FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1520049999> ?p ?o ?g. }

• W1520049999 endingPage "10615" @default.
• W1520049999 startingPage "10611" @default.
• W1520049999 abstract "GM2 activator protein is a protein cofactor which stimulates the enzymatic hydrolysis of both GalNAc and NeuAc from GM2. We have previously isolated two cDNA clones, GM2 activator cDNA and GM2A cDNA, for human GM2 activator protein (Nagarajan, S., Chen, H.-C., Li, S.-C., Li, Y.-T., and Lockyer, J. M.(1992) Biochem. J. 282, 807-813). GM2A mRNA is an RNA alternative splicing product that contains exons 1, 2, 3, and intron 3 of the genomic DNA sequence of GM2 activator protein (Klima, H., Tanaka, A., Schnabel, D., Nakano, T., Schröder, M., Suzuki, K., and Sandhoff, K.(1991) FEBS Lett. 289, 260-264). GM2A cDNA encodes a protein (GM2A protein) containing 1-109 of the 160 amino acids of human GM2 activator protein, plus a tripeptide (VST) encoded by intron 3 at the COOH terminus. Thus, GM2A protein can be regarded as a form (truncated version) of GM2 activator protein. We have expressed GM2A cDNA in Escherichia coli using pT7-7 as the vector. The recombinant GM2A protein was purified to an electrophoretically homogeneous form and was found to stimulate the hydrolysis of NeuAc from GM2 by clostridial sialidase, but not the hydrolysis of GalNAc from GM2 by β-hexosaminidase A. Like GM2 activator protein, GM2A protein also specifically recognized the terminal GM2 epitope in GalNAc-GD1a and stimulated the hydrolysis of only the external NeuAc from this ganglioside by clostridial sialidase. These results enabled us to discern the enzymatic hydrolyses of GalNAc and NeuAc from the GM2 epitope and established that the NeuAc recognition domain of GM2 activator protein is located within amino acids 1-109. The presence of GM2A mRNA in human tissues and the selective stimulation of NeuAc hydrolysis by GM2A protein indicate that this activator protein may be involved in the catabolism of GM2 through the asialo-GM2 pathway. GM2 activator protein is a protein cofactor which stimulates the enzymatic hydrolysis of both GalNAc and NeuAc from GM2. We have previously isolated two cDNA clones, GM2 activator cDNA and GM2A cDNA, for human GM2 activator protein (Nagarajan, S., Chen, H.-C., Li, S.-C., Li, Y.-T., and Lockyer, J. M.(1992) Biochem. J. 282, 807-813). GM2A mRNA is an RNA alternative splicing product that contains exons 1, 2, 3, and intron 3 of the genomic DNA sequence of GM2 activator protein (Klima, H., Tanaka, A., Schnabel, D., Nakano, T., Schröder, M., Suzuki, K., and Sandhoff, K.(1991) FEBS Lett. 289, 260-264). GM2A cDNA encodes a protein (GM2A protein) containing 1-109 of the 160 amino acids of human GM2 activator protein, plus a tripeptide (VST) encoded by intron 3 at the COOH terminus. Thus, GM2A protein can be regarded as a form (truncated version) of GM2 activator protein. We have expressed GM2A cDNA in Escherichia coli using pT7-7 as the vector. The recombinant GM2A protein was purified to an electrophoretically homogeneous form and was found to stimulate the hydrolysis of NeuAc from GM2 by clostridial sialidase, but not the hydrolysis of GalNAc from GM2 by β-hexosaminidase A. Like GM2 activator protein, GM2A protein also specifically recognized the terminal GM2 epitope in GalNAc-GD1a and stimulated the hydrolysis of only the external NeuAc from this ganglioside by clostridial sialidase. These results enabled us to discern the enzymatic hydrolyses of GalNAc and NeuAc from the GM2 epitope and established that the NeuAc recognition domain of GM2 activator protein is located within amino acids 1-109. The presence of GM2A mRNA in human tissues and the selective stimulation of NeuAc hydrolysis by GM2A protein indicate that this activator protein may be involved in the catabolism of GM2 through the asialo-GM2 pathway. It has been well established that the catabolism of ganglioside GM2 1The abbreviations used are: GM2II3NeuAcGgOse3CerGM3II3NeuAcLacCerGalNAc-GD1aIV4GalNAc, IV3NeuAc,II3NeuAcGgOse4CerGA2GgOse3CerPAGEpolyacrylamide gel electrophoresis. requires the assistance of a protein cofactor called GM2 activator protein(1.Li Y.-T. Li S.-C. Dingle J.T. Dean R.T. Sly W. Lysosomes in Biology and Pathology. Elsevier, Amsterdam1984: 99-117Google Scholar, 2.Fürst W. Sandhoff K. Biochim. Biophys. Acta. 1992; 1126: 1-16Crossref PubMed Scopus (248) Google Scholar). The physiological importance of GM2 activator protein has been shown by the presence of an autosomal recessive genetic disease, the AB variant of Tay-Sachs disease, which is caused by the deficiency or the defect of GM2 activator protein(3.Conzelmann E. Sandhoff K. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3979-3983Crossref PubMed Scopus (209) Google Scholar, 4.Hechtman P. Gordon B.A. Ng Ying Kin N.M.K. Pediatr. Res. 1982; 16: 217-222Crossref PubMed Scopus (44) Google Scholar, 5.Hirabayashi Y. Li Y.-T. Li S.-C. J. Neurochem. 1983; 40: 168-175Crossref PubMed Scopus (44) Google Scholar). In addition to GM2 activator protein, four other activator proteins for the catabolism of glycosphingolipids have been reported. These four activator proteins are derived from the proteolytic processing of a single precursor protein, prosaposin(6.O'Brien J.S. Kretz K.A. Dewji N. Wenger D.A. Esch F. Fluharty A.L. Science. 1988; 241: 1098-1101Crossref PubMed Scopus (210) Google Scholar, 7.Fürst W. Machleidt W. Sandhoff K. Biol. Chem. Hoppe-Seyler. 1988; 369: 317-328Crossref PubMed Scopus (85) Google Scholar, 8.Nakano T. Sandhoff K. Stümper J. Christomanou H. Suzuki K. J. Biochem. (Tokyo). 1989; 105: 152-154Crossref PubMed Scopus (111) Google Scholar), and have been named saposin A, B, C, and D according to their placements from the amino terminus of the prosaposin(9.Kishimoto Y. Hiraiwa M. O'Brien J.S. J. Lipid Res. 1992; 33: 1255-1267Abstract Full Text PDF PubMed Google Scholar). Saposin B is also 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(10.Li S.-C. Sonnino S. Tettamanti G. Li Y.-T. J. Biol. Chem. 1988; 263: 6588-6591Abstract Full Text PDF PubMed Google Scholar). Among the five activator proteins, only GM2 activator protein is derived from a separate gene(11.Burg J. Conzelmann E. Sandhoff K. Solomon E. Swallow D.M. Ann. Hum. Genet. 1985; 49: 41-45Crossref PubMed Scopus (47) Google Scholar). We have isolated two distinct cDNA clones for human GM2 activator protein(12.Nagarajan S. Chen H.-C. Li S.-C. Li Y.-T. Lockyer J.M. Biochem. J. 1992; 282: 807-813Crossref PubMed Scopus (20) Google Scholar). One of them, GM2 activator cDNA, which has also been isolated by others(13.Schröder M. Klima H. Nakano T. Kwon H. Quintern L.E. Gärtner S. Suzuki K. Sandhoff K. FEBS Lett. 1989; 251: 197-200Crossref PubMed Scopus (47) Google Scholar, 14.Xie B. McInnes B. Neote K. Lamhonwah A.-M. Mahuran D. Biochem. Biophys. Res. Commun. 1991; 177: 1217-1223Crossref PubMed Scopus (26) Google Scholar), encodes almost the entire amino acid sequence of the native GM2 activator protein isolated from human kidney (15.Fürst W. Schubert J. Machleidt W. Meyer H.E. Sandhoff K. Eur. J. Biochem. 1990; 192: 709-714Crossref PubMed Scopus (48) Google Scholar). The other clone, GM2A cDNA, which was reported only by us, has an identical 5′-terminal sequence as that of GM2 activator cDNA from nucleotides 1 to 302, but different for the next 346 nucleotides toward the 3′ end. Klima et al.(16.Klima H. Tanaka A. Schnabel D. Nakano T. Schröder M. Suzuki K. Sandhoff K. FEBS Lett. 1991; 289: 260-264Crossref PubMed Scopus (41) Google Scholar) isolated the genomic DNA which covered 94% of GM2 activator cDNA, and identified the presence of three introns and four exons. The last exon, exon 4, spanned the segment coding for the carboxyl terminus of GM2 activator protein and the entire 3′-untranslated region of the GM2 activator cDNA. Comparing the sequence of GM2A cDNA with this genomic DNA, we found that the last 346 nucleotides of GM2A cDNA were identical to the sequence of 5′ end of intron 3 (the exons and the introns are defined based on GM2 activator mRNA). Thus, GM2A mRNA is an alternative splicing product of GM2 activator RNA in which the potential 5′ splicing site between exon 3 and intron 3 is not subjected to the splicing process. As shown in Fig. 1, the coding region of GM2 activator cDNA contains the end portion of exon 1, all of exons 2 and 3, and the front portion of exon 4. While the coding region of GM2A cDNA contains the identical exon 1, 2, and 3 as in GM2 activator cDNA, and a stretch of 9 nucleotides encoding a tripeptide, VST, at the COOH terminus which is derived from intron 3. This 9-nucleotide sequence is immediately