Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit
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The 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.
We 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.
We 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.
KeywordsLipid Raft BACE1 Level Human BACE1 BACE1 Cleavage BACE1 Substrate
amyloid precursor protein
amyloid β peptide
voltage-gated sodium channel
voltage-gated sodium channel β2 subunit
β-site APP cleaving enzyme
Navβ2 substrate peptide
Chinese hamster ovary
BACE1/β-secretase is an aspartic protease highly expressed in neuronal cells [1, 2]. Together with presenilin/γ-secretase, BACE1 cleaves the amyloid precursor protein (APP) to generate amyloid β peptides (Aβ). Aβ accumulates in the brains of Alzheimer's disease patients where it promotes disease pathology [3, 4]. In addition to contributing to Aβ generation, BACE1 regulates emotional memory, synaptic function and myelination in mouse brains possibly by cleaving multiple neuronal substrates [5, 6, 7]. More than 60 BACE1 substrates have recently been identified via quantitative proteomics . However, only a few substrates have been investigated and confirmed in vivo [6, 9, 10, 11, 12]. 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.
Voltage-gated sodium channels (Nav) are composed of central α subunits and one or two accessory β subunits . The pore forming α subunits regulate sodium ion transport in neuronal membranes and are therefore essential for neuronal membrane excitability . The β subunits are type I transmembrane proteins with extracellular immunoglobulin and short intracellular C-terminal domains. Interaction of β subunits with α subunits regulates Nav assembly and activity [13, 14, 15]. In particular, the β2 subunit (Navβ2) regulates cell-surface expression and inactivation kinetics of Nav channels in neurons [16, 17]. In addition, β subunits modulate cell adhesion and neurite outgrowth [18, 19, 20].
Previously, we and another group found that ADAM10, BACE1, and γ-secretase cleave Navβ2 in neuronal cells and mouse brains [11, 12]. In a follow-up study, we showed that elevated BACE1 activity increased release of Navβ2-ICD (intracellular domain) through cleavage of Navβ2 resulting in elevated protein and mRNA levels of Nav1.1 α subunits in neuroblastoma cells [21, 22]. Furthermore, processing of endogenous Navβ2 and Nav1.1 protein levels were elevated in BACE1-transgenic mouse brains and eventually resulted in altered sodium current densities in hippocampal neurons. These data strongly suggest that BACE1 can regulate neuronal function, possibly by cleaving Navβ2 in physiological conditions. In order to better understand the role of BACE1 in Nav metabolism, we have identified the BACE1 cleavage site in human Navβ2 in the present study.
Materials and methods
Plasmids, transfection, and reagents
Expression constructs encoding full-length human Navβ2 (GenBank: NM_004588) containing a C-terminal V5-His tag and full-length human BACE1 (GenBank: AF190725) containing a C-terminal myc tag have been described previously . Navβ2 (147LM/VI) and Navβ2 (147LM/AA) were constructed using QuickChange Site-directed Mutagenesis kit (Stratagene) with the following primers: Navβ2 (147LM/VI): 5'-GGCAAGATCCATCTGCAGGTCGTCATTGAAGAGCCCCCTGAGCGG-3' 5'-CCGCTCAGGGGGCTCTTCAATGACGACCTGCAGATGGATCTTGCC-3'; Navβ2 (147LM/AA): 5'-GGCAAGATCCATCTGCAGGTCGCCGCGGAAGAGCCCCCTGAGCGG-3' and 5-'CCGCTCAGGGGGCTCTTCCGCGGCGACCTGCAGATGGATCTTGCC-3'. Effectene (Qiagen) was routinely used for transfecting cell lines. GL189 (Calbiochem) was used in 10 μM concentration.
In vitro cleavage assay of Navβ2 substrate peptide
Navβ2 substrate peptide (β2-peptide) with N-terminal biotin was synthesized by CHI Scientific (M.W. 4049.7, purity = 94.02% determined by HPLC). A biotinylated tyrosine group was added to the N-terminus of the β2-peptide. Reaction mixtures containing 20 mg of β2-peptide, 0.1 M Na-Acetate (pH 4.0), and 2.5 mg human BACE1 (R&D systems), were prepared and incubated at 37°C for 16 h. Reactions were stopped by heating to 95°C with LDS-SDS-PAGE sample loading buffer (Invitrogen) for 5 min.
Reaction samples were then resolved on 12% BisTris gels (Invitrogen), transferred to PDVF membrane for Western blot analysis or fixed directly for silver staining. Vector ABC kit (Vector Labs) was used to detect full-length and N-terminal fragment of β2-peptide in Western Blot while Silver SNAP II kit (Invitrogen) was used to detect total protein in the gel.
