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Factor-inhibiting hypoxia-inducible factor (FIH) catalyses the post-translational hydroxylation of histidinyl residues within ankyrin repeat domains.

Yang M, Chowdhury R, Ge W, Hamed RB, McDonough MA, Claridge TD, Kessler BM, Cockman ME, Ratcliffe PJ, Schofield CJ - FEBS J. (2011)

Bottom Line: However, there are few reports on the selectivity of FIH for the hydroxylation of specific residues.NMR and crystallographic analyses show that the histidinyl hydroxylation occurs at the β-position.The results further expand the scope of FIH-catalysed hydroxylations.

View Article: PubMed Central - PubMed

Affiliation: Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK.

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MS analyses assigning hydroxylation at His 238 and His 553 in tankyrase-2. Tankyrase-2 was purified from transiently transfected 293T cells coexpressing FIH. (A) MS/MS spectra of the tryptic peptide IVQLLLQHGADVHAK derived from tankyrase-2 (residues 226–240) in the hydroxylated ([M + 3H]3+ = m/z 553.28) (upper) and nonhydroxylated ([M + H]3+ = m/z 548.63) (lower) state. The hydroxylated species (upper) exhibits a + 16 Da mass shift on the y-ion series appearing at y3 and assigning hydroxylation to His238. (B) MS/MS of the tankyrase-2 tryptic peptide containing His 553 (VSVVEYLLQHGADVHAK) in hydroxylated ([M + 3H]3+ = m/z 627.64) (upper) and unmodified forms ([M + 3H]3+ = m/z 622.30) (lower). For both hydroxylated spectra, a −2 Da mass shift was commonly observed on fragment ions containing hydroxyhistidine, which is consistent with hydroxylation (+16 Da) followed by dehydration (−18 Da). Because there was no evidence for a −2 Da shift on the precursor ions it is likely that during the collision-induced dissociation process in the MS/MS analyses, the hydroxylated His residue undergoes dehydration to form the conjugated α,β-dehydrohistidine product. Note also there was no evidence for formation of the dehydrohistidine in the NMR analyses (Fig. 4).
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fig02: MS analyses assigning hydroxylation at His 238 and His 553 in tankyrase-2. Tankyrase-2 was purified from transiently transfected 293T cells coexpressing FIH. (A) MS/MS spectra of the tryptic peptide IVQLLLQHGADVHAK derived from tankyrase-2 (residues 226–240) in the hydroxylated ([M + 3H]3+ = m/z 553.28) (upper) and nonhydroxylated ([M + H]3+ = m/z 548.63) (lower) state. The hydroxylated species (upper) exhibits a + 16 Da mass shift on the y-ion series appearing at y3 and assigning hydroxylation to His238. (B) MS/MS of the tankyrase-2 tryptic peptide containing His 553 (VSVVEYLLQHGADVHAK) in hydroxylated ([M + 3H]3+ = m/z 627.64) (upper) and unmodified forms ([M + 3H]3+ = m/z 622.30) (lower). For both hydroxylated spectra, a −2 Da mass shift was commonly observed on fragment ions containing hydroxyhistidine, which is consistent with hydroxylation (+16 Da) followed by dehydration (−18 Da). Because there was no evidence for a −2 Da shift on the precursor ions it is likely that during the collision-induced dissociation process in the MS/MS analyses, the hydroxylated His residue undergoes dehydration to form the conjugated α,β-dehydrohistidine product. Note also there was no evidence for formation of the dehydrohistidine in the NMR analyses (Fig. 4).

Mentions: Having established that His-containing peptides are FIH substrates in vitro, we then investigated whether tankyrase-2 might be subject to FIH-catalysed His hydroxylation in cells. To address this, we transfected plasmids encoding full-length FLAG-tagged tankyrase-2 and FIH into 293T cells, immunopurified the material by FLAG affinity and subjected it to trypsinolysis and MS/MS analysis. Peptides containing both His residues were observed, and MS/MS sequencing assigned hydroxylation at His 238 and His 553 (Fig. 2). As previously observed for hydroxyasparagine-containing peptides [8], under our HPLC conditions, the hydroxyhistidine modification had minimal effect on the peptide chromatographic properties and the hydroxylated and nonhydroxylated peptides coeluted (data not shown). The exact masses and retention times of the His-containing peptides were subsequently used to assign the hydroxylated and nonhydroxylated peptides studied by LC/MS analyses.


