Cathepsin D, but not cathepsin B, releases T cell stimulatory fragments from lysozyme that are functional in the context of multiple murine class II MHC molecules

Abstract

In this study, the major endosomal/lysosomal proteases cathepsin D and cathepsin B were tested on their ability to release T cell stimulatory 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, cathepsin 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 fragments were screened for recognition by HEL-specific T cells from three strains of mice, i.e. B10.A (H-2a), C57BL/6 (H-2b) and BALB/c (H-2d). Peptides in a large number of different HPLC fractions triggered significant T cell responses in all three strains. Interestingly, the response profiles of T cells from the three different strains showed marked similarities. Also, several individual synthetic HEL sequences corresponding to selected cathepsin D-released fragments were recognized by murine T cells in the context of all three major histocompatibility complex (MHC) haplotypes tested. Our data suggest that cathepsin D rather than cathepsin B may play a central role in the initial release of HEL fragments during endosomal/lysosomal processing. The relatively long 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 employed by others for the delineation of HEL epitopes. Extensive documentation of HEL epitopes in previous investigations indicate that this promiscuity cannot be explained by simply assuming that longer peptides contain additional epitopes. Rather, an increased peptide length by itself appears to promote promiscuous MHC binding.

Differential Regulation of the Nature and Functions of Dendritic Cells and Macrophages by Cathepsin E

“…Given that Ag processing is a key determinant of the quality and quantity of a CD4 T cell response, it is of particular importance to identify the specific enzymes that generate MHC class II-bound peptides in vivo. Although previous studies indicated that the aspartic proteinases cathepsins D and/or E would be required for MHC class II peptide presentation (19,20,22), subsequent studies with APC deficient in cathepsin D demonstrated that this protein would be dispensable for this pathway (23, 25, 26). However, it remains unclear whether cathepsin E is essential for MHC class II Ag presentation.…”

Substrate Specificity of Cathepsins D and E Determined by N‐Terminal and C‐terminal Sequencing of Peptide Pools

“…In other cases, where no putative cathepsin D or E motif was found directly at the N-terminal cleavage site of the natural class I1 ligands, potential cathepsin D or E cleavage sites are located in the N-terminal extension of the class I1 ligands (Table 3, bottom). It is possible that in this case, the cleavage by acidic cathepsins is followed by the activity of another protease, most likely with aniiiiopeptidase activity (Werdelin, 1986 ;Falk et al, 1994;van Noort and Jacobs, 1994;Nelson et al, 1997). The knowledge of the substrate specificity of cathepsin D and E is therefore of immunological relevance since this information will be helpful in the process of predicting and identifying MHC class I1 ligands and designing synthetic peptide vaccines.…”

“…The enzymes responsible for antigen processing remain largely undefined, but functional studies suggested that both cathepsins E and D play an important role in the processing of exogenous antigens (Bennett et al, 1992;Rodriguez and Diment, 1992;van Noort and Jacobs, 1994). Several investigations have shown that antigen presentation is inhibited in vitm after treatment with pepstatin, an inhibitor of aspartic proteases (Puri and Factorovich, 1988;Diment, 1990).…”

Functional Requirements for the Lysosomal Thiol Reductase GILT in MHC Class II-Restricted Antigen Processing

“…Hen egg lysozyme (HEL) is an excellent model Ag for evaluating the role of protein structure in Ag processing because it has four intrachain disulfide bonds (11) and is resistant to proteolytic cleavage without prior reduction (12). Intracellular processing of HEL to generate the I-A b -restricted HEL peptide involving residues 74 -88 (HEL 74 -88 ) is entirely dependent on the presence of GILT (8).…”