Study On Isolation, Identification And Immunomodulatory Mechanism Of Bursal-Derived Bioactive Peptides

The bursa of Fabricius (BF) is the only one acknowledged humoral central immune organ, and is critical for normal development of the B lymphocytes responsible for antibody production. The terms of B cell is named from "bursa-derived lymphocyte". In mammals, the B-cell-differentiating organ that is equivalent to the T-cell-differentiating thymus has not been defined. It was acknowledged that birds and mammals evolved from a common reptilian ancestor and has inherited many common immunological systems. Avian immunology research has made seminal contributions to the understanding of fundamental immunological concepts and subsequently explained in mainstream immunology. The avian immune system provides an invaluable model for studies on basic immunology. Therefore, the researches on the bursal-derived active peptides will have important theoretical significance for mechanisms of immune regulation and other physiological activities of the humoral central immune organ of mammals and human. All studies were listed as below:1. Isolation and characterization of the novel bursal-derived peptides from the bursa of FabriciusThe bursa of Fabricius (BF) is the acknowledged central immune organ, which is important to the B cell differentiation and antibody production. However, due to difficult purification, the immunomodulatory peptides from BF were little reported. In this study, the extract samples of BF were taken to a chromatographic analysis by RP-HPLC. Nine novel low molecular peptides were isolated from BF, with amino acid sequence of EPAGSMM, TPSGLVY, LGPGP, MTLTG, YEYAY, RMYEE, GPPAT, AGCCNG, and RRL, and named as Bursal septpeptide (BSP)-Ⅰ,-Ⅱ, and Bursal pentapeptide (BPP)-Ⅰ,-Ⅱ,-Ⅲ,-Ⅳ,-Ⅴ, and Bursal hexapeptide (BHP), and Bursal tripeptide (BTP), respectively. In this paper, we analyzed the chemical formula and characteristics of these nine bursal-derived peptides. The immunization comparative experiment verified the different immunomodulatory activity of these nine bursal peptides on antibody and cytokine productions. BPP-、BPP-Ⅴ、 BPP-Ⅱ、BSP-Ⅰ and BSP-Ⅱ enhanced the levels of antibody and cytokines IL-4and IFN-y. BTP induced the enhancement of the antibody production and IL-4, but not IFN-y, and BPP-Ⅳ could stimulate IL-4production, but not on antibody and IFN-y. However, it was not observed the significant immunomodulatory function of BHP or BPP-Ⅲ. These results provided important orientations for the comprehensively understanding and study of the humoral central immune system of human, and provided a novel insight on the treatment of serious disease and immune improvement of human.2. The immunomodulatory functions of the bioactive bursal-derived peptides in the immunized chickensTo evaluate the immune inducing roles of BSP-Ⅰ, BSP-Ⅱ, BPP-Ⅰ and BPP-Ⅱ in chicken immunization,75day-old chickens were twice co-immunized with these bursal-derived peptides with AIV vaccine or antigen, respectively. These results showed that these bursal-derived peptides were able to significantly induce the immunized chicken to produce the HI antibody especial to the HA of AIV, and potentiate the cytokine responses in different manner. It was observed that three bursal-derived peptides BSP-Ⅰ、 BSP-Ⅱ and BPP-Ⅰ stimulated Thl cytokine (IFN-y) and Th2-type cytokine (IL-4) production after AⅣ vaccine or antigen immunization, whereas BPP-Ⅱ strong increased cytokines IL-4level, not IFN-y. These results indicated that these bursal-derived peptides could induce the humoral immune and cellular mediated immune, and significantly enhance the antigen especial immune responses in the immunized chicken, which proved that these four bursal-derived peptides were the bioactive immunomodulatory peptides from the humoral immune system.3. The immunomodulatory roles of the bursal-derived peptides in immunized mammal miceThe bursa of Fabricius (BF) is the central immune organ, which is important to the B cell differentiation and antibody production. To investigate the immune inducing effects of these bursal-derived peptides, mice were co-immunized with AⅣ vaccine or antigen, respectively. The results showed that BSP-Ⅰ、BSP-Ⅱ、BPP-Ⅰ and BPP-Ⅱ were able to induce the enhancement of the antibody production, and cellular mediated immune responses including Th1and Th2cytokines and T cell phenotyping in different dose-dependent manner, respectively. These observations implied that BSP-Ⅰ, BSP-Ⅱ, BPP-Ⅰ and BPP-Ⅱ might be novel biological active factors with immunomodulatory activities. These results suggested that as the bioactive peptides from humoral immune system, various biological functions of bursal-derived peptides might have far-reaching implication on immune system significance, which might provide novel insight on linking between humoral immune system and development of effective immunotherapeutic strategies for treating human cancers diseases.4. The receptors interacting with the bursal-derived peptides in B lymphocyte screened by T7cDNA phage displayThe bursa of Fabricius (BF) is the acknowledged humoral central immune organ, and is key position for B lymphocyte development and differentiation. However, the mechanism by which the bursal-derived active molecular play role in B lymphocyte is little reported. To investigate the mechanism, in this paper, these interacting proteins with BSP-Ⅰ, BSP-Ⅱ, BPP-Ⅰ, BPP-Ⅱ and BP5were selected by T7B cell cDNA phage display library, respectively. These results showed that three kinds of proteins including N-cadherin (neuronal)、Ⅴ-set and transmembrane domain containing2and v-myc myelocytomatosis viral oncogene homologue interact with BSP-Ⅰ. Four groups of gene sequences interacting with BSP-Ⅱ were selected, which were corresponding to seven kinds of proteins. BPP-Ⅰ interacts with cardiac muscle ryanodine receptor MGC83917protein and BTB/POZ1, respectively, and BPP-Ⅱ interacts with silencing mediator of retinoic acid and thyroi (SMRT), a nuclear transcriptional corepressor, and BP5interacts with cytidylate kinase. Each interacting protein has unique characteristics and plays different functions. These results suggested that these bursal-derived peptides BSP-Ⅰ, BSP-Ⅱ, BPP-Ⅰ, BPP-Ⅱ and BP5might participate in different signal pathways by interacting with various proteins, resulting in a variety of biological functions, including the inducible regulation effect on the immune response and antitumor, which offered a potent insight to study mechanisms on the humoral immune system of mammal and human.5. The mechanism of the bursal-derived peptides on immune regulation in vitroThe bursa of Fabricius (BF) is the only acknowledged central humoral immune organ, and various active factors are presented in BF stimulate antibody production. In this paper, it was observed that five bursal-derived peptides BSP-Ⅰ、BSP-Ⅱ、BPP-Ⅰ、BPP-Ⅱ and BP5could significantly induce antibody production from mouse-derived hybridoma cells in different dose-dependent manner. To understand the underlying intracellular processes of the bursal-derived peptides on immune induction, gene microarrays were used to examine gene expression patterns in hybridoma cells after BSP-Ⅱ, BPP-Ⅱ and BP5treatment, respectively. The results showed that1226genes were regulated after BSP-Ⅱ treatment, and there were2478genes and3223genes differential expressions after BPP-Ⅱ and BP5treatment, respectively. Pathway analysis showed that five pathways were regulated after BSP-II treatment. There were16pathways regulated after BPP-Ⅱ treatment. Also, genes regulated by BP5treatment were involved in26pathways. Most of these regulated pathways are related to the immune response. Additionally, GO analysis showed that the differential expressed genes after BSP-Ⅱ, BPP-Ⅱ and BP5treatment were involved in various immune-related cellular processes, respectively, including T cell activation and proliferation, B cell mediated immunity, and cytokine production and related signal, and so on. These analyses provided the novel informations on the mechanisms of the molecular basis of the bursal-derived peptides on immune induction, which could result in various biological consequences, such as antibody production increase, cellular immunity activation, and antitumor. These results provided a linking between humoral and cellular mediated immune responses, which provide the important molecular foundation for the further research on the humoral immune system of mammal and human.6. The mechanism of the bursal-derived peptides on the pre-B cell in vitroBF, the acknowledged central humoral immune organ, is vital to B cell differentiation. However, the mechanism by which the bursal-derived peptides regulate pre-B cell has not been studied. In this paper, it was proved that bursal-derived peptides BP5, BSP-Ⅱ, BSP-Ⅰ, BPP-Ⅰ and BPP-Ⅱ could regulate the avian DT40cell proliferation in different manner. To investigate the mechanisms of the bursal-derived peptides on pre-B cell, gene microarray was used to determine the gene expression profile and functional analysis in DT40cells after BSP-Ⅱ, BPP-Ⅱ and BP5treatment, respectively. It was identified that the differentially expressed genes after BSP-Ⅱ treatment were involved in various pathways, of which six pathways were associated with signaling transductions, including ErbB signaling, MAPK signaling, Toll-like receptor signaling, Notch signaling, mTOR signaling, and Wnt signaling. BPP-Ⅱ treatment regulated11pathways, in which homologous recombination is a vital mechanism which is involved in antibody Ig gene conversion and diversification during B cell development. BP5treatment regulated9pathways, and one of them, glutathione metabolism plays critical roles on antioxidation. These results provides the potential mechanism of bursal-derived peptides BP5、BSP-Ⅱ and BPP-Ⅱ on pre-B lymphocyte development, which indicated the important molecular foundation and mechanisms by which the humoral central immune organ of mammal and human might play key functions in B cell differentiation and mature, and immune system. These results are valid for the mechanism of the bursa of Fabricius on B lymphocytes development.7. The impact of the bursal-derived peptides on antitumorThe bursa of Fabricius (BF) is the acknowledged central humoral immune organ, and plays key roles on B cell differentiation and antibody production. However, the mechanism of the bursal-derived peptides on antitumor is little reported. In this paper, Hela and MCF-7cells were used as the tumor cells model to investigate the antitumor cell proliferation functions of BP5, BSP-Ⅰ, BSP-Ⅱ, BPP-Ⅰ, and BPP-Ⅱ on tumor cells in vitro. The results showed that BP5and BSP-Ⅰ could significantly reduce the tumor cells proliferations, while BSP-Ⅱ, BPP-Ⅰ, and BPP-Ⅱ reduced tumor cells viability with high doses whereas stimulated tumor cells growth with low doses. It was not observed the inhibition effect on non-tumor cell proliferation after these five bursal-derived peptides treatment. Furthermore, BP5, BSP-Ⅰ, BSP-Ⅱ, BPP-Ⅰ, and BPP-Ⅱ triggered p53activity, and enhanced p53protein expression and stabilization, respectively. These results suggest that these five bursal-derived peptides were the potential immunomodulator of antitumor, which suggested that the humoral immune organ of mammal and human might play function in tumor development.Information generated in this study elucidate further the mechanisms involved in humoral immune system and represent the potential basis of effective immunotherapeutic strategies for treating human tumors and immune improvement.