title: Glycosylation sites and site-specific glycosylation in human Tamm-Horsfall glycoprotein.
abstract: The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex-type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.
authors.affiliation: Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, P.O. Box 80075, NL-3508 TB Utrecht, The Netherlands.
authors.forename: J J
authors.initials: JJ
authors.lastname: van Rooijen
authors.forename: A F
authors.initials: AF
authors.lastname: Voskamp
authors.forename: J P
authors.initials: JP
authors.lastname: Kamerling
authors.forename: J F
authors.initials: JF
authors.lastname: Vliegenthart
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008969:Molecular Sequence Data; D009088:Mucoproteins / Q000737:chemistry* / Q000652:urine*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment; D058949:Uromodulin
chemical_list: D009088:Mucoproteins; D010446:Peptide Fragments; C548612:UMOD protein, human; D058949:Uromodulin; D001216:Asparagine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase