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• W2011883618 abstract "Monoclonal antibodies were raised against hemocytes of the horseshoe crab Tachypleus tridentatus. All of the antibodies obtained reacted with the same protein bands on SDS-PAGE of hemocyte lysate. Flow cytometry and biotinylation of surface substances on the hemocytes indicated that the antigens are major peripheral proteins of hemocytes. The antigens were purified from hemocyte lysate and were good substrates for the horseshoe crab hemocyte transglutaminase (HcTGase). Transglutaminases play an important role during the final stage of blood coagulation in mammals and crustaceans. Although HcTGase did not intermolecularly cross-link a clottable protein coagulogen or its proteolytic product coagulin, HcTGase promoted the cross-linking of coagulin with the surface antigens, resulting in the formation of a stable polymer. We determined the nucleotide sequences for two isoproteins of the antigens. The two proteins containing 271 and 284 residues (66% identity) were composed of tandem repeats of proline-rich segments. We named them proxins-1 and -2 after proline-rich proteins for protein cross-linking. Proxins may form a stable physical barrier against invading pathogens in cooperation with hemolymph coagulation at injured sites. Monoclonal antibodies were raised against hemocytes of the horseshoe crab Tachypleus tridentatus. All of the antibodies obtained reacted with the same protein bands on SDS-PAGE of hemocyte lysate. Flow cytometry and biotinylation of surface substances on the hemocytes indicated that the antigens are major peripheral proteins of hemocytes. The antigens were purified from hemocyte lysate and were good substrates for the horseshoe crab hemocyte transglutaminase (HcTGase). Transglutaminases play an important role during the final stage of blood coagulation in mammals and crustaceans. Although HcTGase did not intermolecularly cross-link a clottable protein coagulogen or its proteolytic product coagulin, HcTGase promoted the cross-linking of coagulin with the surface antigens, resulting in the formation of a stable polymer. We determined the nucleotide sequences for two isoproteins of the antigens. The two proteins containing 271 and 284 residues (66% identity) were composed of tandem repeats of proline-rich segments. We named them proxins-1 and -2 after proline-rich proteins for protein cross-linking. Proxins may form a stable physical barrier against invading pathogens in cooperation with hemolymph coagulation at injured sites. lipopolysaccharides hemocyte transglutaminase enzyme-linked immunosorbent assay reverse-phase high performance liquid chromatography monodansylcadaverine Arthropods lack adaptive immunity, but they are well adapted to diverse environments and effectively defend themselves against invading pathogens by innate immunity. All multicellular organisms have some form of innate immune system. Recent studies (1Lemaitre B. Nicolas E. Michaut L. Reichhart J.M. Hoffmann J.A. Cell. 1996; 86: 973-983Abstract Full Text Full Text PDF PubMed Scopus (3020) Google Scholar, 2Medzhitov R. Preston-Hurlburt P. Janeway Jr., C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4458) Google Scholar) have revealed that insects and mammals conserve a signaling pathway of the innate immune system through cell-surface receptors, Toll and Toll-like receptors. The system is a sensitive non-self-recognizing cascade triggered by microbial cell wall constituents. The target molecules of innate immune systems are not proteins of direct gene products but rather molecular arrays or patterns shared among groups of pathogens. They are called pathogen-associated molecular patterns and include lipopolysaccharides (LPS),1β-1,3-glucans, and peptidoglycans (3Söderhall K. Cerenius L. Curr. Opin. Immunol. 1998; 10: 23-28Crossref PubMed Scopus (1108) Google Scholar, 4Ashida M. Brey P.T. Brey P.T. Hultmark D. Molecular Mechanisms of Immune Responses in Insects. Chapman and Hall Ltd., London1998: 135-172Google Scholar, 5Iwanaga S. Kawabata S. Muta T. J. Biochem. (Tokyo). 1998; 123: 1-15Crossref PubMed Scopus (256) Google Scholar, 6Iwanaga S. Curr. Opin. Immunol. 