Self- hydroxylation of the splicing factor lysyl hydroxylase , JMJD6

Monica Mantri; Celia J. Webby; Nikita D. Loik; Refaat B. Hamed; Michael L. Nielsen; Michael A. McDonough; James S. O. McCullagh; Angelika Böttger; Christopher J. Schofield; Alexander Wolf

doi:10.1039/C1MD00225B

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DOI: 10.1039/C1MD00225B (Concise Article) Med. Chem. Commun., 2012, 3, 80-85

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Self-hydroxylation of the splicing factor lysyl hydroxylase, JMJD6†‡

Monica Mantri ^a, Celia J. Webby ^a, Nikita D. Loik ^a, Refaat B. Hamed ^ab, Michael L. Nielsen ^c, Michael A. McDonough ^a, James S. O. McCullagh ^a, Angelika Böttger ^d, Christopher J. Schofield *^a and Alexander Wolf *^a
^aChemistry Research Laboratory, 12 Mansfield Rd., Oxford, OX1 3TA, United Kingdom. E-mail: christopher.schofield@chem.ox.ac.uk; alexander.wolf@chem.ox.ac.uk; Fax: +44 (0)1865 285022
^bDepartment of Pharmacognosy, Faculty of Pharmacy, Assiut University, 71526, Egypt
^cDepartment of Proteomics, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Faculty of Health Sciences, DK-2200, Copenhagen, Denmark
^dDepartment of Biology II, Ludwig-Maximilians-University, Munich, Großhaderner Str 2, D-82152, Planegg-Martinsried, Germany

Received 1st September 2011 , Accepted 19th October 2011

First published on 18th November 2011

Abstract

The lysyl5S-hydroxylase, JMJD6 acts on proteins involved in RNA splicing. We find that in the absence of substrate JMJD6 catalyses turnover of 2OG to succinate. ¹H-NMR analyses demonstrate that consumption of 2OG is coupled to succinate formation. MS analyses reveal that JMJD6 undergoes self-hydroxylation in the presence of Fe(II) and 2OG resulting in production of 5S-hydroxylysine residues. JMJD6 in human cells is also found to be hydroxylated. Self-hydroxylation of JMJD6 may play a regulatory role in modulating the hydroxylation status of proteins involved in RNA splicing.

JMJD6 has been reported as a lysyl5S-hydroxylase active on proteins involved in RNA splicing.^1–3JMJD6 has also been reported to be a histone methyl arginine demethylase,⁴ though we have not yet observed this activity under our assay conditions.^1,2JMJD6 belongs to the 2-oxoglutarate (2OG) and Fe(II) dependent family of oxygenases which catalyses a diverse range of oxidative reactions.^5,6 Various human 2OG oxygenases are being explored as therapeutic targets via small molecule inhibition, including the hypoxia inducible factor (HIF) hydroxylases and the JmjC domain containing N^ε-methyl lysine histone demethylases.⁷ The JmjC enzymes are of particular interest because of their important role in epigenetics. The 2OG oxygenases utilise 2OG as a co-substrate and share a conserved Fe(II) binding motif. In addition to coupling 2OGoxidation to that of substrate, many 2OG oxygenases catalyse the uncoupled turnover of 2OG, i.e. where 2OG is converted to succinate in the absence of a primary substrate, albeit typically at a substantially lower rate than substrate coupled turnover.^8–12 Several members of the 2OG oxygenase family have also been shown to catalyse self-oxidation in the absence of substrate. Most of the reported self-hydroxylation reactions to date involve hydroxylation of an active site aromatic residue located within ∼10 Å of the active site iron.^9–11

The closest human homologue of JMJD6 is Factor Inhibiting HIF (FIH),¹³ which reduces the transcriptional activity of HIF by post translational asparaginylhydroxylation in the C-terminal transcriptional activation domain of HIF-α.⁵FIH has been reported to undergo self-hydroxylation of an active site tryptophan residue⁹ in the absence of HIF. Other members of the 2OG oxygenase family, taurine dioxygenase (TauD) and 2,4-dichlorophenoxyacetate oxygenase (TfdA) are apparently deactivated by hydroxylation of an active site tyrosine (TauD)¹⁰ and tryptophan (TfdA)¹¹ residues in the absence of their primary substrates, taurine or 2,4-dichlorophenoxyacetate, respectively. In the case of the human AlkBhomologhABH3, there is evidence that a leucine residue (Leu177_hABH3) undergoes oxidation to give an alcohol which can be further oxidised to an aldehyde at the leucine δ-position.¹⁴ Oxidative cleavage has also been reported in case of 1-aminocyclopropane-1-carboxylate (ACC) oxidase, the ethylene forming enzyme in plants which is closely related to 2OG oxygenases.¹⁵

