A novel Kazal-type trypsin inhibitor from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas

The chemical complexity of the defensive skin secretion of the red-eyed leaf frog, (Agalychnis callidryas), has not been elucidated in detail. During a systematic study of the skin secretion peptidomes of phyllomedusine frogs, we discovered a novel Kazal-type protein with potent trypsin inhibitory activity (Ki = 1.9 nM) that displays the highest degree of structural similarity with Kazal proteins from bony fishes. The protein was located in reverse-phase HPLC fractions following a screen of such for trypsin inhibition and subsequent partial Edman degradation of the peak active fraction derived the sequence: ATKPR-QYIVL-PRILRPV-GT. The molecular mass of the major component in this fraction was established by MALDI-TOF MS as 5893.09 Da. This partial sequence (assuming blank cycles to be Cys residues) was used to design a degenerate primer pool that was employed successfully in RACE-PCR to clone homologous precursor-encoding cDNA that encoded a mature Kazal protein of 52 amino acid residues with a computed molecular mass of 5892.82 Da. The protein was named A. callidryas Kazal trypsin inhibitor (ACKTI). BLAST analysis revealed that ACKTI contained a canonical Kazal motif (C-x(7)-C-x(6)-Y-x(3)-C-x(2,3)-C). This novel amphibian skin Kazal trypsin inhibitor adds to the spectrum of trypsin inhibitors of Kunitz- and Bowman Birk-type reported from this amphibian source.     

HV-BBI--a novel amphibian skin Bowman-Birk-like trypsin inhibitor

Here we describe the isolation of a novel C-terminally amidated octadecapeptide—SVIGCWTKSIPPRPCFVK-amide—that contains a disulphide loop between Cys⁵ and Cys¹⁵ that is consistent with a Bowman-Birk type protease inhibitor, from the skin secretion of the Chinese Bamboo odorous frog, Huia versabilis. Named HV-BBI, the peptide is encoded by a single precursor of 62 amino acid residues whose primary structure was deduced from cloned skin cDNA. The precursor exhibits the typical organization of that encoding an amphibian skin peptide with a highly-conserved signal peptide, an intervening acidic amino acid residue-rich domain and a single HV-BBI-encoding domain located towards the C-terminus. A synthetic replicate of HV-BBI, with the wild-type K (Lys-8) residue in the presumed P1 position, was found to be a potent inhibitor of trypsin with a K_i just slightly less than 19 nM. Substitution at this site with R (Arg) resulted in a significant reduction in potency (K_i 57 nM), whereas replacement of K with F (Phe) resulted in the complete abolition of trypsin inhibitory activity. Thus, HV-BBI is a potent inhibitor of trypsin and the lysyl (K) residue that occupies the P1 position appears to be optimal for potency of action against this protease.     

Genetic identification of units for conservation in tomato frogs, genus Dyscophus

Dyscophus antongilii and D. guineti are two morphologically very similar microhylid frogs from Madagascar of uncertain taxonomy. D. antongilii is currently included in Appendix I of the Convention on the International Trade in Endangered Species (CITES) and its exportation is banned completely. In contrast, D. guineti does not receive any legal protection and it is regularly exported. Field data on ecology and behaviour are to a large extent lacking. Here we report on a genetic survey of D. antongilii and D. guineti using nuclear and mitochondrial DNA markers. Sequences of a fragment of 501 bp of the mitochondrial cytochrome b gene from one population of D. antongilii and two populations of D. guineti resulted in a single haplotype network, without haplotype sharing among the populations. However, haplotypes of D.␣guineti were only 1–4 mutational steps from those of D. antongilii, and did not form a clade. The analysis of eight microsatellites newly developed and standardized for D. antongilii revealed an excess of homozygotes and the absence of Hardy–Weinberg equilibrium. The microsatellite data clearly distinguished between D. antongilii and D. guineti, and fixed differences were observed at one locus. Although confirmation of the status of Dyscophus antongilii and D. guineti as separate species requires further data, our study supports the definition of these two taxa as different evolutionary significant units under the adaptive evolutionary conservation concept.     

Evidence for participation of sperm proteinases in fertilization of the solitary ascidian,Halocynthia roretzi: Effects of protease inhibitors

Effects of 15 proteinase inhibitors and an inhibitor against aminopeptidases on fertilization of the solitary ascidian, Halocynthia roretzi were studied in search of lysins. Fertilization of intact eggs was blocked by three trypsin inhibitors, leupeptin, antipain, and soybean trypsin inhibitor, and by two chymotrypsin inhibitors, chymostatin and potato proteinase inhibitor I. On the other hand, the fertilization of naked eggs was not blocked at all by leupeptin and was only partially blocked by chymostatin at the concentrations sufficient for blocking that of intact eggs. This indicates that spermatozoa utilize trypsin-like and chymotrypsin-like proteinases probably as lysins for penetrating through the chorion. The chymotrypsin-like activity appears to be also required for some step besides sperm penetration through the egg investments.     

Phenotypic changes and the fate of digestive enzymes during induction of amber disease in larvae of the New Zealand grass grub (Costelytra zealandica)

Amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) is caused by ingestion of pADAP plasmid carrying isolates of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae) and causes infected larvae to cease feeding and clear their midgut to a pale amber colour where midgut serine protease activities are virtually eliminated. Using bacterial strains and mutants expressing combinations of the anti-feeding (afp) and gut clearance (sep) gene clusters from pADAP, we manipulated the disease phenotype and demonstrated directly the relationship between gene clusters, phenotype and loss of enzyme activity. Treatment with afp-expressing strains caused cessation of feeding without gut clearance where midgut protease activity was maintained at levels similar to that of healthy larvae. Treatment with strains expressing sep-genes caused gut clearance followed by a virtual elimination of trypsin and chymotrypsin titre in the midgut indicating both the loss of pre-existing enzyme from the lumen and a failure to replenish enzyme levels in this region by secretion from the epithelium. Monitoring of enzymatic activity through the alimentary tract during expression of disease showed that loss of serine protease activity in the midgut was matched by a surge of protease activity in the hindgut and frass pellets, indicating a flushing and elimination of the midgut contents. The blocking of enzyme secretion through amber disease appears to be selective as leucine aminopeptidase and α-amylase were still detected in the midgut of diseased larvae.     

Identification and molecular cloning of novel trypsin inhibitor analogs from the dermal venom of the Oriental fire-bellied toad (Bombina orientalis) and the European yellow-bellied toad (Bombina variegata)

The structural diversity of polypeptides in amphibian skin secretion probably reflects different roles in dermal regulation or in defense against predators. Here we report the structures of two novel trypsin inhibitor analogs, BOTI and BVTI, from the dermal venom of the toads, Bombina orientalis and Bombina variegata. Cloning of their respective precursors was achieved from lyophilized venom cDNA libraries for the first time. Amino acid alignment revealed that both deduced peptides, consisting of 60 amino acid residues, including 10 cysteines and the reactive center motif, -CDKKC-, can be affirmed as structural homologs of the trypsin inhibitor from Bombina bombina skin.     

Atomic resolution crystal structure of HV‐BBI protease inhibitor from amphibian skin in complex with bovine trypsin

Protease inhibitors of the Bowman‐Birk (BBI) family are commonly found in plants and animals where they play a protective role against invading pathogens. Here, we report an atomic resolution (1Å) crystal structure of a peptide inhibitor isolated from a skin secretion of a Chinese bamboo odorous frog Huia versabilis (HV‐BBI) in complex with trypsin. HV‐BBI shares significant similarities in sequence with a previously described inhibitor from a diskless‐fingered odorous frog Odorrana graham (ORB). However, the latter is characterized by more than a 16,000 fold higher K_i against trypsin than HV‐BBI. Comparative analysis of trypsin cocrystal structures of HV‐BBI and ORB and additionally that of Sunflower Trypsin Inhibitor (SFTI‐1) together with accessory information on the affinities of inhibitor variants allowed us to pinpoint the inhibitor moiety responsible for the observed large difference in activity and also to define the extent of modifications permissible within the common protease‐binding loop scaffold of BBI inhibitors. We suggest that modifications outside of the inhibitory loop permit the evolution of specificity toward different enzymes characterized by trypsin‐like specificity. Proteins 2015; 83:582–589. © 2014 Wiley Periodicals, Inc.     

Isolation and biochemical characterization of peptides presenting antimicrobial activity from the skin of Phyllomedusa hypochondrialis

Amphibian antimicrobial peptides have been known for many decades and several of them have been isolated from anuran species. Dermaseptins are among the most studied antimicrobial peptides and are found in the skin secretion of tree frogs from the Phyllomedusinae subfamily. These peptides exert a lytic action on bacteria, protozoa, yeast, and filamentous fungi at micromolar concentrations, but unlike polylysines, present little hemolytic activity. In this work, two antimicrobial peptides were isolated from the crude skin secretion of Phyllomedusa hypochondrialis and tested against Gram-positive and Gram-negative bacteria, presenting no hemolytic activity at the tested concentrations. One of them was identified with the recently reported peptide PS-7 belonging to the phylloseptin family, and another was a novel peptide, named DPh-1, which was fully purified, sequenced by ‘de novo’ mass spectrometry and grouped into Dermaseptins (DPh-1).     

Alpha-adrenergic regulation of the secretion of an anticomplementary factor in mouse saliva.

Mouse saliva contains a potent inhibitor of complement activity. The secretion of this inhibitor appears to be regulated by action on alpha-adrenergic receptors for two reasons. First, an alpha-agonist (norepinephrine) elicited saliva with a 260-fold higher specific activity of the inhibitor than that obtained with a cholinergic agent (pilocarpine). Second, the alpha-agonist elicited saliva having 43-foldgreater specific activity than that obtained following administration of a beta-adrenergic agonist (isoproterenol). This anticomplementary factor probably proteolytically degrades one or more of the complement components since it is inhibited by several protease inhibitors. The salivary anticomplementary factor is more potent than trypsin, chymotrypsin, thrombin, or Kallikrein. The anticomplementary factor has a pattern of inhibition like that of Kallikrein but unlike those of trypsin or chymotrypsin.     

The contribution of skin antimicrobial peptides to the system of innate immunity in anurans

Cationic peptides with the propensity to adopt an amphipathic ??-helical conformation in a membrane-mimetic environment are synthesized in the skins of many species of anurans (frogs and toads). These peptides frequently display cytolytic activities against a range of pathogenic bacteria and fungi consistent with the idea that they play a role in the host's system of innate immunity. However, the importance of the peptides in the survival strategy of the animal is not clearly understood. It is a common misconception that antimicrobial peptides are synthesized in the skins of all anurans. In fact, the species distribution is sporadic suggesting that their production may confer some evolutionary advantage to the organism but is not necessary for survival. Although growth inhibitory activity against the chytrid fungus Batrachochytrium dendrobatidis, responsible for anuran population declines worldwide, has been demonstrated in vitro, the ability of frog skin antimicrobial peptides to protect the animal in the wild appears to be limited and there is no clear correlation between their production by a species and its resistance to fatal chytridiomycosis. The low potency of many frog skin antimicrobial peptides is consistent with the hypothesis that cutaneous symbiotic bacteria may provide the major system of defense against pathogenic microorganisms in the environment with antimicrobial peptides assuming a supplementary role in some species.     

Stimulatory effect of an endogenous peptide in rat pancreatic juice on pancreatic enzyme secretion in the presence of atropine: evidence for different mode of action of stimulation from exogenous trypsin inhibitors

A new factor which activated the secretion of pancreatic enzymes was discovered and purified from rat bile-pancreatic juice. A fraction below M.W.10,000 of rat bile-pancreatic juice enhanced trypsinogen secretion by injection into anesthetized rat duodenum. The factor was purified from this fraction using its biological activity as an index by Sephadex G-50, SP Sephadex C-50 and HPLC. This factor was a peptide of which molecular weight was about 6,000 and had trypsin inhibitory activity. From these and some other findings, it was suggested that the peptide was identical with the "Kazal type" inhibitor. In the anesthetized and atropine-treated rat, of which intestinal trypsin was removed by thoroughly washing with saline containing 5 microM soybean trypsin inhibitor (SBTI), pancreatic secretion became basal state, and was not stimulated by injection of SBTI into its duodenum any longer. Under this condition, however, injection of this purified peptide brought about markedly stimulation of pancreatic enzyme secretion. These results suggest that this peptide has a certain function which enhances pancreatic enzyme secretion by the different manner from exogenous trypsin inhibitors such as SBTI.     

Inhibition of Growth of L5178Y Leukemia Cells by a Fungal Folate-deaminating Enzyme

Reactive sites of adzuki bean proteinase inhibitor II were determined by limited hydrolyses with catalytic amounts of trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1] at pH 3.0. Treatment of the trypsin-modified inhibitor with carboxypeptidase B [EC 3.4.12.3] released lysine from the inhibitor and led to complete loss of the activity for trypsin, virtually, without affecting the chymotrypsin-inhibitory activity. Limited hydrolysis with chymotrypsin resulted in a selective cleavage of a single tyrosyi peptide bond in the inhibitor, and treatment of this modified inhibitor with carboxypeptidase A [EC 3.4.12.2] abolished the chymotrypsininhibitory activity, having no effect on the trypsin-inhibitory activity. After reduction and S-carboxymethylation, the trypsin- and the chymotrypsin-modified inhibitors both could be separated into two components by gel-filtration on Sephadex G–50 and DEAE-cellulose chromatography. Amino acid and end group analyses of these components indicated that the reactive sites of inhibitor II are the Lys²⁷-Ser²⁸ bond against trypsin and the Tyr⁵⁴-Ser⁵⁵ against chymotrypsin.

