Isolation and characterization of a cysteine proteinase gene of Plasmodium falciparum.
We have previously identified a 28-kDa cysteine proteinase of Plasmodium falciparum trophozoites that appears to be an essential malarial hemoglobinase and a potential target for antimalarial chemotherapy. The trophozoite cysteine proteinase (TCP) shares a number of biochemical properties with the lysosomal cysteine proteinase cathepsin L. To isolate the gene encoding TCP, we synthesized degenerate oligonucleotides based on two amino acid sequences of cathepsin L that are well conserved among papain-family cysteine proteinases, and used the oligonucleotides to prime the polymerase chain reaction (PCR) with P. falciparum genomic DNA. A 549-bp DNA fragment was amplified by PCR. This fragment was used as a hybridization probe to screen a lambda gt11 library of P. falciparum genomic DNA and isolate a 1.8-kb genomic clone (C1.8) that encoded an intact malarial cysteine proteinase gene. The sequence of C1.8 predicted a 67-kDa protein containing a typical signal sequence, a large pro sequence, and a 26.8-kDa mature proteinase with 37% amino acid identity to cathepsin L. Antisera directed against a peptide encoded by C1.8 recognized a 28-kDa trophozoite protein on immunoblots. In a Northern analysis, C1.8 hybridized predominantly with RNA from rings, the life-cycle stage immediately preceding the trophozoite stage. Taken together, these results strongly suggest that the P. falciparum cysteine proteinase gene we have isolated and characterized encodes TCP.
Rosenthal
P J
PJ
Department of Medicine, San Francisco General Hospital, University of California.
Nelson
R G
RG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003546:Cysteine Endopeptidases / Q000235:genetics*; D016054:DNA, Protozoan / Q000235:genetics; D005796:Genes; D007438:Introns; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid
D016054:DNA, Protozoan; D003546:Cysteine Endopeptidases