title: Detailed characterization of the O-linked glycosylation of the neuropilin-1 c/MAM-domain.
abstract: Neuropilins are involved in angiogenesis and neuronal development. The membrane proximal domain of neuropilin-1, called c or MAM domain based on its sequence conservation, has been implicated in neuropilin oligomerization required for its function. The c/MAM domain of human neuropilin-1 has been recombinantly expressed to allow for investigation of its propensity to engage in molecular interactions with other protein or carbohydrate components on a cell surface. We found that the c/MAM domain was heavily O-glycosylated with up to 24 monosaccharide units in the form of disialylated core 1 and core 2 O-glycans. Attachment sites were identified on the chymotryptic c/MAM peptide ETGATEKPTVIDSTIQSEFPTY by electron-transfer dissociation mass spectrometry (ETD-MS/MS). For highly glycosylated species consisting of carbohydrate to about 50 %, useful results could only be obtained upon partial desialylation. ETD-MS/MS revealed a hierarchical order of the initial O-GalNAc addition to the four different glycosylation sites. These findings enable future functional studies about the contribution of the described glycosylations in neuropilin-1 oligomerization and the binding to partner proteins as VEGF or galectin-1.As a spin-off result the sialidase from Clostridium perfringens turned out to discriminate between galactose- and N-acetylgalactosamine-linked sialic acid.
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190, Vienna, Austria. Markus.Windwarder@boku.ac.at.
authors.forename: Markus
authors.initials: M
authors.lastname: Windwarder
authors.affiliation: Institute of Structural and Molecular Biology, University College London, Gower Street, Darwin Building, London, WC1E 6BT, UK.
authors.forename: Tamas
authors.initials: T
authors.lastname: Yelland
authors.affiliation: Institute of Structural and Molecular Biology, University College London, Gower Street, Darwin Building, London, WC1E 6BT, UK.
authors.forename: Snezana
authors.initials: S
authors.lastname: Djordjevic
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D039942:Neuropilin-1 / Q000737:chemistry* / Q000378:metabolism; D000072417:Protein Domains; D011499:Protein Processing, Post-Translational
chemical_list: D039942:Neuropilin-1; D000117:Acetylglucosamine
keywords: Electron-transfer dissociation; MAM-domain; O-GalNAc glycan; O-glycosylation; neuropilin-1