Agglutination activity of Limulus polyphemus coagulogen following limited proteolysis

Comp. Biochem. Physiol. Vol. 105B, No. I, pp. 79-85, 1993

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A G G L U T I N A T I O N ACTIVITY OF L I M U L U S P O L Y P H E M U S C O A G U L O G E N F O L L O W I N G LIMITED PROTEOLYSIS CONSUELOL. FORTES-DIAs,* CONCEI~.~OA. S. A. MINETTI, YUAN Ll~q and TEH-YuNG LIU~" Division of Biochemistry and Biophysics, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, U.S.A. (Received 14 October 1992; accepted 18 November 1992)

Abstract--l. A 14 kDa protein with cell agglutination properties has been purified from endotoxinactivated L. polyphemus amebocyte lysate. Amino terminal sequence analysis indicates that this protein corresponds to a proteolytically cleaved product (coagulin) of coagulogen. 2. Similar cell agglutination activity can be generated, in vitro, by proteolytic cleavage of the coagulogen with either trypsin, endogenous protease or an a2M/enzyme complex isolated from amebocytes. 3. Studies with [t2~I]-labeledcoagulogen showed that only coagulin, not the intact coagulogen, binds to rabbit erythrocytes and formalin-fixed amebocytes. 4. The cell agglutination activity of coagulin towards erythrocytes was not inhibited by various sugars tested, and was not Ca~+-dependent. 5. These findings suggest that coagulogen and coagulin are reminiscent of their mammalian counterparts, fibrinogen and fibrin, in their clotting and relative adhesive properties.

following cleavage of coagulogen is a substrate for such an enzyme. In contrast to the fibrin clot produced in the mammalian system, the coagulin clot can be dissociated either by simple mechanical disruption or dilution (Roth et al., 1989). Among a variety of biologically active components, a 55 kDa protein from L. polyphemus amebocytes with cell adhesionpromoting properties has been isolated and characterized at both the protein and eDNA levels (Liu et al., 1991). A 24 kDa agglutinin has been isolated, but not fully characterized from the amebocytes of an Indian horseshoe crab, Carcinoscorpius rotundicauda (Srimal et al., 1985a). Agglutination activities have also bccn observed in the hemolymph of other species of horseshoe crabs (Marchalonis and Edelman, 1968; Pistole, 1976; Shishikura and Sekiguchi, 1983). These iectin-like agglutination activities in invertebrates bear some similarities to the vertebrate immune system and could assume a crucial role in the recognition of foreign substances, resulting in their ultimate removal from the circulation (Vasta, 1990). During the course of our studies of proteins with agglutinin/lectin activities from L. polyphemus amebocytes, we have observed that the ability of total amebocyte lysate to promote cell agglutination of rabbit erythrocytes or human leukocytes increases upon activation by endotoxin of the clotting cascade. Several other studies also suggest that agglutination activities observed either in the amebocytes (Srimal et al., 1985a) or in the hemolymph (Shishikura and Sekiguchi, 1983) could arise from activation of the clotting cascade and further granular extrusion via exocytosis. In the present study, we investigated the course of the proteolytic reactions which occur during activation of the clotting cascade with the

INTRODUCTION

Coagulogen is a major protein in the hemolymph of many invertebrates and its role as a substrate in clot formation has been studied extensively (Nakamura et al., 1976; Tai et al., 1977; Moseson et al., 1979; Takagl et al., 1984; Cheng et al., 1986). The complete amino acid sequences of coagulogens have been reported for four species of horseshoe crabs (Tai et al., 1977; Takagi et al., 1984; Cheng et al., 1986; Miyata et al., 1984a,b; Srimai et al., 1985b). In Limulus polyphemus, coagulogen is a 175 amino acid protein containing eight disulfide bonds. During the last step of the clotting process, the endotoxininduced clotting enzyme cleaves coagulogen between amino acids 18-19 and 46-47 to release to peptide fragment of 28 amino acids. The 18 amino acid-peptide at the N-terminus remains linked to the rest of the molecule through a disulfide bond and the resultant cleaved product, coagulin, polymedzes to form an insoluble clot (Miyata et al., 1984b). Previous studies have shown the presence of a transglutaminase-catalyzed covalent cross-linking reaction of polypeptide chains during clot formation (Chung et al., 1977). However, there is no substantial evidence to demonstrate that the coagulin generated

