Formation of a ternary complex between GM2 activator protein, GM2 ganglioside and hexosaminidase A.
The GM2 activator is a 17 kDa protein required for the hydrolysis of GM2 ganglioside by the lysosomal enzyme hexosaminidase A (HexA). The activator behaves as a substrate binding protein, solubilizing GM2 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 (alphabeta) Hex A and GM2 ganglioside. We demonstrated a transient interaction between HexA and the GM2 activator. A chimeric protein containing the FLAG epitope sequence upstream of the GM2 activator was expressed in Escherichia coli and purified using the M1 immunoaffinity (anti-FLAG) column. Binding of the FLAG-GM2 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 GM2 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, HexS, bound weakly, confirming that a hexosaminidase alpha subunit is required for interaction of enzyme and activator.
Yadao
F
F
McGill University-Montreal Children's Hospital Research Institute, Montreal, Canada.
Hechtman
P
P
Kaplan
F
F
D019281:Dimerization; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D049289:G(M2) Activator Protein; D005678:G(M2) Ganglioside / Q000378:metabolism*; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism*
D000939:Epitopes; D049289:G(M2) Activator Protein; D007527:Isoenzymes; D009842:Oligopeptides; D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins; D005678:G(M2) Ganglioside; C085706:FLAG peptide; D054818:Hexosaminidase A; D054819:Hexosaminidase B; D001619:beta-N-Acetylhexosaminidases