Defining the extended substrate specificity of kallikrein 1-related peptidases.
Human kallikrein 1-related peptidases (KLKs) form a subfamily of 15 extracellular (chymo)tryptic-like serine proteases. KLKs 4, 5, 13 and 14 display altered expression/activity in diverse pathological conditions, including cancer. However, their distinct (patho)physiological roles remain largely uncharacterized. As a step toward distinguishing their proteolytic functions, we attempt to define their primary and extended substrate specificities and identify candidate biological targets. Heterologously expressed KLKs 4, 5, 13 and 14 were screened against fluorogenic 7-amino-4-carbamoylmethylcoumarin positional scanning-synthetic combinatorial libraries with amino acid diversity at the P1-P4 positions. Our results indicate that these KLKs share a P1 preference for Arg. However, each KLK exhibited distinct P2-P4 specificities, attributable to structural variations in their surface loops. The preferred P4-P1 substrate recognition motifs based on optimal subsite occupancy were as follows: VI-QSAV-QL-R for KLK4; YFWGPV-RK-NSFAM-R for KLK5; VY-R-LFM-R for KLK13; and YW-KRSAM-HNSPA-R for KLK14. Protein database queries using these motifs yielded many extracellular targets, some of which represent plausible KLK substrates. For instance, cathelicidin, urokinase-type plasminogen activator, laminin and transmembrane protease serine 3 were retrieved as novel putative substrates for KLK4, 5, 13 and 14, respectively. Our findings may facilitate studies on the role of KLKs in (patho)physiology and can be used in the development of selective KLK inhibitors.
Borgoño
Carla A
CA
Department of Laboratory Medicine and Pathobiology, University of Toronto and Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON, Canada.
Gavigan
Julie-Ann
JA
Alves
Juliano
J
Bowles
Ben
B
Harris
Jennifer L
JL
Sotiropoulou
Georgia
G
Diamandis
Eleftherios P
EP
D000595:Amino Acid Sequence; D006801:Humans; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D020840:Tissue Kallikreins / Q000737:chemistry / Q000378:metabolism*
D010447:Peptide Hydrolases; D020840:Tissue Kallikreins