CATHEPSIN-D, BUT NOT CATHEPSIN-B, RELEASES T-CELL STIMULATORY FRAGMENTS FROM LYSOZYME THAT ARE FUNCTIONAL IN THE CONTEXT OF MULTIPLE MURINECLASS-II MHC MOLECULES

In this study, the major endosomal/lysosomal proteases cathepsin D and cathepsin B were tested on their ability to release T cell stimulator y peptides from hen egg white lysozyme (HEL) in vitro. Whereas neither enzyme could cleave unreduced HEL under mild conditions, reduced HEL was readily cleaved by cathepsin D but not by cathepsin B. Instead, ca thepsin B was found to be very active in the trimming of HEL peptides after their release by cathepsin D. Following high-performance liquid chromatography (HPLC) fractionation, cathepsin D-released HEL fragment s were screened for recognition by HEL-specific cells from three strai ns of mice, i.e. B10.A (H-2(a)), C57BL/6 (H-2(b)) and BALB/c (H-2(d)). Peptides in a large number of different HPLC fractions triggered sign ificant T cell responses in all three strains. Interestingly, the resp onse profiles of T cells from the three different strains showed marke d similarities. Also, several individual synthetic HEL sequences corre sponding to selected cathepsin D-released fragments were recognized by murine T cells in the context of all three major histocompatibility c omplex (MHC) haplotypes tested. Our data suggest that cathepsin D rath er than cathepsin B may play a central role in the initial release of HEL fragments during endosomal/lysosomal processing. The relatively lo ng HEL fragments released by cathepsin D, containing about 20-30 amino acid residues, are significantly more promiscuous in murine class II MHC binding than the shorter synthetic HEL sequences previously employ ed by others for the delineation of HEL epitopes. Extensive documentat ion of HEL epitopes in previous investigations indicate that this prom iscuity cannot be explained by simply assuming that longer peptides co ntain additional epitopes. Rather, an increased peptide length by itse lf appears to promote promiscuous MHC binding.