It has been speculated that the self-regulation of some 2OG oxygenases may protect cells from damaging oxygen derived species or may serve as a negative feedback mechanism.⁹ Self-regulation is employed by many enzymes in nature, e.g. kinases autophosphorylate residues on their own protein scaffold to regulate their activity¹⁶ and ubiquitinases are capable of targeting themselves for ubiquitin-dependent degradation.^17,18

Here we demonstrate self-hydroxylation of the JMJD6protein. Unlike some other members of the 2OG oxygenase family that undergo self-hydroxylation involving oxidation of aromatic/active site residues, recombinant JMJD6hydroxylates lysine residues (Lys111_JMJD6 and Lys167_JMJD6) in the same manner as its splicing protein substrates.¹ We further report that endogenous JMJD6 extracted from human cells undergoes self-hydroxylation.

Results

To investigate the uncoupled turnover of 2OG by JMJD6 we began by carrying out assays measuring the conversion of [1-¹⁴C]-2OG into ¹⁴CO₂¹⁹ in the presence and the absence of the human peptide substrate LUC7L2₂₆₇_–278, which is a fragment of splicing regulatory protein LUC7like2.¹ Under standard assay conditions we found that the amount of 2OG consumed was 1.7 fold higher in the presence of the LUC7L2₂₆₇_–278peptide than in its absence (Fig. 1A and Fig. 3 in ref. 29), suggesting that uncoupled turnover by JMJD6 might be relatively high even in the presence of a hydroxylated substrate (albeit only a fraction of a protein). ¹H-NMR analyses verified the uncoupled turnover of 2OG to succinate in the absence of splicing proteinpeptide substrate (Fig. 1B) in an Fe(II) and 2OG dependent manner. We then carried out investigations using a MALDI-MS based assay that monitors substrate hydroxylation by a +16Da mass shift related to the unhydroxylated substrate peptide. The results suggest that when JMJD6 was pre-incubated with 2OG, Fe(II) for 30 min prior to the addition of LUC7L2₂₆₇_–278, a 40–50% decrease in substrate hydroxylation was observed in comparison to when all the components were mixed simultaneously (results not shown). In some but not all cases, coupled turnover of 2OG is proportional to the oxidation of ascorbatee.g. the collagenprolyl-4-hydroxylases^20–22 and references therein. However, in our work the JMJD6 incubations were carried out in the absence of ascorbate. Although the relatively high level of uncoupled turnover in the presence of substrate may reflect use of a non optimal substrate, we considered that JMJD6 might accomplish (some) catalyticoxidationvia a self-hydroxylation process.


	Fig. 1 Radioactive assays, ¹H-NMR and amino acid analysis showing 2OG turnover catalysed by JMJD6. (A) Results of [1-¹⁴C]-turnover assay under standard assay conditions with Fe(II), 2OG and LUC7L2peptide, Values are Mean ± S.D., n = 3; (B) JMJD6 catalysed 2OG turnover in the absence of substrate as observed by ¹H-NMR; (C) and (D) Amino acid analyses of JMJD6 catalysis. After incubation, the peptide product was hydrolysed using trypsin and carboxypeptidase Y. (Ci) Hydroxylsyine product obtained after hydrolysis of LUC7L2₂₆₇_–278peptide that had been incubated with JMJD6, Fe(II) and 2OG; (Cii) Hydroxylsyine product obtained after hydrolysis of JMJD6 that had been incubated with Fe(II) and 2OG; (Ciii) 2S,5R-Hydroxylysine standard; (Civ) Mixture of 2R,5R/2S,5S- and 2R,5S/2S,5R-hydroxylysine standards; (Di) Hydroxylysine product from JMJD6 as purified after hydrolysis, i.e. without Fe(II) and 2OG incubation; (Dii) Hydroxylysine product obtained after incubation of JMJD6 incubated with Fe(II); (Diii) Hydrolysis product obtained after incubation of JMJD6 with 2OG; (Div) Hydroxylysine product obtained after incubation of JMJD6 with Fe(II) and 2OG.