Reaction samples from the β2-peptide in vitro cleavage assay were analyzed by MS using a QStarR Pulsar I (Applied Biosystems) equipped with a nanospray source (in collaboration with Proteomic core at Harvard Partners Center for Genetics and Genomics). Analyst software (Invitrogen) was used to determine the molecular weights of all cleavage products in the reaction mixture.
Western blot analysis
Cell lysates were prepared by directly extracting cells in a buffer containing 10 mM Tris-HCl (pH 6.8), 1 mM EDTA, 150 mM NaCl, 0.25% Nonidet P-40, 1% Triton X-100, and a protease inhibitor cocktail (Roche) followed by a centrifugation at 16,000 g. 20-50 mg of protein were resolved on 12% BisTris gels (Invitrogen). The blots were visualized by enhanced chemiluminescence (ECL). Images were captured using BioMax film (Kodak) or VersaDoc imaging system (Biorad) and quantified with QuantityOne software (Biorad). The followings are antibodies used in this study: anti-V5 (1:5000; Invitrogen), anti-myc (1:2000: Cell Signalling) anti-GAPDH (1:2000; BD Biosciences), and antiflotillin-1 (1:250; BD Biosciences).
In vitro generation of Navβ2-CTFβ
Membrane preparation and in vitro generation of Navβ2-CTFβ were performed as described earlier . In brief, cells were washed with PBS, scraped in 1 ml PBS and centrifuged for min at 8000 rpm. Cell pellets were resuspended in 700 μl buffer H (20 mM HEPES, 150 mM NaCl, 10% glycerol, 5 mM EDTA, pH 7.4) and the solution drawn 20 times through a 3 ml syringe with 20 gauge needle. Unbroken cells were removed by centrifugation at 4000 rpm for 5 min. In order to obtain P2 fractions, the supernatant was centrifuged at 55000 rpm for 1 h and 4°C. Membrane fractions were washed once in 300 μl incubation buffer (0.1 M Na Acetate pH 4.0, 10 μg/ml Leupeptin, 1 μg/ml Aprotinin, 1 mM PNT and 5 mM EDTA) and resuspended in 100 μl incubation buffer in absence or presence of GL189. After incubation for 3 h at either 0°C or 37°C, the samples were loaded on 12% BisTris and investigated by Western blot analysis.
Lipid Raft Fractionation
Cells were grown to 80 - 90% confluency in three 150-mm dishes, washed twice in phosphate buffered saline and scraped into 1.2 ml extraction buffer containing 0.5% Lubrol WX (Lubrol 17A17; Serva), protease inhibitor cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were then homogenized by five passages through a 25-gauge needle. Cell lysates were adjusted to 45% final concentration of sucrose (final volume, 4 ml) and loaded to the bottom of a 12-ml SW41 ultracentrifuge tube. A discontinuous sucrose gradient was established by sequentially layering 35% sucrose (4 ml) and 5% sucrose (4 ml) on top of the sample. Tubes were subjected to ultracentrifugation at 39,000 rpm for 18 h in Beckman SW41 rotor at 4°C. Twelve 1 ml fractions were collected from the top of the gradient and equal volume of each fraction was analyzed by Western blotting. V5 antibody was used to detect Navβ2 and flotillin-1 antibody was used as a lipid raft marker.
Cells were grown on coverslips to 20 - 40% confluency and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were then rinsed three times with PBS and blocked for 1 h in PBS containing 1% BSA (Sigma-Aldrich) 0.1% Triton X-100 (FisherBiotech), 0.1% Gelatin (Sigma-Aldrich) and 0.05% Tween 20 (MP Biomedicals). Cells were incubated with anti V5 antibody for 1 h, washed three times with PBS and incubated for 30 min with rabbit anti-mouse Alexa Fluor 488 antibody (1:200: Serotec) at room temperature. After washing three times with PBS containing 0.1% Triton X-100 and 0.05% Tween-20 for 3 min the coverslips were mounted onto glass slides using Prolong Gold antifade reagent with DAPI (Invitrogen) and analyzed with a Olympus florescence microscope equipped with a confocal disk scanning unit.
