COS CELL EXPRESSION CLONING OF PFG377, A PLASMODIUM-FALCIPARUM GAMETOCYTE ANTIGEN ASSOCIATED WITH OSMIOPHILIC BODIES

We report the deduced protein sequence and preliminary characterizatio n of Pfg377, a novel sexual stage antigen of Plasmodium falciparum. An initial cDNA clone (Pfg377-1) encoding the N-terminal 755 amino acids of Pfg377 was isolated by transfecting a 3D7 gametocyte cDNA library into COS7 cells and selecting using a pool of anti-Pfs230 monoclonal a ntibodies. The protein encoded by Pfg377-1 included an N-terminal hydr ophobic signal sequence, but no apparent transmembrane anchor, Instead , the particular cDNA clone selected was fused in-frame at its 3' end with the coding sequence for the human decay acceleration factor membr ane anchor, which had been deliberately placed downstream of the vecto r polylinker in order to attach potential fusion proteins onto the COS cell surface. Northern blots probed with the Pfg377-1 cDNA demonstrat ed cross-hybridization to a single approximate to 9.5-kb transcript, w hich was present only in sexual stages, and not in asexual stages. DNA hybridization was used to obtain a series of overlapping genomic clon es which collectively yielded the complete DNA sequence for Pfg377. Th ere are no introns within the gene, which contains a 9360-bp open read ing frame and encodes a 377-kDa protein. The Pfg377 protein is highly hydrophilic, and has an essentially non-repetitive structure, with onl y four very limited regions of tandem repeats. The Pfg377 gene resides on chromosome 12, and immunoelectron microscopy with two different an ti-Pfg377 polyclonal antisera raised against two separate recombinant sub-fragments of the protein both indicated that the antigen is locate d in electron-dense organelles of the gametocytes - the osmiophilic bo dies - which are proposed to play a role in parasite emergence from th e erythrocyte during gametocyte maturation in the Anopheles mosquito m idgut. Although it was selected with anti-Pfs230 antibodies, compariso n of the sub-cellular locations and protein sequences of Pfg377 and Pf s230 show them to be completely distinct antigens. We hypothesize that Pfg377-1 was initially isolated because it expresses an epitope which is recognized by (i.e., cross-reacts with) one of the anti-Pfs230 mon oclonal antibodies used to select the original transfected COS cells.