picked and grown in LB media containing 50 pg/ml of ampicillin. Isolation of the plasmid DNA was performed with Qiagen mini-preparation kit

(Qiagen). Digestions of 5 pl of the isolated plasmid DNA with the restriction enzymes BamHI and

Xho I were performed to ensure the plasmid contained the correct insert. Four kb and 2.9 kb Sst I fragments of intron 2 were digested by Sst I and subcloned into the Sst I/ Xho I sites of the same vector and analyzed by similar methods.

2.2.5 Nucleotide seauencing both ends of intron 1 and 2

The nucleotide sequences of the above inserts were determined by the dideoxy chain termination method (Sanger et al. 1977). Plasmid DNA products, 1.S pg, containing each end of the introns, were mixed with 30 ng each of T7 or T3 primers (Stragagene), [a--''s] dATP

(Arnersham) and other components of the sequenase kit as recommended by the manufacturer

(Pharmacia, Cat# 27-1682-01). In order to confirm the sequences of the introns, both antisense and sense were sequenced from PCR products obtained fiom five independent individual genomic DNA samples.

2.2.6 Use of PCR to ampli* al1 of the exons and exod intron iunctions of the GMZA gene

Based on the nucleotide sequences obtained from the 5' and 3' ends of the introns, 1 designed 4 pairs of primers (Table 2-2) using RightPrimer software (BioDisk, San Francisco,

California) to amplify al1 of the exons and their flanking regions by PCR. Fragments made up of exons and their intronic flanking regions were amplified with each pair of primers (Table 2-2) using a GeneAmp PCR systern 2400 (Perkin-Elmer). PCR was performed in a total volume of 100 pl with 0.6 pg genomic DNA, 0.5 FM primers, 0.2 mM each of dNTP, 2.5 mM MgCl* and 2.5 unit

Taq-Gold DNA polymerase (Perkin Elmer). The cycling parameters used were 10 min at 94OC for denaturation, 43 cycles each of 30 sec at 94OC, 30 sec at 54OC and 30 sec at 72OC, then 10 min at

72°C for elongation. With these conditions, PCR amplification of exon 3 and its flanking region produced some nonspecific DNA. Although these nonspecific bands did not interfere with the isolation of the target fragment, it was found that they could be removed by lowering the concentration of MgClz to 2.0 mM. Based on my exonic and intronic sequences and those obtained from previous studies (Xie et al. 199 l), each of the PCR fragments (Exon 1, 2, 3, and 4) was designed to contain a unique restriction site, Xba 1, Sst 1, Pst 1, and EcoR I, respectively (Table 2-2,

Fig 2-5). These digestion sites can be used as controls to confirm the identity of the amplified fragments. Thus each Fragment was digested by their respective restriction endonuclease and analyzed by electrophoresis in a 1% agarose gel. Table 2-2 Primers used to amplify the exons and the exonl intron junctions of GM2A gene

primers (22119~ 5' locationb Tm OC' E 1 GGAAGGCATTTAAAGGACCTCT E I (-69) l AAGGCTGTCTGCATTTTCACTC IVS 1 (256) / 58.1 1 1 I (- 149) 56.2 528bp Sst I:76+452 (2 1 8) 56.2 7 16bp PSI1 :484+232

CATGTCTCTGGATTTGTAAGCC IVS3 (-290) ECOR1 :3 12+352 GGCTATCAAGAACTGTCCAACT E4 (802)

nt = nucleotides Exonic locations are based on the "A" of the first ATG as nucleotide 1 ; intronic locations arc counted from first splice site of 5' end (plus nunibcr) or last nt of 3' splicc site (minus number) Calculation of Tm is bascd on Currcnt Protocol in Molecular Biology, edited by Ausubel et al. (1997). 2.2.7 Direct seauencinq

PCR products containing exons and their flanking regions were purified with the Qiagen

PCR purification kit (Qiagen). In order to confm the nucleotide sequences at both ends of the introns, direct sequencing was performed based on Thermos Sequenase Radiolabeled Terminator

Cycle Sequencing kit (Amersham, Cat# US79750). Up to 250 ng (about 1 pmol) of templates and

30 ng of one PCR primer were mixed with 8 units thermo sequenase polyrnerase and related buffer

(total volume 20 pl), and 4.5 pl of reaction mixtures were transferred to each of 2.5 ~1 [a-33~] ddNTP mixtures (Amersham, Cat# AH 9539). Afier mineral oil was added, cycling termination reactions were performed in Robocycler 40 (Siratagene) with 2 min at 94OC for denaturation, 25 cycles of each 30 sec at 94"C, 30 sec at 55°C and 1 min at 72°C. Reaction solutions were denatured and loaded in glycerol tolerant DNA sequencing gel (Amersham). Sequencing gels were dried and exposed to Kodak Biomax MR autoradiography film.

2.3 RESULTS

The initial primers used to ampli@ both introns were located within exons, allowing their orientation in the GMZA gene to be easily determined. The intron 1 PCR product was approximately 6.7 kb in length (Fig 2-1) and its 5' end contained 96 bp fiom exon 1 and its 3' end contained 146 bp from exon 2. Therefore, the length of intron 1 of the GM2A gene was found to be approximately 6.45 kb. The intron 2 PCR product was approximately 6.9 kb (less than the 7 kb marker, Fig 2-l), and its 5' end contained 133 bp from exon 2 and its 3' end contained 143 bp from exon 3. Therefore, the length of intron 2 of GMZA gene was found to be approximately 6.6 kb.

In addition to the five endonucleases, EcoR i, Bamff 1, Sac 1 (isoschizomer of Sst 4, Kpn 1 and Xba I, which were used in Klima's study (Klima et al. 199 l), 1also digested intron 1 and intron Fig 2- 1. Determination of the length of intron 1 (-6.45kb) and intron 2 (4.6kb) of the GM2A gene:

Intronic sequences were amplified by PCR. Each end of the fragments includes about

150bpexonic sequences. Intron 1,Il; Intron 2,12; 1kb marker, M. 2 with two other endonucleases, Hind 1'1andBo I. The PCR product containing intron 1 was cIeaved by BadI into four smaller fragments, 0.6 kb, 1.2 kb, 1.8 kb and 3.1 kb (Fig 2-2). These

four fragments were digested by six other endonucleases (data not shown). From these data it was concluded that the 0.6 kb and 1.2 kb hgments fkom the BamKI digestion were the 5' and 3' ends of intron 1. The PCR product containing intron 2 was digested by Sst I into tsvo smaller fragments,

4.0 kb and 2.9 kb (Fig 2-2). These eagments were separated and also digested by the other six endonucleases. Two Hind II2 sites in intron 1, one Hind III site in intron 2 and no Xho I site in either introns were identified. These digestion results were used to construct a restriction map of

intron 1 and intron 2 (Fig 2-4).

pBluescript plasmid DNAs containing each end of intron 1 and intron 2 were sequenced.

The sequence results confirmed that the 0.6 kb and 1.2 kb BamH I fragments of intmn 1 contained the 5' and 3' ends of intron 1, respectively, and the 4.0 kb and 2.9 kb Sst I fragments of intron 2 were the 5' and 3' ends of intron 2, respectively. Exonic sequences at cach end of the intron-PCR products were consistent with GM2A cDNA sequences (Klima et al. 199 1; Xie et al. 199 1). Two intronic nucleotides, IVSl (-12)g and IVSl (-lS)c, in the 3' end of intron 1 differed fiom those previously reported by Klima et al (Klima et al. 1991).

Restriction digestion of intron 1 and intron 2 with multiple endonucleases determined their restriction maps while DNA sequencing indicated their 5' to 3' orientation within each intron. My mapping data were combined with the nucleotide sequences of 5' and 3' untranslated regions reported previously (Klima et al. 199 1; Xie et al. 1991) to produce a complete restriction map for the GM2A gene (Fig 2-4).

