Neighboring cysteine residues in human fucosyltransferase VII are engaged in disulfide bridges, forming small loop structures.
Among alpha 3-fucosyltransferases (alpha3-FucTs) from most species, four cysteine residues appear to be highly conserved. Two of these cysteines are located 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 other located in the middle of the catalytic domain. We identified the disulfide bridges in a recombinant, soluble form of human FucT VII. Potential free cysteines were modified with a biotinylated alkylating reagent, disulfide bonds were reduced and alkylated with iodoacetamide, and the protein was digested with either trypsin or chymotrypsin, before characterization by high-performance liquid chromatography/electrospray ionization mass spectrometry. More than 98% of the amino acid sequence for the truncated enzyme (beginning at amino acid 53) was verified. Mass spectrometry analysis also demonstrated that both potential N-linked sites are occupied. All six cysteines in the FucT VII sequence were shown to be disulfide-linked. The pairing of the cysteines was determined by proteolytic cleavage of nonreduced protein and subsequent analysis by mass spectrometry. The results demonstrated that Cys(68)-Cys(76), Cys(211)-Cys(214), and Cys(318)-Cys(321) are disulfide-linked. We have used this information, together with a method of fold recognition and homology modeling, using the (alpha/beta)(8)-barrel fold of Escherichia coli dihydrodipicolinate synthase as a template to propose a model for FucT VII.
de Vries
T
T
Department of Medical Chemistry, Vrije Universiteit Amsterdam, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
Yen
T Y
TY
Joshi
R K
RK
Storm
J
J
van Den Eijnden
D H
DH
Knegtel
R M
RM
Bunschoten
H
H
Joziasse
D H
DH
Macher
B A
BA
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005647:Fucosyltransferases / Q000737:chemistry* / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D004220:Disulfides; D011994:Recombinant Proteins; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase; D003545:Cysteine