Glycosaminoglycans influence enzyme activity of MMP2 and MMP2/TIMP3 complex formation - Insights at cellular and molecular level

The extracellular matrix (ECM) is a highly dynamic network constantly remodeled by a fine-tuned protein formation and degradation balance. Matrix metalloproteinases (MMPs) constitute key orchestrators of ECM degradation. Their activity is controlled by tissue inhibitors of metalloproteinases (TIMPs) and glycosaminoglycans (GAG). Here, we investigated the molecular interplay of MMP2 with different GAG (chondroitin sulfate, hyaluronan (HA), sulfated hyaluronan (SH) and heparin (HE)) and the impact of GAG on MMP2/TIMP3 complex formation using in vitro-experiments with human bone marrow stromal cells, in silico docking and molecular dynamics simulations. SH and HE influenced MMP2 and TIMP3 protein levels and MMP2 activity. Only SH supported the alignment of both proteins in fibrillar-like structures, which, based on our molecular models, would be due to a stabilization of the interactions between MMP2-hemopexin domain and TIMP3-C-terminal tail. Dependent on the temporal sequential order in which the final ternary complex was formed, our models indicated that SH and HA can affect TIMP3-induced MMP2 inhibition through precluding or supporting their interactions, respectively. Our combined experimental and theoretical approach provides valuable new insights on how GAG interfere with MMP2 activity and MMP2/TIMP3 complex formation. The results obtained evidence GAG as promising molecules for fine-balanced intervention of ECM remodeling.


Results and Discussion
For the in vitro experiments with human bone marrow stromal cells (hBMSC), the synthetic low-sulfated hyaluronan (SH; D.S. 1.2, sulfated at C6 of glucosamine), natural low-sulfated chondroitin sulfate (CS; D.S.0.8) and natural high-sulfated heparin (HE; D.S. 2.2) were used and compared to natural non-sulfated hyaluronan (HA) (see materials and methods) and hBMSC cultures without GAG treatment (Ctrl). Since the commercially available CS is a mixture of chondroitin-4-sulfate (C4S, ca. 70%) and chondroitin-6-sulfate (C6S, ca. 30%), the in silico modeling studies were performed with each CS derivative separately.
Influence of GAG on MMP2 and TIMP3 in vitro. All GAG except HE did not significantly alter the gene expression level of mmp2 in hBMSC (Fig. 1A). Despite a slight increase in mmp2 expression by HE, the MMP2 protein content in conditioned medium of hBMSC (released MMP2) was found diminished by SH and HE; the effect, however, was not significant (Fig. 1B). SH and HE significantly reduced MMP2 activity (Fig. 1C). To evaluate whether the decreased MMP2 activity resulted from a decreased amount of enzyme, MMP2 activity was normalized to MMP2 protein. In addition to the reduced MMP2 protein amount, SH and HE significantly reduced MMP2 enzyme activity (Fig. 1D). CS and HA had no significant impact on MMP2 protein and enzyme activity ( Fig. 1B-D). Gelatin zymography was used to detect proMMP2 and MMP2 in conditioned medium of hBMSC at day 22 (Supplemental Fig. S1). The densitometric analysis showed that proMMP2 (72 kDa) was not detectable at this time point, but it confirmed the results for MMP2 protein shown in Fig. 1B.
