Abstract
BackgroundThe voltage-gated sodium channel β2 subunit (Navβ2) is a physiological substrate of BACE1 (β-site APP cleaving enzyme) and γ-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Navβ2 by BACE1 and γ-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navβ2.ResultsWe found a major (147-148 L↓M, where ↓ indicates the cleavage site) and a minor (144145 L↓Q) BACE1 cleavage site in the extracellular domain of human Navβ2 using a cell-free BACE1 cleavage assay followed by mass spectrometry. Next, we introduced two different double mutations into the identified major BACE1 cleavage site in human Navβ2: 147LM/VI and 147LM/AA. Both mutations dramatically decreased the cleavage of human Navβ2 by endogenous BACE1 in cell-free BACE1 cleavage assays. Neither of the two mutations affected subcellular localization of Navβ2 as confirmed by confocal fluorescence microscopy and subcellular fractionation of cholesterol-rich domains. Finally, wildtype and mutated Navβ2 were expressed along BACE1 in B104 rat neuroblastoma cells. In spite of α-secretase still actively cleaving the mutant proteins, Navβ2 cleavage products decreased by ~50% in cells expressing Navβ2 (147LM/VI) and ~75% in cells expressing Navβ2 (147LM/AA) as compared to cells expressing wildtype Navβ2.ConclusionWe identified a major (147-148 L↓M) and a minor (144-145 L↓Q) BACE1 cleavage site in human Navβ2. Our in vitro and cell-based results clearly show that the 147-148 L↓M is the major BACE1 cleavage site in human Navβ2. These findings expand our understanding of the role of BACE1 in voltage-gated sodium channel metabolism.
Highlights
The voltage-gated sodium channel b2 subunit (Navb2) is a physiological substrate of b-site APP cleaving enzyme (BACE1) (b-site amyloid precursor protein (APP) cleaving enzyme) and g-secretase, two proteolytic enzymes central to Alzheimer’s disease pathogenesis
In a follow-up study, we showed that elevated BACE1 activity increased release of Navb2-intracellular domain (ICD) through cleavage of Navb2 resulting in elevated protein and mRNA levels of Nav1.1 a subunits in neuroblastoma cells [21,22]
In order to better understand the role of BACE1 in Nav metabolism, we have identified the BACE1 cleavage site in human Navb2 in the present study
Summary
The voltage-gated sodium channel b2 subunit (Navb2) is a physiological substrate of BACE1 (b-site APP cleaving enzyme) and g-secretase, two proteolytic enzymes central to Alzheimer’s disease pathogenesis. We have found that the processing of Navb by BACE1 and g-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navb. Together with presenilin/g-secretase, BACE1 cleaves the amyloid precursor protein (APP) to generate amyloid b peptides (Ab). In addition to contributing to Ab generation, BACE1 regulates emotional memory, synaptic function and myelination in mouse brains possibly by cleaving multiple neuronal substrates [5,6,7]. Cleavage of substrate proteins may contribute to the important function of BACE1 in development and maintenance of the nervous system but the detailed molecular mechanism is not known
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