A Novel Cyclophilin from Parasitic and Free-living Nematodes with a Unique Substrate- and Drug-binding Domain
2002; Elsevier BV; Volume: 277; Issue: 17 Linguagem: Inglês
10.1074/jbc.m112293200
ISSN1083-351X
AutoresDong Ma, Lewis S. Nelson, Krystel LeCoz, Catherine B. Poole, Clotilde K. S. Carlow,
Tópico(s)Signaling Pathways in Disease
ResumoA highly diversified member of the cyclophilin family of peptidyl-prolyl cis-trans isomerases has been isolated from the human parasite Onchocerca volvulus(OvCYP-16). This 25-kDa cyclophilin shares 43–46% similarity to other filarial cyclophilins but does not belong to any of the groups previously defined in invertebrates or vertebrates. A homolog was also isolated from Caenorhabditis elegans(CeCYP-16). Both recombinant O. volvulus and C. elegans cyclophilins were found to possess an enzyme activity with similar substrate preference and insensitivity to cyclosporin A. They represent novel cyclophilins with important differences in the composition of the drug-binding site in particular, namely, a Glu124 (C. elegans) or Asp123 (O. volvulus) residue present in a critical position. Site-directed mutagenesis studies and kinetic characterization demonstrated that the single residue dictates the degree of binding to substrate and cyclosporin A.CeCYP-16::GFP-expressing lines were generated with expression in the anterior and posterior distal portions of the intestine, in all larval stages and adults. An exception was found in the dauer stage, where fluorescence was observed in both the cell bodies and processes of the ventral chord motor neurons but was absent from the intestine. These studies highlight the extensive diversification of cyclophilins in an important human parasite and a closely related model organism. A highly diversified member of the cyclophilin family of peptidyl-prolyl cis-trans isomerases has been isolated from the human parasite Onchocerca volvulus(OvCYP-16). This 25-kDa cyclophilin shares 43–46% similarity to other filarial cyclophilins but does not belong to any of the groups previously defined in invertebrates or vertebrates. A homolog was also isolated from Caenorhabditis elegans(CeCYP-16). Both recombinant O. volvulus and C. elegans cyclophilins were found to possess an enzyme activity with similar substrate preference and insensitivity to cyclosporin A. They represent novel cyclophilins with important differences in the composition of the drug-binding site in particular, namely, a Glu124 (C. elegans) or Asp123 (O. volvulus) residue present in a critical position. Site-directed mutagenesis studies and kinetic characterization demonstrated that the single residue dictates the degree of binding to substrate and cyclosporin A.CeCYP-16::GFP-expressing lines were generated with expression in the anterior and posterior distal portions of the intestine, in all larval stages and adults. An exception was found in the dauer stage, where fluorescence was observed in both the cell bodies and processes of the ventral chord motor neurons but was absent from the intestine. These studies highlight the extensive diversification of cyclophilins in an important human parasite and a closely related model organism. Cyclophilins belong to a large family of proteins that have been found in most organisms including parasites. It is thought that cyclophilins play an important role in protein folding because of their peptidyl-prolyl cis-trans isomerase (PPIase) 1The abbreviations used are: PPIasepeptidyl-prolyl cis-trans isomeraseCypAhuman cyclophilin ACsAcyclosporin AMBPmaltose-binding proteinGFPgreen fluorescent protein activity, which can be measured in vivo (1.Stamnes M.A. Shieh B.H. Chuman L. Harris G.L. Zuker C.S. Cell. 1991; 65: 219-227Abstract Full Text PDF PubMed Scopus (221) Google Scholar, 2.Galat A. Eur. J. Biochem. 