The lower amount of MMP2 in the conditioned medium in the presence of SH and HE is, as seen, not a result of reduced gene expression. This could be caused either by retardation in the ECM, by accelerated degradation of Figure 1. Influence of CS, HA, SH, and HE on MMP2 and TIMP3 formation and MMP2 activity in hBMSC. 7,000 hBMSC/cm 2 were plated in basic medium and treated with CS, HA, SH, and HE (200 µg/mL each). At day 22 after plating cells and conditioned medium were analyzed. (A) mmp2 expression was assessed by qPCR, normalized to the expression of the house-keeping genes (hkg) gapdh, β-actin, and rps26, and related to untreated control (Ctrl, set to 1) using the comparative quantitation method. Conditioned medium of hBMSC was analyzed for MMP2 protein content using commercially MMP2 ELISA Kit (B) and for MMP2 enzyme activity with fluorogenic peptide (MCA-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH 2 ) as a substrate (C). (D) The relative MMP2 activity per MMP2 protein amount was calculated from fluorescence signals (MMP2 activity) and ELISA data (MMP2 protein). (E) timp3 expression was assessed by qPCR as described. (F) TIMP3 protein amount released into the conditioned medium was determined by ELISA. TIMP3 protein in the pericellular environment of hBMSC was analyzed by Western blotting with chemiluminescence detection. (G) shows a representative Western blot (cropped), according full-length blots presented in Supplemental Fig. S3. www.nature.com/scientificreports www.nature.com/scientificreports/ the enzyme, or by forced endocytotic clearance, e. g. via LRP1 30,31 . The observed MMP2 activity could be influenced by proteolytic activation of proMMP2, endogenous or exogenous activators (e. g. zinc ions or mercurial compounds), and natural (e. g. TIMPs) as well as chemical inhibitors. The interaction with diverse ECM proteins as thrombospondin-2 and fibronectin, cell surface receptors and heparan sulfate proteoglycans can further trigger MMP2 protein level, proMMP2 activation and MMP2 activity 30,32 .
Recent proteomics studies 16,27 reported reduced gelatinolytic activity of MMP2 with osteoblasts cultured on artificial collagen matrices containing high-sulfated hyaluronan (D.S. 3.0), indicating that other sulfated hyaluronan derivatives exhibit comparable effects to SH on MMP2 activity. Van der Smissen et al. 33 showed that MMP1 activity of dermal fibroblasts was decreased by sulfated GAG, indicating that the effects of GAG on MMP are not restricted to MMP2 and to hBMSC.
In the present study we further considered the influence of GAG on TIMP3 in vitro. Quantitative PCR analysis exhibit that GAG did not alter the gene expression level of timp3 in hBMSC (Fig. 1E). TIMP3 protein concentration in the conditioned medium of hBMSC as determined by ELISA was highly induced by SH and HE (Fig. 1F). Compared to Ctrl, CS and HA did not alter TIMP3 concentration (Fig. 1F). The gene expression and protein levels of TIMP1, 2 and 4 (secreted TIMPs) were neither affected by CS, HA, SH nor by HE (Supplemental Fig. S2). TIMP3 content in cell lysates of hBMSC gathering intra-and ECM-associated proteins was analyzed by Western blot (Fig. 1G) and quantified from four independent Western blots by densitometric evaluation of the signals ( Fig. 1H; Full length blots in Supplemental Fig. S3). Exclusively SH increased the amount of cell-associated TIMP3 by ca. 10-fold, whereby CS, HA and HE did not significantly affect cell-associated TIMP3 (Fig. 1H). SH and HE increased the total amount of TIMP3 (sum of TIMP3 in conditioned medium and in the cell-associated portion) formed by hBMSC to nearly the same extent but influenced considerably its distribution (Fig. 1I). Whereas in HE-treated hBMSC about 85% of total TIMP3 was released into conditioned medium, in the case of SH-treated cells, about 55% of total TIMP3 was found to stick in the cell-associated portion (Fig. 1I). SH and HE not only decreased MMP2 amount and enzyme activity, increased TIMP3 level and altered its distribution within ECM-associated and released portion, but these two GAG also significantly reduced the MMP2/TIMP3 protein ratio (Fig. 1J). CS and HA had no effect on released and ECM-associated TIMP3 and changed neither TIMP3 distribution nor the MMP2/TIMP3 ratio ( Fig. 1F-J). The significant increased TIMP3 protein level in the presence of SH and HE is not a result of altered gene expression. As discussed for MMP2, SH and HE seem to be responsible for elevated TIMP3 accumulation in the ECM and, probably, TIMP3 is also rescued from endocytotic clearance. Yu et al. 10 showed that several sulfated GAG as HE, heparan sulfate and diverse CS derivatives closely interact with TIMP3. It has been demonstrated that the interaction of sulfated GAG with TIMP3 blocks its binding to the endocytosis receptor LRP-1 12,26,29 . Therefore, it can be assumed that elevated TIMP3 amounts are owed by the stabilizing effect of SH and HE preventing its removal from the system by LRP1 and sequestering it in the ECM. In line with our results, two previous studies described comparable effects of SH on TIMP3 26,27 . A significantly increased TIMP3/MMP2 protein ratio (Fig. 1J) could be one reason for less MMP2 activity in the presence of SH and HE.
