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 beta-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 (Asn(106)-Tyr(114)) 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.
Highlights
In man, the degradation of the GM2 1 ganglioside requires lysosomal -hexosaminidase A (HexA) and a protein cofactor, the GM2 activator
When the hydrolysis of GA2 (Fig. 3B) was examined in the presence of the same amount of each activator as that used in Fig. 3A, mouse GM2 activator (mM2act) showed a much more pronounced stimulatory activity than that exerted by human GM2 activator (hM2act)
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
Summary
GM2 was isolated from the brain of a Tay-Sachs patient [10]. GA2 was prepared from GM2 by mild acid hydrolysis [11]. The cDNAs encoding human exon 2 and mouse exon 3 were obtained by restriction digestion of the construct ph2m3m4 using KpnI and HindIII to generate the ph2m3 fragment. The construct ph2m3h4 was digested with PvuII and HindIII to yield the 324-bp cDNA fragment encoding Ser103–Glu138 of the mouse sequence followed by the h4 segment. This fragment was digested using SmaI 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 5Ј-GGC-AGC-TGT-ACC-TTT-GAA-CAC-TTC-TGT-GAT-GTG-CTT-GAC-GAA-TAC-ATT-3Ј 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. Expression, refolding, and purification of the human/mouse GM2 activator chimeras were carried out as described previously for the refolding of hM2act [14]
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