Screening And Identification Of Bursopentin (BP5) Interacting Proteins In Tumor Cells

The bursa of Fabricius(BF) is a central humoral immune organ unique to birds which plays important roles in B cell development and antibody production. Bursopentin(BP5) is a novel immunomodulatory peptide isolated from the BF, which with functions of immunomodulatory, antioxidant and antitumor. However, the antitumor mechanisms of BP5 is still unclear. In this study, BP5 interacting proteins in tumor cell were screened and identificated by phage display technology, cDNA microarray method and bioinformatics analysis. All studies were listed as below:1. Screening and identification of specific binding peptides to BP5 by phage display peptide libraryBP5-BSA was used to screen its binding peptides from the 12-mer random phage display peptide library. After four rounds of biopanning, ELISA and competitive inhibition test, three specific peptides binding to BP5 were found with the amino acid sequences of PINMQTLNCMAA(P2-12), GCTLNPMSDLLG(P6-12) and MSSTLNGMLNSL(P7-12), respectively, with the core motif of TLNXM. The effect of P2-12, P6-12 and P7-12 on BP5 anti proliferation of WEHI-231 cells were detected by MTT assay. The results indicated that P2-12(0.2 μg/mL to 20 μg/mL), P6-12(2 μg/mL to 20 μg/m L) and P7-12(0.2 μg/mL to 20 μg/m L) were able to decrease the anti proliferative activity of BP5 in WEHI-231 cells, and the effect of P2-12 at 20 μg/mL was significantly higher than other groups. Moreover, the results of p53 luciferase acticity assay indicated that three BP5 binding peptides had the abilities to downregulate the activity that BP5-induced p53 gene transcription. In conclusion, the three specific peptides binding to BP5 were identified in this study, which provide some reference data for further study on the anti-tumor mechanism of BP5.2. Construction of T7 phage cDNA library of DT40 cells and screening BP5 interacting proteins in DT40 cellsUsing DT40 cells as tumor cell model, T7 phage cDNA library of DT40 cells was constructed. And then, used BP5-BSA as selected molecule to biopan it for the proteins intected with BP5. The results showed that the titers of primary and amplified were 1.2×104 pfu/mL and 1.9×109 pfu/m L, respectively. And the recombination ratio of it was about 91.7%. Combining with determination and bionformatics analysis, we found that trafficking protein particle complex 2(TRAPPC2), vascular endothelial growth factor A(VEGFA) and cyclin E1(CCNE1) interacted with BP5. The bioinformatics analysis results showed that the amino acid homologies of TRAPPC2 in Gallus gallus compared with in Mus musculus and Homo sapiens were 97.9% and 100%, the homologies of VEGFA in Gallus gallus compared with in Mus musculus and Homo sapiens were 76.8% and 74.1%, and the homologies of CCNE1 in Gallus gallus compared with in Mus musculus and Homo sapiens were 67.3% and 71.3%, respectively. VEGFA was an important regulatory factor of angiogensis, and CCNE1 was an important cell cycle regulatory protein of G1/S phase of cell cycle. The abnormal expression of both VEGFA and CCNE1 are closely linked to the occurrence and development of tumor. The results suggested that the antitumor effect of BP5 might be related to VEGFA and CCNE1, which provide a scientific basis for the futher study of molecular mechanism of BP5 anti-tumor.3. Gene expression profiling of DT40 cells after BP5 treatmentIn this study, to preliminary identify the BP5 interacting with tumor cells, we analysised the genomic expression profiling of BP5-treated DT40 cells and detected the effect of BP5 on p53 luciferase activity and the experssion of tumor related proteins in HCT116 cells. The results showed that most pf pathways regulated by BP5 were associated with anti tumor, including p53 signaling pathway. Among these regulated pathways, p53 pathway was involved in five differentially expressed genes that were related to regulation of cell cycle. SIAH1, TP53(p53) and CIP1(p21) were upregulated, while CCNE1 and CHEK2 were down regulated. Then we analyzed BP5 dependent p53 tumor signalling pathway, and the results confirmed that BP5 could significantly increase the p53 lucifease activity at 0.2 μg/mL to 20 μg/mL in HCT116 cells in the transcriptional level and significantly promote the expression level of p21 and p53 and reduced the expression level of CCNE1 and CHEK2 in the protein expression level. Combined with the results of screening BP5 interacting proteins in DT40 cells, CCNE1 was preliminary identified as BP5 candidate interacting protein in tumor cells, which provided an important refernce for further reseaches on the BP5 anti tumor molecular mechanism.