Processing of the Alzheimer's Disease Amyloid Precursor Protein in Pichia pastoris: Immunodetection of α-, β-, and γ-Secretase Products
| Autor: | C Gray, J.E. Tanner, John R. Underwood, Denise Galatis, Konrad Beyreuther, Genevieve Evin, S. Holmes, Roberto Cappai, D. Le Brocque, Colin L. Masters, Qiao-Xin Li, Anna Henry |
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| Rok vydání: | 1998 |
| Předmět: |
Blotting
Western Peptide Biochemistry Pichia Pichia pastoris Amyloid beta-Protein Precursor Neuroblastoma Alzheimer Disease Endopeptidases Tumor Cells Cultured Amyloid precursor protein Aspartic Acid Endopeptidases Humans Cloning Molecular Gamma secretase chemistry.chemical_classification biology Chemistry Hydrolysis biology.organism_classification Precipitin Tests Peptide Fragments Recombinant Proteins Ectodomain Alpha secretase biology.protein Amyloid Precursor Protein Secretases Protein Processing Post-Translational Amyloid precursor protein secretase |
| Zdroj: | Biochemistry. 37:14958-14965 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi981063l |
| Popis: | betaA4 (Abeta) amyloid peptide, a major component of Alzheimer's disease (AD) plaques, is a proteolytic product of the amyloid precursor protein (APP). Endoproteases, termed beta- and gamma-secretase, release respectively the N- and C-termini of the peptide. APP default secretion involves cleavage within the betaA4 domain by alpha-secretase. To study the conservation of APP processing in lower eukaryotes, the yeast Pichia pastoris was transfected with human APP695 cDNA. In addition to the full-length integral transmembrane protein found in the cell lysate, soluble/secreted APP (sAPP) was detected in the culture medium. Most sAPP comprised the N-terminal moiety of betaA4 and corresponds to sAPPalpha, the product of alpha-secretase. The culture medium also contained minor secreted forms detected by a monoclonal antibody specific for sAPPbeta (the ectodomain released by beta-secretase cleavage). Analysis of the cell lysates with specific antibodies also detected membrane-associated C-terminal fragments corresponding to the products of alpha and beta cleavages. Moreover, immunoprecipitation of the culture medium with three antibodies directed at distinct epitopes of the betaA4 domain yielded a 4 kDa product with the same electrophoretic mobility as betaA4 synthetic peptide. These results suggest that the alpha-, beta-, and gamma-secretase cleavages are conserved in yeast and that P. pastoris may offer an alternative to mammalian cells to identify the proteases involved in the generation of AD betaA4 amyloid. |
| Databáze: | OpenAIRE |
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