Vol. 177, No. 3, 1991 June 28, 1991
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ISOLATIONANDEXFRESSIONOFA FULL-LENGTHCDNA ENCODINGTHEHUMANGM~ ACTIVATOR PROTEIN Bei Xie*t, Beth McInnes*, Kuldeep Neote*, Anne-Marie Lamhonwahs,and Don Mahuran*tl *The ResearchInstitute, Hospital For Sick Children, 555 University Ave., Toronto, Ontario, CanadaM5G 1X8 tThe Department of Clinical Biochemistry, University of Toronto, Toronto, Ontario, Canada $The ResearchInstitute, McGill Children’sHospital, Montreal, QuebecCanada Received
May 15,
1991
We report the constructionof a cDNA clone encodinga functional GMZ-activator protein. The sequenceof the complete5’ endof the coding region was determinedby direct nucleotide sequencingof a fragment generatedby multiple RACE PCR proceduresfrom Hela cell cDNA. Specific oligonucleotideswere synthesizedfrom thesedata which allowed usto producea PCR fragment that containedthe completecoding sequenceof the protein. This was then clonedinto a mammalianexpressionvector. The ability of purified hexosaminidase A (P-Nacetylhexosaminidase,EC 3.2.1.52) to hydrolyse labeledGM~ gangliosidewas enhancedlo-fold more by the addition in the assaymix of lysate from transfectedCOS-1 cellsthan by the additionof identical amountsof lysate from mock transfectedcells. Direct sequencingof PCR fragmentsfrom two sourcesalsoidentified three polymorphisms. 0 1991Academic Press,Inc.
Hydrolysis of GMT ganglioside(GM~) to GM~ gangliosideinvolves the removal of the plinked non-reducingterminal GalNAc residue. This reaction requiresthe participation of three separategeneproducts. Two of theseare the (Ysubunit (encodedby the HEXA genemappedto chromosome15) and p subunit (encodedby the HEXE genemappedto chromosome5) of the lysosomalhydrolaseP-hexosaminidase A. The third is a smallso-called“GM;?-activator” protein which transportsGM~ from the lysosomalmembraneto hexosaminidaseA for hydrolysis. The critical in viva role that is played by the activator protein is demonstratedby the occurrenceof a rare autosomalrecessiveform of GM~ gangliosidosis,the AB variant. Patientswith this disease have normal hexosaminidase A levels, but do not synthesizea functional activator protein (reviewed in (1)). Full length cDNA clonesencodingthe a and p subunitsof hexosaminidasehave been isolated(2,3) and their identity confirmed by expressionof hexosaminidase activity in COS cells (4,5). There is only one report of a partial cDNA encodingthe activator protein (6). This cDNA included the deducedsequenceof the matureamino-terminusof the purified activator protein (as
1To whom correspondenceshouldbe addressed. 0006-291X/91
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determined by the authors (7)), however, it lacked the complete signal peptide and the initiation codon and therefore could not be expressed. Using antibodies to their purified activator protein these authors mapped the activator gene to chromosome 5 (8). More recently, Gilliam and colleagues (9) have investigated the possibility that mutations at the activator locus could result in spinal muscular atrophy, also mapped to chromosome 5 (10). Using PCR technology they determined that the fragments generated by two sets of primers corresponding to the published cDNA sequence (6) did not map to chromosome 5 (9). In order to confirm that the published cDNA sequence does indeed encode the GM~activator protein we have isolated a cDNA containing the complete coding sequence, as judged by in-frame stop codons at the 5’ and 3’ ends, and have expressed it in monkey COS-1 cells. Our results demonstrated a consistent IO-fold increase in the ability of purified hexosaminidase A to hydrolyse [~~GMz in the presence of lysate from transfected cells as compared to mock transfected cell lysates. This confirms the identity of the cDNA.
cDNA synthesis Total RNA from Hela cell was isolated by the guanidinium-isothiocyanate method (11) followed by CsCl centrifugation. Reverse transcription was performed in a total volume of 50& in the presence of 6Fg RNA, 2U RNAguard (Pharmacia), O.lM Tris @H 8.3), 0.14M KCl, 1OmM MgC12,28mM P-mercaptoethanol, OSmM each of dNTPs, and 0.5pg oligonucleotide (either oligo dT, 730# (below) or 731# (Fig. 1)). The mixture was incubated with 5U AMV reverse transcriptase (BRL) at 42eC for 60min. l/50 of the synthesized cDNA was used for each PCR. FCR Amplification PCR was performed on cDNA or on an aliquot of a human retinal cDNA library (in Xgt 10, obtained from Dr. Jeremy Nathans) using the conditions recommended by Perkin-Elmer Cetus Corporation using AmpliTaq in a 5Ol.tL reaction volume. Attempts were made to obtain the 5’ end of the activator by PCR of the library (titering 1x10** PFUlmL, usinglul of the library for each PCR). The PCR was performed using the primer complementary to the 3’ end of the hgt 10 vector (Clontech, Cat. No. 541 l-2) and activator specific oligo 731# (shown in Fig. 1) in 30 cycles each of 30s at 94oC, 30s at 58oC, and 90s at 72cC. A second PCR was performed using 1 pL of the first PCR product with the 3’ primer and a second activator specific oligo 732# (Fig. 1) utilizing the same cycling conditions. A 300 bp “middle” fragment encoding additional 44 nucleotides at the 5’ end compared to the published sequence, was obtained. However, no ATG was encoded by this extra sequence. Another tiOObp fragment was also generated by PCR from the cDNA library utilizing two activator