title: Determination of cathepsin S abundance and activity in human plasma and implications for clinical investigation.
abstract: There is strong experimental evidence associating cathepsin S with the pathogenesis of atherosclerosis, with emerging data to support its role in diseases such as abdominal aortic aneurysm, obesity, and type 2 diabetes. To further our understanding of cathepsin S, we have developed a novel sandwich immunoassay to measure the mature form of cathepsin S in plasma (mean values from 12 healthy donors of 53±17ng/ml, range=39-102). We also developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to measure in vitro cathepsin S activity to compare activity levels with the protein mass levels determined by enzyme-linked immunosorbent assay (ELISA). Interestingly, we observed that only 0.4 to 1.1% of circulating cathepsin S was enzymatically active. We subsequently demonstrated that the attenuated activity we observed resulted from binding between cathepsin S and its endogenous inhibitor cystatin C in plasma. These data were obtained through immunoprecipitation coupled with either Western blotting analysis or in-gel tryptic digestion and LC-MS/MS characterization of Coomassie-stained gel bands. Although many laboratories have explored the relationship between cathepsin S and cystatin C, this is the first study to demonstrate their association in human circulation, a finding that could prove to be important in furthering our understanding of cathepsin S biology.
authors.affiliation: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA. coxje@lilly.com
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Cox
authors.forename: Jason S
authors.initials: JS
authors.lastname: Troutt
authors.forename: Michael D
authors.initials: MD
authors.lastname: Knierman
authors.forename: Robert W
authors.initials: RW
authors.lastname: Siegel
authors.forename: Yue-Wei
authors.initials: YW
authors.lastname: Qian
authors.forename: Bradley L
authors.initials: BL
authors.lastname: Ackermann
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Konrad
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002403:Cathepsins / Q000097:blood* / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D055316:Cystatin C / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D047468:Immunoprecipitation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D000911:Antibodies, Monoclonal; D055316:Cystatin C; D011994:Recombinant Proteins; D002403:Cathepsins; C028217:cathepsin S