A Hypodermally Expressed Prolyl 4-Hydroxylase from the Filarial Nematode Brugia malayi Is Soluble and Active in the Absence of Protein Disulfide Isomerase
2003; Elsevier BV; Volume: 278; Issue: 4 Linguagem: Inglês
10.1074/jbc.m210381200
ISSN1083-351X
AutoresAlan D. Winter, Johanna Myllyharju, Antony P. Page,
Tópico(s)Advanced Glycation End Products research
ResumoThe collagen prolyl 4-hydroxylase (P4H) class of enzymes catalyze the hydroxylation of prolines in theX-Pro-Gly repeats of collagen chains. This modification is central to the synthesis of all collagens. Most P4Hs are α2β2 tetramers with the catalytic activity residing in the α subunits. The β subunits are identical to the enzyme protein disulfide isomerase. The nematode cuticle is a collagenous extracellular matrix required for maintenance of the worm body shape. Examination of the model nematode Caenorhabditis elegans has demonstrated that its unique P4Hs are essential for viability and body morphology. The filarial parasite Brugia malayi is a causative agent of lymphatic filariasis in humans. We report here on the cloning and characterization of a B. malayi P4H with unusual properties. The recombinant B. malayi α subunit, PHY-1, is a soluble and active P4H by itself, and it does not become associated with protein disulfide isomerase. The active enzyme form is a homotetramer with catalytic and inhibition properties similar to those of the C. elegans P4Hs. High levels of B. malayi phy-1 transcript expression were observed in all developmental stages examined, and its expression was localized to the cuticle-synthesizing hypodermal tissue in the heterologous host C. elegans. Although active by itself, the B. malayi PHY-1 was not able to replace enzyme function in a C. elegans P4H mutant. The collagen prolyl 4-hydroxylase (P4H) class of enzymes catalyze the hydroxylation of prolines in theX-Pro-Gly repeats of collagen chains. This modification is central to the synthesis of all collagens. Most P4Hs are α2β2 tetramers with the catalytic activity residing in the α subunits. The β subunits are identical to the enzyme protein disulfide isomerase. The nematode cuticle is a collagenous extracellular matrix required for maintenance of the worm body shape. Examination of the model nematode Caenorhabditis elegans has demonstrated that its unique P4Hs are essential for viability and body morphology. The filarial parasite Brugia malayi is a causative agent of lymphatic filariasis in humans. We report here on the cloning and characterization of a B. malayi P4H with unusual properties. The recombinant B. malayi α subunit, PHY-1, is a soluble and active P4H by itself, and it does not become associated with protein disulfide isomerase. The active enzyme form is a homotetramer with catalytic and inhibition properties similar to those of the C. elegans P4Hs. High levels of B. malayi phy-1 transcript expression were observed in all developmental stages examined, and its expression was localized to the cuticle-synthesizing hypodermal tissue in the heterologous host C. elegans. Although active by itself, the B. malayi PHY-1 was not able to replace enzyme function in a C. elegans P4H mutant. prolyl 4-hydroxylase prolyl 4-hydroxylase α subunit protein disulfide isomerase reverse transcriptase PCR untranslated region green fluorescent protein β-galactosidase-encoding gene endoplasmic reticulum hypodermal expressed sequence tag bacterial artificial chromosome rapid amplification of cDNA ends Biosynthesis of vertebrate collagens requires processing by up to eight specific intra- and extracellular posttranslational enzymes (1Myllyharju J. Kivirikko K.I. Ann. Med. 2001; 33: 7-21Crossref PubMed Scopus (568) Google Scholar). The collagen prolyl 4-hydroxylase (P4H)1 class of