AUTHOR=Grillet Bernard , Yu Karen , Ugarte-Berzal Estefania , Janssens Rik , Pereira Rafaela Vaz Sousa , Boon Lise , Martens Erik , Berghmans Nele , Ronsse Isabelle , Van Aelst Ilse , Fiten Pierre , Conings René , Vandooren Jennifer , Verschueren Patrick , Van Damme Jo , Proost Paul , Opdenakker Ghislain TITLE=Proteoform Analysis of Matrix Metalloproteinase-9/Gelatinase B and Discovery of Its Citrullination in Rheumatoid Arthritis Synovial Fluids JOURNAL=Frontiers in Immunology VOLUME=12 YEAR=2021 URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2021.763832 DOI=10.3389/fimmu.2021.763832 ISSN=1664-3224 ABSTRACT=Objectives

To explore posttranslational modifications (PTMs), including proteolytic activation, multimerization, complex formation and citrullination of gelatinases, in particular of gelatinase B/MMP-9, and to detect in gelatin-Sepharose affinity-purified synovial fluids, the presence of specific MMP proteoforms in relation to arthritis.

Methods

Latent, activated, complexed and truncated gelatinase-A/MMP-2 and gelatinase B/MMP-9 proteoforms were detected with the use of zymography analysis to compare specific levels, with substrate conversion assays, to test net proteolytic activities and by Western blot analysis to decipher truncation variants. Citrullination was detected with enhanced sensitivity, by the use of a new monoclonal antibody against modified citrullines.

Results

All MMP-9 and MMP-2 proteoforms were identified in archival synovial fluids with the use of zymography analysis and the levels of MMP-9 versus MMP-2 were studied in various arthritic diseases, including rheumatoid arthritis (RA). Secondly, we resolved misinterpretations of MMP-9 levels versus proteolytic activities. Thirdly, a citrullinated, truncated proteoform of MMP-9 was discovered in archival RA synovial fluid samples and its presence was corroborated as citrullinated hemopexin-less MMP-9 in a small prospective RA sample cohort.

Conclusion

Synovial fluids from rheumatoid arthritis contain high levels of MMP-9, including its truncated and citrullinated proteoform. The combination of MMP-9 as analyte and its PTM by citrullination could be of clinical interest, especially in the field of arthritic diseases.