A particularly labile Asp‐Pro bond in the green mamba muscarinic toxin MTX2. Effect of protein conformation on the rate of cleavage

Ségalas, Isabelle; Thaï, Robert; Vita, Renée Ménez Claudio

doi:10.1016/0014-5793(95)00844-y

Cited by 26 publications

(20 citation statements)

References 20 publications

(36 reference statements)

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“…Interestingly, dSCP2 contains an internal AspPro bond that is not present in M. sexta JHDK. This type of bond is known to be very acid labile (11)(12)(13). Taken together, these results suggest that D. melanogaster JHDK is structurally similar to M. sexta JHDK and dSCP2.…”

Section: Resultsmentioning

confidence: 55%

Juvenile Hormone Diol Kinase

Maxwell¹,

Welch²,

Horodyski³

et al. 2002

Journal of Biological Chemistry

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The gene sequence of Manduca sexta juvenile hormone diol kinase (JHDK) codes for an enzyme that has 59% sequence identity to Drosophila melanogaster sarcoplasmic calcium-binding protein-2 (dSCP2). JHDK and dSCP2 are similar to G-proteins with three conserved sequence elements involved in purine nucleotide binding. Both proteins contain two pairs of EF-hand motifs. Characterization and partial purification of the D. melanogaster homolog of M. sexta JHDK from adult D. melanogaster gave material with JHDK activity. This activity has an experimental pI and molecular mass that are nearly identical to those of dSCP2. Moreover, D. melanogaster phosphotransferase activity has very similar chromatographic retention in three systems compared with M. sexta JHDK. Substrate docking to threedimensional models of JHDK has shown that the three conserved nucleotide-binding elements surround the putative substrate-binding site and align with conserved sequence elements of p21Ras and adenylate kinase. D. melanogaster dSCP2 is a homolog of M. sexta JHDK, and these proteins constitute a novel kinase family that binds nucleotides using the scaffold of an SCP (Protein Data Bank code 2SCP).Endocrine control of embryonic and post-embryonic development in insects is primarily accomplished by juvenile hormone (JH) 1 and ecdysteroids (1). Development proceeds through a series of molts and metamorphosis, where the function of JH is to regulate the character of the molt; metamorphosis is characterized by a sharp decrease in JH, and adult molting by the absence of JH (1). JH levels are regulated by de novo biosynthesis (2) and catabolism (3). Cellular JH metabolism involves a microsomal JH epoxide hydrolase that metabolizes JH to JH diol and a soluble JH diol kinase (JHDK) that catalyzes the formation of JH diol phosphate. JHDK is a homodimer with a subunit molecular mass of 20 kDa that uses MgATP or MgGTP as a phosphate donor to effectively catabolize JH I, II, or III diol by placing a phosphate group on the C-10 hydroxyl, thereby creating JH diol phosphate, the water-soluble end metabolite of JH catabolism (4).In this study, we report the sequencing and cloning of Manduca sexta JHDK, the molecular modeling of JHDK and Drosophila melanogaster sarcoplasmic calcium-binding protein-2 (dSCP2), and the partial purification and characterization of the D. melanogaster homolog of M. sexta JHDK. We show with high probability that the product of the D. melanogaster dSCP2 gene is the D. melanogaster homolog of M. sexta JHDK. Each is a member of a novel kinase family that is structurally similar to SCPs. EXPERIMENTAL PROCEDURES Chemicals and D. melanogaster Protein Preparations-Ampholyteswere obtained from Bio-Rad. 3-Octylthio-1,1,1-trifluoro-2-propanone was from Dr. Bruce D. Hammock (University of California, Davis, CA). Aprotinin, 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin, and high-grade (NH 4 ) 2 SO 4 were purchased from Sigma. Lysyl endopeptidase (Lys-C) was obtained from Wako Bioproducts (Richmond, VA). The synthesis of (10S,11S)- [10-3 H...