Primers and their locations for amplification of exons and their flanking sequences are indicated in Table 2-2. The lengths of the PCR products for exons 1-4 are 426 bp, 528 bp, 716 bp 7kb

3kb 1.6kb lkb

Fig 2-2. Digestion of intron 1 and intron 2 with BamHl and Sstl. the 0.6kb and 1.2kb BamHI

fragments of intron 1 were confimed as the 5' and 3' end of intron 1, respectively, and the 4.0kb

and 2.9kb Sstl fragments of intron 2 as the 5' and 3' end of intron 2, respectively. The lower band

of intron 2 digested by BamHI contains two similar size bands that were confirmed as both ends

of intron 2 (data not shown). A small fiagrnent (4.2kb) of intron 1 produced by digestion with

Ssrl cannot be seen in this gel. Intron 1,11; intron 2,12; undigested. CID; digested by BamHI, B;

digested by Sstl, S; I kb marker, M. and 665 bp, respectively. Each of these four hgments can be digested by unique endonucleases,

Xba I, Sst I, Pst I and EcoR 1, into two srnaller fragments (Table 2-2; Fig 2-3)- The former three restriction sites are located in intronic sequences (Fig 2-S), while the EcoR I site, located in exon 4, is a unique site in the GMtA cDNA meet al. 1991).

Direct sequencing of the PCR product confmed the intronic sequences obtained from sequencing the clones, including IVS 1(- 12)g and IVS 1(-I S)c, which differed fiom NS 1(- 12)t and

IVS(- 1 5)g in the previous report (Klima et al. 1991). Furthermore, an intronic polymorphism,

IVS 1(-92)a/t, was identified. Beside these, three reported exonic polymorphisms, ASSG, G205A and G582A, were confirmed and another novel exonic polymorphisrn, G175A. was found in this study (chapter 3).

2.4 DISCUSSION

This study demonstrates that it is possible to ampli@ long lengths of genomic DNA containing entire introns. Traditionally, genomic DNA was screened, then genomic DNA clones were sequenced to obtain intronic sequence. The screening Iibrary procedwe is time-consuming, and positive genomic clones may not cover the full length of introns. The long PCR technique solves these problems with a simple experïmental technique. Furthermore, electrophoretic anaiysis of the intronic PCR product obtained fiom a long PCR provides a direct measurement of the full length of the intron that is usefui for mapping and further studies of the genes. According to previous reports and this study, long PCR polymerase, primer designing, annealing temperature, and extension time are critical to the long PCR technique (Barnes 1994; Cheng 1994). 1 successfully amplified intron 1 with rTth XL polymerase, but failed to ampli@ intron 2 with the E4 E3 E2 El M E UDP UD S UDXUD

Fig. 2-3. PCR amplification of al1 4 exons and digestion with unique restriction endonudeases.

Exons 1-4 (E 1, E2, E3 and E4) and their intronic junctions were amplified by PCR (see

Table 2-1) and digested by previously identified restriction enzymes that have a unique site

within each fragment. UD, undigested; X, Bal; S, Sstl; P, PstI; E, EcoRI, M, lOObp

marker. Fig 2-4. Restriction map of the GM2A gene. B=BarnHI, E=EcoRI, S=SEtI, X=X6al, K=Kpnl,

H=HindIII. No XhoI site is present in intron 1 and intron2. The numbers with each restriction site indicates the number of times it has appeared reading from 5' to 3' in the gene structure. Intron lengths are given in brackets. The open boxes refer to the exons, and the black boxes refer to the 5' and 3' untranslated region of the cDNA. (II TTAAAGGACCTCTGCCGCCTCAGACCTTGCAGTTAACTCCGCCCTGACCCACCCTTCCCG -1 G ATGCAGTCCCTOATOCAOOCTCCCCTCCTQATCOCCC~TCOCOACCCCT 60 *

ggtctggctgagatatgggggtggcca~t~cgtt~t~tauaattgttctctgcactag120 XbaI gccttccaaagtaactaattatgggattctggtctgtacaatgagggtggcctctaaaga 180

cttgttctgctccaggccctttttggagagattaatctcacgtctgcactctcctgccct 240

Fig 2-5 Nucleotide sequences of the GM2A exons and their 300 bp flanking intronic sequences. The sequence of each exon is given 5' to 3', with the intronic sequence in lowercase letters, the encoding exonic sequence in bold uppercase letters, and the untranslated exonic sequence in regular uppercase letters. The primers for amplification of the 4 exons and their flanking regions are showed in arrows indicating their orientation. The unique restriction sites used to confirm the identity of each fragment are underlined. The numbers in brackets with arrows and restriction sites indicate the exon being amplified. Three reported poIyrnorphisms in exons are indicated with "*": ASSG, G205A and GS82G. One exonic polymorphism G 175A was confinned in this study, as was another intronic polymorphism, IVS 1 (- 92) ah, which are indicated with "#". Two nucleotides, IVS 1(- 12) and IVS l(45) of 3 ' end of intron 1, differ from those previously reported and are indicated with """. ggcctattaggtcagtctcctgtttggaagttccaggtctatcatatcctgccttatagt -241 ttacaatacacttttgggagattatgtcttttgagtcttttagtttagtcctgcctataa -181

gatagtttcttttgtcaacctttttcttcttctccttccttgctgcctgattgtccccag -1

caggacatgtagattcagacactctttcacaggttcatggaatctcaggatcataagatt 180 gaaaggaatctctgatgtcagcq~caqcaacttcctggtgagggcaggagtgacggatac 240 \ (2) cttgcacctggcagaagcgtcctggccttctctgggcctggtggccaactgctcattatt 300 cagtgagccatgatacaaaaaaaaaaaataaagaattctaagtctatgtatagttcagtg -241 (3) tagggggaaaattcacatttgattattaatgtctgccatgggcacaataatacactatac -181

tcacacatgggccacaatgttgccattcctagaacagactatctctaagatctcatccag -121

ttaaaaattctatgattaaaatatattgctgcttttttgaagacagaagagctggtatgt -61

ttgccctggaatttacacttataacctttttcaaacctttgttttatttttttttaccag -1

gtaagtacttagggaggagagagcgttacccctgtggctaaagagatggggtttggagag60/-322 (4) , aagggtctttgcattctccttctacaqatctgcatgtctctggatttgtaagccagtgtg 120/-262 Pst1 acctatcaggaatcacttatcttccgggagcctcagttatccatctacgaaatgggagac 180/-202

ttgaacttagatgtgatcttcagggccctttatccatataatccatgctctacagtgcta 240/-142

tggccgtctctcatcttgtgcggctgttttgagaatgggaagaggggtggtagttcatgg 300/-82 (3) ctgcaatcctagcagtggctctaggagaaagaccccatcagtaggctcccactgactggc 360/-22 ggtccactggctttcccgcag GGAACCTACTCACTGCCCAAGAGC 450

TCCTCTGTTTTGTGTTTGCCAAGGCCAAACTCCCACTCTCTGCCCCCCTTTAATCCCCTT 690 6 1 same polymerase. After designing longer pnmers (42 nucleotides), setting higher denaturing temperature and long extension times (Tellier and Bukh 1996), 1 successfully amplified intron 2 by using another DNA polymerase, Klan Tapl DNA polyrnerase. This result may imply that intron 2 contained a higher percentage of GC pairs (Chenchîk er al. 1996).

The onIy experimental concem for intronic sequences obtained Erom long PCR is related to the fidelity of the polymerases. In my experiment, rTth XL polymerase was optimized for both polymerase and proofieading activity, and the Klan Taq polymerase mix contained the minor component of Deep Vent polymerase, which is believed to contain a 335' exonuclease activity that enhances the fidelity of replication (Mattila et al. 199 1). Therefore, both polymerases produced much less replication error during PCR reaction than does Tuq polymerase alone. Furthemore, 1 directly sequenced the regions in each hgment that were reported in Fig 2-5. 1 also sequenced different individual sampies and the results were consistent among different plasmid clones and among different individual samples, except one heterozygous a/t in IVS 1(-92) found in a normal individual sample (Fig 2-5).

When comparing the sequence and restriction rnapping data obtained in this study with those previously published by Klima et al (Klima et al. 1991)' some differences were found. Klima reported that the length of intron 2 was 7.3 kb. My data clearly indicated the length of intron 2 is approximately 6.6 kb (Fig 2- 1). Two intronic nucleotides also differ, Le. IVS 1(- 12)g and IVS 1(-

15)c in my study, but IVS 1(-12)t and NS(-1S)g in Klima's report. 1 directly sequenced five individual PCR products, including one from an AB variant patient (chapter 3), and 1 obtained the same sequence results. These differences could either be mistakes in Mima et al sequencing or rare polymorphisms.

