Purification and cDNA Cloning of GDP-L-Fuc:N-Acetyl- -D-Glucosaminide: 1-6 Fucosyltransferase ( 1-6 FucT) from Human Gastric Cancer MKN45 Cells

Abstract

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide:alpha1-6 fucosyltransferase (alpha1-6 FucT), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a culture supernatant of human gastric cancer cell line MKN45. The purification procedures included chromatographies on Q-Sepharose Fast Flow, synthetic GDP-hexanolamine-Sepharose, and GnGn-bi-Asn-Sepharose columns. SDS-PAGE of the purified enzyme gave a major band corresponding to an apparent molecular mass of 60 kDa. The enzyme was recovered in a 12% final yield with an approximately 4,600-fold increase in specific activity. The pH optimum was 7.5, and the enzyme was fully active in the presence of 5 mM EDTA and did not require divalent cations, Mg2+ and Ca2+. Oligonucleotide primers designed from partial amino acid sequences were used to amplify and clone alpha1-6 FucT cDNA from a cDNA library of MKN45 cells. The cDNA encodes 575 amino acids in length, and contains the predicted N-terminal and internal amino acid sequences derived on lysyl endopeptidase digestion. The homology to porcine brain alpha1-6 FucT is 92.2% at the nucleotide level and 95.7% at the amino acid level. No putative N-glycosylation sites were found in the predicted amino acid sequence of the human MKN45 cell enzyme or that of porcine brain. Thus, the enzyme is distinct from other fucosyltransferases which catalyze alpha1-2, alpha1-3, and alpha1-4 fucose addition.