By immunofluorescence staining of CS-, HA-, SH-and HE-treated hBMSC, MMP2 and TIMP3 were visualized ( Fig. 2A; Control stainings with only secondary antibodies are depicted in Supplementary Fig. S4). In CS, HA and HE-treated hBMSC, the MMP2 protein, just as TIMP3, was observed in a diffuse pattern around the nuclei, in the perinuclear space and in the cytosol, and it was arranged in a few fibrillar-like structures. Compared to Ctrl and CS-, HA-and HE-treated hBMSC, the morphological arrangement of MMP2 and TIMP3 in the presence of SH was completely different; here both proteins were aligned in fibrillar-like structures. The quantitative analysis of overlapping red and green channel pixels, as verified by Pearson coefficient, indicated only in SH-treated hBMSC a significant colocalization of MMP2 and TIMP3 fluorescence signals aligned in fibrillar-like structures (Fig. 2C).
Although SH and HE increased TIMP3 protein amounts to about the same extent, both GAG substantially differed in their effect on TIMP3 distribution among ECM-bound and released portion (see Fig. 1I). Immunofluorescence images obtained with fluorescent-labelled SH (Fig. 2B) showed that SH itself is part of the MMP2/TIMP3-containing fibrillar-like structures; merged immunofluorescence images showed an overlap of MMP2 and TIMP3, of SH and MMP2, of SH and TIMP3, and hence also of SH, MMP2 and TIMP3. Colocalization analyses gave a Person coefficient of 79% and 70% for the overall overlay of ATTO655-SH with MMP2 and TIMP3, respectively (Fig. 2D). The Manders' coefficients M1 and M2 indicated that ATTO655-SH colocalized with MMP2 to 86% and with TIMP3 to 68% (M1 value), and that 57% of MMP2 pixel and 70% of TIMP3 pixel overlapped with ATTO655-SH (M2 value) ( Fig. 2D-E).
The in vitro-experiments with hBMSC demonstrated a substantial impact of SH and HE on the MMP2/TIMP3 system and gave evidence of a very tight vicinity of SH, MMP2 and TIMP3 in fibrillar-like extracellular structures. Whether the highly increased TIMP3 level, or a direct inhibitory/blocking effect of GAG on MMP2 activity, or the modulation of MMP2/TIMP3 complex formation by GAG could be an explanation for the reduced MMP2 activity seen in the presence of SH and HE was taken apart and elucidated in cell-free assays and by molecular modeling.
The chemiluminescence signals of four independent Western blots were quantified densitometrically and normalized to GAPDH and β-tubulin (Tub) as internal loading controls (H). (I) shows the total TIMP3 content related to Ctrl and the distribution of TIMP3 calculated from Western blot data (pericellular, ECM-associated TIMP3) and ELISA data (released TIMP3). The ratio of released MMP2 protein to released TIMP3 is shown in (J). The results are presented as mean ± SEM. Significant differences of treatment vs. Ctrl were analyzed by oneway ANOVA/Bonferroni's post-test (A-F,H,J), and of SH vs. HE two-way ANOVA/Bonferroni's post-test (I) and are indicated with *(p < 0.05), **(p < 0.01) and ***(p < 0.001), n = 4.
www.nature.com/scientificreports www.nature.com/scientificreports/ Influence of GAG on MMP2 enzyme activity and TIMP3-induced MMP2 inhibition. The plethora of co-players in vitro influencing in parallel MMP2 and TIMP3 makes it difficult to unravel the effects of GAG on MMP2/TIMP3 complex formation. Thus, a cell-free enzyme activity assay with recombinant human MMP2 and TIMP3 and a fluorogenic peptide as a substrate was used to study the influence of CS, HA, SH and HE (200 µg/ mL, each) on MMP2 enzyme activity and TIMP3-mediated MMP2 inhibition ( Fig. 3A-D). The enzymatic reaction was monitored over 5 h. In the first 120 min the proteolytic activity was independent of GAG. Finally, after 5 h, HA, SH and HE induced a slight but significant ca. 10% decrease in MMP2 enzyme activity (Fig. 3E). Varying the concentration of CS, HA and HE from 20-1,000 mg/mL had no influence on their effects on MMP2 activity (significant differences were only seen for control (0 µg GAG/mL) vs. particular GAG concentrations; Supplemental Fig. S5). For SH a marginal dose-dependent effect on MMP2 activity was seen, ranging from 100% for Ctrl (0 µg SH/mL) to 85% of MMP2 activity in the presence of 1,000 µg SH/mL. In a control experiment, the www.nature.com/scientificreports www.nature.com/scientificreports/ fluorescent peptide released by MMP2 activity from the fluorogenic peptide substrate was incubated with CS, HA, SH and HE to exclude a quenching of the fluorescence signal by GAG (see Supplemental Fig. S6).
For TIMP3-mediated MMP2 inhibition experiments, MMP2 and TIMP3 were used in a molecular ratio of 1:1 (appx.), which reduces MMP2 activity to about 15% (Fig. 4A,B). The influence of CS, HA, SH and HE on TIMP3-induced inhibition of MMP2 activity was examined in three different settings in which always two of the three components were pre-incubated for 30 min at 25 °C before adding the third one. Figure 4C-E show GAG-induced differences in TIMP3-mediated MMP2 inhibition compared to the control w/o GAG (=15% residual activity of MMP2 in the presence of TIMP3). When the MMP2/TIMP3 complex was formed at first, HA significantly strengthened the TIMP3-induced inhibition of MMP2 activity leading to about 5% less MMP2 activity (Fig. 4C). CS, SH and HE had no influence on the inhibitory efficiency of such a pre-formed MMP2/ TIMP3 complex. The pre-incubation of either TIMP3 (Fig. 4D) or MMP2 (Fig. 4E) with CS and HA did not alter  www.nature.com/scientificreports www.nature.com/scientificreports/ the inhibitory effect of TIMP3 on MMP2 activity. In the presence of SH and HE, a weakening of TIMP3 inhibitory efficiency was observed. When either TIMP3 or MMP2 were pre-incubated with SH and when TIMP3 was pre-incubated with HE, the effect was highly significant.
Molecular recognition of GAG to MMP2/TIMP3, TIMP3, MMP2, proMMP2 and proMMP2/TIMP3. Molecular docking was used to investigate GAG recognition by the MMP2/TIMP3 complex, TIMP3, MMP2, proMMP2 and the proMMP2/TIMP3 complex. The resulting GAG-protein complexes were refined by molecular dynamics (MD) simulations to obtain binding energies and atomic-detailed information about the intermolecular interactions taking place (see materials and methods).
First, the protein structure of MMP2 catalytic domain (PDB ID 1QIB (2.8 Å)) was used to build a three-dimensional atomic model of its complex with TIMP3. For the in silico calculations, GAG hexamers HA, SH, HE and CS were used. In particular, for CS, both chondroitin-4-(C4S) and chondroitin-6-sulfate (C6S) were taken into account as the in vitro experiments were carried out with a 70:30 mixture of both.
For the pre-formed MMP2/TIMP3 complex, all GAG were predicted to bind to TIMP3. Interestingly, HA was the only GAG disposed along the surface of both proteins (Fig. 4F). After energy refinement by MD simulations, HA was found to slightly strengthen the interaction between both proteins (Table 1, Supplemental Fig. S7) and, thereby, enhancing the ory effect of TIMP3 on MMP2 catalytic domain as stated in the enzyme activity test (Fig. 4C). In the most favorable case (HA-Cluster1), the simultaneous interactions of HA with Arg84 of TIMP3, and Trp147 and Lys158 of MMP2 catalytic domain, among others, could stabilize the MMP2/TIMP3 complex in ca. 4 kcal/mol (ΔG MMP2/TIMP3 = −49.5 ± 9.6 kcal/mol, ΔG MMP2-HA/TIMP3 = −53.1 ± 9.1 kcal/mol, computed with MM-GBSA 34,35 ).
Next, recognition of TIMP3 by C4/6S, HA, SH and HE and its possible influence on MMP2 activity was investigated. Binding poses along β1/β4 and helix 2 were obtained for all GAG, which is in concordance with previous findings 29 . For SH, additional binding poses were predicted along helix 1 at the N-terminus and loop regions between helix 3, β9 and β10 at the C-terminal of TIMP3, which partially overlap with the recognition region of MMP2 catalytic domain (Fig. 4G). As observed experimentally, this weakens TIMP3-induced MMP2 inhibition by SH (Fig. 4D).
Molecular docking calculations suggested that all GAG and TIMP3 (loop regions between β2/β3, β5/β6 and β7/β8) could compete for the same recognition region on MMP2 (Fig. 4H, Supplemental Fig. S8). In particular, the GAG-binding region of the MMP2 catalytic domain partially coincides with the substrate-binding cleft site of MMP2 36 . Therefore, also GAG binding might have a negative impact on the MMP2 enzymatic activity, which is in concordance with the experimental observations (Figs 3 and 4).
Besides, due to the fact that the PEX domain of the proMMP2 has been reported to be involved in the activation of the enzyme by interacting with proteins such as TIMP2-4 5,7,9 and MT1-MMP 5,8,9 , and also with HE 15 , the recognition of GAG by proMMP2 and proMMP2/TIMP3 was further investigated. The proMMP2 structure (PDB ID 1GXD (3.1 Å)) and its modeled complex with TIMP3 (see materials and methods) were used for the in silico experiments.
In the recognition studies of proMMP2, C4/6S, HA, SH and HE were predicted to bind to the PEX domain (Fig. 5). Interestingly, SH, HE and, in lesser extent, C4S were found to bind to the same PEX region at the C-terminal tail of TIMP3 (Fig. 5A,D,E), which would preclude PEX/TIMP3 C-terminal tail recognition. In addition, GAG binding poses bridging the PEX and FNII domains were obtained for C4/6S, HA and SH (Fig. 5A-D). Besides, C4/6S and SH adopted poses bridging the PEX and the catalytic domain (Fig. 5A,B,D). The potential interactions of GAG with some regions of FNII and catalytic domain might affect the interaction of proMMP2 with ECM proteins, which are putative substrates for MMP2 5,37 . In conclusion, the interaction of SH and HE with the PEX domain might affect the activation of proMMP2 leading to different MMP2 activity in vitro (Fig. 1).
Molecular docking of GAG to the proMMP2 (PEX domain)/TIMP3 complex (Fig. 6) indicated that C4/6S, HA and HE only recognized TIMP3. Interestingly, only SH was predicted to bridge the C-terminal tail of TIMP3 and the PEX domain of proMMP2 acting as a kind of "molecular glue" (Fig. 6D). Binding energies obtained with the MD-refined ternary complex indicated that the interaction between both proteins in the presence of SH was further stabilized in ca. 10 kcal/mol (ΔG PEX/TIMP3 = −73.1 ± 8.8 kcal/mol; ΔG PEX/TIMP3-SH = −82.1 ± 2.2 kcal/mol). PEX domain residues Lys547, Asn548, Arg561, Asn582, Ile584, Gln614, Ser615, Leu616 and Lys617 increased binding strength to TIMP3 in the presence of SH (Supplemental Fig. S9). These data strongly support the results from immunofluorescence staining and colocalization analysis in which TIMP3 and MMP2 were found colocalized exclusively in the presence of SH ( Fig. 2A), and where SH was seen to be part of the MMP2/TIMP3 fibrillar-like structures (Fig. 2B).
