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Alpha2-macroglobulin, a protease-binding protein that is reactive with almost all endopeptidases, is present in high concentrations in the plasma of the horseshoe crab, Limulus. Alpha2-macroglobulin was demonstrated by its ability to protect the active site of trypsin from inactivation by the macromolecular active site inhibitor, soybean trypsin inhibitor, and by reaction with an antiserum prepared against purified Limulus α2-macroglobulin. The blood cells also contain α2-macroglobulin in a form that is released when washed cells are stimulated to undergo exocytosis by treatment with the ionophore, A23187. Alpha2-macroglobulin is detected in the materials released from the cells during degranulation both by activity in the soybean trypsin inhibitor-protection assay and by immunochemical staining of Western blots. The subunit molecular weight of the cell-associated form of α2-macroglobulin, 185 kDa, is identical to that of the plasma form. The amount of α2-macroglobulin contained within the cells of a given volume of blood is 0.5-2% of the quantity in solution in that volume of plasma. The distilled water lysates of N-ethylmaleimide-stabilized amebocytes used to detect endotoxin (e.g., Limulus amebocyte lysate or LAL) contain relatively large quantities of active α2-macroglobulin. These preparations are essentially free of the principal plasma protein, hemocyanin, indicating that the cells had been well washed prior to lysis.

The hemolytic activity of Limulus plasma is mediated by the sialic acid-binding plasma lectin, limulin (1). We have recently demonstrated that the hemolytic activity of purified limulin can be potentiated by thiol ester-reacted Limulus Lu>-macroglobulin (LAM)(Z). Proteins of the cyz-m~roglobulinfamily are unique for the presence of an internal thiol ester bond, which in LAM links Cys-999 with Gin-1002 (Ikawi et al., unpub. data). Thiol ester activation hydrolyses this bond, releasing the y-carbonyl ofGIn, which in LAM establishes isopeptide bonds with the t-amino group of Lys-254 (3) and the thiol of Cys. Thiol ester activation is initiated by reaction with proteases and is a key event in protease binding by a2-macroglobulin. Activation can also be initiated by small primary amines, such as methylamine. LAM is a large protein with several different functional domains. We have been interested in identifying the domains that are important in its ability to potentiate limulin-mediated hemolysis. Hemolysis of sheep erythrocytes and purification of limulin and LAM were carried out as described previously ( 1,4,5). The binding of limulin to LAM was quantified with a solid phase assay. Limulin immobilized to microtiter wells was exposed to LAM, and the amount of bound LAM was determined by its subsequent reaction with affinity-purified anti-LAM antibodies and a horseradish peroxidase-conjugated second antibody. Native, un-reacted LAM lacks free thiols (6). The new thiol exposed by reaction of LAM with proteases or methylamine is necessary for the potentiation of hemolysis because its acylation by treatment with iodoacetamide (0.1 M iodoacetamide in 0. I M Tris buffer, pH 8.0, 4 h, room temp.) eliminates the potentiating activity of methylamine-activated LAM (Fig. I). Under the conditions used here, the binding of iodoacetamide was limited to the thiol of Cys-999, because the only [‘“Cl-labeled tryptic peptides of [‘4C]-iodoacetamide-reacted MALAM had the peptide sequence of the thiol ester domain, PTGCGIQNMIK (3). Human and Limulus forms of az-macroglobulin share significant sequence identity at key functional domains but show no homology in the stretch delineated by residues 24-105. Instead, this stretch of LAM shows modest sequence homology with human complement factor-& (C8,). C8, is part of the membrane attack complex generated by the activation of the mammalian complement pathway that inserts in the plasma membrane of targeted foreign cells and that mediates cytolysis. The possibility that this domain of LAM contributes to the potentiation of limulin-mediated hemolysis appears unlikely however, because thiol-reacted, but not native, human N2-mac

Solid surfaces placed in the ocean are targets for colonization by all manner of sessile fouling organisms. Although the carapace of the horseshoe crab presents just such an opportunity for colonization, it is usually surprisingly clean of macroscopic colonizers. The dorsal surfaces of the cephalothorax of 8 of 16 randomly chosen animals from the Marine Resources Center at the Marine Biological Laboratory were completely free of macroscopic fouling organisms and in a second sample of 10 randomly chosen animals, 6.09% of the dorsal surface of the cephalothorax was occupied by macroscopic colonizers. In these samples, the commonest fouling organisms were members of the genera Crepidula (a sessile gastropod) and Balanus (barnacles). Some common sessile organisms, such as bryozoans, tunicates, and green algae, were not present on these animals.

the plasma- based cytolytic system is mediated by a single protein, the sialic acid-binding lectin, limulin (1). Limulin is a member of the pen- traxin protein family and is present in the plasma of the horseshoe crab at 30-50 nM (2). In assays using sheep red cells as the model foreign cell, the entire hemolytic activity of plasma is the province of limulin (1). Hemolysis depends on the sialic acid-binding ac- tivity of limulin, because sialylated glycoconjugates, such as fetuin and the sialic acids N-acetyl neuraminic acid and colominic acid, inhibit hemolysis, and desialylation of the target cells renders them immune to cytolysis (1). Limulin was purified from

In addition to its roles in hemostasis and wound repair, the blood clot plays an underappreciated role in innate immunity, where the established clot serves as a barrier to microbial penetration into the internal milieu and where the early clot entraps and immobilizes microbes that have entered wounds to the integuments. In this report we document the behavior of the pathogenic gram-negative bacterium Vibrio harveyi that has been entrapped in the fabric of the extracellular blood clot of one of its target organisms, the Pacific white shrimp, Litopenaeus vannamei. The freshly entrapped bacteria are held tightly by the clot, losing even Brownian motility, but by 1 h post-entrapment, a fraction of the bacteria have established small domains of fibrinolysis that enlarge progressively, enabling bacteria to escape from the clot's embrace. Escape is dependent on the actions of both serine- and metallo-proteases released from the bacterial cells.

