Activation and enzymatic characterization of recombinant human kallikrein 8

Abstract

Human kallikrein 8 (hK8), whose gene was originally cloned as the human ortholog of a mouse brain protease, is known to be associated with diseases such as ovarian cancer and Alzheimer's disease. Recombinant human pro-kallikrein 8 was activated with lysyl endopeptidaseconjugated beads. Amino-terminal sequencing of the activated enzyme demonstrated the cleavage of a 9-aa propeptide from the pro-enzyme. The substrate specificity of activated hK8 was characterized using synthetic fluorescent substrates. hK8 showed trypsin-like specificity, as predicted from sequence analysis and enzymatic characterization of the mouse ortholog. All synthetic substrates tested containing either arginine or lysine at P1 position were cleaved by hK8. The highest k cat /K m value of 20=10 3 M -1 s -1 was observed with Boc-Val-Pro-Arg-7-amido-4-methylcoumarin. The activity of hK8 was inhibited by antipain, chymostatin, and leupeptin. The concentration for 50% inhibition by the best inhibitor, antipain, was 0.46 mM. The effect of different metal ions on the enzyme activity was analyzed. Whereas Na q had no effect on hK8 activity, Ni 2q and Zn 2q decreased the activity and Ca 2q , Mg 2q , and K q had a stimulatory effect. Ca 2q was the best activator, with an optimal concentration of approximately 10 mM.

Kallikrein-8 Proteolytically Processes Human Papillomaviruses in the Extracellular Space To Facilitate Entry into Host Cells

“…The consensus site for KLK8 has not been elucidated in full detail. However, based on sequence prediction and in vitro peptide proteolysis analysis, it is clear that KLK8 has a trypsin-like specificity (66,68,74,75). The P1 position contains a positively charged amino acid, with Arg preferable over Lys (68,74).…”

“…However, based on sequence prediction and in vitro peptide proteolysis analysis, it is clear that KLK8 has a trypsin-like specificity (66,68,74,75). The P1 position contains a positively charged amino acid, with Arg preferable over Lys (68,74). In addition, a binding site of four amino acids in the substrate confers specificity with hydrophobic residues in P2 and P3 and preferably Ser/Thr or Phe/Tyr at P1= (64).…”

Human Kallikrein 8 Expression in Salivary Gland Tumors

“…Its activity is inhibited by typical serine protease inhibitors [6,27]. This suggests that it is implicated in extracellular matrix protein degradation in the areas surrounding KLK8-producing cells [6].…”

Prostatic trypsin-like kallikrein-related peptidases (KLKs) and other prostate-expressed tryptic proteinases as regulators of signalling via proteinase-activated receptors (PARs)

“…Not detected ND (Rajapakse et al, 2005;Kishi et al, 2006;Stefansson et al, 2008) 9 (Luo et al, 2001a;Petraki et al, 2002Petraki et al, , 2003a 11 Tryptic Arg SF; N; B Up c (Mitsui et al, 2000;) Nakamura et al, 2003; Brought to you by | University of Queensland -UQ Library Authenticated Download Date | 9/14/15 3:38 AM (Memari et al, 2007a; 13 Tryptic Arg)Lys SF; N; B; C Down Kapadia et al, 2004;(Kapadia et al, 2003;Petraki et al, 2003a,b;Borgono et al, 2007a) Shaw and 14 Tryptic Arg)Lys, Tyr, Phe, Tyr, Gly, Leu SF; N Up c (Brattsand et al, 2005;Felber et al, 2005;(Borgono et al, 2007c;Diamandis, 2007) Oikonomopoulou et al, 2006;Borgono et al, 2007a,b,c;Stefansson et al, 2008) …”

“…In prostate cancer, as in other diseases, this dysregulation is mediated by changes in levels of various factors including metal ions (e.g., Zn 2q ) and proteinaceous inhibitors. For example, in prostate and other tissues, Zn 2q ions provide an effective means of regulating the action of several KLK and other trypsin-like enzymes including KLK2 (Lovgren et al, 1999a), KLK4 (Debela et al, 2006a), KLK5 Debela et al, 2007), KLK8 (Kishi et al, 2006), KLK12 (Memari et al, 2007a) and KLK14 (Borgono et al, 2007c), as well as prostasin (Shipway et al, 2004) and uPA (Ishii et al, 2001). Of relevance to normal prostate physiology as well as prostate cancer, Zn 2q levels are up to 10-fold higher in normal prostate than in other tissues (Kavanagh, 1985), while there is a 10-to 20-fold decrease in these levels in patients with advanced prostate cancer (Zaichick et al, 1996(Zaichick et al, , 1997.…”