Sauvagine

PD-sauvagine: a novel sauvagine/corticotropin releasing factor analogue from the skin secretion of the Mexican giant leaf frog, Pachymedusa dacnicolor.[Pubmed:22134582]

Amino Acids. 2012 Sep;43(3):1147-56.

Sauvagine is a potent and broad-spectrum biologically active peptide of 40 amino acid residues originally isolated from the skin of the South American frog, Phyllomedusa sauvagei. Since its discovery, no additional Sauvagine structures have been reported. Following the discovery of Sauvagine, peptides with similar primary structures/activities were identified in mammalian brain [corticotropin-releasing factor (CRF) and urocortin]. Here, we report the identification of a second Sauvagine from the Mexican giant leaf frog, Pachymedusa dacnicolor, which displays primary structural features of both Sauvagine and CRF. A cDNA encoding the peptide precursor was "shotgun" cloned from a cDNA library constructed from lyophilised skin secretion by 3'- and 5'-RACE reactions. From this, the primary structure of a 38-mer peptide was deduced and this was located in reverse phase HPLC fractions of skin secretion and both its mass and structure were confirmed by mass spectrometry. The biological activities of synthetic replicates of PD-Sauvagine and Sauvagine were compared using two different mammalian smooth muscle preparations and the novel peptide was found to be more potent in both. Bioinformatic analyses of PD-Sauvagine revealed that it shared different regional sequence identities with both Sauvagine and CRF.

PH-sauvagine from the skin secretion of Phyllomedusa hypochondrialis: A novel CRF-like peptide with smooth muscle contraction activity.[Pubmed:26216532]

Toxicon. 2015 Sep 15;104:19-25.

Amphibian skin, and particularly that of south/Central American phyllomedusine frogs, is supposed to be "a huge factory and store house of a variety of active peptides". The 40 amino acid amphibian CRF-like peptide, Sauvagine, is a prototype member of a unique family of these Phyllomedusa skin peptides. In this study, we describe for the first time the structure of a mature novel peptide from the skin secretion of the South American orange-legged leaf frog, Phyllomedusa hypochondrialis, which belongs to the amphibian CRF/Sauvagine family. Partial amino acid sequence from the N-terminal was obtained by automated Edman degradation with the following structure: pGlu-GPPISIDLNMELLRNMIEI-. The biosynthetic precursor of this novel Sauvagine peptide, consisted of 85 amino acid residues and was deduced from cDNA library constructed from the same skin secretion. Compared with the standard Sauvagine from the frog, Phyllomedusa sauvagei, this novel peptide was found to exert similar contraction effects on isolated guinea-pig colon and rat urinary bladder smooth muscle preparations.

Characterization of a Pachymedusa dacnicolor-Sauvagine analog as a new high-affinity radioligand for corticotropin-releasing factor receptor studies.[Pubmed:25736419]

J Pharmacol Exp Ther. 2015 May;353(2):307-17.

The corticotropin-releasing factor (CRF) peptide family comprises the mammalian peptides CRF and the urocortins as well as frog skin Sauvagine and fish urophyseal urotensin. Advances in understanding the roles of the CRF ligand family and associated receptors have often relied on radioreceptor assays using labeled CRF ligands. These assays depend on stable, high-affinity CRF analogs that can be labeled, purified, and chemically characterized. Analogs of several of the native peptides have been used in this context, most prominently including Sauvagine from the frog Phyllomedusa sauvageii (PS-Svg). Because each of these affords both advantages and disadvantages, new analogs with superior properties would be welcome. We find that a Sauvagine-like peptide recently isolated from a different frog species, Pachymedusa dacnicolor (PD-Svg), is a high-affinity agonist whose radioiodinated analog, [(125)ITyr(0)-Glu(1), Nle(17)]-PD-Svg, exhibits improved biochemical properties over those of earlier iodinated agonists. Specifically, the PD-Svg radioligand binds both CRF receptors with comparably high affinity as its PS-Svg counterpart, but detects a greater number of sites on both type 1 and type 2 receptors. PD-Svg is also approximately 10 times more potent at stimulating cAMP accumulation in cells expressing the native receptors. Autoradiographic localization using the PD-Svg radioligand shows robust specific binding to rodent brain and peripheral tissues that identifies consensus CRF receptor-expressing sites in a greater number and/or with greater sensitivity than its PS-Svg counterpart. We suggest that labeled analogs of PD-Svg may be useful tools for biochemical, structural, pharmacological, and anatomic studies of CRF receptors.

Residue 17 of sauvagine cross-links to the first transmembrane domain of corticotropin-releasing factor receptor 1 (CRFR1).[Pubmed:18955489]

J Biol Chem. 2008 Dec 19;283(51):35644-51.

Corticotropin-releasing factor receptor 1 (CRFR1) mediates the physiological actions of corticotropin-releasing factor in the anterior pituitary gland and the central nervous system. Using chemical cross-linking we have previously reported that residue 16 of Sauvagine (SVG) is in a close proximity to the second extracellular loop of CRFR1. Here we introduced p-benzoylphenylalanine (Bpa) at position 17 of a Sauvagine analog, [Tyr0, Gln1, Bpa17]SVG, to covalently label CRFR1 and characterize the cross-linking site. Using a combination of receptor mutagenesis, peptide mapping, and N-terminal sequencing, we identified His117 within the first transmembrane domain (TM1) of CRFR1 as the cross-linking site for Bpa17 of 125I-[Tyr0, Gln1, Bpa17]SVG. These data indicate that, within the SVG-CRFR1 complex, residue 17 of the ligand lies within a 9 angstroms distance from residue 117 of the TM1 of CRFR1. The molecular proximity between residue 17 of the ligand and TM1 of CRFR1 described here and between residue 16 of the ligand and the CRFR1 second extracellular loop described previously provides useful molecular constraints for modeling ligand-receptor interaction in mammalian cells expressing CRFR1.

Actions of CRF and its analogs.[Pubmed:10519912]

Curr Med Chem. 1999 Nov;6(11):1035-53.

Corticotropin-releasing factor (CRF), urocortin, Sauvagine and urotensin I form the CRF family. These peptides bind with different affinities to two subtypes of CRF receptor (CRFR), CRFR1 and CRFR2. The latter exists as two splice variants, the neuronal CRFR2a and the peripheral CRFR2b. CRFR is a G protein-dependent receptor which acts mainly through Gs enhancing cAMP production. However, CRFR1 expressed in neutrophils of the spleen in response to immunologic stimulation and psychological stress does not seem to function through Gs, as indicated by the inability of CRF to stimulate the cAMP production of CRFR1+ neutrophils. Besides the two receptors, a 37 kD CRF binding protein (CRF-BP) binds several CRF peptides with high affinity. CRFR and CRF-BP do not share a common amino acid sequence representing the ligand binding site. In view of the unusually slow offrate of CRF-BP, it is proposed that CRF-BP provides an efficient uptake of free extracellular CRF. Thus, the time of exposure of CRFR to CRF or urocortin can be limited. At this time, the fate of the ligand CRF-BP complex is unclear. CRFR1 is not only involved in the hypophyseal stimulation of corticotropin release, but hippocampal CRFR1 mediates enhancement of stress-induced learning. CRFR1 may also be involved in basic anxiety. In contrast, at least in the mouse, CRFR2 of the lateral intermediate septum mediates tonic impairment of learning. In response to stressful stimuli or after local injection of high CRF doses, CRFR2 mediates anxiety. Effects requiring CRFR2 can be blocked specifically by the recently developed peptidic antagonist antiSauvagine-30.

Corticotropin releasing factor receptors and their ligand family.[Pubmed:10816663]

Ann N Y Acad Sci. 1999 Oct 20;885:312-28.

The CRF receptors belong to the VIP/GRF/PTH family of G-protein coupled receptors whose actions are mediated through activation of adenylate cyclase. Two CRF receptors, encoded by distinct genes, CRF-R1 and CRF-R2, and that can exist in two alternatively spliced forms, have been cloned. The type-1 receptor is expressed in many areas of the rodent brain, as well as in the pituitary, gonads, and skin. In the rodent, one splice variant of the type-2 receptor, CRF-R2 alpha, is expressed mainly in the brain, whereas the other variant, CRF-R2 beta, is found not only in the CNS, but also in cardiac and skeletal muscle, epididymis, and the gastrointestinal tract. The poor correlation between the sites of expression of CRF-R2 and CRF, as well as the relatively low affinity of CRF for CRF-R2, suggested the presence of another ligand, whose existence was confirmed in our cloning of urocortin. This CRF-like peptide is found not only in brain, but also in peripheral sites, such as lymphocytes. The broad tissue distribution of CRF receptors and their ligands underscores the important role of this system in maintenance of homeostasis. Functional studies of the two receptor types reveal differences in the specificity for CRF and related ligands. On the basis of its greater affinity for urocortin, in comparison with CRF, as well as its brain distribution, CRF-R2 may be the cognate receptor for urocortin. Mutagenesis studies of CRF receptors directed toward understanding the basis for their specificity, provide insight into the structural determinants for hormone-receptor recognition and signal transduction.

Amino acid composition and sequence analysis of sauvagine, a new active peptide from the skin of Phyllomedusa sauvagei.[Pubmed:7309372]

Int J Pept Protein Res. 1981 Aug;18(2):113-20.

The complete amino acid sequence of Sauvagine, a new active polypeptide from the skin of Phyllomedusa sauvagei, a frog of Central and South America, has been determined by automated liquid-phase procedure after specific removal of the N-terminal pyrrolidonecarboxylic acid, and specific cleavages at the single methionine and at the two arginine residues. The proposed sequence is: Pyr-Gly-Pro-Pro-Ile-Ser-Ile-Asp-Leu-Ser-Leu-Glu-Leu-Leu-Arg-Lys-Met-Ile-Glu-Ile -Glu-Lys-Gln-Glu-Lys-Glu-Lys-Gln-Gln-Ala-Ala-Asn-Asn-Arg-Leu-Leu-Leu-Asp-Thr-Il e-NH2. Sauvagine possesses a number of pharmacological actions on diuresis, the cardiovascular system and endocrine glands; it can be considered the prototype of a new family of amphibian peptides, in addition to the tachykinins, bradykinins, dermorphins, caerulein-like and bombesin-like peptides.