C-reactive protein (CRP) has been conserved throughout evolution. Human native CRP exhibits calcium-dependent binding specificity for phosphocholine. Human CRP in its non-native structure expresses the capability to bind to deposited and conformationally-altered proteins and which can be achieved by several means including treatment of CRP with acidic pH. The ligand-binding property of human CRP in its non-native structure has implications for toxic and inflammatory conditions and favors the conservation of CRP throughout evolution. It is not known, however, whether CRP from invertebrates exhibits structure-based ligand-binding properties similar to that of human CRP. The aim of this study was to investigate the ligand-binding properties of CRP from the American horseshoe crab Limulus polyphemus. We used oxidized low-density lipoprotein (ox-LDL) immobilized on microtiter plates as a model for deposited and conformationally-altered proteins. We found that Limulus CRP binds to ox-LDL at physiological pH, in contrast to human CRP which requires acidic pH to do so. The binding of Limulus CRP to ox-LDL occurred even in the absence of calcium, suggesting that the binding was not mediated through exposed phosphocholine molecules, if any, on ox-LDL. We conclude that the host-defense function of CRP evolved with the development of the immune system to expose a ligand-binding specificity only when needed, that is, an inflammatory microenvironment would have to be sensed by CRP and that CRP would change its structure to execute its function. Limulus CRP also provides us with a tool to investigate the structure-function relationships of human CRP in animal models of inflammation.

Horseshoe crabs provide the important biomedical products, Limulus Amebocyte Lysate (LAL) and Tachypleus Amebocyte Lysate (TAL), which are produced by lysing the blood cells (amebocytes) using endotoxin-free distilled water. LAL/TAL is used to detect and quantify lipopolysaccharide (endotoxin), an important toxin of Gram-negative bacteria. Horseshoe crabs are the sole source for LAL/TAL and their fishery for the production of LAL/TAL is of concern to the sustained management of the species. This study investigated the mortality of the Malaysian horseshoe crab (Tachypleus gigas) related to blood extraction, with the aim of providing a basis for the sustainable management of the local fishery of this animal. We showed that the mortality rate of T. gigas due to the bleeding process is slightly higher than the values which has previously been reported for its conspecific, the American horseshoe crab (Limulus polyphemus). Overall mortalities of bled horseshoe crabs were 17.8% (male = 18.75%; female = 17.2%), w...

The cuticle of the American horseshoe crab, Limulus polyphemus, is largely free of the macroscopic epibionts that rapidly colonize most solid surfaces exposed to sea water. Here, we describe a viscous surface secretion that coats the carapace of the horseshoe crab. We report methods for stimulating production of the substance, identify hemolytic and liposome-permeabilizing activities and provide a partial biochemical characterization. We propose that this secretion functions as an anti-fouling agent protecting the chitinized epithelium of the cuticle from colonization by deleterious epibionts.

The immune system is based on the actions of the collection of specialized immune defense cells and their secreted proteins and peptides that defend the host against infection by parasites. Parasites are organisms that live part or all of their lives in close physical association with the host and extract nutrients from the host and, by releasing toxins and virulence factors, cause disease with the potential for injury and premature death of that host. Parasites of the metazoa can be viruses, eubacteria, fungi, protozoans, and other metazoans. The immune system operates to kill or eliminate parasites and eliminate or detoxify their toxins and virulence factors. Although some of the elements of immune systems are specific to a particular phylum of metazoans, others show extensive evolutionary conservation, being present in several or all major phyla of the metazoa. The pentraxins display this latter character in their roles in immune defense. Pentraxins have been documented in vertebrates, nonvertebrate chordates, arthropods, and mollusks and may be present in other taxa of metazoans. Presumably the pentraxins appeared early in the evolution of metazoa, prior to their evolutionary divergence in the Precambrian epoch into many phyla present today, and have been preserved for the 542 million years since that explosive evolutionary radiation. The fidelity with which these phyla have preserved the pentraxins suggests that the functions of these proteins are important for survival of the members of these diverse taxa of animals.

The immune system is based on the actions of the collection of specialized immune defense cells and their secreted proteins and peptides that defend the host against infection by parasites. Parasites are organisms that live part or all of their lives in close physical association with the host and extract nutrients from the host and, by releasing toxins and virulence factors, cause disease with the potential for injury and premature death of that host. Parasites of the metazoa can be viruses, eubacteria, fungi, protozoans, and other metazoans. The immune system operates to kill or eliminate parasites and eliminate or detoxify their toxins and virulence factors. Although some of the elements of immune systems are specific to a particular phylum of metazoans, others show extensive evolutionary conservation, being present in several or all major phyla of the metazoa. The pentraxins display this latter character in their roles in immune defense. Pentraxins have been documented in vertebrates, nonvertebrate chordates, arthropods, and mollusks and may be present in other taxa of metazoans. Presumably the pentraxins appeared early in the evolution of metazoa, prior to their evolutionary divergence in the Precambrian epoch into many phyla present today, and have been preserved for the 542 million years since that explosive evolutionary radiation. The fidelity with which these phyla have preserved the pentraxins suggests that the functions of these proteins are important for survival of the members of these diverse taxa of animals.

Lipopolysaccharide (LPS, endotoxin) is a component of Gram-negative bacteria and is the principal indicator to the innate immune systems of higher animals of a Gram-negative bacterial invasion. LPS activates the blood clotting system of the American horseshoe crab, Limulus polyphemus. By stimulating blood cell degranulation, LPS triggers the release of the proteins of the clotting system from the cells, and by activating a protease cascade that converts coagulogen, a soluble zymogen, to coagulin, the structural protein of the clot, LPS triggers the production of the fibrillar coagulin blood clot. Although originally thought to be restricted to the Gram-negative bacteria and the cyanobacteria, LPS, or a very similar molecule, has recently been described from a eukaryotic green alga, Chlorella. Here we show that, like LPS from Gram-negative bacteria, the algal molecule stimulates exocytosis of the Limulus blood cell and the clotting of coagulin. The coagulin clot efficiently entraps the cells of Chlorella in a network of fibrils. Invasion and erosion of the carapace by green algae is an important cause of mortality of Limulus, and it is suggested that the cellular response to aLPS may contribute to defense against this pathogen.

The embryonic epidermis of amniotes is a two-cell layer sheet with a periderm positioned superficial to the basal cell layer which, itself, attaches apically to the basal surface of the periderm and basally to the basal lamina. The presence of the periderm is essential to maintain the basal layer as a two-dimensional monolayer. Wounds to the epidermis that remove selectively just the periderm are healed by a stacking of the basal layer cells that constitute the wound bed. Basal cell stacking involves the desertion of the basal lamina by many of the cells so as to increase their contact area with other basal layer cells. This suggests that a preferential adhesion to the planar basal lamina is not important for the monolayered organization of the basal layer but, instead, association with inner surface of the planar periderm is the principal process that maintains the basal layer as a monolayer. The conversion of the basal layer from monolayer to multilayer during wound healing diminishes its planar area, resulting in movement of the wound borders toward the center of the wound. This is a novel scenario for wound healing.

