乳源抗菌肽的分离纯化及部分性质的研究

建立酪蛋白酶解产生抗菌肽的最适条件，对酶解产物进行分离。建立抗菌肽活性测定方法，并对酪蛋白源抗菌肽的理化特性予以评价。本实验采用胰蛋白酶在不同时间条件下水解酪蛋白，经凝胶过滤和RP-HPLC分离纯化制备抗菌肽，酶解液及其分离物质对不同菌株的大肠杆菌和金黄色葡萄球菌的抗菌活性进行了测定。结果表明：2，3h胰蛋白酶的酪蛋白水解液均具有抗菌活性，且有良好的热稳定性。酪蛋白和胰蛋白酶本身无抗菌活性。2h水解液对大肠杆菌ATCC25922和金黄色葡萄球菌CMCC26801的最小抑菌浓度（Minimal Inhibitory Concentration, MIC））分别是1．20mg／ml和0．34mg／ml。2h水解液经SephadexG-15分离收集的5^#组分有抗菌活性，RP-HPLC检测有较单一肽吸收峰。说明酪蛋白经酶水解可以产生抗菌活性肽。     

Solid phase synthesis and characterization of two canine gut gastrin-releasing peptides

Two canine gastrin-releasing peptides originally isolated from gut tissue extracts have been synthesized by solid phase methodology and purified by preparative reverse phase high performance liquid chromatography (RP-HPLC). The synthetic gastrin-releasing peptides GRP1-27 and GRP 5-27 were characterized with regard to homogeneity and composition using nine different RP-HPLC systems, mass spectroscopy, amino acid analysis, Edman degradation, methionine oxidation, and peptide mapping with tryptic, Staph. aureus V8 protease and cyanogen bromide cleavage (the latter two systems performed only with GRP 1-27). Although a scarcity of the natural products prevented quantitative biological comparison of the synthetic and natural peptides, they were found to elute identically on RP-HPLC co-chromatography and similar dose dependent biological potencies were observed in canine antral muscle tissue contraction experiments. Indeed, all the peptides containing the bombesin-like car-boxyl terminal decapeptide sequence studied to date have similar biological activities.     

Comparison of the interaction of methionine and norleucine-containing peptides with phospholipid bilayers

Norleucine is a structural analog of methionine with a methylene group replacing the thio ether. Despite the close structural similarity of these two amino acids, norleucine-containing peptides have markedly different behaviour with phospholipids compared with methionine-containing peptides. For example, HCO-L-Ahx-L-Leu-L-Phe-OMe behaves as a hydrophobic peptide when mixed with dimyristoylphosphatidylcholine. This peptide lowers the enthalpy of the lipid phase transition. The effect is independent of the rate of heating. With the homologous peptide, HCO-L-Met-L-Leu-L-Phe-OMe, the results are markedly dependent on scan rate with a higher enthalpy observed at faster scan rates. Only at a scan rate of 0.2 K min^-1 do the two peptides approach similar behaviour. The higher enthalpy observed for samples with the methionine peptide at higher scan rates can be explained assuming that the peptide aggregates at low temperature. As the phase transition temperature is approached, the more hydrophilic methionine peptide partitions more slowly into the membrane than the norleucine peptide. Partitioning of the peptides between aqueous and lipid phases was measured at 37° by centrifuging down the lipid-bound fraction. At a peptide concentration of 15 μM and a lipid concentration of 1.4 mM, 89% of the HCO-L-Ahx-L-Leu-L-PheOMe and 97% of the HCO-L-Met-L-Leu-L-PheOMe remained in the supernate, indicating a greater tendency of the norleucine-containing peptide to partition into the lipid phase. The peptides Ac-L-Phe-L-Met-L-Arg-L-Phe-NH₂ and Ac-L-Phe-L-Ahx-L-Arg-L-Phe-NH₂ are readily soluble in water. These peptides were deposited with the lipid from a solution in chloroform/methanol. After repeated scanning and/or incubation at the phase transition temperature the effect of the peptide in raising the phase transition temperature of the lipid was lost. Thus these peptides were no longer incorporated into the bilayer after these treatments. The loss of the alterations of the phase transitions caused by these peptides was observed upon immediate rescanning of the methionine-containing samples while for the norleucine-containing ones, incubation for several hours at the phase transition was required. Our results demonstrate that norleucine-containing peptides partition more rapidly into the lipid phase while the methionine peptides are more rapidly extracted from the lipid into the aqueous phase. The results emphasize the importance of determining the effect of scan rate as a criterion of the attainment of equilibrium in lipid-peptide mixtures. We demonstrate how relatively minor changes in structure can alter the behaviour of peptides with lipids. Our results suggest that the pharmacodynamics of peptides in which norleucine has replaced methionine will be altered.     

