Structure prediction for peptides capable of inducing antibody formation in mice

A simple method for the sequence prediction of peptides capable of the in vivo stimulation of antibody production in mice without conjugation with protein carriers was proposed on the basis of literature data on the structure of T-helper epitopes active in vivo. According to this approach, a potentially active peptide should contain a nine-membered sequence with a hydrophobic amino acid residue in the first position and a positively charged residue in the ninth position. The efficiency of this approach was confirmed by the presence of such sequences in the previously described synthetic peptides with immune activities, by the application of this approach to the choice of immunogenic fragments within the sequences of various proteins that exhibited further the specific activity, and by the construction of immunogenic peptides on the basis of inactive natural sequences.     

MHC-associated peptide proteomics enabling highly sensitive detection of immunogenic sequences for the development of therapeutic antibodies with low immunogenicity

ABSTRACT

Immunogenicity is a key factor capable of influencing the efficacy and safety of therapeutic antibodies. A recently developed method called MHC-associated peptide proteomics (MAPPs) uses liquid chromatography/mass spectrometry to identify the peptide sequences derived from a therapeutic protein that are presented by major histocompatibility complex class II (MHC II) on antigen-presenting cells, and therefore may induce immunogenicity. In this study, we developed a MAPPs technique (called Ab-MAPPs) that has high throughput and can efficiently identify the MHC II-presented peptides derived from therapeutic antibodies using magnetic nanoparticle beads coated with a hydrophilic polymer in the immunoprecipitation process. The magnetic beads could identify more peptides and sequence regions originating from infliximab and adalimumab in a shorter measurement time than Sepharose beads, which are commonly used for MAPPs. Several sequence regions identified by Ab-MAPPs from infliximab corresponded to immunogenic sequences reported by other methods, which suggests the method’s high potential for identifying significant sequences involved in immunogenicity. Furthermore, our study suggests that the Ab-MAPPs method can recognize the difference of a single amino acid residue between similar antibody sequences with different levels of T-cell proliferation activity and can identify potentially immunogenic peptides with high binding affinity to MHC II. In conclusion, Ab-MAPPs is useful for identifying the immunogenic sequences of therapeutic antibodies and will contribute to the design of therapeutic antibodies with low immunogenicity during the drug discovery stage.     

Fungal phytopathogens encode functional homologues of plant rapid alkalinization factor (RALF) peptides

In this article, we describe the presence of genes encoding close homologues of an endogenous plant peptide, rapid alkalinization factor (RALF), within the genomes of 26 species of phytopathogenic fungi. Members of the RALF family are key growth factors in plants, and the sequence of the RALF active region is well conserved between plant and fungal proteins. RALF1‐like sequences were observed in most cases; however, RALF27‐like sequences were present in the Sphaerulina musiva and Septoria populicola genomes. These two species are pathogens of poplar and, interestingly, the closest relative to their respective RALF genes is a poplar RALF27‐like sequence. RALF peptides control cellular expansion during plant development, but were originally defined on the basis of their ability to induce rapid alkalinization in tobacco cell cultures. To test whether the fungal RALF peptides were biologically active in plants, we synthesized RALF peptides corresponding to those encoded by two sequenced genomes of the tomato pathogen Fusarium oxysporum f. sp. lycopersici. One of these peptides inhibited the growth of tomato seedlings and elicited responses in tomato and Nicotiana benthamiana typical of endogenous plant RALF peptides (reactive oxygen species burst, induced alkalinization and mitogen‐activated protein kinase activation). Gene expression analysis confirmed that a RALF‐encoding gene in F. oxysporum f. sp. lycopersici was expressed during infection on tomato. However, a subsequent reverse genetics approach revealed that the RALF peptide was not required by F. oxysporum f. sp. lycopersici for infection on tomato roots. This study has demonstrated the presence of functionally active RALF peptides encoded within phytopathogens that harbour an as yet undetermined role in plant–pathogen interactions.     

Biophysical studies of signal peptides: Implications for signal sequence functions and the involvement of lipid in protein export

This review discusses efforts to understand the mode of action of signal sequences by biophysical study of synthetic peptides corresponding to these protein localization signals. On the basis of reports from several laboratories, it is now clear that signal peptides may adopt a variety of conformations, depending on their local environment. In membrane-mimetic systems like detergent micelles or lipid vesicles, they have a high tendency to form helices. Ability to take up a helical conformation appears to be required at some point in the function of a signal sequence, since some peptides corresponding to export-defective signal sequences display reduced helical potential. By contrast, functional signal sequences share a high capacity to adopt helices. High affinity for organized lipid assemblies, like monolayers or vesicles, is also a property of functional signal sequences. This correlation suggests a role for direct interaction of signal sequences with the lipids of the cytoplasmic membranein vivo. Supporting this role are studies of the influence of signal peptides on lipid structure, which reveal an ability of these peptides to pertub lipid packing and to alter the phase state of the lipids. Insertion of the signal sequencein vivo could substantially reduce the barrier for translocation of the mature chain. Lastly, synthetic signal peptides have been added to native membranes and found to inhibit translocation of precursor proteins. This approach bridges the biophysical and the biochemical aspects of protein export and promises to shed light on the functional correlates of the properties and interactions observed in model systems.     

ΔμH+ dependency of in vitro protein translocation into Escherichia coli inner-membrane vesicles varies with the signal-sequence core-region composition

Signal sequences frequently contain α-helix-destabilizing amino acids in the hydrophobic core. Nuclear magnetic resonance studies on the conformation of signal sequences in membrane mimetic environments revealed that these residues cause a break in the α-helix. In the precursor of the Escherichia coli outer membrane protein PhoE (pre-PhoE), a glycine residue at position -10 (Gly^?10) is thought to be responsible for the break in the α-helix. We investigated the role of this glycine residue in the translocation process by employing site-directed mutagenesis. SDS-PAGE analysis showed drastic variations in the electrophoretic mobilities of the mutant precursor proteins, suggesting an important role of the glycine residue in determining the conformation of the signal sequence. In vivo, no drastic differences in the translocation kinetics were observed as compared with wild-type PhoE, except when a charged residue (Arg) was substituted for Gly^?10. However, the in vitro translocation of all mutant proteins into inverted inner-membrane vesicles was affected. Two classes of precursors could be distinguished. Translocation of one class of mutant proteins (Ala, Cys and Leu for Gly^?10) was almost independent of the presence of a ΔμH⁺, whereas translocation of the other class of precursors (wild type or Ser) was strongly decreased in the absence of the ΔμH⁺. Apparently, the ΔμH⁺ dependency of in vitro protein translocation varies with the signal-sequence core-region composition. Furthermore, a proline residue at position -10 resulted in a signal sequence that did not prevent the folding of the precursor in an in vitro trimerization assay.     

Conformation of the metastasis-inhibiting laminin pentapeptide

The binding of cancer cells to the basement membrane glycoprotein laminin appears to be a critical step in the metastatic process. This binding can be inhibited competitively by a specific pentapeptide sequence (Tyr-Ile-Gly-Ser-Arg) of the laminin B1 chain, and this peptide can prevent metastasis formationin vivo. However, other similar pentapeptide sequences (e.g., Tyr-Ile-Gly-Ser-Glu) have been found to be much less active in metastasis inhibition, raising the possibility that such amino acid substitutions produce structural changes responsible for altering binding to the laminin receptor. In this study, conformational energy analysis has been used to determine the three-dimensional structures of these peptides. The results indicate that the substitution of Glu for the terminal Arg produces a significant conformational change in the peptide backbone at the middle Gly residue. These results have important implications for the design of drugs that may be useful in preventing metastasis formation and tumor spread.     

