Abstract
Glycan microarrays prepared by immobilization of amino-functionalized glycans on NHS-activated glass slides have been successfully used to study protein–glycan interactions. Fluorescently tagged glycans with an amino functional group can be prepared from natural glycans released from glycoproteins. These tagged glycans can be enzymatically modified with various glycosyltransferases, phosphotransferases, sulfotransferases, etc., to quickly expand the size and diversity of the tagged glycan libraries (TGLs). The TGLs, presented in the format of microarrays, provide a convenient platform for identifying the glycan ligands of glycan-binding proteins (GBPs). The chapter provides the background to prepare a defined glycan microarray and uses as an example glycans generated as phosphodiesters and phosphomonoesters of high-mannose type N-glycans. The method describes the preparation of high-mannose type glycan-AEAB conjugates (GAEABs), the purification of their phosphodiesters, and the subsequent mild acid hydrolysis to obtain corresponding phosphomonoesters. These GAEABs are covalently printed as a phosphorylated glycan microarray and used for analysis of the glycan ligand specificities of P-type lectins, such as the mannose-6-phosphate receptors (Man-6-P receptors or MPRs).
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References
de Paz JL, and Seeberger PH. (2006) Recent advances in carbohydrate microarrays. QSAR & Combinatorial Science 25, 1027–1032.
Feizi T, and Chai W. (2004) Oligosaccharide microarrays to decipher the glyco code. Nat Rev Mol Cell Biol 5, 582–588.
Hirabayashi J. (2003) Oligosaccharide microarrays for glycomics. Trends Biotechnol 21, 141–143.
Park S, Lee MR, and Shin I. (2008) Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes. Chem Commun (Camb), 4389–4399.
Blixt O, Head S, Mondala T, Scanlan C, Huflejt ME, Alvarez R, Bryan MC, Fazio F, Calarese D, Stevens J, Razi N, Stevens DJ, Skehel JJ, van Die I, Burton DR, Wilson IA, Cummings R, Bovin N, Wong CH, and Paulson JC. (2004) Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc Natl Acad Sci U S A 101, 17033–17038.
Ratner DM, Adams EW, Su J, O’Keefe BR, Mrksich M, and Seeberger PH. (2004) Probing protein-carbohydrate interactions with microarrays of synthetic oligosaccharides. Chembiochem 5, 379–382.
de Boer AR, Hokke CH, Deelder AM, and Wuhrer M. (2007) General microarray technique for immobilization and screening of natural glycans. Anal Chem 79, 8107–13.
Song X, Lasanajak Y, Xia B, Smith DF, and Cummings RD. (2009) Fluorescent glycosylamides produced by microscale derivatization of free glycans for natural glycan microarrays. ACS Chem Biol 4, 741–750.
Song X, Xia B, Stowell SR, Lasanajak Y, Smith DF, and Cummings RD. (2009) Novel fluorescent glycan microarray strategy reveals ligands for galectins. Chem Biol 16, 36–47.
Bohorov O, Andersson-Sand H, Hoffmann J, and Blixt O. (2006) Arraying glycomics: a novel bi-functional spacer for one-step microscale derivatization of free reducing glycans. Glycobiology 16, 21C-27C.
Park S, Lee MR, and Shin I. (2009) Construction of carbohydrate microarrays by using one-step, direct immobilizations of diverse unmodified glycans on solid surfaces. Bioconjug Chem 20, 155–162.
Feizi T, Fazio F, Chai W, and Wong CH. (2003) Carbohydrate microarrays – a new set of technologies at the frontiers of glycomics. Curr Opin Struct Biol 13, 637–645.
Liu Y, Feizi T, Campanero-Rhodes MA, Childs RA, Zhang Y, Mulloy B, Evans PG, Osborn HM, Otto D, Crocker PR, and Chai W. (2007) Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide-protein interactions. Chem Biol 14, 847–859.
Song X, Lasanajak Y, Olson LJ, Boonen M, Dahms NM, Kornfeld S, Cummings RD, and Smith DF. (2009) Glycan microarray analysis of P-type lectins reveals distinct phospho-mannose glycan recognition. J Biol Chem. 284, 35201–35214.
Kudo M, and Canfield WM. (2006) Structural requirements for efficient processing and activation of recombinant human UDP-N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotran sferase. J Biol Chem 281, 11761–11768.
Dahms NM, and Hancock MK. (2002) P-type lectins. Biochim Biophys Acta 1572, 317–340.
Dahms NM, Olson LJ, and Kim JJ. (2008) Strategies for carbohydrate recognition by the mannose 6-phosphate receptors. Glycobiology 18, 664–678.
Ghosh P, Dahms NM, and Kornfeld S. (2003) Mannose 6-phosphate receptors: new twists in the tale. Nat Rev Mol Cell Biol 4, 202–212.
Kornfeld S, and Mellman I. (1989) The biogenesis of lysosomes. Annu Rev Cell Biol 5, 483–525.
Chavez CA, Bohnsack RN, Kudo M, Gotschall RR, Canfield WM, and Dahms NM. (2007) Domain 5 of the cation-independent mannose 6-phosphate receptor preferentially binds phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester). Biochemistry 46, 12604–12617.
Bohnsack RN, Song X, Olson LJ, Kudo M, Gotschall RR, Canfield WM, Cummings RD, Smith DF, and Dahms NM. (2009) Cation-independent mannose 6-phosphate receptor: A composite of distinct phosphomannosyl binding sites. J Biol Chem. 284, 35215–35226.
Gordon JA, Blumberg S, Lis H, and Sharon N. (1972) Purification of soybean agglutinin by affinity chromatography on sepharose-N-epsilon-aminocaproyl-beta-D-galactopyranosylamine. FEBS Lett 24, 193–196.
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Song, X., Heimburg-Molinaro, J., Dahms, N.M., Smith, D.F., Cummings, R.D. (2012). Preparation of a Mannose-6-Phosphate Glycan Microarray Through Fluorescent Derivatization, Phosphorylation, and Immobilization of Natural High-Mannose N-Glycans and Application in Ligand Identification of P-Type Lectins. In: Chevolot, Y. (eds) Carbohydrate Microarrays. Methods in Molecular Biology, vol 808. Humana Press. https://doi.org/10.1007/978-1-61779-373-8_9
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DOI: https://doi.org/10.1007/978-1-61779-373-8_9
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