α1 → 3‐Galactosyltransferase: the use of recombinant enzyme for the synthesis of α‐galactosylated glycoconjugates

Abstract

We have reported the isolation and characterization of a bovine cDNA clone containing the complete coding sequence for UDP‐Gal:Galβ1 → 4GlcNAc α→ 3‐galactosyltransferase [Joziasse, D. H., Shaper, J. H., Van den Eijnden, D. H., Van Tunen, A. J. & Shaper, N. L. (1989) J. Biol. Chem. 264, 14290–14297]. Insertion of this cDNA clone into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) and subsequent infection of Spodoptera frugiperda (Sf9) insect cells with recombinant virus, resulted in high‐level expression of enzymatically active α1 → 3‐galactosyltransferase. The expressed enzyme accounted for about 2% of the cellular protein; the corresponding specific enzyme activity was 1000‐fold higher than observed in calf thymus, the tissue with the highest specific enzyme activity reported to date. The recombinant α1 → 3‐galactosyltransferase could be readily detergent‐solubilized and subsequently purified by affinity chromatography on UDP‐hexanolamine ‐Sepharose. The recombinant α1 → 3‐galactosyltransferase showed the expected preference for the acceptor substrate N‐acetyllactosamine (Galβ1 → 4GlcNAc), and demonstrated enzyme kinetics identical to those previously reported for affinity‐purified calf thymus α1 → 3‐galactosyltransferase [Blanken, W. M. & Van den Eijnden, D. H. (1985) J. Biol. Chem. 260, 12927–12934]. In pilot studies, the recombinant enzyme was examined for the ability to synthesize αl → 3‐galactosylated oligosaccharides, glycolipids and glycoproteins. By a combination of ¹H‐NMR, methylation analysis, HPLC. and exoglycosidase digestion it was established that, for each of the model compounds, the product of galactose transfer had the anticipated terminal structure, Galα1 → 3Galβ1 → 4‐R. Our results demonstrate that catalysis by recombinant α1 → 3‐galactosyltransferase can be used to obtain preparative quantities of various α1 → 3‐galactosylated glycoconjugates. Therefore, enzymatic synthesis using the recombinant enzyme is an effective alternative to the chemical synthesis of these biologically relevant compounds.