Interruptions in the collagen repeating tripeptide pattern can promote supramolecular association

The standard collagen triple‐helix requires a perfect (Gly‐Xaa‐Yaa)_n sequence, yet all nonfibrillar collagens contain interruptions in this tripeptide repeating pattern. Defining the structural consequences of disruptions in the sequence pattern may shed light on the biological role of sequence interruptions, which have been suggested to play a role in molecular flexibility, collagen degradation, and ligand binding. Previous studies on model peptides with 1‐ and 4‐residue interruptions showed a localized perturbation within the triple‐helix, and this work is extended to introduce natural collagen interruptions up to nine residue in length within a fixed (Gly‐Pro‐Hyp)_n peptide context. All peptides in this set show decreases in triple‐helix content and stability, with greater conformational perturbations for the interruptions longer than five residue. The most stable and least perturbed structure is seen for the 5‐residue interruption peptide, whose sequence corresponds to a Gly to Ala missense mutation, such as those leading to collagen genetic diseases. The triple‐helix peptides containing 8‐ and 9‐residue interruptions exhibit a strong propensity for self‐association to fibrous structures. In addition, a small peptide modeling only the 9‐residue sequence within the interruption aggregates to form amyloid‐like fibrils with antiparallel β‐sheet structure. The 8‐ and 9‐residue interruption sequences studied here are predicted to have significant cross‐β aggregation potential, and a similar propensity is reported for ～10% of other naturally occurring interruptions. The presence of amyloidogenic sequences within or between triple‐helix domains may play a role in molecular association to normal tissue structures and could participate in observed interactions between collagen and amyloid.     

Delineation and conformational analysis of two synthetic peptide models of antigenic sites on rodent cytochrome c

Two regions of rodent cytochrome c, one within the first four residues of the molecule, which is N-acetylated, and one at a beta bend around residue 44, are known to be immunogenic and antigenic in rabbits. Using sequential peptide synthesis, we have determined the residues required for linear synthetic peptides within these sequences to bind to antibody raised in rabbits to intact rat cytochrome c. The residues that were important in binding the N-terminal peptides were N-acetylglycine at position 1 and valine at position 3. The smallest peptide sequence around residue 44 that would bind to antibodies was Gln-Ala-Ala-Gly-Phe. A theoretical conformational analysis of these peptides showed that the amino-terminal tetrapeptide adopts a wide statistical ensemble of conformational states and that the addition of residues beyond 41 and 45 in the other sequence does not appear to stabilize longer peptides in the native beta-bend conformation. Thus, the antigenicity conferred by Phe-46 and Gln-42 in this peptide is most likely due to the direct interaction of the side chains of these residues with the antibody binding site. The demonstration here that native conformation is not essential for antigenic peptides to bind to antibodies raised against the whole protein indicates that the association energy between antigen and antibody can be sufficient to induce conformation in conformationally flexible peptides. This supports the concept that anti-protein and anti-peptide antibodies may invoke conformational changes in cross-reactive protein antigens and may explain why longer peptides, which may adopt stable nonnative secondary structure, often do not bind to antibodies raised to the whole molecule.     

Chemical Modification of Neutral Protease from Bacillus subtilis var. amylosacchariticus: Assignment of Tyrosyl Residues Iodinated

The neutral protease of Bacillus subtilis var. amylosacchariticus (B. amylosacchariticus) was iodinated with a 25-fold molar excess of iodine at pH 9.4 for 3 min at 0°C, by which treatment the proteolytic activity toward casein was markedly reduced, while the hydrolytic activity toward an N-blocked peptide substrate was rather increased. The modified enzyme was digested with Staphylococcus aureus V8 protease at pH 8.0 and the amino acid sequences of resultant peptides were compared with those obtained from the native enzyme. One of the peptides was found to have an amino acid sequence of Thr-Ala-Asn-Leu-Ile-Tyr-Glu, which corresponds to residue Nos. 153—159 of the enzyme, where Tyr-158 was identified to be mono-iodotyrosine. The other two peptides were those containing Tyr-21 which was mono- and di-iodinated, respectively. Referring to nitration experiments on the neutral protease and the active site structure of thermolysin, it was concluded that the iodination of Tyr-158 is mainly responsible for the activity changes of B. amylosacchariticus neutral protease.     

