Plasminogen hydrolysis by cathepsin S and identification of derived peptides as selective substrate for cathepsin V and cathepsin L inhibitor

Abstract

Plasminogen is a glycoprotein implicated in angiogenesis and fibrin clot degradation associated with the release of angiostatin and plasmin activation, respectively. We have recently reported that cathepsin V, but not cathepsins L, B, and K, can release angiostatin-like fragments from plasminogen. Here, we extended the investigation to cathepsin S which has been implicated in angiogenesis and tumor cell proliferation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of plasminogen hydrolysis by cathepsin S revealed generation of two fragments (60 and 38 kDa). Amino-terminal sequencing indicated that cleavage occurs at the Leu469-Leu470 peptide bond. In contrast to cathepsin V, which possesses antiangiogenic activity, cathepsin S plasminogen cleavage products were not capable of inhibiting angiogenesis on endothelial cells. Moreover, we explored the different selectivities presented by cathepsins V and S towards plasminogen and synthesized fluorescence resonance energy transfer peptides encompassing the hydrolyzed peptide bonds by both enzymes. The peptide Abz-VLFEKKQ-EDDnp (Abz=ortho-aminobenzoic acid; EDDnp= N-[2,4-dinitrophenyl]ethylenediamine), hydrolyzed by cath-epsin V at the Phe-Glu bond, is a selective substrate for the enzyme when compared with cathepsins B, L, and S, whereas Abz-VLFEKKVYLQ-EDDnp is an efficient cathepsin L inhibitor. The demonstrated importance of the S(3)'-P(3)' interaction indicates the significance of the extended subsites for enzyme specificity and affinity.

Proteomic Identification of Protease Cleavage Sites Characterizes Prime and Non-prime Specificity of Cysteine Cathepsins B, L, and S

“…At the latter pH, slight P1′ preferences for glutamine (2.2 times its natural abundance at pH 7.5 and 1.9 times at pH 6.0) and serine (2.3 times its natural abundance at pH 1.2 times at pH 6.0) are also apparent. Cathepsin S cleavage assays with single proteins did not reveal explicit P1 preferences. , P1′ profiling experiments with peptide libraries indicate a mixed preference for either small or hydrophilic residues or aliphatic amino acids. ,, Similar to cathepsin L and in line with a previous P1′ specificity study, cathepsin S does not share the cathepsin B preference for P1′ phenylalanine, although a structural study illustrates that an aromatic group can be accommodated in the S1′ subsite …”

“…As for cathepsin B and L, the cathepsin S1 subsite has been described as shallow. , Enzyme–substrate interactions are thought to involve only backbone atoms of the peptidic substrate . Additional studies indicate broad P1 specificity. ,,,, …”

“…96 Additional studies indicate broad P1 specificity. 52,68,92,93,98 In P1 0 , cathepsin S displays a preference for glycine that is more pronounced at pH 6.0 than at pH 7.5. At the latter pH, slight P1 0 preferences for glutamine (2.2 times its natural abundance at pH 7.5 and 1.9 times at pH 6.0) and serine (2.3 times its natural abundance at pH 1.2 times at pH 6.0) are also apparent.…”

Papain-like peptidases: structure, function, and evolution

“…Recent research has also identified a plethora of extracellular substrates for cathepsin S, including the basement membrane component nidogen-1 (115) , the epithelial sodium channel (116) , leptin (117) , and plasminogen (118) . Similar to cathepsins B and X, cathepsin S has also been found associated with the cell surface (119) .…”

Cathepsin S-Mediated Negative Regulation of Wnt5a/SC35 Activation Contributes to Ischemia-Induced Neovascularization in Aged Mice

“…[10][11][12] In vitro, CatS inhibition impaired EC tubulogenesis via the prevention of leptin and plasminogen hydrolysis. 20,21 In a mouse model of multistage murine pancreatic islet cell carcinogenesis, CatS deficiency mitigated angiogenesis and neoplastic progression via the modulation of type IV collagen-derived anti-angiogenic peptides and the generation of bioactive pro-angiogenic γ 2 fragments from laminin-5. 22 However, the mechanism by which CatS controls neovascularization in response to ischemia in aged animals and humans is poorly understood.…”