Glycoprotein Analysis

  • Terry D. Butters
Part of the Springer Protocols Handbooks book series (SPH)


In eukaryottc cells, one of the most important posttranslattonal modrficattons of proteins is the co valent addmon of carbohydrate We can consider two major types of modtf-icatron to ammo-acid residues. N-glycosylation of asparagine amme groups and O-glycosylatron of serme or threonme hydroxyl groups (1). N-linked oltgosaccharrdes can be divided mto three maJor classes, the complex type contaming N-acetyl- glucosamme, mannose, galactose, fucose, and stahc acid; the oligomannose type contaming N-acetylglucosamme and mannose only; and the hybrid type that has features common to both complex and olrgomannose chains (Fig. 1) All of these structures are synthesized by a common pathway that begins in the endoplasmtc rettculum (ER) with the assembly of a lipid-lmked donor molecule The preformed oltgosacchartde is transferred to protein cotranslationally in the lumen of the endoplasmlc retrculum and by a serves of glycosrdase (a-glucosrdase and a-mannosrdase)-trmnning reactions is modr- fied as the protein progresses through the ER and Golgi apparatus (2), The diversity of N+lmked ohgosacchartde structure is dictated by the accesstbtllty of these partially processed chains to Golgi resident glycosyltransferases, a group of enzymes able to add monosacchartdes to oltgosacchartdes directly from nucleottde sugar donors. Glycosyltransferases are specrfic for nucleotide sugar donor, anomertctty, glycosrdtc linkage between sugars, and acceptor substrates. Consequently, there are a number of different transferases and each cell, tissue, and species has a unique complement of enzymes that control oltgosaccharrde biosynthesis (3). O-linked ohgosacchartdes con- tain similar residues to N-glycans, but their synthesis has no requirement for en bloc addttton of carbohydrate to the polypepttde chain O-glycosylatron proceeds by glycosyltransferase-catalyzed, stepwtse addition of monosacchartdes to generate, as in thecase of mucm glycoprotems, a diverse number of branched oltgosaccharrdes (4,5) (see Fig. 2).


