An Antimicrobial Peptide from the Australian Native Hardenbergia violacea Provides the First Functionally Characterised Member of a Subfamily of Plant Defensins

1997 ◽  
Vol 24 (5) ◽  
pp. 571 ◽  
Author(s):  
Stuart J. Harrison ◽  
John P. Marcus ◽  
Kenneth C. Goulter ◽  
Jodie L. Green ◽  
Donald J. Maclean ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Antimicrobial Peptide ◽  
Mammalian Cells ◽  
Amino Acid Sequences ◽  
Plant Defensin ◽  
Defensive Role ◽  
Plant Defensins

An antimicrobial peptide (HvAMP1) was isolated from seeds of the Australian native legume Hardenbergia violacea (Schneev.) Stearn. The peptide is 47 amino acid residues in length, contains 8 cysteines, and has a molecular weight of 5392 and a predicted pI of 10.41. HvAMP1 inhibited the growth of several plant pathogenic fungi at concentrations as low as 1 µM in vitro and produced distinct hyphal distortion and increased branching. This antimicrobial activity was greatly diminished in the presence of 1 mM CaCl2 and 50 mM KCl. The purified peptide at 40 µM did not inhibit three different a-amylase enzymes. Aeukaryotic cell-free translation system showed inhibition approaching 50% in the presence of ~100 µM of HvAMP1. The viability of plant and mammalian cells cultured in vitro was not adversely affected by concentrations of HvAMP1 as high as 40 mM. The amino acid sequence of HvAMP1 contained the consensus amino acids that define the plant defensin family of peptides. The HvAMP1 amino acid sequence showed 87% and 57% identity with the amino acid sequences deduced from cDNA sequences from defensins of Vigna unguiculata and Pisum sativum, respectively. Other plant defensin sequences showed less than 33% amino acid identity to the peptide. Therefore, HvAMP1 and the putative plant defensins of cowpea and pea define a distinct sequence subfamily of plant defensins which is at present limited to members of the Fabaceae. HvAMP1 is the first member of this subfamily to be purified and functionally characterised. The antimicrobial activity of HvAMP1 suggests a defensive role for this subfamily of peptides.

scholarly journals Antimicrobial Activity of Small Synthetic Peptides Based on the Marine Peptide Turgencin A: Prediction of Antimicrobial Peptide Sequences in a Natural Peptide and Strategy for Optimization of Potency

2020 ◽  
Vol 21 (15) ◽  
pp. 5460
Author(s):  
Ida K. Ø. Hansen ◽  
Tomas Lövdahl ◽  
Danijela Simonovic ◽  
Kine Ø. Hansen ◽  
Aaron J. C. Andersen ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Amino Acid ◽  
Antimicrobial Peptide ◽  
Mammalian Cells ◽  
Amino Acid Replacement ◽  
The Arctic ◽  
Cytotoxic Activities ◽  
Replacement Strategy ◽  
Bacterial Membranes

Turgencin A, a potent antimicrobial peptide isolated from the Arctic sea squirt Synoicum turgens, consists of 36 amino acid residues and three disulfide bridges, making it challenging to synthesize. The aim of the present study was to develop a truncated peptide with an antimicrobial drug lead potential based on turgencin A. The experiments consisted of: (1) sequence analysis and prediction of antimicrobial potential of truncated 10-mer sequences; (2) synthesis and antimicrobial screening of a lead peptide devoid of the cysteine residues; (3) optimization of in vitro antimicrobial activity of the lead peptide using an amino acid replacement strategy; and (4) screening the synthesized peptides for cytotoxic activities. In silico analysis of turgencin A using various prediction software indicated an internal, cationic 10-mer sequence to be putatively antimicrobial. The synthesized truncated lead peptide displayed weak antimicrobial activity. However, by following a systematic amino acid replacement strategy, a modified peptide was developed that retained the potency of the original peptide. The optimized peptide StAMP-9 displayed bactericidal activity, with minimal inhibitory concentrations of 7.8 µg/mL against Staphylococcus aureus and 3.9 µg/mL against Escherichia coli, and no cytotoxic effects against mammalian cells. Preliminary experiments indicate the bacterial membranes as immediate and primary targets.

