Investigation To The Functional Mechanism Of Cathepsin D As A Protease

Cathepsin D (CD) is an important protease that functions in both lysosome and extracellular matrix (ECM). It is reported that abnormal abundance of CD is often detected in many cancer tissues and cells even in the blood of cancer patients. Although CD is proposed to hydrolyze ECM proteins leading to cancer invasion and metastasis, its cleavage characteristic and substrate selectivity are still largely unknown. In this study, we attempt to identify the cleavage sites of CD in typical CD sensitive/resistant proteins, and to abstract the characteristics of the linear and spatial structures of its target proteins.We constructed an A549 CD-knockdown stable cell line via RNAi. Through analyzing the CD levels in the conditional media of the with/without CD-knockdown A549 cells upon Western blot, two-dimensional gel electrophoresis (2DE) and mass spectrometry, we confirmed that the CD abundance in the A549 CD-knockdown cells was dramatically decreased, and almost no CD was detected in the secretion media for culturing such cells. The results based on the assay of wound scratch and transwell migration revealed that the ability of cell migration of the A549 CD-knockdown cells was attenuated significantly. We thus successfully verified CD performing an influence on tumor cell invasion and migration in our lab, and fully realized a necessity in identifying the proteolytic characteristics of CD.We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the CD cleavage sites in bovine serum albumin (BSA), a typical CD substrate. We found that the hydrophobic residues at P1 (the first residue at the C-ternimal of a peptide) were not only a preferential factor for CD cleavage, but that at P1’(the first residue at the N-ternimal of a peptide) also contributed to CD recognition. For the first time, we proposed the concept of hydrophobic scores of neighbors (HSN) to describe the hydrophobic microenvironment of CD recognition sites, the higher HSN value, the easier cleavability. Ovalbumin (OVA), a typical protein resistant to CD cleavage, was taken for evaluation of its cleavability by CD. In the native state, OVA was not cleavable even incubation with CD until 24 h under an optimal reaction condition, whereas in the denature state, OVA was easily cleaved by CD. Importantly, the features of the cleaved peptides of OVA were quite similar to those found in BSA, in which the CD cleavable sites in OVA held higher HSN values. The results suggested that once CD could access to the cleavage sites of BSA or OVA, HSN was a determinative factor for CD cleavage.We postulated that HSN was globally suitable to evaluate the cleavage possibility of CD to many other proteins. To test the hypothesis, we selected three kinds of proteins as the potential substrate candidates of CD, the proteins as common CD substrates, the protein family members sharing similar protein structures and varying amino acid sequences and the proteins with autolysis. With the same proteomic method as did in the BSA analysis, we identified over 500 cleavage sites of CD in these proteins and constructed a CD-cleaved peptide database (CCPD). On the basis of CCPD, a general characteristic of CD-cleaved peptides is derived, in which the cleavage features of CD in these peptides were very similar to that of found in BSA or OVA, leucine and phenylalanine ranking at top of P1 and HSN as a sensitive indicator for judging the favorable sites of CD cleavage. Furthermore, we found HSN values of 0.5-1.0 as a threshold of CD cleavability to protein.We further conducted 2DE and mass spectrometry to find more proteins that were insensitive to CD cleavage. In the A549 CD-knockdown cells, we identified 4 unique proteins that were relatively resistant to CD hydrolysis, such as nucleoside diphosphate kinase A (NDKA), thioredoxin (Trx), fatty acid-binding protein 5 (FABP5) and coactosin-like protein (COTL1). Bioinformatics analysis towards the secondary and three-dimensional structures of the proteins which were sensitive or insensitive to CD cleavage demonstrated that the proteins with all α-helix structure would be relatively accessible to CD cleavage, whereas the proteins with all β-sheet structure would be insensitive to CD cleavage. We come to a deduction that the spatial access of CD to its target proteins is primary for CD cleavage, while once a protein structure allows CD to be close, HSN values on the scissile sites become critical in CD cleavage.