Characterization of a bacterial beta-1,3-galactosyltransferase with application in the synthesis of tumor-associated T-antigen mimics.
T-Antigen (Gal-beta1,3-GalNAc-alpha-O-Ser/Thr) is an important precursor of mucin-type O-glycans. T-Antigen is found to be closely associated with cancer progression and metastasis and has been used to develop carbohydrate-based anticancer vaccines. Enzymatic synthesis of T-antigen disaccharides have relied on the use of beta-1,3-galactosyltransferases recently cloned and characterized from several eukaryotic organisms. However, its application is limited by the difficulty of obtaining homogeneous enzymes and the strict substrate specificity of enzymes. Recently, a number of bacteria have been found to express carbohydrate structures that mimic host glycans. The corresponding glycosyltransferases have been exploited in the facile synthesis of a number of clinically important glycoconjugate mimics. In this study, we biochemically characterized a bacterial beta-1,3-galactosyltransferase (WbiP) from Escherichia coli O127, which expresses a T-antigen mimic in the lipopolysaccharide (LPS) structure. Substrate study showed that WbiP could readily glycosylate a series of N-acetylgalactosamine (GalNAc) analogues with alpha-substitutions at the reducing end, including glycosylated Ser and Thr (GalNAc-alpha-O-Ser/Thr), which illustrates the use of WbiP for the facile synthesis of T-antigens. Alignment of a group of putative bacterial beta-1,3-galactosyltransferases revealed the presence of two conserved DXD motifs, possibly suggesting a different functional role of each motif. Site-directed mutagenesis, enzyme kinetics as well as UDP-bead binding assays were carried out to investigate the role of each DXD motif in WbiP. The results suggest that 88DSD90 is critical in the binding of sugar donor UDP-Gal, whereas 174DYD176 may participate in the binding of the sugar acceptor. This study expands the scope of using bacterial glycosyltransferases as tools for in vitro synthesis of glycoconjugate mimics with clinical significance.
Yi
Wen
W
Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Perali
Ramu Sridhar
RS
Eguchi
Hironobu
H
Motari
Edwin
E
Woodward
Robert
R
Wang
Peng George
PG
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000096:biosynthesis*; D002240:Carbohydrate Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005700:Galactosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013379:Substrate Specificity
D015295:Antigens, Tumor-Associated, Carbohydrate; D002413:Cations, Divalent; D029968:Escherichia coli Proteins; D005700:Galactosyltransferases; C527372:WbiP protein, E coli