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. 2011 Jul 13;21(10):1382–1388. doi: 10.1093/glycob/cwr097

Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10

Maria Tsachaki 2, Despina Serlidaki 2, Andriana Fetani 2, Vasiliki Zarkou 2, Ismini Rozani 2, Jorge Ghiso 3,4, Spiros Efthimiopoulos 2,1
PMCID: PMC3167477  PMID: 21752865

Abstract

Two different mutated forms of BRI2 protein are linked with familial British and Danish dementias, which present neuropathological similarities with Alzheimer's disease. BRI2 is a type II transmembrane protein that is trafficked through the secretory pathway to the cell surface and is processed by furin and ADAM10 (a disintegrin and metalloproteinase domain 10) to release secreted fragments of unknown function. Its apparent molecular mass (42–44 kDa) is significantly higher than that predicted by the number and composition of amino acids (30 kDa) suggesting that BRI2 is glycosylated. In support, bioinformatics analysis indicated that BRI2 bears the consensus sequence Asn-Thr-Ser (residues 170–173) and could be N-glycosylated at Asn170. Given that N-glycosylation is considered essential for protein folding, processing and trafficking, we examined whether BRI2 is N-glycosylated. Treatment of HEK293 (human embryonic kidney) cells expressing BRI2 with the N-glycosylation inhibitor tunicamycin or mutation of Asn170 to alanine reduced its molecular mass by ∼2 kDa. These data indicate that BRI2 is N-glycosylated at Asn170. To examine the effect of N-glycosylation on BRI2 trafficking at the cell surface, we performed biotinylation and 35S methionine pulse-chase experiments. These experiments showed that mutation of Asn170 to alanine reduced BRI2 trafficking at the cell surface and its steady state levels at the plasma membrane. Furthermore, we obtained data indicating that this mutation did not affect cleavage of BRI2 by furin or ADAM10. Our results confirm the theoretical predictions that BRI2 is N-glycosylated at Asn170 and show that this post-translational modification is essential for its expression at the cell surface but not for its proteolytic processing.

Keywords: Alzheimer's disease, BRI2, familial British dementia, familial Danish dementia, glycosylation

Introduction

BRI2 is a protein involved in two neurodegenerative diseases familial British dementia (FBD) and familial Danish dementia (FDD), since patients with those diseases bear two different mutations in the gene that encodes for BRI2; a stop codon mutation and a 10-nucleotide duplication (Vidal et al. 1999, 2000). The mutant forms of BRI2 in these diseases release upon cleavage by furin the peptides ABri (amyloid Bri) and ADan (amyloid Dan), which form amyloid deposits in the brain of patients with FBD and FDD, respectively. FBD and FDD present many similarities with Alzheimer's disease (AD; Tsachaki et al. 2008). Interestingly, we and others found that BRI2 might also play an important role in AD. More specifically, it interacts with the amyloid precursor protein (APP), which upon cleavage by β- and γ-secretase releases the peptide Aβ that forms amyloid deposits in the brain of AD patients (Fotinopoulou et al. 2005; Matsuda et al. 2005). Furthermore, BRI2 affects APP processing and reduces the production of Aβ both in cellular systems and in the brain of transgenic mice (Fotinopoulou et al. 2005; Matsuda et al. 2005, 2008). Given that FBD/FDD and AD share many neuropathological features, it is possible that common cellular events result in neuronal loss in these diseases. Therefore, the study of FBD/FDD could also give insights into the neurodegenerative mechanism underlying AD.