Comparing restriction mapping in Klima's report, 1 demonstrated one EcoR 1 (&) site located in exon 4, which has been confirmed in cDNA sequences (Xie et al. 1991), and other two 62

EcoR i sites (E2 and E3) located at the 3' end of intron 1. Besides the different distances between some restriction sites 1 obtained and those in Klima's report (Fig 2-4) (Klima et al. 1991), 1 also found one more BaI site (Xis) close to the 5' end of intron 2 and another Xba I (X3) iocated at the

3' end of intron 1. From plasmid sequencing and direct sequencing, 1 confmed the location of the following restriction sites: XI,S2, E2 and E3 (Fig 2-S), which are close to introd exon junctions. As a whole, I completed the nucleotide restriction mapping of the GM2A gene and localized more exactfy the restriction site of seven endonucleases.

In this study, intron 1 and intron 2 were arnplified by long PCR. Four sets of prirners were designed to ampli@ four fragments that contain al1 exons and their flanking regions through a one step PCR reaction using the same parameters and the same concentration of reaction reagents with the Taq-Gold DNA polyrnerase. Because Taq-Gold DNA polymerase produces a T overhang at the

3' end of PCR products, the exon flanking regions cm be directly subcloned into a plasmid vector by using TA cloning technique (Invitrogen) and used for Meranalysis (Clark 1988; Mead et al.

199 1). These four pain of primes cm be used for the diagnosis of any patient suspected of having a disease causing mutation in their GMZA gene. Furthemore, one unique restriction endonuclease was designed to be present in each fragment, Xba I, Ssr I, Pst 1 and EcoR I, which can be used to confirrn each PCR product.

2.5 ACKNOWLEDGMENT

1 would like to thank Dr. Raymond Tellier for his critical advises in the long PCR technique, and Ms. Irené Warren for isolating leukocyte genomic DNA. 2.6 REFERENCES

Bames WM (1994) PCR amplification of up to 35-kb DNA with high fidility and high yield fiom

lamda bacterophage templates. Proc.Natl.Acad.Sci.USA 9 1:22 16-2220

Breathnach R Chambon P (198 1) Organization and expression of eucaryotic split genes coding for

proteins. Annu.Rev.Biochem. 50:349-383

Chenchik A, Diachenko L, Moqadam F, Tarabykin V, Lukyanov S, Siebert PD (1996) Full-length

cDNA cloning and determination of mRNA 5' and 3' ends by amplification of adaptor-

Iigated cDNA. BioTechniques 2 1526-534

Cheng S (1994) Effective amplificaiton of long target fkom cloned inserts and human genomic

DNA. Proc.Nat1.Acad.Sci.USA 9 15695-5699

Clark JM (1988) Novel non-tempiated nucleotide addition reactions catalyzed by procaryotic and

eucaryotic DNA polymerases. Nucl.Acids Res. 16:9677-9686

Grave1 RA, Clarke JTR, Kaback MM, Mahuran D, Sandhoff K, Suzuki K (1995) The GM~

gangliosidoses. In: Scnver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic basis of

inherited disease. McGraw-Hill, New York, pp 2829-2879

Hara T, Ichihara M, Takagi M, Miyajima A (1995) Interleukin-3 (IL-3) poor-responsive inbred

mouse strains carry the identiccal deletion of a branch point in the iL-3 receptor alpha

subunit gene. Blood 852331-2336

Heng HHQ, Xie B, Shi XM,Tsui L-C,Mahuran DI (1993) Refined mapping of the GM~activator

protein (GM2A) locus to 5q3 1-3-q33.1, distal to the spinal muscular atrophy locus.

Genomics 18:429-43 1

Kainz P, Schmiedlechner A, Snack HB (1992) In vitro amplification of DNA fragments > 10 db.

Anal-Biochem. 202:46-49 64

Klima H, Tanaka A, Schnabel D, Nakano T, Schroder M, Suzuki K, Sandhoff K (1991)

Characterization of full-length cDNAs and the gene coding for the human GM~activator

protein. FEBS Lett. 289:260-264

Krawczak M, Reiss J, Cooper DN (1992) The mutational spectrum of single base-pair substitutions

in mRNA splice junctions of human genes:causes and consequences. Hum-Genet. 90:41-54

Lewin B (1997) Genes W. Oxford University Press, Oxford, England, pp 885-920

Maslen C, Babcock D, Raghunath M, Steinmann B (1997) A rare branch-point mutation is

associated with missplicing of fibrillin-2 in a large family with congenital contractural

arachnodactyly. Am.J.Hum.Genet. 60: 1389- 1398

Mattila P, Korpela J, Tenkanen T, Pitkanen K (1991) Fidelity of DNA synthesis by the

Therrnococct~s iïtoralis DNA polymerase -- an extremely heat stable enzyme with

proofreading activity. Nucl. Acids Res. 19:4967-4973

McKeown M (1 992) Alternative mRNA splicing. Annu.Rev.Cel1 Biol. 8: 133- 155

Mead DA, Pey NK, Heimstadt C, Marcil RA, Smith LM (1991) A universal method for the direct

cloning of PCR amplified nucieic acid. Bio/Technology 9:657-663

Nagarajan S, Chen H-C, Li S-C, Li Y-T, Lockyer JM (1992) Evidence for two cDNA clones

encoding human GM~-activatorprotein. Bi0chem.J. 282:807-8 13

OhIer LD, Rose EA (1992) Optimization of Ion-distance PCR using a transposon-based mode1

system. PCR methods & applications 251-59

Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-termination inhibitors.

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Sharp PA (1994) Split genes and RNA splicing. Ce11 77:805-815

Tellier R, Bukh 1 (1996) Long PCR and its application to Hepatitis viruses: amplification of

hepatitis A, hepatitis 9, and hepatitis C virus genomes. 1.Clin.Microbiol. 34:3085-309 1 65

Webb JC, Patel DDTShoulders CC, Knight BL, Soutar AK (1996) Genetic variation at a splicing

branch point in intron 9 of the low dencity Iipoprotein (LDL)-receptor gene: a rare mutation

that dismpts mRNA splicing in a patient with familial hypercholesterolaemia and a common

poiyrnorphisrn. HumMolGenet. 9: 1325- 133 1

Xie B, McInnes B, Neote K, Lamhonwah A-M, Mahuran D (1 99 1) Isolation and expression of a

full-length cDNA encoding the human -2 activator protein. Biochem. Biophys. Res.

Commun. 177: 1217-1223

Xie B, Wang W, Mahuran DJ (1992) A Cys138-to-Arg substitution in the GM2 activator protein is

associated with the AB variant fom of GM2 gangliosidosis. Am.J.Hum.Genet. 50: 1046-

1052

Zhuang Y, Goldstein AM, Weiner AM (1989) UACUAAC is the preferred branch site for

rnammalian mRNA splicing. Proc.Natl.Acad.Sci.USA 86:2752-2756 CHAPTER III

ASSOCIATION OF A NONSENSE MUTATION AT

THE CODON FOR GLU54 IN THE GM2A GENE WITH

ACUTE AB VARIANT C;M2 GANGLIOSIDOSIS 3.1 INTRODUCTION

The GW gangliosidoses are a group of autosomal recessive inherited disorders caused by excessive intralysosomal accumulation of ganglioside Gw. Three variants of these disorders, i.e.

Tay-Sachs disease, Sandhoff disease and the AB variant form, result fiom mutations in the HE- gene, HEBgene and GM2A gene, respectively. Among these three genes, mutations causing Tay-

Sachs disease and Sandhoff disease have been widely characterized (Mahuran 1999). In general, mutations that result in complete absence of enzyme activity, protein and/ or mRNA are associated with an early onset of symptoms and the classic severe acute infantile phenotype. Point mutations, compatible with the production of stable mRNA and some detectable protein and enzyme activity, are associated with a later onset and slower progression of the disease.

To date only the acute AB variant form of GM~gangliosidosis has been described in four patients. These patients were al1 homozygous or hemizygous for distinct mutations. Two of these are missense mutations, a T412 to C (Cysl38Arg) mutation (Schroder et al. 1991; Xie et al. 1992) and a G506 to C (Arg l6gPr0) mutation (Schroder er al. 1993). Recently, an AAG (262-264) deletion, resulting in the loss of Lys88, and an A410 deletion, resulting in substitution of 33 arnino acids and the loss of another 24 amino acid residues afier a premature STOP codon (Schepers et al.

