Identifying And Defining The Key Sequences In MMP26 For Its Subcellular Localization

Matrix metalloproteinases(MMPs) are a family of calcium ions(Ca2 +) and zinc ions(Zn2+)-dependent enzymes. The MMPs mainly functions as a critical factor in the degradation of extracellular matrix, and also play an important role in the processes of cell growth, survival, angiogenesis, invasion, epithelial to mesenchymal transition and inflammation and immune surveillance. MMPs could be divided into membrane-type MMPs and secretory MMPs. Although the transcriptional and activity regulation of MMPs have been studied extensively, the intracellular transport and the underling molecular mechanism of MMPs is not clear.MMP-26(matrilysin 2) is a newly discovered secretory MMP. MMP26 is able to degrade extracellular matrix and part of non-extracellular matrix proteins. The m RNA of MMP-26 has been found with a limited expression in placenta, uterus and kidney tissue. On the contrary, there are a large number of MMP-26’s m RNA in plurality of epithelial cancer cells. Therefore, many previous studies suggest that MMP-26 plays an important role in tumor invasion, angiogenesis and migration. In addition, the MMP-26 is one of the smallest members of MMPs. It contains 261 amino acids, including a signal peptide region, a propeptide region and a catalytic domain. The signal peptide of secretory proteins can affect the amount of protein secreted into the extracellular region, acting as a membrane localization signal. Although MMP-26 contains a signal peptide structure, the previous reports has suggested that, in a variety of cells and the samples, MMP-26 is mainly detected in intracellular region, but not been detected in the cell. Therefore, we hypothesized that except the signal peptide, MMP-26 also contains a sequence which could help it remain in cells.In this dissertation, we applied molecular biology and cell biology methods, such as SOE PCR, Over lop PCR etc. to construct mutants of MMP-26. The mutant was combined with e GFP and the PEI was used to transfer the mutant into Hela cells. The location of mutants was observed by fluorescence microscopy. By constructing MMP-26Δ80-125-e GFP, MMP-261-193Δ80-125-e GF and MMP-26Δ80-193-e GFP, we found that in the case of retention signal peptide, 80-125 amino acids of MMP-26 is the only key sequence in ER( Endoplasmic Reticulum). Furthermore, by constructing MMP26S80-125-e GFP, MMP26S87-125-e GFP, MMP26S88-125-e GFP and MMP26S89-125-e GFP, we found the mutant that deleted to 88 th amino acid from N-terminus could remain in ER, however the mutant that deleted to 89 th amino acid from N-terminus could not remain in ER. On the other hand, by making MMP26S88-124-e GFP, MMP26S88-123-e GFP, MMP-26S88-122-e GFP, and MMP26S88-121-e GFP, we found that the mutant can remain in ER by deleting amino acids down to the 123 th position from C-terminus, while it cannot be remain in ER by deleting to the 122 th position. Therefore, we conclude that MMP-26 is localized in the endoplasmic reticulum by the key sequences of the 88th-123 th amino acid.In summary, this thesis firstly confirmed that the sequence of the 88 th to 123 th amino acid residues is the key sequence for MMP-26’s location in ER, and MMP-26’s location in ER uses a non-KDEL sequence mechanism. This thesis will help us understand the molecular mechanism of non-classical location sequence for ER and its receptors. Currently, the structure and the function of MMP-26 is still unclear. The results in this thesis also provides insight on the molecular mechanism of MMP-26’ location in ER and the normal function of MMP-26 in the cell. Overall, this work also lay the foundation for understanding the relationship between the MMP-26 and cancers.