FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2043143251> ?p ?o ?g. }

• W2043143251 endingPage "28618" @default.
• W2043143251 startingPage "28612" @default.
• W2043143251 abstract "Tay-Sachs disease is an inborn lysosomal disease characterized by excessive cerebral accumulation of GM2. The catabolism of GM2 to GM3 in man requires β-hexosaminidase A (HexA) and a protein cofactor, the GM2 activator. Thus, Tay-Sachs disease can be caused by the deficiency of either HexA or the GM2 activator. The same cofactor found in mouse shares 74.1% amino acid identity (67% nucleotide identity) with the human counterpart. Between the two activators, the mouse GM2 activator can effectively stimulate the hydrolysis of both GM2 and asialo-GM2 (GA2) by HexA and, to a lesser extent, also stimulate HexB to hydrolyze GA2, whereas the human activator is ineffective in stimulating the hydrolysis of GA2 (Yuziuk, J. A., Bertoni, C., Beccari, T., Orlacchio, A., Wu, Y.-Y., Li, S.-C., and Li, Y.-T. (1998) J. Biol. Chem. 273, 66–72). To understand the role of these two activators in stimulating the hydrolyses of GM2 and GA2, we have constructed human/mouse chimeric GM2 activators and studied their specificities. We have identified a narrow region (Asn106–Tyr114) in the mouse cDNA sequence that might be responsible for stimulating the hydrolysis of GA2. Replacement of the corresponding site in the human sequence with the specific mouse sequence converted the ineffective human activator into an effective chimeric protein for stimulating the hydrolysis of GA2. This chimeric activator protein, like the mouse protein, is also able to stimulate the hydrolysis of GA2 by HexB. The mouse model of human type B Tay-Sachs disease recently engineered by the targeted disruption of the Hexa gene showed less severe clinical manifestation than found in human patients. This has been considered to be the result of the catabolism of GM2 via converting it to GA2 and further hydrolysis of GA2 to lactosylceramide by HexB with the assistance of mouse GM2 activator protein. The chimeric activator protein that bears the characteristics of the mouse GM2 activator may therefore be able to induce an alternative catabolic pathway for GM2 in human type B Tay-Sachs patients. Tay-Sachs disease is an inborn lysosomal disease characterized by excessive cerebral accumulation of GM2. The catabolism of GM2 to GM3 in man requires β-hexosaminidase A (HexA) and a protein cofactor, the GM2 activator. Thus, Tay-Sachs disease can be caused by the deficiency of either HexA or the GM2 activator. The same cofactor found in mouse shares 74.1% amino acid identity (67% nucleotide identity) with the human counterpart. Between the two activators, the mouse GM2 activator can effectively stimulate the hydrolysis of both GM2 and asialo-GM2 (GA2) by HexA and, to a lesser extent, also stimulate HexB to hydrolyze GA2, whereas the human activator is ineffective in stimulating the hydrolysis of GA2 (Yuziuk, J. A., Bertoni, C., Beccari, T., Orlacchio, A., Wu, Y.-Y., Li, S.-C., and Li, Y.-T. (1998) J. Biol. Chem. 273, 66–72). To understand the role of these two activators in stimulating the hydrolyses of GM2 and GA2, we have constructed human/mouse chimeric GM2 activators and studied their specificities. We have identified a narrow region (Asn106–Tyr114) in the mouse cDNA sequence that might be responsible for stimulating the hydrolysis of GA2. Replacement of the corresponding site in the human sequence with the specific mouse sequence converted the ineffective human activator into an effective chimeric protein for stimulating the hydrolysis of GA2. This chimeric activator protein, like the mouse protein, is also able to stimulate the hydrolysis of GA2 by HexB. The mouse model of human type B Tay-Sachs disease recently engineered by the targeted disruption of the Hexa gene showed less severe clinical manifestation than found in human patients. This has been considered to be the result of the catabolism of GM2 via converting it to GA2 and further hydrolysis of GA2 to lactosylceramide by HexB with the assistance of mouse GM2 activator protein. The chimeric activator protein that bears the characteristics of the mouse GM2 activator may therefore be able to induce an alternative catabolic pathway for GM2 in human type B Tay-Sachs patients. In man, the degradation of the GM2 1The abbreviations used are:GM2GalNAcβ1→4(NeuAcα2→3) Galβ1→4Glcβ1→1′-ceramideGM3NeuAcα2→3Galβ1→4Glcβ1→1′-ceramideGA2GalNAcβ1→4Galβ1→4Glcβ1→1′-ceramideLacCerlactosylceramideHexA and HexBβ-hexosaminidases A and B, respectivelymM2actmouse GM2 activatorhM2acthuman GM2 activatorPCRpolymerase chain reactionbpbase pair(s) 1The abbreviations used are:GM2GalNAcβ1→4(NeuAcα2→3) Galβ1→4Glcβ1→1′-ceramideGM3NeuAcα2→3Galβ1→4Glcβ1→1′-ceramideGA2GalNAcβ1→4Galβ1→4Glcβ1→1′-ceramideLacCerlactosylceramideHexA and HexBβ-hexosaminidases A and B, respectivelymM2actmouse GM2 activatorhM2acthuman GM2 activatorPCRpolymerase chain reactionbpbase pair(s) ganglioside requires lysosomal β-hexosaminidase A (HexA) and a protein cofactor, the GM2 activator. The physiological importance of the GM2 activator is demonstrated by the severe clinical manifestations and neural accumulation of GM2 in type AB Tay-Sachs disease caused by the deficiency of this protein cofactor (1Li Y.-T. Li S.-C. Lysosomes Biol. Pathol. 1984; 7: 99-117Google Scholar, 2Gravel R.A. Clarke J.T.R. Kaback M.M. Mahuran D. Sandhoff K. Suzuki K. Scriver C.V. Beaudet A.L. Sly W.S. Valle D. Metabolic Basis of Inherited Diseases. McGraw-Hill Book Co., New York1995: 2839-2879Google Scholar). The recent studies of the mouse model of type B Tay-Sachs disease (Hexa −/−), generated via homologous recombination in embryonic stem cells, did not show the severe neurological symptoms characteristic in the same disease found in man (3Yamanaka S. Johnson M.D. Grinberg A. Westphal H. Crawley J.N. Taniike M. Suzuki K. Proia R.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9975-9979Crossref PubMed Scopus (159) Google Scholar, 4Cohen-Tannoudji M. Marchand P. Akli S. Sheardown S.A. Puech J.-P. Kress C. Gressens P. Nassogne M.-C. Beccari T. Muggleton-Harris A.L. Evrard P. Stirling J.L. Poenaru L. Babinet C. Mamm. Genome. 1995; 6: 844-849Crossref PubMed Scopus (57) Google Scholar, 5Sango K. Yamanaka S. Hoffmann A. Okuda Y. Grinberg A. Westphal H. McDonald M.P. Crawley J.N. Sandhoff K. Suzuki K. Proia R.L. Nat. Genet. 1995; 11: 170-176Crossref PubMed Scopus (371) Google Scholar, 6Sango K. McDonald M.P. Crawley J.N. Mack M.L. Tifft C.J. Skop E. Starr C.M. Hoffman A. Sandhoff K. Suzuki K. Proia R.L. Nat. Genet. 1996; 14: 348-352Crossref PubMed Scopus (173) Google Scholar, 7Phaneuf D. Wakamatsu N. Huang J.-Q. Borowski A. Peterson A.C. Fortunato S.R. Ritter G. Igdoura S.A. Morales C.R. Benoit G. Akerman B.R. Leclerc D. Hanai N. Marth J.D. Trasler J.M. Gravel R. Hum. Mol. Genet. 1996; 5: 1-14Crossref PubMed Scopus (184) Google Scholar). In these studies, the mild manifestations were initially attributed to the GM2-degrading activity of mouse HexB as reported by Burg et al. in 1983 (8Burg J. Banerjee A. Conzelmann E. Sandhoff K. Hoppe-Seyler's Z. Physiol. Chem. 1983; 364: 821-829Crossref PubMed Scopus (27) Google Scholar). In contrast, we have shown that the highly purified mouse HexB was not able to convert GM2 to GM3, but was able to slowly catalyze the conversion of GA2 to LacCer in the presence of the mouse GM2 activator (mM2act) (9Yuziuk J.A. Bertoni C. Beccari T. Orlacchio A. Wu Y.-Y Li S.-C. Li Y.-T. J. Biol. Chem. 1998; 273: 66-72Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In the same report, we also showed that mM2act was able to effectively stimulate the hydrolysis of GA2 catalyzed by either human or mouse HexA and that the human GM2 activator (hM2act) was not effective in stimulating the hydrolysis of GA2. GalNAcβ1→4(NeuAcα2→3) Galβ1→4Glcβ1→1′-ceramide NeuAcα2→3Galβ1→4Glcβ1→1′-ceramide GalNAcβ1→4Galβ1→4Glcβ1→1′-ceramide lactosylceramide β-hexosaminidases A and B, respectively mouse GM2 activator human GM2 activator polymerase chain reaction base pair(s) GalNAcβ1→4(NeuAcα2→3) Galβ1→4Glcβ1→1′-ceramide NeuAcα2→3Galβ1→4Glcβ1→1′-ceramide GalNAcβ1→4Galβ1→4Glcβ1→1′-ceramide lactosylceramide β-hexosaminidases A and B, respectively mouse GM2 activator human GM2 activator polymerase chain reaction base pair(s) To better understand the role of hM2act and mM2act in the degradation of GM2 and GA2, we have constructed a series of human/mouse chimeric GM2 activators and studied their ability to stimulate the hydrolysis of GM2 and GA2 by human HexA. Since hM2act is not effective in stimulating the hydrolysis of GA2 by HexA, the specific human/mouse chimeras that elicit this activity should reveal the amino acids that are responsible for stimulating the hydrolysis of GA2. GM2 was isolated from the brain of a Tay-Sachs patient (10Svennerholm L. Methods Carbohydr. Chem. 1972; 6: 464-474Google Scholar). GA2 was prepared from GM2 by mild acid hydrolysis (11Svennerholm L. Månsson J.