enzymes (EC1.14.11.2) catalyze the hydroxylation of prolines in theX-Pro-Gly repeats of collagen chains. This endoplasmic reticulum (ER) resident enzyme is central to collagen synthesis, as collagen triple helices are thermally unstable in the absence of 4-hydroxyproline residues (2Kivirikko K.I. Myllyharju J. Matrix Biol. 1998; 16: 357-368Crossref PubMed Scopus (238) Google Scholar, 3Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 325-400PubMed Google Scholar). P4H also acts as a chaperone in the assembly of collagen ensuring that only correctly folded collagens are released for secretion (4Walmsley A.R. Batten M.R. Lad U. Bulleid N.J. J. Biol. Chem. 1999; 274: 14884-14892Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In vertebrates and Drosophila melanogaster the enzyme is an α2β2tetramer (2Kivirikko K.I. Myllyharju J. Matrix Biol. 1998; 16: 357-368Crossref PubMed Scopus (238) Google Scholar, 3Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 325-400PubMed Google Scholar, 5Annunen P. Koivunen P. Kivirikko K.I. J. Biol. Chem. 1999; 274: 6790-6796Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 6Myllyharju, J. (2003) Matrix Biol., in pressGoogle Scholar), with hydroxylation activity residing in the catalytic α subunits. Two α subunit isoforms, α(I) and α(II), have been characterized in vertebrates (7Helaakoski T. Annunen P. Vuori K. MacNeil I.A. Pihlajaniemi T. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4427-4431Crossref PubMed Scopus (91) Google Scholar, 8Annunen P. Helaakoski T. Myllyharju J. Veijola J. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1997; 272: 17342-17348Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). They become assembled into [α(I)]2β2 and [α(II)]2β2 tetramers with insect cell coexpression data arguing strongly against the formation of mixed α(I)α(II)β2 tetramers (8Annunen P. Helaakoski T. Myllyharju J. Veijola J. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1997; 272: 17342-17348Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The β subunits of P4Hs are identical to the enzyme and chaperone protein disulfide isomerase (PDI) (EC 5.3.4.1) (9Pihlajaniemi T. Helaakoski T. Tasanen K. Myllylä R. Huhtala M.-L. Koivu J. Kivirikko K.I. EMBO J. 1987; 6: 643-649Crossref PubMed Scopus (346) Google Scholar) and are required to maintain the α subunits in a catalytically active nonaggregated conformation (10John D.C.A. Grant M.E. Bulleid N.J. EMBO J. 1993; 12: 1587-1595Crossref PubMed Scopus (95) Google Scholar, 11Vuori K. Pihlajaniemi T. Myllylä R. Kivirikko K.I. EMBO J. 1992; 11: 4213-4217Crossref PubMed Scopus (128) Google Scholar). The P4H is also maintained within its correct subcellular compartment by virtue of an ER retention signal at the C terminus of PDI (11Vuori K. Pihlajaniemi T. Myllylä R. Kivirikko K.I. EMBO J. 1992; 11: 4213-4217Crossref PubMed Scopus (128) Google Scholar). When expressed alone in a recombinant expression system, the α subunits are insoluble and inactive, whereas coexpression with PDI results in the formation of an active, soluble P4H (7Helaakoski T. Annunen P. Vuori K. MacNeil I.A. Pihlajaniemi T. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4427-4431Crossref PubMed Scopus (91) Google Scholar, 12Vuori K. Pihlajaniemi T. Marttila M. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7467-7470Crossref PubMed Scopus (116) Google Scholar, 13Veijola J. Koivunen P. Annunen P. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1994; 269: 26746-26753Abstract Full Text PDF PubMed Google Scholar, 14Myllyharju J. Kukkola L. Winter A.D. Page A.P. J. Biol. Chem. 2002; 277: 29187-29196Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The PDIs from different organisms can often substitute for the authentic partner, such as the human PDI, which can function as a β subunit in mouse andDrosophila P4H tetramers and in a P4H dimer withCaenorhabditis elegans PHY-1 (5Annunen P. Koivunen P. Kivirikko K.I. J. Biol. Chem. 1999; 274: 6790-6796Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 7Helaakoski T. Annunen P. Vuori K. MacNeil I.A. Pihlajaniemi T. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4427-4431Crossref PubMed Scopus (91) Google Scholar, 13Veijola J. Koivunen P. Annunen P. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1994; 269: 26746-26753Abstract Full Text PDF PubMed Google Scholar). In the model nematode C. elegans the cuticular collagen modifying function of P4H is essential for body morphology and viability (15Winter A.D. Page A.P. Mol. Cell. Biol. 2000; 20: 4084-4093Crossref PubMed Scopus (94) Google Scholar, 16Friedman L. Higgin J.J. Moulder G. Barstead R. Raines R.T. Kimble J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4736-4741Crossref PubMed Scopus (61) Google Scholar). In nematodes the exoskeleton (known as the cuticle) is an extracellular matrix composed of small collagen-like molecules (17Page A.P. Kennedy M.W. Harnett W. Parasitic Nematodes. CAB International, Publishing, Wallingford, Oxon, UK2001: 167-193Google Scholar). The nematode cuticle is synthesized by the underlying hypodermal tissue and performs multiple functions including maintenance of worm body shape. Mutations in collagens forming the cuticle and in the enzymes involved in collagen biosynthesis can result in lethality and severe alterations to body shape, as illustrated by the C. elegans sqt-3 (18Priess J.R. Hirsh D.I. Dev. Biol. 1986; 117: 156-173Crossref PubMed Scopus (317) Google Scholar) and bli-4 (19Thacker C. Peters K. Srayko M. Rose A.M. Genes Dev. 1995; 9: 956-971Crossref PubMed Scopus (76) Google Scholar) mutant phenotypes. The P4Hs in C. elegans that are involved in the synthesis of cuticle collagens are formed from the α subunits PHY-1 and PHY-2 and the β subunit PDI-2 (14Myllyharju J. Kukkola L. Winter A.D. Page A.P. J. Biol. Chem. 2002; 277: 29187-29196Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 15Winter A.D. Page A.P. Mol. Cell. Biol. 2000; 20: 4084-4093Crossref PubMed Scopus (94) Google Scholar). The expression, function, and assembly of these subunits have been examined in detail showing that unique P4H forms exist in C. elegans. The most abundant form is a tetramer; this differs, however, from others described, being a mixed PHY-1/PHY-2/(PDI-2)2 tetramer (14Myllyharju J. Kukkola L. Winter A.D. Page A.P. J. Biol. Chem. 2002; 277: 29187-29196Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). PHY-1 and PHY-2 can also each individually associate with PDI-2 to form dimers (14Myllyharju J. Kukkola L. Winter A.D. Page A.P. J. Biol. Chem. 2002; 277: 29187-29196Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Such P4H forms have not been described for any other species to date. Genetic disruption of phy-1 and phy-2simultaneously, or pdi-2 singly, results in embryonic lethality in which embryos develop normally until the first cuticle is required to maintain the elongated worm shape (15Winter A.D. Page A.P. Mol. Cell. Biol. 2000; 20: 4084-4093Crossref PubMed Scopus (94) Google Scholar, 16Friedman L. Higgin J.J. Moulder G. Barstead R. Raines R.T. Kimble J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4736-4741Crossref PubMed Scopus (61) Google Scholar). The weakened cuticle is then unable to maintain this form, after which embryos collapse to a disorganized state and eventually die. The body shape defect and reduced 4-hydroxyproline levels in the cuticle collagens of the viable phy-1 null genetic mutant,dpy-18, underline the importance of collagen modification by P4H for nematode body