Establishment of Structure-Function Relationship of Tissue Inhibitor of Metalloproteinase-1 for Its Interaction with CD63: Implication for Cancer Therapy

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a pleiotropic protein, promoting both tumor-suppressive and tumor-promoting activities. While TIMP-1 is primarily known as an endogenous inhibitor of matrix metalloproteinases (MMPs) and thus associated with tumor cell invasion, clinical studies demonstrated increased expression of TIMP-1 and its association with poor prognosis in cancer. Non-MMP-inhibitory and oncogenic functions of TIMP-1 are mediated by induction of intracellular signaling via its cell surface receptor CD63, a tetraspanin. The present study investigates the structure-function relationship of TIMP-1 for its interaction with CD63, which may eventually help design a novel approach for targeting TIMP-1’s pro-oncogenic activity without interfering its tumor suppressive MMP-inhibitory function. Importantly, our analysis includes TIMP-1/CD63 interactions at the cell surface of live cells. Here, we demonstrate that the 9 C-terminal amino acid residues of TIMP-1 and the large extracellular loop of CD63 are required for their interaction. Considering that the N-terminal half of TIMP-1 is sufficient for TIMP-1’s MMP-inhibitory activity, we propose that those C-terminal amino acid residues are a potentially targetable motif of TIMP-1 oncogenic activity. As a proof of concept, we present the potential for the development of neutralizing antibodies against the C-terminal motif of TIMP-1 for disruption of TIMP-1 interaction with CD63 and the subsequent signal transduction.

protein complementation assay. Modified pEYFP-N1 and pECFP-C1 vectors (Clontech), in which the fluorescent protein genes were replaced by humanized Gaussia Luciferase N-terminal (GLucN) and C-terminal (GLucC) fragments, were obtained from Dr. James Granneman at our institute. The HNF4 vectors were a kind gift of Dr. Todd Leff at our institute. TIMP-1 and CD63 were cloned into these vectors in place of HNF4 (for primers used to make TIMP-1 and CD63 vectors see Supplemental Table 1). For all cases, the GLuc fragments were fused to the protein of interest via a flexible linker consisting of a 10 amino acid sequence (GlyGlyGlyGlySer GlyGlyGlyGlySer) as previously optimized for luciferase-fragment complementation assay 16 .
GLucN and GLucC fusion plasmids were co-transfected in a 1:1 ratio (400 ng DNA total/well) into HEK293FT cells in 24-well plates using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. Transfected cells were given fresh media after 5 hrs and cultured for an additional 17-19 hrs to allow expression of fusion proteins. Medium was exchanged with 220 ul/well of phenol-red free DMEM (Invitrogen) containing protease inhibitor cocktail (Roche; Indianapolis, IN). Cell membranes were disrupted by two cycles of freezing and thawing at −80 °C and room temperature. For each sample, 100 ul was transferred to a white 96-well plate (Thermo Scientific) for luminescence measurements. Next, coelenterazine (a natural substrate for luciferase, purchased from Nanolight Technology) was injected to a final concentration of 10 uM. Signal intensities (integrated over 10 seconds after 2 seconds injection delay) were read on a MicroLumat 96 LB + plate reader (Berthold Technologies; Oak Ridge, TN), or a Glomax 96 microplate luminometer (Promega; Madison, WI).
To perform PCA in live cells, HEK293FT cells were transfected as described above in 96-well clear-bottom white plates (Thermo Scientific, 200 ng DNA total/well). Without rupturing membranes by freeze-thaw cycles, coelenterazine was added into the live cell culture (100 ul/well of phenol-red free DMEM containing protease inhibitor cocktail) and signal intensities were measured as described above with white backing tape (Perkin Elmer; Boston, MA) applied to the bottom of the plate. Statistical analysis. Significant differences in averages of measured enzyme activity were assessed by unpaired two-sided t-tests after data were log-transformed to meet normality assumptions. P-values were adjusted for multiple comparisons using the Dunnett's method if the comparisons were made to single control group and the Holm's method if otherwise. P-values of < 0.05 were considered statistically significant.

Results and Discussion
We initially identified CD63 as a TIMP-1 interacting protein by yeast two-hybrid (Y2H) screening 2 . For the domain mapping study, our current study utilized protein complementation assay (PCA) that allows protein synthesis, post-translational modifications and trafficking in mammalian cells, followed by a quantitative read-out of interactions between the proteins of interest. To measure interactions between TIMP-1 and CD63, we followed the basic design of luciferase bifurcation as optimized by I. Remy and S.W. Michnick 16 . Briefly, the 93 N-terminal amino acid residues of luciferase (GLucN) were fused to the N-terminus of TIMP-1 at Cys 1 (GLucN-TIMP-1), while the C-terminal domain (aa 94-169) of luciferase (GLucC) was fused to the C-terminal region of CD63 truncated at Val 206 (CD63-GLucC), as depicted in Fig. 1A as well as Supplemental Fig. 1. CD63-GLucC was designed not to include the C-terminal internalization motif and most of the 4 th TM domain of CD63 17 so that it allows the GLucC fragment to be extracellular. Immunoblot analysis confirmed stable expression of these fusion-protein products in HEK293FT (Fig. 1C). Upon TIMP-1 interactions with CD63, bi-furcated luciferase fragments were re-combined and restored its activity (Fig. 1A,B). As a positive interaction control, hepatocyte nuclear factor 4α (HNF4), a transcription factor known to readily homodimerize, was used. Briefly, the GLucN (without a secretory signal peptide) and GLucC fragments were appended to the N-terminus and C-terminus of HNF4, respectively. As negative controls, cells were co-transfected with GLucN-TIMP-1 and HNF4-GLucC expression vectors (T1/ HNF4), or GLucN-HNF4 and CD63-GLucC vectors (HNF4/CD63). Results shown in Fig. 1B demonstrated that there is no detectable recombined luciferase activity in the absence of specific interactions between proteins of interest (TIMP-1/CD63 or HNF4/HNF4).
The crystal structure of TIMP-1, complexed with the catalytic domain of MMP-3, revealed two distinct subdomains, the N-terminal wedge-shaped MMP binding domain and the C-terminal domain 18 . We previously www.nature.com/scientificreports www.nature.com/scientificreports/ demonstrated that the C-terminal domain of TIMP-1 (amino acids 126-184) is sufficient for its interaction with CD63 2 . Interestingly, however, the retention of the 2 nd and 3 rd helices (H2 & H3: L 110 -C 124 ) of TIMP-1 is necessary for TIMP-1 activation of CD63-mediated intracellular signaling program 2,3 . Taken together, we hypothesized in this study that the C-terminus of TIMP-1 is the primary binding site for CD63 and the nearby helices H2 and H3 are required for its full engagement with the CD63 signaling complex on the cell surface ( Fig. 2A). To address this hypothesis, we generated deletion mutants of TIMP-1 truncated after L 168 (T1ΔC -deletion of 17 aa) or T 175 (T1ΔC2 -deletion of 9aa) as depicted in Fig. 2A. Stable expression and secretion of these mutants were confirmed by immunoblot analysis using conditioned media (Fig. 2B). In comparison to full-length TIMP-1, these two mutants displayed drastic decreases in their ability to bind CD63 (T1ΔC and T1ΔC2 with loss of ~83% and ~79%, respectively). It is striking that T1ΔC and T1ΔC2, wherein all of TIMP-1's cysteine bridges and its overall structure are expected to remain intact, drastically lost its CD63 binding activity (Fig. 2B). Thus, these results demonstrated that the 9 C-terminal amino acid residues are essential for TIMP-1 interactions with CD63.
Although the crystal structure of CD63 has not been successfully completed to this date, CD63 is thought to have 4 transmembrane domains and two extracellular loops as depicted in Fig. 2C. First, we examined whether the small extracellular loop (SEL, -Q 36 LVLSQTIIQGATPGS 51 ) of CD63 is critical for its interaction with extracellular TIMP-1 by alanine scanning mutagenesis. Amino acid residues in SEL were systematically substituted for alanine at Q 36 -T 42 , I 43 -Q 45 , and T 48 -S 51 by site-directed mutagenesis and the corresponding substitution mutants were named 7AA, IIQ, and TPGS, respectively. Stable expression of those mutants was confirmed by immunoblot analysis (Fig. 2D). When we examined the ability of those SEL substitution mutants to interact with TIMP-1, none of them had significant decrease in its TIMP-1 binding activity; while it was noticed that substitution of IIQ to alanines increased its binding activity (Fig. 2D). This may be associated with a conformational change in CD63, making it more accessible to TIMP-1 binding.
Mutational analysis of the large extracellular loop (LEL) of CD63 is challenging due to its large size and the lack of knowledge about its 3-D structure. To examine the significance of the LEL for CD63's ability to bind   www.nature.com/scientificreports www.nature.com/scientificreports/ TIMP-1, our initial approach was to generate a deletion mutant devoid of LEL after the amino acid F 107 (ΔLEL). As shown in Fig. 2D, CD63 in the absence of LEL significantly lost its ability to interact with TIMP-1. These results demonstrated that LEL, but not SEL, is a critical domain for CD63 interaction with TIMP-1. To narrow down the TIMP-1 interacting domain, the 6 cysteine residues within LEL of CD63 were mutated to serine, individually and in various combinations; two cysteine residues (C145,146 S; C169,170 S; C145,191 S; C146,170 S), three cysteine residues (C145,146,169 S; C146,169,170 S), four cysteine residues (C145,146,169,170 S), or five cysteine residues (C145,146,169,170,177 S). When C145 or C146 was mutated, especially when both C145 and C191 were mutated, CD63 significantly lost its ability to interact with TIMP-1. However, these changes were mostly due to variations in protein stability (data not shown). These results confirmed that the cysteine residues are important to maintain the integrity of the CD63 protein structure as predicted by its homology to other tetraspanins with conserved LEL Figure 4. Antibody against the C-terminus of TIMP-1 interferes with TIMP-1's interaction with CD63 and the C-terminus of TIMP-1 is essential for the activation of intracellular signaling. (A) Anti-TIMP-1 antibodies, EP1549RY and 102 D1, were pre-incubated with or without synthetic peptides corresponding to the 9 C-terminal amino acid residues of TIMP-1. Immunoblot analysis of TIMP-1 was performed using TIMP-1 overexpressing HEK293FT cell lysates. The nitrocellulose membrane was stained with Ponceau S and cut into strips. Each strip was probed with the indicated antibody. β-actin was used for loading control. (B) PCA for TIMP-1/CD63 interactions was performed in the presence or absence of C-terminal and non-C-terminal TIMP-1 Abs (EP1549RY and 102 D1, respectively). Values are shown after normalization to treatment with each antibody buffer alone and are representative of multiple experiments (with 100% at 1.3E07 and 1.6E07). Error bars represent standard deviation. (C) Immunoblot analysis of phopho-and total ERK using cell lysates of MCF10A cells treated with conditioned media collected from HEK293FT cells transfected with control vector (Neo), TIMP-1 (T1), T1ΔC, and T1ΔC2 expression vectors.