Impact of naturally forming human α/β-tryptase heterotetramers in the pathogenesis of hereditary α-tryptasemia

Abstract

Both α-tryptase and β-tryptase are preferentially expressed by human mast cells, but the purpose of α-tryptase is enigmatic, because its tetramers lack protease activity, whereas β-tryptase tetramers are active proteases. The monogenic disorder called hereditary α-tryptasemia, due to increased α-tryptase gene copies and protein expression, presents with clinical features such as vibratory urticaria and dysautonomia. We show that heterotetramers composed of 2α- and 2β-tryptase protomers (α/β-tryptase) form naturally in individuals who express α-tryptase. α/β-Tryptase, but not homotetramer, activates protease-activated receptor-2 (PAR2), which is expressed on cell types such as smooth muscle, neurons, and endothelium. Also, only α/β-tryptase makes mast cells susceptible to vibration-triggered degranulation by cleaving the α subunit of the EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2) mechanosensory receptor. Allosteric effects of α-tryptase protomers on neighboring β-tryptase protomers likely result in the novel substrate repertoire of α/β-tryptase tetramers that in turn cause some of the clinical features of hereditary α-tryptasemia and of other disorders involving mast cells.

Figure 1.

Formation of α/β-tryptase heterotetramers in vitro. (A) Heparin-Sepharose chromatography of homotypic and heterotypic tetramers of natural HMC1-derived β_1/3-tryptases and rH-α1-tryptase. Protryptases β1/3 (top), α (bottom), and β1/3 + α (middle) were activated with human CTSB and separated by heparin-Sepharose chromatography with a linear NaCl gradient into three overlapping protein peaks, labeled H1, H2, and H3. Only H1 and H2 were catalytically active. (B) Peak heparin-Sepharose fractions were reduced, denatured, and analyzed by Western blotting with the G3 anti-tryptase mAb. (C) Peak heparin-Sepharose fractions, nonreduced and nondenatured, were analyzed by gelatin zymography along with labeled mol wt standards (STDS) and IRDye 800–labeled α1-tryptase homotetramers (α*). (D) Gelatin zymography after extended electrophoresis to better separate putative α/β-tryptase heterotetramers from β-tryptase homotetramers. Representative of two independent experiments.

Figure 2.

Separation and stability of tissue-derived β-tryptase homotetramers and α/β-tryptase heterotetramers. (A) B2-agarose–purified tryptase (P0) from tryptase-genotyped donors and subjected to phosphocellulose chromatography. Representative of tryptase prepared from three individuals with each genotype. (B) Western blotting (G3 anti-tryptase mAb) after SDS-gradient PAGE of P0 and P1 and P2 fractions from A, after being ±N-deglycosylated (PNGase F), reduced (DTT), and heat-denatured (SDS). Representative of experiments from two individuals with each genotype. (C) G5-Sepharose immunoaffinity chromatography of mature rH-α1-tryptase (left upper panel), rH-β2-tryptase (right upper panel), and lung tryptase (ββ:ββ genotype, P0, left lower panel; αβ:αβ genotype, P2, right lower panel). Representative of two individuals with each genotype. (D) SDS-PAGE (10–20%) of P2, E1, and E2 fractions (αβ:αβ genotype) obtained as above from one of the two subjects with an αβ:αβ genotype. (E) Stability of α/β-tryptase heterotetramer (P2, ββ:αααβ genotype), β-tryptase (P0, ββ:ββ genotype), and mixtures of α/β-tryptase and β-tryptase (P0, αβ:ββ or αβ:αβ genotypes) to inhibition by B12 anti-tryptase mAb. Mean ± SD. Data reflect tryptase from three different individuals for each of the three genotypes displayed.

Figure 3.

α/β-Tryptase but not β-tryptase makes human MCs susceptible to vibration-triggered degranulation. (A) Treatment of skin MCs adherent to dermatan sulfate, but not to chondroitin sulfate A, degranulate when vibrated if pretreated with α/β-tryptase Hi (3.5 µg/ml), but not with α/β-tryptase Lo (0.35 µg/ml) or with β-tryptase at either concentration. Pretreatment of α/β-tryptase Hi with B12 anti-tryptase IgG, but not with SBTI or B2 anti-tryptase IgG, prevents this degranulation. Triplicate data with cells from one subject are shown, representative of two independent experiments. GAG, glycosaminoglycan; DS, dermatan sulfate; CSA, chondroitin sulfate A. (B) Time for incubating α/β-tryptase (3.5 µg/ml) with skin MCs to make them susceptible to vibration-triggered degranulation during the final 20 min of incubation. Triplicate data with cells from one subject are shown, representative of two independent experiments. *, P < 0.05; ***, P < 0.001; ANOVA followed by a Holm–Sidak test compared with the buffer control. Bars show means, and error bars show the SD.

Figure 4.

α/β-Tryptase targets EMR2α, making MCs susceptible to vibration-triggered degranulation in vitro and urticaria in vivo. (A) EMR2 on skin and CD34-derived MC surfaces by flow cytometry. Histograms representative of two independent experiments. (B) Western blotting of nonreduced/nondenatured, SDS-solubilized EMR2 shows cleavage by P0 α/β-tryptase (αβ:αβ genotype) and negligible cleavage by β-tryptase (αβ:ββ or ββ:ββ genotypes). Purified EMR2 (upper panel, 10–20% acrylamide; Fig. S3) incubated with P0 tryptase (1 µg/ml) from tryptase-genotyped donors or adherent skin MCs (lower panel, 12% acrylamide) incubated with similar tryptase preparations (3.5 µg/ml) for 1 h, and then vibrated or vortexed for 20 min. C, cell extract; S, cell-free medium. Representative of two different subjects with each genotype. (C) EMR2 silencing in CD34⁺ cell-derived MCs with transfected EMR2 siRNA. These MCs (adherent to dermatan sulfate–coated wells, treated with tryptase × 1 h, and vibrated the last 20 min) were denatured, reduced, and assessed by Western blotting (upper panel, 12% acrylamide; representative of two separate experiments) and for degranulation (lower panel, one experiment performed in triplicate). B, buffer; −, negative-control siRNA-A; +, EMR2 siRNA; E, anti-FcεRI. ANOVA followed by a Holm–Sidak test compared with buffer control. Bars show means, and error bars show the SD. (D) Response scores to a cutaneous vibratory challenge (Fig. S4) were analyzed according to the α/β-tryptase gene ratio by Kruskal–Wallis ANOVA on ranks followed by the Dunn’s method, comparing each α-tryptase gene–containing group to the all β-tryptase gene–containing group. Each symbol represents a different subject tested one time. **, P < 0.01; ***, P < 0.001. Bars show median and 25th/75th percentiles, error bars show the 10th/90th percentiles, and the solid black circle shows the 95th percentile.

Figure 5.

PAR2 on Jurkat cells is activated by α/β-tryptase but not by β-tryptase. (A) Calcium response to rH-α1 protryptase (20 nM) or homotetramer (20 nM protomer), and rH-β2 protryptase (20 nM) or homotetramer (20 or 100 nM protomer), trypsin (10 nM), and SLIGKV (50 nM). Representative of two independent experiments. (B) Dose–response calcium signal by α/β-tryptase. Representative of two independent experiments. (C) Desensitization experiments show that the calcium response to α/β-tryptase (50 nM protomer) is due to PAR2 activation, using trypsin (20 nM) or SLIGKV (50 nM), and is prevented by B12 anti-tryptase (400 nM) or PAR2-specific inhibitor FSLLRY (50 nM), while TFLLR activates PAR1. Each uninterrupted line (A, B [left panel], and C) represents an independent experiment.