ORIGINAL ARTICLE JBMR

Topological Mapping of BRIL Reveals a Type II Orientation and Effects of Osteogenesis Imperfecta Mutations on Its Cellular Destination

Alexa Patoine,1,2 Marie‐Hélène Gaumond,1 Prashant K Jaiswal,1,2 François Fassier,1 Frank Rauch,1,3 and Pierre Moffatt1,2 1Shriners Hospital for Children, Montreal, Quebec, Canada 2Department of Human Genetics, McGill University, Montreal, Quebec, Canada 3Department of Pediatrics, McGill University, Montreal, Quebec, Canada

ABSTRACT BRIL/IFITM5 is a membrane present almost exclusively in osteoblasts, which is believed to adopt a type III (N‐out/C‐out) topology. Mutations in IFITM5 cause OI type V, but the characteristics of the mutant protein and the mechanism involved are still unknown. The purpose of the current study was to re‐assess the topology, localization, and biochemical properties of BRIL and compare it to the OI type V mutant in MC3T3 osteoblasts. Immunofluorescence labeling was performed with antibodies directed against BRIL N‐ or C‐terminus. In intact cells, BRIL labeling was conspicuously detected at the plasma membrane only with the anti‐C antibody. Detection of BRIL N‐terminus was only possible after permeabilization, revealing both plasma membrane and Golgi labeling. Trypsinization of live cells expressing BRIL only cleaved off the C‐terminus, confirming that it is a type II protein and that its N‐ terminus is intracellular. A truncated form of BRIL lacking the last 18 residues did not appear to affect localization, whereas mutation of a single leucine to arginine within the transmembrane segment abolished plasma membrane targeting. BRIL is first targeted to the endoplasmic reticulum as the entry point to the secretory pathway and rapidly traffics to the Golgi via a COPII‐dependent pathway. BRIL was found to be palmitoylated and two conserved cysteine residues (C52 and C53) were critical for targeting to the plasma membrane. The OI type V mutant BRIL, having a five residue extension (MALEP) at its N‐terminus, presented with exactly the same topological and biochemical characteristics as wild type BRIL. In contrast, the S42 > L mutant BRIL was trapped intracellularly in the Golgi. BRIL and transcripts were equally detected in bone from a patient with OI type V, suggesting that the cause of the disease is a gain of function mediated by a faulty intracellular activity of the mutant BRIL. © 2014 American Society for Bone and Mineral Research.

KEY WORDS: BRIL; CELL SURFACE PROTEIN; ENDOPLASMIC RETICULUM; GOLGI; IFITM5; OSTEOBLASTS; OSTEOGENESIS IMPERFECTA; PALMITOYLATION; TYPE II

Introduction IFITM5, IFITM1, IFITM2, and IFITM3 are all clustered on chromo- some 11 (in humans); they possess a similar gene architecture one‐restricted IFITM‐like (BRIL), also called IFITM5, is part of (2 coding exons), and they are proposed to encode transmem- Ban evolutionarily conserved family of so‐called small brane proteins that have a similar predicted topology (type III; inducible transmembrane (IFITM) proteins, for which N‐out/C‐out) and 2 transmembrane domains. there are at least 3 closely related members (IFITM1, 2, 3).(1–3) The BRIL, however, is distinct from the other IFITMs in several mouse has 2 other members (IFITM6 and 7), all of which fall under aspects. Unlike IFITM1‐3, BRIL is not responsive to at (6) the larger “dispanin” family of proteins that are predicted to the transcriptional level. Rather, we have shown that it is possess 2 transmembrane passages.(4) Our group discovered regulated by the hedgehog signaling pathway (GLI2) in BRIL using a high throughput screen for cDNAs encoding conjunction with Sp1/Sp3/Sp7, and further controlled by CpG (7) secreted and membrane proteins in osteoblastic cells.(5) The methylation of the promoter region. BRIL is expressed almost (1,8,9) relationship of BRIL to the other members, however, is based on exclusively in osteoblasts, in contrast to the ubiquitous (2) structural rather than functional considerations. For instance, nature of IFITMs. Lastly, BRIL localizes predominantly to the

Received in original form January 9, 2014; revised form March 25, 2014; accepted March 26, 2014. Accepted manuscript online Month 00, 2014. Corresponding author: Pierre Moffatt PhD, Shriners Hospital for Children, 1529 Cedar Ave., Montreal, Quebec, Canada H3G 1A6. E‐mail: [email protected]. Additional Supporting Information may be found in the online version of this article. AP and M‐HG contributed equally to this work. Journal of Bone and Mineral Research, Vol. 29, No. 9, September 2014, pp 2004–2016 DOI: 10.1002/jbmr.2243 © 2014 American Society for Bone and Mineral Research

2004 plasma membrane,(9) whereas IFITMs are targeted mostly to the Antibodies endosomal compartment.(10–12) The antibodies against BRIL were raised in rabbits by immuniza- Functionally, BRIL was ascribed a role as a positive modulator tion with the following mouse peptides: D2TSYPREDPRAPSS16C of mineralization in vitro.(9) IFITM1, 2 and 3 have a prominent role (as described previously(9)); CGS114KLAKDSAAFFSTKFD129; in the inhibition in cell entry and infection by various ,(13) a MALEPMDTGGC. These antibodies will be referred to as anti‐N, function that is dependent on palmitoylation of conserved anti‐C, and anti‐MALEP, respectively. The anti‐MALEP peptide cysteine residues.(14,15) Evaluation of the role of BRIL by genetic included the first 3 residues of the wild type BRIL (bolded) to ablation in mice has not clearly confirmed an in vivo role in improve immunogenicity. Exogenous glycine residues (under- mineralization.(16,17) However, the contribution of BRIL as a major lined) were added to act as neutral spacer. C‐terminal cysteine determinant of skeletal integrity is clearly demonstrated by the was conjugated to activated keyhole limpet hemocyanin with discovery that a mutation in IFITM5 causes OI type V.(18,19) OI maleimide. The peptide/carrier complex was mixed with type V is inherited in an autosomal dominant fashion, and is complete Freund’s adjuvant and injected into rabbits according characterized by distinct clinical features that are not usually to standard protocols (EZBiolab, Carmel, IN, USA). Antibodies observed in any other OI type, such as hyperplastic callus were affinity‐purified using the same peptides coupled to formation, interosseous membrane ossification, and a meshlike SulfoLink Immobilization Resin (Pierce). Anti‐FLAG M2 was from lamellation pattern.(20) All cases reported to date have a single 0 – Sigma, Alexa Fluor‐488 phalloidin, Alexa Fluor (448 and 594) recurrent mutation (c.‐14C > T) in the 5 UTR of IFITM5.(18,19,21 25) coupled goat‐anti‐rabbit or donkey‐anti‐mouse secondary, anti‐ The base change creates a novel in‐frame ATG upstream of the GAPDH (clone 6C5) antibodies were from Life Technologies, and natural coding start of BRIL, resulting in the addition of 5 residues the anti‐58K Golgi protein (clone 58K‐9) was from Abcam. (MALEP) at its N‐terminus. Only one other distinct point mutation in the coding region of BRIL, converting serine 40 into a leucine (S40 > L), has recently been reported to cause severe OI.(26–28) Plasmids and transfection The mechanism by which these BRIL mutants contribute to Plasmids were constructed in the CMV‐promoter driven back- disease is still unclear but likely involves a gain of detrimental bone of pcDNA. Except when the natural 50UTR of Ifitm5 was (27) > function. In one study, the S40 L BRIL mutation was evaluated (Figs. 7 and 8), translation was in the context of an associated with reduced PEDF expression and function, as optimal Kozak consensus sequence (GCCACC) preceding the fl re ected by the increased unmineralized osteoid thickness at the natural ATG of BRIL. A plasmid encoding GFP was used as a histological level. negative control for transfections. Point mutations and deletions ‐ We originally reported that, in HEK293 cells, FLAG tagged BRIL were introduced in BRIL by whole plasmid amplification using is a type III , and thus has both Phusion DNA polymerase (New England Biolabs, Ipswich, MA, (9) extremities extruding into the extracellular milieu. This USA) with phosphorylated primers covering the targeted codons. topology was also previously demonstrated for IFITM1 and The list of oligonucleotides used for all mutagenesis and cloning (15,29) 3. However, more recent investigations of the structural is provided in supplemental Table 1. PCR conditions included an properties of IFITM1 and IFITM3 have challenged this long initial denaturing at 98°C for 2 minutes and 26 cycles at 98°C for (11,14,30–33) assumed model. These new data support a type II 10 seconds, 58°C for 25 seconds, 72°C for 80 seconds. The linear ‐ ‐ fi conformation (N in/C out) and that the predicted rst trans- plasmid products were purified from agarose gels on MinElute “ ”(30) membrane passage could be intramembranous. This new columns (QIAGEN) and religated. Plasmids were prepared using ‐ model prompted us to re evaluate the topology and biochemis- Midiprep Qiafilter Kit (QIAGEN). The plasmid expressing osteocrin try of BRIL. We demonstrate that the bulk of BRIL resides in a type (OSTN) having an N‐terminal FLAG epitope located just after fi II con guration at the plasma membrane and Golgi of its cleavable signal peptide was described previously.(34,35) osteoblasts. Palmitoylation is also necessary for the stability The FLAG‐OSTN‐BRIL construct was made by subcloning a PCR and proper targeting of BRIL to the cell surface. We also present product corresponding to the mouse BRIL entire coding comparative data on the localization of the two OI BRIL mutants sequence (from ATG to STOP), into the blunted HindIII site of > (MALEP and S40 L). the FLAG‐OSTN plasmid. The mouse Sar1 cDNA was amplified by RT‐PCR on RNA from MC3T3 cells and cloned into pCDNA. The Materials and Methods H79G mutation was introduced in SAR1 as described above with primers H79G‐F and H79G‐R. The identity of all constructs was Cell cultures and treatment confirmed by Sanger sequencing on an Applied Biosystem 3730xl DNA Analyzer through the McGill University and Genome Quebec ‐ UMR106 and MC3T3 E1 (subclone #4, hereafter designated Innovation Centre. For transient transfection experiments, cells MC3T3) were obtained from ATCC and used up to passage 20. were seeded at 190,000 cells per well in 6‐well plates. The next a UMR106 were grown in DMEM and MC3T3 in MEM, all day, medium was changed and cells were transfected with supplemented with 10% FBS (Life Technologies). For differentia- plasmid DNA (1mg/well) using XtremeGENE 9 (Roche) at a 1:6 ‐ tion of MC3T3, cells were seeded at 100,000 cells per well in 6 ratio. Cells were collected typically 24 h thereafter. well plates (Sarstedt) and grown for 72h until confluency. From this point on, which was considered day 0, cells were fed Immunofluorescence (IF) and western blotting aMEM þ 10% FBS supplemented with 50mg/ml ascorbic acid (Sigma) and 3mM beta‐glycerophosphate (Sigma). Medium was Cells were processed for IF and western blotting essentially as changed every 2 or 3 days. To assess palmitoylation, cells were described.(35–37) Briefly for IF, cells grown directly in 6‐well culture pre‐incubated for 18h with media containing 10% charcoal plates were washed with phosphate buffered saline (PBS), fixed stripped (CS)‐FBS. Metabolic labelling was performed for 3h in for 10 min with paraformaldehyde (3% w/v in PBS), and left intact CS‐FBS media supplemented with 0.25mCi/ml [9,10‐3H(N)]‐ or permeabilized for 5 min with either Triton X‐100 (0.1% v/v PBS) palmitic acid (PerkinElmer). or digitonin (0.005% w/v in 125 mM sucrose). After washing with

Journal of Bone and Mineral Research BRIL IS A TYPE II MEMBRANE PROTEIN 2005 PBS, cells were blocked for 1 h with 2% skim milk with 0.1% BSA c.2596G > A mutation in COL1A1, respectively. The study was in PBS. Primary and secondary antibodies were diluted in approved by the Institutional Review Board of McGill University blocking solution and incubated sequentially for 1 h at room and informed parental consent was provided. Bone chips were temperature. At the end of each incubation, cells were washed crushed to a powder in liquid nitrogen with a mortar and pestle. three times for 5 min with PBS, and mounted with ProlongGold Total RNA and proteins were extracted with Trizol, as Antifade reagent with DAPI (Life Technologies). Cells were recommended by the manufacturer. RNA was reverse tran- imaged by epifluorescence microscopy on a Leica DMRB scribed with the High Capacity cDNA synthesis kit (Applied equipped with an Olympus DP70 digital camera. All images Biosystems, Inc., Foster City, CA, USA). PCR reactions were set up within each experiment were captured at the same exposure. in 25ml with 5ml of cDNA (corresponding to 0.1 mg of input RNA) The primary antibodies were used at the following dilutions: anti‐ with primers F1 and R1 located in the 50UTR and exon 2, FLAG (1:2000), anti‐N (1:5000), anti‐C (1:1000), anti‐MALEP respectively (see Fig. 8). The 438 bp product corresponded to (1:500), anti‐58K Golgi (1:200), anti‐GAPDH (1:1000). Alexa‐fluor nucleotides 2 to 439 of the human IFITM5 cDNA coupled secondary antibodies were used at 1:1000 and (NM_001025295). The PCR reaction was separated on a 1.5% Alexa488‐phalloidin at 1:100. agarose gel, stained with ethidium bromide, and purified on a For western blotting, cells were washed with PBS and collected MinElute column (QIAGEN). Sanger sequencing was performed by directly scraping in NP‐40 cell lysis buffer (50 mM Tris‐HCl (pH with an internal reverse primer. Proteins were separated on 16% 7.4), 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP‐40) containing SDS‐PAGE and analyzed by western blotting with the anti‐C proteases inhibitor cocktail (Sigma). For assessing the presence antibody. of BRIL at the plasma membrane, live cells were detached for 5 min at 37°C with Trypsin‐EDTA (0.05%‐0.5 mM) (Life Technolo- gies, Inc., Grand Island, NY, USA), mixed with complete media Results with 10% FBS to inactivate trypsin, and centrifuged at 250g for BRIL is predominantly a type II transmembrane protein 2 min at 4°C. After washing twice with PBS, the cell pellet was resuspended with the NP‐40 cell lysis buffer. Total cell extracts The topology of non‐tagged BRIL in osteoblast cells was were incubated on ice for 10 min and the insoluble material and systematically assessed using a novel antibody raised against nuclei were centrifuged at 16000g for 10 min at 4°C. The the C‐terminal end (Fig. 1A) covering residues 114‐129. The supernatant was mixed with 4X Laemmli buffer with 2‐ antibody reacting against the N‐terminal residues 2‐15 was mercaptoethanol, boiled for 2 min, and separated on 16% used in parallel. On intact cells, the anti‐N antibody did not SDS‐PAGE. After transfer to 0.45 mm nitrocellulose (Protran systematically yield significant labeling (Fig. 1B). Occasionally, BA85), equal amount of protein loaded across lanes was however, a punctate staining restricted to discrete focal areas at systematically verified by Ponceau S red staining. Membranes the periphery of cells was visible when using either the anti‐N were blotted in 5% skim milk in PBS‐tween (0.05%) with primary BRIL or anti‐GAPDH antibodies (Supplemental Fig. 1). Incubation antibodies (anti‐N 1:5000; anti‐C 1:1500) overnight at 4°C, and of nonpermeabilized cells with phalloidin, a marker of cytoskel- then for 1 h at room temperature with an HRP‐coupled goat‐anti‐ etal actin fibers,(38,39) perfectly co‐localized with these BRIL‐ and rabbit at 1:30000 (Amersham). Detection was performed with the GAPDH‐positive structures, suggesting that this result was due to chemiluminescent reagent ECL Prime (Amersham). partial access to the intracellular compartment at the cell edges (Supplemental Fig. 1). In sharp contrast, the anti‐C antibody Immunoprecipitation and fluorography detected BRIL uniformly over the entire cell surface and on peripheral extensions (Fig. 1B). After metabolic labeling with 3H‐palmitic acid, NP‐40‐soluble cell Immunolabeling was also carried out in cells permeabilized extracts from GFP or BRIL expressing cells were prepared as with digitonin, which creates pores in the plasma membrane but described for western blotting. An aliquot was kept as the input. not in intracellular organelles (rER/Golgi). Under those con- Extracts were incubated overnight at 4°C with 8 mgofaffinity ditions, the anti‐N antibody yielded conspicuous BRIL labeling on purified anti‐N antibody with gentle mixing. Twenty ml of pre‐ the entire cell surface, including cell processes (Fig. 1B). equilibrated protein G‐Plus agarose 50% beads slurry (Santa Intracellular labeling was also detected over crescent structures Cruz) were added and incubated with mixing for 2 h at 4°C. Beads juxtaposed to the nucleus, reminiscent of Golgi apparatus were centrifuged for 2 min at 200g at 4°C, and the supernatant (Fig. 1B arrows). Co‐localization with a marker of Golgi (Golgi‐ recovered. The bead pellets were washed 3 times with cell lysis 58K),(40,41) testified that BRIL localizes to the Golgi apparatus buffer, mixed with 1X Laemmli sample buffer with 2‐mercap- (Fig. 1B arrows). The signal detected with the anti‐C antibody toethanol, and boiled for 2 min. Equal proportion of samples after digitonin permeabilization was indistinguishable from that (input), unbound (free), and bound were separated on duplicate observed in intact cells except for no Golgi labeling (Fig. 1B). 16% SDS‐PAGE for western blotting and fluorography. For These data indicated that the bulk of BRIL resides at the plasma fluorography, the gel was soaked successively for 1 h each at membrane, with an N‐in and C‐out configuration indicative of a room temperature in 50% methanol/10% acetic acid, En3hance type II topology.(42) In the Golgi, the C‐terminus is lumenal and (Perkin Elmer, Waltham, MA, USA), and in cold 1% glycerol (v/v in the N‐terminus is cytoplasmic. The lack of any specific staining on distilled H O). After drying for 2h at 50°C, gel were exposed to 2 confluent naïve MC3T3 attested to the specificity of the autoradiographic films at 80°C for up to 4 days. antibodies used (Supplemental Fig. 2A). Very similar immunoflu- Human IFITM5 expression in OI type V orescence localization data were obtained in native MC3T3 when they produce high levels of BRIL 8 days after differentiation Bone fragments were obtained after surgical procedures and (Supplemental Fig. 2B–2C). Localization data obtained after snap frozen in liquid nitrogen. Samples were from a 9‐year old transfection and under native conditions also indicates that the girl with OI type V who had the recurrent c.‐14C > T mutation in first hydrophobic segment of BRIL (residues 39‐60) is not IFITM5 and a 8‐year old boy with OI type IV caused by a recognized as a bona fide transmembrane passage. However,

2006 PATOINE ET AL. Journal of Bone and Mineral Research Fig. 1. BRIL is a type II protein present at the plasma membrane and Golgi. (A) Sequence of the mouse BRIL. The peptides used to generate antibodies to the anti‐N and anti‐C regions are bracketed. Conserved cysteine residues at position 52, 53, and 86 are underlined. Downward pointing arrowheads represent predicted trypsin (K, R) cleavage sites. The white on black sequence highlights the predicted transmembrane domain, and the greyed portion corresponds to the extracellular domain. Lysine residues 115 and 118 are labeled in red. (B) MC3T3 cells were transiently transfected with mouse BRIL and processed 24 h later for immunofluorescence staining (B) and western blotting (C). (B) As indicated, cells were left intact (non permeabilized) or permeabilized with digitonin and were incubated with the anti‐N or anti‐C antibody. All pictures are taken at the same magnification; the scale bar represents 50 mm. Co‐immunolabeling for BRIL and Golgi 58K protein in permeabilized cells yield overlapping signal (white arrows in upper right panels). (C) Transfected MC3T3 or native UMR106 cells were treated (þ) or not () with trypsin, and detergent soluble protein extracts separated on 16% SDS‐PAGE. Immunoblotting was performed with the indicated antibodies. Asterisks indicate non‐specific bands.

BRIL can be forced to adopt an unnatural type III configuration BRIL polypeptide contains several potential trypsin cleavage sites (N‐out/C‐out) when a heterologous cleavable signal sequence is (Fig. 1A, arrows), of which only 3 are located in the C‐terminus. fused at its N‐terminus (Supplemental Fig. 3). We reasoned that only the BRIL polypeptide region accessible to The type II topology of BRIL was next verified by determining the extracellular trypsin would be sensitive to cleavage. Forced its susceptibility to trypsin digestion of live transfected cells. The overexpression of BRIL was achieved by transient transfection in

Journal of Bone and Mineral Research BRIL IS A TYPE II MEMBRANE PROTEIN 2007 MC3T3 cells, which do not express in proliferating and non‐ differentiated cells.(7,9) Soluble extracts were prepared from cells detached either by scraping in the NP‐40‐detergent cell lysis buffer or after digestion with trypsin‐EDTA. Proteins were separated on 16% SDS‐PAGE and BRIL was detected by western blotting (Fig. 1C). Trypsin digestion caused BRIL to migrate as a single species of slightly lower molecular mass when probed with the anti‐N antibody (Fig. 1C left). Assuming complete digestion and trimming of the last 19 residues of BRIL at K115, this would correspond to a loss of 2.2kDa, which is in accordance with the decreased mass observed on gel. It is important to note that there was no appreciable loss in signal intensity of the digested BRIL, suggesting that the bulk of the remaining protein is intact and that trypsin only acted on the extracellular surface. However, total loss of the BRIL signal after trypsin digestion was noted when probed with the anti‐C antibody, indicative that the C‐terminal epitope had been destroyed. The same results were obtained when using UMR106 osteosarcoma cells that express BRIL constitutively (Fig. 1C right).

Truncation of the C‐terminal tail of BRIL does not affect localization To corroborate that the trypsin‐digested BRIL corresponded to genuine cleavage at the C‐terminus, two truncated BRIL variants with a stop codon immediately following K115 or K118 were expressed (see Fig. 1A). In permeabilized cells, the two truncated BRIL appeared present at the plasma membrane and Golgi when probed with the anti‐N antibody (Fig. 2A). As expected, no signal was observed in intact cells labeled with the anti‐C antibody (Fig. 2A). Western blotting revealed that the K115 and K118 had masses close to the trypsin‐digested wild type BRIL (Fig. 2B) and were unaffected by trypsin. These data confirmed that trypsin only cleaves the C‐terminus and also that this portion is not essential for plasma membrane targeting. Although the signal observed with the K115 and K118 mutants is suggestive of plasma membrane targeting, the possibility that they did not adopt a transmembrane configuration cannot be formally excluded at present.

Fig. 2. The BRIL C‐terminal tail is not essential for membrane targeting. Mutations in the C‐terminal transmembrane segment MC3T3 cells were transiently transfected with plasmids encoding affect stability and localization truncated mouse BRIL with K115 or K118 being the last residue. Cells fl The type II configuration implies that BRIL crosses the plasma were processed 24 h later for immuno uorescence staining (A) and membrane only once. The trypsin digest experiments suggested western blotting (B). (A) As indicated, cells were left intact (non perm.) or ‐ ‐ that the transmembrane passage likely corresponds to the permeabilized with digitonin, and incubated with the anti C or anti N fi predicted region covering residues 89‐111. To verify that this antibody, respectively. All pictures are taken at same magni cation. The region is important for translocation, two leucine residues (L101 K115 and K118 mutant BRIL proteins are detected at the plasma ‐ and L103) at the center of the transmembrane segment were membrane and Golgi only with the anti N antibody. (B) Transfected þ individually mutated into charged arginine residues, which are MC3T3 were treated ( ) or not ( ) with trypsin, and detergent soluble ‐ expected to disrupt the (Fig. 3A). When expressed in protein extracts separated on 16% SDS PAGE. Immunoblotting was ‐ MC3T3 cells and immunolocalized with the C‐terminal antibody, performed with the anti N antibody, and the bottom panel is the the mutant BRIL L101 > R or L103 > R (data not shown) corresponding Ponceau S red staining of cellular proteins. were entirely absent from the cell surface of intact cells (Fig. 3B). The L101 > R mutant could be detected in the cytoplasmic compartment with the anti‐C after permeabilization of the plasma membrane with digitonin (Fig. 3B). The pattern for the L101 > R appeared far more restricted than the wild type BRIL BRIL is targeted to the ER en route to the plasma in terms of surface area. Expression levels of the mutant proteins membrane were drastically reduced as assessed by western blotting (Fig. 3C). These data demonstrate that the single transmembrane All immunofluorescence labeling demonstrated that BRIL local- passage of BRIL resides in its C‐terminal half and that mutation of izes mostly to plasma membranes and to the Golgi apparatus. critical leucine residues abolished membrane targeting. However, it was unclear whether a significant proportion of BRIL

2008 PATOINE ET AL. Journal of Bone and Mineral Research route to the surface, the last 4 C‐terminal residues (EDYN) were changed to a canonical ER‐retention motif (KDEL) (Fig. 4A). The BRIL‐KDEL mutant was not detectable on the cell surface on intact cells with the anti‐C antibody (Fig. 4B). When probed with the anti‐N on cells permeabilized with digitonin, BRIL‐KDEL staining looked like an ER‐like reticular pattern, was more restricted in terms of surface area, and was absent from the Golgi (Fig. 4B). The trypsin digestion of live cells and western blotting also indicated that BRIL‐KDEL was no longer present at the cell surface (Fig. 4C). These data indicate that the C‐terminus (containing the KDEL motif) gets translocated within the lumen of the rER.

Anterograde trafficking of BRIL is dependent on COPII vesicles To ascertain that native BRIL is targeted to the rER and to investigate whether the transport to the Golgi is mediated through COPII‐coated vesicles, the SAR1‐H79G mutant was utilized. SAR1 is a small GTPase essential for COPII vesicle formation and fission, and the H79G is a dominant negative form locked in a GTP‐bound state which blocks this process.(43) BRIL immunolocalization was normal in the presence of wild type SAR1, being detected at the plasma membrane and Golgi (Fig. 5A). Overexpression of SAR1‐H79G caused BRIL to be retained exclusively in the rER, as depicted by the appearance of a pure reticular staining pattern and the loss of plasma membrane and Golgi labeling (Fig. 5A). Western blotting also indicated that BRIL becomes resistant to trypsin cleavage in the presence of SAR1‐H79G but not with wild type SAR1 (Fig. 5B).

Palmitoylation of BRIL is essential for stability and membrane targeting The BRIL polypeptide contains 3 cysteine residues (C52, C53, C86) that are perfectly conserved among species. The cysteine residues have been shown to be palmitoylated in IFITM3 (15) and IFITM5,(44) but the requirements for membrane localization have not been systematically addressed. We tested the consequence of cysteine to alanine mutants on BRIL localization in MC3T3 cells. Mutation of either C52 > A (Fig. 6A) or C53 > A (not shown) caused a dramatic reduction of BRIL at the plasma Fig. 3. A point mutation in the transmembrane domain of BRIL abolishes membrane. Only a faint plasma membrane and Golgi‐associated membrane targeting. MC3T3 cells were transiently transfected with plasmids signal persisted for those two mutants (Fig. 6A arrowheads). The encoding wild type (WT) or mutant BRIL with a single residue substitution in C86 > A mutant BRIL, however, was still intensely detected at the the middle of the transmembrane segment. Leucine at position 101 or 103 membrane (Fig. 6A), despite a lower expression level (Fig. 6B). was individually substituted by an arginine (L101 > R, L103 > R). (A)Cellswere Because all three mutant proteins accumulated equally less than processed 24 h later for immunofluorescence staining (B)andwestern the wild type BRIL (Fig. 6B), the lack of membrane staining for blotting (C). (B) As indicated, cells were left intact (non perm.) or permeabilized C52 > A and C53 > A is unlikely caused by their low level of with digitonin and were incubated with the anti‐C. All pictures are taken at expression. Consistent with the immunofluorescence labeling same magnification and only pictures for L101 > R are shown. The L101 > R data, trypsin digestion of live cells showed that only the mutant is not detected at the cell surface in non permeabilized cells. In membrane‐localized C86 > A mutant was susceptible to cleav- digitonin treated cells, a patchy signal is visible which is more restricted. age (Fig. 6B). The other two were insensitive to trypsin, indicating (C) Transfected MC3T3 detergent soluble protein extracts separated on 16% that they were not significantly present at the plasma SDS‐PAGE. Immunoblotting was performed with the anti‐N antibody, and the membrane. We next tested whether BRIL is palmitoylated in bottom panel is the corresponding Ponceau S red staining of cellular proteins. vivo by performing metabolic labeling. MC3T3 cells were transfected with BRIL or GFP, as a negative control, and collected after a 4 h pulse labeling with 3H‐palmitate. Detection by localized to the rER at steady state. All proteins destined to the fluorography revealed several palmitoylated products in total plasma membrane must first be targeted to the rER as the lysates, with a band of stronger intensity at the expected size in common entry point of the secretory pathway,(42) and so should BRIL expressing cells compared to GFP (Fig. 6C top, arrow). BRIL. In order to investigate whether BRIL transits in the rER en Immunoprecipitation (bound) with the anti‐N antibody detected

Journal of Bone and Mineral Research BRIL IS A TYPE II MEMBRANE PROTEIN 2009 Fig. 5. Expression of dominant negative SAR1‐H79G causes trapping of BRIL in the ER. MC3T3 cells were co‐transfected with wild type mouse BRIL Fig. 4. Introduction of an ER‐retention signal (KDEL) at the C‐terminus of and either SAR1, SAR1‐H79G mutant, or the empty plasmid (CMV). Cells BRIL prevents plasma membrane targeting. (A) MC3T3 cells were were processed 24 h later for immunofluorescence staining (A) and transiently transfected with plasmids encoding wild type (WT) or a western blotting (B). (A) As indicated, cells were left intact (non perm.) or mutant BRIL having 3 residue changes at its C‐terminus. Cells were permeabilized with digitonin and were incubated with the anti‐C or anti‐ processed 24 h later for immunofluorescence staining (B) and western N antibody, respectively. Pictures shown for anti‐N (left) and anti‐C (right) blotting (C). (B) As indicated, cells were either permeabilized with digitonin were taken at 100X under oil and at 40X, respectively (scale bars ¼ 50 or left intact (non perm.) and were incubated with the anti‐N or the anti‐C mm). Overexpression of SAR1‐H79G causes a total loss of BRIL at the antibody, respectively. BRIL bearing the KDEL motif is restricted to an ER‐ plasma membrane, and complete retention in the ER as revealed by the like reticular pattern in permeabilized cells and is not detectable at the cell reticular pattern and absence of Golgi staining. (B) Transfected MC3T3 surface on intact cells. Arrows point to Golgi‐like staining. (C) Transfected were treated (þ) or not () with trypsin, and detergent soluble protein MC3T3 were treated (þ)ornot() with trypsin, and detergent soluble extracts separated on 16% SDS‐PAGE. Immunoblotting was performed protein extracts separated on 16% SDS‐PAGE. Immunoblotting was with the anti‐N antibody, and the bottom panel is the corresponding performed with the anti‐N antibody, and the bottom panel is the Ponceau S red staining of cellular proteins. Bacterially expressed BRIL corresponding Ponceau S red staining of cellular proteins. (rBRIL) migrates slightly faster than mammalian BRIL.