The immune system is the collection of effector molecules and cells of the host that act against invading parasites and their products. Secreted proteases serve important roles in parasitic metabolism and virulence and the several families of protein protease inhibitors of the plasma and blood cells play an important role in immunity by inactivating and clearing the protease virulence factors of parasites. The protease inhibitors are of two classes, the active-site inhibitors and the alpha2-macroglobulins. Inhibitors for the first class bind and inactivate the active site of the target protease. Proteins of the second class bind proteases by a unique molecular trap mechanism and deliver the bound protease to a receptor-mediated endocytic system for degradation in secondary lysosomes. Proteins of the alpha2-macroglobulin family are present in a variety of animal phyla, including the nematodes, arthropods, mollusks, echinoderms, urochordates, and vertebrates. A shared suite of unique functional characteristics have been documented for the alpha2-macroglobulins of vertebrates, arthropods, and mollusks. The alpha2-macroglobulins of nematodes, arthropods, mollusks, and vertebrates show significant sequence identity in key functional domains. Thus, the alpha2-macroglobulins comprise an evolutionarily conserved arm of the innate immune system with similar structure and function in animal phyla separated by 0.6 billion years of evolution.

Two cell culture models were utilized to characterize the actions of peptide growth factors on the composition of the extracellular matrix of embryonic mesenchymal tissue. To model the three-dimensional architecture of mesenchymal tissue, chick embryonic mesenchymal cells were maintained in organ culture as adherent cell populations in small three-dimensional tissue spheroids and as sparse populations of cells embedded in a mesh of hydrated native collagen fibrils. Cell proliferation was stimulated by a variety of growth factors. All of the growth factors that elicited a mitogenic response in both of these culture systems also stimulated the deposition of an abundant fibronectin-containing extracellular matrix that colocalized with the regions of active cell proliferation. The suggestion that the matrigenic actions of growth factors for intact mesenchymal tissue are an integral part of mitogenic signaling is supported by the observation that surfaces derivatized with ProNectin, an artificial mimic of the RGD attachment domain of fibronectin, stimulated the proliferation of embryonic mesenchyme in the absence of exogenous growth factors. All of the growth factors that activated proliferation and fibronectin matrix accumulation stimulated the transformation of the mesenchymal cells into myofibroblasts that displayed the marker alpha-smooth muscle actin.

The primary structure determination of the dimeric invertebrate alpha(2)-macroglobulin (alpha(2)M) from Limulus polyphemus has been completed by determining its sites of glycosylation and disulfide bridge pattern. Of seven potential glycosylation sites for N-linked glycosylation, six (Asn(275), Asn(307), Asn(866), Asn(896), Asn(1089), and Asn(1145)) carry common glucosamine-based carbohydrates groups, whereas one (Asn(80)) carries a carbohydrate chain containing both glucosamine and galactosamine. Nine disulfide bridges, which are homologues with bridges in human alpha(2)M, have been identified (Cys(228)-Cys(269), Cys(456)-Cys(580), Cys(612)-Cys(799), Cys(657)-Cys(707), Cys(849)-Cys(876), Cys(874)-Cys(910), Cys(946)-Cys(1328), Cys(1104)-Cys(1155), and Cys(1362)-Cys(1475)). In addition to these bridges, Limulus alpha(2)M contains three unique bridges that connect Cys(361) and Cys(382), Cys(1370) and Cys(1374), respectively, and Cys(719) in one subunit with the same residue in the other subunit of the dimer. The latter bridge forms the only interchain disulfide bridge in Limulus alpha(2)M. The location of this bridge within the bait region is discussed and compared with other alpha-macroglobulins. Several peptides identified in the course of determining the disulfide bridge pattern provided evidence for the existence of two forms of Limulus alpha(2)M. The two forms have a high degree of sequence identity, but they differ extensively in large parts of their bait regions suggesting that they have different inhibitory spectra. The two forms (Limulus alpha(2)M-1 and -2) are most likely present in an approximately 2:1 ratio in the hemolymph of each animal, and they can be partially separated on a Mono Q column at pH 7.4 by applying a shallow gradient of NaCl.

Growth of the myocardium involves the completion of a fixed number of rounds of cell division during the embryonic and fetal stages followed by entry into a postmitotic state and hypertrophy of the postmitotic cardiomyocytes during the later stages of heart growth. It has been suggested that at the time of its determination in the early embryo, the embryonic myocardium is programmed for a fixed and limited number of cell divisions, after which the transition to the postmitotic state occurs autonomously. The proliferative response of cultured myocardium of the fetal chick was explored in four culture settings: monolayer cell culture, collagen lattice culture, organ culture of reaggregated cardiomyocyte tissue (cardiomyocyte spheroid culture), and organ culture of pieces of the ventricle wall. Several growth factors were identified by their ability to stimulate DNA synthesis in cardiomyocytes, identified by the incorporation of 5-bromodeoxyuridine (BrdU). The serine proteases thrombin and trypsin, fibroblast growth factor-2 (FGF-2), transforming growth factor-alpha (TGF-alpha), and insulin-like growth factor-II (IGF-II) all show growth factor activity but only for cardiomyocytes cultured in three-dimensional myocardial tissue, and not for cardiomyocytes maintained in monolayer cell culture. Thus, during its proliferation phase, the growth of the fetal myocardium is not controlled solely by internal, cell-autonomous programs, but is subject to external regulation by a family of peptide growth factors. The unconventional setting of three-dimensional culture of the myocardium is required to demonstrate its responsiveness to growth factor challenge.

The mediator of haemolysis in the plasma of the horseshoe crab, Limulus polyphemus, is limulin, a sialic acid-binding lectin. The haemolytic activity of limulin is inhibited by thiol ester-reacted forms of Limulus alpha(2)-macroglobulin, the third-most abundant protein of the plasma. Limulus alpha(2)-macroglobulin that has experienced cleavage of its internal thiol ester bond, consequent to reaction with proteases, or with the small primary amine, methylamine, reduces the haemolytic activity of limulin when present at molar excesses that approximate the relative concentrations of these two proteins in the plasma. Native, unreacted Limulus alpha(2)-macroglobulin has no effect on the haemolytic activity of limulin. Limulin binds thiol ester-reacted forms of Limulus alpha(2)-macroglobulin both in a solid-phase assay and in solution with an avidity 10-25 times higher than native, unreacted Limulus alpha(2)-macroglobulin. Protease-reacted alpha(2)-macroglobulin functions as a marker for the presence of foreign proteases in the blood of Limulus, and thus of pathogenic organisms that release proteases as facilitators of invasion and pathogenicity. Modulation of the haemolytic system represents a novel function for alpha(2)-macroglobulin.

Abnormalities of the cushion tissues lead to atrioventricular septal defects (AVSD) and truncus arteriosus (TA). Bisdiamine exposure in the embryo frequently causes AVSD and TA in the newborn chick, mouse, or rat. We studied the effects of bisdiamine on mesenchymal cells grown in aggregate culture isolated from the developing atrioventricular valves of the stage-36 chick embryo. Fibronectin extracellular matrix formation and cell proliferation in the aggregates were assessed in various media. Chick serum stimulated the cells to produce an extracellular matrix and to divide, and the inclusion of bisdiamine inhibited both responses. If we isolated an extracellular matrix from a monolayer of mesenchymal cells and added the sonicated matrix to the medium containing serum and bisdiamine, the matrix incorporated into the aggregates and the cells entered the mitotic cycle. Our previous work established that cells need to attach to an intact extracellular matrix to begin cell division. Thus, we suggest that bisdiamine inhibits the normal formation of the extracellular matrix, leading to reduced cell proliferation, but it does not affect matrix-cell interaction. The lack of cushion growth in situ may be the cause of AVSD or TA.

A major problem of comparative immunology is the characterization of the internal defense systems that lyse foreign cells, such as bacteria and other microbial pathogens that have gained entry into the body. The plasma cytolytic system of the American horseshoe crab, Limulus polyphemus, is sensitive to treatment with methylamine, which inactivates the abundant plasma defense protein α2-macroglobulin. This has been interpreted to mean that α2-macroglobulin plays an important role in hemolysis, analogous to the role of complement component C3 of the mammalian complement system (Enghild et al., 1990). Sensitivity to methylamine has been suggested to reflect an evolutionary homology with the plasma cytolytic system of mammals, in which the complement system is inactivated by the reaction of methylamine with complement components C3 and C4, C3, C4 and α2-macroglobulin contain an internal thiol ester bond linking cysteinyl and glutamic acid residues and methylamine inactivates all three proteins by reaction with the thiol-esterified glutamic acid. However, we have recently shown that the principal effector of hemolysis in Limulus is the plasma lectin, limulin (Armstrong et al., 1996). In this article we show that native, unreacted α2-macroglobulin is not involved directly in hemolysis but instead that methylamine-reacted α2-macroglobulin inhibits the hemolytic activity of limulin. Thus the thiol ester proteins α2-macroglobulin and C3 operate very differently in the hemolytic systems of Limulus and mammals and are not functionally homologous. Limulus α2-macroglobulin functions indirectly in hemolysis: its inactivation yields an inhibitory molecule for limulin-mediated hemolysis.

The American horseshoe crab Limulus polyphemus contains alpha 2-macroglobulin (alpha 2M) in the hemolymph plasma and hemocytes. alpha 2M from Limulus shows many of the typical characteristics of mammalian alpha 2M, including the presence of an internal thiol-ester, reactivity with a diversity of endopeptidases, a unique proteinase-trapping mechanism, and reactivity with the mammalian alpha 2M receptor. Additionally, Limulus alpha 2M has the unique property that it regulates the limulin-based hemolytic system of the plasma. A cDNA encoding Limulus alpha 2M has been obtained from a hemocyte cDNA library. The open reading frame encodes an N-terminal signal sequence of 25 amino acid residues and a mature protein of 1482 residues. The entire amino acid sequence is similar to those of the mammalian alpha 2Ms (28-29% identity) and contains common features found in mammalian alpha 2Ms. a bait region, an internal thiol-ester site, and a receptor-binding domain. However, the N-terminal portion (positions 24-105) has no sequence similarity with those of mammalian alpha 2Ms, and it is structurally related to that of the human complement factor C8 chain, consistent with a role for Limulus alpha 2M in host defense. The component sugar analysis of Limulus alpha 2M showed the existence of a complex type of oligosaccharide chain similar to those of mammalian alpha 2M. However, unlike mammalian alpha 2M, no sialic acid was detected in Limulus alpha 2M and it contained approximately 3 mol/mol N-acetylgalactosamine, suggesting the presence of O-linked sugar chains, which have not been found in mammalian alpha 2M. Expression of alpha 2M was detected in hemocytes, but not in hepatopancreas, heart, stomach, intestine, coxal gland, brain and skeletal muscle. Furthermore, immunoblotting of large and small granules of the hemocytes with antiserum against alpha 2M indicated the presence of the alpha 2M in large granules. Trypsin-treated Limulus alpha 2M, but not the native alpha 2M, displaced methylamine-treated human 125I-alpha 2M from the human alpha 2M receptor with a Kd of 30 nM, suggesting conservation of the proteinase-clearance mechanisms between mammalian and arthropod evolutionary lineages.

A variety of invertebrates possess plasma lectins with sialic acid recognition capabilities. One of the best studied of these lectins is limulin, which is a member of the pentraxin family of proteins and is found in the plasma of the American horseshoe crab, Limulus polyphemus. We find that limulin is one of several sialic acid-binding lectins of Limulus plasma and is present at a much lower abundance than Limulus C-reactive protein, the other plasma pentraxin. Limulin was purified by sequential affinity chromatography on phosphorylethanolamine-agarose, which isolates the pentraxins and separates limulin from the other sialic acid-binding lectins of the plasma, followed by fetuin-Sepharose, which binds limulin and separates it from Limulus C-reactive protein, the most abundant pentraxin of the plasma. We show here that limulin is the mediator of the Ca+2-dependent hemolytic activity found in the plasma of Limulus. Plasma that was depleted in the pentraxins by passage over phosphorylethanolamine-agarose or was depleted in the sialic acid-binding lectins by passage over fetuin-Sepharose lacked hemolytic activity. Purified limulin was hemolytic at concentrations of 3-5 nM. The other sialic acid-binding lectins of Limulus plasma and Limulus C-reactive protein were nonhemolytic. Foreign cell cytolysis by limulin represents a novel function for a plasma lectin and is the first documented function for limulin.

