Identification of C3 acceptors responsible for complement activation in Crithidia fasciculata.
Crithidia fasciculata, an insect trypanosomatid is readily lysed by normal human serum at concentrations as low as 3%. Lysis occurs in the presence of Mg+2-EGTA and is antibody independent, indicating that the alternative pathway of complement activation is involved. Analysis of [131I]C3 deposition on C. fasciculata cells using C8-deficient serum, revealed that about 4 x 10(5) C3 molecules bound to each cell. Most of the C3 was bound to cells as C3b, part of it forming high molecular weight complexes, which could be dissociated by methylamine treatment at alkaline pH. To characterize the C3 acceptors on C. fasciculata, surface-iodinated cells were incubated with C8D or heat-inactivated serum, extracted and immunoprecipitated with anti-C3 or anti-arabinogalactan antisera. Analysis of the immunoprecipitated material on SDS gels showed high-molecular weight components, which disappeared after methylamine treatment, giving rise to a component of 200 kDa molecular size. This 200-kDa component corresponded to a purified arabinogalactan complex, which was immunoprecipitated from labeled cell extracts, without incubation with C8D, using anti-arabinogalactan antibodies. These results suggest that the arabinogalactan glycoconjugate is a C3 acceptor in C. fasciculata during complement activation. Purified arabinogalactan complexes were able to inactivate C3 in vitro. Solubilization in KOH to cleave the peptide moiety rendered it unable to inactivate C3. Apparently, the aggregated state of the purified arabinogalactan component at the cell surface is important for C3 deposition and activation.
D000818:Animals; D003167:Complement Activation; D003176:Complement C3 / Q000276:immunology; D003170:Complement Pathway, Alternative; D003421:Crithidia / Q000276:immunology*; D004533:Egtazic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D005685:Galactans / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D016177:Macrophage-1 Antigen; D011951:Receptors, Complement / Q000302:isolation & purification / Q000502:physiology*
D003176:Complement C3; D005685:Galactans; D007457:Iodine Radioisotopes; D016177:Macrophage-1 Antigen; D011951:Receptors, Complement; D004533:Egtazic Acid