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Vaspin inhibits kallikrein 7 by serpin mechanism.

ABSTRACT: The molecular target of the adipokine vaspin (visceral adipose tissue-derived serpin; serpinA12) and its mode of action are unknown. Here, we provide the vaspin crystal structure and identify human kallikrein 7 (hK7) as a first protease target of vaspin inhibited by classical serpin mechanism with high specificity in vitro. We detect vaspin-hK7 complexes in human plasma and find co-expression of both proteins in murine pancreatic β-cells. We further demonstrate that hK7 cleaves human insulin in the A- and B-chain. Vaspin treatment of isolated pancreatic islets leads to increased insulin concentration in the media upon glucose stimulation without influencing insulin secretion. By application of vaspin and generated inactive mutants, we find the significantly improved glucose tolerance in C57BL/6NTac and db/db mice treated with recombinant vaspin fully dependent on the vaspin serpin activity and not related to vaspin-mediated changes in insulin sensitivity as determined by euglycemic-hyperinsulinemic clamp studies. Improved glucose metabolism could be mediated by increased insulin plasma concentrations 150 min after a glucose challenge in db/db mice, supporting the hypothesis that vaspin may inhibit insulin degradation by hK7 in the circulation. In conclusion, we demonstrate the inhibitory serpin nature and the first protease target of the adipose tissue-derived serpin vaspin, and our findings suggest hK7 inhibition by vaspin as an underlying physiological mechanism for its compensatory actions on obesity-induced insulin resistance.

SUBMITTER: Heiker JT

PROVIDER: S-EPMC3689916 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Vaspin inhibits kallikrein 7 by serpin mechanism.

Heiker John T JT Klöting Nora N Kovacs Peter P Kuettner E Bartholomeus EB Sträter Norbert N Schultz Stephan S Kern Matthias M Stumvoll Michael M Blüher Matthias M Beck-Sickinger Annette G AG

Cellular and molecular life sciences : CMLS 20130131 14

The molecular target of the adipokine vaspin (visceral adipose tissue-derived serpin; serpinA12) and its mode of action are unknown. Here, we provide the vaspin crystal structure and identify human kallikrein 7 (hK7) as a first protease target of vaspin inhibited by classical serpin mechanism with high specificity in vitro. We detect vaspin-hK7 complexes in human plasma and find co-expression of both proteins in murine pancreatic β-cells. We further demonstrate that hK7 cleaves human insulin in ...[more]

PMID: 23370777

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Project description:Essentials Kallikrein amplifies contact activation and is a potential target for preventing thrombosis. We developed and characterized a kallikrein aptamer using convergent evolution and kinetic assays. Kall1-T4 prolongs intrinsic clotting time by inhibiting factor XIIa-mediated prekallikrein activation. Kall1-T4 decreases high-molecular-weight kininogen cleavage and bradykinin release. SUMMARY:Background Plasma kallikrein is a serine protease that plays an integral role in many biological processes, including coagulation, inflammation, and fibrinolysis. The main function of kallikrein in coagulation is the amplification of activated factor XII (FXIIa) production, which ultimately leads to thrombin generation and fibrin clot formation. Kallikrein is generated by FXIIa-mediated cleavage of the zymogen prekallikrein, which is usually complexed with the non-enzymatic cofactor high molecular weight kininogen (HK). HK also serves as a substrate for kallikrein to generate the proinflammatory peptide bradykinin (BK). Interestingly, prekallikrein-deficient mice are protected from thrombotic events while retaining normal hemostatic capacity. Therefore, therapeutic targeting of kallikrein may provide a safer alternative to traditional anticoagulants with anti-inflammatory benefits. Objectives To isolate and characterize an RNA aptamer that binds to and inhibits plasma kallikrein, and to elucidate its mechanism of action. Methods and Results Using convergent Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we isolated an RNA aptamer that targets kallikrein. This aptamer, Kall1-T4, specifically binds to both prekallikrein and kallikrein with similar subnanomolar binding affinities, and dose-dependently prolongs fibrin clot formation in an activated partial thromboplastin time (APTT) coagulation assay. In a purified in vitro system, Kall1-T4 inhibits the reciprocal activation of prekallikrein and FXII primarily by reducing the rate of FXIIa-mediated prekallikrein activation. Additionally, Kall1-T4 significantly reduces kallikrein-mediated HK cleavage and subsequent BK release. Conclusions We have isolated a specific and potent inhibitor of prekallikrein/kallikrein activity that serves as a powerful tool for further elucidating the role of kallikrein in thrombosis and inflammation.

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Vaspin inhibits kallikrein 7 by serpin mechanism.

Publications

Vaspin inhibits kallikrein 7 by serpin mechanism.

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