The proliferation response of stage 36 chick atrioventricular valve mesenchy- ma1 cells to fibroblast growth factor-2 (FGF-2) was studied in the tissue-like environment of three-di- mensional cell aggregates maintained in organ culture. The mitogenic effects of FGF-2 on mesen- chymal tissue depended on the FGF-%stimulated formation of a fibronectin-containing extracellu- lar matrix. The matrix was absent in unstimulated aggregates, and co-localized with regions of ac- tively proliferating cells in stimulated aggregates. Inhibition of fibronectin matrix formation by the inclusion of Arg-Gly-Asp-containing peptides, which compete with fibronectin for binding to the cell surface (Y&~ integrin receptors, abolished the proliferation effects of FGF-2. Inhibition of sul- fation of cell surface glycosaminoglycans by treat- ment with sodium chlorate significantly reduced both the formation of the fibronectin matrix and cell proliferation in response to FGF-2, suggesting an involvement of the low-affinity sulfated gly- cosaminoglycan FGF receptor system. Thus, the FGF-stimulated growth of embryonic atrioven- tricular valve mesenchyme in vitro involves the production of a fibronectin matrix. We suggest that the stimulation of the fibronectin matrix rep- resents an essential element in growth factor sig- naling of mesenchymal tissue, with the matrix serving as an anchorage substratum for the pro- liferating cells. o

The innate immune system is represented by a complex of cellular and humoral factors and processes that protect the organism against pathogenic attack. Certain of these elements arose early in the evolution of multicellular animals and have been preserved throughout the evolutionary divergence of the different animal phyla. Two mediators that have interested us are α2-macroglobulin and the pentraxins. Both function as elements in the plasma defense systems of arthropods and vertebrates. The ability to purify significant quantities of both proteins from the plasma of the American horseshoe crab Limulus polyphemus has enabled us to investigate their functions at the molecular and organismal levels. In Limulus α2-macroglobulin functions as a broad-spectrum protease-binding molecule that mediates the clearance of proteases from the plasma. Limulus has at least two pentraxins in the plasma, an abundant form designated Limulus C-reactive protein, which is of unknown function, and limulin, a low-abundance form with sialic acid recognition capabilities, which mediates a cytolytic reactivity for the destruction of foreign cells that have contacted the plasma.

Trypsin, thrombin, and peptide analogues of the new amino terminus of the proteolyzed thrombin receptor, SFLLRN and SFLLRNPNDKYEPF, stimulated embryonic fibroblasts cultured as 3-dimensional tissue-like aggregates to elaborate a fibronectin-rich extracellular matrix. Enzymatically inactive thrombin and the control peptide FLLRN failed to stimulate matrix production. The induction of cell proliferation correlated with production of the fibronectin matrix. The regions of active cell proliferation in the fibroblast aggregates co-localized with the matrix and peptide analogues of the RGD cell-adhesion site of fibronectin reversibly inhibited the accumulation of the fibronectin matrix and the stimulation of cell proliferation by SFLLRN. Two different preparations of the fibronectin matrix stimulated cell proliferation in aggregates cultured in growth factor-free medium. We suggest that the stimulation of matrix production is a necessary event for mitogenic signaling in mesenchymal tissue. The tight coupling between the matrigenic and mitogenic activities of growth factors was absent in monolayer cultures of chick embryonic fibroblasts since thrombin and trypsin induced proliferation of monolayer-cultured cells without inducing the production of a fibronectin matrix. 0 1996 Wiley-Liss, Inr.

cxzM in the Horseshoe Crab A Structural and Functional Invertebrate Homologue PETER B. ARMSTRONG, ' LARS SOlTRUP-JENSEN,C ATSUSHI IKA1,d SUBITA SRIMAL, e AND JAMES P. QUIGLEYa,-f.g aMarine Biological Laboratory Woods Hole, Massachusetts 02543 bL.uboratory for Cell Biology Department of Zoology University of California Davis, California 95616 'Department of Molecular Biology University of Aarhus DK-8000 Aarhus, Denmark dLuboratory of Biodynamics Tokyo Institute of Technology Nagatsuta, Midoriku Yokohama, Japan 'Molecular Biophysics Unit Indian Institute of Science Bangalore, 560012 India fDepartment of Pathology Health Sciences Center State University of New York Stony Brook, New York 1 I794 INTRODUCTION Higher animals deploy a variety of defense systems to cope with invading patho- gens that are based on components in the blood cells and plasma. These cellular and humoral components function to neutralize and clear the invading pathogens and the molecules introduced by pathogens. Often, the introduced molecules that are most harmful are toxic proteins and degradative enzymes. In many of the cellular (neutro- phils, lymphocytes, and monocytes) responses and in a majority of the humoral responses (kinin, complement, and coagulation), a variety of proteolytic enzymes are also generated to act on target proteins or to further amplify the response via cascade gSend all correspondence to J. P. Quigley at the State University of New York at Stony Brook.

Clots were allowed to form in samples of whole blood taken from the American horseshoe crab, Limulus polyphemus, in the absence and presence of dansylcadaverine (16), and were analyzed for their contents of N epsilon(gamma-glutamyl)lysine and gamma-glutamyl-dansylcadaverine. Clots obtained without dansylcadaverine yielded significant amounts of N epsilon(gamma-glutamyl)lysine product. Clots formed in the presence of dansylcadaverine yielded only gamma-glutamyl-dansylcadaverine. Formation of these products reflects on the activity of transglutaminase released from the blood cells during coagulation.

The processes responsible for epithelial spreading during wound healing and embryonic morphogenesis were investigated in an organ culture model in which an epithelial tissue (chick embryo pigmented retinal epithelium) spread over the surface of an aggregate of mesenchyme cells (chick embryo cardiac mesenchyme). The heart mesenchyme aggregate is differentiated into a core of stellate cells associated with a fibronectin-poor matrix surrounded by a cortical zone, 2–5 cells in thickness, of flattened cells embedded in a fibronectin-rich extracellular matrix. Envelopment of the mesenchyme aggregate is accompanied by a movement of the cells and the fibronectin-rich extracellular matrix of the cortex over the core tissue in advance of the spreading pigmented retina tissue. Three distinct processes were identified as contributing to epithelial spreading in this system: (1) active migration of the pigmented retinal epithelium; (2) active contraction of the cortical cells of the mesenchyme aggregate to tow the attached epithelial tissue over the mesenchyme aggregate; and (3) ingression of surface-located cells of the mesenchyme aggregate to decrease the exposed surface area by decreasing the number of cells at the surface.

A structural and functional homologue of vertebrate alpha 2-macroglobulin (alpha 2M) has been identified in the hemolymph and blood cells of the arthropod Limulus polyphemus, one of the oldest living fossil invertebrates (Quigley, J. P., and Armstrong, P. B. (1985) J. Biol. Chem. 260, 12715-12719). The subunit molecular mass is 185 kDa. The native molecular mass, determined by scanning transmission electron microscopy (STEM) under conditions in which the linear relationship between the STEM large angle detector signal and specimen mass thickness allows the determination of the total macromolecular mass, was 354 +/- 35 kDa. Sedimentation equilibrium measurements gave a value of 366 kDa, independent of solute concentration. Sedimentation velocity experiments indicated a homogeneous component with a frictional ratio of 1.41. Thus, the native structure appears to be a dimer, with a somewhat extended conformation. The behavior during gel permeation chromatography was anomalous, yielding an apparent molecular mass approximately half-way between that expected for the dimeric and tetrameric configurations. Transmission electron microscopy of negatively stained preparations revealed a dimeric butterfly-like structure that collapsed following reaction with chymotrypsin.

C-reactive protein (CRP) from the American horseshoe crab, Limulus polyphemus, exhibits complex membrane activities. Here, we describe the behavior of protein and lipid as CRP interacts with model liposomes and bacterial membranes. Limulus C-reactive protein (L-CRP) forms extended fibrilar structures that encapsulate liposomes in the presence of Ca2+. We have observed structures consistent in size and shape with these fibers bound to the surface of Gram-negative bacteria. The membranes of Limulus CRP-treated bacteria exhibit significantly different mechano-elastic properties than those of untreated bacteria. In vitro, bilayer lipids undergo a rigidification and reorganization of small domains. We suggest that these interactions reflect the protein's role as a primary defense molecule, functioning in the entrapment and killing of potential pathogens. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and alpha2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

The pentraxins are a family of highly conserved plasma proteins of metazoans known to function in immune defence. The canonical members, C-reactive protein and serum amyloid P component, have been identified in arthropods and humans. Mammalian pentraxins are known to bind lipid bilayers, and a pentraxin representative from the American horseshoe crab, Limulus polyphemus, binds and permeabilizes mammalian erythrocytes. Both activities are Ca2+-dependent. Utilizing model liposomes and planar lipid bilayers, in the present study we have investigated the membrane-active properties of the three pentraxin representatives from Limulus and show that all of the Limulus pentraxins permeabilize lipid bilayers. Mechanistically, Limulus C-reactive protein forms transmembrane pores in asymmetric planar lipid bilayers that mimic the outer membrane of Gram-negative bacteria and exhibits a Ca2+-independent form of membrane binding that may be sufficient for pore formation.

1. Purified alpha 2-macroglobulin (alpha 2M) from the American horseshoe crab, Limulus polyphemus was cleaved with trypsin and 20 of the tryptic peptides were sequenced and compared with the sequences of human alpha 2M, rat alpha 1M, alpha 2M, and alpha 1-inhibitor 3, and human complement proteins C3 and C4. 2. Ten of the peptides (233 residues), including that containing the thiol ester site, could be aligned unambiguously with stretches in mammalian alpha 2M, with a degree of identity greater than 30%. 3. The 12-residue thiol ester-containing peptide of Limulus alpha 2M showed 67% identity with the same stretch of human alpha 2M.

Intercellular invasion is the intrusion of the cells of one tissue into space occupied by a second tissue. The alternative situation to invasion, one characteristic of most coherent tissues, is segregation, with identifiable boundaries existing between contiguous tissues. The interfaces between mesenchymal and myocardial tissues in the developing avian heart show a profoundly different character in different regions of the heart: the interface between epicardial mesenchyme and heart wall myocardium is planar, without intermingling of the two cell types, whereas the interface between endocardial cushion mesenchyme and myocardium is diffuse, with extensive invasion of both tissue types across the border to produce intermingling of the two tissues. Thus, invasion and tissue segregation coexist in different regions of the mesenchyme-myocardium contact zone. Investigation of the involvement of the interstitial matrix in invasion and segregation has been conducted by maintaining the two tissues in mutual contact in organ culture. Investigation of the mechanisms by which the two cell types sort out in randomized chimeric tissue reaggregates has provided insight into the conditions for tissue segregation. We have modeled invasion in organ culture by fusing aggregates of myocardial cells with aggregates of cardiac mesenchymal cells. Cells of both tissues invaded the partner aggregate during a period of 1-3 d of coculture. Both invasion and segregation in the aggregates appear to depend on the presence or absence of a fibronectin-rich interstitial matrix elaborated by the cardiac mesenchyme. During sorting, the matrix appears selectively in regions occupied by the mesenchyme. Under conditions of culture that are nonpermissive for matrix deposition, sorting fails to occur. Stimulation of matrix deposition by addition of serum, transforming growth factor beta, or isolated matrix itself is accompanied by sorting out of the two tissues. Sorting out is blocked reversibly by inclusion of the fibronectin adhesion site peptide, GRGDSP. Invasion of fused aggregates is preceded by a redistribution of the fibronectin-containing matrix of the mesenchymal aggregate such that matrix-poor regions come to occupy the interface with the myocardial partner aggregate. The invasion that ensues involves mesenchymal cells emigrating from, and myocardial cells intruding into, matrix-poor regions of the mesenchymal aggregate.(ABSTRACT TRUNCATED AT 400 WORDS)

Intercellular invasion is the intrusion of one tissue into space occupied by a second tissue. The alternative situation to invasion, one characteristic of most coherent tissues, is segregation, with identifiable boundaries between contiguous tissues. Tissue segregation can be demonstrated as an active process by the sorting-out of the disorganized cell aggregates that are produced when mixed suspensions of dissociated cells are caused to reaggregate. In this situation, active reorganization of the disorganized aggregate restores a segregated tissue arrangement. Investigation of the sorting of the two principal tissues of the chick embryonic heart (namely, the myocardium and the mesenchyme) suggests an involvement of a fibronectin-rich interstitial matrix elaborated by the cardiac mesenchyme in the process of tissue segregation. During sorting the matrix appears selectively in regions occupied by the mesenchyme. Under conditions of culture non-permissive for matrix deposition, sorting fails to occur. Stimulation of matrix deposition by the addition of serum, transforming growth factor-beta, or isolated matrix itself is accompanied by sorting-out of the two tissues. The mutual invasion of the two tissues that occurs when mesenchymal and myocardial aggregates are fused occurs in regions where the mesenchymal matrix is lost. It is suggested that the presence of the fibronectin matrix in the cardiac mesenchyme promotes tissue segregation and organizational stability and that its loss establishes conditions in which invasion will occur.

