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Screening for small peptidic affinity tags for the detection of ubiquitin and ubiquitinated proteins yielded the dodecapeptide amide DPDELRFNAIAL-NH(2) as a specific ubiquitin-interacting ligand. A peptide collection--based on crystal structures with ubiquitin-interacting proteins--was designed and confirmed by sequence comparison of ubiquitin-interacting motifs. Four independent physical detection methods demonstrated that the peptide binds to monomeric ubiquitin with an affinity of about 10 muM and with fast on and off rates. Fluorescence correlation spectroscopy with fluorescent peptides showed specific interaction with ubiquitin. Reflectometric interference spectroscopy with surface-immobilized peptides and isothermal calorimetry measurements confirmed the specific binding of ubiquitin and fast rate constants. (1)H,(15)N heteronuclear NMR localised the interaction site across the beta sheet of ubiquitin. The peptide aligns well with the ubiquitin-interacting motif and represents a lead structure for the rational design of high-affinity tags for targeting ubiquitinated protein in vitro and in vivo.

For the detection of bioanalytes, there is an ongoing search for synthetic sensors to replace enzyme-based assays which are sensitive to contaminants or suboptimal storage conditions. Lipopolysaccharide (LPS), a bacteria-borne endotoxin that may lead to life-threatening conditions such as septic shock, is one such case. Fluorescently labeled analogues of two peptide variants derived from the putative ligand-binding domain of the LPS-binding protein CD14 were developed that detect and discriminate LPS and lipids down to the submicromolar concentration range. Peptides are terminally labeled with carboxyfluorescein and tetramethylrhodamine. For one given peptide, sensitivity and specificity for the detection of LPS and discrimination from other lipids are achieved by spectral signatures that combine changes in the fluorescence resonance energy transfer (FRET) between both dyes and the total emission of tetramethylrhodamine. Alternatively, specificity is obtained by combining the FRET efficiencies of both peptide variants. In comparison to published synthetic LPS sensors, the CD14-derived sensors yield an increase in sensitivity by about 3 orders of magnitude and exhibit specificity for analytes for which the design of synthetic recognition elements is a challenging task. Moreover, one of the sensors enabled the detection of LPS in the presence of up to 50% fetal calf serum, thereby demonstrating the feasibility of this peptide-based approach for clinically relevant samples.

Inside the cell, proteases act in concert in the degradation of proteins and peptides. In order to understand the significance of an individual proteolytic activity within an ensemble of proteases, protocols and probes are required that enable a quantitative determination of the contribution of a protease to the break-down of a given substrate. Here we present a fluorescence resonance energy transfer-based probe and protocols for a quantitative determination of proteolytic activities inside the endolysosomal compartment. A peptide substrate that is readily cleaved by different cathepsins is flanked by fluorescein and tetramethylrhodamine-labeled lysine residues. Efficient endolysosomal targeting of the substrate is achieved by N-terminal elongation with the cell-penetrating peptide nona-arginine. The proteasome inhibitor lactacystin has a small, but significant effect on the break-down of the substrate, thus demonstrating that only a minor fraction of the peptide reaches the cytoplasm in its intact form. Nona-arginine therefore constitutes a highly efficient low-molecular-weight moiety for targeting the endolysosomal compartment.

From the culture broth of the mold Trichoderma viride, strain 63 C-I, the polypeptide antibiotic suzukacillin (SZ) was isolated. A peptide mixture named SZ-A was obtained by crystallization from crude SZ. Individual peptides from SZ-A were isolated by semipreparative HPLC and sequences were determined by HPLC-ESI-MS. The data confirm a general sequence of SZ-A published previously and in addition establish the individual sequences of 15 acetylated eicosa peptides with C-terminal alcohols. The major peptide SZ-A4 (21% of all peptides) shows the sequence:Ac-Aib-Ala-Aib-Ala-Aib-Ala(6)-Gln-Aib-Lx(9)-Aib-Gly-Aib(12)-Aib-Pro-Vx(15)-Aib-Vx(17)-Gln-Gln-Fol. Amino acid exchanges of the peptaibol are located in position 6 (Ala/Aib), 9 (Vx/Lx), 12 (Aib/Lx), 17 (Aib/Vx) and possibly at position15 (Val/Iva) (uncommon abbreviations: Aib (alpha-aminoisobutyric acid); Iva (D-isovaline); Lx (L-leucine or L-isoleucine); Vx (L-valine or D-isovaline); Fol (L-phenylalaninol))., (Copyright (c) 2005 European Peptide Society and John Wiley & Sons, Ltd.)

G protein-coupled receptor kinases (GRKs) are regulatory enzymes involved in the modulation of seven-transmembrane-helix receptors. In order to develop specific inhibitors for these kinases, we synthesized and investigated peptide inhibitors derived from the sequence of the first intracellular loop of the beta2-adrenergic receptor. Introduction of changes in the sequence and truncation of N- and C-terminal amino acids increased the inhibitory potency by a factor of 40. These inhibitors not only inhibited the prototypical GRK2 but also GRK3 and GRK5. In contrast there was no inhibition of protein kinase C and protein kinase A even at the highest concentration tested. The peptide with the sequence AKFERLQTVTNYFITSE inhibited GRK2 with an IC50 of 0.6 microM, GRK3 with 2.6 microM and GRK5 with 1.6 microM. The peptide inhibitors were non-competitive for receptor and ATP. These findings demonstrate that specific peptides can inhibit GRKs in the submicromolar range and suggest that a further decrease in size is possible without losing the inhibitory potency.

Peptide aldehydes are of interest due to their inhibitory properties toward numerous classes of proteolytic enzymes such as caspases or the proteasome. A novel access to peptide aldehydes is described using a combination of solid phase peptide synthesis with polymer-assisted solution phase synthesis based on the oxidation of peptide alcohols with a mild and selective polymer-bound IBX derivative. The oxidation is followed by selective purification via scavenging the peptide aldehyde in a capture-release procedure using threonine attached to an aminomethyl resin. Peptide aldehydes are obtained in excellent purity and satisfying yield. The optical integrity of the C-terminal residue is conserved in a high degree. The procedures are compatible with the use of common side-chain protecting groups. The potential for using the method in parallel approaches is very advantageous. A small collection of new and known peptide aldehydes has been tested for inhibitory activity against caspases 1 and 3., (Copyright (c) 2004 European Peptide Society and John Wiley & Sons, Ltd.)

