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A Peptidyl-prolyl cis/trans-Isomerase (Cyclophilin G) in Regulated Secretory Granules

1997; Elsevier BV; Volume: 272; Issue: 45 Linguagem: Inglês

10.1074/jbc.272.45.28615

1083-351X

Yoshie Takaki, Tatsushi Muta, Sadaaki Iwanaga,

Peptidase Inhibition and Analysis

A 27-kDa protein (p27) in horseshoe crab hemocyte that cross-reacts with antiserum against a β-glucan-sensitive protease zymogen was purified to homogeneity, and its cDNA was cloned. The 1.7-kilobase pair cDNA contains an open reading frame of 660 base pairs, encoding a 23-amino acid signal sequence followed by a mature protein of 197 residues. The sequence of p27 exhibits strong similarity to that of cyclophilin B, a peptidyl-prolylcis/trans-isomerase. p27 exhibits isomerase activity with ak cat /K m of 0.18 μm−1 s−1 for a peptide substrate; this activity is inhibited by cyclosporin A but is not affected by FK506. Although the p27 precursor possesses an amino-terminal secretory hydrophobic signal sequence, unlike other cyclophilin B molecules, it lacks a conserved carboxyl-terminal endoplasmic reticulum retention signal and it contains a central 8-amino acid insertion. Although p27 is secreted into the culture media of transiently expressed COS cells, it is not detected in horseshoe crab hemolymph plasma but rather is localized to the hemocyte large granules, the regulated secretory granules that are exocytosed upon stimulation. These results indicate that p27 is a new peptidyl-prolylcis/trans-isomerase in the regulated secretory granules, and is thus designated cyclophilin G. This first report of a cyclophilin homologue in the secretory granule of the horseshoe crab hemocyte suggests that such chaperon-like proteins may constitute a key quality control system for stored proteins in exocytotic granules. A 27-kDa protein (p27) in horseshoe crab hemocyte that cross-reacts with antiserum against a β-glucan-sensitive protease zymogen was purified to homogeneity, and its cDNA was cloned. The 1.7-kilobase pair cDNA contains an open reading frame of 660 base pairs, encoding a 23-amino acid signal sequence followed by a mature protein of 197 residues. The sequence of p27 exhibits strong similarity to that of cyclophilin B, a peptidyl-prolylcis/trans-isomerase. p27 exhibits isomerase activity with ak cat /K m of 0.18 μm−1 s−1 for a peptide substrate; this activity is inhibited by cyclosporin A but is not affected by FK506. Although the p27 precursor possesses an amino-terminal secretory hydrophobic signal sequence, unlike other cyclophilin B molecules, it lacks a conserved carboxyl-terminal endoplasmic reticulum retention signal and it contains a central 8-amino acid insertion. Although p27 is secreted into the culture media of transiently expressed COS cells, it is not detected in horseshoe crab hemolymph plasma but rather is localized to the hemocyte large granules, the regulated secretory granules that are exocytosed upon stimulation. These results indicate that p27 is a new peptidyl-prolylcis/trans-isomerase in the regulated secretory granules, and is thus designated cyclophilin G. This first report of a cyclophilin homologue in the secretory granule of the horseshoe crab hemocyte suggests that such chaperon-like proteins may constitute a key quality control system for stored proteins in exocytotic granules. The horseshoe crab (or limulus) is an arthropod with a unique innate humoral immune system that differs substantially from the immunoglobulin-based acquired immune system of vertebrates (1Muta T. Iwanaga S. Rinkevich B. Müller W.E.G. Progress in Molecular & Subcellular Biology: Invertebrate Immunology. 15. Springer Verlag, Heidelberg1996: 154-189Google Scholar, 2Muta T. Iwanaga S. Curr. Opin. Immunol. 1996; 8: 41-47Crossref PubMed Scopus (229) Google Scholar). A single type of hemocyte, which comprises approximately 99% of the total, plays a major role in the host defense system of this animal (3Mürer E.H. Levin J. Holme R. J. Cell. Physiol. 1975; 86: 533-542Crossref PubMed Scopus (59) Google Scholar,4Toh Y. Mizutani A. Tokunaga F. Muta T. Iwanaga S. Cell Tissue Res. 1991; 266: 137-147Crossref Scopus (66) Google Scholar). These hemocytes contain two types of granules, larger (L-) 1The abbreviations used are: L-granule, larger granule; S-granule, smaller granule; LPS, lipopolysaccharide; CyP, cyclophilin; PPIase, peptidyl-prolyl cis/trans-isomerase; CyPA, cyclophilin A; CsA, cyclosporin A; CyPB, cyclophilin B; ER, endoplasmic reticulum; GST, glutathione S-transferase; PCR, polymerase chain reaction; Suc-Ala-Ala-Pro-Phe-MCA, succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanine 4-methylcoumaryl-7-amide; CyPG, cyclophilin G; PAGE, polyacrylamide gel electrophoresis. and smaller (S-) granules, both of which are exocytosed by a trace amount of bacterial endotoxin, lipopolysaccharide (LPS), when exposed to Gram-negative bacteria (3Mürer E.H. Levin J. Holme R. J. Cell. Physiol. 