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.

Mass spectrometric characterization of limited proteolysis activity in human plasma samples under mild acidic conditions

“…In-vitro cleavages of HSA by cathepsin D have been published [10,69]. The experimental settings rather match with conditions used for high substrate turnover.…”

Proteolytic Characterization and Lysosomal Localization of Echinoderm Cathepsin D

“…40 µL of each mixture was added with SDS sample buffer and loaded to a 10% SDS PAGE. The cleaved products were visualized using silver nitrate staining [17].…”

“…In addition to this, the hydrophobic scores formed by neighboring amino acids of the cleavage site also add to the efficiency of the cleavage [17]. The active site of the cathepsin D is formed by two aspartic acid residues located at 33 and 231 positions in the order Asp-Thr-Gly [11].…”

Proteolytic resistance of actin but not of myosin heavy chain during processing of Italian PDO (protected designation of origin) dry-cured hams

“…1b, c). Interestingly, at least 5 potential cleavage sites for cathepsin B [48] and two for cathepsin D [49] are present just above Ala 769, the first identified residue of the 135-kDa fragment.…”