Activation of latent protease function of pro-hK2, but not pro-PSA, involves autoprocessing.
BACKGROUND
Human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) are members of an extensive kallikrein family of proteases. Both proteases are secreted as zymogens or proenzymes containing a seven amino acid propeptide that must be proteolytically removed for enzymatic activation. The physiological proteases that activate pro-hK2 and pro-PSA are not known.


METHODS
The pro-hK2 peptide sequence is Val-Pro-Leu-Ile-Gln-Ser-Arg (VPLIQSR). For PSA, the amino acid sequence of the propeptide is Ala-Pro-Leu-Ile-Leu-Ser-Arg (APLILSR). Fluorescent substrates were made by coupling these peptide sequences to 7-amino-4-methylcoumarin (AMC). The hydrolysis of the VPLIQSR-AMC and APLILSR-AMC substrates by hK2, PSA, and a panel of purified proteases was determined.


RESULTS
HK2 readily cleaved the pro-hK2 peptide substrate VPLIQSR-AMC with a rate of hydrolysis that was approximately 8-fold higher than an equimolar amount of purified trypsin. HK2 also had the highest hydrolysis rate from among a group of other trypsin-like proteases. In contrast, neither hK2 nor PSA was able to appreciably cleave the pro-PSA substrate APLILSR-AMC. The pro-PSA substrate was most readily hydrolyzed by urokinase and trypsin.


CONCLUSIONS
HK2 can hydrolyze the pro-hK2 substrate suggesting that maturation of pro-hK2 to enzymatically active hK2 involves autoprocessing. As expected, PSA, a chymotrypsin-like protease, was unable to hydrolyze either of the propeptide substrates. Therefore, it is unlikely that PSA can auto-process its own enzymatic function. HK2 has trypsin-like specificity but was unable to hydrolyze the pro-PSA substrate. These results raise the possibility that an additional processing protease may be required to fully process PSA to an enzymatically active form.
Denmeade
S R
SR
The Johns Hopkins Oncology Center, Bunting Blaustein Cancer Research Building, Baltimore, Maryland 21231, USA. denmeade@jhmi.edu
Lövgren
J
J
Khan
S R
SR
Lilja
H
H
Isaacs
J T
JT
D004790:Enzyme Induction; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008297:Male; D011467:Prostate / Q000201:enzymology*; D017430:Prostate-Specific Antigen / Q000096:biosynthesis* / Q000378:metabolism; D015398:Signal Transduction; D020840:Tissue Kallikreins / Q000096:biosynthesis* / Q000378:metabolism
D020840:Tissue Kallikreins; D017430:Prostate-Specific Antigen