Circulating Truncated Alpha-1 Antitrypsin Glycoprotein in Patient Plasma Retains Anti-Inflammatory Capacity

This information is current as Emer P. Reeves, Danielle M. Dunlea, Karen McQuillan, of October 3, 2021. Ciara A. O'Dwyer, Tomás P. Carroll, Radka Saldova, Prithvi Reddy Akepati, Mark R. Wormald, Oliver J. McElvaney, Vipatsorn Shutchaidat, Michael Henry, Paula Meleady, Joanne Keenan, Derek C. Liberti, Darrell N. Kotton, Pauline M. Rudd, Andrew A. Wilson and Noel G. McElvaney

J Immunol published online 22 February 2019 Downloaded from http://www.jimmunol.org/content/early/2019/02/21/jimmun ol.1801045 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2019/02/21/jimmunol.180104 Material 5.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 22, 2019, doi:10.4049/jimmunol.1801045 The Journal of Immunology

Circulating Truncated Alpha-1 Antitrypsin Glycoprotein in Patient Plasma Retains Anti-Inflammatory Capacity

Emer P. Reeves,* Danielle M. Dunlea,* Karen McQuillan,* Ciara A. O’Dwyer,* Toma´s P. Carroll,† Radka Saldova,‡ Prithvi Reddy Akepati,x Mark R. Wormald,{ Oliver J. McElvaney,* Vipatsorn Shutchaidat,* Michael Henry,‖ Paula Meleady,‖ Joanne Keenan,‖ Derek C. Liberti,x Darrell N. Kotton,x Pauline M. Rudd,† Andrew A. Wilson,x and Noel G. McElvaney*

Alpha-1 antitrypsin (AAT) is an acute phase protein that possesses immune-regulatory and anti-inflammatory functions indepen- dent of antiprotease activity. AAT deficiency (AATD) is associated with early-onset emphysema and chronic obstructive pulmonary disease. Of interest are the AATD nonsense mutations (termed null or Q0), the majority of which arise from premature termination codons in the mRNA coding region. We have recently demonstrated that plasma from an AATD patient homozygous for the Null Downloaded from

Bolton allele (Q0bolton) contains AAT protein of truncated size. Although the potential to alleviate the phenotypic consequences of AATD by increasing levels of truncated protein holds therapeutic promise, protein functionality is key. The goal of this study was

to evaluate the structural features and anti-inflammatory capacity of Q0bolton-AAT. A low-abundance, truncated AAT protein was confirmed in plasma of a Q0bolton-AATD patient and was secreted by patient-derived induced pluripotent stem cell–hepatic cells. Functional assays confirmed the ability of purified Q0bolton-AAT protein to bind neutrophil elastase and to inhibit protease activity. http://www.jimmunol.org/ Q0bolton-AAT bound IL-8 and leukotriene B4, comparable to healthy control M-AAT, and significantly decreased leukotriene B4–induced neutrophil adhesion (p = 0.04). Through a mechanism involving increased mRNA stability (p = 0.007), ataluren treatment of HEK-293

significantly increased Q0bolton-AAT mRNA expression (p =0.03)andQ0bolton-AAT truncated protein secretion (p = 0.04). Results support the rationale for treatment with pharmacological agents that augment levels of functional Q0bolton-AAT protein, thus offering a potential therapeutic option for AATD patients with rare mutations of similar theratype. The Journal of Immunology, 2019, 202: 000–000.

lpha-1 antitrypsin (AAT) is the archetypal member of the AAT is also an acute phase protein, the levels of which become el-

serpin superfamily produced mainly by hepatocytes and evated within hours of developing inflammation or postinfection (2), by guest on October 3, 2021 A is the most abundant endogenous serine protease in- and is known to be raised in a number of conditions, ranging from hibitor in the blood. The predominant role of AAT is as a serine acute community-acquired pneumonia to postsurgery (3, 4). Indeed, protease inhibitor, primarily inhibiting neutrophil elastase (NE) during the resolving phase of community-acquired pneumonia, there but also other proteases, including cathepsin G and proteinase 3 (1). is a significant increase in circulating levels of sialylated negatively The structure of the AAT molecule is critical for its antiprotease charged glycoforms of AAT complexed to potent chemokines, in- activity and is comprised of three b sheets (A, B, and C), nine a cluding IL-8: a regulatory binding event that negates CXCR1 en- helices, and a reactive center loop (RCL) at the C-terminal end. gagement (2). Moreover, AAT has been shown to possess substantial Additionally, AAT undergoes posttranslational modifications with the anti-inflammatory properties independent of its antiprotease activity, addition of N-linked oligosaccharides at asparagines 46, 83, and 247. affecting many cell types, including pancreatic islet b cells (5),

*Irish Centre for Genetic Lung Disease, Department of Medicine, Royal College of R24HL123828 and U01TR001810. This work was also supported by a Boston Uni- Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin 9, versity School of Medicine Department of Medicine pilot grant and NIH Grant Ireland; †Alpha-1 Foundation Ireland, Royal College of Surgeons in Ireland, R01DK101501 (both to A.A.W.). Beaumont Hospital, Dublin 9, Ireland; ‡GlycoScience Group, National Institute for x Address correspondence and reprint requests to Dr. Emer P. Reeves, Irish Centre for Bioprocessing Research and Training, Mount Merrion, Dublin, Ireland; Center for Genetic Lung Disease, Department of Medicine, Royal College of Surgeons in Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA { Ireland, Beaumont Hospital, Dublin 9, Ireland. E-mail address: [email protected] 02118; Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, Oxford OX1 3QU, United Kingdom; and ‖National Institute for Cellular The online version of this article contains supplemental material. Biotechnology, Dublin City University, Dublin 9, Ireland Abbreviations used in this article: AAT, alpha-1 antitrypsin; AATD, AAT deficiency/ ORCIDs: 0000-0002-0912-7361 (K.M.); 0000-0002-0418-1641 (T.P.C.); 0000-0001- deficient; 2AB, 2-aminobenzamide; ACN, acetonitrile; CI, confidence interval; 2D, 5085-5080 (R.S.); 0000-0002-4853-2773 (M.R.W.); 0000-0001-8187-0769 (O.J.M.); two-dimensional; dH2O, distilled H2O; DMD, Duchenne muscular dystrophy; FEV1, 0000-0001-5306-310X (P.M.); 0000-0003-0047-4268 (J.K.); 0000-0003-2991- forced expiratory volume in 1 s; FRET, fluorescence resonance energy transfer; HSA, 9283 (D.C.L.). human serum albumin; IEF, isoelectric focusing; iPSC, induced pluripotent stem cell; LTB , leukotriene B ; NE, neutrophil elastase; NMD, nonsense-mediated mRNA Received for publication July 27, 2018. Accepted for publication January 30, 2019. 4 4 decay; PNGase F, peptide N-glycanase F; PTC, premature termination codon; qRT- This work was supported by the U.S. Alpha-1 Foundation (to E.P.R.) and the Medical PCR, quantitative real-time PCR; RCL, reactive center loop; RT, room temperature; Research Charities Group/Health Research Board Ireland (to N.G.M.). This work UPLC, ultra-performance liquid chromatography. was also supported by the European Union Seventh Framework Programme (FP7/ This article is distributed under The American Association of Immunologists, Inc., 2007-2013) under Grant Agreement 260600 (Glycomics by High Throughput Inte- Reuse Terms and Conditions for Author Choice articles. grated Technologies) and Science Foundation Ireland Starting Investigator Research Grant Agreement 13/SIRG/2164 (both to R.S.). HEK-293 experiments were sup- Ó ported by the Alpha-1 Project as well as National Institutes of Health (NIH) Grants Copyright 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801045 2 MUTANT ALPHA-1 ANTITRYPSIN AND ANTI-INFLAMMATORY ACTIVITY

