The structure of the hemocyanin from the horseshoe crab, Limulus polyphemus. The amino acid sequence of the largest cyanogen bromide fragment

The amino acid sequence of the largest CNBr fragment, CNBrIa, from hemocyanin II of Limulus polyphemus has been determined. This fragment contains 203 residues, has Mr = 23,600, and is the NH2-terminal segment of the molecule. Comparison of this sequence with the first 87 residues of the NH2-terminal sequence of the alpha chain of hemocyanin from the Japanese horseshoe crab, Tachypleus tridentatus, of the Western Pacific (Nemoto, T., and Takagi, T. (1981) Biochim. Biophys. Acta 670, 79-83) shows that 46% of the residues are identical. Residues 172-177, -His-His-Trp-His-Trp-His-, appear to constitute at least part of the copper-binding site. This conclusion is supported by our finding that 40% of the residues in the sequence 172-186 are identical to residues 188-202 in tyrosinase from Neurospora; residues 188 and 193 in tyrosinase are known to be copper-binding histidines (Pfiffner, E., and Lerch, K. (1981) Biochemistry 20, 6029-6035). Model building suggests that histidines 172 and 175 might bind one copper atom and histidines 173 and 177 might bind the second copper. These observations suggest that tyrosinase and hemocyanin may have a common evolutionary origin, but we have not so far seen other correspondences.     

Tachyplesins isolated from hemocytes of Southeast Asian horseshoe crabs (Carcinoscorpius rotundicauda and Tachypleus gigas): identification of a new tachyplesin, tachyplesin III, and a processing intermediate of its precursor

Tachyplesins and their analogs are antimicrobial peptides composed of 17 or 18 amino acid residues present abundantly in acid extracts of hemocyte debris of horseshoe crabs. We purified here tachyplesin isopeptides from hemocytes of two species of Southeast Asian horseshoe crabs, Carcinoscorpius rotundicauda and Tachypleus gigas, and determined their amino acid sequences. The major tachyplesin isolated from both species was identified, respectively, as tachyplesin I, which had previously been found in hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus). The yield from both species was very high (more than 70 mg per 100 g wet weight of hemocytes), i.e., comparable with that from T. tridentatus. In addition to tachyplesin I, a new tachyplesin isopeptide, named tachyplesin III, was also isolated from T. gigas hemocytes, in which an arginine replaced the 15th lysine of tachyplesin I. The carboxyl-terminal residue of the isolated tachyplesins I and III was confirmed, respectively, to be an arginine alpha-amide by chemical analysis. Furthermore, a tachyplesin peptide derivative with a carboxyl-terminal extension of glycine-lysine was newly found in the hemocytes of C. rotundicauda. It appeared to be an intermediate derived from a tachyplesin precursor during processing to the mature form.     

A clottable protein (coagulogen) from amoebocyte lysate of Japanese horseshoe crab (Tachypleus tridentatus). Its isolation and biochemical properties.

A clottable protein, named coagulogen, was highly purified from the amoebocyte lysate of Japanese horseshoe crab (Tachypleus tridentatus) by a method similar to that used for the lysate of Limulus polyphemus amoebocytes. The isolated material gave a single protein band on analytical gel electrophoresis at pH 3.2, gel electrofocusing, and sodium dodecyl sulfate (SDS) gel electrophoresis with or without 2-mercaptoethanol. It was 90 percent coagulable, and the total yield from 10 ml of the amoebocyte lysate was about 40 mg. The sedimentation coefficient of purified coagulogen was 2.6 S and its molecular weight was estimated to be about 15,300 by sedimentation equilibrium analysis. The molecular weight estimated by SDS-gel electrophoretic analysis was 19,500 +/- 1,000. This discrepancy was apparently due to abnormal mobility arising from the basic nature of this protein on electrophoresis. The protein had a high isoelectric point of pH 10.0 +/- 0.2, as measured by the isoelectric focusing technique. It consisted of a total of 132 to 135 amino acid residues and contained high levels of basic amino acids, which accounted for more than 16 per cent of the total amino acid residues. No methionine was detected. High contents of valine, half-cystine, glutamic acid (glutamine), and phenylalanine were found. The N-terminal sequence of the first three residues of the coagulogen was Ala-Asx-Thr, and its C-terminal residues was identified as phenylalanine, indicating that it consists of a single polypeptide chain. It is of interest that the first three N-terminal residues are homologous with those of the Aalpha-chain of non-human primate fibrinogen.     

