Biosynthesis of the lantibiotic Pep5. Isolation and characterization of a prepeptide containing dehydroamino acids

Pep5 is a tricyclic peptide antibiotic which contains the unusual amino acids dehydrobutyrine, lanthionine and 3-methyllanthionine. It is matured from a 60-amino-acid precursor peptide (pre-Pep5) deduced from the sequence of the structural gene pepA. To study the biosynthesis of Pep5 we tried to isolate the primary translation product. We identified a peptide in crude extracts of the Pep5-producing Staphylococcus epidermidis strain using antibodies raised against a synthetic 26-residue peptide representing the leader peptide region of pre-Pep5. The putative precursor was purified by reversed-phase HPLC. The isolated peptide did not react with antibodies directed against a C-terminal fragment of mature Pep5 containing two sulfide bridges. Neither lanthionine nor 3-methyllanthionine was detected in amino acid analysis of the isolated precursor. Its amino acid sequence was identical with the sequence predicted from pepA, but Edman degradation stopped at the first threonine residue of the prolantibiotic region indicating a posttranslational modification at this position. The molecular mass of the isolated peptide was 6575.4 +/- 1.7 Da, determined by ion-spray mass spectrometry. This is in agreement with a molecule being dehydrated at the four threonine and the two serine residues in the propeptide region; such a peptide has a calculated molecular mass of 6576.7 Da. The results strongly suggest that maturation of the lantibiotic Pep5 is initiated by selective dehydration of hydroxyamino acids in the propeptide region of the primary translation product and that thioether ring formation is not closely linked to dehydration.     

Extended reading frame of a ubiquitin gene encodes a stable, conserved, basic protein

Antibodies specific for the 80-amino acid hypothetical protein encoded by the in-frame, 3'-extension of a human ubiquitin gene were produced in rabbits by immunization with a 14-residue synthetic peptide. When used to probe HeLa cell extracts for the non-ubiquitin product of this natural fusion gene, the antipeptide sera detected a protein with an apparent molecular weight of 16,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An immunoreactive protein of identical mobility was detected in organisms ranging from Acanthamoeba to man, indicating that the extension protein, like ubiquitin, is highly conserved. The immunoreactive protein was isolated from calf thymus, and direct sequencing revealed the first 16 amino acids to be identical to those predicted from the extension portion of the human cDNA. Thus, ubiquitin was no longer present at the amino terminus. The purified bovine extension protein failed to react with a ubiquitin-specific antibody indicating the absence of isopeptide-linked ubiquitin as well. Moreover, by denaturing gel permeation chromatography the extension has a molecular weight of 10,000 Da, a value that corresponds more closely to the size of the extension alone (9,000 Da) than to the intact fusion protein (17,500 Da). The extension protein, which was found in both cytoplasmic and nuclear fractions of HeLa cells, persisted at high levels when protein synthesis was blocked with cycloheximide or puromycin. These results show that the 80-residue extension protein is the stable, processed product of the ubiquitin fusion gene.     

Analysis of β-ketothiolase and acetoacetyl-CoA reductase genes of a methylotrophic bacterium, Paracoccus denitrifleans, and their expression in Escherichia coli

Abstract The β-ketothiolase gene ( phaA ) and acetoacetyl-CoA reductase gene ( phaB ) were isolated from Paracoccus denitrificans . Nucleotide sequence analysis showed that they encoded proteins of 391 amino acids with a molecular mass of 40744 Da and of 242 amino acids with a molecular mass of 25614 Da, respectively. The predicted gene products exhibited high amino acid identities with those from other bacteria: 64.4–74.0% for the phaA gene product and 47.6–80.6% for the phaB gene product, respectively. Both genes were co-transcribed in a recombinant Escherichia coli . In addition, promoter activity was detected upstream of the phaA gene. Hence, the two genes are organized as an operon, phaA - phaB , in P. denitrificans . NADH was preferred to NADPH as a cofactor of acetoacetyl-CoA reductase.     

Identification and molecular cloning of yeast homolog of nucleosome assembly protein I which facilitates nucleosome assembly in vitro

Yeast DNA coding for nucleosome assembly protein I (NAP-I), which facilitates nucleosome assembly in vitro at physiological ionic conditions, was cloned and its gene product was characterized. A monoclonal antibody against NAP-I (58 kDa) from human HeLa cells was used to screen a genomic library of Saccharomyces cerevisiae constructed into lambda gt11. A 60-kDa protein was detected by immunoblotting in the extracts of Escherichia coli lysogenized with a positive clone. The 60-kDa protein purified from the extracts had an activity equivalent to that of NAP-I from mouse and human cells. The amino acid sequence deduced from the gene coding for the yeast NAP-I defines a polypeptide of molecular mass 47,848 Da with three negatively charged regions. While the two regions contain 8 and 10 acidic amino acids out of 13 amino acid residues, the longest stretch has 15 glutamic and 13 aspartic acids out of 38 residues. These regions are probably involved in the interaction with histones. Proteins recognized by the anti-NAP-I antibody were also present in Xenopus oocytes and Drosophila cultured cells. Possible roles of NAP-I are discussed in relation to other nucleosome assembly proteins.     

Molecular cloning and expression of the gene encoding family 19 chitinase from Streptomyces sp. J-13-3

The gene encoding chitinase from Streptomyces sp. (strain J-13-3) was cloned and its nucleotide structure was analyzed. The chitinase consisted of 298 amino acids containing a signal peptides (29 amino acids) and a mature protein (269 amino acids), and had calculated molecular mass of 31,081 Da. The calculated molecular mass (28,229 Da) of the mature protein was almost same as that of the native chitinase determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometer. Comparison of the encoded amino acid sequences with those of other chitinases showed that J-13-3 chitinase was a member of the glycosyl-hydrolase family 19 chitinases and the mature protein had a chitin binding domain (65 amino acids) containing AKWWTQ motif and a catalytic domain (204 amino acids). The J-13-3 strain had a single chitinase gene. The chitinase (298 amino acids) with C-terminal His tag was overexpressed in Escherichia coli BL21(DE3) cells. The recombinant chitinase purified from the cell extract had identical N-terminal amino acid sequence of the mature protein in spite of confirmation of the nucleotide sequence, suggesting that the signal peptide sequence is successfully cut off at the predicted site by signal peptidase from E. coli and will be a useful genetic tool in protein engineering for production of soluble recombinant protein. The optimum temperature and pH ranges of the purified chitinase were at 35-40 degrees C and 5.5-6.0, respectively. The purified chitinase hydrolyzed colloidal chitin and trimer to hexamer of N-acetylglucosamine and also inhibited the hyphal extension of Tricoderma reesei.     

Characterization and expression of the cDNA encoding a new kind of phospholipid transfer protein, the phosphatidylglycerol/phosphatidylinositol transfer protein from Aspergillus oryzae: evidence of a putative membrane targeted phospholipid transfer protein in fungi

The full-length cDNA of a phospholipid transfer protein (PLTP) was isolated from Aspergillus oryzae by a RACE-PCR procedure using degenerated primer pool selected from the N-terminal sequence of the purified phosphatidylinositol/phosphatidylglycerol transfer protein (PG/PI-TP). The cDNA encodes a 173 amino acid protein of 18823 Da. The deduced amino acid sequence from position 38 to 67 is 100% identical to the N-terminal sequence (first 30 amino acids) of the purified PG/PI-TP. This amino acid sequence is preceded by a leader peptide of 37 amino acids which is predicted to be composed of a signal peptide of 21 amino acids followed by an extra-sequence of 16 amino acids, or a membrane anchor protein signal (amino acid 5-29). This strongly suggests that the PG/PI-TP is a targeted protein. The deduced mature protein is 138 amino acids long with a predicted molecular mass of 14933 Da. Comparison of the deduced PG/PI-TP sequence with other polypeptide sequences available in databases revealed a homology with a protein deduced from an open reading frame coding for an unknown protein in Saccharomyces cerevisiae (36% identity and 57% similarity). Apart from this homology, the PG/PI-TP is unique and specific to the filamentous fungi on the basis of comparison of PLTP protein sequences. Northern blot analysis of RNA isolated from A. oryzae cultures grown on glucose or glucose supplemented with phospholipids suggests that the PG/PI-TP is transcribed by only one RNA species and allows us to show that expression of the protein is regulated at the messenger RNA level.     

Isolation and identification of the AKH III precursor-related peptide from Locusta migratoria

We have isolated an 8770Da peptide from extracts of corpora cardiaca of adult male and female Locusta migratoria. The N-terminal amino-acid sequence as partially established by Edman degradation is Ala-Leu-Gly-Ala-Pro-Ala-Ala-Gly-Asp. These nine amino acids correspond to the first nine N-terminal amino acids of the adipokinetic hormone precursor-related peptide gamma-chain (APRP-gamma), a peptide that is predicted from the gene encoding the adipokinetic hormone III precursor. The APRP-gamma chain has a monoisotopic mass of 4387Da and contains two cysteine residues. It is known that both AKH I and AKH II precursors occur as dimers. After processing they give rise to the active hormones and three dimeric (two homodimers and one heterodimer) adipokinetic hormone precursor related peptides (APRPs). Based on the mass of 8770Da and the established N-terminal sequence tag, we conclude that the isolated peptide is a homodimer consisting of two APRP-gamma units, covalently linked to each other by two disulphide bounds. In analogy with the previous identified APRPs (APRP-1, APRP-2, and APRP-3), this APRP will be designated as APRP-4.     

Mass spectrometric determination of a novel modification of the N-terminus of histidine-tagged proteins expressed in bacteria.

Two proteins, FKBP, and Spo0F, were expressed in bacteria as histidine-tagged fusion proteins and isolated under native conditions. MALDI-TOF-MS analysis revealed that each protein preparation contained two components, neither of which corresponded to the molecular weights predicted from DNA sequences. The difference in molecular weight between the two FKBP components and two Spo0F components was approximately 178 +/- 14 Da. Site-specific proteolytic cleavage resulted in the release of histidine-tagged peptide from the recombinant proteins. MALDI mass spectra of the cleaved proteins showed a single molecular ion peak for each species with the predicted molecular weights. The histidine-tagged peptide released from both fusion proteins displayed two distinct peaks by MALDI-FT-MS corresponding to monoisotopic molecular weights of 2269. 027 Da and 2447.087 Da, respectively, which were both inconsistent with the predicted peptide sequence M-G-H-H-H-H-H-H-H-H-H-H-S-S-G-H-I-E-G-R of 2400.055 Da. The peptide at 2269.027 Da was sequenced by ESI-MS-MS and found to be a truncated histidine tag resulting from an initiator methionine deletion. ESI-MS-MS analysis of the peptide at 2447.087 Da indicated a moiety of 178.0 Da attached to the second residue glycine of the histidine tag. This alteration of the N-terminus does not fit any known modifications. A synthetic peptide with the identical sequence of the isolated his-tag M-G-H-H-H-H-H-H-H-H-H-H remained unmodified during the protein purification process, suggesting that modification of the initiator methionine was carried out in vivo, rather than the result of a chemical reaction from the isolation procedure.     

Characteristics of transxylosylation by β-xylosidase from Aspergillus awamori K4

The Aspergillus awamori K4 β-xylosidase gene (Xaw1) sequence was deduced by sequencing RT-PCR and PCR products. The ORF was 2,412 bp and the predicted peptide was 804 amino acids long, corresponding to a molecular weight of 87,156 Da. The mature protein was 778 amino acids long with a molecular weight of 84,632 Da. A homology search of the amino acid sequence revealed that it was very similar to the Aspergillus niger β-xylosidase gene with only five amino acid differences. K4 β-xylosidase had the same catalytic mechanism as family 3 β-glucosidases, involving Asp in region A. At an early stage in the reaction with xylobiose and xylotriose, the hydrolysis rate was much lower than the transxylosylation rate, decreasing gradually as the substrate concentration increased, whereas the transxylosylation rate increased greatly. Aspergillus awamori K4 β-xylosidase had broad acceptor specificity toward alcohols, hydroxybenzenealcohols, sugar alcohols and disaccharides. A consensus portion involving the hydroxymethyl group of the acceptor was confirmed in the major transfer products 1(4)-O-β-d-xylosyl erythritol, (2-hydroxyl)-phenyl-methyl-β-d-xylopyranoside, 6S-O-β-d-xylosyl maltitol (S: sorbitol residue) and 6G-O-β-d-xylosyl palatinose (G: glucosyl residue). This might suggest that the methylene in the hydroxymethyl group facilitates base-catalyzed hydroxyl group attack of the anomeric center of the xylosyl–enzyme intermediate.     

Characteristics of transxylosylation by beta-xylosidase from Aspergillus awamori K4

The Aspergillus awamori K4 beta-xylosidase gene (Xaw1) sequence was deduced by sequencing RT-PCR and PCR products. The ORF was 2,412 bp and the predicted peptide was 804 amino acids long, corresponding to a molecular weight of 87,156 Da. The mature protein was 778 amino acids long with a molecular weight of 84,632 Da. A homology search of the amino acid sequence revealed that it was very similar to the Aspergillus niger beta-xylosidase gene with only five amino acid differences. K4 beta-xylosidase had the same catalytic mechanism as family 3 beta-glucosidases, involving Asp in region A. At an early stage in the reaction with xylobiose and xylotriose, the hydrolysis rate was much lower than the transxylosylation rate, decreasing gradually as the substrate concentration increased, whereas the transxylosylation rate increased greatly. Aspergillus awamori K4 beta-xylosidase had broad acceptor specificity toward alcohols, hydroxybenzenealcohols, sugar alcohols and disaccharides. A consensus portion involving the hydroxymethyl group of the acceptor was confirmed in the major transfer products 1(4)-O-beta-D-xylosyl erythritol, (2-hydroxyl)-phenyl-methyl-beta-D-xylopyranoside, 6S-O-beta-D-xylosyl maltitol (S: sorbitol residue) and 6G-O-beta-D-xylosyl palatinose (G: glucosyl residue). This might suggest that the methylene in the hydroxymethyl group facilitates base-catalyzed hydroxyl group attack of the anomeric center of the xylosyl-enzyme intermediate.     

cDNA cloning of the neutrophil bactericidal peptide indolicidin.

A structurally novel, tryptophan-rich antimicrobial tridecapeptide amide, named indolicidin, has recently been purified from bovine neutrophils (Selsted et al. (1992) J. Biol. Chem. 267, 4292-4295). Here we describe the molecular cloning of this endoantibiotic, which is synthesised in bone marrow cells as a 144 amino acid residue precursor. The encoded protein has a predicted mass of 16479 Da and a pI of 6.51. A putative signal peptide of 29 amino acids precedes a 101 residue pro-region. The mature peptide is at the 3' end of the open reading frame. A glycine, not found in purified indolicidin, is present at the carboxyl terminus of the deduced sequence and is very likely involved in post-translational peptide amidation.     

Identification of a cDNA encoding an isoform of human CTP synthetase

A full-length cDNA clone encoding an isoform of human CTP synthetase (type II) was isolated. A 1761-nucleotide open reading frame which corresponds to a protein of 586 amino acids with a predicted molecular mass of 65678 Da was identified. The predicted protein sequence showed 74% identity with the translation product of a previously identified human CTP synthetase cDNA clone (type I). The function of the human cDNA encoding type II CTP synthetase was verified by successful complementation of the cytidine-requiring CTP synthetase deficient mutant JF618 of Escherichia coli. The gene encoding type II CTP synthetase has been localized on chromosome Xp22.     

Structural and immunological characterization of a linear virus-neutralizing epitope of the rabies virus glycoprotein and its possible use in a synthetic vaccine.

We have mapped a linear epitope recognized by the virus-neutralizing monoclonal antibody 6-15C4 within the primary sequence of the G protein from the Evelyn-Rokitnicki-Abelseth strain of rabies virus. This was accomplished by using fragments of the rabies virus G protein and deduced amino acid sequences of neutralization-resistant variant rabies viruses. The monoclonal antibody 6-15C4 specifically recognized a synthetic peptide (peptide G5-24) which resembles the 6-15C4 epitope in structure. In addition, a tandem peptide constructed from the G5-24 peptide and a dominant TH cell epitope of the rabies virus N protein induced protective immunity against lethal rabies virus challenge infection in mice.     

The primary structure and tissue distribution of an amphibian neuropeptide Y.