1993; 216: 689-707Crossref PubMed Scopus (316) Google Scholar) and in vitro (3.Lang K. Schmid F.X. Fischer G. Nature. 1987; 329: 268-270Crossref PubMed Scopus (416) Google Scholar, 4.Takahashi N. Hayano T. Suzuki M. Nature. 1989; 337: 473-475Crossref PubMed Scopus (940) Google Scholar). Most cyclophilins bind the immunosuppressive drug cyclosporin A (CsA), resulting in specific inhibition of their PPIase activity (5.Handschumacher R.E. Harding M.W. Rice J. Drugge R.J. Speicher D.W. Science. 1984; 226: 544-547Crossref PubMed Scopus (1457) Google Scholar, 6.Fischer G. Wittmann-Liebold B. Lang K. Kiefhaber T. Schmid F.X. Nature. 1989; 337: 476-478Crossref PubMed Scopus (1212) Google Scholar). Therefore, CsA may interfere with the correct folding of proline-containing proteins that are the natural substrates for cyclophilins. It remains to be determined whether this is the mechanism by which CsA and its nonimmunosuppressive derivatives exert lethal structural damage on a number of important parasites. For example, subimmunosuppressive levels of CsA cause gross herniation in the gut and blistering of the tegumental surface of Schistosoma mansoni (7.Munro G.H. McLaren D.J. Parasitology. 1990; 100, Pt 1: 19-28Crossref PubMed Scopus (17) Google Scholar). In the case of Litomosoides cariniimicrofilariae, the drug causes shrinkage of the parasite and stiffening of the surrounding sheath (8.Zahner H. Schultheiss K. J. Helminthol. 1987; 61: 282-290Crossref PubMed Scopus (14) Google Scholar). peptidyl-prolyl cis-trans isomerase human cyclophilin A cyclosporin A maltose-binding protein green fluorescent protein Most parasite cyclophilins published to date possess a high degree of similarity to human cyclophilin A (CypA) (5.Handschumacher R.E. Harding M.W. Rice J. Drugge R.J. Speicher D.W. Science. 1984; 226: 544-547Crossref PubMed Scopus (1457) Google Scholar), an 18-kDa cytoplasmic protein that is abundantly expressed in all mammalian tissues (9.Koletsky A.J. Harding M.W. Handschumacher R.E. J. Immunol. 1986; 137: 1054-1059PubMed Google Scholar). Like human CypA, the PPIases described from S. mansoni (10.Bueding E. Hawkins J. Cha Y.N. Agents Actions. 1981; 11: 380-383Crossref PubMed Scopus (89) Google Scholar, 11.Chappell L.H. Liu J.F. Penlington M.C. J. Cell. Biochem. 1993; 17C (suppl.): 114Google Scholar, 12.Munro G.H. McLaren D.J. Parasitology. 1990; 100, Pt 1: 29-34Crossref PubMed Scopus (15) Google Scholar),Toxoplasma gondii (13.Mack D.G. McLeod R. Antimicrob. Agents Chemother. 1984; 26: 26-30Crossref PubMed Scopus (84) Google Scholar, 14.McCabe R.E. Luft B.J. Remington J.S. Transplantation. 1986; 41: 611-615Crossref PubMed Scopus (36) Google Scholar), and Plasmodium falciparum (15.Thommen-Scott K. Agents Actions. 1981; 11: 770-773Crossref PubMed Scopus (62) Google Scholar) increase the rate of isomerization of a standard proline-containing peptide substrate (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide)in vitro, and their PPIase activity is easily inhibited by nanomolar concentrations of CsA (9.Koletsky A.J. Harding M.W. Handschumacher R.E. J. Immunol. 1986; 137: 1054-1059PubMed Google Scholar, 16.High K.P. Joiner K.A. Handschumacher R.E. J. Biol. Chem. 1994; 269: 9105-9112Abstract Full Text PDF PubMed Google Scholar, 17.Bell A. Wernli B. Franklin R.M. Biochem. Pharmacol. 1994; 48: 495-503Crossref PubMed Scopus (116) Google Scholar, 18.Reddy G.R. Mol. Biochem. Parasitol. 