Glycoconjugate Journal

, Volume 26, Issue 2, pp 141–159 | Cite as

Chemo-enzymatic synthesis of poly-N-acetyllactosamine (poly-LacNAc) structures and their characterization for CGL2-galectin-mediated binding of ECM glycoproteins to biomaterial surfaces

  • Birgit Sauerzapfe
  • Karel Křenek
  • Judith Schmiedel
  • Warren W. Wakarchuk
  • Helena Pelantová
  • Vladimir Křen
  • Lothar EllingEmail author


Poly-N-acetyllactosamine (poly-LacNAc) structures have been identified as important ligands for galectin-mediated cell adhesion to extra-cellular matrix (ECM) proteins. We here present the biofunctionalization of surfaces with poly-LacNAc structures and subsequent binding of ECM glycoproteins. First, we synthesized β-GlcNAc glycosides carrying a linker for controlled coupling onto chemically functionalized surfaces. Then we produced poly-LacNAc structures with defined lengths using human β1,4-galactosyltransferase-1 and β1,3-N-acetylglucosaminyltransferase from Helicobacter pylori. These compounds were also used for kinetic characterization of glycosyltransferases and lectin binding assays. A mixture of poly-LacNAc-structures covalently coupled to functionalized microtiter plates were identified for best binding to our model galectin His6CGL2. We further demonstrate for the first time that these poly-LacNAc surfaces are suitable for further galectin-mediated binding of the ECM glycoproteins laminin and fibronectin. This new technology should facilitate cell adhesion to biofunctionalized surfaces by imitating the natural ECM microenvironment.


Poly-LacNAc Chemo-enzymatic synthesis Galectin binding ECM glycoproteins Biomaterials 



The authors thank Prof. Dr. Markus Aebi (ETH Zürich) and Prof. Dr. Eric G. Berger (Zürich University) for providing the CGL2 plasmid and α3GalT plasmid, respectively. L.E. and B.S acknowledge financial support by the DFG Research Training Group 1035 “Biointerface”. Part of this work was supported by a bilateral grant from DAAD-AV ČR project PPP-D7-CZ 26/04-05D/03/44448 (V. K. & L. E.) and by projects MSMT LC06010 and GAAVCR IAA400200503. B.S. thanks the Boehringer Ingelheim Foundation—Travel Allowances for financial support during a stay in Dr. Wakarchuk’s laboratory. Dr. P. Halada (Inst. Microbiol., Prague) is thanked for the MS measurements. The excellent technical assistance (HPLC/ESI-MS and CE) by Dipl.-Ing. Dennis Hirtz (Laboratory for Biomaterials, RWTH Aachen University) is gratefully acknowledged.

Supplementary material

10719_2008_9172_MOESM1_ESM.doc (744 kb)
Supporting Information (DOC 744 KB)


  1. 1.
    Spiro, R.G.: Protein glycosylation: natures, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology 12, 43R–56R (2002)PubMedCrossRefGoogle Scholar
  2. 2.
    Smith, A.E., Helenius, A.: How viruses enter animal cells. Science 304, 237–242 (2004)PubMedCrossRefGoogle Scholar
  3. 3.
    Campbell, C.T., Yarema, K.J.: Large-scale approaches for glycobiology. Genome Biol. 6, 236–244 (2005)PubMedCrossRefGoogle Scholar
  4. 4.
    Paulson, J.C., Blixt, O., Collins, B.E.: Sweet spots in functional glycomics. Nat. Chem. Biol. 2, 238–248 (2006)PubMedCrossRefGoogle Scholar
  5. 5.
    Sasaki, K., Kurata-Miura, K., Ujita, M., Angata, K., Nakagawa, S., Sekine, S., Nishi, T., Fukuda, M.: Expression cloning of cDNA encoding a human b-1,3-N-acetylglucosaminyltransferase that is essential for poly-N-acetyllactosamine synthesis. Proc. Natl. Acad. Sci. U. S. A. 94, 14294–14299 (1997)PubMedCrossRefGoogle Scholar
  6. 6.