The primary structure determination of the dimeric invertebrate alpha(2)-macroglobulin (alpha(2)M) from Limulus polyphemus has been completed by determining its sites of glycosylation and disulfide bridge pattern. Of seven potential glycosylation sites for N-linked glycosylation, six (Asn(275), Asn(307), Asn(866), Asn(896), Asn(1089), and Asn(1145)) carry common glucosamine-based carbohydrates groups, whereas one (Asn(80)) carries a carbohydrate chain containing both glucosamine and galactosamine. Nine disulfide bridges, which are homologues with bridges in human alpha(2)M, have been identified (Cys(228)-Cys(269), Cys(456)-Cys(580), Cys(612)-Cys(799), Cys(657)-Cys(707), Cys(849)-Cys(876), Cys(874)-Cys(910), Cys(946)-Cys(1328), Cys(1104)-Cys(1155), and Cys(1362)-Cys(1475)). In addition to these bridges, Limulus alpha(2)M contains three unique bridges that connect Cys(361) and Cys(382), Cys(1370) and Cys(1374), respectively, and Cys(719) in one subunit with the same residue in the other subunit of the dimer. The latter bridge forms the only interchain disulfide bridge in Limulus alpha(2)M. The location of this bridge within the bait region is discussed and compared with other alpha-macroglobulins. Several peptides identified in the course of determining the disulfide bridge pattern provided evidence for the existence of two forms of Limulus alpha(2)M. The two forms have a high degree of sequence identity, but they differ extensively in large parts of their bait regions suggesting that they have different inhibitory spectra. The two forms (Limulus alpha(2)M-1 and -2) are most likely present in an approximately 2:1 ratio in the hemolymph of each animal, and they can be partially separated on a Mono Q column at pH 7.4 by applying a shallow gradient of NaCl.

guea sporoplasm proteins indicated that, whether whole sporoplasm samples were tested, or only the sporoplasm outer membrane isolates, all four of these peptides were present. Ultrastructural immunolabeling with an antibody-peroxidase probe for p showed the label binding uniformly over the sporoplasm surface (Fig. 1A, B). These results were also supported by immunogold labeling and immunofluorescence (not shown). Since I hypothesized that the dynactin assemblage should be associated with the membrane within unfired spores, tests were made to determine where the dynactin is located in such spores. Spores were prefixed with 1% glutaraldehyde and subsequently partially disrupted by the shearing action of a glass homogenizer. These spores were then subjected to the p antibody probe, and this was visualized with a second antibody coupled to peroxidase. The results showed that the label reacts within the polaroplast domain (Fig. 1C, D). The position of the dynactin within the polaroplast supports an old idea: that the spore discharges a tube through which the membrane of the polaroplast everts to form the sac. The cytoplasm and nucleus are thought to be introduced into the everted polaroplast-derived sac. That the sporoplasm membrane is a secondhand membrane derived from the polaroplast organelle is supported by the absence of cholesterol in the outer envelope (3) and the absence of lectin-binding molecules (unpubl. obs.).

The colonization of a potential host by a parasite requires an ability to cross the integuments and then to escape from the host immune defenses. Proteinases are important virulence factors that assist these processes. Host proteinase inhibitors potentially contribute to immunity by inactivating the proteinase virulence factors of pathogens.

To reproduce successfully, all organisms from bacteria to man must survive the attacks of the myriad of parasites that would otherwise produce fatal episodes of disease and premature death. This is particularly true for long-lived species that require many years to reach reproductive maturity and, presumably, that are required to resist multiple episodes of parasitic infection prior to that happy day. Parasites are species that draw their food resources from the live bodies of a host species and that spend much or all of their life in association with that host species. Typically damage is done to the host by the parasite, resulting in a condition of pathogenesis and disease. Parasites may be unicellular or multicellular, prokaryote, eukaryote, or virus.

Although the surfaces of most solid objects are rapidly colonized by sessile fouling organisms within a short time after immersion in the ocean, macroscopic fouling organisms are surprisingly sparse on the cuticle of healthy adults of the American horseshoe crab, Limulus polyphemus. One of the major identified causes of mortality of adult horseshoe crabs is the erosion of the cuticle that follows its colonization by green or blue-green algae (1). Thus it is in the interest of the animal to maintain the cuticle clear of fouling species. The mechanism(s) responsible for keeping the cuticle clean in an environment liberally stocked with fouling species have not been identified. One likely candidate is a mucous secretion from the hypodermal glands, a system of glands that discharge onto the surface of the cuticle (2). Here we show that this secretion has antibiological activities that may contribute to its ability to deter the colonization of the cuticle of Limulus by fouling organisms. The cuticular secretion can be provoked by housing healthy adults in the presence of decaying fish (3). The secretion from the hypodermal glands was scraped from the dorsal cuticle of the Limulus shell with a rubber scraper and stored at 4°C in the presence of 0.2 mg/ml NaN3 to prevent bacterial contamination. Antibodies to whole cuticular secretion were prepared in rabbits. Immunostaining utilized a 1:1000 dilution of the primary antiserum, a 1:1000 dilution of HRP-anti rabbit IgG, and 4-chloronaphthol using standard methods for staining Western and dot blots (4). Stimulated animals renewed the layer of cuticular secretion within 1–2 h after the cuticle had been scraped clean. Although most of the animals in the Marine Resources Center of the Marine Biological Laboratory, which are collected from the pristine waters of Pleasant Bay, lacked the thick layer of cuticular secretion of the pollution-challenged animal, the secretion was present as a thin layer on their surfaces as well. Anti-cuticular secretion antisera specifically immunostained swipes of the surface of the cephalothorax of these animals, indicating the presence of the secreted products of the hypodermal glands. Control swipes immunostained with non-immune rabbit IgG in place of immune serum failed to stain. It has been proposed that the cuticular secretion contains the secreted products of the Limulus blood cells (2). This apparently is not true because the principal secreted product of the blood cells, the clotting protein coagulogen, is absent from the secretion. Antibodies to coagulogen failed to immunostain dot blots of cuticular secretion, and the anti-cuticular secretion antiserum failed to stain coagulogen or the collective secretion of the blood cells. This latter material was prepared as described previously (5, 6). The anti-coagulogen antiserum did stain dot blots of coagulogen, and the anti-cuticular secretion antiserum did stain dot blots of cuticular secretion. The cuticular secretion showed 15 protein bands by SDS-PAGE (reducing conditions, silver-staining) in the molecular mass range of 143–20 kDa. The anti-biological activity of the cuticular secretion was tested by its cytolytic actions on target cells. Cuticular secretion hemolysed sheep red blood cells at a 1:16 dilution in a standard hemolysis assay (7) (Fig. 1A). Hemolysis was judged to be divalent cation-dependent because the divalent cation chelator, ethylenediaminetetraacetic acid, reduced hemolysis (Fig. 1A). The macromolecular osmolites dextran-8 (Mr 8 –12 kDa) and, to a lesser extent, dextran-4 (Mr 4 – 6 kDa) reduced the extent of hemolysis (Fig. 1B). This suggests that hemolysis is the result of hydrophilic membrane channels established in the plasma membrane of the target red blood cell by the hemolytic protein of the cuticular secretion. Protection by the dextrans is suggested to result from their ability to balance the osmotic pressure across the permeabilized cell membrane, which will reduce the flow of water through the hemolytic pore and into the cell and will prevent swelling and lysis (8). It is difficult to envision ways for macromolecular osmolites to protect the cell if the hemolytic process featured such other possible mechanisms as phospholipase action or detergent-mediated membrane reorganization. It is proposed that the cuticular secretion is one agent that helps maintain the cleanliness of the cuticle of Limulus. Its