Neuropeptide Y (NPY) has been isolated and sequenced from brain extracts of the European common frog, Rana temporaria. Plasma desorption mass spectroscopy of the purified peptide indicated a molecular mass of 4243.3 Da which was in agreement with that deduced from the sequence (4243.7 Da), incorporating a C-terminal amide. The primary structure of frog NPY was established as: YPSKPDNPGEDAPAEDMAKYYSALRHYINLITRQRY-NH2. Frog NPY contains a single, highly-conservative amino acid substitution (Lys for Arg at residue 19) with respect to human NPY. NPY immunoreactivity was localised exclusively in nerves within the brain, pancreas and gastrointestinal tract and reverse-phase HPLC of extracts of these tissues resolved a single immunoreactive peptide of identical retention time in each case. The primary structure of NPY has therefore been highly-conserved over a considerable evolutionary time-span.     

Rainbow trout (Oncorhynchus mykiss) neuropeptide Y.

Neuropeptide Y (NPY) has been isolated from brain extracts of the rainbow trout (Oncorhynchus mykiss) and subjected to structural analyses. Plasma desorption mass spectroscopy estimated the molecular mass of the purified peptide as 4303.9 Da. Automated Edman degradation unequivocally established the sequence of a 36 amino acid residue peptide as: Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Pro-Glu-Glu-Leu-Ala-Lys-Tyr-Tyr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr. The molecular mass calculated from this sequence (4304 Da) is consistent with that obtained by mass spectroscopy. The presence of a C-terminal amide was established by radioimmunoassay. Rainbow trout NPY is identical in primary structure to coho salmon (Oncorhynchus kisutch) pancreatic polypeptide (PP). These data may indicate that, in this group of salmonid fishes, a single member of the NPY/PP peptide family is expressed in both neurons and peripheral endocrine cells.     

A signal peptide secretion-dependent bacteriocin from Carnobacterium divergens.

Divergicin A is a strongly hydrophobic, narrow-spectrum, nonlantibiotic bacteriocin produced by Carnobacterium divergens LV13. This strain of C. divergens contains a 3.4-kb plasmid that mediates production of, and immunity to, the bacteriocin. N-terminal amino acid sequencing of the purified divergicin A was used to locate the structural gene (dvnA). The structural gene encodes a prepeptide of 75 amino acids consisting of a 29-amino-acid N-terminal extension and a mature peptide of 46 amino acids. Directly downstream of dvnA there is a second open reading frame that encodes the immunity protein for divergicin A. Divergicin A has a calculated molecular mass of 4,223.89 Da. The molecular mass determined by mass spectrometry is 4,223.9 Da, indicating that there is no posttranslational modification of the peptide. The N-terminal extension of divergicin A has an Ala-Ser-Ala (positions -3 to -1) cleavage site and acts as a signal peptide that accesses the general export system of the cell (such as the sec pathway in Escherichia coli). This is the first bacteriocin of lactic acid bacteria to be reported that does not have dedicated maturation and secretion genes. Production of divergicin A was observed in heterologous hosts containing only the two genes associated with divergicin A production and immunity. Fusing alkaline phosphatase behind the signal peptide for divergicin resulted in the secretion of this enzyme in the periplasmic space and supernatant of E. coli.     

Purification and molecular cloning of a new galactose-specific lectin from <Emphasis Type="Italic">Bauhinia variegata</Emphasis> seeds

A new galactose-specific lectin was purified from seeds of a Caesalpinoideae plant, Bauhinia variegata, by affinity chromatography on lactose-agarose. Protein extracts haemagglutinated rabbit and human erythrocytes (native and treated with proteolytic enzymes), showing preference for rabbit blood treated with papain and trypsin. Among various carbohydrates tested, the lectin was best inhibited by D-galactose and its derivatives, especially lactose. SDS-PAGE showed that the lectin, named BVL, has a pattern similar to other lectins isolated from the same genus, Bauhinia purpurea agglutinin (BPA). The molecular mass of BVL subunit is 32 871 Da, determined by MALDI-TOF spectrometry. DNA extracted from B. variegata young leaves and primers designed according to the B. purpurea lectin were used to generate specific fragments which were cloned and sequenced, revealing two distinct isoforms. The bvl gene sequence comprised an open reading frame of 876 base pairs which encodes a protein of 291 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to have 263 amino acid residues and 28 963 Da in size.