Substrates of the prostate-specific serine protease prostase/KLK4 defined by positional-scanning peptide libraries.
BACKGROUND
Prostase/KLK4 is a member of the human kallikrein (KLK) gene family that is expressed in prostate epithelial cells under the regulation of androgenic hormones. In this study, we sought to characterize the substrate specificity of KLK4 in order to gain insight into potential physiological roles of the enzyme.


METHODS
A chimeric form of KLK4 was constructed in which the pro-region of KLK4 was replaced with the signal and propeptide sequence of trypsinogen (proT-KLK4) to create an activation site susceptible to enterokinase cleavage. proT-KLK4 was expressed in Drosophila S2 cells, purified, and activated with enterokinase to generate mature KLK4. The extended substrate specificity of KLK4 was defined by screening tetrapeptide positional scanning synthetic combinatorial libraries (PS-SCL).


RESULTS
The preferred P1-P4 positions as determined by PS-SCL were: P1-Arg; P2-Gln/Leu/Val; P3-Gln/Ser/Val; P4-Ile/Val. The trypsin-like specificity of KLK4 was further confirmed using synthetic chromogenic peptides. Based upon the optimal cleavage site residues, a database search for potential KLK4 substrates identified several proteins with potential roles mediating normal prostate physiology or neoplastic growth including KLK3/PSA, parathyroid hormone-related peptide (PTHrP), and members of the bone morphogenetic protein (BMP) family. Recombinant KLK4 was able to activate pro-PSA/KLK3 and degrade members of the insulin-like growth factor (IGF) binding protein (IGFBP) family.


CONCLUSIONS
These results identify potential KLK4 substrates that may serve to define the role of this protease in normal prostate physiology, and facilitate studies of the consequences of KLK4 expression in pathological conditions.
Matsumura
Masazumi
M
Division of Human Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N, Seattle, WA 98109, USA.
Bhatt
Ami S
AS
Andress
Dennis
D
Clegg
Nigel
N
Takayama
Thomas K
TK
Craik
Charles S
CS
Nelson
Peter S
PS
D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D004331:Drosophila melanogaster; D006801:Humans; D007610:Kallikreins / Q000378:metabolism*; D008297:Male; D019151:Peptide Library; D011467:Prostate / Q000502:physiology; D017430:Prostate-Specific Antigen / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000201:enzymology*; D011994:Recombinant Proteins; D013379:Substrate Specificity
D019151:Peptide Library; D011994:Recombinant Proteins; D007610:Kallikreins; C120801:kallikrein 4; D017430:Prostate-Specific Antigen