Steven J. Compton

A Polymorphic Protease-activated Receptor 2 (PAR2) Displaying Reduced Sensitivity to Trypsin and Differential Responses to PAR Agonists

Protease-activated receptor 2 (PAR2) is a trypsin-activated member of a family of G-protein-coupled PARs. We have identified a polymorphic form of human PAR2 (PAR2F240S) characterized by a phenylalanine to serine mutation at residue 240 within extracellular loop 2, with allelic frequencies of 0.916 (Phe240) and 0.084 (Ser240) for the wild-type and mutant alleles, respectively. Elevations in intracellular calcium were measured in permanently transfected cell lines expressing the receptors. PAR2F240S displayed a significant reduction in sensitivity toward trypsin (∼3.7-fold) and the PAR2-activating peptides, SLIGKV-NH2 (∼2.5-fold) and SLIGRL-NH2(∼2.8-fold), but an increased sensitivity toward the selective PAR2 agonist,trans-cinnamoyl-LIGRLO-NH2(∼4-fold). Increased sensitivity was also observed toward the selective PAR-1 agonist, TFLLR-NH2 (∼7-fold), but not to other PAR-1 agonists tested. Furthermore, we found that TLIGRL-NH2 and a PAR4-derived peptide,trans-cinnamoyl-YPGKF-NH2, were selective PAR2F240S agonists. By introducing the F240S mutation into rat PAR2, we observed shifts in agonist potencies that mirrored the human PAR2F240S, suggesting that Phe240 is involved in determining agonist specificity of PAR2. Finally, differences in receptor signaling were paralleled in a cell growth assay. We suggest that the distinct pharmacological profile induced by this polymorphism will have important implications for the design of PAR-targeted agonists/antagonists and may contribute to, or be predictive of, an inflammatory disease.

Human mast cell tryptase stimulates the release of an IL-8-dependent neutrophil chemotactic activity from human umbilical vein endothelial cells (HUVEC)

Tryptase, the major product of human mast cell activation, is a potent stimulus of vascular leakage and neutrophil accumulation in vivo in animal studies, but the mechanisms of action remain unclear. Using HUVEC cultures we have sought to investigate the potential of tryptase to alter monolayer permeability or induce the release of neutrophil chemotactic activity. Tryptase (1–100 mU/ml) failed to alter the permeability of endothelial cell monolayers as assessed by albumin flux over 1 h. However, supernatants from endothelial cells treated with tryptase (1–50 mU/ml) for a 24‐h period induced neutrophil migration across Transwell filters, with maximal migration observed at 10 mU/ml tryptase. Pretreatment of tryptase with the protease inhibitor leupeptin abolished the chemotactic activity, indicating a dependence on the catalytic site. Moreover, this effect was abolished by addition of an IL‐8 neutralizing antibody, suggesting that IL‐8 release makes an important contribution to the chemotactic activity. The interaction of mast cell tryptase with endothelial cells could be important in stimulating the ingress of neutrophils following mast cell activation in inflammatory disease.

Interaction of Human Mast Cell Tryptase with Endothelial Cells to Stimulate Inflammatory Cell Recruitment

125I-albumin flux. The structurally related serine protease, thrombin, increased permeability, though tryptase either in the presence or absence of heparin and with incubation periods up to 1 h was without effect. These observations are consistent with the idea that tryptase-induced microvascular leakage is not mediated by a direct effect of this protease on vascular endothelial cells. The finding that the size of the reactions induced in guinea pig skin may be reduced by pretreating the animals with antihistamines [2] has prompted the suggestion that the increase in microvascular permeability may be a consequence of mast cell degranulation, and tryptase has been demonstrated to stimulate histamine release from both guinea pig and human mast cells [2, 6]. Tryptase was found by RT-PCR to increase expression of mRNA for IL-8 and for IL-1 â though that for GM-CSF, IL-6, IL-15 and RANTES was unaffected. There was a dose-dependent release into the supernatants of IL-8, which was maximal at 24 h with a tryptase concentration of 50 mU/ml. The effect was dependent on an intact catalytic site, being reduced significantly by preincubation of tryptase with the protease inhibitors leupeptin or benzamidine, heat inactivating the enzyme, or depleting samples of tryptase by immunoabsorption with a tryptase-specific antibody AA5. No detectable increases in IL-1â protein (precursor or mature) were detected in the supernatants or lysates of tryptase-treated HUVEC. Incubation of HUVEC with tryptase stimulated the release of neutrophil chemotactic activity in studies of 51Crlabelled neutrophil transmigration through naked Transwell filters. Neutrophil transmigration following incubation of Mast Cell Biology

N-Linked Glycosylation Regulates Human Proteinase-activated Receptor-1 Cell Surface Expression and Disarming via Neutrophil Proteinases and Thermolysin

Proteinase-activated receptor 1 (PAR1) induces activation of platelet and vascular cells after proteolytic cleavage of its extracellular N terminus by thrombin. In pathological situations, other proteinases may be generated in the circulation and might modify the responses of PAR1 by cleaving extracellular domains. In this study, epitope-tagged wild-type human PAR1 (hPAR1) and a panel of N-linked glycosylation-deficient mutant receptors were permanently expressed in epithelial cells (Kirsten murine sarcoma virus-transformed rat kidney cells and CHO cells). We have analyzed the role of N-linked glycosylation in regulating proteinase activation/disarming and cell global expression of hPAR1. We reported for the first time that glycosylation in the N terminus of hPAR1 downstream of the tethered ligand (especially Asn75) governs receptor disarming to trypsin, thermolysin, and the neutrophil proteinases elastase and proteinase 3 but not cathepsin G. In addition, hPAR1 is heavily N-linked glycosylated and sialylated in epithelial cell lines, and glycosylation occurs at all five consensus sites, namely, Asn35, Asn62, Asn75, Asn250, and Asn259. Removing these N-linked glycosylation sequons affected hPAR1 cell surface expression to varying degrees, and N-linked glycosylation at extracellular loop 2 (especially Asn250) of hPAR1 is essential for optimal receptor cell surface expression and receptor stability.

Mast Cell Proteases as New Targets for Therapeutic Intervention in Asthma

Mast cells have long attracted attention for their potential to contribute to the disease process in asthma [1]. These cells are widely distributed throughout the body, but are particularly prevalent in tissues which form an interface with the external environment. In the lower airways mast cells are numerous in the bronchial mucosa [2], submucosa and alveolar walls [3], and are even found free in the lumen [4]. The activation of mast cells by allergen or by other stimuli is associated with the rapid release of a range of potent mediators of inflammation and bronchoconstriction.