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Von Willebrand factor (VWF) is one of the plasma protein carrying ABO(H) blood group antigens, but the combining process of these antigens is not clear. In the present study, we examined whether plasma glycosyltransferase affects the blood group antigens on VWF. VWF expressing H-antigen (H-VWF) from blood group O and bovine serum albumin conjugated with H-antigen (H-BSA) were incubated with recombinant α1-3-N-acetylgalactosaminyltransferase (rA-transferase) and A-plasma with or without an additional UDP-GalNAc. Transformed antigens were detected by western blotting and ELISA, using an anti-A antibody. Both H-VWF and H-BSA acquired the A-antigen after incubation with rA-transferase and UDP-GalNAc. Incubation with A-plasma very weakly converted the H-antigen on BSA and VWF to A-antigen only in the presence of supplemented UDP-GalNAc. This conversion was enhanced on desialylation of H-VWF. These results indicate that sugar chains of plasma VWF can be modified by the external glycosyltransferase, but that plasma glycosyltransferase has no effect on the blood group antigens of VWF due to its low activity and the lack of donor sugars. Further, sialic acid residues of VWF may exert a protective effect against post-translational glycosylation. Our results clearly exclude the possibility that blood group antigens of VWF are constructed extracellularly in plasma.

Endogenous acceptors in a Golgi apparatus-enriched subcellular fraction from rat liver were labeled with UDP-[3H]GalNAc. The great majority of these acceptors were protected from protease degradation in the absence of detergent. These molecules are therefore present in intact vesicles of the correct topological orientation, which are likely to be similar to the Golgi compartments of the intact cell. Several distinct glycoproteins are labeled, but most are different from those labeled with UDP-[3H]GlcNAc. The enzyme peptide-N4(N-acetyl-beta-glucosiminyl)asparagine amidase releases label from a few specific proteins, indicating that [3H]GalNAc is transferred to N-linked oligosaccharides. Both neutral and anionic N-linked oligosaccharides are found, the great majority of which do not bind to ConA-Sepharose. Most of the [3H]GalNAc found in neutral oligosaccharides is terminal and beta-linked. The negative charge on the anionic molecules is due to sialic acid, and phosphate. A major portion of the [3H] GalNAc in this fraction is acid labile, and is released with kinetics consistent with it being in a phosphodiester linkage. These results show the existence of a whole new class of GalNAc-containing N-linked oligosaccharides, and demonstrates that this in vitro approach can detect previously undescribed structures. O-linked oligosaccharide biosynthesis was also studied in the same labeled rat liver Golgi apparatus preparations. beta-Elimination releases approximately 95% of the peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F)-resistant label which, in the absence of other added nucleotides, is almost exclusively [3H] GalNAcitol. If other unlabeled sugar nucleotides and adenosine 3'-phosphate,5'-phosphosulfate are added during the chase period two anionic O-linked oligosaccharides are synthesized, indicating that the UDP-GalNAc:peptide-N-acetylgalactosaminyltransferase is at least in part functionally co-localized with enzymes that extend and modify O-linked oligosaccharides.

We demonstrate that human fibroblasts of the rare Pk phenotype lack globoside, which was identified as the blood group P antigen, and that p cells possess neither globoside nor trihexosyl ceramide, which was identified as Pk antigen. Our investigations indicate also that these glycosphingolipid patterns are most likely caused by inherited preferential biosynthetic pathways in the abnormal phenotypes rather than by excess catabolism of the antigens. Evidence is presented that the fibroblasts of Pk phenotype lack beta-N-acetylgalactosaminyltransferase (globoside synthetase; UDP-N-acetylgalactosamine:trihexosylceramide beta-N-acetylgalactosaminyltransferase; EC 2.4.1.79) activity, and those of p are deficient in alpha-galactosyltransferase (trihexosylceramide synthetase; UDP galactose:lactosylceramide alpha-galactosyltransferase) and possibly also in globoside synthetase. The diminished globoside synthetase activity in p cells, however, is not caused by the defect in the gene coding for this enzyme. It appears, rather, to be caused by a failure in gene expression because one-third of Pk X p hybrids became able to express P antigenicity with a time lag of 3-4 days after cell fusion [Fellous, M., Gerbal, A., Nobillot, G. & Weils, J. (1977) Vox Sang. 32, 262-268].

