Tachyplesin

Introduction

Tachyplesin I, II and III are antimicrobial polypeptide originally detected in the leukocytes of Japanese Horse Shoe Crab. It has been reported to inhibit the growth of bacteria, fungui and viruses. The antimicrobial activity of the polypeptide is contributed by electrostatic interaction between the negatively charged membrane of bacteria and fungi to positively charged part of ^[1] (see the Hydrophobic and Cationic (+) amino acids). Specifically, TP-I shows high affinity for negatively charged lipopolysaccharides (LPS) of gram-negative bacteria, thus neutralizing its effects.

Structural highlights

Tachyplesin I is a 17-residue peptide containing six cationic residues with molecular weight 2,269 and isoelectric point (pI) of 9.93.^[2] The amino acid sequence of the TP-I is NH₂-Lys-Trp-Cys-Phe-Arg-Val-Cys-Tyr-Arg-Gly-Ile-Cys-Tyr-Arg-Arg-Cys-Arg-CONH₂.

Figure 1: Simplified model of Tachyplesin I.

The sequence adapts an antiparallel β-sheet (hairpin) conformation in solution, with a for the centrally located residues , stabilized by two cross-strand between Cys³-Cys¹⁶ and Cys⁷-Cys¹²^[3][4], and C-terminus amidation. In addition there are H-bonds and aromatic rings stacking interactions which helps stabilize the hairpin loop structure of the peptide.

NMR studies have shown that TP-I undergoes a conformational change from to , making it than in the presence of water^[5]. Moreover a docking model suggests the stability of the structure of TP-I is increased in the presence of LPS by the binding of the N and C termini of TP-I to LPS. The conformational change of TP-I seems to be crucial for its antimicrobial activity, since rearrangement of TP-I structure makes it more amphiphilic to negatively charged membrane of bacteria and fungus^[1].

Derivatives or Analogue

Among all the existing interactions, the cysteine bridges were considered as the principal contributors of the hairpin loop structure. To test this, three linear derivatives of TP-I (, TPF4 and TPA4) were created, in which the bridging cysteine residues were systematically replaced with tyrosine, phenylalanine, and alanine, respectively^[1][5]. The linear derivatives of TP-I are mentioned below:

Of these 3 linear derivatives of TP-I, NMR structural investigations had shown that TPA4 was unstructured in solution. Also, TPA4 was inactive in terms of antimicrobial activity. In contrast, TPY4 and TPF4 adapt hairpin loop structure and also retain their antimicrobial properties, typical to TP-I. Therefore, the hairpin properties of the peptide seems to be important for recognition of LPS and its biological activities.

Besides replacement of cysteines, deletions were also performed in TP-I which yielded the surprising result of a hairpin loop that was seen, by NMR structure in LPS, in the (CDT). Thus, CTD with sequence NH₂-Lys-Trp-Phe-Arg-Val-Tyr-Arg-Gly-Ile-Tyr-Arg-Arg-Arg-CONH₂ did not have disulphide linkage, but was found to have broad spectrum of bactericidal activity. Specifically, CDT has been demonstrated to markedly inhibit the growth of Escherichia coli and Listeria monocytogenes akin to TP-I, even with lower minimum inhibitory concentration (MIC) values.

CDT Structure

CDT, like TP-I, has a β-turn with the in its LPS-bound structure.

The β-hairpin topology of CDT is sustained by the between the aromatic ring of Trp2 and the side-chain of nonpolar amino acid of Val5 and the cationic side-chain of residue Arg11. Also, there exists close proximity between residues which is supported by nuclear overhauser effects (NOEs) involving indole ring protons of Trp2 with side-chain proton of Ile9. These packing interactions have rendered an approximate anti-parallel orientation of the hairpin structure of CDT in presence of LPS. The β-hairpin structure displays an extended positively charged surface patch of residues . These positively charged basic residues interacts with the anionic phosphate groups of LPS, that leads to a plausible disruption or fluidization of LPS structures. This facilitates traversal of the peptide through the LPS-outer membrane^[3].

Mode of action

TP-I has high affinity to negatively charged cell membrane containing LPS and also has ability to permeabilize the cell membrane of pathogens. Docking model suggests strong interaction between cationic residues of TP-I with phosphate group and saccharides of LPS, where , acts as hinges ^[1]. Also, interaction between hydrophobic residues of TP-I with acyl chains of LPS was observed which strengthens the TP-I/LPS interaction^[6]. Ultimately, binding of TP-I/LPS neutralizes LPS, which is widely considered as endotoxin, and disrupts membrane function.

In addition to LPS binding, footpriting analysis has revealed the binding of TP-I to DNA by interacting specifically in minor groove of DNA duplex. The interaction between TP-I and DNA is contributed by secondary structure of the peptide which contains an antiparallel beta-sheet constrained by two disulfide bridges and connected by β-turn ^[7]. TP-I on binding to DNA and RNA, inhibits the synthesis of macromolecules.

In summary, three processes might happen upon TP-I exposure: (1) Bacterial cell membranes are penetrated without disruption of the membrane and the peptide reaches the inner structures of the cell, damaging critical intracellular targets and interfering with intracellular functions and normal metabolism. (2) Pores are formed in the cell wall, causing leakage of intracellular content, leading to cell death. (3) DNA, RNA or protein synthesis are inhibited, killing the bacteria.^[6].