Agelaia MP-I: A peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K⁺ (K_ATP) channels, increasing intracellular Ca²⁺ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 μM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 μM diazoxide, a K_ATP channel opener and 10 μM nifedipine, a Ca²⁺ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K_ATP and L-type Ca²⁺ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling.     

Solid-phase synthesis and characterization of carcinoembryonic antigen (CEA) domains

Carcinoembryonic antigen (CEA), a 180,000 dalton cell surface glycoprotein expressed on tumors of the colon, breast, ovary, and lung, has seven predicted immunoglobulin-like domains (N-A1-B1-A2-B2-A3-B3), most of which are recognized by distinct monoclonal antibodies. To study the individual domains, we have prepared several of the domains (N, A3, B3, and A3-B3) by solid-phase peptide synthesis. The syntheses were performed by the Fmoc method using single couplings, elevated temperatures for both the coupling and deblocking reactions, and a flexible solvent system for the coupling reactions. The syntheses were accomplished on an in-house built synthesizer which allowed for temperature control and flexible solvent control during the course of the coupling reactions. Due to the large size of the peptides (84-184 residues), it was anticipated that the overall purity of the final product would not exceed 60% even for an average coupling yield of 99.5%. Therefore, several of the peptides were synthesized with a His₆“tail” at the amino terminus, allowing for purification on a Ni-NTA chelate column. For the most part, the purified peptides exhibited single sharp peaks by RP-HPLC, migrated at their expected molecular weights by gel permeation chromatography, gave correct masses by electrospray ionization or matrix-assisted laser desorption ionization time of flight mass spectrometry, gave the expected amino acid analyses, N-terminal sequences, and tryptic maps, and bound their appropriate monoclonal antibodies. The N-domain was extremely hydrophobic, requiring 6m guanidinium hydrochloride for solubilization, the A3 domain was soluble in dilute acid, and the B3 domain had an intermediate solubility. The affinity constants of the A3 domain and several mutants (also made by peptide synthesis) are reported, along with characterization of the 178 amino acid two-domain peptide, A3-B3. Although there is no evidence for proper folding of these domains by NMR, their ability to bind monoclonal antibodies with high affinity suggests that this is a plausible approach for producing individual domains of CEA.     

Vejovine, a new antibiotic from the scorpion venom of Vaejovis mexicanus

Multidrug resistant bacterial infections are one of the most important health problems in recent years. Resistance to conventional antibiotics limits the therapeutic options causing increase rate in morbid-mortality in hospitals. Therefore, new antibacterial agents with new bacterial targets have been searched and found in many different sources, including scorpion venom and scorpion hemolymph. Here, we report a new anti-microbial peptide named Vejovine. This peptide was isolated from the venom of the Mexican scorpion Vaejovis mexicanus by two steps of reversed phase high performance liquid chromatography (RP-HPLC). It is composed of 47 amino acid residues with no cysteine residues in its sequence, with a molecular weight of 4873 Da. The chemical synthesis of Vejovine was performed by the solid phase method of Merrifield, using fluoren-9-ylmethoxycarbonyl (Fmoc)-amino acids. Both the native and synthetic peptides were shown to have essentially the same activity. Vejovine inhibits growth of clinical isolates of Gram-negative multidrug resistant (Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Acinetobacter baumanii) causing nosocomial infections with a minimum inhibitory concentration (MIC) of 4.4 μM up to 50 μM. This peptide has also hemolytic activity against human erythrocytes with a HC₅₀ value of 100 μM. A cDNA library of the venomous gland of this scorpion provided material for cloning the gene encoding Vejovine. This peptide is a new type of antibiotic, showing less than 50% similarity to other known scorpion peptides. Vejovine is a candidate to be used as a leading compound for future development of an effective peptide against multidrug resistant bacteria.     

Study on the synthesis and characterization of peptides containing phosphorylated tyrosine

Phosphorylation and dephosphorylation are key events in receptor-mediated and post-receptor-mediated signal transduction. Synthetic phosphopeptides have been shown to have dramatic agonist or antagonist effects in several of these signaling pathways. For its 1997 study, the Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Group assessed the ability of member laboratories to synthesize phosphotyrosine peptides. Participating laboratories were requested to synthesize and submit the following crude peptide, H–Glu–Asp–Tyr–Glu–Tyr(PO₃H₂)–Thr–Ala–Arg–Phe–NH₂, for evaluation by amino acid analysis, sequence analysis, RP-HPLC, MALDI-TOF and ESI mass spectrometry. Prior to analysis of submitted peptides from ABRF members, the Peptide Synthesis Research Group synthesized and characterized the nonphosphorylated form of the peptide, the doubly phosphorylated form and the peptides singly phosphorylated on either the first or the second tyrosine. These peptide standards were separated easily by HPLC and capillary electrophoresis and the phosphotyrosine was detected readily by Edman degradation sequence analysis. No differences were seen by amino acid analysis and the expected masses were observed by mass spectrometry. The two singly phosphorylated peptides were easily distinguished by MALDI-PSD. Analysis of the peptides submitted from member facilities revealed that all but four of the 33 samples contained the correct product as determined by HPLC and mass spectrometry. HPLC analysis indicated that 20 of the 33 submitted samples contained greater than 75% correct product, five contained less than 50% correct product and four did not contain any correct product. By ESI/MS, an additional singly charged ion at m/z 535.5 was detected in five of the 33 submitted samples; this ion was subsequently shown to represent Ac-TARF-NH₂. No correlation was found to exist between coupling time and percentage correct product; however, a correlation may exist between a greater percentage of correct product and the use of non-protected phosphotyrosine.     

RP-HPLC tryptic mapping of IgG1 proteins with post-column fluorescence derivatization

Peptide mapping is an important analytical technique widely used to study the primary structure of proteins. In quality control settings, it is employed as an identity test to probe for small changes in protein primary structure. A great challenge in peptide mapping is to minimize the detection limit for peptides due to the low detectability of smaller peptides based on their ultraviolet absorbance. The detection of peptide fragments can be enhanced by pre-or post-column derivatization with fluorescent tags. The use of post-column o-pthalaldehyde (OPA) and fluorescamine chemistries for on-line derivatization of peptide fragments from the RP-HPLC tryptic maps of several IgG1 monoclonal antibodies was explored. This paper describes the simple and sensitive peptide mapping technique for structural confirmation of proteins using picomoles of samples by post-column fluorescence derivatization. A comparison of UV and fluorescence detection of a peptide map is also presented. The method includes post column OPA derivatization of tryptic peptides from RP-HPLC tryptic maps with fluorescence detection. The conclusion reached that fluorescence detection gave relative detectability for tryptic peptides that range from 10- to 100-fold better than those observed with UV detection. The sensitivity of the peptide map increased by about 200-500 fold, i.e. peptide maps could be obtained using 2-5 pmol of digest instead of 1 nmol of digest. A roughly equal fluorescence response for all peptides (equal peak areas) was generally observed.     

Dipeptide derivative synthesis catalyzed by Pseudomonas aeruginosa elastase

Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides. The identity of the products was confirmed by FAB⁺-MS. After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured. With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P′₁ position and it has a wide specificity for the P₁ position. This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH₂). The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P′₁ position. Furthermore, synthesis initial rates were at least 100 times faster with the elastase. To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy. We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups. It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine. Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P₁ position. A synthesis was demonstrated with αAbu, Nle, Nva and Phg.     