Localization and expression is unaltered in the OI type V palmitoylated BRIL (Fig. 6C top, arrowhead). A duplicate gel mutant MALEP‐BRIL blotted with the anti‐N demonstrated expression of BRIL in the lysate, depletion in the unbound material (free) and enrichment A heterozygous C to T transition at position ‐14 in the 50UTR of the in the bound, respectively (Fig. 6C bottom). IFITM5 gene has been shown to be the single recurrent cause of

2010 PATOINE ET AL. Journal of Bone and Mineral Research Fig. 6. Cysteine residues at position 52 and 53 are important for BRIL stability and plasma membrane localization. MC3T3 cells were transfected with plasmids encoding wild type (WT) or BRIL mutants having single residue substitution at the conserved cysteine residues. Cysteines at position 52, 53, and 86 of the cytoplasmic N‐terminal domain were individually substituted for alanine residues (C52 > A, C53 > A, C86 > A). Cells were processed 24 h later for immunofluorescence staining (A) and western blotting (B). (A) As indicated, cells were left intact (non perm.) or permeabilized with digitonin and were incubated with the anti‐C or anti‐N antibody, respectively. Pictures shown are all taken at the same magnification. Intracellular labeling with the anti‐N was restricted to a Golgi‐like pattern in the C52 > A (arrowheads). Cell surface localization with the anti‐C was dramatically reduced for C52 > A (arrows). Localization of the C86 > A mutant was identical to WT. (B) Western blotting revealed that C52 > A and C53 > A were resistant to trypsin cleavage, while C86 > A was similarly sensitive as the WT. (C) Cells were transfected with GFP or mouse BRIL and labeled for 4 h with 3H‐palmitic acid. Cellular proteins were immunoprecipitated with the anti‐N antibody and separated on duplicate SDS‐PAGE gels. One gel was analyzed by fluorography (top panel), and the other by western blotting with the anti‐N (bottom panel). Free represents the unbound material recovered after the immunoprecipitation. Asterisks indicate IgG heavy and light chains. Arrow in top panel points to more intense band in the input which corresponds to BRIL migration. Arrowheads indicate immunoprecipitated palmitoylated BRIL.

type V OI (see Fig. 8A for a schematic). The mutation introduces a BRIL proteins in MC3T3 showed exactly the same characteristics novel translation start site which adds five amino acids (MALEP) to as the mouse MALEP‐BRIL (data not shown). In sharp contrast to the N‐terminus of BRIL. We explored whether this N‐terminal MALEP‐BRIL, a dramatic change in the subcellular distribution of extension had any impact on the characteristics of mouse BRIL. In the mouse BRIL‐S42 > L was observed (Fig. 7D). In this mutant, a every aspect tested, the mouse MALEP‐BRIL did not differ from single point mutation converted serine 42 to a leucine (S42 > L), the wild type BRIL (Fig. 7). MALEP‐BRIL adopted a type II topology which corresponds to the serine at position 40 of human BRIL. The at the plasma membrane and Golgi (Fig. 7A), was sensitive to signal for BRIL‐S42 > L was absent from the plasma membrane trypsin digestion (Fig. 7B), and was palmitoylated (Fig. 7C). and restricted intracellularly to the Golgi apparatus (Fig. 7D). The Because of the five residue extensions at its N‐terminus, MALEP‐ BRIL‐S42 > L was also unaffected by trypsin digestion, supporting BRIL migrated slightly slower than the wild type BRIL on SDS‐ the immunofluorescence data that it was not properly trans- PAGE (Fig. 7B). The migration behavior of MALEP‐BRIL was not located to the plasma membrane (Fig. 7E). Metabolic labeling affected when the natural methionine of BRIL was changed into with 3H‐palmitate showed that the BRIL‐S42 > Lisvery an alanine (MALEP‐M > MALEP‐A), suggesting no internal inefficiently palmitoylated as compared to the wild type translation (Fig. 7B). An anti‐MALEP antibody displayed specificity (Fig. 7F). By comparison, the KDEL and triple C52 > A/C53 > A/ for the MALEP‐BRIL and could only detect it after permeabilization C86 > A mutants completely failed to incorporate palmitate, of cells, indicating that the N‐terminal extension is cytoplasmic while the C52 > A/C53 > A displayed some labeling (Fig. 7F). (Fig. 7A). Overexpression of the human wild type BRIL and MALEP‐ Western blotting indicated all mutants were expressed.

Journal of Bone and Mineral Research BRIL IS A TYPE II MEMBRANE PROTEIN 2011 Fig. 7. Localization and expression of the mouse MALEP‐BRIL and S42 > L‐BRIL mutants. (A) MC3T3 cells were transfected with a plasmid encoding the OI type V MALEP‐BRIL mutant having the single ‐14C > T transition in the 50UTR. Twenty‐four hours later, cells were processed for immunofluorescence staining (A), western blotting (B), and immunoprecipitation after a 4 h metabolic labeling with 3H‐palmitic acid (C). (A) The localization pattern of MALEP‐ BRIL was indistinguishable from wild type (WT), being detected with the anti‐C only in intact cells, and presenting a membrane and Golgi‐like pattern with the anti‐N and anti‐MALEP. (B) MALEP‐BRIL was produced to the same levels as the WT and equally sensitive to trypsin cleavage. The MALEP N‐terminal extension also caused the protein to migrate slightly slower. MALEP‐BRIL in which the natural methionine (MALEP‐M) is converted to an alanine (MALEP‐A) behaved similarly. (C) Metabolic labeling with 3H‐palmitate and immunoprecipitation with the anti‐N antibody indicated that the MALEP‐BRIL is palmitoylated (fluor.: fluorography). (D) BRIL with a single nucleotide change converting serine at position 42 to a leucine (S42 > L) did not reach the plasma membrane but was trapped intracellularly in the Golgi (arrowheads). (E) Western blotting showed that the S42 > L mutant is resistant to trypsin cleavage. (F) 3H‐palmitate labeling and immunoprecipitation indicated that S42 > L BRIL is poorly palmitoylated, like the KDEL and triple C > A mutants. Arrowhead in lower panel point to specific BRIL signal.

