Cleaved SLPI, a novel biomarker of chymase activity

2008 ◽  
Vol 389 (9) ◽  
Author(s):  
Stanley M. Belkowski ◽  
John Masucci ◽  
Andrew Mahan ◽  
Jukka Kervinen ◽  
Matthew Olson ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Disulfide Bonds ◽  
Ex Vivo ◽  
Human Saliva ◽  
Cathepsin G ◽  
Proteinase 3 ◽  
Liquid Chromatography Mass Spectrometry ◽  
Enzymatic Assays ◽  
Reducing Conditions

Abstract Secretory leukocyte protease inhibitor (SLPI) is a protease inhibitor of the whey acidic protein-like family inhibiting chymase, chymotrypsin, elastase, proteinase 3, cathepsin G and tryptase. Performing in vitro enzymatic assays using both Western blotting and liquid chromatography/mass spectrometry techniques we showed that, of the proteases known to interact with SLPI, only chymase could uniquely cleave this protein. The peptides of the cleaved SLPI (cSLPI) remain coupled due to the disulfide bonds in the molecule but under reducing conditions the cleavage can be observed as peptide products. Subsequent ex vivo studies confirmed the presence of SLPI in human saliva and its susceptibility to cleavage by chymase. Furthermore, inhibitors of chymase activity are able to inhibit this cleavage. Human saliva from both normal and allergic individuals was analyzed for levels of cSLPI and a correlation between the level of cSLPI and the extent of allergic symptoms was observed, suggesting the application of cSLPI as a biomarker of chymase activity in humans.

scholarly journals IgE-mediated histamine release from nasal mucosa is inhibited by SLPI (secretory leukocyte protease inhibitor) to the level of spontaneous release

1998 ◽  
Vol 7 (3) ◽  
pp. 217-220 ◽  
Author(s):  
U. Westin ◽  
E. Lundberg ◽  
K. Ohlsson
Keyword(s):  
Protease Inhibitor ◽  
Histamine Release ◽  
Nasal Mucosa ◽  
Secretory Leukocyte Protease Inhibitor ◽  
Cathepsin G ◽  
Low Molecular Weight ◽  
Spontaneous Release ◽  
Ige Mediated ◽  
Dose Dependent

The secretory leukocyte protease inhibitor (SLPI) is a low-molecular-weight inhibitor of proteases, such as elastase and cathepsin G which are released from leukocytes during phagocytosis. The purpose of this study was to determine whether or not SLPI is able to inhibit IgE-mediated histamine release. Nasal mucosa from 11 test subjects without atopic disposition was used for thisin vitrostudy. We found that SLPI inhibited histamine release in a dose-dependent way but was without influence on the spontaneous release.

scholarly journals Structural modeling and thermostability of a serine protease inhibitor belonging to the Kunitz family from the tick Rhipicephalus microplus

2020 ◽  
Author(s):  
Lívia de Moraes Bomediano Camillo ◽  
Graziele Cristina Ferreira ◽  
Adriana Feliciano Alves Duran ◽  
Flavia Ribeiro Santos da Silva ◽  
Wanius Garcia ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Serine Protease ◽  
Disulfide Bonds ◽  
Tertiary Structure ◽  
Dissociation Constants ◽  
Serine Protease Inhibitor ◽  
Rhipicephalus Microplus ◽  
Human Neutrophil Elastase ◽  
Structure Stabilization

ABSTRACTrBmTI-A is a recombinant serine protease inhibitor that belongs to the Kunitz-BPTI family and that was cloned from Rhipicephalus microplus tick. rBmTI-A has inhibitory activities on bovine trypsin, human plasma kallikrein, human neutrophil elastase and plasmin with dissociation constants in nM range. It is characterized by two inhibitory domains and each domain presents six cysteines that form three disulfide bonds, which contribute to the high stability of its structure. Previous studies suggest that serine protease inhibitor rBmTI-A has a protective potential against pulmonary emphysema in mice and anti-inflammatory potential, besides rBmTI-A presented a potent inhibitory activity against in vitro vessel formation. In this study, the tertiary structure of BmTI-A was modeled based on the structure of its Sabellastarte magnifica homologue. The structure stabilization was evaluated by molecular dynamics analysis. Circular dichroism data corroborated the secondary structure found by the homology modeling. Thermostability analysis confirmed the thermostability and the relation between the effects of the temperature in the inhibitor activity. The loss of activity observed was gradual, and, after 60 minutes of incubation at 90°C the inhibitor lost it completely.

Neutrophil elastase enzymatically antagonizes the in vitro action of G-CSF: implications for the regulation of granulopoiesis

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1752-1758 ◽  
Author(s):  
Frank El Ouriaghli ◽  
Hiroshi Fujiwara ◽  
J. Joseph Melenhorst ◽  
Giuseppe Sconocchia ◽  
Nancy Hensel ◽  
...  
Keyword(s):  
Negative Feedback ◽  
Neutrophil Elastase ◽  
Serine Proteases ◽  
Cathepsin G ◽  
Proteinase 3 ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Macrophage Colony ◽  
Serum Free Suspension

There is evidence that neutrophil production is a balance between the proliferative action of granulocyte–colony-stimulating factor (G-CSF) and a negative feedback from mature neutrophils (the chalone). Two neutrophil serine proteases have been implicated in granulopoietic regulation: pro–proteinase 3 inhibits granulocyte macrophage–colony-forming unit (CFU-GM) growth, and elastase mutations cause cyclic and congenital neutropenia. We further studied the action of the neutrophil serine proteases (proteinase 3, elastase, azurocidin, and cathepsin G) on granulopoiesis in vitro. Elastase inhibited CFU-GM in methylcellulose culture. In serum-free suspension cultures of CD34+ cells, elastase completely abrogated the proliferation induced by G-CSF but not that of GM-CSF or stem cell factor (SCF). The blocking effect of elastase was prevented by inhibition of its enzymatic activity with phenylmethylsulfonyl fluoride (PMSF) or heat treatment. When exposed to enzymatically active elastase, G-CSF, but not GM-CSF or SCF, was rapidly cleaved and rendered inactive. These results support a role for neutrophil elastase in providing negative feedback to granulopoiesis by direct antagonism of G-CSF.

Ex Vivo anti-HIV Activity of Human Serum Obtained from Normal Volunteers Following Oral Administration of the HIV-1 Protease Inhibitor CGP 53437: Value as Predictor of Antiviral Efficacy

1997 ◽  
Vol 8 (1) ◽  
pp. 54-59
Author(s):  
JK Lazdins ◽  
JK Walker ◽  
RM Cozens ◽  
G Flesch ◽  
C Czendlik ◽  
...  
Keyword(s):  
Oral Administration ◽  
Protease Inhibitor ◽  
Clinical Studies ◽  
Ex Vivo ◽  
Human Lymphocytes ◽  
Normal Volunteers ◽  
Antiviral Efficacy ◽  
Anti Hiv ◽  
Hiv 1

The aim of the study was to determine whether the concentration of CGP 53437 measured in the sera of normal volunteers following oral administration of a single dose, had retained its anti-HIV activity; and whether such results could be of predictive value for future clinical antiviral efficacy studies. CGP 53437 is an inhibitor of HIV-1 protease that suppresses HIV-1 replication in human lymphocytes in vitro at 100 nM. The in vitro anti-HIV activity of human sera obtained from CGP 53437-treated individuals was compared with that of sera spiked with known concentrations of CGP 53437 (in the presence or absence of α-1 acid glycoprotein). It was found that the concentration of the compound measured in the sera from treated individuals provided the expected in vitro anti-HIV activity. These results not only validate our analytical method for detection of CGP 53437, but also support the notion that interaction of CGP 53437 with plasma proteins does not significantly affect its antiviral activity (shift of the ED90 by a factor of three). In conclusion, ex vivo anti-HIV activity determinations of sera containing an HIV protease inhibitor, in conjunction with the pharmacokinetic evaluation during Phase I clinical studies, can provide valuable information regarding the suitability of such inhibitors for further clinical studies.

