Characterization of the cell adhesion site of Trypanosoma cruzi metacyclicstage surface glycoprotein gp82

The surface glycoprotein gp82, expressed in the insect-stage metacyclic try pomastigotes of Trypanosoma cruzi, has been implicated in mammalian cell in vasion. Here we have characterized the cell adhesion site of gp82 by using recombinant proteins and synthetic peptides based on gp82. The recombinant protein Del-4/8, lacking 65 amino acids of gp82 central domain (at position s 257 to 321), was virtually devoid of cell-binding activity and lacked the ability to inhibit parasite invasion, in contrast to J18, the construct co ntaining the full-length gp82 sequence (amino acids 1 to 516), Constructs w ith shorter deletions, i.e., Del-4 (deleted from 257 to 271) and Del-8 (del eted from 293 to 321), bound to target cells to a significantly lesser degr ee than did J18. The sites deleted in recombinant proteins Del-4 and Del-8 contained acidic amino acids critical for cell adhesion. Thus, the cell-bin ding capacity of protein Del-E/D, lacking the glutamic acid (259/260) and a spartic acid (303/304) pairs, was negligible, as was its capacity to inhibi t parasite internalization. Of a set of synthetic peptides spanning the gp8 2 central domain, a 22-mer hybrid peptide, p4/8, formed by two noncontiguou s sequences (at positions 257 to 273 and 302 to 306) and containing the fou r acidic residues, competed with the binding of J18 protein to target cells and significantly inhibited (similar to 60%) the penetration of parasites. This peptide, generated by the juxtaposition of sequences that are separat ed by a hydrophobic stretch in the linear molecule, appears to be mimicking a conformation-dependent cell-binding site of gp82, Experiments of antibod y competition with a set of 20-mer overlapping peptides mapped the epitope for 3F6, a monoclonal antibody directed to gp82 that inhibits parasite inva sion, to the sequence represented by peptide p3 (244 to 263), which has a p artial overlap with the cell adhesion site.