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. Methods Mol Biol 2012;808:137-48
Date
11/08/2011Pubmed ID
22057522Pubmed Central ID
PMC4668285DOI
10.1007/978-1-61779-373-8_9Scopus ID
2-s2.0-82355183891 (requires institutional sign-in at Scopus site) 12 CitationsAbstract
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).
Author List
Song X, Heimburg-Molinaro J, Dahms NM, Smith DF, Cummings RDAuthor
Nancy M. Dahms PhD Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Fluorescent DyesLectins
Ligands
Mannosephosphates
Microarray Analysis
Phosphorylation
Polysaccharides