Abstract
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.
Highlights
From the Wepartment of Oncology, Nagasaki University School of Medicine, and the §Department of Prosthodontology, Nagasaki University School of Dentistry, Nagasaki, Japan 852, and the ~Memorial
The substrate specificity of fU,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 by extracts from eDNA transfectants
Since specific glycosyltransferases are considered to be needed for the individual combination of sugar donors, acceptors, and modes of linkage, 100-150 or more different glycosyltransferases are required for synthesis of carbohydrate structures present in mammalian cells [1, 2]
Summary
From the Wepartment of Oncology, Nagasaki University School of Medicine, and the §Department of Prosthodontology, Nagasaki University School of Dentistry, Nagasaki, Japan 852, and the ~Memorial. The substrate specificity of fU,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 by extracts from eDNA transfectants Both enzyme sources were capable of producing GM2 and G02, and asialo-GM2, GalNAc-sialylparagloboside, and GalNAc-G018 from appropriate acceptors, the efficiencies were at most 1-3% of those of GMJ!Gn2 The biological significance of these low specificities was studied with transient and stable transfectant cells. Since specific glycosyltransferases are considered to be needed for the individual combination of sugar donors, acceptors, and modes of linkage, 100-150 or more different glycosyltransferases are required for synthesis of carbohydrate structures present in mammalian cells [1, 2]. Regulatory factors governing the carbohydrate structures synthesized by this enzyme in cells were investigated
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