Tyrosinase epitope recognized by an HLA-DR-restricted T-cell line from a Vogt-Koyanagi-Harada disease patient

 Human T-cell-mediated autoimmune diseases are often genetically linked to particular alleles of HLA class II genes. Vogt-Koyanagi-Harada’s (VKH) disease, which is regarded as an autoimmune disorder in multiple organs containing melanocytes, has been found to be associated with HLA-DR4 (DRB1^*0405) and HLA-DR53 (DRB4^*0101). Tyrosinase is a melanoma antigen (Ag) expressed by normal melanocytes as well as melanoma cells against which responses by autologous T cells have been detected. We established a T-cell line from the peripheral blood of a patient with VKH disease which responded to synthetic peptides corresponding to tyrosinase. The T-cell line was generated which recognized the tyrosinase p188 – 208 peptide when presented by the HLA-DR4 (DRB1^*0405) molecule on the surface of HLA class II-expressing L-cell transfectants. The minimal antigenic peptide which induced T-cell responses was an 11-amino-acid sequence and located at tyrosinase p193 – 203 (E-I-W-R-D-I-D-F-A-H-E). This peptide contained the DRB1^*0405-binding peptide motif (hydrophobic residues (Y, F, W) at position 1 as an anchor residue, and negatively charged residues (D, E) at position 9), which corresponded to the W at p195 and the D at p203. These observations demonstrate that tyrosinase peptides are immunogenic, and may be a candidate for an autoantigen in VKH disease, suggesting that probing the T-cell responses against synthetic peptides is a productive approach for identifying the autoantigenic peptides associated with autoimmune diseases including VKH disease. Received: 22 August 1997 / Revised: 7 October 1997     

A catalogue of <Emphasis Type="Italic">Triticum monococcum</Emphasis> genes encoding toxic and immunogenic peptides for celiac disease patients

The celiac disease (CD) is an inflammatory condition characterized by injury to the lining of the small-intestine on exposure to the gluten of wheat, barley and rye. The involvement of gluten in the CD syndrome has been studied in detail in bread wheat, where a set of “toxic” and “immunogenic” peptides has been defined. For wheat diploid species, information on CD epitopes is poor. In the present paper, we have adopted a genomic approach in order to understand the potential CD danger represented by storage proteins in diploid wheat and sequenced a sufficiently large number of cDNA clones related to storage protein genes of Triticum monococcum. Four bona fide toxic peptides and 13 immunogenic peptides were found. All the classes of storage proteins were shown to contain harmful sequences. The major conclusion is that einkorn has the full potential to induce the CD syndrome, as already evident for polyploid wheats. In addition, a complete overview of the storage protein gene arsenal in T. monococcum is provided, including a full-length HMW x-type sequence and two partial HMW y-type sequences. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High selectivity of human tissue transglutaminase for immunoactive gliadin peptides: implications for celiac sprue.

Celiac Sprue is an HLA DQ2 (or DQ8)-associated autoimmune disorder of the human small intestine that is induced by dietary exposure to wheat gliadin and related proteins from barley, rye, and possibly other food grains. Recently, tissue transglutaminase (tTGase)-catalyzed deamidation of gliadin peptides has been shown to increase their potency for activating patient-derived, gliadin-specific T cells, suggesting that tTGase plays a causative role in the onset of an inflammatory response to toxic food grains. To dissect the molecular recognition features of tTGase for gluten derived peptides, the regioselectivity and steady-state kinetics of tTGase-catalyzed deamidation of known immunogenic peptides were investigated. The specificity of recombinant human tTGase for all immunogenic peptides tested was comparable to and, in some cases, appreciably higher than the specificity for its natural substrate. Although each peptide was glutamine-rich, tTGase exhibited a high degree of regioselectivity for a particular glutamine residue in each peptide. This selectivity correlated well with Q --> E substitutions that have earlier been shown to enhance the immunogenicity of the corresponding gliadin peptides. The specificity of tTGase toward homologues of PQPQLPY, a sequence motif found in immunodominant gliadin peptides, was analyzed in detail. Remarkably, the primary amino acid sequences of wheat-, rye-, and barley-derived proteins included many single-residue variants of this sequence that were high-affinity substrates of tTGase, whereas the closest homologues of this sequence found in rice, corn, or oat proteins were much poorer substrates of tTGase. (Rice, corn, and oats are nontoxic ingredients of the Celiac diet.) No consensus sequence for a high-affinity substrate of tTGase could be derived from our data, suggesting that the secondary structures of these food-grain peptides were important in their recognition by tTGase. Finally, under steady-state turnover conditions, a significant fraction of the tTGase active site was covalently bound to a representative high-affinity immunogenic gliadin peptide, suggesting a common mechanism by which cells responsible for immune surveillance of the intestinal tract recognize and generate an antibody response against both gliadin and tTGase. In addition to providing a quantitative framework for understanding the role of tTGase in Celiac Sprue, our results lay the groundwork for the design of small molecule mimetics of gliadin peptides as selective inhibitors of tTGase.     

The diagnostic potential of MPT63‐derived HLA‐A*0201‐restricted CD8+T‐cell epitopes for active pulmonary tuberculosis

MPT63 protein is found only in Mycobacterium tuberculosis complex, including M. tuberculosis and M. bovis. Detection of MPT63‐specific IFN‐γ‐secreting T cells could be useful for the diagnosis of tuberculosis (TB) diseases. In the present study, the HLA‐A*0201 restriction of ten predicted MPT63‐derived CD8 ⁺ T‐cell epitopes was assessed on the basis of T2 cell line and HLA‐A*0201 transgenic mice. The diagnostic potential of immunogenic peptides in active pulmonary TB patients was evaluated using an IFN‐γ enzyme‐linked immunospot assay. It was found that five peptides bound to HLA‐A*0201 with high affinity, whereas the remaining peptides exhibited low affinity for HLA‐A*0201. Five immunogenic peptides (MPT63_18–26, MPT63_29–37, MPT63_20–28, MPT63_5–14 and MPT63_10–19) elicited large numbers of cytotoxic IFN‐γ‐secreting T cells in HLA‐A*0201 transgenic mice. Each of the five immunogenic peptides was recognized by peripheral blood mononuclear cells from 45% to 73% of 40 HLA‐A*0201 positive TB patients. The total diagnostic sensitivity of the five immunogenic peptides was higher than that of a T‐SPOT.TB assay (based on ESAT‐6 and CFP‐10) (93% versus 90%). It is noticeable that the diagnostic sensitivity of the combination of five immunogenic peptides and T‐SPOT.TB assay reached 100%. These MPT63‐derived HLA‐A*0201‐restricted CD8 ⁺ T‐cell epitopes would likely contribute to the immunological diagnosis of M. tuberculosis infection and may provide the components for designing an effective TB vaccine.     

Identification of preferred substrate sequences of microbial transglutaminase from Streptomyces mobaraensis using a phage-displayed peptide library

Microbial transglutaminase (TGase) from Streptomyces mobaraensis (MTG) has been used in many industrial applications because it effectively catalyzes the formation of covalent cross-linking between glutamine residues in various substrate proteins and lysine residues or primary amines. To better understand the sequence preference around the reactive glutamine residue by this enzymatic reaction, we screened preferred peptide sequences using a phage-displayed random peptide library. Most of the peptides identified contained a consensus sequence, which was different from those previously found for mammalian TGases. Of these, most sequences had a specific reactivity toward MTG when produced as a fusion protein with glutathione-S-transferase. Furthermore, the representative sequence was found to be reactive even in the peptide form. The amino acid residues in the sequence critical for the reactivity were further analyzed, and the possible interaction with the enzyme has been discussed in this paper.     

Primary structure of the H-2Db alloantigen

The complete amino acid sequence of the CNBr fragment comprising residues 229–284 of the murine major histocompatibility complex antigen H-2D^b has been determined using radiochemical methodology. The sequence was determined by N-terminal sequence analysis of the intact CNBr fragment and by sequence determinations of peptides derived from this fragment by trypsin and staphylococcal V8 protease cleavage. In addition to the amino acid assignments for H-2D^b, it was possible to assign the linkage position of the third N-linked glycosyl unit to the asparagine at residue 256. Additional amino acid sequence assignments have also been made for three other CNBr fragments that span residues 99–138, 139–228, and 308–331 of the H-2D^b molecule. The total protein sequence information available (222 of 338 residues) agrees in every comparable position with the protein sequence derived from the cDNA clone (pH203) isolated by Reyes and co-workers (1982b), which strongly suggests that this clone encodes H-2D^b. Combination of the protein sequence with that deduced from the cDNA clone provides the complete H-2D^b protein sequence. Comparison of this sequence with other available protein sequence information for murine class I molecules has revealed protein sequences that may be unique to either K or D region molecules.Abbreviations used in this paper HPLC high performance liquid chromatography - V8 Staphylococcus aureus V8 protease - MHC major histocompatibility complex     

Bacteria-based in vivo peptide library screening using biopanning approach

Traditionally, library screening has been performed to identify biologically active agents including small molecules or peptides that inhibit target proteins or molecules with therapeutic interests. Due to its chemical nature, library screening is usually performed under in vitro environments using purified proteins and molecules. However, active agents identified from in vitro screenings often fail to exhibit biological activities in cells. To overcome this inherent limitation, we have developed an in vivo peptide library screening system that allows for the identification of dissociative inhibitors of protein interactions of interest. The screening is based on the reconstitution of the cI repressor from bacteriophage lambda with high-density expression peptide library and is entirely performed in bacteria cells. Furthermore, to enhance the efficacy and sensitivity of the screening, a multiple-round biopanning approach was employed for amplification and enrichment of positive peptides. Overall, this in vivo screening should provide a fast and efficient tool for identification of biologically active peptide molecules against target protein assembly.     

The activity of HDAC8 depends on local and distal sequences of its peptide substrates

This paper introduces a flexible assay for characterizing the activities of the histone deacetylase enzymes. The approach combines mass spectrometry with self-assembled monolayers that present acetylated peptides and enables a label-free and one-step assay of this biochemical activity. The assay was used to characterize the activity of HDAC8 toward peptides taken from the N-terminal tail of the H4 histone and reveals that a distal region of the peptide substrate interacts with the deacetylase at an exosite and contributes to the activity of the substrate. Specifically, a peptide corresponding to residues 8-19 of H4 and having lysine 12 acetylated is an active substrate, but removal of the KRHR (residues 16-19) sequence abolishes activity. Mutation of glycine 11 to arginine in the peptide lacking the KRHR sequence restores activity, demonstrating that both local and distal sequences act synergistically to regulate the activity of the HDAC. Assays with peptides bearing multiply acetylated residues, but in which each acetyl group is isotopically labeled, permit studies of the processive deacetylation of peptides. Peptide substrates having an extended sequence that includes K20 were used to demonstrate that methylation of this residue directly affects HDAC8 activity at K12. This work provides a mechanistic basis for the regulation of HDAC activities by distal sequences and may contribute to studies aimed at evaluating the role of the histone code in regulating gene expression.     

