Identification, molecular cloning and modification of skin-derived bioactive peptides from selected Hylidae and Ranidae

  • Guanzhu Chen

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Amphibians produced a series of adaptations to combat predators and various pathogenic microorganisms in their habitats. One of the greatest adaptations was the peptides secreted from the skin, which are of great importance to maintain their everyday life. Diverse bioactivities and potential pharmaceutical functions of peptides have been confirmed by researchers in recent years. Many peptides show antimicrobial activity to different extents and are less prone to induce bacterial resistance. However, during the evaluation of therapeutic potential, the unfavorable factors of peptides were revealed like modest MIC values relative to conventional antibiotics, higher costs of synthesis/production, stability against proteases, etc. Therefore, a series of approaches have been exploited to design peptide derivatives with improved qualities. Modification based on adjusting the hydrophobicity and hydrophobic moment and ultra-short peptide design are fast and effective means to increase the bioactivity of peptides. Besides, the applications of other approaches, like alanine scanning, W-tagging, unnatural amino acid replacement, introduction of chemical groups, etc., also have achieved great success. Here, different transformation methods were also suggested to generate ideas for future work in Chapter 6. In this thesis, the analysis of peptides was focused on selected species from Hylidae and Ranidae.
With the completion and development of gene sequencing, identification of single peptides from complex mixtures is achievable. In this thesis, the primary structures of peptides, PS-PH, QUB-2492 and QUB-3383, were determined from the skin secretions of Phyllomedusa hypochondrialis, Phyllomedusa baltea and Hylarana guentheri, through ‘shotgun’ cloning. In parallel to this strategy, predicted mature peptides were identified and isolated from skin secretion using HPLC and structures verified by LC-MS. The peptides were then chemically-synthesised to produce the quantities required for biological activity evaluation. NCBI-BLAST searches revealed that PS-PH, QUB-2492 and QUB-3383 belonged to phylloseptin, dermaseptin and brevinin families, respectively. Of note, PS-PH shares high identity with homologues in the phylloseptins, both QUB-2492 and QUB-3383 share limited similarities in primary structure with dermaseptins and brevinins, respectively. However, the conserved preproregions of QUB-2492 and QUB-3383 positively identified their taxonomic relationships.
PS-PH has broad-spectrum antimicrobial activity against S. aureus, E. coli, C. albicans and the drug-resistant bacteria (MRSA, E. faecalis), through the permeability mechanism. The importance of the effect of secondary structure on the antimicrobial activity of PS-PH was explored through designing PS-PHA. The improved antimicrobial activity of PS-PHA against all the bacteria used above was a positive result. QUB-2492 has no antimicrobial activity against S. aureus, E. coli, C. albicans, which is a significant difference from other dermaseptins. However, its anti-proliferation activity on lung cancer cell lines (H157, H460, H838, H23), breast cancer cell lines(MCF-7, MDA-MB-435S,MDA-MB-231S)with negligible cytotoxicity against HMEC-1 was a meaningful discovery. Learning from modifications of other dermaseptins, a truncated analogue QUB-1611, was designed. Limited antimicrobial activity against gram-positive, gram-negative, fungi bacteria and lack of anticancer activity of QUB-1611 against all the cancer cell lines tested revealed the important influence of the structural integrity on anti-proliferation activity of QUB-2492. QUB-3383 was a broad-spectrum antimicrobial molecule. The high MIC value and synthetic difficulty due to its length were shortcomings. Its truncated analogue, QUB-2610, significantly improved the MIC value against S. aureus, E. coli, C. albicans and the drug-resistant bacteria (MRSA, E. faecalis). By replacing the amino acid at position 14 with amino acids that have no adverse effect on the alpha helix, we tried to determine the active position of QUB-2610. The results revealed that the position 14 was irreplaceable for maintaining its bioactivity. With the revealed mechanism, the focus of our future work would be on revealing the intrinsic anticancer mechanism of QUB-2492 and exploring the active positions of QUB-2610, all of which could become very promising small therapeutic proteins.
Date of AwardDec 2019
Original languageEnglish
Awarding Institution
  • Queen's University Belfast
SupervisorTianbao Chen (Supervisor), Lei Wang (Supervisor), Xinping Xi (Supervisor) & Mei Zhou (Supervisor)

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