1996), have been reported. None of these mutations affected mRNA stability, but they al1 lead to a failure to produce a detectable mature Activator in patients' cells, probably owing to abnormal processing or stability of the protein in ER,

Three polymorphisms, A55G, GZ05A and G582A, have been descnbed in the exons of the

GM7A gene through screening and sequencing cDNA libraries (Xie et al. 199 1). The former two poIymorphisms cause an Ala to Thr substitution, and a Val to Met substitution, respectively.

G582A causes the 3' TAA STOP codon to become TAG, another STOP codon. In this study, the GMAgenotype fiom a patient suspected of having & gangliosidosis but

with normal Hex A and Hex B levels was characterized. The fibroblasts of the patient were

deficient in both Activator protein and mRNA. This patient's cells contained two different sizes of

cDNA detectable only by RT-PCR. A G160 to T transversion, causing a Glu54STOP mutation in

the exon 2 of the GM2A gene was found in the larger normal length cDNA, the smalIer cDNA

resulted from the in-frame deletion of exon 2. Further expenments indicated that the patient is

likely hornozygous for the Glu54STOP mutation. However, like the four previously reported

mutations, the possible presence of a second large GM2A deletion allele could not be cornpletely

ruled out.

Finally in the context of the novel GMZA nonsense mutation, 1 examine the controversy

(reviewed in (Maquat 1996)) surrounding the hypothesis that nonsense mutations promote the

skipping of the exon in which they are contained in order to re-estzblish the reading fiame (Dietz er

al. 1993; Mazoyer et aL 1998). This hypothesis would require the presence in the nucleus of a

mechanism for reading the fiame pnor to the splicing out of introns (reviewed in (Dietz and

Kendzior 1994)).

3.2 MATERIALS AND METHODS

3.2.1 Patient information

This patient was a boy with Laotian Hmong ancestry, Le. fiom a geographically isolated

small Laotian hi11 tribe, with no known parental consanguinity. At one-year age he had fiequent

seizures and delayed motor development. Motor seizures, myoclonic jerks, hyperacusis and

exaggerated startle response were seen during his second year. He was admitted to Valley

Children's Hospital in Fresno, California because of pneumonia when he was 2 1/2 years old.

Physical examination revealed that he had lower growth parameters, such as height and weight. 69

Ophthalmologic evaluation r~vealedbilateral macular cherry red spots. A spinal tap was perfomed and total gangliosides were found to be highly elevated. The patient's leukocytes and plasma were assayed for Hex A and Hex B activity and were found to be normal. Two brothers and one sister of the patient did not present with similar clinical symptom, nor was there any family history of individuals with a similar phenotype. Based on the above clinical phenotype and biochemical assay, he was diagnosed with the AB variant form of Gm gangliosidosis.

3.2-2 Cell lines and leukocvte sample

The patient's cultured fibroblast cells were sent fiom Dr. Cynthia Curing, Valley Children's

Hospital, Fresno, California. Two normal fibroblast cel! lines, referred to as N1 and N2, respectively, were obtained fiom the Tissue Culture Facility, Hospital for Sick Children. These fibroblast ce11 lines were grown with a-minimal essential media (crMEM) (Flow Laboratories) supplemented with 10% (v/v) fetal calf serum (FCS), 100 pg/ml streptomycin and 100 pghl penicillin. The normal leukocyte genomic DNA was obtained tiom the method described in chapter

2.

3.2.3 Western blot analvsis

The confluent fibroblast cells fiom N1 and N2, and the patient were harvested with

NaH2P04 lysis buffer (10 mM NaH2P04 with 5% glycerol) and subjected to 5 rounds of freeze- thawing in dry ice and 37OC waterbath, respectively. An equal volume of tetrachloromethane

(CC14) was added and mixed with lysate. After centrifugation, the upper layer, containing the ce11 lysate, was removed for protein assay. The concentration of protein in the lysates was determined using the Lowry method (Lowry et al. 195 1). Fi@ pg and 100 pg of normal and patient protein were mixed with the loading buffer (containing 20 mM DTT and 4% SDS), and boiled for 5 min. The proteins in each sarnple were separated by 12.5% SDS-PAGE and were transferred to a nitro- cellulose membrane (Amersham) over a 16 hour period (Brown et al. 1989). A nitro-cellulose membrane was blocked by exposure to 5% skim milk in Bovine Lacto Transfer Technique

Optimizer (BLOTTO, 10 mM of Tris, 150mM of NaCI, 0.05% of Tween 20, pH 7.5) for 4 hours, then incubated with a I:1500 dilution (1% skim milk in BLOTTO) of goat anti-glutathione-s- transferase/ Activator fusion protein antiserum (Xie et al. 1992). The membrane was washed 4 times for 30 min with 1% skim milk in BLOTTO and incubated with a 1:10,000 dilution (1% skim milk in BLOTTO) of donkey anti-goat IgG/ horse radish peroxidase conjugated for 1 hou. The membrane was then washed 4 times for 15 min with 1% skim milk in BLOTTO, quickly rinsed with BLOTTO (no skim milk), and dned between two filter papers. The membrane was incubated in Detection reagent 1 and 2 (Amersham ECL system) for exactly 1 min, dried briefly on filter papers, covered with Saran wrap, and exposed to Hyperfilm- ECL for 1 min.

3.2.4 Polv A' RNA isolation and Northem Blot analvsis

As the activator mRNA is very rare (Xie et ai. 1991), poly~+RNA must be used for

Northem blot analysis. ~oly~+RNA fiom normal and patient fibroblasts was isolated by the fast mRNA isolation kit (Invitrogen, Cat No. K1593-02). Four pg each of poly~'RNA was separated electrophoretically on a 1.0% agarose gel containing 2% formaldehyde and then transferred to a

Genescreen nylon membrane @EN life science). The probes used were radiolabelled with [a-32~] dCTP (Amersham) using the random primer labeling method (Gibco BELL). These probes, a -600 bp full length Activator cDNA obtained eom RT-PCR cloning product of a normal individual and a

-350 bp cDNA Fragment of cytochrome C oxidase complex IV (Cox6a) in pBluescript vector

(obtained fiom Dr. B. H. Robinson's lab), detected the Activator mRNA and the control mRNA of

Coxoa, respectively. Hybridizations were performed in rotating hybndization oven at 6S°C in a solution containing 100 ng of each probe (10'cpm), 0.5M Na2HP04, 7% SDS, 1% BSA, 50pg/mI salmon sperm DNA and lmM EDTA for 16 hours. The membrane was washed twice for half an hour with 30 mM Na2HP04 and 0.1% SDS at 6S°C. The membrane was fmally exposed to Kodak

X-OMAT AR film for 20 hours and the film developed.

3 -2.5 Total RNA isolation and RT-PCR

Total RNA was isolated fiom 150 mm diameter plates of confluent cultured fibroblasts fiom

NI, N2 and the patient based on the guanidinium thiocyanate method (Chomczynski and Sacchi

1987). Briefly, afrer rinsing with PBS, the cells were harvested in 4 ml denaturing solution (4 M guanidinium thiocyanate, 25 mM sodium citrate pH 7, 0.1 M p-mer~a~toethanoland 0.5%

Sarkosyl). After purification by phenop chloroform extraction, total RNA was precipitated by adding equal volumes of 100% isopropanol. The pellet was then dissolved in diethylpyrocarbonate

(DEPC)-treated H20.

Two primers, 5'-TTGGATCCCACCCTTCCCGATGCAG (# 1680, upstream, - 1S+6

(counting forrn first "A" of the Activator initiating ATG)), and 5'-GGATCCGTGGGA

GTTTGGCCTTGGC (#705, downstream, 666->648, BamH I sites are underlined) were designed for reverse transcription and PCR. Reverse transcription was performed in a total volume 100 pl with 2 pg of total RNA, 0.6 pg of primer #705, 0.5 mM each of dNTP, and 200 units of M-MLV transcriptase (Gibco BRL) in a 37OC waterbath for 1.5 hour. Twenty pl of these transcription solutions were used directly for PCR in Robocycler 40 (Stratagene). ARer a "hot start", 2.5 units of Taq DNA polymerase (Promega) were added in a 50 11 reaction volume with 1.25 pM each of primers #705 and #1680, 0.2 mM each of dNTP, and 1.5 mM MgC12- PCR was performed in 35 cycles each of 2 min at 94"C, 30 sec at 6L°C and 1 min at 72OC, with an addition final cycle of2 min at 94"C, 30 sec at 61°C and 5 min at 72°C. 3-2.6 Cloning; and sequencine of the normal and ~atientGE activator cDNA

Two different sizes of cDNA fiom the patient were separated by agarose gel electrophoresis

and purified by Gene Clean kit (BioICan scientific). One pl each of the cDNA products fiom the patient and 1 pl of fresh RT-PCR product fiom N 1 and N2 were mixed separately with 2 pl of PCR

2.1 vector, 1 pI T4 ligase hthe TA cloning kit (Invitrogen) and ligated at 14°C in a waterbath ovemight. One pl of the ligation reaction was transformed into iNVaF7 One Shot- competent

Ce11 (Invitrogen). White/ blue selection was carried out in 50 pg/ml ampicillin LB plates with the addition of X-Gal, and the white clonies were picked and grown in LB media containing 50 pdml of ampicillin. Isolation of the plasmid DNA was performed with Qiagen mini-preparation kit

(Qiagen). Digestion of 5 pl of the isolated plasmid DNA with BamH I was performed in order to confirm that the plasmid contained a cDNA insert.

