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Cloning of a cysteine proteinase gene from Acanthamoeba culbertsoni
1Department of Parasitology, College of Medicine, Wonkwang University, Iksan, Korea.
Abstract:
Free living amoeba, including pathogenic Acanthamoeba culbertsoni, are widely distributed in soil and fresh water. It has been found that cysteine proteinases are more active in pathogenic strains of amoeba whereas serine proteinases are found in both pathogenic and nonpathogenic strains. Cysteine proteinases thus play important roles in the pathogenesis of several parasitic infections and have been proposed as targets for the structure-based strategy of drug design. As the first step toward applying this strategy to design inhibitors as antiparasitic agents for A. culbertsoni, we isolated and sequenced the full length clone of a cysteine proteinase gene from A. culbertsoni by performing reverse transcription-polymerase chain reaction (RT-PCR) 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 amino acid sequence has the sequence homology with the cysteine proteinase genes of Paragonimus westermani metacercaria, Schistosoma mansoni, human cathepsin L and Fasciola hepatica, each by 45.3%, 45.9%, 57.9% and 50.8% respectively. Sequence analysis and alignment showed significant similarity to other eukaryotic cysteine proteinases, including the conservation of the cysteine, 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 (AcCP2) was found to contain Ex3Rx3Wx2N at the proregion and also a proline/threonine-rich C-terminal extension. Therefore, it has cathepsin L-like characteristics. Phylogenetic analysis based on the amino acid sequences of cysteine proteinase indicated that AcCP2 was closely related with papaya, while it was remotely related with those of Schistosoma.
Insights
Researchers isolated and sequenced a cysteine proteinase gene from pathogenic Acanthamoeba culbertsoni. This gene, AcCP2, shows cathepsin L-like characteristics and is a potential drug target for treating amoebic infections.
Area of Science:
- Molecular Biology
- Parasitology
- Biochemistry
Background:
- Free-living amoebae, including pathogenic Acanthamoeba culbertsoni, are ubiquitous in soil and water.
- Cysteine proteinases are crucial in the pathogenesis of parasitic infections and are potential drug targets.
Purpose of the Study:
- To isolate and sequence the full-length cysteine proteinase gene from Acanthamoeba culbertsoni.
- To characterize the gene and its encoded protein for potential antiparasitic drug design.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) to amplify the gene.
- Rapid amplification of cDNA ends (RACE) to obtain 5' and 3' regions.
- Northern blot analysis to detect mRNA expression.
- Sequence analysis and phylogenetic analysis.
Main Results:
- A full-length cysteine proteinase gene (AcCP2) was isolated with an open reading frame of 1359 bp.
- The deduced amino acid sequence showed homology to various eukaryotic cysteine proteinases, notably human cathepsin L (57.9%).
- AcCP2 exhibits cathepsin L-like characteristics, including a conserved catalytic triad and specific proregion/C-terminal features.
- Phylogenetic analysis revealed AcCP2 is closely related to papaya cysteine proteinase.
Conclusions:
- The identified Acanthamoeba culbertsoni cysteine proteinase gene (AcCP2) is a cathepsin L-like enzyme.
- AcCP2 represents a promising target for developing structure-based antiparasitic drug design strategies against Acanthamoeba infections.