Markaryan A, Morozova I, Lee B S, Kaplan A
Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Biochem Biophys Res Commun. 1999 Aug 19;262(1):263-8. doi: 10.1006/bbrc.1999.1139.
Secretases catalyze the production of important proteolytic products of the amyloid precursor protein. We expressed a fusion protein that contained horseradish peroxidase, fragment 590-695 of amyloid precursor protein, and c-myc and polyhistidine tags in Pichia pastoris. It secreted a 50-kDa N-terminal fragment; a 15-kDa C-terminal fragment accumulated in cells. The N-terminal fragment exhibited peroxidase activity and reacted with antibodies specific for peptides within the sequences -2 to 15 and 21-37 of beta-amyloid peptide. The C-terminal fragment reacted with antibodies that recognize the sequences 649-664 and 676-695 of amyloid precursor protein and the C-terminal c-myc tag. To locate the cut site, the C-terminal fragment was metabolically labeled with either [(35)S]Met or [(3)H]Lys and radiosequenced. A major component, derived from a cleavage at Gly(25)-Ser(26) of beta-amyloid, was detected. Results suggest a predominant atypical cleavage, like that observed in Down Syndrome fibroblasts, occurs between the alpha- and gamma-sites.
分泌酶催化淀粉样前体蛋白重要蛋白水解产物的产生。我们在毕赤酵母中表达了一种融合蛋白，该蛋白包含辣根过氧化物酶、淀粉样前体蛋白的590 - 695片段以及c - myc和多组氨酸标签。它分泌出一个50 kDa的N端片段；一个15 kDa的C端片段在细胞中积累。N端片段表现出过氧化物酶活性，并与针对β - 淀粉样肽序列 - 2至15和21 - 37内肽段的特异性抗体发生反应。C端片段与识别淀粉样前体蛋白序列649 - 664和676 - 695以及C端c - myc标签的抗体发生反应。为了定位切割位点，C端片段用[(35)S]Met或[(3)H]Lys进行代谢标记并进行放射性测序。检测到一个主要成分，其来源于β - 淀粉样肽在Gly(25) - Ser(26)处的切割。结果表明，在α - 位点和γ - 位点之间发生了一种主要的非典型切割，类似于在唐氏综合征成纤维细胞中观察到的情况。