-E. Li Y.-T. J. Biol. Chem. 1973; 248: 740-742Abstract Full Text PDF PubMed Google Scholar). HexA (33.3 units/mg) from human liver was prepared as described previously (12Li 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). The following were purchased from the commercial sources indicated: precoated Silica Gel 60 TLC plates, Merck (Darmstadt, Germany); 4-methylumbelliferyl N-acetylglucosaminide, Coomassie Brilliant Blue R-250, Trizma (Tris base), and glycine, Sigma; 4-methylumbelliferyl N-acetylglucosaminide 6-sulfate, Research Development Corp. (Toronto, Canada); PM-10 ultrafiltration membrane (10,000 molecular weight cutoff), Amicon, Inc.; [32P]dCTP (3000 Ci/mmol), 35S-dATP (1000 Ci/mmol), the multiprime DNA labeling system, the Sequenase sequencing kit, restriction endonucleases, DNA ligase, nitrocellulose membrane, and protein standards for molecular weights, Amersham Pharmacia Biotech; fmol TM DNA sequencing system, andPfu DNA polymerase, Promega; LacCer and GM3, Matreya, Inc.; isopropyl-1-thio-β-d-galactopyranoside, ampicillin, glutathione, yeast extract, and Tryptone, Difco; the T7 sequencing kit (Version 2.0), U. S. Biochemical Corp.; and Escherichia coli strain BL21(DE3), Novagen. The numbering system for the deduced amino acids in the following constructs was based on the alignment of the nucleotide sequences of hM2act and mM2act using the PC/GENE computer software program (Fig. 1). Three gaps in the human sequence and seven gaps in the mouse sequence were inserted to obtain the best alignment. Therefore, the numerical assignment for the amino acids in the human sequence differed from that in the mouse sequence by a factor of +4 amino acids. All the gaps inserted were contained within the first 27 amino acids of the human sequence at the propeptide region, which is removed by proteolysis during the maturation of GM2 activator protein (13Fürst W. Schubert J. Machleidt W. Meyer H.E. Sandhoff K. Eur. J. Biochem. 1990; 192: 709-714Crossref PubMed Scopus (47) Google Scholar). The insertion did not at all affect the construction of the chimeras since all of the cDNA constructs started from Ser32, the N-terminal amino acid of the mature human protein. The first set of cDNA constructs for the activator chimeras were generated from exon swapping (Fig.2 A). For the names of all constructs, the prefix “p” denotes plasmid that contains the designated cDNA.Figure 2Construction of cDNAs for human/mouse chimeric GM2 activators. A, schematic representation of the chimeric GM2 activators obtained by exon swapping of hM2act and mM2act. The right-hand columns summarize the stimulatory activities of the expressed proteins (1 μg/assay) for the hydrolysis of GM2 and GA2 by HexA. The notations h2, h3, and h4 and m2, m3, and m4 indicate the sequences from the human and mouse exons, respectively. B, schematic representation of the subsequent chimeras generated from ph2m3h4 and ph2m3m4. The detailed strategies used to generate these constructs are described under “Experimental Procedures.” Shaded boxes represent the mouse sequence, and white boxes represent the human sequence. In each box, the amino acids of hM2act at the junctions are shown in the upper corners and those of mM2act in the lower corners. The numbering system for the human and mouse sequences follows that shown in Fig. 1. For convenience, the name of each construct without “p” was used to designate the expressed protein. +, >80% hydrolysis; −, <30% or no hydrolysis; +/−, between 30 and 80% hydrolysis under the assay conditions specified in the legends to Figs. 4, 5, and 7.View Large Image Figure ViewerDownload Hi-res image Download (PPT) This