Interaction of G(M2) activator protein with glycosphingolipids

G(M2) activator protein is a protein cofactor that has been shown to stimulate the enzymatic hydrolysis of both GalNAc and NeuAc from G(M2) (Wu, Y. Y., Lockyer, J. M., Sugiyama, E., Pavlova, N.V., Li, Y.-T., and Li, S.-C. (1994) J. Biol. Chem. 269, 16276-16283). To understand the mechanism by which G(M2) activator stimulates the hydrolysis of G(M2), we examined the interaction of this activator protein with GM(2) as well as with other glycosphingolipids by TLC overlay and Sephacryl S-200 gel filtration. The TLC overlay analysis unveiled the binding specificity of G(M2) activator, which was not previously revealed. Under the conditions optimal for the activator protein to stimulate the hydrolysis of G(M2) by beta-hexosaminidase A, G(M2) activator was found to bind avidly to acidic glycosphingolipids, including gangliosides and sulfated glycosphingolipids, but not to neutral glycosphingolipids. The gangliosides devoid of sialic acids, such as asialo-G(M1) and asialo-G(M2), and the G(M2) derivatives whose carboxyl function in the NeuAc had been modified by methyl esterification or reduction, were only very weakly bound to G(M2) activator. These results indicate that the negatively charged sugar residue or sulfate group in gangliosides is one of the important sites recognized by G(M2) activator. For comparison, we also studied in parallel the complex formation between glycosphingolipids and saposin B, a separate activator protein with broad specificity to stimulate the hydrolysis of various glycosphingolipids. We found that saposin B bound to neutral glycosphingolipids and gan gliosides equally well, and there was an exceptionally strong binding to sulfatide. In contrast to previous reports, we found that G(M2) activator formed complexes with G(M2) and other gangliosides in different proportions depending on the ratio between the activator protein and the ganglioside in the incubation mixture prior to gel filtration. We were not able to detect the specific binding of G(M2) activator to G(M2) when G(M2) was mixed with G(M1) or G(M3). Thus, the specificity or the mode of action of G(M2) activator cannot be simply explained by its interaction with glycosphingolipids based on complex formation. The binding of G(M2) activator to a wide variety of negatively charged glycosphingolipids may indicate that this activator protein has functions other than assisting the enzymatic hydrolysis of G(M2).