The DNA sequences of each cDNA were determined by the dideoxy chain termination method (see chapter 2). In order to ensure the accuracy, the full length sequences of three independent clones of each sample were determined using both sense and antisense strands.

3.2.7 PCR amplification of genomic DNA fi-amnents containine; nucleotide 160

Isolation of genomic DNA fiom the patient and N1 fibroblasts was described in chapter 2.

Exon 1, exon 2 and their flanking regions of the patient's genomic DNA were arnplified with the first and second pain of primers in Table 2-2, using the same PCR parameters described in chapter

2. Each of the 528 bp PCR products from the patient and N1 was digested with endonuclease Nla

IVat 37OC for 2 hours and analyzed by electrophoresis on a 1% agarose gel. 3-2.8 Direct seauencine;

Each of the PCR products fiom the patient and normal control containing exon 1, exon 2 and

their flanking regions was purified with Qiagen PCR purification kit and directly sequenced with

Thermos Sequenase radiolabelled terminator cycle sequencing kit (Amersham), as described in chapter 2.

3.2.9 Deterrnination of the length of intron 1 & 2 of the GMZA gene from the vatient's genomic

DNA bv Ionn PCR

A fragment containing intron 1 of the GMZA gene was amplified fiom patient fibroblast

DNA with primer #2833 and #2834 by rTth XL DNA polymerase (see chapter 2). Another fiagment containing intron 2 of the GMZA gene was also amplified fiom the patient fibroblast DNA using primer #805 and #806 by Advantage Klen Taq Polymerase (chapter 2). Each of PCR products was digested with BamH 1, EcoR I and BamH 1 plus EcoR 1, then analyzed by electrophoresis on a 1 % agarose gel.

3.2.1 O Detection of the Activator mRNA lacking exon 2 in normal eenomic DNA

Based on the DNA sequences fiom GMZA, only a single Ninf 1 site exists in the cDNA encoding the Activator, and it resides in exon 2. Therefore, the fiil1 length cDNA can be cleaved by

Kinf 1, while any cDNA lacking exon 2 will remain intact afier digestion. Five pl of the RT-PCR products fiom N 1 and the patient sarnples (Fig 3-3) were digested with Hinf I in a total volume 50 pl at 3 7"C. Two primers, ATCGCCCTGGGCTTGCTT (#1438, upstream, 3 l+48) and

ACAAAACA GAGGAAAAGG (#1968, downstream 642+625), were used to amply the second round PCR. Nested PCR was performed with 1 pl/ 50 FI of the digested RT-PCR products or equal amounts of the undigested RT-PCR products, using 0.2 pM of each ofprimers, 1.O mM MgCl*, and 74

0.2 mM of each dNTP. The cycling panuneters were 10 min at 94OC for denaturation, 35 cycles

each of 30 sec at 94OC, 30 sec at 6I0C and 30 sec at 72OC, then 10 min at 72OC for elongation. Ten

ng of either the cloned wild type or the cDNA lacking exon 2, as well as a Hz0 sample, were

included as controls for the nested PCR amplification.

3.3 RESULTS

The initial diagnosis of the patient as having the AB-variant form of Gw gangliosidosis was

based on his clinical presentation coupled with normal levels of both Hex A and B in his leukocytes

and plasma (see Section 3.2.1 Patient information). Since the four previously descnbed GMZA

disease causing mutations resulted in undetectable levels of Activator cross-reacting material

(CRM) in patient's cells, 1 first analyzed the lysate fiom the patient's fibroblasts by Western

blotting. Whereas lysate from two normal control ce11 lines produced easily detectable immuno-

reactive bands corresponding to the expected Mr of 22,000 (mature form of the Activator), a similar

band was not detectable in lysate samples from the patient's cells containing similar levels of total

protein (Fig 3- 1). This observation confirmed the patient's diagnosis.

To determine the levels of Activator mRNA in the patient's cells, Northern blot analysis was

carried. The results from the positive control Cox6a probe indicated that the normal and patient

samples each contained the same amount of poly~' RNA. The Activator probe produced no

detectable signal in the lane containing the patient sample, in contrast to the lane containing the

normal sample where mRNA of the expected size was detected (Fig 3-2).

To determine if any RNA was transcribed fiom either of the patient's GMZA alleles, RT-

PCR was performed. Normal GM2A cDNA contains a 582 bp region encoding the Activator. In

this study, the PCR pnmers amplified a product containing nucleotide - 13 to 666, plus Fig 3- 1 Western blot analysis. The fibroblast lysate nom a normal individual with 175 G/G (Nl), a

normal individual with 175 NA(N2) and the patient (P) were included. Amount of Iysate

protein loaded in each sarnpIe lane is indicated below. GM2 activator

Fig 3-2. Northem blot analysis. Four pg PolyA RNA nom a normal individual and the patient was

analyzed. The blot was probed with 32~labeled cDNA encoding the Activator and Cox6a

(cytochrome C oxidase complex IV). N, fiom a normal individual; P, fiom the patient. 77

two BumHI sites. Thus the normal full-length RT-PCR product is 69 1 bp. The PCR product nom the patient sample contained two different molecular sizes of cDNA; the larger one corresponded to the normal full-Iength (N1 and N2) (Fig 3-3) and the smaller species was of 529 bp. However, the amount of the smaller cDNA was less than the larger size cDNA (Fig 3-3).

cDNA fragments from Nl, N2 and the two different size cDNA fragments fkom the patient cells were cloned, and their sequences were determined. The fùll-length sequence of the cDNA fiom N1 was determined, and the results were consistent with the previously published sequence

(Xie et al. 199 1). However, the cDNA fiom N2 contained a single G 175+A transition (Fig 3-4) which would encode a Va159IIe substitution in the Activator protein. More significantly the larger cDNA frorn the patient contained G 160 to T transversion in exon 2 (E3g 3-5) which converts the codon Glu54 to a STOP codon. The smaller cDNA was found to be missing exon 2 (AE2) (Fig 3-

6). At least one other clone of each cDNA fiom different RT-PCR products was sequenced with the same primer, and each sequence produced the same results. The complete sequence of each cDNA was determined and found to have no other changes.

Sequencing of the patient's larger cDNA demonstrated an early nonsense mutation was located in exon 2 of at least one allele. Sequencing of the smaller cDNA indicated that exon 2 was skipped which could be the result of a mutation at or near the exon l/ intron 1 or the intron l/ exon

2 junctions. Therefore, exon 1, exon 2 and their flanking regions were amplified and sequenced.

Because the G 160 to T transversion causes genomic DNA to [ose a normal NZa IVsite, each of the

528 bp fragments from the patient and N1 was digested by Ma IVand analyzed by agarose gel elcctrophoresis. The normal fragment (528 bp) contained three Ma IV sites and cleaved into four fragments (47 bp, 159 bp, 29 bp and 293 bp), while the fragment obtained from the patient (528 bp) produced only three hgments, 47 bp, 159 bp and 322 bp (Fig 3-7). Furthemore direct sequencing Fig 3-3. Reverse transcription and PCR analysis. RNA fiom the patient and two normal individuals was included. Arrow's point to the predicted normal size product (69 lbp) and unexpected smaller product fonned in sample from the patient (529bp). P, patient; N1, 175 G/G normal individual; N2, 175 G/A normal individual; M, h DNN HindiII marker. N2 NI GATCGA TC

Fig 3-4. Nucleotide sequence of cDNA fiom two normal individuals. A single G 175jA transition was noted. Patient Normal

G ATCGAT C

Fig 3-5 Nucleotide sequence of larger cDNA fkom the patient. A single G 160 to T transversion is noted. Normal Patient Normal (Exon2Exon3) (ExonlExon3) (ExonlExon2) GATC GA TC GATC

Fig 3-6. Nucleotide sequence of the smaller cDNA from the patient. Exon 2 is missing in the smaller cDNA of the patient. of the patient's exon 1 and its flanking region failed to detect any mermutations. Direct sequencing of the patient's exon 2 and its flanking region detected only the nonsense mutation in the homozygous form with no other sequence difference fiom that of the normal control (Fig 3-9).

