Expression, Purification, and Biochemical Characterization of Human

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Expression, purification and biochemical characterization of human afamin Alessandra Altamirano, Andreas Naschberger, Barbara G. Furnrohr, Radka Saldova, Weston B. Struwe, Patrick M. Jennings, Silvia Millán Martín, Suzana Malic, Immanuel Plangger, Stefan Lechner, Reina Pisano, Nicole Peretti, Bernd Linke, Mario M. Aguiar, Friedrich Fresser, Andreas Ritsch, Tihana Lenac Rovis, Christina Goode, Pauline M. Rudd, Klaus Scheffzek, Bernhard Rupp, and Hans Dieplinger J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00867 • Publication Date (Web): 14 Feb 2018 Downloaded from http://pubs.acs.org on February 15, 2018

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Journal of Proteome Research

Lenac Rovis, Tihana; Center for Proteomics, Faculty of Medicine, University of Rijeka Goode, Christina; Medizinische Universitat Innsbruck Department fur Medizinische Genetik Molekulare und Klinische Pharmakologie, Genetic Epidemiology Rudd, Pauline; University College Dublin, NIBRT Scheffzek, Klaus; Division of Biological Chemistry, Medical University of Innsbruck Rupp, Bernhard; Medizinische Universitat Innsbruck Department fur Medizinische Genetik Molekulare und Klinische Pharmakologie, Genetic Epidemiology Dieplinger, Hans; Medizinische Universitat Innsbruck Department fur Medizinische Genetik Molekulare und Klinische Pharmakologie, Genetic Epidemiology

ACS Paragon Plus Environment

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Expression, purification and biochemical characterization of human afamin Alessandra Altamirano,#,† Andreas Naschberger,#,‡ Barbara G. Fürnrohr,#,†,‡ Radka Saldova,§ Weston B. Struwe,§,x Patrick M. Jennings,§ Silvia Millán Martín,§ Suzana Malic,|| Immanuel Plangger,† Stefan Lechner,‡ Reina Pisano,† Nicole Peretti,† Bernd Linke ,† Mario M. Aguiar,† Friedrich Fresser,⊥ Andreas Ritsch,¶ Tihana Lenac Rovis,|| Christina Goode,† Pauline M. Rudd,§ Klaus Scheffzek,‡ Bernhard Rupp,† Hans Dieplinger†,⊗,* †

Division of Genetic Epidemiology, Department of Medical Genetics and Molecular and

Clinical Pharmacology, ‡Division of Biological Chemistry, Biocenter Innsbruck, Medical University of Innsbruck, Austria; §NIBRT GlycoScience Group, National Institute for Bioprocessing Research & Training, Dublin, Ireland; ||Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia; ⊥Division of Translational Cell Genetics, Department of Medical Genetics and Molecular and Clinical Pharmacology, ¶Department of Internal Medicine I, Medical University of Innsbruck, Austria; ⊗Vitateq Biotechnology GmbH, Innsbruck, Austria x

Current Address: Department of Chemistry, University of Oxford, Oxford, UK

*

Corresponding author at: Division of Genetic Epidemiology, Department of Medical

Genetics, Molecular and Clinical Pharmacology, Medical University of Innsbruck, Schöpfstrasse 41, A-6020 Innsbruck, Austria. Tel +43-512-900370570, Fax +43-512900373570, Email [email protected] #

These authors contributed equally to the work.

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ABSTRACT: Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin’s glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypo-glycosylated forms were expressed in CHO, CHO-Lec1 and HEK293T cells. Fully non-glycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin. Wild-type and hypo/non-glycosylated rhAFM were purified from cell culture supernatants by immobilized metal ion affinity and size exclusion chromatography. Glycan analysis of purified proteins demonstrated differences in micro- and macro-heterogeneity of glycosylation enabling the comparison between hypo-glycosylated, wild-type rhAFM and native plasma afamin. Since antibody fragments can work as artificial chaperones by stabilizing the structure of proteins and consequently enhance the chance for successful crystallization, we incubated a Fab fragment of the monoclonal anti-afamin antibody N14 with human afamin and obtained a stoichiometric complex. Subsequent results showed sufficient expression of various partially or non-glycosylated forms of rhAFM in HEK293T and CHO cells and revealed that glycosylation is not necessary for expression and secretion. KEYWORDS: Afamin; expression in different cellular models; glycosylation heterogeneity

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1. INTRODUCTION The 87 kDa glycoprotein afamin, previously identified as the fourth member of the albumin gene family, is mainly expressed in the liver and secreted into circulating blood. The afamin gene maps to chromosome 4q11-q13 in humans. Afamin’s physiologic function is largely unknown but, due to structural similarity to albumin, probably also related to transport functions for small, hydrophobic molecules.1-3 We could previously assign specific binding properties for vitamin E to afamin by means of in vitro studies.4 Afamin has been shown by suitable in vitro cell culture model systems to carry vitamin E across the blood brain barrier thus acting as an antioxidant against oxidative stress on neurons.5, 6 Afamin has been abundantly quantified not only in human plasma at concentrations of about 60 mg/L but also, at lower concentrations, in follicle and cerebrospinal fluids, suggesting possible roles of afamin for fertility and neuroprotection.7 Most recently, afamin was shown to bind and solubilize various hydrophobic, water-insoluble wnt proteins thereby maintaining their ability to activate the frizzled receptor.8 In search for the (patho)physiological functions of afamin, proteomic profiling, genetically modified mouse models and epidemiological human studies revealed low afamin concentrations in patients with cancer of reproductive organs,9-11 whereas elevated concentrations have been shown associated with prevalent and incident metabolic syndrome and other related diseases.12-15 For a detailed and comprehensive review on our current knowledge on afamin, see 16. While the strong association between afamin and several diseases with epidemic dimensions suggests a potential therapeutic target property for afamin, there is still little knowledge of afamin’s structure-function relationship. Afamin’s recently reported crystal structure will facilitate the discovery of further physiological ligands and subsequent 3


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respective protein-ligand interaction studies will allow a detailed analysis of novel potential drug transport functions and mechanisms of afamin.17 The afamin sequence completely lacks tryptophan,1 in line with its initial description as a tryptophan-poor glycoprotein.18 Afamin is structurally similar to albumin, the wellknown most abundant protein in circulating blood.1 Like other members of the albumin multigene family, afamin consists of three structural domains containing 17 Cys-Cys disulfide bridges. The difference between the calculated molecular weight for afamin of 67 kDa, deduced from its 578 amino acid sequence, and the apparent molecular weight of 87 kDa, observed after electrophoretic separation, is therefore most likely due to glycosylation.1 In contrast to glycan-free albumin, afamin has five predicted N-glycosylation sites at Asn12, Asn88, Asn362, Asn381 and Asn467 carrying an approximately 15% carbohydrate moiety.1, 7, 19

Glycan chains are known to play an important role in protein stability, intracellular folding, quality control and secretion.20, 21 More than 90% of afamin’s glycan chains consist of sialylated bi-antennary complex glycans which may interfere with protein crystallization and protein-ligand interaction studies. Afamin completely lacks O-glycosylation.7 A heterogeneous glycosylation pattern of proteins as often obtained when expressed in mammalian cells is usually a major detriment to crystallization due to lack of surface homogeneity of the molecule. Moreover, the flexibility of the long sugar moieties is entropically unfavorable for the formation of an ordered crystal lattice.22, 23 Crystal formation might be hindered if the oligosaccharide(s) shield the protein surface and prevent or reduce favorable crystal contacts. Complete or partial de-glycosylation prior to crystallization may offer the key to obtaining high quality crystals.24 Alternatively, antibody fragments which recognize and bind the protein of interest are frequently used as crystallization chaperones to 4



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facilitate the crystallization of otherwise ‘uncrystallizable’ target proteins (for comprehensive review, see 25). Therefore, we employed a multi-pronged approach involving a combination of (i) recombinantly expressed wild-type human afamin (rhAFM), (ii) rhAFM expressed in glycosylation-deficient cell lines, (iii) enzymatic deglycosylation of wild-type rhAFM, (iv) cDNA-mutated rhAFM with deleted N-linked glycosylation sites, as well as (v) complexes with Fab anti-afamin antibody fragments. A very similar approach was successfully chosen for crystallization of the ebolavirus envelope glycoprotein.26 With the tools and information regarding the afamin glycan pattern, we finally employed expression experiments to specifically answer the question whether investigate the role of afamin’s glycans are necessary for intracellular trafficking and secretion. 2. MATERIALS AND METHODS 2.1. Expression of wild-type rhAFM Wild-type rhAFM was obtained by stably transfecting the expression vector pEFneo containing C-terminally RGS-His-tagged human afamin cDNA into Chinese Hamster Ovary (CHO) cells, as previously described,7 using Lipofectamin 2000 and OPTI-MEM I according to the manufacturer’s protocol (Invitrogen Life Technologies, Carlsbad, CA). Alternatively, wild-type rhAFM was also expressed by stable transfection in Human Embryonic Kidney (HEK293T) cells using a pLenti-III-EF1α vector conferring puromycin resistance and containing the RGS-His-tagged human afamin gene controlled by elongation factor-1α (EF-1α) promotor. This custom-made vector (Applied Biological Materials Inc., Richmond, BC, Canada) contained a synthetic wild-type afamin gene. Cells were grown at 37°C in 8% CO2 with HEK293T medium (10% FCS, 1% penicillin-streptomycin, 1% glutamine, 1% non-essential amino acids, 1% non-essential amino acids, 1% sodium 5


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pyruvate, 12.5 ml of 1M HEPES buffer) and maintained for several weeks using 2 µg of puromycin for selection. After sufficient cell growth, cells were switched to serum-free CD293 medium (1% L-Glutamine, 0.2% Gentamycin, 0.1% phenol red, 0.02 % puromycin). 2.2. Expression of glycosylation-deficient human afamin Hypoglycosylated rhAFM was obtained by transfecting the glycosylation-deficient cell line CHO-Lec1 lacking the enzyme N-acetylglucosaminyltransferase I (GnTI) which is necessary for conversion of high mannose N-glycans into complex carbohydrate structures.27 This deficiency produces truncated high-mannose N-glycans of reduced size and increased homogeneity.28, 29 CHO-Lec1 cells (ATCC® CRL-1735™) were grown at 37°C with 5% CO2 in D-MEM medium containing 10% heat-inactivated fetal calf serum (FCS). Stable transfections were conducted using a pEF-neo vector containing RGS-His-tagged rhAFM controlled by elongation factor-1a (EF-1a), and conferring neomycin resistance. CHO-Lec1 cells were seeded into 12-well plates at a density of 3x105 cells per well containing 100 µl Ham’s F12 medium (supplemented with 10% FCS), 1 µl vector DNA, 3 µl Metafectene (Biontex, Germany) and 50 µg G418 as selection agent. Lastly, cells were switched to serum-free PFCHOTM LS media containing 1% gentamycin, 500 µg/ml G418, 2 mM butyric acid and 0.1% phenol red. 2.3. Enzymatic de-glycosylation of wild-type rhAFM RhAFM was expressed in HEK293T cells, affinity-purified by Ni-chelating sepharose chromatography (HisTrap excel, GE Healthcare Life Sciences, Vienna, Austria, see 2.6.) and concentrated to a final concentration of 2 mg/ml. To remove N-linked sugar residues purified rhAFM was treated with PNGase F for 4h at 30°C (see 2.3.1). The ratio of glycosidase:glycoprotein was 1:20. 6



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2.3.1. Expression and purification of GST-PNGase F The plasmid pGEX3-PNGaseF was transformed into chemically competent E. coli BL21. Bacteria were cultivated in TB medium containing 100 µg/ml ampicillin at 37°C to an optical density at 600 nm of approximately 0.6. After cooling the culture to room temperature, expression was induced with 1 mM isopropyl-I-thio-P-D-galactoside (IPTG) followed by overnight incubation at 18°C. Cells were harvested by centrifugation (5880 g, 30 min, 4°C) and a 19 g pellet was re-suspended in 100 ml buffer A (50 mM Tris-HCl, pH 8.0, 250 mM NaCl). Cells were first lysed by French Press and genomic DNA was digested by adding 300 µl DNAse (1 mg/ml) supplemented with 3 mM MgCl2. The lysate was cleared by centrifugation for 90 min at 30.000 g and 4°C). A 5 ml GST-Trap column (GE Healthcare) was equilibrated with buffer A and the cleared lysate was circulated over the column for 1.5 hours. The column was washed with 30 ml buffer A and eluted with 20 ml of buffer A supplemented with 15 mM GSH. GST-PNGase F-containing fractions were pooled and applied to size exclusion chromatography using a HiLoad Superdex 200 pg 16/600 column (GE Healthcare Life Sciences, Vienna, Austria) at a flow rate of 0.7 ml/min in buffer B (20 mM HEPES pH 7.5, 150 mM NaCl). Fractions were collected and purity of GST-PNGase F was analyzed and confirmed by SDS-PAGE. GST-PNGase F was concentrated to 6 mg/ml and subsequently used for de-glycosylation of afamin. 2.4. Expression of afamin mutated at glycosylation sites CHO cells were grown at 37°C in α-MEM medium (10% FCS, 1% penicillinstreptomycin, 1% glutamine, 1% non-essential amino acids). Transfections were conducted using a pLenti-III-EF1α vector containing RGS-His-tagged rhAFM controlled by elongation factor-1α (EF-1 α) and conferring puromycin resistance. This custom-made vector (Applied Biological Materials Inc., Richmond, BC, Canada) contained a synthetic afamin gene with 7


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residues Asn12, Asn88, Asn362, Asn381 and Asn467 mutated to the respective Asp residues to eliminate glycosylation. Cells were switched to serum-free CD-CHO medium with 1% Penicillin/Streptomycin, 0.2% Gentamycin, 500 µg/ml G418, 1% L-Glutamine, 0.1% phenol red and supernatants collected for purification. 2.5. Purification of rhAFM The purification protocol included a combination of dialysis, filtration, immobilized metal ion affinity chromatography (IMAC) as well as size exclusion chromatography (SEC) on a fast protein liquid chromatography machine (ÄKTA FPLC) using 1 ml HisTrap excel columns containing Ni-Sepharose and Superdex 200 10/300 Increase columns (GE Healthcare Life Sciences). Serum-free cell culture supernatants were dialyzed for 48 hours at 4°C in equilibration buffer containing 500 mM NaCl and 20 mM Na2HPO4 at pH 7.4, filtered using Durapore PVDF membranes with 0.45 µm pores and applied at a flow rate of 1 ml/min to the IMAC column previously equilibrated with 20 mM imidazole, 500 mM NaCl and 20 mM Na2HPO4 at pH 7.4. Unbound proteins were washed away with 25 ml equilibration buffer. Bound fractions were eluted with 10 mL elution buffer containing a 20-to-500 mM imidazole gradient in 20 mM sodium phosphate and 500 mM NaCl at pH 7.4 for 10 minutes. Fractions containing afamin were collected, pooled and analyzed by SDS-PAGE and immunoblot analysis (see 2.6.). Finally, afamin-containing fractions were polished by SEC on an FPLC machine using a Superdex 200 10/30 column (GE Healthcare Life Sciences) previously equilibrated with 20 mM Hepes, pH 7.4, 150 mM NaCl at 4oC and a flow rate of 0.5 ml/min. 2.6. Gel electrophoresis and immunoblotting Various forms of wild-type, hypo- or non-glycosylated afamin were analyzed by SDSPAGE under reducing conditions using 10% bis-acrylamide gels (Sigma Life Science). 8



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Novex Sharp pre-stained protein standard (Thermo Fisher Scientific, Waltham, MA, USA) was used as protein size marker. Samples were pre-treated for 5 minutes at 95oC and electrophoresis run for 1 hour at 150 V in a Mini-PROTEAN Tetra vertical Cell (Bio-Rad Laboratories, Hercules, CA, USA). Protein bands were visualized by Coomassie staining using Serva Blue G.30 Immunoblotting was performed at room temperature applying a constant electric current of 1 A for 30 minutes in a Trans-Blot Turbo semi-dry blotting transfer system (Biorad) using Amersham 0.45 µm Nitrocellulose membranes (GE Healthcare Life Sciences) previously blocked by incubation with 10 % skim milk in PBS/0.5% NP-40 for 30 minutes at room temperature followed by overnight incubation at 4°C with mouse monoclonal antihuman-afamin antibody N13, diluted 1:3000 in 10% skim milk. The second antibody, antimouse IgG raised in goats and horse-radish-peroxidase-conjugated, was incubated, diluted 1:1000 in 10% skim milk, for 1 hour at room temperature. Afamin-specific bands were visualized using WesternBright ECL Spray (Advansta, Menlo Park, CA, USA). 2.7. Glycan analysis N-glycans were released from the samples by in situ digestion with N-glycosidase F (PNGase F, Prozyme) of in-gel-blocks as described earlier.31 Purified afamin samples were reduced and alkylated, set into SDS-gel blocks, washed and N-glycans were released by PNGase F. Glycans were then fluorescently labelled with 2-aminobenzamide (2AB) by reductive amination32 (LudgerTag 2-AB labeling kit, LudgerLtd., Abingdon, UK). All enzymes were purchased from Prozyme or New England Biolabs (NEB). The 2AB-labelled glycans were digested in a volume of 10 µl for 18 h at 37°C in 50 mM sodium acetate buffer, pH 5.5, (except in the case of jack bean α-mannosidase (JBM) where the buffer was 100 mM sodium acetate, 2 mM Zn2+, pH 5.0), using arrays of the following enzymes: ABS 9


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Arthrobacter ureafaciens sialidase (EC 3.2.1.18), 0.5 U/ml; NAN1-Streptococcus pneumoniae sialidase (EC 3.2.1.18), 1 U/mL; BTG - bovine testes β-galactosidase (EC 3.2.1.23), 1 U/ml; Streptococcus pneumoniae β-galactosidase (SPG, EC 3.2.1.23), 0.4 U/mL; BKF – bovine kidney α-fucosidase (EC 3.2.1.51), 1 U/mL; AMF - Almond meal α-fucosidase (EC 3.2.1.111), 0.4 mU/ ml; GUH – β-N-acetylglucosaminidase cloned from S. pneumonia, expressed in Escherichia coli (EC 3.2.1.30), 8 U/mL (Prozyme) or 400 U/mL (NEB) and JBM (EC 3.2.1.24), 60 U/ mL. After incubation, enzymes were removed by filtration through a protein binding EZ filters (Millipore Corporation, Beford, MA, USA),31 the N-glycans were then analyzed by hydrophilic interaction liquid chromatography (HILIC). HILIC was performed using a TSK-Gel Amide-80 4.6 x 150 mm column (Anachem, Luton, UK) on a 2695 Alliance separations module (Waters, Milford, MA) equipped with a Waters temperature control module and a Waters 2475 fluorescence detector. Solvent A was 50 mM formic acid adjusted to pH 4.4 with ammonia solution. Solvent B was acetonitrile. The column temperature was set to 30°C. Conditions used were as follows (60-min method): a linear gradient of 35 to 47% solvent A over 48 min at a flow rate of 0.4 ml/min, followed by 1 min at 47 to 100% A and 4 min at 100% A, returning to 35% A over 1 min and then finishing with 35% A for 6 min.33 Fluorescence was measured at 420 nm with excitation at 330 nm. The system was calibrated using an external standard of hydrolyzed and 2AB-labeled glucose oligomers to create a dextran ladder, as described previously.31 N-glycans from afamin preparations were also analyzed by mass spectrometry. CHO Lec1 afamin N-glycan spectra were generated using a MALDI-TOF mass spectrometer (Waters, Manchester, UK) equipped with an ultraviolet 337 nm wavelength nitrogen laser. Samples were diluted in 25 µL HPLC grade H2O and 10 µL of methanol and vortexed. 3 µL of reconstituted sample solution was mixed with 1 µL of 2,5-dihydroxybenzoic acid matrix (10 mg/mL in 50% (v/v) acetonitrile aqueous solution) on a stainless steel MALDI target 10