1995; 73: 111-121Crossref PubMed Scopus (40) Google Scholar). Thus far, only CypA homologs have been found in parasites, with the exception of the filarial worms. In addition to this highly conserved form, designated CYP-2 in filarial parasites, Brugia malayi, Onchocerca volvulus, and Dirofilaria immitis express two divergent cyclophilins that are more related to human nuclear-specific cyclophilin (CYP-3/4) and natural killer cell cyclophilin (CYP-1). These cyclophilins are considerably larger than human CypA, prefer other synthetic substrates, and display a reduced sensitivity to CsA (19.Page A.P. Landry D. Wilson G.G. Carlow C.K. Biochemistry. 1995; 34: 11545-11550Crossref PubMed Scopus (38) Google Scholar, 20.Hong X. Ma D. Page A.P. Kumar S. Carlow C.K. Exp. Parasitol. 1998; 88: 246-251Crossref PubMed Scopus (9) Google Scholar, 21.Hong X. Ma D. Carlow C.K.S. Mol. Biochem. Parasitol. 1998; 91: 353-358Crossref PubMed Scopus (9) Google Scholar). We report here the cloning, expression, and characterization of a new class of cyclophilin from the important human parasite O. volvulus (OvCYP-16) and the model organismCaenorhabditis elegans (CeCYP-16). These cyclophilins are distinct from the other cyclophilins present in the data base from C. elegans (designated CeCYP-1 through CeCYP-15, and CeCYP-17) orO. volvulus (designated OvCYP-1,OvCYP-2, OvCYP-4, OvCYP-5, andOvCYP-10). The CYP-16 cyclophilins represent novel, highly diversified cyclophilins with respect to the composition of the drug-binding site and are particularly interesting because, unlike other parasite cyclophilins described thus far, they are apparently not found in mammals. We present molecular and biochemical studies on these new enzymes and use transgenic methodologies in C. elegansto analyze developmental and spatial expression of CeCYP-16 to gain insight into the potential natural substrate(s) for these enzymes. All reagents, kits, and bacterial strains used in cloning, expression, and sequencing (described below) were obtained from New England Biolabs (Beverly, MA) and used as described by the manufacturer, unless otherwise specified. A partial cDNA clone (552 bp) encoding a putative cyclophilin was isolated from an O. volvulus L3 stage Lambda Uni-ZAP XR cDNA library kindly provided by Dr. Steven Williams. The library was screened by hybridization (22.Grandea A.G.d. Tuyen L.K. Asikin N. Davis T.B. Philipp M. Cohen C. McReynolds L.A. Mol. Biochem. Parasitol. 1989; 35: 31-41Crossref PubMed Scopus (27) Google Scholar) with a 1300-bp genomic fragment ofO. volvulus furin (a gift from Dr. Jingmin Jin). The fragment was radiolabeled with [α-32P]dATP by oligonucleotide random priming using the NEBlot kit. Plaque lifts (23.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) were hybridized in 6× SSC at 60 °C overnight with the32P-labeled probe (1 × 106 cpm/ml hybridization solution). After hybridization, the plaque lifts were washed twice at room temperature and once at 60 °C for 30 min each in 2× SSC containing 0.1% SDS. After plaque purification of nine clones, the Bluescript phagemids were excised from Lambda ZAP (Stratagene, La Jolla, CA). After sequencing, the phagemid clone with the smallest insert (552 bp; fragment B) was identified as a fragment of a putative O. volvulus cyclophilin (OvCYP-16) by using the National Center for Biotechnology Information BLAST program. This clone was isolated due to significant homology in a short stretch of sequence (39 nucleotides) encoding part of the catalytic domain of each enzyme (data not shown). To obtain a full-length cDNA of OvCYP-16, two primers, 5′-gcgagtggacattcctttgacc-3′ (antisense) and 5′-ccattcaatgatattgttcc-3′ (sense), were designed using the sequence derived from the 5′ and 3′ ends of the partial cDNA. Two PCR products, designated fragment A (241 bp) and fragment C (322 bp), were obtained by performing thermal cycling on the O. volvulus L3 cDNA library with the following primer pairs: antisense primer/T3 primer, 5′-aattaaccctcactaaaggg-3′; and sense primer/T7 primer, 5′-gtaatacgctcactatagggc-3′. Typical PCR reactions contained 2 μl ofO. volvulus L3 cDNA library stock boiled for 10 min before use, 2 units of Vent polymerase, 1× thermal polymerase buffer, 6 mm MgSO4, 0.2 mm deoxynucleotide triphosphate, and 250 nm of each primer. The reactions were heated at 95 °C for 5 min, followed by 20 cycles of 95 °C for 1 min, 55 °C for 30 s, and 72 °C for 1 min. PCR products were purified with the QIAquick PCR purification kit (Qiagen) and then subcloned into the pGEM-T vector (Promega, Madison, WI) for DNA sequence analysis. Fragments A and C were digested separately withEcoRI/ClaI and BtgI/Xhol, respectively. The 459-bp partial OvCYP-16 cDNA (fragment B) was released from the Bluescript phagemid with ClaI andBtgI. These three fragments (fragments A, B, and C) were ligated into pUC19 digested with EcoRI and SalI. The ligation was performed at 16 °C overnight. Deduced amino acid sequences were aligned and compared using the Clustal method (24.Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar), and searches for homologies to other cyclophilins were performed using the National Center for Biotechnology Information BLAST program. A genomic DNA sequence encoding the putative homolog ofOvCYP-16 was identified in the C. elegans cosmid Y17G7B using the C. elegans BLAST program (Sanger Institute, Cambridge, United Kingdom). This genomic DNA sequence was used to search the C. elegansexpressed sequence tag data base, and a clone (yk648d4) was found that likely represented a full-length cDNA (CeCYP-16) homolog of OvCYP-16. Thermal cycling primers were designed to enable cloning of OvCYP-16 into plasmid pMAL-c2X to generate a fusion with maltose-binding protein (MBP). The forward primer corresponded to the open reading frame of OvCYP-16 and had the sequence 5′-atgtcaaacgttattatcgaattcggc-3′, generating a 5′ blunt end. The reverse primer, 5′-cccaagcttctattcaactttattgaagaccgc-3′, corresponded to the 3′ end of the gene including a downstream termination codon and a HindIII recognition site. 50-μl PCR reactions were carried out using 0.1 μg of the pUC19-OvCYP-16 construct as template, 2 units of Vent DNA polymerase, 5 μl of 10× thermal polymerase buffer, 5 mmMgSO4, 0.2 mm deoxynucleotide triphosphate, and 250 nm of each primer. The thermal cycling conditions used were 95 °C for 5 min, followed by 25 cycles of 95 °C for 1 min, 60 °C for 30 s, and 72 °C for 1 min. The reaction product was purified and digested with HindIII before ligation into pMAL-c2X digested with XmnI and HindIII. Plasmid DNA was isolated, and the insert was sequenced in both directions using the CircumVent thermal cycle dideoxy DNA sequencing kit. Production and purification of the MBP fusion protein were as described by the manufacturer. The cDNA clone yk648d4 (GenBank™ accession numberAV195981) was obtained from Dr. Yuji Kohara, and two thermal cycling primers were designed to subclone CeCYP-16 into pMAL-c2X for protein expression. The forward primer (5′-atgagtaatcaatatatcaacgagccg-3′) corresponded to the open reading frame of CeCYP-16 preceded by an ATG codon. The reverse primer (5′-cccaagcttctaaaccttattaaaaacggcc-3′) corresponded to the 3′ end of the gene and included a downstream termination codon and aHindIII recognition site. PCR reactions (50 μl) were performed as described above using 3 μl of yk648d4 Lambda DNA stock as template. PCR products were purified and digested withHindIII before ligation into pMAL-c2X digested withXmnI and HindIII. The recombinant plasmid DNA was isolated, and the insert was sequenced in both directions to ensure authenticity. Production and purification of the MBP fusion protein were as described by the manufacturer. Site-directed mutagenesis of the PPIase domain of the previously described B. malayi BmCYP-1 (19.Page A.P. Landry D. Wilson G.G. Carlow C.K. Biochemistry. 1995; 34: 11545-11550Crossref PubMed Scopus (38) Google Scholar) was accomplished by the method of Kunkel (25.Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4903) Google Scholar). The histidine residue (132) ofBmCYP-1 was substituted with aspartic acid using the following mutagenic primer: 5′-attactacaacacctgcgccagatctcaatatatccatgtggtatttgg-3′. The bases encoding the mutated amino acid are underlined. Mutagenesis of BmCYP-1 was verified using the CircumVent thermal cycle dideoxy DNA sequencing kit. The protocol used for the production and purification of BmCYP-1 (H132D) was as described previously (26.Ma D. Carlow C.K. Mol. Biochem. Parasitol. 1998; 92: 361-365Crossref PubMed Scopus (6) Google Scholar). The various filarial PPIases were prepared and purified as described previously (19.Page A.P. Landry D. Wilson G.G. Carlow C.K. Biochemistry. 1995; 34: 11545-11550Crossref PubMed Scopus (38) Google Scholar, 21.Hong X. Ma D. Carlow C.K.S. Mol. Biochem. Parasitol. 1998; 91: 353-358Crossref PubMed Scopus (9) Google Scholar, 27.Ma D. Hong X. Raghavan N. Scott A.L. McCarthy J.S. Nutman T.B. Williams S.A. Carlow C.K. Mol. Biochem. Parasitol. 1996; 79: 235-241Crossref PubMed Scopus (23) Google Scholar). DNA sequences were analyzed using the Genetics Computer Group (Madison, WI) software. Pairwise identity comparisons of OvCYP-16 and CeCYP-16 to other cyclophilins were performed using the program GAP. Alignment of the derived amino acid sequence of the enzyme domains ofOvCYP-16, CeCYP-16, and other cyclophilins was made using the program PILEUP (gap weight = 3.0, gap length weight = 0.1). Phylogenetic tree analysis was performed using the Clustal method (24.Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar) with the PAM250 residue weight table in the Magalign program in DNA star. The PPIase activity of OvCYP-16 and CeCYP-16 fusion proteins was determined by measuring thecis-trans conversion of 13 available synthetic peptide substrates of the general structureN-succinyl-Ala-Xaa-Pro-Phe-p-nitroanilide (Bachem), where Xaa is any of the 12 amino acids listed in Table I. Reactions were performed at 10 °C and monitored at 0.3-s intervals at 400 nm using a Beckman DU 640 spectrophotometer. Pseudo-first-order rate kinetics were calculated using the following formula:k obs = (k cat/K m)[E].Table ISubstrate specificity of nematode cyclophilins toward various peptide substratesAla-Xaa-Pro-Phek cat/K mBmCYP-1BmCYP-2DiCYP-3BmCYP-1 (H132D)OvCYP-16CeCYP-16Small -Ala-130,000 ± 24,894610,000 ± 97,045130,000 ± 16,7743400 ± 000 -Gly-11,360 ± 36651,040 ± 10008571 ± 13531190 ± 26900Hydrophobic -Val-116,700 ± 19,450558,200 ± 706671,430 ± 20414365 ± 41202020 ± 110 -Leu-690,000 ± 25,555590,000 ± 112,38048,000 ± 9802778 ± 214516 ± 121010 ± 55 -Ile-258,700 ± 2781733,700 ± 32,46552,380 ± 12,6985532 ± 102412 ± 14758 ± 8 -Nle-195,600 ± 22,678988,900 ± 21,343140,500 ± 52823571 ± 73516 ± 9631 ± 6Aromatic -Phe-41,000 ± 837265,800 ± 019,050 ± 5721389 ± 000 -Trp-6300 ± 1890160,000 ± 13,3338600 ± 2457198 ± 000 *-Phe-9434 ± 30441,400 ± 11193571 ± 1231386 ± 320309 ± 17505 ± 34Acidic and amide -Glu-60,000 ± 5916430,000 ± 494333,000 ± 37872100 ± 75310 ± 15760 ± 23 -Gln-110,410 ± 7066733,700 ± 106,72064,290 ± 17861389 ± 347103 ± 211010 ± 194Basic -His-1894 ± 35885,100 ± 20,023191 ± 29000 -Lys-34,700 ± 890430,600 ± 47,84447,620 ± 1642794 ± 45800Xaa is shown in the first column (Nle is the artificial amino acid norleucine). Asterisk denotes the tripeptide substrate Suc-F-P-F-pNA. First-order rate