RECOMBINANT CATHEPSIN L FROM TRIBOLIUM CASTANEUM: ISOLATION AND PROPERTIES

Klimova, M.A.; Vorotnikova, E.A.; Tereshchenkova, V.F.

Авторы: Klimova M.A., Vorotnikova E.A., Tereshchenkova V.F.
Сборник: Abstracts of the IX International chemistry conference “Mendeleev 2015”
Тезисы
Год издания: 2015
Место издания: Санкт-Петербург, Россия
Аннотация: This research is devoted to recombinant cathepsin L NP_001164001 from Tribolium castaneum, its isolation and properties. The goal is a structure-functional characteristic of the recombinant cathepsin L, cysteine peptidase from papain family C1. The red flour beetle (Tribolium castaneum) is a species of beetle in the family Tenebrionidae and a worldwide pest of stored products, particularly food grains. Analysis of T. castaneum larvae gut transcriptome revealed the presence of twenty-five cysteine peptidases from C1 family. The highest level of expression in the gut of this insect pest was for the major cathepsin L NP_001164001, which is considered as the major digestive enzyme of this insect. [1] The diet of T. castaneum larvae consists mainly of cereals. The major storage proteins in cereals are prolamins that cause celiac disease in susceptible people. Given that, we can assume, that cathepsin L NP_001164001 can be used as part of combination enzyme therapy of coeliac disease. Native cathepsin L isolated from the insect gut is extremely unstable. Therefore, the recombinant protein may facilitate investigation of this enzyme. In this research recombinant T. castaneum cathepsin L was expressed in Pichia pastoris as an inactive proenzyme, therefore the autocatalytic processing of procathepsin L was studied in detail in vitro. Procathepsin L is stable at slightly alkaline pH, and the proregion protects the protein from the denaturing effect of neutral to alkaline pH. Proenzyme activation step consists of proteolytic cleavages of the proregion, autocatalytic processing of procathepsin L occurs under acidic conditions. [2] Based on that, the optimal conditions for processing are determined. Mature enzyme is characterized according to activity profiles in buffers of different pH, mass spectrometry analysis, electrophoretic mobility under native and denaturation conditions, inhibitor sensitivity and substrate specificity. Furthermore, comparative analysis with other cysteine cathepsins will be conducted. References: [1] Martynov A. G., Elpidina E. N., Perkin L., Oppert B. Functional analysis of C1 family cysteine peptidases in the larval gut of Тenebrio molitor and Tribolium castaneum. Martynov et al. BMC Genomics, 2015, vol 16, pp 75 [2] Ménard R., Carmona E., Takebe S., Dufour E., Plouffe C., Mason P., Mort J. S. Autocatalytic Processing of Recombinant Human Procathepsin L. The Journal of Biological Chemistry, 1998, vol 273(8), pp 4478-84 Acknowledgements: We express our deepest sense of gratitude to Benevolensky S. V. and Klyachko E. V. from A. N. Bach Institute of Biochemistry Russian Academy of Sciences for providing all the necessary materials. The research is supported by RFBR grant № 14-04-91167-NSFC_a.
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