Two distinct HLA-A * 0101-specific submotifs illustrate alternative peptide binding modes

 Previous studies have defined two different peptide binding motifs specific for HLA-A ^* 0101. These motifs are characterized by the presence of tyrosine (Y) at the C-termini of 9-mer and 10-mer peptides, and either a small polar or hydrophobic (S, T, M) residue in position 2, or a negatively charged (D or E) residue in position 3. In this study, the structural requirements for peptide binding to A ^* 0101 have been further analyzed by examining the binding capacity of large sets of peptides corresponding to naturally occurring sequences which bore one or the other of these two A ^* 0101-specific motifs. By correlating the presence of specific residue types at each position along the peptide sequence with increased (or decreased) binding affinity, the prominent influence of secondary anchor residues was revealed. In most cases, the two anchors in positions 2 and 3 appear to act synergistically. With the exception of the DE₃ submotif in 9-mer peptides, a positive role for aromatic residues in position 1 and the center of the peptide (positions 4 or 5 of 9- or 10-mer peptides, respectively), and proline at C-3, were also consistently detected. However, secondary anchor residues also appear to differ significantly between the two different submotifs, demonstrating that A ^* 0101 can utilize alternative modes in binding its peptide ligands. According to these analyses, specific refined submotifs were also established, and their merit verified by independent sets of potential A ^* 0101 binding peptides. Besides providing useful insight into the nature of the interaction of the A ^* 0101 allele with its peptide ligands, such refined motifs should also facilitate accurate prediction of potential A ^* 0101-restricted peptide epitopes. Received: 16 July 1996 / Revised: 18 September 1996     

Sequences of stilboflavin C: towards the peptaibiome of the filamentous fungus Stilbella (= Trichoderma) flavipes

Filamentous fungi of the genus Stilbella are recognized as an abundant source of naturally occurring α‐aminoisobutyric acid‐containing peptides. The culture broth of Stilbella (Trichoderma) flavipes CBS 146.81 yielded a mixture of peptides named stilboflavins (SF), and these were isolated and separated by preparative TLC into groups named SF‐A, SF‐B, and SF‐C. Although all three of these groups resolved as single spots on thin‐layer chromatograms, HPLC analysis revealed that each of the groups represents very microheterogeneous mixtures of closely related peptides. Here, we report on the sequence analysis of SF‐C peptides, formerly isolated by preparative TLC. HPLC coupled to QqTOF‐ESI‐HRMS provided the sequences of 10 16‐residue peptides and five 19‐residue peptides, all of which were N‐terminally acetylated. In contrast to the previously described SF‐A and SF‐B peptaibols, SF‐C peptaibols contain Ser‐Alaol or Ser‐Leuol, which are rarely found as C‐termini, and repetitive Leu‐Aib‐Gly sequences, which have not been detected in peptaibols before. Taking the previously determined sequences of SF‐A and SF‐B into account, the entirety of peptides produced by S. flavipes (the ‘peptaibiome’) approaches or exceeds 100 non‐ribosomally biosynthesized peptaibiotics. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Differential expression of protease activity in serum samples of prostate carcinoma patients with metastases

We present here the results from MS peptide profiling experiments of prostate carcinoma patients and controls with a specific focus on protease activity‐related protein fragments. After purification with surface‐active magnetic beads, MALDI‐TOF profiling experiments were performed on tryptic digests of serum samples of prostate cancer patients with metastases (n=27) and controls (n=30). This resulted in the reproducible detection of eight differentially expressed peptides, which were then identified by nanoLC‐MALDI‐TOF/TOF and confirmed by MALDI‐FTMS exact mass measurements. All differentially expressed peptides are derived from two homologous parts of human serum albumin; two of the eight peptides were tryptic and six were nontryptic. The presence of the nontryptic fragments indicates that a proteolysis process occurs which is not mediated by trypsin. Since the nontryptic fragments were found at significantly higher levels in control samples compared with metastases samples, it is proposed that a specific proteolytic inhibition process is in effect in the serum of prostate cancer patients. Experiments using synthetic peptides showed that this proteolytic activity occurs ex vivo and is sequence specific. Importantly, the observed prostate carcinoma‐related inhibition of the proteolysis was reproduced ex vivo using synthetic peptides.