A novel tool for polymer-assisted solution phase (PASP) esterification of amino acid and peptide derivatives has been developed. When treated with carboxylic acids, polymer-bound alkyltriazenes react with a loss of nitrogen and transfer of the alkyl moiety to the carboxylate anion to form the corresponding alkyl esters. There are no limitations with regard to either the protecting groups or the nature of the amino acid. Furthermore no racemization occurs at the chiral centers of the amino acids as demonstrated by chiral GC-MS analyses. Alkyltriazene-resins were also applied successfully to the esterification of peptide acids and other peptidic structures, such as tripalmitoyl-S-glyceryl-cysteine (Pam3Cys). The triazene-mediated esterification reaction is exceptionally mild, and there is no need for prior activation of the carboxy groups. This method is therefore particularly suitable for the alkylation of complex peptidomimetic structures prone to racemization and for acid-sensitive structures.

Most autoimmune diseases are associated with certain MHC class II haplotypes. Autoantigen-based specific immune therapy can lead either to beneficial or, in the context of inflammatory conditions, detrimental outcomes. Therefore, we designed a platform of peptides by combinatorial chemistry selected in a nonbiased Ag-independent approach for strong binding to the rat MHC class II isotype RT1.D(n) allelic product of the RT1(n) haplotype that is presenting autoantigen in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in LEW.1N rats. Peptide p17 (Ac-FWFLDNAPL-NH(2)) was capable of suppressing the induction of and also ameliorated established experimental autoimmune encephalomyelitis. MHC class II isotype and allele specificity of the therapeutic principle were demonstrated in myelin basic protein-induced experimental autoimmune encephalomyelitis in LEW rats bearing the RT1(l) haplotype. A general immunosuppressive effect of the treatment was excluded by allogeneic heart transplantation studies. In vitro studies demonstrated the blocking effect of p17 on autoantigenic T cell responses. We thus demonstrate a rational design of strong MHC class II-binding peptides with absolute isotype and allele specificity able to compete for autoantigenic sequences presented on disease-associated MHC class II molecules.

Tissue transglutaminase (TG2) modifies proteins and peptides by transamidation or deamidation of specific glutamine residues. TG2 also has a central role in the pathogenesis of celiac disease. The enzyme is both the target of disease-specific autoantibodies and generates deamidated gliadin peptides recognized by intestinal T cells from patients. Incubation of TG2 with gliadin peptides also results in the formation of covalent TG2-peptide complexes. Here we report the characterization of complexes between TG2 and two immunodominant gliadin peptides. Two types of covalent complexes were found; the peptides are either linked via a thioester bond to the active site cysteine of TG2 or via isopeptide bonds to particular lysine residues of the enzyme. We quantified the number of gliadin peptides bound to TG2 under different conditions. After 30 min of incubation of TG2 at 1 microm with an equimolar ratio of peptides to TG2, approximately equal amounts of peptides were bound by thioester and isopeptide linkage. At higher peptide to TG2 ratios, more than one peptide was linked to TG2, and isopeptide bond formation dominated. The lysine residues in TG2 that act as acyl acceptors were identified by matrix assisted laser desorption ionization and nanoelectrospray mass spectrometry and tandem mass spectrometry analysis of proteolytic digests of the TG2-peptide complexes. At a high molar excess of gliadin peptides to TG2 altogether six lysine residues of TG2 were found to participate in isopeptide bond formation. The results are relevant to the understanding of how antibodies to TG2 are formed in celiac disease.

The interaction of cyclohexapeptides c(X(1)(1)K(2)X(2)(3)K(4)X(3)(5)K(6)) in water with hydrolysed silicon surfaces were studied by attenuated total reflection Fourier transform infrared (ATR FTIR) spectroscopy and by force field calculations. The band sequences (1800-1500 cm(-1)) for dissolved and adsorbed cyclohexapeptides were recorded and compared with those obtained after flushing with distilled water in order to eliminate the background signal of the peptides in solution. Band analyses and principal component analyses were carried out for the characteristic peptide vibrations in order to evaluate the spectra. In addition, force field calculations were performed to study the binding energies to the surface and to illustrate the possible structures of the cyclohexapeptides. The positively charged lysine side chains of the cyclohexapeptides interact with the OH groups of the surface, as indicated by band shifts. This also was verified by the force field calculations. The bonding stability increases with the number of interacting sites (lysine side chains and other peptide residues) to the surface. These sites are determined by structure and polarity of the cyclohexapeptides.

Capillary liquid chromatography based on particulate and monolithic stationary phases was used to screen complex peptide libraries by fast gradient elution coupled on-line to electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS). A slightly modified commercial electrospray interface consisting of a fused-silica transfer capillary and low dead volume stainless steel union at which the electrospray voltage was grounded enabled the effluent of all the capillary columns to be directly sprayed into the mass spectrometer. Stable electrospray conditions were generated over a wide range of mobile phase compositions, alleviating the need for a tapered end of the spray capillary, pneumatic assistance or preheated nebulizer gas. Since the identification of complex samples containing numerous isobaric substances is facilitated by chromatographic separation prior to mass spectrometry, stationary phase materials have been employed which offer a fast, efficient elution and, due to the complexity of samples, a high loading capacity. Silica-based monolithic capillary columns combine these three characteristics in a unique manner due to a tailored adjustment of both macro- and mesopore sizes in the highly porous silica structure. As we demonstrate by a comparative study of the silica-based monolithic and packed capillaries for LC/MS analysis of complex peptide libraries, silica monoliths show superior performance over packed beds of small-diameter particles with respect to analysis time and separation efficiency. Libraries with more than 1000 different peptides could be screened in less than 20 min., (Copyright 2003 John Wiley & Sons, Ltd.)

