Sj. Compton et al., Glycosylation and the activation of proteinase-activated receptor 2 (PAR(2)) by human mast cell tryptase, BR J PHARM, 134(4), 2001, pp. 705-718
1 Human mast cell tryptase appears to display considerable variation in act
ivating proteinase-activated receptor 2 (PAR(2)). We found tryptase to be a
n inefficient activator of wild-type rat-PAR(2) (wt-rPAR(2)) and therefore
decided to explore the factors that may influence tryptase activation of PA
R(2).
2 Using a 20 mer peptide (P20) corresponding to the cleavage/activation seq
uence of wt-rPAR2, tryptase was as efficient as trypsin in releasing the re
ceptor-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) exp
ressed on the cells was protected from tryptase activation.
3 Three approaches were employed to test the hypothesis that PAR, receptor
glycosylation restricts tryptase activation. (a) pretreatment of wt-rPAR(2)
expressing cells or human embryonic kidney cells (HEK293) with vibrio chol
erae neuraminidase to remove oligosaccharide sialic acid, unmasked tryptase
-mediated PAR(2) activation. (b) Inhibiting receptor glycosylation in HEK29
3 cells with tunicamycin enabled tryptase-mediated PAR(2) activation. (c) W
t-rPAR(2) devoid of the N-terminal glycosylation sequon (PAR(2)T25(-)), but
not rPAR(2) devoid of the glycosylation sequon located on extracellular lo
op-2 (PAR(2)T224A), was selectively and substantially (> 30 fold) more sens
itive to tryptase compared with the wt-rPAR(2).
4 Immunocytochemistry using antisera that specifically recognized the N-ter
minal precleavage sequence of PAR(2) demonstrated that tryptase released th
e precleavage domain from PAR(2)T25(-) but not from wt-rPAR(2).
5 Heparin : tryptase molar ratios of greater than 2: 1 abrogated tryptase a
ctivation 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 regulat
ing receptor activation by tryptase and possibly other proteases.