Chemical modification of inhibitor II with cyanogen bromide had a fatal effect on the inhibitory activity against trypsin but no effect against chymotrypsin.     

BSTI, a trypsin inhibitor from skin secretions of Bombina bombina related to protease inhibitors of nematodes.

From skin secretions of the European frog Bombina bombina, a new peptide has been isolated that contains 60 amino acids, including 10 cysteine residues. Its sequence was determined by automated Edman degradation and confirmed by analysis of the cDNA encoding the precursor. A search in the databanks demonstrated that the pattern of cysteine residues in this skin peptide is similar to the ones found in protease inhibitors from Ascaris and in a segment of human von Willebrand factor. The 3D structure of the trypsin inhibitor from Ascaris suum could be used as a template to build a model of the amphibian peptide. In addition, we have demonstrated that this constituent of skin secretion is indeed an inhibitor of trypsin and thrombin, with K(i) values in the range of 0.1 to 1 microM. The new peptide was thus named BSTI for Bombina skin trypsin/thrombin inhibitor.     

Trypsin inhibitor in mung bean cotyledons: purification, characteristics, subcellular localization, and metabolism

Trypsin inhibitor was purified to homogeneity from seeds of the mung bean (Vigna radiata [L.] Wilczek). The protease inhibitor has the following properties: inhibitory activity toward trypsin, but not toward chymotrypsin; isoelectric point at pH 5.05; molecular weight of 11,000 to 12,000 (sodium dodecyl sulfate gel electrophoresis) or 14,000 (gel filtration); immunological cross-reactivity against extracts of black gram and black-eyed pea, but not against soybean; no inhibitory activity against vicilin peptidohydrolase, the principal endopeptidase in the cotyledons of mung bean seedlings.

The trypsin inhibitor content of the cotyledons declines in the course of seedling growth and the presence of an inactivating factor can be demonstrated by incubating crude extracts in the presence of β-mercaptoethanol. This inactivating factor may be a protease as vicilin peptidohydrolase rapidly inactivates the trypsin inhibitor. Removal of trypsin inhibitory activity from crude extracts by means of a trypsin affinity column does not result in an enhancement of protease activity in the extracts.

The intracellular localization of trypsin inhibitor was determined by fractionation of crude extracts on isopycnic sucrose gradients and by cytochemistry with fluorescent antibodies. Both methods indicate that trypsin inhibitor is associated with the cytoplasm and not with the protein bodies where reserve protein hydrolysis occurs. No convincing evidence was obtained which indicates that the catabolism of trypsin inhibitor during germination and seedling growth is causally related to the onset of reserve protein breakdown.

     

Structural and functional analysis of miraculin-like protein from Vitis vinifera

The so-called miraculin-like proteins (MLPs) are homologous to miraculin, a homodimeric protein with taste-modifying activity that converts sourness into sweetness. The identity between MLPs and miraculin generally ranges from 30% to 55%, and both MLPs and miraculin are categorized into the Kunitz-type soybean trypsin inhibitor (STI) family. MLP from grape (Vitis vinifera; vvMLP) exhibits significant homology to miraculin (61% identity), suggesting that vvMLP possesses miraculin-like properties. The results of size-exclusion chromatography and sensory analysis illustrated that vvMLP exists as a monomer in solution with no detectable taste-modifying activity. Crystal structure determination revealed that vvMLP exists as a β-trefoil fold, similarly as other MLPs and Kunitz-type protein inhibitors. The conformation of the loops, including the so-called reactive loop in the STI family, was substantially different between vvMLP and STI. Recombinant vvMLP had inhibitory activity against trypsin (K_i = 13.7 μM), indicating that the protein can act as a moderate trypsin inhibitor.     

虎纹蛙皮肤分泌物抗氧化二肽——Gln/Lys-Met的纯化及鉴定

目前已自青蛙皮肤分泌物中提取出多种属于“第三套抗氧化系统”的多肽,它们具有较强的抗氧化作用.本文从虎纹蛙皮肤分泌物中提取具有抗氧化活性的多肽物质,并检测其活性.利用电刺激虎纹蛙背部腺和耳后腺获得其皮肤分泌物,利用凝胶过滤色谱Sephadex G-50和反相高效液相色谱（reverse-phase high performance liquid chromatography, RP-HPLC）进行分离纯化.以2,2-二苯基-1-苦肼基（2,2-diphenyl-1- picrylhydrazyl, DPPH·）清除率为指标,测定多肽的抗氧化活性,经ESI- MS（electrospray ionization mass spectrometry）质谱测定该多肽的相对分子质量.结果显示,自虎纹蛙皮肤分泌物中获得一种具有抗氧化活性的二肽,其分子质量为0.277 kD,可能的氨基酸序列为Gln/Lys-Met.虎纹蛙皮肤分泌物中的该二肽是发挥抗氧化作用的重要组成部分.     