Identification of BACE1 cleavage sites in human Navβ2
Mutations of the BACE1 cleavage site decrease BACE1-mediated processing of human Navβ2 in purified CHO cells membrane
Mutations of the BACE1 cleavage site do not affect subcellular localization of human Navβ2
Next, we tested whether the distribution of Navβ2 into cholesterol-rich domains (lipid rafts) is altered by the 147LM/AA mutation. As previously reported, Navβ2 was detected in lipid raft enriched fractions, confirmed by flotillin-1 staining (Figure 3B, fractions 2 to 4, ). We could not detect any significant changes in the levels of Navβ2 (147LM/AA) in lipid-raft fractions. Together, these results suggest that localization of Navβ2 was not affected by mutations of the BACE1 cleavage site.
Mutations of the BACE1 cleavage site decrease processing in cell based models
A recent study identified 68 BACE1 substrates, underscoring the role of BACE1 in various cellular processes . Similarly to Navβ2, the majority of those substrates are, type 1 transmembrane proteins with extracellular N-terminal and intracellular C-terminal ends. However, in addition to Navβ2 only a few BACE1 substrates have been confirmed under physiological conditions. These include APP, Neuregulin 1/3 (NRG-1/3), alpha 2,6-sialyltransferase (ST6GAL1), and P-selectin glycoprotein ligand-1 (PSGL1) [6, 912]. As shown in Figure 5 the BACE1 cleavage sites in these substrates show highly similar sequences. For example, the S1 cleavage position has been suggested as the most important site for determining BACE1-mediated processing based on an in vitro cleavage assay using synthetic substrate peptides . The majority of substrates harbor leucine, phenylalanine, or methionine at the S1 cleavage site. Interestingly, human Navβ2 harbors both leucine and methionine at the S1 and S1' position, which are the most preferable residues at those positions according to in vitro studies . These studies also suggest, that subsites proximal to the scissile bond (S1 and S'1) are more stringent than distal residues which is reflected by the drastic decrease in CTF production in Navβ2 harboring mutations 147LM/AA or 147LM/VI.
In our previous studies, we have found that elevated BACE1-mediated cleavage of human Navβ2 increased mRNA and protein levels of Nav1.1 α subunit by increasing the release of the β2 intracellular domain (Navβ2-ICD) . Therefore, it will be interesting to see whether the blockage of BACE1 cleavage in Navβ2 147LM/IV and Navβ2 147 LM/AA would also decrease Nav1.1 levels by decreasing Navβ2-CTF levels and possibly Navβ2-ICD levels. However, these studies are difficult because α-secretase cleavage of Navβ2 is not affected by the 147 LM/VI and 147 LM/AA mutations. On the contrary, we have observed elevated α-secretase cleavage in CHO cells transiently expressing Navβ2 (147 (LM/AA) (data not shown). The α-secretase cleavage site is distinct from and closer to the membrane than the BACE1 cleavage site, unlikely to be directly affected by the BACE1 cleavage site mutations [21, 25]. It is more likely that the elevated α-secretase cleavage observed only in CHO cells is due to a compensatory αsecretase-mediated cleavage since BACE1 and α-secretase seem to compete for juxtamembrane cleavages as shown in APP processing. Additional mutations completely blocking α-secretase-mediated cleavages will be required to fully address the role of BACE1 in sodium channel metabolism in normal conditions.
BACE1 levels and activities are significantly elevated in AD brains, possibly contributing to the disease progression [26, 27, 28]. BACE1 levels are also increased in some injury conditions including brain trauma  and ischemia [30, 31, 32], suggesting a possible role of BACE1 as a stress-response protein . The fact that BACE1 regulates Nav channel metabolism via Navβ2 suggests the interesting possibility that BACE1 might modulate sodium channel metabolism not only in AD but also in other disease conditions in which BACE1 levels are increased. It will be interesting to test whether the BACE1cleavage mutation in Navβ2 reported here would also alter sodium channel metabolism in various stress conditions including oxidative stress and mitochondrial dysfunctions, which are known to increase BACE1 levels.
We identified a major (147-148 L↓M) and a minor (144-145 L↓Q) BASE1 cleavage site in human Navβ2 by using a synthetic β2-peptide and MS. We also found that mutations of the major BACE1 cleavage site (147LM/VI and 147 LM/AA) dramatically decreased BACE1-mediated cleavage of human Navβ2 in an in vitro assay and a cell based model. Our data clearly demonstrate that the BACE1 cleavage site (147-148 L↓M) is mainly responsible for BACE1 cleavage of human Navβ2.
We would like to thank Drs. Aleister Saunders (Drexel University) and Rudolph E. Tanzi (Massachusetts General Hospital/Harvard Medical School) for the human BACE1 construct. This work is supported by grants from the NIH/NIA.
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