This suggested that both of the patient's alleles likely contained the nonsense mutation.

Genomic DNA fiom N2 (containing G175A transition in its cDNA) was also directly sequenced and was confirmed to have the 175G-A transition presented in both alleles (Fig 3-8).

Interestingly, genomic DNA from another unrelated normal individual (Normal-3, N3) contained the 175 G/A in a heterozygous fom (Fig 3-8). In addition, hvo 175 NAhomozygotes were also detected from the genomic DNA of two normal individuals through PCR amplification and sequencing (data not show). These direct sequencing results indicated that 175 A is a common polymorphism in GM2A gene.

In order to determine whether the skipping of exon 2 in the smaller cDNA from the patient resulted from a partial deletion of GMZA gene, both intron 1 and intron 2 of the patient's GM2A gene were amplified in a single long PCR. The fragment obtained fkom the patient had the same apparent length as that obtained fkom a normal control and produced the same restriction digest pattern as the normal fiagrnent with both BumH I and EcoR I (Fig 3-10). These data suggested that the patient likely is homoygous for the nonsense mutation; however, the presence of a large deletion allele cannot be completely mled out.

Since the activator mRNA in the patient's poly~+RNA was undetectable by Northem blotting, 1 felt it was possible that a similarly low level of the AE2 mRNA might also be present in normal RT-PCR samples, Le. a naturally occumng altematively spliced form of mRNA, but because of the relatively high level of normal Activator mRNA in the controis, AE2 was not detectable. To test this theory 1 developed a nested PCR procedure specific for the AE2 product.

Since only one Hinf I site exists in normal GM2A cDNA (Fig 3-1 LA) and is located in exon 2, UDN PM

SWbp 3Wbp

1Wbp

Normal

Patient

Fig 3-7. (A) Digestion of exon 2 flanking region with NMV. PCR fkagments containing exon 2 and its flanking sequence from a normal individual and the patient were digested by NlolV. UD, undigested 528 bp PCR fragment; N, digestion of the normal fiagrnent; P, digestion of the fiagrnent from the patient; M, lOObp marker. (B) The digestion diagram of exon 2 flanking region with NiuiK The G 160T stop codon mutation in the patient causes Ioss of an NMV site. N2 N1 N3 NA175 G/G175 WA175 GATCGAT CGATC

Fig 3-8 Nucleotide sequence of a PCR arnplified region of the GM2A gene containing nucleotide

175 from three normal individuals (N 1, N2 and N3). Patient Normal GATC GAT C

Fig 3-9. Direct sequencing of PCR products from the patient's genomic DNA. Note that the patient

is apparently homozygous for the G 160 to T nonsense mutation. Fig 3-1 0 Digestion of intron I of the GMZA gene obtained by PCR with EcoiU and BarnWI:

Intron 1 of the GMZA gene from the patient and a normal individual were obtained by long

PCR and digested with EcoRl (E), BamH 1 (B) or both enzymes (E/B).U, undigested; M,

1 kb marker. digestion of the normal RT-PCR hgment with this enzyme should preclude nested-PCR amplification using primers # 1968 and #1438 (Fig 3-1 1B). Without digestion by Ninf 1, the second round PCR products of the normal sample contained predorninantly the fill-length product (lane 3 of Fig 3-12), cornpared with the products fiom the patient's sample (lane 4). However, afier the RT-

PCR products were digested by Hinf 1, both second round PCR products fiom the normal and patient's samples contained predominantly the AE2 fragment in about equal amounts (lane 5 and 6 of Fig 3-12). A small amount of the fûll-length fragment is also detectable (lane 5 and 6 of Fig 3-

12) reflecting the fact that restriction digestions are not 100% efficient (Valentine and Heflich

1997). Significantly, none of AE2 product was detected in the PCR products fiom cloned normal cDNA (lane l), indicating that AE2 cDNA obtained in the normal sample (lane 3, 5) is not due to contamination. This conclusion is strengthened by the negative Hz0 control (lane 7).

3.4 DISCUSSION

The patient showed a classic acute infantile fom of Gm gangliosidosis, but his Hex A and

Hex B levels were normal. In his fibroblast sample, there is no any detectable Activator CRM (Fig

3- 1) and no detectable GM2A mRNA (Fig 3-2). Thus the patient suffered from AB variant form of

GMIgangliosidosis.

Of the four previously reported mutations in the GM2A gene, two are missense mutations, one is a single codon deletion and the other one is a single nucleotide deletion causing a firame shifi and early termination (Schepers et al. 1996; Schr6der et al. 1993; Schroder et al. 1991; Xie et al.

1992). Al1 of these mutations have been located in exon 3 or exon 4 and still produce apparently full-length and normal levels of steady state GMZA rnRNA in patients' cells. In this study, the patient's Activator mRNA of normal size could only be detected through RT-PCR, but in addition a Widetype (one Hinf 1 site) RT-PCR 691bp 705 (=172bp+S19bp) .A1 80 1 1 1 1 nested PCR x4 613bp El E2 E3 Exon 2 skipping (no Hinf 1 site)

nested PCR 7 T 1834 1968 451bp El E3 E4

Fig. 3- 1 1 (A) Digestion of wild type cDNA and the exon 2-lacking cDNA (AES) by Hinf L Wide type cDNA and the exon 2-lacking cDNA (AE2) obtained form RT-PCR (Fig 3-3) are cleaved by Hinf 1. UD, undigested; Hinf 1, Hinf 1digestion; WT, wide type cDNA; AE2, cDNA missing exon 2; M, 100 bp DNA marker. (B) Digestion diagram for fkil length and AE2 cDNA by Hinf 1. Numbers with arrow indicate primers and their orientation. The length of the RT-PCR and nestedPCR product are indicated. One Hinf 1 site in exon 2 of the normal sample is shown with an arrow. RT-PCR product cDNA

Fig. 3-12 Nested PCR amplification of RT-PCR product from the patient and a normal individual. AE2 and WT refer to cDNA lacking exon 2 or containing exon 2, respectively. LJD, undigested; Hinf 1, Hinf 1 digestion prior to nested PCR; P, patient's RT-PCR product; N, normal individual's RT-PCR product; C, H20control; M, lOObp DNA marker. smaller cDNA species was seen at an even lower level (Fig 3-3). Nucleotide sequencing of the

DNAs uncovered a nonsense mutation at the codon for Glu 54 in exon 2 in the normal size cDNA and the inhrne deletion of exon 2 in the smaller cDNA (referred as AE2) (Fig 3-5'6). The Glu54 to

STOP codon mutation, which is located in exon 2, is the €kt nonsense mutation reported in the

GMZA gene. This mutation causes the loss of two thirds of the normal amino acids (54-194) fiom the Activator,

Since the RT-PCR results showed that there were two different sizes of cDNA in the patient's sample, it was suspected that the STOP codon mutation in one allele produced the normal length cDNA and a splicing junction mutation in the other allele produced the AE2 mRNA.