specific oligos 733# and 166# (fig. 1). Both the 5’ and middle PCR fragments were sequenced directly and some polymorphisms were detected. RACE FCR The 5’ end of the GM2 activator cDNA was obtained by the method of Frohman et al (12, 13). The tailed cDNA was synthesized with activator specific oligo 73 l# from Hela Poly (A)+ RNA (prepared using the Fast-Track kit, Invitrogen). Activator oligo 732# and a nonspecific oligo 730# (CG-CGAGATCTTTTTTTTTTTT), complementary to the poly (A)+ track and containing a BamH I “anchor” sequence, was used in PCR for 30 cycles each of lmin at 93oC, lmin at 55oC, and 3min at 72eC. 1pL of the 730#/732# PCR product was used in another PCR with activator specific oligo 602# (Fig. 1) and oligo 730# using same incubation conditions. A ~240 bp fragment which contained two ATGs and an in-frame STOP codon in its 5’ end extension was obtained. Direct Sequencing In order to avoid detection of PCR artifacts (due to Taq errors) which can be contained in individual cloned PCR fragments, we directly sequenced the products of each PCR. PCR 1218
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fragments were isolated by agarose gel electrophoresis and purified with Geneclean Kit (Bio 101). The gel-purified cDNA was sequenced with 5OOng of oligo 602# as a primer, using a method of direct sequencing adapted from the Sequenase protocol (United States Biochemical Corp.) (14). Cloning the GM2 activator cDNA The complete coding region of the GM~ activator was cloned following PCR amplification of the cDNA synthesized with oligo dT from Hela total RNA with two specific oligonucleotides, oligo167# (sequence determined from the direct sequencing of the 5’ end of the 240 bp RACE fragment) and oligo166# (Fig. 1). As the nucleotides at the -3 position of both ATGs at the 5’ end are not A or G and thus do not match Kozak’s rules for efficient translation initiation (15), an “A” was introduced in oligo 167# to change the nucleotide at position -3 of the first ATG from “c” to “A” for more efficient expression (Fig. 1). The G7OObp fragment from 30 cycles of PCR was subcloned to the BamH I site of Bluescript vector (Stratagene). The insert of one of the clones confirmed to contain the correct nucleotide sequence, was subcloned into pSVL-B for expression in COS-1 cells. Prediction of the Cleavage Site of the Activator Signal Peptide The probability of the presence of a signal peptide and the position of its cleavage site was assessed using a weighted matrix method of von Heijne (16). A computer program based on this method for use with the Apple Macintosh was kindly provided to us by Michael Richards. Expression of the cDNA encoding the Activator Protein COS-1 monkey kidney cells (American Type Culture Collection) were maintained in CLminimum essential media (Flow Laboratory) containing streptomycin and penicillin (lOOmg/L), and 10% fetal bovine serum at 37oC in 5% CO2. The 700 bp PCR fragment containing the complete coding sequence of the activator was subcloned into a mammalian expression vector, pSVL (Pharmacia), to yield pAct1. 2Opg of pAct1 was then transfected into monkey COS-1 cells using the calcium phosphate procedure as previously described (17). The identity of the translation product from pAct was confiied utilizing purified hexosaminidase A and GM~ tritiated in the terminal P-linked GalNAc residue (18). 2OOm of [3HGalNAc]GM2 (30,000 dpmnmole) in 80 mM citrate buffer, pH 4.1, containing 0.5% w/v human serum albumin, was incubated with 105 units (nmoles of 4-methylumbelliferyl-~-Nacetylglucosamine hydrolysetihr) of purified placental hexosaminidase A (19) at 37oC for 18 hr. in the presence of varying amounts of total protein from transfected and mock transfected COS-1 cell lysates (total assay volume 1OOpL). GM2 hydrolysis was measured as the amount of [THJGaWAc released with time (20).
RESULTS
Sequence of the GM~ activator cDNA Two 5’ cDNA fragments, sl6Obp and s24Obp were obtained by RACE PCR from Hela cDNA. The sl6Obp fragment was 58 nucleotides longer at the 5’ end than the previously published data and encoded two ATGs. However, no 5’ in-frame STOP codon was found in this shorter 5’ sequence. The sequence of the longer 240bp fragment revealed the expected in-frame STOP codon in its 5’ end extension (Fig. 1). Using the weighted matrix method of von Heijne (16) to analyze the deduced ammo acid sequence of the activator, an amino-terminal signal peptide was strongly predicted with the most probable cleavage site between Ala23 and His24 (Fig.1 hatched underline). Activator-specific
PCR amplification of the human retinal library failed to produce a
fragment containing a candidate full length 5’ end. However, direct sequencing of the most 5’ fragment and a middle fragment revealed three polymorphisms when compared with the sequences obta&d from a) the RACE procedure, b) the previously published partial cDNA clone (6), and c) that deduced from the protein sequence of the purified activator (7). 1) The nucleotide at position 1219
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-60 -90 TTT CTT TGC GTA ACC AAT ACT GGA AGG CAT TTA AAG GAC CTC TGC CGC CTC AGA CCT TGC #167 A -30 BaxmL AGT TAA CTC CGC CCT GAC CCA CCC TTC LCG Stop leu arg pro asp pro pro phe pro 31/11 ATC GCC CTG ile ala leu r,,,//ll///.//l/I/Il.,,,, 91/31 CTC AGT AGC leu ser ser
GGC TTG CTT CTC GCG gly leu leu leu ala
TTT TCC TGG GAT AAC phe ser trp asp am
