title: Isolation of a membrane-associated cathepsin D-like enzyme from the model antigen presenting cell, A20, and its ability to generate antigenic fragments from a protein antigen in a cell-free system.
abstract: The biochemical mechanisms and enzymes involved in the processing of protein antigens for presentation by major histocompatibility complex class II molecules are poorly understood. This work describes the purification of a cathepsin D-like enzyme isolated from the murine B lymphoma cell line A20, a model antigen presenting cell. Two forms of cathepsin D-like enzyme were detected. One is soluble and located in the lysosome-enriched subcellular fraction. The other is membrane-associated and located in the endosome-enriched fraction. The membrane-associated form was purified to apparent homogeneity by affinity chromatography on pepstatin A-Sepharose. Its apparent molecular weight is 48,000, and its pH optimum is pH 4.0. Endosomal cathepsins are known to be involved in antigen processing in vivo, and the purified membrane-associated cathepsin D-like enzyme from A20 cells was used to study antigen processing in vitro. The enzyme cleaved a model protein antigen, Staphylococcus aureus nuclease (Nase) and thereby generated antigenic fragments recognized by a Nase-specific T cell hybridoma. Such studies have allowed us to begin to understand the role of protease specificity and T cell determinant selection.
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston.
authors.forename: K P
authors.initials: KP
authors.lastname: Williams
authors.forename: J A
authors.initials: JA
authors.lastname: Smith
mesh_terms: D000818:Animals; D000938:Antigen-Presenting Cells / Q000201:enzymology*; D000942:Antigens, Bacterial / Q000378:metabolism*; D002402:Cathepsin D / Q000302:isolation & purification*; D002460:Cell Line; D011992:Endosomes / Q000201:enzymology; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D051379:Mice; D008836:Micrococcal Nuclease / Q000276:immunology / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000201:enzymology
chemical_list: D000942:Antigens, Bacterial; D011480:Protease Inhibitors; D008836:Micrococcal Nuclease; D002402:Cathepsin D