Abbreviations--Me2SO: dimethylsulfoxide; PMSF: phenylmethylsulfonylfluoride; HPLC: high-performance liquid chromatography; EDTA: ethylenediaminetetraacetic acid. *Visiting Fellow from Fundae~o Ezequiel Dias, Belo Horizonte, MG, Brazil. Supported by Conselho National de Pesquisa Cientifica e Technologica (CNPq proc. 201124/0). tTo whom correspondence should be addressed. c e r o IO~/~--F

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CONSUELO L. FORTES-DIAS et al.

specific aim of identifying and characterizing molecules that are generated with both agglutination and aggregation properties. MATERIAL AND METHODS

Materials Frozen amebocytes were isolated from blood of

L. polyphemus as previously described (Minetti et al., 1991). Pyrotell L. polyphemus amebocyte lysate was purchased from Associates of Cape Cod, Inc. (Woods Hole, MA) and the LAL chromogenic substrate (Ac-Ile-Glu-Ala-Arg-pNA) was purchased from Whittaker Bioproducts (Walkersville, MD). Sephacryl S-100, CM Sepharose CL-6B, Superose 12, Mono-S and Mono-Q ion exchange columns were from Pharmacia (Uppsala Sweden) and C-4 column from Vydac (Hesperia, CA). Sequencing-grade trypsin was obtained from Promega (Madison, WI). Agarose-immobilized trypsin, concanavalin A type III, mono- and di-saccharides were purchased from Sigma Chemical Co. (St Louis, MO). Iodo beads were from Pierce (Rockford, IL), and NA[~5I] from DuPont-NEN (Wilmington, DE). All other reagents were of the purest grades commercially available.

Isolation and purification of a 14-kDa agglutinin Frozen amebocytes were thawed and homogenized in sterile water, containing 10% Me2SO with a Polytron Tissuemizer. The mixture was centrifuged at 6000 g for 15 min to remove debris. The supernatant was incubated with endotoxin (10mg/ml) for 1 hr and the mixture was centrifuged at 6000 g for 15 min. The supernatant was concentrated by precipitation with 65% ammonium sulfate, solubilized in 0.05 M ammonium acetate buffer, pH 5.5 and applied to a Sephacryl S-100 column (1.6 x 100 cm), equilibrated in the same buffer. Agglutinin-positive fractions were further applied to a Mono-S column (HR 5/5) equilibrated with the same buffer. The bound proteins were eluted with an 0-1 M NaC1 linear gradient and the 14 kDa protein was eluted with 0.25 M NaC1.

Isolation and purification of coagulogen Coagulogen was isolated from L. polyphemus amebocytes as previously described (Tai and Liu, 1977). Further purification was performed according to Donovan and Laue (1991) to ensure the complete removal of a trypsin inhibitor.

Preparation of endogenous protease (s) from amebocytes lysate Frozen amebocytes were sonicated in 50 mM Tris, 0.5 M NaCI, pH 7.4 containing 50 mM PMSF and centrifuged as previously described (Minetti et al., 1991). The clear supernatant was loaded immediately onto a Sephacryl S-100 column (1.6 x 100 cm) equilibrated in the same buffer (without PMSF) with a flow rate of 14 ml/hr. A large molecular weight fraction which was active in hydrolyzing chromogenic

substrate was further purified by a Mono-Q column (HR 5/5) and a Superose 12 column. This fraction was later identified as an ~t2M enzyme complex (see Results). Another endogenous enzyme mixture was prepared by using Pyrotell amebocyte lysate chromatographed on the Sephacryl S-100 column as above and the fractions with amidase activity against LAL chromogenic substrate were pooled and used as such.

Enzymatic cleavage of coagulogen A stock solution of coagulogen (2mg/ml) in 25 mM Tris, pH 7.4, 150 mM NaCI (TBS) was diluted with an equal volume of TBS containing 20 mM CaCI 2 and digested with 1% (v/v) of trypsin (I mg/ml in l mM HCI), endogenous protease mixture (A280n m = 4.0) or a n ~x2M enzyme complex at room temperature for at least 10 min. Alternatively, digestion was performed using immobilized trypsin. Thirty-five percent (v/v) of previously washed trypsin-agarose was added to the stock solution of coagulogen. After 45 min of digestion the beads were washed several times with TBS, followed by a final extraction with 0.1 M glycine pH 2.8 or 0.1% TFA. Samples from every step were analyzed for hemagglutination activity and for composition by HPLC.

Agglutination of rabbit erythrocytes and human leukocyte The agglutination assays were performed as described previously (Srimal et al., 1985a). Leukocytes proliferation assays were conducted in parallel to agglutination studies (Oppenheim and Rosenstreich, 1976).

HPLC analysis Coagulogen and its digestion products were chromatographed on a C-4 column (4.7 x 250 mm) and eluted with a 30-60% gradient of 0.1% TFA in water/0.1% TFA in acetonitrile.

Amino acid sequence analysis Amino acid sequencing was performed by automated Edman degradation in a 477A gas-phase microsequencer (Applied Biosystem, Inc.) connected to an on-line model 120A PTH-amino acid analyzer.

Polyacrylamide gel electrophoresis Gel electrophoresis was carried out using a Pharmacia Phast-gel system with 7.5% gel and native buffer strips.

Binding of [~25I ]coagulin to erythrocytes andformalinfixed amebocytes The iodination of coagulogen was performed as described (Minetti et al., 1991) with Na[~25I] (carrier-free) and Iodo-beads (Markwell, 1982). [~2~I]coagulogen was treated with low concentration of the immobilized trypsin (2% v/v) to generate [t25I]coagulin. The immobilized trypsin was removed

Agglutination property of coagulogen

81

tion mixtures were subjected, at suitable time intervals, to HPLC, amino acid sequence analysis and agglutination assay. Figure 2 illustrates the HPLC pattern of a solution of coagulogen incubated for 10 min at 25°C with 1% trypsin. Peak A is a mixture of fragments 19-27, 18-29, 30-46; Peak B consists of fragment 19-46; Peak C contains fragments 1-18 and 47-175 linked by a disulfide bond (coagulin); peak D is the intact coagulogen residue 1-175; peak E consists of a mixture of two single-cleaved coagulogen species, either at position 18 or at 46. Fragment 19-46 was released upon further digestion with trypsin. After 1 hr of incubation peak C, corresponding to coagulin, was the only major peak (>95%) detected. This pattern remained unchanged for 20 hr. Similar profiles were obtained with the immobilized trypsin. In this instance, however, peak C and E components were adsorbed onto the beads and could be recovered by extraction with 0.1% TFA. Digestion of coagulogen with the endogenous protease(s) under the same conditions as those used for trypsin, yielded cleavage patterns similar to those observed for trypsin digestion. Rabbit erythrocyte hemagglutination assay indicated that neither trypsin, nor the endogenous protease(s) or the untreated coagulog.en exhibits the hemagglutination activity (Fig. 3A). However, hemagglutination activity was observed with cleaved coagulogen at a concentration as low as 3 #g/ml (0.2 # M). Coagulogen with a single cleavage either at position 18 or 46, did not exhibit hemagglutination activity. Fragment 19-45, neither inhibited nor elicited the hemagglutination. When coagulogen was

by centrifugation. Binding studies were conducted as described in Minetti et al. (1991) using TBS-washed erythrocytes (3 x 108 cells/ml). For cell concentration-dependent studies, varying erythrocyte and fixed-amebocyte concentrations (0-8 x 108 cells/cm) were used. RESULTS AND DISCUSSION

