title: Evaluation of an amino acid residue critical for the specificity and activity of human Gb3/CD77 synthase.
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, Pkantigen) 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.
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
authors.forename: Radoslaw
authors.initials: R
authors.lastname: Kaczmarek
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.|Confocal Microscopy Laboratory, Wroclaw Research Centre EIT+, Wroclaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Mikolajewicz
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Szymczak
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
authors.forename: Maria
authors.initials: M
authors.lastname: Duk
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.|Institute of Physiotherapy, Faculty of Physiotherapy and Physical Education, Opole University of Technology, Opole, Poland.
authors.forename: Edyta
authors.initials: E
authors.lastname: Majorczyk
authors.affiliation: Department of Biotechnology and Molecular Biology, University of Opole, Opole, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Krop-Watorek
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.|Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Buczkowska
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland. czerwins@iitd.pan.wroc.pl.|Institute of Physiotherapy, Faculty of Physiotherapy and Physical Education, Opole University of Technology, Opole, Poland. czerwins@iitd.pan.wroc.pl.
authors.forename: Marcin
authors.initials: M
authors.lastname: Czerwinski
mesh_terms: D000116:Acetylgalactosamine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism; D006028:Glycosphingolipids / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D020125:Mutation, Missense; D013379:Substrate Specificity
chemical_list: D034961:Antigens, Nuclear; D006028:Glycosphingolipids; C051963:nucleolar organizer region associated proteins; D005700:Galactosyltransferases; C045097:UDP-galactose-lactosylceramide alpha 1-4-galactosyltransferase; D000116:Acetylgalactosamine
keywords: Gb3/CD77 synthase; Glycopshingolipids; NOR polyagglutination; P1PK blood group system; Site-directed mutagenesis