Abstract
Generation of the amyloid peptide through proteolytic processing of the amyloid precursor protein by beta- and gamma-secretases is central to the etiology of Alzheimer's disease. The highly elusive beta-secretase was recently identified as a transmembrane aspartic proteinase, Asp2 (BACE). The Asp2 homolog Asp1 (BACE2/DRAP) has also been reported to exhibit beta-secretase cleavage of amyloid precursor protein. Most aspartic proteinases are generated as inactive proenzymes, requiring removal of the prodomain to generate active proteinase. Here we show that prodomain processing of Asp1 occurs between Leu(62) and Ala(63) and is autocatalytic. Asp1 cleaved a maltose-binding protein-Asp1 prodomain fusion protein and a synthetic peptide at this site. Mutation of one of the conserved catalytic aspartic acid residues in the active site of Asp1 to asparagine (D110N) abolished this cleavage. Mutation of P(1)' and P(2)' residues in the substrate to phenylalanine reduced cleavage at this site. Asp1 expressed in cells was the mature form, and prodomain processing occurred intramolecularly within the endoplasmic reticulum/early Golgi. Interestingly, a proportion of mature Asp1 was expressed on the cell surface. When full-length Asp1(D110N) was expressed in COS-7 cells, it was not processed, suggesting that no other proteinase can activate Asp1 in these cells.
Highlights
Alzheimer’s disease is a neurodegenerative disorder characterized by the deposition of the amyloid -peptide in the brain as amyloid plaques [1, 2]
Asp1 Cleaves maltose-binding protein (MBP)-Asp1pro-His6 at the Prodomain Cleavage Site—To investigate prodomain processing of Asp1, we generated a recombinant MBP-Asp1 prodomain fusion protein that encodes MBP fused to Asp1 encompassing the prodomain cleavage site, with a His6 tag at the C terminus (MBP-Asp1pro-His6) (Fig. 1a)
We have shown by N-terminal sequencing that both bands have the same MBP N terminus; the difference in the two bands is most likely to be in the number of histidine residues at the C terminus
Summary
Alzheimer’s disease is a neurodegenerative disorder characterized by the deposition of the amyloid -peptide in the brain as amyloid plaques [1, 2]. To determine if Asp1 or Asp2 can mediate cleavage at this site, MBP-Asp1pro-His6 was incubated with pure catalytically active Asp1 or Asp2, both expressed as soluble Fc fusion proteins (Fig. 1d). Incubation of MBP-Asp1pro-His6 with Asp1-Fc resulted in a clear shift in the molecular mass of the prodomain substrate from ϳ51 to ϳ47 kDa (Fig. 2a), consistent with cleavage at the predicted site and loss of the 4-kDa carboxyl-terminal fragment, which was not detected.
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