followed by a stop codon. II3NeuAcGgOse3Cer II3NeuAcLacCer IV4GalNAc, IV3NeuAc,II3NeuAcGgOse4Cer GgOse3Cer polyacrylamide gel electrophoresis. It has been postulated that the function of GM2 activator protein is to extract a single GM2 molecule from the micelles and to present the substrate-activator complex to β-hexosaminidase A(17.Conzelmann E. Sandhoff K. Hoppe-Seyler's Z. Physiol. Chem. 1979; 360: 1837-1849Crossref PubMed Scopus (143) Google Scholar), or to lift GM2 from biological membranes where the sugar chain of GM2 molecules may be shielded by other complex lipids with larger headgroups(18.Meier E.M. Schwarzmann G. Fürst W. Sandhoff K. J. Biol. Chem. 1991; 266: 1879-1887Abstract Full Text PDF PubMed Google Scholar, 2.Fürst W. Sandhoff K. Biochim. Biophys. Acta. 1992; 1126: 1-16Crossref PubMed Scopus (248) Google Scholar). In contrast, we have shown that the action of GM2 activator protein in stimulating the hydrolysis of GM2 by β-hexosaminidase A may be due to its ability to recognize and interact with the branched trisaccharide of GM2, the GM2 epitope(19.Wu Y.-Y. Lockyer J.M. Sugiyama E. Pavlova N.V. Li Y-T. Li S.-C. J. Biol. Chem. 1994; 269: 16276-16283Abstract Full Text PDF PubMed Google Scholar). This view is further supported by the finding that GM2 activator protein also stimulates the hydrolysis of NeuAc from GM2 by clostridial sialidase(19.Wu Y.-Y. Lockyer J.M. Sugiyama E. Pavlova N.V. Li Y-T. Li S.-C. J. Biol. Chem. 1994; 269: 16276-16283Abstract Full Text PDF PubMed Google Scholar). Based on the selective hydrolysis of only the terminal NeuAc residue in GalNAc-GD1a, we postulated that GM2 activator protein can specifically recognize the GM2 epitope in this ganglioside (20.Li S.-C. Wu Y.-Y. Sugiyama E. Taki T. Kasama T. Casellato R. Sonnino S. Li Y-T. J. Biol. Chem. 1995; 270: 24246-24251Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Since the amino acid sequence encoded by GM2A cDNA consists of the segment of GM2 activator protein from amino acid residues 1 to 109 and an additional tripeptide sequence VST at the COOH terminus, the protein encoded by GM2A cDNA (GM2A protein) can be regarded as a truncated form of GM2 activator protein. It is, therefore, important to compare the specificity of GM2A protein with that of GM2 activator protein. We have produced the recombinant GM2A protein and found that this activator protein possesses only one of the two known activities of GM2 activator protein. GM2 from Tay-Sachs brain (21.Svennerholm L. Methods Carbohydr. Chem. 1972; 6: 464-474Google Scholar) and the radioactive GM2(22.Radin N.S. Methods Enzymol. 1972; 28: 300-306Crossref Scopus (55) Google Scholar, 23.Uda Y. Li S.-C. Li Y.-T. McKibbin J.M. J. Biol. Chem. 1977; 252: 5194-5200Abstract Full Text PDF PubMed Google Scholar) were prepared as previously reported. The recombinant GM2 activator protein and the recombinant saposin B were produced in Escherichia coli as described previously(19.Wu Y.-Y. Lockyer J.M. Sugiyama E. Pavlova N.V. Li Y-T. Li S.-C. J. Biol. Chem. 1994; 269: 16276-16283Abstract Full Text PDF PubMed Google Scholar, 20.Li S.-C. Wu Y.-Y. Sugiyama E. Taki T. Kasama T. Casellato R. Sonnino S. Li Y-T. J. Biol. Chem. 1995; 270: 24246-24251Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). β-Hexosaminidase A (specific activity, 33.3 units/mg) (24.Li Y.-T. Mazzotta M.Y. Wan C.-C. Orth R. Li S.-C. J. Biol. Chem. 1973; 248: 7512-7515Abstract Full Text PDF PubMed Google Scholar) was isolated from human liver. GalNAc-GD1a was isolated from the total ganglioside mixture of bovine brain(25.Acquotti D. Cantu L. Ragg E. Sonnino S. Eur. J. Biochem. 1994; 225: 271-288Crossref PubMed Scopus (65) Google Scholar). The following reagents of the highest grade were obtained from commercial sources: clostridial sialidase Type X, isopropyl thio-β-galactoside, ampicillin, and glutathione, Sigma; yeast extract and tryptone, Difco; restriction endonucleases and T4 DNA ligase, Life Technologies, Inc.; Taq DNA polymerase, Promega; T7 sequencing kit version 2.0, U. S. Biochemical Corp.; E. coli strain BL-21(DE3), Novagen; Universol (a scintillation mixture), ICN Biochemicals; the precoated Silica Gel-60 HPTLC plates, Merck (Darmstadt, Germany); Cellex D anion exchange cellulose and the prestained protein standard markers, Bio-Rad. A 23-mer