Abstract
The cerebral deposition of amyloid beta-peptide is an early and critical feature of Alzheimer's disease. Amyloid beta-peptide is released from the amyloid precursor protein by the sequential action of two proteases, beta-secretase and gamma-secretase, and these proteases are prime targets for therapeutic intervention. We have recently cloned a novel aspartic protease, BACE, with all the known properties of beta-secretase. Here we demonstrate that BACE is an N-glycosylated integral membrane protein that undergoes constitutive N-terminal processing in the Golgi apparatus. We have used a secreted Fc fusion-form of BACE (BACE-IgG) that contains the entire ectodomain for a detailed analysis of posttranslational modifications. This molecule starts at Glu(46) and contains four N-glycosylation sites (Asn(153), Asn(172), Asn(223), and Asn(354)). The six Cys residues in the ectodomain form three intramolecular disulfide linkages (Cys(216)-Cys(420), Cys(278)-Cys(443), and Cys(330)-Cys(380)). Despite the conservation of the active site residues and the 30-37% amino acid homology with known aspartic proteases, the disulfide motif is fundamentally different from that of other aspartic proteases. This difference may affect the substrate specificity of the enzyme. Taken together, both the presence of a transmembrane domain and the unusual disulfide bond structure lead us to conclude that BACE is an atypical pepsin family member.
Highlights
The cerebral deposition of amyloid -peptide is an early and critical feature of Alzheimer’s disease
We show that BACE is an Nglycosylated integral membrane protein that undergoes constitutive N-terminal processing in the Golgi apparatus
BACE Is a Glycosylated Integral Membrane Protein That Is N-terminally Processed in the Golgi Apparatus—Analysis of the BACE protein sequence suggests that BACE is a single transmembrane domain protein (7), and it has been shown that active enzyme can be released from membrane fractions after treatment with 0.2% Triton (9)
Summary
Despite the conservation of the active site residues and the 30 –37% amino acid homology with known aspartic proteases, the disulfide motif is fundamentally different from that of other aspartic proteases This difference may affect the substrate specificity of the enzyme. Studies with intact cells expressing APP and the endogenous secretases have led to conclusions about the properties of the - and ␥-secretases, e.g. their tissue distribution, subcellular localization, substrate requirements (see e.g. Ref. 6) etc., but until recently the identity of both - and ␥-secretase was unknown. This changed when we very recently identified the novel transmembrane aspartic protease BACE as the major -secretase (7). Our results demonstrate that BACE is an unusual member of the pepsin family
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