New type of linkage between a carbohydrate and a protein: C-glycosylation of a specific tryptophan residue in human RNase Us

Hofsteenge, Jan; Müller, Dieter; Beer, Tonny de; Löffler, Andreas; Richter, Wilhelm J.; Vliegenthart, Johannes F.G.

doi:10.1021/bi00250a003

Cited by 270 publications

(246 citation statements)

References 43 publications

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“…Furthermore, digestion of the thermolytic peptide with elastase yielded the peptides 5-8 and 9-10, whose absorbance ratio at 280 nm was 1.62 and 1.58, for the peptides obtained from hp-and lp-RNase 2, respectively. The value of this ratio was in good agreement with the one previously found for C-mannosylated and unmodified Trp, i.e., 1.56 (Hofsteenge et al, 1994), which suggests that the peptides from both proteins are identical and also that Trp-7 in the peptide from hp-RNase 2 is modified.…”

Section: Stability Of the O-mannosylated Syntheticsupporting

confidence: 90%

“…Recently, it was found that Trp-7 in human RNase 2 1 is modified by C-glycosidic attachment of an R-mannosyl residue (Hofsteenge et al, 1994;de Beer et al, 1995). In contrast to the previously known N-and O-glycosidic linkages to asparagine and serine, or threonine, the mannopyranosyl C1′ atom is in this case directly linked to the C2 atom of the tryptophan indole ring.…”

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confidence: 88%

“…High-molecular mass isoforms, eluting at the lower NaCl concentration, were extensively dialyzed against 10 mM Bistris (pH 6.0) and subsequently rechromatographed on heparin Sepharose using a gradient of 0 to 350 mM NaCl. Final purification of the active fractions, eluting at approximately 250 mM NaCl, was achieved by reversed-phase HPLC as described for lp-RNase 2 (Hofsteenge et al, 1994).…”

Section: Purification Of Rnase 2 and High-molecular Massmentioning

confidence: 99%

“…First, is the mannopyranosyl residue on Trp-7 originally present in the protein, or is (C 2 -Man-)Trp 2 possibly formed during purification or analysis of the protein and/or its peptides? So far the evidence for the presence of (C 2 -Man-)Trp in the intact protein relies on the detection of the same PTH derivative(s) during Edman degradation of the protein and the peptides derived from it (Hofsteenge et al, 1994). However, this procedure employs highly acidic conditions under which the indolic tryptophan side chain can undergo a variety of modifications (Fontana & Tonioli, 1976).…”

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confidence: 99%

“…3 We thank one of the referees of one of our previous papers (Hofsteenge et al, 1994) for suggesting this possibility.…”

mentioning

confidence: 99%

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Spectroscopic and Protein Chemical Analyses Demonstrate the Presence of C-Mannosylated Tryptophan in Intact Human RNase 2 and Its Isoforms

et al. 1996

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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 -R-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 posttranslationally 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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Section: Stability Of the O-mannosylated Syntheticsupporting

confidence: 90%

mentioning

confidence: 88%

Section: Purification Of Rnase 2 and High-molecular Massmentioning

confidence: 99%

mentioning

confidence: 99%

“…3 We thank one of the referees of one of our previous papers (Hofsteenge et al, 1994) for suggesting this possibility.…”

mentioning

confidence: 99%