B cells (6), neutrophils (7, 8), and macrophages (9), and has been a mean forced expiratory volume in 1 s (FEV1)of1036 14.6% predicted, implicated in modulating cellular processes as diverse as endothelial showed no evidence of any disease, were all nonsmokers, and none were cell apoptosis (10) and fibroblast-mediated cytokine expression (11). taking medication. This group were defined as having an MM phenotype by IEF, with serum AAT concentrations within the normal range (20–50 mM). AAT deficiency (AATD) is a hereditary disorder characterized by AAT measurements were performed by a rate immune nephelometric low circulating levels of AAT and is associated with the development method (Array 360 System; Beckman Coulter) or by immune turbidimetry of chronic obstructive pulmonary disease often by the third or fourth (AU5400; Beckman Coulter). IEF for phenotyping AAT from plasma decade, liver disease, and in rare cases, skin panniculitis (12). Studies was performed using the HYDRASYS platform (Sebia). A nonsmoking ZZ-AATD patient who was not receiving augmentation therapy (mean FEV indicate that neutrophilic inflammation plays a major role in the 1 of 73% predicted) and a heterozygous MZ-AATD individual (mean FEV1 of pathogenesis of AATD-associated emphysema, with increased lung 103% predicted) were recruited. A patient (never smoker, mean FEV1 neutrophil burden described in AATD subjects even with mild of 59% predicted) homozygous for the Q0bolton mutation was recruited, functional lung impairment (13). Infusion of plasma-purified AAT as determined by sequencing all coding exons (II-V) of the AAT gene protein (augmentation therapy) has proven therapeutic benefit in (SERPINA1, RefSeq: NG_008290) as previously described (33), using the CEQ 8800 Genetic Analysis System (Beckman Coulter) or the Big Dye AATD (14, 15) and has been explored in a number of disease models, Terminator Cycle Sequencing Kit 3.1 (Applied Biosystem) with the 3130 including diabetes (16, 17) and arthritis (6, 11) and airway diseases, Genetic Analyzer. Following sequencing of all coding exons (II-V) of the including bronchiectasis/chronic obstructive pulmonary disease and SERPINA1 gene, the patient was identified as homozygous for the Q0bolton cystic fibrosis (18, 19). Most recently, the Randomized, Placebo- mutation, with two PTCs at aa 373 and 374 on exon V. controlled Trial of Augmentation Therapy in a-1 Proteinase Inhibi- Isolation of plasma and purification of active AAT tor Deficiency trial demonstrated the benefit of AAT augmentation therapy as compared with placebo (14), and the Randomized, Placebo- Blood was obtained from consenting volunteers in 7.5-ml heparinized a S-Monovette tubes (10 U/ml; Sarstedt, Germany). Plasma was immediately Downloaded from controlled Trial of Augmentation Therapy in -1 Proteinase Inhibitor isolated by centrifugation of the blood (1000 3 g, 10 min at room tem- Deficiency open-label extension trial further supported the continued perature [RT]) and was then aliquoted and stored at 280˚C until required. efficacy of AAT in decelerating the progression of AATD lung disease AAT was isolated from human plasma by use of Alpha-1 Antitrypsin over 4 y (15). However, plasma-purified AAT is not a limitless re- Select Resin packed into a Tricorn column (GE Healthcare Life Sciences, Buckinghamshire, U.K.) and chromatographed by fast protein liquid source, and alternative sources of augmentation therapy, in addition to chromatography on an ӒKTAprime Plus (GE Healthcare Life Sciences). novel therapeutics, are being sought. Plasma was diluted with Buffer A (20 mM Tris, 150 mM NaCl, pH 7.4) at The common normal AAT allele is M, which accounts for 95% of a ratio of 1:3 and then loaded onto the column at a flow rate of 0.5 ml/min. http://www.jimmunol.org/ alleles, and in healthy individuals leads to AAT plasma levels .1.04 g/L Bound AAT was eluted from the resin with a gradient of 0–100% Buffer B or 20 mM. The AATD Z allele accounts for 1–2% of alleles in the (2 M MgCl2) over 20 ml. Fractions containing AAT were concentrated using Amicon Ultra centrifugal filters (Millipore) and desalted into PBS population and is caused by the substitution of glutamic acid for lysine using NAP-10 desalting columns (GE Healthcare Life Sciences). AAT was at position 342. AATD associated with the ZZ phenotype is well de- quantified by ELISA using the SERPINA1 DuoSet (R&D Systems) scribed and understood; conversely, little is known about the family of according to manufacturer’s instructions, and the plate was recorded using SERPINA1 mutations termed null or Q0, such as the Q0 and a Spectra Max M3 (Molecular Devices). bolton Following this, the ability of purified AAT to inhibit NE (Athens Re- Q0hongkong mutations. Null mutations arise from a number of different search) activity was evaluated by an NE-specific fluorescence resonance types of mutations, including nonsense and frameshift mutations (20),

energy transfer (FRET; substrate Abz-APEEIMRRQ-EDDnp; assay buffer by guest on October 3, 2021 resulting in a premature termination codon (PTC) in the mRNA coding [0.5 M NaCl, 0.1% (v/v) Brij-35, 0.1 M HEPES, pH 7.6]) assay. Fixed 26 region and undetectable serum levels of AAT by routine nephelometry volumes of NE (containing 1 3 10 M active NE) were incubated with a and isoelectric focusing (IEF) methods. Recently, we identified the range of concentrations of AAT (0–5 nM) at 37˚C for 30 min. NE with no AAT acted as a control for each experiment. Postincubation, each sample presence of truncated AAT protein in plasma of a patient homozygous was mixed with FRET substrate in NE assay buffer, and fluorescence was for the Q0bolton mutation (21), which represents an attractive therapeutic recorded at excitation 320 nm and emission 420 nm at 20 s intervals at target. Drugs such as geneticin (G418), amikacin, and ataluren 28˚C. The percentage of remaining NE was calculated by converting the (PTC124) force the ribosome to read through early stop codons (22) values into percentages and taking the value obtained for the AAT (2 nM) samples at 5.4 min away from the value obtained from the control NE and can suppress disease-causing PTCs in mammalian cells in vitro (4 nM) samples. IC50 measurements for AAT were calculated from the plot and in vivo (23–25), with therapeutic potential demonstrated in Du- of the percentage of remaining activity versus the AAT concentrations, chenne muscular dystrophy (DMD) (26) and cystic fibrosis (27–29). using GraphPad Software (Prism 5.0), as was the amount of AAT required 3 26 Alternatively, several pharmacological agents inhibit nonsense- to completely inhibit NE activity. The IC50 for AAT against 1 10 M NE activity was 5.41 3 1027 M. To completely inhibit 1 3 1026 M active mediated mRNA decay (NMD), and this is particularly beneficial 2 NE, 1 3 1.09 6 M-AAT was required. Reactions were run on SDS-PAGE when the truncated protein encoded by PTC mRNAs retain normal and immunoblotted for AAT (34). function (30, 31). NMD inhibition (referred to as PTC suppression therapy) has the potential to alleviate the phenotypic consequences Protein electrophoresis and Western blot analyses of a wide range of genetic diseases by increasing levels of truncated, For one-dimensional gel electrophoresis, SDS-PAGE sample buffer (103 yet functional, protein to the protective threshold level (32), with containing 0.2% (w/v) bromophenol blue, 50% (w/v) sucrose, 1% (w/v) associated potential benefit for a range of disorders, including SDS, 1% (w/v) DTT, 200 mM EDTA, 3 M Tris-HCL) was added to protein samples, boiled for 3 min, and then subjected to 12.5% (w/v) SDS-PAGE haemophilia and several cancers (25). In this article, we extend under denaturing conditions. Alternatively, samples were subjected to these concepts to AATD and characterize the structural and anti- NuPAGE Novex 4–12% Bis-Tris gel electrophoresis (Bio-Sciences Limited). inflammatory properties of the circulating truncated AAT protein arising Two-dimensional (2D) PAGE was performed as previously described (34). because of the Q0bolton mutation. Moreover, we extend our tests on the Gels were stained with Coomassie Blue stain for visualization of protein circumvention of translation-dependent mRNA surveillance to enhance banding patterns, or, alternatively, proteins were transferred onto poly- vinylidene difluoride membrane for Western blotting. Polyvinylidene Q0bolton-AAT protein production. difluoride membranes were incubated with 1 mg/ml polyclonal goat AAT Ab or 1 mg/ml polyclonal rabbit AAT Ab (Abcam). The secondary Abs Materials and Methods were HRP-linked anti-rabbit (Cell Signaling) or HRP-linked anti-goat Study design (Santa Cruz). Visualization of immune-reactive protein bands was achieved using Immobilon Western Chemiluminescent HRP Substrate Ethical approval was obtained from Beaumont Hospital institutional review (Millipore) and the Syngene G:Box Chemi XL gel documentation board, and written informed consent was obtained from all study partici- system. Densitometry was performed using the GeneSnap SynGene pants. Healthy control individuals (n = 10, mean age = 31.55 6 7.05 y) had program (Synoptics). The Journal of Immunology 3

AAT leukotriene B4 and IL-8 binding and activity assays using an external standard of hydrolyzed and 2AB-labeled glucose olig- omers to create a dextran ladder, as previously described (37). UV spectra were recorded on a Jenway 6405 for the in vitro binding of For exoglycosidase digestions, all enzymes were purchased from Prozyme leukotriene B4 (LTB4) to AAT or human serum albumin (HSA; control (San Leandro, CA). The labeled glycans were digested in a volume of 10 ml plasma protein) as previously reported (8). The ratio of ligand/protein was for 16 h at 37˚C in 50 mM sodium acetate buffer, pH 5.5 (except in the m m 5 M:1 M, and UV spectra values were recorded between 230- and 330-nm case of jack bean a-mannosidase, in which the buffer was 100 mM sodium wavelengths (5-nm intervals) in a quartz cuvette. The molar absorptivity acetate, 2 mM Zn2+, pH 5), using the following enzymes: 0.5 U/ml (ε) at 270 nm was calculated as described by Gill and von Hippel (35), Arthrobacter ureafaciens Streptococcus 21 21 sialidase (EC 3.2.1.18), 1 U/ml with a path length of 1 cm and represented as M cm . A molar ab- pneumonia sialidase (NAN1, EC 3.2.1.18), 1 U/ml bovine testes 3 4 21 21 sorptivity of 4.3 10 M cm at 270 nm (in PBS) was adopted for b-galactosidase (EC 3.2.1.23), 1 U/ml bovine kidney a-fucosidase (EC 3.2.1.51), LTB4. The interaction of LTB4 with fast protein liquid chromatography– 8U/mlb-N-acetylglucosaminidase cloned from S. pneumonia expressed purified M-AAT or Q0bolton-AAT was analyzed with solutions prepared in PBS in Escheria coli (EC 3.2.1.30), 60 U/ml jack bean a-mannosidase (EC (8). Neutrophil adhesion assays in response to LTB4 (100 nM) were performed 3.2.1.24), and 0.4 mU/ml almond meal a-fucosidase (EC 3.2.1.111). by measuring the number of calcein AM (5 mM; Life Technologies)–loaded After incubation, enzymes were inactivated by incubation at 65˚C for neutrophils adhering to a fibronectin-coated plate in the presence or absence of 15 min. The enzymes were then removed by filtration through a 10-kDa AAT, HSA (control plasma protein, 27.5 mM), or the BLT1 antagonist protein-binding EZ filter (Millipore). N-glycans were then dried by U-75302 (1 mM; Enzo Life Sciences, Exeter, U.K.) as previously described (8). vacuum centrifuge and analyzed by UPLC (38). The Whatman Minifold I Slot-Blot System (GE Healthcare) was Liquid chromatography tandem mass spectrometry was performed on employed to assess binding of AAT to IL-8 using 0.4 mm of nitrocellulose. individual gel pieces so as to confirm the identity of AAT, using Proteome AAT or HSA (protein control, 5 mg) was added to each well in a volume Discoverer Software (v1.4; Thermo Fisher Scientific) using a comple- m of 200 l of PBS under vacuum. Human recombinant carrier-free IL-8 mentary two stream algorithm search with SEQUEST and MASCOT m (0.1 g/ml; Cambridge Bioscience) or PBS was incubated for 1 h at RT, against a human subset from the UniProtKB/Swiss-Prot database. Carba- without vacuum. The unbound IL-8 was removed by washing with PBS midomethylation of cysteine residues was selected as a fixed modification, (3 times), and then each well was blocked with 1% (w/v) gelatin in PBS and oxidation of methionine was considered as a variable modification also Downloaded from m for 1 h. The wells were then incubated with 1 g/ml IL-8 mAb (R&D allowing for two missed cleavages. The following SEQUEST filters were Systems) for 1 h, followed by horse anti-mouse secondary HRP-linked Ab applied: for charge state 1, Xcorr . 1.9; 2, Xcorr . 2.2; 3, Xcorr . 3.75 and (Cell Signaling). Controls for this experiment excluded IL-8 but included peptide Delta correlation (maximum delta Cn0.1). The following MASCOT primary and secondary Abs. A final wash step was carried out prior to filters were applied: MASCOT threshold score of 40 and MASCOT signi- visualization of the nitrocellulose using Immobilon Western Chemilumi- ficance threshold of 0.05. nescent HRP Substrate (Millipore).

Generation and differentiation of induced pluripotent stem http://www.jimmunol.org/ Preparation of AAT glycans for ultra-performance cell–derived hepatic cells liquid chromatography To isolate human dermal fibroblasts for reprogramming, 6-mm full- For removal of N-linked glycans, all AAT gel spots were excised from 2 thickness skin biopsies were performed and fibroblasts were expanded Coomassie Blue–stained 2D gels. Each gel spot was cut into 1-mm pieces using previously described methods (Supplemental Fig. 1) (39). Fibroblasts and placed into a polypropylene 96-well microplate. The gels were first (1 3 105) were transduced with the excisable hSTEMCCA reprogramming washed with acetonitrile (ACN) for 10 min followed by 20 mM NaHCO3 vector, and emerging colonies were picked and characterized to confirm (pH 7.2); this was repeated three times, with the buffers vacuumed to waste normal karyotype and expression of markers of pluripotency before pro- each time. N-glycans were removed by employing the high-throughput ceeding to additional experiments (Supplemental Fig. 1) (40). Wild-type method described by Royle et al. (36). The N-linked glycans were re- control induced pluripotent stem cell (iPSC) experiments were performed leased using peptide N-glycanase F (PNGase F), which was prepared using using the previously published human iPSC WT lines, BU-1 and BU-3 by guest on October 3, 2021 m 50 l of 100 mU/ml PNGase F (GKE-5006D; Prozyme) in 20 mM (41). Human iPSCs were adapted to feeder-free conditions and cultured on NaHCO3. Each gel piece was allowed to soak for 5 min and was then matrigel in mTeSR1 media (StemCell Technologies) prior to initiation of m further covered with 50 l of 20 mM NaHCO3 buffer alone and transferred directed differentiation experiments. Directed hepatic differentiation was to a 2-aminobenzamide (2AB) collection block. The plate was sealed with performed using a previously described differentiation protocol with slight adhesive film and incubated overnight at 37˚C. The following day, the re- modifications. Briefly, undifferentiated iPSCs were dissociated with Gentle m leased glycans were collected. Distilled H2O(dH2O; 200 l) was added to Cell Dissociation reagent (StemCell Technologies) and 1 3 106 cells each well and placed on the plate mixer for 10 min. This was then vacuumed plated per well of a matrigel-coated six-well plate. Twenty-four hours later, m to the 2AB collection block. This was followed by addition of 200 lof culture media was aspirated and cells were cultured using the STEMdiff ACN for 10 min and subsequent vacuuming to the collection block. This Definitive Endoderm Kit according to the manufacturer’s instructions. m cycle was repeated 4 times. Once the samples were dry, 20 lof1%(v/v) After 6 d of culture in these media conditions, cells were differentiated formic acid was added to the samples and then incubated at RT for 40 min. according to a previously published protocol (40). The samples were dried overnight in a vacuum centrifuge (SpeedVac). Glycans were fluorescently labeled with 2AB by a reductive amination Lentiviral production and cell culture of HEK-293 cells reaction. In brief, 2AB labeling mixture (LudgerTag kit, U.K.) was added to the samples and incubated at 65˚C for 2 h with gentle agitation. Excess Q0bolton-AAT cDNA was amplified by PCR from differentiated Q0bolton 2AB reagent was removed on Whatman 3-mm paper (Clifton, NJ) in ACN. iPSC-hepatic cells using primers including Not I and Bgl II restriction Pieces of Whatman paper were cut (1 cm3) and washed in a beaker of sequences at the 59 and 39 ends, respectively. Amplified cDNA was dH2O 3 times. They were then placed in aluminum foil and dried at 65˚C. digested and cloned into the first position of the previously generated The pieces were folded into quarters and placed in the wells of a Whatman pHAGE lentiviral backbone, lenti CMV-AAT-UBC-GFP (42). To induce protein precipitation plate (prewashed with ACN followed by dH2O). The expression of transgenes, HEK-293 cells were transduced using the samples in 5 ml of 2AB labeling solution were then added to the center of aforementioned lentiviral vectors to coexpress the enhanced GFP together the folded paper in the wells and allowed to dry for 15 min. The excess with either M or Q0bolton-AAT cDNA under control of the CMV or UBC 2AB was removed using ACN with agitation for 15 min; this was then promoter element. GFP+ cells were sorted using a MoFlo cell sorter vacuumed to waste and repeated 4 times. The 2AB-labeled glycans were (DakoCytomation) to 98.5% purity for use in further experiments. HEK- then eluted from the paper by adding 2 3 900 mlofdH2O and agitating for 293 cells were cultured in DMEM (Life Technologies) supplemented with 30 min and were then collected by vacuum in a 96-well plate. Finally, this 10% (v/v) FBS (Biosciences), 1% (v/v) L-Glutamine (Life Technologies), was dried overnight in a vacuum centrifuge. The dried glycans were then and 0.2% (v/v) primocin (Invivogen). Once the cells were 80% confluent, redissolved in a known volume of dH2O for analysis by ultra-performance media was removed and discarded, and wells were washed with Dulbecco’s liquid chromatography (UPLC). The glycans were rehydrated in 9 mlof PBS before detaching the adherent cells using 0.05% (w/v) trypsin-EDTA H2O, and 21 ml of ACN was additionally added and agitated for 1 min. The (Life Technologies) at 37˚C for 2–3 min or until cellular detachment oc- 30-ml sample was then transferred to an analysis plate. UPLC was per- curred. After this period, 7 ml of growth medium was added to inhibit the formed using a BEH Glycan 1.7 mm, 2.1 3 150 mm column (Waters, activity of trypsin, and the cells were centrifuged at 300 3 g for 5 min. Cells Milford, MA) on an Acquity UPLC (Waters). Solvent A was 50 mM at a density of 1 3 105 were seeded in 12-well plates and allowed to adhere formic acid adjusted to pH 4.4 with ammonia solution. Solvent B was overnight before culturing in serum-free media containing either gentamicin ACN. The column temperature was set as 30˚C. Fluorescence was mea- (0.5 mg/ml) or ataluren (0.1, 0.5, 2.5, 12.5, or 62.5 mg/ml). The employed sured at 420 nm with excitation at 330 nm. The system was calibrated doses were based on that of a study with gentamicin and ataluren (PTC124) 4 MUTANT ALPHA-1 ANTITRYPSIN AND ANTI-INFLAMMATORY ACTIVITY on fibroblast cells (43). The supernatants were collected after this time, and Confocal microscopy RNA was collected by subjecting the cells to TRI Reagent. Protein was isolated from the TRI Reagent sample using four times the volume of 100% Cells were fixed with 4% (w/v) paraformaldehyde on a polysine glass slide, (v/v) acetone with incubation on ice for 30 min. The resultant pellet was and cell membranes were permeabilized using 0.2% (v/v) Triton x-100 in washed briefly with acetone and solubilized with 23 SDS-PAGE sample PBS for 5 min at RT. Nonspecific binding was prevented by blocking with buffer and then analyzed by Western blotting. 4% (w/v) BSA in PBS. To determine protein colocalization, cells were m Throughout the experiments, cell viability was assessed as per manu- incubated with 1 g/ml FITC-labeled goat polyclonal anti-AAT (Abcam, facturer’s instructions using the CellTiter 96 AQueous One Solution Cell Cambridge, U.K.). Cells were mounted using Vectashield mounting me- Proliferation Assay (Promega) by pipetting 20 ml of CellTiter96 reagent dium with DAPI for nuclear staining (Vectashield Lab, Burlingame, CA). into wells of a clear 96-well plate containing 100 ml of samples in culture The controls for these experiments included cells alone and nonspecific isotype control IgG (Santa Cruz, TX). All immunofluorescence was medium. This was incubated at 37˚C for 1–4 h in a humidified 5% CO2 atmosphere. The absorbance was recorded at 490 nm on a Spectra Max viewed and images were acquired using a Zeiss LSM 710 confocal im- M3 (Molecular Devices, U.K.). Toxicity was determined by comparing the munofluorescence microscope (Zeiss, Germany). Images were captured viability of treated cells to untreated control cells. with excitation wavelengths for DAPI and FITC of 364 and 488 nm, re- spectively. The images were represented as two-and-a-half–dimensional RNA isolation and quantitative real-time PCR reconstructions using Zen software (2011 Edition; Zeiss). Total RNA was extracted from HEK-293 cells using TRI Reagent as per the Statistical analysis manufacturer’s instructions. In brief, 500 ml of TRI Reagent was added to Data sets were analyzed for statistical significance using GraphPad Prism cell pellets and subsequently, 100 ml of chloroform was added, vortexed 5.0 software package (GraphPad Software) with significance determined at briefly, and incubated at RT for 10 min. Centrifugation at 12,000 3 g at p , 0.05. For data sets with three or more paired groups, a repeated 4˚C separated the sample into three phases. The aqueous RNA fraction at measures ANOVA was employed followed by post hoc Bonferroni mul- the top was taken off and transferred into a fresh tube with 250 mlof tiple comparisons test. To determine mRNA stability experiments, a isopropanol and incubated for 5 min at RT. A pellet was formed by cen- Downloaded from nonlinear fit one-phase exponential decay curve was employed. For the trifugation at 12,000 3 g at 4˚C for 10 min. The supernatant was dis- m glycosylation data set, the 95% confidence intervals (CI) were calculated. carded, and the pellet was washed with 700 l of 75% (v/v) ethanol and Results are expressed as mean 6 SEM of biological replicates or inde- 3 g centrifuged for 7500 at 4˚C for 5 min. The RNA pellet was subse- pendent experiments, as stated in the figure legends. quently air dried, and 25 ml of diethyl pyrocarbonate dH2O was added. The RNA was stored at 280˚C until ready to use. The RNA content was quantified using a Nanodrop 8000 Spectrophotometer (Thermo Scientific, Results Ireland). The isolated RNA was considered free of contaminants when the Q0 -AAT mutation is associated with truncated http://www.jimmunol.org/ . bolton A260/280 was 1.7. AAT protein Prior to cDNA synthesis, contaminating genomic DNA was removed m from 500 ng of RNA in a total volume of 14 l using genomic DNA A patient was identified as homozygous for the Q0bolton mutation as Wipeout Buffer from the QuantiTect Reverse Transcription Kit (Qiagen). a result of two PTCs at aa 373 and 374 on exon V (21). High- This was heated at 42˚C for 2 min. The RNA was then reverse transcribed to cDNA with 1 ml of RTase, 4 mlof53 reverse transcriptase buffer, and resolution computerized tomography scan of the thorax demon- 1 ml of reverse transcriptase primer mix per 2-ml sample. An RTase-free strated panlobular emphysema with bibasal predominance. sample was prepared by supplementing RNase-free H2O for RTase. cDNA Pulmonary function testing subsequently confirmed an obstructive was prepared on a PTC-200 Thermo Cycler (MJ Research, MN) with the pattern of moderate severity (FEV1 = 59% predicted; FEV1/forced following cycles: 30 min at 42˚C, 3 min at 95˚C, and at 4˚C for infinity. For vital capacity (FVC) = 0.55) with a minimal bronchodilator re- quantitative real-time PCR (qRT-PCR) analyses, the Lightcycler 480 by guest on October 3, 2021 (Roche Diagnostics) was employed, and the following program was used: sponse and impaired diffusion capacity of the lung for carbon preincubation (95˚C, 3 min); amplification, 40 cycles consisting of dena- monoxide (56% predicted; Fig. 1A). AAT phenotype testing by turation (10 s, 95˚C), annealing (with an optimized annealing temperature IEF revealed no discernable AAT protein banding pattern in of 56˚C for 10 s), and elongation (72˚C for 10 s); melting curve analysis plasma of the patient homozygous for the Q0 mutation (95˚C, 5 s; 65˚C, 1 min; and 97˚C continuous acquisition); and final bolton cooling step at 4˚C. The expression of target genes relative to GAPDH was (Fig. 1B). Of note, IEF used for AAT phenotyping has a lower determined using the 22ΔΔcycle threshold method (44). AAT forward limit of sensitivity of 0.05 g/L (46). Controls for the latter assay primer was 59-39:59-TGGATTTGGTCAAGGAGCTT-39, and reverse primer include a sample from an individual homozygous for the common was 59-39:59-CATGCCTAAACGCTT CATCA-39. GAPDH forward primer normal AAT M allele. The resultant M-AAT protein has five 9 9 9 9 was 5 -3 :5 -CATGAGAAGTATGACAACAGCCT-3 , and reverse primer was distinct bands on IEF gels termed M2, M4, M6, M7, and M8. The 59-39:59-AGTCCTTCCACGATACCAAAGT-39. For mRNA stability assays, experiments were carried out as previously ZZ-AATD protein demonstrates three IEF bands, Z2, Z4, and Z6, described (45). In brief, HEK-293 cells at a density of 1 3 105 were seeded as well as the classic cathodal shift (Fig. 1B). To understand po- in 12-well plates, allowed to adhere overnight, and then were serum tential conformational changes to the tertiary structure of the starved for 16 h. After this time, the media was removed, and the wells Q0bolton-AAT molecule, molecular modeling was performed were washed with Dulbecco’s PBS before the addition of serum-free media with or without ataluren (62.5 mg/ml) for 30 min, followed by actinomycin (Fig. 1C). The native AAT structure is in a kinetically trapped D (10 mg/ml) for 30 min. After 1, 2, or 3 h, the cells were washed with meta-stable state, with the rapid folding of residues 383–400 PBS before being lysed in TRIzol reagent. Following this, total RNA was (18 residues, strands 1c and 4b) responsible for kinetic trapping extracted from cells as per the manufacturer’s instructions. RNA was (47). Of these 18 residues, 14 remain in the Q0bolton-AAT mole- quantified using the Nanodrop 8000 Spectrophotometer (Fisher Scientific, cule; however, it is also missing a further 18 residues (strand 5b). Ireland). RNA (100 ng) was reverse transcribed to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen, U.K.) as per manufac- Molecular dynamics simulations of Q0bolton-AAT based on the turer’s instructions. qRT-PCR was performed using a Lightcycler 480. meta-stable AAT form (Fig. 1C, missing residues in orange) did Q0bolton-AAT was detected using the PCR primers 59-TGGATTTGGT- not show any significant changes to residues 383–396, suggesting CAAGGAGCTT-39 and 59-CATGCCTAAACGCTTCATCA-39,andRPLPO that there are sufficient interactions involving these residues to 9 9 were detected using the 5 -GGCAGCATCTACAACCCTGA-3 and stabilize a native-like structure should it form during the early 59-GGCAGCATCTACAACCCTGA-39 primers (MWG Eurofins). qRT-PCR was performed using the following protocol: preincubation (95˚C for 3 min); steps of protein folding, and thus these residues may be sufficient amplification (50 cycles consisting of denaturation, annealing, elonga- to kinetically trap a meta-stable form. Ensuing experiments used tion [10 s at 95˚C, 10 s at 55˚C, and 10 s at 72˚C]); melting curve conventional column chromatography to purify Q0bolton-AAT. analysis (95˚C for 5 s, 65˚C for 1 min, and 97˚C for five continuous acquisitions); and a final cooling step to 4˚C. Expression of AAT relative Q0bolton-AAT is produced by, and secreted from, hepatocytes to RPLPO was determined using the 22DD cycle threshold method. The , t1/2 was determined by comparing the percentage of Q0bolton-AAT mRNA The reported amount of circulating AAT in the proband was 0.1 g/L remaining at each time point with that at time 0. as measured by immune nephelometry, a commonly used standard The Journal of Immunology 5

