ABSTRACT
A novel peptide with antimicrobial activity was isolated from leukocytes of the European pond turtle Emys orbicularis and purified to homogeneity by preparative gel electrophoresis followed by reversed phase chromatography. It was highly active in vitro against Escherichia coli, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus, and Candida albicans. The isolated peptide was sequenced de novo by tandem mass spectrometry using both collision-induced and electron-transfer dissociation in combination with different chemical derivatization techniques. The 40-residue peptide, called TBD-1 (turtle beta-defensin 1), represents the first defensin isolated from reptilian leukocytes. It contains three disulfide bonds and shows high structural similarities to beta-defensins isolated from birds and mammals.
Subject(s)
Leukocytes/immunology , Reptilian Proteins/genetics , Reptilian Proteins/isolation & purification , Turtles , beta-Defensins/genetics , beta-Defensins/isolation & purification , Amino Acid Sequence , Animals , Birds , Female , Hemolysis/drug effects , Male , Mammals , Mass Spectrometry , Molecular Sequence Data , Reptilian Proteins/chemistry , Reptilian Proteins/immunology , Sequence Alignment , beta-Defensins/chemistry , beta-Defensins/immunologyABSTRACT
A novel 40-residue antimicrobial peptide, aurelin, exhibiting activity against Gram-positive and Gram-negative bacteria, was purified from the mesoglea of a scyphoid jellyfish Aurelia aurita by preparative gel electrophoresis and RP-HPLC. Molecular mass (4296.95 Da) and complete amino acid sequence of aurelin (AACSDRAHGHICESFKSFCKDSGRNGVKLRANCKKTCGLC) were determined. Aurelin has six cysteines forming three disulfide bonds. The total RNA was isolated from the jellyfish mesoglea, RT-PCR and cloning were performed, and cDNA was sequenced. A 84-residue preproaurelin contains a putative signal peptide (22 amino acids) and a propiece of the same size (22 amino acids). Aurelin has no structural homology with any previously identified antimicrobial peptides but reveals partial similarity both with defensins and K+ channel-blocking toxins of sea anemones and belongs to ShKT domain family.
Subject(s)
Anti-Infective Agents/chemistry , Antimicrobial Cationic Peptides/chemistry , Amino Acid Sequence , Animals , Anti-Infective Agents/isolation & purification , Anti-Infective Agents/pharmacology , Antimicrobial Cationic Peptides/isolation & purification , Antimicrobial Cationic Peptides/pharmacology , Base Sequence , Cloning, Molecular , Defensins/chemistry , Escherichia coli/drug effects , Listeria monocytogenes/drug effects , Marine Toxins/chemistry , Molecular Sequence Data , Scyphozoa/chemistryABSTRACT
Two novel 21-residue antimicrobial peptides, arenicin-1 and arenicin-2, exhibiting activity against Gram-positive and Gram-negative bacteria and fungi, were purified from coelomocytes of marine polychaeta Arenicola marina (lugworm) by preparative gel electrophoresis and RP-HPLC. Molecular masses (2758.3 and 2772.3 Da) and complete amino acid sequences (RWCVYAYVRVRGVLVRYRRCW and RWCVYAYVRIRGVLVRYRRCW) were determined for each isoform. Each arenicin has one disulfide bond (Cys3-Cys20). The total RNA was isolated from the lugworm coelomocytes, RT-PCR and cloning were performed, and cDNA was sequenced. A 202-residue preproarenicin contains a putative signal peptide (25 amino acids) and a long prodomain. Arenicins have no structure similarity to any previously identified antimicrobial peptides.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Peptides , Polychaeta/chemistry , Amino Acid Sequence , Animals , Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides , Base Sequence , Chromatography, High Pressure Liquid , Cloning, Molecular , DNA Primers , Electrophoresis, Polyacrylamide Gel , Helminth Proteins , Molecular Sequence Data , Molecular Weight , RNA/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino AcidABSTRACT
Escherichia coli protease Lon (La) is an adenosine triphosphate (ATP)-regulated homo-oligomeric proteolytic complex responsible for the recognition and selective degradation of abnormal and unstable proteins. Each subunit of the protease Lon appears to consist of three functional domains: the C-terminal proteolytic containing a serine active site, the central displaying the ATPase activity, and the N-terminal with still obscure function. We have used limited proteolysis to probe the domain structure and nucleotide-induced conformational changes in the enzyme. Limited proteolysis of the native protease Lon generated a low number of stable fragments roughly corresponding to its functional domains. Conformational changes in the wild-type enzyme and its mutant forms in the presence or absence of adenine and guanine nucleotides were investigated by limited proteolysis. The nucleotide character was shown to play a key role for susceptibility of the protease Lon to limited proteolysis, in particular, for resistance of the ATPase functional domain. ATP and adenosine diphosphate displayed a protective effect of the ATPase domain of the enzyme. We suggest that these nucleotides induce conformational changes of the enzyme, transforming the ATPase domain from the most vulnerable part of the molecule into a spatially inaccessible one. Both limited proteolysis and autolysis demonstrate that the most stable part of the protease Lon molecule is its N-terminal region. Obvious resistance of the protease Lon C-terminus to proteolysis indicates that this region of the enzyme molecule including its substrate-binding and proteolytic domains has a well folded structure.
