Julio C. Calvo

Plasmodium falciparum AMA-1 erythrocyte binding peptides implicate AMA-1 as erythrocyte binding protein

The role of AMA-1 during merozoite invasion has not yet been determined. However, reported experimental evidence suggests that this protein can be used, in particular as erythrocyte-binding protein, since, Fab fragments against this protein are able to block merozoite invasion. Using a previously described methodology, eight peptides with high binding activity to human erythrocyte, scattered along the different domains and having around 130 nM affinity constants, were identified in the Plasmodium falciparum AMA-1 protein. Their binding activity was sialic acid independent. Some of these peptides showed homology with the erythrocyte binding domains of one of the apical organelle protein family, MAEBL, identified in rodent malarial parasites. One of these peptides shares amino acid sequence with a previously reported B-cell epitope which induces antibodies to block parasite growth. The critical residues were identified for erythrocyte binding conserved peptides 4313 (DAEVAGTQYRLPSGKCPVFG), 4321 (VVDNWEKVCPRKNLQNAKFG), 4325 (MIKSAFLPTGAFKADRYKSH) and 4337 (WGEEKRASHTTPVLMEKPYY). All conserved peptides were able to block merozoite invasion of new RBC and development, suggesting that these peptides are involved in P. falciparum invasion.

Conformationally Restricted Peptides from Rice Proteins Elicit Antibodies That Recognize the Corresponding Native Protein in ELISA Assays

The rice hoja blanca virus (RHBV), transmitted by the planthopper insect Tagosodes orizicolus, is a disease that attacks rice and generates significant production losses in Colombia. Fedearroz 2000 and Colombia I commercial rice varieties, which have different resistance levels to the disease, were selected in this study. To identify proteins associated to the insect and virus signaling, a comparative proteomics study was performed. By comparing proteomic profiles, between virus-infected and control group plants in two-dimensional electrophoresis, proteins exhibiting significant changes in abundance were found. In another test, peptide dendrimers containing sequences conformationally restricted to α-helix from four of those rice proteins were synthesized. In the experiment, sera from mice inoculated with peptide dendrimers could recognize the corresponding native protein in ELISA assays. Reported comparative proteomic results provide new insights into the molecular mechanisms of plant response to the RHBV and comprehensive tools for the analysis of new crop varieties. Besides, results from conformational peptide dendrimer approach are promising and show that it is feasible to detect proteins as markers, and may have biological applications by decreasing the susceptibility to proteolytic degradation.

Reduced amide bond pseudopeptides induce antibodies against native proteins of Plasmodium falciparum

The malaria peptide coded 1513 whose primary structure is was designed from the amino acid sequence of Plasmodium falciparum protein MSP-1. The primary sequence of the binding motif KeKMVL of 1513 to RBCs is also contained in the structure of the SPf-66 synthetic malaria vaccine[l]. In order to explore the immunogenicity of 1513 peptide analogues containing reduced amide bonds, a set of peptide mimetics was synthesized and biochemically characterized. The analogues were synthesized by systematically replacing one CO-NH peptide bond at a time by a reduced amide isostere according to the Coy strategy [2]. To determine the bidimensional structure of each 1513 analogue studies were performed in a mixture of in a ratio 90/10 as well as in aqueous 30%TFE. NMR analysis performed for the Pse-437 analogue containing the bond between and showed features of secondary structure not yet detected for the parent non modified 1513 peptide. The recorded Pse-437 NMR data were processed for molecular modeling on an Indigo-II computer provided with a graphic package from Biosym/MSI. In the present work we propose a preliminary tridimensional molecular model for Pse-437. Polyclonal antibodies were obtained after immunization of BALB/c mice and New Zealand rabbits with oxidized pseudopeptide analogues of 1513 containing a cysteine residue at the N and C termini. Monoclonal antibodies to Pse-437 were obtained by standard cellular fusion of mice spleen cells to X-63Ag8 myeloma cells [3]. Seven immunoglobulin producing hybridomas to Pse-437 showed strong reactivity by ELISA and Western blot against Plasmodium falciparum protein MSP-1 as well as against the SPf-66 vaccine. Each reactive hybrid cell was cloned to obtain cross-reacting mAbs to 195 kDa, and 83 kDa proteins and SPf-66. We propose these second generation novel peptides as possible tools for the development of chemically synthesized Plasmodium falciparum malaria vaccines.