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  • Title: Antibodies against the C2 COOH-terminal region discriminate the active and latent forms of the multicatalytic proteinase complex.
    Author: Arribas J, Arizti P, Castaño JG.
    Journal: J Biol Chem; 1994 Apr 29; 269(17):12858-64. PubMed ID: 8175701.
    Abstract:
    The mouse cDNA homologues of the rat C2, C9, and C5 subunits of the multicatalytic proteinase have been cloned and expressed in bacteria. The respective recombinant proteins were purified and used to produce specific anti-subunit antibodies. Immunoblotting of two-dimensional gels of purified rat liver multicatalytic proteinase showed that the C2 (32-kDa) and C9 (29-kDa) polypeptides are resolved into three and two isoelectric variants, respectively, likely due to post-translational modifications, i.e. phosphorylation, and the presence of two anti-C5 reacting polypeptides (25.5 and 23 kDa). Epitope mapping of the anti-C2-specific antibody with different constructs of the recombinant C2 protein allowed us to determine that one major epitope of this anti-C2 antibody is located within the last 9-11 amino acids of the C2 polypeptide. Affinity purified antibodies directed against the C2 COOH-terminal were able to discriminate the active and latent forms of the multicatalytic proteinase, supporting the conclusion that the C2 protein found in the active form of the enzyme is a polypeptide of 28 kDa, produced by the loss, at least, of the last 9-13 amino acids (DEPAEKADEPMEH) of the intact C2 (32-kDa) component. By in vitro treatment of the latent form of the enzyme with elastase, we show the conversion of the C2 (32-kDa) component to a 28-kDa protein with loss of recognition by the anti-C2 COOH-terminal affinity purified antibodies, but this limited degradation of the C2 component did not have any significant effect on the proteolytic activity (assayed with myelin basic protein and fluorogenic peptides) of the multicatalytic proteinase. It is suggested that the proteolytic cleavage of the C2 COOH-terminal region may be involved in the regulation of the interaction of the multicatalytic proteinase with other cellular proteins and/or in the translocation of the complex to the nucleus.
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