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plate. The sample/matrix mixture was then recrystallized with 0.7 µL ethanol. All samples were ionized with 50-60% of the maximum power while rastering on the plate surface. The acceleration voltage was 20 kV; the pulse voltage was 1980 V; the delay for the delayed extraction ion source was 500 ns. Spectra processing was performed with Masslynx software. Ions were observed as [M+Na]+ adducts. Nano-electrospray mass spectrometry was performed with a Waters-Micromass quadrupole-time-of-flight (Q-Tof) Ultima Global instrument. Samples in 1:1 (v:v) methanol:water containing 0.5 mM ammonium phosphate were infused through Proxeon (Proxeon Biosystems, Odense, Denmark) nanospray capillaries. The ion source conditions were: temperature, 120oC; nitrogen flow 50 L/hr; infusion needle potential, 1.2 kV; cone voltage 100 V; RF-1 voltage 150 V. Spectra (2 sec scans) were acquired with a digitization rate of 4 GHz and accumulated until a satisfactory signal/noise ratio had been obtained. For MS/MS data acquisition, the parent ion was selected at low resolution (about 4 m/z mass window) to allow transmission of isotope peaks and fragmented with argon. The voltage on the collision cell was adjusted with mass and charge to give an even distribution of fragment ions across the mass scale. Typical values were 80-120 V. HEK afamin preparations were analyzed by UPLC-FLR-QTOF MS. The samples were dried down and reconstituted in 3 µL of water and 9 µL acetonitrile. Online coupled fluorescence (FLR)-mass spectrometry detection was performed using a Waters Xevo G2 QTof (YCA 219) with Acquity UPLC (Waters Corporation, Milford, MA, USA) and BEH Glycan column (1.0 x 150mm, 1.7 µm particle size). For MS acquisition data, the instrument was operated in negative-sensitivity mode with a capillary voltage of 1.80kV. The ion source block and nitrogen desolvation gas temperatures were set at 120 °C and 400 °C, respectively. The desolvation gas was set to a flow rate of 600 L/h. The cone voltage was maintained at 11


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50V. Full-scan data for glycans were acquired over m/z range of 450 to 2500. Data collection and processing were controlled by MassLynx 4.1 software (Waters Corporation, Milford, MA, USA). Fluorescence was measured at 420 nm with excitation at 330 nm; data rate was 1pts/s and a PMT gain = 10. Sample injection volume was 8 µL. The flow rate was 0.150 mL/min and column temperature was maintained at 60 °C; solvent A was 50 mM ammonium formate in water (pH 4.4) and solvent B was acetonitrile. A 40-min linear gradient was used and was as follows: 28% A for 1 min, 28-43% A for 30 min, 43-70 % A for 1 min, 70 % A for 3 min, 70-28% solvent A for 1 min and finally 28 % A for 4 min. Samples were diluted in 75% acetonitrile prior to analysis. The weak wash solvent was 80% acetonitrile and the strong wash solvent was 20% acetonitrile. To avoid contamination of the MS system, the flow was sent to waste for the first 1.2 min and again after 32 min. The following glycan nomenclature was used throughout all analyses: all N-glycans have two core N-acetylglucosamines (GlcNAc) and a tri-mannosyl core; F at the start of the abbreviation indicates a core fucose linked α1–6 to the core GlcNAc; A[y]a, represents the number a of antenna (GlcNAc) on the trimannosyl core linked to the α1-y mannose arm; B, bisecting GlcNAc linked β1–4 to β1–4 core mannose; Fb after Aa, represents the number b of fucose linked α1–3 to antenna GlcNAc; Gc, represents the number c of galactose linked β1-4 on antenna; S(z)d, represents number d of sialic acids linked α2-z to the galactose; Lac(x), number (x) of lactosamine (Galβ1-4GlcNAc) extensions. 2.8. Complex formation between rhAFM and anti-afamin Fab fragment 2.8.1. Production of Fab fragments from monoclonal anti-afamin antibody Monoclonal antibody N14 against human afamin was obtained with conventional hybridoma technology34 by immunizing Balb/c mice with purified human afamin dissolved in phosphate buffered saline (PBS) solution, pH 7.4, as described in the supplemental material section of Dieplinger et al.35 12



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Affinity-purified mouse monoclonal antibody N14 was concentrated to 2 mg/ml in PBS and cleaved into Fab and Fc fragments according to previously described protocols.36 In brief, immunoglobulins were dissolved in equal volumes of freshly prepared 2x digestion buffer (0.035 M EDTA, 0.04 M L-cystein in PBS). Papain (0.1 mg/ml) was also freshly prepared in 2x digestion buffer and equal volume of antibody and papain were mixed and incubated for 2 hours at 37oC. The reaction was stopped by adding iodoacteamide at a final concentration of 30 mM. Fab fragments were separated from Fc fragments which left remaining un-cleaved immunoglobulins on Äkta HPLC equipped with a Protein A sepharose column. Fab fragments were concentrated from flow-through fractions in PBS using centrifugal filter concentrators using a MW cut-off of 10 kDa. Papain was removed by size exclusion chromatography (SEC) using a Superdex 200-10/300 column on an Äkta purifier 100 FPLC system with a flow rate of 0.5 ml/min (SEC buffer: 20 mM HEPES pH 7.5, 150 mM NaCl). The Fab solution was concentrated with a centrifugal filter concentrator (Vivaspin VS2021, 30 kDa cut-off) to a final concentration of 10 mg/ml. The purity of the Fab fragment was assessed by Coomassie-stained SDS PAGE. 2.8.2. Complex formation with rhAFM and subsequent purification by SEC Four mg of rhAFM (purified from transfected HEK293T cell culture supernatants, as described in 2.1. and 2.5., dissolved in 17 ml PBS) were incubated for 30 mins in a screwcapped glass bottle at room temperature under gentle shaking with 4 mg purified Fab fragments (dissolved in 37 ml PBS) obtained by papain cleavage of mouse monoclonal antiafamin antibody N14 as described above. Afamin-Fab complexes were then concentrated by centrifugal filter concentrator and purified with SEC, as described in 2.5. 3. RESULTS AND DISCUSSION 3.1. Expression, purification and characterization of wild-type, hypo- and nonglycosylated afamin 13


