Selectin ligands and tumor-associated carbohydrate structures: specificities of alpha 2,3-sialyltransferases in the assembly of 3'-sialyl-6-sialyl/sulfo 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 alpha 2,3-sialyltransferases (ST). The first enzyme, porcine liver ST, was 57, 37, and 79% active (Km: 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 approximately 40,000) containing approximately 40 T-haptens and asialo Cowper's gland mucin (MW approximately 200,000) containing approximately 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 (Km: 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 1,4GlcNAc beta- and its 6-sulfo derivative were approximately 20% active; the Lewis a structure, Gal beta 1,3- (Fuc alpha 1,4)GlcNAc beta-, was not an acceptor. The acrylamide copolymers containing approximately 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 Colo 205 Lewis type (alpha 1, 3/4) fucosyltransferase catalyzed the formation of 3'-sialyl-6-sulfo Lewis a from [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta-O-Allyl and copolymer containing [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta- units, using GDP[14C]Fuc as fucosyl donor. The third enzyme, HL-60 ST, was 103% active with Gal beta 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 3'-sialyl LacNAc and 3'-sialyl Lewis x units in leukosialin of HL60.(ABSTRACT TRUNCATED AT 400 WORDS)
Chandrasekaran
E V
EV
Department of Gynecologic Oncology, Roswell Park Cancer Institute, Buffalo, New York 14263.
Jain
R K
RK
Larsen
R D
RD
Wlasichuk
K
K
Matta
K L
KL
D000818:Animals; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry / Q000378:metabolism*; D002240:Carbohydrate Sequence; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D006001:Glycoconjugates / Q000737:chemistry / Q000378:metabolism; D006241:Haptens / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D019041:L-Selectin; D007983:Lewis Blood Group Antigens / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry / Q000378:metabolism; D012794:Sialic Acids / Q000737:chemistry; D012799:Sialyltransferases / Q000378:metabolism*; D013379:Substrate Specificity; D013431:Sulfates / Q000737:chemistry; D013552:Swine; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
D015295:Antigens, Tumor-Associated, Carbohydrate; D015815:Cell Adhesion Molecules; D006001:Glycoconjugates; D006241:Haptens; D007983:Lewis Blood Group Antigens; D008024:Ligands; D009077:Mucins; D012794:Sialic Acids; D013431:Sulfates; D019041:L-Selectin; C075185:sulfated glycoprotein p50; D012799:Sialyltransferases; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase