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Proteome analysis of mast cell releasates reveals a role for chymase in the regulation of coagulation factor XIIIA levels via proteolytic degradation 

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Presentation on theme: "Proteome analysis of mast cell releasates reveals a role for chymase in the regulation of coagulation factor XIIIA levels via proteolytic degradation "— Presentation transcript:

1 Proteome analysis of mast cell releasates reveals a role for chymase in the regulation of coagulation factor XIIIA levels via proteolytic degradation  Nicholas J. Shubin, PhD, Veronika A. Glukhova, PhD, Morgan Clauson, BSc, Phuong Truong, BSc, Magnus Abrink, PhD, Gunnar Pejler, PhD, Nathan J. White, MD, MS, Gail H. Deutsch, MD, Stephen R. Reeves, MD, PhD, Tomas Vaisar, PhD, Richard G. James, PhD, Adrian M. Piliponsky, PhD  Journal of Allergy and Clinical Immunology  Volume 139, Issue 1, Pages (January 2017) DOI: /j.jaci Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Differentially produced extracellular proteins from bone marrow compared with peritoneal-derived mast cells. A, A schematic overview of the approach used to define the releasates generated by BMCMCs and PCMCs following IgE-mediated activation. B, A Venn diagram showing the number of shared and exclusive proteins among the BMCMC and PCMC releasates. DNP, 2, 4-Dinitrophenyl. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Mast cells produce and release FXIIIA. BMCMCs were sensitized with IgE mAb to DNP (2 μg/mL) overnight and then were challenged with DNP-HSA (100 ng/mL) for 6 hours (A) or for the indicated time points (B). Fig 2, A, FXIIIA (83 kDa) was observed by Western blot analysis in BMCMC protein lysate and their extracellular supernatant. The data are representative of 2 independent experiments. Fig 2, B, FXIIIA expression was analyzed by quantitative PCR. The FXIIIA versus GAPDH mRNA expression level for the BMCMCs that were incubated in media alone was assigned a value of 1.0 for calculation of the corresponding IgE/antigen-stimulated BMCMC results. The data were pooled from 3 independent experiments. C, Immunofluorescence of fixed and permeabilized BMCMCs labeled first with anti-FXIIIA antibody and then with Alexa Fluor 594-conjugated antimouse donkey polyclonal antibody (red). The cells were mounted with antifade reagent containing DAPI for nuclear staining (blue). The images are representative of 2 experiments. DAPI, 4′-6-Diamidino-2-phenylindole, dihydrochloride; DNP-HAS, 2, 4-dinitrophenyl-human serum albumin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Cpa3-Cre; ROSA DTA/DTA mast cell– and basophil-deficient mice exhibit increased plasma FXIII amounts and activity, as well as reduced bleeding time. Western blot (A) and densitometry analyses (B) for plasma FXIIIA levels in Cpa3-Cre; ROSA+/+ (n = 4) and Cpa3-Cre; ROSADTA/DTA mice (n = 7). FXIIIa activity levels (C) and tail tip bleeding times (D) in Cpa3-Cre; ROSA+/+ (n = 11-13) and Cpa3-Cre; ROSADTA/DTA mice (n = 7-20). Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Mouse chymase (mMCP-4) downregulates FXIIIA plasma levels and activity via proteolytic degradation and contributes to prolonged bleeding times under homeostatic conditions. A, Wild-type (Mctp4+/+) (n = 5) and mMCP-4–deficient (Mcpt4−/−) (n = 3) mouse PCMCs and Mcpt4+/+ BMCMCs (n = 6) were sensitized with IgE mAb to DNP (2 μg/mL) overnight and then were challenged with DNP-HSA (100 ng/mL) for 6 hours. The cell supernatants were collected, concentrated, and subjected to shotgun proteomics. The FXIIIA abundance is expressed in spectral counts. B, PCMC lysates were incubated with human FXIIIA for the indicated time points at 37°C. The FXIIIA levels and their fragments were assessed with Western blot analysis. The data are representative of those obtained in 2 independent experiments. Western blot analysis (C) and densitometry values (D) for plasma FXIIIA levels in Mcpt4+/+ (n = 5) and Mcpt4−/− (n = 5) mice. E and F, FXIIIa activity levels (Fig 4, E) and tail tip bleeding times (Fig 4, F) in Mcpt4+/+ (n = 8-9) and Mcpt4−/− (n = 5-8) mice. DNP-HSA, 2, 4-Dinitrophenyl-human serum albumin. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Human chymase degrades FXIIIA and diminishes its activity. A, Human FXIIIA was incubated with human skin chymase for 6 hours at 37oC. FXIIIA fragmentation was assessed by silver stain analysis. The data are representative of those obtained from 3 independent experiments. B, A human FXIIIA domain map, including the conserved transglutaminase regions and the location of the detected chymase cleavage sites. C, FXIIIA was incubated with increasing concentrations of human skin chymase for 6 hours at 37oC and then subjected to LC-MS/MS. Six chymase cleavage sites in FXIIIA produced a marked dose-response to the increasing chymase levels, as exemplified for cleavage site F574. D, An approximation of the consensus peptide sequence alignment patterns containing the detected chymase cleavage sites. Chymase cleavage occurs between P1 and P1′. The peptide sequences shown were sorted in order of their negatively charged and aliphatic residues. E and F, ROTEM results are reported as maximal clot firmness (MCF) (Fig 5, E), which represents fibrin polymerization, FXIIIa activity, and the lysis index 60 (LI60) (Fig 5, F), which represents the percentage of the remaining clot in relation to the MCF value at 60 minutes after clot onset time. The data were pooled from 3 independent experiments. FBG, Fibrinogen; ROTEM, rotational thromboelastometry. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 mMCP-4 diminishes FXIIIA amounts and FXIII activity levels in mice during experimental sepsis. Western blot analysis (A) and densitometry values (B) for FXIIIA plasma levels, FXIIIa plasma activity levels (C), and bleeding times at 24 hours after CLP (D) in Mcpt4+/+ (n = 5-9) and Mcpt4−/− mice (n = 3-8). Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E1 Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E2 Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E3 Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E4 Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E5 Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 Fig E6 Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

14 Fig E7 Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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