Catabolism of Tay-Sachs ganglioside: Studies of the recombinant GM2-activator proteins
Description
GM2-Activator protein is essential for the in vivo catabolism of GM2, the Tay-Sachs ganglioside, by $\beta$-hexosaminidase A. Progress in understanding the mechanism of the action of GM2-activator protein has been hampered by difficulty in isolating sufficient amounts of the pure GM2-activator protein from human tissues. Therefore, we expressed human GM2-activator protein in E. coli. The functionally active recombinant GM2-activator protein in pure form was obtained by denaturing extraction, refolding, and purification. We also expressed an alternatively spliced and truncated version of GM2-activator protein, GM2A protein. Studies of the specificities of these two activator proteins led to the following conclusions: (a) in addition to stimulating the hydrolysis of GM2 by $\beta$-hexosaminidase A, GM2-activator protein can also stimulate the liberation of NeuAc from GM2 by clostridial sialidase. The specificity of this action was demonstrated by specific stimulation of the cleavage of the terminal NeuAc from GalNAc-GD1a by GM2-activator protein. In contrast, saposin B, a nonspecific activator protein with a detergent-like activity, caused a nonspecific stimulation of the hydrolysis of both internal and external NeuAc from GalNAc-GD1a. Thus, GM2-activator protein may recognize and interact with the GM2 epitope branched trisaccharide structure GalNAc$\beta1\to 4$ (NeuAc$\alpha2\to 3)$ Gal. (b) GM2A protein was found to stimulate the enzymatic hydrolysis of NeuAc, but not GalNAc, from GM2 and also to specifically recognize the GM2 epitope in GalNAc-GD1a. These results suggest that in the presence of GM2A protein the catabolism of GM2 may alternatively go through the GM2 $\to$ asialo GM2 pathway. Since GM2A protein contains mainly the same 1-109 amino acids as the N-terminus of GM2-activator protein and both of these activator proteins could stimulate the hydrolysis of NeuAc from GM2, the NeuAc recognition domain of GM2-activator protein is located within amino acids 1-109. (c) The hydrophobicity, not the specific structure, of the lipid portion in GM2 is required for the stimulatory effects of these two activator proteins. This was based on the facts that GM2-activator protein stimulates the hydrolysis of II$\sp3$NeuAcGgOse$\sb3$-dipal-mitoylphosphatidyl ethanolamine, but not II$\sp3$NeuAcGgOse$\sb3$, by $\beta$-hexosaminidase A, and that GM2-activator protein and GM2A protein stimulate the hydrolysis of II$\sp3$NeuAcGgOse$\sb3$-dipal-mitoylphosphatidyl ethanolamine, but not II$\sp3$NeuAcGgOse$\sb3,$ by clostridial sialidase. We also expressed, purified and characterized mouse GM2-activator protein. The recombinant mouse GM2-activator protein was found to be as active as the human GM2-activator protein