Cloning and Characterization of a Low Molecular Weight Prolyl 4-Hydroxylase from Arabidopsis thaliana

• DOI: 10.1074/jbc.m201865200
• Publication Date: 2002-07-28T22:26:46Z
• Authors: Reija Hieta; Johanna Myllyharju
• Keywords: Cloning (programming)

4-Hydroxyproline is found in collagens and collagen-like proteins animals many glycoproteins plants. Animal prolyl 4-hydroxylases (P4Hs) have been cloned characterized from sources, but no plant P4H has so far. We report here that the genome of Arabidopsis thaliana encodes six P4H-like polypeptides, one which, a 283-residue soluble monomer, was as recombinant protein. Catalytically critical residues identified animal P4Hs are conserved this P4H, their mutagenesis led to complete or almost inactivation. The effectively hydroxylated poly(l-proline) synthetic peptides corresponding proline-rich repeats present other proteins. Surprisingly, were also good substrates, V max with (Pro-Pro-Gly)10 being similar poly(l-proline). enzyme acted peptide preferentially on prolines Y positions theX-Y-Gly triplets. Correspondingly, (Gly-Pro-4Hyp)5 (Pro-Ala-Gly)5 poor values less than 5 20% obtained (Pro-Pro-Gly)10, respectively, K m for latter high. Peptides representing N- C-terminal hydroxylation sites hypoxia-inducible transcription factor α served substrates. As these contain only proline residue, type II conformation clearly not required hydroxylation. 4-hydroxylase P. bursaria chlorella virus-1 high performance liquid chromatography dithiothreitol collagens, elastin, more 15 additional domains tissues (1Prockop D.J. Kivirikko K.I. Annu. Rev. Biochem. 1995; 64: 403-434Crossref PubMed Scopus (1379) Google Scholar, 2Myllyharju J. Ann. Med. 2001; 33: 7-21Crossref (552) 3Kivirikko Pihlajaniemi T. Adv. Enzymol. Related Areas Mol. Biol. 1998; 72: 325-398PubMed Scholar). Its formation catalyzed by (P4Hs),1 which act within lumen endoplasmic reticulum hydroxylate -X-Pro-Gly- sequences. reaction requires Fe2+, 2-oxoglutarate, O2, ascorbate involves an oxidative decarboxylation 2-oxoglutarate (for reviews, see Refs. Scholar 4Kivirikko Myllyharju Matrix 16: 357-368Crossref (235) vertebrate enzymes α2β2 tetramers molecular weight approximately 240,000, β subunit identical chaperone protein-disulfide isomerase (3Kivirikko Two isoforms catalytic extensively several sources shown form [α(I)2]β2 [α(II)2]β2 5Helaakoski Annunen Vuori K. MacNeil I.A. Proc. Natl. Acad. Sci. U. S. A. 92: 4427-4431Crossref (90) 6Annunen Helaakoski Veijola Chem. 1997; 272: 17342-17348Abstract Full Text PDF (110) Caenorhabditis elegans (7Veijola Koivunen 1994; 269: 26746-26753Abstract 8Veijola Page A.P. 1996; 317: 721-729Crossref (31) 9Winter A.D. Cell. 2000; 20: 4084-4093Crossref 10Friedman L. Higgin J.J. Moulder G. Barstead R. Raines R.T. Kimble 97: 4736-4741Crossref (61) 11Riihimaa Nissi Winter Keskiaho 2002; 277: 18238-18243Abstract (23) Scholar) andDrosophila melanogaster (12Annunen 1999; 274: 6790-6796Abstract (34) In addition, family cytoplasmic -Leu-X-X-Leu-Ala-Pro- sequences very recently play role regulation (HIF) (13Ivan M. Kondo Yang H. Kim W. Valiando Ohh Salic Asara J.M. Lane W.S. Kaelin W.G., Jr. Science. 292: 464-468Crossref (3893) 14Jaakkola Mole D.R. Tian Y.-M. Wilson M.I. Gielbert Gaskell S.J. von Kriegsheim Hebestreit H.F. Mukherji Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. 468-472Crossref (4456) 15Epstein A.C.R. Gleadle McNeill L.A. Hewitson K.S. O'Rourke Metzen E. Dhanda Masson N. Hamilton D.L. Jaakkola Hodgkin 107: 43-54Abstract (2736) 16Bruick R.K. McKnight S.L. 294: 1337-1340Crossref (2114) glycoproteins, especially extensins, arabinogalactan (17Showalter A.M. Plant 1993; 5: 9-23Crossref (843) 18Sommer-Knudsen Bacic Clarke A.E. Phytochemistry. 47: 483-497Crossref (135) 19Cassab G.I. Physiol. 49: 281-309Crossref (424) 20Serpe M.D. Nothnagel E.A. Bot. Res. 30: 207-289Crossref (65) unicellular multicellular green algae 60-kDa monomers (21Kaska D.D. Günzler V. Myllylä 1987; 241: 483-490Crossref (32) 22Kaska Gibor 1988; 256: 257-263Crossref (24) Those higher plants likely be monomers, although variable presence polypeptide reported partially purified preparations (23Bolwell G.P. Robbins M.P. Dixon R.A. 1985; 229: 693-699Crossref (25) Scholar,24Wojtaszek Smith C.G. Bolwell Int. Cell 31: 463-477Crossref (20) No detail far, however. require same cosubstrates enzymes, they differ them primarily poly(l-proline)-like may helix review, Ref. 25Kivirikko Harding Crabbe M.J.C. Post-Translational Modifications Proteins. CRC Press, Boca Raton, FL1992: 1-51Google Very low rates random-coil triple-helical forms (Pro-Pro-Gly)5 however (25Kivirikko Recently, viral Paramecium (PBCV-1) (26Eriksson J., Tu, Hellman 22131-22134Abstract (58) This 242-amino acid monomer resembles it hydroxylates much coded Our sequence homology searches thalianagenome indicated contains open reading frames coding polypeptides. now these, 283-amino polypeptide. expressed insect cells peptides. (Ala-Pro-Gly)5with those P4Hs. Furthermore, thalianaP4H resembled preceding glycines (X-Y-Gly) n A search (27Altschul S.F. Madden T.L. Schäffer A.A. Zhang Z. Miller Lipman Nucleic Acids 25: 3389-3402Crossref (59933) genes encoding polypeptides 280–332 amino acids (GenBankTM accession nos. AAC64297, AAB80790, AAF88161, NP_197391, AAF08583, BAB02864) similarity halves human α(I) α(II) subunits (6Annunen 28Helaakoski 1989; 86: 4392-4396Crossref (103) These aligned (29Thompson J.D. Higgins D.G. Gibson T.J. 22: 4673-4680Crossref (55767) PBCV-1 Scholar), cleavage signal predicted (30Nielsen Engelbrecht Brunak Hejne Protein Eng. 10: 1-6Crossref (4934) PCR primers 5′-GCGGGATCCCTCCTTGTTACAATTGGCCTTTA-3′ 5′-CGGGATCCTCAAGAAGTAGCTTTTTGCCTCAT-3′ synthesized based gene AAC64297 (named At-P4H-1) used obtain 783-base pair product whole cDNA library (Stratagene). template prepared incubating 2 μl 200-μl final volume 1% Nonidet P-40, 100 μg/ml proteinase K, 1 mm EDTA, 10 Tris, pH 8.0, at 55 °C 45 min, followed min 95 °C, centrifuged 12,000 rpm 50-μl reaction. Hot-start preincubation 94 72 before addition Pfupolymerase (Promega) used, after 30 cycles performed follows: denaturation annealing 65 extension 3 °C. To increase amount product, second 1:50 diluted first template. above, exception temperature 58 fragment Ser23–Ser283 At-P4H-1 had BamHI restriction both ends (underlined primer sequences) cytosine codon Ser23, into BamHI-digested baculovirus vector pACGP67-A (Invitrogen). verified automated DNA sequencer (Abi Prism 377, Applied Biosystems). cotransfected Spodoptera frugiperda Sf9 BaculoGold (PharMingen) calcium phosphate transfection, viruses amplified (31Crossen Gruenwald Baculovirus Expression Vector System; Instruction Manual. PharMingen, San Diego, CA1998Google High Five (Invitrogen) cultured monolayers TNM-FH medium (Sigma) supplemented 10% fetal bovine serum (BioClear) suspension Sf900IISFM serum-free seeded density × 106 cells/100-mm plate 106/ml infected multiplicity virus At-P4H-1. harvested h infection, washed solution 0.15 NaCl 0.02m phosphate, 7.4, homogenized 0.1 mNaCl, glycine, μm (DTT), 0.1% Triton X-100, 0.01 Tris buffer, 7.8, 50% glycerol, 0.6 NaCl, DTT, 0.06m 10,000 ×g 20 min. pellets further solubilized SDS, aliquots all fractions analyzed 12% SDS-PAGE under reducing conditions. 