Abstract
Human tissue kallikreins (hKs) are a family of fifteen serine proteases. Several lines of evidence suggest that hKs participate in proteolytic cascade pathways. Human kallikrein 5 (hK5) has trypsin-like activity, is able to self-activate, and is co-expressed in various tissues with other hKs. In this study, we examined the ability of hK5 to activate other hKs. By using synthetic heptapeptides that encompass the activation site of each kallikrein and recombinant pro-hKs, we demonstrated that hK5 is able to activate pro-hK2 and pro-hK3. We then showed that, following their activation, hK5 can internally cleave and deactivate hK2 and hK3. Given the predominant expression of hK2 and hK3 in the prostate, we examined the pathophysiological role of hK5 in this tissue. We studied the regulation of hK5 activity by cations (Zn2+, Ca2+, Mg2+, Na2+, and K+) and citrate and showed that Zn can efficiently inhibit hK5 activity at levels well below its normal concentration in the prostate. We also show that hK5 can degrade semenogelins I and II, the major components of the seminal clot. Semenogelins can reverse the inhibition of hK5 by Zn2+, providing a novel regulatory mechanism of its serine protease activity. hK5 is also able to internally cleave insulin-like growth factor-binding proteins 1, 2, 3, 4, and 5, but not 6, suggesting that it might be involved in prostate cancer progression through growth factor regulation. Our results uncover a kallikrein proteolytic cascade pathway in the prostate that participates in seminal clot liquefaction and probably in prostate cancer progression.
Highlights
Proteolytic cascade pathways are implicated in many physiological functions such as blood coagulation, fibrinolysis, apoptosis, digestion, among others [1]
It is known that hK2, hK4, and hK15 can activate pro-hK3 in vitro and that they may be involved in a proteolytic cascade in prostate tissue and seminal plasma [15,16,17,18,19]
We have previously shown that Human kallikrein 5 (hK5) has trypsin-like activity with strong pref
Summary
Materials—The synthetic heptapeptides N-Ile-Gln-Ser-Arg-IleVal-Gly-C, N-Ile-Leu-Ser-Arg-Ile-Val-Gly-C, N-Ser-Cys-Ser-Gln-Ile-IleAsn-C, N-Ser-Ser-Ser-Arg-Ile-Ile-Asn-C, N-Glu-Gln-Asn-Lys-Leu-ValHis-C, N-Gln-Gly-Asp-Lys-Ile-Ile-Asp-C, N-Gln-Glu-Asp-Lys-Val-LeuGly-C, N-Asp-Thr-Arg-Ala-Ile-Gly-C, N-Asn-Asp-Thr-Arg-Leu-AspPro-C, N-Glu-Thr-Arg-Ile-Ile-Lys-C, N-Ala-Thr-Pro-Lys-Ile-Phe-Asn-C, N-Glu-Ser-Ser-Lys-Val-Leu-Asn-C, N-Asp-Glu-Asn-Lys-Ile-Ile-Gly-C, and N-Asp-Gly-Asp-Lys-Leu-Leu-Glu-C were purchased from Genemed Synthesis, San Francisco, CA. To determine the malignant or benign nature of the tissue samples, a histological analysis was performed as described previously [36]. Effect of Cations (Zn2ϩ, Ca2ϩ, Mg2ϩ, Naϩ, and Kϩ) and Citrate on hK5 Activity—To determine the effect of the cations (solutions made from salts of ZnCl2, NaCl, KCl, MgCl2, and CaCl2) and citrate on hK5 activity, reactions mixtures were set up as follows: purified recombinant hK5 (final concentration of 12 nM) was incubated with each cation and citrate (final concentrations of 0, 12, 60, 120, 1,200, and 12,000 nM) diluted in the assay buffer (0.1 mM Tris-HCl, 0.1 mM NaCl, 0.01% Tween 20, pH 8.0) at a final volume of 100 l in microtubes for 10 min at 37 °C with gentle agitation.
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