Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes.

Cytotoxic T lymphocytes (CTLs) recognize peptides presented by HLA class I molecules on the cell surface. The C terminus of these CTL epitopes is considered to be produced by the proteasome. Here we demonstrate that the cytosolic endopeptidases nardilysin and thimet oligopeptidase (TOP) complemented proteasome activity. Nardilysin and TOP were required, either together or alone, for the generation of a tumor-specific CTL epitope from PRAME, an immunodominant CTL epitope from Epstein-Barr virus protein EBNA3C, and a clinically important epitope from the melanoma protein MART-1. TOP functioned as C-terminal trimming peptidase in antigen processing, and nardilysin contributed to both the C-terminal and N-terminal generation of CTL epitopes. By broadening the antigenic peptide repertoire, nardilysin and TOP strengthen the immune defense against intracellular pathogens and cancer.

The human peptidylarginine deiminases type 2 and type 4 have distinct substrate specificities.

Human peptidylarginine deiminases (hPADs) have been implicated in several diseases, particularly in rheumatoid arthritis. Since hPAD2 and hPAD4 are the isotypes expressed in the inflamed joints of RA patients and protein citrullination by PADs has been proposed to play a pathophysiological role, they represent unique therapeutic targets. To facilitate the development of substrate-based PAD inhibitors the substrate specificity of hPAD2 and hPAD4 was determined. Recombinant hPADs were expressed in bacteria or mammalian cell lines and allowed to citrullinate proteins in cell lysates, as well as a series of synthetic peptides. The citrullinated residues in proteins and the efficiency of peptide citrullination were determined by mass spectrometry. In total 320 hPAD2 and 178 hPAD4 citrullination sites were characterized. Amino acid residues most commonly found in citrullination sites for both isotypes are Gly at +1 and Tyr at +3 relative to the target arginine. For hPAD4 several additional amino acids were observed to be preferred at various positions from -4 to +4. The substrate motifs determined by amino acid substitution analysis partially confirmed these preferences, although peptide context dependent differences were also observed. Taken together, our data show that the enzyme specificity for cellular substrates and synthetic peptides differs for hPAD2 and hPAD4. hPAD4 shows more restrictive substrate specificity compared to hPAD2. Consensus sequences, which can be used as the basis for the development of PAD inhibitors, were derived for the citrullination sites of both hPAD2 and hPAD4.

Aminopeptidase-resistant peptides are targeted to lysosomes and subsequently degraded.

Most cytoplasmic and nuclear proteins are degraded via the ubiquitin-proteasome system into peptides, which are subsequently hydrolyzed by downstream aminopeptidases. Inefficient degradation can lead to accumulation of protein fragments, and subsequent aggregation and toxicity. Whereas the role of the proteasome and the effect of its impairment on aggregation have been intensively studied, little is known about how cells deal with peptides that show resistance to degradation by aminopeptidases. Here, we introduced peptidase-resistant peptides into living cells and show that these peptides rapidly and irreversibly accumulate into puncta in the perinuclear region of the cell. Accumulation appears to be independent of peptide sequence but is less efficient for longer peptides. The puncta colocalize with autophagosomal and lysosomal markers, suggesting that these peptides end up within lysosomes via macroautophagy. Surprisingly, the peptides still accumulate within lysosomes when macroautophagy is impaired, suggesting a trafficking route independent of macroautophagy. Upon lysosomal uptake, peptides are degraded, suggesting that cells can clear peptidase-resistant proteasomal products by an alternative pathway, which targets them to lysosomes.

Specificity of tissue transglutaminase explains cereal toxicity in celiac disease.

Celiac disease is caused by a selective lack of T cell tolerance for gluten. It is known that the enzyme tissue transglutaminase (tTG) is involved in the generation of T cell stimulatory gluten peptides through deamidation of glutamine, the most abundant amino acid in gluten. Only particular glutamine residues, however, are modified by tTG. Here we provide evidence that the spacing between glutamine and proline, the second most abundant amino acid in gluten, plays an essential role in the specificity of deamidation. On the basis of this, algorithms were designed and used to successfully predict novel T cell stimulatory peptides in gluten. Strikingly, these algorithms identified many similar peptides in the gluten-like hordeins from barley and secalins from rye but not in the avenins from oats. The avenins contain significantly lower percentages of proline residues, which offers a likely explanation for the lack of toxicity of oats. Thus, the unique amino acid composition of gluten and related proteins in barley and rye favors the generation of toxic T cell stimulatory gluten peptides by tTG. This provides a rationale for the observation that celiac disease patients are intolerant to these cereal proteins but not to other common food proteins.

Expression, purification, and in vitro activity of an arterivirus main proteinase.

To allow the biochemical and structural characterization of the chymotrypsin-like "main proteinase" (non-structural protein 4; nsp4) of the arterivirus prototype Equine Arteritis Virus (EAV), we developed protocols for the large-scale production of recombinant nsp4 in Escherichia coli. The nsp4 proteinase was expressed either fused to maltose binding protein or carrying a C-terminal hexahistidine tag. Following purification, the nsp4 moiety of MBP-nsp4 was successfully used for structural studies [Barrette-Ng, I.H., Ng, K.K.S., Mark, B.L., van Aken, D., Cherney, M.M., Garen, C, Kolodenko, Y., Gorbalenya, A.E., Snijder, E.J., James, M.N.G, 2002. Structure of arterivirus nsp4-the smallest chymotrypsin-like proteinase with an alpha/beta C-terminal extension and alternate conformations of the oxyanion hole. J. Biol. Chem. 277, 39960-39966]. Furthermore, both forms of the EAV proteinase were shown to be proteolytically active in two different trans-cleavage assays. Recombinant nsp4 cleaved the cognate nsp6/7- and nsp7/8 site in in vitro synthesized substrates. In a synthetic peptide-based activity assay, the potential of the recombinant proteinase to cleave peptides mimicking the P9-P7' residues of six nsp4 cleavage sites was investigated. The peptide representing the EAV nsp7/8 junction was used to optimize the reaction conditions (pH 7.5, 25mM NaCl, 30% glycerol at 30 degrees C), which resulted in a maximum turnover of 15% of this substrate in 4h, using a substrate to enzyme molar ratio of 24:1. The assays described in this study can be used for a more extensive biochemical characterization of the EAV main proteinase, including studies aiming to identify inhibitors of proteolytic activity.

Competition-based cellular peptide binding assays for 13 prevalent HLA class I alleles using fluorescein-labeled synthetic peptides.

We report the development, validation, and application of competition-based peptide binding assays for 13 prevalent human leukocyte antigen (HLA) class I alleles. The assays are based on peptide binding to HLA molecules on living cells carrying the particular allele. Competition for binding between the test peptide of interest and a fluorescein-labeled HLA class I binding peptide is used as read out. The use of cell membrane-bound HLA class I molecules circumvents the need for laborious biochemical purification of these molecules in soluble form. Previously, we have applied this principle for HLA-A2 and HLA-A3. We now describe the assays for HLA-A1, HLA-A11, HLA-A24, HLA-A68, HLA-B7, HLA-B8, HLA-B14, HLA-B35, HLA-B60, HLA-B61, and HLA-B62. Together with HLA-A2 and HLA-A3, these alleles cover more than 95% of the Caucasian population. Several allele-specific parameters were determined for each assay. Using these assays, we identified novel HLA class I high-affinity binding peptides from HIVpol, p53, PRAME, and minor histocompatibility antigen HA-1. Thus these convenient and accurate peptide-binding assays will be useful for the identification of putative cytotoxic T lymphocyte epitopes presented on a diverse array of HLA class I molecules.

Enhanced cross-presentation and improved CD8+ T cell responses after mannosylation of synthetic long peptides in mice.