peptide, PFKEGTYSLPKSEFVVPDLELPS-amide, which is identical to amino acids 106-128 of the mature GM2 activator protein, was synthesized using a solid phase peptide synthesizer (Milligen 9050) by the Core Laboratories of Louisiana State University Medical Center. The fragment of GM2A cDNA, which encodes amino acids 1-109 of the human GM2 activator protein plus the tripeptide VST at the COOH terminus, was obtained by polymerase chain reaction using GM2A-KS Bluescript as template. The upstream primer was: 5′-CGC-TCT-AGA-CGG-ATC-CCA-TAT-GTT-TTC-CTG-GGA-TAA-CTG-TGA-T-3′ and the downstream primer was 5′-TCA-TCT-AGA-GGA-TCC-AAG-CTT-AGC-CAC-AGG-GGT-AAC-GCT-CTC-3′. This cDNA fragment was subcloned into pT7-7 expression vector at BamHI and HindIII sites, and was verified for its sequence. The recombinant GM2A protein was expressed and purified according to the method described previously(19.Wu Y.-Y. Lockyer J.M. Sugiyama E. Pavlova N.V. Li Y-T. Li S.-C. J. Biol. Chem. 1994; 269: 16276-16283Abstract Full Text PDF PubMed Google Scholar). The production of GM2A protein was assessed by SDS-PAGE of Laemmli (26.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207123) Google Scholar) using silver staining and by Western blot analysis using polyclonal anti-GM2 activator antibodies(5.Hirabayashi Y. Li Y.-T. Li S.-C. J. Neurochem. 1983; 40: 168-175Crossref PubMed Scopus (44) Google Scholar). The NH2-terminal amino acid sequence of the purified GM2A protein was confirmed by a gas-phase peptide sequencer (Applied Biosystems Model 477A). Each ganglioside substrate in micellar form was incubated with the appropriate enzyme and the designated activator protein in a final volume of 100 μl at 37°C. For the conversion of GM2 into GA2, 40 μM GM2 was incubated with 10 units of clostridial sialidase in 10 mM acetate buffer, pH 5.5, in the presence of activator protein. For the hydrolysis of NeuAc from GalNAc-GD1a, 20 μM GalNAc-GD1a was used. For quantitative analysis of the hydrolysis of NeuAc from GM2, the incubation conditions were identical to that described above, except three different concentrations of [3H]GM2 were used. For the hydrolysis of GalNAc from GM2, 20 μM GM2 was incubated at 37°C with 0.2 units of β-hexosaminidase A in 10 mM acetate buffer, pH 4.6, in the presence of the indicated amount of an activator protein. The quantitative analysis of the conversion of GM2 to GM3 was carried out by using [3H]GM2 as substrate at the indicated concentrations and measuring the release of [3H]GalNAc. For analyzing the reaction products by TLC, the reaction was stopped by heating the tube in a bath of boiling water for 3 min, followed by adsorption of gangliosides on C18 beads as described previously(27.Williams M.A. McCluer R.H. J. Neurochem. 1980; 35: 266-269Crossref PubMed Scopus (405) Google Scholar). The beads were then extracted by 0.5 ml of methanol and followed by 0.5 ml of chloroform/methanol (2:1, v/v)(27.Williams M.A. McCluer R.H. J. Neurochem. 1980; 35: 266-269Crossref PubMed Scopus (405) Google Scholar). The extracts were combined, dried, and analyzed by TLC using the following solvents: chloroform/methanol/water (60:35:8, v/v/v) for the separation of GM2 and GM3; chloroform/methanol/water (65:25:4, v/v/v) for the separation of GM2 and GA2; methyl acetate, 1-propanol, chloroform, methanol, 0.25% KCl (25:20:20:20:17) for the separation of GalNAc-GM1a, GalNAc-GM1b, GalNAc-GA1, and GalNAc-GD1a. The plates were sprayed with diphenylamine reagent (28.Harris, G., MacWilliams, I. C. (1954) Chem. & Ind. (Lond.), 249Google Scholar) and heated at 110°C for 15-20 min to reveal the glycosphingolipids. When [3H]GM2 was used, the reaction mixtures were evaporated to dryness under vacuum, then redissolved in 1 ml of chloroform/methanol (2:7) and passed through a DEAE-cellulose column (0.5 × 4 cm) which had been equilibrated with the same solvent. The column was washed with 4 ml of chloroform/methanol (2:7) and then eluted with 5 ml of chloroform/methanol (2:7) containing 20 mM sodium acetate. The breakthrough and the eluted fractions were separately collected, evaporated to dryness, dissolved in 0.5 ml of water, and mixed with 5 ml of Universol. The radioactivity was measured by using a Packard 1600CA liquid scintillation counter. As in the case of the recombinant GM2 activator protein(19.Wu Y.