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Section: Resultsmentioning

confidence: 55%

Juvenile Hormone Diol Kinase

Maxwell¹,

Welch²,

Horodyski³

et al. 2002

Journal of Biological Chemistry

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“…During purification of closticin 574, such acidic conditions are encountered in the HPLC step when the sample is eluted in the presence of trifluoroacetic acid, a very acidic compound. Cleavage of Asp-Pro bonds during protein purification has been observed previously (16,54,55,57). The cysteines present in closticin 574 are not involved in the formation of a disulfide bridge required for activity since DTT treatment did not inactivate the protein.…”

Section: Discussionmentioning

confidence: 71%

Identification and Characterization of Two Novel Clostridial Bacteriocins, Circularin A and Closticin 574

Kemperman

Kuipers

Karsens

et al. 2003

Appl Environ Microbiol

145

122

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Two novel antibacterial peptides of clostridial species were purified, N-terminally sequenced, and characterized. Moreover, their structural genes were identified. Closticin 574 is an 82-amino-acid bacteriocin produced by Clostridium tyrobutyricum ADRIAT 932. The supernatant of the producing strain showed a high level of activity against the indicator strain C. tyrobutyricum. The protein is synthesized as a preproprotein that is possibly secreted via the general secretion pathway, after which it is hydrolyzed at an Asp-Pro site. Circularin A is produced by Clostridium beijerinckii ATCC 25752 as a prepeptide of 72 amino acids. Cleavage of the prepeptide between the third leucine and fourth valine residues followed by a head-to-tail ligation between the N and C termini creates a circular antimicrobial peptide of 69 amino acids. The unusually small circularin A leader peptide of three amino acids is cleaved off in this process. The supernatant of C. beijerinckii ATCC 25752 showed a broad antibacterial activity range.Clostridia have a negative image as many species are known as pathogens (50), toxin producers (50), and food-spoilage bacteria (36). Various clostridia produce bacteriocins, but despite their long history as a means of typing clostridia (33, 39), only little sequence, functional, or structural information is available on these antibacterial peptides. The few clostridial bacteriocins that have been further characterized are BCN5 and boticin B (12, 18). BCN5 is a large (97-kDa) UV-inducible protein produced by Clostridium perfringens (18). Boticin B is produced by Clostridium botulinum as a small peptide with a predicted size of 50 amino acid residues (12). Little is known about the regulation of production or secretion of these bacteriocins. Generally, bacteriocins are small ribosomally synthesized antimicrobial peptides (27,35). They are mostly membrane permeabilizing and cationic, and they typically comprise fewer than 50 amino acid residues (35). Bacteriocins can be used as an additive to food products to prevent the growth of spoilage bacteria (10). Klaenhammer divided bacteriocins into four classes (35). Class I bacteriocins, known as lantibiotics, contain posttranslationally modified residues such as lanthionine, ␤-methyl lanthionine, and dehydrated residues. Class II bacteriocins lack these modifications and are linear peptides (35,44). Class III contains the large, heat-labile bacteriocins. Class IV bacteriocins are complex molecules composed of protein and chemical moieties. Both class I and class II bacteriocins have been intensively studied, and more than 20 and 100 representatives are known, respectively.The subclass IIc (class II bacteriocins other than the pediocin-like bacteriocins or the two-peptide bacteriocins) contains a few examples of bacteriocins that are produced as circular molecules. This circularization is the result of a head-to-tail peptide bond formation of a prepeptide. Bacteriocins with this typical structure are microcin J25, gassericin A, and AS-48/ Bac21 (6,31,40,59). Mi...

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“…The reaction of PH (3) yielded the X-peptide [PH (2-11)-NH 2 (3a)] as a main product, demonstrating the cleavage of pGlu 1 -Ala 2 peptide bond occurred to a greater extent than the internal peptide bond of Asp 3 -Pro 4 , which has previously been reported to be one of the most susceptible bonds to acidic hydrolysis. [16][17][18] Treatment of BPP-5a (4) of the internal peptide bonds or the deamidation at the side chain of Asn at the position 5 in 70% MSA, resulting in the appearance of numerous peaks on HPLC chromatogram (Fig. 9A), with the peak of NT (2-13) (5a) decidedly the largest.…”

Section: Resultsmentioning

confidence: 99%

Hydrolytic Cleavage of Pyroglutamyl-peptide Bond. V. Selective Removal of Pyroglutamic Acid from Biologically Active Pyroglutamylpeptides in High Concentrations of Aqueous Methanesulfonic Acid

