Alpha 1----3-galactosyltransferase: the use of recombinant enzyme for the synthesis of alpha-galactosylated glycoconjugates.
We have reported the isolation and characterization of a bovine cDNA clone containing the complete coding sequence for UDP-Gal:Gal beta 1----4GlcNAc alpha 1----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 alpha 1----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 alpha 1----3-galactosyltransferase could be readily detergent-solubilized and subsequently purified by affinity chromatography on UDP-hexanolamine-Sepharose. The recombinant alpha 1----3-galactosyltransferase showed the expected preference for the acceptor substrate N-acetyllactosamine (Gal beta 1----4GlcNAc), and demonstrated enzyme kinetics identical to those previously reported for affinity-purified calf thymus alpha 1----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 alpha 1----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, Gal alpha 1----3Gal beta 1----4-R. Our results demonstrate that catalysis by recombinant alpha 1----3-galactosyltransferase can be used to obtain preparative quantities of various alpha 1----3-galactosylated glycoconjugates. Therefore, enzymatic synthesis using the recombinant enzyme is an effective alternative to the chemical synthesis of these biologically relevant compounds.
Joziasse
D H
DH
Department of Medical Chemistry, Vrije Universiteit, Amsterdam, The Netherlands.
Shaper
N L
NL
Salyer
L S
LS
Van den Eijnden
D H
DH
van der Spoel
A C
AC
Shaper
J H
JH
D000818:Animals; D001212:Asialoglycoproteins; D015139:Blotting, Southern; D002240:Carbohydrate Sequence; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000378:metabolism*; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D005915:Globosides / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis*; D009961:Orosomucoid / Q000031:analogs & derivatives / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D001212:Asialoglycoproteins; D005915:Globosides; D009844:Oligosaccharides; D009961:Orosomucoid; D011994:Recombinant Proteins; C017030:asialoorosomucoid; C036363:paragloboside; D005700:Galactosyltransferases; C038887:N-acetyllactosamine alpha-D-galactosyltransferase; D005690:Galactose