In this study, the effect of HA, SH, HE and CS on ECM remodeling by the means of gene expression, protein levels and function of MMP2 and TIMP3 in hBMSC was investigated. The expression of mmp2 and timp3 was www.nature.com/scientificreports www.nature.com/scientificreports/ not significantly influenced by CS, HA, SH, and HE. Both, SH and HE significantly decreased MMP2 protein content and additionally reduced MMP2 enzyme activity. This effect could be caused by direct interaction of MMP2 and/or proMMP2 with SH and HE, which in principle might shield the catalytic site or block the activation of the proenzyme. The latter could be neither excluded nor confirmed in vitro since proMMP2 was under the detection limit of gelatin zymography. Another reason, which is supported by experimental data, could be the elevated TIMP3 protein levels induced by SH and HE. Nevertheless, both GAG have the same effect on MMP2 and TIMP3 protein activity, but they distinguished themselves in their respective effects on TIMP3 distribution within the ECM. The tight vicinity of MMP2 and TIMP3 obtained only in the presence of SH, as seen in immunofluorescence staining, was the trigger to study at molecular level the interaction of single components in the ternary system of GAG/MMP2/TIMP3 using a combination of a simplified cell-free assay involving recombinant human proteins and molecular docking calculations. Recent proteomics studies have shown that SH and HE, in comparison to other GAG, alter MMP2 and TIMP3 not only in hBMSC and in their pericellular space, but also in hBMSC-derived matrix vesicles 38 . Opposite effects of HE and HA and C6S as well on MMP2 activity are described for human dermal fibroblasts 39 and for corneal explant cultures 40,41 , respectively. One consequence of the diminished MMP2/TIMP3 ratio and the reduced MMP2 activity is a substantial stabilization and accumulation of several ECM proteins (e.g. fibronectin), in particular in the presence of SH 22,27 .
In a previous study, Rother et al. 29 reported that the interaction of native and chemically modified GAG with TIMP3 had a negligible influence on MMP1 and MMP2 recognition and enzyme activity. In our studies, we observed that the temporal sequence in which the ternary complex of GAG/MMP2/TIMP3 is built determines the impact of GAG on TIMP3-induced MMP2 activity, especially for HA, SH and HE. These results are in good agreement with previous findings on the interaction of TIMP3 with MMP2 and MMP9 42 . To unravel the molecular mechanisms underlying GAG-mediated TIMP3 function, in silico docking and MD simulations were www.nature.com/scientificreports www.nature.com/scientificreports/ performed. The inhibitory capacity of TIMP3 was weakened when either the complex of SH with MMP2 or with TIMP3 was pre-formed, while HA was able to slightly enhance MMP2 inhibition by TIMP3 in the preformed complex between both proteins. Hence, SH is able to impair TIMP3/MMP2 recognition, whereas HA could slightly stabilize the interaction between both proteins once the complex is formed.
In addition, the interplay of GAG on the molecular recognition of proMMP2 and its complex with TIMP3 was examined. Morgunova et al. 7 reported that the MMP2 PEX domain is necessary for the first activation cleavage of proMMP2 and recognition by TIMP2, TIMP3 and TIMP4 in an alternative activation mechanism of proMMP2 in cells. In particular, the interaction of the C-terminal tail of TIMP2 with the MMP2 PEX domain has been proven to be crucial to initiate this latent activation mechanism 9,43 . Therefore, we investigated in silico whether different GAG could preclude TIMP3 recognition and modulate cell-mediated proMMP2 activation, as well as their interplay once the proMMP2/TIMP3 complex is formed. Our findings suggested that SH, HE and in certain extent C4S could preclude TIMP3 C-terminal tail binding. It has been previously reported that HE and C4S bind to the PEX domain and promote proMMP2 activation by autolysis 15 and by cell-bound membrane type-3 MMP (MT3-MMP) 44 , respectively. Taking into account the role of TIMP3 in assisting the localization of proMMP2 on the cell surface previous activation of the zymogen, GAG could play a similar role on the activation of proMMP2. Our molecular models indicated that SH could act as a "molecular glue" by strengthening the interaction between the PEX domain of proMMP2 and the C-terminal tail of TIMP3, which could efficiently assist the colocalization of both proteins as experimentally demonstrated in fluorescence images.

Conclusions
The results obtained from our combined experimental and molecular modeling approach provide very valuable new insights on how GAG can influence ECM remodeling via interactions with TIMP3, MMP2 or both simultaneously. SH and HA could tightly modulate TIMP3-induced MMP2 inhibition through precluding or supporting the interactions between both proteins, respectively, in a temporal sequential manner. In addition, it was shown that only SH and none of the other GAG (HA, HE or CS) supported the alignment of TIMP3 and MMP2 in www.nature.com/scientificreports www.nature.com/scientificreports/ hBMSC. Recent studies gave evidence that SH is the only GAG that significantly enhances osteogenic differentiation of hBMSC 19,45 . It still remains to be elucidated whether any link between the two facts (enhanced osteogenic differentiation by SH, increased ECM stability by SH) exists. Taken altogether, defined GAG are able to fine-tune the MMP2/TIMP3 system and so allow a discreet adjustment of ECM remodeling. Through the establishment of atomic-detailed three-dimensional molecular models, and in combination with experimental work, we have been able to provide cellular and mechanistic insights on GAG recognition by TIMP3 and the proMMP2 PEX domain, as well as to characterize their functional impact on MMP2 activity.
