Isolation, purification, characterization and antigenic evaluation of GPI-anchored membrane proteins from Leishmania (Viannia) braziliensis.
GPI-anchored proteins from the plasma membrane of Leishmania (Viannia) braziliensis promastigotes were isolated, characterized and their migration pattern compared with those from other Leishmania species. In all cases the SDS-PAGE migration patterns were obtained under reducing and non-reducing conditions, using DL-dithiothreitol (DTT) as a reducer agent. Our results reveal that under reducing conditions the SDS-PAGE migration pattern is modified as a consequence of the disruption of disulphur-bonds and protein transformation. This is demonstrated when in non-reducing conditions the L. (V.) braziliensis-GPI-anchored proteins pattern showed a group of bands over the 100kDa, and two more bands of 52kDa and 50kDa in four different isolates, whereas under reducing conditions the major GPI-anchored protein fractions were detected as bands of 63kDa, 50kDa and an increase of peptides between 34kDa and 22kDa. Similar modifications were detected in the SDS-PAGE migration patterns of GPI-anchored protein fractions from L. (Leishmania) donovani, L. (L.) mexicana and L. (L.) amazonensis run under the same reducing conditions. Antigenic evaluation carried out by Western blot revealed the presence of two very specific L. (V.) braziliensis-GPI-anchored protein bands of 50kDa and 28kDa. These bands were specifically recognized by anti-L. (V.) braziliensis-GPI-anchored protein serum from experimentally immunized animals. These two peptides were not detected when GPI-anchored protein fractions from L. (L.) donovani, L. (L.) mexicana and L. (L.) amazonensis, were challenged with the same anti-serum. The present results lead us to suggest the use of these two peptides as biochemical markers to identify and differentiate leishmaniasis caused by L. (V.) braziliensis. The lack of immunogenicity observed here with the peptide gp63, a very common protein detected in Leishmania species, is considered.
Rojas
Agustina
A
Investigaciones Parasitológicas J.F. Torrealba, Facultad de Ciencias, Departamento de Biología, Universidad de Los Andes, Mérida 5101, Venezuela.
García-Lugo
Pablo
P
Crisante
Gladys
G
Añez-Rojas
Néstor
N
Añez
Néstor
N
D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood* / Q000276:immunology; D000953:Antigens, Protozoan / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D015415:Biomarkers; D004591:Electrophoresis, Polyacrylamide Gel; D017261:Glycosylphosphatidylinositols / Q000737:chemistry* / Q000378:metabolism; D007892:Leishmania braziliensis / Q000254:growth & development / Q000276:immunology* / Q000378:metabolism; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis / Q000276:immunology / Q000469:parasitology; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D011817:Rabbits
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D015415:Biomarkers; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins