Synthetic Glycosides as Primers of Oligosaccharide Biosynthesis and Inhibitors of Glycoprotein and Proteoglycan Assembly

Jeffrey D. Esko1, Rebecca I. Montgomery2

1 University of Alabama at Birmingham, Birmingham, Alabama, 2 Northwestern University, Chicago, Illinois
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.11
DOI:  10.1002/0471142727.mb1711s32
Online Posting Date:  May, 2001
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Abstract

With the exception of hyaluronic acid, all mammalian saccharides assemble while attached to a lipid or protein primer. Several cases are now known in which oligosaccharide synthesis will occur on synthetic glycoside primers added to cells. A protocol is described in this unit in which b‐D‐xylosides initiate glycosaminoglycan (GAG) synthesis by substituting for endogenous xylosylated core proteins. At high concentration xylosides will also prime oligosaccharides that resemble glycolipids. N‐acetyl‐a‐D‐galactosaminides initiate the synthesis of O‐linked oligosaccharides found on mucins and other glycoproteins in an analogous manner. Even disaccharides, such as peracetylated N‐acetyllactosaminide, can act as primers. Because these primers compete with endogenous substrates, they also act as inhibitors of proteoglycan (PG) and glycoprotein synthesis. Thus, primers have utility for studying the biological activity of glycoconjugates in cells, tissues, and animals. This unit describes procedures for using glycoside primers in cell culture.

     
 
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Table of Contents

  • Basic Protocol 1: Xyloside Initiation of Gag Synthesis and Inhibition of PG Assembly
  • Basic Protocol 2: N‐Acetyl‐α‐D‐Galactosaminide Initiation of Oligosaccharide Synthesis and Inhibition of O‐Linked Glycoprotein Assembly
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Xyloside Initiation of Gag Synthesis and Inhibition of PG Assembly

  Materials
  • recipe0.2 M p‐nitrophenyl‐β‐D‐xyloside in DMSO (see recipe)
  • Tissue culture growth medium appropriate for cell line
  • Cultured cells, in suspension or adherent
  • Dimethylsulfoxide (DMSO, C 2H 6SO; mol. wt. 78.13)
  • Radioactive precursors: H 235SO 4, [6‐3H]GlcNH 2, or [1‐3H]Gal
  • Additional reagents and equipment for metabolic radiolabeling (unit 17.4), isolating proteoglycans and glycosaminoglycans (unit 17.3), and gel filtration (unit 17.20) and reversed‐phase chromatography (unit 17.21)
NOTE: All incubations should be carried out in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: N‐Acetyl‐α‐D‐Galactosaminide Initiation of Oligosaccharide Synthesis and Inhibition of O‐Linked Glycoprotein Assembly

  • recipe0.2 M p‐nitrophenyl‐N ‐acetyl‐α‐D‐galactosaminide in DMSO (see recipe)
NOTE: All incubations should be carried out in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Figures

