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PubMed2000

A T-cell epitope determined with random peptide libraries and combinatorial peptide chemistry stimulates T cells specific for cutaneous T-cell lymphoma.

BACKGROUND: Mycosis fungoides is the most frequent T-cell lymphoma of the skin. Despite numerous attempts, no tumour antigens have yet been identified. Only in one case has an idiotype-derived peptide been found to trigger CTL of the respective patient. The identification of natural antigens requires the cultivation of large amounts of tumour cells in vitro, which has been possible in two exceptional cases. The identification of synthetic epitopes for tumour-specific CTL with random peptide libraries can overcome this limitation and is a powerful tool for application in the development of immune therapies for a wide range of patients. MATERIALS AND METHODS: The critical amino acids for the construction of epitopes for the CTCL-specific CTL clone My-La CTL were determined with synthetic peptide libraries in positional scanning OX8 format in a standard 61chromium release assay. Sixteen different peptides could be synthesized from the combinatoric of these amino acids with the canonical anchor amino acids for MHC binding. These peptides were tested for their capacity to stimulate My-La CTL and PBMC of an HLA-matched CTCL patient. RESULTS: A synthetic epitope could be identified for My-La CTL, which was recognized in a HLA-restricted manner. The response towards this epitope was comparable to the response towards their natural target My-La. Using these synthetic epitopes, T cells of a HLA-matched patient could be induced in vitro and led to the establishment of different cell lines and clones. Some of these lines recognized the peptides as well as the allogenic but HLA-matched tumour cell line My-La, indicating that they are specific for a naturally expressed tumour antigen. CONCLUSIONS: The identification of synthetic epitopes for tumour-specific CTL clones can be used for the development of vaccines for immune therapies of cancer; such peptides can be applied inter-individually. Synthetic epitopes must not correspond to the natural ones, but they can be even more potent as stimulation of specific T cells and can be fine-tuned to increase the success of the therapy.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2000

Coexistence of C-type natriuretic peptide and atrial natriuretic peptide systems in the bovine cornea.

PURPOSE: To determine whether the cornea synthesizes natriuretic peptides and contains their receptors. METHODS: The synthesis of the natriuretic peptides, C-type natriuretic peptide (CNP) and atrial natriuretic peptide (ANP), in the bovine cornea was determined by high-performance liquid chromatography (HPLC) with radioimmunoassay and Southern blot analysis. The presence of natriuretic peptide receptor (NPR)-A and -B and their localizations were measured by reverse transcription-polymerase chain reaction (RT-PCR), in vitro autoradiography, and the activation of particulate guanylyl cyclase by natriuretic peptides in the corneal membrane. RESULTS: The serial dilution curves of corneal extracts were parallel to the standard curves of CNP and ANP. With reversed-phase HPLC, a major immunoreactive peak of CNP or ANP was observed at the elution time corresponding with synthetic CNP(1-53) or atriopeptin III (APIII), respectively. The presence of mRNAs of CNP and ANP was also detected in the cornea by RT-PCR and/or Southern blot analysis. Production of 3',5'-cyclic guanosine monophosphate (cGMP) by the activation of particulate guanylyl cyclase in the corneal membrane was stimulated by ANP, BNP, and CNP. More cGMP was produced by CNP than by the other natriuretic peptides. Specific 125I-[Tyr0]-CNP(1-22) binding sites were localized in the endothelial cell layer of cornea. The apparent dissociation constant (Kd) value of the cornea was 3.06 +/- 0.73 nM and the maximum binding capacity was 3.40 +/- 0.63 femtomoles/mm2. Both NPR-A and NPR-B mRNAs were detected by RT-PCR. CONCLUSIONS: The cornea synthesizes CNP and ANP and contains their receptors. These results suggest that the CNP and ANP systems coexist in the bovine cornea.

Animals↗
PubMed2001

Ferrocenoyl amino acids and peptides: probing peptide structure.

