Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum.

Leucocin A-UAL 187 is a bacteriocin produced by Leuconostoc gelidum UAL 187, a lactic acid bacterium isolated from vacuum-packaged meat. The bacteriocin was purified by ammonium sulfate or acid (pH 2.5) precipitation, hydrophobic interaction chromatography, gel filtration, and reversed-phase high-performance liquid chromatography with a yield of 58% of the original activity. Leucocin A is stable at low pH and heat resistant, and the activity of the pure form is enhanced by the addition of bovine serum albumin. It is inactivated by a range of proteolytic enzymes. The molecular weight was determined by mass spectrometry to be 3,930.3 +/- 0.4. Leucocin A-UAL 187 contains 37 amino acids with a calculated molecular weight of 3,932.3. A mixed oligonucleotide (24-mer) homologous to the sequence of the already known N terminus of the bacteriocin hybridized to a 2.9-kb HpaII fragment of a 7.6-MDa plasmid from the producer strain. The fragment was cloned into pUC118 and then subcloned into a lactococcal shuttle vector, pNZ19. DNA sequencing revealed an operon consisting of a putative upstream promoter, a downstream terminator, and two open reading frames flanked by a putative upstream promoter and a downstream terminator. The first open reading frame downstream of the promoter contains 61 amino acids and is identified as the leucocin structural gene, consisting of a 37-amino-acid bacteriocin and a 24-residue N-terminal extension. No phenotypic expression of the bacteriocin was evident in several lactic acid bacteria that were electrotransformed with pNZ19 containing the 2.9-kb cloned fragment of the leucocin A plasmid.     

Identification of the lantibiotic nisin Q,a new natural nisin variant produced by Lactococcus lactis 61-14 isolated from a river in Japan

Lactococcus lactis 61-14 isolated from river water produced a bacteriocin active against a wide range of Gram-positive bacteria. N-terminal amino acid sequencing, mass spectral analysis of the purified bacteriocin, and genetic analysis using nisin-specific primers showed that the bacteriocin was a new natural nisin variant, termed nisin Q. Nisin Q and nisin A differ in four amino acids in the mature peptide and two in the leader sequence.     

Design,synthesis and evaluation of antimicrobial activity of N-terminal modified Leucocin A analogues

Class IIa bacteriocins are potent antimicrobial peptides produced by lactic acid bacteria to destroy competing microorganisms. The N-terminal domain of these peptides consists of a conserved YGNGV sequence and a disulphide bond. The YGNGV motif is essential for activity, whereas, the two cysteines involved in the disulphide bond can be replaced with hydrophobic residues. The C-terminal region has variable sequences, and folds into a conserved amphipathic α-helical structure. To elucidate the structure–activity relationship in the N-terminal domain of these peptides, three analogues (1–3) of a class IIa bacteriocin, Leucocin A (LeuA), were designed and synthesized by replacing the N-terminal β-sheet residues of the native peptide with shorter β-turn motifs. Such replacement abolished the antibacterial activity in the analogues, however, analogue 1 was able to competitively inhibit the activity of native LeuA. Native LeuA (37-mer) was synthesized using native chemical ligation method in high yield. Solution conformation study using circular dichroism spectroscopy and molecular dynamics simulations suggested that the C-terminal region of analogue 1 adopts helical folding as found in LeuA, while the N-terminal region did not fold into β-sheet conformation. These structure–activity studies highlight the role of proper folding and complete sequence in the activity of class IIa bacteriocins.     

Characterization of the Lactococcus lactis nisin A operon genes nisP, encoding a subtilisin-like serine protease involved in precursor processing, and nisR, encoding a regulatory protein involved in nisin biosynthesis.

Biosynthesis of the lantibiotic peptide nisin by Lactococcus lactis NIZO R5 relies on the presence of the conjugative transposon Tn5276 in the chromosome. A 12-kb DNA fragment of Tn5276 including the nisA gene and about 10 kb of downstream DNA was cloned in L. lactis, resulting in the production of an extracellular nisin precursor peptide. This peptide reacted with antibodies against either nisin A or the synthetic leader peptide, suggesting that it consisted of a fully modified nisin with the nisin leader sequence still attached to it. This structure was confirmed by N-terminal sequencing and 1H-nuclear magnetic resonance analysis of the purified peptide. Deletion studies showed that the nisR gene is essential for the production of this intermediate. The deduced amino acid sequence of the nisR gene product indicated that the protein belongs to the family of two-component regulators. The deduced amino acid sequence of NisP, the putative product of the gene upstream of nisR, showed an N-terminal signal sequence, a catalytic domain with a high degree of similarity to those of subtilisin-like serine proteases, and a putative C-terminal membrane anchor. Cell extracts of Escherichia coli overexpressing nisP were able to cleave the nisin precursor peptide, producing active, mature nisin. A similar activation was obtained with whole cells but not with membrane-free extracts of L. lactis strains carrying Tn5276 in which the nisA gene had been inactivated. The results indicate that the penultimate step in nisin biosynthesis is secretion of precursor nisin without cleavage of the leader peptide, whereas the last step is the cleavage of the leader peptide sequence from the fully maturated nisin peptide.     

An X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis: cloning, expression in Escherichia coli, and application for removal of N-terminal Pro-Pro from recombinant proteins

A novel pepX gene was cloned from isolated DNA of Lactococcus lactis by PCR. The deduced amino acid sequence of the 89-kDa protein showed 94, 93, 65, and 44% identity with the pepX protein from Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactobacillus delbruecki subsp. bulgaricus, and Lactobacillus helveticus, respectively, and contained a serine protease G-K-S-Y-L-G consensus motif. The pepX gene has been cloned into pET17b and was expressed at a high level in Escherichia coli BL21 (DE3) LysS. PepX was purified to approximate homogeneity with ammonium sulfate precipitation and DEAE Sephadex A-50 chromatography. Optimal pepX activity was observed at pH 8.0 and 37 degrees C. According to SDS-PAGE analysis, pepX has a molecular mass of approximately 89 kDa. The peptidase can remove completely the unwanted X-Pro from the N-terminal of the target protein, releasing the naturally active protein and peptide, revealing a prospective application of pepX in large-scale production of pharmaceutical protein and peptide products.     

Hipposin,a histone-derived antimicrobial peptide in Atlantic halibut (Hippoglossus hippoglossus L.)

A novel 51-residue antimicrobial peptide (AMP) from the skin mucus of Atlantic halibut (Hippoglossus hippoglossus L.) was isolated using acid extraction, and cationic exchange and reversed phase chromatography. The complete amino acid sequence of the AMP, termed hipposin, was determined by automated Edman degradation and mass spectrometry to be SGRGKTGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAHRVGAGAPVYL. The N-terminal amino group was acetylated. The theoretical mass of hipposin was calculated to be 5458.4 Da, which was in good agreement with the mass of 5459 Da determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Hipposin was shown to be derived from histone H2A by PCR amplifying the encoding sequences from Atlantic halibut genomic DNA. The peptide showed sequence similarity with the 39-mer AMP buforin I of Asian toad and the 19-mer AMP parasin I of catfish. Fifty of the fifty-one residues in hipposin were identical to the N-terminal region of histone H2A from rainbow trout. Hipposin showed strong antimicrobial activity against several Gram-positive and Gram-negative bacteria and activity could be detected down to hipposin concentrations of 0.3 microM (1.6 microg/ml). Hipposin without N-terminal acetylation was prepared by solid-phase peptide synthesis and shown to have the same antimicrobial activity as the natural acetylated peptide. Thus, hipposin is a new broad-spectrum histone-derived AMP found in the skin mucus of Atlantic halibut.     

Molecular cloning, enhancement of expression efficiency and site-directed mutagenesis of rat epidermal cystatin A.

A rat cystatin A cDNA clone was isolated from a lambda ZAP library representing newborn rat skin mRNA by screening with a synthetic oligonucleotide designed from amino acid sequence 15-23 of the cysteine proteinase inhibitor. The obtained clone contained a partial coding region of the inhibitor, lacking the 5'-untranslated region and coding sequence for the NH(2)-terminal 13 residues. The amino acid sequence deduced from the base sequence, Glu14-Phe103, coincided with that determined at the amino acid level. To obtain the recombinant cystatin A protein, the DNA was fused with a synthetic linker encoding its missing N-terminal 17 residues and introduced into an expression vector, pMK2. In Escherichia coli, however, the expression level of the semi-synthetic gene was low, 0. 5 mg of the purified recombinant protein per 1 liter culture being produced. Changing of the codon usage of the N-terminal region in a pET-15b expression system led to an increase in the yield depending on the instability of the putative secondary structure around an initiation codon of the mRNA. The expressed cystatin A showed identical characteristics with the authentic form except for the absence of the N-terminal acetyl blocking group. Using the expression system, two kinds of point mutation, the conservative Val54 in the first loop QxVxG region being changed to Lys and Glu, were introduced, but there was almost no effect on the inhibitory activity toward papain. This suggests that the conserved Val in the reactive site is not restricted and that the hydrophobicity of the position is not essential for the activity of rat cystatin A.     