The contribution of blood-group-active glycolipids and glycoproteins to the blood-group-ABH character of human erythrocytes was investigated. For that purpose the blood-group-H sites of human O cells were converted in vitro into group-A sites by transfer of alpha-N-acetyl-D-[14C]galactosamine residues with the aid of the blood-group-A gene-dependent alpha-N-acetylgalactosaminyl transferase prepared from human A1 plasma. Upon partition of the red cell membranes between water and organic solvent, about 5% of the label was found in the organic phase and about 20% in the water phase, thus reflecting the distribution of blood-group antigenic sites between glycosphingolipids with short carbohydrate chains and polyglycosylceramides, respectively. The fact that about 70% of the radioactivity remained tightly bound to the membranes and could only be released by treatment with pronase provided good evidence that the bulk of blood-group-H determinants is bound to glycoprotein material. Following these results it can thus be assumed that blood-group-ABH activity of human erythrocytes is determined preferentially by group-specific glycoproteins rather than glycolipids.

Porcine A blood group-specific N-acetylgalactosaminyl-transferase required either Mn2+, Cd2+, or Zn2+ for activity and 2'-O-alpha-fucosylgalactosides as acceptor substrates. The presence of detergent stabilizes the enzyme but is not essential for catalysis. To obtain information about the kinetic mechanism of the transferase reaction, initial rate parameters have been determined using 2'-fucosyllactose or A--mucin as acceptors, and Mn2+ or Cd2+ as cosubstrates. 2'-Fucosyllactose is a competitive inhibitor with respect to A--mucin and a noncompetitive inhibitor with respect to UDP-N-acetylgalactosamine. UDP inhibits noncompetively with respect to acceptor; thus UDP-N-acetylgalactosamine or acceptor can bind to the transferase via an equilibrium random pathway. The transferase converts human O blood type erythrocytes of A blood types. After exhaustive glycosylation, 3 X 10(6) N-acetylgalactosaminyl residues were incorporated per cell. Gel electrophoretic analysis of the labeled erythrocyte membranes indicates that glycoproteins with apparents molecular weights from 30,000 to 100,000 have been glycosylated; glycolipids account for only 15% of the labeled material, although pure H-glycolipid is a good acceptor. The transferase, with its strict acceptor specificity, can thus be used as a tool to study the biosynthesis and function of glycolipids and glycoproteins.

The membrane-bound N-acetylgalactosaminyltransferase from porcine submaxillary glands which provides A blood group specificity to mucin has been purified 38,000-fold by affinity chromatography on UDP-hesanolamine-agarose in aqueous Triton X-100. Design of a suitable purification procedure was developed by assessing the strength of interaction between enzyme and affinity adsorbent using batch desorption. The pure transferase has an apparent molecular weight of 100,000 as judged by zonal centrifugation and by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of a reducing agent. The reduced and carboxymethylated protein has an apparent molecular weight of 46,000 and 57,000 as judged by sedimentation equilibrium and sodium dodecyl sulfate polyacrylamide gel electrophoresis, respectively, suggesting that the native enzyme contains two subunits. It is a glycoprotein with a specific activity of 30 micronmol/min/mg of enzyme, which is 55,000 times that reported for the same enzyme isolated from human serum.

UDP-N-acetylgalactosamine:globoside alpha-N-acetylgalactosaminyltransferase (EC 2.4.1.-) synthesizing Forssman hapten was solubilized from dog spleen microsomes by a combination of Triton X-100 treatment and sonication. The solubilized enzyme was partially purified by calcium phosphate gel, ammonium sulfate fractionation and then DEAE-cellulose column chromatography. The enzymatic activity of the purified preparation was stimulated by exogenously added phosphatidylserine, as found in the particulate enzyme. When the properties of the purified enzyme were examined in the presence of exogenous phosphatidylserine, the enzyme had an absolute requirement for Mn2+; this was not substituted by Ca2+ or Mg2+. Apparent Km values for UDP-N-acetylgalactosamine and globoside were 1-10(-5) and 5-10(-4) M, respectively. It had a pH optimum of 6.55 regardless of the presence or absence of exogenous lipids. Since the partially purified enzyme was completely free of uridine diphosphatase which was found in the particulate preparaton, the effect of UDP on the transferase activity could be studied. Thus, UDP inhibited 85% of the activity at a concentration of 1.5 mM. p-Cholormercuribenzoate inhibited over 90% of the activity at 2 mM, indicating the transferase to be SH-enzyme.