Glycosylation and the activation of proteinase‐activated receptor 2 (PAR₂) by human mast cell tryptase

Abstract

1 Human mast cell tryptase appears to display considerable variation in activating proteinaseactivated receptor 2 (PAR 2 ). We found tryptase to be an ine cient activator of wild-type rat-PAR 2 (wt-rPAR 2 ) and therefore decided to explore the factors that may in¯uence 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 e cient 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 Nterminal glycosylation sequon (PAR 2 T25 7 ), but not rPAR 2 devoid of the glycosylation sequon located on extracellular loop-2 (PAR 2 T224A), was selectively and substantially (430 fold) more sensitive to tryptase compared with the wt-rPAR 2 . 4 Immunocytochemistry using antisera that speci®cally recognized the N-terminal precleavage sequence of PAR 2 demonstrated that tryptase released the precleavage domain from PAR 2 T25 7 but not from wt-rPAR 2 . 5 Heparin : tryptase molar ratios of greater than 2 : 1 abrogated tryptase activation of PAR 2 T25 7 . 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.

A major role for proteolytic activity and proteinase-activated receptor-2 in the pathogenesis of infectious colitis

“…Cell Culture. Previously described Kirsten virus-transformed rat kidney cells (KNRK; American Type Culture Collection, Manassas, VA) permanently vector-transfected with rat PAR 2 (KNRK-PAR 2 ) were grown to 90% confluence with antibioticcontaining 10% FBS growth medium (Invitrogen), supplemented with 0.6 mg͞ml geneticin to maintain vector-selective pressure (13)(14)(15).…”

“…The procedure used was similar to that described in ref. 13. Trypsin (25 ng), granzyme A (450 ng), and C. rodentium-or sham-infected luminal fluid (3 l) was incubated with P20 peptide (200 M), which corresponds to the cleavage͞activation site of the wtrPAR 2 N terminus [ 30 GPNSKGR2SLIGRLDT 45 P-YGGC (2, trypsin cleavage site, -YGGC was added for affinity column coupling for another project)] in 50 mM Hepes͞150 mM NaCl, pH 7.5 (75 l).…”

Protease-Activated Receptor 2 Mediates Eosinophil Infiltration and Hyperreactivity in Allergic Inflammation of the Airway

“…The explanation of this discrepancy is unknown. However, alterations in the extent of receptor glycosylation dramatically affect the ability of tryptase to cleave and activate PAR2 (28). Thus, deglycosylation in vivo could regulate tryptase activation of PAR2.…”

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

“…In addition, the alanine mutations were created at amino acid positions Phe 55 -Trp 56 or Val 72 -Ser 73 of N62Q/ N75Q to generate cathepsin G mutant (F55A/W56A) or proteinase 3 mutant (V72A/S73A). KNRK and CHO cells permanently expressing wild-type or mutant hPAR 1 cell lines were generated and cultured as described previously (Table 1) (19,20).…”

“…Our previous pharmacological studies have reported that activation of hPAR 2 by mast cell tryptase can be regulated by receptor N-terminal glycosylation (19,20). To our knowledge, hPAR 1 contains a total of five predicted sites for N-linked glycosylation; three sequons are located on the receptor N terminus (Asn 35 -Ala 36 -Thr 37 , Asn 62 -Glu 63 -Ser 64 , and Asn 75 -Lys 76 -Ser 77 ), and two are located within extracellular loop 2 (ECL2; Asn 250 -Ile 251 -Thr 254 , and Asn 259 -Glu 260 -Thr 261 ) ( Fig.…”