Characterization of the cell adhesion site of Trypanosoma cruzi metacyclic stage surface glycoprotein gp82.
The surface glycoprotein gp82, expressed in the insect-stage metacyclic trypomastigotes of Trypanosoma cruzi, has been implicated in mammalian cell invasion. 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 positions 257 to 321), was virtually devoid of cell-binding activity and lacked the ability to inhibit parasite invasion, in contrast to J18, the construct containing the full-length gp82 sequence (amino acids 1 to 516). Constructs with shorter deletions, i.e., Del-4 (deleted from 257 to 271) and Del-8 (deleted from 293 to 321), bound to target cells to a significantly lesser degree 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-binding capacity of protein Del-E/D, lacking the glutamic acid (259/260) and aspartic acid (303/304) pairs, was negligible, as was its capacity to inhibit parasite internalization. Of a set of synthetic peptides spanning the gp82 central domain, a 22-mer hybrid peptide, p4/8, formed by two noncontiguous sequences (at positions 257 to 273 and 302 to 306) and containing the four acidic residues, competed with the binding of J18 protein to target cells and significantly inhibited ( approximately 60%) the penetration of parasites. This peptide, generated by the juxtaposition of sequences that are separated by a hydrophobic stretch in the linear molecule, appears to be mimicking a conformation-dependent cell-binding site of gp82. Experiments of antibody competition with a set of 20-mer overlapping peptides mapped the epitope for 3F6, a monoclonal antibody directed to gp82 that inhibits parasite invasion, to the sequence represented by peptide p3 (244 to 263), which has a partial overlap with the cell adhesion site.
Manque
P M
PM
Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
Eichinger
D
D
Juliano
M A
MA
Juliano
L
L
Araya
J E
JE
Yoshida
N
N
D000268:Adhesiveness; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000737:chemistry* / Q000502:physiology
D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins