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

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Topological Mapping of BRIL Reveals a Type II Orientation and Effects of Osteogenesis Imperfecta Mutations on Its Cellular Destination ORIGINAL ARTICLE JBMR Topological Mapping of BRIL Reveals a Type II Orientation and Effects of Osteogenesis Imperfecta Mutations on Its Cellular Destination Alexa Patoine,1,2Ã Marie‐Hélène Gaumond,1Ã 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 protein 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 cell 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 proteins 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; interferon 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 interferons 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. ÃAP 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 viruses,(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 transmembrane protein, 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.
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