FIGURE 1. PTC in the SERPINA1 gene encoding truncated AAT protein. (A) Chest high-resolution computerized tomography scan demonstrating pan- acinar emphysema and bronchiectatic changes of the lower lobes. (B) IEF patterns of AAT phenotypes. MM-AAT glycoforms (M2–M8) are denoted on the left and ZZ (Z2, Z4, and Z6) are shown by the broken arrow. Plasma from an individual homozygous for the Q0bolton mutation is presented on the right. Levels of plasma AAT (grams per liter) for each phenotype are indicated. (C) Molecular model of glycosylated

Q0bolton-AAT. Green, peptide; blue, glycans; red, RCL Downloaded from (residues M382–S383); pink, altered amino acid se- quence (residues 386–396); orange, deleted amino acid sequence (residues 396–418). http://www.jimmunol.org/

clinical assay for serum protein determinations. This suggests Next, we explored whether iPSC-derived hepatic cells could Q0 -AAT is present in extremely low concentrations that are serve as an appropriate cellular model to study the Q0 -AAT

bolton bolton by guest on October 3, 2021 below the lower limit of detection for this standard clinical assay. In protein (Supplemental Fig. 1) and to confirm that the vascular turn, however, truncated Q0bolton-AAT protein was purified from the origin of truncated Q0bolton-AAT was via hepatic secretion. patient’s plasma by use of Alpha-1 Antitrypsin Select, a resin with high Confocal imaging of both healthy MM and Q0bolton iPSC-hepatic selectivity for AAT. From whole plasma (200 ml), we purified ∼190 mg cells stained with anti-human AAT Abs revealed a punctuate of AAT from the MM plasma and 2 mg (0.01 g/L) of protein from the pattern throughout the cell, confirming the production of AAT Q0bolton plasma, as determined by BCA assay. Coomassie-stained one- protein by hepatic cells despite the nonsense mutation, albeit dimensional SDS-PAGE bands from the peak fraction containing the with considerably reduced intensity (Fig. 2D). In accordance AAT protein is shown in Fig. 2A. 2D-PAGE staining revealed eight with low plasma levels of Q0bolton-AAT in vivo, we found se- glycoforms of M-AAT and seven glycoforms of the Q0bolton-AAT creted levels of Q0bolton-AAT from patient-derived iPSC-hepatic protein run over a pH range of 4–7 (Fig. 2B), with Q0bolton-AAT cells to be ∼7% of controls (581 6 40 ng/ml versus 8522 6 demonstrating an anodal shift compared with M-AAT. To further con- 674 ng/ml; mean 6 SD; p , 0.0001) after 25 d in culture (Fig. firm the identity of the protein that was extracted from whole plasma, 2E). Collectively, these results confirm the hepatic production both the spots representing purified M-AAT and the Q0bolton-AAT and secretion of Q0bolton-AAT and its presence in the plasma of a protein were excised from the gel and assessed by LC-MS/MS. Mass null AATD homozygote. spectrometry confirmed each of the protein spots as AAT with a high MASCOT score achieved for both samples (mean 63.64 and 54.50% Q0bolton-AAT maintains antiprotease and anti-inflammatory capacity coverage for M-AAT and Q0bolton-AAT, respectively). Western blot analysis with a polyclonal goat AAT Ab (Fig. 2A, middle panel) con- The main function of AAT is to act as an antiprotease, and pre- firmed the presence of AATand a size shift for Q0bolton-AAT. We further viously we demonstrated the ability of Q0bolton-AAT to inhibit NE investigated the reduced molecular mass of Q0bolton-AAT by running (21), a result confirmed in this study. AAT readily forms a covalent 8% (w/v) SDS-PAGE and visualized the 49-kDa truncated protein on a complex with NE at approximately a 1:1 molar ratio, yielding a Western blot using a rabbit Ab for AAT (Fig. 2A, bottom panel). protein complex of a combined molecular mass of 81 kDa (48). Glycosylation of proteins can result in a heterogeneous migratory pat- The Western blot in Fig. 3A depicts the AAT complex that occurs tern on gels; thus, to confirm the reduced molecular mass, we treated when M-AAT (52 kDa) and NE (29 kDa) interact at a 1:1 ratio for purified M-AAT and the Q0bolton protein with PNGase F to remove the 30 min, with minimal levels of free AAT (52 kDa) remaining. entire N-glycan structure and then subjected the treated protein to Although less active than M-AAT, Q0bolton-AAT successfully Western blot analysis. All of the 52-kDa M-AAT was reduced to 44 reacted with the protease, as indicated by an increase in the in- kDa, and the 49-kDa Q0bolton-AAT protein was reduced to 41 kDa upon tensity of an immunoband of ∼78 kDa (Fig. 3A, lower panel). We PNGase F treatment (Fig. 2C). These results confirm the successful extended this set of experiments and employed FRET analysis to isolation of Q0bolton-AAT as a low-m.w. truncated glycosylated protein. demonstrate that both M-AAT and Q0bolton-AAT can inhibit NE 6 MUTANT ALPHA-1 ANTITRYPSIN AND ANTI-INFLAMMATORY ACTIVITY Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 2. Isolation of truncated AAT protein from Q0bolton plasma. (A) Coomassie Blue–stained SDS-PAGE showing AAT purified from MM and Q0bolton plasma at 1 mg (lane 1 and 3) and 0.1 mg (lane 2 and 4) loadings. Purified MM and Q0bolton-AAT ran at 52 and 49 kDa, respectively (top panel). To confirm identity of AAT, purified protein was subjected to Western blot analyses with goat polyclonal (Gt anti-AAT, middle panel) or rabbit polyclonal Ab to AAT (Rb anti-AAT, bottom panel). Molecular mass markers are indicated at the left margin in kilodaltons. (B) Coomassie Blue–stained 2D-PAGE run over a linear pH range of 4–7. AAT glycoforms are numbered. (C) Western blot of purified AAT protein was treated with PNGase, and electrophoretic mobility of glycosylated 52-kDa M-AAT and 49-kDa truncated Q0bolton-AAT compared with deglycosylated (degly) protein (44 and 41 kDa, respectively) is demonstrated. (D) MM control and Q0bolton patient-derived iPSC-hepatic cells exhibit intracellular AAT. By confocal microscopy, the distribution of AAT (green) in MM and Q0bolton patient-derived iPSC-hepatic cells presented as punctuate staining throughout the cell. Cell nuclei are stained blue with DAPI. (E) MM control and Q0bolton patient-derived iPSC-hepatic cells exhibit extracellular AAT. ELISA demonstrated that MM and Q0bolton iPSC-hepatic cells secrete ∼8500 and 600 ng/ml of AAT, respectively (ANOVA, p , 0.0001; n = 3 technical repeats). Experiments illustrated in (A)–(C) are each repre- sentative gels and blots of three separate experiments. Confocal analysis in (D) is a representative result of three experiments. activity (Fig. 3B). The percentage of NE (4 nM) remaining after effects shown in this study do not simply reflect the nonspecific treatment with AAT (2 nM) for 5.4 min was recorded at excitation effect of a protein, in the assay, we extended this experiment to 320 nm and emission 420 nm. M-AAT was shown to reduce NE include HSA as a control plasma protein. Results revealed that, activity by 58%, and although significantly less, Q0bolton-AAT compared with HSA, Q0bolton-AAT bound significantly higher reduced NE activity by 13% (n =3,p = 0.04) (Fig. 3B), suggesting levels of IL-8 (p = 0.007), equivalent to that of M-AAT (Fig. 3C). In that it may provide lung tissue protection if present in sufficient a subset of control experiments, IL-8 was omitted from reactions, levels. with no nonspecific interaction between AAT, or HSA, and the anti– In subsequent experiments, we investigated the anti-inflammatory IL-8 Abs detected. capacity of Q0bolton-AAT. It has previously been shown that M-AAT As the glycan moieties of AAT are essential for not only the binds IL-8 via glycan sites on the molecule, thereby modulating serum t1/2 of AAT but also its capacity to bind IL-8, it was es- neutrophil migration (34). To confirm the competence of Q0bolton- sential to investigate the glycosylation pattern of Q0bolton-AAT. AAT as an immunoregulatory molecule, its ability to bind IL-8 was Consequently, glycoanalysis was performed on plasma-purified assessed in vitro by use of slot blot analysis. To confirm that the M-AAT and Q0bolton-AAT cut from Coomassie Blue–stained 2D The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 3. Q0bolton-AAT maintains antiprotease and anti-inflammatory properties. (A) Formation of AAT/NE inhibitory complexes. M-AAT (top panel) was incubated with NE at an AAT/NE molar ratio of 1:1 for 10 min (M-AAT). Reaction products were subjected to Western blot analysis employing a goat anti-human AAT Ab for AAT or AAT/NE complexes. M-AAT incubated with NE resulted in the formation of an 81-kDa AAT/NE complex. Q0bolton-AAT incubated with NE for 0, 5, 10, or 20 min resulted in the formation of a 78-kDa AAT/NE complex. Experiments illustrated are a representative of three separate experiments. (B) FRET analyses of the percentage of NE (4 nM) activity remaining after treatment with AAT (2 nM) for 5.4 min. M-AAT significantly reduced the level of active NE compared with Q0bolton-AAT (n = 3, two-tailed Student t test). (C)Q0bolton-AAT binds IL-8. Slot blot of 5 mgof M-AAT, Q0bolton-AAT, or HSA (positive control) for IL-8 binding. Blots were incubated with carrier-free IL-8, and the binding event was detected by employing an IL-8 mAb. Control (Con) for nonspecific binding excluded IL-8 but included primary and secondary Ab. Total immobilized protein was visualized by Ponceau S Stain (bottom panel). Densitometry values of IL-8 binding to both AAT forms were normalized to the HSA control and dem- onstrate the ability of M-AAT (p = 0.005) and Q0bolton-AAT (p = 0.007) to bind IL-8 (Student t test; n = 3 independent experiments). (D) Glycoanalysis of AAT protein. Q0bolton-AAT and M-AAT were excised from Coomassie Blue–stained gels and analyzed by UPLC. Values indicate individual biological replicates (n = 3) of the M-AAT.

gels. The N-glycans of purified M-AAT and Q0bolton-AAT were Core fucosylation for M-AAT was calculated to be 5.35% (95% CI: analyzed by UPLC in combination with exoglycosidase digestions 4.69–6.02) compared with 8.3% for the Q0bolton-AAT sample, and and structural assignments. Typical chromatograms of hydrophilic outer-arm fucosylation for M-AAT was reported to be 9.51% (95% interaction liquid chromatography columns of undigested AAT from CI: 6.43–12.60) compared with 16.32% for the Q0bolton-AAT. Total a healthy MM individual and the Q0bolton AATD patient are shown in fucosylation was demonstrated to be 14.36% (95% CI: 11.11– Supplemental Fig. 2, and the list of identified Q0bolton-AAT glycans 17.61) in M-AAT samples, whereas it was found to be increased to is presented in Supplemental Table I. The core (a1-6 linked), 23.86% in the Q0bolton-AAT samples. Furthermore, there appears outer-arm (a1-3 linked), and total fucosylation as well as amounts to be a difference in the percentage of branched glycans between of biantennary, triantennary, and tetra-antennary glycans were cal- the two sample types, with M-AAT reporting a higher percentage culated in all samples from the percentages of peak areas after of biantennary glycans (77.62%; 95% CI: 71.67–83.56) compared A. ureafaciens sialidase digest, as presented in Supplemental with the 62.81% reported in the Q0bolton-AAT samples. Additionally, Table II. The 95% CI were calculated for each of the M-AAT there was a decrease in the percentage of both tri- (19.84%; 95% samples. For all except biantennary, the percentage of glycans in CI: 15.20–24.48) and tetra-antennary (2.54%; 95% CI: 0.63–4.45) the Q0bolton-AAT sample was greater than the 95% CI (Fig. 3D). glycans in M-AAT samples compared with Q0bolton-AAT samples 8 MUTANT ALPHA-1 ANTITRYPSIN AND ANTI-INFLAMMATORY ACTIVITY

(32.01 and 5.16%, respectively) (Fig.3D).Thistrendtowardincreased was to evaluate additional readthrough compounds, over a range core and outer-arm fucosylation differentiates Q0bolton-AAT from of concentrations, for their ability to overcome the PTC in the M-AAT and is consistent with persistent inflammation (49), although SERPINA1 gene. To achieve this, HEK-293 cells transduced with these differences do not appear to impact the binding capacity of lentiviral vectors containing M-AAT or Q0bolton-AAT were gener- Q0bolton-AAT for IL-8. ated (Fig. 5A). A 2-fold reduction in basal mRNA expression was Moreover, the capacity of AAT to disable the stimulating effect apparent in the PTC containing Q0bolton-AAT mRNA compared of LTB4 has been previously shown, with the mechanisms of in- with M-AAT mRNA (p = 0.01) (Fig. 5B), and the level of trun- hibition involving direct binding of LTB4 via a hydrophobic cated AAT secreted from Q0bolton-AAT–expressing cells was of pocket on the surface of the molecule (8). Additionally, a previous lower abundance and molecular mass compared with M-AAT study has detailed the ability of LTB4 to interact with HSA (50). (p = 0.0001) (Fig. 5C). The in vitro data indicate that the pro- For this analysis, the structure of LTB4 is key, as it comprises a tein levels detected are reduced and out of proportion to the triene moiety that can be recorded by UV absorption at 262, 270, mRNA levels. To understand why this was the case, we assessed and 282 nm (50), and when protein conjugated, the triene structure whether the Q0bolton-AAT protein was susceptible to degradation. of LTB4 becomes planar. In control experiments of the current To analyze this, M-AAT and Q0bolton-AAT protein was subjected study, the UV spectrum of free LTB4 gave rise to the characteristic to two freeze-thaw cycles. Robustness of a protein against freezing vibrational structure in PBS solution (Fig. 4A) (51). By use of a and thawing depends on its conformational stability, and results of 5:1 mixed ratio of LTB4 to HSA or M-AAT, it was confirmed that the current study demonstrate that although M-AAT remained LTB4 interacts with HSA and M-AAT, demonstrating a reduction intact after two cycles of freeze-thaw (280˚C), the level of in εmax at 270 nm compared with that of LTB4 alone (Fig. 4A). Q0bolton-AAT protein detected by Western blot analysis was re- Downloaded from Moreover, an LTB4/AAT molar ratio of 5:1 resulted in an ∼45% duced (Fig. 5D). decrease compared with LTB4 alone, with area under the curve Given that endogenous levels of mRNA are reduced in the analysis of UV profiles of three technical replicates performed on Q0bolton-AAT–expressing HEK-293 cells, we next assessed the three separate days revealing a significant decrease in the presence ability of readthrough compounds to augment the levels of pro- of M-AAT when compared with the control unbound free LTB4 tein. We first investigated the effect of two readthrough com- or HSA bound LTB4 (p = 0.006 and p = 0.001, respectively) pounds, using two previously described drug concentrations (43). http://www.jimmunol.org/ (Fig. 4A). Subsequent experiments explored the ability of Q0bolton- Neither gentamicin (0.5 and 1 mg/ml) nor ataluren (30 and AAT to bind LTB4. In the current study, Q0bolton-AAT protein 62.5 mg/ml) had a significant impact on the viability of HEK-293 retained the ability to bind LTB4 as evaluated by a reduction in εmax cells expressing M-AAT (Fig. 6A). On examination of mRNA at 270 nm, similar to that of M-AAT. An LTB4/Q0bolton-AAT molar levels in response to readthrough compounds, no significant ratio of 5:1 resulted in a significant 40% decrease compared with change in mRNA levels in either the M-AAT or Q0bolton-AAT the LTB4 profile alone (p = 0.004), as deduced by area under the expressing cells was observed in response to treatment with curve analysis (Fig. 4C). Q0bolton-AAT bound to LTB4 to the same gentamicin (0.5 mg/ml) (Fig. 6B). In contrast, however, treatment extent as M-AAT at equivalent concentration. of Q0bolton-AAT expressing cells with ataluren (62.5 mg/ml) A number of studies have documented the activating effects of resulted in a modest but significant increase in mRNA levels by guest on October 3, 2021 LTB4 on neutrophil adhesion (52), and exposure of cells to LTB4 (p = 0.03) (Fig. 6B). Moreover, by Western blot analyses, it was in the presence of AAT has been shown to reduce neutrophil ad- observed that ataluren treatment induced a significant 1.5-fold hesion (8). Accordingly, ensuing experiments examined the effect increase in AAT protein secretion by both M-AAT (p = 0.03) and of Q0bolton-AAT on LTB4-induced neutrophil adhesion. In control Q0bolton-AAT treated cells (p = 0.04) (Fig. 6C). As Western blot experiments, calcein AM–loaded neutrophils were stimulated with analysis is only semiquantitative, we performed an ELISA for LTB4 (100 nM) in the presence and absence of AAT or HSA secreted AAT in response to ataluren. For this experiment, (control plasma protein, 27.5 mM), and adherence to fibronectin- Q0bolton-AAT or M-AAT cells were treated with ataluren (62.5 mg/ml) coated surfaces was assessed after 30 min incubation at 37˚C for 48 h. After this time, secreted AAT levels were measured by (Fig.4C).LTB4 significantly increased neutrophil adhesion ELISA. At baseline, Q0bolton-AAT untreated cells secreted 2.8-fold compared with unstimulated control cells (p = 0.01). A physio- less AAT (p , 0.001) compared with M-AAT cells. Both cell lines logical concentration of 27.5 mM AAT significantly reduced the treated with ataluren (62.5 mg/ml) secreted significantly higher LTB4 response to that of unstimulated control cells (p = 0.02), and levels of AAT compared with respective untreated cells (p , 0.001); AAT significantly reduced cell adhesion compared with the effect however, 1.85-fold less Q0bolton-AAT was detected compared with of HSA (p = 0.01) (Fig. 4C). Moreover, as illustrated in Fig. 4D, M-AAT (p , 0.001) (Fig. 6D). 27.5 mMQ0bolton-AAT (p = 0.04) significantly inhibited LTB4- An ataluren dose response was next studied, with a significant induced cell adhesion, comparable to the effect of M-AAT. increase in Q0bolton-AAT secretion apparent at low concentra- U-75302 (1 mM) was employed as a positive control (p = 0.04, tions of ataluren employed at 0.1 and 0.5 mg/ml (p = 0.001 and Student t test, n = 3). Collectively, these results demonstrate that p = 0.002, respectively) and up to 62.5 mg/ml (p = 0.04) (Fig. 7A). Q0bolton-AAT, despite its truncated size, maintains antiprotease We further investigated by Western blot analysis whether ataluren activity and normal ability to interact with the proinflammatory treatment of Q0bolton-AAT–expressing HEK-293 cells led to molecules IL-8 and LTB4, thereby modulating cell adhesion. translation of the full-length AAT protein. By Western blot anal- yses, it was confirmed that ataluren employed at a concentration of AAT production is increased in response to 62.5 mg/ml increased levels of secreted Q0bolton-AAT protein readthrough compounds compared with 12.5 mg/ml treatment, although the reduced mo- The fundamental objective of theratyping is to partner medications lecular mass of Q0bolton-AAT (49 kDa) compared with M-AAT to specific mutations. Therapies for AATD individuals with non- (52 kDa) was still apparent (Fig. 7B). sense mutations could include readthrough compounds, leading to One explanation for the increased protein expression in response translation of the full-length AAT protein. iPSC-derived Q0bolton to ataluren could be through stabilization of Q0bolton-AAT mRNA hepatic cells treated with single-dose ataluren demonstrated in- levels. To analyze mRNA stability, we blocked cellular tran- creased AAT protein secretion (21). With this in mind, our aim scription with the inhibitor actinomycin D, which interferes with The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 4. Q0bolton-AAT binds LTB4.(A) The triene chromophore structure of LTB4 (solid line) with the addition of M-AAT (dashed line) or HSA (dotted line). The molar ratio of ligand to protein was 5:1. UV spectra were recorded between 230 and 320 nm. The three arrows indicate covalently bound 21 21 atoms in the LTB4 structure at 262, 270, and 283 nm. The molar absorptivity (ε) at 270 nm was calculated and represented as M cm with a path length of 1 cm. Area under the curve analysis demonstrates the ability of M-AAT (p = 0.006) to bind LTB4 (Student t test; n = 3 independent experiments). (B) The triene chromophore structure of LTB4 (solid line) with the addition of M-AAT or Q0bolton-AAT (broken line). Area under the curve analysis demonstrates the ability of M-AAT (p = 0.0006) and Q0bolton-AAT (p = 0.004) to bind LTB4 compared with uncomplexed LTB4 (Student t test; n = 3 independent 6 experiments). (C) Neutrophil adhesion. Neutrophils (5 3 10 cell/ml) were loaded with calcein AM dye and stimulated with LTB4 (100 nM) in the presence or absence of AAT (27.5 mM) or HSA (27.5 mM). AAT treatment significantly reduces neutrophil adhesion compared with HSA-treated cells (p = 0.01).

