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-transfera se (alpha 1-6FucT; EC 2.4.1.68), which catalyzes the transfer of fucos e from GDP-Fuc to N-linked type complex glycopeptides, was purified fr om a Triton X-100 extract of porcine brain microsomes. The purificatio n procedures included sequential affinity chromatographies on GlcNAc b eta 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-Sepharo se 4B columns. The enzyme was recovered in a 12% final yield with a 44 0,000-fold increase in specific activity. SDS-polyacrylamide gel elect rophoresis of the purified enzyme gave a major band corresponding to a n apparent molecular mass of 58 kDa, The alpha 1-6FucT has 575 amino a cids and no putative N-glycosylation sites. The cDNA was cloned in to pSVK3 and was then transiently transfected into COS-l cells. alpha 1-6 FucT activity was found to be high in the transfected cells, as compar ed with non- or mock-transfected cells. Northern blotting analyses of rat adult tissues showed that alpha 1-6FucT was highly expressed in br ain. No sequence homology was found with other previously cloned fucos yltransferases, but the enzyme appears to be a type II transmembrane p rotein like the other glycosyltransferases.