Biosynthesis in vitro of SA-Lex and SA-diLex by α1–3 fucosyltransferases from colon carcinoma cells and embryonic brain tissues

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Abstract
The sialyl-fucosyl-lactosamine-epitope present in sialyl (SA)-Lex (NeuAcα2–3Galβ1–4 [Fucα1–3]GlcNAαβ1–3Galβ1–4Gk-Cer), a carcinoembryonic antigen, has been recognized recently as a ligand for the binding of leukocyte-endothelial cell adhesion molecule 1 (LECAM-1) to myeloid and tumour cell surfaces. We have recently detected the presence of an αl-3 fucosyltransferase (FucT-3) activity in both embryonic chicken brain (ECB) and human colon carcinoma cells (Colo-205) which catalyses the biosynthesis in vitro of SA-Lex and SA-diLex. Fucosyltransferase activities from both sources are stimulated in the presence of divalent cations (Mn2+, Mg2+, Ca2+, Co2+ and Fe2+), although absolute metal requirement is not observed. Substrate specificity studies with this partially purified (ECB, 3000-fold; Colo-205, 100-fold) novel FucT-3 indicate the preference for terminally sialyl-sub-stituted glycolipid acceptors, as observed by the lower Km values when sialyl-neolactotetraosyl ceramide, LM1, (Neu-Gcα2–3Galβ1–4GlcNAcβ1–3Galcβ1–3Gk-Cer; Km = 0.048 mM) and sialyl-norhexaosylceramide, NeuGc-nLc6, (Neu-Gcα2–3Gαlβ1–4 GlcNAcβ1–3Gαlβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4Glc-Cer; Km = 0.032 mM) were used as substrates. Fucosyltransferase from Colo-205 requires the presence of the acyl group of the ceramide moiety and an acetyl group on glucosamine in the acceptor glycolipid since lyso-LM1 was found to be completely inactive. FucT-3-catalysed radioactive reaction products, SA-[14C]Lex and SA-[14C]diLex, from both enzyme sources were isolated, chemically characterized and further identified by ELISA, using specific monoclonal antibodies CSLEX1 and FH6, respectively. This is the first report to characterize αl-3 fucosyltransferase activities from ECB as well as human colon carcinoma Colo-205 cells, which catalyse the biosynthesis in vitro of oncofetal cell surface antigens, SA-Lex and SA-diLex from LM1 (NeuGc-nLc4) and NeuGc-nLc6, respectively.

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