The mechanism of clotting in horseshoe crabs involves the degranulation of amebocytes and the activation of an enzyme cascade. The degranulation can be initiated by minute quantities of endotoxin which interacts with the amebocyte membrane to induce the release of clotting zymogens. Activation of zymogens triggers a chain of events which leads to the proteolytic cleavage of the coagulogen and the subsequent clot formation. During the course of our studies, we have observed that the ability of the amebocyte lysate to promote cell agglutination increases with the activation of the clotting enzymes. In this study, using gel-permeation and ion-exchange chromatographic procedures, we have isolated and characterized a 14 kDa agglutinin after the activation of the lysate by endotoxin. Amino terminal sequence analysis established that the isolated protein corresponds to two fragments of coagulogen, residues, 1-18 and 47-175, linked by a disulfide bond (Fig. 1). To study the kinetics and the molecular mechanism of the generation of cell agglutination activity accompanying the proteolytic cleavage of coagulogen, purified coagulogen was incubated with either trypsin or preparations of amebocyte protease(s). The rea¢-

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Fig. 2. HPLC pattern of a solution of coagulogen incubated for I0 min at 25°C with 1% trypsin. The column used was a Vydac C4, 4.7 x 200mm. Solution A, 0.1% TFA; Solution B, 0.08% TFA in 80% acetonitrile. Gradient, 30-60% at 40°C. incubated over 1 hr with a high concentration of the immobilized trypsin (35% v/v) (Fig. 3B), the resulting coagulin was completely adsorbed onto the beads. This was confirmed by the detection of hemagglutination activity in the agarose beads but not in the supernatant. Extraction of the beads with 0.1% T F A followed by HPLC analysis revealed that the eluted material corresponded to the 14 kDa coagulin. The supernatant contained a mixture of cleavage peptides

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(see Figs 1 and 2). Coagulin also has the ability to agglutinate human leukocytes (Fig. 4) but, unlike many other lectins, it is devoid of mitogenic activity (data not shown). Fractionation of amebocyte lysate yielded a high molecular weight component which contained an • 2M sequence as shown by N-terminal amino acid analysis. When preparations of ~t2M from serum and amebocyte were analyzed on a native polyacrylamide

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Fig. 3. A. Hemagglutination of rabbit erythrocytes. Controls--la, 3a--TBS, 2a---coagulogen, 3b--trypsin. 2b-coagulogen digested by trypsin, 2c---coagulogen digested by endogenous protease. Each sample is run in duplicate wells. B. Adsorption of rabbit erythrocytes to trypsin-agarose beads. Top: negative control beads with erythrocytes. Bottom: trypsin beads cleaved coagulogen with erythrocytes.

Agglutination property of coagulogen

83

Fig. 4. Agglutination of human leukocytes in culture. A, control; B, in the presence of coagulin (100 x magnification).

gel electrophoresis, both fast- and slow-moving bands were observed in the amebocyte preparation while only the slow-moving species was observed in serum ~2M (Fig. 5). The fast-moving band derives from a conformational change of ~2M resulting from the trapping of a protease or the interaction with primary amines (Quigley et al., 1991). The relative amounts of the fast and slow bands in the amebocyte lysate preparations vary in different isolations. It is likely that during the isolation a portion of the clotting enzyme was activated and trapped by e~zM since the isolation procedure was not carried out under sterile conditions. The fast-moving species was able to cleave both the chromogenic substrate and coagulogen while the slow moving species, which presumably does not contain enzyme does not cleave either substrate (data not shown). This is similar to the human ~2M/plasmin complex which remains active towards its substrate fibrinogen (Gonias and Pizzo, 1983). Figure 6 demonstrates that [t25I]coagulin binds to erythrocytes. Saturation kinetics could not be approached due to the self-aggregation of coagulin in addition to its binding to erythrocytes. Indeed, the high affinity of coagulin for the cell surface of erythrocytes, coupled with the ability to selfaggregate, may account for the strong agglutination activity of the coagulin. Similar results have been observed for fibrinogen, which becomes much more

Fig. 5. Polyacrylamide gel electrophoresis of A, serum ~tzM and B, ameboeyte.

