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.
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.
Williams
K P
KP
Department of Molecular Biology, Massachusetts General Hospital, Boston.
Smith
J A
JA
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
D000942:Antigens, Bacterial; D011480:Protease Inhibitors; D008836:Micrococcal Nuclease; D002402:Cathepsin D