title: 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.
authors.affiliation: Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China.
authors.forename: Huiying
authors.initials: H
authors.lastname: Sun
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Lou
authors.forename: Qiang
authors.initials: Q
authors.lastname: Shan
authors.forename: Ju
authors.initials: J
authors.lastname: Zhang
authors.forename: Xu
authors.initials: X
authors.lastname: Zhu
authors.forename: Jia
authors.initials: J
authors.lastname: Zhang
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: Yingying
authors.initials: Y
authors.lastname: Xie
authors.forename: Ningzhi
authors.initials: N
authors.lastname: Xu
authors.forename: Siqi
authors.initials: S
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002402:Cathepsin D / Q000737:chemistry*; D002417:Cattle; D045744:Cell Line, Tumor; D002645:Chickens; D016384:Consensus Sequence; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D017433:Protein Structure, Secondary; D059748:Proteolysis; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013379:Substrate Specificity; D034421:Sus scrofa
chemical_list: D010446:Peptide Fragments; D012710:Serum Albumin, Bovine; D010047:Ovalbumin; C533922:CTSD protein, human; D002402:Cathepsin D