Characterization of hK4 (prostase), a prostate-specific serine protease: activation of the precursor of prostate specific antigen (pro-PSA) and single-chain urokinase-type plasminogen activator and degradation of prostatic acid phosphatase.
hK4 (prostase, KLK4), a recently cloned prostate-specific serine protease and a member of the tissue kallikrein family, is a zymogen composed of 228 amino acid residues including an amino-terminal propiece, Ser-Cys-Ser-Gln-. A chimeric form of hK4 (ch-hK4) was constructed in which the propiece of hK4 was replaced by that of prostate-specific antigen (PSA) to create an activation site susceptible to trypsin-type proteases. ch-hK4 was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified with an overall yield of 25%. The zymogen was readily self-activated during the refolding process to generate an active form (21 kDa) of hK4 (rhK4). rhK4 cleaved the chromogenic substrates Val-Leu-Arg-pNA (S-2266), Pro-Phe-Arg-pNA (S-2302), Ile-Glu-Gly-Arg-pNA (S-2222), and Val-Leu-Lys-pNA (S-2251), indicating that rhK4 has a trypsin-type substrate specificity. The rhK4 was inhibited by aprotinin (6 kDa), forming an equimolar 27 kDa complex. rhK4 readily activated both the precursor of PSA (pro-PSA) and single chain urokinase-type plasminogen activator (scuPA, pro-uPA). rhK4 also completely degraded prostatic acid phosphatase but failed to cleave serum albumin, another protein purified from human seminal plasma. These results indicate that hK4 may have a role in the physiologic processing of seminal plasma proteins such as pro-PSA, as well as in the pathogenesis of prostate cancer through its activation of pro-uPA.
Takayama
T K
TK
Departments of Biochemistry and Urology, University of Washington, Box 357350, Seattle, Washington 98195-7350, USA. tomt@u.washington.edu
McMullen
B A
BA
Nelson
P S
PS
Matsumura
M
M
Fujikawa
K
K
D000135:Acid Phosphatase; D000595:Amino Acid Sequence; D001665:Binding Sites; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007610:Kallikreins; D008297:Male; D011467:Prostate / Q000201:enzymology*; D017430:Prostate-Specific Antigen / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000209:etiology; D011498:Protein Precursors / Q000378:metabolism*; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029589:Seminal Plasma Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D011498:Protein Precursors; D011994:Recombinant Proteins; D029589:Seminal Plasma Proteins; D000135:Acid Phosphatase; C428439:prostatic acid phosphatase; D017027:Protein Tyrosine Phosphatases; D007610:Kallikreins; D012697:Serine Endopeptidases; C120801:kallikrein 4; D014568:Urokinase-Type Plasminogen Activator; D017430:Prostate-Specific Antigen