Nuclear Magnetic Resonance Acceptor Substrate Reporter Group Exoglycosidase Digestion Unique Complement 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Allen, H. J. and Krsailus, E C (1992) Glycoconjugates Composrtron, Structure and Function Marcel Dekker, New YorkGoogle Scholar
  2. 2.
    Kornfeld, R and Kornfeld, S (1985) Assembly of asparagine-lmked ohgosaccharrdes Annu Rev Blochem 54, 631–664CrossRefGoogle Scholar
  3. 3.
    Kleene, R and Berger, E G (1993) The molecular and cell btology of glycosyltransferases Blochlm Blophys Acta 1154,283–325Google Scholar
  4. 4.
    Can-away, K L and Hull, S R (1991) Cell surface mucm-type glycoprotems and mucmlike domains Glycobiology 1, 131–138CrossRefGoogle Scholar
  5. 5.
    Corfield, T (1992) Mucus glycoprotems, super glycoforms how to solve a stocky problem? GlycoconJugate J 9, 217–221CrossRefGoogle Scholar
  6. 6.
    Helemus, A (1994) How N-lmked ollgosaccharrdes affect glycoprotem folding in the endoplasmrc retrculum. Mel Biol Cell 5, 253–265Google Scholar
  7. 7.
    Varki, A (1993) Brologrcal roles of ohgosacchartdes—All of the theortes are correct Glycobzology 3, 97–130CrossRefGoogle Scholar
  8. 8.
    Kobata, A (1992) Structures and functrons of the sugar chains of glycoprotems Eur J Biochem 209,483–501PubMedCrossRefGoogle Scholar
  9. 9.
    O’Shannessy, D J and Quarles, R H. (1987) Labeling of the ohgosaccharrde moretres of mnnunoglobulms J Immunol Methods 99, 153–161PubMedCrossRefGoogle Scholar
  10. 10.
    Edge, A S, Faltynek, C R, Hof, L, Rerchert, L E, Jr, and Weber, P (1981) Deglycosylation of glycoprotems by trifluoromethanesulfonic acid Anal Biochem 118, 131–137.PubMedCrossRefGoogle Scholar
  11. 11.
    Tretter, V, Altmann, F, and Marz, L (1991) Peptrde-N4-(N-acetyl-beta-glucosammyl) asparagine amldase-F cannot release glycans with fucose attached α-l → 3 to the aspar-agine-linked N-acetylglucosamme restdue Eur J Biochem 199, 647–652.PubMedCrossRefGoogle Scholar
  12. 12.
    Tremble, R B and Tarentmo, A L (1991) Identrficatron of drstmct endoglycosrdase (Endo) actlvltles in Flavobactenum-mentngoseptlcum—Endo-Fl, Endo-F2, and Endo-F3—Endo-Fl and Endo-H hydrolyze only high mannose and hybrtd glycans J Biol Chem 266, 1646–1651Google Scholar
  13. 13.
    LIS, H and Sharon, N (1986) Lectms as molecules and as tools Annu Rev Biochem 55, 35–67PubMedCrossRefGoogle Scholar
  14. 14.
    Kijimoto-Ochial, S, Katagm, Y U, and Ochlai, H (1985) Analysis of N-linked ohgosaccharlde chams of glycoprotems on mtrocellulose sheets using lectm-peroxldase reagents Anal Biochem 147, 222–229CrossRefGoogle Scholar
  15. 15.
    Cummings, R D (1994) Use of lectins in analysis of glycoconjugates Methods Enzymol 230,66–86PubMedCrossRefGoogle Scholar
  16. 16.
    Tachlbana, H, Sekl, K, and Murakaml, H (1993) Identification of hybrid-type carbohydrate chains on the hght cham of human monoclonal antibody specific to lung adenocarcmoma Biochzm Biophys Acta 1182,257–263Google Scholar
  17. 17.
    Leonards, K S and Kutchal, H (1985) Coupling of Ca2+ transport to ATP hydrolysis by the Ca2+-ATPase of sarcoplasmlc reticulum potential role of the 53-kllodalton glycoprotem Biochemwy 24, 4876–4884CrossRefGoogle Scholar
  18. 18.
    Ogawa, H, Ueno, M, Uchlbon, H, Matsumoto, I, and Seno, N (1990) Direct carbohydrate analysis of glycoprotems electroblotted onto polyvmylldene dlfluorlde membrane from sodium dodecyl sulfate-polyacrylamlde gel Anal Biochem 190, 165–169PubMedCrossRefGoogle Scholar
  19. 19.
    Weltzhandler, M, Kadlecek, D, Avdalovlc, N, Forte, J G, Chow, D, and Townsend, R R (1993) Monosaccharide and ollgosaccharlde analysis of proteins transferred to polyvmylldene fluoride membranes after sodium dodecyl sulfate-polyacrylamlde gel electrophoresis J Biol Chem 268,5121–5130Google Scholar
  20. 20.
    Takasakl, S, Mlzuochl, T, and Kobata, A (1982) Hydrazmo lysls of asparagine-lmked sugar chains to produce free ohgosaccharldes Methods Enzymol 83, 263–268CrossRefGoogle Scholar
  21. 21.
    Patel T, Bruce, J, Merry, A, Blgge, C, Wormald, M, Jaques, A, and Parekh, R (1993) Use of hydrazme to release in intact and unreduced form both N-lmked and O-linked ohgosaccharldes from glycoprotems Biochemzstry 32, 679–693CrossRefGoogle Scholar
