Human Gb3/CD77 synthase (alpha 1,4-galactosyltransferase, P-k synthase), encoded by A4GALT gene, is known for synthesis of Gal(alpha 1-4)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(alpha 1-4)GalNAc moiety, which constitutes the defining terminal disaccharide of the NOR antigen (carried by two glycosphingolipids: NOR1 and NOR2). Terminal Gal(alpha 1-4)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 alpha 1,4-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. (C) 2016 Elsevier Inc. All rights reserved.