    Ujita, M., McAuliffe, J., Suzuki, M., Hindsgaul, O., Clausen, H., Fukuda, M.N., Fukuda, M.: Regulation of I-branched poly-N-acetyllactosamine synthesis—concerted actions by i-extension enzyme, I-branching enzyme, and b1,4-galactosyltransferase I. J. Biol. Chem. 274, 9296–9304 (1999)PubMedCrossRefGoogle Scholar
  7. 7.
    Leffler, H., Carlsson, S., Hedlund, M., Qian, Y., Poirier, F.: Introduction to galectins. Glycoconjugate J. 19, 433–440 (2004)CrossRefGoogle Scholar
  8. 8.
    Di Virgilio, S., Glushka, J., Moremen, K., Pierce, M.: Enzymatic synthesis of natural and 13C enriched linear poly-N-acetyllactosamines as ligands for galectin-1. Glycobiology 9, 353–364 (1999)PubMedCrossRefGoogle Scholar
  9. 9.
    Stowell, S.R., Dias-Baruffi, M., Penttila, L., Renkonen, O., Nyame, A.K., Cummings, R.D.: Human galectin-1 recognition of poly-N-acetyllactosamine and chimeric polysaccharides. Glycobiology 14, 157–167 (2004)PubMedCrossRefGoogle Scholar
  10. 10.
    Leppanen, A., Stowell, S., Blixt, O., Cummings, R.D.: Dimeric galectin-1 binds with high affinity to {alpha}2,3-sialylated and non-sialylated terminal N-acetyllactosamine units on surface-bound extended glycans. J. Biol. Chem. 280, 5549–5562 (2005)PubMedCrossRefGoogle Scholar
  11. 11.
    Patnaik, S.K., Potvin, B., Carlsson, S., Sturm, D., Leffler, H., Stanley, P.: Complex N-glycans are the major ligands for galectin-1, -3, and -8 on Chinese hamster ovary cells. Glycobiology 16, 305–317 (2006)PubMedCrossRefGoogle Scholar
  12. 12.
    Wu, A.M., Singh, T., Wu, J.H., Lensch, M., Andre, S., Gabius, H.-J.: Interaction profile of galectin-5 with free saccharides and mammalian glycoproteins: probing its fine specificity and the effect of naturally clustered ligand presentation. Glycobiology 16, 524–537 (2006)PubMedCrossRefGoogle Scholar
  13. 13.
    Hughes, R.C.: Galectins as modulators of cell adhesion. Biochimie 83, 667–676 (2001)PubMedCrossRefGoogle Scholar
  14. 14.
    Hughes, R.C.: Galectins in kidney development. Glycoconjugate J. 19, 621–629 (2004)CrossRefGoogle Scholar
  15. 15.
    Sharon, N., Lis, H.: History of lectins: from hemagglutinins to biological recognition molecules. Glycobiology 14, 53R–62R (2004)PubMedCrossRefGoogle Scholar
  16. 16.
    Blixt, O., Vasiliu, D., Allin, K., Jacobsen, N., Warnock, D., Razi, N., Paulson, J.C., Bernatchez, S., Gilbert, M., Wakarchuk, W.: Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides GD3, GT3, GM2, GD2, GT2, GM1, and GD1a. Carbohydr. Res. 340, 1963–1972 (2005)PubMedCrossRefGoogle Scholar
  17. 17.
    Vasiliu, D., Razi, N., Zhang, Y., Jacobsen, N., Allin, K., Liu, X., Hoffmann, J., Bohorov, O., Blixt, O.: Large-scale chemoenzymtic synthesis of blood group and tumor-associated poly-N-acetyllactosamine antigens. Carbohydr. Res. 341, 1447–1457 (2006)PubMedCrossRefGoogle Scholar
  18. 18.
    Unverzagt, C., André, S., Seifert, J., Kojima, S., Fink, C., Srikrishna, G., Freeze, H., Kayse, K., Gabius, H.-J.: Structure–activity profiles of complex biantennary glycans with core fucosylation and with/without additional a2,3/a2,6 sialylation: synthesis of neoglycoproteins and their properties in lectin assays, cell binding, and organ uptake. J. Med. Chem. 45, 478–491 (2002)PubMedCrossRefGoogle Scholar
  19. 19.
