Identification of monomeric and oligomeric forms of a major Leishmania infantum antigen by using monoclonal antibodies.
Ten monoclonal antibodies (MAbs) produced against isolated Leishmania infantum membranes were used as probes of L. infantum membrane antigens. Western blots of L. infantum membranes, sodium dodecyl sulfate solubilized and heated at 100 degrees C before analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed that all 10 MAbs recognized a band at 58 kilodaltons (kDa). However, when solubilized membranes were not heated, 2 of the 10 MAbs recognized, in addition to the 58-kDa band, bands of higher molecular weight. Limited digestion of heated or nonheated membranes showed that both groups of MAbs (i.e., not capable or capable of binding to the high-molecular-weight bands) recognized the same proteolytic digests. Hydrophilic forms of the above proteins, possessing proteolytic activity, were detected and isolated by gel filtration. Protein staining of the isolated monomer analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reducing and heating conditions, revealed incomplete reduction of the 58-kDa protein. The reduced form of the 58-kDa protein migrated at 63 to 65 kDa and was not recognized by the MAbs. These results suggest the existence of a monomeric and an oligomeric form of the 58-kDa antigen. The observed inhibition of Leishmania promastigote-macrophage binding caused by MAbs representative of the two groups (capable of oligomeric and/or monomeric antigen recognition) suggest that the 58-kDa monomer and oligomer play an important role in promastigote-macrophage interaction. We suggest that the 58-kDa L. infantum antigen is the major surface Leishmania antigen (p63) identified by others.
Soteriadou
K P
KP
Laboratory of Biochemistry, Hellenic Pasteur Institute, Athens, Greece.
Tzinia
A K
AK
Hadziantoniou
M G
MG
Tzartos
S J
SJ
D000818:Animals; D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan / Q000276:immunology*; D000953:Antigens, Protozoan / Q000276:immunology*; D000954:Antigens, Surface / Q000276:immunology*; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D066298:In Vitro Techniques; D007891:Leishmania / Q000276:immunology*; D046911:Macromolecular Substances; D008264:Macrophages / Q000276:immunology; D051379:Mice; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000276:immunology; D010587:Phagocytosis
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D000954:Antigens, Surface; D046911:Macromolecular Substances; D010447:Peptide Hydrolases