Proc. Natl. Acad. Sci. USA Vol. 96, pp. 1965–1970, March 1999 Biochemistry The crayfish plasma clotting protein: A vitellogenin-related protein responsible for clot formation in crustacean blood i MARTIN HALL*†,RUIGONG WANG*†,RIK VAN ANTWERPEN‡§,LARS SOTTRUP-JENSEN¶, AND KENNETH SO¨DERHA¨LL* *Department of Comparative Physiology, Evolutionary Biology Centre, Uppsala University, Villava¨gen6, S-752 36 Uppsala, Sweden; ‡Department of Biochemistry, University of Arizona, Tucson, AZ 85721; and ¶Department of Molecular and Structural Biology, University of Aarhus, DK-8000 Århus C, Denmark Communicated by Russell F. Doolittle, University of California, San Diego, La Jolla, CA, December 29, 1998 (received for review November 13, 1998) ABSTRACT Coagulation in crayfish blood is based on the recognize the coagulogen as a substrate (8, 9), and its role transglutaminase-mediated crosslinking of a specific plasma during clotting is unclear. clotting protein. Here we report the cloning of the subunit of The clotting reaction also is characterized at the molecular this clotting protein from a crayfish hepatopancreas cDNA level in crustaceans, where it depends on the TGase-mediated library. The ORF encodes a protein of 1,721 amino acids, crosslinking of a specific plasma clotting protein (CP) (10–13). including a signal peptide of 15 amino acids. Sequence anal- The crayfish CP, cloning of which is reported in this article, has ysis reveals that the clotting protein is homologous to vitel- previously been biochemically and functionally characterized logenins, which are proteins found in vitellogenic females of (12, 14). It is a very high density lipoprotein (VHDL) (14) egg-laying animals. The clotting protein and vitellogenins are consisting of two identical 210-kDa subunits held together by all lipoproteins and share a limited sequence similarity to disulfide bonds (12). Each one of the 210-kDa subunits has certain other lipoproteins (e.g., mammalian apolipoprotein B both lysine and glutamine sidechains, which are recognized and microsomal triglyceride transfer protein) and contain a and become covalently linked to each other by TGases (12). stretch with similarity to the D domain of mammalian von Clotting is induced when a TGase is released from hemocytes Willebrand factor. The crayfish clotting protein is present in or tissue, becomes activated by the Ca21-content in plasma, both sexes, unlike the female-specific vitellogenins. Electron and starts crosslinking the plasma CP molecules into large microscopy was used to visualize individual clotting protein aggregates. The hemocytes also contain components of the molecules and to study the transglutaminase-mediated clot- so-called prophenoloxidase activating system (proPO system), ting reaction. In the presence of an endogenous transglutami- which constitutes an important part of the immediate immune nase, the purified clotting protein molecules rapidly assemble response in crustaceans (15, 16). Components of the proPO into long, flexible chains that occasionally branch. system cause degranulation and lysis of hemocytes, and as a result more proPO components and TGase are released (15, Vertebrate and invertebrate animals have evolved efficient 16). In this way, the proPO system could affect the clotting molecular mechanisms to immediately form clots of blood reaction by causing the release of TGase activity. However, the components. This is necessary to prevent loss of blood in case proPO system and the clotting reaction do not appear to share of injury. All vertebrates have similar coagulation systems a common activation pathway, as the proPO system is activated based on the proteolytically induced aggregation of fibrinogen by a proteolytic cascade [triggered by microbial polysaccha- into insoluble fibrin (1–3). The fibrin aggregates, which initially rides (15, 16)], and the initiation of the clotting reaction 1 are noncovalently associated, are further stabilized by inter- requires no proteolytic processing [only Ca2 , which activates molecular covalent crosslinks formed by a proteolytically the TGase (11, 12)]. activated transglutaminase (TGase), factor XIIIa. TGases (EC The N terminus of the lobster fibrinogen (the CP homologue 2.3.2.13) are Ca21-dependent enzymes capable of forming in lobster) was reported to have sequence similarity to vitel- covalent bonds between the side chains of specific lysine and logenins (VTGs) (17), which are proteins expressed only in glutamine residues on certain proteins (4–6). Among the females of egg-laying animals [vertebrates as well as inverte- invertebrates, which is a more diverse group, different coag- brates (18–20)]. Besides having similar functions, the CP does ulation mechanisms seem to have evolved, and detailed infor- not appear to share any characteristics with fibrinogen or mation on the coagulatory mechanisms at the molecular level coagulogen, the proteins forming clots in vertebrate animals is lacking in most groups. One exception is the hemocyte and horseshoe crabs, respectively. This indicates that the (blood cell)-derived clotting cascade in horseshoe crabs, which crayfish CP (12, 14), and its homologues in other crustaceans has been characterized in detail (7). This horseshoe crab (10, 11, 13, 17, 21) constitute a separate group of blood CPs. (Limulus) clotting system is activated by microbial lipopoly- To establish the evolutionary origin of the crustacean CPs, saccharides or b-1,3-glucans, and it has some resemblance to we have cloned the crayfish CP. Our results show that the the vertebrate coagulation system, as it is based on a proteo- crustacean CPs constitute a distinct group of proteins unre- lytic cascade that leads to the conversion of a soluble protein (coagulogen) into an insoluble aggregate (coagulin). However, Abbreviations: CP, clotting protein; TGase, transglutaminase; UTR, the proteins participating in the Limulus clotting system are all untranslated region; VHDL, very high density lipoprotein; VTG, from the hemocytes and are not homologous to the vertebrate vitellogenin; vWF, von Willebrand factor; proPO, prophenol oxidase; plasma coagulation proteins. A TGase has been characterized HLS, hemocyte lysate supernatant. Data deposition: The sequence reported in this paper has been and cloned from Limulus hemocytes, but it does not appear to deposited in the GenBank database (accession no. AF102268). †M.H. and R.W. contributed equally to this work. § The publication costs of this article were defrayed in part by page charge Present address: Department of Biochemistry and Molecular Bio- physics, Virginia Commonwealth University, Richmond, VA 23298- payment. This article must therefore be hereby marked ‘‘advertisement’’ in 0614. accordance with 18 U.S.C. §1734 solely to indicate this fact. iTo whom reprint requests should be addressed. e-mail: Kenneth. PNAS is available online at www.pnas.org. [email protected]. 1965 Downloaded by guest on September 25, 2021 1966 Biochemistry: Hall et al. Proc. Natl. Acad. Sci. USA 96 (1999) lated to proteins involved in clotting reactions in other groups 15 s, 30 s, 45 s, 1 min, or 2 min at room temperature. The of animals. samples were analyzed by using electron microscopy. The ability of CP-containing samples to form stabilized clots was MATERIALS AND METHODS examined in a similar way by mixing CP, HLS, and CaCl2 (or H2O in a control) as described above. After1hatroom Animals. Freshwater crayfish, Pacifastacus leniusculus, were temperature, the presence of stabilized clots was examined by kept in tanks in aerated tap water. Only intermoult animals shaking and tilting the tubes. were used in the experiments. Protein Determination. Protein concentrations were deter- Protein Purification and Sequencing. For protein sequenc- mined according to Bradford (23), using BSA as standard. ing, CP from crayfish hemolymph was isolated by precipitation Electron Microscopy. The samples were analyzed by nega- at low ionic strength according to Kopacek et al. (12). Purified tive staining, essentially according to the method of Valentine CP (20 mg) was reduced, alkylated, and digested with trypsin. et al. (24). A thin film of carbon was evaporated onto a piece Tryptic peptides were isolated by anion exchange chromatog- of freshly cleaved mica. The carbon-coated mica was inserted raphy on a DEAE-Sephacel column followed by reversed in a solution of clotting protein (diluted to '50 mgyml in PBS) phase HPLC on a Nucleosil C18 column. The N-terminal in such a way that the carbon film partly detached from the amino acid sequences of the intact protein and internal tryptic mica and floated on the solution surface. After 30 s, the carbon peptides were determined on an Applied Biosystem 470A film was retracted from the solution of clotting protein, floated automated gas-phase sequencer. for a few seconds on deionized water, and transferred to a 2% For electron microscopy study (see below), CP was isolated solution of methylamine tungstate in deionized water (pH 7.5). by repeated KBr density gradient ultracentrifugation using a Subsequently, the carbon film was picked up from the tung- different rotor and different KBr gradients compared with the state solution with a 300 mesh copper grid coated with lacey previously described procedure (14). Ultracentrifugation tubes substrate (Ted Pella, Redding, CA). Excess methylamine (13 3 51 mm polyallomer Quickseal tubes, Beckman) were tungstate solution was removed with filter paper, after which filled with crayfish KBr-plasma (0.445 g of KBr per ml of the preparation