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.

Figure 1

Inability of tryptase to activate either wt-rPAR₂ (upper) or wt-hPAR₂ (lower) in contrast to its ability to release the PAR₂-AP, SLIGRL... from the synthetic cleavage/activation peptide, P20. Upper, (i – iv) Wt-rPAR₂: activation by tryptase hydrolyzed P20, and by trypsin, but not by tryptase alone. Arrows indicate when test agents were added to the cells. Controls are shown in the right panel of each individual figure, except for (iii). Cells were lifted with non-enzymatic cell dissociation fluid and loaded with Fluo-3 (22 μM) prior to incubation for 25 min at room temperature. Cells were challenged with different agents and responses were monitored by fluorescence spectrophotometry (excitation 480 nm, emission 530 nm). Results are representative of three separate experiments using separately grown crops of cells. Lower, (i – ii) Wt-hPAR₂: activation by tryptase hydrolyzed P20 and by trypsin, but not by tryptase alone. Arrows indicate when test agents were added to the cells. Cell monolayers were rinsed and loaded with fura-2AM at room temperature for 40 min. Fluorescence was measured in individual cells using an ICCD video camera and video microscopy acquisition program. The fluorescence ratio (340/380 nm) was measured. Twenty cells were analysed for each coverslip of cells treated. TPZ, tryptase; TPN, trypsin; SL-NH₂, SLIGRL-NH₂.

Figure 2

The sialidase, neuraminidase, unmasks PAR₂ to activation by tryptase. (i & ii), enhancement of tryptase, (but not trypsin) activation of PAR₂ on wt-rPAR₂ and HEK cells by treatment with neuraminidase. Wt-rPAR₂ or HEK cells were lifted with non-enzymatic cell dissociation fluid and incubated either with or without neuraminidase (10 mU ml⁻¹) for 30 min at room temperature. Cells were washed then loaded with Fluo-3 (22 μM) prior to incubation for 25 min at room temperature. Cells were challenged with test agonists and responses were monitored by fluorescence spectrophotometry (excitation 480 nm, emission 530 nm). Responses were normalized to the peak height obtained with 2 μM calcium ionophore. Each treatment represents the mean±s.e.mean (bars) from 3 – 6 independent experiments from separately grown crops of cells. NS, no signal detected. (iii & iv) Cross-desensitization of tryptase induced calcium signalling in neuraminidase treated HEK cells. Arrows indicate when test agents were added to the cells. Control traces are shown in the right panel of each individual figure. Results are representative of two separate experiments using separately grown crops of cells. TPZ, tryptase; TPN, trypsin; SL-NH₂, SLIGRL-NH₂.

Figure 3

Inhibition of protein glycosylation in HEK cells by tunicamycin results in the expression of tryptase responsive PAR₂. (i) tryptase activation of PAR₂ following pretreatment of HEK cells with tunicamycin for 48 h. Semi-confluent HEK cells in 75 cm² culture flasks were propagated with or without tunicamycin (1 μg ml⁻¹) for 48 h. Cells were lifted with non-enzymatic cell dissociation fluid and washed, then loaded with Fluo-3 (22 μM) prior to incubation for 25 min at room temperature. Cells were challenged with test agonists and responses were monitored by fluorescence spectrophotometry (excitation 480 nm, emission 530 nm). Responses were normalized to the peak height obtained with 2 μM calcium ionophore. Each treatment represents the mean±s.e.mean (bars) from three independent experiments from separately grown crop of cells. NS, no signal detected. (ii & iii) Cross-desensitization of tryptase induced calcium signalling in tunicamycin treated HEK cells. Arrows indicate when test agents were added to the cells. Control traces are shown in the right panel of each individual figure. Results are representative of two separate experiments using separately grown crops of cells. TPZ, tryptase; TPN, trypsin; SL-NH₂, SLIGRL-NH₂; Tunica, tunicamycin.

Figure 4

Tryptase activation of rPAR₂ is dramatically enhanced by deletion of the N-linked glycosylation motif on the receptor N-terminus. Upper, (i and ii) Calcium signalling in the wt-rPAR₂ and PAR₂T25⁻ cells in response to tryptase and trypsin. Concentration effect curves are shown for tryptase (i) and trypsin (ii) in wt-rPAR₂ and PAR₂T25⁻. Cells were lifted with non-enzymatic cell dissociation fluid and loaded with Fluo-3 (22 μM) prior to incubation for 25 min at room temperature. Cells were challenged with different agents and responses were monitored by fluorescence spectrophotometry (excitation 480 nm, emission 530 nm). Each data point for PAR₂T25⁻ and wt-rPAR₂ represents the mean±s.e.mean (bars) of three separate experiments respectively from separately grown crops of cells, except for tryptase (300 nM) on wt-rPAR₂, n=11. Lower, (i – iv) Cross-desensitization of tryptase induced calcium signalling in PAR₂T25⁻. Arrows indicate when test agents were added to the cells. Control traces are shown in the right panel of each individual figure. Results are representative of two separate experiments using separately grown crops of cells. TPZ, tryptase; TPN, trypsin; SL-NH₂, SLIGRL-NH₂.

Figure 5

Tryptase removes the precleavage/activation domain on PAR₂T25⁻ but not on wt-rPAR₂ cells. Immunostaining with the SLAW antiserum on wt-rPAR₂ and PAR₂T25⁻ cells following: (A,B) no treatment, (C,D) tryptase 300 nM, and (E,F) trypsin 10 nM at room temperature for 10 min. (G,H): Histograms showing quantitative morphometric counting analysis of stained wt-rPAR₂ and PAR₂T25⁻ cells (e.g. arrows, A,B) demonstrating the degree of immunostaining for SLAW (G) and B5 (H) antiserum following various treatments. Arrows in A, B and C, show cell surface staining, whilst D, E and F show a loss of cell surface SLAW immunoreactivity. The insets in A and B show the elimination of immunostaining by peptides preabsorbed with the SLAW antiserum. Calcium responses of cells to test agonists were confirmed in the calcium assay (as described in the Methods) before cytospins were prepared. Immunostaining with the SLAW antiserum was conducted as described in the Methods. Results are expressed as the mean counts±s.e.mean from 10 different fields (>100 cells/field) for each treatment. Bar=25 μm.

Figure 6

Inhibition of tryptase induced calcium signalling in PAR₂T25⁻ by PIM heparin and Fraxiparine®. Concentration-effect curves are shown for PIM heparin (MW, 16.5 kDa) and Fraxiparine® (MW, 4.3 kDa). Cells were lifted with non-enzymatic cell dissociation fluid and loaded with Fluo-3 (22 μM) prior to incubation for 25 min at room temperature. Cells were challenged with tryptase (50 nM) containing increasing concentrations of heparin and increases in intracellular calcium were monitored by fluorescence spectrophotometry (excitation 480 nm, emission 530 nm). Responses were normalized to the peak height obtained with 2 μM calcium ionophore. For PIM heparin each data point represents the mean±s.e.mean (bars) of four separate experiments, and for Fraxiparine® each data point represents the mean±s.e.mean. TPZ, tryptase; PIM heparin, porcine intestinal mucosa heparin.