Preparation Of Recombinant Human ADAM15 Dinsintegrin Domain And Its Anticancer Mechanisms

A Disintegrin And Metalloproteases (ADAM) proteins are a family of multifunctional proteins containing disintegrin and metalloproteinase domains that perform both adhesive and proteolytic functions in cell-cell and cell-matrix interactions. ADAM15 is unique among these proteins in having an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain. This dissertation chose the B16 murine melanoma cell as a model, to investigate the effect of the recombinant human ADAM15 disintegrin domain (rhddADAM15) on B16 cells gowth, and the target protein of rhddADAM15 on B6 cells, and further study the involvement of the target protein in the anti-cancer effect of rhddADAM15. The main results were described as follows:1) In order to enhance the production and bioactivity of rhddADAM15 in Escherichia coli, two different strategies were examined based on the genetic analysis of coding sequence of rhddADAM15: to select the suitable host strain, and to optimize the rare codons and delete the amino acids residues. It was found that (1) when choosing E. coli Rosetta (DE3) as the expression host that supplys additional tRNA for rare codons, 298 mg/L GST-ADAM15 and 42 mg/L rhddADAM15 were achieved under the optimal expression and thrombin digestion conditions, respectively; (2) when introducing E. coli preferred codon GGC and deleting"Pro-Glu-Phe"residues by PCR-based site-directed mutagenesis, 326 mg/L GST-△-ADAM15 and 68 mg/L rhddADAM15 were achieved under the same condition, respectively, higher 19.4% and 61.9% than that of the wide type strain. The data presented here demonstrated that high expression of heterologous protein could be achieved by releasing rare codon usage and amino acids residues restriction.2) The effect of rhddADAM15 on the proliferation of B16, MCF-7/W and HMEC-1 cells were examined by MTT assay. The effect of rhddADAM15 on B16 cell migration, adhesion and invention were tested by the wound migration assay, cell-Matrigel adhesion assay and the Transwell chambers, respectively; The growth and activity of B16 cells were explored by using trypan blue staining. The cell cycle and apoptosis were determined by flow cytometry. The results demonstrated that rhddADAM15 could inhibit the proliferation of normal cells, but exhibit stronger inhibitory effect on the tumor cells. Moreover, rhddADAM15 inhibited B16 cell migration, adhesion and invasion in the dose/time-dependent mode and the necrosis could not be observed in the low concentration of rhddADAM15. The apoptotic peaks were not found in cell cycle analyses and the cells are arrested in G2/M phase.3) The protein pull-down assay using the biotin-avidin system supported by the immol/Lunomagnetic beads was used to capture the binding protein of rhddADAM15 from the cell lysis buffer of B16. The mixture of the binding protein was concentrated and separated by the two-dimensional eletrophoresis (2DE) and eight of the protein spots were identified by the ESI-MS/MS. By using the software BIOWORKS and the protein database ipi MOUSE v3.26, the binding proteins were mainly identified as follows: (1) Tumor markers in the clinical application: malate dehydrogenase, phosphate dehydrogenase and glutamate dehydrogenase; (2) Enzymes in glycolytic pathway: alpha-enolase and triosephosphate isomerase; (3) The important components in cytoskeleton: tropomyosin-3; (4) Proteins in oxygen transportation: hemoglobin subunit beta-1; (5) Members in signal transduction: p38MAPK. In addition, the interaction modes between rhddADAM15 and p38MAPK were preliminarily analyzed using the molecular docking program, molsoft ICM-PRO-3.5.4) The target peptides of rhddADAM15 were explored by the phage display technique. Firstly, the rhddADAM15 was biotinylated and immol/Lobilized on the immol/Lunomagnetic beads with avidin labeled. Secondly, four specific binding peptides (peptide A, B, C and D) were screened from a phage display-12 peptide library by a screening protocol towards immol/Lobilized rhddADAM15. By using the software BLAST and the relevant protein database, some target proteins were screened, such as the Cdc25 phosphatase, tyrosine kinase p56, integrinαvβ3, and the p38 mitogen activated protein kinase (p38MAPK), related with the cell cycles and the signal transduction.5) The p38MAPK was selected for further investigation of the involvement in the anti-proliferative effect of rhddADAM15 on melanoma cells. Firstly, the effect of rhddADAM15 on activity of p38 MAPK in vitro was observed. Secondly, phosphorylation of p38 kinase in melanoma cells was detected by Western blot analysis upon treatment with rhddADAM15 (0-10μg/mL). Thirdly, the specific inhibitor of p38 kinase (SB203580) was employed to partly suppress the activity of p38 kinase in B16 cells, and the inhibitory effect of rhddADAM15 was then observed. The results were demonstrated as follows: (1) rhddADAM15 could bind to the recombinant p38 kinase at 4 oC but had no effect on the activity of p38 kinase in vitro; (2) rhddADAM15 had inhibitory effect on the activity of p38 kinase in B16 cells; (3) A decrease in the rhddADAM15 inhibitory effect on melanoma cell proliferation, and the value of IC50 was increased from 9.5μg/mL to11.4μg/mL, when the activity of p38 kinase was partly suppressed; (4) A release in the cell cycle arrest, the cell ratio in G2/M phase was decreased from 28.77% to16.59%, under the condition of suppressing the p38 kinase. These results provided evidences that rhddADAM15 inhibiting the proliferation and the cell cycle of the melanoma cells was partly through p38 kinase activation and the p38MAPK signal transduction pathway was involved in the effect of rhddADAM15 on B16 cells.