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. 2019 Jul 23;9(1):10625.
doi: 10.1038/s41598-019-47140-5.

Tyrosine 51 residue of the syndecan-2 extracellular domain is involved in the interaction with and activation of pro-matrix metalloproteinase-7

Bohee Jang  1 , Ji-Hye Yun  2 , Sojoong Choi  1 , Jimin Park  3 , Dong Hae Shin  3 , Seung-Taek Lee  2 , Weontae Lee  4 , Eok-Soo Oh  5
Affiliations

Tyrosine 51 residue of the syndecan-2 extracellular domain is involved in the interaction with and activation of pro-matrix metalloproteinase-7

Bohee Jang et al. Sci Rep. .

Abstract

Although syndecan-2 is known to interact with the matrix metalloproteinase-7 (MMP-7), the details of their interaction were unknown. Our experiments with a series of syndecan-2 extracellular domain deletion mutants show that the interaction is mediated through an interaction of the extracellular domain of syndecan-2 (residues 41 to 60) with the α2 helix-loop-α3 helix in the pro-domain of MMP-7. NMR and molecular docking model show that Glu7 of the α1 helix, Glu32 of the α2 helix, and Gly48 and Ser52 of the α2 helix-loop-α3 helix of the MMP-7 pro-domain form the syndecan-2-binding pocket, which is occupied by the side chain of tyrosine residue 51 (Tyr51) of syndecan-2. Consistent with this notion, the expression of a syndecan-2 mutant in which Tyr51 was changed to Ala diminished the interaction between the syndecan-2 extracellular domain and the pro-domain of MMP-7. Furthermore, HT-29 colon adenocarcinoma cells expressing the interaction-defective mutant exhibited reductions in the cell-surface localization of MMP-7, the processing of pro-MMP-7 into active MMP-7, the MMP-7-mediated extracellular domain shedding of both syndecan-2 and E-cadherin, and syndecan-2-mediated anchorage-independent growth. Collectively, these data strongly suggest that Tyr51 of the syndecan-2 extracellular domain mediates its interaction with and activating processing of pro-MMP-7 and regulates MMP-7-dependent syndecan-2 functions. VSports手机版.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
The N-terminus of syndecan-2 interacts with the pro-domain of MMP-7. (A) Schematic representation of the syndecan-2 core protein (SDC2) and MMP-7. The signal peptide (SP), the extracellular domain (EC), the transmembrane domain (TM), and the cytoplasmic domain (CT) of syndecan-2 are noted, and the various deletion mutants are indicated. A peptide corresponding to residues 41–60 of the syndecan-2 extracellular domain (S2-P) was synthesized. Syndecan-2 is labeled with amino acid numbers to show the location of each deletion (left). Schematic representation of MMP-7. The pre-domain (Pre), pro-domain (PD), and catalytic domain are shown (right top). Purified GST-SDC2 mutants and the His-tagged pro-domain of MMP-7 were separated by 15% SDS-PAGE and stained with Coomassie Blue (right bottom). (B) Purified GST or GST-SDC2 mutants were incubated with His-tagged pro-domain of MMP-7 (His-PD). Bound materials were subjected to immunoblotting with an anti-His tag antibody (top). The membranes were then stripped and re-probed with an anti-GST antibody (bottom). (C) Purified GST-SDC2 was incubated with purified His-PD MMP-7 plus the indicated amounts of S2-P for 2 h at 4 °C. Bound materials were subjected to immunoblotting with an anti-His tag antibody (top). The membranes were then stripped and re-probed with an anti-GST antibody (bottom). M and D indicate monomer and dimer of syndecan-2, respectively. (D) Fluorescence spectroscopy indicates the binding affinity between His-PD and S2-P peptide. Titration between His-PD and S2-P peptide was performed up to 1 by 200 molar ratios and the Kd value was calculated as 1.586 ± 0.012 mM.
Figure 2
Figure 2
