Substrate specificity of cathepsins D and E determined by N-terminal and C-terminal sequencing of peptide pools

被引:41
作者
Arnold, D
Keilholz, W
Schild, H
Dumrese, T
Stevanovic, S
Rammensee, HG
机构
[1] Department of Immunology, Institute for Cell Biology, Eberhard-Karls-University, Tübingen
[2] Department of Immunologie, Institute for Cell Biology, D-72076 Tübingen
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1997年 / 249卷 / 01期
关键词
cathepsin D; cathepsin E; substrate specificity; N-terminal and C-terminal pool sequencing of peptides; antigen processing;
D O I
10.1111/j.1432-1033.1997.t01-1-00171.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Degradation of protein antigens by cellular proteases is a crucial step in the initiation of a T-cell-mediated immune response. But still little is known about the enzymes responsible fur the processing of antigens, including their specificity. In this paper, we show that the combination of automated N-terminal sequencing with a newly developed method for C-terminal sequencing of peptide pools generated by the aspartic proteases cathepsins D and E is a fast and easy method to obtain detailed information of the substrate specificity of these endopeptidases. Using a 15-residue synthetic peptide library and a native protein as substrates, we confirm and extend the knowledge about the cleavage motif of cathepsin E where positions P1 and P1' of the substrate must be occupied exclusively by hydrophobic amino acids with aromatic or aliphatic side chains. However, Val and Ile residues are not allowed at position P1. Position P2' accepts a broad range of amino acids, including charged and polar ones. Additional requirements concerning the substrate positions P3' and P4' were also defined by pool sequencing. Furthermore, analysis of melittin digests with the aspartic proteases cathepsin D and E provided evidence that both enzymes share the same cleavage motif, identical to the one derived from the peptide library and the native protein. Therefore, pool sequencing analysis is a valuable and fast tool to determine the substrate specificity of any endopeptidase.
引用
收藏
页码:171 / 179
页数:9
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