Mutant recombinant serpins as highly specific inhibitors of human kallikrein 14

Felber, Loyse M.; Kündig, Christoph; Borgoño, Carla A.; Chagas, Jair Ribeiro; Tasinato, Andrea; Jichlinski, Patrice; Gygi, Christian M.; Leisinger, H.-J.; Diamandis, Eleftherios P.; Deperthes, David; Cloutier, Sylvain M.

doi:10.1111/j.1742-4658.2006.05257.x

Cited by 29 publications

(27 citation statements)

References 43 publications

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“…To this end, we have already constructed highly specific recombinant serpins for KLK14 by replacing the RSL of AT and ACT with KLK14-selected pentapeptides from our phage-displayed library screen (85), which may be useful in future functional studies on KLK14 and in assessing its utility as a therapeutic target.…”

Section: Discussionmentioning

confidence: 99%

Expression and Functional Characterization of the Cancer-related Serine Protease, Human Tissue Kallikrein 14

Borgoño

Michael

Shaw

et al. 2007

Journal of Biological Chemistry

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104

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Human tissue kallikrein 14 (KLK14) is a novel extracellular serine protease. Clinical data link KLK14 expression to several diseases, primarily cancer; however, little is known of its (patho)-physiological role. To functionally characterize KLK14, we expressed and purified recombinant KLK14 in mature and proenzyme forms and determined its expression pattern, specificity, regulation, and in vitro substrates. By using our novel immunoassay, the normal and/or diseased skin, breast, prostate, and ovary contained the highest concentration of KLK14. Serum KLK14 levels were significantly elevated in prostate cancer patients compared with healthy males. KLK14 displayed trypsin-like specificity with high selectivity for P1-Arg over Lys. KLK14 activity could be regulated as follows: 1) by autolytic cleavage leading to enzymatic inactivation; 2) by the inhibitory serpins ␣ 1 -antitrypsin, ␣ 2 -antiplasmin, antithrombin III, and ␣ 1 -antichymotrypsin with second order rate constants (k ؉2 /K i ) of 49.8, 23.8, 1.48, and 0.224 M ؊1 min ؊1 , respectively, as well as plasminogen activator inhibitor-1; and 3) by citrate and zinc ions, which exerted stimulatory and inhibitory effects on KLK14 activity, respectively. We also expanded the in vitro target repertoire of KLK14 to include collagens I-IV, fibronectin, laminin, kininogen, fibrinogen, plasminogen, vitronectin, and insulinlike growth factor-binding proteins 2 and 3. Our results indicate that KLK14 may be implicated in several facets of tumor progression, including growth, invasion, and angiogenesis, as well as in arthritic disease via deterioration of cartilage. These findings may have clinical implications for the management of cancer and other disorders in which KLK14 activity is elevated.

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Section: Discussionmentioning

confidence: 99%

Expression and Functional Characterization of the Cancer-related Serine Protease, Human Tissue Kallikrein 14

Borgoño

Michael

Shaw

et al. 2007

Journal of Biological Chemistry

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104

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“…including KLK2, -3, -4, -5, and -12, were not inhibited by this protein (40). 3 Although KLK14 inhibition considerably delayed semen liquefaction, it did not completely block the process.…”

Section: Discussionmentioning

confidence: 99%

“…The mutant inhibitor ACT G9 used in this study is highly potent and selective toward KLK14 (40). ACT G9 contains mutations at the reactive center loop of the biological inhibitor ACT, converting the natural reactive center loop to the phage display-selected KLK14 substrate G9 (40).…”

Section: Discussionmentioning

confidence: 99%

“…HUK-IgG antibody recognizing KLK1 and purified Sg proteins were kindly provided by Prof. J. Chao (Medical University of South Carolina) and Dr. J. Malm (Malmö University Hospital, Sweden), respectively. ACT G9 , a KLK14-specific recombinant mutant inhibitor, was developed in collaboration with Dr. D. Deperthes (Med-Discovery) by replacing the scissile bond of the reactive center loop of the ␣1-antichymotrypsin (ACT) inhibitor with KLK14 phage display-selected G9 (TVDYA) substrate, as described in detail elsewhere (40).…”

Section: Methodsmentioning

confidence: 99%

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Major Role of Human KLK14 in Seminal Clot Liquefaction

Emami

Deperthes

Malm

et al. 2008

Journal of Biological Chemistry

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Liquefaction of human semen involves proteolytic degradation of the seminal coagulum and release of motile spermatozoa. Several members of human kallikrein-related peptidases (KLKs) have been implicated in semen liquefaction, functioning through highly regulated proteolytic cascades. Among these, KLK3 (also known as prostate-specific antigen) is the main executor enzyme responsible for processing of the primary components of semen coagulum, semenogelins I and II. We have recently identified KLK14 as a potential activator of KLK3 and other KLKs. This study aims to elucidate the cascade-mediated role of KLK14 ex vivo. KLK14 expression was significantly lower (p ‫؍‬ 0.0252) in individuals with clinically delayed liquefaction. Concordantly, KLK14 expression was significantly (p ‫؍‬ 0.0478) lower in asthenospermic cases. Specific inhibition of KLK14 activity by the synthetic inhibitor ACT G9 resulted in a significant delay in semen liquefaction, a drop in the "early" (30 min postejaculation) "chymotrypsin-like" and KLK1 activity, and an increase in the "late" (90 min postejaculation) chymotrypsinlike activity. Conversely, the addition of recombinant active KLK14 facilitated the liquefaction process, augmented the early chymotrypsin-like activity, and lowered late chymotrypsin-like activity. Given that the observed chymotrypsin-like activity was almost completely attributed to KLK3 activity, KLK3 seems to be regulated bidirectionally. Accordingly, a higher level of KLK3 fragmentation was observed in KLK14-induced coagula, suggesting an inactivation mechanism via internal cleavage. Finally, semenogelins I and II were directly cleaved by KLK14. Semenogelins were also able to reverse KLK14 inhibition by Zn 2؉ , providing a novel regulatory mechanism for KLK14 activity. Our results show that KLK14 exerts a significant and dose-dependent effect in the process of semen liquefaction.

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“…This region was replaced by two pentapeptides, previously selected by kallikrein 14 using phage-display technology. In this manner inhibitors with high reactivity towards the enzyme were generated (Fleber et al, 2006). Sensing the binding site of chosen proteinase by studying structure of bound regions of its inhibitors and substrates is a classical tool for the design of new inhibitors of these enzymes.…”

Section: Proteinous Inhibitorsmentioning

confidence: 99%