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Cell culture supernatants were collected from the respective transfected cell lines and purified via dialysis and IMAC to obtain sufficient amounts of rhAFM. Subsequently, SDSPAGE under reducing conditions was conducted to generate a qualitative comparison between the various methods of rhAFM de-glycosylation (Fig. 1A). RhAFM expression in wild-type CHO cells, at an apparent molecular weight of approximately 90 kDa, was considered reference standard. RhAFM expression in CHO-Lec1 cells appeared at a considerably lower molecular weight at an apparent molecular weight of approximately 70 kDa indicating a reduction in glycan content. RhAFM treated with PNGase F also appeared at a slightly lower molecular weight at approximately 75 kDa indicating incomplete enzymatic de-glycosylation with a clear reduction in glycan material. rhAFM expression in HEK293T cells resulted in a slightly lower molecular weight compared to rhAFM expressed in CHO cells suggesting qualitative differences in glycosylation between expression lines originating from human kidney and hamster ovaries. These observed size difference were further investigated by glycan analysis (see 3.2). As expected, cDNA-mutated AFM exhibited the lowest molecular weight band at around 64 kDa. Since the N-glycosylation sites were mutated, there is no indication of glycan material on the protein, therefore explaining the lower molecular weight bands compared to those seen in CHO-Lec1 and PNGase treated rhAFM exhibiting partially glycosylated proteins. Coomassie-stained gels suggested that afamin preparations were pure enough to proceed with glycan analysis and ultimately, crystallization trials. Next, afamin was identified by immunoblotting using anti-afamin antibody N13 (Fig 1B). A)

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Figure 1. Coomassie-stained (A) and immunoblotted (B) SDS-PAGE of various forms of hypo- and de-glycosylated afamin in comparison to wild-type afamin. Left- and right-most lanes indicate molecular size markers (MSM) for molecular size evaluation of afamin species. Lane 1 indicates CHO-expressed afamin, lane 2, afamin expressed in glycosylation-deficient CHO-Lec1 cells, lane 3, PNGase-treated rhAFM, lane 4, glycosylation-free mutant rhAFM expressed in HEK293T cells, and lane 5, wild-type rhAFM expressed in HEK293T cells. Immunoblotting was performed using the afamin-specific monoclonal antibody N13. 15


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3.2. Glycan analysis of various rhAFM forms Glycan analysis was necessary for evaluation of the glycan complexity level of the variously expressed afamin forms. As expected, all results yielded different traces as seen in Fig. 2. Afamin from human plasma contained mostly biantennary digalactosylated disialylated and monosialylated glycans, other glycans were bi- and tri-antennary, mono-, diand trigalactosylated and mono-, di- and tri-sialylated with some outer arm fucosylated.7 Afamin expressed in HEK293T cells contained mostly core fucosylated biantennary digalactosylated bisected and core fucosylated triantennary digalactosylated glycans, other glycans were core-fucosylated, mono-, di-, tri- and tetra-antennary, non-, mono-, di-, tri- and tetragalactosylated, neutral, mono-, di- and trisialylated, some with polylactosamine extensions and some bisected glycans (Table 1). Afamin expressed in HEK293T cells and treated with PNGase F had lower amounts of glycans than untreated afamin, though they were not completely removed. Afamin expressed in CHO cells contained mostly core-fucosylated biantennary digalactosylated monosialylated glycans, while other glycans were mostly corefucosylated, mono-, bi-, tri- and tetra-antennary, non-, mono-, di-, tri- and tetragalactosylated and neutral, mono- and disialylated, some bisected and high-mannosylated glycans (Table 1). Afamin expressed in CHO-Lec1 cells contained mostly high-mannosylated and hybrid glycans, other glycans were bi-and triantennary, mono-, di-, trigalactosylated, neutral, mono-, di-and trisialylated (Table 1). CHO Lec1 has decreased activity of GlcNAcT-I; therefore increased high-mannosylated glycans compared to CHO were expected.37 Finally, cDNA mutated afamin showed no peaks indicating complete removal of glycans.

16



Page 18 of 31

A2G2S2

A2G2S1

Human plasma FA2BG2/FA3G2

HEK 1 2 3 45678 91011121314 16 18 20 21 23 252627 28 293031 15 17 19 22 24

FA2BG2/FA3G2

HEK-PNGase F digested 1 2 345678 9 1011121314 16 18 20 21 23 252627 28 293031 15 17 19 22 24

Mutant HEK FA2G2S[6]1

FA2G2S[3]1

CHO 1

2 3 4 5 6 78 91011 121314 15 16 1718 19

M5/M4A1

CHO Lec 1 1

2

3

4

5

6

7 8 9 10 11

Blank 4

5

6

7

8

9

10

11

12 GU

Fig. 2. HILIC chromatograms of released N-glycans from human plasma afamin (taken from previous work7), wild-type rhAFM expressed in CHO or HEK cells, and the different forms of hypo-glycosylated as well as fully glycan-free mutant rhAFM. Glucose units suggest the magnitude of complexity of the glycan chains. Peaks are scaled to the highest one in the particular chromatogram. Main N-glycans are shown and all assigned N-glycans from wildtype HEK, CHO and CHO Lec1 are presented in Table 1. 3.3. Characterization of Fab-rhAFM complexes Wild-type rhAFM was incubated with Fab fragments from monoclonal antibody N14. The formed antigen-antibody complex was further purified by size exclusion chromatography to separate complexes from un-complexed Fab N14 and rhAFM (Fig. 3). A)

