Bioconjugation and Detection of Lactosamine Moiety using α1,3-Galactosyltransferase Mutants That Transfer C2-Modified Galactose with a Chemical Handle

• Published in: Bioconjugate chemistry 2009-03, Vol.20 (3), p.608-618
• Main Authors: Pasek, Marta, Ramakrishnan, Boopathy, Boeggeman, Elizabeth, Manzoni, Maria, Waybright, Timothy J, Qasba, Pradman K
• Format: Article
• Language: English
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc800534r

Studies on wild-type and mutant glycosyltransferases have shown that they can transfer modified sugars with a versatile chemical handle, such as keto or azido group, that can be used for conjugation chemistry and detection of glycan residues on glycoconjugates. To detect the most prevalent glycan epitope, N-acetyllactosamine (LacNAc (Galβ1−4GalNAcβ)), we have mutated a bovine α1,3-galactosyltransferse (α3Gal-T) enzyme which normally transfers Gal from UDP-Gal to the LacNAc acceptor, to transfer GalNAc or C2-modified galactose from their UDP derivatives. The α3Gal-T enzyme belongs to the α3Gal/GalNAc-T family that includes human blood group A and B glycosyltransferases, which transfer GalNAc and Gal, respectively, to the Gal moiety of the trisaccharide Fucα1−2Galβ1−4GlcNAc. On the basis of the sequence and structure comparison of these enzymes, we have carried out rational mutation studies on the sugar donor-binding residues in bovine α3Gal-T at positions 280 to 282. A mutation of His280 to Leu/Thr/Ser/Ala or Gly and Ala281 and Ala282 to Gly resulted in the GalNAc transferase activity by the mutant α3Gal-T enzymes to 5−19% of their original Gal-T activity. We show that the mutants 280SGG282 and 280AGG282 with the highest GalNAc-T activity can also transfer modified sugars such as 2-keto-galactose or GalNAz from their respective UDP-sugar derivatives to LacNAc moiety present at the nonreducing end of glycans of asialofetuin, thus enabling the detection of LacNAc moiety of glycoproteins and glycolipids by a chemiluminescence method.