Molecular cloning and functional characterization of an aspartic protease from the hard tick Haemaphysalis longicornis.

Autor(es): Boldbaatar Damdinsuren; Sikalizyo Sikasunge Chummy; Battsetseg Badgar; Xuan Xuenan; Fujisaki Kozo

Resumo: Haemaphysalis longicornis cDNA encoding an aspartic protease (longepsin) was identified from a midgut cDNA library. The longepsin cDNA contains 1176bp that code for 392 amino acid residues with a predictable molecular weight of 39.3kDa. The cDNA has a signal peptide sequence associated with the N-terminal domains and domain structure analysis revealed that the deduced protein has two aspartic acid residues that are characteristic of a single active site for aspartic proteases. This novel longepsin cDNA exhibits 57% identity to the lysosomal aspartic protease of Aedes aegypti, 52% to Bombyx mori cathepsin D, 38% to Ancylostoma caninum, 44% to Schistosoma mansoni and 28% to Boophilus microplus aspartic proteases. The DNA fragment coding for longepsin was cloned into a pGEX-4T-3 vector and expressed in Escherichia coli. The recombinant longepsin, once activated was able to hydrolyze casein substrate as well as hemoglobin (Hb) under acidic conditions (pH 3.5). RT-PCR analysis showed that the longepsin mRNA transcripts were expressed in salivary glands and midgut and not in the ovary. Northern blot analysis revealed that longepsin (1.5kb) was expressed in unfed and partially fed ticks and expression levels increased during feeding. The finding that longepsin is expressed in the midgut and salivary glands, proteolytic activity occurs under acidic conditions and longepsin can be gene silenced of longepsin provides compelling support for the hypothesis that longepsin plays an integral role in the proteolysis of erythrocyte Hb obtained from a host blood meal.

Palavras-Chave: Tick; Haemaphysalis longicornis; Aspartic protease; Longepsin; Cdna; Hemoglobin; Proteolysis

Imprenta: Insect Biochemistry and Molecular Biology, v. 36, n. 1, p. 25-36, 2006

Identificador do objeto digital: 10.1016/j.ibmb.2005.10.003

Descritores: Aedes aegypti - DNA ; Aedes aegypti - Molecular Structure ; Aedes aegypti - Pathogenesis ; Aedes aegypti - RNA ; Aedes aegypti - RT-PCR

Data de publicação: 2006