To test for self-hydroxylation of JMJD6, we first carried out amino acid analyses on recombinant JMJD6 that had been incubated with or without Fe(II) and/or 2OG. The JMJD6hydrolysis procedure employed was the one which we had previously reported for splicing regulatory peptideLUC7L2₂₆₇_–278 product analysis, i.e. trypsin and carboxypeptidase Y were used rather than acid hydrolysis in order to avoid isomerisation of the hydroxylysine product.² The results reveal that JMJD6 undergoes self-hydroxylation on one or more lysine residues in a manner that is stimulated by 2OG. Notably, we found that as purified, recombinant JMJD6 had already undergone a degree of self-hydroxylation presumably in Escherichia colicells (Fig. 1D). The extent of hydroxylation was not substantially increased by incubation with Fe(II) (Fig. 1Dii), likely reflecting co-purification of JMJD6 with Fe(II). However, the extent of hydroxylation was increased substantially by addition of 2OG (Fig. 1Diii). Importantly, the stereochemistry of the hydroxylysine product was found to be 2S,5S, i.e. the same as that observed for JMJD6 catalysed LUC7L2hydroxylation (Fig. 1C). The lack of evidence for 5R-lysylhydroxylation as shown in Scheme 1 supports the proposal that the self-hydroxylation of lysines in JMJD6 occurs at the active site in a manner similar to splicing protein hydroxylation.


	Scheme 1 JMJD6 catalysed self-hydroxylation gives the 2S,5S-hydroxylysine product as observed for hydroxylation of splicing regulatory protein substrates.

We then investigated the site(s) of lysinehydroxylation in recombinant JMJD6 that had been incubated with Fe(II) and 2OG using LysCdigestion followed by LC–MS/MS analyses.²³ These analyses support hydroxylation of Lys111_JMJD6 and Lys167_JMJD6 (Fig. 2). No hydroxylation at Lys167_JMJD6 was observed in the absence of 2OG and Fe(II) (Fig. S1‡). Hydroxylation at Lys167_JMJD6 was also observed to occur in the presence of recombinant U2AF65 substrate, when incubated with Fe(II) and 2OG (Fig. S2‡). We cannot rule out the possibility of hydroxylations at other sites because the proteomic analyses were limited (at least for confident assignment) to fragments containing residues Lys91, Lys100, Lys111, Lys113, Lys115, Lys142, Lys151, Lys154, Lys167, Lys219, Lys317 and Lys397. Indeed the observation that hydroxylysine is present in recombinant JMJD6 as determined by amino acid analysis reveals that hydroxylation at other sites is likely (Fig. 1D).


	Fig. 2 MS/MS spectra showing hydroxylation of recombinant JMJD6 in the presence of 2OG and Fe(II). LC-MS/MS analyses supports hydroxylation at Lys111_JMJD6 (a′) and Lys167_JMJD6 (b′) of recombinant JMJD6 in the presence of Fe(II) and 2OG. Hydroxylation was not observed to be complete and the spectra corresponding to unhydroxylated Lys111_JMJD6 (a) and Lys167_JMJD6 (b) are also shown. The MH²⁺peptide that was fragmented to give the resulting MSspectra is shown on the right hand side of each spectrum. The fragmented peptide sequence of resulting MS is shown on right hand side and MH²⁺ precursor ion is shown on left hand side of each spectra.

We then made a set of peptides as shown in Table 1 containing the identified hydroxylation sites at Lys111_JMJD6 (with recombinant protein) and Lys167_JMJD6 (with recombinant and endogenous protein). Out of the 10 peptides analysed, only two were substrates as observed by MALDI-MS analysis, corresponding to JMJD6₁_–14 and JMJD6₃₀₁_–314. Notably the two peptides, JMJD6₁₀₅_–120, JMJD6₁₆₇_–176 containing the sites of hydroxylation identified in JMJD6protein (Lys111_JMJD6 and Lys167_JMJD6) were not observed to be substrates (Fig. S4‡). Although we cannot be certain, it is possible that the hydroxylation of lysines corresponding to JMJD6₁_–14 and JMJD6₃₀₁_–314 observed in peptide work, may reflect the hydroxylation of purified recombinant JMJD6 as obtained by amino acid analysis.

