Nature of T lymphocyte recognition of macrophage-associated antigens. V. Contribution of individual peptide residues of human fibrinopeptide B to T lymphocyte responses.

Guinea pig T lymphocyte responses to a decapeptide antigen (NH1-Asp5-Ans6-Glu7-Glu8-Gly9-Phe10-Phe11-Ser12-Ala13-Arg14-OH) of human fibrinopeptide B (hFPB) were examined using various synthetic peptide analogues containing single residue substitutions. Each analogue was examined for antigenicity as determined by an in vitro proliferative responses of hFPN-immune strain 2 guinae pig T cells. In addition, both strain 2 and strain 13 animals were immunized with each analogue and immunogenicity assessed by in vitro T cell-proliferative responses with the homologous immunizing analogue and the parent peptide. Replacement of arginine14 with lysine formed an immunogenic analogue which showed no antigenic cross-reactivity with the native peptide in strain 2 T cell responses. In addition, substitution of arginine14 with blocked lysine again produced a unique immunogenic analogue that showed little or no antigenic identity with the intact lysine analogue or the native peptide. In similar fashion, substitution of resideu phenylalanie10 with tyrosine or Phe(4-NO2) created unique immunogenic analogues with little or no antigenic identity to the native peptide with strain 2 T cells. By contrast, replacement of phenylalanine11 with either tyrosine or Phe(4-NO2) resulted in analogues with a total loss of immunogenicity and antigenicity in strain 2 T cell responses. An analogue in which glutamic acid7,8 were replaced with glutamine retained a small degree of antigenicity with hFPB-immune T cells, but T cells from strain 2 animals immunized with the Gln analogue responded only marginally to the Gln analogue while producing good proliferative responses with the native peptide. On the other hand, an analogue in which asparatic acid5 was replaced with asparagine retained most of the antigenic identity with hFPB for strain 2 T cell responses. None of thee analogues were immunogenic for strain 13 guinea pigs. These observations are discussed with respect to the contribution of each substituted residue to T cell respones, mechanism of Ir gene function, and a model for T cell recognition of small peptide antigens.

Binding and degradation of elastin by the staphylolytic enzyme lysostaphin.

Lysostaphin is a bacterial zinc metalloproteinase that degrades staphylococcal cell wall peptidoglycans. We have shown that lysostaphin also binds tightly to elastin and contains elastolytic activity. The objective of this investigation was to further characterize the biochemical mechanism of elastolysis by lysostaphin. Binding of lysostaphin to elastin was demonstrated with elastin peptide affinity chromatography. Elastolysis by lysostaphin was studied using a tritium release assay with tritium borohydride-reduced elastin as substrate. Proteolysis of elastin by lysostaphin was maximal at neutral to slightly basic pH and proceeded linearly for 10 hr before reaching saturation. The elastolytic activity was not affected by inhibitors of cysteine or serine proteinases, but was inhibited by o-phenanthroline, EDTA, and by the addition of exogenous zinc. Inhibition by zinc was not due to an alteration in enzyme-ligand interaction since zinc-containing buffers did not impair the ability of lysostaphin to bind elastin. Lysostaphin elastolysis was not inhibited by trypsin treatment of the enzyme, which has been shown to inactivate the enzyme's staphylolytic properties. It is therefore concluded that: (1) lysostaphin binds and degrades elastin; (2) the elastolytic activity of lysostaphin is distinct from its staphylolytic activity; (3) sequence similarities with matrix metalloproteinases suggest the Ala-Ala-Thr-His-Glu sequence in the amino terminus of lysostaphin to be involved in elastin degradation; (4) our studies are important in establishing that metalloproteinases from staphylococci can participate in elastin degradation.

Fibrin Polymerization. 1. Alkylating peptide inhibitors of fibrin polymerization.

A series of analogues relating to the NH2-terminal region of the fibrin alpha chain, i.e., Gly-Pro-Arg-Pro, were prepared by stepwise solid-phase synthesis, and their abilities to inhibit fibrin polymerization and to prolong thrombin-initiated clotting time were evaluated. Among the analogues systematically modified at different positions, replacement of the NH2-terminal three residues of Gly-Pro-Arg-Pro by either chlorambucil, p-nitrophenyl-L-alanine, or p-aminophenyl-L-alanine gave inactive compounds in the thrombin time assay, whereas similar substitution or extension of the COOH terminus produced the highly active analogues Gly-Pro-Arg-Phe(4-NH2), 22%; Gly-Pro-Arg-Pro-Phe(4-NO2), and Gly-Pro-Arg-Pro-Phe(4-NH2), 105%; relative to Gly-Pro-Arg-Pro = 100% in the fibrin polymerization inhibitory assay. As potential photoaffinity labeling probes, analogues containing a nitrophenylalanine residue in position 4 or 5 underwent photolysis under the experimental photoactivation conditions. As a potential alkylating probe, Chl-Pro-Arg-Pro was selectively effective in inhibiting thrombin amidolysis and fibrin polymerization. In the latter assay, Chl-Pro-Arg-Pro was approximately 20 times more potent than Gly-Pro-Arg-Pro in inhibiting fibrin aggregation.

