Activation of human neutrophil gelatinase by endogenous serine proteinases.
Ontology highlight
ABSTRACT:
SUBMITTER: Vissers MC
PROVIDER: S-EPMC1148706 | biostudies-other | 1988 Jan
REPOSITORIES: biostudies-other
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Project description:BackgroundDermatomyositis and polymyositis are the best known idiopathic inflammatory myopathies (IIMs). Classic histopathologic findings include the infiltration of inflammatory cells into muscle tissues. Neutrophil serine proteinases (NSPs) are granule-associated enzymes and play roles in inflammatory cell migration by increasing the permeability of vascular endothelial cells. In this study, we aimed to find the roles of NSPs in pathogenesis of IIMs.MethodsRNA and DNA were isolated to measure the relative expression of NSPs and their methylation levels. The expression of NSPs in serum and muscle tissues was tested by enzyme-linked immunosorbent assay, immunohistochemistry, and immunofluorescence, respectively. Serum from patients was used to culture the human dermal microvascular endothelial cells (HDMECs), and then we observed the influence of serum on expression of VE-cadherin, endothelial cell tube formation, and transendothelial migration of peripheral blood mononuclear cells (PBMCs).ResultsWe found that the expression of NSPs was increased in PBMCs, serum, and muscle tissues of IIM patients; these NSPs were hypomethylated in the PBMCs of patients. Serum NSPs were positively correlated with clinical indicators of IIM patients, including lactic dehydrogenase, erythrocyte sedimentation rate, C-reactive protein, immunoglobulin G, immunoglobulin M, and immunoglobulin A. Patients with anti-Jo-1, with anti-Ro-52, or without interstitial lung disease had lower levels of proteinase 3. Serum NSPs degraded the VE-cadherin of HDMECs, and serum NSP application increased the permeability of HDMECs.ConclusionsOur studies indicate, for the first time, that NSPs play an important role in muscle inflammatory cell infiltration by increasing the permeability of vascular endothelial cells in IIM patients.
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Project description:Cancer remains one of the major cause of death in the Western world. Although, it has been demonstrated that new therapies can improve the outcome of cancer patients, still many patients relapse after treatment. Therefore, there is a need to identify novel factors involved in cancer development and/or progression. Recently, neutrophil gelatinase-associated lipocalin (NGAL) has been suggested as a key player in different cancer types. Its oncogenic effect may be related to the complex NGAL/MMP-9. In the present study, NGAL was analyzed at both transcript and protein levels in different cancer types by analysing 38 public available microarray datasets and the Human Protein Atlas tool. NGAL transcripts were significantly higher in the majority of solid tumors compared to the relative normal tissues for every dataset analyzed. Furthermore, concordance of NGAL at both mRNA and protein levels was observed for 6 cancer types including bladder, colorectal, liver, lung, ovarian, and pancreatic. All metastatic tumors showed a decrease of NGAL expression when compared to matched primary lesions. According to these results, NGAL is a candidate marker for tumor growth in a fraction of solid tumors. Further investigations are required to elucidate the function of NGAL in tumor development and metastatic processes.
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Project description:Alpha-defensins are mammalian antimicrobial peptides expressed mainly by cells of myeloid lineage or small intestinal Paneth cells. The peptides are converted from inactive 8.5-kDa precursors to membrane-disruptive forms by post-translational proteolytic events. Because rhesus myeloid pro-alpha-defensin-4 (proRMAD-4((20-94))) lacks bactericidal peptide activity in vitro, we tested whether neutrophil azurophil granule serine proteinases, human neutrophil elastase (NE), cathepsin G (CG), and proteinase-3 (P3) have in vitro convertase activity. Only NE cleaved proRMAD-4((20-94)) at the native RMAD-4 N terminus to produce fully processed, bactericidal RMAD-4((62-94)). The final CG cleavage product was RMAD-4((55-94)), and P3 produced both RMAD-4((55-94)) and RMAD-4(57-94). Nevertheless, NE, CG, and P3 digests of proRMAD4 and purified RMAD-4((62-94)), RMAD-4((55-94)), and RMAD-4(57-94) peptides had equivalent in vitro bactericidal activities. Bactericidal peptide activity assays of proRMAD-4((20-94)) variants containing complete charge-neutralizing D/E to N/Q or D/E to A substitutions showed that (DE/NQ)-proRMAD-4((20-94)) and (DE/A)-proRMAD-4((20-94)) were as active as mature RMAD-4((62-94)). Therefore, proregion Asp and Glu side chains inhibit the RMAD-4 component of full-length proRMAD-4((20-94)), perhaps by a combination of charge-neutralizing and hydrogen-bonding interactions. Although native RMAD-4((62-94)) resists NE, CG, and P3 proteolysis completely, RMAD-4((62-94)) variants with disulfide pairing disruptions or lacking disulfide bonds were degraded extensively, evidence that the disulfide array protects the alpha-defensin moiety from degradation by the myeloid converting enzymes. These in vitro analyses support the conclusion that rhesus macaque myeloid pro-alpha-defensins are converted to active forms by serine proteinases that co-localize in azurophil granules.
