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• W2008541833 abstract "Three hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the HIFs by hydroxylating prolines at two separate sites in the oxygen-dependent degradation domain (ODDD) of their α subunits. We compared in vitro hydroxylation by purified recombinant human HIF-P4Hs of 19–20- and 35-residue peptides corresponding to the two sites in HIF-αs and purified recombinant HIF-1α and HIF-2α ODDDs of 248 and 215 residues. The increase in the length of peptides representing the C-terminal site from 19 to 20 to 35 residues reduced the Km values to 90–800 nm, i.e. to 0.7–11% of those for the shorter peptides, whereas those representing the N-terminal site were 10–470 μm, i.e. 10–135%. The Km values of HIF-P4H-1 for the recombinant HIF-α ODDDs were 10–20 nm, whereas those of HIF-P4H-2 and -3 were 60–140 nm, identical values being found for the wild-type HIF-1α ODDD and its N site mutant. The Km values for the C site mutant were about 5–10 times higher but only 0.2–3% of those for the 35-residue N site peptides, and this marked difference suggested that the HIF-P4Hs may become bound first to the C-terminal site of an ODDD and that this binding may enhance subsequent binding to the N-terminal site. The Km values of HIF-P4H-2 for oxygen determined with the HIF-1α ODDD and both its mutants as substrates were all about 100 μm, being 40% of those reported for the three HIF-P4Hs with a 19-residue peptide. Even this value is high compared with tissue O2 levels, indicating that HIF-P4Hs are effective oxygen sensors. Three hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the HIFs by hydroxylating prolines at two separate sites in the oxygen-dependent degradation domain (ODDD) of their α subunits. We compared in vitro hydroxylation by purified recombinant human HIF-P4Hs of 19–20- and 35-residue peptides corresponding to the two sites in HIF-αs and purified recombinant HIF-1α and HIF-2α ODDDs of 248 and 215 residues. The increase in the length of peptides representing the C-terminal site from 19 to 20 to 35 residues reduced the Km values to 90–800 nm, i.e. to 0.7–11% of those for the shorter peptides, whereas those representing the N-terminal site were 10–470 μm, i.e. 10–135%. The Km values of HIF-P4H-1 for the recombinant HIF-α ODDDs were 10–20 nm, whereas those of HIF-P4H-2 and -3 were 60–140 nm, identical values being found for the wild-type HIF-1α ODDD and its N site mutant. The Km values for the C site mutant were about 5–10 times higher but only 0.2–3% of those for the 35-residue N site peptides, and this marked difference suggested that the HIF-P4Hs may become bound first to the C-terminal site of an ODDD and that this binding may enhance subsequent binding to the N-terminal site. The Km values of HIF-P4H-2 for oxygen determined with the HIF-1α ODDD and both its mutants as substrates were all about 100 μm, being 40% of those reported for the three HIF-P4Hs with a 19-residue peptide. Even this value is high compared with tissue O2 levels, indicating that HIF-P4Hs are effective oxygen sensors. The hypoxia-inducible factors (HIFs) 2The abbreviations used are: HIF, hypoxia-inducible transcription factor; ODDD, oxygen-dependent degradation domain; VHL, von Hippel-Lindau; HIF-P4H, HIF prolyl 4-hydroxylase; C-P4H, collagen prolyl 4-hydroxylase; FIH, HIF asparaginyl hydroxylase. 2The abbreviations used are: HIF, hypoxia-inducible transcription factor; ODDD, oxygen-dependent degradation domain; VHL, von Hippel-Lindau; HIF-P4H, HIF prolyl 4-hydroxylase; C-P4H, collagen prolyl 4-hydroxylase; FIH, HIF asparaginyl hydroxylase. are master regulators of the transcription of more than 100 hypoxia-regulated genes and play central roles in cellular oxygen homeostasis. HIFs are heterodimers that consist of an oxygen-regulated α subunit (HIF-α) and a stable β subunit (HIF-β), and both types of subunits are members of the basic helix-loop-helix Per-Arnt-Sim protein family. The human α subunit has three isoforms, HIF-1α to HIF-3α (for reviews, see Refs. 1Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5204) Google Scholar, 2Schofield C.J. Ratcliffe P.J. Nat. Rev. Mol. Cell. Biol. 2004; 5: 343-354Crossref PubMed Scopus (1581) Google Scholar, 3Kaelin Jr., W.G. Annu. Rev. Biochem. 2005; 74: 115-128Crossref PubMed Scopus (358) Google Scholar). HIF-1α and HIF-2α are synthesized continuously, and hydroxylation of at least one of two critical proline residues in their oxygen-dependent degradation domain (ODDD), Pro402 and Pro564 in HIF-1α, generates a binding site for the von Hippel-Lindau (VHL) ubiquitin-protein isopeptide ligase complex that targets them for rapid proteasomal degradation under normoxic conditions (4Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lanie W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3828) Google Scholar, 5Jaakkola P. Mole D.R. Tian Y.-M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A.V. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4369) Google Scholar, 6Yu F. White S. Zhao Q. Lee F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9630-9635Crossref PubMed Scopus (635) Google Scholar, 7Masson N. William C. Maxwell P.H. Pugh C.W. Ratcliffe P.J. EMBO J. 2001; 20: 5197-5206Crossref PubMed Scopus (846) Google Scholar). This hydroxylation is catalyzed in humans by three recently identified cytoplasmic and nuclear HIF prolyl 4-hydroxylases (HIF-P4Hs 1–3, also named PHD1–3, HPH3-1, and EGLN2, -1, and -3, respectively) (8Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2088) Google Scholar, 9Epstein A.C.R. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.-M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2697) Google Scholar, 10Ivan M. Haberberger T. Gervasi D.C. Michelson K.S. Günzler V. Kondo K. Yang H. Sorokina I. Conaway R.C. Conaway J.W. Kaelin Jr., W.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13459-13464Crossref PubMed Scopus (486) Google Scholar). These are distinct from the well characterized collagen prolyl 4-hydroxylases (C-P4Hs) that reside within the lumen of the endoplasmic reticulum and likewise have three human isoenzymes (11Myllyharju J. Matrix Biol. 2003; 22: 15-24Crossref PubMed Scopus (321) Google Scholar, 12Kukkola L. Hieta R. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 47685-47693Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 13Van Den Diepstraten C. Papay K. Bolder Z. Brown A. Pickering G. Circulation. 2003; 108: 508-511Crossref PubMed Scopus (41) Google Scholar). All P4Hs are 2-oxoglutarate dioxygenases and require Fe2+, 2-oxoglutarate, O2, and ascorbate (2Schofield C.J. Ratcliffe P.J. Nat. Rev. Mol. Cell. Biol. 2004; 5: 343-354Crossref PubMed Scopus (1581) Google Scholar, 3Kaelin Jr., W.G. Annu. Rev. Biochem. 2005; 74: 115-128Crossref PubMed Scopus (358) Google Scholar, 11Myllyharju J. Matrix Biol. 2003; 22: 15-24Crossref PubMed Scopus (321) Google Scholar, 14Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 325-398PubMed Google Scholar). The lack of oxygen inhibits these hydroxylations so that HIF-αs are no longer recognized by the VHL protein and degraded but instead dimerize with HIF-β. These dimers then become bound to the HIF-responsive elements in various hypoxia-regulated genes (1Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5204) Google Scholar, 2Schofield C.J. Ratcliffe P.J. Nat. Rev. Mol. Cell. Biol. 2004; 5: 343-354Crossref PubMed Scopus (1581) Google Scholar, 3Kaelin Jr., W.G. Annu. Rev. Biochem. 2005; 74: 115-128Crossref PubMed Scopus (358) Google Scholar).Hydroxylation of a specific asparagine in the C-terminal transactivation domain of an HIF-α prevents its interaction with the transcriptional coactivator p300, thus inhibiting its full transcriptional activity (15Lando D. Peet D.J. Whelan D.A. Gorman J.J. Whitelaw M.L. Science. 2002; 295: 858-861Crossref PubMed Scopus (1259) Google Scholar). The asparaginyl hydroxylase responsible for this modification is identical to a previously identified factor inhibiting HIF (FIH) (16Hewitson K.S. McNeill L.A. Riordan M.V. Tian Y.-M. Bullock A.N. Welford R.W. Elkins J.M. Oldham N.J. Bhattacharya S. Gleadle J.M. Ratcliffe P.J. Pugh C.W. Schofield C.J. J. Biol. Chem. 2002; 277: 26351-26355Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 17Lando D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. Bruick R.K. Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1201) Google Scholar). It is also one of the 2-oxoglutarate dioxygenases (16Hewitson K.S. McNeill L.A. Riordan M.V. Tian Y.-M. Bullock A.N. Welford R.W. Elkins J.M. Oldham N.J. Bhattacharya S. Gleadle J.M. Ratcliffe P.J. Pugh C.W. Schofield C.J. J. Biol. Chem. 2002; 277: 26351-26355Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 17Lando D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. Bruick R.K. Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1201) Google Scholar), but its catalytic properties are distinct from those of the HIF-P4Hs (18Koivunen P. Hirsilä M. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2004; 279: 9899-9904Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar).The proline residues to be hydroxylated in HIF-αs are located in -Leu-X-X-Leu-Ala-Pro-sequences. It was therefore initially suggested that the HIF-P4Hs may require this conserved core motif (1Semenza G.L. Nat. Rev. Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5204) Google Scholar, 2Schofield C.J. Ratcliffe P.J. Nat. Rev. Mol. Cell. Biol. 2004; 5: 343-354Crossref PubMed Scopus (1581) Google Scholar, 3Kaelin Jr., W.G. Annu. Rev. Biochem. 2005; 74: 115-128Crossref PubMed Scopus (358) Google Scholar). Early mutagenesis experiments supported this suggestion (8Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2088) Google Scholar, 10Ivan M. Haberberger T. Gervasi D.C. Michelson K.S. Günzler V. Kondo K. Yang H. Sorokina I. Conaway R.C. Conaway J.W. Kaelin Jr., W.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13459-13464Crossref PubMed Scopus (486) Google Scholar), but subsequent studies indicated that the two leucines can be replaced by many residues (19Huang J. Zhao Q. Mooney S.M. Lee F.S. J. Biol. Chem. 2002; 277: 39792-39800Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 20Li D. Hirsilä M. Koivunen P. Brenner M.C. Xu L. Yang C. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2004; 279: 55051-55059Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), alanine being the only relatively but not absolutely strict requirement in addition to the proline itself (20Li D. Hirsilä M. Koivunen P. Brenner M.C. Xu L. Yang C. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2004; 279: 55051-55059Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The HIF-P4Hs require long substrates, the shortest HIF-α-like peptide hydroxylated by all three recombinant human isoenzymes having 11 residues (21Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). All three isoenzymes hydroxylated 19-residue peptides with sequences corresponding to those around the C-terminal hydroxylation site in HIF-αs, with Km values of about 5–15 μm for HIF-1α and HIF-3α-like peptides and 10–30 μm for an HIF-2α-like peptide (21Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). A subsequent study indicated that a leucine located 10 residues downstream from the proline influences its hydroxylation by HIF-P4Hs (22Kageyama Y. Koshiji M. To K.K.W. Tian Y.-M. Ratcliffe P.J. Huang L.E. FASEB J. 2004; 18: 1028-1030Crossref PubMed Scopus (62) Google Scholar), which agrees with the previous finding that deletion of two residues, a glutamine and a leucine, from the C terminus of a 19-residue peptide corresponding to the C-terminal hydroxylation site in HIF-1α increased its Km values for HIF-P4H-1 and -2 but not for HIF-P4H-3 (21Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). As the 19-residue peptide used to study hydroxylation of the C-terminal site in HIF-2α ended just before this leucine (21Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar), it is unknown