Synthesis and properties of cholecystokinin-releasing peptide (monitor peptide), a 61-residue trypsin inhibitor

A 61-residue cholecystokinin-releasing peptide (monitor peptide), which was obtained from rat pancreatic juice and found to stimulate pancreatic enzyme secretion, was recently reported to inhibit bovine trypsin and to possess epidermal growth factor (EGF)-like activities, at a concentration of about 10nM. However, monitor peptide is structurally different from the EGF family of growth factors. To investigate whether monitor peptide contains the supposed EGF-like activities, it has been synthesized together with its [Ala23, Ala47] analog. The purified peptides, which were fully characterized by a range of methods including Cf-252 ionization mass spectrometry and enzymatic digestion to establish the locations of disulfide linkages, were shown to belong to the pancreatic secretory trypsin inhibitor family and not to the EGF family. Neither synthetic monitor peptide nor its analog were able to compete with ¹²⁵I-EGF in A-431 cells or to stimulate growth of Swiss 3T3 and NRK 49F cells, up to 1 μM concentration. However, synthetic monitor peptide was as effective as the native product in the inhibition of trypsin. Replacement of the essential Arg23 in the [Ala23, Ala47]-analog led to loss of trypsin inhibition activity.     

Regeneration of native structure and biological activity by air oxidation of the reduced bovine trypsin inhibitor (Kunitz)*

Derivatives of the tetraguanidinated bovine trypsin-kallikrein inhibitor lacking up to four amino-acid residues have been prepared by Edman degradation of the amino-terminal Arg-Pro-Asp-Phe sequence. Comparative studies on the native inhibitor, tetraguanidinated inhibitor, and truncated sequences demonstrated that none of the four N-terminal amino-acid residues are essential for the regeneration of native structure and biological activity by air oxidation of the reduced molecule. However residues 2, 3, and 4, although to a different extent, are important for the protein stability and significantly affect the conformation of the tetraguanidinated inhibitor and the rate of the reactivation process. Removal of the terminal positive charge from Arg 1 by diazotization gave a fully active deaminated, tetraguanidinated inhibitor but the lack of the salt bridge interaction between the amino and the carboxyl terminus of the protein prevented the correct folding of the reduced molecule by air oxidation as shown by activity measurements and conformational studies. The stability constants and the standard free energies of binding of the complexes between trypsin and the different inhibitor derivatives have been determined.     

Synthesis and secondary structure of loop 4 of myelin proteolipid protein: effect of a point mutation found in Pelizaeus‐Merzbacher disease

Abstract: To study the effects of a point mutation found in Pelizaeus‐Merzbacher disease (PMD) on the physicochemical and structural properties of the extracellular loop 4 of the myelin proteolipid protein (PLP), we synthesized the peptide PLP(181–230)Pro²¹⁵ and one mutant PLP(181–230)Ser²¹⁵ with regioselective formation of the two disulphide bridges Cys²⁰⁰‐Cys²¹⁹ and Cys¹⁸³‐Cys²²⁷. As conventional amino acid building blocks failed to give crude peptides of good quality we had to optimize the synthesis by introducing pseudoproline dipeptide building blocks during the peptide elongation. In peptide Pro²¹⁵ the first bridge Cys²⁰⁰–Cys²¹⁹ was obtained after air oxidation, but in peptide Ser²¹⁵ because of aggregation, dimethyl sulfoxide (DMSO) oxidation had to be used. The second bridge Cys¹⁸³–Cys²²⁷ was obtained by iodine oxidation of both Cys (acetamidomethyl, Acm)‐protected peptides. The secondary structures of the parent and mutant loops were analysed by circular dichroism (CD) in the presence of trifluoroethanol (TFE) and sodium dodecyl sulphate (SDS) as a membrane mimetic. Analysis of the spectra showed that the content of α‐helix and β‐sheet varied differently for both peptides in TFE and SDS solutions, demonstrating the sensitivity of their conformation to the environment and the differences in their secondary structure. The ability of both peptides to insert into the SDS micelles was assayed by intrinsic tryptophan fluorescence.     

Disulfide pairing and secondary structure of ASP1, an olfactory‐binding protein from honeybee (Apis mellifera L)

Abstract: In insects, the transport of airborne, hydrophobic odorants and pheromones through the sensillum lymph is accomplished by olfactory‐binding proteins (OBPs). We report the structural characterization of a honeybee OBP called ASP1 found in workers and drones, previously observed to bind queen pheromone components. A novel method based on ion‐spray mass spectrometry analysis of cyanylation‐induced cleavage products of partially reduced protein with Tris(2‐carboxyethyl)phosphine was needed to determine the recombinant ASP1 disulfide bond pairing. It was observed to be Cys(I)‐Cys(III), Cys(II)‐Cys(V), Cys(IV)‐Cys(VI), similar to those already described for other OBPs from honeybee and Bombyx mori suggesting that this pattern occurs commonly throughout the diverse family of insect OBPs. Circular dichroism revealed that ASP1 is an all‐alpha protein in accordance with NMR preliminary data, but unlike lipocalin‐like vertebrate OBPs.     

Relationship between temporary inhibition and structure of disulfide‐linkage analogs of marinostatin,a natural ester‐linked protein protease inhibitor

Abstract: A 12‐residue marinostatin [MST(1–12): ¹FATMRYPSDSDE¹²] which contains two ester linkages of Thr³–Asp⁹ and Ser⁸–Asp¹¹ strongly inhibits subtilisin. In order to study the relationship between the inhibitory activity, structure, and stability of MST, MST analogs were prepared by changing ester linkages to a disulfide linkages. The analogs without the disulfide linkage between 3 and 9 positions lost their inhibitory activity. The K_i value of 1SS(C³–C⁹) (¹FACMRYPSCSDE¹²), which has a single disulfide linkage of Cys³–Cys⁹ was comparable with those of MST(1–12) and MST‐2SS (¹FACMRYPCCSCE¹²), a doubly linked analog of Cys³–Cys⁹ and Cys⁸–Cys¹¹. However, 1SS(C³–C⁹) and MST‐2SS showed temporary inhibition, but not MST(1–12): These analogs were inactivated after incubation with subtilisin for 30 min, and were specifically hydrolyzed at the reactive site. ¹H NMR study showed that 1SS(C³–C⁹) has two conformations, which contain a cis‐ (70%) or trans‐ (30%) Pro residue, while MST‐2SS as well as MST(1–12) takes a single conformation containing only a cis‐Pro residue. Hydrogen–deuterium exchange rate of the Arg⁵ (P1′) NH proton of the MST analogs was about 100 times faster than that of MST(1–12). These results indicate that the linkage between the positions 8 and 11 plays a role for fixing the cis‐conformation of the Pro⁷ residue, and that the linkage between 3 and 9 is indispensable for the inhibition, but not enough for stable protease‐inhibitor complex.     

Anti-convulsive and anti-epileptic properties of brivaracetam (ucb 34714), a high-affinity ligand for the synaptic vesicle protein, SV2A

BACKGROUND AND PURPOSE: Screening of 12,000 compounds for binding affinity to the synaptic vesicle protein 2A (SV2A), identified a high-affinity pyrrolidone derivative, brivaracetam (ucb 34714). This study examined its pharmacological profile in various in vitro and in vivo models of seizures and epilepsy, to evaluate its potential as a new antiepileptic drug. EXPERIMENTAL APPROACH: The effects of brivaracetam and levetiracetam on epileptiform activity and seizure expression were examined in rat hippocampal slices, corneally kindled mice, audiogenic seizure-susceptible mice, maximal electroshock and pentylenetetrazol seizures in mice, hippocampal-kindled rats, amygdala-kindled rats and genetic absence epilepsy rats. KEY RESULTS: Brivaracetam and levetiracetam reduced epileptiform responses in rat hippocampal slices, brivaracetam being most potent. Brivaracetam also differed from levetiracetam by its ability to protect against seizures in normal mice induced by a maximal electroshock or maximal dose of pentylenetetrazol. In corneally kindled mice and hippocampal-kindled rats, brivaracetam induced potent protection against secondarily generalized motor seizures and showed anti-kindling properties superior to levetiracetam. In amygdala-kindled rats, brivaracetam induced a significant suppression in motor-seizure severity and, contrary to levetiracetam, reduced the after-discharge at a higher dose. Audiogenic seizure-susceptible mice were protected more potently against the expression of clonic convulsions by brivaracetam than by levetiracetam. Brivaracetam induced a more complete suppression of spontaneous spike-and-wave discharges in genetic absence epilepsy rats than levetiracetam. CONCLUSIONS AND IMPLICATIONS: Brivaracetam has higher potency and efficacy than levetiracetam as an anti-seizure and anti-epileptogenic agent in various experimental models of epilepsy, and a wide therapeutic index.     

Synthesis,characterization and inhibitory activities of (4-N3[3,5-3H]Phe10)PKI(6–22)amide and its precursors: photoaffinity labeling peptides for the active site of cyclic AMP-dependent protein kinase

PKI(6–22)amide is a 17 residue peptide corresponding to the active portion of the heat-stable inhibitor of cAMP-dependent protein kinase. The peptide is a potent (K_i= 1.6 nM), competitive inhibitor of the enzyme. The photoreactive peptide analog (4-azidophenylalanine¹⁰)PKI(6–22)amide was synthesized in both its non-radiolabeled and tritiated forms by chemical modification of precursor peptides that were prepared by stepwise solid-phase synthesis. (4-Amino[3,5-³H]phenylalanine¹⁰)PKI(6–22)amide, the precursor for the radiolabeled arylazide peptide, was obtained by catalytic reduction of the corresponding peptide containing the 3,5-diiodo-4-aminophenylalanine residue at position 10. The purified PKI peptides were analyzed by HPLC, amino acid analysis, and U.V. spectra. In the dark, (4-azidophenylalanine¹⁰)PKI(6–22)amide inhibited the catalytic subunit of CAMP-dependent protein kinase with a K_i value of 2.8 nM. The photoreactivity of the arylazide peptide was demonstrated by time-dependent U.V. spectral changes on exposure to light. Photolysis of the catalytic subunit.(4-azido[3,5-³H]phenylalanine¹⁰)PKI(6–22)amide complex resulted in specific covalent labeling of the enzyme. The data indicate that this peptide is a useful photoaffinity labeling reagent for the active site of the protein kinase.     

1H‐NMR determination of the solution structure and absolute configuration of FR134043, a novel inhibitor of human leukocyte elastase

Abstract: FR134043 is a semisynthetic disulfonated derivative of the natural product FR901277, is isolated from the culture filtrate of Streptomyces resistomicificus and has potent inhibitory activity against human leukocyte elastase. Although the chemical structure of FR134043 was determined to be a unique bicyclic peptide‐like compound consisting of seven amino acids by using several spectroscopic analytical methods, the chiralities of three centers were unknown. A simple simulated annealing protocol to determine the structure was applied to the eight possible stereoisomers, and the one that best satisfied the NOE distance constraints was determined to be the true stereoconfiguration of FR134043. The solution structure showed that all Cα atoms existed in the l configuration and six of the seven side chains were located towards the outside of the bicyclic framework, even though most of them are highly hydrophobic moieties. The simulated annealing calculation described here is a frequently used method for the determination of the solution structure of peptides or small proteins. We show here that it is also applicable to the determination of the absolute configuration of macrocyclic compounds produced from natural sources.     