2002; 14: 87-95Crossref PubMed Scopus (212) Google Scholar). The major host defense system in the horseshoe crab Tachypleus tridentatus is carried by hemolymph that contains a kind of granular hemocytes, which make up 99% of the total hemocytes (7Toh Y. Mizutani A. Tokunaga F. Muta T. Iwanaga S. Cell Tissue Res. 1991; 266: 137-147Crossref Scopus (66) Google Scholar). The granular hemocyte is filled with two types of secretory granules, L-granules and S-granules, which selectively store defense molecules such as coagulation factors, protease inhibitors, lectins, and antimicrobial peptides (5Iwanaga S. Kawabata S. Muta T. J. Biochem. (Tokyo). 1998; 123: 1-15Crossref PubMed Scopus (256) Google Scholar, 6Iwanaga S. Curr. Opin. Immunol. 2002; 14: 87-95Crossref PubMed Scopus (212) Google Scholar). The hemocyte is highly sensitive to LPS, and the defense molecules stored are secreted by exocytosis in response to stimulation by LPS. This response is very important for host defense involving the engulfing and killing of invading microbes, in addition to preventing the leakage of hemolymph. To identify the cell surface proteins of hemocytes involved in the innate immunity, monoclonal antibodies were raised against the hemocytes of T. tridentatus. Here we purified, characterized, and determined the cDNA sequences of the major surface antigens on hemocytes. Coagulogen (8Nakamura S. Iwanaga S. Harada T. Niwa M. J. Biochem. (Tokyo). 1976; 80: 1011-1021Crossref PubMed Scopus (60) Google Scholar, 9Nakamura S. Takagi T. Iwanaga S. Niwa M. Takahashi K. Biochem. Biophys. Res. Commun. 1976; 72: 902-908Crossref PubMed Scopus (37) Google Scholar, 10Takagi T. Hokama Y. Miyata T. Morita T. Iwanaga S. J. Biochem. (Tokyo). 1984; 95: 1445-1457Crossref PubMed Scopus (16) Google Scholar) and 8.6-kDa protein (11Tokunaga F. Yamada M. Miyata T. Ding Y.L. Hiranaga-Kawabata M. Muta T. Iwanaga S. Ichinose A. Davie E.W. J. Biol. Chem. 1993; 268: 252-261Abstract Full Text PDF PubMed Google Scholar) were prepared from hemocyte lysates of the horseshoe crab T. tridentatus as described. LPS from Salmonella minnesotaR595 was from List Biological Laboratories, Inc. (Campbell, CA). Hemolymph was collected into 5 volumes of 2% paraformaldehyde in 10 mm sodium phosphate, pH 7.5, containing 0.5 m NaCl and 0.05% NaN2(PBS) and allowed to stand for 10 min at 25 °C. The fixed hemocytes were collected by centrifugation at 800 rpm (100 × g) at 4 °C for 5 min and washed three times with PBS. The cell numbers were counted by a Coulter Z1 cell counter (Coulter Electronics, Ltd., Luton, UK). The immunization and hybridoma preparation were carried out at Panapharm Laboratories, Co., Ltd., Kumamoto, Japan. Five mice were immunized by injection of the fixed hemocyte suspension containing 2 × 106 cells emulsified with complete Freund's adjuvant. Hybridoma cells were plated in microculture plates, and positive hybridomas were screened by enzyme-linked immunosorbent assay (ELISA). Positive hybridomas selected were further analyzed by a FACScan flow cytometer (BD Biosciences). Briefly, each culture supernatant was incubated with the fixed hemocytes at 105 cells/ml in PBS containing 0.1% bovine serum albumin on ice for 30 min. After washing with the same buffer, the hemocytes were incubated with a fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG (DAKO, Glostrup, Denmark) on ice for 30 min. After washing, the labeled hemocytes were analyzed using the flow cytometer. For each sample, 1.0 × 104 cells were analyzed using Cellquest software. The fixed hemocytes were incubated with a monoclonal antibody at 1.0 μg/ml on ice for 30 min. After washing with PBS, the hemocytes were incubated with an Alexa FluorTM 488-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR) at 20 μg/ml on ice for 30 min. After washing with PBS, fluorescence microscopy was done using an Olympus fluorescence microscope, model BX-FLA (Olympus Optical Co., Ltd., Tokyo, Japan). Surface substances exposed on hemocytes were biotinylated using the water-soluble biotinylation reagent, sulfo-N-hydroxysuccinimide long chain biotin (Pierce). Briefly, hemolymph was collected into PBS in a sterilized plastic plate and allowed to stand for 30 min at 4 °C. Hemocytes that adhered to the plate were washed three times with PBS and then incubated with the biotinylation reagent (0.5 mg/ml) in PBS at 4 °C for 30 min with rocking. The biotinylation reaction was stopped by incubating with 100 mm glycine in PBS at 4 °C for 20 min. No morphological change of the hemocytes that adhered to the plate was observed microscopically after biotinylation. The hemocytes biotinylated were lysed with 50 mm Tris-HCl, pH 7.5, containing 0.15m NaCl, 1 mm EDTA, and 1% Nonidet P-40 at 4 °C for 30 min with rocking. The cell lysate, 20 μl, was mixed with a monoclonal antibody (12.5 μg/ml) and incubated at 4 °C for 4 h. Protein A-Sepharose (Amersham Biosciences) was then added and further incubated at 4 °C for 2 h. The resulting immunocomplex bound to protein A-Sepharose was collected by centrifugation and washed three times with 25 mm Tris-HCl, pH 8.0, containing 0.5m NaCl. The pellet was subjected to SDS-PAGE, and the antigens were visualized by streptavidin-biotin-horseradish peroxidase. Protein samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes overnight at 20 V using an electroblot apparatus (Bio-Rad). The membrane was stained with Coomassie Brilliant Blue R-250, and the desired protein bands were excised (12LeGendre N. Matsudaira P.T. Matsudaira P.T. A Practical Guide to Protein and Peptide Purification for Microsequencing. Academic Press Inc., San Diego1989: 49-69Google Scholar). The proteins on the membrane were reduced, S-alkylated with iodoacetamide, and digested with Asp-N protease (Roche Molecular Biochemicals) or chymotrypsin (Worthington) (enzyme/substrate = 1:100, w/w) in 30 mmNH4HCO3 containing 5 mmCaCl2 and 10% acetonitrile at 40 (for Asp-N protease) or 37 °C (for chymotrypsin) for 20 h. The resulting peptides were separated by reverse-phase high performance liquid chromatography (rpHPLC) using a Cosmosil 5C18-MS column (2.0 × 150 mm, Nacalai Tesque, Inc., Kyoto, Japan). Peptides were eluted from the column with a linear gradient of 0–72% acetonitrile in 0.1% trifluoroacetic acid for 120 min at a flow rate of 0.2 ml/min. The effluent was monitored at 210 nm. The degenerate nucleotide sequences of primers used for PCR were based on the amino acid sequences of the peptides derived from chymotrypsin digestion (HDHQHK and ENKPQD). Sense and antisense nucleotides were synthesized with an EcoRI site at the 5′ end. Reactions for PCR contained the cDNA template (corresponding to 0.1 μg of poly(A)+ RNA), and 100 pmol of each primer was carried out using a Takara PCR thermal cycler. The PCR products were treated with EcoRI and purified using agarose gel electrophoresis. Fragments of interest were then ligated into plasmid Bluescript II SK+ (Stratagene, La Jolla, CA) for sequence analysis as described by Sambrook et al. (13Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). One clone that contained the protein sequence was used as a probe. A cDNA library prepared with the λ ZipLox system (Invitrogen) was screened by using the probe, as described previously (14Okino N. Kawabata S. Saito T. Hirata M. Takagi T. Iwanaga S. J. Biol. Chem. 1995; 270: 31008-31015Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). SDS-PAGE was performed according to Laemmli (15Laemmli U.K. Nature. 1970; 277: 680-685Crossref Scopus (207537) Google Scholar). The gels were stained with Coomassie Brilliant Blue R-250. For immunoblotting, the proteins were transferred to nitrocellulose membranes overnight at 20 V using an electroblot apparatus. The membranes were then treated with the first antibody and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG and visualized using an ECL kit, according to the protocol provided by the manufacturer (Amersham Biosciences). Total RNA was extracted from various tissues of T. tridentatus according to Chomczynski and Sacchi (16Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). First-strand cDNA synthesis from 0.1 μg of poly(A)+ RNA was performed using SuperScriptTM II RNase H− reverse transcriptase (Invitrogen) and random primers. One two-hundredth of the first strand of cDNA and 20 pmol of each primer were subjected to PCR (30 cycles) with denaturation at 94 °C for 45 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min 30 s. PCR products were analyzed on a 2% agarose gel and visualized following ethidium bromide staining. Freshly prepared hemocytes were lysed with 50 mm Tris-HCl, pH 7.5, containing 1% Nonidet P-40 at 4 °C for 30 min, and the supernatant was obtained by centrifugation. The transglutaminase reaction of the supernatant was started by adding 10 mm CaCl2and 10 mm dithiothreitol in the presence or absence of 0.5 mm DCA and incubated at 37 °C. The reaction was terminated by adding 100 mm EDTA. An aliquot of the reaction mixture was subjected to SDS-PAGE, and the fluorescence-labeled proteins were visualized by a trans-illuminator. HcTGase was partially purified as described (11Tokunaga F. Yamada M. Miyata T. Ding Y.L. Hiranaga-Kawabata M. Muta T. Iwanaga S. Ichinose A. Davie E.W. J. Biol. Chem. 1993; 268: 252-261Abstract Full Text PDF PubMed Google Scholar) with some modifications. The freshly prepared hemocyte lysate was dialyzed against 50 mm Tris acetate, pH 7.5, containing 1 mm EDTA. The dialyzed sample was applied to an SP-Sepharose FF column, followed by a DEAE-Sepharose CL-6B column. The transglutaminase activity of each fraction was detected by DCA incorporation into the 8.6-kDa protein. The partially purified HcTGase (13 μg/ml) was incubated with the hemocyte surface antigens (400 μg/ml) in the presence of 10 mm CaCl2 and 10 mm dithiothreitol at 37 °C for 1 h. An aliquot of the reaction mixture was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue R-250. Coagulogen (2 mg/ml in 50 mm Tris-HCl, pH 7.5, containing 0.15 m NaCl) was incubated with trypsin (Worthington) at 37 °C for 1 h (enzyme/substrate = 1:250, w/w). The resulting coagulin gel was dissolved by 20-fold dilution with the same buffer to a final concentration of 0.1 mg/ml, and no clot formation was observed under these conditions. The purified hemocyte surface antigens (50 μg/ml) in 50 mm Tris acetate, pH 7.5, containing 10 mm CaCl2 and 10 mm dithiothreitol, were preincubated with coagulogen, coagulin, or 8.6-kDa protein (50 μg/ml) on ice for 20 min. Partially purified transglutaminase (7 μg/ml) was then added to the mixture and incubated at 37 °C for 1 h. An aliquot of the reaction mixture was subjected to electrophoresis using 1% agarose gel, not polyacrylamide gel, under the same conditions of SDS-PAGE, followed by immunoblotting. Microtiter plates were coated with 10 μg/ml coagulogen or coagulin in 50 mmTris-HCl, pH 7.5, containing 0.15 m NaCl, by incubating overnight at 4 °C. After washing with the same buffer, the plates were blocked with 1% bovine serum albumin, and serial dilutions of samples were added, incubated at 37 °C for 2 h, and then washed. A monoclonal antibody was added and incubated at 37 °C for 1 h and washed. Horseradish peroxidase-conjugated goat anti-mouse IgG was added and incubated at 37 °C for 1 h. The enzyme activity of horseradish peroxidase was detected witho-phenylenediamine at 490 nm, using a microplate reader, model 3550 (Bio-Rad). Hemolymph, 2 ml, was collected into the sterile tube, and the hemocytes were collected by centrifugation at 800 rpm (100 × g) at 4 °C for 5 min and washed twice with 10 mm HEPES-NaOH, pH 7.0, containing 0.5 m NaCl. Then the hemocytes were suspended in the same buffer containing 50 mmMgCl2 and various concentrations of LPS and incubated at 30 °C for 30 min. HcTGase in the supernatant obtained by centrifugation at 800 rpm (100 × g) at 4 °C for 5 min was detected by ELISA, using polyclonal antibody against HcTGase and the horseradish peroxidase-conjugated goat anti-rabbit IgG. To determine whether significant membrane damage of hemocytes occurred because of LPS, the activity of lactate dehydrogenase, a cytosolic enzyme, was measured by using a colorimetric cytotoxicity assay kit according to the protocol provided by the manufacturer (Oxford Biomedical Research, Inc., Oxford, MI). Amino acid analysis was analyzed by an AccQ-Tag system (Waters Associates, Milford, MA). Amino acid sequence analysis was carried out using an Applied Biosystems 491 protein sequencer. Protein concentrations for determining extinction coefficients of the cell surface antigens were calculated from the amino acid mass/A 280. An internal standard, norleucine, was added to the protein hydrolysates to allow for the collection of losses. Fifteen hybridomas producing monoclonal antibodies reacting with surface substances of the horseshoe crab hemocytes were selected by the two screening methods, ELISA and the flow cytometric analysis. Immunoblotting showed that all the monoclonal antibodies produced by these hybridomas reacted with the protein bands of apparent molecular masses of 90 and 120 kDa in the 1% SDS extract of hemocytes (data not shown). One of the hybridomas cloned by limiting dilution, named 6C1-1F, produced an antibody with the highest affinity to the antigens. Flow cytometric analysis confirmed that antibody 6C1-1F reacts with the antigens expressed on the surface of hemocytes (Fig. 1 A). The antigens on the hemocytes were visualized by a fluorescence-labeled second antibody, Alexa 488-conjugated goat anti-mouse IgG (Fig. 1 B). On the other hand, the cell surface substances of hemocytes were biotinylated and then extracted by 1% SDS and subjected to SDS-PAGE, followed by detection with streptavidin-biotinylated horseradish peroxidase. At least six protein bands were detected with apparent molecular masses ranging from 48 to 170 kDa (Fig.2, lane 1). The biotinylated cell surface proteins were immunoprecipitated by antibody 6C1-1F and subjected to SDS-PAGE, followed by detection with streptavidin-biotinylated horseradish peroxidase (Fig. 2, lane 2). As a result, the proteins of 90 and 120 kDa were immunoprecipitated, indicating that antibody 6C1-1F recognizes the major hemocyte surface antigens, tentatively named band-90 and band-120. The hemocyte lysate (33.6 g wet weight) was fractionated with solid ammonium sulfate, and bands-90 and -120 were precipitated at the saturation of 30%. The precipitate was dissolved in 50 mm Tris acetate, pH 7.5, containing 8m urea and 1 mm EDTA, and applied to a Sepharose CL-6B column (3.5 × 90 cm) equilibrated with the same buffer (Fig. 3 A). Immunoblotting of every five tubes showed the presence of bands-90 and -120 in the fraction, as indicated by a solid bar. The pooled fraction was dialyzed against 50 mm Tris acetate, pH 7.5, containing 0.03 m NaCl and 1 mm EDTA and applied to a DEAE-Sephacel column (2.5 × 10 cm) equilibrated with the same buffer. After washing with the equilibration buffer, the proteins were eluted with a linear gradient of 0.03–0.6 mNaCl in the same buffer (Fig. 3 B). Immunoblot analysis indicated that bands-90 and -120 were eluted in the flow-through fraction. Bands-90 and -120 could not be separated by these purification procedures (Fig. 3 C). The NH2-terminal sequence analysis of bands-90 and -120 transferred to a membrane proved to be identical at least up to 23 residues. The transferred proteins were digested with Asp-N protease or chymotrypsin, and their peptide mappings were done by rpHPLC. As far as they were examined, the peptide sequences derived from band-90 were identical to those from band-120, and the amino acid compositions of bands-90 and -120 were indistinguishable (TableI). Band-120 is possibly a post-translationally modified protein of band-90.Table IAmino acid compositions of band-90 and band-120AnalysisSequenceBand-90Band-120Proxin-1Proxin-2mol %Asp11.511.911.4 (31)9.9 (28)Ser5.14.94.8 (13)4.9 (14)Glu13.613.517.3 (47)13.0 (37)Gly6.96.84.4 (12)4.9 (14)His6.56.35.2 (14)5.6 (16)Arg6.46.45.5 (15)5.3 (15)Thr1.71.54.1 (11)4.2 (12)Ala0.20.30.0 (0)0.7 (2)Pro17.418.217.7 (48)19.7 (56)1/2CysNDND0.7 (2)0.7 (2)Tyr2.42.33.0 (8)2.8 (8)Val6.86.57.0 (19)7.4 (21)Met0.00.00.0 (0)0.4 (1)Lys6.77.06.6 (18)6.3 (18)Ile5.75.73.7 (10)6.3 (18)Leu8.88.48.5 (23)7.8 (22)Phe0.10.20.0 (0)0.0 (0)GlcNH2−−GalNH2++Total100100100 (271)100 (284)The values in parentheses are the residue numbers deduced from the nucleotide sequences. ND indicates not determined; − indicates not detectable; and + indicates detectable. Open table in a new tab The values in parentheses are the residue numbers deduced from the nucleotide sequences. ND indicates not determined; − indicates not detectable; and + indicates detectable. The NH2-terminal sequence of bands-90 and -120 showed significant sequence similarity with that of the 80-kDa protein previously identified in T. tridentatus hemocytes (11Tokunaga F. Yamada M. Miyata T. Ding Y.L. Hiranaga-Kawabata M. Muta T. Iwanaga S. Ichinose A. Davie E.W. J. Biol. Chem. 1993; 268: 252-261Abstract Full Text PDF PubMed Google Scholar). The partial NH2-terminal sequence reported for the 80-kDa protein (SVXTLQV) was identical to that of band-90 or band-120 (HVKTLQV), except for the NH2 terminus. The 80-kDa protein is a substrate for HcTGase that catalyzes the DCA incorporation into the 80-kDa protein in a Ca2+-dependent manner. To test whether DCA is incorporated into the surface antigens, the hemocytes lysate was incubated with DCA in the presence of CaCl2, and aliquots were subjected to SDS-PAGE at the indicated times. DCA incorporation into the several specific proteins was observed by UV illumination of the gel, and the proteins of 90- and 120-kDa were most reactive among these DCA-labeled ones (Fig.4 A). The incorporation reaction proceeded rapidly (<5 min), and it was completely inhibited by EDTA, indicating that the intrinsic HcTGase catalyzes the DCA incorporation. Furthermore, the 90- and 120-kDa proteins labeled with DCA were immunoprecipitated by antibody 6C1-1F (Fig. 4 B), indicating that bands-90 and -120 are substrates for HcTGase and function as amine acceptors. To test whether bands-90 and -120 are cross-linked with endogenous proteins, the freshly prepared hemocyte lysate was incubated in the presence of CaCl2, and aliquots were immunoprecipitated by antibody 6C1-1F and then subjected to immunoblotting. Two protein bands with apparent molecular masses of 140 and 200 kDa newly appeared after a 5-min incubation. These bands were not observed in the presence of EDTA (data not shown). Furthermore, the incubation of the purified bands-90 and -120 with HcTGase resulted in the cross-linked products of 140 and 200 kDa (data not shown). These results indicate that bands-90 and -120 are cross-linked intermolecularly by HcTGase. The final stage of the blood coagulation cascade in mammals (17Lorand L. Credo R.B. Janus T.J. Methods Enzymol. 1981; 80: 333-341Crossref PubMed Scopus (99) Google Scholar) and the hemolymph coagulation reactions in crustaceans (18Kopacek P. Hall M. Söderhall K. Eur. J. Biochem. 1993; 213: 591-597Crossref PubMed Scopus (99) Google Scholar) depends on the transglutaminase-mediated cross-linking of specific clotting proteins. However, HcTGase dose not cross-link a clottable protein coagulogen of the horseshoe crab (11Tokunaga F. Yamada M. Miyata T. Ding Y.L. Hiranaga-Kawabata M. Muta T. Iwanaga S. Ichinose A. Davie E.W. J. Biol. Chem. 1993; 268: 252-261Abstract Full Text PDF PubMed Google Scholar). Coagulogen is converted to an insoluble gel known as coagulin polymer through the proteolytic cascade triggered by LPS. HcTGase neither catalyzed DCA incorporation into coagulin nor cross-linked it intermolecularly (data not shown). Interestingly, HcTGase promoted cross-linking of coagulin with bands-90 and -120, resulting in the high molecular weight products located at the top of the gel by 1% agarose gel electrophoresis in the presence of SDS (data not shown). Coagulogen or 8.6-kDa protein, a previously identified substrate for HcTGase (11Tokunaga F. Yamada M. Miyata T. Ding Y.L. Hiranaga-Kawabata M. Muta T. Iwanaga S. Ichinose A. Davie E.W. J. Biol. Chem. 1993; 268: 252-261Abstract Full Text PDF PubMed Google Scholar), did not result in the high molecular weight products cross-linked with bands-90 and -120 by HcTGase. In the absence of HcTGase bands-90 and -120 non-covalently bound to coagulin but not coagulogen-coated on microtiter plates, suggesting that bands-90 and -120 have specific binding affinity to coagulin (Fig.5). To determine whether HcTGase could be released from hemocytes in response to external stimuli, hemocytes were treated with LPS. HcTGase was released from hemocytes into the extracellular fluid in response to the stimulation by LPS, as detected by ELISA (data not shown). The lactate dehydrogenase activity in the extracellular fluid was not detectable under the same conditions. The specific probe of 0.45 kb was identified with oligonucleotide primers corresponding to peptides derived from band-90 or band-120, using PCR and DNA sequence analyses. Screening a hemocyte cDNA library with the probe