scholarly journals In vitro and In Silico Approach For Characterization of Antimicrobial Peptide From Probiotics Against Staphylococcus Aureus and Escherichia Coli

2021 ◽  
Author(s):  
Amrutha Bindu ◽  
Lakshmi Devi
Keyword(s):  
Amino Acid ◽  
Antimicrobial Peptide ◽  
Lactobacillus Plantarum ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Proteinase K ◽  
Kinetic Assay ◽  
Wide Range

Abstract The focus of present study was to characterize antimicrobial peptide produced by probiotic cultures, Enterococcus durans DB-1aa (MCC4243), Lactobacillus plantarum Cu2-PM7 (MCC4246) and Lactobacillus fermentum Cu3-PM8 (MCC4233) against Staphylococus aureus and E. coli. The growth kinetic assay revealed 24 h of incubation to be optimum for bacteriocin production. The partially purified compound after ion-exchange chromatography was found to be thermoresistant and stable under wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, a-amylase and lipase. The apparent molecular weight of bacteriocin from MCC4243 and MCC4246 was found to be 3.5 KDa. Translated partial amino acid sequence of plnA gene in MCC4246 displayed 48 amino acid sequences showing 100% similarity with plantaricin A of Lactobacillus plantarum (WP_0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns. The functions on cytoplasm show 10.82 isoelectric point and 48.6% hydrophobicity. The molecular approach of using Geneious Prime software and protein prediction data base for characterization of bacteriocin is novel and predicts “KSSAYSLQMGATAIKQVKKLFKKWGW” as peptide responsible for antimicrobial activity. The study provides information about broad spectrum bacteriocin in native probiotic culture and paves a way towards its application in functional foods as biopreservative agents.

scholarly journals Identification and Characterization of a Hexapeptide with Activity Against Phytopathogenic Fungi That Cause Postharvest Decay in Fruits

2000 ◽  
Vol 13 (8) ◽  
pp. 837-846 ◽  
Author(s):  
Belén López-García ◽  
Luis González-Candelas ◽  
Enrique Pérez-Payá ◽  
Jose F. Marcos
Keyword(s):  
Antimicrobial Activity ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Plant Protection ◽  
Phytopathogenic Fungi ◽  
Bacterial Strains ◽  
General Sequence ◽  
Postharvest Decay ◽  
Structure Conservation

A hexapeptide of amino acid sequence Ac-Arg-Lys-Thr-Trp-Phe-Trp-NH 2 was demonstrated to have antimicrobial activity against selected phytopathogenic fungi that cause postharvest decay in fruits. The peptide synthesized with either all D- or all L-amino acids inhibited the in vitro growth of strains of Penicillium italicum, P. digitatum, and Botrytis cinerea, with MICs of 60 to 80 μM and 50% inhibitory concentration (IC50) of 30 to 40 μM. The inhibitory activity of the peptide was both sequence- and fungus-specific since (i) sequence-related peptides lacked activity (including one with five residues identical to the active sequence), (ii) other filamentous fungi (including some that belong to the genus Penicillium) were insensitive to the peptide's antifungal action, and (iii) the peptide did not inhibit the growth of several yeast and bacterial strains assayed. Experiments on P. digitatum identified conidial germination as particularly sensitive to inhibition although mycelial growth was also affected. Our findings suggest that the inhibitory effect is initially driven by the electrostatic interaction of the peptide with fungal components. The antifungal peptide retarded the blue and green mold diseases of citrus fruits and the gray mold of tomato fruits under controlled inoculation conditions, thus providing evidence for the feasibility of using very short peptides in plant protection. This and previous studies with related peptides indicate some degree of peptide amino acid sequence and structure conservation associated with the antimicrobial activity, and suggest a general sequence layout for short antifungal peptides, consisting of one or two positively charged residues combined with aromatic amino acid residues.

scholarly journals MALDI-MS Analysis of Peptide Libraries Expands the Scope of Substrates for Farnesyltransferase

2021 ◽  
Vol 22 (21) ◽  
pp. 12042
Author(s):  
Garrett L. Schey ◽  
Peter H. Buttery ◽  
Emily R. Hildebrandt ◽  
Sadie X. Novak ◽  
Walter K. Schmidt ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Solid Phase ◽  
Amino Acid Sequences ◽  
Maldi Ms ◽  
Yeast Cells ◽  
Spectrometric Analysis ◽  
Ionization Mass ◽  
Post Translational Modification

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This modification typically causes proteins to associate with the membrane and allows them to participate in signaling pathways. In the canonical understanding of FTase, the isoprenoids are attached to the cysteine residue of a four-amino-acid CaaX box sequence. However, recent work has shown that five-amino-acid sequences can be recognized, including the pentapeptide CMIIM. This paper describes a new systematic approach to discover novel peptide substrates for FTase by combining the combinatorial power of solid-phase peptide synthesis (SPPS) with the ease of matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The workflow consists of synthesizing focused libraries containing 10–20 sequences obtained by randomizing a synthetic peptide at a single position. Incubation of the library with FTase and farnesyl pyrophosphate (FPP) followed by mass spectrometric analysis allows the enzymatic products to be clearly resolved from starting peptides due to the increase in mass that occurs upon farnesylation. Using this method, 30 hits were obtained from a series of libraries containing a total of 80 members. Eight of the above peptides were selected for further evaluation, reflecting a mixture that represented a sampling of diverse substrate space. Six of these sequences were found to be bona fide substrates for FTase, with several meeting or surpassing the in vitro efficiency of the benchmark sequence CMIIM. Experiments in yeast demonstrated that proteins bearing these sequences can be efficiently farnesylated within live cells. Additionally, a bioinformatics search showed that a variety of pentapeptide CaaaX sequences can be found in the mammalian genome, and several of these sequences display excellent farnesylation in vitro and in yeast cells, suggesting that the number of farnesylated proteins within mammalian cells may be larger than previously thought.

scholarly journals Passaging impact of H9N2 avian influenza virus in hamsters on its pathogenicity and genetic variability

2014 ◽  
Vol 8 (05) ◽  
pp. 570-580 ◽  
Author(s):  
Houssam A. Shaib ◽  
Nelly Cochet ◽  
Thierry Ribeiro ◽  
Afif M Abdel Nour ◽  
Georges Nemer ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Avian Influenza ◽  
Mammalian Cells ◽  
Pcr Amplification ◽  
Amino Acid Sequences ◽  
Influenza Viruses ◽  
H9n2 Virus ◽  
World Health ◽  
The Impact

Introduction: Avian influenza viruses of the H9N2 subtype have been reported to cause human infections. This study demonstrates the impact of nasal viral passaging of avian H9N2 in hamsters on its cross species-pathogenic adaptability and variability of amino acid sequences of the hemagglutinin (HA) and neuraminidase (NA) stalk. Methodology: Three intranasal passagings of avian H9N2 in hamsters P1, P2, and P3 were accomplished. Morbidity signs and lesions were observed three days post viral inoculation. The HA test was used for presumptive detection of H9N2 virus in the trachea and lungs of the hamsters challenged with the differently passaged viruses. Different primers were used for PCR amplification of the HA1 and NA stalk regions of the differently passaged H9N2 viruses, followed by sequence alignment. Results: The morbidity signs indicated low pathogenicity of the differently passaged H9N2 viruses in hamsters. The frequency of gross and microscopic lesions in the tracheas and lungs were insignificantly different among hamsters challenged with the differently passaged H9N2 viruses (p > 0.05). There was 100% similarity in the amino acid sequence of the HA gene of most passaged viruses. The amino acid sequence of the neuraminidase in the third passaged H9N2 virus recovered from lungs showed a R46P mutation that might have a role in the pathogenic adaptability of P3 viruses in hamsters’ lungs. Conclusions: The apparent adaptation of avian H9N2 virus to mammalian cells is in agreement with the World Health Organization’s alertness for a possible public health threat by this adaptable virus.