morphology (15Winter A.D. Page A.P. Mol. Cell. Biol. 2000; 20: 4084-4093Crossref PubMed Scopus (94) Google Scholar, 16Friedman L. Higgin J.J. Moulder G. Barstead R. Raines R.T. Kimble J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4736-4741Crossref PubMed Scopus (61) Google Scholar). Applying the knowledge of P4H function in C. elegans, we examined a P4H in the filarial parasitic nematode Brugia malayi. B. malayi along with Brugia timoriand Wuchereria bancrofti are the causative agents of lymphatic filariasis in humans, with over 120 million people infected and over 1 billion people at risk of infection worldwide (20World Health Organization (2000) WHO Monogr. Ser.Fact Sheet No. 102Google Scholar). Lymphatic filariasis is a debilitating disease, with approximately one-third of those infected being incapacitated and/or disfigured by the infection (21Ottesen E.A. Duke B.O.L. Karam M. Behbehani K. Bull. W. H. O. 1997; 75: 491-503PubMed Google Scholar). Commercially available inhibitors of P4H have been shown to be toxic to B. malayi adults, producing associated cuticular defects (22Merriweather A. Guenzler V. Brenner M. Unnasch T.R. Mol. Biochem. Parasitol. 2001; 116: 185-197Crossref PubMed Scopus (17) Google Scholar). These observations and the requirement inC. elegans for P4H activity highlight this enzyme class as a potential drug target in the control of human and veterinary parasitic nematode infections. In this article we describe the identification of a P4Hphy-1 gene from B. malayi, characterize the molecular and enzymatic properties of the recombinant B. malayi P4H produced in an insect cell expression system, and examine the expression profile and putative function of the B. malayi phy-1 gene by heterologous expression in the model nematodeC. elegans. Unusually, the B. malayi PHY-1 is a soluble and active P4H when expressed alone in a recombinant system in the absence of PDI, and it does not associate with PDIs from other organisms, including C. elegans. The developmental temporal expression pattern of the B. malayi phy-1 gene was analyzed by RT-PCR using stage-specific mRNA samples. Reporter gene experiments showed that the B. malayi phy-1 promoter directs tissue-specific spatial expression to the hypodermal cells of C. elegans. C. elegans strains were cultured as described elsewhere (23Brenner S. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar). The wild type Bristol N2, CB364 (dpy-18(e364)), and DR96 (unc-76(e911)) C. elegans strains were provided by the Caenorhabditis Genetics Centre. The B. malayi nematodes were provided by Rick Maizels (University of Edinburgh). ESTs SW3D9CA480SK, MBAFCX8G05T3, and MBAFCZ7H09T3 (Fig. 1 A) were received from the Filarial Genome Project, subcloned, and sequenced. The primers X8G5F1 (5′-CAGTCGCTCAACACCGG-3′) and BMNPHYR (5′-CCAATAGTATTTAAGCAC-3′) were designed from the EST sequences and used to obtain a 312-bp PCR product (Fig. 1 A) from B. malayi adult stage cDNA prepared as described previously (15Winter A.D. Page A.P. Mol. Cell. Biol. 2000; 20: 4084-4093Crossref PubMed Scopus (94) Google Scholar). The purified PCR product was labeled with [α-32P]dCTP and used to screen a B. malayi adult male cDNA library, SAW94NLBmAm (from Steven Williams, Filarial Genome Project, Northampton, MA). Eleven positive clones, all representing the same cDNA, named B. malayi phy-1, were identified from a total of 2 × 104 recombinants. Amplification of the 5′ end of theB. malayi phy-1 cDNA was performed using the Invitrogen 5′-RACE system. Two 418-bp products from independent 5′-RACE PCR reactions (Fig. 1 A) were sequenced to generate a consensus 5′ sequence. The primers BMPHY-1RESF(BamHI), and BMPHY-1RESR(NotI) (for primer sequences, see “dpy-18 Rescue Experiments with