whether its slightly higher Km value relative to those of the HIF-1α and HIF-3α peptides (above) is simply because of the lack of this residue. A 19-residue peptide corresponding to the N-terminal hydroxylation site in HIF-1α had much higher Km values for HIF-P4H-1 and -2 than the C-terminal site peptide and was not hydroxylated by HIF-P4H-3 to any significant extent at all, whereas a 19-residue peptide corresponding to the N-terminal site in HIF-2α was hydroxylated by all three isoenzymes, but again with distinctly higher Km values than the corresponding C-terminal site peptide (21Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar).As the Km values determined for the 19-residue HIF-α-like peptides are relatively high (21Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar), and as residues such as a leucine located 10 residues downstream of the proline to be hydroxylated (22Kageyama Y. Koshiji M. To K.K.W. Tian Y.-M. Ratcliffe P.J. Huang L.E. FASEB J. 2004; 18: 1028-1030Crossref PubMed Scopus (62) Google Scholar) and also some of the acidic residues present in various parts of the 19-residue peptides (19Huang J. Zhao Q. Mooney S.M. Lee F.S. J. Biol. Chem. 2002; 277: 39792-39800Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 20Li D. Hirsilä M. Koivunen P. Brenner M.C. Xu L. Yang C. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2004; 279: 55051-55059Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) have been shown to have distinct effects, we studied here whether the Km values for 35-residue HIF-α-like peptides and recombinant HIF-1α and HIF-2α fragments of 248 and 215 residues, respectively, differ from those determined for shorter peptides. The HIF-α fragments correspond to the ODDDs and contain both proline hydroxylation sites in the same polypeptide, which made it possible to study whether this situation differs from the presence of the two sites in separate peptides. Hydroxylation of only one of the two sites in the HIF-1α ODDD could be studied using its two mutants, P402A and P564A. A recent study has indicated that Pro564 in HIF-1α is hydroxylated before Pro402 in cultured cells and that hydroxylation of Pro402 is inhibited at higher oxygen tensions than that of Pro564 (23Chan D.A. Sutphin P.D. Yen S.-E. Giaccia A.J. Mol. Cell. Biol. 2005; 25: 6415-6426Crossref PubMed Scopus (188) Google Scholar). We therefore also determined the Km values of HIF-P4H-2 for O2 in the hydroxylation of the wild-type and the two mutant HIF-1α ODDDs. Our data indicate that increasing the peptide length from 19 to 20 to 35 residues has a marked effect on the Km values for peptides representing the C-terminal hydroxylation site in all three HIF-α isoforms, whereas more modest or no effects are seen in the case of peptides representing the N-terminal site. The Km values determined for the C-terminal hydroxylation site in the recombinant HIF-1α ODDD are in most cases still somewhat lower than those for the corresponding 35-residue peptides, whereas the Km values for its N-terminal site are markedly lower than those for the 35-residue N site peptides, and this site had a low Km value even for HIF-P4H-3, which had very high Km values for the corresponding 20- and 35-residue peptides. The Km value of HIF-P4H-2 for O2 determined with the HIF-1α ODDD was lower than that reported with a 19-residue peptide, but was still high, about 100 μm, and no difference was found in this Km value when determined with the wild-type or the two mutant ODDDs.EXPERIMENTAL PROCEDURESExpression and Purification of Recombinant HIF-P4Hs—FLAG His-tagged HIF-P4H-1–3 (24Hirsilä M. Koivunen P. Xu L. Seeley T. Kivirikko K.I. Myllyharju J. FASEB J. 2005; 19: 1308-1310Crossref PubMed Google Scholar) were expressed in H5 insect cells cultured in suspension or on plates in Sf900IISFM serum-free medium (Invitrogen). The cells, seeded at a density of 1 × 106/ml, were infected with recombinant baculoviruses at a multiplicity of 5, harvested 72 h after infection, and then were