-Y. Lockyer J.M. Sugiyama E. Pavlova N.V. Li Y-T. Li S.-C. J. Biol. Chem. 1994; 269: 16276-16283Abstract Full Text PDF PubMed Google Scholar), the recombinant GM2A protein produced in E. coli was also found to accumulate in the inclusion bodies. The same scheme used for the extraction, refolding, and purification of the recombinant GM2 activator protein (19.Wu Y.-Y. Lockyer J.M. Sugiyama E. Pavlova N.V. Li Y-T. Li S.-C. J. Biol. Chem. 1994; 269: 16276-16283Abstract Full Text PDF PubMed Google Scholar) was used for the preparation of the recombinant GM2A protein. As shown in the SDS-PAGE profile (Fig. 2), the purified recombinant GM2A protein (lane 2) moved at the position corresponding to 13.5 kDa, the calculated molecular size of GM2A protein. It is considerably smaller than the recombinant GM2 activator protein (Fig. 2, lane 3). The identity of GM2A protein was also verified by the microsequencing of the NH2-terminal amino acid sequence. In Western blot analysis, GM2A protein was also recognized by the polyclonal antibodies against GM2 activator protein(5.Hirabayashi Y. Li Y.-T. Li S.-C. J. Neurochem. 1983; 40: 168-175Crossref PubMed Scopus (44) Google Scholar). Our previous results indicate that GM2 activator protein can stimulate the hydrolysis of both GalNAc and NeuAc from GM2 by β-hexosaminidase A and clostridial sialidase, respectively, and that it may be able to recognize the branched trisaccharide structure GalNAcβ1→4(NeuAcα2→3)Gal-, the GM2 epitope(19.Wu Y.-Y. Lockyer J.M. Sugiyama E. Pavlova N.V. Li Y-T. Li S.-C. J. Biol. Chem. 1994; 269: 16276-16283Abstract Full Text PDF PubMed Google Scholar). Since GM2A protein contains only the NH2-terminal 109 amino acids of GM2 activator protein without the COOH terminus 110-160 amino acids which were derived from exon 4, it would be important to examine whether or not this short version of the activator protein possesses the two known biological activities expressed by GM2 activator protein. The TLC analysis of the conversion of GM2 to GA2 (Fig. 3A) showed that, as in the case of GM2 activator protein (lane 4), GM2A protein (lane 5) also stimulated the hydrolysis of NeuAc from GM2 by clostridial sialidase. However, GM2A protein was found to be slightly less effective than GM2 activator protein. In order to compare the stimulatory potency of these two activator proteins, quantitative analysis was performed using [3H]GM2 at three different substrate concentrations instead of triplicates of one concentration. As shown in Fig. 3B, at all three substrate concentrations tested, the conversion of GM2 to GA2 was greatly enhanced by the presence of 2.5 μM of either GM2 activator protein or GM2A protein. GM2A protein was about 20% less effective than GM2 activator protein. Previously we have shown that GM2 activator protein specifically recognized and stimulated the hydrolysis of the NeuAc residue in the GM2 epitope of GalNAc-GD1a by clostridial sialidase(20.Li S.-C. Wu Y.-Y. Sugiyama E. Taki T. Kasama T. Casellato R. Sonnino S. Li Y-T. J. Biol. Chem. 1995; 270: 24246-24251Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). We, therefore, examined the possible recognition of the same NeuAc residue in GalNAc-GD1a by GM2A protein. As shown in Fig. 4, GM2A protein did preferentially stimulate the hydrolysis of the external NeuAc in GalNAc-GD1a. In the presence of 2.5 μM GM2A protein, the clostridial sialidase produced GalNAc-GM1a as the major product and GalNAc-GA1 as the minor product from GalNAc-GD1a (Fig. 4A, lane 6). The same products were produced from GalNAc-GD1a by the clostridial sialidase in the presence of 2.5 μM GM2 activator protein as seen in Fig. 4A, lane 5. The products GalNAc-GM1a, GalNAc-GM1b, and GalNAc-GA1 were analyzed by secondary ion mass spectrometry as described previously(20.Li S.-C. Wu Y.-Y. Sugiyama E. Taki T. Kasama T. Casellato R. Sonnino S. Li Y-T. J. Biol. Chem. 1995; 270: 24246-24251Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The strict specificity of GM2 activator protein and GM2A protein toward the hydrolysis of the terminal NeuAc from GalNAc-GD1a was further demonstrated by the comparison of this result with that of the parallel experiments carried out in the presence of 10 or 20 μM saposin B. The concentrations of saposin B were chosen according to our previous experience(20.Li S.