Purification and characterization of the clotting protein from the white shrimp Penaeus vannamei

Comparative Biochemistry and Physiology Part B 122 (1999) 381 – 387

Purification and characterization of the clotting protein from the white shrimp Penaeus 6annamei Karla Montan˜o-Pe´rez, Gloria Yepiz-Plascencia, Inocencio Higuera-Ciapara, Francisco Vargas-Albores * CIAD, Marine Biotechnology, P.O. Box 1735, Hermosillo, Son, 83000, Mexico Received 12 February 1998; received in revised form 29 December 1998; accepted 4 January 1999

Abstract The protein responsible for clot formation was isolated from plasma of the white shrimp Penaeus 6annamei by affinity chromatography in a heparin–agarose column. The protein, named clotting protein (CP), was found to be a lipoglycoprotein, composed of two 210-kDa subunits covalently bound by disulfide bridges. CP formed large polymers when incubated with hemocyte lysate. Dansylcadaverine can be incorporated into CP by a hemocyte lysate or guinea pig transglutaminase mediated reaction. The amino acid composition and the amino terminal sequence were determined and compared with the clotting protein of the crayfish and the spiny lobster. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Defense system; Crustacea; Invertebrate coagulation; Clotting protein; Shrimp immunity; Transglutaminase; Coagulogen; Hemocyte lysate

1. Introduction Studies related to invertebrates immunity are scarce mainly due to the idea that they do not require an efficient immune system because of their short life period and high reproductive rate. However, recent studies show the opposite . The economical importance of some crustaceans has prompted the necessity to study their immune components. Invertebrate animals, which lack adaptive immune responses, have developed defense systems that respond to common antigens located on the surface of potential pathogens [17]. These defense systems include hemolymph coagulation, cellular and hemolytic factors, agglutinins and proPO system [9,10,17,18,20,22,23].Hemolymph coagulation is an essential defense response of crustaceans that prevents loss of hemolymph through breaks in the exoskeleton * Corresponding author. Tel: + 52-62-80-00-57 (ext. 524); fax: +52-62-80-00-55. E-mail address: [email protected] (F. Vargas-Albores)

and the dissemination of bacteria throughout the body [16]. The clotting system in crustaceans involves a cellular component [4] identified as a Ca2 + -dependent transglutaminase (TGase) from the hemocytic granules [4,16]. The key plasma protein which constitute the clot has been named clotting protein or CP [12], although it is sometimes called fibrinogen or coagulogen [6,15,16] probably to emphasize its functional analogy to vertebrate or Limulidae clotting protein. In spite of CP having been reported to be present in relatively high concentrations, it has been purified and characterized only in the spiny lobster, Panulirus interruptus [6], in the crayfish, Pacifastacus leniusculus [12] and in the sand crayfish, Ibacus ciliatus [11]. In all cases, CP was reported as a lipoglycoprotein with a molecular mass of approximately 400 kDa, composed of two identical subunits that are bound by disulfide bridges. In addition to its participation in the clotting process, CP has been reported to have other functions such as pigment, lipid and carbohydrate transport and protein reserves during starvation [4,11]. From the very earliest studies,

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evidence has steadily accumulated that distinguishes the crustacean coagulation from the vertebrate clotting process. Apparently, proteolysis is not involved in the crustacean event; the same N-terminal sequence is present before and after gelation [7]. However, the basis of coagulation is a TGase-catalyzed reaction that results in the formation of intermolecular o-(g-glutamyl)lysine cross-links between the side chain of a glutamine residue on one protein and the side chain of a lysine residue on another protein [14]. In the sand crab (O6alipes bipustulatus), a 70-kDa cellular protein called coagulogen, was reported to clot in the presence of human thrombin (factor XII), that is a TGase [15].In this paper we report the purification of CP from the white shrimp (Penaeus 6annamei ) by heparin-affinity chromatography as well as the biochemical characteristics of the purified protein.

2. Materials and methods

2.1. Solutions All chemicals were purchased from Sigma. Shrimp anticoagulant solution (450 mM NaCl, 10 mM KCl, 10 mM EDTA.Na2, 10 mM Hepes, pH 7.3, 850 mOsm/kg) was used to collect the shrimp hemolymph [24]. Buffer A, containing 20 mM sodium phosphate and 1 mM EDTA (pH 6), was used during purification. Buffer B, containing 50 mM Tris – HCl and 0.15 M NaCl (pH 8.0), was used for the clotting tests. Columns (5 ml) of heparin immobilized in agarose (Sigma) were used.

2.2. Animals Juvenile white shrimp (P. 6annamei ), were grown in an experimental tide pond (Center for Biological Research, La Paz, B.C.S, Mexico) and maintained in an aquarium (249 4°C, salinity: 36 parts per thousand) for at least 2 days before bleeding. Only hemolymph from intermolt, apparently healthy shrimp was used. Hemolymph was extracted from the pleopod base of the first abdominal segment into 2 volumes of precooled (10°C) shrimp anticoagulant solution [24].

2.3. Isolation of white shrimp CP The freshly collected hemolymph was centrifuged at 800× g for 5 min and the cell-free plasma separated. The plasma was dialyzed overnight against buffer A, and centrifuged at 3000×g for 20 min at 4°C, to eliminate the precipitated material. Supernatant containing CP (1.5 ml) was applied to a heparin – agarose column (5 ml) previously equilibrated with buffer A. The column was washed with buffer A until the protein content was zero, as indicated by the Bradford reaction

[1]. Bound protein was eluted by using 10 ml buffer A plus 200 mM NaCl. The CP-rich fractions were pooled and contaminating hemocyanin was eliminated by either electroelution or immunoaffinity. For electroelution, the sample was applied to a preparative 5–8% polyacrylamide gradient gel (14× 14× 0.1 cm). Electrophoresis was run for 12 h, at 20 mA, 10°C, and the 400-kDa band was excised and placed in an electroeluter (model 422 Bio-Rad) for 4 h using elution buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). Hemocyanin present in the CP-rich fraction was also eliminated by using antibodies against shrimp hemocyanin [5] immobilized to sepharose (Pharmacia). The sample (1.5 ml) was applied to the column (3 ml) equilibrated with buffer A plus 200 mM NaCl. Purified shrimp CP was recovered in the flow through fraction (10 ml), while hemocyanin was eluted from the column by using glycine–HCl (0.2 M, pH 2.5). For some experiments, CP purified by immunoaffinity was concentrated by dialyzing against deionized water overnight at 4°C that resulted in precipitation of CP. The protein was resuspended in buffer A or in 0.1 M NaCl at 0.7 mg/ml.

2.4. SDS-polyacrylamide gel electrophoresis (PAGE) Analytical PAGE was performed using a 5% polyacrylamide gel in the presence of SDS, and run in a Mini Protean II system (Bio-Rad) under either reducing or non-reducing conditions [13]. Samples and molecular weight markers (Sigma) were dissolved in 5× sample buffer (312 mM Tris–HCl, 10% SDS, 25% glycerol, pH 6.8). For reducing conditions, 50% (v/v) 2-mercaptoethanol (2-ME) was added to the 5× sample buffer and the samples were boiled for 5 min. The gels were stained with 0.1% Coomassie brilliant blue. Immobilization of the proteins was performed by soaking the gel in transfer buffer (48 mM Tris, 39 mM glycine, 20% methanol, SDS 0.035%, pH 8.8) for 10 min and electrotransferred (1 h at 18 V) to a nitrocellulose membrane (Millipore) [21]. After transfer, the membrane was incubated in blocking buffer (PBS containing 5% non fat dry milk and 0.05% Tween 20) for 2 h.

2.5. Chemical Analysis Protein concentrations were determined by using the Bradford method [1]. In samples containing SDS, the BCA method (bicinchoninic acid, Pierce) [19] was preferred over the Bradford method. In both cases, bovine serum albumin (BSA) was used as standard protein. To detect glycosylation, the purified CP was reduced by 2-ME, run on SDS-PAGE, electrotransferred and blocked as described above. The membrane was incubated with 10 mg/ml biotinylated lectins (BK-1000 Vector Laboratories) from: jack bean (Concanavalin A), Pisum sati6um (PSA), peanut (PNA), wheat germ

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(WGA), soy bean (SBA), horse gram (DBA) and castor bean (RCA). After washing, the reaction with the lectins was detected by using avidin – peroxidase and 4-Cl, 1-naphtol as substrate. To detect lipids, 60 ml of purified CP (0.7 mg/ml) were incubated with 20 ml Sudan black (20%) for 30 min at 22°C as described in Ref. [27]. The sample was run in a 5–8% gradient native PAGE. Lanes containing the molecular weight markers and CP were stained by Coomassie, while the lane containing CP incubated with Sudan black was preserved.

2.6. Amino acid analysis and N-terminal sequence Purified white shrimp CP was reduced by 2-ME and electrotransferred from a 5% polyacrylamide gel to an Immobilon P membrane as described above. The membrane was stained with 0.1% Coomassie brilliant blue and washed extensively with distilled water. The amino acid composition of the CP was determined by acid hydrolysis and HPLC. The N-terminal amino acid sequence was obtained by automated Edman degradation on an Applied Biosystem protein sequencer (model 477A). Amino acid composition and N-terminal sequence analyses were performed by the Division of Biotechnology, University of Arizona. Sequence comparisons were performed at the NCBI (National Center for Biotechnology Information) using the BLAST network service.

2.7. Fluorescent probes into the clotting protein Hemocyte supernatant lysate (HSL) were prepared as in Ref. [10]. Either plasma (30 ml) (2 mg protein/ml) or CP (0.6 mg/ml), in buffer B, were mixed with 10 ml dansylcadaverine (5 mM), then 10 ml HSL (15 mg of protein/ml) or guinea pig TGase (EC.2.3.2.13) (1 U/ml) and 10 ml 200 mM CaCl2 were added. Controls with 200 mM EDTA instead of CaCl2 and without TGase were included. The samples were incubated at 37°C for 45 min. The reaction was stopped by adding 12 ml PAGE 5× sample buffer and kept on ice. The samples were run on a 2 – 10% gel, without stacking, in the presence of SDS. After electrophoresis, the gel was fixed (50% ethanol, 10% acetic acid, 40% water) for 15 min. Fluorescent labeled proteins were visualized by UV light (302 nm) and photographed using a Polaroid camera. Afterwards, the gel was stained with Coomassie brilliant blue and the patterns were compared.

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ing an IEF 3–9 gel. IEF standards (Sigma) were used as reference. The gel was run and silver stained following the manufacturer instructions (Pharmacia, Uppsala).

3. Results and discussion The initial step in the procedure for the isolation of CP from crayfish [12] and lobster [6] used dialysis of plasma against a solution with low ionic strength and slightly acidic pH. This permitted the precipitation of CP and the elimination of the most abundant plasma protein, hemocyanin. However, dialysis of shrimp plasma under these conditions did not precipitate CP. Instead, the main protein precipitated from shrimp plasma was b-1,3-glucan binding protein, BGBP [25,26] and only a small amount of CP was afterwards detected in the precipitated material. However, when BGBP-depleted plasma was applied to a heparin–agarose column, most of the plasma proteins were not retained. CP showed affinity towards heparin and it was eluted from the column by 200 mM NaCl in buffer A (Fig. 1). CP-containing fractions were analyzed by SDS-PAGE using a 5% gel and small quantities of contaminating proteins were detected. Apparently these contaminants were hemocyanin monomers and polymers which were eliminated by either electroelution or affinity chromatography. Electroelution was performed by excising the corresponding band after preparative SDS-PAGE. The presence of SDS in the electroeluted CP prevented its use for some experiments. Therefore, another approach was considered to remove hemocyanin, the contaminating protein. Using immobilized antibodies against white shrimp hemocyanin [5] the purification conditions were mild and the sample was SDS-free. The anti-hemocyanin

2.8. Electrofocousing The isoelectric point was determined using a sample of CP resuspended in 0.15 M NaCl. The sample was run in a electrophoresis Phast System (Pharmacia) us-

Fig. 1. Isolation of white shrimp (P. 6annamei ) CP by affinity chromatography in a heparin – agarose column (5 ml). Fractions of 1 ml were collected and the protein content was determined by the Bradford method.

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Fig. 2. SDS-PAGE of the clotting protein from white shrimp (P. 6annamei ) plasma. Lane 1, molecular weight markers; lane 2, shrimp plasma; lane 3, shrimp serum; lane 4, reduced plasma; lane 5, reduced monomeric CP subunit; and lane 6, purified clotting protein. The gel was stained with Coomassie brilliant blue.

column eliminated all contaminant proteins, confirming that such material was hemocyanin-related. In both cases, purified CP was detected as a single 400-kDa band by SDS-PAGE in 4-15% gels (Fig. 2). Thus, white shrimp CP has a molecular mass similar to freshwater crayfish, P. leniusculus, CP [12], the so-called fibrinogen from spiny lobster, P. interruptus [2] and a clottable lipoprotein protein from sand crayfish [11]. The plasma protein responsible for clot formation in these crustaceans has a molecular mass of approximately 400 kDa. By SDS-PAGE under reducing conditions a band of approximately 200 kDa was identified, indicating that CP is a homodimeric protein. When shrimp plasma was obtained without anticoagulant, the characteristic 400 kDa band was not detected in the supernatant after clot formation (serum), indicating that this protein was eliminated during clot formation (Fig. 2). In addition, by electrofocousing a pI of 5.6 was determined for the purified CP. To determine amino acid composition and N-terminal sequence, white shrimp CP was reduced and the analysis was performed using the 200-kDa monomer. The amino acid composition of the white shrimp CP (Table 1) is similar to its homologous in crayfish [12], lobster [6] and sand crab [15], even though the last one has a different type of coagulation [8]. Likewise, the N-terminal sequence of the white shrimp CP is identical to sand crayfish VHDL, and very similar to crayfish CP and lobster fibrinogen (Fig. 3). It has been shown that the N-terminal of this protein has some sequence similarity to vitellogenin from the nematode Caenorhabditis elegans [3] and this vitellogenin has some sequence

similarity to human von Willebrand factor, a protein involved in the human blood clotting process. A affinity-blot test, using biotinylated lectins, revealed that CP is a glycoprotein that was strongly recognized by Con A. The lectins from wheat germ and castor bean also recognized sugar residues in the CP molecule, although the reaction was weaker than for Con A (Table 2). The highly positive reaction with Con Table 1 Amino acid composition (mol%) of CP from white shrimp (P. 6annamei ), crayfish (P. leniusculus) [12], lobster (P. interruptus) [6] and sand crab (O. bipustulatus) [15] Amino acid

P. 6annamei

P. leniusculus

P. interruptus

O. bipustulatus

Asx Thr Ser Glx Pro Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg

10.39 7.68 7.92 11.61 5.64 7.17 5.44 n.d.a 6.97 1.38 5.69 8.79 3.28 4.65 3.77 5.02 4.56

9.05 8.72 7.65 12.30 4.99 5.84 5.72 1.53 6.48 1.49 6.31 8.32 3.13 5.19 3.18 6.28 3.84

9.90 7.12 8.19 10.91 5.25 6.17 5.46 1.31 6.90 1.69 5.12 9.60 3.18 4.10 4.16 4.18 4.75

11.94 6.66 6.75 12.22 5.74 7.40 6.01 1.27 6.66 1.75 5.09 8.14 2.31 3.98 3.61 5.64 4.81

a

n.d., Not determined.

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Fig. 3. Alignment of clotting protein N-terminal sequences of P. 6annamei, P. leniusculus, P. interruptus and I. ciliatus. Identical sequences are boxed.

