Dentin Sialoprotein is a Novel Substrate of Matrix Metalloproteinase 9 in vitro and in vivo

Dentin sialoprotein (DSP) is essential for dentinogenesis and processed into fragments in the odontoblast-like cells and the tooth compartments. Matrix metalloproteinase 9 (MMP9) is expressed in teeth from early embryonic to adult stage. Although MMP9 has been reported to be involved in some physiological and pathological conditions through processing substrates, its role in tooth development and whether DSP is a substrate of MMP9 remain unknown. In this study, the function of MMP9 in the tooth development was examined by observation of Mmp9 knockout (Mmp9−/−) mouse phenotype, and whether DSP is a substrate of MMP9 was explored by in vitro and in vivo experiments. The results showed that Mmp9−/− teeth displayed a phenotype similar to dentinogenesis imperfecta, including decreased dentin mineral density, abnormal dentin architecture, widened predentin and irregular predentin-dentin boundary. The distribution of MMP9 and DSP overlapped in the odontoblasts, the predentin, and the mineralized dentin, and MMP9 was able to specifically bind to DSP. MMP9 highly efficiently cleaved DSP into distinct fragments in vitro, and the deletion of Mmp9 caused improper processing of DSP in natural teeth. Therefore, our findings demonstrate that MMP9 is important for tooth development and DSP is a novel target of MMP9 during dentinogenesis.


Mmp9−/− mouse teeth show severe cusp wear and increased reactionary dentin (RD) formation.
To address the role of MMP9 for tooth development, we first examined the morphology of Mmp9− /− teeth from 1.5 months old (M1.5) to M13.5 by stereomicroscopy and histology. At M1.5, only a very slight wear of molar cusp was seen in the control and Mmp9− /− molars, and no RD was seen in either group (Fig. 1A,B). At M4.5 and M7.5, Mmp9− /− mice showed more severe wear of molar cusps ( Fig. 1C-I). In response to severe wear, the amount of RD in Mmp9− /− molars was apparently increased compared to the control (Fig. 1J-O). At M13.5, the cusp wear progressed to the depth of RD in Mmp9− /− mice, but thick dentin tissue remained on the top of RD in the control mice (Fig. 1P,Q).

Abnormal mineralization and structures of Mmp9−/− teeth.
To further understand why severe cusp wear occurred in Mmp9− /− molars, possible changes in the mineralization and structures of Mmp9− /− teeth were examined by radiography and SEM. Decreased mineral density of the dentin and the enamel in the Mmp9− /− molars was seen from M0.5 to M4.5 by radiography ( Fig. 2A-F). SEM analysis showed that the molar enamel surfaces in the M4.5 Mmp9− /− mice had abnormal fissures (Fig. 2G,H,G' ,H'). Meanwhile, the dentinal tubules and inter-tubular dentin were uniformly distributed in the wild-type mice, but sparsely scattered at an obviously impaired density in Mmp9− /− mice. Numerous abnormal "holes" were also observed in the Mmp9− /− dentin ( Fig. 2I-L). Then, NMR proton spin-spin (T2) relaxation time measurement was applied to analyze the porosity and pore size of molars at M1.5 and M2.5 23 . Inversion NMR T2 relaxation time spectra showed that Mmp9− /− molars had larger porosity and pore size compared to the wild-type control (Fig. 2M,N). These results indicated that teeth in Mmp9− /− mice had not only reduced mineral content but also abnormal architecture.
In addition, examination of the mandibles by radiography also identified alveolar bone defects in Mmp9− /− mice ( Fig. 2C-F). The mineral density in the furcation region of mandibular molars decreased in Mmp9− /− mice, which is most notable when comparing the 4.5-month (Fig. 2E,F) samples. The significant alveolar bone loss was confirmed by hematoxylin and eosin (HE)-stained tissue sections ( Supplementary Fig. 1).

Delayed differentiation of odontoblasts, widened predentin, and irregular mineralization front
in Mmp9−/− mice. To further observe the function of MMP9 during the odontoblast differentiation and secretion at earlier stages, Mmp9− /− teeth at D1 and D19 were analyzed using histological method. In wild-type mice at D1, the odontoblasts were well polarized and elongated at the cusp tip region, where the deposition of the predentin matrix was clearly discernible (Fig. 3A). In contrast, the polarization and elongation of odontoblasts were delayed, and the thickness of predentin at the cusp area was reduced in Mmp9− /− molars at D1 (Fig. 3B). At D19, increased predentin width and irregular predentin-dentin boundary was seen in Mmp9− /− molars versus the control (Fig. 3C,D).
Overlapping expression of MMP9 and DSP in the odontoblast-like cells and the developing teeth. MMP9 and MMP2 belong to gelatinase subgroup of MMP family and share many common Scientific RepoRts | 7:42449 | DOI: 10.1038/srep42449 substrates 24 . DSP is critical for dentinogenesis and a substrate of MMP2 in the porcine molars 7,12 . Therefore, we hypothesized that DSP might act as a substrate of MMP9, which may explain the dentin phenotype of Mmp9− /− mice. To assess whether MMP9 is able to cleave DSP, we first confirmed co-distribution of DSP and MMP9 proteins in the mouse odontoblast-like (MO6-G3) cells and tooth tissues. Double immunofluorescence experiments showed that expression of MMP9 and DSP overlapped in the cytoplasm of MO6-G3 cells ( Fig. 4A-D). Immunohistochemistry showed that in molars at D1, DSP and MMP9 were both highly expressed in the odontoblasts (Fig. 4E,F). At D5 and D15, signals of DSP and MMP9 were intense in the odontoblasts and the predentin, and mild in the mineralized dentin ( Fig. 4G-J,G'-J').
Binding between DSP and MMP9. We next determined whether MMP9 was able to bind to DSP. First, rDSP fusion protein was generated, purified, and confirmed by Coomassie Blue staining and Western blot assays using anti-DSP and anti-GST antibodies as described previously 16 . Then the rDSP protein was labeled with biotin and serial dilutions of the biotinylated rDSP were incubated with either mut-rMMP9 or BSA. The results showed that rDSP bound specifically to MMP9 in the dose-and time-dependent manner whereas there was no binding effect on control BSA (Fig. 5A,B).

DSP processed by MMP9 in vitro.
To verify that rMMP9 was successfully activated and had the ability to cleave its substrates, gelatin zymography was first performed with rMMP9 and mut-rMMP9. Our data showed that rMMP9 efficiently digested gelatin with two clear bands at the molecular weight of 92 kDa and 86 kDa, whereas mut-rMMP9 failed to do so (Fig. 5C). Next, to determine whether the activated rMMP9 was able to process DSP protein, the rDSP was incubated alone or with rMMP9 for different time periods. The results showed that rDSP was cleaved by rMMP9 into three major fragments, whereas rDSP alone remained intact and stable ( Fig. 5D-F). Western blot analyses demonstrated that the intact rDSP and the two cleaved fragments at high molecular weight (HMW) were recognized by anti-DSP-NH 2 antibody (Fig. 5E), and the intact rDSP and the smallest cleaved rDSP product were detected by anti-DSP-COOH antibody (Fig. 5F). The cleaved products were quantified when digested for 0, 0.5, 1, 2, 3 and 6 h with initial rDSP substrate as 100%. The results showed that approximately 50% of the substrate was cleaved after 30 min of incubation, and the cleavage reaction was almost complete after 2 h. The products reached their maximum amount at 1 h, and were further processed by rMMP9 when incubated longer resulting in reduced remnant quantity (Fig. 5G).  The dentin tubules (arrowheads) and the intertubular dentins were well-distributed in the wild-type mice, but in the Mmp9− /− mice the distribution of the dentine tubules (arrowheads) and the intertubular dentins were not uniform with decreased number of dentin tubules and numerous "holes" (*) in the intertubular dentins. K and L are higher magnifications of the rectangles in (I and J), respectively. (M,N) Inversion T2 relaxation time spectra for the first mandibular molars of the wild-type and the Mmp9− /− mice at M1.5 and M2.5. The lines with "dots" are for the Mmp9− /− molars and the lines with "triangles" are for the wild-type molars. Longer T2 relaxation time and larger area under the curve was found in the Mmp9− /− molars. The longer T2 relaxation time is corresponding to larger pores, and the larger area is corresponding to larger porosity (larger amount of holes). WT, wild-type.   To assess the catalytic efficiency of MMP9, steady state cleavage velocities were measured with a constant amount of rMMP9 and varying amounts of DSP substrate. As expected, the enzymatic reaction displayed reaction kinetics within a Michaelis-Menten analysis. An Eadie-Hofstee plot of these results showed Michaelis-Menten parameters were K m = 1.57 mM and kcat = 230 (s −1 ), yielding a relative catalytic efficiency (kcat/K m ) of 146, 500 M −1 s −1 (Fig. 6A). Similar to rDSP, MMP9 also had high efficiency in catalyzing a known fluorescent MMP9 substrate as a positive control (Fig. 6B) 25 . These results demonstrated that DSP was a novel substrate of MMP9.
