ASSOCIATED COMPLEMENT C3B - TOWARDS AN UNDERSTANDING OF ITS INTRACELLULAR MODIFICATIONS

Covalent Superose microspheres-bound C3b was used as a model system to simplify the analysis of antigen-bound C3b modifications during antig en processing. The model was set up using purified C3 and Superose-bou nd trypsin. C3b was covalently bound to Superose through an ester link , as indicated by lability to hydroxylamine treatment at alkaline pH. C3b-Superose was incubated with L subcellular fraction, enriched in en dosomes/lysosomes, purified from U937 cell line. Two types of limited activities on the C3b-Superose model system were detected: (i) a prote olytic activity cleaving C3b into mainly a C3c-like fragment which was released and a C3d-like fragment of apparent M(r) 32 kDa which remain ed bound to Superose through the original ester link; (ii) an esteroly tic activity cleaving the ester bond and releasing C3b. Inhibition exp eriments pointed to the involvement of serine, aspartyl and cysteine p roteases. Cathepsin B appeared most probably as one of the major prote ases of L fraction catalysing the proteolysis of the C3b-bound. Kineti c studies were in favour of a good stability of the ester bond, suppor ting an effective role of C3b as a chaperone during the extracellular and intracellular travel of C3b-bound antigen.