Expression of Blood Group Lewis b Determinant from Lewis a: Association of this Novel α(1,2)-L-Fucosylating Activity with the Lewis Type α(1,3/4)-L-Fucosyltransferase

Blood group H type 1 [Fuca(1,2)Galβ(1,3)GlcNAcβ→] is known as the precursor structure of the blood group determinant, Lewis b [Fucα(1,2)Galβ(1,3)(Fuca(1,4))GlcNAcβ→]. Recently, a new biosynthetic route for Lewis b from Lewis a [Galβ(1,3)(Fucα(1,4))GlcNAc→] was identified in human gastric carcinoma cells, colon carcinoma Colo 205, and ovarian tumor. The present study demonstrates the association of this new type of α(1,2)-L-fucosyltransferase (FT) activity with the Lewis-type α(1,3/4)-L-FT as follows: (i) the α(1,4)- and novel α(1,2)-FT activities of Colo 205 were much less inhibited than the α(1,3)-FT activity by N-ethylmaleimide [K_i (μM) = 714.0, 119.0, and 6.5 respectively]. (ii) The α(1,4)- and novel α(1,2)-FT activities emerged from a Sephacryl S-200 column in identical positions, (iii) A specific inhibitor (copolymer from 3-sulfo-Galβ(1,3)GlcNAcβ-O-allyl and acrylamide) of α(1,4)-FT activity inhibited both α(1,4)- and α(1,2)-FT activities in Sephacryl S-200 column effluent to almost the same extent (~80%); (iv) separation of the Lewis-type α(1,3/4)-FT from the plasma-type α(1,3)-FT by specific elution of the affinity column (bovine IgG glycopep-Sepharose) with lactose and further purification on a Sephacryl S-100 HR column showed that (a) the α(1,3)-FT activity was the inherent capacity of the Lewis-type FT (Colo 205 fraction L) since ~90% of both the α(1,4)- and α(1,3)-FT activities is inhibited by the copolymer, (b) the unique ability of catalyzing the α(1,2)-L-fucosylation of Gal in Lewis a structure and also the α(1,3)-L-fucosylation of Glc in lactose-based structure belonged to the Lewis-type enzyme (Colo 205 fraction L), (c) a measurement of the [¹⁴C]fucosyl products arising from the two acceptors Galβ(1,3)(4,6-di-O-Me)GlcNAcβ-O-Bn and 3-sulfo-Galβ(1,3)GlcNAcβ-O-Al (specific for α(1,2) and α(1,4), respectively) taken in the same incubation mixture showed mutual inhibition by the acceptors [K_m for the α(1,4)-specific acceptor, 3-sulfo-Galβ(1,3)GlcNAcβ-O-Al, increased from 32 to 50 μM in the presence of 7.5 mM Galβ(1,3)(4,6-di-O-Me)GlcNAcβ-O-Bn, whereas K_i for the mutual inhibition of α(1,2)-FT activity by the former was 102 μM], and (d) the Lewis-type FT, in contrast to the plasma-type FT, was highly effective in fucosylating complex glycopeptides, (iv) A cloned FT (FT III: Lewis type) and the Colo 205 Lewis-type FT (fraction L) showed similar activities toward various acceptors; the enzymatic product resulting from the action of cloned FT on Galβ(1,3)(Fucα(1,4))GlcNAc-β-O-Bn was identified by FAB mass spectrometry as the difucosyl compound, (v) An examination of six human cell lines indicated that the novel α(1,2)-FT activity associates with the α(1,4)-FT activity.