Spectroscopic and Protein Chemical Analyses Demonstrate the Presence of C-Mannosylated Tryptophan in Intact Human RNase 2 and Its Isoforms
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- 作者：Andreas Lö ; ffler ; Marie-Agnè ; s Doucey ; Anita M. Jansson ; Dieter R. Mü ; ller ; Tonny de Beer ; Daniel Hess ; Morten Meldal ; Wilhelm J. Richter ; Johannes F. G. Vliegenthart ; and Jan Hofsteenge
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：September 17, 1996
- 年：1996
- 卷：35
- 期：37
- 页码：12005 - 12014
- 全文大小：527K
- 年卷期：v.35,no.37(September 17, 1996)
- ISSN：1520-4995
文摘
Recently, the C-mannosylation of a specific tryptophan residue inRNase 2 from human urinehas been reported [Hofsteenge, J., et al. (1994)Biochemistry 33, 13524-13530; de Beer, T., etal. (1995)Biochemistry 34, 11785-11789]. In those studies,identification of this unusual modification wasaccomplished by mass spectrometric and NMR spectroscopic analysis ofpeptide fragments. The evidencefor the occurrence of C2-
-mannosyltryptophan[(C2-Man-)Trp] in the intact proteinrelied exclusively onthe detection of the same phenylthiohydantoin derivatives during Edmandegradation. In this paper, wehave (1) excluded the possibility that(C2-Man-)Trp arose artificially under theacidic conditions previouslyemployed for protein and peptide isolation and analysis, by maintainingthe pH >5 throughout theseprocedures, (2) demonstrated the occurrence of(C2-Man-)Trp in the intact protein, by NMRspectroscopy,(3) showed that (C2-Man-)Trp is not uniquefor RNase 2 from urine but that it is also present in theenzyme isolated from erythrocytes, and (4) found also thathigh-molecular mass isoforms of urinary RNase2 are C-mannosylated. These observations firmlyestablish C-mannosylation as a novel way of post-translationally attaching carbohydrate to protein, in addition to thewell-known N- and O-glycosylations.Furthermore, the NMR data, in combination with molecular dynamicscalculations, indicate that in thenative protein the mannopyranosyl residue is in a differentconformation than in the glycopeptide ordenatured protein, due to protein-carbohydrateinteractions.
-mannosyltryptophan[(C2-Man-)Trp] in the intact proteinrelied exclusively onthe detection of the same phenylthiohydantoin derivatives during Edmandegradation. In this paper, wehave (1) excluded the possibility that(C2-Man-)Trp arose artificially under theacidic conditions previouslyemployed for protein and peptide isolation and analysis, by maintainingthe pH >5 throughout theseprocedures, (2) demonstrated the occurrence of(C2-Man-)Trp in the intact protein, by NMRspectroscopy,(3) showed that (C2-Man-)Trp is not uniquefor RNase 2 from urine but that it is also present in theenzyme isolated from erythrocytes, and (4) found also thathigh-molecular mass isoforms of urinary RNase2 are C-mannosylated. These observations firmlyestablish C-mannosylation as a novel way of post-translationally attaching carbohydrate to protein, in addition to thewell-known N- and O-glycosylations.Furthermore, the NMR data, in combination with molecular dynamicscalculations, indicate that in thenative protein the mannopyranosyl residue is in a differentconformation than in the glycopeptide ordenatured protein, due to protein-carbohydrateinteractions.