The physiological role of BRI2 is largely unknown, although there are studies implicating the protein in different cellular processes, such as neurite outgrowth (Choi et al. 2004) and apoptosis (Fleischer et al. 2002; Fleischer and Rebollo 2004). More recently, we found that BRI2 forms disulfide-linked homodimers that appear at the cell surface, a property that is often very important for the function of cell surface receptors (Tsachaki et al. 2010). Understanding the biological properties of BRI2 is of particular significance for the elucidation of its role in neurodegeneration. In accordance with this line of thought, we performed bioinformatics analysis to identify post-translational modifications of BRI2 that may play a role in its properties and function. We used NetNGlyc 1.0 Server to identify N-glycosylation sites and NetO-Glyc3.1 Server to predict O-glycosylation sites. This analysis indicated that of all three post-translation modifications, N-glycosylation is the most probable since BRI2 has a potential N-glycosylation site at asparagine residue 170, part of the consensus sequence Asn-X-Ser. This prediction is supported by the fact that BRI2 follows the secretory pathway from the endoplasmic reticulum (ER) and Golgi to the cell surface, and it is now known that many transmembrane cell surface proteins are glycosylated. In addition, the predicted molecular mass of BRI2 based on its amino acid sequence is around 30 kDa, whereas the protein migrates in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) at ∼42–43 kDa, indicating that post-translational modifications render the protein heavier. Based on these facts, we examined the possibility that BRI2 is post-translationally modified by the addition N-linked sugars because N-glycosylation is thought to play an important role in the folding of proteins, their trafficking through the secretory pathway to the cell surface as well as their metabolism. In the current paper, we show that ectopically expressed BRI2 in cell culture is indeed subjected to N-glycosylation at asparagine residue 170 (N170). Glycosylation affects the expression of BRI2 at the cell surface, but not its cleavage by furin or ADAM10 (a disintegrin and metalloproteinase domain 10). As a next step, unraveling the role of N-glycosylation for the cell surface function of BRI2 could broaden our understanding about the physiological role of this protein.

Results

BRI2 is glycosylated at asparagine residue 170

To examine whether BRI2 is N-glycosylated, we used the specific N-glycosylation inhibitor tunicamycin. This inhibitor blocks the formation of dolicholpyrophosphate N-acetylglucosamine, which is the first step in the N-glycosylation process. mycBRI2 was expressed in HEK293 cells and the cells were treated with 12 μg/mL of tunicamycin for 24 h prior to lysis. We observed (Figure 1A) that upon tunicamycin treatment mycBRI2 migrates at a molecular mass corresponding to 42 kDa (lane 2), whereas without any treatment it migrates to 44 and 42 kDa (lane 1), indicating that N-glycosylation confers further weight to the protein. To verify this result and prove that BRI2 is glycosylated at asparagine residue 170 (N170), we mutated this residue to alanine (Figure 5A). This mutation is not expected to cause major changes in the overall conformation of the protein, since alanine is a small hydrophobic amino acid, generally regarded to be rather inactive. When mutated mycBRI2 at asparagine residue 170 (mycBRI2/N170A) was expressed in HEK293 cells, it migrated at a molecular mass similar to that observed after tunicamycin treatment of cells (Figure 1B). Wild-type mycBRI2 migrated at the previously observed molecular masses of 44 and 42 kDa. Thus, BRI2 is subjected to N-glycosylation at N170.

Fig. 1.

Fig. 1.

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BRI2 is N-glycosylated at asparagine 170 residue. (A) HEK293 cells overexpressing wild-type mycBRI2 were incubated with N-glycosylation inhibitor tunicamycin (lane 2) or without (lane 1). Cell extracts were analyzed with western blot against myc with 9B11 antibody. (B) Wild-type mycBRI2 (mycBRI2, lane 1) or BRI2 with the mutation of asparagine 170 into alanine (mycΒRI2/N170, lane 2) were expressed in HEK293 cells. Cell extracts were analyzed by western blot using the 9B11 antibody against the myc epitope.

Fig. 5.

Fig. 5.

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Schematic representation of BRI2 proteolytic processing (A) and constructs used in this study (B and C). (A) BRI2 is cleaved by furin after amino acid 243 to release the secreted peptide p23. In addition, it is cleaved extracellularly by the metalloproteinase ADAM10, which leads to the release of an extracellular fragment and the production of a membrane-bound NTF. Upon subsequent cleavage of the NTF by the sPPLs, the intracellular domain of BRI2 is released intracellularly. The asparagine residue at amino acid position 170 (N170) is depicted by an asterisk. mycBRI2/N170A contains a substitution of this residue to an alanine residue. (B) mycBRI2-V5 contains a V5 epitope tag after the C-terminus for detection of the p23 peptide in the conditioned media. mycBRI2-V5/N170 bears a substitution of N170 by an alanine residue. (C) In BRI2ΔFC-V5, the V5 epitope tag is inserted after amino acid 240. BRI2ΔFC-V5/N170 is identical but bears substitution of N170 to alanine.

