James Travis

α1-Antitrypsin and the serpins: variation and countervariation

Abstract α 1-Antitrypsin is a plasma protein which protects elastic tissue from proteolytic attack. Consequently, genetic deficiency, or the oxidation of its reactive centre in cigarette smokers can result in the degenerative lung disease emphysema. Structural studies explain the mechanisms involved and have also drawn attention to a new family of serine proteinase inhibitors. The specificity of each of these inhibitors is primarily dependent on a single amino acid at its reactive centre. Site-directed mutagenesis is enabling the production of specifically designed inhibitors for therapeutic use, including an improved replacement for α 1-antitrypsin deficiency.

Selective removal of albumin from plasma by affinity chromatography

Abstract The removal of albumin from human plasma or serum can be easily accomplished by passage through a column of sepharose-blue dextran conjugate, without significant binding of any other proteins. The albumin-free plasma should be useful as a starting material for the isolation of many plasma proteins by classical procedures.

New method for typing Staphylococcus aureus strains: multiple-locus variable-number tandem repeat analysis of polymorphism and genetic relationships of clinical isolates.

ABSTRACT The PCR-based methodology applied to multiple-locus variable numbers of tandem repeat (VNTR) analysis was recently shown to be a useful technique for the molecular typing of clinical isolates of several bacterial species. We have adopted this method for the molecular typing of methicillin-resistant Staphylococcus aureus. Five staphylococcal VNTR loci (sdr, clfA, clfB, ssp, and spa) were subjected to analysis, and it was shown that the method allows typing of S. aureus strains with the discriminatory power and reproducibility of pulsed-field gel electrophoresis while at the same time being rapid and applicable to analysis of large numbers of isolates.

Enzymatic inactivation of human alpha-1-proteinase inhibitor by neutrophil myeloperoxidase

Human myeloperoxidase, in the presence of H2O2 and halide ion, can catalytically inactivate human alpha-1-proteinase inhibitor (α-1-PI), the major plasma inhibitor of elastolytic activity. The rate of inactivation is directly proportional to both the myeloperoxidase and α-1-PI concentrations and inversely proportional to the H2O2 concentration. Amino acid analysis of the oxidized α-1-PI reveals that the only modified amino acid is methionine, which is converted to the sulfoxide form during the course of the reaction. Significantly, this system has no effect on either α-2-macroglobulin (α2M) or α-1-antichymotrypsin (α-1-Achy), two other important plasma proteinase inhibitors, nor can the system be replaced with horseradish peroxidase. Since it has been shown recently that methionine occupies part of the reactive site of α-1-PI, it is possible that the release of myeloperoxidase by leukocytes during phagocytosis inactivates this inhibitor through oxidation of this particular residue, thereby indirectly augmenting the proteolytic activity released by these same cells.

Arginine-Specific Protease from Porphyromonas gingivalis Activates Protease-Activated Receptors on Human Oral Epithelial Cells and Induces Interleukin-6 Secretion

ABSTRACT Periodontitis is a chronic inflammatory disease affecting oral tissues. Oral epithelial cells represent the primary barrier against bacteria causing the disease. We examined the responses of such cells to an arginine-specific cysteine proteinase (RgpB) produced by a causative agent of periodontal disease, Porphyromonas gingivalis. This protease caused an intracellular calcium transient in an oral epithelial cell line (KB), which was dependent on its enzymatic activity. Since protease-activated receptors (PARs) might mediate such signaling, reverse transcription-PCR was used to characterize the range of these receptors expressed in the KB cells. The cells were found to express PAR-1, PAR-2, and PAR-3, but not PAR-4. In immunohistochemical studies, human gingival epithelial cells were found to express PAR-1, PAR-2, and PAR-3 on their surface, but not PAR-4, indicating that the cell line was an effective model for the in vivo situation. PAR-1 and PAR-2 expression was confirmed in intracellular calcium mobilization assays by treatment of the cells with the relevant receptor agonist peptides. Desensitization experiments strongly indicated that signaling of the effects of RgpB was occurring through PAR-1 and PAR-2. Studies with cells individually transfected with each of these two receptors confirmed that they were both activated by RgpB. Finally, it was shown that, in the oral epithelial cell line, PAR activation by the bacterial protease-stimulated secretion of interleukin-6. This induction of a powerful proinflammatory cytokine suggests a mechanism whereby cysteine proteases from P. gingivalis might mediate inflammatory events associated with periodontal disease on first contact with a primary barrier of cells.

Gingipains, the Major Cysteine Proteinases and Virulence Factors of Porphyromonas gingivalis: Structure, Function and Assembly of Multidomain Protein Complexes

Gingipains, extracellular cysteine proteinases of Porphyromonas gingivalis, constitute the major virulence factor of this periodontopathogenic bacterium. They are the product of three genes, two coding for an Arg-specific (RgpA and RgpB) and one for a Lys-specific proteinase (Kgp). Proteinase domains of RgpA and RgpB are virtually identical; however, the gene encoding the former enzyme is missing a large segment coding for hemaglutinin / adhesin (HA) domains. The latter domains are present also in Kgp. The tertiary structure of RgpB revealed that the proteinase domain of gingipains has a protein fold referred to as the caspase-hemoglobinase fold. On this basis, they are also evolutionary related to other highly specific proteinases including clostripain, caspases, legumains and separase (clan CD of cysteine peptidases). Gingipains are produced as large preproproteins and are subject to elaborate, not yet fully understood, secretion, glycosylation, activation, and maturation processes. How they traverse the outer membrane is unknown, although it can be hypothesized that they use an autotransporter pathway. Apparently during transport through the periplasm the LPS-like glycan moiety is added at the conserved C-terminal portion of progingipains. At the cell surface pro-gingipains fold into partially active, single-chain zymogens and undergo autocatalytic, intermolecular processing. Two sequential cleavages within the profragment domain enhance zymogen activity and in the case of RgpA and Kgp are followed by excision of the individual HA domains. These domains are further truncated at the C-terminus by concerted action of Kgp and carboxypeptidase and form a non-covalent multidomain, multifunctional complex anchored into the outer membrane by the glycated, C-terminal HA domain. This hypothetical scenario is a reasonable explanation for the occurrence of many forms of gingipains.