The Intracellular Serpin Proteinase Inhibitor 6 Is Expressed in Monocytes and Granulocytes and Is a Potent Inhibitor of the Azurophilic Granule Protease, Cathepsin G

Blood ◽  
1999 ◽  
Vol 93 (6) ◽  
pp. 2089-2097 ◽  
Author(s):  
Fiona L. Scott ◽  
Claire E. Hirst ◽  
Jiuru Sun ◽  
Catherina H. Bird ◽  
Stephen P. Bottomley ◽  
...  
Keyword(s):  
Proteinase Inhibitor ◽  
Sodium Dodecyl ◽  
Serine Proteinase ◽  
Cathepsin G ◽  
Stable Complex ◽  
Proteinase 3 ◽  
Azurophilic Granule ◽  
Caspase 7 ◽  
Extracellular Proteolysis

The monocyte and granulocyte azurophilic granule proteinases elastase, proteinase 3, and cathepsin G are implicated in acute and chronic diseases thought to result from an imbalance between the secreted proteinase(s) and circulating serpins such as 1-proteinase inhibitor and 1-antichymotrypsin. We show here that the intracellular serpin, proteinase inhibitor 6 (PI-6), is present in monocytes, granulocytes, and myelomonocytic cell lines. In extracts from these cells, PI-6 bound an endogenous membrane-associated serine proteinase to form an sodium dodecyl sulfate (SDS)-stable complex. Using antibodies to urokinase, elastase, proteinase 3, or cathepsin G, we demonstrated that the complex contains cathepsin G. Native cathepsin G and recombinant PI-6 formed an SDS-stable complex in vitro similar in size to that observed in the extracts. Further kinetic analysis demonstrated that cathepsin G and PI-6 rapidly form a tight 1:1 complex (ka = 6.8 ± 0.2 × 106mol/L−1s−1 at 17°C;Ki = 9.2 ± 0.04 × 10−10 mol/L). We propose that PI-6 complements 1-proteinase inhibitor and 1-antichymotrypsin (which control extracellular proteolysis) by neutralizing cathepsin G that leaks into the cytoplasm of monocytes or granulocytes during biosynthesis or phagocytosis. Control of intracellular cathepsin G may be particularly important, because it has recently been shown to activate the proapoptotic proteinase, caspase-7.

Novel recombinant serpin, LEX-032, attenuates myocardial reperfusion injury in cats

1996 ◽  
Vol 270 (3) ◽  
pp. H881-H887 ◽  
Author(s):  
J. A. Delyani ◽  
T. Murohara ◽  
A. M. Lefer
Keyword(s):  
Protease Inhibitor ◽  
Reperfusion Injury ◽  
Serine Proteases ◽  
Marked Reduction ◽  
Ex Vivo ◽  
Treated Group ◽  
Cathepsin G ◽  
The Novel ◽  
Amino Acid Residues ◽  
Phosphate Buffered Saline

We studied the potential cardioprotective effects of the novel recombinant serine protease inhibitor (serpin), LEX-032, which inhibits the serine proteases elastase and cathepsin G. LEX-032 is a recombinant construct of human alpha 1-antichymotrypsin in which six amino acid residues were replaced around the active center with those of human alpha 1-protease inhibitor. Cats were subjected to 90 min of left anterior descending coronary artery (LAD) occlusion and 270 min of reperfusion (MI/R). Either LEX-032 or its vehicle (i.e., phosphate-buffered saline) was administered intravenously 10 min before reperfusion. Control cats were subjected to sham MI/R. Cats treated with LEX-032 demonstrated a marked reduction in cardiac necrosis after MI/R compared with cats receiving only vehicle (10 +/- 3 vs. 31 +/- 3%, P < 0.01). In addition, relaxation of LAD rings to the endothelium-dependent dilators (e.g., acetylcholine and A23187) was greater in the LEX-032-treated group than in cats receiving vehicle (72 +/- 5 vs. 52 +/- 7%, P < 0.05, and 74 +/- 8 vs. 50 +/- 8%, P < 0.05, respectively), indicating that endothelial function was preserved by LEX-032. Moreover, LEX-032 administration resulted in a marked reduction of polymorphonuclear leukocyte (PMN) adherence to ex vivo coronary vascular endothelium compared with vehicle (33 +/- 4 vs. 86 +/- 7 PMNs/mm2, P < 0.01). These data indicate that LEX-032 is a significant cardioprotective agent exerting its protective effect by inhibition of PMN-mediated cellular injury, and this agent represents a novel means of attenuating PMN-mediated reperfusion injury.

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