Preece AF, Strahan KM, Devitt J, Yamamoto F, Gustafsson K
Blood •
No definitive biologic function has been associated with the human ABO histo-blood group polymorphism, or any other terminal carbohydrate differences within or between closely related species. We have experimentally addressed the question of whether viral particles can become glycosylated as determined by the glycosylation (eg, ABO) status of the producer cell and as a result be affected by human serum containing specific natural antibodies (NAbs). Measles virus was produced in cells transfected with cDNA encoding, either human A-transferase, B-transferase, an inactive "O-transferase," or a pig alpha1-3galactosyltransferase (alpha1-3GT) synthesizing the Galalpha1-3Gal structure. The viruses were shown to carry the same ABO structures as the cells; that is, A but not B if produced in A-type cells, and B but not A if produced in B-type cells. Only O was detected on the virus produced from O-type cells, whereas reduced amounts of O appeared on the A- and B-type viral particles. In addition, the Galalpha1-3Gal structure was transferred onto measles only when grown in human cells expressing this structure. When subjected to human preimmune sera, the A-type, the B-type, and the Galalpha1-3Gal viral particles were partially neutralized in a complement-dependent manner. However, the O-type or the Galalpha1-3Gal-negative viral particles were not neutralized. The neutralization appeared to be mediated by specific NAb, as judged by specific inhibition using synthetic A and Galalpha1-3Gal oligosaccharides. Such viral glycosylation may thus partly explain why the ABO antigens and other similar intraspecies as well as interspecies polymorphic carbohydrates have evolved and been maintained over long evolutionary periods.
The accompanying article (Manzi, A., Salimath, P. V., Spiro, R. C., Keifer, P. A., and Freeze, H. H. (1995) J. Biol. Chem. 270, 9154-9163) reported the complete structure of a novel molecule made by human melanoma cells incubated with 1 mM 4-methylumbelliferyl-beta Xyl (Xyl beta MU). The product resembles a common pentasaccharide core region found in chondroitin/dermatan sulfate glycosaminoglycans, except that a terminal alpha-Gal-NAc residue is found in a location normally occupied by beta-GalNAc in these chains or alpha-GlcNAc in heparan sulfate chains. In this paper we show that several other human cancer cell lines and Chinese hamster ovary cells also make alpha-GalNAc-capped xylosides. The [6-3H]galactose-labeled Xyl beta MU product binds to immobilized alpha-GalNAc-specific lectin from Helix pomatia and the binding is competed by GalNAc, but not by Glc. Binding to the lectin is destroyed by digestion with alpha-N-acetylgalactosaminidase, but not beta-hexosaminidase. The nature of the aglycone influences the amount and relative proportion of this material made, with p-nitrophenyl-beta-xyloside being a better promoter of alpha-GalNAc-terminated product than Xyl beta MU. This novel oligosaccharide accounts for 45-65% of xyloside-based products made by both human melanoma and Chinese hamster ovary cells when they are incubated with 30 microM Xyl beta MU, but at 1 mM both the total amount and the proportion decreases to only 5-10%. In both cell lines this product is replaced by a corresponding amount of Sia alpha 2,3Gal beta 4Xyl beta MU. Preferential synthesis of the alpha-GalNAc-capped material at very low xyloside concentration argues that it is a normal biosynthetic product and not an experimental artifact. This pentasaccharide may be a previously unrecognized intermediate in glycosaminoglycan chain biosynthesis. Since this alpha-GalNAc residue occurs at a position that determines whether chondroitin or heparan chains are added to the acceptor, it may influence the timing, type, and extent of further chain elongation.
Manzi A, Salimath PV, Spiro RC, Keifer PA, Freeze HH
The Journal of biological chemistry •
beta-Xylosides compete with endogenous proteoglycan core proteins and act as alternate acceptors for synthesizing protein-free glycosaminoglycan chains. Their assembly on these alternate acceptors utilizes the same glycosyltransferases that make the protein-bound chains. Most studies using alternate acceptors focus on the production of sulfated glycosaminoglycan chains that are thought to be the major products. However, we previously showed that labeling melanoma cells with [6-3H]galactose in the presence of 4-methylumbelliferyl (MU) or p-nitrophenyl (pNP) beta-xylosides led to the synthesis of mostly di- to tetrasaccharide products including incomplete core structures. We have solved the structure of one of the previously unidentified products as, GalNAc alpha(1,4)GlcA beta(1,3)Gal beta(1,3)Gal beta(1,4)Xyl beta MU, based on compositional analysis by high performance liquid chromatography, fast atom bombardment, electrospray mass spectrometry, and one-dimensional and two-dimensional 1H NMR spectroscopy. The novel aspect of this molecule is the presence of a terminal alpha-Gal-NAc residue at a position that is normally occupied by beta-GalNAc in chondroitin/dermatan sulfate or by alpha-Glc-NAc in heparin or heparan sulfate chains. An alpha-GalNAc residue at this critical location may prevent further chain extension or influence the type of chain subsequently added to the common tetrasaccharide core.
