Heat shock cognate 70 is a prominent immunogen in Brugian filariasis.
A cDNA expression library constructed from RNA derived from adult stage Brugia pahangi (mixed sexes) was screened with pooled sera from chronic, amicrofilaremic cases of human lymphatic filariasis from the Indonesian island of Tanjungpinang, where Brugia malayi is endemic. Polyclonal antisera raised to purified beta-galactosidase fusion proteins from two of the most highly reactive clones identified a protein of Mr 70,000 in all stages examined (microfilariae, L3 and adults) of both B. malayi and Brugia pahangi. Derivation of the amino acid sequence from these two overlapping cDNAs identified the encoded protein as a member of the heat shock protein 70 family, and showed the closest similarity to the constitutively expressed "heat shock cognate 70" (hsc70) protein. Hybridization of hsc70 cDNAs to RNA and DNA from B. pahangi under stringent conditions identified a major transcript of 2.4 kb and revealed the existence of a family of related genes. In vitro culture of larval stages of B. pahangi at elevated temperatures (43 degrees C) resulted in increased expression of hsc70, and a classic heat shock response in which five proteins (mr 18,500, 22,000, 62,000, 70,000, and 85,000) were exclusively synthesized in microfilariae. Analysis of cross-reactivities by Western blotting implied that antibody generated by infection with B. malayi was directed at filarial-specific determinants of Brugia hsc70. However, ELISA with recombinant fusion proteins for both Plasmodium falciparum and Schistosoma mansoni hsc70 indicated that some individuals with Brugian or Bancroftian filariasis did produce antibodies which cross-reacted with plasmodial and schistosomal homologs. Thus filarial-specific antibody responses were not generated in all individuals, indicating that this molecule would not be suitable for diagnostic purposes. ELISA with a purified beta-galactosidase fusion protein from B. pahangi showed antibody responses to hsc70 across the clinical spectrum of filariasis. Alignment of the derived amino acid sequences from B. pahangi, P. falciparum, S. mansoni and rat hsc70 homologs, and comparison of the immunologic reactivity of the products of the two cDNA clones by Western blotting and ELISA suggested that these determinants were located primarily at the C terminus of the protein.
Selkirk
M E
ME
Department of Biochemistry, Imperial College of Science and Technology, London, UK.
Denham
D A
DA
Partono
F
F
Maizels
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000276:immunology; D000918:Antibody Specificity; D000947:Antigens, Helminth / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D001483:Base Sequence; D002009:Brugia / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D004605:Elephantiasis, Filarial / Q000276:immunology*; D005260:Female; D005368:Filariasis / Q000276:immunology*; D005849:Gerbillinae; D006360:Heat-Shock Proteins / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000276:immunology; D012550:Schistosoma mansoni / Q000276:immunology; D012689:Sequence Homology, Nucleic Acid
D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D006360:Heat-Shock Proteins