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Cited by 127Mast cells and histamineProtease and Inhibitor MechanismsCoagulation, Bradykinin, Polyphosphates, and Angioedema

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

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Le, Quang T., Lyons, Jonathan J., Naranjo, Andrea N., Olivera, Ana, Lazarus, Robert A., Metcalfe, Dean D., Milner, Joshua D., Schwartz, Lawrence B.·2019·Journal of Experimental Medicine
DOI 10.1084/jem.20190701PMC PMC6780998
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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.

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Figures

Figure 1. Formation of α/β-tryptase heterotetramers in vitro. (A) Heparin-Sepharose chromatography of homotypic and heterotypic tetramers of natural HMC1-derived β1/3-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 1. Formation of α/β-tryptase heterotetramers in vitro. (A) Heparin-Sepharose chromatography of homotypic and heterotypic tetramers of natural HMC1-derived β1/3-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.CC BY-NC-SA · PubMed Central
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 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.CC BY-NC-SA · PubMed Central
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 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.CC BY-NC-SA · PubMed Central
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 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.CC BY-NC-SA · PubMed Central
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.
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.CC BY-NC-SA · PubMed Central

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Metadata

Authors
Le, Q. T., Lyons, J. J., Naranjo, A. N., Olivera, A., Lazarus, R. A., Metcalfe, D. D., Milner, J. D., & Schwartz, L. B.
Affiliations
  • Virginia Commonwealth University (US)
  • National Institutes of Health (US)
  • Genentech (US)
Corresponding author
Schwartz, Lawrence B.
ORCID
Venue
Journal of Experimental Medicine
Publisher
Rockefeller University Press
Published
2019-07-23
Volume
216
Issue
10
Pages
2348-2361
ISSN
  • 0022-1007 (ISSN-L)
  • 1540-9538
DOI
10.1084/jem.20190701
PMC
PMC6780998
OpenAlex
W2964163570
Cited by
126
References
54
Citations by year
2026: 12 · 2025: 17 · 2024: 13 · 2023: 18 · 2022: 17 · 2021: 24 · 2020: 16 · 2019: 8
FWCI
6.48
Type
journal article
Language
en
Open access
Licence
Locations
  • The Journal of Experimental Medicine (publishedVersion, cc-by-nc-sa)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Funders
  • Charles Thomas Research Fund
  • Medical College of Virginia Foundation
  • National Institutes of Health (U18AI77435, R0127517)
  • Division of Intramural Research of the National Institute of Allergy and Infectious Diseases
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Mast cells and histamine
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Protease and Inhibitor Mechanisms
  • Health Sciences › Medicine › Hematology › Coagulation, Bradykinin, Polyphosphates, and Angioedema
Keywords
hereditary alpha-tryptasemia, beta-tryptase, heterotetramer, mast cells, protease-activated receptor 2, mast cell degranulation, autonomic dysfunction, allosteric effects, protease specificity, mast cell disease
MeSH terms
Adult, Allosteric Regulation, Female, Humans, Male, Mast Cells, Vibration, Cell Degranulation, Genetic Diseases, Inborn, Receptors, G-Protein-Coupled, Receptor, PAR-2, Tryptases, Protein Multimerization

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