Purification and characterization of human kallikrein 11, a candidate prostate and ovarian cancer biomarker, from seminal plasma

Liu Ying Luo, Shannon J.C. Shan, Marc B. Elliott, Antoninus Soosaipillai, Eleftherios P. Diamandis

Research output: Contribution to journalArticle

Abstract

Purpose: Preliminary data suggest that hK11 is a novel serum biomarker for prostate and ovarian cancer. To examine the enzymatic characteristics of hK11, we purified and functionally characterized native hK11 from seminal plasma. Experimental Design: hK11 was purified from seminal plasma by immunoaffinity chromatography and characterized by kinetic analysis, electrophoresis, Western blots, and mass spectrometry. Results: hK11 is present in seminal plasma at concentrations ranging from 2 to 37 μg/mL. Using immunoaffinity chromatography and reverse-phase high-performance liquid chromatography, we purified hK11 to homogeneity. In seminal plasma, hK11 is present as a free enzyme of ∼40 kDa. About 40% of hK11 is enzymaticalty active, whereas the rest is inactivated by internal cleavage after Arg156 (Genbank accession no. AF164623), which generates two peptides of ∼20 kDa, connected by internal disulfide bonds. Purified hK11 possesses trypsin-like activity and cleaves synthetic peptides after arginine but not lysine residues. It does not cleave chymotrypsin substrates. Antithrombin, α1- antichymotrypsin, α2-antiplasmin, and α1- antitrypsin have no effect on hK11 activity and do not form complexes with hK11 in vitro. The strongest inhibitor, APMSF, completely inhibited hK11 activity at a concentration of 2.5 mmol/L. Aprotinin and an hK11-specific monoclonal antibody inhibited hK11 activity up to 40%. Plasmin is a strong candidate for cleaving hK11 at Arg156. Conclusion: This is the first report on purification and characterization of native hK11. We speculate that hK11, along with other kallikreins, proteases, and inhibitors, participates in a cascade enzymatic pathway responsible for semen liquefaction after ejaculation.

Original languageEnglish (US)
Pages (from-to)742-750
Number of pages9
JournalClinical Cancer Research
Volume12
Issue number3 I
DOIs
StatePublished - Feb 1 2006

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ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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