Glycosylphosphatidylinositol-anchored Proteins Play an Important Role in the Biogenesis of the Alzheimer's Amyloid β-Protein

Abstract

The Alzheimer's amyloid protein (A␤) is released from the larger amyloid ␤-protein precursor (APP) by unidentified enzymes referred to as ␤-and ␥-secretase. ␤-Secretase cleaves APP on the amino side of A␤ producing a large secreted derivative (sAPP␤) and an A␤-bearing C-terminal derivative that is subsequently cleaved by ␥-secretase to release A␤. Alternative cleavage of the APP by ␣-secretase at A␤16/17 releases the secreted derivative sAPP␣. In yeast, ␣-secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases. To examine the role of GPIanchored proteins, we specifically removed these proteins from the surface of mammalian cells using phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC treatment of fetal guinea pig brain cultures substantially reduced the amount of A␤40 and A␤42 in the medium but had no effect on sAPP␣. A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of A␤40, A␤42, and sAPP␤ than its parental line (GPI؉). When this parental line was treated with PI-PLC, A␤40, A␤42, and sAPP␤ decreased to levels similar to those observed in the mutant line, and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one or more GPI-anchored proteins play an important role in ␤-secretase activity and A␤ secretion in mammalian cells. The cell-surface GPI-anchored protein(s) involved in A␤ biogenesis may be excellent therapeutic target(s) in Alzheimer's disease.The amyloid that is invariably deposited in Alzheimer's disease (AD) 1 is composed of an approximately 4-kDa peptide (amyloid ␤-peptide, A␤) that is derived from a larger protein referred to as the amyloid ␤-protein precursor (APP) (1, 2). APP is a type I integral membrane glycoprotein with a large Nterminal extracellular domain, a single transmembrane domain, and a short cytoplasmic tail. The A␤ peptide begins 99 amino acids from the C terminus of APP, and it extends from the extracellular region to a point half-way through the APP membrane-spanning domain (1). A␤ is released from APP by cleavage on its N-and C-terminal ends by ␤-and ␥-secretase, respectively. ␤-Secretase cleavage before residue 1 of A␤ (672 of APP770) also releases the secreted derivative sAPP␤, whereas an alternative cleavage before residue 17 by ␣-secretase releases sAPP␣. In most culture systems tested, the predominant cleavage product is sAPP␣, and this may serve to prevent the production of A␤ (1). The proteolytic processing of APP to sAPP and A␤ is regulated by protein kinase C (3), protein tyrosine kinase (4), muscarinic receptors (5), and estrogens (6). The regulatory pathways involved are cell type-dependent, have little or no effect in some cell types, and normally stimulate the secretion of sAPP␣ while simultaneously reducing the secretion of A␤ (2). A metalloproteinase related to the tumor necrosis factor-␣ converting enzyme (7,8) can cleave APP to sAPP␣ upon activation of PKC by phorbol esters 2 (9, 10). Strong evidence that A␤ plays...

Internalization of Exogenously Added Memapsin 2 (β-Secretase) Ectodomain by Cells Is Mediated by Amyloid Precursor Protein

“…The fact, that the removal of GPI-anchored proteins from the cell surface resulted in the diminished M2 ED internalization and the cleavage of APP, is most probably related to the previous observation that the treatment of cells with PI-PLC reduced the production of A␤ (36). Our current data suggest that the interaction between APP and M2 ED is facilitated by GPIanchored proteins.…”

“…Being located in the lipid raft at the plasma membrane (19,20), memapsin 2 endocytosis is influenced by other components having direct or indirect contact with the protease. Such components include the glycosylphosphatidylinositol (GPI)-anchored proteins (19,36), scramblase (21), heparan sulfate (22), and cholesterol (20). It has been shown recently that memapsin 2 and APP are in close contact on the cell surface and throughout the endocytic process (23).…”

A Novel γ-Secretase Assay Based on Detection of the Putative C-terminal Fragment-γ of Amyloid β Protein Precursor

“…For comparison of treatments between experiments, treated samples were compared with controls adjusted to 100%. A␤ was measured as reported earlier by a specific and sensitive sandwich ELISA using a monoclonal antibody BNT77 against A␤11-28 for capture and horseradish peroxidase-labeled end-specific antibodies BA27 (A␤40) or BC05 (A␤42) for detection (12,31).…”

“…Additional reagents such as pepstatin A (Peptides International), 1,10-phenanthroline (PNT; J. T. Baker Co.), thiorphan (ICN), phosphatidylinositol-specific phospholipase C (PI-PLC; Molecular Probes), human recombinant caspase-3, Z-Val-Ala-Asp(OMe)-CH 2 F (Z-VAD-FMK), MG132, phenylmethylsulfonyl fluoride, E64, leupeptin, N-acetyl-LeuLeu-Nle-CHO (ALLN), CHAPSO, and Big CHAP (Calbiochem) were purchased as indicated. The Chinese hamster ovary (CHO) cell line, G9PLAP, expressing the human placental alkaline phosphatase (PLAP) cDNA, a kind gift from Dr. Victoria Stevens (Emory University, Atlanta, GA), was maintained in F12HAMS medium supplemented with 10% fetal calf serum and 200 g/ml geneticin (31).…”

“…In addition to BACE, thimet oligopeptidase, a metalloprotease, was shown to be involved in ␤-secretase activity in COS7 cells (30). We recently showed that GPI-anchored proteins play an important role in ␤-secretase activity (31). Recently, a family of disintegrin metalloproteases, the adamalysins such as tumor necrosis factor ␣-converting enzyme and ADAM 10, has been implicated in ␣-secretase cleav-age of APP (32)(33)(34).…”