| S-EPMC2583298 | biostudies-other
Project description:Neutrophil gelatinase associated lipocalin (NGAL), was originally identified in neutrophil granules as a heterodimer complex with gelatinase B (matrix metalloproteinase 9, MMP9), but more recently has been found to be secreted by damaged epithelial cells. Ngal is a member of the lipocalin family and subsequently named as lipocalin 2 on the basis of structural similarity with other members of the lipocalin family and its potential association with hydrophobic retinol and cholesterol oleate more strongly than their hydrophilic counterparts. In 2002, a landmark paper suggested that Ngal is a bacteriostatic agent which blocks iron acquisition by interacting with a number of bacterial siderophores, especially enterobactin. Since then, more siderophore-carrying functions have been reported than the possibility of hydrophobic ligand transport. In this setting, Ngal was renamed Siderocalin. Functions of siderocalin include not only bacteriostatic activity but potentially as a mediator of cell growth and differentiation; some of these functions appear to be referable to the holo siderocalin:siderophore:iron complex and recent work suggests that metabolic products may act as mammalian siderophores bound by Ngal. While still speculative, it may be that the mammalian siderophores can establish the missing link between Ngal and a number of its functions in vivo. This review provides an overview of the discoveries of the different ligands of Ngal and consequently related functions. Hydrophobic ligands, bacterial siderophores as well as their modified structures (synthetic siderophores), and mammalian siderophores are summarized.
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Project description:Two metallo-proteinases of human neutrophil leucocytes, collagenase and gelatinase, were studied. Collagenase specifically cleaved native collagen into the TCA and TCB fragments, whereas gelatinase degraded denatured collagen, i.e. gelatin, and the TCA fragments produced by collagenase. On subcellular fractionation by zonal sedimentation, collagenase was found to be localized in the specific granules, separate from gelatinase, which was recovered in smaller subcellular organelles known as C-particles. Neither enzyme was present in the azurophil granules, which contain the two major serine proteinases of neutrophils, elastase and cathepsin G. Collagenase and gelatinase were separated by gel filtration from extracts of partially purified granules. Both enzymes were found to occur in latent forms and were activated either by trypsin or by 4-aminophenylmercuric acetate. Gelatinase was also activated by cathepsin G, which, however, destroyed collagenase. Both enzymes were destroyed by neutrophil elastase. Activation resulted in a decrease by 25 000 in the apparent mol. wt. of both latent metallo-proteinases.
| S-EPMC1162366 | biostudies-other
Project description:An e.l.i.s.a. was developed using specific polyclonal rabbit antibodies against human neutrophil gelatinase. This assay, in contrast to the functional assay, is independent of activation of gelatinase, and is specific for the detection of gelatinase in both its reduced and unreduced forms. Using this assay, we were able to demonstrate a difference between the subcellular localization of gelatinase on the one hand, and the subcellular localization of vitamin B-12-binding protein, lactoferrin and cytochrome b558 on the other hand. The latter three co-localized in fractions of slightly higher density than gelatinase on a two-layer Percoll density gradient. Furthermore, the release of gelatinase exceeded the release of vitamin B-12-binding protein as well as lactoferrin by a factor of 3-6 following stimulation with formylmethionyl-leucyl-phenylalanine, leukotriene B4 and other soluble stimuli. Thus, although gelatinase has previously been found to co-localize with lactoferrin on immuno-electron microscopy, we confirm the existence of gelatinase-rich and lactoferrin- and vitamin B-12-binding-protein-poor granules, that are lighter and mobilized more easily than specific granules. These gelatinase-containing granules are not the store of cytochrome b558.
| S-EPMC1133208 | biostudies-other
Project description:BackgroundWe have recently reported the expression of murine Implantation Serine Proteinase genes in pre-implantation embryos (ISP1) and uterus (ISP1 and ISP2). These proteinases belong to the S1 proteinase family and are similar to mast cell tryptases, which function as multimers.ResultsHere, we report the purification and initial characterization of ISP1 and 2 with respect to their physico-chemical properties and physiological function. In addition to being co-expressed in uterus, we show that ISP1 and ISP2 are also co-expressed in the pre-implantation embryo. Together, they form a heterodimer with an approximate molecular weight of 63 kD. This complex is the active form of the enzyme, which we have further characterized as being trypsin-like, based on substrate and inhibitor specificities. In addition to having a role in embryo hatching and outgrowth, we demonstrate that ISP enzyme is localized to the site of embryo invasion during implantation and that its activity is important for successful implantation in vivo.ConclusionOn the basis of similarities in structural, chemical, and functional properties, we suggest that this ISP enzyme complex represents the classical hatching enzyme, strypsin. Our results demonstrate a critical role for ISP in embryo hatching and implantation.