scholarly journals Amino Acid Sequences of Lactoferrin from Red Deer (Cervus elaphus) Milk and Antimicrobial Activity of Its Derived Peptides Lactoferricin and Lactoferrampin

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1305
Author(s):  
Ye Wang ◽  
James D. Morton ◽  
Alaa EL-Din A. Bekhit ◽  
Alan Carne ◽  
Susan L. Mason
Keyword(s):  
Amino Acids ◽  
Antimicrobial Activity ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Positive Charge ◽  
Antimicrobial Activities ◽  
Amino Acid Sequences ◽  
Bovine Lactoferrin ◽  
E Coli ◽  
Net Positive Charge

Although the bioactivities of bovine lactoferrin have been extensively investigated, little is known about deer milk lactoferrin bioactivity and its amino acid sequence. This research investigated the amino acid sequence of deer lactoferrin and the antimicrobial activities of two lactoferrin-encrypted peptides; lactoferricin (Lfcin) and lactoferrampin (Lfampin). Deer lactoferrin was found to have a molecular weight of 77.1 kDa and an isoelectric point of 7.99, which are similar to that of bovine lactoferrin, 78 kDa and pI 7.9. Deer lactoferrin contains 707 amino acids, one amino acid less than bovine lactoferrin, and has 92% homology with bovine lactoferrin. Deer lactoferricin exhibited strong antimicrobial activity against E. coli American Type Culture Collection (ATCC) 25922 and L. acidophilus ATCC 4356. The antimicrobial activities of deer and bovine Lfcin and Lfampin were compared. Based on MIC, deer Lfcin was found to be a more effective inhibitor of L. acidophilus ATCC 4356 than bovine Lfcin, but bovine Lfcin and Lfampin were more effective against E. coli ATCC 25922 than deer Lfcin and Lfampin. The deer Lfcin sequence differed at seven amino acids from bovine Lfcin and this decreased the net positive charge and increased the hydrophobicity. Deer Lfampin contained two differences in amino acid sequence compared to bovine Lfampin which decreased the net positive charge. These amino acid sequence differences likely account for differences in antibacterial activity. Positive charge and hydrophobic residues provide the amphipathic character of these helical peptides, and are considered important for binding of antimicrobial peptides. In silico modelling of deer Lfcin indicated an identical α-helical structure compared to bovine Lfcin.

scholarly journals The amino acid sequence of cytochromes c-551 from three species of Pseudomonas

1973 ◽  
Vol 131 (3) ◽  
pp. 485-498 ◽  
Author(s):  
R. P. Ambler ◽  
Margaret Wynn
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Cytochrome C ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Mitochondrial Cytochrome ◽  
Cytochromes C ◽  
Lending Library ◽  
Single Peptide

The amino acid sequences of the cytochromes c-551 from three species of Pseudomonas have been determined. Each resembles the protein from Pseudomonas strain P6009 (now known to be Pseudomonas aeruginosa, not Pseudomonas fluorescens) in containing 82 amino acids in a single peptide chain, with a haem group covalently attached to cysteine residues 12 and 15. In all four sequences 43 residues are identical. Although by bacteriological criteria the organisms are closely related, the differences between pairs of sequences range from 22% to 39%. These values should be compared with the differences in the sequence of mitochondrial cytochrome c between mammals and amphibians (about 18%) or between mammals and insects (about 33%). Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50015 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.

scholarly journals Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

2001 ◽  
Vol 183 (6) ◽  
pp. 1954-1960 ◽  
Author(s):  
Grit Zarnt ◽  
Thomas Schräder ◽  
Jan R. Andreesen
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Tertiary Structure ◽  
Ralstonia Eutropha ◽  
Signal Sequence ◽  
Substrate Inhibition ◽  
Catalytic Efficiency ◽  
Gene Encoding

ABSTRACT The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparentk cat/Km andK i values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k cat/Km value of 788 × 104 M−1 s−1. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.