B. malayiphy-1”) were used to generate the B. malayi phy-1 genomic sequence from the translation start codon ATG to the stop codon TAA usingB. malayi genomic DNA as a template. Three identical full-length clones were then fully sequenced to generate a consensus genomic sequence. Computer prediction for analysis of conceptual translation of B. malayi phy-1, protein alignments, signal peptide analysis, and predicted posttranslational modifications were performed on the ExPASy proteomics tools data base (www.expasy.ch). The full-length coding sequence of B. malayi phy-1 cDNA was cloned from B. malayi adult stage cDNA by PCR using Pfu polymerase (Stratagene) with the primers BMPHY-1BVF(NotI) (5′-gagcggccgcATGATAGCTACCGTGGTGTTC-3′) and BMPHY-1BVR(XbaI) (5′-gctctagaTTAAGCACTTAGATCGCCCAC-3′) (artificial restriction sites are set in lowercase and underlined). The PCR product was cloned into a NotI-XbaI-digested transfer vector pVL1392 (BD Pharmingen) and sequenced. The recombinant vector was cotransfected into Spodoptera frugiperda Sf9 cells with a modified Autographa californica nuclear polyhedrosis virus DNA (BaculoGold, BD Pharmingen) by calcium-phosphate precipitation. Sf9 or High Five (Invitrogen) insect cells were cultured as monolayers in TNM-FH (modified Grace's insect cell medium) medium (Sigma) supplemented with 10% fetal bovine serum (BioClear) or in suspension in Sf900IISFM serum-free medium (Invitrogen). The cells were seeded at a density of 5 × 106cells/100-mm plate or 1 × 106 cells/ml and infected at a multiplicity of 5 with the virus coding for the B. malayi PHY-1 alone or together with viruses coding for C. elegans PDI-1, PDI-2, or human PDI. In control experiments, the cells were coinfected with the viruses coding for C. elegansPHY-1, PHY-2, and PDI-2, with PHY-1 and human PDI, or with the various PDI viruses alone. The cells were harvested 72 h after infection, washed with a solution of 0.15 m NaCl and 0.02m phosphate, pH 7.4, homogenized in a 0.1 mNaCl, 0.1 m glycine, 10 μm dithiothreitol, 0.1% Triton X-100, and 0.01 m Tris buffer, pH 7.4, and centrifuged at 10,000 × g for 20 min. The pellets were further solubilized in 1% SDS. Aliquots of the samples were analyzed by 8% SDS-PAGE under reducing conditions and by nondenaturing PAGE followed by Western blotting with polyclonal antibodies againstB. malayi PHY-1 (see below), C. elegans PDI-1 or PDI-2 (14Myllyharju J. Kukkola L. Winter A.D. Page A.P. J. Biol. Chem. 2002; 277: 29187-29196Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), or a monoclonal antibody against human PDI (5B5, Dako). P4H activity was assayed by a method based on the hydroxylation-coupled decarboxylation of 2-oxo-[1-14C]glutarate, andK m and K i values were determined as described previously (24Kivirikko K.I. Myllylä R. Methods Enzymol. 1982; 82: 245-304Crossref PubMed Scopus (331) Google Scholar). The molecular weight of the recombinantB. malayi P4H was analyzed by applying the Triton X-100-soluble fraction of insect cells expressing B. malayiPHY-1 to a calibrated HiPrep Sephacryl S-200 HR gel filtration column (Amersham Biosciences), equilibrated and eluted with 0.1 mNaCl, 0.1 m glycine, 10 μm dithiothreitol, and 0.01 m Tris buffer, pH 7.4, and P4H activity was assayed in the eluted fractions. Purified recombinant human type I P4H (12Vuori K. Pihlajaniemi T. Marttila M. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7467-7470Crossref PubMed Scopus (116) Google Scholar) was used as a control in gel filtration experiments. Extracts fromB. malayi were made by disrupting 100 adult females with a hand-held glass homogenizer in the following buffer: 0.1m NaCl, 0.1 m glycine, 10 μmdithiothreitol, 0.1% Triton-X100, and 10 mm Tris, pH 8.0, supplemented with protease inhibitors 1 mmphenylmethylsulfonyl fluoride, 1 mm EDTA, 1 mmEGTA, 2 μm E64, and 0.1 μm pepstatin. The soluble extracts were analyzed by reducing SDS-PAGE and nondenaturing PAGE in 4–12% NuPAGE BisTris polyacrylamide gels and 4–12% Tris-glycine gels (Invitrogen), respectively, followed by Western blotting. N-glycosidase F (PNGase F, New England Biolabs) treatment was performed according to the manufacturer's recommendations. Polyclonal antiserum was raised in two rabbits against a synthetic peptide corresponding to the C-terminal region of B. malayiPHY-1. The peptide CRRPCGLSRSVEEQFVGDLSA was conjugated to keyhole limpet hemocyanin (Sigma GenoSys) via an added cysteine residue (underlined) and used for immunization. A 2.8-kbPstI-BamHI C. elegans phy-1 promoter fragment from the pPD95–03-phy-1 construct (15Winter A.D. Page A.P. Mol. Cell. Biol. 2000; 20: 4084-4093Crossref PubMed Scopus (94) Google Scholar) was cloned into pBluescript SKM (Stratagene). The C. elegans phy-13′-UTR sequence was generated by PCR from C. elegans N2 genomic DNA using the primers CEPHY-1 3′-UTRF(SacI) (5′-gcggagctcCTCTAAGCATTGGTTTTCATTG-3′) and CEPHY-13′-UTRR(SacI) (5′-gcggagctcACTAGGGAATTGTCGGCTGC-3′) with Vent polymerase (New England Biolabs) and cloned into the pBluescript-Cephy-1 promoter construct to generate the plasmid pAW1 (Fig. 8 B). The coding sequence of B. malayi phy-1 cDNA and the genomic sequence of B. malayi phy-1 from the translation initiation codon to the stop codon were generated by PCR using the primers BMPHY-1RESF(BamHI) (5′-gcggatccGATGATAGCTACCGTGGTGTTC-3′ and BMPHY-1RESR(NotI) (5′-gagcggccgcTTAAGCACTTAGATCGCCCAC-3′) with Pfuand Pfu Turbo (Stratagene) polymerases, respectively. The PCR products were cloned into BamHI-NotI-digested vector pAW1 to generate B. malayi phy-1 cDNA and genomic rescue constructs (Fig. 8 B). A synthetic intron (5′-GTAAGTTTAAACTATTCGTTACTAACTAACTTTAAACATTTAAATTTTCAG-3′) was inserted into the B. malayi phy-1 cDNA rescue construct by ligating a double-stranded oligo into a StuI blunt-ended restriction site. The B. malayi phy-1 cDNA (± a synthetic intron) and genomic rescue constructs were microinjected into the syncytial gonad of C. elegans phy-1 null,dpy-18(e364), nematodes at concentrations of 10 and 100 μg/ml. A marker plasmid with a dpy-7 cuticle collagen promoter in the green fluorescent protein (GFP) fusion vector pPD95-67 (from Iain Johnstone, University of Glasgow) was coinjected at 5 μg/ml, and the injection mixes were made up to a final concentration of 150 μg/ml with pBluescript SKM. Transformants were selected by GFP fluorescence, and more than five semi-stable transmitting lines were examined for each concentration. PCR was performed on cDNA samples generated from daily extracts of B. malayiinfected jirds, up to day 14 post-infection, after which extracts from 2–4-day intervals were taken (25Gregory W.F. Atmadja A.K. Allen J.E. Maizels R.M. Infect. Immun. 2000; 68: 4174-4179Crossref PubMed Scopus (150) Google Scholar). Two sets of primers were used for each PCR, BMPHY1.1IS1F (5′-GCTTCTGGTGTTCAACCG-3′) and BMPHY1.1IS2R (5′-GGTATGATGCTGTTTCAAG-3′), corresponding to the B. malayi phy-1, and BMTUBA (5′-AATATGTGCCACGAGCAGTC-3′) and BMTUBB (5′-CGGATACTCCTCACGAATTT-3′), corresponding to the control B. malayi tubulin gene. To identify the putative promoter region, a genomic B. malayi BAC library was screened with a 1.7-kb biotin-labeled probe generated by PCR using the primers T7PL (5′-CTCACTATAGGGCGAATTGG-3′) (New England Biolabs) and BMPHY1.1IS3R(B) (5′-GCGTGGATGATTTGGATC-3′) and a plasmid containing a T7 site and the 5′ genomic coding sequence from B. malayi phy-1 as a template. The gridded BAC filters were hybridized using NEBlot Phototope and Phototope-star (New England Biolabs) detection