Maturation and Pro-peptide Cleavage of β-Secretase

Anja Capell,Harald Steiner,Michael Willem,Hartmut Kaiser,Carmen Meyer,Jochen Walter,Sven Lammich,Gerd Multhaup,Christian Haass

doi:10.1074/jbc.m003202200

Anja Capell, Harald Steiner + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m003202200

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Abstract

Amyloid beta-peptide is generated by two sequential proteolytic cleavages mediated by beta-secretase (BACE) and gamma-secretase. BACE was recently identified as a membrane-associated aspartyl protease. We have now analyzed the maturation and pro-peptide cleavage of BACE. Pulse-chase experiments revealed that BACE is post-translationally modified during transport to the cell surface, which can be monitored by a significant increase in the molecular mass. The increase in molecular mass is caused by complex N-glycosylation. Treatment with tunicamycin and N-glycosidase F led to a BACE derivative with a molecular weight corresponding to an unmodified version. In contrast, the mature form of BACE was resistant to endoglycosidase H treatment. The cytoplasmic tail of BACE was required for efficient maturation and trafficking through the Golgi; a BACE variant lacking the cytoplasmic tail undergoes inefficient maturation. In contrast a soluble BACE variant that does not contain a membrane anchor matured more rapidly than full-length BACE. Pro-BACE was predominantly located within the endoplasmic reticulum. Pro-peptide cleavage occurred immediately before full maturation and trafficking through the Golgi.

Highlights

Alzheimer’s disease is the most common age-dependent dementia
Senile plaques are predominantly composed of amyloid ␤-peptide (A␤),1 which is derived from the ␤-amyloid precursor protein (␤APP) [2]. ␤APP is a type 1 transmembrane protein that maturates during its transport to the cell surface by several post-translational modifications including N- and O-glycosylation [3], phosphorylation [3,4,5], sulfation [3], and endoproteolysis [2]
We studied the maturation of BACE with a particular emphasis on glycosylation, pro-peptide cleavage, and trafficking as a function of the cytoplasmic tail

Summary

The abbreviations used are

A␤, amyloid ␤-peptide; ␤APP, ␤-amyloid precursor protein; BACE, ␤-site APP-cleaving enzyme; ER, endoplasmic reticulum; Tricine, N-tris(hydroxymethyl)methylglycine; wt, wild-type; endo H, endoglycosidase H; HEK, human embryonic kidney. During its transport to the cell surface, ␤APP undergoes endoproteolytic cleavage. ␣-Secretase cleaves ␤APP within its A␤ domain leading to the secretion of APPs-␣ in biological fluids and conditioned medium of cultured cells [3, 13]. ␤-Secretase (BACE is the ␤-site APP-cleaving enzyme) has recently been identified [21,22,23,24,25] and demonstrated to be an aspartyl protease. Because very little is known about the maturation and proteolytic activation of BACE, it was necessary to investigate the maturation of BACE during its transport from the endoplasmic reticulum (ER) to the cell surface

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION