An unusual tryptophan-rich domain characterizes two secreted antigens of Plasmodium yoelii-infected erythrocytes

Previously, we reported the characterization of pypAg-1, a novel protective membrane protein of Plasmodium yoelii-infected erythrocytes. Immunization studies indicated that pypAg-1 contained at least two protective epitopes. One of these determinants was associated with the N-terminal portion of pyp Ag-1, that also included a 220 amino acid domain unusually rich in tryptoph an residues. Using sera from mice immunized against P. yoelii, we have iden tified a second related antigen, pypAg-3. The pypag-3 cDNA encodes a 43 kDa blood-stage protein that is also characterized by the presence of a 220 re sidue tryptophan-rich domain. Of particular interest, sequence comparisons revealed that 24 tryptophan residues are positionally conserved between pyp Ag-1 and pypAg-3. Otherwise, the two antigens share limited sequence simila rity. Full-length recombinant pypAg-3 was expressed, purified and used to p roduce a high titer polyclonal rabbit antiserum. As with pypAg-1, immunoflu orescence studies showed that pypAg-3 is expressed in the cytoplasm and ass ociated with the membrane of P. yoelii infected erythrocytes. In addition, pypAg-1 and pypAg-3 appear to be secreted proteins, as both were detected i n culture supernatants of P. yoelii-infected erythrocytes. Finally, metabol ically labeled pypAg-1 and pypAg-3 secreted from parasitized cells bind to the surface of uninfected, normal mouse erythrocytes. As such, the conserva tion of the unusual tryptophan-rich domain between two blood-stage malarial proteins with similar biological properties suggests that it may be import ant for protein export and/or function. (C) 2000 Elsevier Science B.V. All rights reserved.