scholarly journals Factor D̄ of the alternative pathway of human complement. Purification, alignment and N-terminal amino acid sequences of the major cyanogen bromide fragments, and localization of the serine residue at the active site

1980 ◽  
Vol 187 (3) ◽  
pp. 863-874 ◽  
Author(s):  
D M Johnson ◽  
J Gagnon ◽  
K B Reid
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Alternative Pathway ◽  
Amino Acid Sequences ◽  
Serine Residue ◽  
Amino Acid Sequence Analysis ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

The serine esterase factor D of the complement system was purified from outdated human plasma with a yield of 20% of the initial haemolytic activity found in serum. This represented an approx. 60 000-fold purification. The final product was homogeneous as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (with an apparent mol.wt. of 24 000), its migration as a single component in a variety of fractionation procedures based on size and charge, and its N-terminal amino-acid-sequence analysis. The N-terminal amino acid sequence of the first 36 residues of the intact molecule was found to be homologous with the N-terminal amino acid sequences of the catalytic chains of other serine esterases. Factor D showed an especially strong homology (greater than 60% identity) with rat ‘group-specific protease’ [Woodbury, Katunuma, Kobayashi, Titani, & Neurath (1978) Biochemistry 17, 811-819] over the first 16 amino acid residues. This similarity is of interest since it is considered that both enzymes may be synthesized in their active, rather than zymogen, forms. The three major CNBr fragments of factor D, which had apparent mol.wts. of 15 800, 6600 and 1700, were purified and then aligned by N-terminal amino acid sequence analysis and amino acid analysis. By using factor D labelled with di-[1,3-14C]isopropylphosphofluoridate it was shown that the CNBr fragment of apparent mol.wt. 6600, which is located in the C-terminal region of factor D, contained the active serine residue. The amino acid sequence around this residue was determined.

scholarly journals The Designer Antimicrobial Peptide A-hBD-2 Facilitates Skin Wound Healing by Stimulating Keratinocyte Migration and Proliferation

2018 ◽  
Vol 51 (2) ◽  
pp. 647-663 ◽  
Author(s):  
Bobin Mi ◽  
Jing Liu ◽  
Yi Liu ◽  
Liangcong Hu ◽  
Yukun Liu ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Wound Healing ◽  
Antimicrobial Peptide ◽  
Structural Stability ◽  
Cell Counting ◽  
Sprague Dawley ◽  
Skin Wound Healing ◽  
High Sodium

Background/Aims: Antimicrobial peptides are effective promoters of wound healing but are susceptible to degradation. In this study, we replaced the GIGDP unit on the N-terminal of the endogenous human antimicrobial peptide hBD-2 with APKAM to produce A-hBD-2 and analyzed the effect on wound healing both in vitro and in vivo. Methods: The effects of A-hBD-2 and hBD-2 on cytotoxicity and proliferation in keratinocytes were assessed by Cell Counting Kit-8 assay. The structural stability and antimicrobial activity of hBD-2 and A-hBD-2 were evaluated against Staphylococcus aureus. RNA and proteins levels were evaluated by real-time PCR and western blotting, respectively. Cell migration was evaluated using a transwell assay. Cell cycle analysis was performed by flow cytometry. Wound healing was assessed in Sprague-Dawley rats. Epidermal thickness was evaluated by hematoxylin and eosin staining. Results: We found that hBD-2 exhibited cytotoxicity at high concentrations and decreased the structural stability in the presence of high sodium chloride concentrations. A-hBD-2 exhibited increased structural stability and antimicrobial activity, and had lower cytotoxicity in keratinocytes. A-hBD-2 increased the migration and proliferation of keratinocytes via phosphorylation of EGFR and STAT3 and suppressed terminal differentiation of keratinocytes. We also found that A-hBD-2 elicited mobilization of intracellular Ca2+ and stimulated keratinocytes to produce pro- and anti-inflammatory cytokines and chemokines via phospholipase C activation. Furthermore, A-hBD-2 promoted wound healing in vivo. Conclusion: Our data suggest that A-hBD-2 may be a promising candidate therapy for wound healing.

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