SPECTROSCOPIC AND PROTEIN CHEMICAL-ANALYSES DEMONSTRATE THE PRESENCE OF C-MANNOSYLATED TRYPTOPHAN IN INTACT HUMAN RNASE-2 AND ITS ISOFORMS

Recently, the C-mannosylation of a specific tryptophan residue in RNas e 2 from human urine has been reported [Hofsteenge, J., et al. (1994) Biochemistry 33, 13524-13530; de Beer, T., et al. (1995) Biochemistry 34, 11785-11789]. In those studies, identification of this unusual mod ification was accomplished by mass spectrometric and NMR spectroscopic analysis of peptide fragments. The evidence for the occurrence of C-2 -alpha-mannosyltryptophan [(C-2-Man-)Trp] in the intact protein relied exclusively on the detection of the same phenylthiohydantoin derivati ves during Edman degradation. In this paper, we have (1) excluded the possibility that (C-2-Man-)Trp arose artificially under the acidic con ditions previously employed for protein and peptide isolation and anal ysis, by maintaining the pH >5 throughout these procedures, (2) demons trated the occurrence of ((C-2-Man-)Trp in the intact protein, by NMR spectroscopy, (3) showed that (C-2-Man-)Trp is not unique for RNase 2 from urine but that it is also present in the enzyme isolated from ery throcytes, and (4) found also that high-molecular mass isoforms of uri nary RNase 2 are C-mannosylated. These observations firmly establish C -mannosylation as a novel way of post-translationally attaching carboh ydrate to protein, in addition to the well-known N- and O-glycosylatio ns. Furthermore, the NMR data, in combination with molecular dynamics calculations, indicate that in the native protein the mannopyranosyl r esidue is in a different conformation than in the glycopeptide or dena tured protein, due to protein-carbohydrate interactions.