Markus Schirle

Dermcidin: A novel human antibiotic peptide secreted by sweat glands

Antimicrobial peptides are an important component of the innate response in many species. Here we describe the isolation of the gene Dermcidin, which encodes an antimicrobial peptide that has a broad spectrum of activity and no homology to other known antimicrobial peptides. This protein was specifically and constitutively expressed in the sweat glands, secreted into the sweat and transported to the epidermal surface. In sweat, a proteolytically processed 47–amino acid peptide was generated that showed antimicrobial activity in response to a variety of pathogenic microorganisms. The activity of the peptide was maintained over a broad pH range and in high salt concentrations that resembled the conditions in human sweat. This indicated that sweat plays a role in the regulation of human skin flora through the presence of an antimicrobial peptide. This peptide may help limit infection by potential pathogens in the first few hours following bacterial colonization.

Two new proteases in the MHC class I processing pathway.

The proteasome generates exact major histocompatibility complex (MHC) class I ligands as well as NH2-terminal-extended precursor peptides. The proteases responsible for the final NH2-terminal trimming of the precursor peptides had, until now, not been determined. By using specific selective criteria we purified two cytosolic proteolytic activities, puromycin-sensitive aminopeptidase and bleomycin hydrolase. These proteases could remove NH2-terminal amino acids from the vesicular stomatitis virus nucleoprotein cytotoxic T cell epitope 52–59 (RGYVYQGL) resulting, in combination with proteasomes, in the generation of the correct epitope. Our data provide evidence for the existence of redundant systems acting downstream of the proteasome in the antigen-processing pathway for MHC class I molecules.

Contribution of Proteasomal β-Subunits to the Cleavage of Peptide Substrates Analyzed with Yeast Mutants

Proteasomes generate peptides that can be presented by major histocompatibility complex (MHC) class I molecules in vertebrate cells. Using yeast 20 S proteasomes carrying different inactivated β-subunits, we investigated the specificities and contributions of the different β-subunits to the degradation of polypeptide substrates containing MHC class I ligands and addressed the question of additional proteolytically active sites apart from the active β-subunits. We found a clear correlation between the contribution of the different subunits to the cleavage of fluorogenic and long peptide substrates, with β5/Pre2 cleaving after hydrophobic, β2/Pup1 after basic, and β1/Pre3 after acidic residues, but with the exception that β2/Pup1 and β1/Pre3 can also cleave after some hydrophobic residues. All proteolytic activities including the “branched chain amino acid-preferring” component are associated with β5/Pre2, β1/Pre3, or β2/Pup1, arguing against additional proteolytic sites. Because of the high homology between yeast and mammalian 20 S proteasomes in sequence and subunit topology and the conservation of cleavage specificity between mammalian and yeast proteasomes, our results can be expected to also describe most of the proteolytic activity of mammalian 20 S proteasomes leading to the generation of MHC class I ligands.

Identification of tumor-associated MHC class I ligands by a novel T cell-independent approach.

Specific immunotherapy of cancer utilizes tumor‐directed cytotoxic T lymphocytes (CTL) that lyse tumor cells presenting MHC class I‐associated peptides derived from tumor‐associated proteins. Many tumor‐associated gene products are known, but corresponding T cell epitopes are only known for relatively few of these. The most commonly used approaches to identify such antigens require pre‐existing CTL lines or clones. By using a CTL‐independent high performance liquid chromatography mass spectrometry (HPLC MS)–based approach we identified HLA‐A2‐presented peptides from carcinoembryonic antigen and wild‐type p53 with a copy number as low as eight molecules per cell. Potential epitopes were predicted from the sequences of known tumor antigens and the corresponding synthetic peptides were analyzed by nanocapillary HPLC MS. In parallel, peptides were extracted from fresh, solid tumor tissue or tumor cell lines and analyzed in the same way. Upon co‐elution of a natural peptide with a predicted peptide of the same mass, the peptide sequence was confirmed by on‐line tandem MS. This approach allows rapid screening of large numbers of tumor‐associated gene products for naturally processed peptides presented by different MHC class I molecules as a prerequisite for efficient epitope identification and rapid transfer to therapeutic vaccine trials.

Combining computer algorithms with experimental approaches permits the rapid and accurate identification of T cell epitopes from defined antigens

The identification of T cell epitopes from immunologically relevant antigens remains a critical step in the development of vaccines and methods for monitoring of T cell responses. This review presents an overview of strategies that employ computer algorithms for the selection of candidate peptides from defined proteins and subsequent verification of their in vivo relevance by experimental approaches. Several computer algorithms are currently being used for epitope prediction of various major histocompatibility complex (MHC) class I and II molecules, based either on the analysis of natural MHC ligands or on the binding properties of synthetic peptides. Moreover, the analysis of proteasomal digests of peptides and whole proteins has led to the development of algorithms for the prediction of proteasomal cleavages. In order to verify the generation of the predicted peptides during antigen processing in vivo as well as their immunogenic potential, several experimental approaches have been pursued in the recent past. Mass spectrometry-based bioanalytical approaches have been used specifically to detect predicted peptides among isolated natural ligands. Other strategies employ various methods for the stimulation of primary T cell responses against the predicted peptides and subsequent testing of the recognition pattern towards target cells that express the antigen.

The HLA-A*6601 peptide motif: prediction by pocket structure and verification by peptide analysis

F. H. Seeger 7 M. Schirle 7 J. Gatfield 7 D. Arnold W. Keilholz 7 P. Nickolaus 7 H.-G. Rammensee Stefan Stevanović (Y) Eberhard-Karls-Universität Tübingen, Interfakultäres Institut für Zellbiologie, Abteilung Immunologie, Auf der Morgenstelle 15 (Verfügungsgebäude), D-72076 Tübingen, Germany E-mail: Stefan.Stevanovic6uni-tuebingen.de, Tel.: c49-7071-2987645, Fax: c49-7071-295653

A Naturally Processed Rat Major Histocompatibility Complex Class I-associated Viral Peptide as Target Structure of Borna Disease Virus-specific CD8+ T Cells

The first naturally processed peptide synthesized by a virus and recognized by classical CD8+ T cells in association with the RT1.Al major histocompatibility complex class I molecule of the Lewis rat is reported. Borna disease virus-specific CD8+ T cells recognize syngeneic target cells pulsed with peptides extracted from Borna disease virus-infected cells. The predicted peptide sequence ASYAQMTTY from the viral p40 protein coeluted with the cytotoxic T-lymphocyte-reactive fraction was identified among natural ligands by tandem mass spectrometry. Numerous naturally processed peptides derived from intracellular bacteria, viruses, or tumors and recognized by CD8+ T cells of man and mice are known, leading to a better understanding of cellular immune mechanisms against pathogens in these two species. In contrast, for the rat little information exists with regard to the function and role of CD8+ T cells as part of their cellular immune defense system. This first naturally processed viral epitope in the rat contributes to the understanding of the rat cellular immune response and might trigger the identification of more cytotoxic T-lymphocyte epitopes in this animal.