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

Kumar Sambamurti,Daniel Sevlever,Thillai Koothan,Lawrence M Refolo,Inga Pinnix,Swetal Gandhi,Luisa Onstead,Linda Younkin,Christian M Prada,Debra Yager,Yasumasa Ohyagi,Christopher B Eckman,Terrone L Rosenberry,Steven G Younkin

doi:10.1074/jbc.274.38.26810

Kumar Sambamurti, Daniel Sevlever + Show 12 more

Open Access

https://doi.org/10.1074/jbc.274.38.26810

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Abstract

The Alzheimer's amyloid protein (Abeta) is released from the larger amyloid beta-protein precursor (APP) by unidentified enzymes referred to as beta- and gamma-secretase. beta-Secretase cleaves APP on the amino side of Abeta producing a large secreted derivative (sAPPbeta) and an Abeta-bearing C-terminal derivative that is subsequently cleaved by gamma-secretase to release Abeta. Alternative cleavage of the APP by alpha-secretase at Abeta16/17 releases the secreted derivative sAPPalpha. In yeast, alpha-secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases. To examine the role of GPI-anchored 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 Abeta40 and Abeta42 in the medium but had no effect on sAPPalpha. A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of Abeta40, Abeta42, and sAPPbeta than its parental line (GPI+). When this parental line was treated with PI-PLC, Abeta40, Abeta42, and sAPPbeta 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 beta-secretase activity and Abeta secretion in mammalian cells. The cell-surface GPI-anchored protein(s) involved in Abeta biogenesis may be excellent therapeutic target(s) in Alzheimer's disease.

Highlights

The amyloid that is invariably deposited in Alzheimer’s disease (AD)1 is composed of an approximately 4-kDa peptide
phosphatidylinositol-specific phospholipase C (PI-PLC) Treatment Reduces the Secretion of A␤ but Not sAPP␣ in Guinea Pig Mixed Brain Cultures—Guinea pig mixed brain cultures were treated with PI-PLC for 1 and 3 days in triplicate
Since PI-PLC treatment is known to release GPI-anchored proteins from the cell surface, these data suggest that one or more GPI-anchored proteins are involved in A␤ biogenesis

Summary

EXPERIMENTAL PROCEDURES

Purification of PI-PLC—PI-PLC was expressed in Bacillus subtilis cultures transformed with a plasmid containing the Bacillus thuringiensis gene and was purified from 8 liters of the culture media [28]. The free inositol released by PI-PLC was measured in the aqueous fraction after stopping the reaction in 10:5:0.1 chloroform:methanol:HCl. The protein concentration was determined by a BCA assay (Pierce). The gpi mutant is deficient in N-acetylglucosaminylphosphatidylinositol deacetylase which is responsible for the second step in the biosynthesis of the GPI anchor [32] These cultures were maintained in Ham’s F-12 media supplemented with 10% fetal bovine serum and 100 ␮g/ml G418. Nunc immunomax dishes were coated with capture antibodies BAN50 (guinea pig) or BNT77 (CHO) in 0.1 M carbonate buffer, pH 9.6, and blocked with 0.5% block ace (Wako) in PBS, pH 7.4. After washing twice with PBS and once with PBS ϩ 0.05% Tween 20 horseradish peroxidase activity was detected using a Pierce ELISA kit as described by the manufacturer. Total sAPP in the medium was detected using the 22C11 antibody (Roche Molecular Biochemicals)

RESULTS

46 Ϯ 3 13 Ϯ 2a

12 Ϯ 5 11 Ϯ 4

DISCUSSION