My supervisory committee members: Dr. Ann Herscovics and Dr. Ted Meighen for their guidance and good ... Rachel Harnmonds. Michael Prokaziuk, and Sheri.
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Analysis of Chimeric Human Hexosaminidases
By
Emmanuelle Denis
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Department of Biology McGill University. Montreal November. 2000
A thesis submitted in partial fultillment of the requirements for the degree of
Master of Science
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© Emmanuelle Denis. 2000
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ABSTRACT
The major f3-hexosaminidase isozymes in humans are Hex A (deficient in Tay-Sachs disease, TSO), an af3 heterodimer and Hex B (deficient in Sandhoff disease) a
pp
homodimer. Hex S, the aa homodimer is physiologically unstable. Mature a and subunits share 60% sequence identity. The
p
p subunit active site hydrolyzes neutral
substrates. The (l subunit active site hydrolyzes neutral (4MUG) and charged substrates (4MUGS, G M2 ganglioside). Only Hex A hydrolyzes the natural substrate, G M2 ganglioside, in the presence of the G M2 activator protein (AP).
We investigated regions of the
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(l
and
p subunits involved in AP binding. subunit
dimerization, and substrate specificity. We constructed four chimeric cONAs: (l1-259~h92-5+4, (l1-118P 152-5+4,
P1-IISa 387-529. and f3 1-151(l119-25CJP292-544 (subscripts refer to
amino acid residues). Chimeric cDNAs were expressed in a TSO neuroglial cellline. which produces no endogenous a subunits. The chimeric isozymes were chromatofocused and assayed for hydrolysis afa) 4MUG.
b)
4MUGS and c) GM2
ganglioside.
Transfection of the cONA constructs lead to expression of homodimeric and heterodimeric chimeric proteins, albeit at lower yields than transfection of wild a-cONA. Ali of the chimeric proteins hydrolyzed 4MUG but none \vere active to\vards 4MUGS or GM 2 ganglioside. These results suggest that a) ail constructs contained sufficient •
information to form both heterodimeric and homodimeric chimeric proteins b) the
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chimeras Iacked the a-subunit sequence necessary for the hydrolysis of charged substrates.
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RÉSUMÉ
Chez les humains. les principaux isozymes de rhexosaminidase sont la Hex A (qui est déficiente dans les cas de la maladie Tay-Sachs (MTS). un hétérodimer aa et Hex B (qui est déficient dans les cas de maladie Sandhoft), un homodimer
pp.
Le Hex S, le
homodimer au' est physiologiquement instable. La séquence des sous-unités matures est identique à 60%. La sous-unité a. hydrolise les substrats neutres. La sous-unité (3 hydrolise les substrats neutres (4MUG) et chargés (4MUGS. ganglioside G~u). C'est seulement la Hex A. en présence de la protéine activatrice (PA) G M2 • qui hydrolise le substrat naturel, ganglioside G M2 · Nous avons examiné les régions des sous-unités a et J3 impliquées dans la liaison par protéine activatrice (PA). la dimérisation des sous-unités et
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la spécificité des substrats.
Nous avons construit quatre cAONs chimériques: PI-4IS a 387-529,
UI-25lJP2IJ:-S+h UI-IISPI52-544,
et ~1-15IaI19-259P292-544 (les numéros représentent la position des
aminoacides). par moyen d'une technique de fusion génique à base de peRo Les cAONs ont été exprimés dans une lignée cellulaire neurogliale provenant de la MTS. qui ne produit pas de sous-unités a endogènes. Les isozymes chimériques ont été chromatofocalisés et analysés pour leur capacité d'hydroliser: (a) le 4MUG. (b) le 4 MUGS et (c) les gangliosides
G~12.
La transfection des constructions cADN donne l'expression des protéines chimériques
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homodimériques et hétérodimériques, bien qu'à des quantités moindres que celles
iii
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produites par la transfection des a-cAON du type sauvage. Toutes les protéines chimériques hydrolisaient le 4MUG mais aucune ne se montrait active envers le 4MUGS ou les gangliosides
GM2.
Ces résultats suggèrent que (a) toutes les constructions
contenaient suffisamment d'infonnation pour former les protéines chimériques hétérodimériques aussi bien qu'homodimériques et que (b) les chimères manquaient la séquence a. nécessaire pour hydroliser les substrats chargés .
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ACKNO~EDGEMENTS
1wouId like to thank the tollowing individuaIs whose support and assistance made this thesis possible: Dr. Peter Hechtrnan and Dr. Feige Kaplan. my co-supervisors. for their guidance and encouragement. Their vast scientific knowledge and witty repartee helped ta make my graduate studies experience not only educational but entertaining~ as weIl. Special thanks ta Dr. Hechtman for helping me ta write this thesis and ta Dr. Kaplan for her aid in the preparation of my poster for the 1999 ASHG conference.
My supervisory committee members: Dr. Ann Herscovics and Dr. Ted Meighen for their guidance and good advice. Dr. Don Mahuran for providing the AP-pQE-9 vector used to produce His-tagged AP. Paulo Cordeiro. my partner in crime, for listening to my whining and putting up with my mess without ever complaining. Gail Dunbar for her excellent tissue culture training.
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Bernard Boulay for his invaluable technical assistance. AlI of the other wonderful people in our lab whose helpful advice. friendship and support kept me going: Bev Ackerman. Angéline Boulay, Claudia Carriles. Julie Comber. Hannah Hoag. Andrew Jones. Ferhat Kassamali. Pierre Ledoux. Tara Macreae. Lynne Prevost. Huguette Rizziero. Shannon Ryan, Yves Sabbagh. Christineh Sarkissian, Pierre Savoie, Saeed Teebi. Paula Waters, Dan Wilson. Ping Zhao and many others. My wonderful parents and my Nana whose encouragement. prayers and Turtle deprivation helped to keep me on track. Marna. an extra-special thanks for ail ofyour help and support and for translating my abstract. Ali ofmy incredible. \vonderful and patient friends: Lisa Bracegirdle. Sandrine Campeau. Melissa Clark. Kelly Davison. Rachel Harnmonds. Michael Prokaziuk, and Sheri Zernentsch. Keith Marchand for keeping me laughing and for showing me that there is litè beyond science.
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TABLE OF CONTENTS
Abstract Résumé Acknowledgements. . . . . . . . . . .. . . . . . . . . . . .. . . . . .. . .. . . . .. . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . ... Table of Contents............................................................................ Abbreviations List of Figures 1. INTRODUCTION 1.1. Lysosomal storage disorders and the G M2 gangliosidoses 1. 1.1. Historical background 1.2. Biochemistry and genetics of the G M2 gangliosidoses 1.2.1. Gangliosides 1.2.2. The hexosarnindase isozymes 1.2.3. GM2 activator protein 1.3. Mutations causing the GM2 gangliosidoses 1.4. Diagnosis of the G M 2 gangliosidoses 1.5. Clinical features and pathology of the GM2 gangliosidoses 1.6. Treatment of the G M2 gangliosidoses 1.7. Research Project Objectives
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i iii v VI
viii IX
1
,.
1 3 3 5 7 8 Il 13 14 17
2. MATERIALS AND METHODS 2.1. Construction of chimeric cDNAs 22 2.1.1. Construction of UI,2J33,4 25 26 2.1.2. Construction of al P2.3,4 2.1.3. Construction OfPI.:UU4 , 27 2.1.4. Construction of P l.2a3,4 28 2.1.5. Construction of f3la2f33,4 ,. 28 2.2. Cloning of chimeric constructs into the pCMV vector 29 2.3. Expression and purification of recombinant activator protein 30 2.4. Purification OfG M2 ganglioside 32 2.5. Transfection of chimeric cDNA constructs into NG 125 neurogliaI cells .. 33 2.6. Chromatofocusing of transfected cell Iysates 34 ? 7 E d ' _. . nzyme an protern assays -,) 2.8. Western blot analysis 36 2.9. GM2 ganglioside hydrolysis assay 36 "'1-
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3. RESULTS 3.1. Construction of chimeric cDNAs 3.2. Expression and purification of recombinant activator protein 3.3. Expression of chimeric cDNA constructs in NG 125 neuroglial cells ., 3.4. Chromatofocusing oftransfected celllysates 3.5. Activity of chimeric hexosaminidases towards synthetic substrates 3.6. G M2 ganglioside hydrolysis assay
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38 39 39 41 46
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4. DISCUSSION 4.1. Expression of chimeric proteins 4.2. Substrate specificity of chimeric proteins 4.3. Subunit dimerization 4.4. Summary ofresearch and future prospects
51 52 54 55
5. REFERENCES
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ABBREVIATIONS
AP
G M2 Activator Protein
BSA
Bovine serum albumin
C/M
Chioro torm/Methano1
GaINAc
{l- D-N-acety19a1actosamine
GleNAc
p-D-N-acetylglucosamine
Hex
p-N-acetylhexosaminidase
HSA
Hunlan serum albumin
IPTG
isopropylthio-p-galactoside
MCB
Membranous curvilincar bodies
4MUG
4-methylumbelliteryl-f3-N-acetylglucosaminide
4MUGS
4-methylumbell iteryl-GIcNAc-6-sulfate
NANA
N-acetylneuraminic acid
PBS
Phosphate buffèred saline
SD
Sandhoffs Disease
TBS
Tris buffered saline
TCA
Trichloroacetic acid
TLC
Thin-layer chromatography
TSD
Tay-Sachs Disease
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LIST OF FIGURES
Figure 1: Structure of GM:! ganglioside.
4
Figure 2: Madel for the lysosomal degradation of G M2 ganglioside.
9
Figure 3: Schematic representation of fusion constructs.
18
Figure 4: Schematic illustration of PCR overlap extension technique.
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Table 1: PCR primers used to generate chimeric constructs.
24
Figure 5: SOS-PAGE analysis of His-AP purification.
40
Table 2: Expression of Hex chimeras in NG 125 cells
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Figure 6: Chromatofocusing of untransfected and pCMVa transfected NG f 25 extracts.
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Figure 7: Chromatofocusing ofNG 125 extracts transfected with auf33A and PI.2.J ap > aa. However. when the a-
subunit is greatly overexpressed (such as when NG 125 neuroglial cells are transfected with a-subunit cDNA. see Figure 6), aa dimers predominate. We hypothesized that a region of the p-chain contains a dimerization motif or signal, which if inserted into the a-subunit sequence would drive the dimerization reaction to produce homodimers rather than heterodimers. All of the constructs tested produced a higher ratio of heterodimers to homodimers than \Vas the case for the control transfection (\vild-type a-subunit cDNA) (Figures 6. 7. 8). This result is not unexpected since, as shown in Table 2~ aIl mutant constructs produced fewer Hex subunits than did
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the wild-type transfection. In other wards, the expression level of the chimeras was not
54
high enough to drive the formation of chimeric homodinlers and the fusion proteins •
instead dimerized preferentially with the native p-subunits. Nevertheless. there were no signifieant differences observed in the ratios of homodimers to heterodimers bet\veen the four ehimeric transfections. This. despite the faet that eaeh of the four segments of the p-ehain were present in at least one construct and eaeh of the six possible pairings of 13 segments \Vere represented in at least one of the
possessed the p-subunit dinlerization motif we would have expected to see a shift to the fomlation ofmore chimeric homodimers. Il is very likely. therefore. that the stronger dimerization signal of the f3-chain is a non-linear array of umino-acid residues which is distributed on different regions of the polypeptides.
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4.4.
Summary of research and future prospects
We have succeeded in generating four chimeric cDNA constructs. which can be expressed in TSO neuroglial cells to yield chimeric hexosaminidase heterodimers and homodimers. Ail of our tùsion proteins had 4MUG activity but none were active toward 4MUGS. the negatively charged synthetic substrate. We were unable to test the chimeras for activator protein binding because our assay was dependent on having an intact asubunit active site (i.e. the ability to hydrolyze negatively charged substrates). None of the chimeric constructs showed an increased ratio of homodimers to heterodimers. indicating that none possess a stronger dimerization motif. Ail of the possible pairings of
•
p-subunit regions were represented in the eonstructs suggesting that that the stronger
55
dimerization signal of the fl-chain is non-linear and distributed on different regions of the •
polypeptides. Other groups have aiso done experiments with chimeric human hexosaminidases and their results suggest sorne possible avenues for future research. As mentioned in section 4.2. comparison of the activity of the Fusion 2 construct (Tse et al.. 1996) with our a1.2fl3.4 construct identified two a-subunit praline residues (Pro:!68 and Pro l71 ) which warrant tùrther investigation. Site-directed mutagenesis cauld be llsed to change their flsubunit counterparts. LYS:!88 and Lys291 ta prolines. together and individually. These aitered subunits could then be tested for 4MUGS and G M 2 ganglioside activity. Similarly. these lysine residues could be changed ta pralines in the al.:!J33A construct ta see ifthis conferred the ability to hydrolyze negatively charged substrates.
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Pennybacker el al created a construct. ap 1 (comprised of a-subunit amino acids 1 to 191 fused with
p amino acids 225 to 556) which hydrolyzed GM2 ganglioside in the
presence of activator protein when it was co-expressed with wild-type a-subunit (Pennybacker et al.. 1996). This construct is similar ta our a1.2(33A chimera which is composed of a-subunit amino acids 1 to 259 tùsed \vith J3 amino acids 292 to 556. It would be interesting ta do a similar co-transtèction \Vith al .2J33A and wild-type asubunits. However. such an experiment could not be pertormed in NG 125 neuroglial cells due ta the presence of native. human J3-subunits. It would be very difficult to separate Hex A. formed fonn the transfected a-subunits and native J3-subunits from chimeric aI.2J33.4/a heterodimers. Instead~ a baculovirus systenl (which has no
•
endogenous Hex) such as that described by Pennybacker el al might he employed.
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Reference List Acorazza~ H.D., Fla.x.l.D.. Snyder, E.Y. and Jendoubi. M. Expression ofhuman bhexosaminidase a-subunit gene (the gene detèct of Tay-Sachs disease) in mouse brains upon engraftment of transduced progenitor cells. Nature ,\;fedicine 2:424-429. 1996.
Arpaia. E.• Dumbrille-Ross. A.. Maler~ T.~ et al. Identification of an altered splice site in Ashkenazi Tay-Sachs disease. lVature 333:85-86, 1988. Bayleran~ 1.. Hechtman. P. and Saray, W. Synthesis of 4-methylumbelliferyl-beta-D-Nacetylglucosamine-6-sultàte and its use in classification of GM2 gangliosidosis genotypes. C/in.Chim.Acta 143 :73-89. 1984.
Bierfreund. U.. Kolter. T. and Sandhof[ K. Sphingolipid hydrolases and activator proteins. i\l/ethods En=ymol. 31 1:255-276. 2000. Burg, J.• Conzelmann. E.. Sandhoff. K.. Solomon. E. and Swallow. D.M. Mapping of the gene coding for the human GM2 activator protein to chromosome 5. Annal.\· ofHuman Genelics 49:41 -45. 1985. Chavany, C. and Jendoubi. M. Biology and potential strategies for the treatment ofGM2 gangliosidoses. [Review] [36 rets]. Mo/ecu/ar l'v1edicine Today 4: 158-165. 1998.
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Chen, B., Rigat, B.. Curry. C. and Mahuran. D.1. Structure of the GM2A gene: identification of an exon 2 nonsense mutation and a naturally occurring transcript with an in-frame deletion ofexon 2. Am .J. Hum. Genel. 65:77-87. 1999. Chern. C.l.. Kennett. R.. Engel. E.. Mellman. W.l. and Croce. C .M. Assignment of the structural genes for the alpha subunit of hexosaminidase A. mannosephosphate isomerase. and pyruvate kinase ta the region q22-qter of hunlan chromosome 15. Somalie Ce!! Genelics 3:553-560. 1977. Conzelmann. E., Burg. J.• Stephan. G. and Sandhoff. K. COl11plexing of glycolipids and their transfer between membranes by the activator protein t{)r degradation of lysosomal ganglioside GM2. ElIr.J. Biochem. 123 :455-464, 1982. Conzelmann. E., Kytzia. [-1.1.. Navon, R. and SandhotI K. Ganglioside GM2 N-acetylbeta-D-galactosaminidase activity in cultured tïbroblasts of late-intàntile and adult GM2 gangliosidasis patients and of healthy probands with law hcxosanlinidase level. Am .J Hum. Genet. 35:900-913. 1983. Conzelmann. E. and Sandhoff. K. AB variant of infantile GM2 gangliosidosis: deficiency of a factor necessary for stimulation ofhexosaminidase A-catalyzed degradation of ganglioside GM2 and glycolipid GA2. Proc. Nat!. Aead. Sei. [!.SA. 75:3979-3983. 1978.
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Dana, S.L. and Wasmuth.l.J. Selective linkage disruption in hunlan-Chinese hampster cell hybrids: deletion mapping on the leuS. hexB. emtB. and chr genes on human chromosome 5. Mo/ecular & Cellular Biochemislry 2: 1220- 1228. 1982.