washed with a solution of 0.15 m NaCl and 0.02 m phosphate, pH 7.4, and homogenized in a buffer containing 0.15 m NaCl, 0.1 m glycine, 10 μm dithiothreitol, 0.1% Triton X-100, 1 or 5 μm FeSO4, and 0.01 m Tris, pH 7.8, supplemented with Complete EDTA-free protease inhibitor mixture (Roche Applied Science). The recombinant HIF-P4Hs were purified from the soluble fractions with an anti-FLAG M2 affinity gel (Sigma) (24Hirsilä M. Koivunen P. Xu L. Seeley T. Kivirikko K.I. Myllyharju J. FASEB J. 2005; 19: 1308-1310Crossref PubMed Google Scholar).Expression and Purification of Recombinant ODDDs—The primer pairs 5′-GCGCATATGCAAACAGAATGTGTCCTTAAACCGG-3′ and 5′-GCGCTCGAGCTGGAATACTGTAACTGTGCTTTGAGG-3′, and 5′-GCGCATATGCAGACTGAATCCCTGTTCAAGCCCC-3′ and 5′-GCGCTCGAGCTGGAAGATGTTTGTCATGGCACTGAAGC-3′ (NdeI and XhoI sites underlined) were used to amplify cDNA fragments encoding the ODDDs of HIF-1α and HIF-2α, residues Gln356–Gln603 and Gln358–Gln572, respectively, by PCR using the plasmids d38e6 and d28e6 containing full-length HIF-1α and HIF-2α cDNAs (a gift from FibroGen Inc.) as templates. The NdeI-XhoI-digested PCR fragments were cloned into a similarly cut pET-22b(+) Escherichia coli expression vector (Novagen) in-frame with a C-terminal histidine tag. Site-directed mutagenesis of the HIF-1α ODDD to P402A and P564A mutant ODDDs and to P402A/P564A double mutant ODDD was performed using the QuikChange™ kit (Stratagene). The expression plasmids were transformed into the E. coli BL21(DE3) strain (Novagen), and the cells were grown at 37 °C to an absorbance of 0.4 at 600 nm, and expression was induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside. Cells were harvested after a 3-h induction, solubilized, and boiled in the SDS-PAGE sample buffer for 5 min, and expression of the recombinant ODDDs was analyzed by 12% SDS-PAGE under reducing conditions followed by Coomassie Blue staining.The recombinant ODDDs were purified from 150–300-ml cultures on a chelating Sepharose column charged with Ni2+ (ProBond, Invitrogen). The cells were harvested after a 3-h induction, suspended in 1/20 volume of a 0.5 m NaCl, 0.1% Triton X-100, and 20 mm Tris-HCl buffer, pH 8, supplemented with Complete EDTA-free protease inhibitor mixture (Roche Applied Science), disrupted by sonication, and centrifuged at 17,000 × g for 20 min, and the soluble fractions were applied to a chelating Sepharose column stabilized with a solution of 0.5 m NaCl and 20 mm Tris-HCl, pH 8. The column was washed with the same buffer containing 0.02 m imidazole and eluted with a 150-ml linear imidazole gradient (0.02–0.3 m), and the fractions were analyzed by 12% SDS-PAGE. The fractions containing the recombinant ODDD polypeptides were pooled and concentrated using Amicon ultracentrifugal devices with Mr 5,000 cut-off membrane (Millipore) and applied to a Superdex S-200 column (Amersham Biosciences) in a 0.3 m NaCl, 5 mm dithiothreitol, 20 mm Tris buffer, pH 8.0. Fractions of 2 ml were collected and analyzed by 12% SDS-PAGE. Those containing the pure ODDD polypeptides were pooled, and their buffer was changed to 50 mm Tris-HCl, pH 8, by concentrating as above, and the protein concentrations were measured by RotiQuant (Carl Roth GmbH).HIF-P4H Activity Assays—HIF-P4H activity was assayed by a method based on measurement of the hydroxylation-coupled stoichiometric release of 14CO2 from 2-oxo[1-14C]glutarate in a final reaction volume of 0.5 ml (21Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). The Km values of the purified enzymes for the synthetic HIF-1α, HIF-2α, and HIF-3α peptides (Table 1), the recombinant ODDD polypeptides, and of HIF-P4H-2 for O2 with the wild-type HIF-1α ODDD and its P402A and P564A mutants as substrates were determined as described previously (21Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). The purities of the synthetic HIF-α peptides (Innovagen) were about 80%. The Km values for the 19–20-residue and 35-residue peptides and the ODDD polypeptides were determined