N-Glycosylation of BRI2 affects its cell surface expression

Next, we sought to identify whether glycosylation is important for cell surface expression of BRI2, as is the case for many transmembrane proteins. For that purpose, we expressed mycBRI2 or mycBRI2/N170A in HEK293 cells and labeled cell surface proteins using a non-cell permeable biotin ester. Biotinylated cell surface proteins were precipitated with streptavidin and subjected in western blot using the anti-myc antibody 9B11. Our data show that the levels of mycBRI2/N170A protein at the cell surface are significantly lower than those of mycBRI2 protein (Figure 2A), although the expression levels of both proteins are the same (Figure 2B). To examine whether this is due to reduced rate of transport of the non-glycosylated mycBRI2/N170A protein to the cell surface, we expressed mycBRI2 or mycBRI2/N170A in HEK293 cells and performed 35S radiolabeling for 2 h at 16°C (pulse) and chased the newly synthesized proteins for 0′, 20′, 40′ and 60′. After that, we labeled cell surface proteins with biotin followed by precipitation with streptavidin beads. Precipitated cell surface proteins were eluted from the streptavidin beads, subjected to immunoprecipitation using the 9B11 antibody against the myc epitope of the BRI2 proteins and analyzed on SDS–PAGE followed by autoradiography (Figure 3A). We observed that mycBRI2 was present at the cell surface 20 min after the initiation of chase, whereas mycBRI2/N170A failed to appear at the cell surface even after 60 min. To verify the proper expression of BRI2 proteins, we immunoprecipitated the cell extracts with 9B11 without prior biotinylation (Figure 3B). The above results indicate that N-glycosylation of BRI2 is important for its transfer at the cell surface.

Fig. 2.

Fig. 2.

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Inhibition of N-glycosylation of BRI2 inhibits its expression at the cell surface. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. Cell surface proteins were labeled with biotin (lanes 1 and 2) or were not labeled (lanes 3 and 4), as a control for biotinylation specificity. (A) Cell extracts were precipitated with streptavidin beads and analyzed with western blot against myc with 9B11 antibody. (B) Cell extracts were directly analyzed with western blot as a control for protein expression. The two immunoreactive bands of BRI2 proteins correspond to the furin-cleaved and the non-cleaved wild-type mycBRI2 or mycBRI2/N170A.

Fig. 3.

Fig. 3.

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The rate of cell surface expression/appearance/transport of BRI2 is reduced in the absence of N-glycosylation. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. The newly synthesized proteins were labeled with 35S in radiolabeling medium for 2 h (pulse) at 16°C and then were incubated in non-radiolabeling medium for 0′, 20′, 40′ and 60′ (chase). (A) Cell surface proteins were labeled with biotin and precipitated with streptavidin beads. Precipitated cell surface proteins were eluted from the beads and immunoprecipitated with 9B11 antibody against the myc epitope before electrophoresis and autoradiography. (B) Immunoprecipitation of cell extracts with 9B11, electrophoresis and autoradiography were performed to verify the expression levels of BRI2.

Inhibition of BRI2 N-glycosylation leads to its accumulation in intracellular compartments

To further prove that glycosylation of BRI2 affects its trafficking at the cell surface, we expressed mycBRI2 or mycBRI2/N170A proteins in HEK293 cells. Twenty-four hours post-transfection, we fixed the cells and labeled them with the 9E10 antibody against the myc-epitope of BRI2 proteins and with a secondary anti-mouse antibody conjugated with CF™488. Confocal microscope analysis (Figure 4A higher magnification, B lower magnification) indicated that mycBRI2/N170A accumulated in intracellular compartments, which most likely represent the ER and Golgi. In contrast, mycBRI2 was distributed in vesicle-like structures covering the whole cytoplasm and the cell membrane. The aberrant distribution of mycBRI2/N170A compared with the wild-type protein could account for its absence from the cell surface observed before.

Fig. 4.

Fig. 4.

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Non-N-glycosylated BRI2 accumulates in intracellular compartments. HEK293 cells were transfected with the cDNAs that encode for wild-type mycBRI2 or mycBRI2/N170A. Twenty-four hours post-transfection, the cells were fixed and incubated with the 9E10 anti-myc antibody and with a secondary anti-mouse antibody conjugated with CF™488. Following, mounting with Vectashield mounting medium containing the fluorescent dye DAPI for nuclear staining, cells were viewed under a confocal microscope (A: higher magnification, B: lower magnification).