    Murata, T., Honda, H., Hattori, T., Usui, T.: Enzymatic synthesis of poly-N-acetyllactosamines as potential substrates for endo-b-galactosidase-catalysed hydrolytic and transglycosylation reactions. Biochim. Biophys. Acta. 1722, 60–68 (2005)PubMedGoogle Scholar
  20. 20.
    Blixt, O., Razi, N.: Strategies for synthesis of an oligosaccharide library using a chemo-enzymatic approach. In: Wang, P.G., Ichikawa, Y. (eds.) Synthesis of Carbohydrate through Biotechnology, pp. 93–112. American Chemical Society, Washington D. C. (2004)Google Scholar
  21. 21.
    Sears, P., Wong, C.-H.: Toward automated synthesis of oligosaccharides and glycoproteins. Science 291, 2344–2350 (2001)PubMedCrossRefGoogle Scholar
  22. 22.
    Nicolaou, K.C., Watanabe, N., Li, J., Pastor, J., Winssinger, N.: Solid-phase synthesis of oligosaccharides: construction of a dodecasaccharide. Angew. Chem. Int. Ed. Engl. 37, 1559–1561 (1998)CrossRefGoogle Scholar
  23. 23.
    Plante, O.J., Palmacci, E.R., Seeberger, P.H.: Automated solid-phase synthesis of oligosaccharides. Science 291, 1523–1527 (2001)PubMedCrossRefGoogle Scholar
  24. 24.
    Werz, D.B., Seeberger, P.H.: Carbohydrates as the next frontier in pharmaceutical research. Chem. Eur. J. 11, 3194–3206 (2005)CrossRefGoogle Scholar
  25. 25.
    Koeller, K.M., Wong, C.-H.: Synthesis of complex carbohydrates and glycoconjugates: enzyme-based and programmable one-pot strategies. Chem. Rev. 100, 4465–4493 (2000)PubMedCrossRefGoogle Scholar
  26. 26.
    Daines, A.M., Maltman, B.A., Flitsch, S.L.: Synthesis and modifications of carbohydrates, using biotransformations. Curr. Opin. Cem. Biol. 8, 106–113 (2004)CrossRefGoogle Scholar
  27. 27.
    Zervosen, A., Elling, L.: A novel three-enzyme reaction cycle for the synthesis of N-acetyllactosamine with in situ regeneration of uridine 5′-diphosphate glucose and uridine 5′-diphosphate galactose. J. Am. Chem. Soc. 118, 1836–1840 (1996)CrossRefGoogle Scholar
  28. 28.
    Blixt, O., vanDie, I., Norberg, T., vandenEijnden, D.H.: High-level expression of the Neisseria meningitidis lgtA gene in Escherichia coli and characterization of the encoded N-acetylglucosaminyltransferase as a useful catalyst in the synthesis of GlcNAc beta 1 -> 3Gal and GalNAc beta 1-3Gal linkages. Glycobiology 9, 1061–1071 (1999)PubMedCrossRefGoogle Scholar
  29. 29.
    Blixt, O., Brown, J., Schur, M.J., Wakarchuk, W., Paulson, J.C.: Efficient preparation of natural and synthetic galactosides with a recombinant b-1,4-Galactosyltransferase-/UDP-4′-Gal epimerase fusion protein. J. Org. Chem. 66, 2442–2448 (2001)PubMedCrossRefGoogle Scholar
  30. 30.
    Niemelä, R., Natunen, J., Majuri, M.-L., Maaheima, H., Helin, J., Lowe, J.B., Renkonen, O., Renkonen, R.: Complementary acceptor and site specificities of Fuc-TIV and Fuc-TVII allow effective biosynthesis of Sialyl-TriLex and related polylactosamines present on glycoprotein counterreceptors of selectin. J. Biol. Chem. 273, 4021–4026 (1998)PubMedCrossRefGoogle Scholar
  31. 31.
