Membrane pore formation by pentraxin proteins from Limulus, the American horseshoe crab.

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 Ca(2+)-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 Ca(2+)-independent form of membrane binding that may be sufficient for pore formation.

Histochemical evidence for lipid A (endotoxin) in eukaryote chloroplasts.

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.

Proteases and protease inhibitors: a balance of activities in host-pathogen interaction.

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.

Comparative biology of the pentraxin protein family: evolutionarily conserved component of innate immune system.

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.

Interaction of pathogenic vibrio bacteria with the blood clot of the pacific white shrimp, Litopenaeus vannamei.

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.

Capture of lipopolysaccharide (endotoxin) by the blood clot: a comparative study.

In vertebrates and arthropods, blood clotting involves the establishment of a plug of aggregated thrombocytes (the cellular clot) and an extracellular fibrillar clot formed by the polymerization of the structural protein of the clot, which is fibrin in mammals, plasma lipoprotein in crustaceans, and coagulin in the horseshoe crab, Limulus polyphemus. Both elements of the clot function to staunch bleeding. Additionally, the extracellular clot functions as an agent of the innate immune system by providing a passive anti-microbial barrier and microbial entrapment device, which functions directly at the site of wounds to the integument. Here we show that, in addition to these passive functions in immunity, the plasma lipoprotein clot of lobster, the coagulin clot of Limulus, and both the platelet thrombus and the fibrin clot of mammals (human, mouse) operate to capture lipopolysaccharide (LPS, endotoxin). The lipid A core of LPS is the principal agent of gram-negative septicemia, which is responsible for more than 100,000 human deaths annually in the United States and is similarly toxic to arthropods. Quantification using the Limulus Amebocyte Lysate (LAL) test shows that clots capture significant quantities of LPS and fluorescent-labeled LPS can be seen by microscopy to decorate the clot fibrils. Thrombi generated in the living mouse accumulate LPS in vivo. It is suggested that capture of LPS released from gram-negative bacteria entrapped by the blood clot operates to protect against the disease that might be caused by its systemic dispersal.

Membrane activity of a C-reactive protein.

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 Ca(2+). 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.

Crystal structures of Limulus SAP-like pentraxin reveal two molecular aggregations.

The serum-amyloid-P-component-like pentraxin from Limulus polyphemus, a recently discovered pentraxin species and important effector protein of the hemolymph immune system, displays two distinct doubly stacked cyclic molecular aggregations, heptameric and octameric. The refined three-dimensional structures determined by X-ray crystallography, both based on the same cDNA sequence, show that each aggregate is constructed from a similar dimer of protomers, which is repeated to make up the ring structure. The native octameric form has been refined at a resolution of 3 A, the native heptameric form at 2.3 A, and the phosphoethanolamine (PE)-bound octameric form at 2.7 A. The existence of the hitherto undescribed heptameric form was confirmed by single-particle analysis using cryo-electron microscopy. In the native structures, the calcium-binding site is similar to that in human pentraxins, with two calcium ions bound in each subunit. Upon binding PE, however, each subunit binds a third calcium ion, with all three calcium ions contributing to the binding and orientation of the bound phosphate group within the ligand-binding pocket. While the phosphate is well-defined in the electron density, the ethanolamine group is poorly defined, suggesting structural and binding variabilities of this group. Although sequence homology with human serum amyloid P component is relatively low, structural homology is high, with very similar overall folds and a common affinity for PE. This is due, in part, to a "topological" equivalence of side-chain position. Identical side chains that are important in both function and fold, from different regions of the sequence in human and Limulus structures, occupy similar space within the overall subunit fold. Sequence and structure alignment, based on the refined three-dimensional structures presented here and the known horseshoe crab pentraxin sequences, suggest that adaptation and refinement of C-reactive-protein-mediated immune responses in these ancient creatures lacking antibody-based immunity are based on adaptation by gene duplication.

Response of the blood clotting system of the American horseshoe crab, Limulus polyphemus, to a novel form of lipopolysaccharide from a green alga.

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.

A novel form of epithelial wound healing of the embryonic epidermis.

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.

Time-lapse cinemicrographic studies of cell motility during morphogenesis of the embryonic yolk sac of Fundulus heteroclitus (Pisces: Teleosti).

The active motility of the cells of the yolk sac of the living Fundulus embryo was studied by time-lapse cinemicrography with phase contrast optics. In the teleost, the yolk sac lies peripheral to the body of the embryo proper and consists of a fluid-filled space bounded above by a superficial epithelium, the enveloping layer (EVL), and below by the yolk syncytial layer (YSL). The cell types treated in the present study are the enveloping layer epithelial cell, the stellate cell which lies in a layer flattened on the inner surface of the EVL, the epithelioid deep cell, the yolk sac amoebocyte, the yolk sac endothelial cell and the yolk sac melanoblast. The most actively motile cells examined in the present study are the yolk sac amebocyte and the melanoblast, which emigrates from the embryo proper at stages 19-21. The amoebocytes are compact rounded cells that move very rapidly by the extension of lamellipods with scalloped margins. The amoebocytes wander over the yolk sac in an apparently undirected fashion and invade the embryo proper when they happen to encounter it, moving between cells of the lateral mesoderm. The melanoblasts migrate by the gradual extension of elongated branching processes. Cells are sometimes monopodial, with movement being parallel to the long axis of the cell. Alternatively, movement may be perpendicular to the predominant long axis, with processes being extended alternatively from opposite ends of the cell obliquely forward, so the path described is a zig-zag to either side of the overall direction of movement. Although the melanoblasts show irregularity in their movement, the predominant direction of initial movement is away from the embryo proper. The major yolk sac blood vessels form in situ by the collective activities of presumptive endothelial cells that enclose volumes of the yolk sac space with sheet-like processes from the cell body and from the extensions that connect cells into networks.

Light and electron microscope studies of cell sorting in combinations of chick embryo neural retina and retinal pigment epithelium.

A recent hypothesis, proposed by A. S. G. Curtis, asserts that the formation of homogeneous tissues within heterotypic aggregates occurs during the initial phases of cellular aggregation rather than by a process of cell sorting in mixed aggregates. The present studies of the early stages of aggregation of mixed suspensions of dissociated chick embryo pigmented retinal epithelial and neural retinal cells do not support Curtis' hypothesis. The early aggregates formed from these mixed cell suspensions are disordered mixtures of the two cell types. Establishment of homogeneous neural retinal and pigmented retinal epithelial tissues occurs much later in the aggregates. The hypothesis of Moscona that the process of formation of type-specific tissues in mixed aggregates requires a specificity of cell adhesion was examined by attempting to demonstrate cell contact specificity at the electron microscope level in heterotypic aggregates undergoing cell sorting. Specificity of cell contact was not observed. Instead, fine structural studies of cell contact interactions demonstrate that pigmented retinal epithelial cells have broad areas of cell contact and specialized contact junctions with neural retinal cells within the aggregates.

Growth factor modulation of the extracellular matrix.

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.

Localization of carbohydrate attachment sites and disulfide bridges in limulus alpha 2-macroglobulin. Evidence for two forms differing primarily in their bait region sequences.

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.