1975; 86: 533-542Crossref PubMed Scopus (59) Google Scholar, 4Toh Y. Mizutani A. Tokunaga F. Muta T. Iwanaga S. Cell Tissue Res. 1991; 266: 137-147Crossref Scopus (66) Google Scholar, 5Levin J. Bang F.B. Bull. Johns Hopkins Hosp. 1964; 115: 265-274PubMed Google Scholar, 6Ornberg R.L. Reese T.S. Prog. Clin. Biol. Res. 1979; 29: 125-130PubMed Google Scholar). These granules contain LPS- and β-glucan-sensitive coagulation factors, lectins, and antimicrobial proteins, which after exocytosis participate in the establishment of proteolytic cascades that ultimately immobilize and kill invading pathogens (1Muta T. Iwanaga S. Rinkevich B. Müller W.E.G. Progress in Molecular & Subcellular Biology: Invertebrate Immunology. 15. Springer Verlag, Heidelberg1996: 154-189Google Scholar, 7Kawabata S. Muta T. Iwanaga S. Söderhäll K. Iwanaga S. Vasta G.R. New Direction in Invertebrate Immunology. SOS Publications, Fair Haven, NJ1996: 255-283Google Scholar). Recently, we purified a serine protease zymogen factor G from hemocytes (8Muta T. Seki N. Takaki Y. Hashimoto R. Oda T. Iwanaga A. Tokunaga F. Iwanaga S. J. Biol. Chem. 1995; 270: 892-897Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The autocatalytic activation of factor G by a trace amount of (1,3)-β-d-glucan on the surface of fungi triggers a protease cascade that culminates in hemolymph coagulation. Factor G is a heterodimeric protein composed of subunits α and β. Subunit α is a novel mosaic protein involved in β-glucan recognition, whereas subunit β is a 37-kDa serine protease zymogen (9Seki N. Muta T. Oda T. Iwaki D. Kuma K. Miyata T. Iwanaga S. J. Biol. Chem. 1994; 269: 1370-1374Abstract Full Text PDF PubMed Google Scholar). During immunoblotting analyses of horseshoe crab hemocyte lysate with subunit β-specific antisera, we found a 27-kDa protein (p27) that cross-reacts with subunit β antiserum. Thus, anticipating a related serine protease, we initiated a study of p27. In this study, we purified p27 and cloned its cDNA. The deduced amino acid sequence of p27 unexpectedly revealed that it was a cyclophilin (CyP) homologue with peptidyl-prolylcis/trans-isomerase (PPIase) activity. A PPIase, which catalyzes the cis/trans-isomerization of Xaa-Pro bonds, was first isolated from porcine kidney cortex (10Fischer G. Bang H. Mech C. Biomed. Biochim. Acta. 1984; 43: 1101-1111PubMed Google Scholar), and sequence analysis revealed that it was identical to cyclophilin A (CyPA), a cytosolic protein isolated from bovine thymocytes which binds to the immunosuppressant cyclosporin A (CsA) (11Handschumacher R.E. Harding M.W. Rice J. Drugge R.J. Speicher D.W. Science. 1984; 226: 544-547Crossref PubMed Scopus (1457) Google Scholar, 12Takahashi N. Hayano T. Suzuki M. Nature. 1989; 337: 473-475Crossref PubMed Scopus (940) Google Scholar, 13Fischer G. Wittmann-Liebold B. Lang K. Kiefhaber T. Schmid F.X. Nature. 1989; 337: 476-478Crossref PubMed Scopus (1212) Google Scholar). CyPA, by making a complex with CsA, suppresses immune reactions by inhibiting a protein phosphatase, calcineurin (14Liu J. Farmer Jr., J.D. Lane W.S. Friedman J. Weissman I. Schreiber S.L. Cell. 1991; 66: 807-815Abstract Full Text PDF PubMed Scopus (3627) Google Scholar). Although CsA inhibits the PPIase activity of CyPA, its calcineurin inhibitory activity is independent of its PPIase activity. Other non-cytosolic CyP homologues with PPIase activity have been found to participate in the processing of secretory proteins (15Galat A. Eur. J. Biochem. 1993; 216: 689-707Crossref PubMed Scopus (316) Google Scholar). Cyclophilin B (CyPB) is mainly found in the endoplasmic reticulum (ER) and is thought to have a role in the folding of nascent peptide chains (16Hasel K.W. Glass J.R. Godbout M. Sutcliffe J.G. Mol. Cell. Biol. 1991; 11: 3484-3491Crossref PubMed Scopus (113) Google Scholar, 17Freskgard P.O. Bergenhem N. Jonsson B.H. Svensson M. Carlsson U. Science. 1992; 258: 466-468Crossref PubMed Scopus (185) Google Scholar). The ninaA gene product, a CyP homologue inDrosophila photoreceptor cells, co-localizes with rhodopsin in secretory transport vesicles, suggesting a role in protein trafficking and macromolecular assembly (18Baker E.K. Colley N.J. Zuker C.S. EMBO J. 1994; 13: 4886-4895Crossref PubMed Scopus (276) Google Scholar). This study characterizes a new 27-kDa CyP homologue, cyclophilin G, which is located in the L-granule, one of two types of regulated secretory granules in horseshoe crab hemocytes. This is the first report demonstrating the presence of a PPIase in regulated secretory granules, and suggests an expanded role for CyPs in maintaining the integrity of stored secretory proteins. Hemocyte lysate of the Japanese horseshoe crabs (Tachypleus tridentatus) was prepared as described previously (19Nakamura T. Morita T. Iwanaga S. J. Biochem. (Tokyo). 1985; 97: 1561-1574Crossref PubMed Scopus (73) Google Scholar). L- and S-granules were purified from the hemocytes by the method described in Ref. 20Shigenaga T. Takayenoki Y. Kawasaki S. Seki N. Muta T. Toh Y. Ito A. Iwanaga S. J. Biochem. (Tokyo). 1993; 114: 307-316Crossref PubMed Scopus (44) Google Scholar. Sephadex G-150, CM-Sepharose CL-6B, Superdex 75 HR 10/30, and glutathione-Sepharose 4B were products of Pharmacia Fine Chemicals (Uppsala, Sweden). α-Chymotrypsin was obtained from Worthington and succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanine 4-methylcoumaryl-7-amide (Suc-Ala-Ala-Pro-Phe-MCA) was from Peptide Institute Inc. (Osaka, Japan). Oligonucleotides were synthesized by Sawady Technology (Tokyo, Japan). Cyclosporin A and FK506 were kindly provided by Sandoz AG (Basel, Switzerland) and Fujisawa Pharmaceutical Co., Ltd. (Ibaraki, Japan). λgt22A horseshoe crab hemocyte cDNA library was prepared by using a Superscript™ Lambda system (Life Technologies, Inc.). The rabbit antiserum against factor G subunits and p27 was prepared by using glutathione S-transferase (GST)-fusion proteins of a factor G fragment and recombinant p27 as antigens, respectively. All other chemicals were of analytical grade or the highest quality commercially available. Dextran sulfate-Sepharose CL-6B column (inner diameter, 5.0 × 14 cm) chromatography of the lysate (590 ml) derived from 97.5 g (wet weight) of the horseshoe crab hemocytes was performed as described (8Muta T. Seki N. Takaki Y. Hashimoto R. Oda T. Iwanaga A. Tokunaga F. Iwanaga S. J. Biol. Chem. 1995; 270: 892-897Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The 2.0 m NaCl fractions containing p27 were lyophilized, and applied to a Sephadex G-150 column (inner diameter, 3.2 × 98 cm) in 20 mmsodium acetate (pH 5.5) containing 0.5 m NaCl. Fractions containing p27 were pooled, diluted 10-fold with 20 mmsodium acetate (pH 5.5), and then applied to a CM-Sepharose CL-6B (inner diameter, 1.0 × 5.0 cm) equilibrated with the same buffer. After washing with 20 mm sodium acetate (pH 5.5) containing 0.05 m NaCl, bound proteins were eluted by a linear salt gradient from 0.05 m to 0.4 m NaCl in 20 mm sodium acetate (pH 5.5). Further purification of p27 was performed by a Superdex 75 HR 10/30 chromatography in 50 mmTris-HCl (pH 8.0) containing 0.1 m NaCl. SDS-PAGE was performed according to the method of Laemmli (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207478) Google Scholar). Immunoblotting was carried out by the standard method with antiserum against factor G subunit β or p27 (22Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 471-510Google Scholar). The immunoreactive proteins were visualized using horseradish peroxidase-conjugated goat anti-rabbit IgG (Nippon Bio-Rad Laboratories) and an ECL™ Western blotting detection system (Amersham Japan, Tokyo). p27 was further purified by reversed-phase high performance liquid chromatography on a Phenyl-5PW RP column (inner diameter, 4.6 × 75 mm) (Tosoh, Tokyo, Japan) for sequence analysis. Purified p27 was reduced,S-alkylated by iodoacetamide, and digested with lysyl endopeptidase (Wako Pure Chemical Industries Ltd., Tokyo, Japan). The generated peptides were separated on a Chemcosorb 5-ODS-H column (inner diameter, 2.1 × 150 mm) (Chemco Scientific Co., Ltd., Osaka, Japan). The amino-terminal sequence analyses of each peptide and intact p27 were carried out with an Applied Biosystems 477A or 473A gas-phase protein sequencer. Amino acid analysis was performed on a PICO·TAG system (Waters). Two degenerated oligonucleotide primers (5′-CTGAATTCGTNTAYTTYGAYATHAC-3′ and 5′- GCGAATTCTTRAARTTYTCRTCNGG-3′), each of which contained an EcoRI site (underlined), were synthesized based on the partial amino acid sequences of p27. A partial cDNA fragment for p27 was amplified by polymerase chain reaction (PCR) with the first strand cDNA derived from the horseshoe crab hemocyte as a template, 100 pmol each of the oligonucleotide primers, and 2.5 units ofPfu polymerase (Stratagene). The generated DNA fragment containing a partial p27 cDNA fragment was used as a probe for the screening of λgt22A horseshoe crab hemocytes cDNA library (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A positive clone was isolated and its cDNA fragment was subcloned into pBluescript II SK(+) (Stratagene). The nucleotide sequence analysis was carried out on ABI PRISM™ 373A or 377 DNA sequencer using ABI PRISM™ dye terminator cycle sequencing ready reaction kit. All sequences were confirmed by sequencing overlapping fragments. A computer-assisted homology search of p27 was performed in the DDBJ homology search system using the programs FASTA version 3.0 (24Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9391) Google Scholar) and BLAST version 1.4.9 (25Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71339) Google Scholar). PPIase assay was performed by the chymotrypsin-coupled assay (12Takahashi N. Hayano T. Suzuki M. Nature. 1989; 337: 473-475Crossref PubMed Scopus (940) Google Scholar, 26Heitman J. Koller A. Cardenas M.E. Hall M.N. Methods: Companion Methods Enzymol. 1993; 5: 178-187Crossref Scopus (22) Google Scholar). The sample was preincubated at 10 °C for 2 min with 6.7 μm Suc-Ala-Ala-Pro-Phe-MCA in 0.2 m Tris-HCl (pH 8.0), in the presence and absence of CsA or FK506, in a total volume of 1.485 ml. The reaction was started by adding 15 μl of 0.1 mm α-chymotrypsin, and the fluorescence was monitored for 5 min with excitation at 380 nm and emission at 440 nm. Under these assay conditions, the rate constant for the cisto trans isomerization can be obtained by measuring the rate constant of the substrate hydrolysis at the slow phase. The apparent first order rate constant (k app =k H2O +k enz) was determined from the slope of the Guggenheim plot (27Guggenheim E.A. Phil. Mag. Ser. VII. 1926; 2: 538-543Crossref Google Scholar). k H2O is the rate constant in the absence of PPIase, andk enz is that catalyzed by PPIase. One unit of PPIase activity was defined as k enz(s−1). Thek cat /K m was calculated from the equation, k enz/[E], where [E] is the concentration of PPIase. A cDNA fragment encoding the mature p27 was created by a PCR and inserted into aBamHI site of pGEX-2T expression vector (Pharmacia Biotech Inc.). The expression and purification of the GST-p27 fusion protein was carried out with glutathione-Sepharose 4B according to the manufacturer's protocol. The purified GST-p27 was digested by trypsin (N-tosyl-l-phenylalanyl chloromethyl ketone-treated, Worthington), and then released p27 was purified by passing it through benzamidine-Sepharose (28Holmberg L. Bladh B. Astedt B. Biochim. Biophys. Acta. 1976; 445: 215-222Crossref PubMed Scopus (169) Google Scholar) and glutathione-Sepharose 4B columns. The amino acid composition and amino-terminal sequence of the prepared recombinant p27 was confirmed. COS7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. A plasmid containing the full-length cDNA for p27 in pcDNAI/Amp (Invitrogen) was transfected by the calcium phosphate method (29Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4823) Google Scholar). Forty-eight hours after the transfection, the medium was changed to a serum-free medium and the culture was continued for another 24 h. The cell lysate and the culture supernatants were analyzed by immunoblotting using anti-p27 antiserum. The horseshoe crab hemolymph were fixed, immediately after bleeding, in 2% paraformaldehyde EM (Taab Laboratories Equipment Ltd., Berkshire, UK) for 10 min at room temperature. The washed hemocytes were mounted on a glass coverslip. The cells, permeabilized with ice-cold methanol for 10 min, were incubated with primary antibody for 2 h at room temperature. After the washing with phosphate-buffered saline containing 0.1% saponin, the cells were incubated with rhodamine-conjugated swine anti-rabbit IgG secondary antibody (1:50 dilution, DAKO) for 1 h, followed by washing with phosphate-buffered saline. The specimen was mounted in Entellan neu (Merck) and viewed with a Carl Zeiss LSM 410 confocal laser scanning microscope. Anti-p27 antiserum, preimmune serum, or anti-horseshoe crab tachylectin 1 (L6) (30Saito T. Kawabata S. Hirata M. Iwanaga S. J. Biol. Chem. 1995; 270: 14493-14499Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) was used as primary antibody at a dilution of 1:100. Western blotting analysis with antiserum against factor G revealed two bands of 37 and 27 kDa in the horseshoe crab hemocyte lysate, which reacted specifically with anti-subunit β but not with anti-subunit α antisera (Fig.1). The 37-kDa band was indistinguishable from