However, the experirnental data did not support this hypothesis. a) Direct sequencing (Fig 3-9) and restriction digestion (Fig 3-7) indicated that the patient was Iikely homozygous for the nonsense mutation. b) Abnormal altematively spliced mRNA is usually caused by nucleotide mutations in exon/ intron junctions (Lewin 1997) (Chapter 2). However, no mutations were found in these sites in intron Il exon 2 or exon 2/ intron 2 junction regions through direct sequencing. c) If the smaller

BE2 cDNA was produced fiom a second unidentified deletion allele, the deletion would have to encompass al1 of exon 2, and either >149bp of the 3' end of IVS 1 or >218bp of the 5' end of IVS 2, othewise it would have been detected by long PCR with the exonic primers or by conventional

PCR with the intronic primers I subsequently designed to ampl* exon 2 and its flanking sequences

(Chapter 2). It is unlikely that such a large deletion would result in the exact inframe deletion of only exon 2 from the allele's transcribed RNA product. d) Since no early STOP codon is present in the AE2 RNA, its stability should be similar to that of the wild type transcription product (reviewed in (Maquat 1996)). However, levels of AE2 RNA were much lower than that of normal RNA and even lower than that of the patient's RNA containing the early STOP codon. Thus the above data strongly indicate that the patient is likely homozygous for GluS4STOP mutation in his GA4ïA gene and the AE2 RNA is not transcribed by a second unidentified deletion allele. Nonetheless a near total deletion of the GM2A gene can not be completely ruled out, which would make the patient hemizygous for the nonsense mutation. This possibility has also not been ruled out for any of the other 4 patients previously described with AB variant Gm gangliosidosis.

Another observation also supports the likelihood that the patient is homozygous for nonsense mutation. The patient's family belongs to a deme, the Hmong, a small Laotian hi11 tribe, and it is unlikely that 2 GM2A mutant alleles would exist in such a small population. For example, the HEAB mutations responsible for Sandhoff disease (also a rare disease, but still much more common than the AB-variant) in an Argentinean deme have been characterized. It was concluded that there was only a single, novel, high fiequency, splice site mutation present in this population

(Kleiman et al. 1994).

Premahire STOP codons can be generated directly through a nonsense point mutation or indirectly through a Me-shift. The latter can be generated through abnormal mRNA splicing, deletions, or insertions. There are nurnerous cases of fiame-shift mutation causing GM2 gangliosidosis (reviewed in (Gravel et al. 1995) (Mahuran 1999)). Of those in the HE= or HEM) genes where the steady state mRNA levels have been reported, only 1 out of 13 have not been associated with a dramatically reduced amount of transcript (reviewed in (Gravel er al. 1995;

Mahuran 1999)). This Ione exception also produces the most C-terminal STOP codon, AC15 10 in exon 13 of 14 in HEX4, and results in the loss of only 22 residues (Zokaeem er al. 1987). In contrast to the effect of early STOP codons causing reduction of steady state levels of mutant mRNA, the 4 infiame deletion or insertion mutations that have been characterized in the HEXA and

HED genes al1 produce normal steady state levels of mutant mRNA (reviewed in (Mahuran in press)). An example of this type of mutation is the major HEXA mutation among Japanese Tay- Sachs patients, a '@t" substitution in intron 5 resulting in the inhedeletion of exon 6 (Tanaka et al. 1993). Based on the above case reports, in-hune deletion usually results in normal steady state levels of mutant &A. In my study, there is no detectable -A in patient's sampfe as determined by Northern Biot; thus it is unlikely that the AE2 mRNA (an in-fiame deletion) is transcribed dominantly in either allele.

There has been a great deal of previously reported data concluding that shortened reading frames, i.e. early STOP codons, can iead not only to mRNA instability, but also to the inframe skipping of the constitutive exon in which the mutation is found (Dietz and Kendzior 1994; Dietz et ai. 1993; Mazoyer et al. 1998; Ronce et al. 1997). There remains a controversy over whether this is caused by a mechanism in the nucleus that can sense the lack of an open reading frarne and affect normal splicing (Maquat 1996). In this study, RT-PCR results indicated that in a normal sample only fbll length mRNA was produced, while in the patient's sample both fiill-length mRNA and

AE2 mRNA was produced, the former one containing G160T (Glu54 to STOP codon) nonsense mutation in exon 2. At this level it would seem my results are consistent with the conclusion that a nonsense mutation induces exon skipping to restore the reading fiame. However, nested PCR amplification of RT-PCR products suggested that AE2 cDNA also existed in normal samples (Iane 3 of Fig 3-12). This conclusion was confirmed by digestion of RT-PCR product with an exon 2- unique restriction endonuclease, Hinf l, and nested PCR amplification (Iane 5, 6 of Fig. 3-12)

(Valentine and Heflich 1997). Digestion of RT-PCR products with Hinf l eliminated most of the full-length of cDNA (Fig 3-1 l), thus AE2 cDNA become the predominant form of cDNA and was amplified equally in both normal and patient samples by the second nested PCR. From this data it can be concluded that AE2 GM2A mRNA indeed exists in the normal fibroblasts.

Sirnilar study has been reported in the hypoxanthine phosphoribosyl transferase (hprt) gene by Valentine and Heflich (Valentine and Heflich 1997). They exarnined a homozygous nonsense mutation associated with exon skipping in hprr mRNA of Chinese hamster ovary cells and concluded that the apparent increase in exon skipping was a RT-PCR artifact. Tnis artifact was due to the instability and thus, very low steady state levels, of the normal size RNA containing the nonsense mutation, coupled with the normal stability of the smaller AE-RNA in which the nonsense mutation had been deleted and the reading t'rame restored. This small AE-RNA was found to be constitutively produced at very 1ow levels in normal as wetl as mutant cells. However it could only be amplified to a level of detectability in normal ceils afier the removal of the hl1 length, wild type

RT-PCR-generated cDNA by digestion with a specific restriction enzyme, followed by a second nested PCR.

In summary, as premature STOP codon mutation usually causes rapid mRNA degradation

(Muhlrad and Parker 1994), the infiame skipping of the constitutive exon, in which the STOP codon mutation is found as detected by RT-PCR, is due to a decreasing abundance of the full-length mRNA (Maquat 1995; Valentine and Heflich 199?), not due to a nuclear mechanism that causes the increasing abundance of the AE mRNA species. Thus this study indicates that there is likely no mechanism in the nucleus that can read the fiame of precursor mRNA and affect final splicing events as previously reported (Dietz and Kendzior 1994; Dietz et al. 1993).

1 would like to thank Dr. C.-C. Hui for his help in Northem blot analysis and Ms. Amy

Leung for her technical assistance in Western blot analysis. 3.6 REFERENCES

Brown CA, Neote K, Leung A, Gravel RA, Mahwan DJ (1989) introduction of the alpha subunit

mutation associated with the B 1 variant of Tay-Sachs disease into the beta subunit produces

a beta-hexosarninidase B without catalytic activity. J.Biol.Chem. 264:2 1705-2 1710

Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium

thiocyanate-phenol-chloroform extraction. Anal-Biochem. 162: 1 56- 159

Dietz HC, Kendzior RJ, Jr. (1994) Maintenance of an open reading hmeas an additional level of

scrutiny during splice site selection. Nat Genet 8: 183-8

Dietz HC, Valle D, Francomano CA, Kendzior RJ, Jr., Pyentz RE, Cutting GR (1993) The skipping

of constitutive exons in vivo induced by nonsense mutations. Science 259:680-3

Gravel RA, Clarke JTR, Kaback MM, Mahuran D, Sandhoff K, Suzuki K (1995) The GM~

gangliosidoses. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic basis of

inherited disease. McGraw-Hill, New York, pp 2829-2879

Kleiman FE, De Kremer RD, De Ramirez AO, Gravel RA, Argaraiia CE (1994) Sandhoff disease in

Argentina: High fiequency of a splice site mutation in the HEXB gene and correlation

between enzyme and DNA-based tests for heterozygote detection. Hum Genet 94:279-282

Lewin B (!997) Genes VI. Oxford University Press, Oxford, England, pp 885-920

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (195 1) Protein measurement with the Folin phenol

reagent. J.B iol.Chem. 193 :265-275

Mahuran DJ (1999) Biochernical consequences of mutations causing the GM2 gangliosidoses.

Biochem Biophys Acta :in press

Maquat LE (1995) When cells stop making sense: effects of nonsense codons on RNA metabolism

in vertebrate cells. Rna 1:453-65 Maquat LE (1996) Defects in RNA splicing and the consequence of shortened translationai reading

&es [editorial] [see comments]. Am J Hum Genet 59:279-86

Mazoyer S, Puget N, Perrin-Vidoz L, Lynch HT, Serova-Sinilnikova OM, Lenoir GM (1998) A

BRCA 1 nonsense mutation causes exon skipping [letter]. Am J Hum Genet 62:7 13-5

Muhlrad D, Parker R (1 994) Prernature translational termination triggers mRNA decapping. Nature

370578-58 1

Schepers U, Glombitza G, Lemm T, Hoffmann A, Chabas A, Ozand P, Sandhoff K (1996)

MoIecuIar analysis of a GM2 activator deficiency in two patients with GM2 gangliosidosis

AB variant. Am. J.Hum.Genet. 59: 1048- 1O56

Schroder M, Schnabel D, Hurwitz R, Young E, Suzuki K, Sandhoff K (1993) Molecular genetics of

GM2 gangliosidosis AB variant: a novel mutation and expression in BHK cells. HumGenet.

