ovalbumin and pepstatin

Allergenic potential of food proteins is associated with stability to gastric and pancreatic digestive enzymes. However, much attention has not been focused on intracellular digestion of protein antigens during the passage through intestinal epithelia. We report here the degradation and survival of a bis-phosphorylated protein, ovalbumin (OVA), in the course of passage through Caco-2 cell monolayers cultured on porous membrane. SDS-PAGE in combination with phosphoprotein staining showed that OVA, which had passed through the cell layers, was almost intact in its polypeptide chain but partly dephosphorylated. By contrast, quantitative analysis using ELISA indicated that complete dephosphorylation in advance by an alkaline phosphatase markedly reduced the OVA passage. The reduced passage was restored in the presence of cathepsin inhibitors, leupeptin and pepstatin-A. Moreover, the complete dephosphorylation increased susceptibility of OVA to in vitro digestion with cathepsin B, which cleaved near an OVA phosphorylation site, Ser345. The susceptibility of OVA to lysosomal proteases may affect its passage through the intestinal epithelia, leading to determination of allergic sensitization and elicitation in egg allergy.

Topics: Alkaline Phosphatase; Animals; Antigens; Caco-2 Cells; Cathepsins; Cysteine Proteinase Inhibitors; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Female; Humans; Intestinal Mucosa; Leupeptins; Mice; Mice, Inbred Strains; Ovalbumin; Pepstatins; Phosphorylation; Protease Inhibitors

Cathepsin E is an aspartic proteinase that has been implicated in Ag processing within the class II MHC pathway. In this study, we document the presence of cathepsin E message and protein in human myeloid dendritic cells, the preeminent APCs of the immune system. Cathepsin E is found in a perinuclear compartment, which is likely to form part of the endoplasmic reticulum, and also a peripheral compartment just beneath the cell membrane, with a similar distribution to that of Texas Red-dextran within 2 min of endocytosis. To investigate the function of cathepsin E in processing, a new soluble targeted inhibitor was synthesized by linking the microbial aspartic proteinase inhibitor pepstatin to mannosylated BSA via a cleavable disulfide linker. This inhibitor was shown to block cathepsin D/E activity in cell-free assays and within dendritic cells. The inhibitor blocked the ability of dendritic cells from wild-type as well as cathepsin D-deficient mice to present intact OVA, but not an OVA-derived peptide, to cognate T cells. The data therefore support the hypothesis that cathepsin E has an important nonredundant role in the class II MHC Ag processing pathway within dendritic cells.

Topics: Animals; Antigen Presentation; Aspartic Acid Endopeptidases; Cathepsin D; Cathepsin E; Cells, Cultured; Dendritic Cells; Down-Regulation; Humans; Intracellular Fluid; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Transgenic; Ovalbumin; Pepstatins; RNA, Messenger; T-Lymphocytes

Development of type-II collagen (CII)-induced arthritis (CIA) is dependent on activation of CII-reactive T cells. Dendritic cells (DCs) are believed to play a crucial role in antigen-specific priming of T cells but it is still unclear how the CII-reactive T cells are primed since Langerhans cells (LCs) are poor antigen-presenting cells for CII. In the present study we show that LCs, treated with cysteine protease inhibitors, are able to process and present CII to T-cell hybridomas specific for the immune-dominant glycosylated 259-270 peptide bound to the MHC class II molecule A(q). Interestingly, the self (mouse) CII peptide could also now be efficiently presented. The poor presentation by LCs is a peptide-specific effect, since both bovine CII (bCII) (presenting a different peptide on H-2(r)) and ovalbumin could be efficiently presented, and blockage of cysteine proteases did not enhance antigen presentation. The enhanced CII-presentation by cysteine protease inhibition is seen mainly in LCs and not in antigen-primed B cells or macrophages. B cell and macrophage presentation of CII occur even without protease inhibition and are only to a minor extent influenced by cysteine protease inhibition. These data suggest that a LC deficiency in processing of the immune-dominant CII epitope in both CIA and RA may limit the exposure of this self-antigen to T cells, but that presentation can be overcome by modulation of the peptide proteolysis during CII processing.

Topics: Animals; Antigen Presentation; Autoantigens; B-Lymphocytes; Cartilage; Collagen Type II; Cysteine Endopeptidases; Dendritic Cells; Genes, MHC Class II; Langerhans Cells; Leucine; Macrophages; Mice; Ovalbumin; Pepstatins; Peptides; Rats; T-Lymphocytes

The effect of highly selective inhibitors of cathepsins on the processing of ovalbumin (OVA) and the presentation of an OVA-derived antigenic peptide (OVA323-339) by antigen presenting cells (APC) was investigated. Both CA-074 (a specific inhibitor of cathepsin B) and pepstatin A (a specific inhibitor of cathepsin D) showed an inhibitory effect on the IL-2 production from an OVA-specific, I-Ad-restricted helper T (Th) cell clone upon stimulation with OVA presented by the I-Ad-positive APC. In contrast, the presentation of the antigenic epitope, OVA323-339, to the same Th clone was not inhibited by either CA-074 or pepstatin A alone, nor even by the mixture of both inhibitors. When APC were treated with cathepsin inhibitor for 24 h, and then antigen and Th were added to the culture, the presentation of not only OVA but also an OVA-derived antigenic peptide was inhibited by either cathepsin inhibitor alone. In addition, the expression of invariant chain on APC was significantly augmented by the pretreatment of APC with either cathepsin inhibitor. Two main conclusions are drawn from these results. First, not only aspartyl protease, such as cathepsin D, but also thiol protease, such as cathepsin B, is involved in antigen processing by APC. Second, both cathepsin B and cathepsin D are necessary for degradation of the invariant chain (Ii) from the MHC class II alpha beta heterodimer in endosomes in order to express functional MHC class II molecules for binding antigenic peptides.

