Anti-infection peptidomics of amphibian skin

Peptidomics and genomics analyses were used to study an anti-infection array of peptides of amphibian skin. 372 cDNA sequences of antimicrobial peptides were characterized from a single individual skin of the frog Odorrana grahami that encode 107 novel an

Antioxidant Peptidomics Reveals Novel Skin Antioxidant System

It is generally agreed that reactive oxygen species (ROS) contribute to skin aging, skin disorders, and skin diseases. Skin possesses an extremely efficient antioxidant system. This antioxidant activity is conferred by two systems: antioxidant enzymes and

Peptidomics and genomics analysis of novel antimicrobial peptides from the frog, Rana nigrovittata

Much attention has been paid on amphibian peptides for their wide-ranging pharmacological properties, clinical potential, and gene-encoded origin. More than 300 antimicrobial peptides (AMPs) from amphibians have been studied. Peptidomics and genomics analysis combined with functional test including microorganism killing, histamine-releasing, and mast cell degranulation was used to investigate antimicrobial peptide diversity. Thirty-four novel AMPs from skin secretions of Rana nigrovittata were identified in current work, and they belong to 9 families, including 6 novel families. Other three families are classified into rugosin, gaegurin, and temporin family of amphibian AMP, respectively. These AMPs share highly conserved preproregions including signal peptides and spacer acidic peptides, while greatly diversified on mature peptides structures. In this work, peptidomics combined with genomics analysis was confirmed to be an effective way to identify amphibian AMPs, especially novel families. Some AMPs reported here will provide leading molecules for designing novel antimicrobial agents. (C) 2009 Elsevier Inc. All rights reserved

Novel dermaseptin, adenoregulin and caerin homologs from the Central American red-eyed leaf frog, Agalychnis callidryas, revealed by functional peptidomics of defensive skin secretion

By integrating systematic peptidome and transcriptome studies of the defensive skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas, we have identified novel members of three previously described antimicrobial peptide families, a 27-mer dermaseptin-related peptide (designated DRP-AC4), a 33-mer adenoregulin-related peptide (designated ARP-AC1) and most unusually, a 27-mer caerin-related peptide (designated CRP-AC1). While dermaseptin and adenoregulin were originally isolated from phyllomedusine leaf frogs, the caerins, until now. had only been described in Australian frogs of the genus, Litoria. Both the dermaseptin and adenoregulin were C-terminally amidated and lacked the C-terminal tripeptide of the biosynthetic precursor sequence. In contrast, the caerin-related peptide, unlike the majority of Litoria analogs. was not C-terminally amidated. The present data emphasize the need for structural characterization of mature peptides to ensure that unexpected precursor cleavages and/or post-translational modifications do not produce mature peptides that differ in structure to those predicted from cloned biosynthetic precursor cDNA. Additionally, systematic study of the secretory peptidome can produce unexpected results such as the CRP described here that may have phylogenetic implications. It is thus of the utmost importance in the functional evaluation of novel peptides that the primary structure of the mature peptide is unequivocally established - something that is often facilitated by cloning biosynthetic precursor cDNAs but obviously not reliable using such data alone. (C) 2008 Elsevier Masson SAS. All rights reserved.

Functional peptidomics of amphibian skin secretion: A novel Kunitz-type chymotrypsin inhibitor from the African hyperoliid frog, Kassina senegalensis

Amphibian skin secretions are, for the most part, complex peptidomes. While many peptide components have been biologically- and structurally-characterised into discrete "families", some of which are analogues of endogenous vertebrate regulatory peptides, a substantial number are of unique structure and unknown function. Among the components of these secretory peptidomes is an array of protease inhibitors. Inhibitors of trypsin are of widespread occurrence in different taxa and are representative of many established structural classes, including Kunitz, Kazal and Bowman-Birk. However, few protease inhibitors with activity against other specific proteases have been described from this source. Here we report for the first time, the isolation and structural characterisation of an inhibitor of chymotrypsin of Kunitz-type from the skin secretion of the African hyperoliid frog, Kassina senegalensis. To this end, we employed a functional peptidomic approach. This scheme involves fractionation of the peptidome, functional end-point screening, structural characterisation of resultant actives followed by molecular cloning of biosynthetic precursor-encoding cDNA(s). The novel mature and active polypeptide identified consisted of 62 amino acid residues (average molecular mass 6776.24 Da), of which 6 were positionally-conserved cysteines. The P(1) position within the active site was occupied by a phenylalanyl residue. Bioinformatic analysis of the sequence using BLAST, revealed a structural similarity to Kunitz-type chymotrypsin inhibitors from other organisms, ranging from silkworms to snakes.

Salivary proteomics and peptidomics of type 1 diabetes Mellitus

A Diabetes Mellitus (DM) compreende um conjunto de desordens metabólicas comuns caracterizadas por hiperglicemia, que afeta diferentes órgãos do organismo. Ao longo do tempo, ocorrem danos microvasculares no glomérulo renal, retina e nervos periféricos, bem como doença macrovascular nas artérias. A composição da saliva também é afetada pela DM, com consequências na homeostasia oral. No entanto, o proteoma e o peptidoma salivar têm sido pouco explorados na DM tipo 1 e nas suas complicações crónicas. Tendo em conta o crescente interesse na saliva como fluido diagnóstico, o objetivo principal deste trabalho foi avaliar os eventos proteolíticos subjacentes à DM tipo 1 e às suas complicações microvasculares, bem como, caracterizar as alterações induzidas pela DM tipo 1 no proteoma e peptidoma salivar. A DM tipo 1 e particularmente as complicações microvasculares associadas modulam o perfil proteolítico dos fluidos biológicos, com diferenças significativas de atividade observadas na urina e saliva, atribuídas principalmente ao complexo Metaloproteinase da Matriz (MMP)-9/lipocalina associada à gelatinase de neutrófilos, aminopeptidase N, azurocidina e calicreína 1. O aumento da atividade proteolítica observado na saliva total dos diabéticos resultou no aumento da percentagem de péptidos, principalmente de um número acrescido de fragmentos de colagénio do tipo I, refletindo possivelmente um estado inflamatório crónico dos tecidos orais e periodontais. O peptidoma também corrobora uma maior suscetibilidade das proteínas salivares, especificamente, das proteínas ricas em prolina básicas (bPRP) 1, bPRP2 e proteínas ricas em prolina ácidas (aPRP) à proteólise, evidenciando a geração de fragmentos de proteínas associadas à ligação a bactérias. A análise do proteoma salivar baseada em iTRAQ mostrou uma sobre-expressão de L-plastina, fator do adenocarcinoma do pâncreas e das proteínas S100-A8 e S100-A9, enfatizando a importância do sistema imune inato na patogénese da DM tipo 1 e das complicações microvasculares associadas. A análise integrada de todas as proteínas expressas diferencialmente entre os pacientes diabéticos com ou sem complicações microvasculares e indivíduos saudáveis foi realizada com o STRING, onde se observam três conjuntos funcionalmente ligados, um compreende a interação entre o colagénio tipo I, colagénio tipo II e MMP-9, um segundo conjunto envolve a MMP-2 e o colagénio de tipo I e um terceiro conjunto composto por proteínas salivares e inflamatórias. Estes conjuntos estão associados com as vias Kegg de interação recetor-matriz extracelular, de adesão focal e migração transendotelial dos leucócitos. Por outro lado, a análise do proteoma e peptidoma salivar destacou potenciais biomarcadores para o diagnóstico e prognóstico da DM tipo 1 e das suas complicações.

Analysis of peptides in prohormone convertase 1/3 null mouse brain using quantitative peptidomics

P>Neuropeptides are produced from larger precursors by limited proteolysis, first by endopeptidases and then by carboxypeptidases. Major endopeptidases required for these cleavages include prohormone convertase (PC) 1/3 and PC2. In this study, quantitative peptidomics analysis was used to characterize the specific role PC1/3 plays in this process. Peptides isolated from hypothalamus, amygdala, and striatum of PC1/3 null mice were compared with those from heterozygous and wild-type mice. Extracts were labeled with stable isotopic tags and fractionated by HPLC, after which relative peptide levels were determined using tandem mass spectrometry. In total, 92 peptides were found, of which 35 were known neuropeptides or related peptides derived from 15 distinct secretory pathway proteins: 7B2, chromogranin A and B, cocaine- and amphetamine-regulated transcript, procholecystokinin, proenkephalin, promelanin concentrating hormone, proneurotensin, propituitary adenylate cyclase-activating peptide, proSAAS, prosomatosatin, provasoactive intestinal peptide, provasopressin, secretogranin III, and VGF. Among the peptides derived from these proteins, similar to 1/3 were decreased in the PC1/3 null mice relative to wild-type mice, similar to 1/3 showed no change, and similar to 1/3 increased in PC1/3 null. Cleavage sites were analyzed in peptides that showed no change or that decreased in PC1/3 mice, and these results were compared with peptides that showed no change or decreased in previous peptidomic studies with PC2 null mice. Analysis of these sites showed that while PC1/3 and PC2 have overlapping substrate preferences, there are particular cleavage site residues that distinguish peptides preferred by each PC.

Peptidomics of Three Bothrops Snake Venoms: Insights Into the Molecular Diversification of Proteomes and Peptidomes

Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 L-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from L-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4' sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed different outcomes in the peptidome characterization and suggested that degradomic-peptidomic analysis of snake venoms is highly sensitive to the conditions of sampling procedures. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019331, 1245-1262, 2012.

Potentials and pitfalls of clinical peptidomics and metabolomics

Clinical peptidomics and metabolomics are two emerging "-omics" technologies with the potential not only to detect disease-specific markers, but also to give insight into the disease dependency of degradation processes and metabolic pathway alterations. However, despite their rapid evolution and major investments, a clinical breakthrough, such as the approval of a major cancer biomarker, is still out of sight. What are the reasons for this failure? In this review we focus on three important factors: sensitivity, specificity and the avoidance of bias. The way to clinical implementation of peptidomics and metabolomics is still hampered by many of the problems that had to be solved for genomics and proteomics in the past, as well as new ones that require the creation of new analytic, computational and interpretative techniques. The greatest challenge, however, will be the integration of information from different "-omics" subdisciplines into straightforward answers to clinical questions, for example, in the form of new, superior "meta-markers".

Toward an understanding of the molecular mechanism for successful blood feeding by coupling proteomics analysis with pharmacological testing of horsefly salivary glands

Horseflies are economically important blood-feeding arthropods and also a nuisance for humans and vectors for filariasis. They rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands; especially no horsefly immune suppressants have been reported. By proteomics or peptidomics and coupling transcriptome analysis with pharmacological testing, several families of proteins or peptides, which act mainly on the hemostatic system or immune system of the host, were identified and characterized from 30,000 pairs salivary glands of the horsefly Tabanus yao (Diptera, Tabanidae). They are: (i) a novel family of inhibitors of platelet aggregation including two members, which possibly inhibit platelet aggregation by a novel mechanism and act on platelet membrane, (ii) a novel family of immunosuppressant peptides including 12 members, which can inhibit interferon-gamma production and increase interleukin-10 secretion, (iii) a serine protease inhibitor with 56 amino acid residues containing anticoagulant activity, (iv) a serine protease with anticoagulant activity, (v) a protease with fibrinogenolytic activity, (vi) three families of antimicrobial peptides including six members, (vii) a hyaluronidase, (viii) a vasodilator peptide, which is an isoform of vasotab identified from Hybomitra bimaculata, and interestingly (ix) two metallothioneins, which are the first metallothioneins reported from invertebrate salivary glands. The current work will facilitate the understanding of the molecular mechanisms of the ectoparasite-host relationship and help in identifying novel vaccine targets and novel leading pharmacological compounds.

无指盘臭蛙皮肤抗感染多肽组学研究

在过去的一个多世纪里，两栖类动物皮肤分泌液作为它们的第一道防御屏障引起了研究者们极大的兴趣，同时也开展了相关的许对研究，到目前为止，已从中分离鉴定出了百余种的活性物质。无指盘臭蛙是我国的一种特有两栖动物，初步的活性检测发现，无指盘臭蛙皮肤分泌液具有很强的抗菌，溶血以及蛋白酶抑制剂活性。 在本论文中，我们利用多肽组学与基因组学的方法对无指盘臭蛙皮肤的抗感染多肽组进行了研究。 通过三步分离纯化过程：一步Sephadex G-50分子筛和两步反相高压液相（RP-HPLC）的方法，从无指盘臭蛙皮肤分泌液中分离纯化得到了21条新的抗菌肽，它们分别属于17个不同的抗菌肽家族，其中8个分别属于已知的5个抗菌肽家族，它们是：Brevinin-1E(2个)、Brevinin-2E（1个）、Esculentin-1（1个）、Esculentin-2（1个）和Nigrocin（3个）抗菌肽家族。另外的13个抗菌肽与已发现的抗菌肽表现出较低的相似性，我们将它们归类到12种新的抗菌肽家族，它们是：Odorranain-A (1个)、Odorranain-B(1个)、 Odorranain-C(1个)、 Odorranain-G (1个)、Odorranain-H (2个)、 Odorranain-J (1个)、Odorranain-L (1个)、Odorranain-M (1个)、 Odorranain-N (1个)、 Odorranain-O (1个)、Odorranain-Q(1个)、 Odorranain-T(1个)。 从单个无指盘臭蛙皮肤里面，我们克隆得到了372条抗菌肽序列，它们编码107条新的抗菌肽，这一发现使得目前发现的两栖类抗菌肽的数目几乎增加了1倍。这也是目前发现的抗菌肽最为丰富的物种。这107条抗菌肽分别属于30个不同的抗菌肽家族，其中有24个为新的抗菌肽家族。这些抗菌肽的多样性可能是通过点突变、碱基的插入或删除、结构域的穿梭以及拼接等多种机制形成的。这些抗菌肽多样性的形成可能与无指盘臭蛙生活环境中微生物的组成有关。30个家族抗菌肽前体序列的SPD区域（包括信号肽和前导肽序列，Signal and Propiece Domain, SPD）非常保守，表明它们可能起源于同一个祖先基因。对7个抗菌肽家族的非同义碱基替代率Dn与同义碱基替代率Ds进行检测发现，它们可能经受着不同选择压力的作用。无指盘臭蛙皮肤抗菌肽在二级结构和功能上都表现出丰富的多样性。在一个两栖类个体里面发现如此丰富的抗菌肽甚是让人惊讶。这一发现也使得我们不得不重新认识先天性免疫在两栖类动物防御系统中的重要性，对两栖类生态环境的分子基础以及那种认为一种两栖类只需要20-30种抗菌肽就足以抵御环境中的微生物的看法重新审视。我们的研究还显示：无指盘臭蛙抗菌肽之间还存在着协同效用。 我们对无指盘臭蛙皮肤抗菌肽的去极化作用进行了研究，发现所检测的7个抗菌肽都可使金黄色葡萄球菌发生去极化，但是它们使细菌发生去极化的能力不同。对12种抗菌肽的抗菌机制研究发现，它们通过多种不同的机制发挥作用：有些在细菌内形成片层样的囊泡状结构，有些导致细胞质壁的分离，有些在细菌的膜上形成穿孔，有些则导致了细菌染色质的固缩。 总之，这些研究为设计新型的抗菌肽提供了有用的参考资料。

Nigrocin-2 peptides from Chinese Odorrana frogs – integration of UPLC/MS/MS with molecular cloning in amphibian skin peptidome analysis

Peptidomics is a powerful set of tools for the identification, structural elucidation and discovery of novel regulatory peptides and for monitoring the degradation pathways of structurally and catalytically important proteins. Amphibian skin secretions, arising from specialized granular glands, often contain complex peptidomes containing many components of entirely novel structure and unique site-substituted analogues of known peptide families. Following the discovery that the granular gland transcriptome is present in such secretions in a PCR-amenable form, we designed a strategy for peptide structural characterization involving the integration of ‘shotgun’ cloning of cDNAs encoding peptide precursors, deduction of putative bioactive peptide structures, and confirmation of these structures using tandem MS/MS sequencing. Here, we illustrate this strategy by means of elucidation of the primary structures of nigrocin-2 homologues from the defensive skin secretions of four species of Chinese Odorrana frogs, O. schmackeri, O. livida, O. hejiangensis and O. versabilis. Synthetic replicates of the peptides were found to possess antimicrobial activity. Nigrocin-2 peptides occur widely in the skin secretions of Asian ranid frogs and in those of the Odorrana group, and are particularly well-represented and of diverse structure in some species. Integration of the molecular analytical technologies described provides a means for rapid structural characterization of novel peptides from complex natural libraries in the absence of systematic online database information.