Biochemical and Biophysical Research Communications
Characterization of the enzymatic activity of human kallikrein 6: autoactivation, substrate specificity, and regulation by inhibitors☆
Section snippets
Materials and methods
Production and purification of recombinant prohK6 protein. Human embryonic kidney 293 cells, stably transfected with a plasmid containing the 1.4-Kb prohK6 cDNA, were grown as previously described [15]. Their supernatant was collected and concentrated 20 times by using Centricon ultafiltration devices (Millipore, Waltham, MA). Purification of prohK6 was achieved by ion-exchange chromatography (FPLC). Concentrated supernatant was diluted 1:2 with running buffer (50 mM acetate buffer, pH 5.3) and
hK6 autoactivation and autolysis
N-terminal sequencing of purified prohK6 revealed the sequence Glu–Glu–Gln–Asn–Lys–Leu–Val–His–Gly; the first five amino acids (in bold) correspond to the propeptide. The proform of the protein was, as expected, enzymatically inactive. After incubation of prohK6 at 37 °C for approximately 5 min, enzymatic activity was detected, as shown in Fig. 1. Prolonged incubation led to decrease of the enzymatic activity (Fig. 2A) due to autolysis, as revealed by the presence of two bands on SDS–PAGE gel (
Discussion
The crystal structures of the pro- and the mature forms of hK6 were recently resolved [28], [29]. The first study suggested that prohK6 is activated in vivo by limited proteolysis between Lys15 and Leu16, either autolytically or by another, yet unknown, protease. Both studies identified an autolysis-sensitive point, located at Arg76–Glu77 which, when processed, led to an inactive species.
We produced the proform of hK6, which, after purification and concentration, was auto-activated by a short
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Abbreviations: KLK, kallikrein gene; hK, kallikrein protein; PSA, prostate-specific antigen; MSP, myelencephalon-specific protease; CNS, central nervous system; ELISA, Enzyme-linked immunosorbent assay; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; AMC, 7-amino-4-methyl-coumarin; ACT, α1-antichymotrypsin; API, α1-protease inhibitor; AAT, α1-antitrypsin; ATIII, antithrombin III; α2M, α2-macroglobulin; PCI, protein C inhibitor; α2AP, α2-antiplasmin; APP, amyloid precursor protein; BSA, bovine serum albumin; ECM, extracellular matrix; uPA, urokinase-type plasminogen activator; Aβ, amyloid β peptide.