摘要
膜I型基质金属蛋白酶(Membrane Type I Matrix Metalloproteinases, MT1-MMP)是膜型MMPs中最重要的成员,在损伤修复,脂肪细胞分化,骨的生长及重塑,关节炎,肿瘤发生及发展等众多生理和病理过程中起了重要的作用。实验室以MT1-MMP表面一段特殊序列(MT1-160p)为靶点,通过噬菌体肽库筛选得到了一系列特异性亲和短肽(MT1-AFps),为了最终作为MT1-MMP的成像探针或者药物靶向运输载体,我们对亲和肽与MT1-MMP的分子间相互作用进行了细致的研究。
首先,我们通过核磁共振方法确定MT1-AF3p、MT1-AF7p与MT1-160p有明显的相互作用,通过圆二色谱方法证明它们相互作用时不形成二级结构。其次,我们通过分子模拟、核磁共振和原子力方法对MT1-AF7p与MT1-160p的相互作用位点进行了研究,结果显示MT1-AF7p的6位His和7位Asn都参与了与MT1-160p的相互作用。最后,我们构建并表达了MT1-AF7p与M13噬菌体衣壳蛋白PIII的融合蛋白,通过ELISA方法确定与MT1-MMP的KD值缩小了8-10倍。
这些结果为MT1-MMP亲和肽的进一步应用提供了理论基础,同时也为MT-loop的功能研究提供了新的平台。
The matrix metalloproteinases (MMPs) comprise a family of zinc-dependent endopeptidases that consist of 24 human MMPs and seven of which are membrane bound. MMPs participate in degradation and remodeling of extracellular matrix (ECM), synthesis and secretion of growth factor, cytokine, hormone and cell surface adhesion receptors, regulation of morphogenesis, proliferation and evolution, and other physiological processes. Moreover, MMPs play important roles in ostarthritis, cardiovascular disease, atherosclerotic plaque, tumorigenesis, tumor growth and other pathological processes.
Membrane type-1 matrix metalloproteinase (MT1-MMP) is the most prominent member of the MT-MMP family with multiple biological and pathological significances. It is intrinsically associated with the plasma membrane of normal and tumor cells to remodel the ECM. MT1-MMP displays a broad spectrum of activity against ECM components such as type I and II collagens, fibronectin, vitronectin, laminin, fibrin and proteoglycan. It also activates pro-MMP-2 and pro-MMP-13 (pro-collagenase 3), therefore promotes invasion and metastasis. It is directly involved in the cleavage of cell surface receptors including tissue transglutaminase, CD44, pro-αv integrin, syndecan-1, low-density lipoprotein receptor-related protein and L-glycan, and thus affects adhesion between cells, adhesion between cells and ECM, angiogenesis, expression and clearance of protein, and cell differentiation. MT1-MMP is highly expressed in different cancers, but shows very low level in normal tissues. In recent years, it has been demonstrated that the MT1-MMP activity is regulated by endocytosis through both clathrin-dependent and caveolae-mediated pathways. The drug can be delivered to the cell surface of MT1-MMP and then internalized with this molecule and function within the cell. This results in high killing of tumor, but decreases the side effect of normal organs. As MT1-MMP possess many crucial biological and pathological functions, molecules with high affinity against MT1-MMP could be used for imaging and targeted drug delivery in MT1-MMP over-expressed diseases such as cancers. In our lab, we targeted a unique peptide (MT1-160p) on the surface of MT1-MMP and got a series of peptides that bind to MT1-MMP by in vitro panning of phage display library. In the present study, we focus on the in vitro study of the interactions between MT1-MMP and its affinity peptides.
In the present study, we synthesized MT1-AFps and MT1-160p through solid phase synthesis method and the interaction between two peptides was investigated using nuclear magnetic resonance (NMR). The results suggest that MT1-AF3p and MT1-AF7p showed marked interaction with MT1-160p. We also demonstrated that there is no formation of secondary structure when MT1-AFps interact MT1-160p by Circular Dichroism Spectrum (CD). This suggests MT1-AFps will not change the structure and function of MT1-MMP severely in vivo. Furthermore, we study the interaction sites of one of the affinity peptides, MT1-AF7p, and MT1-160p. First, molecule modeling revealed that MT1-AF7p forms three hydrogen bonds with MT1-MMP: Asn7 of MT1-AF7p with Tyr166 of MT1-MMP, Asn7 of MT1-AF7p with Ile167 of MT1-MMP, and His6 of MT1-AF7p with Glu169 of MT1-MMP. And this is in line with the collective motif (His-X-His) of MT1-AFps. Then we mutated the His6 or Asn7 of MT1-AF7p to Ala respectively (M1-AF7p and M2-AF7p), and synthesized a scrambled peptide (Scr-AF7p) with an identical amino acid composition as MT1-AF7p as negative control. We studied the interaction between MT1-AF7p, M1-AF7p, M2-AF7p, Scr-AF7p and MT1-160p by NMR and atomic force microscope (AFM). The results of NMR suggested that one residue substitution did not abolish the interactions between peptides and Scr-AF7p showed no specific interaction with MT1-160p. The results of AFM suggested that the force between MT1-AF7p and MT1-160p is conspicuous (304pN or 186pN), but the interaction between M1-AF7p, M2-AF7p, Scr-AF7p and MT1-160p showed nearly no force. According to all the results above, it is considered that the His6 and Asn7 of MT1-AF7p both participate in the interaction with MT1-160p, but more precise interaction site still need to be research. In addition, we successfully construct a recombinant protein fusing MT1-AF7p with PIII, a M13 phage coat protein, to improve the affinity to target molecule. And the KD value from ELISE indicates eight to ten times decrease compared with the peptide itself. The fusion protein can be used for targeted drug delivery in MT1-MMP over-expressed diseases such as cancers.
In conclusion, we demonstrated that MT1-AF3p and MT1-AF7p shows marked interaction with MT1-160p by NMR, and there is no formation of secondary structure when they interact. Then we indicated that His6 and Asn7 of MT1-AF7p participate in the interaction with MT1-160p. In addition, a fusion protein of MT1-AF7p and PIII showed eight to ten times increase of affinity to MT1-MMP. All the results warrant further use of the affinity peptides as ligand for this important protease.
引文
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