Processing of the Alzheimer's Disease Amyloid Precursor Protein in Pichia pastoris: Immunodetection of -, 详细信息    查看全文

• 作者：Darren Le Brocque ; Anna Henry ; Roberto Cappai ; Qiao-Xin Li ; Jane E. Tanner ; Denise Galatis ; Carol Gray ; Steven Holmes ; John R. Underwood ; Konrad Beyreuther ; Colin L. Masters ; and Geneviè ; ve Evin
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：October 20, 1998
• 年：1998
• 卷：37
• 期：42
• 页码：14958 - 14965
• 全文大小：113K
• 年卷期：v.37,no.42(October 20, 1998)
• ISSN：1520-4995

文摘

A4 (A) amyloid peptide, a major component of Alzheimer's disease (AD) plaques, is aproteolytic product of the amyloid precursor protein (APP). Endoproteases, termed - and -secretase,release respectively the N- and C-termini of the peptide. APP default secretion involves cleavage withinthe A4 domain by -secretase. To study the conservation of APP processing in lower eukaryotes, theyeast Pichia pastoris was transfected with human APP₆₉₅ cDNA. In addition to the full-length integraltransmembrane protein found in the cell lysate, soluble/secreted APP (sAPP) was detected in the culturemedium. Most sAPP comprised the N-terminal moiety of A4 and corresponds to sAPP, the productof -secretase. The culture medium also contained minor secreted forms detected by a monoclonal antibodyspecific for sAPP (the ectodomain released by -secretase cleavage). Analysis of the cell lysates withspecific antibodies also detected membrane-associated C-terminal fragments corresponding to the productsof and cleavages. Moreover, immunoprecipitation of the culture medium with three antibodies directedat distinct epitopes of the A4 domain yielded a 4 kDa product with the same electrophoretic mobility asA4 synthetic peptide. These results suggest that the -, -, and -secretase cleavages are conserved inyeast and that P. pastoris may offer an alternative to mammalian cells to identify the proteases involvedin the generation of AD A4 amyloid.