Comparison of the binding of α-helical and β-sheet peptides to a hydrophobic surface

The induction and stabilisation of secondary structure for a series of amphipathic α-helical and β-sheet peptides upon their binding to lipid-like surfaces has been characterised by reversed phase high-performance liquid chromatography (RP-HPLC). In addition, a series of peptides which have been shown to switch from β-sheet to α-helical conformation upon transfer from a polar to a non-polar solution environment also have been studied. Binding parameters related to the hydrophobic contact area and affinity for immobilised C18 chains were determined at temperatures that ranged from 5 to 85°C, allowing conformational transitions for the peptides during surface adsorption to be monitored. The results demonstrated that all peptides which adopt secondary structure in solution also exhibited large changes in their interactive properties. Overall, this study demonstrates that the hydrophobic face of each amphipathic peptide dominates the binding process and that hydrophobic interactions are a major factor controlling the surface induction of secondary structure.     

Peptide synthesis catalyzed by papain at alkaline pH values

The synthesis of peptides in the presence of papain at pH 8–9.5 is described. Starting substances are acylamino acid alkyl esters (the carboxyl component) and amides or tert.-butylesters of amino acids, as well as peptide (the amino component). Under such conditions secondary hydrolysis is not essential, making the synthesis of peptides soluble in aqueous medium. The yield of peptides is 50–94%. The effect of different factors (temperature, solvents, reagent concentrations) on the result of the reaction has been studied. It has been found that an excess of the carboxyl component is expedient to increase the yield of peptides.     

The mammalian tachykinin receptors

The tachykinins (TKs) are a family of small peptides which share the common C-terminal sequence Phe-X-Gly-Leu-MetNH₂. Three peptides of this family, substance P, neurokinin A and neurokinin B, have an established role as neurotransmitters in mammals.

• 1.2. Three receptors for TKs have been cloned: they are G-protein coupled receptors with seven putative transmembrane spanning segments and have been termed NK₁ (substance P-preferring), NK₂ (neurokinin A-preferring) and NK₃ (neurokinin B-preferring).
• 2.3. Synthetic agonists are available to selectively stimulate only one receptor, while natural TKs can act as full agonist at each one of the three receptors, albeit at different concentrations.
• 3.4. A number of potent and selective antagonists, both peptide and nonpeptide in nature, have recently been developed.
• 4.5. The introduction of these ligands has revealed an unforeseen pharmacological heterogeneity of NK₁, NK₂ and NK₃ receptors which appears largely, if not exclusively, linked to the existence of species homologues of the three receptors.

     

Structure,Size, and Solubility of Antigen Arrays Determines Efficacy in Experimental Autoimmune Encephalomyelitis

Presentation of antigen with immune stimulating “signal” has been a cornerstone of vaccine design for decades. Here, the antigen plus immune “signal” of vaccines is modified to produce antigen-specific immunotherapies (antigen-SITs) that can potentially reprogram the immune response toward tolerance of an autoantigen. The codelivery of antigen with a cell adhesion inhibitor using Soluble Antigen Arrays (SAgAs) was previously shown to slow or halt experimental autoimmune encephalomyelitis (EAE), a murine form of multiple sclerosis (MS). SAgAs are comprised of a hyaluronic acid backbone with cografted intercellular cell adhesion molecule-1 ligand derived from α_L-integrin (CD11a_237–246, “LABL”) and an encephalitogenic epitope peptide of proteolipid protein (PLP_139–151, “PLP”). Here, the physical characteristics of the carrier were investigated to evaluate how structure, size, and solubility drive the immune response when treating EAE. A bifunctional peptide (small, soluble), SAgAs (large, soluble), and PLGA nanoparticles (large, insoluble) all displaying PLP and LABL in equimolar ratios were compared. Maximum EAE suppression was achieved with coincident display of both peptides on a soluble construct.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-014-9654-z) contains supplementary material, which is available to authorized users.KEY WORDS: experimental autoimmune encephalomyelitis, multivalency, proteolipid peptide, scaffold, soluble antigen array     

