Proteolytic characteristics of cathepsin D related to the recognition and cleavage of its target proteins

PLoS One. 2013 Jun 20;8(6):e65733. doi: 10.1371/journal.pone.0065733. Print 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cathepsin D / chemistry*
  • Cattle
  • Cell Line, Tumor
  • Chickens
  • Consensus Sequence
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Molecular Sequence Data
  • Ovalbumin / chemistry
  • Peptide Fragments / chemistry
  • Protein Structure, Secondary
  • Proteolysis*
  • Serum Albumin, Bovine / chemistry
  • Substrate Specificity
  • Sus scrofa

Substances

  • Peptide Fragments
  • Serum Albumin, Bovine
  • Ovalbumin
  • CTSD protein, human
  • Cathepsin D

Grant support

This work was supported by the National Basic Research Program of China (973) (2010CB912703 and 2011CB910704) and the National Natural Science Foundation of China (30800568 and 30901717). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.