Peptide Leucine Arginine, a Potent Immunomodulatory Peptide Isolated and Structurally Characterized from the Skin of the Northern Leopard Frog, Rana pipiens

Abstract

On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens). This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGC-WTKSYPPKPCFVR, in which Cys 5 and Cys 15 are disulfide bridged. The peptide was named peptide leucinearginine (pLR), reflecting the N-and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions. pLR was synthesized and elicited rapid, noncytolytic histamine release that had a 2-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin. pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action. pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i.e. mature neutrophils. pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.Studies on the array of biologically active substances present in amphibian skin have been reported in the scientific literature for almost 40 years (1). In particular, skin is a rich source of bioactive peptides, many of which are present in copious amounts (mg/g wet weight skin) (2). Many peptides occur at concentrations that are several orders of magnitude higher than the circulating levels in the frog, indicating that a major source of biosynthesis is in the skin (3). These peptides are putative components in the defense of the frog against predation or invading microorganisms (1).Several peptides have been isolated from the skins of ranid frog species, which have structural analogs in mammalian neuroendocrine systems. Bradykinin and related family members have been identified in several Rana species (4 -8) and others include the bombesin-like peptide, ranatensin (9), thyrotropin-releasing hormone (10, 11), caerulein (12), xenopsinrelated peptide, margaratensin (13), and the tachykinin ranamargarin (14). The hemolytic/chemotactic ranid frog peptides (7, 12) and the family of temporin peptides (15) demonstrate structural similarity to the mast cell-activating and phospholipase A2-facilitating peptide, crabrolin, from the venom of the wasp, Vespa crabro (16).Many peptides activate rat peritoneal mast cells; thus, histamine release can be utilized as a screen for putative bioactive peptides. There are several frog skin peptides that have been identified in this manner, namely, peptide XO-4 from Kassina maculata (17), granuliberin R from Rana rugosa (18), and the pipinins from Rana pipiens (19). A number of peptides have also been isolated from the venoms of hornets, wasps, and bees on the basis of histam...

Dermaseptin DA4, although closely related to dermaseptin B2, presents chemotactic and Gram‐negative selective bactericidal activities

“…The first antimicrobial peptide shown to exhibit immunological properties was DRS‐S1, a 34 amino acid cationic antimicrobial peptide, which stimulates the microbicidal activity of PMN leukocytes [12]. A few other frog peptides, temporin A, rana‐6 and pLR‐like peptides and, recently, another dermaseptin‐related peptide, DRS‐S9, have been shown to be microbicidal as well as immunomodulatory [13,27–29]. Here, we report the isolation, by screening for chemotactic activity, of a new member of the dermaseptin superfamily, DRS‐DA4, from the defensive skin secretion of the Mexican leaf frog P. dacnicolor .…”

pLR‐HL: A Novel Amphibian Bowman–Birk‐type Trypsin Inhibitor from the Skin Secretion of the Broad‐folded Frog, Hylarana latouchii

“…Here, we describe the ‘shotgun’ molecular cloning of a BBI biosynthetic precursor‐encoding cDNA, identification and structural characterization/functional analysis of its encoded novel Bowman–Birk‐type trypsin inhibitor of the pLR/ranacyclin family, which was named pLR‐HL, from the skin secretion of the broad‐folded frog, Hylarana latouchii . This peptide contains an inhibitory loop region demonstrating high homology with that of pLR identified from the skin of the northern leopard frog, Rana pipiens and with that of ranacyclins isolated from the skin of several species of Ranidae frogs. In addition, preliminary investigation of its structure–activity relationship was performed by comparing the protease inhibitory potency of a synthetic P 1 ‐substituted analogue (Phe 8 ‐pLR‐HL) with that of wild‐type pLR‐HL.…”

“…Identification and structural analysis of pLR-HL in skin secretion A second sample of 5 mg of lyophilized H. latouchii skin secretion was dissolved in 1 mL of trifluoroacetic acid (TFA)/water (0.05/99.95, v/v) and clarified by centrifugation. The clear supernatant was decanted and subjected to reverse-phase HPLC fractionation using a Cecil Adept CE4200 HPLC system (Amersham Biosciences, Buckinghamshire, UK) fitted with a Jupiter C-5 semi-preparative Table 1: Comparison of Bowman-Birk-type trypsin inhibitors on the primary structures, trypsin inhibitory and antimicrobial activities (14,15,20,24) NE, not effective. Identical amino acid resides are shaded black.…”

Bi-functional peptides with both trypsin-inhibitory and antimicrobial activities are frequent defensive molecules in Ranidae amphibian skins

“…They belong to the peptide family of Ranacyclin, which is originally found in the skin secretions of R. pipiens (Salmon et al 2001) and first named by Mangoni et al (2003). By cDNA cloning, 16 Ranacyclin B-like peptides were identified from these Ranidae amphibian skins (Fig.…”

“…pLR and pYR were originally identified as mammalian immune system regulatory peptides (Salmon et al 2001;Graham et al 2005). Ranacyclins from R. esculenta were originally identified as antimicrobial peptides.…”