title: Human Gb3/CD77 synthase reveals specificity toward two or four different acceptors depending on amino acid at position 211, creating P(k), P1 and NOR blood group antigens.
abstract: Human Gb3/CD77 synthase (α1,4-galactosyltransferase, P(k) synthase), encoded by A4GALT gene, is known for synthesis of Gal(α1-4)Gal moiety in globotriaosylceramide (Gb3Cer, CD77, P(k) blood group antigen), a glycosphingolipid of the globo series. Recently, it was shown that c.631C > G mutation in A4GALT, which causes p.Q211E substitution in the open reading frame of the enzyme, broadens the enzyme specificity, making it able also to synthesize Gal(α1-4)GalNAc moiety, which constitutes the defining terminal disaccharide of the NOR antigen (carried by two glycosphingolipids: NOR1 and NOR2). Terminal Gal(α1-4)Gal disaccharide is also present in another glycosphingolipid blood group antigen, called P1, which together with P(k) and NOR comprises the P1PK blood group system. Despite several attempts, it was never clearly shown that P1 antigen is synthesized by Gb3/CD77 synthase, leaving open an alternative hypothesis that there are two homologous α1,4-galactosyltransferases in humans. In this study, using recombinant Gb3/CD77 synthase produced in insect cells, we show that the consensus enzyme synthesizes both the P(k) and P1 antigens, while its p.Q211E variant additionally synthesizes the NOR antigen. This is the first direct biochemical evidence that Gb3/CD77 synthase is able to synthesize two different glycosphingolipid antigens: P(k) and P1, and when p.Q211E substitution is present, the NOR antigen is also synthesized.
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114, Wroclaw, Poland. Electronic address: radoslaw.kaczmarek@iitd.pan.wroc.pl.
authors.forename: Radoslaw
authors.initials: R
authors.lastname: Kaczmarek
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114, Wroclaw, Poland.
authors.forename: Maria
authors.initials: M
authors.lastname: Duk
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114, Wroclaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Szymczak
authors.affiliation: Laboratory of Carbohydrate Chemistry, Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997, Moscow, GSP-7, Russian Federation.
authors.forename: Elena
authors.initials: E
authors.lastname: Korchagina
authors.affiliation: Department of Molecular Virology, Intercollegiate Faculty of Biotechnology of the University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822, Gdansk, Poland.
authors.forename: Jolanta
authors.initials: J
authors.lastname: Tyborowska
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114, Wroclaw, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Mikolajczyk
authors.affiliation: Laboratory of Carbohydrate Chemistry, Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997, Moscow, GSP-7, Russian Federation.
authors.forename: Nicolai
authors.initials: N
authors.lastname: Bovin
authors.affiliation: Department of Molecular Virology, Intercollegiate Faculty of Biotechnology of the University of Gdansk and Medical University of Gdansk, Kladki 24, 80-822, Gdansk, Poland.
authors.forename: Boguslaw
authors.initials: B
authors.lastname: Szewczyk
authors.affiliation: Department of Molecular Biology, Faculty of Biological Sciences, University of Zielona Góra, Szafrana 1, 65-516, Zielona Gora, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Jaskiewicz
authors.affiliation: Laboratory of Glycoconjugate Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114, Wroclaw, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Czerwinski
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D034961:Antigens, Nuclear / Q000096:biosynthesis* / Q000737:chemistry; D001665:Binding Sites; D002460:Cell Line; D004789:Enzyme Activation; D004795:Enzyme Stability; D005700:Galactosyltransferases / Q000737:chemistry* / Q000378:metabolism*; D007313:Insecta; D011485:Protein Binding; D061987:Sf9 Cells; D018411:Spodoptera; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D034961:Antigens, Nuclear; C051963:nucleolar organizer region associated proteins; D005700:Galactosyltransferases; C045097:UDP-galactose-lactosylceramide alpha 1-4-galactosyltransferase
keywords: Blood group antigens; Enzyme promiscuity; Glycosphingolipids; Glycosyltransferase specificity; P(k) antigen; P1 antigen; α1,4-Galactosyltransferase