Identification of a domain of Trypanosoma cruzi metacyclic trypomastigote surface molecule gp82 required for attachment and invasion of mammalian cells.
Recombinant proteins and synthetic peptides representing various sequences of gp82, a surface glycoprotein of Trypanosoma cruzi metacyclic trypomastigotes implicated in mammalian cell invasion, were used in this study aiming at the identification of the domain(s) of this molecule required for interaction with target cells. Invasion of cultured HeLa cells by metacyclic trypomastigotes was inhibited by about 80% in the presence of native gp82 or the corresponding recombinant construct J18. Inhibition by recombinant proteins J18a and J18b, containing respectively the N-terminal and the C-terminal portions of gp82, was on the order of 30% and 65%. As compared to J18b (amino acids 224-516), the truncated gp82 fragments J18b1 (amino acids 303-516) and J18b2 (amino acids 357-516) displayed lower inhibitory effect (approximately 40% and approximately 15%, respectively). Compatible with these observations, we found that the recombinant protein J18b, but not J18a or J18b2, binds to HeLa cells in a dose-dependent and saturable fashion. Experiments with ten overlapping synthetic peptides, representing the gp82 portion spanning amino acids 224-333, showed that peptides 4 (amino acids 254-273) and 8 (amino acids 294-313) have significant inhibitory activity on HeLa cell invasion by metacyclic forms. All these results indicate that the portion of gp82 required for mammalian cell attachment and invasion is located in the central domain of the molecule.
Santori
F R
FR
Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, Brazil.
Dorta
M L
ML
Juliano
L
L
Juliano
M A
MA
da Silveira
J F
JF
Ruiz
R C
RC
Yoshida
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002448:Cell Adhesion / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology*
C038790:GP72 antigen, Trypanosoma; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins