Leptoglycin: A new Glycine/Leucine-rich antimicrobial peptide isolated from the skin secretion of the South American frog Leptodactylus pentadactylus (Leptodactylidae)
Introduction
Antimicrobial peptides (AMPs) are relevant components of innate immunity that is the first-line defense for a wide range of organisms against invading pathogens (Hancock, 2001, Kimbrell and Beutler, 2001, Brown and Hancock, 2006). Classic AMPs are mainly cationic and amphipathic peptides with generally 12–50 amino acids in length, have a net positive charge due an excess of basic lysine and arginine residues over acid residues, and contain around 50% hydrophobic amino acids (Hancock, 2001, Bulet et al., 2004, Brogden, 2005, Brown and Hancock, 2006). These peptides, also know as host-defense peptides, have been isolated and characterized from diverse organisms (Sitaram and Nagaraj, 2002, Zasloff, 2002, Bulet et al., 2004) such as plants (Condit, 1993, Pelegrini and Franco, 2005, Pelegrini et al., 2008), arthropods (Bulet et al., 1999, Lorenzini et al., 2003, Herbinière et al., 2005, Dubovskii et al., 2006), amphibians (Zasloff et al., 1988, Bevins and Zasloff, 1990, Apponyi et al., 2004, Conceição et al., 2006, Pukala et al., 2006) and mammals (Mallow et al., 1996, Johansson et al., 1998, Rieg et al., 2004, Chromek et al., 2006, Zasloff, 2006).
Among amphibians, different antimicrobial peptides have been isolated from the granular gland skin secretion of anurans, such as Leptodactylus genus. It contains 64 species, spread out from South America, mainly in Brazil and Antilles (Frost, 2009). Biochemical analyses using the skin secretions of five Leptodactylus species revealed a family of eight structurally homologous AMPs that tend to adopt α-helical conformations in a membrane-mimetic system (King et al., 2005, Nielsen et al., 2007, Nascimento et al., 2007). Thus, the AMPs isolated from Leptodactylus ocellatus, ocellatins 1, 2 and 3, displayed growth-inhibitory activity against the Gram-negative bacterium Escherichia coli (Nascimento et al., 2004), and ocellatin 4 isolated from the same species presented cytolytic properties and growth-inhibitory activity against the Gram-negative E. coli and Gram-positive Staphylococcus aureus bacteria (Nascimento et al., 2007). The antibacterial peptide fallaxin, isolated from West Indian mountain chicken frog Leptodactylus fallax showed growth-inhibitory activity against several Gram-negative bacteria strains, such as E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterobacter cloacae, but not toward to Gram-positive bacterium S. aureus or the yeast Candida albicans (Rollins-Smith et al., 2005). Other peptide, termed pentadactylin, purified from Leptodactylus pentadactylus skin secretions, presented a better inhibition against the growth of several Gram-negative bacteria than Gram-positive bacteria and showed a very weak hemolytic activity (King et al., 2005). Laticeptin (Leptodactylus laticeps) (Conlon et al., 2006) and syphaxin (Leptodactylus syphax) (Dourado et al., 2007) peptides were tested against both Gram-positive and Gram-negative bacteria showing a high growth-inhibitory activity. More recently, AMPs with very low antimicrobial potency, named ocellatins-V1, 2 and 3, were isolated from Leptodactylus validus (King et al., 2008).
In the present study, we describe the isolation, biological characterization and amino acid sequencing of a novel neutral Glycine/Leucine-rich antimicrobial peptide, containing a central proline, active against Gram-negative bacteria, named Leptoglycin, from skin secretion of L. pentadactylus. This peptide was screened through biological assays and presented no hemolytic activity at the MIC levels. Its unique amino acid sequence was deduced by mass spectrometric de novo sequencing and confirmed by Edman degradation.
Section snippets
Collection of skin secretion
Adult specimens, both male and female (n = 6), of L. pentadactylus were collected in Paraipaba, Ceará, Brazil. The L. pentadactylus skin secretions were obtained by mild electrical stimulation (Conlon et al., 1999) and collected in a cooled beaker by washing the skin surface with cold bi-distilled water. This solution was lyophilized and stored at −25 °C. All the procedures involving animals were in accordance with the guidelines provided by the Brazilian College Animal Experimentation.
Reagents
All
Purification and characterization of leptoglycin
The supernatant fraction of the centrifuged L. pentadactylus crude skin secretion was fractionated by semi-preparative RP-HPLC, as shown in Fig. 1, panel A. Initially, peaks were tested for antibacterial activity against P. aeruginosa (LP01), C. freundii (LP02) and S. aureus LP03. Significant biological activity against Gram-negative bacteria could be found for the peak containing leptoglycin (LPG, indicated by the arrow, Fig. 1, panel A). The peptide was further purified as observed by
Discussion
In the present study, we report the isolation, amino acid sequence and antimicrobial effects of Leptoglycin, a novel antimicrobial peptide purified from the skin secretion of L. pentadactylus (Anura, Leptodactylidae).
In spite of the deduced (and chemically confirmed) amino acid sequence (Gly-Leu-Leu-Gly-Gly-Leu-Leu-Gly-Pro-Leu-Leu-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Leu-Leu), leptoglycin would not be an ideal candidate for MS/MS sequencing due to the hydrophobicity and repetitiveness of the
Acknowledgments
Supported by funds provided by FUNCAP, CAPES, FAPESP, Lab. Cristália, FAPEMIG (Edital 24000/01[MR]) and CNPq (grants 303516/2005-4 [DCP] & 473614/2006-5 [DCP]). Parts of this work were developed at the CAT/CEPID, a FAPESP grant. We are grateful to Rosa G.S. Oliveira for HPLC assistance.
References (59)
- et al.
Antimicrobial peptides isolated from skin secretions of the diploid frog, Xenopus tropicalis (Pipidae)
Biochim. Biophys. Acta
(2001) - et al.
Host-defense peptides of Australian anurans: structure, mechanism of action and evolutionary significance
Peptides
(2004) - et al.
Antimicrobial properties of two purified skin peptides from the mink frog (Rana septentrionalis) against bacteria isolated from the natural habitat
Comp. Biochem. Physiol. Part C: Toxicol. Pharmacol.
(2007) - et al.
Phenotypic characterization and in vitro antifungal sensitivity of Candida spp. and M. pachydermatis strains from dogs
Vet. J.
(2007) - et al.
Cationic host defense (antimicrobial) peptides
Curr. Opin. Immunol.
(2006) - et al.
Antimicrobial peptides in insects: structure and function
Dev. Comp. Immunol.
(1999) - et al.
Isolation and biochemical characterization of peptides presenting antimicrobial activity from the skin of Phyllomedusa hypochondrialis
Peptides
(2006) - et al.
Antimicrobial peptide from the skin secretion of the frog Leptodactylus syphax
Toxicon
(2007) Cationic peptides: effectors in innate immunity and novel antimicrobials
Lancet. Infect. Dis.
(2001)- et al.
Armadillidin: a novel glycine-rich antibacterial peptide directed against gram-positive bacteria in the woodlouse Armadillidium vulgare (Terrestrial Isopod, Crustacean)
Dev. Comp. Immunol.
(2005)
Conformation-dependent antibacterial activity of the naturally occurring human peptide LL-37
J. Biol. Chem.
Pentadactylin: an antimicrobial peptide from the skin secretions of the South American bullfrog Leptodactylus pentadactylus
Comp. Biochem. Physiol. Part C
Purification and characterization of antimicrobial peptides from the Caribbean frog, Leptodactylus validus (Anura: Leptodactylidae)
Peptides
Acanthoscurrin: a novel glycine-rich antimicrobial peptide constitutively expressed in the hemocytes of the spider Acanthoscurria gomesiana
Dev. Comp. Immunol.
Human enteric defensins: gene structure and developmental expression
J. Biol. Chem.
Purification, characterization and homology analysis of ocellatin 4, a cytolytic peptide from the skin secretion of the frog Leptodactylus ocellatus
Toxicon
Identification of putative palytoxin as the cause of clupeotoxism
Toxicon
A novel antimicrobial peptide from Bufo bufo gargarizans
Biochem. Phys. Res. Commun.
Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions
Biochem. Biophys. Res. Commun.
Plant gamma-thionins: novel insights on the mechanism of action of a multi-functional class of defense proteins
Int. J. Biochem. Cell. Biol.
Identification of a novel storage glycine-rich peptide from guava (Psidium guajava) seeds with activity against gram-negative bacteria
Peptide
Cryptides: buried secrets in proteins
Peptides
An antimicrobial peptide from the skin secretions of the mountain chicken frog Leptodactylus fallax (Anura: Leptodactylidae)
Regul. Pept.
Activity of antimicrobial skin peptides from ranid frogs against Batrachochytrium dendrobatidis, the chytrid fungus associated with global amphibian declines
Dev. Comp. Immunol.
The BPI/LBP family of proteins: a structural analysis of conserved regions
Protein Sci.
Peptides from frog skin
Annu. Rev. Biochem.
Antifungal susceptibility and genotypical pattern of Microsporum canis strains
Can. J. Microbiol.
Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?
Nat. Rev. Microbiol.
Anti-microbial peptides: from invertebrates to vertebrates
Immunol. Rev.
Cited by (59)
Isolation and functionalities of bioactive peptides from fruits and vegetables: A reviews
2022, Food ChemistryCitation Excerpt :Antimicrobial peptides are shorts chains of cationic and hydrophobic amino acids, facilitating interaction with bacterial cells whose cell wall is rich in negatively charged phospholipids (Matsuzaki, 1999; de Castro & Sato, 2015). Due to their hydrophobicity, glycine and leucine-rich BPs have shown high potency, also the presence of arginine residues contributes to antimicrobial activity, as they are cationic (Sousa et al., 2009; Amadou et al., 2013; Tang et al., 2015). Antimicrobial peptides have been classified based on their structure as: Linear peptides with alpha helices, disulfide bridged cyclic and open-ended cyclic peptides, and peptides rich in certain amino acid residues (Sánchez & Vázquez, 2017).
NMR structures in different membrane environments of three ocellatin peptides isolated from Leptodactylus labyrinthicus
2018, PeptidesCitation Excerpt :It is well known that these molecules exert their activities by disturbing the integrity of the microorganism membrane, although it is also known that some of these compounds may interact with internal targets after crossing the membrane barrier [14,15]. Ocellatins are antimicrobial peptides that have been discovered in the skin secretions of anurans of the Leptodactylus genus [16–22], which is composed of 74 species that inhabit South America, mainly Brazil and the Antilles [23]. We have recently isolated the peptides ocellatin-LB1, −LB2 and −F1 (Table 1) from the skin secretion of the frog species Leptodactylus labyrinthicus, commonly known as pepper frog, and we have investigated their biological activities, as well as their secondary structural preferences and membrane interactions by using different biophysical approaches [24].
Skin secretions of Leptodactylidae (Anura) and their potential applications
2024, Journal of Venomous Animals and Toxins Including Tropical Diseases
- 1
These authors contributed equally to the paper.