(Student t test; n = 5 technical repeats). (D)Q0bolton-AAT significantly reduced cell adhesion (p = 0.04). The BLT1 antagonist U-75302 served as a positive control (Student t test, n = 3 independent experiments).

transcription (Fig. 7C). HEK-293 cells expressing Q0bolton-AAT demonstrate increased AAT protein production in the presence were treated with ataluren (62.5 mg/ml) for 30 min, followed by of ataluren, and this is particularly useful in this disorder as actinomycin D (10 mg/ml) for 30 min, and the amount of Q0bolton- the truncated protein encoded by Q0bolton mRNAs retains anti- AAT mRNA remaining at 1 h of treatment was calculated. The t1/2 inflammatory function. of Q0bolton-AAT mRNA in cells that had not received ataluren treatment was determined to be 40 min. This was significantly Discussion increased to 1.3 h in the presence of ataluren, accounting for a ThefirstAATDcasestudyduetoanull mutation was described 1.95-fold increase in message stability (p = 0.007) (Fig. 7C). This in a 24-y-old patient with undetectable serum levels of AAT result may account for the heightened expression of AAT fol- (53). Currently, at least 32 AATD null mutations have been lowing ataluren treatment, amplified translation of Q0bolton- reported (54). Historically, it has been thought that no AAT AAT, and increased protein secretion. Collectively, these data protein arises from null mutations, but as part of this study, we 10 MUTANT ALPHA-1 ANTITRYPSIN AND ANTI-INFLAMMATORY ACTIVITY Downloaded from http://www.jimmunol.org/

FIGURE 5. HEK-293 M-AAT and Q0bolton-AAT protein production. (A) Schematic of lenti CMV-AAT-UBC-GFP. The enhanced GFP reporter gene is constitutively expressed in the second position together with either M-AAT or Q0bolton-AAT under control of the human UBC promoter. (B) Q0bolton-AAT HEK-293 cells express significantly less AAT mRNA than the cells expressing M-AAT (p = 0.011, Student t test; n = 3). Cells not transduced with AAT acted as a negative control for AAT mRNA expression (Con). (C) AAT protein produced by M-AAT– or Q0bolton-AAT–expressing HEK-293 cells. Cell culture supernatants were harvested and the cells lysed for protein analysis. Supernatants and cell lysates were run on an SDS-PAGE gel (12% [w/v]) and by guest on October 3, 2021 Western blotted for AAT. Densitometry analysis was performed, and the bar graph represents AAT relative expression. M-AAT is secreted to a greater extent than Q0bolton-AAT (p = 0.0001, Student t test; n = 3). (D) M-AATand Q0bolton-AAT protein was subjected to two freeze-thaw cycles (cycle 1 = F/T 1, cycle 2 = F/T 2) and detected by Western blot analysis (n = 3 technical repeats).

purified truncated AAT protein because of the Q0bolton null targeted for destruction. It is noteworthy to mention that research mutation from human plasma and evaluated its capacity as an developments in this area suggest that NMD can further contribute anti-inflammatory mediator. Furthermore, we used transduced to disease by preventing the translation of truncated proteins that HEK-293 cells expressing Q0bolton-AAT to study the efficacy of may retain function (56). readthrough compounds on successfully augmenting secreted Proteomic and glycomic analyses were performed to charac- protein levels. terize the plasma-purified Q0bolton-AAT protein. Using 2D-PAGE, The discovery of the truncated Q0bolton-AAT protein of 49 kDa a significant anodal shift of the Q0bolton-AAT protein compared is expected due to the position of the PTC on the mRNA sequence. with the native M-AAT form was observed. This is in contrast to However, why we detected Q0bolton-AAT protein within the cir- the cathodal shift that occurs as a result of the point mutation culation, whereas studies of other null variants have not, is of (Glu342Lys) in the SERPINA1 gene that results in the ZZ-AAT interest. One possible explanation for this may be the unfolded protein (38). The results of this study also indicate that the protein response, a response observed associated with the null glycosylation of Q0bolton-AAT protein is altered, comprising a variant Q0hongkong, which leads to degradation of the protein be- trend toward increased branching of glycans with a decrease of fore it can enter the circulation (55). However, the fact that we glycans containing biantennary branching and a concomitant identified circulating truncated Q0bolton-AAT may be due to the increase in those containing tri- and tetra-antennary branching. different locations of the PTC; Q0hongkong is located 39 aa up- A similar degree of altered branching has been reported for the stream of the Q0bolton PTC, before the RCL, which is in contrast to acute phase protein a-1 acid glycoprotein in asthmatic patients the Q0bolton mutation transcripts, which retain the active site of the and this correlates with FEV1 and eosinophil numbers (57). Re- molecule. Consequently, in contrast to Q0hongkong, which is char- sults of the current study also demonstrate increased fucosylation acterized by a complete absence of secreted protein, the concen- of Q0bolton-AAT protein, a phenomenon previously shown for tration of Q0bolton-AAT protein purified was ∼1% of the healthy AAT in patients with rheumatoid arthritis and chronic joint in- MM control level. From confocal microscopy and Western blot flammation (58). Of particular interest, the low level of plasma analyses of HEK-293 cells, it would appear that Q0bolton-AAT is Q0bolton-AAT detected may indicate that the mutant protein is not retained intracellularly and that the Q0bolton-AAT protein that more susceptible to clearance (or alternatively, degradation) we have purified from plasma represents protein that was not compared with M-AAT. Indeed, glycans on AAT are important not The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on October 3, 2021

FIGURE 6. Increased M-AAT and Q0bolton-AAT production in response to ataluren. (A) Viability of HEK-293 cells expressing M-AAT exposed to either gentamicin (GT, 0.5 or 1 mg/ml) or ataluren (Atal, 30 or 62.5 mg/ml) for 48 h. Results revealed no significant impact on cell viability (n = 3, Student t test).