The use of synthetic long peptides (SLP) has been proven to be a promising approach to induce adaptive immune responses in vaccination strategies. Here, we analyzed whether the efficiency to activate cytotoxic T cells by SLP-based vaccinations can be increased by conjugating SLPs to mannose residues. We could demonstrate that mannosylation of SLPs results in increased internalization by the mannose receptor (MR) on murine antigen-presenting cells. MR-mediated internalization targeted the mannosylated SLPs into early endosomes, from where they were cross-presented very efficiently compared to non-mannosylated SLPs. The influence of SLP mannosylation was specific for cross-presentation, as no influence on MHC II-restricted presentation was observed. Additionally, we showed that vaccination of mice with mannosylated SLPs containing epitopes from either ovalbumin or HPV E7 resulted in enhanced proliferation and activation of antigen-specific CD8+ T cells. These findings demonstrate that mannosylation of SLPs augments the induction of a cytotoxic T cell response in vitro and in vivo and might be a promising approach to induce cytotoxic T cell responses in e.g. cancer therapy and anti-viral immunity.

Reduced amyloid-ß degradation in early Alzheimer's disease but not in the APPswePS1dE9 and 3xTg-AD mouse models.

Alzheimer's disease (AD) is hallmarked by amyloid-ß (Aß) peptides accumulation and aggregation in extracellular plaques, preceded by intracellular accumulation. We examined whether intracellular Aß can be cleared by cytosolic peptidases and whether this capacity is affected during progression of sporadic AD (sAD) in humans and in the commonly used APPswePS1dE9 and 3xTg-AD mouse models. A quenched Aß peptide that becomes fluorescent upon degradation was used to screen for Aß-degrading cytoplasmic peptidases cleaving the aggregation-prone KLVFF region of the peptide. In addition, this quenched peptide was used to analyze Aß-degrading capacity in the hippocampus of sAD patients with different Braak stages as well as APPswePS1dE9 and 3xTg-AD mice. Insulin-degrading enzyme (IDE) was found to be the main peptidase that degrades cytoplasmic, monomeric Aß. Oligomerization of Aß prevents its clearance by IDE. Intriguingly, the Aß-degrading capacity decreases already during the earliest Braak stages of sAD, and this decline correlates with IDE protein levels, but not with mRNA levels. This suggests that decreased IDE levels could contribute to early sAD. In contrast to the human data, the commonly used APPswePS1dE9 and 3xTg-AD mouse models do not show altered Aß degradation and IDE levels with AD progression, raising doubts whether mouse models that overproduce Aß peptides are representative for human sAD.

Enhancing sensitivity of detection of immune responses to Mycobacterium leprae peptides in whole-blood assays.

Although worldwide leprosy prevalence has been reduced considerably following multidrug therapy, new case detection rates remain relatively stable, suggesting that transmission of infection still continues. This calls for new efforts, among which is development of assays that can identify subclinical/early-stage Mycobacterium leprae-infected subjects, a likely source of transmission. Areas in which leprosy is endemic often lack sophisticated laboratories, necessitating development of field-friendly immunodiagnostic tests for leprosy, like short-term whole-blood assays (WBA). In classical, peripheral blood mononuclear cell (PBMC)-based gamma interferon (IFN-gamma) release assays, M. leprae peptides have been shown to discriminate in a more specific fashion than M. leprae proteins between M. leprae-exposed contacts and patients as opposed to healthy controls from the same area of endemicity. However, peptides induced significantly lower levels of IFN-gamma than did proteins, particularly when whole blood was used. Therefore, possibilities of specifically enhancing IFN-gamma production in response to M. leprae peptides in 24-h WBA were sought by addition of various cytokines and antibodies or by mannosylation of peptides. In addition, other cytokines and chemokines were analyzed as potential biomarkers in WBA. We found that only interleukin 12 (IL-12), not other costimulants, increased IFN-gamma production in WBA while maintaining M. leprae peptide specificity, as evidenced by lack of increase of IFN-gamma in control samples stimulated with IL-12 alone. The IL-12-induced increase in IFN-gamma was mainly mediated by CD4+ T cells that did not produce IL-2 or tumor necrosis factor (TNF). Mannosylation further allowed the use of 100-fold-less peptide. Although not statistically significantly, macrophage inflammatory protein 1beta (MIP-1beta) and macrophage c protein 1 (MCP-1) levels specific for M. leprae peptide tended to be increased by IL-12. IP-10 production was also found to be a useful marker of M. leprae peptide responses, but its production was enhanced by IL-12 nonspecifically. We conclude that IFN-gamma-based WBA combined with IL-12 represents a more sensitive and robust assay for measuring reactivity to M. leprae peptides.