Low molecular weight hyaluronan (HA) was produced by thermal degradation 47 . Low-sulfated hyaluronan (SH) was synthesized as described 19,22 . Fluorescence labelling of SH was carried out by side-on functionalization with ATTO655-NH 2 resulting in a dye content of 0.41 µg/mg 22,48 . Chemical structures of GAG are shown in For the experiments, 7,000 hBMSC/cm 2 were plated in basic medium (Dulbecco's modified essential medium (DMEM, 1 g glucose/l) with 10% heat-inactivated fetal calf serum and 20 U penicillin/20 µg streptomycin/mL) on tissue culture polystyrene (TCPS). 24 h after plating, hBMSC were treated with GAG derivatives, 200 µg/mL each.
Medium change and adding of GAG was carried out twice a week. Cells were prepared according to the assay procedures; the conditioned medium was stored at −20 °C until analyses.

Quantitative PCR analyses.
For the analysis of gene expression, RNA was directly prepared from cell lysates and real time PCR analysis was performed as previously described 26 . Primers were synthesized by Eurofins MWG Operon for β-actin, gapdh, rps26 26 , mmp2 (88 bp, 5′-3′-sequences: forward agaaggctgtgttctttgcag, reversed aggctggtcagtggcttg) and timp3 (101 bp, 5′-3′-sequences: forward gtgcaacttcgtggagaggt, reversed agcaggacttgatcttgcagt). The relative expression values were calculated using the comparative quantification method of the RotorGene software release 6.0. For quantitation, values are normalized to the house-keeping genes and related to Ctrl.
ELISA. MMP2 and TIMP3 protein concentration in conditioned medium of hBMSC was determined using DuoSet ELISA development kits (DY902 for MMP2, and DY973 for TIMP3, BioTechne, Wiesbaden, Germany) www.nature.com/scientificreports www.nature.com/scientificreports/ according to manufacturer's instructions. If required, samples were diluted in 1% of bovine serum albumin (BSA, from Calbiochem/Merck, Darmstadt, Germany) in phosphate buffered saline (PBS). MMP2 enzymatic activity. Fluorometric MMP2 enzymatic activity assay was performed accordingly to 50 . In general, 20 µL of sample (conditioned medium or rh-proMMP2 (BioTechne, 200 µg/mL in 20 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl 2 , and 0.05% Brij-35 TM were diluted in assay buffer (50 mM Tris-HCl, 10 mM CaCl 2 , 175 mM NaCl, 1% dimethyl sulfoxide (DMSO), and 0.05% Brij-35 TM , pH 7.5) to 500 ng/mL, in the assay a final concentration of 100 rh-proMMP2 ng/mL was applied) were mixed with 20 µL of 1.25 mM 4-aminophenylmercuric acetate (APMA, proMMP2 activator, Sigma, diluted in assay buffer from 1 M stock solution in DMSO to 1.25 mM, final concentration in the assay 0.25 mM) in a black 96 well plate for 20 min at 25 °C. Further, depending on the test set-up, either 20 µL of GAG (diluted with assay buffer, final concentration in the assay 200 µg/mL) or 20 µL of rhTIMP3 (BioTechne, 100 µg/mL in water, diluted with assay buffer, final concentration 40 ng/mL), and 20 µl of assay buffer, or 20 µl of both rhTIMP3 and GAG, or 40 µl of assay buffer were added. In particular tests, the order of adding of the components was varied: For that either rhMMP2 and rhTIMP3, or rhMMP2 and GAG, or rhTIMP3 and GAG were pre-incubated for 30 min at 25 °C before adding the other components. To study the dose-dependent influence of GAG on MMP2 activity, the concentration of GAG was varied from 20-1000 µg/mL, each in 20 µl (see Supplemental Fig. S5). In each case the enzyme reaction was started by adding 20 µL of fluorogenic MMP2 peptide substrate (MCA-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH 2 , Calbiochem/ Merck, diluted in assay buffer from 1 mM stock solution in DMSO to 50 µM, final concentration 10 µM). The assay blank was made from 80 µL of assay buffer and 20 µL of substrate solution. The fluorescence signal detection was performed in a fluorimeter plate reader (Fluorostar Galaxy, BMG LABTECH, Ortenberg, Gemany) at 320 nm excitation and 405 nm emission in kinetics mode for 300 min in total with 5 min measurement intervals at 37 °C. To exclude that GAG quenched the fluorescence signal, 20 µl of OMMNIMMP fluorogenic control peptide (MCA-Pro-Leu-OH, Enzo Life Science Lörrach, Germany, diluted in assay buffer from 1 mM stock solution in DMSO to 50 µM, final concentration 10 µM) were mixed with 20 µl of GAG, and 60 µl of assay buffer and measured as described above.
Immunofluorescence staining. Immunofluorescence staining was performed as described before 22 .
Colocalization analysis of immunofluorescence signals was performed as previously described 22 . The immunofluorescence signals of ATTO655-SH and MMP2 respectively TIMP3 or MMP2 and TIMP3 were quantified with software Fiji setting threshold automatically by Otsu filter. Colocalization values (Pearson, Manders' coefficients) were calculated from eight representative images using the colocalization plugin JACoP (Just another colocalization plugin) 51 .

Statistics. All in vitro-experiments were performed with four biological independent samples (donors).
Fluorometric MMP2 activity determination was performed in quadruplets in four independent experiments. All statistical analyses were performed with GraphPad Prism 7.02 software. Statistical significance was analyzed by one-or two-way ANOVA with Bonferroni's post-test. More detailed information about the applied tests is given in the figure captures.
Molecular dynamics simulations. The complexes proMMP2(PEX domain)/TIMP3, MMP2/TIMP3 and the selected from the docking studies were further refined by Molecular Dynamics (MD) simulations in AMBER14 55 . GAG charges and parameters were taken from GLYCAM 06-j 56 . Charges for sulfates were taken from literature 57 . Protein parameters were obtained from ff14SB 55 . Each GAG/protein complex was solvated in a TIP3P truncated octahedral box and neutralized with Cl − counterions. MD simulations were preceded by two energy-minimization steps: i) only solvent and ions were relaxed with position restraints for solute (500 kcal/ mol·Å 2 ) by 1000 steps of steepest descendent followed by 500 of conjugate gradient; ii) the entire system without restraints with 3000 cycles of steepest descendent and 3000 of conjugate gradient. The system was heated up from 200 K to 300 K in 20 ps with 10 kcal/mol·Å 2 position restraints. Langevin temperature coupling with a collision frequency γ = 1 ps −1 was used. Three equilibration steps of 500 ps each with 10, 5, and 2 kcal/mol·Å 2 consecutively decreased position restraints for the Zn 2+ and Ca 2+ were applied when considering MMP2. Isothermal-isobaric ensemble (NPT) with Langevin thermostate and Berendsen barostat were applied under periodic boundary conditions. The system was further equilibrated 500 ps without restraints at 300 K under same conditions. 20 ns simulation was carried out at 300 K NPT conditions. SHAKE was used to constrain all bonds involving hydrogens with a time step of 2 fs per step. 8 Å cutoff was used for nonbonded interactions, and PME was used to treat long-range electrostatic interactions. MD trajectories were recorded every 10 ps. GAG pyranose rings were harmonically restrained. Trajectories were visualized in VMD 58 and intermolecular H-bonds evaluated with CPPTRAJ in AMBER. At least 10% occupancy was taken as H-bond formation criterion. Energy decomposition per residue, pairwise and binding free energy analysis of 200 simulation frames were obtained with MM-GBSA (gb = 2) in AMBER14 34,35 . R 59 was used for data analysis and PyMOL 60 for preparation of figures.

Data Availability
The datasets generated and analyzed during the current study are available from the corresponding author on request.