Application Of MT1-MMP Affinity Peptide Labeling In Biological Imaging

Matrix metalloproteinases(MMPs) are a kind of cysteine endopeptidase which dependent on the zinc ions, calcium ions, since they are named as they require a condition where zinc and calcium ions exist to catalyze substrate. Matrix metalloproteinase family members are currently found in a total of 25, with 24 species existing in mammals. They are associated with a variety of reactions under physiological and pathological conditions. Matrix metalloproteinase can be roughly divided into two categories,namely membrane type and soluble matrix metalloproteinase. Membrane-type 1 matrix metalloproteinase also called matrix metalloproteinase MMP-14 or MT1-MMP, is the one which is studied more and clearly.MT1-MMP is the membrane-anchored collagenase which can degrade the extracellular matrix, promote cell invasion and migration and plays an important role in a variety of major diseases especially in cancer of human.It can lead to cancer occurrence, growth,invasion and angiogenesis.It also has a protease-activated function, and play a role to facilitate degradation of extracellular matrix indirectly. It exposure to the cell surface,so can be easy to combinate.Thus there will be an increasing number of researchers who focus on early cancer detection and treatment though targeting to MT1-MMP. Thus finding a kind of probes to identity MT1-MMP in vivo efficiently can provide important theoretical basis with the clinical treatment of MT1-MMP-related diseases. Optical imaging instrumentation in vivo are widely used as its mature,high sensitivity, fast imaging and non-destructive detection, etc. The use of polypeptides probe is the most successful strategie in bioimaging and attract widespread attention as its good specificity, non-immunogenic, little toxicity,fast marking, clear image, etc.Based on the these theorise, our laboratory found a amino acid sequence with low homology which is exposed to the extracellular surface with ring-shaped by a way of amino acid sequence alignment and a way of homology modeling. This sequence is called MT-LOOP. MT1-MMP’s MT-LOOP(MT1-LOOP) structure is located in its catalytic domain and plays a role in the process of activating pro MMP-2, gelatin, hydrolyzing collagen and cell invasion in collagen.Then we used phage display technologytake to find out the specific affinity peptides for the fifteen amino acid sequence which include MT1-LOOP region.In order to further improve the affinity of peptide, in this thesis we mutated selected residues of affinity polypeptide to specified types.Then the molecular docking was carried out with Protein Docking Module of DS and one peptide was selected for the optimal interaction energy by molecular simulation. We also designed another peptide according existing peptides as the main research objects in the nest series of experiments.First, we used atomic force microscope single molecule force spectroscopy technology to detect interaction forces range and highest frequency interaction force between affinity peptide and the target peptide; and used isothermal titration calorimetry measurement to detect the binding force, binding site, binding constants of interactions between affinity peptide and the target peptide. Then we used the affinity peptide conjugated by FITC to stain human fibrosarcoma cells HT1080 and human breast cancer cells MDA-MB-231 in which MT1-MMP level is high.Optical imaging showed that the mutant peptides and their mother peptide had good specificity, it is expected to develop as an effective probes for diagnosis and treatment of diseases in which MT1-MMP has a high expression.In summary, we had achieved reaction parameters between affinity peptide and MT1-160 p and results of optical imaging at the cellular level using affinity peptide targeting MT1-LOOP which we had filterd and designed.Which provided the solid foundation for Whole-body small animal optical imaging.A series of remolding methods of peptides and detection methods of peptides’ affinity, which we took in this process are practical for filtering and designing other synthetic peptide probe.