Table 1 Results of MALDI-TOF analyses of peptides synthesised containing possible lysinehydroxylation sites within JMJD6

No.	JMJD6 Sequence	Peptide sequence	Molecular weight	+16 Da Shift in MS analysis
1	JMJD6 1–14	MNHKSKKRIREAKR	1782.15	YES
2	JMJD6 14–30	RSARPELKDSLDWTRHN	2081.27	NO
3	JMJD6 78–95	GWSAQEKWTLERLKRKYR	2335.69	NO
4	JMJD6 86–104	TLERLKRKYRNQKFKCGED	2412.80	NO
5	JMJD6 105–120	NDGYSVKMKMKYYIEY	2032.36	NO	Hydroxylation at K111 observed in protein.
6	JMJD6 135–150	SSYGEHPKRRKLLEDY	1978.19	NO
7	JMJD6 160–176	LFQYAGEKRRPPYRWFV	2213.57	NO	Hydroxylation at K167 observed in protein.
8	JMJD6 301–314	TVRGRPKLSRKWYR	1803.14	YES
9	JMJD6 363–379	SGSEGDGTVHRRKKRRT	1921.15	NO
10	JMJD6 363–381	SGSEGDGTVHRRKKRRTCS	2116.07	NO

Finally, we investigated whether self-hydroxylation of endogenous JMJD6 in human cells occurs. JMJD6 was immunoprecipitated from HeLa cells using an anti-JMJD6 antibody and was isolated by SDS-PAGE. The band corresponding to the expected molecular weight of JMJD6 was excised from an acrylamide gel, digested with LysCprotease and analysed by LC-MS/MS. The fragment ion series was consistent with hydroxylation of Lys167_JMJD6 (Fig. 3). As mentioned before we cannot rule out the possibility of hydroxylation at other sites.


	Fig. 3 MS/MS analysis of endogenous JMJD6 supports hydroxylation of Lys167_JMJD6. JMJD6 was immunoprecipitated from HeLa cells, digested with LysCprotease and analysed by LC-MS/MS. The MH²⁺peptide that was fragmented to give the resulting MSspectra is shown on the right hand side of the spectrum (in green). The co-elution of two different peptides is shown by use of blue and red colours.

Discussion

Overall our results demonstrate that JMJD6 can undergo self-hydroxylation in recombinant form when produced in bacteria, and that self-hydroxylation is stimulated by 2OG addition. Evidence for hydroxylation at one site Lys167_JMJD6 was also seen for JMJD6 isolated from human cells.

The amino acid analysis studies imply that the self-hydroxylation proceeds to give the product with the 5S-stereochemistry, i.e. the same as that observed for JMJD6 catalysed hydroxylation of arginine serine rich domain proteins (e.g.U2AF65).^1,2 Thus, the JMJD6protein product differs from the 5R-hydroxylation stereochemistry observed for collagenlysylhydroxylation which is also catalysed by a 2OG dependent oxygenase.^2,24,25 It should be noted that the combined amino acid analysis and proteomic studies imply that there are self-hydroxylation sites additional to Lys111_JMJD6 and Lys167_JMJD6. We thus suggest that wherever possible assignments of post-translational modification based on proteomic MS-based methods should be supplemented by amino acid or equivalent analyses. The amino acid analysis results also imply that the JMJD6 catalysed self-hydroxylation is active site mediated and is not a result of reactive oxidising species ‘leaking’ away from the iron centre. In this regard the JMJD6 catalysed self-hydroxylation is probably distinguished from that observed for other 2OG oxygenases to date, where the oxidised residues differ from the normal substrates. For example, in the case of FIH, a tryptophan residue is oxidised by self-oxidation,⁹ but a tryptophan residue has not been reported to be a substrate for FIH,²⁶ (though this cannot be ruled out because FIH does catalyse hydroxylation of residues other than aspargine^26,27). Thus it would seem that the JMJD6 catalysed self-hydroxylation is more likely to be biologically relevant than the self-oxidations so far observed for other 2OG oxygenases, in particular because the JMJD6 self-hydroxylation is observed in human cells.

Interestingly, peptides containing the hydroxylation sites Lys111_JMJD6 and Lys167_JMJD6 identified in JMJD6 recombinant protein were not observed to be substrates under our current assay conditions. Instead, two other peptides (corresponding to JMJD6₁_–14 and JMJD6₃₀₁_–314) that were not observed to be hydroxylated in the JMJD6protein were observed to be substrates. Together these results reveal that factors other than the immediate sequence around the lysylhydroxylation site are important in determining selectivity as has been found for FIH,²⁶ where relevant factors include secondary structure and overall protein stability.²⁸ It should also be noted that for the self- hydroxylation of JMJD6, it is unclear if the reaction is intra- or inter-molecular (for either of the Lys111_JMJD6, Lys167_JMJD6 or other hydroxylation sites), and the available JMJD6 crystal structures^29,30 do not rule out either possibility (Fig. 4).


