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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 6, 2004 as doi:10.1096/fj.03-0966fje. |
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,2
* Division of Geriatrics, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA; and
Craniofacial and Skeletal Diseases Branch, National Institutes of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA
2 Correspondence: Room 5B-79 JHAAC, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA. E-mail: ndarko{at}jhmi.edu
SPECIFIC AIMS
Objectives were to identify proteins that co-purified with members of the small integrin-binding ligand N-linked glycoprotein (SIBLING) gene family and characterize structural and functional consequences of their binding interactions.
PRINCIPAL FINDINGS
1. Specific MMPs co-purify with SIBLINGs
Individual SIBLING family members BSP, OPN and DMP1 were subcloned into an adenovirus system and expressed in human bone marrow stromal cells. Each SIBLING was purified from the serum-free media to 
95% purity by anion exchange chromatography under nondenaturing conditions. When purity was assessed by zymography, each HPLC purified SIBLING exhibited a single band of proteolytic activity. Bands originally visible on the zymogram did not appear in gels treated with 1,10-phenanthroline, showing that co-purifying proteolytic activity arose from metalloproteinases. Identity of proteolytic bands was determined by Western blotting and probing with specific antibodies against MMPs. MMP-2 co-purified with BSP, MMP-3 with OPN and MMP-9 with DMP1.
Specificity observed was confirmed by showing that purified SIBLINGs could be used to affinity purify their respective MMPs from conditioned media containing several different MMPs. When aliquots of eluted fractions were analyzed by zymography, positive bands for multiple MMPs were visible in the flow through peak. Fractions that eluted at 
0.3 M salt were analyzed by Western blot and immunoreactive MMP-2 (from the BSP affinity column) and immunoreactive MMP-3 (from the OPN affinity column) were identified. A DMP1 affinity column was not made due to insufficient amounts of highly purified DMP1.
2. SIBLING and MMP binding specificity
Co-purification from similar media of a single but different MMP with each SIBLING demonstrated that specific interactions were occurring between the proteins. Binding interactions between recombinant MMP-2, MMP-3 and MMP-9 and purified recombinant SIBLINGs were investigated. Relative abundance of tryptophan residues in the MMPs was exploited by carrying out intrinsic fluorescence studies of purified, authentic MMP protein binding to SIBLINGs. Titration of proMMP-2 with BSP yielded a quenching of the MMP’s tryptophan emission spectra and a saturable binding curve. Addition of OPN to proMMP-3 and DMP1 to proMMP-9 also yielded fluorescent signal quenching and saturable binding functions. Stoichiometry of binding between SIBLINGs and their respective proMMPs was 1:1. Scatchard analysis indicated binding constants in the nM range. Quenching of the tryptophan fluorescent signal is consistent with a gross conformational change as a result of binding. Fluorescent binding studies were also carried out using mixed pairs of SIBLINGs and pro- and active-MMPs. SIBLINGs and MMPs showed consistent specificity in their partnering, with BSP binding to pro- and active MMP-2, OPN with pro- and active MMP-3, and DMP1 with pro- and active-MMP-9. Other combinations of SIBLINGs and MMP’s exhibited either no saturable binding or binding that was orders of magnitude weaker.
3. SIBLING-MMP complexes modify the protease activity
Fluorescence spectroscopy observations suggesting that SIBLING binding induces conformational changes in their corresponding MMP partner led to an investigation of whether SIBLING binding affected MMP structure. Addition of SIBLING to pro-MMP did not appear to promote autocatalysis to the active form. An increased susceptibility of SIBLING-proMMP complexes to cleavage and activation by plasmin (a protease that is normally an inefficient activator) was seen and was consistent with SIBLING binding altering proMMP structure.
To measure potential biological effects, a fluorescent substrate assay was employed to screen SIBLING modulation of proMMP activity. Pro-MMP-2, -3, and -9 were reacted in combination with increasing concentrations of SIBLINGs (either BSP, OPN, or DMP1) and enzyme activity was measured by increased fluorescence signal. Increased proteolytic activity was observed for all three strong proMMP+SIBLING binding pairs (Fig. 1
A–C). When the strong binding SIBLING was added to its corresponding active MMP, enzymatic activity was not significantly changed indicating that binding of the SIBLING with its active MMP partner did not interfere with normal proteolytic activity (Fig. 1D-F
). Properly matched SIBLING-proMMP pairs showed a dose-response increase in the rate of substrate digestion (Fig. 1G, H, I
). Incubation of SIBLING alone with substrate was no different from pro-MMP alone or mismatched SIBLING MMP pairs, showing the increase in activity in the proMMP + SIBLING was not caused by any residual proteolytic activity that co-purified with the SIBLING (Fig. 1J-L
). Given that there was no observed increase in the amount of propeptide-free enzyme in all of these SIBLING-proMMP pairs, it is reasonable to hypothesize that the increase in activity is due to a conformational change in the protease which allows its propeptide to be removed from the active site and thereby permit digestion of both small and large macromolecular substrates.
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4. SIBLINGs restore activity to inhibited MMPs
Quenching of tryptophan fluorescence and increase in activity caused by SIBLING binding to proMMP is consistent with an alteration in the local structure near the active site. The effect of SIBLINGs on the ability of small molecular weight inhibitors to modulate MMP activity was investigated next. SIBLINGs were able to increase proMMP activity in the presence of specific small molecular weight inhibitors of MMPs, but not in the presence of 1,10 phenanthroline (which disrupts MMP activity by chelating and removing the active site required zinc ion). Active forms of MMPs also exhibited quenching of tryptophan fluorescence emission upon binding their specific SIBLING partner. The possibility that SIBLING binding also altered inhibitor interaction with active MMPs was investigated. Specific low molecular weight inhibitors were used to block active MMP activity. Addition of the corresponding SIBLING, however, rescued much of the original activity even in presence of equimolar amounts of specific inhibitor. As was the case for proMMPs, SIBLINGs were not able to restore activity to active MMPs treated with 1,10 phenanthroline. When the complex of equimolar active MMP + SIBLING was treated with increasing concentrations of the inhibitor, significant loss of activity was observed but only at substantially higher concentrations.
Because MMPs occur in vivo associated with inhibitors (tissue inhibitors of matrix metalloproteinases, TIMPs), the effect of SIBLINGs on the activity of MMP + TIMP complexes was also investigated. As expected, presence of TIMPs reduced the enzymatic activity of propeptide-free, active MMP. Addition of the correctly matched SIBLING to active MMP + TIMP complex caused a restoration of proteolytic activity. It is a reasonable hypothesis that conformational change in the active MMP upon binding its SIBLING partner lowers affinity of the TIMP (and low molecular weight inhibitors) for the active site of the MMP thereby enabling substrate access.
5. Reversal of SIBLING-induced activity by factor H
BSP, OPN and DMP1 have previously been shown to bind to factor H with high affinity, 10–100-fold higher than that just described for their partner MMPs. Gelatin and casein fluorescein conjugate assays were used to investigate whether factor H can compete with MMPs for SIBLING binding and thereby affect each SIBLING’s interactions with its respective proMMP and active MMPs. TIMP-inhibited MMPs which had regained enzymatic activity by the addition of their corresponding SIBLING were treated with factor H and a significant reduction in the SIBLING-induced recovered activity for MMPs was observed. Higher affinity of factor H for SIBLING protein appears to promote removal of SIBLING from the SIBLING-MMP complex thereby permitting MMP to reverse its conformation and allow TIMP to again bind to the active site and re-inhibit the enzyme. The action of factor H on the SIBLING-mediated activation of proMMPs was also investigated. Addition of factor H caused the rate of substrate digestion by SIBLING-activated proMMP complex to decrease suggesting that removal of SIBLING from proMMP resulted in re-inactivation of catalytic activity by still-attached propeptide. These results support the hypothesis that propeptide is not removed in order to create enzymatic activity in proMMP-SIBLING pairs.
CONCLUSIONS AND SIGNIFICANCE
Results describe a novel, alternative method of MMP modulation (Fig. 2
). SIBLING binding was associated with activation of latent pro-MMPs though this activation did not require cleavage of the propeptide. However, SIBLING binding did increase susceptibility of the propeptide to cleavage by plasmin. SIBLINGs and MMPs showed specificity in their partnering, with BSP binding to and "activating" proMMP-2, OPN with proMMP-3, and DMP1 with proMMP-9. Restoration of activity to TIMP-inhibited MMPs upon SIBLING binding demonstrates that even in presence of TIMPs, MMPs may be enzymatically active in regions of locally high concentrations of specific SIBLINGs.. The observation that complement factor H can compete with MMPs for SIBLINGs and successfully strip the SIBLING from the MMP complex suggests that activation of proMMP or reactivation of TIMP-inhibited MMPs by simple binding of their respective SIBLINGs will be limited to short distances from their sites of secretion due to abundance of factor H in the body.
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SIBLING expression has been correlated with cancer progression and severity and it is interesting to consider that these proteins may be locally activating their corresponding proteases in vivo. From a clinical standpoint, SIBLINGs were found to restore activity to propeptide-free MMPs whose activity had been blocked by both natural and synthetic inhibitors. SIBLINGs are induced by neoplasms in vivo and their modulation of MMP activity might contribute to the relative lack of efficacy seen in recent clinical trials of MMP inhibitors in numerous cancers.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0966fje; doi: 10.1096/fj.03-0966fje 
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