kits. BLASTX analysis was performed on the ExPASy proteomics tools data base. A 2.2-kb putative promoter fragment of the B. malayi phy-1 gene was amplified from B. malayi genomic DNA using Pfu polymerase and the primers BMPHY-1PF(SphI) (5′-ggcgcatgcGAATGAGACAATTGCACAAG-3′ and BMPHY-1PR(BamHI) (5′-ggcggatccGCTATCATCACTGGCTCTGGA-3′). This fragment, extending from −2189 to +8 relative to the translation start site, was cloned into SphI-BamHI-digestedC. elegans reporter gene vector, pPD96-04. This was microinjected into the syncytial gonad of the C. elegansstrain DR96(unc-76) together with the unc-76rescue plasmid (p7616B), both at 100 μg/ml. Six semi-stable transgenic lines were identified and examined for reporter gene expression by viewing GFP expression in live worms and by sensitive staining of fixed worms for β-galactosidase activity (26Fire A. Genet. Anal. Biomol. Eng. 1992; 9: 151-158Crossref Scopus (97) Google Scholar). Live nematodes were transferred to 2% agarose, 0.065% sodium azide pads, and images were taken with an Axioskop 2 microscope using a Hamamatsu digital camera and Improvision Openlab processing software. Three ESTs from the B. malayi data base were identified that encode amino acid sequences homologous to C. elegans PHY-1 and PHY-2. MBAFCX8G05T3 (AA509222) and MBAFCZ7H09T3 (AA406985) were both derived from aB. malayi adult female cDNA library and SW3D9CA480SK (AA585698) from a B. malayi L3 cDNA library (GenBankTM accession numbers in parentheses). Sequencing of the ESTs established that they were all derived from a single gene, termed B. malayi phy-1 (Fig. 1 A). A 312-bp PCR probe was generated from B. malayi adult stage cDNA, based on the EST sequences, and used to screen a B. malayi adult male cDNA library (Fig. 1 A). Eleven positive clones were identified; full-length sequencing was performed on two 1.6-kb clones, and both were found to represent the B. malayi phy-1cDNA. The nine other identified clones contained inserts with sizes ranging from 0.6 to 1.6 kb, and all corresponded to the B. malayi phy-1 gene. The 1669-bp sequence generated from the library screening contained a 1515-bp open reading frame and 154-bp of 3′-UTR sequence (Fig. 1 A). The 3′ untranslated region did not contain a consensus polyadenylation signal (AATAAA). A divergent poly(A) signal sequence (GATAAA) was located, however, 11-bp upstream of the poly(A) tail, representing a variant poly(A) signal sequence also found in ∼5% of the C. elegans genes examined (27Blumenthal T. Steward K. Riddle D.L. Blumenthal T. Meyer B.J. Priess J.R. C. elegans II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 117-145Google Scholar). A comparison of the amino acid sequences encoded by the 1515-bpB. malayi phy-1 cDNA sequence with known P4H α subunits signified that the 5′ coding sequence was incomplete. Additionally, according to the signal sequence prediction program, SignalP, the N terminus did not contain a characteristic signal peptide. The 5′-UTR, the trans-spliced leader sequence, and the coding sequence for 37 additional N-terminal amino acids was obtained using the 5′-RACE system for rapid amplification of cDNA ends (Fig. 1 A). These data were assembled with the 1669-bp B. malayi phy-1 sequence obtained from the cDNA libraries to give the full-length cDNA sequence, which was confirmed by the sequencing of a full-length Pfu-generated PCR product (Fig. 1 A). The complete B. malayi phy-1 cDNA sequence contains a consensus 22-bp SL1trans-spliced leader sequence, an 8-bp 5′-UTR, a single open-reading frame of 1626-bp that encodes a 541 amino acid polypeptide (GenBankTM accession No. AJ297845), and a 154-bp 3′-UTR (Fig. 1 A). The 4596-bp consensus genomic sequence of the B. malayi phy-1, determined from three individual full-length PCR products, contains 12 exons and 11 introns (Fig. 1 B) (GenBankTM accession No. AJ421993). The intron sizes range from 119 to 479 bp and have an average size of 270 bp. A signal peptide cleavage site between Ala-17 and Asp-18 was predicted by the SignalP program, and thus the processed B. malayi PHY-1 consists of 524 amino acids. The highest amino acid sequence homology was found between the processedB. malayi PHY-1 and C. elegans PHY-1 sequences (13Veijola J. Koivunen P. Annunen P. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1994; 269: 26746-26753Abstract Full Text PDF PubMed Google Scholar), the identity being 59% and similarity 76%, whereas the identity and similarity between the B. malayi PHY-1 and C. elegans PHY-2 (15Winter A.D. Page A.P. Mol. Cell. Biol. 2000; 20: 4084-4093Crossref PubMed Scopus (94) Google Scholar, 16Friedman L. Higgin J.J. Moulder G. Barstead R. Raines R.T. Kimble J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4736-4741Crossref PubMed Scopus (61) Google Scholar) are 53 and 71%, respectively (Fig. 2). The amino acid sequence homology between the B. malayi and C. elegans PHY polypeptides is slightly higher than that between the B. malayi PHY-1 and the PHY-1 from a closely related filarial nematode, Onchocerca volvulus (22Merriweather A. Guenzler V. Brenner M. Unnasch T.R. Mol. Biochem. Parasitol. 2001; 116: 185-197Crossref PubMed Scopus (17) Google Scholar), with the B. malayi and O. volvulus PHY-1 polypeptides being 49% identical and 70% similar. The amino acid sequence identities between the B. malayi PHY-1 and the human α(I) and α(II) subunits are 45 and 44%, and the similarities are 62 and 63%, respectively. The cysteine residues essential for intrachain disulfide bonding (28John D.C.A. Bulleid N.J. Biochemistry. 1994; 33: 14018-14025Crossref PubMed Scopus (35) Google Scholar, 29Lamberg A. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1995; 270: 9926-9931Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and the active site histidine, aspartic acid, and lysine residues (29Lamberg A. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1995; 270: 9926-9931Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 30Myllyharju J. Kivirikko K.I. EMBO J. 1997; 16: 1173-1180Crossref PubMed Scopus (168) Google Scholar) are all conserved in B. malayiPHY-1 (Fig. 2). The extended C-terminal regions present in the C. elegans and O. volvulus PHY-1 polypeptides are not found in B. malayi PHY-1 (Fig. 2). The B. malayi phy-1 cDNA was cloned into the baculovirus expression vector pVL1392, a recombinant baculovirus was generated and used to infect insect cells. The cells were harvested 72 h after infection, homogenized in a 0.1% Triton X-100-containing buffer, and centrifuged. The remaining pellet was solubilized in 1% SDS, and the samples were analyzed by reducing SDS-PAGE followed by Western blotting (Fig. 3 A). The C. elegansPHY polypeptides (13Veijola J. Koivunen P. Annunen P. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1994; 269: 26746-26753Abstract Full Text PDF PubMed Google Scholar, 14Myllyharju J. Kukkola L. Winter A.D. Page A.P. J. Biol. Chem. 2002; 277: 29187-29196Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) and vertebrate P4H α subunits (7Helaakoski T. Annunen P. Vuori K. MacNeil I.A. Pihlajaniemi T. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4427-4431Crossref PubMed Scopus (91) Google Scholar, 12Vuori K. Pihlajaniemi T. Marttila M. Kivirikko K.I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7467-7470Crossref PubMed Scopus (116) Google Scholar) require association with PDI to form soluble and active P4Hs. When expressed alone in a recombinant system, these polypeptides f
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