Serum amyloid A-derived peptides, present in human rheumatic synovial fluids, induce the secretion of interferon-gamma by human CD(4)(+) T-lymphocytes.

Serum amyloid A (SAA) is a major acute-phase protein whose biochemical functions remain largely obscure. Human rheumatic synovial fluids were screened by high performance liquid chromatography mass spectrometry for SAA-derived peptides, specifically the sequence AGLPEKY (SAA(98-104)) which was previously shown to modulate various leukocyte functions. Two such fluids were found to contain a truncated version of SAA(98-104). Synthetic SAA(98-104) and several of its analogs were shown capable of binding isolated human CD(4)(+) T-lymphocytes and stimulating them to produce interferon-gamma. Given the high acute-phase serum level of SAA and its massive proteolysis by inflammatory related enzymes, SAA-derived peptides may be involved in host defense mechanisms.

IL-2 induces T cell adherence to extracellular matrix: inhibition of adherence and migration by IL-2 peptides generated by leukocyte elastase.

Migration of inflammatory cells requires cell adhesion and their subsequent detachment from the extracellular matrix (ECM). Leukocyte activation and migration must be terminated to stop inflammation. Here, we report that IL-2 enhances human T cell adherence to laminin, collagen type IV, and fibronectin (FN). In contrast, neutrophil elastase, an enzyme activated during inflammation, degrades IL-2 to yield IL-2 fractions that inhibit IL-2-induced T cell adhesion to FN. The amino acid composition of two of these IL-2 fractions, which appear to block T cell adherence to FN, were analyzed, and three peptides were consequently synthesized. The three peptides IVL, RMLT, and EFLNRWIT, but not the corresponding inversely synthesized peptides, inhibited T cell adhesion to FN induced by a variety of activators: IL-2, IL-7, macrophage inflammatory protein (MIP)-1beta, and PMA, as well as anti-CD3 and anti-beta1 integrin-activating mAb. Moreover, these IL-2 peptides inhibited T cell chemotaxis via FN-coated membranes induced by IL-2 and MIP-1beta. Inhibition of T cell adherence and migration apparently involves abrogation of the rearrangement of the T cell actin cytoskeleton. Thus, the migrating immune cells, the cytokines, and the ECM can create a functional relationship in which both inflammation-inducing signals and inhibitory molecules of immune responses can coexist; the enzymatic products of IL-2 may serve as natural feedback inhibitors of inflammation.

Peptides derived from human C-reactive protein inhibit the enzymatic activities of human leukocyte elastase and cathepsin G: use of overlapping peptide sequences to identify a unique inhibitor.

Ten overlapping 15-mer peptides, spanning the entire inner disulfide loop of human C-reactive protein (residues 36-97), were used to isolate a potent inhibitor of the enzymes human leukocyte elastase and human leukocyte cathepsin G, which are associated with chronic inflammatory tissue damage. In contrast to the inability of intact C-reactive protein to inhibit both enzymes, the synthetic peptide E62ILIFWSKDIGYSFT76 inhibited leukocyte elastase (Ki = 0.18 microM) and cathepsin G (Ki = 0.25 microM) at concentrations far lower than the acute-phase concentration of C-reactive protein. Several peptide-enzyme binding motifs were elucidated by structure-function studies, with the Glu62 residue being crucial in establishing long-range subsite interactions. Peptides derived from C-reactive protein, which may be generated in vivo by neutrophil-mediated proteolysis as part of a complex regulatory homeostatic mechanism, may play an important role in regulating the activity of matrix-degrading enzymes, specifically at sites of inflammation. The present results thus may shed additional insight on the physiological functions of the major acute-phase reactant C-reactive protein, and perhaps be used as a basis for the design of novel therapeutic substances.

Synthetic peptides derived from the sequence of human C-reactive protein inhibit the enzymatic activities of human leukocyte elastase and human leukocyte cathepsin G.

Peptides derived from the primary sequence of the acute phase reactant C-reactive protein (CRP) are shown to inhibit in vitro the enzymatic activities of human leukocyte elastase (hLE) and human leukocyte cathepsin G (hCG), which are associated with tissue damage occurring in the course of several chronic inflammatory conditions. CRP-derived peptides were synthesized based on their sequence similarity to domains within the natural inhibitors of hLE and hCG. The octapeptide Val89-Thr-Val-Ala-Pro-Val-His-Ile96 (CRP 89-96) is shown to inhibit hLE and hCG to a larger extent than peptides of similar chain lengths corresponding to the active sites of their natural inhibitors, alpha 1-protease inhibitor and alpha-antichymotrypsin, respectively. Several additional peptides containing this core sequence were synthesized and shown to be inhibitors, in contrast to peptides derived from other regions of CRP as well as the intact protein, which are totally inactive. The inhibitory capability of CRP-derived peptides, which may be generated in vivo by neutrophil-mediated proteolysis as part of a complex regulatory homeostatic mechanism, may now be used as a basis for the design of novel therapeutic substances. The present finding may shed some light on the enigmatic physiological functions of CRP.

Binding pockets on the surface of human leukocyte elastase and human leukocyte cathepsin G. Implications to the design of inhibitors derived from human C-reactive protein.

Analogs of the peptide Val-Thr-Val-Ala-Pro-Val-His-Ile, derived from the primary sequence of the acute phase reactant CRP, i.e. amino acid residues 89-96, were optimized to inhibit the enzymatic activities of human leukocyte elastase (hLE) and human leukocyte cathepsin G (hCG), which are associated with tissue damage occurring in the course of several chronic inflammatory conditions. hLE's major S1 pocket, lined mostly by hydrophobic amino acid residues, was shown by theoretical electrostatic potential calculations to possess some negative charge. This pocket was found to be extremely sensitive towards modifications in the P1 position of CRP derived inhibitors, with valine being the preferred amino acid. In contrast, the corresponding S1 pocket of hCG is large and accepts the positively charged 'aromatic' side chain of histidine, which increases most significantly the capability of CRP derived inhibitors. A prominent positive pocket was observed in the distant S7 region of hLE, which is generated by two exposed positive residues, Arg177 and Arg217, on the enzymes surface. This long range subsite was utilized to increase the hLE inhibitory activity of CRP derived peptide using the natural sequence of CRP, which contains a unique glutamic acid moiety in the P7 position. In contrast to the charged nature of hLE's S7 pocket, the corresponding pocket on the surface of hCG appears to be less prominent. Additional hydrophobic N-terminus modifications of CRP89-96 increased the inhibitory activity towards both enzymes, provided that residues P1 and p7, were designed according to the individual preferences of hLE and hCG. The unique interaction between the negative amino acid side chain of CRP with the positive S7 pocket of hLE as elucidated in this study, and additional subsite preferences may now be used in the design of novel therapeutic substances.