cells such as mammalian erythrocytes, may contribute to this activity. Under normal conditions, the volume of cuticular secretion is low, but it can be detected by immunological assays. Under conditions of challenge by a polluted environment, the volume of the secretion is augmented. In addition to its anti-biological activity, the continuous production of the cuticular secretion can be expected to exert a mechanical action, entrapping and sweeping potential fouling organisms away from the solid surface of the cuticle. Contrary to previous suggestions (2), the cuticular secretion does not contain secretion products of the blood cells. Supported by Grant No. MCB-97-26771 from the National Science Foundation. We thank Ms. Yvonne Coursey for the anticoagulogen antiserum. Literature Cited

All animals and plants have immune systems that protect them from the diversity of pathogens that would otherwise threaten their survival. The different components of the immune system may inactivate the pathogens themselves or promote the inactivation and clearance of toxic products produced by the pathogens. An important category of virulence factors of bacterial and prokaryotic pathogens are the proteases, which act to facilitate the invasion of the pathogens and to promote their destructive growth in the host organism. The present review concentrates on the comparative biology of an evolutionarily conserved arm of the immune system, the protein, alpha2-macroglobulin. alpha2-Macroglobulin is an abundant protein of the plasma of vertebrates and members of several invertebrate phyla and functions as a broad-spectrum protease-binding protein. Protease-conjugated alpha2-macroglobulin is selectively bound by cells contacting the body fluids and alpha2-macroglobulin and its protease cargo are then internalized and degraded in secondary lysosomes of those cells. In addition to this function as an agent for protease clearance, alpha2-macroglobulin binds a variety of other ligands, including several peptide growth factors and modulates the activity of a lectin-dependent cytolytic pathway in arthropods.

Lipopolysaccharide (LPS) (a.k.a., endotoxin) is an essential component of the outer leaflet of the outer membrane of gram-negative bacteria and is a potent activator of the innate immune system of animals. Lipid A, the glycolipid core of LPS, is the agent responsible for disease and death from gram-negative sepsis, an important cause of human mortality and morbidity. Although it is generally accepted that lipid A is restricted to the prokaryotes, recent efforts to purify molecules from green algae with structural features unique to lipid A have met with success. Furthermore, the vascular plant Arabidopsis thaliana has been found to contain genes that encode all of the enzymes of the biosynthetic pathway for lipid A. It is not known whether vascular plants synthesize lipid A or where lipid A might be located in the tissues. For the present study, we used affinity reagents for lipid A to probe green alga and tissues of the garden pea for a light microscopic localization of lipid A in these eukaryote cells. We find staining for lipid A in free-living and endosymbiotic green algae and in the chloroplasts of vascular plants, indicating that this molecule is not restricted to prokaryotes, but is found also in select eukaryotes.

The properties of the thiol ester-containing alpha-macroglobulin (alphaM) from the horseshoe crab (Limulus polyphemus) have been compared with those of the human analogue (alpha2M). Thiol ester accessibility was more restricted in Limulus alphaM than in human alpha2M. Fluorescent probes attached to the thiol ester cysteine indicated very similar local environments in the cleaved state of the two alphaMs. The separation between the two thiol ester cysteines in the cleaved state, determined by fluorescence resonance energy transfer, was also very similar for the two alphaMs. Differences were found in the oligomerization state and conformational changes of the two proteins. Whereas human alpha2M appears to be exclusively a dimer of dimers, Limulus alphaM can exist in both tetrameric and dimeric forms, although with marked preference for the dimer. Conformational change within a dimeric trapping unit, monitored by 6-(p-toluidino)-2-napthalene-sulfonic acid fluorescence change, showed that each monomer of the Limulus alphaM dimer appears to change conformation independently, whereas human alpha2M requires both thiol esters within a functional unit to be cleaved before the conformational change occurs. Taken together, these findings indicate that, whereas individual thiol esters in both types of alphaM are similar in properties, differences in subunit-subunit interaction result both in differences in state of oligomerization and in cooperativity of conformational change, which may reflect a fundamentally different organization of the subunits within a dimer in the two alphaMs.

When alpha 2-macroglobulin (alpha 2M) from the American horseshoe crab, Limulus polyphemus, reacts with proteinases, its thiol esters, like those of other alpha-macroglobulins, become activated, leading to the formation of covalently crosslinked species that can be detected as high molecular weight bands in reducing SDS-PAGE. While other alpha-macroglobulins extensively form crosslinks to the reacting proteinase, Limulus alpha 2M does not. It rather becomes internally crosslinked. It was found from N-terminal sequence analysis of purified [14C]carboxymethylated peptides from Limulus alpha 2M-trypsin complexes that an isopeptide bond formed in approx. 60% yield from the thiol esterified Gln-1002 specifically to Lys-254 in the opposing monomer of the disulphide bridged dimer is the main cause of the internal crosslinking.