A and other lectins indicated that CP is a glycosylated protein, containing mainly a-D-mannose and a-D-glucose. To determine the presence of lipids, purified CP obtained by affinity chromatography was incubated with the lipophilic stain Sudan black. By native PAGE (5 – 8%) gradient gel, CP was identified as a stained band with low relative mobility (Fig. 4). The fluorescent probe dansylcadaverine (DC) showed that white shrimp CP is a substrate for an enzyme present in HSL inasmuch as DC was bound to the CP molecule (Fig. 5). Polymerization of CP is also clearly observed in the gel, indicating that this mechanism could occur in vivo conditions. A similar pattern was observed by using guinea pig TGase instead of HLS (Fig. 5). The polymerization of CP in the presence of either HSL or TGase, and CaCl2 occurs very fast and the polymers appear almost immediately after calcium addition. The 400-kDa band transformation to high molecular mass aggregates is clearly observed by the presence of CP precipitated on the top of the gel, even when using 2–5% polyacrylamide gels. The tests were also performed with a reduced and alkylated CP subunit, but during the incubation time, CP was partially degraded. This phenomenon has been also reported for crayfish CP [12], but in shrimp CP subunits the degradation was seen in a shorter period of time, even under refrigerated conditions. The reason for this degradation remains to be investigated. The rate of in vitro shrimp CP polymerization, as well as shrimp hemolymph coagulation are very fast. Special care must be taken during bleeding and cell separation in order to avoid the release of the cellular Ca2 + -dependent TGase. By using the shrimp anticoagulant containing EDTA [24] clot formation did not occur during hemolymph extraction. The centrifugation

of the hemolymph extracted with shrimp anticoagulant at 700× g did not result in cell damage and the cell-free plasma never showed coagulation, even after dialysis with EDTA-free phosphate buffer. For clot formation a cellular component is necessary and it is a TGase-type. This is in agreement with Martin et al., [16] who reported that, in crustaceans, a TGase is responsible for CP polymerization. In vertebrate systems, proteolysis of fibrinogen produces fibrin, which is the target substrate for Factor XIII (a TGase), to produce an insoluble clot. If previous proteolysis is required for crustacean CP polymerization is still unknown. In the sand crab, O. bipustulatus, polymerization of the clottable protein required a limited proteolysis by thrombin, in addition to TGase and Ca2 + . However, this clottable protein has several differences to shrimp CP: it was purified from circulating cells (amebocytes), has different molecular mass and amino acid composition. On the other hand, the well characterized CP from crayfish [12] is similar to shrimp CP in molecular and functional characteristics and there is no evidence that previous proteolysis could be required. In shrimp as well as in crayfish, in addition to a Ca2 + -dependent TGase, apparently, other cellular fac-

Table 2 Reaction of white shrimp CP with biotinylated lectins: Con A, WGA and RCA Lectin

Specificity

Reaction

Con A WGA RCA

a-D-mannose/a-D-glucose glucosamine b-D-galactosamine

++++ ++ ++

D-N-acetyl

Fig. 4. Sudan black staining of white shrimp CP. PAGE was run under non-denaturing conditions in a 5% polyacrylamide gel before staining with either Coomassie (A) or Sudan black (B). MWMs were included as reference.

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References

Fig. 5. Fluorescent labeling of shrimp CP. DC incorporation catalyzed by hemocyte lysate in presence (1) or absence (2) of calcium, and by guinea pig TGase without (3) and with (4) calcium. The bands were visualized under UV light, after running the samples in a 5% polyacrylamide gel.

tors are not required for clot formation. Since polymerization of purified shrimp CP occurs with guinea pig liver TGase, it is possible that these proteins are the only factors involved in clot formation in shrimp. Thus, in spite of structural or probable origin differences, vertebrate and invertebrate clot formation is very similar, but using different strategies. In both cases the clot is formed by the action of a Ca2 + -dependent TGase on a clottable plasma protein, fibrinogen or clotting protein, respectively. However, in vertebrates, a previous proteolytic processing reaction is required (fibrinogen to fibrin) to obtain a substrate for TGase which is found in plasma. On the other hand, in invertebrates TGase is compartmentalized into hemocytes and acts immediately after its release because previous CP proteolysis is not required.

Acknowledgements This research was partially supported by CONACyT (Mexico) Grant 3932P-B. We are grateful to T. ReyesIzquierdo (CIAD) for helping with the fluorescent tests and to F. Magallo´n and G. Portillo (CIBNOR, Mexico) for supplying the experimental animals.

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