Inactivation of tissue inhibitor of metalloproteinases by neutrophil elastase and other serine proteinases

Tissue inhibitor of metalloproteinases (TIMP) from cultured bovine dental pulp inhibits human rheumatoid synovial matrix metalloproteinase 3 (MMP‐3) with a stoichiometry of 1:1 on a molar basis. Among the serine proteinases examined, human neutrophil elastase, trypsin and α‐chymotrypsin destroyed the inhibitory activity of TIMP against MMP‐3 by degrading the inhibitor molecule into small fragments. In contrast, the inhibitory activity of TIMP was not significantly reduced by the actions of cathepsin G, pancreatic elastase and plasmin. These data indicate that neutrophils which infiltrate tissues in various inflammatory conditions may play an important role in regulating TIMP activity in vivo through the action of neutrophil elastase.

The Mitogen-activated Protein Kinase and JAK-STAT Signaling Pathways Are Required for an Oncostatin M-responsive Element-mediated Activation of Matrix Metalloproteinase 1 Gene Expression

Both astrocytes in the central nervous system and fibroblasts in somatic tissues are not only the major sources of extracellular matrix components but also of matrix metalloproteinases (MMPs), a family of enzymes directly involved in extracellular matrix breakdown. We have analyzed the regulation of the expression of MMPs and TIMPs (tissue inhibitors of metalloproteinases) in human primary astrocytes stimulated with oncostatin M (OSM) and other extracellular mediators in comparison with normal human dermal fibroblasts. It was found that OSM induced/enhanced transcription of MMP-1 (interstitial collagenase) and MMP-3 (stromelysin 1) in astrocytes, and MMP-1, MMP-9 (gelatinase B), and TIMP-1 in fibroblasts. Analysis of the signal transduction leading to activation of the MMP-1 gene revealed the presence of an OSM-responsive element (OMRE) encompassing the AP-1 binding site and the signal transducer and activator of transcription (STAT) binding element, which mediate activation by OSM. OMRE is also present in the TIMP-1 gene promoter and, although there are some differences in these two motifs, both appear to be targets for the simultaneous action of OSM-induced nuclear effectors. The induced enhancement of transcription by synergistically acting AP-1 and STAT binding elements in response to OSM is Raf-dependent. Cross-talk between the mitogen-activated protein kinase and JAK-STAT pathways is required to achieve maximal induction of the OMRE-driven transcription by OSM.

Pathogenesis of periodontitis: a major arginine-specific cysteine proteinase from Porphyromonas gingivalis induces vascular permeability enhancement through activation of the kallikrein/kinin pathway.

To elucidate the mechanism of production of an inflammatory exudate, gingival crevicular fluid (GCF), from periodontal pockets in periodontitis, we examined the vascular permeability enhancement (VPE) activity induced by an arginine-specific cysteine proteinase, Arg-gingipain-1 (RGP-1), produced by a major periopathogenic bacterium, Porphyromonas gingivalis. Intradermal injections into guinea pigs of RGP-1 (> 10(-8) M), or human plasma incubated with RGP-1 (> 10(-9) M), induced VPE in a dose- and activity-dependent manner but with different time courses for the two routes of production. VPE activity induced by RGP-1 was augmented by kininase inhibitors, inhibited by a kallikrein inhibitor and unaffected by an antihistamine drug. The VPE activity in human plasma incubated with RGP-1 also correlated closely with generation of bradykinin (BK). RGP-1 induced 30-40% less VPE activity in Hageman factor-deficient plasma and no VPE in plasma deficient in either prekallikrein (PK) or high molecular weight kininogen (HMWK). After incubation with RGP-1, plasma deficient in PK or HMWK, reconstituted with each missing protein, caused VPE, as did a mixture of purified PK and HMWK, but RGP-1 induced no VPE from HMWK. The VPE of extracts of clinically isolated P. gingivalis were reduced to about 10% by anti-RGP-1-IgG, leupeptin, or tosyl-L-lysine chloromethyl ketone, which paralleled effects observed with RGP-1. These results indicate that RGP-1 is the major VPE factor of P. gingivalis, inducing this activity through PK activation and subsequent BK release, resulting in GCF production at sites of periodontitis caused by infection with this organism.

Cleavage and activation of proteinase‐activated receptor‐2 on human neutrophils by gingipain‐R from Porphyromonas gingivalis

Gingipain‐R, the major arginine‐specific proteinase from Porphyromonas gingivalis, a causative agent of adult periodontal disease, was found to cleave a model peptide representing the cleavage site of proteinase‐activated receptor‐2 (PAR‐2), a G‐protein‐coupled receptor found on the surface of neutrophils. The bacterial proteinase was also shown to induce an increase in the intracellular calcium concentration of enzyme‐treated neutrophils, most probably due to PAR‐2 activation. This response by neutrophils to gingipain‐R may be a mechanism for the development of inflammation associated with periodontal disease.