construct contains human exons 2 and 3 plus mouse exon 4. A T7-7 plasmid vector containing the cDNA (p513) encoding only the 162 amino acids of mature hM2act (14Wu 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 as template to generate, by polymerase chain reaction (PCR), a 445-bp cDNA fragment. This human cDNA fragment encodes 9 amino acids of the pT7-7 expression vector (Met-Ala-Arg-Ile-Arg-Ala-Arg-Gly-Ser) plus human exons 2 and 3. The upstream primer was 5′-TAA-TAC-GAC-TCA-CTA-TAG-GGA-GA-3′ (T7 primer) within the pT7 vector region, and the downstream primer (noncoding) was 5′-GAG-TAG-GTA-CCT-TCT-TTG-3′ with a built-inKpnI restriction site (underlined). This cDNA segment was digested with BamHI and KpnI. The remaining 164-bp cDNA fragment encoding mouse exon 4 was obtained by restriction digestion of pMact with KpnI andHindIII. The two cDNA segments were subcloned into the pT7-7 expression vector at BamHI and HindIII sites. This construct contains human exon 2 and mouse exons 3 and 4. A 177-bp DNA fragment encoding the 9 amino acids of the pT7-7 expression vector plus the first 50 amino acids (Ser32–Lys81) of the mature hM2act sequence (13Fürst W. Schubert J. Machleidt W. Meyer H.E. Sandhoff K. Eur. J. Biochem. 1990; 192: 709-714Crossref PubMed Scopus (47) Google Scholar) encoded by exon 2 of the human sequence (also see Fig. 1) was generated by PCR amplification using p513 as template. The upstream primer was the T7 primer as described above. The downstream primer (noncoding) was 5′-ACG-GTG-AGC-TCC-ACC-TTC-3′, which contained aSacI restriction site (underlined). The remaining 112 amino acids encoded by mouse exons 3 and 4 were obtained by restriction digestion of the mM2act cDNA (pMact) (9Yuziuk J.A. Bertoni C. Beccari T. Orlacchio A. Wu Y.-Y Li S.-C. Li Y.-T. J. Biol. Chem. 1998; 273: 66-72Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) with SacI andHindIII to yield a 344-bp cDNA fragment encoding Val78–Arg189 of mM2act. This mouse cDNA segment and the above PCR fragment of human exon 2, which had been digested with BamHI and SacI, were subcloned into a pT7-7 expression vector at BamHI and HindIII sites. This construct contains human exon 2, mouse exon 3, and human exon 4. The cDNAs encoding human exon 2 and mouse exon 3 were obtained by restriction digestion of the construct ph2m3m4using KpnI and HindIII to generate the ph2m3 fragment. The remaining cDNA of the 164-bp fragment encoding human exon 4 was generated by PCR using p513 as template. The upstream primer was 5′-GAA-GGT-ACC-TAC-TCA-3′, in which the KpnI site is underlined. The downstream noncoding primer was the T7-7 primer 5′-GAT-GAT-AAG-CTT-GGG-CTG-3′, corresponding to the 3′-untranslated region of the pT7-7 vector. The amplified human fragment (207 bp) was digested with KpnI and HindIII and ligated into ph2m3 at KpnI and HindIII sites. This construct contains mouse exons 2 and 3 plus human exon 4. The construct ph2m3h4 was digested withPvuII and HindIII to yield the 324-bp cDNA fragment encoding Ser103–Glu138 of the mouse sequence followed by the h4 segment. This fragment was subcloned into mouse pMact that had been digested by PvuII and HindIII. From the initial experiments, the m3 segment appeared to be important for eliciting the stimulatory activity for the enzymatic hydrolysis of GA2. Therefore, we subsequently modified the m3 segment in ph2m3h4by including more human sequence and generated the following constructs (also see Fig. 2 B). This construct contains an extended human sequence (25 amino acids from Val82 to Cys106) at the N terminus of m3. The 226-bp fragment encoding Ser32–Cys106 of hM2act was excised from p513 by restriction digestion with BamHI and PvuII. The segment of mouse exon 3 encoding Ser103–Glu138 plus human exon 4 encoding Gly143-Ile193 was obtained by digestion of ph2m3h4 with PvuII andHindIII. The two fragments were ligated into the pT7-7 vector at its BamHI and HindIII sites. This construct contains an extended human sequence (36 amino acids from Thr107 to Glu142) at the C terminus of m3. The 323-bp cDNA fragment encoding Thr107–Ile193 of the human sequence was generated by PCR using p513 as template. The upstream primer was 5′-GGC-AGC-TGT-ACC-TTT-GAA-3′, containing