Ovarian cancer α1,3-L-fucosyltransferase: Differentiation of distinct catalytic species with the unique substrate, 3′-sulfo-N-acetyllactosamine in conjunction with other synthetic acceptors

Several N-acetyllactosamine (LacNAc) derivatives were tested as acceptors for α1,3-L-fucosyltransferase present in human ovarian cancer sera and ovarian tumor. The enzyme of the soluble fraction of tumor was purified to apparent homogeneity by chromatography on bovine IgG glycopeptide-Sepharose followed by Sephacryl S-200 (M_r < 67,000). As compared with 2′-methyl LacNAc, 3′-sulfo LacNAc was about 5-fold more sensitive in measuring α1,3-fucosyltransferase in sera (K_m, 3′-sulfo LacNAc, 0.12 mM; 2′-methyl LacNAc, 6.67 mM). When ovarian cancer serum was the enzyme source, either the sulfate group or a sialyl moiety at C-3′ of LacNAc enhanced the acceptor ability (341 and 242%, respectively), whereas the sulfate group at C-2′ or C-6′ reduced the activity (22-36%); sulfate at C-6 or fucose at C-2′ increased the activity (172 and 253%). The β-benzylation of the reducing end, in general, increased the activity 2-3-fold. The enzyme of the soluble fraction of tumor exhibited more activity toward 3′-sulfo LacNAc (447%), 2′-fucosyl-LacNAc (436%), and 6-sulfo LacNAc (272%). Very low activity was observed with 3′-sialyl LacNAc (12.4%), 2′-sulfo LacNAc (33%), and 6′-sulfo LacNAc (5%); Fucα1,2Galβ1,3GlcNAcβ-O-p-nitrophenyl (166%), 2-methyl Galβ1,3GlcNAcβ-O-benzyl (204%), and 3-sulfo Galβ1,3GlcNAc (415%) also acted as acceptors, indicating the coexistence of α1,3- and α1,4-fucosyltransferase. The tumor particulate enzyme behaved entirely different, exhibiting low activity with 3′-sulfo LacNAc (39%) and 2′-fucosyl-LacNAc (148%); 3′-sialyl, 6′-sulfo, 6-sulfo, or 2′-sulfo LacNAc were 3, 43, 53, and 10% active, respectively. Thus, the ovarian cancer serum α1,3-fucosyltransferase acts equally well on H-type 2,3′-sialyl LacNAc and 3′-sulfo LacNAc, but not on H-type 1. The enzyme of soluble tumor fraction acts on H-type 2,3′-sulfo LacNAc as well as H-type 1 but poorly on 3′-sialyl LacNAc. The tumor particulate enzyme acts on H-type 2 but poorly on 3′-sulfo or 3′-sialyl LacNAc and is inactive with H-type 1. When normal serum was examined with synthetic acceptors, >80% activity was found as α1,2-fucosyltransferase and the rest as α1,3-fucosyltransferase. A screening of 21 ovarian cancer and 3 normal sera (3′-sulfo LacNAc as acceptor) showed 17-572% increase (average increase, 188%) of α1,3-fucosyltransferase activity in cancer. A parallel testing of Triton X-100 extracts of normal ovarian tissue and ovarian tumor with synthetic acceptors revealed a high level of α1,3-fucosyltransferase in tumor (2.17-fold increase when assayed with 3′-sulfo LacNAc and 0.42-0.56-fold increase with other specific acceptors). An examination of four ovarian tumors along with one normal ovarian tissue indicated that three tumors had 0.37-2.36-fold increase of α1,3-fucosyltransferase activity, whereas all four tumors had 2.06-139.6-fold increase of α1,4-fucosyltransferase activity using 2-O-methyl Galβ1→ 3GlcNAcβ1→O-benzyl as an acceptor.