W:43 7-440

Schroder M, Schnabel D, Suzuki K, Sandhoff K (1991) A mutation in the gene of a glycolipid-

binding protein (GM2 activator) that causes GM2 gangliosidosis variant AB. FEBS letters

290: 1-3

Valentine CR, Heflich RH (1997) The association of nonsense mutation with exon-skipping in hprt

mRNA of Chinese hamster ovary cells results fiom an artifact of RT-PCR.Rna 3:660-76

Xie B, McInnes B, Neote K, Larnhonwah A-M, Mahuran D (1991) Isolation and expression of a

full-length cDNA encoding the human GM~activator protein. Biochem. Biophys. Res.

Commun. 177: 12 17-1223

Xie B, Wang W, Mahuran DI (1992) A Cysl38-to-Arg substitution in the GM2 activator protein is

associated with the AB variant fonn of GM2 gangliosidosis. Am.J.Hum.Genet. 50: 1046-

1 OS2 %

Zokaeem G, Bayleran I, Kaplan P, Hechtman P, Neufeld EF (1987) A shortened f3-hexosaminidase

a-chain in an Italian patient with infantile Tay-Sachs disease. Am. J. Hum. Genet. 40537-

547 CHAPTER IV

FUTURE WORK The exon 2-lacking cDNA (AE2) fkom the GMZA gene has been cloned in this sîudy.

Because AE2 mRNA also exists in normal fibroblasts, it is possible that the AE2 mRNA can translate a shorter protein, which lacks residues between Pro28 and Lys80 (referred as the AE2 activator). This protein would have the signal peptide intact but the single N-linked giycosylation site would be missing. The question to be answered is whether or not the AE2 activator has a physiological function in any hurnan tissue. Therefore, the AE2 cDNA will be expressed in E coii in order to obtain a large amount of the AE2 activator for antibody production, and to determine if its lipid transport and Hex A binding firnctions still exist. [t will also be transfected into mammalian cells to elucidate the biosynthetic and intra/ inter cellular transportation pathway in virro. Since the level of alternative splicing may Vary in different tissues if it is biologically sipificant, it will also be necessary to determine the level of the AE2 mRNA in a series of other human tissues.

4.1 Expression of exon 2-lacking activator in E coli

The wild type and AE2 cDNAs inserted into pBluescript obtained in this study wilt be subcloned into an E coli expression vector, pQE-12 with 6xHis tag (Qiagen) (Klima et al. 1993).

After expression in E coli, the fusion protein will be purified on Ni-NTA resin (Qiagen) and analyzed by Western blot analysis (method in Chapter 2). If no CRM were detected using our present anti-Activator antibody, it would indicate that the AE2 activator has lost the epitopes recognized by the present anti-Activator antibody. In this case a specific polyclonal antibody against the AE2 activator will be produced. Briefly, the purified and refolded AE2 activator will be mixed with the Freunds adjuvant and injected into rabbits. After two booster injections, the anti-

AE2 activator IgG will be isolated frorn rabbit serum (Harlow and Lane 1988). The anti-AE2 activator antibody will be used to detect the AE2 activator in CHO cells transfected with AE2 cDNA.

4.2 Testing the lipid binding function of the exon 2-lacking activator

The localization of hydrophobic binding site of the Activator has not been fblly elucidated, and some controversy still exists in the literature. Li and colleagues suggested that the hydrophobic binding site is located in residue 34-142 (Nagarajan et al. 1992; Wu et al. 1996), however, fluorescent dequenching assay in our iaboratory demonstrated that the hydrophobic binding site is located in the C-terminus of the Activator (Smiljanic-Georgijev et al. 1997). In order to confinn the

Iocalization of hydrophobic binding site of the Activator, the AE2 activator obtained fkom E cati expression cm be used for dequenching assay. In this assay, the AE2 activator protein, wild type activator and a tnincated negative control will be tested for their ability to bind and transport R-18

(Smiljanic-Georgijev et al. 1997) (chapter 1). If this functional test is positive for AES, Gm gangiioside will be added to the Iiposome mixture to test whether it inhibits the fluorescence dequenching of the AE2 activator. If both functions are normal, it will localize the carbohydrate and lipid binding sites to the C-terminus of the Activator. Given positive results in these assays, the

AE2 activator will be tested as a CO-factorfor Hex A. Negative R-18 transport results will argue against any biological significance for the alternatively spliced transcript.

4.3 Establishing a permanently exon 2-lacking cDNA transfected CHO cell line

The methods used for transfection of AE2 cDNA into CHO cells is based on those previously reported (Rigat et al. 1997). Bnefly, the AE2 transfection fragment will be obtained through PCR amplification of AE2 cDNA with specific primers, and will be ligated with pEF-NE0 vector to form a constnict, pEF-NEO-AE2. pEF-NEO-AE2 will be transfected into CHO cells using 100 lipofectamine (Gibco BRL). The cells will be rnaintained in selection media containing G418.

After a control line of cells completely dies out due to G418 processing, some of the remaining transfected cells and culture medium will be analyzed by Western blotting using anti AE2 antibody

(see section 4.1). If no CRM is seen with anti AE2 activator antibody in transfected cells or media, two possibilities may exist. L) AE2 mRNA is not being transcribed; thus Northern blot analysis will be performed to detect AE2 mRNA. 2) The AE2 RNA is transcribed but the protein is rapidly degraded in ER. Pulse-chase experiments will be used to determine if this is true. if the latter is true, the AE2 is likely not a physiologically significant protein. If CRM with anti-activator or anti-

AE2 activator is detected, the localization of the AE2 activator can be determined by immunofluroscence (Hinek et al. 1996).

4.4 Determination of the levels of exon 2-lacking mRNA in variant tissue

In this study a smail amount of AE2 mRNA was detected in fibroblasts, thus it rnay also exist in other tissues and cells, perhap in larger amounts. Total mRNA corn other tissues, e.g.

Iiver, kidney, intestine, and muscle, will be isolated. Afier reverse transcription, the primers #1680 and #705 (Chapter 3) will be used to ampli& mRNA. The RT-PCR products will be cleaved by exon 2-unique restriction enzyme, Hinf 1, and the second round of nested PCR will be applied based on the method described in chapter 3. The poly (A) RNA will also be isolated from different tissues. Northem blot analysis will be applied to more quantitatively detect any AE2 mRNA with a specific AE2 probe in tissues that appear by RT-PCR to produce larger amounts than do fibroblasts. 4.5 REFERENCES

Harlow E, Lane D (1988) Antiiodies - A laboratory manual. Cold Spring Harbor Laboratory, Cold

Spring Harbor, New York, USA

Einek A, Molossi S, Rabinovitch M (1996) Funçtional interplay between interleukin-1 receptor and

elastin binding protein regulates fibronectin production in coronary smooth muscle cells.

Exp. CeIl Res. 225:122-13 1

Klima H, Klein A, van Echten G, Schwacpnatm GySuzuki K, Sandhoff K (1993) Over-expression

of a functionally active human GM2 activator protein in Escherichia coli. E3iochem.J.

29257 1-576

Nagarajan S, Chen H-C,Li S-C, Li Y-T,Lockyer SM (1992) Evidence for two cDNA clones

encoding human Gm-activator protein. Biochem J. 2822307-8 13

Rigat B, Wang W, Leung A, Mahuran DJ (1 997) Two mechanisms for the recapture of extracellular

Gm2 activator protein: evidence for a major secretory form of the protein. Biochemistry

36:8325-833 1

Smiijanic-Georgijev N, Rigat B, Xie B, Wang W, Mahuran D (1997) Characterization of the

affinity of the GM2 activator protein for glycolipids by a fluorescence dequenching assay.

Biochim. Biophys. Acta 1339: 192-202

Wu YY, Sonnino S, Li Y-T, Li S-C (1996) Characterization of an alternatively spliced GM2

activator protein, GMîA protein. J.Biol.Chem. 27 1: 106 1 1-1 O6 15