The MALEP‐BRIL is expressed and produced in OI type V exons (Fig. 8A) yielded the expected 438bp product (Fig. 8B). bone Sanger sequencing proved that the amplified IFITM5 cDNA had a doublet G/A peak (reverse strand) of equal intensity at position ‐ Lastly, a human bone sample obtained from a child with OI 14 of the 50UTR (Fig. 8C). Proteins extracted from bone samples of type V was analyzed to detect IFITM5 expression and production OI type V and type IV patients were analyzed by western blotting (Fig. 8). RT‐PCR amplification with primers spanning the two with the anti‐C antibody. The OI type V material had two

2012 PATOINE ET AL. Journal of Bone and Mineral Research Fig. 8. The mutant MALEP‐BRIL is expressed in OI type V. (A) Schematic representation of the human IFITM5 cDNA with the 2 exons. Primers used for PCR amplification (B) and sequencing (C) are indicated. The F1 Fig. 9. BRIL is a type II membrane protein that is palmitoylated. BRIL 0 ‐ oligonucleotide sequence lies in the 5 UTR (underlined) upstream of the contains a cytoplasmic N terminus (N), a single transmembrane domain ‐ c.‐14C > T mutation known to cause OI type V. (B) Total RNA was (cylinder), and an extracellular/luminal C terminal tail (C). After transla- ‐ extracted from bone fragments obtained from a patient with OI‐V. RNA tion, BRIL is targeted to the endoplasmic reticulum (ER), possibly by a tail fi was reverse transcribed and cDNAs were used for PCR amplification with anchored mechanism. Traf cking of BRIL from the ER to the Golgi is ‐ primers F1 and R1, generating a 438bp product (þRT). A negative control dependent on COPII vesicles. This step is presumed to be rapid as BRIL is reaction was run in parallel in which the reverse transcriptase had been not readily detected in the ER at steady state. BRIL is palmitoylated on omitted (‐RT). (C) The PCR product was gel extracted and sequenced cysteines at position 52, 53, and 86, either in the Golgi, and/or the ER. using the internal reverse primer R2. The chromatogram representing the Palmitoylation at C52 and C53 is necessary for further targeting to the ‐ reverse complement of the sequence greyed in (A), displays a G/A read at plasma membrane. The OI type V BRIL mutant (MALEP BRIL) is also 0 fi > position ‐14 in the 5 UTR. (D) Following RNA extraction, protein were localized at the cell surface in a type II con guration, but not the S42 L obtained from the same samples, separated on 16% SDS‐PAGE, and which is trapped in the Golgi. analyzed by western blotting using the anti‐C antibody. The left panel is the Ponceau S red staining of the western results (right panel), indicating the presence of two individual BRIL bands only in the OI‐V patient. Bone proteins extracted from a type IV patient having a COL1A1 mutation only display the lower wild type BRIL (arrow). signal is due to partial permeabilization by the fixation procedure. This would potentially explain why our group had originally inferred a type III topology (N‐out/C‐out) from transfection studies in HEK293 cells with 3xFLAG‐tagged versions of BRIL.(9) The putative first hydrophobic region of BRIL is not immunoreactive bands of equal intensity exactly at the recognized as a genuine transmembrane segment, and we have migration size of BRIL (Fig. 8D). In comparison, in proteins no direct evidence at present that this conformation bears any from bones of an age‐matched child with OI type IV, only the physiological relevance. It remains to be established, however, faster migrating wild type BRIL species was detected (Fig. 8D, whether this hydrophobic stretch with the two palmitoylated arrow). cysteines in its center, forms an intramembrane configuration in the inner plasma membrane leaflet, as was proposed recently for Discussion IFITM3.(30) Just like what we found here for BRIL, the topology of IFITM3 was recently revised from a type III to a type II.(31) The N‐ In the present study, two antibodies directed against BRIL terminus of IFITM3 was found in rare instances on the extremities were used for a systematic reassessment of its extracellular surface, and this was cell‐type dependent, coinci- topology by immunofluorescence labeling and western blotting. dentally being observed only in HEK293.(31) Trypsin digestion of live cells was also employed as a surrogate Although BRIL was not significantly detected in the rER at technique to evaluate the accessibility to the extracellular steady state, all proteins destined to the plasma membrane must compartment. We provide evidence that in transfected MC3T3 go through the rER as the entry point to the secretory osteoblasts BRIL is a type II (N‐in/C‐out) transmembrane protein pathway.(42) This is true also for BRIL, as introduction of a KDEL (schematized in Fig. 9). This type II topology was also validated in retention motif in the C‐terminal end resulted in an exclusive rER wild type differentiated MC3T3 cells and in UMR106, which staining, as was previously shown for other type II proteins such express BRIL constitutively.(9) On intact cells, the BRIL C‐terminus as dipeptidyl peptidase IV(45) and IFITM3.(31) The same result was was detected conspicuously on the plasma membrane surface. observed after overexpression of the SAR1‐H79G. These experi- The N‐terminus was localized predominantly on the cytoplasmic ments confirmed that the C‐terminus is lumenal in the secretory side of the plasma membrane and the Golgi. The N‐terminus was pathway and that trafficking of BRIL involves a COPII‐mediated only rarely observed at the periphery of intact cells, and the vesicular transport. Although rates of transport have not been