-C. Wu Y.-Y. Sugiyama E. Taki T. Kasama T. Casellato R. Sonnino S. Li Y-T. J. Biol. Chem. 1995; 270: 24246-24251Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Our results clearly showed that in addition to GalNAc-GM1a and GalNAc-GA1, GalNAc-GM1b was also produced from GalNAc-GD1a in the presence of saposin B (Fig. 4A, 20 μM in lane 7 and 10 μM in lane 8). This indicates that saposin B can stimulate the hydrolyses of both NeuAc residues from GalNAc-GD1a. Since the concentrations of saposin B used in Fig. 4A were much higher than that of the two other activator proteins, we repeated the experiment using 20 μM of each activator protein. As shown in Fig. 4B, in the presence of GM2A protein (lane 2′) or GM2 activator protein (lane 3′), only GalNAc-GM1a and GalNAc-GA1 were produced. However, in the presence of saposin B (lane 1′), GalNAc-GM1b was also produced in addition to GalNAc-GM1a and GalNAc-GA1. These results suggest that GM2 activator protein and GM2A protein have the same specificity in recognizing the external NeuAc residue of GalNAc-GD1a. It is of interest to note that in the presence of 10 μM saposin B (Fig. 4A, lane 8), slightly more GalNAc-GM1a was produced than GalNAc-GM1b, while in the presence of 20 μM saposin B (Fig. 4A, lane 7), the reverse was observed. Thus, the concentration of saposin B appeared to influence the ratio of GalNAc-GM1a to GalNAc-GM1b produced from GalNAc-GD1a. In contrast, the production of only GalNAc-GM1a in the presence of GM2 activator protein or GM2A protein was not affected by the activator protein concentration. In spite of being different in their molecular sizes (18.5 kDa for GM2 activator protein and 13.5 kDa for GM2A protein), both GM2 activator protein and GM2A protein were able to stimulate the hydrolysis of the same NeuAc from GalNAc-GD1a. The above results strongly suggest that GM2A protein is also able to recognize the NeuAc residue in the GM2 epitope and its mode of action in stimulating the liberation of NeuAc from GM2 by clostridial sialidase is identical to that of GM2 activator protein. In view of the facts that the amino acid sequence of GM2A protein (except VST) is identical to the sequence of GM2 activator protein from 1 to 109 and that both proteins have the same stimulatory activity for the hydrolyses of the NeuAc from GM2 and GalNAc-GD1a, it is logical to assign the NeuAc recognition domain of GM2 activator protein to be within amino acids 1-109. As shown in Fig. 5A, while GM2 activator protein was an effective activator for the enzymatic hydrolysis of GalNAc from GM2 (0.5 μM in lane 3, and 2.5 μM in lane 4), GM2A protein did not stimulate the hydrolysis of GalNAc from GM2 by β-hexosaminidase A (0.5 μM in lane 5 and 2.5 μM in lane 6). The inability of GM2A protein to stimulate the conversion of GM2 to GM3 was further confirmed by examining the reaction at three different substrate concentrations (Fig. 5B). These results indicate that whatever enables GM2 activator protein to exert the stimulatory activity toward the hydrolysis of GalNAc from GM2 by β-hexosaminidase A is absent in GM2A protein. It is reasonable to conclude that, in GM2 activator protein, the portion of the peptide sequence encoded by exon 4 may govern the recognition of the GalNAc residue in GM2 epitope and/or contribute to the formation of the essential conformation required for the specific recognition of the GalNAc residue. Thus, the 51 amino acids at the COOH terminus of GM2 activator protein must be crucial to make the GalNAc residue accessible to β-hexosaminidase A. The functional importance of the COOH-terminal segment of GM2 activator protein is supported by the fact that a case of type AB Tay-Sachs disease was found to be caused by a Arg → Pro mutation at the exon 4 coding region(29.Schröder M. Schnabel D. Hurwitz R. Young E. Suzuki K. Sandhoff K. Hum. Genet. 1993; 92: 437-440Crossref PubMed Scopus (36) Google Scholar). The Arg → Pro mutation may disrupt the tertiary structure of GM2 activator protein which is vital for its activity and/or stability. Although our results suggest that amino acids 1-109 of GM2 activator protein is sufficient for the stimulation of the cleavage of NeuAc from GM2 by clostridial sialidase and the 51 amino acids at the COOH terminus is essential for the stimulation of GalNAc cleavage from GM2 by β-hexosaminidase A, it is not possible at this point to ascertain if GM2 activator protein requires a simultaneous interaction with both GalNAc and NeuAc residues in the GM2 epitope for the stimulation of hydrolysis of GalNAc from GM2. The fact that GM2 activator protein is not as effective in stimulating the hydrolysis of GA2 as that of GM2 by β-hexosaminidase A (19.Wu Y.