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Erzberger, A .. Conzelmann. E. and Sandhoff, K. Assay of ganglioside GM2-N-acetylbeta-D-galactosaminidase activity in human fibroblasts employing the natural activator protein--diagnosis of variant forms of GM2 gangliosidosis. Clin.Chim.Aeta 108:361 -368. 1980. Fernandes, M.J.. Yew. S.. Leclerc. D.. et al. Identification of candidate active site residues in lysosomal beta-hexosaminidase A. J Biol. Chem. 272:814-820. 1997. Gilbert, F.. Kucherlapati. R.. Creagan, R.P., Mumane. M.J .. Darlinton. G.J. and Ruddle. F.H. Tay-Sachs' and Sandhoff Diseases: The Assignment of Genes tor Hexosaminidase A and 8 to Individual Human Chromosomes. Proe.1Vall.AeadSci. U.S.A. 72:263-267. 1975. Gravel, R.A .. Clarke. J.T.R.. Kaback, M.M.. Mahuran. D.. Sandhoff. K. and Suzuki. K. The G M2 Gangliosidoses. In: The :'vIelabolie Basis oflnherited Disease. edited by Scriver. C.R.. Beaudet. A.L .. Sly. W.S. and Valle. D. New York: tvfcGra\v Hill. 1995. p. 28392879. Griffiths, G., Hoflack. B.. Simons. K.. Mellman. 1. and Korntèld. S. The mannose 6phosphate receptor and the biogencsis of lysosomes. Ce Il 52:329-341. 1988. Guidotti, J.E.. Mignon. A.• Haase. G.. et al. Adenoviral genc therapy of the Tay-Sachs disease in hexosaminidase A-deficient knock-out mice. flwllan i\loleeular Genelïes 8:831-838. 1999.
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Hanai. N.. Norest. G.A.. MacLeod. C., Torres-Mendez. C.-R. and Hakomori. S. Ganglioside-mediated modulation of celi growth - specifie effects ofG M3 and lyso-G~B in tyrosine phosphorylation of the epidermal gro\vth factor receptor. .J.Biol. Chem. 263: 10915-1 0923. 1988. Hasilik. A.. Waheed. A. and von Figura. K. Enzymatic phosphorylation of lysosomal enzymes in the presence of UDP-N-acetylglucosamine. Absence of the activity in I-cell fibroblasts. Biochemic.:al & Biophysical Researeh Communications 98:761-767 ~ 1981. Hechtman. P. Characterization of an activating factor required tor hydrolysis of Gm2 ganglioside catalyzed by hexosaminidase A. Canadian Journal (~lBiochemisI1Jl 55 :315324. 1977. Hechtman. P. and Kachra. Z. Interaction of activating protein and surfactants \vith human liver hexosaminidase A and GM2 ganglioside. Bioehemic.:alJournal 185:583-591. 1980. Hoffman, L.M., Amsterdam. D.. Brooks. S.E. and Schneck. L. Glycosphingolipids in fetal Tay-Sachs disease brain and lung cultures. J1Vcurochem. 29:551-559. 1977.
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Hoffman, L.M .. Brooks. S.E.. Stein, M.R., Adachi, 1\11. and Schneck, L. Gangliosides in SV-40-transfonned cells derived from Tay-Sachs disease retal brain. Arfetaholic Brain Disease 4:87-93, 1989.
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Horton. R.M.. Caio Z .. Ho. S. and Pease. L. Gene splicing by overlap extension: Tailormade genes using the Polymerase Chain Reaction. Bio Techniques 8:528-535. 1990. Hou. Y.. Tse. R. and Mahuran. O.J. Direct determination of the substrate specificity of the alpha-active site in heterodimeric beta-hexosaminidase A. Biochemistry 35:39633969, 1996. Huang, J.Q.. Trasler. J.M.. Igdoura, 5., Michaud, J.. HanaL N. and Grave!. R.A. Apoptotic cell death in mouse models of GM2 gangliosidosis and observations on human Tay-Sachs and Sandhoff diseases. Human Nlolecular Gene/ies 6: [879-1885. 1997. Hubbes. M.. Callahan. J.. Gravel. R. and Mahuran. O. The amino-terminal sequences in the pro-alpha and -beta polypeptides of human lysosomal bcta-hexosaminidase A and 8 are retained in the nlature isozymes. FEBS Lel/ers 249:316-320. 1989. Iber. H.. van Echten. G. and Sandhoff, K. Fractionation of primary cultured cerebellar neurons: distribution of sialyltransferases involved in ganglioside biosynthesis. Journal ofNeurochemistry 58: 1533-1537. 1992. Igdoura. S.A.. Mertineit. C .. Trasler, J.M. and Gravel. R.A. Sialidase-mediated depletion ofGM2 ganglioside in Tay-Sachs neuroglia ceIls. Human .vIolecula,. Genetics 8:11111116. 1999.