Effect of N-glycosylation on BRI2 processing

To investigate if N-glycosylation of BRI2 is significant in any other aspect of BRI2 cell biology, we examined whether it affects BRI2 processing (for a depiction of BRI2 processing view, Figure 5A). BRI2 is subjected to extracellular cleavage by furin and releases a C-terminal secreted peptide of 23 amino acids (p23; Kim et al. 1999). The mycBRI2 used before expresses the myc epitope N-terminally. Therefore, in order to be able to detect the p23 peptide, we fused the cDNA of mycBRI2 with that encoding for the V5 epitope (mycBRI2-V5), appearing C-terminally in the expressed protein (Figure 5B). In another construct, we mutated the triplet encoding for N170 to that encoding for alanine (mycBRI2-V5/N170A). We expressed in cells mycBRI2-V5 or mycBRI2-V5/N170A and incubated the conditioned media with an anti-V5 in order to immunoprecipitate the V5-tagged p23 peptide (p23-V5). Immunoprecipitates were then analyzed by western blot in order to detect p23-V5 peptide. We observed that the amount of peptides released following furin cleavage is unaltered between mycBRI2-V5 and mycBRI2-V5/N170A (Figure 6A). The cellular levels of both proteins were similar (Figure 6B). This result shows that N-glycosylation is not a pre-requisite for furin cleavage.

Fig. 6.

Fig. 6.

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N-Glycosylation does not affect BRI2 cleavage by furin. HEK293 cells were transfected with the cDNAs encoding for wild-type mycBRI2-V5, mycBRI2-V5/N170A or PRK5 vector. (A) Forty-eight hours post-transfection conditioned medium samples were incubated with the anti-V5 antibody to immunoprecipitate p23V5 peptides. Immunoprecipitates were analyzed by western blot using the anti-V5 antibody. (B) Cell extracts were analyzed by western blot using the anti-myc antibody to confirm the expression of transfected proteins.

More recently (Martin et al. 2008), it was shown that BRI2 is cleaved extracellularly by the metalloproteinase ADAM10 for the release of a secreted fragment of 25 kDa and the production of a membrane-bound N-terminal fragment (NTF), which is subjected to intramembrane regulated proteolysis by the aspartyl-proteases sPPLa/b (signal peptide peptidase-like proteases), to release the intracellular domain of BRI2. Given that these two cleavage events were shown to be independent of furin cleavage, we investigated whether N-glycosylation is essential for ADAM10 cleavage of BRI2. To specifically detect the fragment released extracellularly after ADAM10 cleavage, we used a construct in which the sequence 240–266 (includes the cleavage site of furin which is between arginine 243 and glutamic acid 244) was replaced by the V5 epitope tag (BRI2ΔFC-V5), so that this protein is not cleaved by furin (Figure 5C). In another construct, we mutated the triplet that encodes for N170 to a triplet that encodes for alanine (BRI2ΔFC-V5/N170A). BRI2ΔFC-V5 or BRI2ΔFC-V5/N170A was expressed in cells and the conditioned media were incubated with anti-V5 in order to immunoprecipitate the V5-tagged C-terminal fragment (CTF) of BRI2 produced following ADAM10 cleavage. After western blot of the immunoprecipitates using the anti-V5 antibody, no difference in the amount of the secreted (sBRI2-V5) CTFs was observed, indicating that ADAM10 cleavage of BRI2 is not dependent on N-glycosylation (Figure 7A). The sBRI2-V5 that derives from the processing of BRI2ΔFC-V5/N170A has a lower molecular mass than the one that derives from the processing of BRI2ΔFC-V5, since the site of N-glycosylation resides within the fragment that is released after ADAM10 cleavage. Figure 7B shows expression of BRI2ΔFC-V5 and BRI2ΔFC-V5/N170A which due to the deletion of 26 amino acids and the insertion of the V5 epitope migrate at ∼42 and 40 kDa, respectively.

Fig. 7.

Fig. 7.

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Non-N-glycosylated BRI2 is efficiently processed by ADAM10. HEK293 cells were transfected with the cDNAs encoding for wild-type mycBRI2-ΔFC-V5 or mycBRI2-ΔFC-V5/N170A. (A) Forty-eight hours post-transfection conditioned medium samples were incubated with the anti-V5 antibody to immunoprecipitate the proteolytic fragments that derive following processing of BRI2 by ADAM10. Immunoprecipitates were analyzed by western blot using the anti-V5 antibody. (B) Cell extracts were analyzed by western blot using the anti-V5 antibody to confirm the expression of transfected proteins.