gave two types of positive clones that code the full length of the protein sequences. They were subjected to restriction mappings followed by sequence determinations of both strands. One cDNA included an open reading frame of 874 nucleotides with a mature protein of 271 residues, and the other included an open reading frame of 926 nucleotides with a mature protein of 284 residues; the overall sequence identity between the two proteins was 66% (Fig. 6,A and B). The intriguing feature of these sequences was the presence of four tandem repeats with an extremely high content of proline, accounting for 18 and 20% of the total residues, respectively (Fig. 7). Therefore, we named them proxin-1 and proxin-2, respectively, after proline-rich proteins for protein cross-linking. There were glutamine-rich regions containing Gln-Gln dipeptides at the NH2-terminal regions of proxins-1 and -2 and the COOH-terminal region of proxin-1.Figure 7Sequence comparisons of proxins-1 and -2. Alignment of the amino acid sequence of proxins-1 and -2. Amino acid residues repeated more than three times in the tandem repeats are shown in boldface letters. All glutamine residues are crowned with dots.View Large Image Figure ViewerDownload (PPT) The calculated molecular weights from the deduced sequences of proxins-1 and -2 were 30,899 and 31,890, respectively. Peptide fragments corresponding to only proxin-1 or proxin-2 were obtained from the proteolytic digest of band-90 and from that of band-120 by rpHPLC, indicating that not only band-90 but also band-120 contains both proxins-1 and -2. Several peptide fragments were isolated with low yields, and their sequences were homologous but not identical to those of proxins-1 and -2, indicating the presence of an unidentified isoprotein(s) for the proxins. The proxins did not have putative transmembrane domains or glycosylphosphatidylinositol anchor sites in their sequences (19Cross G.A. Annu. Rev. Cell Biol. 1990; 6: 1-39Crossref PubMed Scopus (403) Google Scholar). Immunoblotting with antibody 6C1-1F detected the proxins only in hemocytes but not in other tissues, including heart, skeletal muscle, hepatopancreas, and stomach (Fig. 8 A). Furthermore, reverse transcription PCR showed that the cDNA fragments corresponding to proxins were highly amplified only in hemocytes but not in other tissues, thus indicating the specific expression of proxins in hemocytes (Fig. 8 B). In mammals, the coagulation system is based on the proteolytically induced aggregation of fibrinogen into insoluble fibrin (20Doolittle R.F. Putnam F.W. 2nd Ed. The Plasma Proteins. 10. Academic Press, San Diego1984: 421-544Google Scholar, 21Furie B. Furie B.C. Cell. 1988; 53: 505-518Abstract Full Text PDF PubMed Scopus (995) Google Scholar, 22Daivie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1636) Google Scholar). Initially, fibrins are non-covalently associated and are further stabilized through the intermolecular cross-linking of ε-(γ-glutamyl)lysine bonds by the plasma transglutaminase. On the other hand, in crustaceans, hemolymph coagulation depends on the transglutaminase-mediated cross-linking of a specific plasma-clotting protein without the proteolytic cascade (18Kopacek P. Hall M. Söderhall K. Eur. J. Biochem. 1993; 213: 591-597Crossref PubMed Scopus (99) Google Scholar, 23Doolittle R.F. Riley M. Biochem. Biophys. Res. Commun. 1990; 167: 16-19Crossref PubMed Scopus (47) Google Scholar). In the horseshoe crab, the coagulation cascade, whose components have structural similarity to those of the morphogenetic cascade for determining embryonic dorsal-ventral polarity in Drosophila, is activated by LPS or β-1,3-glucans, leading to the conversion of a soluble coagulogen into an insoluble coagulin gel (5Iwanaga S. Kawabata S. Muta T. J. Biochem. (Tokyo). 1998; 123: 1-15Crossref PubMed Scopus (256) Google Scholar, 6Iwanaga S. Curr. Opin. Immunol. 2002; 14: 87-95Crossref PubMed Scopus (212) Google Scholar). No transglutaminase activity has been found in horseshoe crab plasma. However, the clots of whole blood yielded significant amounts of ε-(γ-glutamyl)lysine products, suggesting the activity of transglutaminase released from the hemocytes during coagulation (24Wilson J. Rickles F.R. Armstrong P.B. Lorand L. Biochem. Biophys. Res. Commun. 