Entactin expression by rat lung and rat alveolar epithelial cells.

Although the composition of the subepithelial basement membrane of the alveolar septum has been studied in detail, there is relatively little information about which cells produce it. We examined intact rat lung and isolated rat alveolar type II cells for the expression of entactin, an integral basement membrane component that binds laminin and type IV collagen. By Northern analysis, late gestation and early neonatal rat lungs expressed high levels of entactin mRNA whereas lungs from adult animals had only minimal levels of entactin mRNA. These latter findings were confirmed by in situ hybridization, which showed prominent signal for entactin mRNA in cells in the alveolar walls of neonatal animals and no signal for entactin mRNA in the alveolar walls of lungs from adult animals. The entactin mRNA throughout the alveolar walls of neonatal animals was not limited to cells that expressed surfactant-associated protein C mRNA, a marker of alveolar type II cells. Freshly harvested adult alveolar type II cells and alveolar type II cells in culture for < 6 days expressed none to minimal entactin mRNA or protein. However, with longer periods in culture, both entactin mRNA and entactin protein synthesis were evident and progressively increased. In situ hybridization indicated that >60% of the alveolar epithelial cells expressed entactin mRNA with increasing time in culture. When cultured on Engelbreth-Holm-Swarm matrix, alveolar type II cells showed the same time course of entactin mRNA expression as cells cultured on plastic. Neonatal lung mesenchymal cells produced abundant entactin in culture, consistent with the likelihood that these cells are the principal source of entactin in alveolar walls in the developing lung. These results indicate that entactin production in the normal alveolar wall occurs primarily during lung development and that mesenchymal cells are probably the principal source of production. However, because adult alveolar epithelial cells synthesize entactin in culture, it is possible that alveolar epithelium contributes to the entactin in the alveolar subepithelial basement membrane.

Localization of a chemotactic domain in human thrombin.

The cyanogen bromide fragment CB67-129 of human prethrombin 1, corresponding to residues 54-116 of the thrombin B chain, is a potent chemotaxin for human peripheral blood monocytes and the murine macrophage like cell line, J774. Both of these cell types have been shown to respond chemotactically to alpha-thrombin and iPr2P-alpha-thrombin. Effective concentrations for stimulating directed cell movement with the fragment vary from 10(-11) to 10(-7) M. Moreover, CB67-129 and its parent protein compete for the same chemotactic receptor site. Fragment CB67-129, representing residues 54-116 of the human thrombin B chain sequence, contains a nine-residue insertion ("loop B") that is absent in homologous sequences derived from the closely related proteases chymotrypsin and trypsin. Unlike iPr2P-alpha-thrombin, iPr2P derivatives of these latter enzymes possess little or no chemotactic activity, suggesting a relationship between the insertion sequence and thrombin chemotactic activity. The loop B sequence is unique insofar as it contains all of the carbohydrate moieties known to reside in alpha-thrombin. However, chemotactic activity is only minimally reduced subsequent to hydrolysis by both neuraminidase and beta-galactosidase, indicating that receptor recognition and stimulated cell movement are mainly a function of structure of the cyanogen bromide derived fragment rather than of asparagine-linked carbohydrates.

Expression of laminin alpha3, alpha4, and alpha5 chains by alveolar epithelial cells and fibroblasts.

Laminins are principal components of basement membranes. Eleven laminin isoforms are known, each a heterotrimer composed of polypeptide chains designated alpha, beta, and gamma. Five alpha chains have been identified to date: alpha1, alpha2, alpha3, alpha4, and alpha5. Recent studies of fetal and adult mouse lung show prominence of alpha3, alpha4, and alpha5 in alveolar tissue, and point to differences in the cellular expression of these alpha chains in the developing alveolus. We examined isolated rat alveolar type II cells and lung fibroblasts for expression of laminins alpha3, alpha4, and alpha5. We found that laminin alpha3 was expressed only by alveolar epithelial cells, that laminin alpha4 was expressed only by lung fibroblasts, and that laminin alpha5 was expressed primarily by alveolar epithelial cells. Metabolic labeling and immunoprecipitation confirmed the production of laminin alpha4 by fibroblasts and laminin alpha5 by alveolar epithelial cells in culture. These studies indicate that different alveolar cell types contribute different laminin alpha chains to the laminin isoforms in alveolar basement membranes. Immunohistochemistry showed colocalization of these laminin alpha chains with the laminin beta1, beta2, and gamma1 chains, indicating the likelihood that laminins 6 to 11 are present in alveolar basement membranes.