Recent studies clearly show the utility of the carbodiimide protocol to attach the redox-active Fc moiety to the N-terminal side of amino acids and peptides under mild conditions, resulting in stable and often crystalline products that afford themselves to structural analysis by X-ray crystallography. Electrochemical studies of Fc-peptides show that there is a significant influence of the redox potential depending on the amino acid sequence. The Fc moiety is sensitive to structural changes that occur in the peptide to which it is attached. For helical Fc-oligoprolines, the redox potential of the Fc group makes it easier to oxidize as the oligoproline chain increases in length. Nonhelical peptides, having a similar primary but different secondary structure, give rise to very different redox potentials. The ramifications of these findings to biological systems are significant in that they provide further evidence that the redox properties of a metal center are influenced by factors that go beyond the primary ligand sphere and thus for the involvement of long-range interactions. The Fc group is clearly sensitive to the shape of the peptide. These effects are currently under more detailed investigation [45] in order to gain further insight into the electronic structure of these ferrocenoyl peptides. Although we are not yet in a position to distinguish between "sensing" coordination or "sensing" conformation changes, this effect is of interest because it may allow the development of peptidic sensors.

Amino Acids↗
PubMed1992

A structural homologue of the N-formyl peptide receptor. Characterization and chromosome mapping of a peptide chemoattractant receptor family.

Phagocytic cells of many higher species express calcium mobilizing G protein-coupled receptors for bacterial N-formyl peptides which mediate chemotaxis, degranulation, and the respiratory burst. cDNA encoding an N-formyl peptide receptor (FPR) has been reported. We now report the isolation of a closely related cDNA, 2.6 kilobase pairs in length, which we have designated as the FPRL1 receptor cDNA (FPRL1 = formyl peptide receptor like-1). FPR and the FPRL1 receptor derive from small, single-copy genes, both of which are located on human chromosome 19. The gene loci are designated FPR1 and FPRL1, respectively. Both FPR and FPRL1 cDNA cross-hybridize under high stringency conditions with a third gene, designated as FPRL2, which does not appear to be expressed in neutrophils. In contrast, transcripts for both the FPRL1 receptor and FPR are detected only in differentiated myeloid cells; the distribution of N-formyl peptide binding sites is also restricted to mature myeloid cells. FPRL1 cDNA encodes a 351-amino acid polypeptide whose sequence is 69% identical to that of FPR. G protein-coupled receptors that exhibit this degree of structural similarity typically possess a conserved ligand specificity. However, the FPRL1 receptor does not bind prototype N-formyl peptide ligands when expressed in heterologous cell types. These results suggest that FPR1 may be the only gene that is expressed by neutrophils that encodes a receptor capable of binding prototype N-formyl peptides. Moreover, discovery of the FPRL1 receptor indicates the existence of another as yet unidentified peptide that may recruit neutrophils to sites of inflammation.

Amino Acid Sequence↗
PubMed1992

Antibody responses to non-immunogenic synthetic peptides induced by co-immunization with immunogenic peptides.

Chimeric peptides comprising B- and T-helper cell epitopes from the proteins of infectious agents represent immunogens with potential for use as new vaccines. However, it has become clear that the orientation of the epitopes, the presence of spacer residues and the number of copies of the epitopes influence the specificity, levels and affinity of the antibody produced following immunization with such constructs. Furthermore, the response to peptides is under genetic control leading to major histocompatibility complex (MHC)-linked non-responsiveness. In this study, we have investigated the potential of co-immunization of immunogenic peptides (to provide T-cell help) with non-immunogenic peptides (representing B-cell epitopes) to overcome the non-response to the latter. For this purpose, we have employed peptides representing T- and B-cell epitopes derived from the sequences of the fusion and haemagglutinin glycoproteins of measles virus. The results obtained show that simple co-immunization of a B-cell epitope with a T-cell epitope results in the production of antibody to the B-cell epitope without the requirement for covalent linkage of the two peptides. This approach could thus be used to overcome the problem of poor immunogenicity of peptides and will be of potential value in the design of immunization strategies using synthetic immunogens.

