Proteoform Analysis of Matrix Metalloproteinase-9/Gelatinase B and Discovery of Its Citrullination in Rheumatoid Arthritis Synovial Fluids

Abstract

ObjectivesTo 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.MethodsLatent, 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.ResultsAll 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.ConclusionSynovial 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.

Citrullination of matrisomal proteins in health and diseases

“…Citrullination can modulate protease activity in cis (the left model of figure 3 b ). Several MMPs are substrates of PADs [29,30]. For example, MMP1, MMP13 and MMP9 can be citrullinated by PAD2 or PAD4 in vitro and citrullinated MMP9 has been detected in synovial fluid from RA.…”

How to place the duality of specific MMP-9 inhibition for treatment of inflammatory bowel diseases into clinical opportunities?

“…Another so far neglected post-translational modification in IBD is citrullination ( 70 ). Recently, citrullination of MMPs was discovered ( 71 ) and demonstrated to be relevant in acute and chronic inflammations ( 71 , 72 ). Whether posttranslational modifications of MMPs, including trimerization of MMP-9 and citrullination of various MMPs, have impacts on the processes of acute or chronic inflammation or fibrosis in IBD is not known, yet deserves attention.…”

Novel Matrix Metalloproteinase-9 (MMP-9) Inhibitors in Cancer Treatment

“…MMP-9 is engaged in several pathophysiological processes, such as extracellular matrix (ECM) degradation, tissue remodeling, and normal tissue turnover. The contribution of MMP-9 in the progression of several diseases was reported in extracranial arteriovenous malformations (AVMs) [10], rheumatoid arthritis [11,12], several neurological diseases and inflammatory processes [13,14], cancer [15][16][17], and ischemic stroke [18,19]. The suppression of MMP-9 activity is achieved by the binding of matrix metalloproteinase inhibitors (MMPIs) to the zinc (Zn 2+ ) ion at the catalytic site [20].…”