l/l > ATG CAG TCC CTG ATG CAG GCT CCC met gin ser leu met gin ala pro ,1/////1//////,/,/,//l//l////// #602 A 61/21 GCC CCT GCG CAA GCC CAC CTG AAA AAG CCA ala pro ala gin ala his leu lys lys pro P ,/,//II//,//./l 121/41 TGT GAT GAA GGG AAG GAC CCT GCG GTG ATC cys asp glu gly lys asp pro ala val ile
CTC CTG leu leu
TCC CAG ser gin
AGA AGC arg ser
A 181/61 C CCC ATC GTC GTT CCT GGA AAT GTG ACC CTC AGT GTC GTG leu thr leu glu pro asp pro ile val val pro gly am val thr leu ser val Y&L met 241/81 211/71 AGC ACC AGT GTC CCC CTG AGT TCT CCT CTG AAG GTG GAT TTA GTT TTG GAG AAG GAG ser thr ser val pro leu ser ser pro leu lys val asp leu val leu glu lys glu
GGC gly
GTG val
271191 301/101 GCT GGC CTC TGG ATC AAG ATC CCA TGC ACA GAC TAC ATT GGC AGC TGT ACC TTT GAA CAC ala gly leu trp ile lys ile pro cys thr asp tyr ile gly ser cys thr phe glu his 361/121 331/111 TTC TGT GAT GTG CTT GAC ATG TTA ATT CCT ACT GGG GAG CCC TGC CCA GAG CCC CTG CGT phe cys asp val leu asp met leu ile pro thr gly glu pro cys pro glu pro leu arg 391/131 421/141 ACC TAT GGG CTT CCT TGC CAC TGT CCC TTC AAA GAA GGA ACC TAC TCA CTG CCC AAG AGC thr tyr gly leu pro cys his cys pro phe lys glu gly thr tyr ser leu pro lys ser 4 #731 #-l32 451/151 481/161 GAA TTC GTT GTG CCT G~CTG GAG CTG CCC AGT TGG CTC ACC ACC GGG AAC TAC CGC ATA glu phe val val pro asp leu glu leu pro ser trp leu thr thr gly am tyr arg ile 511/1?1 541/181 GAG AGC GTC CTG AGC AGC AGT GGG AAG CGT CTG GGC TGC ATC AAG ATC glu ser val leu ser ser ser gly lys arg leu gly cys ile lys ile
GCT GCC ala ala
571/191 601 G AAG GGC ATA TAA CAT GGC ATC TGC CAC AGC AGA ATG GAG CGG TGT GAG GM lys gly ile Stop
stop 631 TCC 691 TCT 751 GGG 811 CTG
#166
fifil
TCT GTT TTG TGT TTG CCA AGG CCA AAC TCC 721 ACA GTG AGT CCA CTA CCC TCA CTG AAA ATC 781 CAA GCA GCC CTG ACC TAA GGG AGA ATG AGT 841 CTG GGC TGA CCA CGT TX TCA TCC CCG TTA
TCT CTA ser leu
GGT ccc
TTT
a CAC TCT CTG CCC CCC TTT AAT CCC CTT ATT TTG TAC CAC TTA CAT TTT AGG CTG TGG ACA GTT CTT GAT AGC CCA GGG CAT ACA TTC TCT CTA RAG AGC CT
Figure 1. Nucleotide and deduced ammo acid sequence of the human @2 activator protein (the “A” of the first ATG is labeled as nucleotide 1 and the corresponding “met” as ammo acid 1). The previously reported partial sequence began at our nucleotide #46 (6) The various oligonucleotides used in this study and their 5’ to 3’ direction of extension are shown as bold arrows. The single mismatch in oligo #167 to produce a more efficient translation initiation sequence is shown above the underlined wild type nucleotide. BamH I anchors were added to oligonucleotides #I67 and #166 to facilitate cloning of the FCR fragment The predicted signal peptide sequence is indicated by a hatched underline (residues I-23). The amino-terminus of the mature protein (7) is double underlined. The three polymorphisms identified in this study are shown above the corresponding nucleotide in the cloned sequence, which was expressed in transfected COS cells. The change in the deduced sequence resulting from these polymorphisms is also indicated below the deduced ammo acid. 1220
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Protein
Figure. 2. The effects of increasing amounts of lysate protein from two independent transfections of COS cells with pActI (Transfection 1 and Transfection 2), on the ability of purified hexosaminidase A to hydrolyse the labeled GalNAc residue from GM~ ganglioside. These arecompared to the effect of identical amounts of protein from the lysate of mock
transfected cells(COS(-)).
55 (Fig. 1) is “G”, which is different from the previously publisheddata (“A”) (6), and changes Thrl9 to Ala in the predicted signalpeptidesequence(Fig. 1). This “G” wasfound in both of the directly sequencedzl60bp RACE PCR fragment and in the &OObp PCR Send fragment from the retina cDNA library asdescribedin “MATERIALS AND ~~ETHODS”, andin the sequence of the clone that we expressedcontaining the activator’scoding region. 2) A “G582” to “A” substitution wasalsofound in the STOP codon at the 3’ end of the coding region in the clone (which still produceda STOP codon), but not in the z5OObpPCR fragment from the retina library. 3) Finally, the nucleotideat position 205 wasfound to be “A” in the PCR fragment from the retina cDNA library insteadof “G” aswasreportedin the previously publishedpartial sequenceandalso found in our clone from Hela cDNA containing the coding region. This resultsin a Valgg to Met substitution (Fig. 1). Interestingly, Met was alsofound at this position when the purified activator protein was sequenceddirectly (7). This data is consistentwith the highly polymorphic nature of the activator coding sequence(21). Expressionof the @,,,fzactivator The nucleotide sequencebetweenoligos 167#and 166#in Fig 1 is the sequenceobtained from the cDNA clone of GM~ activator usedfor expressionin COS cells. Fig 2 showsthe difference in the ability of lysatesfrom transfectedandmock transfectedcells to enhancethe hydrolysis of IsHGalNAc]GMz gangliosideby purified hexosaminidaseA. Two independent transfectionexperimentswere carried out and a seriesof assaysperformedusingincreasing amountsof total lysate protein for each. The enhancementof hexosaminidase A activity was linear with respectto the amountof lysate protein addedto eachreaction. Lysatesfrom COS cells transfectedwith pAct1 consistentlyproduceda lo-fold better enhancementof activity than identical amountsof lysatesfrom mock aansfectedcells (Fig. 2).