Animals↗
PubMed2004

Immunological evaluation of vaccination with pre-designated peptides frequently selected as vaccine candidates in an individualized peptide vaccination regimen.

We previously conducted a trial of an 'individualized' regimen, in which cancer patients were vaccinated with peptides after the confirmation of pre-existing peptide-specific cytotoxic T lymphocyte (CTL) precursors. In this study, we performed a new trial of 'pre-designated' regimen, in which cancer patients were vaccinated with peptides that were frequently selected as vaccine candidates in the preceding individualized regimen. Eighteen cancer patients (10 with uterine cervical cancer and 8 with gastric cancer) were enrolled in the new regimen. The pre-designated regimen was well tolerated by all patients. Although peptide-specific CTL precursors and humoral responses increased in the majority of patients with the pre-designated regimen, neither of the responses correlated with clinical outcome. Three patients had long stable disease, and their pre-vaccination peripheral blood mononuclear cells contained peptide-specific CTL precursors reactive to more than 2 of 4 peptides. With the pre-designated regimen, the levels of pre-existing immunoglobulin G reactive to non-vaccinated peptides decreased in 5 of 15 patients with progressive disease, and their time to progression was very short, whereas such a decrease was rarely observed in the preceding individualized regimen. These results suggest that the pre-designated regimen can elicit a primary immune response, but may incidentally suppress pre-existing immune responses.

Adult↗
PubMed1991

Design of bioactive peptides based on antibody hypervariable region structures. Development of conformationally constrained and dimeric peptides with enhanced affinity.

The variable regions of antibody molecules bind antigens with high affinity and specificity. This binding is imparted largely by the hypervariable portions of the variable region. Hypervariable regions typically fold into reverse turn or loop structures. Peptides derived from antibody hypervariable region sequences can bind antigens with similar specificity, albeit with markedly lower affinity. In this study, cyclic and dimeric peptide analogs of an anti-idiotypic/antireceptor antibody hypervariable region were developed. This antibody (87.92.6) binds to reovirus type 3 receptors on cells as well as to a neutralizing anti-reovirus type 3 monoclonal antibody (9B.G5). The cyclic peptides were utilized to probe the optimal conformation for binding to both the receptor and 9B.G5. By dimerizing or constraining the conformation of these peptides, higher affinity binding was produced. By utilizing several different cyclic peptides, the optimal conformation for binding was established. The conformationally optimized cyclic peptide possessed greater than 40-fold higher affinity for the receptor and the idiotype than the linear analog. This study suggests that conformationally constrained and dimeric peptides derived from antibody hypervariable loop sequences can bind antigens (including receptors) with reasonable affinity. hypervariable loop sequences can bind antigens (including

Amino Acid Sequence↗
PubMed1989

Deletion of a highly conserved tetrapeptide sequence of the proinsulin connecting peptide (C-peptide) inhibits proinsulin to insulin conversion by transfected pituitary corticotroph (AtT20) cells.

The biological function of the connecting peptide (C-peptide) of proinsulin is unknown. Comparison of all known C-peptide sequences reveals the presence of a highly conserved peptide sequence, Glu/Asp-X-Glu/Asp (X being a hydrophobic amino acid), adjacent to the Arg-Arg doublet at the B chain/C-peptide junction. Furthermore, the next amino acid in the C-peptide sequence is also acidic in many animal species. To test the possible involvement of this hydrophilic domain in insulin biosynthesis, we constructed a mutant of the rat proinsulin II gene lacking the first four amino acids of the C-peptide and expressed either the normal (INS) on the mutated (INSDEL) genes in the AtT20 pituitary corticotroph cell line. In both cases immunoreactive insulin (IRI) was stored by the cells and released upon stimulation by cAMP. In the INS expressing cells, the majority of IRI, whether stored or released in response to a secretagogue, was mature insulin. By contrast, most of the stored and releasable IRI in the INSDEL expressing cells appeared to be (mutant) proinsulin or conversion intermediate with little detectable native insulin. Release of the mutant proinsulin and/or conversion intermediates was stimulated by cAMP. These results suggest that the mutant proinsulin was appropriately targeted to secretory granules and released predominantly via the regulated pathway, but that the C-peptide deletion prevented its conversion to native insulin.

