Bioconjugation and detection of lactosamine moiety using alpha1,3-galactosyltransferase mutants that transfer C2-modified galactose with a chemical handle.
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 (Galbeta1-4GalNAcbeta)), we have mutated a bovine alpha1,3-galactosyltransferse (alpha3Gal-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 alpha3Gal-T enzyme belongs to the alpha3Gal/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 Fucalpha1-2Galbeta1-4GlcNAc. 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 alpha3Gal-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 alpha3Gal-T enzymes to 5-19% of their original Gal-T activity. We show that the mutants (280)SGG(282) and (280)AGG(282) 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.
Pasek
Marta
M
Structural Glycobiology Section, Nanobiology Program, NCI-Frederick, Frederick, MD 21702, USA.
Ramakrishnan
Boopathy
B
Boeggeman
Elizabeth
E
Manzoni
Maria
M
Waybright
Timothy J
TJ
Qasba
Pradman K
PK
D000595:Amino Acid Sequence; D000606:Amino Sugars / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D005690:Galactose / Q000378:metabolism*; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment
D000606:Amino Sugars; C000458:N-acetyllactosamine; D005700:Galactosyltransferases; D005690:Galactose