To further identify the specific cleavage sites of DSP by MMP9, the three cleaved rDSP products were analyzed by mass spectrometry. The sites of DSP cleavage by MMP9 were determined as indicated in Fig. 6C-F. These results indicated that MMP9 selectively cleaved DSP protein.

DSP processed by MMP9 in vivo.
To analyze whether DSP was a substrate of MMP9 in vivo, immunohistochemistry with anti-DSP-NH 2 and -COOH specific antibodies were performed in Mmp9− /− first mandibular molars at D20 and M1.5 with wild-type first mandibular molars in parallel as control. Our results showed that immunoreactions for anti-DSP-NH 2 antibody were intense in the predentin matrix and odontoblasts, but mild in the mineralized dentin ( Fig. 7A-D,A'-D'); for anti-DSP-COOH antibody, immunoreactions were apparently strong in the mineralized dentin, but weak in the predentin and odontoblasts ( Fig. 7E-H,E'-H'). DSP signals recognized by both antibodies were more prominent in Mmp9− /− than control molars ( Fig. 7A-H,A'-H').
To further identify protein profiles of DSP fragments, proteins were isolated from wild-type and Mmp9− /− mouse teeth at D15 and Western blots were conducted using anti-DSP-NH 2 and -COOH antibodies. The results showed different expression patterns of DSP in the wild-type and Mmp9− /− teeth. For anti-DSP-NH 2 antibody, several high HMW bands (ranging from 95 to over 250 kDa) were seen in the Mmp9− /− but not in wild-type tooth proteins. Several low molecular weight (LMW) DSP bands (lower than 95 kDa) were detected in the wild-type and the Mmp9− /− teeth, but at a higher molecular weight in the Mmp9− /− teeth (Fig. 7I). For anti-DSP-COOH antibody, one additional HMW DSP band was observed in Mmp9− /− teeth but not in wild-type teeth (Fig. 7J). The ratio of the LMW fragments to the HMW DSP was significantly lower in the Mmp9− /− teeth compared to the control (Fig. 7K). Thus, MMP9 activity was responsible for DSP processing in natural dental tissues.
Osteopontin (OPN) has been reported to be localized in tooth tissues including predentin, dentin and RD 11 . And OPN is known as a substrate of MMP9 26 . Therefore, OPN expression changes in the Mmp9− /− mice from D17 to M7.5 were investigated by immunohistochemistry. OPN showed a higher expression level in the predentin, dentin and RD in the Mmp9− /− molars ( Supplementary Fig. 2).

Discussion
Heterogeneous mutations in DSP coding domain are associated with human hereditary dentin defects 7 . MMP9 and DSP are both highly expressed by the odontoblasts and secreted to the dentin ECM (Fig. 4) 11,22 . In this study, to gain insights of the role of MMP9 in the tooth development and whether DSP acts as a substrate of MMP9, we  Our results showed that Mmp9− /− mice displayed phenotype similar to human DGI, including severe tooth wear, decreased dentin mineralization, delay of the odontoblast differentiation, and abnormal dentin structures. MMP9 was able to bind to and specifically process DSP into given fragments in vitro. In vivo DSP processing was affected in the Mmp9− /− teeth, which may mainly account for the dentin defects in the Mmp9− /− mice.
Previous studies have reported developmental abnormality of long bone in the mice deficient of Mmp9 and in humans carrying Mmp9 mutations [19][20][21] . Bone and dentin share many similarities in composition and mechanisms of formation 2 . Therefore, we hypothesized that dentin formation might be affected by deletion of Mmp9. In this study, we investigated the tooth phenotype of the Mmp9− /− mice by multiple methods including stereomicroscopy, histology, radiography, SEM and NMR. Mmp9− /− molars from M4.5 to M13.5 displayed more severe tooth cusp wear by stereomicroscopy and histology (Fig. 1), suggesting that the resistance to masticatory mechanical strength of Mmp9− /− molars diminished. SEM and radiography experiments did show abnormal appearance of dentin structure, and reduced dentin mineral density in the Mmp9− /− mice. In addition, the enamel in the Mmp9− /− molars was observed abnormal with fissures on the surface and showed reduced mineral density. Meanwhile, larger porosity and pore size were detected in the Mmp9− /− teeth by NMR (Fig. 2). Therefore, reduced mineral content of the enamel and abnormal tooth architecture may explain why Mmp9− /− teeth had lower mechanical resistance to masticatory forces. In this study, we focused on the dentin formation of the Mmp9− /− mice.
In response to severe tooth wear, more amount of RD was formed in Mmp9− /− molars from M4.5 to M13.5 compared to control molars at the corresponding ages (Fig. 1), suggesting that odontoblasts in Mmp9− /− molars are active in secreting dentin matrix to protect themselves from exposure to bacterial environment in oral cavity. This indicates that the capability of odontoblasts to synthesize and secret dentin ECM under masticatory force stimuli was not inhibited by deficiency of Mmp9.
The expression of MMP9 in the early embryonic dental epithelium and mesenchyme suggests a potential role of MMP9 in tooth morphogenesis 27 . However, Mmp9− /− molars and incisors did not show obvious morphological changes, indicating that MMP9 is dispensable for tooth morphogenesis. The delayed differentiation of odontoblasts in the Mmp9− /− molars (Fig. 3) is consistent with the expression of MMP9 in presecretory and secretory odontoblasts (Fig. 4). Later, MMP9 is present in the predentin and the dentin matrix ( Fig. 4) 22 , suggesting that MMP9 may be involved in the dentin mineralization through processing and degrading dentin ECM, in accordance with which widened predentin and irregular boundary between predentin and dentin were detected in Mmp9− /− molars at D19 (Fig. 3).
The above experiments indicated the necessity of MMP9 activity for odontoblast differentiation and dentin formation, and showed that Mmp9− /− mice exhibited similar tooth phenotype to humans carrying DSP mutations [28][29][30] . Since DSP is processed into fragments in the odontoblast-like cells and the tooth compartment 11,13 , further studies were carried out to determine whether MMP9 participated in the DSP processing. Immunostaining experiments confirmed the overlapping distribution of MMP9 and DSP in the odontoblast-like MO6-G3 cells, and in the odontoblasts and dentin ECM of mouse molars (Fig. 4). DSP but not BSA bound to MMP9 in the dose-and time-dependent manner (Fig. 4). The overlapping localization and direct physical interaction between DSP and MMP9 are two prerequisites for processing of DSP by MMP9. Next, in vitro studies revealed that MMP9 selectively cleaved DSP protein into three major fragments (Fig. 5). Enzyme kinetics showed that MMP9 had a high efficiency for processing DSP with a turnover of (kcat/K m ) of 146,000 M −1 s −1 (Fig. 6). This value is comparable to other substrates cleaved by MMP9 31 . For in vivo study, Mmp9− /− molars showed elevated expression levels of both DSP NH 2 -terminal and COOH-terminal fragments by immunohistochemistry (Fig. 7), suggesting that the processing of DSP was reduced in Mmp9− /− molars. Western blot assay was further performed and confirmed that expression patterns of DSP fragments were different between the control and the Mmp9− /− teeth. Several HMW DSP fragments were present in Mmp9− /− teeth but not in control molars and the ratio of the LMW DSP fragments to the HMW DSP was lower in the Mmp9− /− teeth (Fig. 7), assuring that DSP processing in vivo was affected by deletion of Mmp9. DSP is well known as a critical protein for proper dentin formation. Thus, the lack of appropriate DSP processing is likely the most important contributor to the dentin phenotype in Mmp9− /− teeth.
In addition to dentin defects, we revealed severe loss of alveolar bone surrounding Mmp9− /− molar roots, especially in the furcation region of the mandibular molars ( Fig. 2 and Supplementary Fig. 1). The alveolar bone phenotype in Mmp9− /− mice is similar to Dspp− /− mice 10 . Like in dentin, DSPP is also proteolytically processed into DSP and DPP in bone. Processing of DSPP is essential to its biological function in alveolar bone, for disruption of DSPP processing into the given fragments failed to rescue the alveolar bone defect of Dspp− /− mice 9,10 . Therefore, in addition to dentin, the further cleavage of DSP by MMP9 might also exist in the alveolar bone, which is critical for alveolar bone remodeling. Further studies are needed to verify this possible underlying mechanism of alveolar bone loss in Mmp9− /− mice in the future.
OPN and DSP are both members of SIBLING (Small Integrin-Binding Ligand N-linked Glycoproteins) family. OPN shares many common characteristics with DSP 32 and is known as a substrate of MMP9 26 . OPN has been reported in predentin, dentin and RD by our previous study 11 . As expected, improper processing and degradation of OPN in Mmp9− /− molars was documented by remarkably elevated protein level of OPN ( Supplementary Fig. 2). Both in vitro and in vivo studies have shown that OPN is a potent inhibitor of hydroxyapatite crystal formation and growth 33,34 . However, the teeth of Opn deficient mice were found morphologically normal at the level of light and electron microscopy 35 . Therefore, compared to DSP, increased OPN protein might play a minor role for the dentin defects in Mmp9− /− molars. The decreased processing of OPN acts as a positive control for the improper processing of DSP in Mmp9− /− teeth.
Taken together, DSP is critical for tooth development, and DSP is a novel target of MMP9 during dentinogenesis. Like other MMPs, MMP9 was originally discovered to function in the breakdown of ECM. However, Scientific RepoRts | 7:42449 | DOI: 10.1038/srep42449 recent studies have identified intracellular proteins as MMP9 substrates [36][37][38] . The overlapping prominence of DSP and MMP9 in the cytoplasm of MO6-G3 cells and the existence of multiple LMW DSP fragments in MO6-G3 cell lysate suggest that the cleavage of DSP by MMP9 may be initiated inside odontoblasts 11 . Further studies are needed to determine the mechanisms of intracellular MMP9 activation and its cleavage of DSP to provide deeper insights into the processing of DSP by MMP9 during dentinogenesis.

Methods
Animal preparation. The protocol of animal use was approved by the Animal Welfare Committee at the University of Texas Health Science Center at San Antonio (UTHSCSA, Protocol No. 05012x). All experiments were performed in accordance with the relevant guidelines and regulations. The Mmp9− /− mice used in this study were originally generated and described by Vu and colleagues 19 . Genotyping was performed by PCR of genomic DNA extracted from tail snips (Supplementary Fig. 3). The tissue sections from the wild-type and Mmp9− /− mice were immunostained with anti-MMP9 antibody. MMP9 expression was seen in the wild-type tooth tissues, but not in Mmp9− /− mice (Supplementary Fig. 3). At least three mice for each time point were sacrificed and taken for the following analyses.
Stereomicroscopy, X-radiography and low-field nuclear magnetic resonance (NMR). To compare the overall morphology of wild-type and Mmp9− /− teeth, mice at the ages from M0.5 to M7.5 were put to death. Mandibles at M4.5 and M7.5 were observed under stereomicroscope. Mandibles at M4.5 were incubated in lysis buffer (2 × SSC, 0.2% SDS, 10 mM EDTA, 10 mg/ml proteinase K) for 2 d. After muscles surrounding teeth were digested, the molars were extracted and observed under stereomicroscope.
To compare the mineralization of teeth in Mmp9− /− mice with that in wild-type mice, mandibles from M0.5 to M4.5 were examined using a Faxitron radiograph inspection unit (Field X-ray Corporation, Lincolnshire, IL, USA).
To analyze the structural changes of Mmp9− /− teeth, the extracted first mandibular molars at M1.5 and M2.5 were processed for NMR measurement and compared with control as described previously 23 .

Histology and immunohistochemistry.
To evaluate the histological features of Mmp9− /− teeth, fresh mandibles at ages from D1 to M13.5 were fixed in 4% paraformaldehyde for 1-2 d at 4 °C, and demineralized in 8% EDTA for 1 d to 8 wk depending on age. Then, the tissues were processed for paraffin embedding and 5-μ m serial sections were prepared and stained with HE.
To detect the distribution of MMP9, DSP, and OPN in mouse mandibular first molars, immunohistochemistry was performed as previously described 11 . Primary antibodies included goat anti-MMP9 antibody (Santa Cruz Biotechnology, CA, USA), two rabbit anti-DSP antibodies specific to the NH 2 and COOH domains described previously 11 , and goat anti-OPN antibody (R & D systems, MN, USA). Normal goat or rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology, CA, USA) was employed as negative control (data not shown).
For double immunofluorescence, mouse odontoblast-like (MO6-G3) cells were grown on glass slides, rinsed with ice-cold phosphate-buffered saline (PBS), and fixed for 10 min on ice with methanol/acetone (1:1). Then, the cells were processed with blocking buffer for 1 h at room temperature (RT), and two primary antibodies were incubated simultaneously overnight at 4 °C (1:200 for rabbit anti-DSP antibody (M-300), 1:50 for goat anti-MMP9 antibody). After washes, slides were incubated with the secondary antibodies conjugated with Alexa Fluo ® 486 green and Alexa Fluo ® 568 red (1:500; Molecular Probes, OR, USA) for 1 h at RT. For nuclear staining, the cells were treated with Hoechst (Sigma-Aldrich, MO, USA).

Scanning electron microscopy (SEM). Mandibular incisors from
Mmp9− /− and wild-type mice were fractured at the level of the labial alveolar crest. The first mandibular molars and the fractured incisor surfaces were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer. After washes, the samples were dehydrated in an ascending alcohol series, allowed to air dry, and then sputter-coated with gold for conventional SEM (JEOL, JSM 6610 LV; JEOL, Inc., Peabody, Mass., USA).

Extraction of proteins from mouse teeth and detection of DSP.
Molars from wild-type and Mmp9− /− mice were extracted as described above and tooth proteins were isolated by procedures described previously 6 . Extracted molars were crushed and incubated with 4 M guanidine HCl solution containing protease inhibitor (Sigma-Aldrich, MO, USA) overnight. The residue was then demineralized with 4 M guanidine HCl, 0.5 M EDTA plus protease inhibitors for 2 d. The solution was dialyzed against 4 M guanidine for 1 d, and against distilled water for another 2 d. Dialyzed solution was lyophilized and dissolved into distilled water. Protein concentrations were then measured.
Same amount of protein from wild-type and Mmp9− /− molars was loaded and subjected to Western blot assay.
Expression and purification of recombinant DSP and MMP9. The cDNA coding for recombinant DSP (rDSP) was subcloned into pGEX-6P1 vector tagged with a NH 2 -terminal GST (GE Healthcare Biosciences, NJ, USA), termed GST-rDSP. To obtain mutant recombinant MMP9 protein (mut-rMMP9) with ligand-binding property but without catalytic activity, point mutation of the active site Glu 402 of proMMP9 to alanine residue was generated as we previously described 39 . The constructs of GST-rDSP and mut-rMMP9 were transformed to E. coli BL21 (DE3). GST-rDSP and mut-rMMP9 proteins were induced and purified 16  Biotinylation of rDSP and substrate binding assay. For probing protein-protein interactions, rDSP protein at the concentration of 300 μ g/ml was dialyzed against 0.1 M NaHCO 3 and then reacted with 100 μ g/ ml Sulfo-NHS (N-hydroxysuccinimido)-LC (long-chain)-biotin (Pierce, Rockford, IL, USA) for 20 min at RT, followed by 2 h at 4 °C. Free biotin was removed by dialysis against 50 mM Tris-HCl and 150 mM NaCl, pH 7.4. To characterize the relative rDSP interaction with MMP9, substrate binding assay was performed as described earlier 40 . Briefly, 96-microwell plates were coated with 1 μ g/well of mut-rMMP9 or bovine serum albumin (BSA) as control overnight at 4 °C and non-specific binding were blocked with 1% BSA (Sigma-Aldrich, MO, USA) for 1 h at RT. After thorough rinses with PBS, the biotinylated rDSP ranged from 0-18 fold molar excesses in PBS with 1% BSA was added into the plates and incubated for 30 min. Bound rDSP was reacted with AP-conjugated streptavidin diluted 1:10,000 in PBS for different time points using 1 mg/ml PNPP (ρ -nitrophenyl phosphate disodium) as substrate (Pierce, Rockford, IL, USA) and quantified at 405 nm (Opsys MR, Dynex, Chantilly, VA, USA). BSA was used as control group and the binding of rDSP to mut-rMMP9 was expressed as relative binding activity compared to the control group. The experiments were performed three times in triplicate.

Processing of rDSP by rMMP9 in vitro.
To quantify MMP9 activity, rMMP9 and mut-rMMP9 were To assay the role of rMMP9 on rDSP processing, rDSP was incubated alone or in combination with P-aminophenylmercuric acetate (APMA)-activated rMMP9 (enzyme : substrate ratio of 1:20) in reaction buffer (50 mM Tris/HCl, 200 mM NaCl, 5 mM CaCl 2 , 20 mM ZnSO 4 , 0.05% Brij 35, pH 7.6). The reactions were carried out at 37 °C for various time intervals and stopped by adding 2 x SDS-PAGE sample buffer containing 2-mercaptoethanol. Samples were boiled for 5 min and subjected to SDS-PAGE and stained with Sypro Ruby dye. To detect DSP fragments in the digestion mixture more specifically, Western blot assay was performed using anti-DSP-NH 2 and -COOH antibodies.
Analysis of enzyme kinetics. Kinetics constants Km and kcat were determined from an Eadie-Hofstee plot of initial velocities under multiple turnover conditions 31 . Mixtures of the rMMP9 (13.6 nM) and substrate rDSP (0.25, 0.5, 1.0, 2.5, 5.0, 10 μ M) were incubated in reaction buffer for 30 min at 37 °C. The reaction products were run on SDS-PAGE gel, stained with silver dye, and quantified with ImageJ. All experiments were performed in triplicate.
Mass spectrometry. Twelve μ g of purified rDSP were incubated with 0.6 μ g of APMA-activated rMMP9 at 37 °C for 1 h in reaction buffer. Products were run on SDS-PAGE gel and visualized by Sypro Ruby dye staining. Then the products were excised from the gel, digested with trypsin, and the peptide mixtures were extracted from the gel as described by Kerkhoff et al. 41 . MALDI-mass spectrometry was performed using a TofSpec-2E instrument (Micromass, Manchester, UK) provided by Protein Core Facility of UTHSCSA. Statistical analysis. Quantitative data were presented as means + S. D. from three independent experiments and analyzed with Student' s t test. The differences between groups were statistically significant when P < 0.05. For the Western blot, densitometry of immunoblot bands was collected and relative quantification was processed with the ImageJ.