EXPRESSION AND SPECIFICITY OF HUMAN G(M2) ACTIVATOR PROTEIN

The cDNA encoding G(M2) activator was expressed in the Escherichia col i/pT7-7 system. The yield of the G(M2) activator with greater than 99% purity was about 3 mg per liter culture. The recombinant G(M2) activa tor was found to be as active as that isolated from human kidney. The availability of the recombinant G(M2) activator enabled us to critical ly examine the specificity of this activator protein. Our results show that the specificity of G(M2) activator is not as strict as that repo rted previously. Although G(M2) activator stimulates most efficiently the degradation of G(M2) carried out by beta-N-acetylhexosaminidase A (Hex A), this activator also stimulates the following reactions: (a) c onversion of G(M2) to G(A2) by clostridial sialidase; (b) hydrolysis o f GalNAc from lmitoylphosphatidylethanolamine-II(3)NeuAcGgOse(3) by He x A; and (c) liberation of Gal from G(M1) by beta-galactosidase at a h igh activator concentration. Thus, this activator does not differentia te between G(M2) and lmitoylphosphatidylethanolamine-II(3)NeuAcGgOse(3 ) or between Hex A and clostridial sialidase. The micellar forms of G( D2) and GalNAc-G(D1a) were found to be more readily hydrolyzed by Hex A than G(M2) in the absence of G(M2) activator. Our results also show that saposin B can enhance the stimulatory activity of G(M2) activator , but it cannot promote the stimulatory activity of sodium taurodeoxyc holate. Taken together, our results suggest that the mechanism of acti on of G(M2) activator is different from saposin B, and the action of G (M2) activator is more than to solubilize lipid substrates. The effect iveness of G(M2) activator in stimulating the hydrolysis of G(M2) may be due to its abil ity to recognize the specific trisaccharide structu re of the G(M2) epitope, GalNAc beta 1 --> 4(NeuAc alpha 2 --> 3)Gal-, and to modify the GalNAc-NeuAc interaction in this structure.