The sequence in the assembly of the functional unit of selectin ligands containing sulfate, sialic acid, and fucose and also tumor-associated O-glycan structures was studied by examining the specificities of alpha 2,3-sialyltransferases (ST). The first enzyme, porcine liver ST, was 57, 37, and 79% active (K-m: 0.105, 0.420, and 0.200 mM), respectively, toward 6-sulfo, 6-sialyl, or 6-O-methyl derivatives of the Gal beta 1,3GalNAc alpha- unit; C-3 or C-6 substitution on Gal abolished sialylation. An acrylamide copolymer (MW similar to 40 000) containing similar to 40 T-haptens and asialo Cowper's gland mucin (MW similar to 200 000) containing similar to 48 T-haptens was -5-fold more active as an acceptor as compared to Gal beta 1, 3GalNAc alpha-O-Al on a molecular weight basis. The second enzyme, a cloned alpha-2,3-ST specific for lactose-based structure, was 70, 102, and 108% active (K-m: 0.500, 0.210, and 0.330 mM), respectively, toward 6-sialyl, 6-sulfo, or 6-O-methyl derivatives of the Gal beta 1,3GlcNAc beta- unit; C-3 and C-6 substitution on Gal abolished sialylation. Gal beta 1,4FGlcNAc beta- and its 6-sulfo derivative were similar to 20% active; the Lewis a structure, Gal beta 1,3(Fuc alpha 1,4)GlcNAc beta-, was not an acceptor. The acrylamide copolymers containing similar to 40 units of Gal beta 1, 3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, or fetuin triantennary asialo or bovine IgG diantennary glycopeptides were respectively 5.9-, 5.4-, 0.7-, and 0.1-fold as active. A transfer of 7-9 mol of NeuAc per mole of the above copolymers was catalyzed by this ST, the sialyl linkage being susceptible to alpha 2,3-specific sialidase. A partially purified Cole 205 Lewis type (alpha 1, 3/4) fucosyltransferase catalyzed the formation of 3'-sialyl-6-sulfo Lewis a from [9-H-3]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta-O-Allyl and copolymer containing [9-H-3]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta- units, using GDP[C-14]Fuc as fucosyl donor. The third enzyme, HL-60 ST, was 103% active with Gal beta 1,3(6-sulfo)GalNAc alpha- but was only 8% active with 6-sialo compound; it showed 11.6-fold greater activity with the copolymer of T-hapten. Further, we observed the alpha 2,3 sialylation of Gal beta 1,4GlcNAc beta- but not Gal beta 1,3GlcNAc beta- by HL60-ST, consistent with the occurrence of S'-sialyl LacNAc and 3'-sialyl Lewis x units in leukosialin of HL60. The present results indicating that C-6 sulfation or sialylation of Gal beta 1,3GlcNAc beta- could precede C-3' sialylation, which is followed by C-4 fucosylation in the biosynthetic pathway, are of importance, considering the occurrence of the 6-sulfoglucosamine moiety as a major constiuent of GLYCAM-I and human immunodeficiency virus envelope glycoproteins. In addition, since C-6' sulfation in Gal beta 1, 3GlcNAc beta- or Gal beta 1,3GalNAc alpha- abolishes the enzymatic C-3' sialylation, it is imperative that 3'-sialylation must precede B-sulfation in the assembly of the major capping group of GLYCAM-I.
