Formation of a ternary complex between GM(2) activator protein, GM(2) ganglioside and hexosaminidase A

The GM(2) activator is a 17 kDa protein required for the hydrolysis of GM, ganglioside by the lysosomal enzyme hexosaminidase A (Hex A). The activator behaves as a substrate binding protein, solubilizing GM(2) ganglioside monomers from micelles (in vitro) or membranes (in vivo), However, the activator also shows a high order of specificity for activation of lysosomal hydrolases and has been predicted to form a ternary complex with the heterodimeric enzyme (alpha beta) Hex 4 and GM(2) ganglioside. We demonstrated a transient interaction between Hex A and the GM(2) activator. A chimeric protein containing the FLAG epitope sequence upstream of the GM(2) activator was expressed in Escherichia coli and purified using the M1 immunoaffinity (anti-FLAG) column. Binding of the FLAG-GM(2) activator (FLAG-AP) fusion protein to the M1 column led to the specific retardation of Hex A applied to the column. Other proteins were not retarded by the column nor did they compete with Hex A for binding to FLAG-AP. Hex A and GM(2) ganglioside could be simultaneously bound to the column, but the binding of each ligand was independent of the other. The homodimeric (beta beta) isozyme Hex B did not bind to the immobilized activator, The alpha alpha homodimer, Hex S, bound weakly, confirming that a hexosaminidase alpha subunit is required for interaction of enzyme and activator.