(B) AAT gene expression in HEK-293 cells expressing M-AAT or Q0bolton-AAT. HEK-293 cells overexpressing M-AAT or Q0bolton-AAT were exposed to GT (0.5 mg/ml), Atal (62.5 mg/ml), or vehicle control (Con) for 24 h. M-AAT and Q0bolton-AAT mRNA expression is significantly increased in the presence of Atal compared with Con cells. (C) AAT protein expression in response to readthrough compounds. Culture supernatants of HEK-293 exposed to GT (0.5 mg/ml) or Atal (62.5 mg/ml) for 48 h were Western blotted for secreted AAT. Protein levels were normalized to that of respective Con. An increase in

AAT protein secretion post-Atal treatment was observed for HEK-293 cells expressing M-AAT (p = 0.03) and Q0bolton-AAT (p = 0.04, Student t test, three independent experiments). (D)Q0bolton-AAT or M-AAT cells were treated with Atal (62.5 mg/ml) for 48 h. Secreted AAT levels were measured by ELISA. An increase in AAT protein secretion post-Atal treatment was observed for M-AAT cells and Q0bolton-AAT cells (p , 0.001, n = 4 independent exper- iments, ANOVA). only for conformational stability of AAT by decreasing the energy role of AAT in modulation of cell activity, facilitating resolution of level of the native state protein but also for the plasma t1/2. The inflammation (2). In contrast, glycosylation does not affect binding plasma t1/2 of M-AAT is 4–5 d, which is in contrast to non- of AAT to LTB4, but instead, the lipid locates to a hydrophobic glycosylated AAT that is rapidly eliminated from plasma (59). pocket along the protein surface against the s6A and s5A strands (8). Furthermore, AAT can be removed from the circulation via ga- AAT-LTB4 complex formation modulates BLT1 engagement and lactose residues that, when exposed, trigger removal of AAT neutrophil adherence (8). Results of the current study revealed that through endocytosis-mediated uptake by asialoglycoprotein Q0bolton-AAT bound IL-8 and LTB4 to the same extent as M-AAT, receptors on hepatocytes (60, 61). Although the results of confirming the potential immune-regulatory ability of this truncated this study indicate that the glycosylation of Q0bolton-AAT protein. Glycosylation is also unrelated to the antielastase capacity of protein is altered compared with M-AAT, the recorded changes the protein, as demonstrated by the retained antiproteasefunctionina are most likely not the main cause of the low levels of plasma recombinant, aglycosylated AAT molecule (62). In the current study, Q0bolton-AAT, but may possibly be due to the observed reduced the Q0bolton-AAT protein retained anti-NE capacity at a lower level protein stability. than M-AAT but nevertheless may provide antiprotease protection if Crucially, AAT glycans are important for controlling IL-8 (2). present in sufficient levels. Epidemiological studies have revealed that Indeed, a recent study demonstrated the enhanced anti-inflammatory although normal circulating levels of M-AAT are ∼27.5 mM, a quality of sialylated AAT involving superior inhibitory influence on concentration of ∼11 mM may be sufficient to protect the lung from neutrophil IL-8–induced chemotaxis. During the resolving phase of an increased risk of emphysema (63). As such, if adequate infection, there was a significant increase in circulating levels of amounts of Q0bolton-AAT were released from hepatocytes and sialylated AAT/chemokine complexes, in keeping with a pivotal other AAT-producing cells, the resulting protein in its truncated 12 MUTANT ALPHA-1 ANTITRYPSIN AND ANTI-INFLAMMATORY ACTIVITY Downloaded from http://www.jimmunol.org/

FIGURE 7. Stabilization of mRNA leads to increased Q0bolton-AAT truncated protein (A) AAT protein production by Q0bolton-expressing cells in re- sponse to increasing concentrations of ataluren (Atal). Atal employed at 0.1, 0.5, 2.5, 12.5, and 62.5 mg/ml induced a significant increase in levels of secreted AAT protein compared with untreated cells (control [Con]) (Student t test, three independent experiments). (B) AAT protein size in response to by guest on October 3, 2021 readthrough compounds. Electrophoretic mobility of AAT secreted by Q0bolton-expressing HEK-293 cells in response to Atal (12.5 and 62.5 mg/ml) was reduced (49 kDa) compared with AAT produced by HEK-293 cells expressing M-AAT (42 kDa), indicating that Atal caused increased expression but not an increase in size. Representative image of three separate experiments. In (A) and (B), postnuclear supernatants were prepared of untreated and treated cells, and Western blots for actin demonstrated equal protein loading, supporting the use of equal cell numbers per reaction. (C) Effect of Atal (62.5 mg/ml) on

Q0bolton-AAT mRNA stability over 3 h. HEK cells were cultured in the presence or absence of Atal for 30 min and actinomycin D (10 mg/ml) for 30 min. mRNA levels were normalized to RPLP0 mRNA levels and expressed as a percentage of AAT/RPLP0 mRNA levels at time 0. Pretreatment with Atal resulted in an increased t1/2 (1.3 h) of Q0bolton-AAT mRNA compared with mRNA from untreated cells (40 min) (p = 0.007, n = 3, nonlinear-fit one-phase exponential decay curve). form may provide a layer of protection against neutrophil-mediated concentration of ataluren employed minimally affected cell viability. damage by, first, preventing neutrophil migratory responses to In vivo, two separate studies have explored the effect of ataluren in LTB4 and IL-8 and, second, as an antielastase screen to protect patients with DMD. The first recruited patients who received ataluren the lungs. Supportive studies in line with this theory include the orally three times daily (10–40 mg/kg) (65), and the second involved W12823 CFTR PTC, a C-terminal CFTR mutation that exhibits a multicenter, randomized, double-blind, placebo-controlled, phase-3 partial channel activity and is moderately affected via ivacaftor trial in patients (40 mg/kg per day) (26). In both cases, the dose of therapy (64). ataluren was generally well tolerated and within the range used in the We next assessed the ability of readthrough compounds to current study. Moreover, our data demonstrate variations in the ata- augment the levels of AAT protein, by treating HEK-293 M-AAT– and lureneffectbydose,andinlinewiththis observation, a bell-shaped Q0bolton-AAT–expressing cells with gentamicin and ataluren. Genta- dose response with the use of ataluren has been documented. In this micin, as the progenitor drug in this category, has been shown to regard, by use of cultured myotubes from a murine model of DMD, cause suppression of PTC in a number of in vitro cell models (32). and also patients with DMD, a bell-shaped response curve for dys- Ataluren has the ability to induce the readthrough of stop codons, trophin production following ataluren treatment was observed with readthrough highest in the genetic code UGA, followed by UAG (25, 66). In addition to these findings, a bell-shaped dose response and then UAA. The mutation to the SERPINA1 gene resulting in the relationship to ataluren was apparent in a zebra fish DMD model Q0bolton phenotype causes UAA in the coding region, suggesting that (67). It was clear from our data that ataluren did not cause read- ataluren may potentially trigger readthrough and production of full- through to full-length protein but augmented levels of both length protein. Results of the current study demonstrate that AAT M-AATand truncated Q0bolton-AAT protein, a result confirming protein levels were boosted in HEK-293 M-AAT– and Q0bolton-AAT– data obtained with the use of iPSC-hepatic cells (21). Although the expressing cells treated with ataluren, with a dose between 0.1 and main function performed by readthrough compounds is suppression 62.5 mg/ml causing significant increase in protein levels. The highest of PTC, recent data suggest that some of the compounds may The Journal of Immunology 13 additionally act as NMD inhibitors (68). Moreover, it has been 11. Grimstein, C., Y. K. Choi, M. Satoh, Y. Lu, X. Wang, M. Campbell-Thompson, and S. Song. 2010. Combination of alpha-1 antitrypsin and doxycycline sup- proposed that ataluren can affect premature translation termination presses collagen-induced arthritis. J. Gene Med. 12: 35–44. by altering the competition between regulatory factors and near- 12. Franciosiz, A. N., C. McCarthy, T. P. Carroll, and N. G. McElvaney. 2015. cognate tRNAs for binding to the ribosomal A site (69). Results of Unusual acute sequelae of a1-antitrypsin deficiency: a myriad of symptoms with one common cure. Chest 148: e136–e138. the current study indicate an alternative explanation for the increased 13. Rouhani, F., G. Paone, N. K. Smith, P. Krein, P. Barnes, and M. L. Brantly. 2000. protein expression in response to ataluren, involving stabilization of Lung neutrophil burden correlates with increased pro-inflammatory cytokines AAT mRNA levels, a documented effect of agents that induce and decreased lung function in individuals with alpha(1)-antitrypsin deficiency. Chest 117(5 Suppl. 1): 250S–251S. readthrough of PTC (70). 14. Chapman, K. R., J. G. Burdon, E. Piitulainen, R. A. Sandhaus, N. Seersholm, In conclusion, results of this study demonstrate the possible use J. M. Stocks, B. C. Stoel, L. Huang, Z. Yao, J. M. 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