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1.  Sulfated Glycosaminoglycans Control the Extracellular Trafficking and the Activity of the Metalloprotease Inhibitor TIMP-3 
Chemistry & Biology  2014;21(10):1300-1309.
Tissue inhibitor of metalloproteinase 3 (TIMP-3) is an important regulator of extracellular matrix (ECM) turnover. TIMP-3 binds to sulfated ECM glycosaminoglycans or is endocytosed by cells via low-density lipoprotein receptor-related protein 1 (LRP-1). Here, we report that heparan sulfate (HS) and chondroitin sulfate E (CSE) selectively regulate postsecretory trafficking of TIMP-3 by inhibiting its binding to LRP-1. HS and CSE also increased TIMP-3 affinity for glycan-binding metalloproteinases, such as adamalysin-like metalloproteinase with thrombospondin motifs 5 (ADAMTS-5), by reducing the dissociation rate constants. The sulfation pattern was crucial for these activities because monosulfated or truncated heparin had a reduced ability to bind to TIMP-3 and increase its affinity for ADAMTS-5. Therefore, sulfation of ECM glycans regulates the levels and inhibitory activity of TIMP-3 and modulates ECM turnover, and small mimicries of sulfated glycans may protect the tissue from the excess destruction seen in diseases such as osteoarthritis, cancer, and atherosclerosis.
Graphical Abstract
•The metalloprotease inhibitor TIMP-3 binds to sulfated extracellular glycans•This inhibits cellular uptake of TIMP-3 by the endocytic receptor LRP-1•Glycans also increase TIMP-3 affinity for selected target proteases•The sulfation of matrix glycans therefore modulates TIMP-3 activity and ECM turnover
Tissue inhibitor of metalloproteinase (TIMP)-3 regulates extracellular matrix turnover. Troeberg et al. show that TIMP-3 levels are regulated by the balance between its binding to matrix glycans and receptor-mediated endocytosis and that glycan sulfation regulates this equilibrium.
PMCID: PMC4210636  PMID: 25176127
2.  Proteases involved in cartilage matrix degradation in osteoarthritis 
Biochimica et biophysica acta  2011;1824(1):133-145.
Osteoarthritis is a common joint disease for which there are currently no disease-modifying drugs available. Degradation of the cartilage extracellular matrix is a central feature of the disease and is widely though to be mediated by proteinases that degrade structural components of the matrix, primarily aggrecan and collagen. Studies on transgenic mice have confirmed the central role of Adamalysin with Thrombospondin Motifs 5 (ADAMTS-5) in aggrecan degradation, and the collagenolytic matrix metalloproteinase MMP-13 in collagen degradation. This review discusses recent advances in current understanding of the mechanisms regulating expression of these key enzymes, as well as reviewing the roles of other proteinases in cartilage destruction.
PMCID: PMC3219800  PMID: 21777704
osteoarthritis; proteinase; cartilage; aggrecanase; collagenase
3.  Microvesicles shed by oligodendroglioma cells and rheumatoid synovial fibroblasts contain aggrecanase activity 
Matrix Biology  2012;31-20(4):229-233.
Membrane microvesicle shedding is an active process and occurs in viable cells with no signs of apoptosis or necrosis. We report here that microvesicles shed by oligodendroglioma cells contain an ‘aggrecanase’ activity, cleaving aggrecan at sites previously identified as targets for adamalysin metalloproteinases with disintegrin and thrombospondin domains (ADAMTSs). Degradation was inhibited by EDTA, the metalloproteinase inhibitor GM6001 and by tissue inhibitor of metalloproteinases (TIMP)-3, but not by TIMP-1 or TIMP-2. This inhibitor profile indicates that the shed microvesicles contain aggrecanolytic ADAMTS(s) or related TIMP-3-sensitive metalloproteinase(s). The oligodendroglioma cells were shown to express the three most active aggrecanases, namely Adamts1, Adamts4 and Adamts5, suggesting that one or more of these enzymes may be responsible for the microvesicle activity. Microvesicles shed by rheumatoid synovial fibroblasts similarly degraded aggrecan in a TIMP-3-sensitive manner. Our findings raise the novel possibility that microvesicles may assist oligodendroglioma and rheumatoid synovial fibroblasts to invade through aggrecan-rich extracellular matrices.
► Microvesicles are shed by numerous cell types. ► We isolated microvesicles shed by oligrodendroglioma and rheumatoid synovial fibroblasts. ► These microvesicles contained an aggrecan-degrading proteolytic activity. ► Microvesicles may thus assist cells in degrading aggrecan-rich extracellular matrices.
PMCID: PMC3391679  PMID: 22406378
ADAM, adamalysin; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; ECM, extracellular matrix; MEF, mouse embryonic fibroblasts; MMP, matrix metalloproteinase; MVs, microvesicles; RA, rheumatoid arthritis; TIMP, tissue inhibitor of metalloproteinase; Membrane vesicles; Aggrecan; Metalloproteinase; ADAMTS
4.  Pentosan polysulfate increases affinity between ADAMTS-5 and TIMP-3 through formation of an electrostatically driven trimolecular complex 
Biochemical Journal  2012;443(Pt 1):307-315.