Amino Acid Sequence↗
PubMed1988

Sequence of peptides from Saccharomyces cerevisiae glutamine synthetase. N-terminal peptide and ATP-binding domain.

Sequences of seven tryptic peptides derived from Saccharomyces cerevisiae glutamine synthetase have been determined. The amino terminus of yeast enzyme is acetylated and has the following sequence: acetyl-Ala-Glu-Ala-Ser-Ile-Glu-Lys. Neither higher eukaryotic nor bacterial glutamine synthetase contain sequences homologous to this yeast amino terminus. 8-Azidoadenosine 5'-triphosphate [( alpha-32P]8-N3ATP) has been used to photolabel the ATP-binding site in yeast glutamine synthetase. Only one 32P-labeled tryptic peptide was obtained as a major fraction and its sequence is Glu-Gly-Tyr-Gly-X-Phe-Glu-Asp-Arg. Similar photolabeling experiments with bovine glutamine synthetase yielded a tryptic peptide whose sequence is Gly-X-Phe-Glu-Asp-Arg, where X is likely Tyr covalently attached by nitrene derived from [alpha-32P]8-N3ADP. Sequences very homologous to this nucleotide-binding site can be found in other eukaryotic enzymes but not in prokaryotic enzymes. In addition, the sequences of two cysteine-containing peptides and three other tryptic peptides were established. Sequences homologous to all these five peptides can be found in mammalian and plant enzymes. The homology between yeast and higher eukaryotic glutamine synthetases was sufficiently strong to suggest that the overall tertiary and quaternary structures of these enzymes must be similar. The sequences presented here, particularly the amino terminus sequence will be valuable in identifying the structural gene of yeast glutamine synthetase, thereby making it possible to study its transcriptional regulation. In addition, the sequences of the cysteine-containing peptides will be useful in determining whether or not the covalent modification of a sulfhydryl group(s) is responsible for the modulation of glutamine synthetase activity.

Adenosine Triphosphate↗
PubMed1994

Generation of cytotoxic T-lymphocytes to a self-peptide/class I complex: a model for peptide-mediated tumor rejection.

Cytotoxic T-lymphocytes (CTL) typically recognize foreign peptides bound to class I products of the major histocompatibility complex. A function of CTL is to identify and eliminate tumor cells that bear inappropriately expressed peptide/class I complexes (i.e., mutated self-peptides or self-peptides that are expressed at abnormally high levels). The processes that result in tolerance to self-antigens can undermine the effectiveness of this system by deleting or inactivating T-cells that might potentially be reactive with tumor-associated antigens. To up-regulate the response to tumor antigens it will be useful to develop methods whereby CTL responses to specific self-peptides can be elicited without damage to normal tissue. In this report a CTL response was generated in BALB/c mice against the ubiquitous self-peptide p2Ca (LSPFPFDL), which binds to Ld and is derived from the mitochondrial enzyme alpha-ketoglutarate dehydrogenase. CTL derived in vitro recognize specifically the p2Ca/Ld complex and use V beta 8 regions predominantly. The cultured cells lysed target cells with lower levels of p2Ca than the levels used for induction. This result suggests that it may be possible to use peptides at high concentrations to elicit CTL react with endogenous levels of a peptide/class I complex. The in vivo potential of the response was demonstrated by the observation that BALB/c mice, coinjected with a syngeneic BALB/c myeloma and exogenous p2Ca, are able to reject the tumor. The p2Ca/Ld system may thus provide a model for evaluating the parameters for effective immunotherapy with tumor-associated peptides.

Animals↗
PubMedDate not supplied

Simultaneous synthesis of peptide libraries on single resin and continuous cellulose membrane supports: examples for the identification of protein, metal and DNA binding peptide mixtures.

Peptide libraries were simultaneously synthesized on single supports by double coupling 0.8 equivalents of an equimolar acylating amino acid mixture consisting of 19 amino acids (cysteine omitted) at randomized sites, thus compensating for the different coupling rates of the amino acids. Peptide epitope mixtures, as well as very complex mixtures such as a completely randomized hexapeptide, were prepared and analyzed by HPLC and amino acid analysis. The results obtained indicate that this method can be applied to the synthesis of peptide libraries. Parts of a simultaneously synthesized solution phase combinatorial library XXB1B2XX were successfully used for the detection of the linear epitope HFND of transforming growth factor-alpha (TGF alpha) recognized by the monoclonal antibody Tab2. Furthermore, novel combinatorial peptide libraries XXB1B2XX were prepared on continuous cellulose membrane supports, also allowing the identification of TGF alpha epitope sequences. In addition, peptide mixtures that bound to a double-stranded DNA (15mer) and silver were identified. These preliminary results indicate that cellulose-bound combinatorial peptide libraries can be used for the rapid and inexpensive screening of millions of peptides to identify single molecules that bind any given ligand such as proteins, nucleic acids and metals.

Amino Acid Sequence↗
PubMed1996

Enhanced tumor outgrowth after peptide vaccination. Functional deletion of tumor-specific CTL induced by peptide vaccination can lead to the inability to reject tumors.

CTL can play an important role in the defense against tumors. Protective CTL-mediated immunity can be established in animal tumor models after vaccination with synthetic peptides representing CTL epitopes. We now report that immunization with synthetic peptides can also lead to CTL tolerance associated with the inability to reject tumors. B6 tumor cells transformed by the human adenovirus early region 1 (Ad5E1) present an Ad5E1A- and an Ad5E1B-encoded CTL epitope to the immune system. CTL clones directed against either of these epitopes are able to eradicate established Ad5E1-induced tumors, showing that these CTL epitopes are targets of CTL that can mediate tumor regression. Here, we show that protective immunity against Ad5E1-expressing tumor cells can be established by immunization with Ad5E1-transformed cells and with an adenovirus vector containing the Ad5E1 region. Protective immunity, in either case, is associated with specific CTL memory. To test whether vaccination with synthetic peptides leads to protection against Ad5E1-expressing tumor cells, we vaccinated mice s.c. with a low dose of the Ad5E1B peptide. This peptide was chosen because the CTL response against the Ad5E1B-encoded CTL epitope contributes most to the antitumor response in B6 mice after vaccination with Ad5E1-transformed cells. Ad5E1B peptide-vaccinated mice were not protected against the outgrowth of Ad5E1-expressing tumor cells, but instead were no longer able to reject a tumor inoculum that was rejected by nonvaccinated mice. Moreover, the protection induced by tumor cell vaccination against Ad5E1B-expressing tumors was gone when the Ad5E1B-encoded CTL epitope was injected a few days before tumor challenge. This is associated with peptide-induced tolerance of Ad5E1B-specific CTL activity. These findings are relevant for the design of therapeutic approaches against both malignancies and T cell-mediated autoimmune diseases.

Adenovirus E1A Proteins↗
PubMedDate not supplied

Substitution of peptide bond 53-54 of HEL(52-61) with an ethylene bond rather than reduced peptide bond is tolerated by an MHC-II restricted T cell.