factor G subunit β, whereas the 27-kDa band did not correspond with any fragments of factor G or activated factor G (8Muta T. Seki N. Takaki Y. Hashimoto R. Oda T. Iwanaga A. Tokunaga F. Iwanaga S. J. Biol. Chem. 1995; 270: 892-897Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). We started the purification of this protein (p27) by using anti-subunit β antiserum as a probe. The hemocyte lysate was initially fractionated with dextran sulfate-Sepharose CL-6B chromatography (Fig.2 A). Whereas the 37-kDa subunit β of factor G eluted from the column with 0.25 mNaCl corresponding to factor G activity (8Muta T. Seki N. Takaki Y. Hashimoto R. Oda T. Iwanaga A. Tokunaga F. Iwanaga S. J. Biol. Chem. 1995; 270: 892-897Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), p27 was found to elute in the 2.0 m NaCl fractions. The fractions containing p27 were pooled and subjected to gel filtration on Sephadex G-150 at pH 5.5 in the presence of 0.5 m NaCl (Fig. 2 B). The p27-containing fractions were pooled, diluted to reduce NaCl concentration, and then applied to a CM-Sepharose CL-6B column at pH 5.5 (Fig. 2 C). p27 bound to the column was eluted with a NaCl gradient and was further purified by gel filtration on a Superdex 75 HR column (Fig. 2 D). The purified p27 gave a single band (mass = 27 kDa) on SDS-PAGE under reducing conditions (Fig.2 D, inset, fraction 15), and its mobility did not change under non-reducing conditions (data not shown). Forty μg of p27 was obtained from 97.5 g (wet weight) of hemocytes (Table I).Table IPurification of p27 from the horseshoe crab hemocyte lysateStepVolumeTotal protein1-aEstimated from absorbance assumingA 280 nm1% of 10.0.Total activitySpecific activityPurificationYieldmlmgunits1-bSee “Experimental Procedures” for the unit definition.units/mg-fold%Hemocyte lysate59021,0001-cEstimated by the method of Bradford (44).7.800.00041.0100Dextran sulfate-Sepharose CL-6B240762.400.0318330Sephadex G-1501105.31.30.2567017CM-Sepharose CL-6B500.451.22.7720015Superdex 75 HR1.00.0400.143.286001.81-a Estimated from absorbance assumingA 280 nm1% of 10.0.1-b See “Experimental Procedures” for the unit definition.1-c Estimated by the method of Bradford (44Schneuwly S. Shortridge R.D. Larrivee D.C. Ono T. Ozaki M. Pak W.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5390-5394Crossref PubMed Scopus (151) Google Scholar). Open table in a new tab To obtain a partial amino acid sequence, amino-terminal sequences of the purified p27 and its proteolytic fragments were determined as described under “Experimental Procedures” (Fig. 3,underlined). Two degenerated primers were synthesized based on the partial sequences (Fig. 3, boxed) and were used in reverse transcriptase-PCR with hemocyte poly(A)+ RNA as a template. A PCR fragment of 280 bp was obtained, and sequencing confirmed that it encoded p27. Using this fragment as a probe, one clone was isolated from 500,000 clones of the hemocyte cDNA library. Sequence analysis of this clone showed that the cDNA contained an open reading frame of 660 bp followed by a TAA termination codon, and that it was flanked by a 104-bp 5′-noncoding sequence containing in-frame termination codons and 914 bp of 3′-noncoding sequence. A poly(A)+ signal was missing. The open reading frame encoded a p27 precursor of 220 amino acid residues. All the partial amino acid sequences were consistent with the deduced sequence, confirming that it encoded p27 (Fig. 3, underlined). The amino-terminal sequence of the purified p27 was located at the 24th amino acid of the deduced sequence, consistent with a 23-residue pro-sequence, which exhibited the characteristics of a typical hydrophobic ER signal sequence. Thus, mature p27 was composed of 197 amino acids with a calculated molecular mass of 21,614 Da. The amino acid compositions calculated from the mature protein of 197 amino acids agreed well with the analytical values obtained from the purified protein (TableII). Although one potential glycosylation site (Asn148) for an N-linked carbohydrate chain (Asn-Xaa-Ser/Thr) was present in the sequence, no amino sugar was detected in the purified p27 (Table II).Table IIAmino acid composition of p27Amino acidResidues/molecule2-aObtained by 20-h hydrolysis with 6 m HCl. Values in parentheses are from the cDNA sequence.Asp25.8(20)Glu17.6(13)Ser10.7(11)Gly22.7(23)His4.5(4)Arg7.9(7)Thr15.6(18)Ala9.8(8)Pro8.9(8)Tyr5.3(5)Val14.8(17)Met1.5(3)1/2CysND2-bND, not determined.(1)Ile14.2(15)Leu6.3(6)Phe11.2(11)TrpND(2)Lys14.2(25)Total(197)GlcNH22-cFrom 24-h hydrolysis with 3 mmercaptoethanesulfonic acid.