Granzyme B cleaves tenascin-C to release its C-terminal domain in rheumatoid arthritis
Alexandre Aubert, Amy Liu, Martin Kao, Jenna Goeres, Katlyn C. Richardson, Lorenz Nierves, Karen Jung, Layla Nabai, Hongyan Zhao, Gertraud Orend, Roman Krawetz, Philipp F. Lange, Alastair Younger, Jonathan Chan, David J. Granville
Alexandre Aubert, Amy Liu, Martin Kao, Jenna Goeres, Katlyn C. Richardson, Lorenz Nierves, Karen Jung, Layla Nabai, Hongyan Zhao, Gertraud Orend, Roman Krawetz, Philipp F. Lange, Alastair Younger, Jonathan Chan, David J. Granville
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Research Article Inflammation

Granzyme B cleaves tenascin-C to release its C-terminal domain in rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is a common autoimmune disorder characterized by exacerbated joint inflammation. Despite the well-documented accumulation of the serine protease granzyme B (GzmB) in RA patient biospecimens, little is understood pertaining to its role in pathobiology. In the present study, tenascin-C (TNC) — a large, pro-inflammatory extracellular matrix glycoprotein — was identified as a substrate for GzmB in RA. GzmB cleaves TNC to generate 3 fragments in vitro: a 130 kDa fragment that remains anchored to the matrix and 2 solubilized fragments of 70 and 30 kDa. Mass spectrometry results suggested that the 30 kDa fragment contained the pro-inflammatory TNC C-terminal fibrinogen-like domain. In the synovial fluids of patients with RA, soluble levels of GzmB and TNC were significantly elevated compared with healthy controls. Further, immunoblotting revealed soluble 70 and 30 kDa TNC fragments in the synovial fluids of patients with RA, matching TNC fragment sizes generated by GzmB cleavage in vitro. Granzyme K (GzmK), another serine protease of the granzyme family, also cleaves TNC in vitro; however, the molecular weights of GzmK-generated TNC fragments did not correspond to TNC fragment sizes detected in patients. Our data support that GzmB, but not GzmK, contributes to RA through the cleavage of TNC.

Authors

Alexandre Aubert, Amy Liu, Martin Kao, Jenna Goeres, Katlyn C. Richardson, Lorenz Nierves, Karen Jung, Layla Nabai, Hongyan Zhao, Gertraud Orend, Roman Krawetz, Philipp F. Lange, Alastair Younger, Jonathan Chan, David J. Granville

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Figure 3

GzmB cleaves TNC substratum to impair fibroblast spreading and viability.

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GzmB cleaves TNC substratum to impair fibroblast spreading and viability...
(A) Schematic representation of the experimental procedure used to investigate how GzmB digestion of a TNC substratum impacts fibroblast behaviors. (B) Western blot analysis of the presence of soluble TNC fragments after 24-hour digestion of the immobilized TNC substratum as indicated in A. (C) Phase contrast microscopy performed on primary human dermal fibroblasts cultured for 72 hours onto noncoated (NC) wells, wells coated with nondigested (ND) rhTNC, or wells coated with rhTNC and digested with 50 nM rhGzmB as indicated in A. Bars, 400 μm. (D) Percentage of cell viability obtained by MTS assay performed on primary human dermal fibroblasts cultured for 72 hours onto NC wells, wells coated with ND rhTNC, or wells coated with rhTNC and digested with 50 nM rhGzmB as indicated in A. Results were normalized compared to the NC condition and are represented as means ± SD from 6 independent experiments. Statistical analyses were performed using nonparametric ANOVA (Kruskal-Wallis rank-sum test) followed by Dunn’s multiple comparisons test with Bonferroni’s P value adjustment. ***=P < 0.001. Images shown are representative of at least 3 independently repeated experiments.