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

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.

Figure 1. The cleavage efficiency and cleavage characteristics of CD in BSA.

A, The sensitivity of BSA and OVA to CD cleavage was examined via SDS-PAGE after the two proteins were incubated with/without CD and pepstatin A. B, The CD specificity profile for BSA is depicted using sequence logos. Six amino acids are represented as P6…P1 and P1’…P6’, which are located at N- and C-termini near the scissile site, respectively. The sequence logo ordinate is scaled in bits based on Schneider method . Color coding: acidic residues in red, basic residues in blue, polar residues in green and hydrophobic residues in black. C, The hydrophobicity distributions of all the residues at 12 positions around the termini of the CD-cleaved BSA peptides were estimated with box plots. D, Comparison of the median values of T-P1+P1’, C-P1+P1’ and U-P1+P1’ in BSA. The analysis was performed for the six amino acids whose occurrence rates at P1 received the top ranks. E, Comparison of the HSN distributions between the cleaved and undetected peptides from BSA. The HSN values were broadly divided into 12 intervals.

Figure 2. Analysis of CD cleavage characteristics in denatured OVA.

A, Sequence logos of the CD cleavage sites in native OVA (left) and denatured OVA (right). B, The sensitivity of denatured OVA to CD cleavage was examined via SDS-PAGE, in which the denatured protein was incubated with/without CD and pepstatin A for 3 h. C, The median T-HSNs, C-HSNs and U-HSNs in denatured OVA are depicted using box plots. D, Comparison of the HSN distributions between the cleaved and undetected peptides from denatured OVA.

Figure 3. Analysis of hydrophobicity distributions in the CCPD.

A, The hydrophobicity distributions of all the residues at 12 positions around the termini of the peptides in the CCPD were estimated with box plots. B, The HSN profile in the CCPD. The occurrence frequencies of all the HSNs in the CCPD were plotted against the HSN axis, which was divided into 14 intervals. C, Comparison of the HSN distributions between the cleaved and undetected peptides in the CCPD. The HSN values were estimated for the top 6 amino acids.

Figure 4. Screening of proteins that were insensitive to CD cleavage in A549-CR cells.

A, The expression of CD in A549 cells was evaluated by Western blotting. The control was transfected with empty vector. B, Comparison of the 2DE images obtained from lysates of A549-CR cells treated with/without CD. The insets represent the typical 2DE images showing spots that were sensitive or insensitive to CD cleavage.