Label-free optical detection methods are of particular value for the investigation of biomolecular interactions. A label-free method based on reflectometric interference spectroscopy is described which enables both the on-line monitoring of solid phase peptide syntheses and subsequently the determination of antibody binding to these peptides without cleavage from the support. The method uses SiO(2) transducers that were modified with diaminopolyethylene glycol. The stepwise coupling of different amino acids to the transducer surface was investigated and the complete monitoring of the synthesis of a viral epitope was performed. The success of the synthesis was proven via binding of a specific monoclonal antibody to the transducer-bound product. In order to demonstrate specificity the binding was inhibited with the same peptide epitope added in solution. The approach is attractive especially in the field of high throughput screening since both the synthesis and the interaction with the biological receptor can be monitored using the same technique., ((c)2001 Elsevier Science.)

Peptidomimetics of the major histocompatibility complex (MHC) class I-restricted ovalbumin-derived T cell epitope SIINFEKL were generated by replacing parts of the peptide backbone by a poly-N-acylated amine (PAA) backbone with aromatic, heteroaromatic, and pseudoaromatic side chains that branch off of the main chain at the amine nitrogen. The structure of the PAAs was designed to position this side chain in the central epitope anchor pocket of the MHC molecule. A number of biologically active PAAs were found that induced cytolysis by the mouse cytotoxic T cell clone 4G3. Competition experiments with independent peptides that are known to bind to the restricting MHC molecule H-2K(b) suggest that the PAAs are bound by the MHC molecules at the same site as conventional peptide epitopes. The PAAs were active also in vivo and induced primary cytotoxic T cell responses in mice.

Among the numerous forms of chemical degradation of peptides or proteins, deamidation is one of the alterations observed most frequently. In this irreversible reaction, a glutamine or an asparagine side chain is hydrolyzed to glutamic acid or aspartic acid, respectively (conversion of NH2 to OH). Besides its influence in the deterioration of biotechnological and food products, deamidation represents a defined posttranslational modification reaction with respect to proteomics. Here mass spectrometric techniques play a leading role in determining posttranslational modifications. However, not all mass spectrometers are able to resolve signal differences of 0.0193 Da (mass difference of 12CO vs 13CNH) for singly charged molecules, the mass difference between the first isotopic signal of an asparagine/glutamine-containing peptide and the monoisotopic signal of the corresponding partially deamidated aspartate/glutamate derivative. To detect partial deamidation within peptides, advantage has been taken of the ability of Fourier transform ion cyclotron resonance mass spectrometry to perform very high mass resolution. In this work, we investigated up to triply charged ions produced by electrospray ionization using direct infusion. Although the special heterodyne detection mode enables higher mass resolution than the routinely used broadband detection, often only a small mass window can be investigated. Using broadband detection, we were able to resolve ions with a difference of m/z 0.0064 to detect partially deamidated peptides formed either enzymatically or under acidic and basic conditions.

Polypeptide constructs containing up to four copies of the T cell epitope 306-318 of influenza virus haemagglutinin have been synthesized on solid phase. Between the copies, a non-natural PEG-based spacer amino acid has been introduced. The oligomeric epitopes were analysed by RP-HPLC and ES-MS. The arrangement of the epitopes within the peptide constructs was either linear or comb-like. The proliferative response in a T helper cell assay induced by these oligomerized epitopes has been tested, showing that the linearly arranged epitopes are more effective than the comb-like oligomers.

Model lipid layers are very promising in investigating the complex network of recognition, transport and signaling processes at membranes. We have developed a novel and generic approach to create supported lipid membranes tethered by metal-affinity binding. By self-assembly we have generated various interfaces that display histidine sequences (6xHis) via polymer spacers. These histidine-functionalized interfaces are designed to allow specific docking and fusion of vesicles containing metal-chelating lipids. By means of surface plasmon resonance and atomic force microscopy we analyzed the formation and subsequently the structure of these solid-supported membranes. Although the affinity constant of single ligand-receptor pairs is only in the micromolar range, very stable immobilization of these membranes was observed. This behavior can be explained by multivalent interactions resembling many features of cell adhesion. The process is highly specific, because vesicle docking and bilayer formation are strictly dependent on the presence of metal-affinity ligand-receptor pairs. The surface accessibility and geometry of these tethered membranes were probed by binding of histidine-tagged polypeptides. The supported membranes show adsorption kinetics and values similar to planar supported monolayers. Using various combinations of metal-chelating and histidine-tagged lipids or thiols these metal-affinity-tethered membranes should make a great impact on probing and eventually understanding the dynamic dialog of reconstituted membrane proteins.

Specificities of three mouse major histocompatibility complex (MHC) class I molecules, Kb, Db, and Ld, were analyzed by positional scanning using combinatorial peptide libraries. The result of the analysis was used to create a scoring program to predict MHC-binding peptides in proteins. The capacity of the scoring was then challenged with a number of peptides by comparing the prediction with the experimental binding. The score and the experimental binding exhibited a linear correlation but with substantial deviations of data points. Statistically, for approximately 80% of randomly chosen peptides, MHC-binding capacity could be predicted within one log concentration of peptides for a half-maximal binding. Known cytotoxic T-lymphocyte epitope peptides could be predicted, with a few exceptions. In addition, frequent findings of MHC-binding peptides with incomplete or no anchor amino acid(s) suggested a substantial bias introduced by natural antigen processing in peptide selection by MHC class I molecules.

Staphylococcus equorum WS 2733 was found to produce a substance exhibiting a bacteriostatic effect on a variety of gram-positive bacteria. The metabolite was purified to homogeneity by ammonium sulfate precipitation and semipreparative reversed-phase high-performance liquid chromatography. Electrospray mass spectrometry confirmed the high purity of the compound and revealed a molecular mass of 1,143 Da. By two-dimensional nuclear magnetic resonance spectroscopy the substance was identified as micrococcin P(1) which is a macrocyclic peptide antibiotic that has not yet been reported for the genus Staphylococcus. A total of 95 out of 95 Listeria strains and 130 out of 135 other gram-positive bacteria were inhibited by this substance, while none of 37 gram-negative bacteria were affected. The antilisterial potential of this food-grade strain as a protective starter culture was evaluated by its in situ application in cheese-ripening experiments under laboratory conditions. A remarkable growth reduction of Listeria monocytogenes could be achieved compared to control cheese ripened with a nonbacteriocinogenic type strain of Staphylococcus equorum. In order to prove that inhibition was due to micrococcin P(1), a micrococcin-deficient mutant was constructed which did not inhibit L. monocytogenes in cheese-ripening experiments.

Bacterial-derived antimicrobial polypeptides enjoy a large degree of structural and chemical diversity. Two well-studied examples of such polypeptides are the lanthionine-containing lantibiotics produced by a variety of Gram-positive bacteria, and their Gram-negative counterparts, the microcins. Both groups are produced as gene-encoded precursor peptides and undergo post-translational modification to generate the active moieties. Structure elucidation of novel lantibiotics and microcins has recently uncovered further novel structural and chemical features and, combined with the generation of analogue peptides by genetic manipulation, new insights into structure-function relationships have been gained. Furthermore, study of the mode of action of the lantibiotics nisin and mersacidin has revealed their use of a 'docking molecule' in the target cell to facilitate their biological activities. Meanwhile, in vitro studies with microcin B17 have helped to uncover the molecular mechanisms by which post-translational modification results in the formation of heterocyclic oxazole and thiazole rings. From a practical standpoint, both groups of polypeptides represent new lead structures for future development of antimicrobial agents, whilst the identification of the 'docking molecules' represents a step forward in the search for novel targets for future antibiosis.

Serum amyloid A (SAA) is a major acute-phase protein whose biochemical functions remain largely obscure. Human rheumatic synovial fluids were screened by high performance liquid chromatography mass spectrometry for SAA-derived peptides, specifically the sequence AGLPEKY (SAA(98-104)) which was previously shown to modulate various leukocyte functions. Two such fluids were found to contain a truncated version of SAA(98-104). Synthetic SAA(98-104) and several of its analogs were shown capable of binding isolated human CD(4)(+) T-lymphocytes and stimulating them to produce interferon-gamma. Given the high acute-phase serum level of SAA and its massive proteolysis by inflammatory related enzymes, SAA-derived peptides may be involved in host defense mechanisms.

An N-hydroxylated peptide bond was found to be cleaved faster by an endopeptidase than the corresponding peptide bond. This preferred enzymatic cleavage was detected during proteolytic studies of the N-hydroxy peptide SIINFpsi[CO-N(OH)]GKL in the presence of the serine protease alpha-chymotrypsin in comparison with the natural SIINFEKL epitope and related analogs. For the first time, the replacement of the peptide bond by another motif afforded an oligomer which is degraded faster than the natural peptide. The N-hydroxy peptide is also more sensitive to the enzymatic degradation than the Gly-containing analog SIINFGKL. A tentative explanation for the unexpected higher cleavage rate of the CO-N(OH) bond is given on the basis of the N-OH intramolecular H-bonding capacity as indicated by NMR experiments. This property of the hydroxamate group may be of particular advantage for the introduction of a specific cleavage site within peptidomimetics or in prodrugs.

The tremendous progress in the field of basic immunology and immunochemistry made in the last decade has significantly advanced our understanding of antigen processing and presentation by MHC class I and II proteins. In this review different techniques to study peptide interaction with MHC class II molecules are summarized and their impact on the elucidation of quantitative parameters, like affinities or kinetic data, is discussed. A recently introduced method based on synthetic combinatorial peptide libraries allows to quantify the binding contribution of each amino acid residue in a class II ligand and is presented in more detail. As this knowledge is fundamental for current investigations in modern medicine, e.g. for novel immune system based therapy concepts, further aspects like the design of new high affinity MHC class II ligands and the prediction of peptide antigens are discussed., (Copyright 1999 Academic Press.)

Novel 5-ring heterocyclic building blocks are synthesized. These can be incorporated into analogs of peptide antibiotics such as microcin B17, which is a potent DNA-gyrase inhibitor that exhibits eight thiazole and oxazole moieties. In particular, the syntheses of imidazole and bisoxazole amino acids as novel peptidomimetics are reported, this includes a new procedure for the oxidative conversion of the intermediates oxazoline, imidazoline as well as oxazole-oxazoline into the corresponding heteroaromatic compounds. A mixture of 1,8-diazabicyclo-[5.4.0.]-undec-7-ene carbon tetrachloride/acetonitrile and pyridine proved to be a very effective and mild agent.

2-(Pyrrolidinyl)thiazole-4-carboxylic acid 5d, 2-(1-aminoalkyl)thiazole-4-carboxamides and hydrazides 8, 10 have been synthesized using alanine, valine, and proline as educts. In addition oxazole amino acids derived from leucine 20a and alanine 20b and some peptides 13, 14, 16 containing the 5-ring heterocyclic backbone modifications have been prepared. The thiazole and oxazole containing amino acids and peptides showed moderate antibacterial activity in vitro against various Gram-positive (Staphylococcus aureus, Bacillus cereus, etc.) and Gram-negative (Escherichia coli, Protens vulgaris, etc.) bacteria, fungi (Candida albicans), and yeast (Saccharomyces cerevisae, etc.).

Class II MHC molecules are known to exist in conformational variants. "Floppy" and "compact" forms of murine MHC molecules, for example, are discriminated by their migration behavior on SDS/PAGE and represent empty and ligand-loaded forms. Here we show that formation of distinctly faster-migrating ligand complexes (F-forms) rather than the normal compact (C-) forms of HLA-DR1 or -DR4 results from extensions of minimal peptide epitopes (such as HA306-318 or IC106-120) by approximately 10 amino acids at either the N or the C terminus. Two similar but distinct F-forms (FI and FII) were detected, depending on the site of the extension. Both F-forms were characterized by increased surface hydrophobicity and reduced SDS-stability. Native gel separations and size exclusion chromatography indicated that the F-forms had increased hydrodynamic radii compared with the C-form and an apparent size similar to that of empty MHC molecules. The regions on the ligand overhangs responsible for the effect began at a distance of approximately 5 amino acids on either side of the epitopes, comprised 4-8 amino acids (i.e., a total overhang of 9-14), and did not have a particular sequence preference. The possible functional significance of these forms is discussed.

The determination of the primary structure of peptides and proteins is routine in many laboratories; however, many of the obtained sequences are incomplete or can be misinterpreted when the samples contain unusual amino acids. Here we report the development of an automated peptide sequenator coupled to an electrospray-ionization (ESI) mass spectrometer (MS) that, in conjunction with minor modifications to the sequencing conditions and, in some cases, prior derivatization of amino acids, allows the detection of the phenylthiohydantoin (PTH) derivatives of a number of unusual amino acids. Using the coupled sequenator-ESI-MS system we were able to determine the complete sequence of the lantibiotic gallidermin, a partial sequence of the calcium-dependent peptide antibiotic CDA2 as well as the pool sequence of a mixture of synthetic peptides containing nonproteinogenic amino acids. In addition to the 20 proteinogenic amino acids, the procedure was able to detect PTH derivatives of hydroxyphenylglycine, 2,3-didehydroasparagine, 3-methylglutamic acid, oxytryptophan, ornithine, N-methylglycine, dihydroxyphenylalanine, and alpha-aminoisobutyric acid. Similarly, after a simple derivatization procedure, we were also able to correctly identify educts of 2,3-didehydroalanine, 2,3-didehydrobutyrine, lanthionine, and 3-methyllanthionine.

Positively charged antimicrobial peptides with membrane-damaging activity are produced by animals and humans as components of their innate immunity against bacterial infections and also by many bacteria to inhibit competing microorganisms. Staphylococcus aureus and Staphylococcus xylosus, which tolerate high concentrations of several antimicrobial peptides, were mutagenized to identify genes responsible for this insensitivity. Several mutants with increased sensitivity were obtained, which exhibited an altered structure of teichoic acids, major components of the Gram-positive cell wall. The mutant teichoic acids lacked D-alanine, as a result of which the cells carried an increased negative surface charge. The mutant cells bound fewer anionic, but more positively charged proteins. They were sensitive to human defensin HNP1-3, animal-derived protegrins, tachyplesins, and magainin II, and to the bacteria-derived peptides gallidermin and nisin. The mutated genes shared sequence similarity with the dlt genes involved in the transfer of D-alanine into teichoic acids from other Gram-positive bacteria. Wild-type strains bearing additional copies of the dlt operon produced teichoic acids with higher amounts of D-alanine esters, bound cationic proteins less effectively and were less sensitive to antimicrobial peptides. We propose a role of the D-alanine-esterified teichoic acids which occur in many pathogenic bacteria in the protection against human and animal defense systems.

Epicidin 280 is a novel type A lantibiotic produced by Staphylococcus epidermidis BN 280. During C18 reverse-phase high-performance liquid chromatography two epicidin 280 peaks were obtained; the two compounds had molecular masses of 3,133 +/- 1.5 and 3,136 +/- 1.5 Da, comparable antibiotic activities, and identical amino acid compositions. Amino acid sequence analysis revealed that epicidin 280 exhibits 75% similarity to Pep5. The strains that produce epicidin 280 and Pep5 exhibit cross-immunity, indicating that the immunity peptides cross-function in antagonization of both lantibiotics. The complete epicidin 280 gene cluster was cloned and was found to comprise at least five open reading frames (eciI, eciA, eciP, eciB, and eciC, in that order). The proteins encoded by these open reading frames exhibit significant sequence similarity to the biosynthetic proteins of the Pep5 operon of Staphylococcus epidermidis 5. A gene for an ABC transporter, which is present in the Pep5 gene cluster but is necessary only for high yields (G. Bierbaum, M. Reis, C. Szekat, and H.-G. Sahl, Appl. Environ. Microbiol. 60:4332-4338, 1994), was not detected. Instead, upstream of the immunity gene eciI we found an open reading frame, eciO, which could code for a novel lantibiotic modification enzyme involved in reduction of an N-terminally located oxopropionyl residue. Epicidin 280 produced by the heterologous host Staphylococcus carnosus TM 300 after introduction of eciIAPBC (i.e., no eciO was present) behaved homogeneously during reverse-phase chromatography.

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease with a predominantly non-hereditary etiology that results in a destruction of pancreatic beta cells by autoaggressive T lymphocytes. Neither the mechanism of initial stimulation of these T cells nor the nature of the environmental factors implicated in the disease have so far been identified. However, both issues are taken into account by the hypothesis of initial T cell activation by viral or bacterial mimicry peptides with sequence similarities to pancreatic self antigens. We determined sequential epitope motifs to search for mimicry peptides stimulating T cell lines specific for two epitopes derived from the IDDM autoantigen 65-kDa glutamic acid decarboxylase (GAD65). These were GAD65 (88-99), presented by HLA-DRB1*0101, and GAD65 (248-257), presented by HLA-DRB5*0101. T cell stimulation by peptides with substitutions in HLA anchor or T cell contact positions was analyzed to establish degenerate epitope motifs for database searching. Out of 28 tested candidate mimicry peptides derived from bacterial, viral and human proteins, 3 stimulated T cell lines and a T cell clone specific for epitope GAD65 (248-257). Our results demonstrate that mono- and polyclonal GAD65-specific T cells from IDDM patients can be stimulated by viral and bacterial peptides with little apparent sequence homology with autoantigenic epitopes. Moreover, in a synopsis with related published studies, our findings suggest that simple degenerate search motifs comprising principal T cell contacts plus HLA class II binding motifs may suffice to identify most mimicry peptides.

Siderophores are low molecular weight iron chelators, produced by virtually all bacteria, fungi and some plants. They serve to deliver the essential element iron, barely soluble under aerobic conditions, into microbial cells. Siderophores are therefore important secondary metabolites which are very often based on amino acids and their derivatives. Biosynthesis, transport, regulation and chemical synthesis of natural siderophores and their analogues is of considerable interest for the protein and peptide chemist. This review gives an overview of the structural classes of peptidic siderophores, along with data on their biosynthesis. On a number of representative examples, strategies and schemes of their chemical synthesis are described.