5′-GGAATTCCATATGTCCTTGTTACAATTGGCCTTTAT-3′ amplify without flankingNdeI NdeI-BamHI-digested expression pET15b (Novagen). plasmid transformed coliBL21(DE3) strain grown 37 optical 0.5 600 nm, incubated induced mmisopropyl-1-thio-β-d-galactopyranoside. induction, suspended 50 Tris-HCl, sonicated, 17,000 g insoluble SDS-PAGE. Histidines 180 260 converted individually glutamate (codon GAA) alanine (GCT), Asp182 (GCT) (GAA), Lys270 arginine (AGG) (GCG), Ser272 Arg278 (GCG) histidine (CAC). reactions containing full-length using QuikChangeTM site-directed kit mutant cDNAs flanking (above), products digested pACGP67-A. Recombinant baculoviruses generated infect above. activity assayed method hydroxylation-coupled 2-oxo-[1-14C]glutarate (32Kivirikko Methods 1982; 82: 245-304Crossref (324) Poly(l-proline) purchased Sigma, whereas Innovagen. All except HIFα denatured heating rapid cooling mixture. some experiments 4-hydroxyproline formed determined colorimetric samples hydrolyzed 6 HCl 120 overnight (33Kivirikko Laitinen O. Prockop Anal. 1967; 19: 249-255Crossref (971) others mixture HiTrap Q-Sepharose (Amersham Biosciences) reverse phase HPLC, manual gas-phase hydrolysis method, Biosystems 421A analyzer. N-terminal sequencing (Pro-Pro-Gly)10peptide 492 ProciseTM protein sequencer. Typically, third each cycle carried over next cycle. corrected carry over, significant background level, could quantitated accurately. described previously (34Myllyharju EMBO 1173-1180Crossref (164) gel filtration calibrated HiPrep Sephacryl S-100 HR column Biosciences). theA. (Fig.1) show identity 21–27% catalytically important regions 33–81% (Fig. 1). two histidines aspartate bind Fe2+ atom site 35Lamberg 270: 9926-9931Abstract (64) lysine binds C-5 carboxyl group collagen thalianasequences fifth probably involved binding C-1 cosubstrate five sequences, replaced position occupied Drosophila suggesting However, like enzyme, substrate domain located between 140 240 (36Myllyharju 18: 306-312Crossref noncleavable cleavable ones NP_197391 AAF08583 AAB80790 BAB02864 highest, 25 27%, respectively. polypeptide, named At-P4H-1, recombinantly expressed. four cysteine residues, fourth, +3 respect Fe2+-binding histidine, subunits. cysteines thalianapolypeptides region homologous counterpart potential N-glycosylation sites, such sites. PCR, frame GP67 sequence, generate virus. buffer centrifuged. remaining pellet Coomassie Blue staining (Fig.2). little 29-kDa extracted X-100 2, lane 1), most remained fraction SDS 4). Therefore, various means extracting efficiently tested, including sonication, use buffers salt concentrations, detergents, concentrations urea (details shown). slightly improved solubility 2), consisting 7.8 (37Pirskanen Kaimio A.-M. 271: 9398-9402Abstract (53) best solubilizing among tested 3), even enzyme. pET15bE. coli tag. insoluble, however, accumulated inclusion bodies (data study whether recombinantA. any activity, cell homogenate When mg/ml (M r 5,000) substrate, observed sample (typically 7000 cpm 200–300 cpm), readily detected when Blue-stained efficiently, levels ranging up 30,000 (an example Table II). Gel showed eluted corresponded calculated 29,252, monomer.Table IIP4H activities extracts expressing wild-type polypeptidesRecombinant enzyme2-aMutant numbers converted.P4H activitycpm/50 μl%At-P4H-131,500100His180 → Ala<100<1His180 Glu<100<1Asp182 Ala<100<1Asp182 Glu<100<1His260 Ala<100<1His260 Glu<100<1Lys270 Ala<100<1Lys270 Arg<100<1Ser272 Ala540017Arg278 Ala<100<1Arg278 His810026P4H given cpm/50 extractable poly(l-proline),M 5,000, substrate.2-a Mutant converted. Open table new tab substrate. expected, TheK (TableI) close Chlamydomonas reinhardtii Volvox carteri 40-fold (Table I) algal 6-fold 34Myllyharju lysyl hydroxylase isoenzymes 38Passoja Rautavuoma Ala-Kokko Kosonen 95: 10482-10486Crossref (91) algal, PBCV-1, 21Kaska 26Eriksson 5–10-fold lower poly(l-proline), M 7,000, 20,000, 35-fold 31,000, 2500-fold 13,000, 4 40 value r6,000 30,000, case