17


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B)

Fig. 3. A) Elution profile of size exclusion chromatography (SEC) of a rhAFM/N14 Fab complex using FPLC (blue line). Elution profile of unbound afamin (green line) corresponds to a protein size of 67 kD, whereas unbound fab fragments correspond to a protein size of 30 kD. B) Coomassie-stained SDS-PAGE, run under reducing conditions, identified proteins eluting at fractions A11-A14 as rhAFM/Fab complex and at fractions B1-B5 as unbound Fab

18



Page 20 of 31

fractions. First lane: molecular weight standard, second lane: loaded afamin/fab mixture before SEC. The crystal structure of a Fab fragment of N14 had recently been described by Naschberger et al.38 The authors identified a rare N-glycosylation site at the binding loop of the variable region of Fab N14. It will be interesting to structurally investigate Fab-afamin complexes whether these rare glycans play a functional role in antibody binding to the respective epitopes. 4. CONCLUSIONS Several attempts to express various recombinant forms of wild-type, hypo- and nonglycosylated human afamin where conducted in this work. Three different approaches were pursued for obtaining hypo- and de-glycosylated afamin. First, partial deglycosylation of rhAFM was obtained using a glycosylation-deficient cell line (CHO-Lec1) lacking the enzyme N-acetylglucosaminyltransferase I (GnTI), which is necessary for conversion of high mannose N-glycans into complex glycan structures. This deficiency produces truncated highmannose N-glycans of reduced size and increased homogeneity.28, 29 It is assumed that such truncated glycosylation could improve structural microheterogeneity possibly leading to the successful growth of afamin crystals. Second, hypo-glycosylated rhAFM was also obtained by enzymatic removal using peptide-N-Glycosidase F (PNGase F). PNGase catalyzes the cleavage of N-linked oligosaccharides between the innermost N-acetylglucosamine and asparagine residues. Consequently, accessible glycans are removed, yet inaccessible glycans may remain. Third, fully non-glycosylated rhAFM was obtained by cDNA mutated rhAFM where all asparagines involved in coding for N-glycosylation were replaced with aspartic acid. This cDNA was virally transfected and expressed in both CHO and HEK293T cells. By inducing such chemical changes to rhAFM, our goal was to express deglycosylated rhAFM in 19


Page 21 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


sufficient quantities in order to determine the effect that glycosylation has on expression and secretion. Our results indicate that afamin expressed in CHO-Lec1 cells and PNGase F-treated afamin yielded only partially (hypo)-glycosylated products. The degree of hypo-glycosylation of afamin expressed in CHO-Lec1 cells were higher than that obtained after enzymatic deglycosylation, thereby confirming results from the glycan analysis. These results are most likely due to PNGase F’s limited accessibility to glycosylation sites for to complete removal of glycan moieties of afamin. Various de-glycosylation experiments led to pertinent insight regarding the function of the carbohydrate moieties of afamin. Partially and fully de-glycosylated, recombinantly expressed afamin were able to be secreted from their respective host cell lines confirming that glycosylation of afamin is obviously not mandatory for intracellular trafficking and secretion. Many secretory proteins possess more or less substantial amounts of carbohydrates and are therefore called glycoproteins. These glycan moieties have multiple intracellular functions generally referred to as “quality control” consisting of regulating proper folding, sorting, and signaling to mention the most important ones. However, not all secretory proteins are glycoproteins and genetically or enzymatically de-glycosylated glycoproteins have been shown to fully retain their secretory properties (see review39). According to our results, afamin seems to be one of those few proteins whose large glycan moiety is obviously not necessary for proper intracellular folding, processing and final secretion. The function of the glycan moiety of afamin is thus currently completely unknown. Although highly speculative, afamin’s glycan decoration could interfere with its postulated binding and chaperone properties to members of the wnt signaling family.8 Further structurefunction studies will be required to investigate this exciting hypothesis. 20



Page 22 of 31

ACKNOWLEDGMENTS We thank G. Baier for helpful discussions and L. Fineder and R. Berberich for excellent technical assistance. The pGEX3-PNGaseF plasmid was provided by Dr. Joel Sussman, Weizmann Institute of Science, Rehovot, Israel. This work has been financially supported by grants from the Austrian Science Funds (P28395-B26) to B.R. and the European Union FP7 Marie Curie People Action [PIIF-GA-2011-300025 (SAXCESS)] to H.D. R.S. acknowledges funding from the European Union Seventh Framework Programme (FP7/2007747 2013) under grant agreement number 260600 (GlycoHIT) and funding from the Science Foundation Ireland Starting Investigator Research grant (SFI SIRG) under grant number 13/SIRG/2164. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have declared that no competing interests exist. REFERENCES 1.