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Kaback. M.. Miles. J.. Yaffee. M.. et al. Hexosaminidase-A (Bex A) deticiency in early adulthood: a new type of 01\,12 gangliosidosis. Am J Hum. Genet. 30:31 A 1978. Klima, H.. Klein. A.. van Echten. G.. Schwarznlann. G .. Suzuki. K. and Sandhoff. K. Over-expression of a functionally active human GM2-acti\'ator protein in Escherichia coli. Biochemical Journal 292:571-576. 1993. Komfeld, S. Trafficking of lysosomal enzymes in normal and disease states. .J.CIin./nvesl. 77: 1-6. [986. Kytzia. H.J. and Sandhoff. K. Evidence for two different active sites on hUlnan betahexosaminidase A. Interaction of GM2 activator protein with beta-hexosaminidase A. J.Biol.Chem. 260:7568-7572. 1985. Leaback. D.H. and Walker. P.G. The fluorometric assay ofN-acetyl- b-glucosaminidase. Biochemical Journal 78: 151-156. 1961. Li, S.C., Hirabayashi. Y. and Li. Y.T. A protein activator tor the enzymic hydrolysis of GM2 ganglioside. J. Biol. Chem. 256:6234-6240. 1981. Li't S.C. and Li. Y.T. An activator stimulating the enzymic hydrolysis of sphingoglycolipids. .J. Biol. Chem. 251: 1159-1163. 1976.
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Li, S.C.. Wu. Y. Y., Sugiyama. E.• et al. Specifie recognition ofN-acetylneuraminic acid in the GM2 epitope by human GM2 activator protein. .J.BioI.C'hem. 270:24246-24251 . 1995. Li, Y.T.• Li, S.C.. Hasegawa. A.. et al. Structural basis tor the resistance of Tay-Sachs ganglioside GM2 to enzymatic degradation. .l.Biol.Chem. 274: 10014-10018, 1999. Li. Y.T.• Mazzotta, M.Y.. Wan. C.C., Ortho R. and Li. S.C. Hydrolysis of Tay-Sachs ganglioside by b-hexosaminidase A ofhuman liver and urine. J.Biol.Chem. 248:75127515,1973. Little. L.E., Lau. M.M.. Quon. D. V.. Fowler. A. V. and Neuteld. E.F. Proteolytic processing of the alpha-chain of the Iysosomal enzyme. bera-hexosaminidase. in nonnal human fibroblasts. Journal ofBiological Chemistry 263:4288-4292. 1988. Mahuran. D. and Lowden. J.A. The subunit and polypeptide structure ofhexosaminidases from human placenta. Canac/ian Journal ofBiochemistlY 58:287-294. 1980. Mahuran. D.. Novak. A. and Lowden, J.A. The lysosomal hexosaminidase isozymes. [Review] [220 refs]. Isozymes Cur,.. Top. Biol. Afed Res. 12:229-288. 1985. Mahuran, D.J. The GM2 activator protein. its roles as a co-factor in GM2 hydrolysis and as a general glycolipid transport protein. [Review] [110 rets 1. 8iochimica el Biophysica Acta 1393: 1-18. 1998.
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Mahuran, D.J. Biochemical consequences of mutations causing the GM2 gangliosidoses. [Review] [239 refs]. Biochimica el Biophysica Acta 1455: 105-138. 1999. Martinsson. A.. Raal. A. and Svennerholm. L. Isolation of N-acetylsialic acid l'rom normalliver. 8iochimica et Biophysica Acla 24:604-609. 1957. Myerowitz. R. Splice junction mutation in sorne Ashkenazi Jews \vith Tay-Sachs disease: evidence against a single defect within this ethnic group. Proc.iVatl.AcadSci. U.S.A. 85 :3955-3959. 1988. Myerowitz. R. and Costigan. F.C. The major defect in Ashkenazi Jews \Vith Tay-Sachs disease is an insertion in the gene for the alpha-chain of beta-hexosaminidase. .J.Biol.Chem. 263: 18587-18589. 1988. Myerowitz. R. and Hogikyan. N.D. Different mutations in Ashkenazi Jewish and nonJewish French Canadians with Tay-Sachs disease. Science 232: 1646-1648. 1986. Myerowitz, R.. Piekarz. R.. Neuteld, E.F .. Shows. T.B. and Suzuki. K. Human bhexosaminidase a chain: Coding sequence and homology \Vith the b chain. Proc.NatI.Acad.Sci. U.S.A. 82:7830-7834, 1985.
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Navon. R., Argov, Z. and Frisch. A. Hexosaminidase A deficiency in adults. Am J.MedGenet. 24: 179-196, 1986.
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