An investigation of the stability of the Dde protecting group for amines, used in solid-phase peptide synthesis, shows that an unprotected epsilon-NH2 group of lysine can acquire the Dde protection from another epsilon-NH2 group or from an alpha-NH2 group. An unprotected alpha-NH2, however, cannot remove Dde from an epsilon-NH2 function. This migration takes place during Fmoc removal from the epsilon-NH2 with piperidine and/or during the subsequent washing steps. The Dde migration is also possible in neat dimethylformamide by a direct nucleophilic attack of the free epsilon-NH2 group. Addition of piperidine to the reaction medium accelerates the side reaction, probably because of the formation of an unstable piperidine-Dde adduct. Dde migration can be prevented if the 9-fluorenylmethyloxycarbonyl is cleaved with 1,8-diazabicyclo[5.4.0]undec-7-ene for a short reaction time (2%, 3 x 3 min). Finally, this rearrangement is shown to occur both as an intra- and intermolecular reaction between peptides on the same resin bead.

Integrin receptor-targeted transfer of oligodeoxynucleotides (ODNs) by small synthetic peptides was used for improving delivery of antisense ODNs. An 18-mer phosphodiester bond containing ODN complementary to c-myb-encoded mRNA was complexed with several transfer peptides, containing as their parts two modules: (a) an RGD-motif as targeting sequence for integrin receptor and (b) nucleocapsid protein (NCp) 7 of HIV-1 or NCp7-derived peptides for complex formation with the ODNs. The amount of antisense ODN required for the inhibition of proliferation of human myeloid cell line HL-60 in vitro can be more than 50-fold reduced by complexing with transfer peptides. The efficiency of antisense delivery was increased by multimerization of the targeting sequence for the integrin receptor. Competition with integrin peptide abolished the effect, indicating that the integrin receptor is indeed responsible for the reaction.

Boc-resin-bound alpha-hydroxy-beta-amino-aldehydes are accessible starting from N-terminally bound amino acid esters by using Dondoni's C1-homologation reaction sequence. The conversion of these synthons to two different peptide mimetics--2-hydroxy-1,3-ethyl-diamines and gamma-hydroxy-delta-amino-vinyl sulfones--has been investigated. The successful transfer of the complex alpha-amino acid homologation reaction sequence into solid-phase chemistry demonstrates the potentials of the Boc-resin for synthesis of peptidomimetics.

Polymorphism of the genes coding for the human histocompatibility leukocyte antigen class II DR and DQ molecules makes the single largest genetic contribution to the risk of developing insulin-dependent diabetes mellitus (IDDM) and can be associated with highly elevated as well as decreased disease frequency. The mechanism of IDDM risk modification by HLA polymorphism is likely to involve differential presentation of autoantigenic peptides by HLA class II proteins. We have generated T cell lines (TCL) with specificity for the IDDM autoantigen 65 kDa glutamic acid decarboxylase (GAD65) from lymphocytes of two patients carrying HLA class II alleles associated with distinct risk of IDDM (DRB1*0101/0401 and 1302/1501). For both patients, TCL generated at various time points all recognized single epitopes mapped as GAD 88-99 and 248-257, respectively. These epitopes are presented by the DRB1*0101 and DRB5*0101, HLA class II molecules associated with a moderately elevated risk of IDDM, or carried in a strongly protective haplotype, respectively. In an HLA/peptide binding assay, epitope GAD 248-257 was shown to possess high affinity for DRB5*0101. This epitope overlaps with a central GAD peptide binding to the high risk allele DQB1*0302 and containing a Coxsackie P2C-identical mimicry sequence, raising the possibility of competition of DRB5*0101 and DQB1*0302 for binding of a central GAD65 fragment.

The three-dimensional solution structure of the lantibiotic actagardine was determined at high resolution by homonuclear and heteronuclear two-dimensional and three-dimensional NMR spectroscopy in [2H3]acetonitrile/H2O (7:3). 133 non-trivial distance and 22 torsional-angle constraints were derived from the NMR data. An ensemble of 15 low-energy structures was calculated by distance geometry followed by an iterative relaxation-matrix-refinement procedure. The rmsd of the backbone coordinates with respect to the average structure was 17 pm. The two distinct thioether ring systems 1-6 and 7-19 were even better defined, with backbone rmsd of 10 pm and 14 pm, respectively. Actagardine shows a rigid compact globular shape based on the constraining bridging pattern, which is composed of an N-terminal lanthionine ring from residues 1-6 and three intertwined C-terminal methyllanthionine rings comprising residues 7-12, 9-17 and 14-19. In addition, this C-terminal ring system is stabilised by a short antiparallel beta sheet. A feature of the actagardine structure is the presence of two putative binding pockets. A pocket is generated by the covalent constraints of the C-terminal thioether ring system. The rim of this pocket is built up by a loop structure comprising residues 12-19, whose backbone amide protons are all directed to the centre of the pocket. The second pocket is formed by an L-shaped orientation of the N-terminal and C-terminal thioether ring systems. The only two hydrophilic amino acid residues of actagardine, Glu11 and Ser2, are directed to this pocket. A region of high sequence similarity with the related lantibiotic mersacidin is located exactly at the position of the second pocket (residues 3-12). This suggests that the second pocket is responsible for the antibiotic mode of action of actagardine and mersacidin as inhibitors of the murein biosynthesis of gram-positive bacteria.

To generate conventional or monoclonal antibodies for the serological detection of drugs, antibiotics, toxins and other low molecular mass substances, a suitable and effective adjuvant is needed. Lipopeptides derived from a major component of the bacterial cell wall constitute potent nontoxic and nonpyrogenic immunoadjuvants when mixed with conventional antigens. Here we demonstrate that the synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-(R)-cysteinyl- serine (P3CS) coupled to a Th-cell epitope (P3CS-Th) can efficiently enhance the specific immune response against low molecular weight compounds in different species. In the presence of the synthetic lipopeptide P3CS-Th, the peptides which are per se non-immunogenic stimulated a specific humoral immune response in mice after intraperitoneal application. Mixtures containing adjuvants without the Th sequence showed no significant antibody induction. A marked enhancement of the humoral immune response was obtained with the low molecular mass antigens Iturin AL, Herbicolin A and Microcystin (MLR) coupled to poly-l-lysin (MLR-PLL), in rabbits and in chickens. Lipopeptide-Th cell epitope conjugates also constituted adjuvants for the in vitro immunization of either human mononuclear cells or mouse B-cells with MLR-PLL; after fusion of the immunized cultures with the heteromyeloma cell lines CB-F7 or the mouse myeloma cell line SP 2/0, respectively, we observed a significantly increased yield of antibody secreting hybridomas.

Lantibiotics are lanthionine-containing antibiotic peptides which are synthesized from ribosomal prepeptides by post-translational modification. In order to elucidate the function of a conserved motif in the N-terminal leader sequence of lantibiotic prepeptides, three amino acids were exchanged in the leader peptide sequence of the lantibiotic Pep5. Exchanging Phe-19 for Ser and Glu-16 for Lys in the FDLEI-motif, reduced Pep5 production to 35 and 38% of the control whereas, after exchanging Asp-6 for Ser and Glu-16 for Lys in the FDLEI-motif, reduced Pep5 production to 35 and 38% of the control whereas, after exchanging ASp-6 for Lys, the production was decreased only to 82%. Proteolytic fragments of Pep5 or incorrectly modified Pep5 molecules, indicative of incorrect modifications, were not found in the culture supernatant. Thus, in contrast to the biosynthesis of the lantibiotic nisin, the FDLEI-motif is not essential for biosynthesis of Pep5 and has no influence on correct ring formation or processing, but seems to be important for optimal biosynthesis rates.

A new approach to T cell epitope determination is presented. Critical amino acids for the induction of cytotoxic T cell responses were identified using synthetic peptide libraries with single defined sequence positions combined with randomized sequence positions. Sequences for potential T cell epitopes were deduced from scan profiles using combinations of the active amino acids. Highly potent epitopes for cytotoxic T lymphocytes were obtained. Epitopes defined by this approach are, as shown in this communication, not necessarily the natural epitopes and, therefore, were named synthetic epitopes. They can serve effectively for the development of vaccines or for the determination of T cell receptor antagonists.

Rheumatoid arthritis (RA), one of the most common autoimmune disorders, is believed to be mediated via. T lymphocytes and genetic studies have shown that it is strongly associated with HLA-DR4. The DR4 subtypes DR4Dw4, DR4Dw14 and DR4Dw15 represent increased risk factors for RA, whereas DR4Dw10 is not associated with the disorder. Our study determines and compares the natural ligand motifs of these MHC class II molecules and identifies 60 natural ligands. At relative position 4 (P4), only the RA-associated DR4 molecules allow, or even prefer, negatively charged amino acids, but do not allow those which are positively charged (Arg, Lys). In the case of DR4Dw10 the preference for these amino acids is reversed. The results predict features of the putative RA-inducing peptide(s). A remarkable specificity, almost exclusively for negative charges (Asp, Glu), is found at P9 of the DR4Dw15 motif. This specificity can be ascribed to amino acid beta57 of the DR beta chain, and gives an important insight into the beta57-association of another autoimmune disease, insulin-dependent diabetes mellitus type I.

During the last decade, Nuclear Magnetic Resonance has played an important role in the unravelling of the primary and tertiary structures of lantibiotics. A short overview of these studies, together with typical spatial structures obtained, is presented.

Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases be a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibiotics were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.

Pep5 is a 34-amino-acid antimicrobial peptide, produced by Staphylococcus epidermidis 5, that contains the thioether amino acids lanthionine and methyllanthionine, which form three intramolecular ring structures. In addition, two didehydrobutyrines are present in the central part of the lantibiotic and an oxobutyryl residue is located at the N terminus. All rare amino acids are introduced by posttranslational modifications of a ribosomally made precursor peptide. To elucidate the function of the modified residues for the antimicrobial action of Pep5, mutant peptides, in which single modified residues had been eliminated, were produced by site-directed mutagenesis. All of these peptides showed a reduced antimicrobial activity. In addition, those peptides from which the ring structures had been deleted became susceptible to proteolytic digest. This demonstrates that the ring structures serve as stabilizers of conformations essential for activity, e.g., amphiphilicity, as well as for protecting Pep5 against proteases of the producing strains. In addition, residues that could serve as precursors of new modified amino acids in lantibiotics were introduced into the Pep5 precursor peptide. This way, a novel methyllanthionine and a didehydroalanine were inserted into the flexible central part of Pep5, demonstrating that biosynthesis of modified amino acids is feasible by protein engineering and use of the lantibiotic modification system.

To evaluate the ability of the lipotripeptide P3CSS to increase peptide-specific immune responses in vivo, we immunized mice from different inbred strains (BALB/c, C3H/HeJ, C57BL/6) with the 22-mer lipopeptide conjugates P3CSS-[RT-(522-543)] and P3CSS-[RT-(528-549)] of HIV-1 reverse transcriptase (RT) which included an immunodominant Th epitope [i.e. RT-(528-543)] characterized previously. Analysis of T and B cell responses to these lipopeptide conjugates indicated that specific Th responses could be readily induced in vivo. The peptide segments could also efficiently prime mice for secondary recognition of native RT. The use of shorter peptides permitted a delineation of the minimal T cell recognition site of this RT C-terminal region [i.e. RT-(528-540)]. Close to this T cell epitope we identified a B cell determinant containing the motif EQVD [RT-(546-549)] which was recognized in three different strains of mice (H-2b, H-2d and H-2k). A comparison with X-ray analysis of the C-terminal region of HIV-1 reverse transcriptase indicated exposed positions of these Th and B cell epitopes. Both the presence of T and B cell sites and its limited polymorphism make the region RT-(528-549) a promising candidate for vaccine design. The use of the P3CSS adjuvant/carrier principle as a nontoxic adjuvant may be of major importance in the development of vaccines applicable to humans.

