Abstract
Kunitz-type serine protease inhibitors are involved in various physiological processes, such as ion channel blocking, blood coagulation, fibrinolysis, and inflammation. While spider-derived Kunitz-type proteins show activity in trypsin or chymotrypsin inhibition and K+ channel blocking, no additional role for these proteins has been elucidated. In this study, we identified the first spider (Araneus ventricosus) Kunitz-type serine protease inhibitor (AvKTI) that acts as a plasmin inhibitor and an elastase inhibitor. AvKTI possesses a Kunitz domain consisting of a 57-amino-acid mature peptide that displays features consistent with Kunitz-type inhibitors, including six conserved cysteine residues and a P1 lysine residue. Recombinant AvKTI, expressed in baculovirus-infected insect cells, showed a dual inhibitory activity against trypsin (Ki 7.34 nM) and chymotrypsin (Ki 37.75 nM), defining a role for AvKTI as a spider-derived Kunitz-type serine protease inhibitor. Additionally, AvKTI showed no detectable inhibitory effects on factor Xa, thrombin, or tissue plasminogen activator; however, AvKTI inhibited plasmin (Ki 4.89 nM) and neutrophil elastase (Ki 169.07 nM), indicating that it acts as an antifibrinolytic factor and an antielastolytic factor. These findings constitute molecular evidence that AvKTI acts as a plasmin inhibitor and an elastase inhibitor and also provide a novel view of the functions of a spider-derived Kunitz-type serine protease inhibitor.
Highlights
Kunitz-type serine protease inhibitors are ubiquitous, exist in multiple forms, and are found in numerous tissues of many organisms, including animals, plants, and microbes
AvKTI is a Spider Kunitz-type Serine Protease Inhibitor To characterize the spider-derived Kunitz-type serine protease inhibitor, we identified an expressed sequence tags (ESTs) for a gene encoding a Kunitz-type serine protease inhibitor (AvKTI) in an A. ventricosus cDNA library
Our results show that AvKTI inhibits the plasmin-mediated degradation of fibrin to fibrin degradation products (FDPs), which is consistent with an antifibrinolytic activity for AvKTI
Summary
Kunitz-type serine protease inhibitors are ubiquitous, exist in multiple forms, and are found in numerous tissues of many organisms, including animals, plants, and microbes. These protease inhibitors consist of approximately 60 amino acid residues that display features such as three disulfide bridges, which contribute to the stable nature of the folded mature peptide, and a P1 site, which corresponds to the specificity of their cognate enzymes [1,2,3]. Kunitz-type serine protease inhibitors are involved in various physiological processes, such as ion channel blocking, blood coagulation, fibrinolysis, and inflammation [3,7,17,18,19,20,21]. These Kunitz-type serine protease inhibitors appear to have a strong potential for pharmaceutical development [7,14,17,18,19,20,21]
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