EXPRESSION OF BLOOD-GROUP LEWIS-B DETERMINANT FROM LEWIS-A - ASSOCIATION OF THIS NOVEL ALPHA(1,2)-L-FUCOSYLATING ACTIVITY WITH THE LEWIS TYPE ALPHA(1,3/4)-L-FUCOSYL-TRANSFERASE

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