title: Enhanced Cross-Linking of Diazirine-Modified Sialylated Glycoproteins Enabled through Profiling of Sialidase Specificities.
abstract: Sialic-acid-mediated interactions play critical roles on the cell surface, providing an impetus for the development of methods to study this important monosaccharide. In particular, photo-cross-linking sialic acids incorporated onto cell surfaces have allowed covalent capture of transient interactions between sialic acids and sialic-acid-recognizing proteins via cross-linking. However, natural sialic acids also present on the cell surface compete with photo-cross-linking sialic acids in binding events, limiting cross-linking yields. In order to improve the utility of one such photo-cross-linking sialic acid, SiaDAz, we examined a number of sialidases, enzymes that remove sialic acids from glycoconjugates, to find one that would cleave natural sialic acids but remain inactive toward SiaDAz. Using this sialidase, we improved SiaDAz-mediated cross-linking of an antisialyl Lewis X antibody and of endoglin. This protocol can be applied generally to sialic-acid-mediated interactions and will facilitate identification of sialic acid binding partners.
authors.affiliation: Department of Biochemistry, The University of Texas Southwestern Medical Center , Dallas, Texas 75390-9038, United States.
authors.forename: Janet E
authors.initials: JE
authors.lastname: McCombs
authors.affiliation: Alberta Glycomics Centre, Department of Chemistry, University of Alberta , Edmonton, Alberta T6G 2G2, Canada.
authors.forename: Chunxia
authors.initials: C
authors.lastname: Zou
authors.affiliation: Department of Biochemistry, The University of Texas Southwestern Medical Center , Dallas, Texas 75390-9038, United States.
authors.forename: Randy B
authors.initials: RB
authors.lastname: Parker
authors.affiliation: Alberta Glycomics Centre, Department of Chemistry, University of Alberta , Edmonton, Alberta T6G 2G2, Canada.
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Cairo
authors.affiliation: Department of Biochemistry, The University of Texas Southwestern Medical Center , Dallas, Texas 75390-9038, United States.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Kohler
mesh_terms: D000906:Antibodies / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000737:chemistry; D003978:Diazomethane / Q000737:chemistry*; D000071063:Endoglin; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D009439:Neuraminidase / Q000737:chemistry* / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D012794:Sialic Acids / Q000737:chemistry*; D013379:Substrate Specificity
chemical_list: D000906:Antibodies; D015703:Antigens, CD; D003432:Cross-Linking Reagents; C064206:ENG protein, human; D000071063:Endoglin; D006023:Glycoproteins; D011956:Receptors, Cell Surface; D012794:Sialic Acids; D003978:Diazomethane; D009439:Neuraminidase