Abstract
Human Gb3/CD77 synthase (α1,4-galactosyltransferase) is the only known glycosyltransferase that changes acceptor specificity because of a point mutation. The enzyme, encoded by A4GALT locus, is responsible for biosynthesis of Gal(α1–4)Gal moiety in Gb3 (CD77, Pk antigen) and P1 glycosphingolipids. We showed before that a single nucleotide substitution c.631C > G in the open reading frame of A4GALT, resulting in replacement of glutamine with glutamic acid at position 211 (substitution p. Q211E), broadens the enzyme acceptor specificity, so it can not only attach galactose to another galactose but also to N-acetylgalactosamine. The latter reaction leads to synthesis of NOR antigens, which are glycosphingolipids with terminal Gal(α1–4)GalNAc sequence, never before described in mammals. Because of the apparent importance of position 211 for enzyme activity, we stably transfected the 2102Ep cells with vectors encoding Gb3/CD77 synthase with glutamine substituted by aspartic acid or asparagine, and evaluated the cells by quantitative flow cytometry, high-performance thin-layer chromatography and real-time PCR. We found that cells transfected with vectors encoding Gb3/CD77 synthase with substitutions p. Q211D or p. Q211N did not express Pk, P1 and NOR antigens, suggesting complete loss of enzymatic activity. Thus, amino acid residue at position 211 of Gb3/CD77 synthase is critical for specificity and activity of the enzyme involved in formation of Pk, P1 and NOR antigens. Altogether, this approach affords a new insight into the mechanism of action of the human Gb3/CD77 synthase.
Highlights
Glycosyltransferases are a large group of enzymes that synthesize carbohydrate moieties of glycoproteins, glycosaminoglycans and glycosphingolipids
We showed before that c.631C > G mutation in A4GALT resulting in substitution of glutamine with glutamic acid at position 211
We found that cells transfected with the vector encoding Gb3/CD77 synthase with substitutions p
Summary
Glycosyltransferases are a large group of enzymes that synthesize carbohydrate moieties of glycoproteins, glycosaminoglycans and glycosphingolipids. These molecules are involved in many processes, such as cell differentiation, signal transduction, immune response and pathogen infection [1]. They contribute to diversity of many human blood group antigens belonging to blood group systems such as ABO, Lewis or P1PK [2, 3]. The reactions catalyzed by glycosyltransferases involve transferring sugar residues from the sugar donors to the acceptor molecules, forming glycosidic bonds. While change in donor specificity is a well described phenomenon and has been shown for several enzymes, such as ABO transferase [6] or β1,4galactosyltransferase [7], the change of acceptor specificity has been demonstrated for only one enzyme, Gb3/ CD77 synthase, which is a glycosphingolipid-specific glycosyltransferase [8]
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