Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from onehealthy male donor have been characterized, based on an approach usingendoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and acombination of chromatographic techniques, automated Edman sequencing, andfast atom bombardment mass spectrometry. Seven out of the eight potentialN-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298,Asn372, and Asn489, turned out to be glycosylated, and the potentialglycosylation site at Asn14, being close to the N-terminus, is not used.The carbohydrate microheterogeneity on three of the glycosylation sites wasstudied in more detail by high-pH anion-exchange chromatographic profilingand 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainlydi- and tri-charged oligosaccharides which comprise, among others, theGalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2toMan8GlcNAc2, in addition to a small amount of complex- type structures.Profiling of the carbohydrate moieties of Asn208 indicates a largeheterogeneity, similar to that established for native human Tamm-Horsfallglycoprotein, namely, multiply charged complex-type carbohydratestructures, terminated by sulfate groups, sialic acid residues, and/or theSda-determinant.