Atypical processing of amyloid precursor fusion protein by proteolytic activity in Pichia pastoris.
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.
Markaryan
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A
Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
Morozova
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I
Lee
B S
BS
Kaplan
A
A
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D017077:Culture Media, Conditioned; D019281:Dimerization; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006031:Glycosylation; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D017421:Sequence Analysis; D012694:Serine / Q000378:metabolism
D016564:Amyloid beta-Protein Precursor; D000906:Antibodies; D017077:Culture Media, Conditioned; D000939:Epitopes; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D012694:Serine; D006735:Horseradish Peroxidase; D005998:Glycine