using 2-oxoglutarate with a specific activity of 1100 or 11,100, 11,100 or 55,500, and 55,500 dpm/nmol, respectively. The relative Vmax values of each HIF-P4H for the various peptide substrates were determined with respect to that obtained with the 19-residue peptide representing the C-terminal hydroxylation site of HIF-1α in the same experiment.TABLE 1Amino acid sequences of the synthetic peptides used as substratesPeptideAmino acid sequenceHIF-1αC19DLDLEMLAPYIPMDDDFQLHIF-1αC35KNPFSTGDTDLDLEMLAPYIPMDDDFQLRSFDQLSHIF-2αC19ELDLETLAPYIPMDGEDFQHIF-2αC20ELDLETLAPYIPMDGEDFQLHIF-2αC35QCSTQTDFNELDLETLAPYIPMDGEDFQLSPICPEHIF-3α20DALDLEMLAPYISMDDDFQLHIF-3α35DLDIAQDADALDLEMLAPYISMDDDFQLNASEQLPHIF-1αN20DALTLLAPAAGDTIISLDFGHIF-1αN20A566PDALTLLAPAPGDTIISLDFGHIF-1αN20A558EDELTLLAPAAGDTIISLDFGHIF-1αN35SLFDKLKKEPDALTLLAPAAGDTIISLDFGSNDTEHIF-2αN20EPEELAQLAPTPGDAIISLDHIF-2αN35FLFTKLKEEPEELAQLAPTPGDAIISLDFGNQNFE Open table in a new tab In additional experiments HIF-P4H-3 activity was assayed by using l-[2,3,4,5-3H]proline-labeled substrates and measuring the amount of 4-hydroxy[3H]proline formed. The cDNAs for the wild-type, P402A and P564A mutant, and the P402A/P564A double mutant HIF-1α ODDDs in pET-22b(+) were translated in the presence of l-[2,3,4,5-3H]proline (85 mCi/mmol; PerkinElmer Life Sciences) in rabbit reticulocyte lysate using a TnT® Quick-coupled transcription/translation system (Promega). The translation products were analyzed by 12% SDS-PAGE followed by fluorography. Aliquots of 45 μl of the translation products containing ∼2 × 106 cpm of incorporated radioactive proline were used as substrates for the purified recombinant HIF-P4H-3 in a final reaction volume of 1 ml under the reaction conditions described previously (21Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar), except that the 2-oxoglutarate was nonlabeled. In control experiments the recombinant HIF-P4H-3 was omitted. The enzyme reactions were dialyzed extensively to remove any remaining free l-[2,3,4,5-3H]proline, and the 4-hydroxy[3H]proline formed in the substrate was analyzed by a specific radiochemical procedure (25Juva K. Prockop D.J. Anal. Biochem. 1966; 15: 77-83Crossref PubMed Scopus (513) Google Scholar).RESULTSKm Values of the Three HIF-P4Hs for 35-Residue Peptides Representing the C-terminal Hydroxylation Site in HIF-1α, HIF-2α, and HIF-3α Are Markedly Lower than Those for the Corresponding 19–20 Residue Peptides—The human HIF-P4H-1–3 were expressed as FLAG His-tagged recombinant enzymes in insect cells and purified to near homogeneity by anti-FLAG affinity chromatography (24Hirsilä M. Koivunen P. Xu L. Seeley T. Kivirikko K.I. Myllyharju J. FASEB J. 2005; 19: 1308-1310Crossref PubMed Google Scholar). The 35-residue peptides were designed to contain 17 amino acids on each side of the proline to be hydroxylated (Table 1). The Km values of the HIF-P4Hs were determined by a method based on measurement of the hydroxylation-coupled stoichiometric release of 14CO2 from 2-oxo[1-14C]glutarate (21Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 24Hirsilä M. Koivunen P. Xu L. Seeley T. Kivirikko K.I. Myllyharju J. FASEB J. 2005; 19: 1308-1310Crossref PubMed Google Scholar). These values were compared with those determined previously for the corresponding 19- or 20-residue peptides (21Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar), and additional values were determined for some of the short peptides. As the 19-residue HIF-2α peptide studied previously ended just before a leucine subsequently reported to influence hydroxylation (22Kageyama Y. Koshiji M. To K.K.W. Tian Y.