Inhibition of hemorrhagic and edematogenic activities of snake venoms by a broad-spectrum protease inhibitor, murinoglobulin; the effect on venoms from five different genera in Viperidae family.

In order to obtain basic data on the effect of broad-spectrum protease inhibitor against local symptoms of Viperidae snake envenomation, inhibitory capacity of rat murinoglobulin on local hemorrhagic and edematogenic activities of venoms from Crotalus atrox, Bothrops jararaca, Lachesis muta muta, Trimeresurus flavoviridis and Echis carinatus sochureki were examined. Murinoglobulin, pre-incubated with the crude venoms at 37 degrees C for 15 min, inhibited hemorrhagic activity of all five venoms to various extents. The activity of C. atrox was almost completely inhibited at the murinoglobulin/venom ratio (w/w) of 20. The activity of B. jararaca, Lachesis muta muta and T. flavoviridis venoms was considerably inhibited at the ratio of 20 (77.2, 80.0 and 86.2% inhibition, respectively), however some of the activity still remained even at the ratio of 40 (84.2, 79.8 and 86.2% inhibition, respectively). Among the five venoms, E. c. sochureki venom is quite resistant to murinoglobulin treatment and statistically significant inhibition was only found at the ratio of 40 (64.1% inhibition). Fibrinolytic and gelatinase activities were more susceptible to murinoglobulin inhibition. The treatment at the ratios of 10 and 20 almost completely inhibited respectively the fibrinolytic and the gelatinase activities of all the venoms. Murinoglobulin treatment also significantly inhibited the edematogenic activity of L. muta muta, T. flavoviridis and Echis carinatus sochureki. The treatment of murinoglobulin at the ratio of 40 considerably suppressed the swelling up to 60 min after subcutaneous injection of L. muta muta and E. c. sochureki venoms, and up to 30 min after T. flavoviridis venom injection. Murinoglobulin is a potent inhibitor against local effects of multiple snake venoms in Viperidae family.     

Purification and characterization of a new platelet aggregation inhibitor with dissociative effect on ADP-induced platelet aggregation, from the venom of Protobothrops elegans (Sakishima-habu)

A platelet aggregation inhibitor, named snake venom platelet aggregation dissociator (SV-PAD)-1, with a dissociative reaction of ADP-induced platelet aggregation, was purified from the venom of Protobothrops elegans (Sakishima-habu) by gel-filtration employing Sephadex G-100, and ion-exchange chromatographies using DEAE-Sepharose Fast Flow, CM-Sepharose Fast Flow, and Mono S. By this procedure, about 1.5 mg of purified protein was obtained from 1.0 g of P. elegans venom. The purified protein showed a single protein band and the molecular weight was about 110 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The pI of purified protein showed four-bands of 7.7, 7.8, 7.95, and 8.15. This protein strongly inhibited ADP-induced platelet aggregation in rabbit platelet-rich plasma (PRP), and its IC₅₀ was about 58 nM. It inhibited ristocetin-induced platelet aggregation in rabbit PRP (IC₅₀: 100 nM), but hardly blocked collagen-induced platelet aggregation. This protein promptly dissociated platelet aggregation in rabbit PRP stimulated by high-concentration ADP.     

Molecular cloning, expression and immunological properties of LiD1, a protein from the dermonecrotic family of Loxosceles intermedia spider venom.

The present report describes the identification and molecular characterization of LiD1, a protein expressed in the venom gland of the brown spider Loxosceles intermedia. LiD1 belongs to a family of proteins with dermonecrotic activity and members of this family have been found in spiders from the genus Loxosceles. The necrotic lesions caused by this group of proteins may lead to serious socio-economic problems such as surgical tissue reconstitution and even patient death. LiD1 was cloned using a cDNA library constructed from the venom gland of L. intermedia and antibodies against proteins with dermonecrotic activity isolated from the crude venom of this spider. The amino acid sequence deduced from the cDNA revealed a mature protein of approximately 31 kDa, with a pI of 7.37. The cDNA also revealed the existence of a signal peptide, a propeptide and also an untranslated 3′ region with 218 nucleotides. LiD1 was expressed as a protein fused with β-galactoside protein using the vector pBK-CMV, resulting in the recombinant protein recLiD1 with important immunological properties. recLiD1 was strongly recognised by anti-dermonecrotic antibodies and was also able to generate reactive antibodies against native dermonecrotic proteins isolated from the venom of L. intermedia.     

