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


METHODS
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 microg/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 approximately 40 kDa. About 40% of hK11 is enzymatically active, whereas the rest is inactivated by internal cleavage after Arg156 (Genbank accession no. AF164623), which generates two peptides of approximately 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, alpha1-antichymotrypsin, alpha2-antiplasmin, and alpha1-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.


CONCLUSIONS
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.
Luo
Liu-Ying
LY
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada.
Shan
Shannon J C
SJ
Elliott
Marc B
MB
Soosaipillai
Antoninus
A
Diamandis
Eleftherios P
EP
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D014408:Biomarkers, Tumor / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010051:Ovarian Neoplasms / Q000201:enzymology* / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000201:enzymology* / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012661:Semen / Q000201:enzymology* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013997:Time Factors
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D015842:Serine Proteinase Inhibitors; C114880:trypsin-like serine protease; D012697:Serine Endopeptidases