  22. 22.
    Cooper, C A, Packer, N H, and Redmond, J W (1994) The ellmmatlon of Olmked glycans from glycoprotems under non-reducing condltlons GlycoconJugate J 11, 163–167CrossRefGoogle Scholar
  23. 23.
    Takahashl, N and Muramatsu, T (1992) Handbook of Endoglycoszdases and Glycoamtdases CRC, FloridaGoogle Scholar
  24. 24.
    Yamashlta, K, Mlzuochl, T, and Kobata, A (1982) Analysis of ohgosaccharrdes by gel filtration Methods Enzymof 83, 105–126CrossRefGoogle Scholar
  25. 25.
    Kobata, A, Yamashlta, K, and Takasakl, S (1987) BioGel P-4 column chromatography of oligosaccharldes effective size of ollgosaccharldes expressed in glucose units Methods Enzymol 138, 84–94PubMedCrossRefGoogle Scholar
  26. 26.
    Hase, S, Ikenaka, K, Mlkoshlba, K, and Ikenaka, T (1988) Analysis of tissue glycoprotem sugar chams by two dlmenslonal high-performance hquld chromatographlc mapping J Chromatog 434, 51–60CrossRefGoogle Scholar
  27. 27.
    Lee, Y C (1990) High-performance amon-exchange chromatography for carbohydrate analysis. Anal Biochem 189, 151–162PubMedCrossRefGoogle Scholar
  28. 28.
    Townsend, R R and Hardy, M R (1991) Analysis of glycoprotein ollgosaccharldes using high-pH amon exchange chromatography Glycobzology 1, 139–147CrossRefGoogle Scholar
  29. 29.
    Merkle, R K and Cummings, R D (1987) Lectm affinity chromatography of glycopeptides Methods Enzymol 138, 232–259PubMedCrossRefGoogle Scholar
  30. 30.
    Osawa, T and Tsuji, T (1987) Fractlonatlon and structural assessment of ollgosaccharldes and glycopeptldes by use of lmmoblltsed lectins Annu Rev Biochem 56, 21–42PubMedCrossRefGoogle Scholar
  31. 31.
    Jackson, P (1990) The use of polyacrylamide-gel electrophoresls for the high-resolution separation of reducing saccharides labelled with the fluorophore 8-ammonaphthalene-1,3,6-tnsulphomcacid Biochem J 270,705–713PubMedGoogle Scholar
  32. 32.
    Jackson, P (1991) Polyacrylamlde gel electrophorests of reducing saccharides labeled with the fluorophore 2-ammoacridone—subptcomolar detection using an rmaging system based on a cooled charge-coupled device Anal Biochem 196,238–244PubMedCrossRefGoogle Scholar
  33. 33.
    Kenne, L and Stromberg, S (1990) A method for the microanalysis ofhexoses inglycoprotems Curb Res 198, 173–179CrossRefGoogle Scholar
  34. 34.
    Montreutl, J, Bouquelet, S., Debray, H, Fournet, B, Spik, G, and Strecker, G (1986) Glycoprotems, in Carbohydrate Analysis A Practical Approach (Chaplm, M F and Kennedy, J F, eds), IRL, Oxford, UK, pp 143–204Google Scholar
  35. 35.
    Dell, A, Khoo, K-H, Panico M, McDowell, R A, Ettenne, A T, Reason, A J, and Morris, H R (1993) FAB-MS and ES-MS of glycoprotems, in Glycobzology A Practzcal Approach (Fukuda, M and Kobata, A, eds), IRL, Oxford, UK, pp 187–222Google Scholar
  36. 36.
    Harvey, D J (1992) Theroleofmassspectrometryinglycobiology GlycoconJugate J 9, 1–12CrossRefGoogle Scholar
  37. 37.
    Medzthradszky, K F, Maltby, D A, Hall, S C, Settmeri, C A, and Burlingame, A L (1994) Characterlsatlon of protem N-glycosylatton by reversed-phase mtcrobore liquid chromatography/electrospray mass spectrometry, complementary mobile phases, and sequenttal exoglycosidase digestion J Am Sot Mass Spectrom 5, 350–358CrossRefGoogle Scholar
  38. 38.
    Leroy, Y, Lemome, J, Rmart, G, Mtchalskt, J-C, Montreuil, J, and Fournet, B (1990) Separation of ohgosacchartdes by capillary supercritical fluid chromatography and analysts by dtrect coupling to high-resolution mass spectrometer—applmation to analysts of oltgomannostdic N-glycans Anal Biochem 184,235–243PubMedCrossRefGoogle Scholar
  39. 39.
    Kelly, J F, Locke, S J, and Thtbault, P (1993) Analysis of protein glycoforms by captllary electrophoresls-electrospray mass spectrometry Dlscovely Newsletter (Beckman) 2, 1–6Google Scholar
  40. 40.
    Dwek, R A, Edge, C J, Harvey, D J, Wormald, M R, and Parekh, R B (1993) Analysis of glycoprotem-associated olrgosacchartdes Annu Rev Biochem 62,65–100PubMedCrossRefGoogle Scholar
  41. 41.
    Jacob, G S and Scudder, P (1994) Glycostdases in structural analysts Methods Enzymol 230, 280–299PubMedCrossRefGoogle Scholar
  42. 42.