—2-d—, not detected.GalNH22-cFrom 24-h hydrolysis with 3 mmercaptoethanesulfonic acid.—2-a Obtained by 20-h hydrolysis with 6 m HCl. Values in parentheses are from the cDNA sequence.2-b ND, not determined.2-c From 24-h hydrolysis with 3 mmercaptoethanesulfonic acid.2-d —, not detected. Open table in a new tab A sequence homology search indicated that p27 had extensive similarity with proteins belonging to CyPB family (Fig.4). p27 showed the highest similarity with human and mouse CyPBs (70% identity). p27 also showed more than 60% sequence identity with other vertebrate CyPBs and CyP homologues in Caenorhabditis elegans (CyP-5 and 6). A CyP homologue in yeast and an eye-specific CyP homologue in Drosophila, NinaA, had 50% and 36% identity, respectively. The 15 residues conserved in all CyPs, which are involved in close contact with substrates or CsA, were also found in the p27 sequence (Fig. 3,boldface). Two distinct differences were found between p27 and other members of the CyPB family. All the members of CyPB family contain a conserved 10-residue ER retention signal at their carboxyl termini, which differs from a typical motif, K/HDEL. This signal was missing in p27. In addition, p27 had an insertion of 8 residues in the middle of the molecule, which has not been observed in any other CyPs. Since p27 showed high sequence similarity with CyPs having PPIase activity, the isomerase activity of p27 was assessed at each purification step (Fig. 2). At each step, the elution profile of p27 antigen correlated well with PPIase activity. However, p27 represented only a minor part of total PPIase activity in the hemocytes, since most of the activity was eluted in the flow-through fractions of the dextran sulfate-Sepharose CL-6B column chromatography, the first step of the purification procedure (Fig.2 A). The chymotrypsin-coupled assay for PPIase activity showed that p27 increased the hydrolysis of a peptide substrate by α-chymotrypsin by catalyzing cis to trans isomerization of Pro at the P2 site (Fig. 5 A). Thek cat /K m of purified p27 was estimated to be 0.18 μm−1 s−1, which was 5–50-fold lower than the other CyPBs. The PPIase activity of p27 was inhibited by CsA, an inhibitor for CyP family (Fig.5 B). The CsA concentration (IC50) required for 50% inhibition of the p27 (1.3 nm) activity was 8.3 nm, similar to that reported for other eukaryotic CyPs (31Schonbrunner E.R. Mayer S. Tropschug M. Fischer G. Takahashi N. Schmid F.X. J. Biol. Chem. 1991; 266: 3630-3635Abstract Full Text PDF PubMed Google Scholar). On the other hand, another PPIase inhibitor, FK506, did not show any inhibitory activity on p27 (data not shown). Recombinant p27 was prepared by excising an expressed insert from a GST-p27 fusion protein. Its amino-terminal sequence and amino acid composition showed that recombinant p27 started with the 4th Lys residue, indicating that the first 4 residues were removed after trypsin digestion (data not shown). This recombinant p27 showed a similar mobility with the purified protein on SDS-PAGE and reacted with the anti-subunit β antiserum (Fig.6 A). The recombinant p27 also exhibited a PPIase activity as the purified p27 (Fig. 6 B), ruling out the possibility that p27 was co-purified with minor contaminant that has isomerase activity. CyPBs have been reported to localize in the ER through their carboxyl-terminal ER retention signal (32Arber S. Krause K.H. Caroni P. J. Cell Biol. 1992; 116: 113-125Crossref PubMed Scopus (98) Google Scholar). We tested whether p27, which lacks this retention signal, would also localize to the ER when expressed in COS cells. COS7 cells were transfected with the full-length cDNA for p27, and cell lysate and culture supernatant of the transfected cells were analyzed by using anti-p27 antiserum. p27 was detected in the culture supernatant, but not in the cells (Fig. 7). We next analyzed the distribution of p27 in the horseshoe crab hemocytes. The fixed and permeabilized hemocytes were stained with anti-p27 antiserum, followed by rhodaminelabeled secondary antibody. Confocal laser scanning microscopic analyses indicated that intracellular granular structures of the hemocyte were stained with anti-p27 (Fig. 8 A) but not with preimmune serum (Fig. 8 B). The staining pattern with anti-p27 antiserum was essentially same as that of antiserum against tachylectin-1 (L6), a lectin abundantly present in the L-granule (20Shigenaga T. Takayenoki Y. Kawasaki S. Seki N. Muta T. Toh Y. Ito A. Iwanaga S. J. Biochem. (Tokyo). 