title: Cloning, characterization and transmission blocking potential of midgut carboxypeptidase A in Anopheles stephensi.
abstract: Transmission-blocking vaccines (TBV) interrupt malaria parasite transmission and hence form an important component for malaria eradication. Mosquito midgut exopeptidases such as aminopeptidase N & carboxypeptidase B have demonstrated TBV potential. In the present study, we cloned and characterized carboxypeptidase A (CPA) from the midgut of an important malarial vector, Anopheles stephensi. ClustalW amino acid alignment and in silico 3-dimensional structure analysis of CPA predicted the presence of active sites involved in zinc and substrate binding that are conserved among all the known mosquito species. Real-time PCR analysis demonstrated that CPA is predominantly expressed in the midgut throughout the mosquito life cycle and that this gene is significantly elevated in P. berghei-infected mosquitoes compared to uninfected blood-fed controls. The high midgut CPA activity correlated with the prominent mRNA levels observed. Peptide-based anti-CPA antibodies were raised that cross-reacted specifically to ∼48kDa and ∼37kDa bands, which correspond to zymogen and active forms of CPA. Further, the addition of CPA-directed antibodies to P. berghei-containing blood meal significantly reduced the mosquito infection rate in the test group compared to control and blocked the parasite development in the midgut. These results support further development of A. stephensi CPA as a candidate TBV.
authors.affiliation: Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
authors.forename: V
authors.initials: V
authors.lastname: VenkatRao
authors.affiliation: Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.
authors.forename: Surendra K
authors.initials: SK
authors.lastname: Kumar
authors.affiliation: Department of Biotechnology, Indira Gandhi National Tribal University, Amarkantak, 484224, India.
authors.forename: P
authors.initials: P
authors.lastname: Sridevi
authors.affiliation: Centre for Computational Science, School of Basic & Applied Sciences, Central University of Punjab, Bhatinda 151001, India.
authors.forename: Vijaykumar Yogesh
authors.initials: VY
authors.lastname: Muley
authors.affiliation: Centre for Animal Sciences, School of Basic & Applied Sciences, Central University of Punjab, Bhatinda, 151001, India. Electronic address: chaitanyark@gmail.com.
authors.forename: R K
authors.initials: RK
authors.lastname: Chaitanya
mesh_terms: D000818:Animals; D000852:Anopheles / Q000033:anatomy & histology / Q000201:enzymology* / Q000235:genetics / Q000469:parasitology; D043422:Carboxypeptidases A / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D005260:Female; D041981:Gastrointestinal Tract / Q000201:enzymology* / Q000276:immunology / Q000469:parasitology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007303:Insect Vectors / Q000033:anatomy & histology / Q000201:enzymology* / Q000469:parasitology; D008288:Malaria / Q000469:parasitology / Q000517:prevention & control / Q000635:transmission; D017780:Malaria Vaccines; D008297:Male; D010962:Plasmodium berghei / Q000254:growth & development; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D017780:Malaria Vaccines; D043422:Carboxypeptidases A
keywords: Anopheles stephensi; Carboxypeptidase A; Midgut; Plasmodium berghei; Transmission blocking vaccine (TBV)