Discussion

FBD and FDD are two neurodegenerative diseases genetically linked with mutations in the gene that encodes for BRI2 protein (Vidal et al. 1999, 2000). These diseases present many similarities with AD, the most prevalent form of dementia. Therefore, their study could reveal aspects of the underlying mechanisms of neurodegeneration, which could be in part common among the different brain diseases. It has been hypothesized that the function of the proteins involved in neurodegeneration could be compromised in certain disease conditions. For example, tau is a protein that normally associates with and stabilizes the microtubules of axons. In FBD/FDD and AD, tau is hyperphosphorylated, dissociates from the microtubules and forms intraneuronal aggregates, known as neurofibrillary tangles, which are believed to play a pivotal role in neurite degeneration and neuronal loss (Buee et al. 2010).

BRI2 is a central protein in the pathogenesis of FBD/FDD, since mutated forms of the protein appear in patients with FBD/FDD. Through the proteolytic cleavage of these mutated proteins, the peptides ABri or ADan are released, forming amyloid deposits in the brain. So far, no physiological property directly related to neuronal dysfunction or neuronal loss has been attributed to BRI2. Therefore, more elaborate work needs to be done on the study of the physiological properties of BRI2 and its cellular role.

N-Glycosylation is one of the most important post-translational modifications and has been involved in different aspects of the physiology of proteins, such as for proper folding and quality control in the ER, for targeting in intracellular compartments of the secretory pathway or cell surface expression (Helenius and Aebi 2001). Given the significance of N-glycosylation, we studied whether BRI2 is subjected to this modification, since it has a potential N-glycosylation site and its electrophoretic molecular mass is ∼12–14 kDa greater than that predicted by its amino acid constitution.

BRI2 is indeed N-glycosylated at asparagine residue 170 (N170), since tunicamycin treatment of cells or substitution of that residue with an alanine residue, caused a shift in the molecular mass of the protein of ∼2 kDa (Figure 1). To gather further information regarding the post-translational modifications of BRI2, bioinformatics analysis of the BRI2 sequence (Q9Y287) was done using the NetO-Glyc3.1 Server and the NetC-Glyc1.0 Server to predict O-GalNac glycosylation sites and C-mannosylation sites, respectively. This analysis gave us low prediction scores indicating that the probability of BRI2 to accept other than N-linked sugars is rather low. However, by using the NetGlycate Server, which predicts glycation sites (non-enzymatic glycosylation) of epsilon amino groups of lysines in mammalian proteins, and the NetPhos2.0 Server, which predicts Ser, Thr and Tyr phosphorylation sites in eukaryotic proteins, we found that BRI2 has eight potential glycation and eight phosphorylation sites in its sequence. Therefore, the reduction in the molecular mass of BRI2 by only 2 kDa in the absence of N-glycosylation and the fact that in this case the apparent molecular mass of BRI2 is still 10 kDa more than that predicted by its amino acid composition indicate the need for more detailed analysis of all the possible post-translational modifications of BRI2.

The N-glycosylation mutant (BRI2/N170A) exhibited similar expression levels with the wild-type protein indicating that N-glycosylation is not essential for the stability of BRI2. N-Glycosylation is involved in the trafficking of many transmembrane proteins at the cell surface, as, for example, is the case for the prolactin receptor (Buteau et al. 1998), the angiotensin II receptor 1 (AT1; Deslauriers et al. 1999), the water channel aquaporin-2 (Hendriks et al. 2004) and the neuronal glycine transporter (Martınez-Maza et al. 2001). Similarly, biotinylation experiments showed that the levels of mycBRI2/N170A at the cell surface are significantly lower that those of the wild-type mycBRI2 (Figure 2). Furthermore, pulse-chase experiments (Figure 3) showed that although wild-type mycBRI2 appears at the cell surface 20 min after its biosynthesis, mycBRI2/N170A fails to reach the cell surface even at 60 min after its biosynthesis. These results indicate that trafficking of the non-glycosylated BRI2 at the cell surface is drastically disturbed. In support, immunocytochemistry experiments showed that (Figure 4) wild-type mycBRI2 is present in vesicular structures throughout the cytoplasm while the non-glycosylated mutant mycBRI2/N170A accumulates intracellularly most probably in compartments of the ER and Golgi apparatus. These results indicate that N-glycosylation is essential for the proper trafficking of BRI2 through the membrane compartments of the cell.

It has been shown that BRI2 is processed by furin between amino acids 243 and 244 resulting in the release of a secreted CTF of ∼2.5 kDa (Kim et al. 1999). Furthermore, it has been shown that BRI2 is cleaved extracellularly by ADAM10 to release a secreted CTF of ∼25 kDa (Martin et al. 2008). The NTF that remains membrane bound is subjected to intramembrane proteolysis by signal peptidases like 2a/2b proteases (Martin et al. 2008). Given that the N-glycosylation mutant of BRI2 displays aberrant intracellular accumulation and reduced levels at the cell surface, it is reasonable to assume that its proteolytic processing will be also affected. However, the N-glycosylation mutant of BRI2 was found to be processed normally by both furin and ADAM10 (Figures 6 and 7). The efficient processing of the N-glycosylation-deficient BRI2 is a surprising result, indicating that cleavage by ADAM10 can also take place intracellularly.

N-Glycosylation has been shown to be important for the function of many proteins. For instance, branched N-glycans are involved in the adhesion properties of E-cadherin and integrins, which in turn affect cell migration and cell–cell and cell–matrix interactions (Zhao et al. 2008). Also, N-glycosylation reduces the uptake of dopamine by the dopamine transporter (Li et al. 2004). N-Glycosylation of BRI2 could also be associated with the biological function of the protein, which until now remains enigmatic. Unraveling the importance of N-glycosylation for the function of BRI2 at the cell surface will contribute in our understanding of its normal function and the aberrance from this function of the mutants of BRI2 protein that lead to disease.

Materials and methods

Chemicals

All common chemicals were purchased from Sigma-Aldrich (Athens, Greece) unless otherwise indicated.

Plasmids and antibodies

The cDNA of human BRI2 was subcloned in myc-pRK5 vector (kind gift of Dr. PF Worley, John Hopkins, Baltimore, MD, USA) (mycBRI2). mycBRI2-V5 was created by subcloning the cDNA of myc BRI2 in the pcDNA 3.1/V5-His vector (Invitrogen, Carlsbad, CA) between the sites XhoI and HindIII. BRI2 ΔFC-V5 is described elsewhere (Martin et al. 2008) and was a kind gift of Prof. Christian Haass (Center for Integrated Protein Science Munich and the Adolf Butenandt Institute, Department of Biochemistry, Laboratory for Neurodegenerative Disease Research, Ludwig Maximilians University, Munich, Germany). The constructs mycBRI2/N170A, mycBRI2-V5/N170A and BRI2ΔFC-V5/N170A, in which the triplet that encodes for N170 has been substituted with that encoding for alanine, were generated with polymerase chain reaction using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, CA). Primers are available upon request.

The anti-myc monoclonal antibody 9B11 (Cell Signaling Technology, MA) was used for western blot and immunoprecipitation of myc-tagged BRI2. The monoclonal antibody against the V5 epitope was from AbD Serotec (Morphosys UK Ltd, Oxford, UK). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were supplied from Invitrogen. The secondary goat anti-mouse antibody conjugated with CF™488A Green fluorescent dye was obtained from Biotium, Inc. (Hayward, CA).

Cell culture and transfections

HEK293 cells were purchased from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with heat inactivated 10% fetal bovine serum (both from PAA Laboratories GmbH, Linz, Austria) and 100 g/mL of penicillin/streptomycin. Transfections were performed with Escort IV reagent (Sigma-Aldrich) according to the manufacturer's instructions.

Protein extraction and western blotting

Forty-eight hours after transfection, conditioned media were removed and cells were lysed at 4°C in lysis buffer [50 mM Tris–HCl, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), pH 7.6, 1% Triton X-100 (v/v)] supplemented with complete protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). Samples were incubated for 30 min on ice and protein extracts were clarified by centrifugation at 15,000 × g for 30 min at 4°C. The supernatants were quantified for protein content using the BCA Assay Kit (Pierce, Rockford, IL). Samples from cell extracts were prepared in SDS Laemmli sample buffer (1% SDS, 0.4 M Tris, 40 mM EDTA, 50% glycerol, Bromophenol blue) containing 5% β-mercaptoethanol. Extracts were separated by SDS–PAGE and transferred to polyvinyldene difluoride membranes (Roth GmbH, Karlsruhe, Germany). Membranes were immunoblotted with the primary antibody diluted in a blocking solution containing 10% new-born calf serum supplemented with NaN3 overnight at 4°C. After washing with tris-buffered saline - Tween-20 (TBS-T) [20 mM Tris–HCl, pH 7.6, 137 mM NaCl, 0.05% (v/v) Tween-20], filters were incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies in blocking solution (1:5000 dilution) without NaN3 for 1 h at room temperature. Protein bands were detected by chemiluminescence using the enhanced chemiluminescence system (Amersham, Upsala, Sweden) on a Fluorochem 8800 imaging station (AlphaInnotech, CA).

Immunoprecipitation

For the immunoprecipitation experiments, cell extracts (1000–1500 g of total protein) or conditioned media were incubated with appropriate dilutions of the IP antibody overnight at 4°C. Following incubation for 1 h at 4°C with protein G agarose beads (Millipore, Billerica, MA), antibody-bound protein complexes were collected by centrifugation at 15,000 × g for 10 min at 4°C. Pellets were washed three times with washing buffer containing 50 mM Tris, 150 mM NaCl, 2 mM EDTA, pH 7.6, 0.5% Triton X-100. The beads were resuspended in SDS Laemmli sample buffer and the recovered proteins were analyzed by immunoblot.

Metabolic labeling and biotinylation

Twenty-four hours post-transfection the newly synthesized proteins were labeled with 35S in serum-free DMEM that did not contain cysteine and methionine for 2 h (pulse) at 16°C, to prevent membrane trafficking of the newly synthesized proteins. The labeling medium was removed and replaced with non-radiolabeling medium for 0′, 20′, 40′ and 60′ (chase). For biotinylation of cell surface proteins, cells were washed three times with ice-cold phosphate buffered saline (PBS) containing 0.5 mM CaCl2 and 1 mM MgCl2 (PBS-CM). Then, cells were biotinylated with 0.5 mg/mL Sulfo-NHS-LC-biotin (Pierce) for 30 min on ice-cold water. Unbound biotin was quenched with 50 mM NH4Cl for 10 min. Cells were rinsed twice with ice-cold PBS-CM and lysed as described in “Protein extraction and western blotting” within the “Materials and Methods” section. The cell extracts were centrifuged at 15,000 × g for 30 min and supernatants were incubated with 50 μL of streptavidin–agarose beads (Millipore) for 1 h at 4°C.

Immunofluorescence

Cells were cultured in slides and transfected as described in “Cell culture and transfections” within the “Materials and Methods” section. Twenty-four hours post-transfection, the supernatant media were removed and the cells were rinsed twice with PBS. Cells were incubated for 15 min at −20°C with cold methanol for fixation and permeabilization, and then rinsed once with PBS. Cell cultures were incubated with 1% new-born calf serum blocking solution supplemented with NaN3 for 30 min at 37°C and with the primary antibody diluted in the same blocking solution overnight. Cell cultures were rinsed twice with TBS-T, and the secondary goat anti-mouse antibody CF™488A was added for 1 h. Cell cultures were rinsed twice with TBS-T and mounted with Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA) containing the fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining. The fluorescent signal was detected using a confocal microscope (Leica TCS SP5).

Funding

This research was funded by an Empeirikion Foundation grant, the University of Athens grant “Kapodistrias”, the Alzheimer's Association grant (IIRG-09-133340) and in part by the National Institutes of Health grant AG030539. M.T. is supported by a fellowship from the “Greek State Scholarships Foundation”.

Conflict of interest

None declared.

Abbreviations

ABri, amyloid Bri; AD, Alzheimer's disease; ADan, amyloid Dan; ADAM10, a disintegrin and metalloproteinase domain 10; APP, amyloid precursor protein; CTF, C-terminal fragment; DAPI, 4',6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; EDTA, ethylenediaminetetraacetic acid; ER, endoplasmic reticulum; FBD, familial British dementia; FDD, familial Danish dementia; HEK, human embryonic kidney; NTF, N-terminal fragment; PBS, phosphate buffered saline; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; sPPLs, signal peptide peptidase-like proteases; TBS-T, tris-buffered saline - Tween-20.

Acknowledgements

We thank Prof. Christian Haass for kindly providing us with the BRI2 ΔFC-V5 DNA construct.

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