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Project description:BMP-1/tolloid-like proteinases (BTPs) are major players in tissue morphogenesis, growth and repair. They act by promoting the deposition of structural extracellular matrix proteins and by controlling the activity of matricellular proteins and TGF-β superfamily growth factors. They have also been implicated in several pathological conditions such as fibrosis, cancer, metabolic disorders and bone diseases. Despite this broad range of pathophysiological functions, the putative existence of a specific endogenous inhibitor capable of controlling their activities could never be confirmed. Here, we show that procollagen C-proteinase enhancer-2 (PCPE-2), a protein previously reported to bind fibrillar collagens and to promote their BTP-dependent maturation, is primarily a potent and specific inhibitor of BTPs which can counteract their proteolytic activities through direct binding. PCPE-2 therefore differs from the cognate PCPE-1 protein and extends the possibilities to fine-tune BTP activities, both in physiological conditions and in therapeutic settings.
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Project description:Periodontitis is a chronic destructive infection of the tooth-supportive tissues, which is caused by pathogenic bacteria such as Actinobacillus actinomycetemcomitans. A severe form of periodontitis is found in Papillon-Lefèvre syndrome (PLS), an inheritable disease caused by loss-of-function mutations in the cathepsin C gene. Recently, we demonstrated that these patients lack the activity of the polymorphonuclear leukocyte (PMN)-derived serine proteinases elastase, cathepsin G, and proteinase 3. In the present study we identified possible pathways along which serine proteinases may be involved in the defense against A. actinomycetemcomitans. Serine proteinases are capable to convert the PMN-derived hCAP-18 into LL-37, an antimicrobial peptide with activity against A. actinomycetemcomitans. We found that the PMNs of PLS patients released lower levels of LL-37. Furthermore, because of their deficiency in serine proteases, the PMNs of PLS patients were incapable of neutralizing the leukotoxin produced by this pathogen, which resulted in increased cell damage. Finally, the capacity of PMNs from PLS patients to kill A. actinomycetemcomitans in an anaerobic environment, such as that found in the periodontal pocket, seemed to be reduced. Our report demonstrates a mechanism that suggests a direct link between an inheritable defect in PMN functioning and difficulty in coping with a periodontitis-associated pathogen.
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Project description:Extracellular serine proteinase pathways control immune and homeostatic processes in insects. Our current knowledge of their components is limited-prophenoloxidase-activating proteinases (PAPs) are among the few hemolymph proteinases (HPs) with known functions. To identify components of proteinase systems in the hemolymph of Manduca sexta, we amplified cDNAs from larval fat body or hemocytes using degenerate primers coding for two conserved regions in S1 family serine proteinases. PCR yielded fragments encoding seven known (HP1-HP4, PAP-1, PAP-2 and PAP-3) and 18 unknown (HP5-HP22) serine proteinases. We screened cDNA libraries and isolated clones for 17 of the newly discovered HPs (HP5-HP22 except for HP11) and prepared antibodies to 14 recombinant proteins (HP6, HP8-HP10, HP12, HP14-HP19, HP21 and HP22). Fourteen of the HPs contain regulatory clip domain(s) at their amino-terminus--HP1, HP2, HP6, HP8, HP13, HP17, HP18, HP21, HP22 and PAP-1 have one, whereas HP12, HP15, PAP-2 and PAP-3 have two clip domains. Multiple sequence alignment of catalytic domains in these and other arthropod serine proteinases provided useful clues for future functional analysis. Northern blot and reverse transcription PCR (RT-PCR) analyses showed increases in HP2, HP7, HP9, HP10, HP12-HP22 mRNA levels at 24h after a bacterial challenge, and immunoblot analysis confirmed elevated concentrations of HP12, HP14-HP19, HP21 and HP22 proteins in plasma in response to injected bacteria. Hemocytes express HP13 and HP18; fat body produces HP12, HP20-HP22; both tissues synthesize the other HPs. These results collectively indicate the existence of a complex serine proteinase network in M. sexta hemolymph, predicted to mediate rapid defense responses upon wounding and/or microbial infection.
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