DISCUSSION
Expressionof a functional GM~ activator in transfectedCOS cells both confirms the identity of the previously publishedpartial cDNA, anddemonstratesthat we have isolateda cDNA 1221
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that contains the complete coding sequence of the protein. The deduced sequence predicts a prepro-polypeptide chain of 20,808 daltons (residues l-193), a pro-polypeptide of 18,463 daltons (residues 24-193) and a mature chain of 17,531 daltons (residues 32-193). Since the protein requires N-linked glycosylation for transport to the lysosome (reviewed in (22)), the activator must be glycosylated at its single Asnbg-Val-Thr
site. These predicted polypeptide chain weights plus
the effect of an oligosaccharide on the apparent weight determined by SDS-PAGE, are consistent with previously published biosynthetic data indicating a 24 kDa precursor which is processed to its 22 kDa mature lysosomal form (23). Our data indicate that the activator mRNA must be relatively rare. We were forced to use a “nested’ RACE procedure in order to generate a single PCR fragment from Hela cDNA. Further, we screened several cDNA libraries for the presence of an activator cDNA using two specific oligonucleotides without success (data not shown). Kozak has shown that while more than 50% of 211 translation initiation sequences analyzed match three or four of the nucleotides in the consensus sequence, CCIA or GICCATG 5’ to the ATG, only 6 failed to have a purine at the -3 position (15). The rareness of the activator mRNA coupled with the poor match to the translation initiation consensus at either of the 5’ ATGs in the native sequence, TCCCGATG or CCCTGATG, suggests that the protein is either very long lived or is rarer than has previously been reported (24). The three variations in the nucleotide sequences we have found in this study confirm that the activator protein is highly polymorphic. Previously we found two polymorphisms in Southern blot analyses using a 300bp coding region fragment as a probe (21). This may reflect the fact that the activator performs no actual catalytic function. Thus, its structuremay be more flexible to evolutionary changesthan would be an enzyme. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Sandhoff, K., Conzelmann, E., Neufeld, E.F., Kaback, M.M., and Suzuki, K. (1989) In The Metabolic Basisof Inherited Disease(C.V. Striver, A.L. Beaudet, W.S. Sly, and D. Valle, eds.), pp. 1807-1839,McGraw-Hill, New York. Komeluk, R.G., Mahuran, D.J., Neote, K., Klavins, M.H., O’Dowd, B.F., Tropak, M., Willard, H.F., Anderson, M.-J., Lowden, J.A., and Gravel, R.A. (1986) J. Biol. Chem. 261, 8407-8413. Myerowitz, R., Piekarz, R., Neufeld, E.F., Shows, T.B., and Suzuki, K. (1985) Proc. Natl. Acad. Sci. USA 82, 7830-7834. Tanaka, A., Ohno, K., Sandhoff, K., Maire, I., Kolodny, E.H., Brown, A., and Suzuki, K. (1990) Amer. J. Hum. Gen. 46,329-339. Brown, CA., Neote, K., Leung, A., Gravel, R.A., and Mahuran, D.J. (1989) .I. Biol. Chem. 264,21705-21710. Schroder, M., Klima, H., Nakano, T., Kwon, H., Quintem, L.E., GBi-tner, S., Suzuki, K., and Sandhoff, K. (1989) FEBS. Lett. 251, 197-200. Furst, W., Schubert, J., Machleidt, W., Meyer, H.E., and Sandhoff, K. (1990) Eur. J. Biochem. 192,709-714. Burg, J., Conzelmann, E., Sandhoff, K., Solomon, E., and Swallow, D.M. (1985) Ann. Hum. Genet. 49, 41-45. Kleyn, P.W., Brzustowicz, L.M., Wilhelmsen, K.C., Freimer, N.B., Miller, J.M., Munsat, T.L., and Gilliam, T.C. Neurology in press. Brzustowicz, L.M., Lehner, T., Castilla, L.H., Penchaszadeh,G.K., Wilhelmsen, K.C., Daniels, R., Davies, K.E., Leppert, M., Ziter, F., Wood, D., Dubowitz, V., Zerres, K., Hausmanowa-Petrusewicz,I., Ott, J., Munsat, T.L., and Gillian, T.C. (1990) Nature 344,540-541. 1222
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Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., and Rutter, W.J. (1979) Biochemistry. 18,5294-5299. Frohman, M.A. (1990) In PCR Protocols (M.A. Innis, D.H. Gelfand, J.J. &in&y, and T.J. white, eds.), pp. 28-38, Academic Press, Inc., San Diego, Calf. Frohman, M.A., Dush, M.K., and Martin, G.R. (1988) Proc. Natl. Acad. Sci. (USA) 85, 8998-9003. Winship, P.R. (1989) Nucleic Acids Res. 17, 1266-1266. Kozak, M. (1984) Nucleic Acid Research. 12, 857-872. von Heijne, G. (1986) Nucleic Acids Res. 14,4683-4690. Rutherford, M.N., Hannigan, G.E., and Williams, R.G. (1988) Embo J. 7, 751-759. Novak, A., Lowden, J.A., Gravel, Y.L., and Wolfe, L.S. (1979) J. Lipid Res. 20, 678680. Mahuran, D.J., and Lowden, J.A. (1980) Can. J. Biochem. 58, 287-294. Novak, A., and Lowden, J.A. (1980) Can. J. Biochem. 58, 82-88. Bapat, B., Bei, X., Mahuran, D., and Ray, P.N. (1991) Nucleic Acids Res 19, 683-683. Kornfeld, S. (1990) Biochemical Society Transactions 18, 367-374. Burg, J., Banerjee, A., and Sandhoff, K. (1985) Biol. Chem. Hoppe-Seyler 366, 887891. Banerjee, A., Burg, J., Conzelmann, E., Carroll, M., and Sandhoff, K. (1984) Biol. Chem. Hoppe-Seyler 365, 347-356.
