Related Experiment Videos
The basic isoform of profilin in pathogenic Entamoeba histolytica. cDNA cloning, heterologous expression, and
1Institute for Specific Prophylaxis and Tropical Medicine, University of Vienna, Austria.
Abstract:
In the human parasite Entamoeba histolytica, components of the cytoskeleton are involved in the pathogenicity by their contribution to immune evasion by antibody capping and shedding. In this study, we focus on profilin as a central regulatory component of the cytoskeleton. Profilin was isolated from trophozoites of the pathogenic E. histolytica strain SFL-3, and partial amino acid sequences were used to devise a probe for isolating a profilin cDNA. The deduced complete primary structure was divergent: plant profilins with amino acid sequence identities in the range 33-38% were more closely related than the mammalian profilins with sequence identities 21-28%. The cDNA was expressed as a nonfusion protein in Escherichia coli. Isoelectric focussing of the natural profilin isolated from E. histolytica showed two isoforms with different isoelectric points; the recombinant profilin migrated with the basic isoform. In a blot overlay experiment, purified 125I-labeled recombinant profilin bound not only to plant actin, but also to mammalian actin, demonstrating that cytoskeletal components from distantly related organisms with divergent primary structures can be compatible.
Insights
This study isolated profilin from the parasite Entamoeba histolytica, revealing its divergent structure compared to plant and mammalian profilins. The research demonstrates functional compatibility of E. histolytica profilin with actin from various species.
Area of Science:
- Parasitology
- Molecular Biology
- Biochemistry
Background:
- Cytoskeletal components in Entamoeba histolytica contribute to pathogenicity via immune evasion mechanisms like antibody capping.
- Profilin, a key cytoskeleton regulator, is investigated for its role in the parasite's virulence.
Purpose of the Study:
- To isolate and characterize profilin from the pathogenic parasite Entamoeba histolytica.
- To determine the evolutionary relationship of E. histolytica profilin to profilins from other organisms.
- To assess the functional compatibility of E. histolytica profilin with actin.
Main Methods:
- Isolation of profilin from E. histolytica trophozoites.
- cDNA cloning and sequencing to determine the primary structure of profilin.
- Expression of recombinant profilin in Escherichia coli.
- Isoelectric focusing to analyze profilin isoforms.
- Blot overlay assays using labeled recombinant profilin and actin from various sources.
Main Results:
- The deduced primary structure of E. histolytica profilin showed greater sequence identity to plant profilins (33-38%) than to mammalian profilins (21-28%).
- Two natural profilin isoforms were identified in E. histolytica, with recombinant profilin matching the more basic isoform.
- Recombinant E. histolytica profilin demonstrated binding to both plant and mammalian actin.
Conclusions:
- E. histolytica profilin exhibits significant structural divergence, with closer evolutionary ties to plant profilins.
- Despite structural differences, E. histolytica profilin retains functional compatibility with actin from evolutionarily distant organisms.
- This finding highlights the conserved nature of actin-binding domains in profilins across diverse species.