Cloning of a cysteine proteinase gene from Acanthamoeba culbertsoni

Free living amoeba, including pathogenic Acanthamoeba culbertsoni, are wide ly distributed in soil and fresh water. It has been found that cysteine pro teinases are more active in pathogenic strains of amoeba whereas serine pro teinases are found in both pathogenic and nonpathogenic strains. Cysteine p roteinases thus play important roles in the pathogenesis of several parasit ic infections and have been proposed as targets for the structure-based str ategy of drug design. As the first step toward applying this strategy to de sign inhibitors as antiparasitic agents for A. culbertsoni, we isolated and sequenced the full length clone of a cysteine proteinase gene from A. culb ertsoni by performing reverse transcription-polymerase chain reaction (RT-P CR) with degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5' and the 3' regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends (RACE). It has an open reading frame of 1359 bp. The deduced amin o acid sequence has the sequence homology with the cysteine proteinase gene s of Paragonimus westermani metacercaria, Schistosoma mansoni, human cathep sin L and Fasciola hepatica, each by 45.3%, 45.9%, 57.9% and 50.8% respecti vely. Sequence analysis and alignment showed significant similarity to othe r eukaryotic cysteine proteinases, including the conservation of the cystei ne, histidine, and asparagine residues that form the catalytic triad. A 1.5 kbp mRNA was detected on Northern blot analysis using full-length cysteine proteinase cDNA as a probe. The A. culbertsoni cysteine proteinase gene (A cCP2) was found to contain Ex3Rx3Wx2N at the proregion and also a proline/t hreonine-rich C-terminal extension. Therefore, it has cathepsin L-like char acteristics. Phylogenetic analysis based on the amino acid sequences of cys teine proteinase indicated that AcCP2 was closely related with papaya, whil e it was remotely related with those of Schistosoma.