Volume 272, Number 5, Issue of January 31, 1997 pp. 2828-2833
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Interaction of G_M2 Activator Protein with Glycosphingolipids

(Received for publication, September 3, 1996, and in revised form, October 29, 1996)

From the Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112

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 G_M2 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 -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 gangliosides 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.

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