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.
Löffler
A
A
Friedrich Miescher-Institut, Basel, Switzerland.
Doucey
M A
MA
Jansson
A M
AM
Müller
D R
DR
de Beer
T
T
Hess
D
D
Meldal
M
M
Richter
W J
WJ
Vliegenthart
J F
JF
Hofsteenge
J
J
D000595:Amino Acid Sequence; D015153:Blotting, Western; D004722:Endoribonucleases / Q000737:chemistry* / Q000302:isolation & purification / Q000652:urine; D005260:Female; D006031:Glycosylation; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000302:isolation & purification / Q000652:urine; D009682:Magnetic Resonance Spectroscopy; D008358:Mannose / Q000032:analysis*; D013058:Mass Spectrometry; D008593:Menopause; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011247:Pregnancy; D011487:Protein Conformation; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000032:analysis
C101500:C(2)-mannosyltryptophan; D007527:Isoenzymes; D010446:Peptide Fragments; D014364:Tryptophan; D004722:Endoribonucleases; C029494:ribonuclease U; D008358:Mannose