1992; 188: 655-661Crossref PubMed Scopus (7) Google Scholar). We identified here the proline-rich surface antigens of the horseshoe crab hemocytes, named proxins, that function as substrates for protein cross-linking with clotting protein coagulin. These results clearly indicate that the horseshoe crab also uses the protein cross-linking reaction at the final stage of the coagulation system as an important host defense system. The apparent molecular masses of proxins on SDS-PAGE are definitively higher than those deduced from the cDNA sequences. Proxins-1 and -2 contained small amounts of galactosamine and no glucosamine in hydrolysates of bands-90 and -120 by amino acid analysis (Table I). Therefore, the glycosylation may not explain the big difference between the molecular weights of proxins estimated by the sequence and those estimated by SDS-PAGE. Bands-90 and -120 may consist of cross-linked oligomers of proxins-1 and -2. In mammals, proline-rich proteins such as cornifins and small proline-rich proteins are involved in the formation of the cornified cell envelope, a highly insoluble structure at the cell periphery of the stratum corneum (25Steinert P.M. Marekov L.N. J. Biol. Chem. 1995; 270: 17702-17711Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 26Robinson N.A. Lapic S. Welter J.F. Eckert R.L. J. Biol. Chem. 1997; 272: 12035-12046Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). The stratum corneum of the skin serves as a forefront physical barrier for invading pathogens. The envelope is composed of multiple membrane-associated and cytosolic proteins, including members of the cornifin/small proline-rich protein family and various other proteins. These proteins are cross-linked into an insoluble mesh by the keratinocyte transglutaminase, a membrane-bound enzyme. Cornifins have no significant sequence similarity to proxins, but cornifins also consist of an NH2-terminal glutamine-rich portion and proline-containing tandem repeats of octa- or nonapeptides (27Austin S.J. Fujimoto W. Marvin K.W. Vollberg T.M. Lorand L. Jetten A.M. J. Biol. Chem. 1996; 271: 3737-3742Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Cornifins function as amine acceptors through glutamine residues of the NH2-terminal portion. The similar glutamine cluster is also present at the NH2-terminal regions of proxins-1 and -2 and the COOH-terminal region of proxin-1 (Fig. 7). Possibly, these glutamine residues of proxins function as amine acceptors. HcTGase cross-linked proxins with coagulin but did not catalyze DCA incorporated into coagulin. Therefore, glutamine residues functioning as amine acceptors are not present on coagulin, but several lysine residues on coagulin function as amine donors for protein cross-linking with proxins. The three-dimensional structural and protein chemical studies of coagulogen suggest a possible polymerization mechanism in which the release of the helical peptide C of coagulogen would expose a hydrophobic cove on the head, which interacts with the hydrophobic tail of a second molecule, resulting in the formation of coagulin gel (28Kawasaki H. Nose T. Muta T. Iwanaga S. Shimohigashi Y. Kawabata S. J. Biol. Chem. 2000; 275: 35297-35301Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar,29Bergner A. Oganessyan V. Muta T. Iwanaga S. Typke D. Huber R. Bode W. EMBO J. 1996; 15: 6789-6797Crossref PubMed Scopus (69) Google Scholar). A hypothetical scheme of the cross-linked coagulin polymer is shown in Fig. 9. Furthermore, coagulin fibers tend to aggregate laterally to form a thicker fiber, probably through other hydrophobic patches on the coagulin surface, resulting in the formation of a reticulate (29Bergner A. Oganessyan V. Muta T. Iwanaga S. Typke D. Huber R. Bode W. EMBO J. 1996; 15: 6789-6797Crossref PubMed Scopus (69) Google Scholar). Proxins may participate in forming the stable reticulate structure, a casting net, and an important physical barrier for invading microbes. We thank W. Kamada, M. Hiranga-Kawabata, and N. Ichinomiya for expert technical assistance with peptide sequencing and amino acid analysis." @default.
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