Gallidermin (Gdm) and epidermin (Epi) are highly homologous tetracyclic polypeptide antibiotics that are ribosomally synthesized by a Staphylococcus gallinarum strain and a Staphylococcus epidermidis strain, respectively. These antibiotics are secreted into media and are distinguished by the presence of the unusual amino acids lanthionine, 3-methyllanthionine, didehydrobutyrine, and S-(2-aminovinyl)-D-cysteine, which are formed by posttranslational modification. To study the substrate specificities of the modifying enzymes and to obtain variants that exhibit altered or new biological activities, we changed certain amino acids by performing site-specific mutagenesis with the Gdm and Epi structural genes (gdmA and epiA, respectively). S. epidermidis Tü3298/EMS6, an epiA mutant of the Epi-producing strain, was used as the expression host. This mutant synthesized Epi, Gdm, or analogs of these antibiotics when the appropriate genes were introduced on a plasmid. No Epi or Gdm analogs were isolated from the supernatant when (i) hydroxyamino acids involved in thioether amino acid formation were replaced by nonhydroxyamino acids (S3N and S19A); (ii) C residues involved in thioether bridging were deleted (delta C21, C22 and delta C22); or (iii) a ring amino acid was replaced by an amino acid having a completely different character (G10E and Y20G). A strong decrease in production was observed when S residues involved in thioether amino acid formation were replaced by T residues (S16T and S19T). A number of conservative changes at positions 6, 12, and 14 on the Gdm backbone were tolerated and led to analogs that had altered biological properties, such as enhanced antimicrobial activity (L6V) or a remarkable resistance to proteolytic degradation (A12L and Dhb14P). The T14S substitution led to simultaneous production of two Gdm species formed by incomplete posttranslational modification (dehydration) of the S-14 residue. The fully modified Dhb14Dha analog exhibited antimicrobial activity similar to that of Gdm, whereas the Dhb14S analog was less active. Both peptides were more sensitive to tryptic cleavage than Gdm was.

Antigenic peptides are translocated into the lumen of the endoplasmic reticulum by the action of the transporter associated with antigen processing (TAP), where they are subsequently needed for the correct assembly of major histocompatibility complex molecules. The transport function was reconstituted in insect cells by expression of both TAP genes. On the basis of this over-expression system, substrate selection was analyzed in detail by a direct biomolecular peptide binding assay. Competition assays with peptide variants, including substitutions of residues with alanine or structurally related amino acids, underline the broad peptide specificity of the human TAP complex. Steric requirements of the substrate-binding pocket were mapped using elongated peptides and scans with bulky, hydrophobic amino acids. Complex nonapeptide libraries were used to determine the contribution of each residue to stabilize peptide-TAP complexes. For the first time, this approach lets us directly evaluate the importance of peptide selection for the overall process of antigen presentation on the level of the peptide transporter.

Various lipopeptides representing the N-terminal part of the cytochrome subunit of the photosynthetic reaction centre from the purple bacterium Rhodopseudomonas virdis were prepared by solid-phase peptide synthesis. These lipopeptides consisted of a S-[2,3-dihydroxypropyl]-cysteinyl (Dhc) residue N-terminally coupled to the nonapeptide FEPPPATTT. Different numbers of palmitoyl (Pam) chains were attached to Dhc via ester and/or amide bonds. The lipopeptide Dhc(Pam)2-FEPPPATTT containing two ester-bonded palmitoyl residues and a free N-terminus was a potent polyclonal activator of murine (BALB/c) spleen cells at subnanomolar concentrations. The lipopeptide Pam-Dhc(Pam)2-FEPPPATTT containing three palmitoyl residues, the two-chain lipopeptide Pam-Dhc(Pam)-FEPPPATTT containing one amide- and one ester-bonded palmitoyl residue, and the N-terminally elongated lipopeptide SLVAG-Dhc(Pam)2-FEPPPATTT were less active. The nonapeptide FEPPPATTT and the decapeptide Dhc-FEPPPATTT were only marginal splenocyte activators, even at concentrations as high as 1 microM. Thus, lipopeptide Dhc(Pam)2-FEPPPATTT constitutes the first potent splenocyte stimulation Dhc-lipopeptide described so far that contains only two fatty acid residues.

Cleavage and kinetic studies have been carried out using commercially obtained H-Tyr(tBu)-5-(4'-aminomethyl-3',5'-dimethoxyphenoxy)valeric acid-TentaGelS (H-Tyr(tBu)-4-ADPV-TentaGelS) and H-Tyr (tBu)-4-ADPV-Ala-aminomethyl-resin (H-Tyr(tBu)-4-ADPV-AM-resin) prepared from commercially available resin and loaded with commercially available Fmoc-4-ADPV-OH amide anchor. Cleavage with pure trifluoroacetic acid (TFA) gave the intermediate H-Tyr-4-ADPV-NH2, which was then degraded to H-Tyr-NH2, and cleavage with TFA/dichloromethane (1:9) yielded H-Tyr-4-ADPV-NH2 which could be isolated in preparative amounts. Cleavage reactions with 15N-labelled H-Ala-4-ADPV-(15N)-Gly-AM-resin yielded the intermediate H-Ala-4-ADPV-NH2, which contained no 15N as demonstrated by 1H-NMR. The analysis of the commercial Fmoc-4-ADPV-OH amide anchor showed the presence of Fmoc-4-ADPV-4-ADPV-OH as an impurity in high amounts. This dimeric anchor molecule is the cause of formation of the anchor-linked peptide intermediate obtained during the cleavage from the resin. The particularly high acid-lability of the amide bond between the two ADPV moieties was utilized to synthesize sidechain and C-terminally 4-ADPV protected pentagastrin on a double-anchor resin, and to cleave it using 5% trifluoroacetic acid in dichloromethane. This method may offer a new way for the synthesis of protected peptide amides with improved solubility to be used in fragment condensation.