Degradation Signals in ErbB-2 Dictate Proteasomal Processing and Immunogenicity and Resist Protection by cis Glycine–Alanine Repeat

• Published in: Cellular immunology 2001-09, Vol.212 (2), p.138-149
• Main Authors: Piechocki, Marie P., Pilon, Shari A., Kelly, Carmen, Wei, Wei-Zen
• Format: Article
• Language: English
• Subjects: alanine; Alanine - chemistry; Animals; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal, Humanized; antigen processing and presentation; Antineoplastic Agents - pharmacology; Benzoquinones; Cancer Vaccines - therapeutic use; Cell Membrane - metabolism; CTL; Cysteine Endopeptidases - metabolism; Cytosol - metabolism; Cytotoxicity, Immunologic; Drug Design; ErbB-2; ErbB-2 protein; Female; GA repeat; glycine; Glycine - chemistry; Her-2; Humans; Immunotherapy, Active; Lactams, Macrocyclic; Mammary Neoplasms, Experimental - immunology; Mammary Neoplasms, Experimental - pathology; Mammary Neoplasms, Experimental - therapy; Membrane Proteins - immunology; Membrane Proteins - metabolism; Mice; Mice, Inbred BALB C; Multienzyme Complexes - metabolism; Mutagenesis, Insertional; Neoplasm Proteins - immunology; Neoplasm Proteins - metabolism; Neoplasm Transplantation; Neu; proteasome; Proteasome Endopeptidase Complex; proteasome inhibitors; Quinones - pharmacology; Receptor, ErbB-2 - immunology; Receptor, ErbB-2 - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - immunology; Repetitive Sequences, Amino Acid; Species Specificity; Structure-Activity Relationship; Transfection; Trastuzumab; Tumor Cells, Cultured - immunology; Ubiquitin - metabolism; Vaccines, DNA - therapeutic use
• ISSN: 0008-8749; 1090-2163
• DOI: 10.1006/cimm.2001.1853

ErbB-2 is ubiquitinated and degraded when dissociated from its membrane chaperone or bound by specific antibody. Reagents which induce such degradation have demonstrated antitumor activity and may impact ErbB-2 immunogenicity. To further understand ErbB-2 degradation and immunogenicity, a glycine–alanine repeat (GAr) or the reverse proline–alanine repeat (PAr) which protects certain proteins from proteasome degradation, was inserted after amino acid 5 (GAr5/PAr5) or 55 (GAr55/PAr55) of ErbB-2. When dissociated from the membrane with geldanamycin, E2-GAr5 and E2-PAr5 were not protected and still ubiquitinated and degraded by the proteasome, despite the presence of GAr. Insertional mutagenesis with GAr sequences at a.a. 55 of E2 enhanced proteasome degradation rendering E2-GAr55 and E2-PAr55 unstable on the membrane, but rescued in the cytosol by proteasome inhibitors. Immunization with E2-GAr induced antitumor immunity and CTL which lysed tumor cells expressing chimeric E2-GAr or wild-type E2 proteins, demonstrating efficient presentation through MHC I pathway. Improved understanding of the strong degradation signals in ErbB-2 may facilitate the development of anticancer agents or vaccines.