Functional and structural studies of protease inhibitor cistatin type identified in midgut of Rhipicephalus (Boophilus) microplus tick.

• Grant number: 11/09340-3
• Support Opportunities: Scholarships in Brazil - Doctorate
• Effective date (Start): December 01, 2011
• Effective date (End): November 30, 2014
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Aparecida Sadae Tanaka
• Grantee: Thyago Hermylly Santana Cardoso
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil

Abstract

Ticks are important vectors of diseases to humans and other vertebrates. The Rhipicephalus (Boophilus) microplus is an exclusive bovine ectoparasite, being responsible for infections caused by ricketsia, Anaplasma sp, Babesia sp. The R. microplus causes heavy damage to livestock, resulting in considerable losses in the production of meat, leather and milk. In Brazil, losing has reached two billion dollars. Some proteins such as enzymes and inhibitors have been identified and characterized in saliva, but little is known about the proteins identified in the gut of the tick. Recently, it was performed a gut transcriptome of R. microplus in our laboratory, and among the genes found were identified a cisteine protease inhibitor of the cystatin family. In an attempt to understand the mechanisms of action of this cystatin, the sequence of the cystatin gene fragment will be confirmed by cloning in pGEMT-easy vector, confirmed the sequence, it will be sub-cloned into the expression vector and protein will be expressed in bacteria. The recombinant protein will be produced and purified for biochemical characterization and evaluation of its selectivity towards different cisteine proteases, among them BmCL1 enzyme, digestive enzyme from R. microplus. Having the cystatin purified, we will conduct tests in an attempt to solve crystallographic three dimensional structure, at the same time, we are going to produce Bmcistatina-1 (LIMA et al., 2006) that has been characterized in our laboratory as well as BmCL1 (CLARA et al., 2011 accepted for publication), for assays of crystallization of the cystatin and the enzyme-inhibitor complex. This will be performed to increase the probability to obtain protein crystals, which will allow diffraction and possibly the resolution of three-dimensional structure of a cystatin of tick by our group.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

CARDOSO, THYAGO H. S.; LU, STEPHEN; GONZALEZ, BORIS R. G.; TORQUATO, RICARDO J. S.; TANAKA, APARECIDA S.. Characterization of a novel cystatin type 2 from Rhipicephalus microplus midgut. Biochimie, v. 140, p. 117-121, SEP 2017. (12/03657-8, 11/09340-3)