Anti-elastolytic activity of a honeybee (Apis cerana) chymotrypsin inhibitor

The honeybee is an important insect species in global ecology, agriculture, and alternative medicine. While chymotrypsin and trypsin inhibitors from bees show activity against cathepsin G and plasmin, respectively, no anti-elastolytic role for these inhibitors has been elucidated. In this study, we identified an Asiatic honeybee (Apis cerana) chymotrypsin inhibitor (AcCI), which was shown to also act as an elastase inhibitor. AcCI was found to consist of a 65-amino acid mature peptide that displays ten cysteine residues. When expressed in baculovirus-infected insect cells, recombinant AcCI demonstrated inhibitory activity against chymotrypsin (K_i 11.27 nM), but not trypsin, defining a role for AcCI as a honeybee-derived chymotrypsin inhibitor. Additionally, AcCI showed no detectable inhibitory effects on factor Xa, thrombin, plasmin, or tissue plasminogen activator; however, AcCI inhibited human neutrophil elastase (K_i 61.05 nM), indicating that it acts as an anti-elastolytic factor. These findings constitute molecular evidence that AcCI acts as a chymotrypsin/elastase inhibitor.     

A protease inhibitor of the Kunitz family from skin secretions of the tomato frog, Dyscophus guineti (Microhylidae)

Norepinephrine-stimulated skin secretions of the tomato frog, Dyscophus guineti, contained a trypsin inhibitor whose primary structure was established as: SPAEVCF LPK(10) ESGLCRARAL(20) RYYYDRGDGK(30) CEEFIYGGCG(40) GNGNNY KSLL(50) TCKISCE. This amino acid sequence identifies the peptide as a member of the Kunitz/bovine pancreatic trypsin inhibitor (BPTI) family and demonstrates that selective evolutionary pressure has acted to conserve those domains in the molecule (corresponding to positions 12-18 and 34-39 in BPTI) that interact with trypsin. Extracellular proteases produced by pathogenic microorganisms play important roles in facilitating invasion of the host and broad spectrum antimicrobial activity of BPTI has been described. Cationic, amphipathic alpha-helical antimicrobial peptides of the magainin type, important in the defense strategy of several species of frog, were not detected in the skin secretions. We speculate, therefore, that synthesis of a proteinase inhibitor in the skin of the tomato frog may be a component of an alternative strategy of this animal to defend itself against microorganisms.     

Identification and characterization of protease inhibitors in Diplotaxis species

PCR analysis of the genomes of two wild Brassicaceae plants, Diplotaxis muralis and Diplotaxis tenuifolia, demonstrated the presence of several genes coding for potential protease inhibitors, classifiable within the mustard inhibitor family (MSI). This is a small family of plant protease inhibitors named after the mustard trypsin inhibitor MTI-2, the first protease inhibitor characterized in Brassicaceae. From identified sequences two recombinant inhibitors were expressed in Pichia pastoris. In comparison with MTI-2, they show a reduced activity against bovine trypsin. However, when tested against trypsin-like proteases present in the guts of Helicoverpa zea larvae, the Diplotaxis inhibitors and MTI-2 show similar activities, indicating that the usually adopted procedure of reporting activity of plant protease inhibitors against bovine trypsin may lead to wrong estimation of their effect on insect proteases. This issue is of particular relevance when planning the use of PI genes for developing insect resistant plants.     

A Kazal-Type Serine Protease Inhibitor from the Defense Gland Secretion of the Subterranean Termite Coptotermes formosanus Shiraki

Coptotermes formosanus is an imported, subterranean termite species with the largest economic impact in the United States. The frontal glands of the soldier caste termites comprising one third of the body mass, contain a secretion expelled through a foramen in defense. The small molecule composition of the frontal gland secretion is well-characterized, but the proteins remain to be identified. Herein is reported the structure and function of one of several proteins found in the termite defense gland secretion. TFP4 is a 6.9 kDa, non-classical group 1 Kazal-type serine protease inhibitor with activity towards chymotrypsin and elastase, but not trypsin. The 3-dimensional solution structure of TFP4 was solved with nuclear magnetic resonance spectroscopy, and represents the first structure from the taxonomic family, Rhinotermitidae. Based on the structure of TFP4, the protease inhibitor active loop (Cys⁸ to Cys¹⁶) was identified.