Molecular cloning, expression and immunological properties of LiD1, a protein from the dermonecrotic family of Loxosceles intermedia spider venom

The present report describes the identification and molecular characterization of LiD1, a protein expressed in the venom gland of the brown spider Loxosceles intermedia. LiD1 belongs to a family of proteins with dermonecrotic activity and members of this family have been found in spiders from the genus Loxosceles. The necrotic lesions caused by this group of proteins may lead to serious socio-economic problems such as surgical tissue reconstitution and even patient death. LiD1 was cloned using a cDNA library constructed from the venom gland of L. intermedia and antibodies against proteins with dermonecrotic activity isolated from the crude venom of this spider. The amino acid sequence deduced from the cDNA revealed a mature protein of approximately 31 kDa, with a pI of 7.37. The cDNA also revealed the existence of a signal peptide, a propeptide and also an untranslated 3′ region with 218 nucleotides. LiD1 was expressed as a protein fused with β-galactoside protein using the vector pBK-CMV, resulting in the recombinant protein recLiD1 with important immunological properties. recLiD1 was strongly recognised by anti-dermonecrotic antibodies and was also able to generate reactive antibodies against native dermonecrotic proteins isolated from the venom of L. intermedia.     

Purification and characterization of abelesculin,a novel ribosome-inactivating protein from the mature seeds of <Emphasis Type="Italic">Abelmoschus esculentus</Emphasis>

A novel, type-1 ribosome-inactivating protein (RIP), abelesculin, from the mature seeds of Abelmoschus esculentus was successively purified to homogeneity using ammonium sulfate precipitation, ion-exchange chromatography and size-exclusion chromatography. Abelesculin was found to have a molecular mass of 30 kDa and a pI of more than 10.1. The protein depurinates 28S rRNA in the ribosomes of rat liver with an EC₅₀ (concentration causing 50% cleavage) of 62.8 pM. Study of the sequence of the 26 N-terminal amino acids of the protein revealed a close relationship with other RIPs. This study reports for the first time that RIPs exist in Malvaceae plants.     

Understanding the Increased Aggregation Propensity of a Light-Exposed IgG1 Monoclonal Antibody Using Hydrogen Exchange Mass Spectrometry,Biophysical Characterization,and Structural Analysis

This work compares the conformational stability, backbone flexibility, and aggregation propensity of monomer and dimer fractions of an IgG1 monoclonal antibody (mAb) generated on UVA light exposure for up to 72 h collected by preparative size-exclusion chromatography, compared with unstressed control. UVA light exposure induced covalent aggregation, and fragmentation as measured by size-exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and extensive oxidation of specific methionine residues (Met 257, Met 433, and Met 109) in both size fractions identified by reverse phase chromatography coupled to mass spectrometry. Compared with unstressed mAb, both the monomer and dimer fractionated from 72 h UVA light–exposed mAb had decreased thermal melting temperatures (T_m1) by 1.4°C as measured by differential scanning calorimetry, minor changes in tertiary structure as measured by near-UV CD, increased monomer loss, and aggregation on accelerated storage at 35°C. Hydrogen/deuterium exchange mass spectrometry identified local segments with increased flexibility in C_H2 and C_H3 domains of both size fractions, and decreased flexibility in few segments of F_ab and C_H1 domains in the dimer fraction. Segment 247-256 in heavy chain, an established aggregation hotspot in IgG1 mAbs had large increase in flexibility in both size fractions compared with unstressed mAb.