-M. Ratcliffe P.J. Huang L.E. FASEB J. 2004; 18: 1028-1030Crossref PubMed Scopus (62) Google Scholar), we also determined the Km values for a 20-residue peptide containing this leucine (Table 1). Its Km values were about 50–70% of those for the 19-residue peptide, indicating that the leucine had a distinct although relatively modest effect (Table 2).TABLE 2Km and Vmax values of the HIF-P4H isoenzymes for peptides representing the C-terminal hydroxylation site in HIF-1α, HIF-2α, and HIF-3αPeptideHIF-P4H-1HIF-P4H-2HIF-P4H-3KmVmaxaThe Vmax values are expressed relative to that obtained for each enzyme with the HIF-1αC19 peptide.nKmVmaxaThe Vmax values are expressed relative to that obtained for each enzyme with the HIF-1αC19 peptide.nKmVmaxaThe Vmax values are expressed relative to that obtained for each enzyme with the HIF-1αC19 peptide.nμm%μm%μm%HIF-1αC19bThe values shown for these peptides include those determined previously (21), in most cases together with some additional values determined in this study.10 ± 5100107 ± 210055 ± 310010HIF-1αC350.3 ± 0.06100 ± 1060.8 ± 0.295 ± 430.09 ± 0.0390 ± 306HIF-2αC19bThe values shown for these peptides include those determined previously (21), in most cases together with some additional values determined in this study.30 ± 1070 ± 15730 ± 1080 ± 25310 ± 685 ± 104HIF-2αC2020 ± 5105 ± 10315 ± 2105 ± 1536 ± 380 ± 153HIF-2αC350.15 ± 0.0580 ± 1540.4 ± 0.275 ± 1030.2 ± 0.0760 ± 203HIF-3αC20bThe values shown for these peptides include those determined previously (21), in most cases together with some additional values determined in this study.8 ± 5115 ± 3320 ± 6140 ± 2034 ± 3120 ± 103HIF-3αC350.2 ± 0.06120 ± 2560.6 ± 0.1110 ± 1530.2 ± 0.07110 ± 203a The Vmax values are expressed relative to that obtained for each enzyme with the HIF-1αC19 peptide.b The values shown for these peptides include those determined previously (21Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar), in most cases together with some additional values determined in this study. Open table in a new tab The Km values of the three isoenzymes for the 35-residue peptides were markedly lower than those for the corresponding 19–20-residue peptides, ranging in all but one case from 90 to 600 nm and being only about 0.7–5% of those for the shorter peptides, the only exception being the Km of HIF-P4H-2 for the 35-residue HIF-1α peptide, which was 800 nm and 11% (Table 2; Fig. 1A). The Km values of HIF-P4H-2 for the three 35-residue peptides were higher than those of the other two isoenzymes (at least p < 0.01), the lowest Km being that of HIF-P4H-3 for the 35-residue HIF-1α peptide, 90 nm (Table 2). Interestingly, the increase in peptide length from 19–20 to 35 residues had no significant effect on the Vmax values, although there was a tendency for slightly lower Vmax values with the longer peptides in some cases (Table 2).An Increase in the Peptide Length from 19–20 to 35 Residues Has Relatively Small Effects on the Km Values for Peptides Representing the N-terminal Hydroxylation Site in HIF-1α and HIF-2α—The Km values for 19–20-residue peptides representing the N-terminal hydroxylation site in HIF-1α and HIF-2α are much higher than those for the 19-residue peptides representing the C-terminal site (21Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). As the 19-residue peptide representing the N-terminal site in HIF-1α was subsequently found to erroneously lack an aspartate located in position +10 with respect to the proline to be hydroxylated, a new 20-residue peptide containing this aspartate (Table 1) was used here as the reference peptide (Table 3). The Km values of HIF-P4H-1 and -2 for this 20-residue peptide were about 50 μm, i.e. 5–7 times those for the corresponding C-terminal site peptide, whereas the Km of HIF-P4H-3 was 620 μm, i.e. more than 100 times that for the C site peptide (Tables 2 and 3). The Km values of the three HIF-P4Hs for the 20-residue HIF-2α N site peptide were about 60–110 μm (21Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). The Vmax values for the 20-residue HIF-1α N site peptide were similar to those for the HIF-1α C site pept" @default.
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• W2008541833 title "The Length of Peptide Substrates Has a Marked Effect on Hydroxylation by the Hypoxia-inducible Factor Prolyl 4-Hydroxylases" @default.
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