Specific recognition of N-acetylneuraminic acid in the GM2 epitope by human GM2 activator protein.
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.
Li
S C
SC
Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA.
Wu
Y Y
YY
Sugiyama
E
E
Taki
T
T
Kasama
T
T
Casellato
R
R
Sonnino
S
S
Li
Y T
YT
D001483:Base Sequence; D002240:Carbohydrate Sequence; D002855:Chromatography, Thin Layer; D017931:DNA Primers; D000939:Epitopes / Q000032:analysis*; D049289:G(M2) Activator Protein; D005678:G(M2) Ganglioside / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis / Q000378:metabolism*; D006028:Glycosphingolipids / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000378:metabolism*; D049231:Saposins; D012794:Sialic Acids; D049128:Sphingolipid Activator Proteins; D013379:Substrate Specificity; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D017931:DNA Primers; D000939:Epitopes; D049289:G(M2) Activator Protein; D006023:Glycoproteins; D006028:Glycosphingolipids; C483345:PSAP protein, human; D011506:Proteins; D049231:Saposins; D012794:Sialic Acids; D049128:Sphingolipid Activator Proteins; D005678:G(M2) Ganglioside; D009439:Neuraminidase; D001619:beta-N-Acetylhexosaminidases; D019158:N-Acetylneuraminic Acid