Antigen processing for presentation by class II major histocompatibility complex requires cleavage by cathepsin E

Abstract

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.

Human cathepsin E produced in E. coli

“…Values for k~a, were derived from Vma X = k,.,, " [E] where the active concentration of individual preparations of enzyme was determined by active site titration against pepstatin as described previously [18]. The protein inhibitor from Ascaris lumbricoides and the synthetic inhibitor H-77 described previously [4,7,17], were kindly provided by Dr. R.J. Peanasky, University of South Dakota, USA and Dr. P.A. Charlton (formerly) of Glaxo, Greenford, UK, respectively.…”

“…inhibitor from the parasitic worm, Ascaris lumbricoides [4,7,14,18]. Consequently, an inhibition constant (Ki) was determined for the interaction of this protein with the recombinant enzyme.…”

“…The most recently characterised enzyme is cathepsin E [4], which has been implicated in the biogenesis of the vasoconstrictor peptide, endothelin [5] and to have a possible role in enabling infection of cells by the human immunodeficiency virus [6]. Recently, it has also been identified, for the first time unequivocally, to be the enzyme responsible for processing of a defined antigen (ovalbumin) in a defined lymphocyte system [7].…”

“…Production (of the precursors) of the secretory enzymes, pepsin and renin, and the membrane-encapsulated lysosomal cathepsin D in recombinant form [8][9][10] has greatly facilitated investigations into structure/function relationships in addition to providing sufficient material for 3-dimensional structural analysis by X-ray crystallography [11][12][13]. Cathepsin E is neither a secretory nor a lysosomal enzyme [7,14]; thus this intracellular aspartic proteinase is difficult to isolate in reasonable amounts. With its potential involvement in the processing reactions itemised above, it was considered of interest to produce cathepsin E in recombinant form to facilitate its molecular characterisation.…”

Rabbit procathepsin E and cathepsin E

“…Cathepsin E has been suggested to play an important role in the intracellular processing or degradation of proteins, for example, in the processing of antigen in tissues associated with the immune system [17], in self-destruction of erythrocytes [lo] and the surface epithelial cells of the stomach [7]. However, it remains to be determined whether or not cathepsin E functions under physiological conditions, since the enzyme has been shown to be maximally active against protein substrates at around pH 2 [5, 8, 9, 11, 161.…”