Expression and specificity of human GM2 activator protein.
The cDNA encoding GM2 activator was expressed in the Escherichia coli/pT7-7 system. The yield of the GM2 activator with greater than 99% purity was about 3 mg per liter culture. The recombinant GM2 activator was found to be as active as that isolated from human kidney. The availability of the recombinant GM2 activator enabled us to critically examine the specificity of this activator protein. Our results show that the specificity of GM2 activator is not as strict as that reported previously. Although GM2 activator stimulates most efficiently the degradation of GM2 carried out by beta-N-acetylhexosaminidase A (Hex A), this activator also stimulates the following reactions: (a) conversion of GM2 to GA2 by clostridial sialidase; (b) hydrolysis of GalNAc from dipalmitoylphosphatidylethanolamine-II3NeuAcGgOse3 by Hex A; and (c) liberation of Gal from GM1 by beta-galactosidase at a high activator concentration. Thus, this activator does not differentiate between GM2 and dipalmitoylphosphatidylethanolamine-II3NeuAcGgOse3 or between Hex A and clostridial sialidase. The micellar forms of GD2 and GalNAc-GD1a were found to be more readily hydrolyzed by Hex A than GM2 in the absence of GM2 activator. Our results also show that saposin B can enhance the stimulatory activity of GM2 activator, but it cannot promote the stimulatory activity of sodium taurodeoxycholate. Taken together, our results suggest that the mechanism of action of GM2 activator is different from saposin B, and the action of GM2 activator is more than to solubilize lipid substrates. The effectiveness of GM2 activator in stimulating the hydrolysis of GM2 may be due to its ability to recognize the specific trisaccharide structure of the GM2 epitope, GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal-, and to modify the GalNAc-NeuAc interaction in this structure.
Wu
Y Y
YY
Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112.
Lockyer
J M
JM
Sugiyama
E
E
Pavlova
N V
NV
Li
Y T
YT
Li
S C
SC
D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005677:G(M1) Ganglioside / Q000378:metabolism; D049289:G(M2) Activator Protein; D005678:G(M2) Ganglioside / Q000378:metabolism*; D005732:Gangliosides / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006028:Glycosphingolipids / Q000378:metabolism*; D054818:Hexosaminidase A; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D049289:G(M2) Activator Protein; D005732:Gangliosides; D006023:Glycoproteins; D006028:Glycosphingolipids; C483345:PSAP protein, human; D011506:Proteins; D011994:Recombinant Proteins; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D005678:G(M2) Ganglioside; C038244:ganglio-N-triaosylceramide; D005677:G(M1) Ganglioside; D001616:beta-Galactosidase; D054818:Hexosaminidase A; D001619:beta-N-Acetylhexosaminidases