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ISOLATIONANDEXFRESSIONOFA FULL-LENGTHCDNA ENCODINGTHEHUMANGM~ ACTIVATOR PROTEIN Bei Xie*t, Beth McInnes*, Kuldeep Neote*, Anne-Marie Lamhonwahs,and Don Mahuran*tl *The ResearchInstitute, Hospital For Sick Children, 555 University Ave., Toronto, Ontario, CanadaM5G 1X8 tThe Department of Clinical Biochemistry, University of Toronto, Toronto, Ontario, Canada $The ResearchInstitute, McGill Children’sHospital, Montreal, QuebecCanada Received
May 15,
1991
We report the constructionof a cDNA clone encodinga functional GMZ-activator protein. The sequenceof the complete5’ endof the coding region was determinedby direct nucleotide sequencingof a fragment generatedby multiple RACE PCR proceduresfrom Hela cell cDNA. Specific oligonucleotideswere synthesizedfrom thesedata which allowed usto producea PCR fragment that containedthe completecoding sequenceof the protein. This was then clonedinto a mammalianexpressionvector. The ability of purified hexosaminidase A (P-Nacetylhexosaminidase,EC 3.2.1.52) to hydrolyse labeledGM~ gangliosidewas enhancedlo-fold more by the addition in the assaymix of lysate from transfectedCOS-1 cellsthan by the additionof identical amountsof lysate from mock transfectedcells. Direct sequencingof PCR fragmentsfrom two sourcesalsoidentified three polymorphisms. 0 1991Academic Press,Inc.
Hydrolysis of GMT ganglioside(GM~) to GM~ gangliosideinvolves the removal of the plinked non-reducingterminal GalNAc residue. This reaction requiresthe participation of three separategeneproducts. Two of theseare the (Ysubunit (encodedby the HEXA genemappedto chromosome15) and p subunit (encodedby the HEXE genemappedto chromosome5) of the lysosomalhydrolaseP-hexosaminidase A. The third is a smallso-called“GM;?-activator” protein which transportsGM~ from the lysosomalmembraneto hexosaminidaseA for hydrolysis. The critical in viva role that is played by the activator protein is demonstratedby the occurrenceof a rare autosomalrecessiveform of GM~ gangliosidosis,the AB variant. Patientswith this disease have normal hexosaminidase A levels, but do not synthesizea functional activator protein (reviewed in (1)). Full length cDNA clonesencodingthe a and p subunitsof hexosaminidasehave been isolated(2,3) and their identity confirmed by expressionof hexosaminidase activity in COS cells (4,5). There is only one report of a partial cDNA encodingthe activator protein (6). This cDNA included the deducedsequenceof the matureamino-terminusof the purified activator protein (as
1To whom correspondenceshouldbe addressed. 0006-291X/91
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determined by the authors (7)), however, it lacked the complete signal peptide and the initiation codon and therefore could not be expressed. Using antibodies to their purified activator protein these authors mapped the activator gene to chromosome 5 (8). More recently, Gilliam and colleagues (9) have investigated the possibility that mutations at the activator locus could result in spinal muscular atrophy, also mapped to chromosome 5 (10). Using PCR technology they determined that the fragments generated by two sets of primers corresponding to the published cDNA sequence (6) did not map to chromosome 5 (9). In order to confirm that the published cDNA sequence does indeed encode the GM~activator protein we have isolated a cDNA containing the complete coding sequence, as judged by in-frame stop codons at the 5’ and 3’ ends, and have expressed it in monkey COS-1 cells. Our results demonstrated a consistent IO-fold increase in the ability of purified hexosaminidase A to hydrolyse [~~GMz in the presence of lysate from transfected cells as compared to mock transfected cell lysates. This confirms the identity of the cDNA.
