A Novel Metalloprotease in Rat Brain Cleaves the Amyloid Precursor Protein of Alzheimer's Disease Generating Amyloidogenic Fragments

Abstract

The amyloid protein (A beta or beta A4) is the major constituent of amyloid plaques in the Alzheimer's disease brain. A beta is cleaved from the amyloid precursor protein (APP) by a mechanism which is poorly understood. Cell culture studies suggest that APP may be cleaved by secretases within the late Golgi compartment. Studies performed so far have mainly used exogenous APP and synthetic peptides as substrates. For this study, a Golgi and plasma membrane-enriched fraction was isolated from rat brain and incubated at 37 degrees C at pH 7.2 to study the degradation of endogenous APP. The breakdown of APP was accompanied by the concomitant generation of A beta-containing C-terminal fragments, in a time-dependent fashion. The metal ion chelators EDTA and 1,10-phenanthroline inhibited this degradation. The inhibition by EDTA was reversed by 50 microM Zn2+ but not by other metal ions. The protease activity was not inhibited by cysteine, serine or aspartic protease inhibitors nor was it inhibited by compounds which are inhibitors of known metalloendopeptidases and matrix metalloproteinases (cFP, phosphoramidon and TIMP-2). Our data suggest that a novel Zn(2+)-dependent metalloprotease activity associated with a Golgi and plasma membrane-enriched fraction can degrade endogenous APP to generate A beta containing C-terminal fragments. This protease may generate amyloidogenic fragments of APP which may serve as precursors for A beta.

ZnT4 provides zinc to zinc-dependent proteins in the trans-Golgi network critical for cell function and Zn export in mammary epithelial cells

“…The Golgi apparatus plays a critical role in the process of secretion (8,24,32) and numerous resident proteins such as galactosyltransferase and metalloproteases are Zn dependent (30,33). We found that changes in ZnT4 abundance modulated the activity of galactosyltransferase.…”

Wheat germ agglutinin‐binding glycoproteins are decreased in Alzheimer's disease cerebrospinal fluid

“…Membranes were blocked in 0.5% hydrolysed casein in phosphate‐buffered saline (PBS), pH 7.4, for 1 h. The blocked membranes were then incubated with 12.5 µg/mL biotinylated lectin in Tris‐buffered saline–Tween 20 (TBST) for 1 h at room temperature (22 °C). After washing the membranes with TBST, glycoproteins were visualized using (1 : 1000 dilution) NeutrAvidin‐conjugated to alkaline phosphatase and developed with a mixture of Fast Red/naphthol AS‐MX substrate (Mok et al . 1997).…”

The amyloid β‐protein precursor of Alzheimer's disease is degraded extracellularly by a Kunitz protease inhibitor domain‐sensitive trypsin‐like serine protease in cultures of chick sympathetic neurons

“…A number of proteases can cleave APP in vitro including the cathepsins D and G [32], calpain [33], thrombin [34], metalloprotease (EC 3.4.24.15) [35], gelatinase A [36,37], calcium‐activated serine protease [38], and Factors XIa [39] and Xa [40]. Our studies have identified a novel metalloprotease in a Golgi‐enriched fraction from rat brain which cleaves APP in vitro [41]. This Zn 2+ ‐dependent enzyme generates several potentially amyloidogenic fragments ranging in molecular mass from 14 kDa to 80 kDa.…”