Kobayashi

Ohki

Okimura

et al. 2006

CHEMICAL & PHARMACEUTICAL BULLETIN

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Biologically active peptides bearing pyroglutamic acid residue (pGlu) at the N-terminal are widely known. The removal of pGlu residue from a pGlu-X-peptide (X, amino acid residue at position 2) is required for the primary structure determination by Edman degradation. Our previous studies indicated that pGlu-X peptide bond is highly sensitive to mild acidic conditions, generating not only the ring opened product (H-Glu-X-peptide) at the pyrrolidone moiety of pGlu, but also the cleavage product (H-X-peptide) at pGlu-X linkage.1,2) High selectivity to remove pGlu-OH from synthetic model pGlu-X-peptides was attained in concentrated hydrochloric acid 3) and in 70% aqueous methanesulfonic acid (MSA). 4,5) The aim of this study was to compare the selectivity of the cleavage reaction at pGlu-X bond in aqueous MSA with various internal peptide bonds in five biologically active pGlu-X-peptides, containing well-known acid sensitive amide bonds. The sequence of these peptides, namely, gonadotropin-releasing hormone (Gn-RH; 1), 6) dog neuromedin U-8 (d-NMU-8; 2), 7) physalaemin (PH; 3), 8) a bradykinin potentiating peptide (BPP-5a; 4), 9) and neurotensin (NT; 5), 10) are shown in Fig. 1. ExperimentalGeneral and Apparatus HPLC analysis was performed on a module consisted of a 7125 injector (Rheodyne Inc., U.S.A.), a 616 pump, a 600 s controller, a 486 tunable absorbance detector, a 717 plus autosampler, and an SDM solvent degas module (all from Waters Corp., Milford, MA, U.S.A.).Amino acid analysis was conducted on a 7300 Model amino acid analyzer system (Beckman Instruments Ltd., Fullerton, CA, U.S.A.). The hydrolysis of a synthetic peptide was performed by 6 M HCl vapor at 130°C for 3 h. Fast-atom bombardment mass spectra (FAB-MS) were obtained on a JMS-DX300 mass spectrometer (JEOL Ltd., Tokyo, Japan). Optical rotations of the peptides were measured with a DIP-370 digital polarimeter (Nippon Bunko Co., Ltd., Tokyo, Japan). HP-TLC analysis was carried out on precoated silica gel plates (Kieselgel 60; Merck, Darmstadt, Germany).Peptides Syntheses of Gn-RH (1), PH (3), BPP-5a (4) and NT (5) (Fig. 1) and various fragment peptides related to these biologically active peptides (Figs. 2,5,6,8,9) were carried out according to a method previously reported for d-NMU-8 (2).11) Protected peptide resins were constructed by Boc strategy either on a benzhydrylamine resin or a chloromethylated polystyrene resin (Peptide Institute Inc., Osaka, Japan) using a peptide synthesizer (ABI 433A; Applied Biosystems, Foster City, CA, U.S.A.). Side chain protected Boc-amino acid derivatives (Watanabe Chemical Industries Ltd., Hiroshima, Japan) were Tyr(BrZ), Arg(Tos), Asp(OcHex), Lys(Cl-Z), Ser(Bzl), His(Bom) and Glu(OBzl). The protected peptide resins were treated with anhydrous liquid hydrogen fluoride (HF) containing 10% anisole under ice cooling for 45 min in a Teflon HF apparatus (Peptide Institute Inc., Osaka, Japan). After evaporation of HF in vacuo, the residual peptides were purified by HPLC, using a column of YMC-pack ODS-AM S-5 120 Å (20...

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A particularly labile Asp‐Pro bond in the green mamba muscarinic toxin MTX2. Effect of protein conformation on the rate of cleavage

Cited by 26 publications

References 20 publications

Juvenile Hormone Diol Kinase

Juvenile Hormone Diol Kinase

Identification and Characterization of Two Novel Clostridial Bacteriocins, Circularin A and Closticin 574

Hydrolytic Cleavage of Pyroglutamyl-peptide Bond. V. Selective Removal of Pyroglutamic Acid from Biologically Active Pyroglutamylpeptides in High Concentrations of Aqueous Methanesulfonic Acid

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