Spot synthesis: observations and optimizations

Abstract: Positionally addressable syntheses of peptides on continuous cellulose membranes (spot synthesis) have often been reported in detail, but important questions dealing with synthesis quality, reproducibility and subsequent binding assays have largely been under-emphasized. In this report we have investigated some of these problems. The most important results were: (i) the signal intensity of ligate binding to cellulose-bound peptides and the affinity of the corresponding soluble peptides show good correlation, illustrated by three different ligate binding assays; (ii) reducing peptide density on the cellulose avoids the ‘ring spot’ effect, i.e. where less binding is observed in the spot-center compared to the rim. We recommend a peptide density of 10 nmol/cm² as a reasonable starting point for further optimization; (iii) statistical analysis of binding assay reproducibility with more than 15 000 peptides resulted in a mean standard signal deviation of 0.18; and (iv) optimization of side-chain deprotection revealed that a 30-min pretreatment of the cellulose with 90% trifluoroacetic acid followed by the standard deprotection protocol resulted in higher purity of the synthesized products.     

Mechanism and kinetics of secretion degradation in aqueous solutions

In order to clarify the mechanism of secretin degradation in aqueous solutions, the formation of degradation products from secretin, aspartoyl3 secretin and beta-aspartyl3 secretin was investigated; the stabilities of these three peptides were investigated as well. Aspartoyl3 secretion and beta-aspartyl3 secretin, degradation peptides produced during the storage of secretin in aqueous solutions, were isolated by preparative reversed-phase HPLC (RP-HPLC). The amounts of secretin and its two degradation peptides resulting from storage of secretin in various buffer solutions (pH 2.3 to 10.0, mu = 0.5 M, 60 degrees C) were determined by analytical RP-HPLC. Secretin and the isolated degradation peptides were stored separately in various aqueous buffer solutions resulting in the degradation of each peptide. A mixture of secretin and its degradation or cleavage peptides was formed in each solution. The observed degradation rates for each peptide approximately followed first-order kinetics. The pH-rate profiles for conversion of secretin and beta-aspartyl3 secretin were similar, while that for aspartoyl3 secretin was different from these two. Aspartoyl3 secretin was more stable than the others at pH 2.3 to 4.0, but it was easily degraded between pH 5.0 and 10.0. Investigation of aspartoyl3 secretin degradation showed that its degradation was related to the pH value of the solution, and that hydroxide ion catalyzes the ring opening of the aspartoyl peptide. Secretin was most stable in pH 7.0 buffer solution and more stable in acidic solutions than in alkaline solutions. Secretion was mainly degraded through the following pathways: cleavage peptides reversible secretin in equilibrium aspartoyl peptide in equilibrium beta-aspartyl peptide vector cleavage peptides.     

Targeted delivery of a proapoptotic peptide to tumors in vivo

RGD peptides recognize the α_vβ₃ integrin, a receptor that is overexpressed on the surface of both tumor blood vessels and cancerous cells. These peptides are powerful tools that act as single antiangiogenic molecules, but recently also have been used for tumor imaging and drug targeting. We designed the molecule RAFT-(c[-RGDfK-])₄, a constrained and chemically defined entity that can be produced at clinical-grade quality. This scaffold was covalently coupled via a labile bridge to the proapoptotic peptide (KLAKLAK)₂ (RAFT-RGD-KLA). A fluorescent, activatable probe was also introduced, allowing intracellular localization. At 2.5 µM, this molecule induced the intracellular release of an active KLA peptide, which in turn caused mitochondrial depolarization and cell death in vitro in tumor cells. In a mouse model, the RAFT-RGD-KLA peptide was found to prevent the growth of remote subcutaneous tumors. This study demonstrated that the antitumor peptide is capable of killing tumor cells in an RGD-dependent manner, thus lowering the nonspecific cytotoxic effects expected to occur when using cationic cytotoxic peptides. Thus, this chemistry is suitable for the design of complex, multifunctional molecules that can be used for both imaging and therapeutics, representing the next generation of perfectly controlled, targeted drug-delivery systems.     