Videos

Literature Cited

Literature Cited
   Esko, J.D., Weinke, J.L., Taylor, W.H., Ekborg, G., Rodén, L., Anantharamaiah, G., and Gawish, A. 1987. Inhibition of chondroitin sulfate and heparan sulfate synthesis in a Chinese hamster ovary cell mutant defective in Galactosyltransferase I. J. Biol. Chem. 262:12189‐12195.
   Farach, M.C., Tang, J.P., Decker, G.L., and Carson, D.D. 1988. Differential effects of p‐nitrophenyl‐D‐xylosides on mouse blastocysts and uterine epithelial cells. Biol. Reprod. 39:443‐455.
   Fransson, L.‐A., Havsmark, B., Sakurai, K., and Suzuki, S. 1992. Sequence analysis of p‐hydroxyphenyl‐O‐β‐D‐xyloside initiated and radio‐iodinated dermatan sulfate from skin fibroblasts. Glycoconj. J. 9:45‐55.
   Freeze, H.H., Sampath, D., and Varki, A. 1993. α‐ and β‐xylosides alter glycolipid synthesis in human melanoma and Chinese hamster ovary cells. J. Biol. Chem. 268:1618‐1627.
   Fritz, T., Lugemwa, F.N., Sarkar, A.K., and Esko, J.D. 1994. Priming of heparan sulfate on β‐D‐xylosides depends on aglycone structure. J. Biol. Chem. 296:300‐308.
   Galligani, L., Hopwood, J., Schwartz, N.B., and Dorfman, A. 1975. Stimulation of synthesis of free chondroitin sulfate chains by β‐D‐xylosides in cultured cells. J. Biol. Chem. 250:5400‐5406.
   Kanwar, Y.S., Rosenzweig, L.J., and Jakubowski, M.L. 1986. Xylosylated‐PG‐induced Golgi alterations. Proc. Natl. Acad. Sci. U.S.A. 83:6499‐6503.
   Kojima, N., Handa, K., Newman, W., and Hakomori, S.‐I. 1992. Inhibition of selectin dependent tumor cell adhesion to endothelial cells and platelets by blocking O‐glycosylation of these cells. Biochem. Biophys. Res. Comm. 182:1288‐1295.
   Kolset, S.O., Sakurai, K., Ivhed, I., Overvatn, A., and Suzuki, S. 1990. The effect of β‐D‐xylosides on the proliferation and PG biosynthesis of monoblastic U‐937 cells. Biochem. J. 265:637‐645.
   Kuan, S.‐F., Byrd, J.C., Busbaum, C., and Kim, Y.S. 1989. Inhibition of mucin glycosylation by aryl‐N‐acetyl‐α‐galactosaminides in human colon cancer cells. J. Biol. Chem. 264:19271‐19277.
   Lugemwa, F.N. and Esko, J.D. 1991. Estradiol β‐D‐xyloside, an efficient primer for heparan sulfate biosynthesis. J. Biol. Chem. 266:6674‐6677.
   Montgomery, R.I., Lidholt, K., Flay, N.W., Liang, J., Vertel, B., Lindahl, U., and Esko, J.D. 1992. Stable heparin‐producing cell lines from the Furth murine mastocytoma. Proc. Natl. Acad. Sci. U.S.A. 89:11327‐11331.
   Neville, D.C.A., Field, R.A., and Ferguson, M.A.J. 1995. Hydrophobic glycosides of N‐acetylglucosamine can act as primers for polylactosamine synthesis and can affect glycolipid synthesis in vivo. Biochem. J. 307:791‐797.
   Okayama, M., Kimata, K., and Suzuki, S. 1973. The influence of p‐nitrophenyl‐β‐D‐xyloside on the synthesis of proteochondroitin sulfate by slices of embryonic chick cartilage. J. Biochem. 74:1069‐1073.
   Robinson, H.C., Brett, M.J., Tralaggan, P.J., Lowther, D.A., and Okayama, M. 1975. The effect of D‐xylose, β‐D‐xylosides and β‐D‐galactosides on chondroitin sulphate biosynthesis in embryonic chicken cartilage. Biochem. J. 148:25‐34.
   Robinson, J.A. and Robinson, H.C. 1981. Control of chondroitin sulphate biosynthesis. β‐D‐xylopyranosides as substrates for UDP‐galactose: D‐xylose transferase from embryonic‐chicken cartilage. Biochem. J. 194:839‐846.
   Robinson, J.A. and Robinson, H.C. 1985. Initiation of chondroitin sulphate synthesis by β‐D‐galactosides. Substrates for galactosyltransferase II. Biochem. J. 227:805‐814.
   Salimath, P.V., Spiro, R.C., and Freeze, H.H. 1995. Identification of a novel glycosaminoglycan core‐like molecule II. α‐GalNAc‐capped xylosides can be made by many cell types. J. Biol. Chem. 270:9164‐9168.
   Sarkar, A.K., Fritz, T.A., Taylor, W.H., and Esko, J.D. 1995. Disaccharide uptake and priming in animal cells: Inhibition of sialyl Lewis X by acetylated Galβ1→4GlcNAcβ‐O‐naphthalenemethanol. Proc. Natl. Acad. Sci. U.S.A. In press.
   Schwartz, N.B., Galligani, L., Ho, P.L., and Dorfman, A. 1974. Stimulation of synthesis of free chondroitin sulfate chains by β‐D‐xylosides in cultured cells. Proc. Natl. Acad. Sci. U.S.A. 71:4047‐4051.
   Sobue, M., Habuchi, H., Ito, K., Yonekura, H., Oguri, K., Sakurai, K., Kamohara, S., Ueno, Y., Noyori, R., and Suzuki, S. 1987. β‐D‐xylosides and their analogues as artificial initiators of GAG chain synthesis. Aglycone‐related variation in their effectiveness in vitro and in ovo. Biochem. J. 241:591‐601.
   Zhuang, D., Grey, A., Harris‐Brandts, M., Higgins, E., Kashem, M.A., and Dennis, J.W. 1991. Characterization of O‐linked oligosaccharide biosynthesis in cultured cells using para‐nitrophenyl‐α‐D‐GalNAc as an acceptor. Glycobiology 1:425‐433.
Key References
   Fritz et al., 1994. See above.
  Demonstrates that the type of glycoaminoglycan chain produced on a β‐D‐xyloside depends on the structure of the aglycone.
   Kuan et al., 1989. See above.
  First description of N‐acetyl‐α‐galactosaminides as primers and inhibitors of O‐linked glycosylation of glycoproteins.
   Salimath et al., 1995. See above.
   β‐D‐xylosides will also prime unusual oligosaccharides—e.g., GalNAcα1‐4GlcAβ1‐3Galβ1‐3Galβ1‐4Xyl‐R, GM3‐like compounds, and other oligosaccharides.
   Sarkar et al., in press. See above.
  Demonstrates that cells will take up suitably modified disaccharides and use them as primers, opening up the possibility of designing more complex and selective glycosylation inhibitors.
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