Peptide mapping of conformational epitopes in a human malarial parasite heat shock protein.
A protein of 75 kDa is found in large quantities throughout the blood stages of the human malarial parasite, Plasmodium falciparum. Based on a partial amino acid sequence for p75, previously deduced from a cDNA clone encoding approximately 40% of the molecule, secondary structural predictions were made. The potential role of long range effects on the tertiary structure of the protein stabilized by disulfide bridges was determined by reduction and alkylation of the fusion protein. Five regions were then chosen for peptide modeling. Peptides of 16, 28, 49, 64, and 76 residues were synthesized and used to immunize rabbits. All but the 16-residue peptides were capable of stimulating boostable IgG antibody responses in rabbits, but the antibody produced against the 49 mer did not react with the native parasite protein. Thus, the 28, 64, and 76 residue peptides represent good immunologic models for portions of the P. falciparum 75-kDa protein capable of stimulating both T and B cells in rabbits. The peptides were also used to probe whether any of the selected regions contain epitopes which react with antibodies from owl monkeys immune to P. falciparum. Of these peptides, two were found to be consistently recognized in ELISA by four owl monkey antisera raised in response to malarial infection. Because these two peptides model a cysteine-containing region of the protein, owl monkey sera were also used as probes of the importance of disulfide bonding in maintaining the native structure. The results obtained were consistent with a folding pattern for p75 that incorporates a disulfide bond between cysteines 161 and 194. These results also suggest that most of the epitopes recognized in this part of p75 by the immune system of the monkey are created by folding of the molecule.
Richman
S J
SJ
Agouron Institute, La Jolla, CA 92037.
Vedvick
T S
TS
Reese
R T
RT
D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan; D000937:Antigen-Antibody Reactions; D000953:Antigens, Protozoan / Q000276:immunology / Q000302:isolation & purification*; D001029:Aotus trivirgatus; D000939:Epitopes / Q000276:immunology / Q000302:isolation & purification*; D006360:Heat-Shock Proteins / Q000302:isolation & purification*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000008:administration & dosage / Q000138:chemical synthesis / Q000276:immunology; D010449:Peptide Mapping / Q000379:methods; D010963:Plasmodium falciparum / Q000276:immunology*; D011487:Protein Conformation; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000939:Epitopes; D006360:Heat-Shock Proteins; D010446:Peptide Fragments