aPvuII restriction site (underlined); and the downstream primer was the T7-7 primer as described above. The PCR product was digested with PvuII and HindIII and subcloned into ph2m3h4 that had been cut byPvuII and HindIII. This construct contains an extended human sequence from both ends of m3 and keeps only Ser103–Pro117 in the mouse sequence. The p513 clone was used as template to generate, by PCR, a cDNA fragment covering Thr121–Ile193 of hM2act. The upstream primer was 5′-CCT-CCC-GGG-GAG-CCC-TGC-3′, containing a point mutation in the Thr121 codon to give Pro117 of mM2act and also to generate a SmaI restriction site (underlined). The T7-7 primer as described above was used as the downstream noncoding primer. This fragment was digested usingSmaI and HindIII. Another 374-bp cDNA fragment encoding Ser32–Cys106 of the human sequence followed by Ser103–Pro116 of the mouse sequence was generated from the ph2m3h4-a clone by PCR. The upstream primer was the T7 primer as described above, and the downstream noncoding primer was 5′-GCT-CTC-CCC-GGG-AGG-AAT-3′, in which the SmaI restriction site is underlined. This fragment was digested withBamHI and SmaI. Then, both cDNA fragments were inserted at the BamHI and HindIII sites of the pT7-7 vector. This construct contains an additional extension of 7 amino acids (Thr107–Asp113) of the human sequence in ph2m3h4-a-SH, leaving only Leu110–Pro117 in the mouse sequence. The p513 clone was used as template to generate a cDNA fragment by PCR to span from Thr107 to Ile193 of the human sequence. The upstream primer was 5′-GGC-AGC-TGT-ACC-TTT-GAA-CAC-TTC-TGT-GAC-CTG-ATA-GAC-GAA-TAC-ATT-3′ with a built-in PvuII site (underlined), and the T7-7 primer described above was used as the downstream noncoding primer. Nine point mutations (indicated in boldface) were introduced into the upstream primer to generate the fragment encoding Leu110, Ile111, Glu113, Tyr114, and Pro117 of the mouse sequence. The amplified cDNA fragment was digested with PvuII and HindIII and subcloned into p513 at the corresponding restriction site. This construct contains the human sequence from Ser32 to Asp113 followed by the mouse sequence from Leu110 to Arg189. The portion from Ser32 to Asp113 of the human sequence plus Leu110–Pro117 of the mouse sequence was generated by digestion of ph2m3h4-a-NI with BamHI and SmaI. This cDNA fragment was subcloned into mouse pMact that was digested with BamHI and SmaI. Since ph2m3h4-a-SH contains only 5 amino acids (Asn106, Ile107, Glu113, Tyr114, and Pro117) that are different from the human sequence, the following constructs were prepared to evaluate the importance of these amino acids (see Fig.6). This construct is basically the human sequence except with the changes of Met117 and Leu118to Glu113 and Tyr114, respectively, to match the mouse sequence. The upstream primer 5′-GGC-AGC-TGT-ACC-TTT-GAA-CAC-TTC-TGT-GAT-GTG-CTT-GAC-GAA-TAC-ATT-3′ with a built-in PvuII site (underlined) was used along with the T7-7 primer described above to generate, by PCR, the cDNA encoding the amino acid sequence from Thr107 to Ile193 of the human sequence. The p513 clone served as template. The five point mutations in the upstream primer (indicated in boldface) were introduced to create the replacement of human Met117 and Leu118 with Glu113 and Tyr114, respectively, at the corresponding positions of the mouse sequence. The amplified fragment was digested withPvuII and HindIII and subcloned into the p513 clone at its equivalent restriction site. This construct is essentially the same as p513ML with an additional change of Thr121 of the human sequence to Pro117 at the corresponding position of the mouse sequence. The primers used to generate p513ML were also used to construct p513MLT. However, the template for PCR was ph2m3h4-a-NI instead of p513. This construct contains the human sequence except with the replacement of 5 amino acids with the corresponding Asn106, Ile107, Glu113, Tyr114, and Pro117 from the mouse sequence. p513MLT was used as template to generate a cDNA fragment by PCR. The upstream primer was 5′-GGC-AGC-TGT-ACC-TTT-GAA-AAC-ATC-TGT-3′ with a built-in PvuII site (underlined). Two point mutations (indicated in boldface) were introduced to provide Asn106and Ile107 of the mouse sequence. The noncoding downstream primer was the T7-7 primer as described above. The PCR product was digested with PvuII and HindIII and subcloned into p513 at its corresponding restriction sites. Each construct was verified by sequencing the cDNA prior to transforming the competent E. coli BL21(DE3) cells. TheE. coli transformants were inoculated into 150 ml of LB medium containing 1 mg/ml ampicillin and incubated overnight at 37 °C. The overnight culture was diluted at a ratio of 1:33 with fresh LB/ampicillin medium (30 ml/1 liter) and grown for ∼4 h at 37 °C. Expression of GM2 activator protein was then induced by addition of isopropyl-1-thio-β-d-galactopyranoside at a final concentration of 1 mm, and the culture was grown for an additional 6 h. The cells were harvested by centrifugation at 6000 rpm for 15 min using a GS3 rotor in a Sorvall RC5C centrifuge. Expression, refolding, and purification of the human/mouse GM2 activator chimeras were carried out as described previously for the refolding of hM2act (14Wu 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). Human HexA activity was determined using fluorogenic substrates (4-methylumbelliferyl N-acetylglucosaminide and 4-methylumbelliferyl N-acetylglucosaminide 6-sulfate) according to Potier et al. (15Potier M. Mameli L. Bélisle M. Dallaire L. Melançon S.B. Anal. Biochem. 1979; 94: 287-296Crossref PubMed Scopus (735) Google Scholar). An appropriate amount of HexA was incubated with 1.5 mm 4-methylumbelliferylN-acetylglucosaminide or 4-methylumbelliferylN-acetylglucosaminide 6-sulfate in 50 mm sodium citrate buffer (pH 5.0) in a total volume of 50 μl at 37 °C. After a preset time, 1.5 ml of 0.2 m sodium borate buffer (pH 9.8) was added to stop the reaction. The released 4-methylumbelliferone was determined using a Sequoia-Turner Model 450 fluorometer. One unit of enzyme activity is defined as the amount that liberates 1 μmol of 4-methylumbelliferone/min at 37 °C. This fluorogenic assay was used only to standardize the amount of HexA for each experiment. For the hydrolysis of GM2 and GA2, the reaction mixture contained 3 nmol of substrate in 40 μl of 10 mm sodium acetate buffer (pH 5.0). The reactions were initiated by adding 20 milliunits of human HexA and terminated by adding 40 μl of ethanol. The mixtures were dried under vacuum using a SpeedVac, redissolved in 20 μl of chloroform/methanol (2:1, v/v), and applied to a TLC plate. The plate was developed with chloroform/methanol/water (60:35:8, v/v/v), sprayed with diphenylamine reagent (16Harris G. MacWilliams I.C. Chem. Ind. (Lond.). 1954; : 249Google Scholar), and heated at 115 °C for 15–20 min to visualize the glycolipids. The quantitative analysis of the glycolipid bands on the TLC plate was carried out using a Scan Jet 2C/ADF scanner (Hewlett-Packard Co.) and the NIH Image 1.41 program. The recombinant human/mouse chimeric GM2 activators were analyzed by SDS-polyacrylamide gel electrophoresis using 15% gel (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). Proteins were electrophoretically transferred onto a nitrocellulose membrane in 20 mm Tris and 150 mm glycine buffer (pH 8.0) containing 20% methanol at 70 V for 1 h using a Bio-Rad transfer apparatus. The nitrocellulose membrane was first soaked with 1% milk powder and then overlaid with rabbit anti-hM2act antibodies (1:1500) (18Li S.-C. Hirabayashi Y. Li Y.-T. J. Biol. Chem. 1981; 256: 6234-6240Abstract Full Text PDF PubMed Google Scholar) or rabbit anti-mM2act antibodies (produced by Cocalico Biological, Inc.) as the primary antibody followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000) as the secondary antibody. The membrane was then developed in 3.4 mm 4-chloro-1-naphthol containing 0.01% hydrogen peroxide to produce purple bands. The nucleotide sequences of hM2act and mM2act downstream from the initiation codon were analyzed using the PC/GENE program (Fig.1). With 10 gap insertions in exon 1 (three in the human sequence and seven in the mouse sequence), the two sequences obtained the best alignment and showed 67% identity in their nucleotides. These gaps are upstream of the sequence encoding the mature form of hM2act (13Fürst W. Schubert J. Machleidt W. Meyer H.E. Sandhoff K. Eur. J. Biochem. 