Cloning, molecular analysis and differential cell localisation of the p36 RACK analogue antigen from the parasite protozoon Crithidia fasciculata

The family of the RACK molecules (receptors for activated C kinases) are pr esent in all the species studied so far. In the genus Leishmania, these mol ecules also induce a strong immune reaction against the infection. We have cloned and characterised the gene that encodes the RACK analogue from the p arasite trypanosomatid Crithidia fasciculata (CACK), The molecule stems to be encoded by two genes, The sequence analysis of the cloned open reading f rame indicates the existence of a high degree of conservation not only with other members of the Trypanosomatidae but also with mammalians. The study of the protein kinase C phosphorylation sites shows the presence of three o f them, shared with the mammalian species, additional to those present in t he other protozoa suggesting a certain phylogenetic distance between the pr otozoon Crithidia fasciculata and the rest of the Trypanosomatidae. The CAC K-encoded polypeptide shows an additional sequence of four amino acids at t he carboxy-terminal end,,which produces a different folding of the fragment with the presence of an alpha-helix instead of the beta-sheet usual in all the other species studied. A similar result is elicited at the amino-termi nal end by the change of three amino acid residues, The immunolocalisation experiments show that the CACK displays a pattern with a distribution mainl y at the plasma membrane, different from that of the related leishmania spe cies used as control, that displays a distribution close to the nucleus. Al together, the data suggest that the existence of the structural differences found may have functional consequences. (C) 1999 Federation of European Bi ochemical Societies.