The semi-synthetic sulfated polysaccharide PPS (pentosan polysulfate) increases affinity between the aggrecan-degrading ADAMTSs (adamalysins with thrombospondin motifs) and their endogenous inhibitor, TIMP (tissue inhibitor of metalloproteinases)-3. In the present study we demonstrate that PPS mediates the formation of a high-affinity trimolecular complex with ADAMTS-5 and TIMP-3. A TIMP-3 mutant that lacks extracellular-matrix-binding ability was insensitive to this affinity increase, and truncated forms of ADAMTS-5 that lack the Sp (spacer) domain had reduced PPS-binding ability and sensitivity to the affinity increase. PPS molecules composed of 11 or more saccharide units were 100-fold more effective than those of eight saccharide units, indicating the involvement of extended or multiple protein-interaction sites. The formation of a high-affinity trimolecular complex was completely abolished in the presence of 0.4 M NaCl. These results suggest that PPS enhances the affinity between ADAMTS-5 and TIMP-3 by forming electrostatically driven trimolecular complexes under physiological conditions.
PMCID: PMC3369482  PMID: 22299597
adamalysin with thrombospondin motifs 5 (ADAMTS-5); aggrecanase; extracellular matrix; glycosaminoglycan; osteoarthritis; tissue inhibitor of metalloproteinases 3 (TIMP-3); ADAMTS, adamalysin with thrombospondin motifs; ATIII, antithrombin III; CysR, cysteine-rich; Dis, disintegrin; DMEM, Dulbecco's modified Eagle's medium; dp, degree of polymerization; HEK, human embryonic kidney; MMP, matrix metalloproteinase; OA, osteoarthritis; PPS, pentosan polysulfate; Bio-PPS, biotinylated PPS; Sp, spacer; TBA, tetrabutyl ammonium; TIMP, tissue inhibitor of metalloproteinases; N-TIMP-3, N-terminal domain of TIMP-3; TS, thrombospondin
5.  The C-terminal domains of ADAMTS-4 and ADAMTS-5 promote association with N-TIMP-3 
We investigated whether the affinity of tissue inhibitor of metalloproteinases (TIMP)-3 for adamalysins with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 is affected by the non-catalytic ancillary domains of the enzymes. For this purpose, we first established a novel method of purifying recombinant FLAG-tagged TIMP-3 and its inhibitory N-terminal domain (N-TIMP-3) by treating transfected HEK293 cells with sodium chlorate to prevent heparan sulfate proteoglycan-mediated TIMP-3 internalization. TIMP-3 and N-TIMP-3 affinity for selected matrix metalloproteinases and forms of ADAMTS-4 and -5 lacking sequential C-terminal domains was determined. TIMP-3 and N-TIMP-3 displayed similar affinity for various matrix metalloproteinases as has been previously reported for E. coli-expressed N-TIMP-3. ADAMTS-4 and -5 were inhibited more strongly by N-TIMP-3 than by full-length TIMP-3. The C-terminal domains of the enzymes enhanced interaction with N-TIMP-3 and to a lesser extent with the full-length inhibitor. For example, N-TIMP-3 had 7.5-fold better Ki value for full-length ADAMTS-5 than for the catalytic and disintegrin domain alone. We propose that the C-terminal domains of the enzymes affect the structure around the active site, favouring interaction with TIMP-3.
PMCID: PMC2835468  PMID: 19643179
aggrecanase; inhibition kinetics; MMPs
6.  MT1-MMP is a crucial promotor of synovial invasion in human rheumatoid arthritis 
Arthritis and rheumatism  2009;60(3):686.
A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage and this step requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen-degrading matrix metalloproteinases (MMPs), membrane-type 1 MMP (MT1-MMP), in synovial pannus invasiveness.
Expression and localization of MT1-MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemistry of RA joints specimens. The functional role of MT1-MMP was analyzed by 3D collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, or GM6001. The effect of adenoviral expression of a dominant negative MT1-MMP construct lacking a catalytic domain was also examined.
MT1-MMP was highly expressed at the pannus-cartilage junction of RA joints. Freshly isolated rheumatoid synovial tissues and isolated RA synovial fibroblasts invaded into a 3D collagen matrix in an MT1-MMP-dependent manner. Invasion was blocked by TIMP-2 and GM6001, but not by TIMP-1. It was also inhibited by the over-expression of a dominant negative MT1-MMP which inhibits collagenolytic activity and proMMP-2 activation by MT1-MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1-MMP-dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix.
MT1-MMP is an essential collagen-degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1-MMP-dependent invasion may form a novel therapeutic strategy for RA.
PMCID: PMC2819053  PMID: 19248098
MT1-MMP; synovial pannus; rheumatoid arthritis

Results 1-6 (6)