1990; 192: 709-714Crossref PubMed Scopus (47) Google Scholar). A higher degree of similarity (74.1% identical and 9.9% similar) was found in the deduced amino acid sequences when they were compared as mature proteins from Ser32 to the C terminus of the human sequence (Fig. 1). The comparison based on the mature protein is appropriate in this study since all the chimeras were expressed only as the mature proteins. Although hM2act and mM2act share a very high degree of homology and both are active in stimulating the enzymatic hydrolysis of GM2, their stimulatory activities for the hydrolysis of GA2 are distinctly different. Fig.3 A shows the time course of GM2 hydrolysis by human HexA in the presence of either hM2act or mM2act. Under the same condition, 1 μg each of hM2act and mM2act showed comparable stimulatory activity for the hydrolysis of GM2 throughout the 10-, 20-, and 30-min incubations. This indicates that these two activator proteins have similar potency in stimulating the hydrolysis of GM2. However, when the hydrolysis of GA2 (Fig.3 B) was examined in the presence of the same amount of each activator as that used in Fig. 3 A, mM2act showed a much more pronounced stimulatory activity than that exerted by hM2act. This result corroborates our previous report (9Yuziuk J.A. Bertoni C. Beccari T. Orlacchio A. Wu Y.-Y Li S.-C. Li Y.-T. J. Biol. Chem. 1998; 273: 66-72Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) that mM2act was able to effectively stimulate the hydrolysis of GA2 and that hM2act was ineffective in stimulating this reaction. Image scanning of the TLC plate showed that the stimulatory activity of hM2act for GA2 hydrolysis was only ∼15–18% of that of mM2act. Based on this difference, we reasoned that any human/mouse chimeric activator that can elicit stimulatory activity similar to that of mM2act for the hydrolysis of GA2 should be attributed to the specific mouse sequence in the chimeric protein. All chimeras were therefore examined for their stimulatory activities for the hydrolyses of GM2 and GA2 using 1 μg of the protein. Our strategy was to exchange selected parts of the human sequence with the corresponding mouse sequence and to examine the chimeras for their increased ability to stimulate the hydrolysis of GA2. This strategy was proven to be effective. Alternatively, one can start from the mouse sequence and substitute it with certain parts of the human sequence. This strategy would result in chimeras with diminished stimulatory activity for GA2 hydrolysis. Because the stimulatory activity of the activator protein can also be attenuated by factors such as poor protein refolding, the second approach might not provide clear results. Taking advantage of the fact that the cDNAs of hM2act and mM2act share a high degree of homology and identical intron/exon junctions (19Klima H. Tanaka A. Schnabel D. Nakano T. Schröder M. Suzuki K. Sandhoff K. FEBS Lett. 1991; 289: 260-264Crossref PubMed Scopus (40) Google Scholar, 20Bertoni C. Apolloni M.G. Stirling J.L. Li S.-C. Li Y.-T. Orlacchio A. Beccari T. Mamm. Genome. 1997; 8: 90-93Crossref Scopus (7) Google Scholar), we first constructed four chimeric constructs (ph2h3m4, ph2m3m4, ph2m3h4, and pm2m3h4) according to the four possible exchanges of exons (Fig. 2). All four proteins expressed by these chimeric constructs were fully active in stimulating the hydrolysis of GM2 (Fig. 4 A,lanes 4–7). These results indicate that the exchange of the corresponding exon segments between the human and mouse sequences did not alter the stimulatory activity of the chimeric proteins for the hydrolysis of GM2. These results were not unexpected since both hM2act and mM2act could effectively stimulate this reaction. We were intrigued, however, by the differences in the ability of these chimeras to stimulate the hydrolysis of GA2 (Fig. 4 B). Under the same incubation conditions, those proteins encoded by ph2m3m4, ph2m3h4, and pm2m3h4, but not that by ph2h3m4, showed significant activity to stimulate the hydrolysis of GA2 (Fig. 4 B,lanes 4–7; also summarized in Fig. 2 A). The protein encoded by ph2h3m4 and the parent hM2act showed on" @default.
• W2043143251 created "2016-06-24" @default.
• W2043143251 creator A5022177255 @default.
• W2043143251 creator A5022226408 @default.
• W2043143251 creator A5041762599 @default.
• W2043143251 date "1999-10-01" @default.
• W2043143251 modified "2023-10-10" @default.
• W2043143251 title "Catabolism of Asialo-GM2 in Man and Mouse" @default.
• W2043143251 cites W1514518095 @default.
• W2043143251 cites W1520626903 @default.
• W2043143251 cites W1527790448 @default.
• W2043143251 cites W1536736330 @default.
• W2043143251 cites W1987800452 @default.
• W2043143251 cites W1994644330 @default.
• W2043143251 cites W1997270657 @default.
• W2043143251 cites W2023641192 @default.
• W2043143251 cites W2042512150 @default.
• W2043143251 cites W2044970570 @default.
• W2043143251 cites W2048701007 @default.
• W2043143251 cites W2089486887 @default.
• W2043143251 cites W2094353124 @default.
• W2043143251 cites W2100837269 @default.
• W2043143251 cites W2151880210 @default.
• W2043143251 cites W2171981447 @default.
• W2043143251 cites W2320435053 @default.
• W2043143251 cites W2505104519 @default.
• W2043143251 cites W2413390034 @default.
• W2043143251 doi "https://doi.org/10.1074/jbc.274.40.28612" @default.
• W2043143251 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10497228" @default.
• W2043143251 hasPublicationYear "1999" @default.
• W2043143251 type Work @default.
• W2043143251 sameAs 2043143251 @default.
• W2043143251 citedByCount "14" @default.
• W2043143251 countsByYear W20431432512013 @default.
• W2043143251 countsByYear W20431432512016 @default.
• W2043143251 countsByYear W20431432512020 @default.
• W2043143251 countsByYear W20431432512021 @default.
• W2043143251 crossrefType "journal-article" @default.
• W2043143251 hasAuthorship W2043143251A5022177255 @default.
• W2043143251 hasAuthorship W2043143251A5022226408 @default.
• W2043143251 hasAuthorship W2043143251A5041762599 @default.
• W2043143251 hasBestOaLocation W20431432511 @default.
• W2043143251 hasConcept C185592680 @default.
• W2043143251 hasConcept C55493867 @default.
• W2043143251 hasConcept C62231903 @default.
• W2043143251 hasConcept C70721500 @default.
• W2043143251 hasConcept C86803240 @default.
• W2043143251 hasConcept C96942376 @default.
• W2043143251 hasConceptScore W2043143251C185592680 @default.
• W2043143251 hasConceptScore W2043143251C55493867 @default.
• W2043143251 hasConceptScore W2043143251C62231903 @default.
• W2043143251 hasConceptScore W2043143251C70721500 @default.
• W2043143251 hasConceptScore W2043143251C86803240 @default.
• W2043143251 hasConceptScore W2043143251C96942376 @default.
• W2043143251 hasIssue "40" @default.
• W2043143251 hasLocation W20431432511 @default.
• W2043143251 hasOpenAccess W2043143251 @default.
• W2043143251 hasPrimaryLocation W20431432511 @default.
• W2043143251 hasRelatedWork W1517983644 @default.
• W2043143251 hasRelatedWork W1528720586 @default.
• W2043143251 hasRelatedWork W1535356922 @default.
• W2043143251 hasRelatedWork W1597363043 @default.
• W2043143251 hasRelatedWork W1610514370 @default.
• W2043143251 hasRelatedWork W2055011390 @default.
• W2043143251 hasRelatedWork W2056593986 @default.
• W2043143251 hasRelatedWork W2316189360 @default.
• W2043143251 hasRelatedWork W2383301737 @default.
• W2043143251 hasRelatedWork W2775077500 @default.
• W2043143251 hasVolume "274" @default.
• W2043143251 isParatext "false" @default.
• W2043143251 isRetracted "false" @default.
• W2043143251 magId "2043143251" @default.
• W2043143251 workType "article" @default.