Molecular characterization of a calcium-binding protein from the filarial parasite Dirofilaria immitis

A full length D. immitis cDNA (nDiCal) encoding a protein with significant similarity to the calreticulin protein family was isolated from a 6-day fou rth-stage larval cDNA expression library by immunoscreening, using serum fr om 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 lead er sequence SL1 at the 5' end. nDiCal encodes for a protein (pDiCal) with a predicted molecular mass of 46 kDa. pDiCal sequence analysis revealed simi larities with calreticulin, a protein that typically resides in the endopla smic reticulum. pDiCal possesses three consensus sequences of the calreticu lin 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-te rminal region. A Ca-45(2+)-overlay assay showed that recombinant pDiCal (rD iCal) is a Ca2+-binding protein. Antibodies to rDiCal identified a 56 kDa n ative antigen in all developmental stages including the excretory-secretory products derived from larvae and adult worms. Localization studies demonst rated the ubiquitous presence of pDiCal with intense expression in the hypo dermis and syncitial muscle cells in both male and female adult worms. Labe ling was also seen in the developing embryos within the uterus of the femal e worms. Sera from immune as well as chronically-infected microfilaremic do gs contained antibodies that bind rDiCal. In addition, immunoblot analysis showed that serum from a rabbit immunized with L3 cuticles reacted with rDi Cal. (C) 1998 Elsevier Science B.V. All rights reserved.