kinetics were calculated using the formula:k obs = (k cat/K m)[E]. All enzymes are fusion proteins with MBP. Open table in a new tab Xaa is shown in the first column (Nle is the artificial amino acid norleucine). Asterisk denotes the tripeptide substrate Suc-F-P-F-pNA. First-order rate kinetics were calculated using the formula:k obs = (k cat/K m)[E]. All enzymes are fusion proteins with MBP. To determine inhibition of enzyme activity by CsA (Sigma), recombinant enzyme (30 nm to 10 μm) was preincubated for 1 h at 4 °C with CsA (1 nm to 5 μm), and the assay was performed as described above. Data were fitted into the following equation: k obs =k obs*/(1 + [CsA]/IC50), wherek obs* is k obs in the absence of CsA (19.Page A.P. Landry D. Wilson G.G. Carlow C.K. Biochemistry. 1995; 34: 11545-11550Crossref PubMed Scopus (38) Google Scholar). Wild-type C. elegans were obtained from the Caenorhabditis Genetics Center (St. Paul, MN). Worms were maintained on nematode growth medium agar plates withEscherichia coli (OP50) as a food source (28.Brenner S. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar). A CYP-16::GFP expression construct (pIP10) was generated as follows: A 1213-bp region upstream of the CYP-16 start site was PCR-amplified from C. elegans genomic DNA using the following primers: 5′-aaggcgtctagacgccggctgaaatattcac-3′ and 5′-cagtcgccaagcttctcctgaaatagtcgtttcg-3′. Amplification products were subcloned using the TOPO TA® Dual Promotor Cloning Kit (Invitrogen) according to the manufacturer's instructions. Restriction sites near the 5′ ends of the primers (XbaI and HindIII) were then used to excise the insert and clone it into a multicloning site upstream of GFP in the pPD95.75 vector (29.Fire A. Harrison S.W. Dixon D. Gene (Amst.). 1990; 93: 189-198Crossref PubMed Scopus (529) Google Scholar). 50–75 ng/μl pIP10 was microinjected into the gonad of adult wild-type worms along with 100 ng/μl pRF-4 plasmid containing the dominant marker, rol-6 (su1006). Two independent worm lines demonstrating the rolling marker phenotype were isolated and designated IP102: nbEx3[rol-6(su1006)CYP-16::GFP] and IP103: nbEx4[rol-6(su1006)CYP-16::GFP]. Rolling worms of all stages were observed by fluorescence microscopy to determine where GFP was expressed. The complete cDNA of OvCYP-16 encoding an O. volvuluscyclophilin is 823-bp long and possesses an open reading frame of 669 bp, with a putative initiation codon at position 41 (GenBank™ accession number AF017738). The conserved nematode-specific 22-nucleotide spliced leader sequence (30.Takacs A.M. Denker J.A. Perrine K.G. Maroney P.A. Nilsen T.W. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7932-7936Crossref PubMed Scopus (59) Google Scholar) is located at the 5′ end, 27 nucleotides upstream of the putative start codon. The 3′-untranslated region is 114-bp long with a putative polyadenylation signal (AATAAA) at position 784. The translated protein (223 amino acids) has a predicted molecular mass of 25.2 kDa with a pI of 6.75 and contains a PPIase domain (Figs. 1 and3).Figure 3Alignment of the deduced amino acid sequences of various cyclophilins. The amino acid sequences of various cyclophilins are denoted as follows (GenBank™accession numbers are indicated in parentheses): D. immitis, DiCYP-1 (U70884), DiCYP-2 (U47813),DiCYP-3 (AF000668); B. malayi, BmCYP-1 (L37292),BmCYP-2 (U47811), and BmCYP-4 (AJ000916);O. volvulus, OvCYP-1 (U70827), OvCYP-2 (U47812), OvCYP-4 (AJ000917), and OvCYP-16 (AF017738); human nuclear-specific cyclophilin, HuCYP60 (U37219); human cyclophilin A, HuCYPA (X52851); human natural killer cell cyclophilin, HuCYPNK (L04288); and C. elegans, CeCYP-4 (U36187), CeCYP-7 (U27559), CeCYP-8 (U31078), and CeCYP-16 (AF393636). C-terminal asterisks indicate translational terminations. In the CYP-1 and CYP-3 sequences, the additional C-terminal residues of CYP-1 and the additional N- and C-terminal residues of CYP-3 are not shown. Dashes indicate residues identical to the corresponding residue in OvCYP-16.Dots denote gaps. The residues important in CsA binding (33.Pflugl G. Kallen J. Schirmer T. Jansonius J.N. Zurini M.G. Walkinshaw M.D. Nature. 