cDNA synthesis Total RNA from Hela cell was isolated by the guanidinium-isothiocyanate method (11) followed by CsCl centrifugation. Reverse transcription was performed in a total volume of 50& in the presence of 6Fg RNA, 2U RNAguard (Pharmacia), O.lM Tris @H 8.3), 0.14M KCl, 1OmM MgC12,28mM P-mercaptoethanol, OSmM each of dNTPs, and 0.5pg oligonucleotide (either oligo dT, 730# (below) or 731# (Fig. 1)). The mixture was incubated with 5U AMV reverse transcriptase (BRL) at 42eC for 60min. l/50 of the synthesized cDNA was used for each PCR. FCR Amplification PCR was performed on cDNA or on an aliquot of a human retinal cDNA library (in Xgt 10, obtained from Dr. Jeremy Nathans) using the conditions recommended by Perkin-Elmer Cetus Corporation using AmpliTaq in a 5Ol.tL reaction volume. Attempts were made to obtain the 5’ end of the activator by PCR of the library (titering 1x10** PFUlmL, usinglul of the library for each PCR). The PCR was performed using the primer complementary to the 3’ end of the hgt 10 vector (Clontech, Cat. No. 541 l-2) and activator specific oligo 731# (shown in Fig. 1) in 30 cycles each of 30s at 94oC, 30s at 58oC, and 90s at 72cC. A second PCR was performed using 1 pL of the first PCR product with the 3’ primer and a second activator specific oligo 732# (Fig. 1) utilizing the same cycling conditions. A 300 bp “middle” fragment encoding additional 44 nucleotides at the 5’ end compared to the published sequence, was obtained. However, no ATG was encoded by this extra sequence. Another tiOObp fragment was also generated by PCR from the cDNA library utilizing two activator specific oligos 733# and 166# (fig. 1). Both the 5’ and middle PCR fragments were sequenced directly and some polymorphisms were detected. RACE FCR The 5’ end of the GM2 activator cDNA was obtained by the method of Frohman et al (12, 13). The tailed cDNA was synthesized with activator specific oligo 73 l# from Hela Poly (A)+ RNA (prepared using the Fast-Track kit, Invitrogen). Activator oligo 732# and a nonspecific oligo 730# (CG-CGAGATCTTTTTTTTTTTT), complementary to the poly (A)+ track and containing a BamH I “anchor” sequence, was used in PCR for 30 cycles each of lmin at 93oC, lmin at 55oC, and 3min at 72eC. 1pL of the 730#/732# PCR product was used in another PCR with activator specific oligo 602# (Fig. 1) and oligo 730# using same incubation conditions. A ~240 bp fragment which contained two ATGs and an in-frame STOP codon in its 5’ end extension was obtained. Direct Sequencing In order to avoid detection of PCR artifacts (due to Taq errors) which can be contained in individual cloned PCR fragments, we directly sequenced the products of each PCR. PCR 1218
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fragments were isolated by agarose gel electrophoresis and purified with Geneclean Kit (Bio 101). The gel-purified cDNA was sequenced with 5OOng of oligo 602# as a primer, using a method of direct sequencing adapted from the Sequenase protocol (United States Biochemical Corp.) (14). Cloning the GM2 activator cDNA The complete coding region of the GM~ activator was cloned following PCR amplification of the cDNA synthesized with oligo dT from Hela total RNA with two specific oligonucleotides, oligo167# (sequence determined from the direct sequencing of the 5’ end of the 240 bp RACE fragment) and oligo166# (Fig. 1). As the nucleotides at the -3 position of both ATGs at the 5’ end are not A or G and thus do not match Kozak’s rules for efficient translation initiation (15), an “A” was introduced in oligo 167# to change the nucleotide at position -3 of the first ATG from “c” to “A” for more efficient expression (Fig. 1). The G7OObp fragment from 30 cycles of PCR was subcloned to the BamH I site of Bluescript vector (Stratagene). The insert of one of the clones confirmed to contain the correct nucleotide sequence, was subcloned into pSVL-B for expression in COS-1 cells. Prediction of the Cleavage Site of the Activator Signal Peptide The probability of the presence of a signal peptide and the position of its cleavage site was assessed using a weighted matrix method of von Heijne (16). A computer program based on this method for use with the Apple Macintosh was kindly provided to us by Michael Richards. Expression of the cDNA encoding the Activator Protein COS-1 monkey kidney cells (American Type Culture Collection) were maintained in CLminimum essential media (Flow Laboratory) containing streptomycin and penicillin (lOOmg/L), and 10% fetal bovine serum at 37oC in 5% CO2. The 700 bp PCR fragment containing the complete coding sequence of the activator was subcloned into a mammalian expression vector, pSVL (Pharmacia), to yield pAct1. 2Opg of pAct1 was then transfected into monkey COS-1 cells using the calcium phosphate procedure as previously described (17). The identity of the translation product from pAct was confiied utilizing purified hexosaminidase A and GM~ tritiated in the terminal P-linked GalNAc residue (18). 2OOm of [3HGalNAc]GM2 (30,000 dpmnmole) in 80 mM citrate buffer, pH 4.1, containing 0.5% w/v human serum albumin, was incubated with 105 units (nmoles of 4-methylumbelliferyl-~-Nacetylglucosamine hydrolysetihr) of purified placental hexosaminidase A (19) at 37oC for 18 hr. in the presence of varying amounts of total protein from transfected and mock transfected COS-1 cell lysates (total assay volume 1OOpL). GM2 hydrolysis was measured as the amount of [THJGaWAc released with time (20).