To probe the interactions between major histocompatibility class-II molecules and the amide bonds of the antigenic peptide main chain, we synthesized ethylenic and reduced analogues of HEL(52-61), an immunogenic peptide for murine major histocompatibility class-II IA k restricted T-cell clones. The synthesis of the corresponding ethylenic analogue of HEL(52-61) in position 53-54 was performed by coupling the Fmoc-protected tripeptide Asp-Tyr-psi [E, CH = CH]Gly with HEL(55-61). Biological tests showed that the ethylenic peptide was presented by major histocompatibility class-II IA kappa molecule and recognized by HEL(52-61)-specific T-cell clones. The corresponding reduced peptide of HEL(52-61) at position 53-54 neither stimulated T-cell clones nor competed with the natural peptide. These results show that, while reduced pseudopeptides might not be appropriate, ethylenic pseudopeptides may be used as probes to dissect the role of hydrogen bonding between the peptide main chain and MHC residues and also help in the design of more stable immunogenic peptides.

Animals↗
PubMed1997

Functional mapping of the orientation for TCR recognition of an H2-Kb-restricted ovalbumin peptide suggests that the beta-chain subunit can dominate the determination of peptide side chain specificity.

T cells recognize a complex of antigenic peptide bound to the class I or class II products of the MHC. Crystallographic analysis of the interaction between MHC class I-bound peptide fragments and specific TCR have recently been described and highlight the importance of the CDR3 in determining peptide specificity. The results presented here show functional data for TCR recognition of the H2-Kb class-I restricted determinant derived from OVA (SIINFEKL) that are consistent with the TCR orientation defined by these crystal structures. In addition, we also found that the beta-chain CDR3 dominates side chain specificity for the most exposed regions within this peptide. The data also suggest that this orientation and pattern of beta-chain dominance may extend to the recognition of a second H2-Kb-restricted peptide from the herpes simplex virus type 1 glycoprotein B (SSIEFARL), which shares a common alpha-chain contact with the OVA peptide. These results are discussed in terms of a common orientation for TCR-ligand interaction and the greater potential for TCR beta-chain CDR3 diversity in determining peptide side chain specificity.

Amino Acid Sequence↗
PubMed1998

A viral peptide with limited homology to a self peptide can induce clinical signs of experimental autoimmune encephalomyelitis.

Molecular mimicry has been suggested as a mode of autoreactive T cell stimulation in autoimmune diseases. Myelin basic protein (MBP) peptide 1-11 induces experimental autoimmune encephalomyelitis (EAE) in susceptible strains of mice. Here we show that a herpesvirus Saimiri (HVS) peptide, AAQRRPSRPFA, with a limited homology to MBP1-11 peptide, ASQKRPSQRHG (underlined letters showing homology), can stimulate a panel of MBP-11-specific T cell hybridomas and more importantly cause EAE in mice. We demonstrate that this is due to cross-recognition of these two peptides by TCRs. Results presented in this communication are the first demonstration that a viral peptide with homology at just 5 amino acids with a self peptide can induce clinical signs of EAE in mice. These findings have important implications in understanding the breakdown of T cell tolerance to self Ags in autoimmune diseases by means of cross-reactivity with unrelated peptides.

Animals↗
PubMed1998

Structural basis of specificity and degeneracy of T cell recognition: pluriallelic restriction of T cell responses to a peptide antigen involves both specific and promiscuous interactions between the T cell receptor, peptide, and HLA-DR.