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hydrophobic and less soluble when subjected to limited proteolysis (Van Oss, 1990). A number of monosaccharides (N-acetylneuraminic acid, N- acetyl - t) - galactosamine, N - acetyl- t)- glucosamine, ~t- methyl- ~)-galactoside, fl - methyl - ~)- glucoside, t)- fucose, r~- mannose), disaccharide (t)(+)-cellobiose) and polymers of sialic acids (Meningococcal A and C polysaccharide) at a concentration of 0.1 M did not interfere with the hemagglutination activity of coagulin, indicating that this property is most likely not due to its lectin-like activity. In contrast to endotoxin-induced clot formation, the agglutination activity of coagulin is not Ca 2+dependent. Moreover, the binding of coagulin to erythrocytes was not reversible with l0 mM EDTA. These findings are in accordance with those observed for fibrinogen binding to platelets, where the binding reaction is independent of Ca 2+ (Budzynski, 1986) and cannot be reversed by EDTA (Peerschke and Wainer, 1985). The counterpart of the horseshoe crab coagulogen in the mammalian system is fibrinogen and the major function of fibrinogen involves the formation of a fibrin clot after limited proteolysis. Fibrin has been considered as a molecular scaffold (Hangtan, 1987), since it binds to platelets and plasma proteins to act as a hemostatic plug. These properties appear to resemble those in L. polyphemus where coagulogen and coagulin, corresponding to fibrinogen and fibrin, serve respectively as substrate for clot and as adhesive and aggregating factors. In this study, we have demonstrated that coagulin derived from proteolysis of the coagulogen binds to rabbit erythrocytes, human leukocytes, and L. polyphemus amebocytes, and a number of other cells such as bacteria and Leishmania (data not shown), in a concentration-dependent manner. The consequence of this binding is the entrapping and sequestering of cell particles for their eventual removal from the circulation. Over 10 years ago, Bang (1979) observed a timeand dose-dependent reversible coagulation of the whole hemolymph upon injection of endotoxin into

young horseshoe crabs. While the mechanism of the clot formation is to a large extent understood, the mechanism of clot dissolution and removal in the L. polyphemus hemolymph remains to be clarified. Acknowledgements We thank John B. Ewell for protein sequence determination, and S. Unger for her expert assistance in the preparation of this manuscript. REFERENCES

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agglutinin of horse erythrocytes from the hemolymph of Tachypleus tridentatus, the Japanese horseshoe crab. J. Biochem. 93, 1539-1546. Srimal S., Dorai D. T., Somasundaran M., Bachhawat B. M. and Mitata T. (1985a) A new haemagglutinin from the amoebocytes of the horseshoe crab Carcinoscorpiu~ rotundicauda. Biochem. J. 230, 321-327. Srimal S., Miyata T., Kawabata S., Miyata T. and Iwanaga S. (1985b) The complete amino acid sequence ofcoagulogen isolated from southeast Asian horseshoe crab, Carcinoscorpius rotundicauda J. Biochem. 98, 302-318. Takagi T., Hokama Y., Myiata T., Morita T. and Iwanaga S. (1984) Amino acid sequence of Japanese horseshoe crab (Tachypleus tridentatus) coagulogen B. chain: completion of the coagulogen sequence. J. Biochem. 95, 1445-1457. Tai J. Y. and Liu T.-Y. (1977) Studies on Limulus amebocyte lysate. J. biol. Chem. 252, 2178-2181. Tai J. Y., Seid R. C., Huhn R. D. and Liu T.-Y. (1977) Studies on Limulus amebocyte lysate II. J. biol. Chem. 252, 4773-4776. Van Oss C. J. (1990) Surface properties of fibrinogen and fibrin. J. protein Chem. 9, 487-491. Vasta G. R. (1990) Defense molecules In UCLA Symposia on Molecular and Cellular Biology: New Series, Vol. 121, pp. 183-199. Alan R. Liss, New York.