Purification and partial amino acid sequence of fuctinin, an endogenous inhibitor of fucosyltransferase activities.

A powerful endogenous protein inhibitor of fucosyltransferase activities, called fuctinin, was purified to homogeneity from rat small-intestinal mucosa. The purification scheme involved DEAE-cellulose ion-exchange chromatography, ammonium sulfate fractionation, hexyl-agarose hydrophobic chromatography and size-exclusion HPLC. Active native fuctinin has an isoelectric point of 4.55 and apparent molecular mass approximately 66 kDa, whereas a single protein band with a molecular mass of approximately 24 kDa was obtained by denaturing polyacrylamide gel electrophoresis, suggesting that fuctinin is an oligomeric protein. Two-dimensional polyacrylamide gel electrophoresis displayed eight spots in this single band. Comparisons of the N-terminal amino acid sequences of each spot support the idea of the existence of three related polypeptides and suggest a proteolytic N-terminal cleavage despite the use of an efficient protease inhibitor throughout the purification. In spite of the presence of an N-glycosylation site, fuctinin is not glycosylated. One of the three polypeptides, peptide 3, possesses two consensus sequences for phosphorylation and a consensus sequence for myristoylation. The sequences of functinin-related peptides, especially peptide 3, exhibit high similarity to the N-terminal domain of the Set protein and a putative human leukocyte antigen-associated protein. The possible implications of these results are discussed.