ABSTRACT
Scorpine is an antimicrobial peptide whose structure resembles a hybrid between a defensin and a cecropin. It exhibits antibacterial activity and inhibits the sporogonic development of parasites responsible for murine malaria. In this communication we report the production of scorpine in a heterelogous system, using a specific vector containing its cloned gene. The recombinantly expressed scorpine (RScp) in (Anopheles gambie) cells showed antibacterial activity against (Bacillus subtilis) and (Klebsiella pneumoniae), at 5 and 10 microM, respectively. It also produced 98% mortality in sexual stages of (Plasmodium berghei) at 15 microM and 100% reduction in (Plasmodium falciparum) parasitemia at 5 microM. RScp also inhibited virus dengue-2 replication in C6/36 mosquito cells. In addition, we generated viable and fertile transgenic (Drosophila) that overexpresses and correctly secretes RScp into the insect hemolymph, suggesting that the generation of transgenic mosquitoes resistant to different pathogens may be viable.
Subject(s)
Anti-Infective Agents , Antimicrobial Cationic Peptides/pharmacology , Bacillus subtilis/drug effects , Defensins/pharmacology , Klebsiella pneumoniae/drug effects , Recombinant Proteins/pharmacology , Amino Acid Sequence , Animals , Animals, Genetically Modified , Anopheles , Anti-Infective Agents/metabolism , Anti-Infective Agents/pharmacology , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/metabolism , Cells, Cultured , Defensins/genetics , Defensins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Microbial Sensitivity Tests , Molecular Sequence Data , Plasmodium berghei/drug effects , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Scorpion Venoms/genetics , Scorpion Venoms/metabolism , Scorpion Venoms/pharmacologyABSTRACT
Salivary glands of female mosquitoes produce proteins, not completely described yet, that participate in carbohydrate and blood feeding. Here, we report an acidic glycoprotein of 35 kDa (GP35 ANOAL) secreted in the saliva of the malaria vector mosquito Anopheles albimanus. GP35 ANOAL is produced exclusively in the distal lateral lobes of adult female salivary glands, it has a pI of 4.45 and is negatively stained by regular silver stain. An 888 bp cDNA clone encoding a predicted product of 240 amino acids has a signal peptide, potential post-translational modification sites, and a disintegrin signature RGD. The GP35 ANOAL sequence depicts high similarities with the 30 kDa saliva allergen of Aedes aegypti, 30 kDa allergen-like hypothetical proteins, and GE-rich proteins present in several Anopheles species, as well as in Ae. albopictus and Culex pipiens quinquefasciatus. The function of this protein family is still unknown.
Subject(s)
Anopheles/metabolism , Glycoproteins/metabolism , Insect Proteins/metabolism , Amino Acid Sequence , Animals , Anopheles/genetics , Anopheles/growth & development , Base Sequence , Female , Glycoproteins/genetics , Insect Proteins/genetics , Molecular Sequence Data , RNA, Messenger/metabolism , Salivary Glands/metabolismABSTRACT
A group of salivary-gland-specific proteins, designated gp65, were identified in the mosquito Anopheles albimanus. Two-dimensional gel electrophoresis resolved this group into at least four molecules with pI 6.4-6.5. The N-terminal amino acid sequence was determined for the major species, gp65-1, and degenerate oligonucleotide primers were used to amplify a specific probe for library screening. A 1312 bp cDNA clone encoding a predicted translation product of 386 amino acids was recovered. gp65-1 is expressed abundantly in the medial and distal-lateral lobes of the adult female glands, and is secreted in the saliva. The amino acid sequence has potential sites for N-glycosylation, phosphorylation and myristylation, and is similar to a number of proteins of unknown function from other mosquito species.