Tertiary peptide bond containing-oligo(Leu)s

In order to elucidate the role of tertiary peptide bonds in the conformational development and solubility improvement of peptides, the conformational properties of oligo(Leu)s with the Pro residue and the Gly-(Dmob)Leu sequence were investigated in solution using i.r. absorption, CD, and molar rotation measurements. The i.r. absorption spectroscopy indicated that the peptides soluble in inert solvents such as CCl₄ and toluene had a predominantly β-sheet structure in these solvents. The conformations of the peptides in CCl₄ and toluene were essentially the same as those in a solid state, whereas in THF and in MeOH, the peptides examined were efficiently subjected to solvation, and a randomly coiled structure was predominant. In order to confirm the randomly coiled structure, measurements have been made of the molar rotations of the peptides in a variety of strong proton acceptor and donor solvents. CD measurements are also carried out in MeOH. Through the investigations, it was shown that the protection of peptide bonds and the insertion of the Pro residue had the same effect on conformational and solubilizing behaviors and induced onset of an unordered structure and easy solvation of the peptides in medium and high polarity-solvents.     

Structure-Based Grafting,Mutation, and Optimization of Peptide Inhibitors to Fit in the Active Pocket of Human Secreted Phospholipase A2: Find New Use of Old Peptide Agents with Anti-Inflammatory Activity

Phospholipase A₂ (PLA₂) is a key enzyme in the production of diverse mediators of inflammatory conditions, which possesses an open active pocket that is physicochemically compatible with a variety of small-molecule substrates and peptide inhibitors. Although various peptides and peptide analogues have been identified to have inhibitory activity against PLA₂ originated from animals and plants, only very few were designed for human secreted PLA₂ (hsPLA₂), an attractive target of inflammatory arthritis. Considering that the catalytic domains of PLA₂ family members across different species are highly conserved in primary sequence, advanced structure, and biological function, in this study, we proposed a synthetic pipeline to implement structure-based grafting, mutation, and optimization of peptide ligands from the snake PLA₂–peptide complex crystal structures into the active pocket of apo hsPLA₂ structure to computationally generate a large number of potential peptide inhibitors for hsPLA₂, and the hsPLA₂ inhibitory potency of few highly promising candidates arising from the theoretical analysis was determined. As might be expected, three peptides FLSFK, FLVYK, and FISYR showed relatively high inhibitory capability against hsPLA₂, and other three ALSYK, LVFYA, and KGAILGFM were also modestly potent as they can suppress the enzymatic activity with observable doses. Further, the designed peptide FLVYK with highest potency was carried out with structure-guided modification based on its atomic interactions with hsPLA₂ using the computationally modeled structure data, consequently resulting in a dual-point mutant E L I YK with significantly increased activity.     

Engineering and Structural Insights of a Novel BBI-like Protease Inhibitor Livisin from the Frog Skin Secretion

The Bowman–Birk protease inhibitor (BBI) family is a prototype group found mainly in plants, particularly grasses and legumes, which have been subjected to decades of study. Recently, the discovery of attenuated peptides containing the canonical Bowman–Birk protease inhibitory motif has been detected in the skin secretions of amphibians, mainly from Ranidae family members. The roles of these peptides in amphibian defense have been proposed to work cooperatively with antimicrobial peptides and reduce peptide degradation. A novel trypsin inhibitory peptide, named livisin, was found in the skin secretion of the green cascade frog, Odorrana livida. The cDNA encoding the precursor of livisin was cloned, and the predicted mature peptide was characterized. The mature peptide was found to act as a potent inhibitor against several serine proteases. A comparative activity study among the native peptide and its engineered analogs was performed, and the influence of the P₁ and P_2′ positions, as well as the C-terminal amidation on the structure–activity relationship for livisin, was illustrated. The findings demonstrated that livisin might serve as a potential drug discovery/development tool.