Glycosyltransferases involved in type 1 chain and Lewis antigen biosynthesis exhibit glycan and core chain specificity.
Sialyl Lewis A (SLe(a)), Lewis A (Le(a)), and Lewis B (Le(b)) have been studied in many different biological contexts, for example in microbial adhesion and cancer. Their biosynthesis is complex and involves beta1,3-galactosyltransferases (beta3Gal-Ts) and a combined action of alpha2- and/or alpha4-fucosyltransferases (Fuc-Ts). Further, O-glycans with different core structures have been identified, and the ability of beta3Gal-Ts and Fuc-Ts to use these as substrates has not been resolved. Therefore, to examine the in vivo specificity of enzymes involved in SLe(a), Le(a), and Le(b) synthesis, we have transiently transfected CHO-K1 cells with relevant human glycosyltransferases and, on secreted reporter proteins, detected the resulting Lewis antigens on N- and O-linked glycans using western blotting and Le-specific antibodies. beta3Gal-T1, -T2, and -T5 could synthesize type 1 chains on N-linked glycans, but only beta3Gal-T5 worked on O-linked glycans. The latter enzyme could use both core 2 and core 3 precursor structures. Furthermore, the specificity of FUT5 and FUT3 in Le(a) and Le(b) synthesis was different, with FUT5 fucosylating H type 1 only on core 2, but FUT3 fucosylating H type 1 much more efficient on core 3 than on core 2. Finally, FUT1 and FUT2 were both found to direct alpha2-fucosylation on type 1 chains on both N- and O-linked structures. This knowledge enables us to engineer recombinant glycoproteins with glycan- and core chain-specific Lewis antigen substitution. Such tools will be important for investigations on the fine carbohydrate specificity of Le(b)-binding lectins, such as Helicobacter pylori adhesins and DC-SIGN, and may also prove useful as therapeutics.
Holgersson
Jan
J
Division of Clinical Immunology, Karolinska Institutet, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden.
Löfling
Jonas
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000951:Antigens, Neoplasm / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007983:Lewis Blood Group Antigens; D051379:Mice; D009844:Oligosaccharides / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology; D011134:Polysaccharides / Q000378:metabolism; D012709:Serum Albumin / Q000737:chemistry; D013379:Substrate Specificity; D000097631:Galactoside 2-alpha-L-fucosyltransferase
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D005647:Fucosyltransferases; D005700:Galactosyltransferases; D006023:Glycoproteins; D016695:Glycosyltransferases; D007983:Lewis Blood Group Antigens; D009844:Oligosaccharides; D011134:Polysaccharides; D012709:Serum Albumin; D000097631:Galactoside 2-alpha-L-fucosyltransferase; C500822:3-aminopropyl fucopyranosyl-1-2-galactopyranosyl-1-3-(fucopyranosyl-1-4)-2-acetamido-2-deoxyglucopyranosyl-1-3-galactopyranosyl-1-4-glucopyranoside; C042317:DU-PAN-2 antigen, human; C494219:Lewis a oligosaccharide; C479886:B3GALT5 protein, human