RESULTS
Sequence of the GM~ activator cDNA Two 5’ cDNA fragments, sl6Obp and s24Obp were obtained by RACE PCR from Hela cDNA. The sl6Obp fragment was 58 nucleotides longer at the 5’ end than the previously published data and encoded two ATGs. However, no 5’ in-frame STOP codon was found in this shorter 5’ sequence. The sequence of the longer 240bp fragment revealed the expected in-frame STOP codon in its 5’ end extension (Fig. 1). Using the weighted matrix method of von Heijne (16) to analyze the deduced ammo acid sequence of the activator, an amino-terminal signal peptide was strongly predicted with the most probable cleavage site between Ala23 and His24 (Fig.1 hatched underline). Activator-specific
PCR amplification of the human retinal library failed to produce a
fragment containing a candidate full length 5’ end. However, direct sequencing of the most 5’ fragment and a middle fragment revealed three polymorphisms when compared with the sequences obta&d from a) the RACE procedure, b) the previously published partial cDNA clone (6), and c) that deduced from the protein sequence of the purified activator (7). 1) The nucleotide at position 1219
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-60 -90 TTT CTT TGC GTA ACC AAT ACT GGA AGG CAT TTA AAG GAC CTC TGC CGC CTC AGA CCT TGC #167 A -30 BaxmL AGT TAA CTC CGC CCT GAC CCA CCC TTC LCG Stop leu arg pro asp pro pro phe pro 31/11 ATC GCC CTG ile ala leu r,,,//ll///.//l/I/Il.,,,, 91/31 CTC AGT AGC leu ser ser
GGC TTG CTT CTC GCG gly leu leu leu ala
TTT TCC TGG GAT AAC phe ser trp asp am
l/l > ATG CAG TCC CTG ATG CAG GCT CCC met gin ser leu met gin ala pro ,1/////1//////,/,/,//l//l////// #602 A 61/21 GCC CCT GCG CAA GCC CAC CTG AAA AAG CCA ala pro ala gin ala his leu lys lys pro P ,/,//II//,//./l 121/41 TGT GAT GAA GGG AAG GAC CCT GCG GTG ATC cys asp glu gly lys asp pro ala val ile
CTC CTG leu leu
TCC CAG ser gin
AGA AGC arg ser
A 181/61 C CCC ATC GTC GTT CCT GGA AAT GTG ACC CTC AGT GTC GTG leu thr leu glu pro asp pro ile val val pro gly am val thr leu ser val Y&L met 241/81 211/71 AGC ACC AGT GTC CCC CTG AGT TCT CCT CTG AAG GTG GAT TTA GTT TTG GAG AAG GAG ser thr ser val pro leu ser ser pro leu lys val asp leu val leu glu lys glu
GGC gly
GTG val
271191 301/101 GCT GGC CTC TGG ATC AAG ATC CCA TGC ACA GAC TAC ATT GGC AGC TGT ACC TTT GAA CAC ala gly leu trp ile lys ile pro cys thr asp tyr ile gly ser cys thr phe glu his 361/121 331/111 TTC TGT GAT GTG CTT GAC ATG TTA ATT CCT ACT GGG GAG CCC TGC CCA GAG CCC CTG CGT phe cys asp val leu asp met leu ile pro thr gly glu pro cys pro glu pro leu arg 391/131 421/141 ACC TAT GGG CTT CCT TGC CAC TGT CCC TTC AAA GAA GGA ACC TAC TCA CTG CCC AAG AGC thr tyr gly leu pro cys his cys pro phe lys glu gly thr tyr ser leu pro lys ser 4 #731 #-l32 451/151 481/161 GAA TTC GTT GTG CCT G~CTG GAG CTG CCC AGT TGG CTC ACC ACC GGG AAC TAC CGC ATA glu phe val val pro asp leu glu leu pro ser trp leu thr thr gly am tyr arg ile 511/1?1 541/181 GAG AGC GTC CTG AGC AGC AGT GGG AAG CGT CTG GGC TGC ATC AAG ATC glu ser val leu ser ser ser gly lys arg leu gly cys ile lys ile
GCT GCC ala ala
571/191 601 G AAG GGC ATA TAA CAT GGC ATC TGC CAC AGC AGA ATG GAG CGG TGT GAG GM lys gly ile Stop
stop 631 TCC 691 TCT 751 GGG 811 CTG
#166
fifil
TCT GTT TTG TGT TTG CCA AGG CCA AAC TCC 721 ACA GTG AGT CCA CTA CCC TCA CTG AAA ATC 781 CAA GCA GCC CTG ACC TAA GGG AGA ATG AGT 841 CTG GGC TGA CCA CGT TX TCA TCC CCG TTA
TCT CTA ser leu
GGT ccc
TTT
a CAC TCT CTG CCC CCC TTT AAT CCC CTT ATT TTG TAC CAC TTA CAT TTT AGG CTG TGG ACA GTT CTT GAT AGC CCA GGG CAT ACA TTC TCT CTA RAG AGC CT
Figure 1. Nucleotide and deduced ammo acid sequence of the human @2 activator protein (the “A” of the first ATG is labeled as nucleotide 1 and the corresponding “met” as ammo acid 1). The previously reported partial sequence began at our nucleotide #46 (6) The various oligonucleotides used in this study and their 5’ to 3’ direction of extension are shown as bold arrows. The single mismatch in oligo #167 to produce a more efficient translation initiation sequence is shown above the underlined wild type nucleotide. BamH I anchors were added to oligonucleotides #I67 and #166 to facilitate cloning of the FCR fragment The predicted signal peptide sequence is indicated by a hatched underline (residues I-23). The amino-terminus of the mature protein (7) is double underlined. The three polymorphisms identified in this study are shown above the corresponding nucleotide in the cloned sequence, which was expressed in transfected COS cells. The change in the deduced sequence resulting from these polymorphisms is also indicated below the deduced ammo acid. 1220
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Protein
Figure. 2. The effects of increasing amounts of lysate protein from two independent transfections of COS cells with pActI (Transfection 1 and Transfection 2), on the ability of purified hexosaminidase A to hydrolyse the labeled GalNAc residue from GM~ ganglioside. These arecompared to the effect of identical amounts of protein from the lysate of mock
transfected cells(COS(-)).