The pro-domain of MMP-7 is involved in its interaction with the syndecan-2 extracellular domain. (A) Schematic representations of the pro-domain of MMP-7 (PD) and the deletion mutants lacking the pro-domain, N-terminus (ΔN), C-terminus (ΔC) and both N- and C-terminus (ΔNC) (top). His-tagged MMP-7 pro-domain was purified with Ni-NTA agarose beads, separated by SDS-PAGE and stained with Coomassie Blue (bottom). (B) Purified GST-S2E, S2E-C and S2E-NII were incubated with His-tagged PD, or ΔN, ΔC or ΔNC MMP-7 pro-domain. Materials bound to glutathione-agarose beads were immunoblotted with an anti-His tag antibody (top). The membranes were then stripped and re-probed with an anti-GST antibody (bottom). (C) ELISA plates coated with 600 ng of S2-P were incubated with the indicated His-tagged MMP-7 pro-domains (500 ng/well) for 2 h at 20 °C. The plates were washed, incubated with an anti-His tag antibody followed by IgG-HRP, and developed with TMB-ELISA. Absorbance was measured at 450 nm. Data are shown as mean ± S.D. (n = 3), *p < 0.05 versus blank. (D) Purified GST-SDC2 was mixed with His-tagged ΔNC for 2 h at 4 °C., separated by SDS-PAGE, and immunoblotted with anti-His or -GST antibodies. (E) The secondary structures of the MMP-7 pro-domain mutants of ΔN, ΔC and ΔNC were diluted to 50 μM in a buffer consisting of 10 mM HEPES and 20 mM NaCl, pH 8, and kept at 20 °C. The TFE-induced helicity curves were obtained by recording the CD signal for each independent sample from 0% to 40% TFE (100 mM NaCl, pH 6, 20 °C) with far-UV.
Figure 3
Figure 3
Chemical shift perturbations of the 1HN and 15N resonances of proMMP-7 ΔNC upon binding the human syndecan-2 peptide ligand. (A) 1H-15N HSQC spectrum of proMMP-7 ΔNC in the absence and presence of S2-P. The spectra were collected at pH 6 using Bruker 850 and 900 MHz spectrometers, and NMR titrations were performed with 15N-labeled proMMP-7 ΔNC and S2-P at a molar ratio of 1:15. The backbone resonance assignments are shown in red (1:0) and blue (1:15). (B) The chemical shift perturbations of proMMP-7 ΔNC in the absence and presence of S2-P were assessed by overlapping the three titration points and performing interaction-site mapping of the proMMP-7/S2-P peptide complex using NMR titration (top). The average chemical-shift changes were calculated using the following formula: ΔδAV = [(Δδ1H)2 + (Δδ15N/5)2]1/2, where ΔδAV, Δδ1H, and Δδ15N represent the average chemical shift value, proton chemical shifts, and nitrogen chemical shift changes, respectively. The highly perturbed residues, Gln 7, Glu 32, Gly 48, and Ser 52, are marked in red. The chemical-shift movements of these residues were plotted in overlapped HSQC analyses (bottom). (C) The NMR structure of the pro-domain of human MMP-7 (PDB; 2MZE) (shown in magenta) and the modeled structure of proMMP-7 ΔNC (shown in cyan) were overlapped and visualized using a ribbon diagram (left). The side-chains of the four highly perturbed residues (Gln 7, Glu 32, Gly 48, and Ser 52) are shown as balls (center). HADDOCK model obtained for the proMMP-7/S2-P complex based on our NMR titration data, including the predicted binding site for S2-P on the pro-domain of proMMP-7 (right).
Figure 4
Figure 4
The α2–3 helix of the MMP-7 pro-domain is involved in its interaction with the syndecan-2 extracellular domain. (A) Structure of the MMP-7 pro-domain (PDB; 2MZE) and schematic diagram of the MMP-7 pro-domain (PD) and its deletion mutants. The indicated MMP-7 pro-domain mutants were purified with Ni-NTA agarose beads, separated by SDS-PAGE. Purified MMP-7 pro-domain mutants were incubated with GST-SDC-2. Materials bound to glutathione-agarose beads were immunoblotted with an anti-His tag antibody and the membranes were stripped and re-probed with an anti-GST antibody. Image is of a single membrane cropped to remove intervening lanes. (B) Purified GST-SDC2 mutants were incubated with His-tagged α2–3 helix of the MMP-7 pro-domain. Materials bound to glutathione-agarose beads were immunoblotted with an anti-His tag antibody (top) and the membranes were then stripped and re-probed with an anti-GST antibody (bottom). (C) The secondary structure of the α2–3 helix of the MMP-7 pro-domain was analyzed using CD in the presence of different concentrations of TFE (top). Quantitative estimations of the secondary-structure content were made with the CDPro software package, which includes the programs CDSSTR, CONTIN, and SELCON3. The α-helical fractions were extracted from the CDPro calculations based on empirical methods with ellipticities set at 208 or 222 nm (bottom). Smaller values of NRMSD indicate closer correspondence between calculated structures and the experimental data.
Figure 5
Figure 5
Tyrosine 51 of the syndecan-2 extracellular domain is involved in the interaction of syndecan-2 with the pro-domain of MMP-7. (A) A docking structure for the complex formed between ΔNC (sky blue) and S2-P (dodger blue), as generated by the HEX 6.3 program. Docking states were generated using electrostatic and shape interactions (a), or with shape interactions alone (b). Purified GST-SDC2 or its mutants were incubated with His-ΔNC for 2 h at 4 °C. Materials bound to glutathione-agarose beads were immunoblotted with the indicated antibodies (a or b right). (B) The HADDOCK program was used to generate the docking structure of ΔNC and S2-P. The residues identified as being important matched those identified in our NMR titration experiments. The residues lining the hydrophobic cavity are drawn with a stick model (left). Purified GST-SDC2 and the Y51A mutant were incubated with His-ΔNC for 2 h at 4 °C and the materials that bound to the glutathione-agarose beads were immunoblotted with the indicated antibodies (right). (C) Control HT-29 cells (VEC) and HT-29 cells transfected with vectors encoding SDC2 or mutants were immunostained with anti-syndecan-2 or anti-MMP-7. The results were visualized with Texas Red-conjugated goat anti-rabbit (red) or FITC-conjugated goat anti-mouse (green). DAPI was used to stain nuclei (blue). Scale bar, 20 μm.
Figure 6
Figure 6
Tyrosine 51 of the syndecan-2 extracellular domain is involved in regulating pro-MMP-7 activation. (A) HT-29 cells were transiently transfected with 1 µg of vectors encoding SDC2 or the interaction-defective syndecan-2 mutant, Y51A, and the mRNA expression levels of SDC2 and MMP-7 were evaluated by RT-PCR (top). Conditioned media (CM) were collected and proteolytic activity was measured using quenched fluorescence peptide cleavage assay. The relative activity was normalized versus the fluorescence of a vector control (bottom). Data are shown as mean ± S.D. (n = 3), **p < 0.01 versus VEC or SDC2. (B) HT-29 cells were stably transfected with vectors encoding SDC2 or Y51A. The expression levels of the target mRNAs were analyzed by RT-PCR and quantitative real-time PCR (q-PCR) of three independent experiments was performed and normalized to GAPDH expression. Data are shown as mean ± S.D. (n = 3), *p < 0.05, **p < 0.01 versus VEC or SDC2 (left). Flow-cytometric analysis was used to examine membrane-bound SDC2 and Y51A (right top). CM were collected and proteolytic activity was measured using quenched fluorescence peptide cleavage assay (right bottom). (C) Control HT-29 cells (VEC) and HT-29 cells stably expressing SDC2 or Y51A were immunostained with anti-syndecan-2 or anti-MMP-7. The results were visualized with Texas Red-conjugated goat anti-rabbit (red) or FITC-conjugated goat anti-mouse (green). DAPI was used to stain nuclei (blue). Scale bar, 20 μm. (D) The indicated cells were treated with 1 ng/ml of interleukin-1α (IL-1α). The mRNA expression levels of SDC2 and MMP-7 were evaluated with RT-PCR (left top). CM were collected from the indicated cells and immunoblotted with an anti-MMP-7 antibody (left bottom) or subjected to quenched fluorescence peptide cleavage activity assay (right). Data are shown as mean ± S.D. (n = 3), *p < 0.05, **p < 0.01 versus VEC or SDC2.
Figure 7
Figure 7
The Tyrosine 51-mediated interaction regulates syndecan-2-mediated tumorigenic activities. (A) CM were collected from the indicated cells, immunoblotted with anti-syndecan-2 (left) or anti-E-cadherin (right) antibodies, and subjected to RT-PCR. (B) Cells were immunostained with anti-E-cadherin antibody and the results were visualized with Texas Red-conjugated goat anti-rabbit. DAPI was used to stain nuclei (blue). Scale bar, 20 μm. (C) The number of cells were evaluated with MTT assay as described in ‘Materials and Methods’. Data are shown as mean ± S.D. (n = 3), *p < 0.05, **p < 0.01 versus VEC or SDC2. (D) The indicated cells (1 × 105 cells/well) were seeded on soft agar. After 17 days, colonies were stained with 0.005% crystal violet and counted. Data are shown as mean ± S.D. (n = 3); *p < 0.05 versus VEC or SDC2. (E) HT-29 cells stably expressing syndecan-2 were treated with 0, 5, and 50 nM S2-P peptide. At 24 h post-treatment the expression levels of the target mRNAs were analyzed by RT-PCR. GAPDH was used as a control (top). Cells (1 × 105 cells/well) were seeded in soft agar with or without 50 nM S2-P peptide, allowed to grow for 17 days, and the number of viable colonies was counted. Data are shown as mean ± S.D. (n = 3), *p < 0.05 versus VEC or un-treatment S2-P peptide.
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