Substrate specificity of beta 1,4-N-acetylgalactosaminyltransferase in vitro and in cDNA-transfected cells. GM2/GD2 synthase efficiently generates asialo-GM2 in certain cells.
The substrate specificity of beta 1,4-N-acetylgalactosaminyltransferase has been analyzed using a fusion enzyme which consisted of the catalytic domain of the enzyme and the IgG binding domain of protein A, and also by extracts from cDNA transfectants. Both enzyme sources were capable of producing not only GM2 and GD2, but also asialo-GM2, GalNAc-sialylparagloboside, and Gal-NAc-GD1a from appropriate acceptors, although the efficiencies were at most 1-3% of those of GM2/GD2. The biological significance of these low specificities was studied with transient and stable transfectant cells. From the results of transient expression of the cDNA, asialo-GM2 expression appeared to inversely correlate with GM2 synthase levels in those lines. Consequently, GM2 seemed to be preferentially synthesized when both GM3 and lactosylceramide are available, and asialo-GM2 is synthesized in the absence of GM3 synthesis. However, the results of double immunostaining of CHO transfectants with anti-GM2 and anti-asialo-GM2 antibodies indicated that another factor may be involved in asialo-GM2 synthesis. From the in vitro assay using mixed acceptors, it was concluded that the presence of certain levels of GM2 might enhance the asialo-GM2 synthesis. These results suggest that even acceptors showing low efficiencies in vitro might be used in certain cells depending on the availability of precursors, expression levels of other gangliosides, as well as the kinetic properties of the enzyme, and the compartmentation of the glycosylation machineries in the cells.
Yamashiro
S
S
Department of Oncology, Nagasaki University School of Medicine, Japan.
Haraguchi
M
M
Furukawa
K
K
Takamiya
K
K
Yamamoto
A
A
Nagata
Y
Y
Lloyd
K O
KO
Shiku
H
H
Furukawa
K
K
D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006224:Cricetinae; D005732:Gangliosides / Q000096:biosynthesis*; D015870:Gene Expression; D006017:Glycolipids / Q000096:biosynthesis; D006801:Humans; D007700:Kinetics; D007739:L Cells; D008546:Melanoma, Experimental; D051379:Mice; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000096:biosynthesis / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000096:biosynthesis; D013379:Substrate Specificity; D013997:Time Factors; D014162:Transfection; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D005732:Gangliosides; D006017:Glycolipids; D017350:N-Acetylgalactosaminyltransferases; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C023220:(N-acetylneuraminyl)-galactosylglucosylceramide N-acetylgalactosaminyltransferase