    Sauerzapfe, B., Namdjou, D.-J., Schumacher, T., Linden, N., Krenek, K., Kren, V., Elling, L.: Characterization of recombinant fusion constructs of human b1,4-galactosyltransferase 1 and the lipase pre-propeptide from Staphylococcus hyicus. J. Mol. Catal. B: Enzym. 50, 128–140 (2008)CrossRefGoogle Scholar
  32. 32.
    Logan, S.M., Altman, E., Mykytczuk, O., Brisson, J.-R., Chandan, V., Michael, F.S., Masson, A., Leclerc, S., Hiratsuka, K., Smirnova, N., Li, J., Wu, Y., Wakarchuk, W.W.: Novel biosynthetic functions of lipopolysaccharide rfaJ homologs from Helicobacter pylori. Glycobiology 15, 721–733 (2005)PubMedCrossRefGoogle Scholar
  33. 33.
    Boulianne, R.P., Liu, Y., Aebi, M., Lu, B.C., Kues, U.: Fruiting body development in Coprinus cinereus: regulated expression of two galectins secreted by a non-classical pathway. Microbiology 146, 1841–1853 (2000)PubMedGoogle Scholar
  34. 34.
    Walser, P.J., Haebel, P.W., Künzler, M., Sargent, D., Kues, U., Aebi, M., Ban, N.: Structure and functional analysis of the fungal galectin CGL2. Structure 12, 689–702 (2004)PubMedCrossRefGoogle Scholar
  35. 35.
    Shin, H., Jo, S., Mikos, A.G.: Biomimetic materials for tissue engineering. Biomaterials 24, 4353–4364 (2003)PubMedCrossRefGoogle Scholar
  36. 36.
    Lutolf, M.P., Hubbell, J.A.: Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat. Biotechnol. 23, 47–55 (2005)PubMedCrossRefGoogle Scholar
  37. 37.
    Krist, P., Vannucci, L., Sadalapure, K., Patel, A., Bezouška, K., Pospíšil, M., Kuzma, M., Lindhorst, T.K., Petruš, L., Kren, V.: Fluorescent labeled thiourea-bridged glycodendrons. ChemBioChem. 5, 445–452 (2004)PubMedCrossRefGoogle Scholar
  38. 38.
    Ujita, M., Misra, A.K., McAuliffe, J., Hindsgaul, O., Fukuda, M.: Poly-N-acetyllactosamine extension in N-Glycans and core 2- and core 4-branched O-glycans is differentially controlled by i-extension enzyme and different members of the beta 1,4-galactosyltransferase gene family. J. Biol. Chem. 275, 15868–15875 (2000)PubMedCrossRefGoogle Scholar
  39. 39.
    Alvarez, R.A., Blixt, O.: Identification of ligand specificities for glycan-binding proteins using glycan arrays. Meth. Enzymol. 415, 292–310 (2006)PubMedCrossRefGoogle Scholar
  40. 40.
    de Paz, J.L., Horlacher, T., Seeberger, P.H.: Oligosaccharide microarrays to map interactions of carbohydrates in biological systems. Meth. Enzymol. 415, 269–292 (2006)PubMedCrossRefGoogle Scholar
  41. 41.
    Nakamura-Tsuruta, S., Uchiyama, N., Hirabayashi, J.: High throughput analysis of lectin oligosaccharide interactions by automated frontal affinity chromatography. Meth. Enzymol. 415, 311–325 (2006)PubMedCrossRefGoogle Scholar
  42. 42.
    Liu, Y., Chai, W., Childs, R.A., Feizi, T.: Preparation of neoglycolipids with ring closed cores via chemoselective oxime ligation for microarray analysis of carbohydrate–protein interactions. Meth. Enzymol. 415, 326–340 (2006)PubMedCrossRefGoogle Scholar
  43. 43.
    Uchiyama, N., Kuno, A., Koseki-Kuno, S., Ebe, Y., Horio, K., Yamada, M., Hirabayashi, J.: Development of a lectin microarray based on an evanescent field fluorescence principle. Meth. Enzymol. 415, 341–351 (2006)PubMedCrossRefGoogle Scholar
  44. 44.
