The contribution of proteinase inhibitors to immune defense.

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.

Regulation of proliferation of the fetal myocardium.

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.

Binding of alpha2-macroglobulin and limulin: regulation of the plasma haemolytic system of the American horseshoe crab, Limulus.

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.

Intercellular invasion and the organizational stability of tissues: a role for fibronectin.

Intracellular invasion is the movement of cells of one type into the fabric of other, contiguous tissues. Invasion is a signature behavior of the malignant tumor and also is found as part of the normal behavior of inflammatory blood cells and tissues engaged in the morphogenetic movements of normal embryogenesis and in a number of instances of normal and pathological tissue remodeling in the adult. Informed by the view that the underlying mechanisms of invasion will be similar for tumor cells and invasive blood and embryonic cells, this review adopts a comparative approach to the analysis of invasion. Invasion results in the development of a diffuse interface between contiguous tissues. Its alternative is the maintenance of stable, planar tissue boundaries. This is the more usual condition for contiguous tissues in the animal. This review will focus on the processes that, on the one hand, stabilize planar contact interfaces between tissues, and, on the other, promote the destabilization of tissue integrity by fostering intercellular invasion. Particular attention is devoted to a role for adhesive interactions mediated by the matrix adhesion molecule, fibronectin. In certain instances, fibronectin in the matrix promotes invasion whereas in others, the presence of fibronectin prevents invasion. The distinction appears to depend on whether the invasive tissue is migrating into an acellular extracellular matrix or whether invasion involves densely cellular tissues. In the first instance, fibronectin promotes invasion, whereas in the second, it stabilizes the interface of the contacting tissues and prevents invasion.

Alpha2-macroglobulin: an evolutionarily conserved arm of the innate immune system.

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.

Bisdiamine inhibits extracellular matrix formation and cell proliferation of atrioventricular mesenchyme from developing chick heart valves.

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.

Alpha2-macroglobulin does not function as a C3 homologue in the plasma hemolytic system of the American horseshoe crab, Limulus.

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 alpha2-macroglobulin. This has been interpreted to mean that alpha2-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 alpha2-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 alpha2-macroglobulin is not involved directly in hemolysis but instead that methylamine-reacted alpha2-macroglobulin inhibits the hemolytic activity of limulin. Thus the thiol ester proteins alpha2-macroglobulin and C3 operate very differently in the hemolytic systems of Limulus and mammals and are not functionally homologous. Limulus alpha2-macroglobulin functions indirectly in hemolysis: its inactivation yields an inhibitory molecule for limulin-mediated hemolysis.

Comparison of Limulus alpha-macroglobulin with human alpha2-macroglobulin: thiol ester characterization, subunit organization, and conformational change.

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.

Molecular cloning of Limulus alpha 2-macroglobulin.

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.

Localisation of the major reactive lysine residue involved in the self-crosslinking of proteinase-activated Limulus alpha 2-macroglobulin.

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.