The midgut of Aedes albopictus females expresses active trypsin-like serine peptidases

Particle and Fibre Toxicology

Jose Batista De Jesus²  Geovane Dias-Lopes³  Constança Britto³  Nathália Pinho de Souza¹  Andre Borges-Veloso³  Patricia Cuervo¹  Leonardo Saboia-Vahia³ 

[1] Laboratório de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil;Universidade Federal de São João del Rei, São João del Rei, MG, Brazil;Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil

关键词: Mass spectrometry;    Two-dimensional electrophoresis;    Proteomics;    Zymography;    Midgut;    Culicidae;    Aedes albopictus;   

Others  :  1183578
DOI  :  10.1186/1756-3305-7-253

 received in 2013-08-26, accepted in 2014-05-06,  发布年份 2014

【 摘 要 】

Background

Aedes albopictus is widely distributed across tropical and sub-tropical regions and is associated with the transmission of several arboviruses. Although this species is increasingly relevant to public health due its ability to successfully colonize both urban and rural habitats, favoring the dispersion of viral infections, little is known about its biochemical traits, with all assumptions made based on studies of A. aegypti. In previous studies we characterized the peptidase profile of pre-imaginal stages of A. albopictus and we reported the first proteomic analysis of the midgut from sugar-fed females of this insect species.

Methods

In the present work, we further analyzed the peptidase expression in the midgut of sugar-fed females using 1DE-substrate gel zymography, two-dimensional electrophoresis (2DE), mass spectrometry (MS), and protein identification based on similarity.

Results

The combination of zymography, in solution assays using fluorescent substrates and 2DE-MS/MS allowed us to identify the active serine peptidase “fingerprint” in the midgut of A. albopictus females. Zymographic analysis revealed a proteolytic profile composed of at least 13 bands ranging from ~25 to 250 kDa, which were identified as trypsin-like serine peptidases by using specific inhibitors of this class of enzymes. Concomitant use of the fluorogenic substrate Z-Phe-Arg-AMC and trypsin-like serine protease inhibitors corroborated the zymographic findings. Our proteomic approach allowed the identification of two different trypsin-like serine peptidases and one chymotrypsin in protein spots of the alkaline region in 2DE map of the A. albopictus female midgut. Identification of these protein coding genes was achieved by similarity to the A. aegypti genome sequences using Mascot and OMSSA search engines.

Conclusion

These results allowed us to detect, identify and characterize the expression of active trypsin-like serine peptidases in the midgut of sugar-fed A. albopictus females. In addition, proteomic analysis allowed us to confidently assign the expression of two trypsin genes and one chymotrypsin gene to the midgut of this mosquito. These results contribute to the gene annotation in this species of unknown genome and represent a small but important step toward the protein-level functional and localization assignment of trypsin-like serine peptidase genes in the Aedes genus.

【 授权许可】

   
2014 Saboia-Vahia et al.; licensee BioMed Central Ltd.

【 图 表 】

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