55 (Fig. 1) is “G”, which is different from the previously publisheddata (“A”) (6), and changes Thrl9 to Ala in the predicted signalpeptidesequence(Fig. 1). This “G” wasfound in both of the directly sequencedzl60bp RACE PCR fragment and in the &OObp PCR Send fragment from the retina cDNA library asdescribedin “MATERIALS AND ~~ETHODS”, andin the sequence of the clone that we expressedcontaining the activator’scoding region. 2) A “G582” to “A” substitution wasalsofound in the STOP codon at the 3’ end of the coding region in the clone (which still produceda STOP codon), but not in the z5OObpPCR fragment from the retina library. 3) Finally, the nucleotideat position 205 wasfound to be “A” in the PCR fragment from the retina cDNA library insteadof “G” aswasreportedin the previously publishedpartial sequenceandalso found in our clone from Hela cDNA containing the coding region. This resultsin a Valgg to Met substitution (Fig. 1). Interestingly, Met was alsofound at this position when the purified activator protein was sequenceddirectly (7). This data is consistentwith the highly polymorphic nature of the activator coding sequence(21). Expressionof the @,,,fzactivator The nucleotide sequencebetweenoligos 167#and 166#in Fig 1 is the sequenceobtained from the cDNA clone of GM~ activator usedfor expressionin COS cells. Fig 2 showsthe difference in the ability of lysatesfrom transfectedandmock transfectedcells to enhancethe hydrolysis of IsHGalNAc]GMz gangliosideby purified hexosaminidaseA. Two independent transfectionexperimentswere carried out and a seriesof assaysperformedusingincreasing amountsof total lysate protein for each. The enhancementof hexosaminidase A activity was linear with respectto the amountof lysate protein addedto eachreaction. Lysatesfrom COS cells transfectedwith pAct1 consistentlyproduceda lo-fold better enhancementof activity than identical amountsof lysatesfrom mock aansfectedcells (Fig. 2).
DISCUSSION
Expressionof a functional GM~ activator in transfectedCOS cells both confirms the identity of the previously publishedpartial cDNA, anddemonstratesthat we have isolateda cDNA 1221
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that contains the complete coding sequence of the protein. The deduced sequence predicts a prepro-polypeptide chain of 20,808 daltons (residues l-193), a pro-polypeptide of 18,463 daltons (residues 24-193) and a mature chain of 17,531 daltons (residues 32-193). Since the protein requires N-linked glycosylation for transport to the lysosome (reviewed in (22)), the activator must be glycosylated at its single Asnbg-Val-Thr
site. These predicted polypeptide chain weights plus
the effect of an oligosaccharide on the apparent weight determined by SDS-PAGE, are consistent with previously published biosynthetic data indicating a 24 kDa precursor which is processed to its 22 kDa mature lysosomal form (23). Our data indicate that the activator mRNA must be relatively rare. We were forced to use a “nested’ RACE procedure in order to generate a single PCR fragment from Hela cDNA. Further, we screened several cDNA libraries for the presence of an activator cDNA using two specific oligonucleotides without success (data not shown). Kozak has shown that while more than 50% of 211 translation initiation sequences analyzed match three or four of the nucleotides in the consensus sequence, CCIA or GICCATG 5’ to the ATG, only 6 failed to have a purine at the -3 position (15). The rareness of the activator mRNA coupled with the poor match to the translation initiation consensus at either of the 5’ ATGs in the native sequence, TCCCGATG or CCCTGATG, suggests that the protein is either very long lived or is rarer than has previously been reported (24). The three variations in the nucleotide sequences we have found in this study confirm that the activator protein is highly polymorphic. Previously we found two polymorphisms in Southern blot analyses using a 300bp coding region fragment as a probe (21). This may reflect the fact that the activator performs no actual catalytic function. Thus, its structuremay be more flexible to evolutionary changesthan would be an enzyme. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
Sandhoff, K., Conzelmann, E., Neufeld, E.F., Kaback, M.M., and Suzuki, K. (1989) In The Metabolic Basisof Inherited Disease(C.V. Striver, A.L. Beaudet, W.S. Sly, and D. Valle, eds.), pp. 1807-1839,McGraw-Hill, New York. Komeluk, R.G., Mahuran, D.J., Neote, K., Klavins, M.H., O’Dowd, B.F., Tropak, M., Willard, H.F., Anderson, M.-J., Lowden, J.A., and Gravel, R.A. (1986) J. Biol. Chem. 261, 8407-8413. Myerowitz, R., Piekarz, R., Neufeld, E.F., Shows, T.B., and Suzuki, K. (1985) Proc. Natl. Acad. Sci. USA 82, 7830-7834. Tanaka, A., Ohno, K., Sandhoff, K., Maire, I., Kolodny, E.H., Brown, A., and Suzuki, K. (1990) Amer. J. Hum. Gen. 46,329-339. Brown, CA., Neote, K., Leung, A., Gravel, R.A., and Mahuran, D.J. (1989) .I. Biol. Chem. 264,21705-21710. Schroder, M., Klima, H., Nakano, T., Kwon, H., Quintem, L.E., GBi-tner, S., Suzuki, K., and Sandhoff, K. (1989) FEBS. Lett. 251, 197-200. Furst, W., Schubert, J., Machleidt, W., Meyer, H.E., and Sandhoff, K. (1990) Eur. J. Biochem. 192,709-714. Burg, J., Conzelmann, E., Sandhoff, K., Solomon, E., and Swallow, D.M. (1985) Ann. Hum. Genet. 49, 41-45. Kleyn, P.W., Brzustowicz, L.M., Wilhelmsen, K.C., Freimer, N.B., Miller, J.M., Munsat, T.L., and Gilliam, T.C. Neurology in press. Brzustowicz, L.M., Lehner, T., Castilla, L.H., Penchaszadeh,G.K., Wilhelmsen, K.C., Daniels, R., Davies, K.E., Leppert, M., Ziter, F., Wood, D., Dubowitz, V., Zerres, K., Hausmanowa-Petrusewicz,I., Ott, J., Munsat, T.L., and Gillian, T.C. (1990) Nature 344,540-541. 1222
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