1993; 361: 91-94Crossref PubMed Scopus (201) Google Scholar) are indicated with a #.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The CeCYP-16 cDNA is 669 bp in length (GenBank™ accession number AF393636) and codes for a 223-amino acid protein with a predicted molecular mass of 25.2 kDa with a pI of 7.44 and contains a PPIase domain (Figs. 1 and 3). The derived amino acid sequence (Figs. 1 and 3) and phylogenetic tree analyses (Fig. 2) indicate thatOvCYP-16 and CeCYP-16 are novel cyclophilins and homologs. The proteins are the same size (223 amino acids), possess a short tail that is KR-rich, and share 75% similarity and 64% identity. OvCYP-16 possesses only 48%, 43%, and 46% similarity, respectively, to the previously described cyclophilinsOvCYP-1 (20.Hong X. Ma D. Page A.P. Kumar S. Carlow C.K. Exp. Parasitol. 1998; 88: 246-251Crossref PubMed Scopus (9) Google Scholar), OvCYP-2 (27.Ma D. Hong X. Raghavan N. Scott A.L. McCarthy J.S. Nutman T.B. Williams S.A. Carlow C.K. Mol. Biochem. Parasitol. 1996; 79: 235-241Crossref PubMed Scopus (23) Google Scholar), andOvCYP-4 (31.Page A.P. Winter A.D. Mol. Biochem. Parasitol. 1998; 95: 215-227Crossref PubMed Scopus (24) Google Scholar) from O. volvulus. CeCYP-16 is a novel C. elegans cyclophilin and shares 35–56% similarity to the 17 documented cyclophilins in the C. elegans genome. It does not belong to any of the previously characterized groups defined by Page et al. (32.Page A.P. MacNiven K. Hengartner M.O. Biochem. J. 1996; 317: 179-185Crossref PubMed Scopus (60) Google Scholar). A data base search revealed an absence of mammalian homologs of theOvCYP-16 and CeCYP-16 cyclophilins (Figs. 2 and3). In contrast, homologs of the filarial CYP-1, CYP-2, and CYP-3/4 proteins are present in humans (Figs. 2 and3). There are 13 residues that constitute the CsA-binding site of human cyclophilin A (33.Pflugl G. Kallen J. Schirmer T. Jansonius J.N. Zurini M.G. Walkinshaw M.D. Nature. 1993; 361: 91-94Crossref PubMed Scopus (201) Google Scholar) (Fig. 3, #), and one of these residues (tryptophan 121) is essential for drug binding (34.Liu J. Chen C.M. Walsh C.T. Biochemistry. 1991; 30: 2306-2310Crossref PubMed Scopus (154) Google Scholar). In the OvCYP-16 andCeCYP-16 cyclophilins, 9 and 11, respectively, of the 13 residues are conserved. However, unlike any other cyclophilins described to date, the tryptophan residue is substituted with a Glu124 (C. elegans) or Asp123(O. volvulus) amino acid (Fig. 3). The characteristics of recombinant OvCYP-16- andCeCYP-16-MBP fusion proteins were examined using 13 different synthetic peptides of the general structureN-succinyl-Ala-Xaa-cis-Pro-Phe-p-nitroanilide, where Xaa is any of the 12 amino acids listed in Table I. The tripeptide substrate Suc-Phe-Pro-Phe-pNA was also evaluated. MutantBmCYP-1 (H132D) and the previously characterized filarial cyclophilins BmCYP-1, BmCYP-2, andDiCYP-3 were included for comparison as MBP fusion proteins.BmCYP-1, BmCYP-2, and DiCYP-3 are active PPIases, both as a MBP fusion protein and in a purified (minus MBP) form (19.Page A.P. Landry D. Wilson G.G. Carlow C.K. Biochemistry. 1995; 34: 11545-11550Crossref PubMed Scopus (38) Google Scholar, 21.Hong X. Ma D. Carlow C.K.S. Mol. Biochem. Parasitol. 