Role of cathepsin D in antigen presentation of ovalbumin.
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.
Rodriguez
G M
GM
Michael Heidelberger Division of Immunology, Department of Pathology, New York University Medical Center, NY 10016.
Diment
S
S
D000818:Animals; D000938:Antigen-Presenting Cells / Q000276:immunology*; D002401:Cathepsin B; D002402:Cathepsin D / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D066298:In Vitro Techniques; D007376:Interleukin-2 / Q000096:biosynthesis; D007976:Leupeptins / Q000494:pharmacology; D008264:Macrophages / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008808:Mice, Inbred CBA; D010047:Ovalbumin / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D013601:T-Lymphocytes / Q000276:immunology*
D000949:Histocompatibility Antigens Class II; D007376:Interleukin-2; D007976:Leupeptins; D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D010047:Ovalbumin; D002401:Cathepsin B; D002402:Cathepsin D; C032854:leupeptin; C031375:pepstatin