Purification and cDNA cloning of porcine brain GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1->6fucasyltransferase

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1 --> 6fucosyl-transferase (alpha 1-6FucT; EC 2.4.1.68), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a Triton X-100 extract of porcine brain microsomes. The purification procedures included sequential affinity chromatographies on GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-2)-Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn-Sepharose 4B and synthetic GDP-hexanolamine-Sepharose 4B columns. The enzyme was recovered in a 12% final yield with a 440,000-fold increase in specific activity. SDS-polyacrylamide gel electrophoresis of the purified enzyme gave a major band corresponding to an apparent molecular mass of 58 kDa, The alpha 1-6FucT has 575 amino acids and no putative N-glycosylation sites. The cDNA was cloned in to pSVK3 and was then transiently transfected into COS-l cells. alpha 1-6FucT activity was found to be high in the transfected cells, as compared with non- or mock-transfected cells. Northern blotting analyses of rat adult tissues showed that alpha 1-6FucT was highly expressed in brain. No sequence homology was found with other previously cloned fucosyltransferases, but the enzyme appears to be a type II transmembrane protein like the other glycosyltransferases.