	Fig. 4 Positions of self-hydroxylation sites within various 2OG oxygenases and ACCO. Ribbons representation of 2OG oxygenases and ACCO showing the distances in Angstorms (black dashes) between the iron centre (white sticks for protein derived ligands, green for 2OGcofactors and orange for iron or surrogate metal) and C-alpha atom of the self-hydroxylated residues (yellow sticks). a. JMJD6 (PDB ID3K2O), b. FIH (PDB ID1H2K), c. TauD (PDB ID1GQW), d. ACCO (PDB ID1W9Y), e. ABH3 (PDB ID2IUW), f. AlkB (PDB ID2FD8). In the case of ACCOhydroxylation was not observed directly but is proposed to lead to backbone fragmentation. Note that the distances to the two residues as being hydroxylated in JMJD6 are substantially longer than in the other structures and other JMJD6 residues (than Lys111_JMJD6 or Lys167_JMJD6) are likely hydroxylated (see text).

For other 2OG oxygenases^9–11,14 and the related enzymeACC oxidase,¹⁵ the identified oxidative modifications have all been observed to occur within ∼10 Å of the active site iron (Fig. 4), implying that they are formed directly by reaction of active site iron bound oxidising species or leakage of reactive oxidising species (e.g.H₂O₂, O₂˙⁻) away from the active site. However, in the case of JMJD6 the identified sites of hydroxylation at Lys111_JMJD6, Lys167_JMJD6 are both ∼22 Å away from the active site iron as shown in Fig. 4. Lys111_JMJD6 and Lys167_JMJD6 are conserved in JMJD6 across higher eukaryotes (Fig. S6‡), with deviations in residues observed in the corresponding Lys167_JMJD6 position in lower organisms. Both Lys111_JMJD6 and Lys167_JMJD6 are located in loop regions that on the basis of the crystallographic analysis are likely to be relatively flexible which may explain how these residues are able to fit into the active site to be hydroxylated.

We also observed that self-hydroxylation and uncoupled turnover occur in the presence of a JMJD6 substrate, U2AF65. With the available techniques, it is difficult to study the effects of, at least, several self-hydroxylation events on substrate hydroxylation; however, self-hydroxylation may reduce the extent of substrate hydroxylation by simple competition. It is now of interest to see if the extent of self-hydroxylations varies with different JMJD6 substrates and if self-hydroxylation has any biological role. As suggested for other 2OG oxygenases, it is possible that self-hydroxylation of JMJD6 is involved in (likely negative) feedback mechanisms, though at this stage there is no evidence for the in vivo relevance of self hydroxylation.⁹JMJD6 has been shown to play a role in the regulation of splicing of genes including vascular endothelial growth factor (VEGF) receptor 1,³ but the exact role of JMJD6 catalysed hydroxylation is unclear. At present the available evidence suggests that, as proposed for hydroxylation catalysed by FIH,^{26–28,31,32}JMJD6 may have multiple hydroxylation substrates and that the sum of many hydroxylation events may be regulatory in a manner that is context dependent. It is of interest that hydroxylation mediated modulation of FIH and ankyrin repeat domain substrate interactions is proposed to regulate the role of FIH in hydroxylation of C-terminal transcriptional activity domain of HIF.^31–33 The occurrence of self-hydroxylation by JMJD6 introduces another layer of complexity, potentially applying to other 2OG oxygenases.

Thus, it is now of interest to study self-hydroxylation by other human 2OG oxygenases including the collagenlysyl hydroxylase, and the JmjC containing histoneN^ε-methyl lysine demethylases. N^ε-Methyl lysine formation is a common post-translational modification as observed by proteomic studies,³⁴ thus self-demethylation is also a possibility for some 2OG oxygenases. If indeed self-oxidation plays a regulatory role, its modulation should be taken into consideration when considering the phenotypic effects of small molecule inhibitors targeting 2OG oxygenases subject to self-oxidation.

Acknowledgements

We thank Holger Kramer, Benedikt Kessler and Matthias Mann for helpful discussions on interpretation of mass spectrometric results. (Michael Nielsen and Matthias Mann performed the mass spectrometry reported in this work.) We thank Timothy D. W. Claridge for discussion on NMR work. This work was funded by DFG grant BO1748–3 awarded to A. B. and the Biotechnology and Biological Sciences Research Council, the European Union and the Wellcome Trust. C. W. was funded by a Glasstone Fellowship.

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Footnotes

† This article is part of a MedChemComm web themed issue on epigenetics.

‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/c1md00225b