The Mutation Of MT-loop Of MMP-14and Screening Of Affinity Peptides To MT-loop

Matrix metalloproteinases (MMPs) are a family of zinc-dependentendopeptidases, which can degrade a variety of extracellular matrix components andbiologically active molecules such as chemokines, growth factors. MMPs playimportant roles in many diseases such as cancer. Type I membrane matrixmetalloproteinase (MMP-14, MT1-MMP) was the first one to be identified in themembrane-type matrix metalloproteinases.MMP-14overexpressives in almost allkinds of tumors, it can be located to the cell surface and recycled tointracellular.MMP-14plays important roles in many diseases, particularly tumorsgrowth, invasion, metastasis and angiogenesis. The development of a ligand (peptide)holding high specific and binding affinity to MMP-14as an inhibitor or probe oftumor imaging has an important significance. The MT-loop (163PYAYIREG170) ofMMP-14is a unique amino acid sequence in the catalytic domain of MMP14(cdMMP-14) and located on the surface of the molecule. These make MT-loop as is agood target for the screening of peptide affinity to MMP-14. Besides, the mutation ofMT-loop has little impact on the conformation of the cdMMP-14outside the MT-loopregion.In this paper, we aimed to screen peptides which hold high specific and bindingaffinity to the MT-loop of MMP-14with the Phage Display Libraries Kit. Ourexperimental scheme was based on the difference of the MT-loop conformationbetween the wild-type cdMMP-14(WT-cdMMP-14) and the mutant of cdMMP-14。First of all，we made three mutants of cdMMP-14. The first one was namedD-cdMMP-14, which lacked the MT-loop region. The other two were L1-cdMMP-14and L2-cdMMP-14, which had the sequence of160EPRQHEYARIEVKYG174and160IPEKQAEYVGERRYH174respectively from the random sequence of160REVPYAYIREGHEKQ174of the cdMMP-14. We reconstructed the mutant genesand the WT-cdMMP-14gene to the expression vector of pET21a (+) and expressedthe recombinant proteins as inclusion body with the BL21(DE3). Then we purifiedand refolded the recombinant proteins, and we found the D-cdMMP-14-His6hadactivity against DQ-gelatin and gelatin zymography activity just like the WT-cdMMP-14-His6. Computer simulation results showed that the difference of3Dstructure between the D-cdMMP-14-His6and the WT-cdMMP-14-His6only happenedin the region of MT-loop.Then, we designed our experimental scheme for screening of phage displaypeptide libraries according to the difference of the MT-loop conformation between theD-cdMMP-14-His6and the WT-cdMMP-14-His6. Every round of panning, we firstused the D-cdMMP-14-His6as the target incubating with the phage (peptides on thesurface of the phage, the same below) and discarded the binding phage. Then thenonbinding phage was incubated with the WT-cdMMP-14-His6, the specific bindingphage to the WT-cdMMP-14-His6was amplified for the next round of panning. Afterfour rounds, we selected thirty specifically-bound phage of WT-cdMMP-14-His6todetermine the binding affinity to both of the two targets by ELISA. But all of thethirty phages shared similar binding affinity to the D-cdMMP-14-His6and theWT-cdMMP-14-His6. It meant no one of the thirty phages bound to the MT-loop ofMMP-14, because if the phage bound to the MT-loop, it should not bound to theD-cdMMP-14-His6(had no MT-loop).In summary，we made three mutants of cdMMP-14. One of the mutants had thesimilar activity to the WT-cdMMP-14. Computer simulation results showed that thedifference of3D structure between the D-cdMMP-14-His6and theWT-cdMMP-14-His6only appeared in the MT-loop area.On this basis，we conductedthe screening of phage display peptide libraries and we obtained some phages withaffinity to the WT-cdMMP-14-His6，but the binding sites were not in the region ofMT-loop.