Journal of Bone and Mineral Research BRIL IS A TYPE II MEMBRANE PROTEIN 2013 formally investigated, the lack of significant rER BRIL at steady least not when tested in the “homozygote” transfection state implies that the rER‐Golgi movement would be rapid. conditions. We also provide the first demonstration that BRIL Another implication of our study is the mechanism by which is expressed and produced in OI type V bone tissue. Based on this BRIL is being targeted to the secretory pathway. We showed that evidence, it can be put forward that OI type V is caused by a gain the first putative hydrophobic stretch in BRIL is not normally of function. The N‐terminal extension might generate a novel acting as a transmembrane passage or signal anchor. As the interface on the cytoplasmic side of the plasma membrane that is genuine transmembrane segment is located in the C‐terminus, deleterious to osteoblast activity. during BRIL translation it would only emerge out of the ribosome Another de novo mutation in IFITM5, substituting serine 40 by only once the nascent polypeptide is almost fully synthesized. It a leucine, was found in patients presenting extremely severe OI is reasonable to propose that this would prevent targeting manifestations(26–28) but not the OI type V characteristics.(20) BRIL through the canonical co‐translational signal peptide recogni- containing this S40 > L change was poorly palmitoylated and tion particle pathway,(42) and perhaps occur in a post‐ trapped in the Golgi, at least when studied in the context of the translational manner. These characteristics would be compatible mouse BRIL protein (S42 > L), which is a much more dramatic with targeting through the tail‐anchored machinery,(42,46) which change than what was observed with MALEP‐BRIL. Although recognizes membrane proteins having their first transmembrane they remain to be investigated, the mechanisms would appear to domain usually more than 40 residues away from the initiator be distinct in the two subtypes of OI with IFITM5 mutations. methionine. Although this was not directly addressed, some of Irrespective of the mechanisms involved, it would argue that our results would indirectly support this possibility. For instance, both mutations are caused by dominant negative effects we have consistently observed low levels of expression for some occurring in the cytosolic compartment but not by haploinsuffi- of the mutants, particularly the L101 > R and L103 > R, which ciency. Further studies will be required to verify the subcellular accumulated in the cytoplasm. Misfolded or improperly targeted localization of the two OI BRIL mutants when produced, like in transmembrane proteins are usually rapidly degraded in order to the patients, in equal (heterozygote) abundance. Our data avoid aggregation in the cytoplasm and ensuing toxicity.(47,48) further suggest that key residues within the cytoplasmic We also showed that BRIL is palmitoylated. Mutation of either N‐terminal domain, in addition to the cysteines, are critical for cysteine residues at position 52 and 53 attenuated targeting of targeting BRIL to the proper sub‐cellular compartment. BRIL at the plasma membrane. Cellular levels of either mutant In conclusion, BRIL is a type II transmembrane protein, the bulk were also significantly reduced. The other highly conserved of which resides at the cell surface, with some in the Golgi. One cysteine at position 86 did not significantly alter BRIL localization important remaining question is about the functional role of BRIL and production, although it is palmitoylated. Our results are in in osteoblast activity and the impact of the BRIL mutants. accord with and extend those of a recent report showing Ongoing and future studies are required to shed light on the palmitoylation of BRIL at C52, C53, and C86.(44) In that study, BRIL mechanisms at play, which will hopefully provide a handle to was reported to migrate as a doublet on SDS‐PAGE, representing develop new and/or more targeted therapeutic strategies for OI. the top palmitoylated and bottom non‐modified forms, In that regard, our findings clearly indicate that an intracellular respectively. Although we have observed occasional double approach would need to be envisioned for blocking the bands on western blots, this was not consistent even though we pathogenic activity of MALEP‐BRIL. took precaution to not overheat samples prior to loading, as this (49) may cause a loss of the palmitate moiety. The increased mass Disclosures for BRIL carrying two palmitate residues would correspond to less than 0.5 kDa, and this difference would be difficult to discrimi- Frank Rauch: Genzyme Inc: Advisory Board member; Novartis Inc: nate on gel. Tsukamoto et al.(44) further showed by co‐ Study grant to institution; Alexion Inc: Study grant to institution. immunoprecipitation that palmitoylation of BRIL was required All other authors state that they have no conflicts of interest. for its interaction with another transmembrane protein, FKBP19 (the product of the Fkbp11 gene), but did not provide cellular localization data. Given that FKBP19 resides predominantly in the Acknowledgments rER (data not shown), it is at present unclear how and if this (transient) interaction would impact on BRIL structure or We thank Ms. Liljana Lalic for performing the Sanger sequencing function. A direct peptidyl‐prolyl‐isomerase action of FKBP19 reactions. This work was supported in part by grants from the on the BRIL polypeptide appears unlikely as the enzymatic Shriners of North America and CIHR, and by a Recruitment Aid domain is predicted to be lumenal,(50) facing the opposite program from The Network of Oral and Bone Health Research of compartment to the proline‐rich N‐terminal cytoplasmic end of the Fonds de Recherche du Québec en Santé. BRIL. Whether it would serve another role is presently unknown. Authors’ roles: Study design: MHG, PM. Study conduct: AP, OI type V is caused by a single recurrent heterozygous MHG, PKJ, PM. Data collection: AP, MHG, PKJ, PM. Data analysis: 0 – mutation in the 5 UTR of IFITM5.(18,19,21 25) The c.‐14C > T change AP, MHG, FF, FR, PM. Data interpretation: MHG, FR, PM. Drafting leads to the creation of an in‐frame ATG with the addition of five manuscript: FR, PM. Revising manuscript content: AP, MHG, PKJ, amino acids (MALEP) at the N‐terminus of BRIL. At present it still FF, FR, PM. Approving final version of manuscript: AP, MHG, PKJ, remains unknown how the mutant MALEP‐BRIL protein causes FF, FR, PM. PM takes responsibility for the integrity of the data the OI type V phenotype. Because OI‐V is transmitted in an analysis. autosomal dominant fashion, mutated BRIL could a priori either lead to haploinsufficiency or have a dominant negative effect. In order to shed light on this possibility, we studied the mouse References ‐ fi version of MALEP BRIL. However, we did not nd any obvious 1. Hickford D, Frankenberg S, Shaw G, Renfree MB. 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2016 PATOINE ET AL. Journal of Bone and Mineral Research