    Bülter, T., Schumacher, T., Namdjou, D.-J., Gutiérrez Gallego, R., Clausen, H., Elling, L.: Chemo-enzymatic synthesis of biotinylated nucleotide sugars as substrates for glycosyltransferases. ChemBioChem. 2, 884–894 (2001)PubMedCrossRefGoogle Scholar
  45. 45.
    Namdjou, D.-J., Sauerzapfe, B., Schmiedel, J., Dräger, G., Bernatchez, S., Wakarchuk, W.W., Elling, L.: Combination of UDP-Glc(NAc) 4′-epimerase and galactose oxidase in a one-pot synthesis of biotinylated nucleotide sugars. Adv. Synth. Catal. 349, 314–318 (2007)CrossRefGoogle Scholar
  46. 46.
    Stults, C., Macher, B., Bhatti, R., Srivastava, O., Hindsgaul, O.: Characterization of the substrate specificity of alpha1,3galactosyltransferase utilizing modified N-acetyllactosamine disaccharides. Glycobiology 9, 661–668 (1999)PubMedCrossRefGoogle Scholar
  47. 47.
  48. 48.
    Hirabayashi, J., Hashidate, T., Arata, Y., Nishi, N., Nakamura, T., Hirashima, M., Urashima, T., Oka, T., Futai, M., Muller, W.E.G., Yagi, F., Kasai, K.-I.: Oligosaccharide specificity of galectins: a search by frontal affinity chromatography. Biochim. Biophys. Acta. 1572, 232–254 (2002)PubMedGoogle Scholar
  49. 49.
    Knibbs, R.N., Perini, F., Goldstein, I.J.: Structure of the major concanavalin a reactive oligosaccharides of the extracellular matrix component laminin. Biochemistry 28, 6379–6392 (1989)PubMedCrossRefGoogle Scholar
  50. 50.
    Cooper, D.N.W.: Galectin-1: secretion and modulation of cell interactions with laminin. Trends Glycosci. Glycotechnol. 9, 57–67 (1997)Google Scholar
  51. 51.
    Barboni, E.A.M., Bawumia, S., Hughes, R.C.: Kinetic measurements of binding of galectin 3 to a laminin substratum. Glycoconjugate J. 16, 365–373 (1999)CrossRefGoogle Scholar
  52. 52.
    Tisi, D., Talts, J.F., Timpl, R., Hohenester, E.: Structure of the C-terminal laminin G-like domain pair of the laminin a2 chain harbouring binding sites for a-dystroglycan and heparin. EMBO J. 19, 1432–1440 (2000)PubMedCrossRefGoogle Scholar
  53. 53.
    Ido, H., Harada, K., Futaki, S., Hayashi, Y., Nishiuchi, R., Natsuka, Y., Li, S., Wada, Y., Combs, A.C., Ervasti, J.M., Sekiguchi, K.: Molecular dissection of the a-dystroglycan- and integrin-binding sites within the globular domain of human laminin-10. J. Biol. Chem. 279, 10946–10954 (2004)PubMedCrossRefGoogle Scholar
  54. 54.
    Tajiri, M., Yoshida, S., Wada, Y.: Differential analysis of site-specific glycans on plasma and cellular fibronectins: application of a hydrophilic affinity method for glycopeptide enrichment. Glycobiology 15, 1332–1340 (2005)PubMedCrossRefGoogle Scholar
  55. 55.
    Hörmann, H., Richter, H., Jelinic, V.: Evidence for a cryptic lectin site in the cell-binding domain of plasma fibronectin. Hoppe Seylers Z. Physiol. Chem. 365, 517–524 (1984)PubMedGoogle Scholar
  56. 56.
    Horton, D.: 2-Acetoamido-3,4,6-tri-O-acetyl-2-deoxy-a-d-glucopyranosyl chloride. Org. Synth. 46, 1 (1966)Google Scholar
  57. 57.
    Benalil, A., Carboni, B., Vaultier, M.: Synthesis of 1,2-Aminoazides. Conversion to unsymmetrical vicinal diamines by catalytic hydrogenation or reductive alkylation with dichloroboranes. Tetrahedron 47, 8177–8194 (1991)CrossRefGoogle Scholar
  58. 58.