1H NMR structural and functional characterisation of a cAMP-specific phosphodiesterase-4D5 (PDE4D5) N-terminal region peptide that disrupts PDE4D5 interaction with the signalling scaffold proteins, beta-arrestin and RACK1

The unique 88 amino acid N-terminal region of cAMP-specific phosphodiesterase-4D5 (PDE4D5) contains overlapping binding sites conferring interaction with the signaling scaffold proteins, betaarrestin and RACK1. A 38-mer peptide, whose sequence reflected residues 12 through 49 of PDE4D5, encompasses the entire N-terminal RACK1 Interaction Domain (RAID1) together with a portion of the beta-arrestin binding site. (1)H NMR and CD analyses indicate that this region has propensity to form a helical structure. The leucine-rich hydrophobic grouping essential for RACK1 interaction forms a discrete hydrophobic ridge located along a single face of an amphipathic alpha-helix with Arg34 and Asn36, which also play important roles in RACK1 binding. The Asn22/Pro23/Trp24/Asn26 grouping, essential for RACK1 interaction, was located at the N-terminal head of the amphipathic helix that contained the hydrophobic ridge. RAID1 is thus provided by a distinct amphipathic helical structure. We suggest that the binding of PDE4D5 to the WD-repeat protein, RACK1, may occur in a manner akin to the helix-helix interaction shown for G(gamma) binding to the WD-repeat protein, G(beta). A more extensive section of the PDE4D5 N-terminal sequence (Thr11-Ala85) is involved in beta-arrestin binding. Several residues within the RAID1 helix contribute to this interaction however. We show here that these residues form a focused band around the centre of the RAID1 helix, generating a hydrophobic patch (from Leu29, Val30 and Leu33) flanked by polar/charged residues (Asn26, Glu27, Asp28, Arg34). The interaction with beta-arrestin exploits a greater circumference on the RAID1 helix, and involves two residues (Glu27, Asp28) that do not contribute to RACK1 binding. In contrast, the interaction of RACK1 with RAID1 is extended over a greater length of the helix and includes Leu37/Leu38, which do not contribute to beta-arrestin binding. A membrane-permeable, stearoylated Val12-Ser49 38-mer peptide disrupted the interaction of both beta-arrestin and RACK1 with endogenous PDE4D5 in HEKB2 cells, whilst a cognate peptide with a Glu27Ala substitution selectively failed to disrupt PDE4D5/RACK1 interaction. The stearoylated Val12-Ser49 38-mer peptide enhanced the isoprenaline-stimulated PKA phosphorylation of the beta(2)-adrenergic receptors (beta(2)AR) and its activation of ERK, whilst the Glu27Ala peptide was ineffective in both these regards.     

Insights on the amino acid side-chain interactions of a synthetic T-cell determinant.

The effect of single amino acid substitutions at positions 18 and 20 on the T-cell determinant (TD) character of peptide p12-26 from lambda repressor protein and on its recognition by a monoclonal antibody was studied by means of 40 synthetic peptides of a length of 15 amino acids. ELISA competition experiments showed that the identity of amino acid at position 20 is very important for antibody recognition, whereas that of amino acid at position 18 is much less important. In contrast, both Leu 18 and Ala 20 are important residues in defining the TD character of peptide p12-26. The most tolerated replacements, ordered in increasing disrupting power are: Ala 20 by Cys, Ser or Gly and Leu 18 by Ile or Val. Any other amino acid replacement completely abolishes the TD capacity of peptide p12-26. The peptides used in this study were synthesized using a multiple solid-phase peptide synthesizer newly designed. Their purity was very high as shown by amino acid sequence experiments.     

Isolation, amino acid sequence, and synthesis of dermaseptin, a novel antimicrobial peptide of amphibian skin

A 34-residue antimicrobial peptide named dermaseptin was purified to homogeneity from amphibian skin by a 3-step protocol involving molecular sieve filtration, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The complete amino acid sequence of dermaseptin, ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ, was determined by automated Edman degradation of the peptide and of fragments generated by trypsin. Fast atom bombardment mass spectra of dermaseptin gave a protonated molecular ion m/z 3455.4 which matched the theoretical molecular weight predicted from the amino acid sequence. Dermaseptin was synthesized by the solid-phase method. The synthetic replicate was shown to be indistinguishable from natural dermaseptin with respect to chromatographic properties, amino acid sequence determination, and mass spectrometry analysis. Dermaseptin is a water-soluble, thermostable, and nonhemolytic peptide endowed with highly potent antimicrobial activity against pathogenic fungi at micromolar concentration. Circular dichroism spectra of dermaseptin in hydrophobic media indicated 80% alpha-helical conformation, and predictions of secondary structure suggested that dermaseptin can be configured as an amphiphatic alpha-helix spanning over residues 1-27, a structure that perturbs membrane functions regulating water flux.     

Monoclonal antibodies distinguish synthetic peptides that differ in one chemical group

By using human calcitonin (hCT), human calcitonin-gene-related peptide (hCGRP), and a synthetic peptide with a sequence analogous to the 34 C-terminal amino acids of human preprocalcitonin (designated as PQN-34) as haptens in the generation of monoclonal antibodies, we assessed the role of amido and amino groups in paratope-epitope binding. By using peptide inhibition experiments and solid-phase immunoassays, monoclonal anti-hCT antibody CT07 and monoclonal anti-hCGRP antibody CGR01 were found to bind to an antigenic determinant located in the C-terminal segment of the hormones. These epitopes comprise the seven C-terminal amino acids of the hormones, and the presence of the hormone-ending carboxamide group was found to be essential for antibody binding. The corresponding heptapeptides, either bearing a carboxyl group or else linked to a glycine residue at their C-terminal part, failed to react with the antibodies. Moreover, these monoclonal antibodies did not bind to synthetic peptides analogous to the C-terminal region of the hormone precursor molecules that comprised the epitope site flanked by a peptide sequence. In an attempt to assess whether amido groups when present on the side-chain of amino acids may also modulate antibody binding, a monoclonal antibody referred to as QPO1 was produced and was found to recognize an antigenic determinant localized in the N-terminal region of the PQN-34 peptide bearing a glutamine residue as the N-terminal amino acid. The epitope was found to correspond to a topographic assembled site, and binding of QPO1 was found to be substantially dependent on the presence of the free amino and the side-chain amido groups borne by the N-terminal glutamine residue of this peptide PQN-34. In contrast to these findings, an antigenic determinant located in the internal sequence of calcitonin and recognized by monoclonal anti-hCT antibody CT08 was found to be expressed on the mature form of the hormone, as well as on synthetic peptides with sequence mimicking that of preprocalcitonin. These data should guide the choice of synthetic peptide haptens for the production of anti-protein antibodies.     

Kenojeinin I, antimicrobial peptide isolated from the skin of the fermented skate, Raja kenojei

An antimicrobial peptide was purified from fermented skate skin extract using the solid-phase extraction and separation on HPLC reversed-phase chromatography. Amino acid sequence of the purified peptide (Peak A) having an antimicrobial activity revealed the presence of many cationic residues of the total 28 amino acids. Its molecular mass was found to be 3059 Da. This result was in excellent agreement with the theoretical molecular mass calculated from the amino acid sequence. The synthetic kenojeinin I had inhibitory effects on B. subtilis (MIC, 12 microg/ml), E. coli (28 microg/ml), and S. cerevisiae (12 microg/ml). These results indicate that fermented skate skin is potentially antimicrobial.     

Purification, characterization, gene cloning, sequencing, and overexpression of aminopeptidase N from Streptococcus thermophilus A.

The general aminopeptidase PepN from Streptococcus thermophilus A was purified to protein homogeneity by hydroxyapatite, anion-exchange, and gel filtration chromatographies. The PepN enzyme was estimated to be a monomer of 95 kDa, with maximal activity on N-Lys-7-amino-4-methylcoumarin at pH 7 and 37 degrees C. It was strongly inhibited by metal chelating agents, suggesting that it is a metallopeptidase. The activity was greatly restored by the bivalent cations Co2+, Zn2+, and Mn2+. Except for proline, glycine, and acidic amino acid residues, PepN has a broad specificity on the N-terminal amino acid of small peptides, but no significant endopeptidase activity has been detected. The N-terminal and short internal amino acid sequences of purified PepN were determined. By using synthetic primers and a battery of PCR techniques, the pepN gene was amplified, subcloned, and further sequenced, revealing an open reading frame of 2,541 nucleotides encoding a protein of 847 amino acids with a molecular weight of 96,252. Amino acid sequence analysis of the pepN gene translation product shows high homology with other PepN enzymes from lactic acid bacteria and exhibits the signature sequence of the zinc metallopeptidase family. The pepN gene was cloned in a T7 promoter-based expression plasmid and the 452-fold overproduced PepN enzyme was purified to homogeneity from the periplasmic extract of the host Escherichia coli strain. The overproduced enzyme showed the same catalytic characteristics as the wild-type enzyme.     

