Selectin Ligands and Tumor-Associated Carbohydrate Structures: Specificities of α2,3-Sialyltransferases in the Assembly of 3'-Sialyl-6-sulfo/sialyl Lewis a and x, 3'-Sialyl-6'-sulfo Lewis x, and 3'-Sialyl-6-sialyl/sulfo Blood Group T-hapten

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 α2,3-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 Galβ1,3GalNAca- unit; C-3 or C-6 substitution on Gal abolished sialylation. An acrylamide copolymer (MW~ 40 000) containing ~40 T-haptens and asíalo Cowper's gland mucin (MW ~ 200 000) containing ~48 T-haptens was -5-fold more active as an acceptor as compared to Galβ1, 3GalNAcα-0- A1 on a molecular weight basis. The second enzyme, a cloned α-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 Galβ1,3GlcNAcβ- unit; C-3 and C-6 substitution on Gal abolished sialylation. Galβ1,4GlcNAcβ- and its 6-sulfo derivative were ~20% active; the Lewis a structure, Galβ1,3- (Fucal,4)GlcNAcβ-, was not an acceptor. The acrylamide copolymers containing ~40 units of Galβ1,- 3GlcNAcβ-, Galβ1,3(6-sulfo)GlcNAcβ-, or fetuin triantennary asíalo 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 (12,3- specific sialidase. A partially purified Colo 205 Lewis type (αl, 3/4) fucosyltransferase catalyzed the formation of 3'-sialyl-6-sulfo Lewis a from [9-³H]NeuAcα2, 3Galβl, 3(6-sulfo)GlcNAcβ-0-Allyl and copolymer containing [9-³H]NeuAcα2, 3Galβl, 3(6-sulfo)GlcNAcβ- units, using GDP[¹⁴C]Fuc as fucosyl donor. The third enzyme, HL-60 ST, was 103% active with Galβ1,3(6-sulfo)GalNAcα- 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 α2,3 sialylation of Galβ1,4GlcNAcβ- but not Galβ1,3GlcNAcβ- by HL60-ST, consistent with the occurrence of 3'-sialyl LacNAc and 3'-sialyl Lewis x units in leukosialin of HL60. The present results indicating that C-6 sulfation or sialylation of Galβ1,3GlcNAcβ- 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 Galβ1,- 3GlcNAcβ- or Galβ1,3GalNAcα- abolishes the enzymatic C-3’ sialylation, it is imperative that 3'-sialylation must precede 6'-sulfation in the assembly of the major capping group of GLYCAM-I.