-Y. Lockyer J.M. Sugiyama E. Pavlova N.V. Li Y-T. Li S.-C. J. Biol. Chem. 1994; 269: 16276-16283Abstract Full Text PDF PubMed Google Scholar, 30.Li S.-C. Hirabayashi Y. Li Y.-T. J. Biol. Chem. 1981; 256: 6234-6240Abstract Full Text PDF PubMed Google Scholar) suggests that the binding of both the NeuAc and the GalNAc residues in the GM2 epitope may be necessary for the action of GM2 activator protein on the hydrolysis of GalNAc from GM2. We have synthesized a 23-mer peptide (see “Experimental Procedures”) which covers amino acids 106 through 128 of the GM2 activator protein. This segment of the peptide is encoded mostly by exon 4. The synthetic 23-mer peptide showed neither the stimulatory activity for the hydrolysis of GalNAc nor for the hydrolysis of NeuAc from GM2. When this peptide was mixed with GM2A protein, it did not enable GM2A protein to stimulate the hydrolysis of GalNAc from GM2. Taken together, our results indicate that the recognition of both the NeuAc and GalNAc residues in the GM2 epitope is a unique function of GM2 activator protein. As in the case of GM2 activator protein, GM2A protein also requires the hydrophobic lipid moiety of the substrate to express the stimulatory activity, since the oligosaccharide derived from GM2 was not hydrolyzed by β-hexosaminidase A or clostridial sialidase in the presence of GM2A protein. As reported previously, GM2A mRNA was found in both human placenta and fibroblasts, although in a much lower abundance than the mRNA of GM2 activator protein(12.Nagarajan S. Chen H.-C. Li S.-C. Li Y.-T. Lockyer J.M. Biochem. J. 1992; 282: 807-813Crossref PubMed Scopus (20) Google Scholar). This suggests that the existence of an alternative splicing for GM2 activator RNA may be physiologically important. Nature may use the alternative splicing of GM2 activator RNA to direct the catabolism of GM2. The production of GM2 activator mRNA or GM2A mRNA should lead to the production of GM2 activator protein or GM2A protein, respectively. In the presence of GM2 activator protein, the catabolism of GM2 may preferentially go through the cleavage of GalNAc residue, which is a well established pathway for the catabolism of GM2. This pathway explains the biochemical bases of Tay-Sachs diseases caused by the deficiency or the defect of β-hexosaminidase A, or GM2 activator protein. However, in the presence of GM2A protein the catabolism of GM2 might shift to a possible alternate pathway, GM2→GA2, since GM2A protein can only stimulate the hydrolysis of NeuAc from GM2 by clostridial sialidase, but not the hydrolysis of GalNAc. This would mean that the control of the production of GM2 activator mRNA and GM2A mRNA could be the branching point to direct the GM2 hydrolysis to GM2→GM3 or GM2→GA2 pathways. The possible in vivo pathway for the conversion of GM2 to GA2 has been proposed by Riboni et al.(31.Riboni L. Caminiti A. Bassi R. Tettamanti G. J. Neurochem. 1995; 64: 451-454Crossref PubMed Scopus (15) Google Scholar) in studies on the Neuro2a cell line. The authors suggested that the GA2 pathway was carried out by a specific sialidase which could convert GM2 to GA2. It has also been suggested by Fingerhut et al.(32.Fingerhut R. Van Der Horst G.T.J. Verheijen F.W. Conzelmann E. Eur. J. Biochem. 1992; 208: 623-629Crossref PubMed Scopus (37) Google Scholar) that the degradation of gangliosides by lysosomal sialidase also required an activator protein. The question of the physiological role of GM2A protein will remain unanswered until the native GM2A protein is isolated. By SDS-PAGE and Western blotting analysis, we have observed the presence of a protein band corresponding to 13 kDa which reacted with the antibody against GM2 activator protein in the partially purified placental GM2 activator protein preparation. At the present time, the isolation of GM2A protein is hampered by the lack of an assay method which can distinguish GM2A protein from GM2 activator protein." @default.
