Spectroscopic and protein chemical analyses demonstrate the presence of C-mannosylated tryptophan in intact human RNase 2 and its isoforms

被引:64
作者
Loffler, A
Doucey, MA
Jansson, AM
Muller, DR
deBeer, T
Hess, D
Meldal, M
Richter, WJ
Vliegenthart, JFG
Hofsteenge, J
机构
[1] FRIEDRICH MIESCHER INST,CH-4002 BASEL,SWITZERLAND
[2] DEPT CHEM,CARLSBERG LAB,DK-2500 VALBY,DENMARK
[3] CIBA GEIGY LTD,CENT RES SERV,CH-4002 BASEL,SWITZERLAND
[4] UNIV UTRECHT,DEPT BIOORGAN CHEM,BIJVOET CTR,NL-3584 CH UTRECHT,NETHERLANDS
关键词
D O I
10.1021/bi9610515
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 C-2-alpha-mannosyltryptophan [(C-2-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 (C-2-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 ((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 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.
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页码:12005 / 12014
页数:10
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