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 antigen processing. The model was set up using purified C3 and Superose-bound 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 endosomes/lysosomes, purified from U937 cell line. Two types of limited activities on the C3b-Superose model system were detected: (i) a proteolytic activity cleaving C3b into mainly a C3c-like fragment which was released and a C3d-like fragment of apparent M(r) 32 kDa which remained bound to Superose through the original ester link; (ii) an esterolytic activity cleaving the ester bond and releasing C3b. Inhibition experiments pointed to the involvement of serine, aspartyl and cysteine proteases. Cathepsin B appeared most probably as one of the major proteases of L fraction catalysing the proteolysis of the C3b-bound. Kinetic studies were in favour of a good stability on the ester bond, supporting an effective role of C3b as a chaperone during the extracellular and intracellular travel of C3b-bound antigen.
Rey-Millet
C A
CA
Laboratoire d'Immunochimie, DBMS/ICH-INSERM U.238, Grenoble, France.
Chesne
S
S
Colomb
M G
MG
D002401:Cathepsin B / Q000378:metabolism; D002402:Cathepsin D / Q000378:metabolism; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism; D003167:Complement Activation / Q000187:drug effects / Q000502:physiology*; D003179:Complement C3b / Q000187:drug effects / Q000378:metabolism*; D003180:Complement C3b Inactivator Proteins / Q000494:pharmacology; D003546:Cysteine Endopeptidases; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D004792:Enzyme Precursors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015151:Immunoblotting; D008247:Lysosomes / Q000378:metabolism; D008863:Microspheres; D013997:Time Factors; D014407:Tumor Cells, Cultured
D003180:Complement C3b Inactivator Proteins; D004792:Enzyme Precursors; D003179:Complement C3b; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; D002402:Cathepsin D