Neighboring cysteine residues in human fucosyltransferase VII are engaged in disulfide bridges, forming small loop structures

Among alpha3-fucosyltransferases (alpha3-FucTs) from most species, four cys teine residues appear to be highly conserved. Two of these cysteines are lo cated at the N-terminus and two at the C-terminus of the catalytic domain. FucT VII possesses two additional cysteines in close proximity to each othe r located in the middle of the catalytic domain. We identified the disulfid e bridges in a recombinant, soluble form of human FucT VII, Potential free cysteines were modified with a biotinylated alkylating reagent, disulfide b onds were reduced and alkylated with iodoacetamide, and the protein was dig ested with either trypsin or chymotrypsin, before characterization by high- performance liquid chromatography/ electrospray ionization mass spectrometr y, More than 98% of the amino acid sequence for the truncated enzyme (begin ning at amino acid 53) was verified. Mass spectrometry analysis also demons trated that both potential N-linked sites are occupied. All six cysteines i n the FucT VII sequence were shown to be disulfide-linked, The pairing of t he cysteines was determined by proteolytic cleavage of nonreduced protein a nd subsequent analysis by mass spectrometry, The results demonstrated that Cys(68)-Cys(76) Cys(211)-Cys(214), and Cys(318)-Cys(321) are disulfide-link ed. We have used this information, together with a method of fold recogniti on and homology modeling, using the (alpha/beta)(8)-barrel fold of Escheric hia coli dihydrodipicolinate synthase as a template to propose a model for FucT VII.