Factor-inhibiting hypoxia-inducible factor (FIH) catalyses the post-translational hydroxylation of histidinyl residues within ankyrin repeat domains.

Yang M, Chowdhury R, Ge W, Hamed RB, McDonough MA, Claridge TD, Kessler BM, Cockman ME, Ratcliffe PJ, Schofield CJ - FEBS J. (2011)

MS analyses assigning hydroxylation at His 238 and His 553 in tankyrase-2. Tankyrase-2 was purified from transiently transfected 293T cells coexpressing FIH. (A) MS/MS spectra of the tryptic peptide IVQLLLQHGADVHAK derived from tankyrase-2 (residues 226–240) in the hydroxylated ([M + 3H]3+ = m/z 553.28) (upper) and nonhydroxylated ([M + H]3+ = m/z 548.63) (lower) state. The hydroxylated species (upper) exhibits a + 16 Da mass shift on the y-ion series appearing at y3 and assigning hydroxylation to His238. (B) MS/MS of the tankyrase-2 tryptic peptide containing His 553 (VSVVEYLLQHGADVHAK) in hydroxylated ([M + 3H]3+ = m/z 627.64) (upper) and unmodified forms ([M + 3H]3+ = m/z 622.30) (lower). For both hydroxylated spectra, a −2 Da mass shift was commonly observed on fragment ions containing hydroxyhistidine, which is consistent with hydroxylation (+16 Da) followed by dehydration (−18 Da). Because there was no evidence for a −2 Da shift on the precursor ions it is likely that during the collision-induced dissociation process in the MS/MS analyses, the hydroxylated His residue undergoes dehydration to form the conjugated α,β-dehydrohistidine product. Note also there was no evidence for formation of the dehydrohistidine in the NMR analyses (Fig. 4).
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Related In: Results  -  Collection

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fig02: MS analyses assigning hydroxylation at His 238 and His 553 in tankyrase-2. Tankyrase-2 was purified from transiently transfected 293T cells coexpressing FIH. (A) MS/MS spectra of the tryptic peptide IVQLLLQHGADVHAK derived from tankyrase-2 (residues 226–240) in the hydroxylated ([M + 3H]3+ = m/z 553.28) (upper) and nonhydroxylated ([M + H]3+ = m/z 548.63) (lower) state. The hydroxylated species (upper) exhibits a + 16 Da mass shift on the y-ion series appearing at y3 and assigning hydroxylation to His238. (B) MS/MS of the tankyrase-2 tryptic peptide containing His 553 (VSVVEYLLQHGADVHAK) in hydroxylated ([M + 3H]3+ = m/z 627.64) (upper) and unmodified forms ([M + 3H]3+ = m/z 622.30) (lower). For both hydroxylated spectra, a −2 Da mass shift was commonly observed on fragment ions containing hydroxyhistidine, which is consistent with hydroxylation (+16 Da) followed by dehydration (−18 Da). Because there was no evidence for a −2 Da shift on the precursor ions it is likely that during the collision-induced dissociation process in the MS/MS analyses, the hydroxylated His residue undergoes dehydration to form the conjugated α,β-dehydrohistidine product. Note also there was no evidence for formation of the dehydrohistidine in the NMR analyses (Fig. 4).
Mentions: Having established that His-containing peptides are FIH substrates in vitro, we then investigated whether tankyrase-2 might be subject to FIH-catalysed His hydroxylation in cells. To address this, we transfected plasmids encoding full-length FLAG-tagged tankyrase-2 and FIH into 293T cells, immunopurified the material by FLAG affinity and subjected it to trypsinolysis and MS/MS analysis. Peptides containing both His residues were observed, and MS/MS sequencing assigned hydroxylation at His 238 and His 553 (Fig. 2). As previously observed for hydroxyasparagine-containing peptides [8], under our HPLC conditions, the hydroxyhistidine modification had minimal effect on the peptide chromatographic properties and the hydroxylated and nonhydroxylated peptides coeluted (data not shown). The exact masses and retention times of the His-containing peptides were subsequently used to assign the hydroxylated and nonhydroxylated peptides studied by LC/MS analyses.

Bottom Line: However, there are few reports on the selectivity of FIH for the hydroxylation of specific residues.NMR and crystallographic analyses show that the histidinyl hydroxylation occurs at the β-position.The results further expand the scope of FIH-catalysed hydroxylations.

View Article: PubMed Central - PubMed

Affiliation: Oxford Centre for Integrative Systems Biology, University of Oxford, Oxford, UK.

Show MeSH
Related in: MedlinePlus