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Table 1 Glycosylation analysis of afamin expressed in CHO, CHO-Lec1 and HEK cells

CHO afamin Peak

CHO lec1 afamin

GU % Area Structure

Peak

GU % Area Structure

1

4,90

0,67 A1

1

6,28

33,76 *M5, M4A1

2

5,34

0,51 FA1

2

6,75

7,27 FA2G1

3

5,65

0,57 A2B

3

7,16

5,99 *A2G2

4

5,80

1,22 FA2

4

7,73

9,49 *A3G2/FA2G2

5

6,16

3,04 M5, FA1G1

5

8,12

3,09 A2G2S(6)1

6

6,36

0,86 A3B

6

8,58

6,36 *A3G3

7

6,62

0,52 FA2G[6]1

7

9,12

6,64 *A2G2S(3,6)2

8

6,75

1,64 FA2G[3]1

8

9,41

8,56 *A2G2S(6,6)2

9

7,07

2,93 M6/A2G2

9

9,81

7,91 *A3G3S(3,6)2

10

7,23

3,65 FA2G1S1

10 10,28

6,24 *A3G3S(3,3,6)3

11

7,57

5,80 A2G2S1, FA2G2

11 10,68

4,67 *A3G3S(3,6,6)3

12

8,00

13

8,13

14

8,51

15

8,96

4,88 FA3BG3

16

9,26

3,27 A3BG3S2

17

9,64

3,16 FA3BG3S(3)1

18,69 FA2G2S(3)1/M7D3 2,05 M7D1 41,48 FA2G2S(6)1

28



18

9,76

19

10,17

Page 30 of 31

1,26 FA3BG3S(6)1 3,81 FA4G4

HEK293T afamin A) wild-type and B) partially digested with PNGase F A Peak

B

GU

Structure % Area

% Area

1

5,84

0,56

0,23 *FA2, *FA1B

2

6,20

2,16

0,48 *FA2B, *FA3

3

6,51

0,32

0,08 *FA1BG1

4

6,65

0,39

0,10 *FA2[6]G1

5

6,80

0,53

0,12 *FA2[3]G1

6

6,92

4,97

1,36 *FA2[6]G1, *FA2B[6]G1, *FA4

7

7,06

2,74

0,41 *FA2[3]G1, *FA2B[3]G1

8

7,26

1,29

0,51 FA3B[6]G1

9

7,45

0,83

0,31 FA3B[3]G1

10

7,63

2,09

1,10 *FA2G2

11

7,77

16,62

12

8,06

6,72

5,19 *FA2G2S1

13

8,19

4,85

2,00 *FA2G1Lac1, *FA2BG2S1, *FA3G2S1

14

8,54

8,57

6,07 *FA1G1Lac1S1

15

8,76

5,59

4,92 *FA2G2S2, *FA3BG2S1, *FA2G1Lac1S1

9,90 *FA2BG2, *FA3G2, *FA2BG1S1,*FA3G1S1, *FA1G1Lac1

29


Page 31 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


16

8,91

1,11

1,25 *FA3G3

17

9,00

3,03

3,12 *FA2BG2S2

18

9,19

6,00

7,02 *FA2G2Lac1, *FA3G2S2

19

9,39

2,83

3,06 *FA3BG3, *FA4G3, *FA3G2Lac1

20

9,59

4,79

5,59 *FA3G3S1

21

9,97

2,82

3,71 *FA3BG3S1, *FA2G2Lac1S1

22 10,14

2,16

3,37 *FA4G3S1

23 10,32

1,60

2,58 *FA3G3S2

24 10,50

2,63

5,52 *FA3BG3S2

25 10,69

3,35

6,41 *FA4G3S2, *FA2G2Lac1S2

26 10,87

3,75

7,14 *FA4G4S1, *FA3G3S3, *FA3G3Lac1S1, *FA3G2Lac1S2

27 11,23

3,05

6,33 *FA4BG4S1

28 11,63

3,06

7,39 *FA4G4S2, *FA3G3Lac1S2

29 12,02

1,18

3,65 *FA4BG4S2

30 12,29

0,26

0,64 *FA4G4S3

31 12,63

0,14

0,40 *FA3G3Lac1S3

All sialic acids without specific linkage indicated are linked both α2-3 and α2-6. *Compositions corresponding to these assignments were identified by mass spectrometry.

30



Page 32 of 31

ToC - Graphic

Various recombinantly expressed forms of afamin – purified from culture supernatants

A2G2S2

A2G2S1

Human plasma FA2BG2/FA3G2

Wild-type HEK293T cells

HEK 1

2 3 456 78 9 1011121314 16 18 20 21 23 25262 7 28 29303 1 15 17 19 22 24

FA2BG2/FA3G2

HEK293T cells – PNGase-digested

HEK-PNGase F digested 1

2 3 456 78 9 1011121314 16 18 20 21 23 25262 7 28 293031 15 17 19 22 24

HEK293T cells – afamin glycosylation sites mutated

Mutant HEK FA2G2S[6]1

FA2G2S[3]1

Wild-type CHO cells

CHO 1

2 3 4 5 6 7 8 9 1011 12 1314 15 16 171 8 19

M5/M4A1

CHO cells – glycosylation-deficient

CHO Lec 1 1

2

3

4

5

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Glycan analysis of various recombinantly expressed forms of afamin compared to afamin from human plasma

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Expression, Purification, and Biochemical Characterization of Human

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