Functional genomic study and bioinformatic analysis on natural bioactive peptides

Abstract

An extraordinary diverse components of animal toxins have emerged along with the long-term evolution of animals, which have unique functions that protect them to survive in the wild. Specifically, the granular glands of amphibians contain multiple chemical compounds, among which, peptides are one of the major types of the constituents.This thesis is divided into six chapters. Chapter 1, as general introduction, describes the detailed background on peptide-based therapeutic agents, the promising therapeutic application of animal toxins, and a brief review on anuran skins and their secretions. Chapter 2 presents the materials and methods employed in this project. In chapter 3, a novel bradykinin-related peptide was isolated and identified from the skin secretion of odorrana livida using shotgun cloning and tandem mass spectrometry fragmentation sequencing approach. This peptide exhibited a dose-dependent contractile property on rat bladder and ileum and increased the contraction frequency on rat uterus ex vivo smooth muscle preparations. It also showed vasorelaxant activity on rat tail artery smooth muscle. In addition, the peptide was modified by substituting the penultimate amino acid in the amino terminus from phenylalanine to leucine. Theanalogue completely abolished parental peptide activity, but showed an inhibition effects on bradykinin-induced rat tail artery smooth muscle relaxation. By using specific antagonists for bradykinin B1 and B2 receptors, we found that bradykinin b2receptor is highly likely to be involved in the rat tail artery related effects caused bythis novel bradykinin-related peptide and its analogue. Chapter 4 and 5 are about thediscovery of two pairs of novel antimicrobial peptides belonging to Bombinin and VIBombinin H families, respectively, from the skin secretion of Bombina genus. In chapter 4, the sequence modification was applied on bombinin HL by replacing Lisomer-leucine to D-isomer leucine from the second position of the amino terminus.Both the wild type and modified peptides displayed well-defined α-helical structure in bacterial membrane mimicking environment. BHL-bombinin displayed broad-spectrum bactericidal activity against a wide range of microorganisms, while bombinin H only exhibited a mild bacteriostatic effect on gram positive bacteria. The synergistic antimicrobial effects were observed between BHL-bominin with bombininH and between bombinin H with ampicillin. In addition, haemolytic and cytotoxic examination exhibited a highly synergistic selectivity and low cytotoxicity on mammalian cells of these three peptides. In chapter 5, the sequence modification was employed in the BHK-bombinin by replacing the glutamic acid with lysine at the 23rdposition from amino terminus, which increased the net charge and expanded thenonpolar face of the original peptide. According to the results from in vitro function alanalysis, this modification strategy significantly improved the selectivity index of thepeptide with increased antimicrobial activity and decreased haemolysis activity. The combined antimicrobial evaluation on both natural and modified peptides showed synergistic inhibition activity against both gram positive bacteria and fungi yeast.In summary, this thesis reveals the combined strategy of using a molecular cloning technique and mass spectrometric method for novel host-defensive peptides identification from amphibian skin secretions. In vitro and ex vivo functionalevaluations were subsequently employed which not only bring us a better understanding on the diversity of natural sourced bioactive peptides but also emphasized the research value for characterizing their in depth mechanisms

Date of Award	2017
Original language	English
Awarding Institution	Queen's University Belfast
Supervisor	Tianbao Chen (Supervisor), Lei Wang (Supervisor) & Mei Zhou (Supervisor)