1993; 114: 307-316Crossref PubMed Scopus (44) Google Scholar,30Saito T. Kawabata S. Hirata M. Iwanaga S. J. Biol. Chem. 1995; 270: 14493-14499Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) (Fig. 8 C). We further examined subcellular localization of p27 in the horseshoe crab hemocytes, which contain two types of secretory granules (L- and S-granules) (4Toh Y. Mizutani A. Tokunaga F. Muta T. Iwanaga S. Cell Tissue Res. 1991; 266: 137-147Crossref Scopus (66) Google Scholar). The L- and S-granules were purified from the cells (20Shigenaga T. Takayenoki Y. Kawasaki S. Seki N. Muta T. Toh Y. Ito A. Iwanaga S. J. Biochem. (Tokyo). 1993; 114: 307-316Crossref PubMed Scopus (44) Google Scholar) and were analyzed by immunoblotting (Fig.8 D). The anti-p27 antiserum identified p27 protein in the L-granules in addition to the hemocyte lysate, but not in the S-granules. p27 was not detected in the hemolymph plasma. Similar results were obtained with anti-factor G subunit β antiserum. 2Y. Takaki and T. Muta, unpublished data. Although the cross-reactivity of p27 with antiserum against subunit β of the horseshoe crab β-glucan-sensitive serine protease zymogen factor G (Fig. 1) led us to initially speculate that it might be a new serine protease, p27 instead exhibits an amino acid sequence most similar to that of CyPs and PPIases (Figs. 3 and 4). The amino acid sequence of p27 showed little similarity with that of factor G. p27 and factor G subunit β should share a conformation-specific epitope(s), although its biological significance is unknown. Representing only a small portion of total hemocyte isomerase activity, this protein was found to localize to the hemocyte L-granules and was purified approximately 8600-fold with an overall yield of 1.8% (TableI). Comparative sequence analysis revealed that p27 was most homologous with members of the CyPB family (Fig. 4), and functional data supported this conclusion; in a chymotrypsin-coupled assay, p27 exhibited isomerase activity that was inhibited by CsA, but not by FK506 (Fig.5). These results indicate that p27 is a CyP-type enzyme, distinct from the FK506/rapamycin-binding protein (FKBP)-type enzyme (15Galat A. Eur. J. Biochem. 1993; 216: 689-707Crossref PubMed Scopus (316) Google Scholar). But unlike other cyclophilins, which are proteins functioning in cytosol or ER, p27 is instead found only in the L-granules of horseshoe crab hemocytes. Thus, p27 is a new type of CyPB, hereby designated as cyclophilin G (CyPG). A sequence comparison between CyPG and other CyPs offers insight into the mechanisms underlying its unique biological activities. Similar to other CyPBs, the CyPG precursor contains an amino-terminal hydrophobic pro-peptide, but unlike them it lacks a well conserved 10-amino acid carboxyl-terminal sequence, VEKPFAIAKE, which functions as an ER retention signal (Fig. 4) (32Arber S. Krause K.H. Caroni P. J. Cell Biol. 1992; 116: 113-125Crossref PubMed Scopus (98) Google Scholar). When transiently expressed, CyPG is not retained in the cells but instead is secreted into the medium (Fig. 7), indicating that the amino-terminal pro-peptide functions as a signal peptide targeting the polypeptide to the ER, but because CyPG lacks the ER retention signal, it is not retained. The specific signal(s) which target proteins to the regulated secretory granules in hemocytes are not yet known. The signal(s) for targeting CyPG to the L-granule might be present in the unique central 8-amino acid insertion, which is not found in any known CyPBs. Alternatively, it may be present in the amino-terminal pro-peptide, or CyPG might instead localize through a conformation-specific signal patch(es). It might be noted that the amino-terminal signal sequences of CyPG and factor G subunit β (8Muta T. Seki N. Takaki Y. Hashimoto R. Oda T. Iwanaga A. Tokunaga F. Iwanaga S. J. Biol. Chem. 1995; 270: 892-897Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 9Seki N. Muta T. Oda T. Iwaki D. Kuma K. Miyata T. Iwanaga S. J. Biol. Chem. 1994; 269: 1370-1374Abstract Full Text PDF PubMed Google Scholar), both of which are localized in the L-granules, are similar: FLVFVTTLS and FLVFITLS, respectively, although the mature proteins exhibit little sequence similarity. In addition, both CyPG and C. elegans CyP-5 share similar carboxyl-terminal and amino-terminal signal sequences; an analysis of the subcellular localization of CyP-5, which is not currently known, may provide a key insight into this question (33Page A.P. MacNiven K. Hengartner M.O. Biochem. J. 1996; 317: 179-185Crossref PubMed Scopus (60) Google Scholar). The specific function of CyPG in the hemocyte granule is not known. The L-granule, one of two regulated secretory granules in the hemocytes, is known to contain many proteins involved in the defense system, such as coagulation factors, lectins, and antimicrobial proteins (1Muta T. Iwanaga S. Rinkevich B. Müller W.E.G. Progress in Molecular & Subcellular Biology: Invertebrate Immunology. 