Characterization of a complementary deoxyribonucleic acid for the coagulogen of Limulus polyphemus

An 869-nucleotide-long cDNA clone for the coagulogen from Limulus amebocyte has been isolated and its nucleotide sequence has been determined. The deduced amino-acid sequence revealed a signal peptide, 20 amino acids long, and a mature protein of 175 amino acids. The amino-acid sequence of the coagulogen was compared to all known proteins by two computer programs. Using these programs, Limulus coagulogen showed 70% homology with the coagulogen of Tachypleus tridentatus (Japanese horseshoe crab). Further computer analysis showed no statistically significant homology to support an evolutionary origin of the horseshoe crab coagulogen common to other protein families. These results place horseshoe crab coagulogen in a new superfamily unrelated to any other proteins investigated. RNA blot analysis of Limulus RNA indicated that the coagulogen mRNA was about 900 bases long and represented an abundant species in the amebocyte while detected only in small quantities in the hepatopancreas. Besides mature RNA, high-molecular-weight forms of coagulogen RNA were also observed. Southern blot analysis of Limulus DNA digested with restriction endonucleases suggested that the Limulus coagulogen gene contains at least three introns, or belongs to a multigene family.     

Two types of coagulogen mRNAs found in horseshoe crab (Tachypleus tridentatus) hemocytes: molecular cloning and nucleotide sequences

The complete cDNA sequence coding for the coagulogen present in the horseshoe crab (Tachypleus tridentatus) hemocytes was determined. Clones carrying cDNA fragments for coagulogen were isolated from a cDNA library of the hemocyte mRNA using synthetic oligodeoxyribonucleotides as probes. The nucleotide sequence analyses of the cloned cDNAs revealed that the hemocyte coagulogen consists of 175 amino acids with 20 amino acids in a presegment, and that there are two types of mRNAs for coagulogen. The two mRNAs exhibited three nucleotide substitutions, two of which were in their protein-coding regions, resulting in two amino acid replacements. Subsequently, two molecular species of coagulogen, named coagulogens type I and type II, were identified by tryptic peptide mapping of the mature proteins isolated from the hemocyte lysate. These results suggest that the two types of coagulogens are first synthesized as preproteins and are incorporated into the granules that are abundantly present in the hemocytes with liberation of the signal peptides.     

Primary structure of anti-lipopolysaccharide factor from American horseshoe crab, Limulus polyphemus

The complete amino acid sequence of anti-lipopolysaccharide (LPS) factor purified from the hemocytes lysate of the American horseshoe crab, Limulus polyphemus, was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, clostripain, and Staphylococcus aureus V8 protease. Upon sequencing the peptides by the automated Edman method, the following primary structure was obtained: (Sequence: in text). During the sequence analysis, two species of the protein, which differed from each other at one locus, were found and characterized. L. polyphemus anti-LPS factor was a basic protein consisting of a single polypeptide chain of 101 residues and a calculated molecular weight of 11,786 or 11,800. The hydrophobic NH2-terminal sequence and the clustering of positive charges found in the disulfide loop yielded a typical amphipathic character of this protein. Moreover, L. polyphemus anti-LPS factor showed 83% sequence identity with the Tachypleus tridentatus protein, and the sequence similar to that observed in the EF-hand structure was found to contain in the COOH terminal portions of these proteins, although its function is unknown.     

Lipopolysaccharide-sensitive serine-protease zymogen (factor C) of horseshoe crab hemocytes. Identification and alignment of proteolytic fragments produced during the activation show that it is a novel type of serine protease

The horseshoe crab clotting factor, factor C, present in the hemocytes is a serine-protease zymogen activated with lipopolysaccharide. It is a two-chain glycoprotein (Mr = 123,000) composed of a heavy chain (Mr = 80,000) and a light chain (Mr = 43,000) [T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511-521]. In our continued study of this zymogen, we have now also found a single-chain form of factor C (Mr = 123,000) in the hemocyte lysate. The heavy chain had the NH2-terminal sequence of Ser-Gly-Val-Asp-, consistent with that of the single-chain factor C, indicating that the heavy chain is derived from the NH2-terminal part of the molecule. The light chain had an NH2-terminal sequence of Ser-Ser-Gln-Pro-. Incubation of the two-chain zymogen with lipopolysaccharide resulted in the cleavage of a Phe-Ile bond between residues 72 and 73 of the light chain. Concomitant with this cleavage, the A (72 amino acid residues) and B chains derived from the light chain were formed. The complete amino acid sequence of the A chain was determined by automated Edman degradation. The A chain contained a typical segment which is similar in sequence to a family of repeats in human beta 2-glycoprotein I, complement factors B, protein H, C4b-binding protein, and coagulation factor XIII b subunit. The NH2-terminal sequence of the B chain was Ile-Trp-Asn-Gly-. This chain contained the serine-active site sequence-Asp-Ala-Cys-Ser-Gly-Asp-Ser-Gly-Gly-Pro-. These results indicate that horseshoe crab factor C exists in the hemocytes in a single-chain zymogen form and is converted to an active serine protease by hydrolysis of a specific Phe-Ile peptide bond.     

Antimicrobial peptides, isolated from horseshoe crab hemocytes, tachyplesin II, and polyphemusins I and II: chemical structures and biological activity

Tachyplesin is an antimicrobial peptide recently found in the acid extract of hemocytes from the Japanese horseshoe crab (Tachypleus tridentatus) [Nakamura, T. et al. (1988) J. Biol. Chem. 263, 16709-16713]. In our continuing studies on the peptide, we have found an isopeptide, tachyplesin II, and also polyphemusins I and II in hemocytes of the American horseshoe crab (Limulus polyphemus). The complete primary structures of these peptides, which are very similar to that of the previously isolated peptide, now named tachyplesin I, were determined to be as follows: (Table: see text). The isopeptide, tachyplesin II, consists of 17 residues with a COOH-terminal arginine alpha-amide. On the other hand, both polyphemusins I and II were found to contain 18 residues due to an additional Arg residue at the NH2-terminal end as well as a COOH-terminal arginine alpha-amide. The disulfide linkages for polyphemusin I consisted of two bridges between Cys-4 and Cys-17 and between Cys-8 and Cys-13, which was identical to in the case of tachyplesin I. Moreover, all of these peptides inhibited the growth of not only Gram-negative and -positive bacteria but also fungi, such as Candida albicans M9. Furthermore, complex formation between these peptides and bacterial lipopolysaccharides was also observed in a double diffusion test. These results suggest that tachyplesins and polyphemusins are probably located in the hemocyte membrane, where they act on antimicrobial peptides as a self-defense mechanism in the horseshoe crab against invading microorganisms.     

The complete amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda

The complete amino acid sequence of coagulogen purified from the hemocytes of the horseshoe crab Carcinoscorpius rotundicauda was determined by characterization of the NH2-terminal sequence and the peptides generated after digestion of the protein with lysyl endopeptidase, Staphylococcal aureus protease V8 and trypsin. Upon sequencing the peptides by the automated Edman method, the following sequence was obtained: A D T N A P L C L C D E P G I L G R N Q L V T P E V K E K I E K A V E A V A E E S G V S G R G F S L F S H H P V F R E C G K Y E C R T V R P E H T R C Y N F P P F V H F T S E C P V S T R D C E P V F G Y T V A G E F R V I V Q A P R A G F R Q C V W Q H K C R Y G S N N C G F S G R C T Q Q R S V V R L V T Y N L E K D G F L C E S F R T C C G C P C R N Y Carcinoscorpius coagulogen consists of a single polypeptide chain with a total of 175 amino acid residues and a calculated molecular weight of 19,675. The secondary structure calculated by the method of Chou and Fasman reveals the presence of an alpha-helix region in the peptide C segment (residue Nos. 19 to 46), which is released during the proteolytic conversion of coagulogen to coagulin gel. The beta-sheet structure and the 16 half-cystines found in the molecule appear to yield a compact protein stable to acid and heat. The amino acid sequences of coagulogen of four species of limulus have been compared and the interspecies evolutionary differences are discussed.     

Further studies on lipopolysaccharide-sensitive serine protease zymogen (factor C): its isolation from Limulus polyphemus hemocytes and identification as an intracellular zymogen activated by alpha-chymotrypsin, not by trypsin

An intracellular serine protease zymogen, factor C, is an initiator in the hemolymph coagulation system of horseshoe crab. We purified this zymogen from the hemocytes of the American horseshoe crab, Limulus (L.) polyphemus, the objective being to compare its properties with those of the Japanese horseshoe crab, Tachypleus (T.) tridentatus, factor C. The purified zymogen L.-factor C showed similar properties to those of T.-factor C, in terms of molecular mass (123,000), amino acid composition (1,011 residues), subunit structure (two chains), and antigenicity. Like the zymogen T.-factor C, this zymogen was also activated autocatalytically in the presence of bacterial lipopolysaccharide (LPS) and its synthetic lipid A analogue. A most interesting finding is that both protease zymogens are rapidly activated by alpha-chymotrypsin or rat mast cell chymase, but not by trypsin. The active enzyme factor C showed alpha-thrombin-like specificity toward synthetic tripeptide substrates. This factor C was also strongly inhibited by an alpha-thrombin inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. Thus, the enzymatic properties of factor C are similar to those of mammalian alpha-thrombin. On the other hand, the coagulation cascade system present in the hemocyte lysate was activated when chymotrypsin, free from LPS, was added to the lysate used to detect the endotoxins. The implication of our findings is that the chymotrypsin-catalyzed initiation of the horseshoe crab coagulation system is unique, since all known mammalian coagulation, fibrinolysis and complement systems are initiated by trypsin-like enzymes.     

Isolation and Characterization of a Sialic Acid-specific Lectin from Hemolymph of the Southeast Asian Horseshoe Crab Tachypleus gigas

A lectin was isolated from hemolymph of the Southeast Asian horseshoe crab Tachypleus gigas by using glycophorin HA affinity chromatography and Sephacryl S-300 gel filtration. The purified lectin had a molecular mass of approximately 396 kDa and was composed of 13 identical subunits with molecular masses of 31 kDa. The serological specificity of the purified lectin was specifically inhibited by sialic acids sialoglycoproteins, but not by neutral sugars, hexosamines, N-acetylhexosamines, or asialoglycoproteins. Although the N-terminal amino acid sequence of the lectin from T. gigas was identical to that from American horseshoe crab (liphemin) by the same purification method and cross reacted with the anti-liphemin serum, the calcium concentration of hemagglutinating activity of the purified lectin showed a smaller optimal concentration than that of liphemin.     

Reproductive behavior in horseshoe crabs:Does density matter?

While the four species of horseshoe crabs share many common reproductive traits with respect to their reproductive systems,they do differ with respect to their mating behavior (monogamy vs.polygynandry).Past research has attributed these differences to a number of factors including:spawning densities,operational sex ratios (OSR's),male condition (or age),environmental and/or genetic factors,or a combination thereof.Mating behaviors in the three Asian horseshoe crab species (Tachypleus gigas,T.tridentatus,an...     

Molecular characterization of limulin, a sialic acid binding lectin from the hemolymph of the horseshoe crab, Limulus polyphemus.

The sialic acid binding lectin, limulin, was isolated by gel filtration and ion-exchange chromatography from the hemolymph of Limulus polyphemus. When the purified protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of beta-mercaptoethanol, two major protein bands were observed. These two bands, subsequently found to contain carbohydrate as well, corresponded to molecular weights of 25 000 and 27 000. Amino acid sequence analyses were performed on both the intact protein and isolated cyanogen bromide fragments. The following primary structural features were noted in the amino-terminal region of limulin: (1) the absence of histidine and alanine from the NH2-terminal 50 residues; (2) the presence of five of the total eight prolines of the molecule between positions 13 and 30; and (3) a possible carbohydrate attachment site consisting of only the amino acids proline and serine between residues 13 and 19. The resultsof cyanogen bromide cleavage studies confirmed the presence of 2 methionine residues per subunit, at positions 25 and 58 respectively. No sequence heterogeneity was observed in this study. While it is quite possible that limulin plays some role in the defense mechanisms of the horseshoe crab, there is no obvious sequence homology between this invertebrate lectin and vertebrate immunoglobulins.     

Amino acid sequence of horseshoe crab, Tachypleus tridentatus, striated muscle troponin C

The amino acid sequence of troponin C obtained from horseshoe crab, Tachypleus tridentatus, striated muscle was determined by sequence analysis and alignments of chemically and enzymatically cleaved peptides. Troponin C is composed of 153 amino acid residues with a blocked N-terminus and contains no tryptophan or cysteine residue. The site I, one of the four Ca2+-binding sites, is considered to have lost its ability to bind Ca2+ owing to the replacements of certain amino acid residues.     

Acrosomal actin bundles of Asian and American horseshoe crab sperm differ in protein composition

Acrosomal actin bundles were isolated from the sperm of horseshoe crabs from four different sources, three from Asia and one from North America, and their protein constituents and structures were compared. The bundle from the American Limulus polyphemus sperm was composed of actin and two associated proteins of MW 95,000 and MW 52,000, as reported previously (Tilney, L. G. (1975) J. Cell Biol. 64, 289-310). However, those from the three Asian species (Tachypleus tridentatus, T. gigas, and Carcinoscorpius rotundicauda) were composed only of actin and the protein of MW 95,000. Electron microscopic and optical diffraction studies indicated that both the helical structures and the interfilament spacing of the actin filaments composing the acrosomal bundle were indistinguishable among the four species. These results suggest that the MW 95,000 protein crosslinks actin filaments in the bundle. Moreover, they support the idea that Limulus and the three Asian species have evolved independently from a common ancestor.     

Head-to-tail polymerization of coagulin, a clottable protein of the horseshoe crab

A clottable protein coagulogen of the horseshoe crab Tachypleus tridentatus is proteolytically converted into an insoluble coagulin gel through non-covalent self-polymerization. Here we identified binding sites for the polymerization. A tryptic fragment, derived from the coagulin polymer chemically cross-linked by a bifunctional cross-linker, was isolated. Amino acid sequence analysis indicated that the fragment consists of two peptides cross-linked between Lys(85) and Lys(156). The two lysine residues are oppositely located at the head and tail regions of the elongated molecule separated by a much greater distance than the length of the cross-linker, which suggests that the cross-linking occurs intermolecularly. Based on the x-ray structural analysis, exposure of a hydrophobic cove on the head in response to the release of peptide C has been postulated (Bergner, A., Oganessyan, V., Muta, T., Iwanaga, S., Typke, D., Huber, R., and Bode, W. (1996) EMBO J. 15, 6789-6797). An octapeptide containing Tyr(136), which occupies the tail end of coagulin, was found to inhibit the polymerization. Replacement of Tyr(136) of the peptide with Ala resulted in loss of the inhibitory activity. These results indicated that the polymerization of coagulin proceeds through the interaction between the newly exposed hydrophobic cove on the head and the wedge-shaped hydrophobic tail.     

Purification and amino acid sequence of Kunitz-type protease inhibitor found in the hemocytes of horseshoe crab (Tachypleus tridentatus)

A low molecular weight protein protease inhibitor was purified from Japanese horseshoe crab (Tachypleus tridentatus) hemocytes. It consisted of a single polypeptide with a total of 61 amino acid residues. This protease inhibitor inhibited stoichiometrically the amidase activity of trypsin (Ki = 4.60 X 10(-10) M), and also had inhibitory effects on alpha-chymotrypsin (Ki = 5.54 X 10(-9) M), elastase (Ki = 7.20 X 10(-8) M), plasmin, and plasma kallikrein. However, it had no effect on T. tridentatus clotting enzyme and factor C, mammalian blood coagulation factors (activated protein C, factor Xa and alpha-thrombin), papain, and thermolysin. The complete amino acid sequence of this inhibitor was determined and its sequence was compared with those of bovine pancreatic trypsin inhibitor (BPTI) and other Kunitz-type inhibitors. It was found that the amino acid sequence of this inhibitor has a high homology of 47 and 43% with those of sea anemone inhibitor 5-II and BPTI, respectively. Thus, this protease inhibitor appeared to be one of the typical Kunitz-type protease inhibitors.     

Identification and characterization of a phospholipid-binding site of bovine factor Va

Coagulation factor Va is a cofactor which combines with the serine protease factor Xa on a phospholipid surface to form the prothrombinase complex. The phospholipid-binding domain of bovine factor Va has been reported to be located on the light chain of the molecule and more precisely on a fragment of Mr = 30,000 which is obtained after digestion of factor Va light chain by factor Xa. This proteolytic fragment is located in the NH2-terminal part of factor Va light chain (residues 1564-1765). In order to further characterize the lipid-binding domain of bovine factor Va, isolated bovine light chain was preincubated with synthetic phospholipid vesicles (75% phosphatidylcholine, 25% phosphatidylserine) and digested with trypsin, chymotrypsin, and elastase. Two peptide regions protected from proteolytic cleavage were identified and characterized from each proteolytic digestion. A comparison of the NH2-terminal sequence and amino acid composition of the two tryptic peptides with the deduced sequence of human factor V indicates a match with residues 1657-1791 of the light chain of human factor V for one peptide and residues 1546-1656 for the other peptide. When chymotrypsin or elastase were used for digestion, the NH2-terminal sequence of one peptide showed a match with residues 1667-1797 of the light chain, while the other peptide presented an NH2-terminal sequence identical with the previously described for the bovine factor Va light chain. When these peptides were assayed for direct binding to phospholipid vesicles, only the tryptic and the chymotryptic peptides covering the middle region of the A3 domain of the bovine factor Va light chain demonstrated an ability to interact with phospholipid vesicles. Thus, knowing that the factor Xa cleavage site on the factor Va light chain is located between residues 1765 and 1766 of the light chain this lipid-binding region of the bovine factor Va is further localized to amino acid residues 1667-1765.