Article: article from journal or magazin.
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 RNase 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 modification was accomplished by mass spectrometric and NMR spectroscopic analysis of peptide fragments. The evidence for the occurrence of C2-alpha-mannosyltryptophan [(C2-Man-)Trp] in the intact protein relied exclusively on the detection of the same phenylthiohydantoin derivatives during Edman degradation. In this paper, we have (1) excluded the possibility that (C2-Man-)Trp arose artificially under the acidic conditions previously employed for protein and peptide isolation and analysis, by maintaining the pH > 5 throughout these procedures, (2) demonstrated the occurrence of (C2-Man-)Trp in the intact protein, by NMR spectroscopy, (3) showed that (C2-Man-)Trp is not unique for RNase 2 from urine but that it is also present in the enzyme isolated from erythrocytes, and (4) found also that high-molecular mass isoforms of urinary RNase 2 are C-mannosylated. These observations firmly establish C-mannosylation as a novel way of post-translationally attaching carbohydrate to protein, in addition to the well-known N- and O-glycosylations. Furthermore, the NMR data, in combination with molecular dynamics calculations, indicate that in the native protein the mannopyranosyl residue is in a different conformation than in the glycopeptide or denatured protein, due to protein-carbohydrate interactions.
Amino Acid Sequence, Blotting, Western, Endoribonucleases/chemistry, Endoribonucleases/isolation & purification, Female, Glycosylation, Humans, Isoenzymes/chemistry, Isoenzymes/isolation & purification, Magnetic Resonance Spectroscopy, Mannose/analysis, Mass Spectrometry, Menopause, Models, Molecular, Molecular Sequence Data, Molecular Weight, Peptide Fragments/chemistry, Peptide Fragments/isolation & purification, Pregnancy, Protein Conformation, Tryptophan/analogs & derivatives, Tryptophan/analysis
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