High level expression of monomeric and dimeric human alpha1,3-fucosyltransferase V.
alpha3/4-Fucosyltransferases play a crucial role in inflammatory processes and tumor metastasis. While several human fucosyltransferases (FucTs) with different acceptor substrate specificities have been identified, the design of specific inhibitors for therapeutic approaches is hampered by the lack of structural information. In this study, we evaluated the expression of different constructs of human fucosyltransferase V to generate the large amounts required for structural studies. The truncated constructs lacking the transmembrane region and the cytosolic N-terminus, were expressed in baculovirus-infected Trichoplusia ni (Tn) insect cells and in two non-lytic expression systems, stably transfected human HEK 293 and T. ni cells. Since secretion of some glycosyltransferases is controlled by formation of dimeric molecules via disulfide bonds, one of the fucosyltransferase V constructs contained the N-terminal cysteine residue 64 for dimerization, whereas this residue was replaced in the other construct by serine. In both human and insect cells dimerization did not prove to be essential for efficient expression and secretion. On the basis of enzymatic activity, the yield of secreted fucosyltransferase V was approximately 10-fold higher in stably transfected insect cells than in HEK 293 cells. In particular the monomeric form of the enzyme provides a valuable tool for structural analyses to elucidate the fine specifity of fucosyltransferase V-mediated fucosylation of Lewis type glycans.
Münster
Jan
J
Institut für Biochemie und Lebensmittelchemie, Abteilung für Biochemie und Molekularbiologie, Universität Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany.
Ziegelmüller
Patrick
P
Spillner
Edzard
E
Bredehorst
Reinhard
R
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D019281:Dimerization; D005647:Fucosyltransferases / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D018411:Spodoptera / Q000235:genetics; D013379:Substrate Specificity
D011994:Recombinant Proteins; D005647:Fucosyltransferases