Topics: Animals; Antigen Presentation; Antigen-Presenting Cells; Antigens, Differentiation, B-Lymphocyte; B-Lymphocytes; Cathepsin B; Cathepsin D; Cell Line; Cysteine Proteinase Inhibitors; Dipeptides; Histocompatibility Antigens Class II; Humans; Interleukin-2; Leucine; Major Histocompatibility Complex; Mice; Ovalbumin; Pepstatins; T-Lymphocytes, Helper-Inducer; Tumor Cells, Cultured

Studies on the kinetics of antigenic peptide binding to major histocompatibility complex class II molecules have been used extensively to probe major histocompatibility complex (MHC) structure as well as to investigate the molecular mechanism of peptide recognition. Previous experiments have frequently been carried out in the presence of a cocktail of protease inhibitors to inhibit the proteolysis of MHC heterodimers. By using high performance size exclusion chromatography to measure fluorescent peptide binding to MHC protein, we have found that the addition of a commonly used mixture of protease inhibitors leads to a significant reduction in peptide binding to the class II heterodimer.

Topics: Amino Acid Sequence; Animals; Chickens; Histocompatibility Antigens Class II; Humans; In Vitro Techniques; Kinetics; Molecular Sequence Data; Ovalbumin; Pepstatins; Peptide Fragments; Peptides; Protease Inhibitors; Protein Binding

Modification of protein Ag by proteolysis is one of the principal steps in the presentation of Ag to Th cells. However, little is known about the enzymes participating in these events, their specificity or the characteristics of the natural fragments that they produce. Cathepsin D (CD) is an aspartyl protease identified in endosomes of APC. In this report, the role of CD in the processing of OVA has been investigated. OVA digested in vitro with purified CD was able to stimulate IL-2 secretion by three different OVA-specific I-Ad restricted Th cell hybridomas when it was presented by fixed APC. The digest of OVA was recognized in the context of I-Ad, but not by I-Ak-restricted OVA-specific Th cells. No difference was observed in the ability of OVA digested with CD to stimulate Th cells in the absence of FCS or in the presence of protease inhibitors indicating that extracellular proteases were not likely to contribute to processing of OVA. Taken together, these results suggest that CD is necessary and sufficient for the generation of an antigenic epitope from OVA. A fragment containing the epitope was isolated from the OVA digest by reverse phase HPLC. This fragment, which migrates in SDS-PAGE as a 10-kDa polypeptide, is a potent epitope. Its capacity to activate Th cells is compared to that of the tryptic peptide OVA323-339.

Topics: Animals; Antigen-Presenting Cells; Cathepsin B; Cathepsin D; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Histocompatibility Antigens Class II; In Vitro Techniques; Interleukin-2; Leupeptins; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred CBA; Ovalbumin; Pepstatins; T-Lymphocytes

Proteolytic degradation (processing) of antigen by antigen-presenting cells is a major regulatory step in the activation of a T lymphocyte immune response. However, the enzymes responsible for antigen processing remain largely undefined. In this study we show that cathepsin E, and not the ubiquitous lysosomal cathepsin D, is the major aspartic proteinase in a murine antigen-presenting cell line, A20. This enzyme is localized to a non-lysosomal compartment of the endosomal system in these cells. Functional studies using a highly specific inhibitor of cathepsin E show that this enzyme is essential for the processing of ovalbumin by this cell line. Thus, cathepsin E, whose function was hitherto unknown, may play a major role in antigen processing.

Topics: Animals; Antigen-Presenting Cells; Aspartic Acid Endopeptidases; B-Lymphocytes; Cathepsin E; Cathepsins; Cell Fractionation; Chromatography, Gel; Dose-Response Relationship, Immunologic; Fluorescent Antibody Technique; Histocompatibility Antigens Class II; Interleukin-2; Macrophages; Mice; Mice, Inbred CBA; Molecular Weight; Ovalbumin; Pepstatins; T-Lymphocytes

By using the model Ag, chicken OVA, the proteolytic events required for effective presentation of the antigenic epitope, OVA323-339 to H-2d-restricted Th cells were investigated. First, the ability of aspartyl and thiol proteases to generate antigenic fragments of Ova in vitro was determined. It was found that cathepsin D, an aspartyl protease, digested OVA to fragments that could be recognized by Th cells without further processing by APC. Cathepsin B, a thiol protease, was unable to generate antigenic fragments of OVA in vitro. These results provide evidence that APC do not require thiol protease activity for processing OVA. In contrast, APC were unable to present OVA to Th cells when thiol protease inhibitors were added to the incubation. Taken together, these observations indicate that thiol proteases may be important, not for processing, OVA, but for presentation of processed fragments by APC. This conclusion is supported by evidence obtained from experiments in which APC were treated with thiol protease inhibitors before addition of the antigenic peptide, OVA323-339. Under these conditions, the capacity of I-Ad at the cell surface to present OVA323-339 to Th cells was reduced. The results of these experiments provide evidence that Ag presentation of OVA may be achieved through the action of two different classes of proteases: aspartyl proteases such as cathepsin D, which process OVA to antigenic fragments, and thiol proteases such as cathepsin B, which are important for expression of functional MHC II molecules by APC.

Topics: Animals; Antigen-Presenting Cells; Aspartic Acid Endopeptidases; B-Lymphocytes; Cathepsins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Endopeptidases; Epitopes; H-2 Antigens; In Vitro Techniques; Leupeptins; Macrophages; Mice; Ovalbumin; Pepstatins; Peptide Fragments; T-Lymphocytes, Helper-Inducer