Importance and relevance

Plants and Agriculture

Evidences suggest that TP-I has ability to permeabilize the cell membranes of pathogens.^[1]. Also, LPS and DNA being the potential biological targets of the peptide, its antimicrobial activity might be exploited. Eyeing the potential of TP-I, it has been insetred successfully in genome of Ornithogalum dubium and Ornithogalum thyrsoides. These ornamentals plants were originally sensitive to soft rot erwinias (SREs) and insertion of TP-I in the plants has successfully protected them without affecting their normal physiology ^[8][9].

Clinical Importance

Unlike mammalian cell membrane, bacterial cell membrane are negatively charged. Escherichia coli and Listeria monocytogenes being most common pathogenic bacteria in animals and humans, studying the effect of TP-I on E. coli and S. aureus will be valuable in guiding clinical practice. The study in E. coli has shown membrane disruption upon treatment with TP-I. Also macromolecule leakage into the cytoplasm and the release of potassium ions was observed that ultimately killed E. coli^[6].

Possible Function as anti-tumor peptide

The cationic nature of Tachyplesin allows it to interact with anionic phospholipids present in the bacterial membrane and thereby disrupting membrane function. Besides this, the structural nature of Tachyplesin also highlights its antitumor properties. Since it can interact with the membrance of prokaryotic cell, it is likely that TP-I can also interact with the mitochondrial membrane of eukaryotic cells. Mitochondria are widely believed to have evolved from prokaryotic cells, that have established a symbiotic relationship with the primitive eukaryotic cell which signifies the structural similarity of mitochondrial and prokaryotic membranes.

It was found that the synthetic Tachyplesin conjugated to the integrin homing domain (RGD-Tachyplesin) can inhibit the proliferation of TSU tumor cells prostate cancer and B16 melanoma cells as well as endothelial cells in a dose-dependent manner in vitro and reduce tumor growth in vivo by inducing apoptosis.^[2]. Besides this RGD-Tachyplesin can activate caspases and induce Fas ligand, which are the markers for programmed cell death (PCD)^[10].

Collectively, suppression of tumor associated cell and induction of programmed cell death will eventually act as therapy for cancer and tumor cells.

3D structure of tachyplesin

Updated on 18-August-2024

Quiz

References

1. ↑ ^{1.0 1.1 1.2 1.3 1.4} Laederach A, Andreotti AH, Fulton DB. Solution and micelle-bound structures of tachyplesin I and its active aromatic linear derivatives. Biochemistry. 2002 Oct 15;41(41):12359-68. PMID:12369825
2. ↑ ^{2.0 2.1} Chen, Yixin, et al. "RGD-Tachyplesin inhibits tumor growth." Cancer research 61.6 (2001): 2434-2438.‏
3. ↑ ^{3.0 3.1} Saravanan R, Mohanram H, Joshi M, Domadia PN, Torres J, Ruedl C, Bhattacharjya S. Structure, activity and interactions of the cysteine deleted analog of tachyplesin-1 with lipopolysaccharide micelle: Mechanistic insights into outer-membrane permeabilization and endotoxin neutralization. Biochim Biophys Acta. 2012 Mar 23;1818(7):1613-1624. PMID:22464970 doi:10.1016/j.bbamem.2012.03.015
4. ↑ Nakamura, Takanori, et al. "Tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (Tachypleus tridentatus). Isolation and chemical structure." Journal of Biological Chemistry 263.32 (1988): 16709-16713
5. ↑ ^{5.0 5.1} Kushibiki T, Kamiya M, Aizawa T, Kumaki Y, Kikukawa T, Mizuguchi M, Demura M, Kawabata SI, Kawano K. Interaction between tachyplesin I, an antimicrobial peptide derived from horseshoe crab, and lipopolysaccharide. Biochim Biophys Acta. 2014 Jan 2;1844(3):527-534. doi:, 10.1016/j.bbapap.2013.12.017. PMID:24389234 doi:http://dx.doi.org/10.1016/j.bbapap.2013.12.017
6. ↑ ^{6.0 6.1 6.2} Hong, Jun, et al. "Mechanism of Tachyplesin I injury to bacterial membranes and intracellular enzymes, determined by laser confocal scanning microscopy and flow cytometry." Microbiological research (2014)
7. ↑ Yonezawa A, Kuwahara J, Fujii N, Sugiura Y. Binding of tachyplesin I to DNA revealed by footprinting analysis: significant contribution of secondary structure to DNA binding and implication for biological action. Biochemistry. 1992 Mar 24;31(11):2998-3004. PMID:1372516
8. ↑ Lipsky A, Cohen A, Ion A, Yedidia I. Genetic transformation of Ornithogalum via particle bombardment and generation of Pectobacterium carotovorum-resistant plants. Plant Sci. 2014 Nov;228:150-8. doi: 10.1016/j.plantsci.2014.02.002. Epub 2014 Feb, 12. PMID:25438795 doi:http://dx.doi.org/10.1016/j.plantsci.2014.02.002
9. ↑ Lipsky A, Joshi JR, Carmi N, Yedidia I. Expression levels of antimicrobial peptide tachyplesin I in transgenic Ornithogalum lines affect the resistance to Pectobacterium infection. J Biotechnol. 2016 Nov 20;238:22-29. doi: 10.1016/j.jbiotec.2016.09.008. Epub, 2016 Sep 14. PMID:27639550 doi:http://dx.doi.org/10.1016/j.jbiotec.2016.09.008
10. ↑ Ellerby, H. Michael, et al. "Anti-cancer activity of targeted pro-apoptotic peptides." Nature Medicine(1999)‏