    Butters, T D, Scudder, P, Rotsaert, J, Petursson, S, Fleet, G W J., Wtllenbrock, F W, and Jacob, G S (1991) Purtfication to homogeneity of Charonza lampas α-fucostdase by using sequenttal hgand-affinity chromatography Biochem J 279, 189–195PubMedGoogle Scholar
  43. 43.
    Clarke, V A, Platt, N, and Butters, T D (1995) Cloning and expression of the B-N-acetylglucosamimdase gene from Streptococcus pneumonlae —generatton of truncated enzymes wtth modified aglycon specificity J Biol Chem 270,8805–8814PubMedCrossRefGoogle Scholar
  44. 44.
    Kobata, A (1979) Use of endo-and exoglycostdases for structural studies of glycocomugates Anal Biochem 100, 1–14PubMedCrossRefGoogle Scholar
  45. 45.
    Guile, G R, Wong, S Y and Dwek, R A (1994) Analytical and preparative separation of anionic oltgosaccharides by weak amon-exchange high-performance ltqmd chromatography on an inert polymer column Anal Biochem 222,231–235PubMedCrossRefGoogle Scholar
  46. 46.
    Edge, C J, Rademacher, T W, Wormald, M R, Parekh, R B, Butters, T D, Wing, D R, and Dwek, R A (1992) Fast sequencing of ohgosacchartdes—the reagent-array analysis method Proc Natl Acad Scz USA 89, 6338–6342CrossRefGoogle Scholar
  47. 47.
    Rademacher, T W, Parekh, R B, and Dwek, R A (1988) Glycobiology Annu Rev Biochem 57,785–838PubMedCrossRefGoogle Scholar
  48. 48.
    Yeh, J C, Seals, J R, Murphy, C I, Vanhalbeek, H, and Cummings, R D (1993) Sitespecific N-glycosylatton and ohgosaccharrde structures of recombmant HIV-1 gp120 derived from a baculovtrus expression system Biochemzstry 32, 11,087–11,099CrossRefGoogle Scholar
  49. 49.
    Rohrer, J S, Cooper, G A, and Townsend, R R (1993) Identification, quantification, and characterizatton of glycopeptldes in reversed-phase HPLC separations of glycoprotem proteolytm digests Anal Biochem 212,7–16PubMedCrossRefGoogle Scholar
  50. 50.
    Elbem, A D (1987) Glycosylation inhibttors for N-linked glycoprotems Methods Enzymol 138,661–709CrossRefGoogle Scholar
  51. 51.
    Fleet, G W J (1988) Ammo-sugar derivatives and related compounds as glycosidase mhibitors Spec Pub1-RSot Chem 65, 149–162Google Scholar
  52. 52.
    Jacob, G S, Scudder, P, Butters, T D, Jones, I, and Tiemeter, D C (1992) Ammosugar attenuation of HIV Infection, in Natural Products as Antwral Agents (Chu, C K and Cutler, H G, eds), Plenum, New York, pp 137–152Google Scholar
  53. 53.
    Karlsson, G B, Butters, T D, Dwek, R A, and Platt, F M (1993) Effects of the ammo sugar N-butyldeoxynoJtrimycm on the N-glycosylation of recombinant gp120 Bzol Chem 268,570–576Google Scholar
  54. 54.
    Davis, S J, Davies, E A, Barclay, A N, Daenke, S, Bodian, D L., Jones, E Y, Stuart, D I, Butters, T D, Dwek, R A, and Vandermerwe, P A (1995) Ligand bmding by the nnmunoglobulm superfamrly recognmon molecule CD2 is glycosylation-independent J Biol Chem 270,369–375PubMedCrossRefGoogle Scholar
  55. 55.
    Lee, W.-R, Syu, W-J, Du, B, Matsuda, M, Tan, S, Wolf, A, Essex, M, and Lee, T-H (1992) Nonrandom distribution of gpl20 N-lmked glycosylation sites important for mfectivlty of human immunodeficiency vnus type 1 Proc Natl Acad SCI USA 89, 2213–2217PubMedCrossRefGoogle Scholar
  56. 56.
    Paulson, J C and Rogers, G N (1987) Restalylated erythrocytes for assessment of the specificity of sialylohgosaccharide binding protems Methods Enzymol 138, 162–168PubMedCrossRefGoogle Scholar
  57. 57.
    Whtteheart, S W., Passamtt, A, Retchner, J S, Holt, G D, Haltlwanger, R S, and Hart, G. W (1989) Glycosyltransferase probes Methods Enzymol 179, 82–95CrossRefGoogle Scholar
  58. 58.
    Natsuka, S and Lowe, J B (1994) Enzymes involved in mammahan ohgosaccharide biosynthests Curr Open Strut Biol 4, 683–691CrossRefGoogle Scholar
  59. 59.
    Mizuochl, T, Matthews, T J, Kato, M, Hamako, J, Titani, K, Solomon, J, and Feizi, T. (1990) Diversity of ohgosaccharide structures on the envelope glycoprotem GP 120 of Human Immunodefictency Vn-us-1 from the lymphoblastoid cell lme H9—presence of complex-type ohgosaccharides with bisecting N-acetylglucosamme residues J Bzol Chem 265,8519–8524Google Scholar

Copyright information

© Humana Press Inc , Totowa, NJ. 1998

Authors and Affiliations

  • Terry D. Butters
    • 1
  1. 1.Department of BtochemutryUntverstty of OxfordUK

Personalised recommendations