COS cell expression cloning of Pfg377, a Plasmodium falciparum gametocyte antigen associated with osmiophilic bodies.
We report the deduced protein sequence and preliminary characterization 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 antibodies. The protein encoded by Pfg377-1 included an N-terminal hydrophobic 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 membrane anchor, which had been deliberately placed downstream of the vector polylinker in order to attach potential fusion proteins onto the COS cell surface. Northern blots probed with the Pfg377-1 cDNA demonstrated cross-hybridization to a single approximately 9.5-kb transcript, which was present only in sexual stages, and not in a sexual stages. DNA hybridization was used to obtain a series of overlapping genomic clones which collectively yielded the complete DNA sequence for Pfg377. There are no introns within the gene, which contains a 9360-bp open reading frame and encodes a 377-kDa protein. The Pfg377 protein is highly hydrophilic, and has an essentially non-repetitive structure, with only four very limited regions of tandem repeats. The Pfg377 gene resides on chromosome 12, and immunoelectron microscopy with two different anti-Pfg377 polyclonal antisera raised against two separate recombinant sub-fragments of the protein both indicated that the antigen is located in electron-dense organelles of the gametocytes--the osmiophilic bodies--which are proposed to play a role in parasite emergence from the erythrocyte during gametocyte maturation in the Anopheles mosquito midgut. Although it was selected with anti-Pfs230 antibodies, comparison of the sub-cellular locations and protein sequences of Pfg377 and Pfs2 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 monoclonal antibodies used to select the original transfected COS cells.
Alano
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Laboratorio di Biologia Cellulare, Istituto Superiore di Sanirtá, Roma, Italy.
Read
D
D
Bruce
M
M
Aikawa
M
M
Kaido
T
T
Tegoshi
T
T
Bhatti
S
S
Smith
D K
DK
Luo
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C
Hansra
S
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Carter
R
R
Elliott
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D017125:Genes, Protozoan; D006801:Humans; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D015388:Organelles / Q000276:immunology / Q000648:ultrastructure; D010963:Plasmodium falciparum / Q000235:genetics* / Q000254:growth & development / Q000276:immunology*; D015800:Protozoan Proteins / Q000235:genetics* / Q000276:immunology*; D012091:Repetitive Sequences, Nucleic Acid
D000953:Antigens, Protozoan; D017931:DNA Primers; D018076:DNA, Complementary; D016054:DNA, Protozoan; D015800:Protozoan Proteins