Proteolytic Characteristics of Cathepsin D Related to the Recognition and Cleavage of Its Target Proteins

Huiying Sun,Jia Zhang,Siqi Liu,Xu Zhu,Ju Zhang,Ningzhi Xu,Yang Wang,Yingying Xie,Qiang Shan,Xiaomin Lou,Sonja Hess

doi:10.1371/journal.pone.0065733

Abstract

Cathepsin D (CD) plays an important role in both biological and pathological processes, although the cleavage characteristics and substrate selection of CD have yet to be fully explored. We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the CD cleavage sites in bovine serum albumin (BSA). We found that the hydrophobic residues at P1 were not only a preferential factor for CD cleavage but that the hydrophobicity at P1’ also contributed to CD recognition. The concept of hydrophobic scores of neighbors (HSN) was proposed to describe the hydrophobic microenvironment of CD recognition sites. The survey of CD cleavage characteristics in several proteins suggested that the HSN was a sensitive indicator for judging the favorable sites in peptides for CD cleavage, with HSN values of 0.5–1.0 representing a likely threshold. Ovalbumin (OVA), a protein resistant to CD cleavage in its native state, was easily cleaved by CD after denaturation, and the features of the cleaved peptides were quite similar to those found in BSA, where a higher HSN value indicated greater cleavability. We further conducted two-dimensional gel electrophoresis (2DE) to find more proteins that were insensitive to CD cleavage in CD-knockdown cells. Based on an analysis of secondary and three-dimensional structures, we postulated that intact proteins with a structure consisting of all α-helices would be relatively accessible to CD cleavage.

Highlights

Cathepsins are a class of lysosomal proteases that play important roles in proteolysis during physiological processes
In accordance with previous observations, we found that Cathepsin D (CD) exhibited the highest bovine serum albumin (BSA) cleavage activity at pH 3.5
Considering that CD cleavage is likely correlated with the hydrophobic microenvironment contributed by P1 and P1’, we introduce a new concept to describe the hydrophobicity of neighboring residues referred to as the hydrophobic scores of neighbors (HSN), where an HSN value represents the sum of the hydrophobic scores of two neighboring residues

Summary

Introduction

Cathepsins are a class of lysosomal proteases that play important roles in proteolysis during physiological processes. They are reportedly involved in a number of diseases, such as cancer [1,2,3], atherosclerosis [4], arthritis [5] and neurodegenerative diseases [6,7]. Identification of the native substrates and cleavage sites of cathepsins is necessary to understand their physiological and pathological roles. CD recognizes its substrate with a relatively low selectivity It does not function on some proteins under certain circumstances, such as hen egg white lysozyme and ovalbumin (OVA) [22,23]. In the field of CD research, it remains to be systematically evaluated which cleavage sites of target proteins, for native forms, are favored for CD action

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Results

Conclusion