    Römer, U., Schrader, H., Günther, N., Nettelstroth, N., Frommer, W.B., Elling, L.: Expression, purification and characterization of recombinant sucrose synthase 1 from Solanum tuberosum L. for carbohydrate engineering. J. Biotechnol. 107, 135–149 (2004)PubMedCrossRefGoogle Scholar
  59. 59.
    Bernatchez, S., Szymanski, C.M., Ishiyama, N., Li, J., Jarrell, H.C., Lau, P.C., Berghuis, A.M., Young, N.M., Wakarchuk, W.W.: A single bifunctional UDP-GlcNAc/Glc 4-Epimerase supports the synthesis of three cell surface glycoconjugates in Campylobacter jejuni. J. Biol. Chem. 280, 4792–4802 (2005)PubMedCrossRefGoogle Scholar
  60. 60.
    Wakarchuk, W.W., Cunningham, A., Watson, D.C., Young, N.M.: Role of paired basic residues in the expression of active recombinant galactosyltransferases from the bacterial pathogen Neisseria meningitidis. Protein Eng. 11, 295–302 (1998)PubMedCrossRefGoogle Scholar
  61. 61.
    Shibatani, S., Fujiyama, K., Nishiguchi, S., Seki, T., Maekawa, Y.: Production and characterization of active soluble human [beta]1,4-galactosyltransferase in Escherichia coli as a useful catalyst in synthesis of the Gal [beta]1->4 GlcNAc linkage. J. Biosci. Bioeng. 91, 85–87 (2001)PubMedCrossRefGoogle Scholar
  62. 62.
    Wakarchuk, W.W., Watson, D., St Michael, F., Li, J., Wu, Y., Brisson, J.-R., Young, N.M., Gilbert, M.: Dependence of the bi-functional nature of a sialyltransferase from Neisseria meningitidis on a single amino acid substitution. J. Biol. Chem. 276, 12785–12790 (2001)PubMedCrossRefGoogle Scholar
  63. 63.
    Gilbert, M., Karwaski, M.-F., Bernatchez, S., Young, N.M., Taboada, E., Michniewicz, J., Cunningham, A.-M., Wakarchuk, W.W.: The genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, campylobacter jejuni. Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide. J. Biol. Chem. 277, 327–337 (2002)PubMedCrossRefGoogle Scholar
  64. 64.
    Brinkmann, N., Malissard, M., Ramuz, M., Römer, U., Schumacher, T., Berger, E.G., Elling, L., Wandrey, C., Liese, A.: Chemo-enzymatic synthesis of the galili epitope Gal[alpha](1->3)Gal[beta](1->4)GlcNAc on a homogeneously soluble PEG polymer by a multi-enzyme system. Bioorg. Med. Chem. Lett. 11, 2503–2506 (2001)PubMedCrossRefGoogle Scholar
  65. 65.
    Wakarchuk, W.W., Cunningham, A.M.: Capillary electrophoresis as an assay method for monitoring glycosyltransferase activity. Meth. Mol. Biol. 213, 263–274 (2003)Google Scholar
  66. 66.
    Uhrín, D., Barlow, P.N.: Gradient-enhanced one-dimensional proton chemical-shift correlation with full sensitivity. J. Magn. Reson. 126, 248–255 (1997)CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Birgit Sauerzapfe
    • 1
  • Karel Křenek
    • 2
  • Judith Schmiedel
    • 1
  • Warren W. Wakarchuk
    • 3
  • Helena Pelantová
    • 2
  • Vladimir Křen
    • 2
  • Lothar Elling
    • 1
    Email author
  1. 1.Laboratory for Biomaterials, Institute of Biotechnology and Helmholtz-Institute for Biomedical EngineeringRWTH Aachen UniversityAachenGermany
  2. 2.Institute of MicrobiologyAcademy of Sciences of the Czech RepublicPrague 4Czech Republic
  3. 3.Institute for Biological SciencesNational Research Council of CanadaOttawaCanada

Personalised recommendations