TCR engagement of peptide-MHC class II ligands involves specific contacts between the TCR and residues on both the MHC and peptide molecules. We have used molecular modeling and assays of peptide binding and T cell function to characterize these interactions for a CD4+ Th1 cell clone, ESL4.34, which recognizes a peptide epitope of the herpes simplex type 2 virus virion protein, VP16 393-405, in the context of several HLA-DR alleles. This clone responded to VP16 393-405 in proliferation and cytotoxicity assays when presented by DRB1*0402, DRB1*1102, and DRB1*1301, which share a common amino acid sequence, ILEDE, at residues 67-71 in the alpha-helical portion of the DRbeta polypeptide, but not when presented by other DR4, DR11, and DR13 alleles that are negative for this sequence. Using a panel of APCs expressing DR4 molecules that were mutagenized in vitro at individual residues within this shared epitope and using peptide analogues with single amino acid substitutions of predicted MHC and TCR contact residues, a unit of recognition was identified dependent on DRbeta residues 67-71 and relative position 4 (P4) of the VP16 393-405 peptide. The interactions of this portion of the peptide-DR ligand with the ESL4.34 TCR support a structural model for MHC-biased recognition in some Ag-specific and alloreactive T cell responses and suggest a possible mechanism for autoreactive T cell selection in rheumatoid arthritis.

Alleles↗
PubMed1998

Coloured peptides: synthesis, properties and use in preparation of peptide sub-library kits.

Several methods were developed for the solid-phase synthesis (SPPS) of coloured peptides and peptide libraries. At first a bifunctional red compound, 4-(4-(N-ethyl-N-(3-(tert-butyloxycarbonyl)aminopropyl)amino)phenylazo)be nzoic acid (Boc-EPAB), was coupled with chloromethyl resin to obtain a new solid support suitable for SPPS using Boc chemistry. Peptides synthesized on this coloured resin had the chromophore at their C-termini. N-terminally coloured peptides were synthesized on a traditional solid support, coupled with chromophoric carboxylic acid before cleavage. A model pentapeptide, Phe-Ala-Val-Leu-Gly, and its ten derivatives were synthesized and their properties studied. It was found that the presence of chromophores decreases the water solubility of peptides. However, insertion of solubilizing tags (penta-lysine sequences or polyoxyethyl chains) into the molecule of any coloured derivative resulted in enhancement of the solubility. The RP-HPLC hydrophobicity indexes (phi0) of the coloured peptides were also determined because phi0 values are closely related to their water solubility. A coloured pentapeptide library was synthesized using the portioning-mixing method. Each component of this library contained the red azo dye (EPAB) and the penta-lysine tag. Before the last coupling step the samples were not mixed. All of the 19 sub-libraries obtained after cleavage were readily soluble in water, giving intense red solutions. The effect of chromophore (EPAB) and/or penta-lysine solubilizing tag on the biological activity was also studied. Potencies of the bovine neurotensin 8-13 fragment and its different coloured and penta-lysine derivatives were compared in isolated longitudinal muscle strips of guinea pig ileum. It was shown that the hexapeptide with penta-lysine tag had almost the same activity as the 8-13 fragment itself. The activity of the EPAB-derivative was found to be rather low. However, the presence of the solubilizing tag in the coloured hexapeptide compensated the negative effect of the chromophore.

Aminobenzoates↗
PubMed1998

Genetic evidence for a role of thioesterase domains, integrated in or associated with peptide synthetases, in non-ribosomal peptide biosynthesis in Bacillus subtilis.

Next to almost all prokaryotic operons encoding peptide synthetases, which are involved in the nonribosomal synthesis of peptide antibiotics, distinct genes have been detected that encode proteins with strong sequence similarity to type II fatty acid thioesterases of vertebrate origin. Furthermore, sequence analysis of bacterial and fungal peptide synthetases has revealed a region at the C-terminal end of modules that are responsible for adding the last amino acid to the peptide antibiotics; that region also exhibits significant similarities to thioesterases. In order to investigate the function of these putative thioesterases in non-ribosomal peptide synthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis, srfA fragments encoding the thioesterase domain of the surfactin synthetase 3 and the thioesterase-like protein SrfA-TE were deleted. This led to a 97 and 84% reduction of the in vivo surfactin production, respectively. In the double mutant, however, no surfaction production was detectable. These findings demonstrate for the first time that the C-terminal thioesterase domains and the SrfA-TE protein are directly involved in nonribosomal peptide biosynthesis.

Bacillus subtilis↗