Abstract
GM2 Activator is a low molecular weight protein cofactor that stimulates the enzymatic conversion of GM2 into GM3 by human beta-hexosaminidase A and also the conversion of GM2 into GA2 by clostridial sialidase (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). Among the five known activator proteins for the enzymatic hydrolysis of glycosphingolipids, only GM2 activator is effective in stimulating the hydrolysis of GM2. However, the mechanism of action of GM2 activator is still not well understood. Using a unique disialosylganglioside, GalNAc-GD1a, as the substrate, we were able to show that in the presence of GM2 activator, GalNAc-GD1a was specifically converted into GalNAc-GM1a by clostridial sialidase, while in the presence of saposin B, a nonspecific activator protein, GalNAc-GD1a was converted into both GalNAc-GM1a and GalNAc-GM1b. Individual products generated from GalNAc-GD1a by clostridial sialidase were identified by thin layer chromatography, negative secondary ion mass spectrometry, and immunostaining with a monoclonal IgM that recognizes the GM2 epitope. Our results clearly show that GM2 activator recognizes the GM2 epitope in GalNAc-GD1a. Thus, GM2 activator may interact with the trisaccharide structure of the GM2 epitope and render the GalNAc and NeuAc residues accessible to beta-hexosaminidase A and sialidase, respectively.
Highlights
Sugar chains in glycosphingolipids of higher animals are catabolized by lysosomal glycosidases, and some of the hydrolytic steps have been shown to require the assistance of protein cofactors called activator proteins [1,2,3]
We have shown that the effectiveness of GM2 activator in stimulating the hydrolysis of GM2 may be due to its ability to recognize and interact with the specific trisaccharide structure of the GM2 epitope, GalNAc134(NeuAc␣233)-Gal, and make the GalNAc residue in GM2 accessible to -N-acetylhexosaminidase A [18]
We have shown that saposin B stimulates the hydrolysis of GM1 by human hepatic -galactosidase and that GM2 activator stimulates the hydrolysis of GM2 by -N-acetylhexosaminidase A [19], the mechanisms of action of these two activator proteins are still not well understood
Summary
Sugar chains in glycosphingolipids of higher animals are catabolized by lysosomal glycosidases, and some of the hydrolytic steps have been shown to require the assistance of protein cofactors called activator proteins [1,2,3]. Analysis of Glycosphingolipids from TLC Blotting by SIMS—The glycosphingolipid products generated from GalNAc-GD1a by clostridial sialidase in the presence of GM2 activator or saposin B were first resolved on a HPTLC plate and transferred to a PVDF membrane by TLC blotting.
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