1998; 91: 353-358Crossref PubMed Scopus (9) Google Scholar, 27.Ma D. Hong X. Raghavan N. Scott A.L. McCarthy J.S. Nutman T.B. Williams S.A. Carlow C.K. Mol. Biochem. Parasitol. 1996; 79: 235-241Crossref PubMed Scopus (23) Google Scholar). The catalytic efficiency (k cat/K m) of the substrates varied, and a distinct profile was obtained for each filarial andC. elegans cyclophilin (Table I). Both OvCYP-16 and CeCYP-16 proteins were found to possess a low level of PPIase activity that was only detectable using relatively large amounts of protein (namely, 9.7 μm OvCYP-16 and 4 μm CeCYP-16, respectively) in the presence of specific substrates (Fig. 4). The highest level of PPIase activity (k cat/K m) forOvCYP-16 and CeCYP-16 was 5.2 × 102 (Ala-Leu-Pro-Phe or Ala-Nle-Pro-Phe) and 2 × 103 (Ala-Val-Pro-Phe), respectively. The profile observed for the mutant PPIase (5 μm BmCYP-1, H132D) was more similar to OvCYP-16 and CeCYP-16 and was dramatically reduced (2.7 × 103 Ala-Leu-Pro-Phe) compared with wild-type (6.9 × 105) (Table I). To determine the sensitivity of OvCYP-16,CeCYP-16, and mutant BmCYP-1 (H132D) PPIases to CsA, recombinant enzyme (10 μm) was preincubated with varying concentrations of CsA (10 nm to 5 μm) at 4 °C for 1 h before the assays were performed as described above. The previously characterized filarial PPIases,BmCYP-1, BmCYP-2, and DiCYP-3, were included for comparison. Appropriate synthetic substrates were used in the assays, namely,N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide forOvCYP-16 and CeCYP-16 andN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide forBmCYP-1, BmCYP-1 (H132D), BmCYP-2, andDiCYP-3, respectively. No inhibition of the PPIase activities of DiCYP-3, OvCYP-16, CeCYP-16, or mutant BmCYP-1 (H132D) was observed, even at CsA concentrations as high as 5 μm (Fig. 5). In contrast, nanomolar concentrations of CsA are sufficient to inhibit 50% of the activities ofBmCYP-1 and BmCYP-2. Transgenic lines IP102 and IP103 were obtained after coinjection of pIP10 and pRF-4 into the gonad of adult worms. Consistent GFP expression patterns were observed in these lines throughout the intestine, with particularly strong fluorescence in the anterior and posterior ends, in all the larval stages and adults (Fig. 6). An exception was found in the dauer stage, where fluorescence was observed in both the cell bodies and processes of the ventral chord motor neurons but was absent from the intestine (Fig. 6). Cyclophilins appear to have undergone proliferation and extensive diversification in filarial worms. In contrast to other parasites that appear to only express homologs of human CypA, the filariae possess, in addition, highly distinctive cyclophilins. The three filarial PPIases (CYP-1, CYP-2, and CYP-3) analyzed in detail thus far differ in size and display unique substrate preferences and a range of sensitivity to inhibition with CsA (21.Hong X. Ma D. Carlow C.K.S. Mol. Biochem. Parasitol. 1998; 91: 353-358Crossref PubMed Scopus (9) Google Scholar, 27.Ma D. Hong X. Raghavan N. Scott A.L. McCarthy J.S. Nutman T.B. Williams S.A. Carlow C.K. Mol. Biochem. Parasitol. 1996; 79: 235-241Crossref PubMed Scopus (23) Google Scholar, 32.Page A.P. MacNiven K. Hengartner M.O. Biochem. J. 1996; 317: 179-185Crossref PubMed Scopus (60) Google Scholar). The other cyclophilins (designated CYP-4, CYP-5, and CYP-10) identified from O. volvulus as potential vaccine and drug target candidates after immunoscreening of cDNA libraries and expressed sequence tag analysis (35.Lizotte-Waniewski M. Tawe W. Guiliano D.B. Lu W. Liu J. Williams S.A. Lustigman S. Infect. Immun. 2000; 68: 3491-3501Crossref PubMed Scopus (82) Google Scholar) belong to previously defined groups (19.Page A.P
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