15. Springer Verlag, Heidelberg1996: 154-189Google Scholar, 20Shigenaga T. Takayenoki Y. Kawasaki S. Seki N. Muta T. Toh Y. Ito A. Iwanaga S. J. Biochem. (Tokyo). 1993; 114: 307-316Crossref PubMed Scopus (44) Google Scholar). These components are stored in the granules until they are released from the cells after being stimulated by bacterial endotoxin. Since CyPs generally function as “molecular chaperones,” CyPG might similarly be involved in the quality control of stored protein components,i.e. in the refolding or repair of partially denatured proteins. For instance, a serine protease zymogen called factor C triggers an LPS-sensitive coagulation cascade, and it contains autocatalytic cleavage sites: -Lys(P3)-Pro(P2)-Arg(P1)-Ser(P1′)- and -Ser(P3)-Pro(P2)-Phe(P1)-Ile(P1′)- (34Tokunaga F. Miyata T. Nakamura T. Morita T. Kuma K. Miyata T. Iwanaga S. Eur. J. Biochem. 1987; 167: 405-416Crossref PubMed Scopus (51) Google Scholar, 35Muta T. Miyata T. Misumi Y. Tokunaga F. Nakamura T. Toh Y. Ikehara Y. Iwanaga S. J. Biol. Chem. 1991; 266: 6554-6561Abstract Full Text PDF PubMed Google Scholar). The cleavage of the latter site can be catalyzed by α-chymotrypsin (36Tokunaga F. Nakajima H. Iwanaga S. J. Biochem. (Tokyo). 1991; 109: 150-157Crossref PubMed Scopus (26) Google Scholar) and is essential for the activation (37Nakamura T. Morita T. Iwanaga S. Eur. J. Biochem. 1986; 154: 511-521Crossref PubMed Scopus (121) Google Scholar). In each cleavage site, a proline is located at the P2 site, and an appropriate cis/trans conformation of theseX-Pro bonds may be critical for the autocatalytic activation of the zymogen. CyPG might function to maintain the correct conformation of such X-Pro bonds. Similarly, 7 out of 8 well defined proline residues in the crystal structure of coagulogen, a clottable protein that is the final target of the hemolymph clotting cascade, have the trans-configuration, implying that thecis/trans conformation of prolines is strictly regulated in these granules (38Bergner A. Oganessyan V. Muta T. Iwanaga S. Typke D. Huber R. Bode W. EMBO J. 1996; 15: 6789-6797Crossref PubMed Scopus (69) Google Scholar). Moreover, CyPG was not detected in the S-granules (Fig. 8 D). The protein/peptide components in the S-granule are much smaller and rich in cystines, compared with those in the L-granule (20Shigenaga T. Takayenoki Y. Kawasaki S. Seki N. Muta T. Toh Y. Ito A. Iwanaga S. J. Biochem. (Tokyo). 1993; 114: 307-316Crossref PubMed Scopus (44) Google Scholar). Those S-granule components could be stabilized by disulfide bond(s), thus the PPIase activity to repair peptide folding might not be required. Maintaining the conformational integrity of the proline-rich L-granule proteins may be essential to sustain the integrity of the organism's host defense, and CyPG may thereby provide a key immunological function. In summary, this report demonstrates the purification, cloning, expression, and characterization of CyPG (p27), a unique PPIase that is present in the regulated secretory granules of horseshoe crab hemocytes. This is the first report to establish the existence of such “quality control” molecules in regulated secretory granules, and although the exact function of CyPG is not known, the recognized function of cyclophilins as molecular chaperones suggests that it may play a role in maintaining the conformational integrity of stored granular proteins. Indeed, the presence of at least 11 isoforms of CyPs in C. elegans (33Page A.P. MacNiven K. Hengartner M.O. Biochem. J. 1996; 317: 179-185Crossref PubMed Scopus (60) Google Scholar) has suggested that CyPs may exhibit more varied biological activities than previously recognized. Further elucidation of the function of CyPG in the horseshoe crab hemocyte granule may therefore offer interesting insights into the diverse functions of this interesting group of molecules. We thank Dr. N. Takahashi (Tonen Corp.) and Dr. K. Tawada (Kyushu University) for helpful advice in the PPIase assay, H. Iwanari (Institute of Immunology Inc.) for preparing anti-factor G subunit β antiserum, Dr. H. Nishimura (Hokkaido University) for providing COS7 cells, Dr. M. Sakaguchi (Kyushu University) for comments on immunofluorescence staining, and Dr. D. L. Sylvestre (HAART, Inc.) for critical comments on the manuscript. We are also grateful to C. Yano for technical assistance with the amino acid analyses.