Yadao F, Hechtman P, Kaplan F
McGill University-Montreal Children's Hospital Research Institute, Montreal, Canada.
Biochim Biophys Acta. 1997 Jun 20;1340(1):45-52. doi: 10.1016/s0167-4838(97)00027-7.
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.
GM2激活蛋白是一种17 kDa的蛋白质，溶酶体酶己糖胺酶A（HexA）水解GM2神经节苷脂时需要该蛋白。该激活蛋白表现为一种底物结合蛋白，可使GM2神经节苷脂单体从胶束（体外）或膜（体内）中溶解出来。然而，该激活蛋白对溶酶体水解酶的激活也表现出高度的特异性，并且预计会与异二聚体酶（αβ）Hex A和GM2神经节苷脂形成三元复合物。我们证明了HexA与GM2激活蛋白之间存在瞬时相互作用。一种在GM2激活蛋白上游含有FLAG表位序列的嵌合蛋白在大肠杆菌中表达，并使用M1免疫亲和（抗FLAG）柱进行纯化。FLAG-GM2激活蛋白（FLAG-AP）融合蛋白与M1柱的结合导致应用于该柱的Hex A出现特异性滞留。其他蛋白质不会被该柱滞留，也不会与Hex A竞争与FLAG-AP的结合。Hex A和GM2神经节苷脂可以同时与该柱结合，但每个配体的结合相互独立。同二聚体（ββ）同工酶Hex B不与固定化的激活蛋白结合。αα同二聚体HexS结合较弱，这证实了酶与激活蛋白相互作用需要己糖胺酶α亚基。
