Molecular characterization of a calcium-binding protein from the filarial parasite Dirofilaria immitis
N Tsuji1, T H Morales, V V Ozols
1Heska Corporation, Fort Collins, CO 80525, USA.
Insights
Researchers identified a novel protein, Dirofilaria immitis calreticulin (nDiCal), in heartworm larvae. This protein is expressed across all life stages and binds calcium, suggesting a role in heartworm development and potential diagnostic applications.
Area of Science:
- Parasitology
- Molecular Biology
- Immunology
Background:
- Dirofilaria immitis is the causative agent of heartworm disease in canids.
- Calreticulin is a conserved protein family involved in calcium homeostasis and immune modulation.
- Understanding parasite-specific proteins is crucial for developing diagnostics and therapeutics.
Purpose of the Study:
- To isolate and characterize a novel calreticulin-like protein from Dirofilaria immitis.
- To investigate the expression and function of this protein in different life stages of the parasite.
- To assess its potential as a diagnostic target.
Main Methods:
- cDNA expression library screening using specific antibodies.
- Sequence analysis of the isolated cDNA and predicted protein.
- Recombinant protein expression and characterization (e.g., calcium binding assays).
- Immunoblotting and immunolocalization studies using antibodies against the native and recombinant proteins.
Main Results:
- Isolation of a full-length cDNA (nDiCal) encoding a calreticulin-like protein (pDiCal).
- pDiCal exhibits structural similarities to known calreticulins and binds calcium ions.
- A native antigen of 56 kDa, recognized by anti-rDiCal antibodies, is present in all D. immitis developmental stages and excretory-secretory products.
- pDiCal is ubiquitously expressed in adult worms, particularly in hypodermis and muscle cells, and in developing embryos.
- Antibodies against rDiCal were detected in sera from infected dogs and rabbits immunized with parasite components.
Conclusions:
- nDiCal represents a novel Dirofilaria immitis calreticulin homolog.
- The calcium-binding protein pDiCal is expressed throughout the parasite's life cycle.
- pDiCal is a potential target for serodiagnostic assays for heartworm infection.
Abstract:
A full length D. immitis cDNA (nDiCal) encoding a protein with significant similarity to the calreticulin protein family was isolated from a 6-day fourth-stage larval cDNA expression library by immunoscreening, using serum from a rabbit immunized by repeated injection of small numbers of third-stage larvae. nDiCal is 1538 bp long and contains the 21 bp nematode splice leader sequence SL1 at the 5' end. nDiCal encodes for a protein (pDiCal) with a predicted molecular mass of 46 kDa. pDiCal sequence analysis revealed similarities with calreticulin, a protein that typically resides in the endoplasmic reticulum. pDiCal possesses three consensus sequences of the calreticulin family of proteins: a neutral N-terminal region with a putative signal sequence; a proline- and tryptophan-rich P region; and a highly acidic C-terminal region. A 45Ca2+-overlay assay showed that recombinant pDiCal (rDiCal) is a Ca2+-binding protein. Antibodies to rDiCal identified a 56 kDa native antigen in all developmental stages including the excretory-secretory products derived from larvae and adult worms. Localization studies demonstrated the ubiquitous presence of pDiCal with intense expression in the hypodermis and syncitial muscle cells in both male and female adult worms. Labeling was also seen in the developing embryos within the uterus of the female worms. Sera from immune as well as chronically-infected microfilaremic dogs contained antibodies that bind rDiCal. In addition, immunoblot analysis showed that serum from a rabbit immunized with L3 cuticles reacted with rDiCal.