• W1520049999 created "2016-06-24" @default.
• W1520049999 creator A5022226408 @default.
• W1520049999 creator A5035014869 @default.
• W1520049999 creator A5041762599 @default.
• W1520049999 creator A5086892811 @default.
• W1520049999 date "1996-05-01" @default.
• W1520049999 modified "2023-09-26" @default.
• W1520049999 title "Characterization of an Alternatively Spliced GM2 Activator Protein, GM2A Protein" @default.
• W1520049999 cites W1514204573 @default.
• W1520049999 cites W1514518095 @default.
• W1520049999 cites W1523310946 @default.
• W1520049999 cites W1527790448 @default.
• W1520049999 cites W1534074292 @default.
• W1520049999 cites W1536736330 @default.
• W1520049999 cites W1964437175 @default.
• W1520049999 cites W1966555141 @default.
• W1520049999 cites W1984560166 @default.
• W1520049999 cites W1987800452 @default.
• W1520049999 cites W1994566293 @default.
• W1520049999 cites W1997270657 @default.
• W1520049999 cites W1997467660 @default.
• W1520049999 cites W2015126957 @default.
• W1520049999 cites W2043054344 @default.
• W1520049999 cites W2043502619 @default.
• W1520049999 cites W2054251756 @default.
• W1520049999 cites W2060153238 @default.
• W1520049999 cites W2077909420 @default.
• W1520049999 cites W2079114605 @default.
• W1520049999 cites W2100837269 @default.
• W1520049999 cites W2107227103 @default.
• W1520049999 cites W2109314031 @default.
• W1520049999 cites W2110663159 @default.
• W1520049999 cites W2135354472 @default.
• W1520049999 cites W2203299862 @default.
• W1520049999 cites W2322338818 @default.
• W1520049999 cites W2326860060 @default.
• W1520049999 cites W38492059 @default.
• W1520049999 doi "https://doi.org/10.1074/jbc.271.18.10611" @default.
• W1520049999 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/8631864" @default.
• W1520049999 hasPublicationYear "1996" @default.
• W1520049999 type Work @default.
• W1520049999 sameAs 1520049999 @default.
• W1520049999 citedByCount "12" @default.
• W1520049999 countsByYear W15200499992022 @default.
• W1520049999 crossrefType "journal-article" @default.
• W1520049999 hasAuthorship W1520049999A5022226408 @default.
• W1520049999 hasAuthorship W1520049999A5035014869 @default.
• W1520049999 hasAuthorship W1520049999A5041762599 @default.
• W1520049999 hasAuthorship W1520049999A5086892811 @default.
• W1520049999 hasConcept C104317684 @default.
• W1520049999 hasConcept C185592680 @default.
• W1520049999 hasConcept C55493867 @default.
• W1520049999 hasConcept C70721500 @default.
• W1520049999 hasConcept C86803240 @default.
• W1520049999 hasConcept C88045685 @default.
• W1520049999 hasConcept C95444343 @default.
• W1520049999 hasConceptScore W1520049999C104317684 @default.
• W1520049999 hasConceptScore W1520049999C185592680 @default.
• W1520049999 hasConceptScore W1520049999C55493867 @default.
• W1520049999 hasConceptScore W1520049999C70721500 @default.
• W1520049999 hasConceptScore W1520049999C86803240 @default.
• W1520049999 hasConceptScore W1520049999C88045685 @default.
• W1520049999 hasConceptScore W1520049999C95444343 @default.
• W1520049999 hasIssue "18" @default.
• W1520049999 hasLocation W15200499991 @default.
• W1520049999 hasOpenAccess W1520049999 @default.
• W1520049999 hasPrimaryLocation W15200499991 @default.
• W1520049999 hasRelatedWork W1972345237 @default.
• W1520049999 hasRelatedWork W2022333369 @default.
• W1520049999 hasRelatedWork W2025794474 @default.
• W1520049999 hasRelatedWork W2034109154 @default.
• W1520049999 hasRelatedWork W2055762486 @default.
• W1520049999 hasRelatedWork W2187784320 @default.
• W1520049999 hasRelatedWork W2248239788 @default.
• W1520049999 hasRelatedWork W2320604543 @default.
• W1520049999 hasRelatedWork W3007422624 @default.
• W1520049999 hasRelatedWork W4242851021 @default.
• W1520049999 hasVolume "271" @default.
• W1520049999 isParatext "false" @default.
• W1520049999 isRetracted "false" @default.
• W1520049999 magId "1520049999" @default.
• W1520049999 workType "article" @default.