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Glycosylation and the activation of proteinase-activated receptor 2 (PAR(2)) by human mast cell tryptase.

Abstract：

:1. Human mast cell tryptase appears to display considerable variation in activating proteinase-activated receptor 2 (PAR(2)). We found tryptase to be an inefficient activator of wild-type rat-PAR(2) (wt-rPAR(2)) and therefore decided to explore the factors that may influence tryptase activation of PAR(2). 2. Using a 20 mer peptide (P20) corresponding to the cleavage/activation sequence of wt-rPAR(2), tryptase was as efficient as trypsin in releasing the receptor-activating sequence (SLIGRL.). However, in the presence of either human-PAR(2) or wt-r PAR(2) expressing cells, tryptase could only activate PAR(2) by releasing SLIGRL from the P20 peptide, suggesting that PAR(2) expressed on the cells was protected from tryptase activation. 3. Three approaches were employed to test the hypothesis that PAR(2) receptor glycosylation restricts tryptase activation. (a) pretreatment of wt-rPAR(2) expressing cells or human embryonic kidney cells (HEK293) with vibrio cholerae neuraminidase to remove oligosaccharide sialic acid, unmasked tryptase-mediated PAR(2) activation. (b) Inhibiting receptor glycosylation in HEK293 cells with tunicamycin enabled tryptase-mediated PAR(2) activation. (c) Wt-rPAR(2) devoid of the N-terminal glycosylation sequon (PAR(2)T25(-)), but not rPAR(2) devoid of the glycosylation sequon located on extracellular loop-2 (PAR(2)T224A), was selectively and substantially (>30 fold) more sensitive to tryptase compared with the wt-rPAR(2). 4. Immunocytochemistry using antisera that specifically recognized the N-terminal precleavage sequence of PAR(2) demonstrated that tryptase released the precleavage domain from PAR(2)T25(-) but not from wt-rPAR(2). 5. Heparin : tryptase molar ratios of greater than 2 : 1 abrogated tryptase activation of PAR(2)T25(-). 6. Our results indicate that glycosylation of PAR(2) and heparin-inhibition of PAR(2) activation by tryptase could provide novel mechanisms for regulating receptor activation by tryptase and possibly other proteases.

journal_name

Br J Pharmacol

journal_title

British journal of pharmacology

authors

Compton SJ,Renaux B,Wijesuriya SJ,Hollenberg MD

doi

10.1038/sj.bjp.0704303

subject

Has Abstract

pub_date

2001-10-01 00:00:00

pages

705-18

issue

eissn

0007-1188

issn

1476-5381

journal_volume

134

pub_type

杂志文章

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Glycosylation and the activation of proteinase-activated receptor 2 (PAR(2)) by human mast cell tryptase.