A major role for TPPII in trimming proteasomal degradation products for MHC class I antigen presentation.

Intracellular proteins are degraded by the proteasome, and resulting peptides surviving cytoplasmic peptidase activity can be presented by MHC class I molecules. Here, we show that intracellular aminopeptidases degrade peptides within seconds, almost irrespectively of amino acid sequence. N- but not C-terminal extension increases the half-life of peptides until they are 15 amino acids long. Beyond 15 amino acids, peptides are exclusively trimmed by the peptidase TPPII, which displays both exo- and endopeptidase activity. Surprisingly, most proteasomal degradation products are handled by TPPII before presentation by MHC class I molecules. We define three distinct proteolytic activities during antigen processing in vivo. Proteasome-generated peptides relevant for antigen presentation are mostly 15 amino acids or longer. These require TPPII activity for further trimming before becoming substrates for other peptidases and MHC class I. The heterogeneous pool of aminopeptidases will process TPPII products into MHC class I peptides and beyond.

Competition-based cellular peptide binding assay for HLA class I.

This unit describes a competition assay to determine binding of unlabeled test peptides to thirteen of the most prevalent HLA class I molecules. It uses cells expressing the HLA class I molecule of interest on their surface, fluorescently labeled reference peptides, and unlabeled test peptides. Cells of interest are stripped from their natural HLA-bound peptides using acid treatment and subsequently incubated with a mixture of labeled reference peptide and titrating concentrations of test peptide. Subsequently, FACS analysis is performed to determine the amount of bound reference peptide, which is a measure of the ability of test peptide to compete for binding to HLA. The assay provides IC50 values for binding of test peptides to HLA molecules. It can be performed in a normally equipped cellular laboratory, requires no additional equipment besides a flow cytometer (FACS), and is relatively easy to perform. Assay-specific parameters for several HLA alleles are provided.

The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides.

BACKGROUND & AIMS: Gluten (GLU)-specific T-cell responses in HLA-DQ2 positive adult celiac disease (CD) patients are directed to an immunodominant alpha-gliadin (GLIA) peptide that requires deamidation for T-cell recognition. The aim of the current study was to determine which GLU peptide(s) are involved early in disease. METHODS: We have characterized the GLU-specific T-cell response in HLA-DQ2 positive children with recent onset CD. RESULTS: We found that 50% of these patients do not respond to the alpha-GLIA peptide but to a diverse set of GLIA and glutenin (GLT) peptides, including 6 novel epitopes. Moreover, individual patients respond to distinct (combinations of) GLU peptides. T-cell cross-reactivity toward homologous GLIA and GLT peptides was observed, which might play a role in the initial spreading of the GLU-specific T-cell response. Although all pediatric patients displayed deamidation-dependent responses, deamidation-independent responses were found in the majority of patients as well. Finally, T-cell responses to 3 of these novel GLU peptides were found in adult CD patients. CONCLUSIONS: The diversity of the GLU-specific T-cell response is far greater than was previously appreciated. Both adult and young CD patients can respond to a diverse repertoire of GLU peptides. The observation that T-cell responses to 3 of the novel peptides are independent of deamidation indicates that T-cell responses can be initiated toward native GLU peptides. The possibility that deamidation drives the GLU response toward immunodominant T-cell stimulatory peptides after disease initiation is discussed.