Cloning, nucleotide sequence, and expression of Achromobacter protease I gene

Achromobacter protease I (API) is a lysine-specific serine protease which hydrolyzes specifically the lysyl peptide bond. A gene coding for API was cloned from Achromobacter lyticus M497-1. Nucleotide sequence of the cloned DNA fragment revealed that the gene coded for a single polypeptide chain of 653 amino acids. The N-terminal 205 amino acids, including signal peptide and the threonine/serine-rich C-terminal 180 amino acids are flanking the 268 amino acid-mature protein which was identified by protein sequencing. Escherichia coli carrying a plasmid containing the cloned API gene overproduced and secreted a protein of Mr 50,000 (API') into the periplasm. This protein exhibited a distinct endopeptidase activity specific for lysyl bonds as well. The N-terminal amino acid sequence of API' was the same as mature API, suggesting that the enzyme retained the C-terminal extended peptide chain. The present experiments indicate that API, an extracellular protease produced by gram-negative bacteria, is synthesized in vivo as a precursor protein bearing long extended peptide chains at both N and C termini.     

Propionicin SM1, a bacteriocin from Propionibacterium jensenii DF1: isolation and characterization of the protein and its gene

We purified a bacteriocin from the cell-free supernatant of Propionibacterium jensenii DF1 isolated from Swiss raw milk, and named it propionicin SM1. The heat-stable protein was strongly bactericidal against P. jensenii DSM20274. On the basis of the N-terminal amino acid sequence of the purified protein, a degenerate oligonucleotide probe was designed to locate and clone the corresponding gene of P. jensenii DF1. It hybridized exclusively with the DF1l-resident plasmid pLME106, but not with chromosomal DNA. Sequencing of the 6.9-kb plasmid revealed the targeted amino acid sequence within an open reading frame (ORF4) of 207 amino acids (molecular mass, 22,865 Da). The corresponding gene was named ppnA. It encodes the prepeptide PpnA that is processed to the mature protein (19,942 Da) propionicin SM1. No sequence homology is detectable with known proteins. However, the proposed leader peptide sequence containing 27 amino acids has typical signal peptide features and shows good homology to the leader peptide of Usp45, a protein excreted from Lactococcus lactis (VAN ASSELDONK et al., 1993). Plasmid pLME106 contains at least 9 ORFs, some exhibiting significant homologies to plasmid-encoded functions from other bacteria. The highest identity values were found for ORF1 with the theta replicase (acc. no. U39878) of Brevibacterium linens (58.8%) and ORF6 with the recombinase/invertase (acc. no. AF060871) found in Rhodococcus rhodochrous (46.4%).     

Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici.

A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80,000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43-44 amino acid residues. The predicted M(r) and isolectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules--17 of the first 19 residues were common--indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.     

Sequence of an intestinal cDNA encoding human motilin precursor

A cDNA clone encoding the human motilin precursor was isolated from an intestinal library using synthetic oligonucleotide probes. The predicted amino acid sequence indicates that the motilin precursor consists of 115 amino acids and includes a 25-residue N-terminal signal peptide followed by the 22-amino-acid motilin sequence and a long, 68-residue C-terminal peptide. The amino acid sequence of human motilin predicted from the cDNA sequence is identical to its porcine counterpart, which has been determined by protein sequencing. Proteolytic processing of promotilin to motilin occurs at the sequence, Lys-Lys, this being the first reported instance of processing occurring at a pair of Lys residues. In other precursors it occurs at Lys-Arg, Arg-Arg, Arg, or very rarely Lys.     

Cloning and sequence analysis of the gene encoding Lactococcus lactis malolactic enzyme: Relationships with malic enzymes

Abstract Malolactic enzyme is the key enzyme in the degradation of L-malic acid by lactic acid bacteria. Using degenerated primers designed from the first 20 N-terminal amino acid sequence of lactococcal malolactic enzyme, a 60-bp DNA fragment containing part of the mleS gene was amplified from Lactococcus lactis in a polymerase chain reaction. This specific probe was used to isolate two contiguous fragments covering the gene as a whole. The 1.9-kb region sequenced contains an open reading frame of 1623 bp, coding a putative protein of 540 amino acids. The deduced amino acid sequence reveals that lactococcal putative protein (Mlep) is highly homologous to the malic enzyme of other organisms. Expression of the mleS gene in Escherichia coli results in malolactic activity.