Identification of a new alpha1,2-fucosyltransferase involved in O-antigen biosynthesis of Escherichia coli O86:B7 and formation of H-type 3 blood group antigen.
Escherichia coli O86 possesses high human blood group B activity because of its O-antigen structure, sharing the human blood group B epitope. In this study, the wbwK gene of E. coli O86:B7 was expressed and purified as the GST fusion protein. Thereafter, the wbwK gene was biochemically identified to encode an alpha1,2-fucosyltransferase through radioactivity assays, as well as mass spectrometry and NMR spectroscopy. WbwK shows strict substrate specificity and only recognizes Gal beta1,3GalNAc alpha-OR (T-antigen and derivatives) as the acceptor to generate the H-type 3 blood group antigen. In contrast to other alpha1,2-fucosyltransferases, WbwK does not display activity toward the simple substrate Gal beta-OMe. Comparison with another recently characterized alpha1,2-fucosyltransferase (WbsJ) of E. coli O128:B12 indicates a low level of amino acid identity between them; however, they share a common acceptor substrate, Gal beta1,3GalNAc alpha-OR. Domain swapping between WbwK and WbsJ revealed that the smaller variable domains located in the C-terminus determine substrate specificity, whereas the larger variable domain in the N-terminus might play a role in forming the correct conformation for substrate binding or for localization of the alpha1,2-fucosyltransferase involved in O-antigen biosynthesis. In addition, milligram scale biosynthesis of the H-type 3 blood group antigen was explored using purified recombinant WbwK. WbwK may have potential applications in masking T-antigen, the tumor antigen, in vivo.
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Mei
M
Li
Jie
J
Shen
Xianwei
X
Liu
Jun
J
Shao
Wen
W
Yi
Christine S
CS
Chow
Peng G
PG
Wang
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000737:chemistry; D002240:Carbohydrate Sequence; D054308:Enteropathogenic Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000276:immunology*; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D019081:O Antigens / Q000096:biosynthesis* / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000097631:Galactoside 2-alpha-L-fucosyltransferase
D000942:Antigens, Bacterial; D005647:Fucosyltransferases; D019081:O Antigens; D011993:Recombinant Fusion Proteins; D000097631:Galactoside 2-alpha-L-fucosyltransferase; C022863:H antigen