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Protein expression and purification procedures
Vector: pFB-LIC-Bse (available from The Addgene Nonprofit Plasmid Repository)
Cell line: DH10Bac, Sf9 cells
Tags and additions: N-terminal, 6-His, TEV protease cleavable tag
Wild-type sequence:
MGHHHHHHSSGVDLGTENLYFQSMWAFSELPMPLLINLIVSLLGFVATVTLIPAFRGHFIAARLCGQDLNKTSRQQIPESQGVISGAVFLIILFCFIPFPFLNCFVKEQCKAFPHHEFVALIGALLAICCMIFLGFADDVLNLRWRHKLLLPTAASLPLLMVYFTNFGNTTIVVPKPFRPILGLHLDLGILYYVYMGLLAVFCTNAINILAGINGLEAGQSLVISASIIVFNLVELEGDCRDDHVFSLYFMIPFFFTTLGLLYHNWYPSRVFVGDTFCYFAGMTFAVVGILGHFSKTMLLFFMPQVFNFLYSLPQLLHIIPCPRHRIPRLNIKTGKLEMSYSKFKTKSLSFLGTFILKVAESLQLVTVHQSETEDGEFTECNNMTLINLLLKVLGPIHERNLTLLLLLLQILGSAITFSIRYQLVRLFYDV
The Val264Gly mutant:
MGHHHHHHSSGVDLGTENLYFQSMWAFSELPMPLLINLIVSLLGFVATVTLIPAFRGHFIAARLCGQDLNKTSRQQIPESQGVISGAVFLIILFCFIPFPFLNCFVKEQCKAFPHHEFVALIGALLAICCMIFLGFADDVLNLRWRHKLLLPTAASLPLLMVYFTNFGNTTIVVPKPFRPILGLHLDLGILYYVYMGLLAVFCTNAINILAGINGLEAGQSLVISASIIVFNLVELEGDCRDDHVFSLYFMIPFFFTTLGLLYHNWYPSRVFVGDTFCYFAGMTFAGVGILGHFSKTMLLFFMPQVFNFLYSLPQLLHIIPCPRHRIPRLNIKTGKLEMSYSKFKTKSLSFLGTFILKVAESLQLVTVHQSETEDGEFTECNNMTLINLLLKVLGPIHERNLTLLLLLLQILGSAITFSIRYQLVRLFYDV
Expression: The full length gene for human DPAGT1 was obtained from the Mammalian gene collection and the Val264Gly mutant gene was obtained from Prof. David Beeson, WIMM, Oxford. This gene was cloned into the pFB-LIC-Bse vector and baculoviruses were produced by transformation of DH10Bac cells. Spodoptera frugiperda (Sf9) insect cells in Sf-900 II SFM medium (Life Technologies) were infected with recombinant baculovirus and incubated for 65 h at 27°C in shaker flasks.
Cell Lysis and detergent extraction of membrane proteins:
Extraction Buffer, EXB: 50 mM HEPES (pH 7.5), 5mM MgCl2, 200 mM NaCl, 5mM Imidazole, 2mM TCEP, 5% Glycerol, Roche protease inhibitors
The cell pellet from 1 litre of insect cell culture was resuspended in 40ml of lysis buffer in warm water, mixing constantly to keep the sample cold. Cells were lysed with a EmulsiFlex-C3 homogenizer (Aventin) (at room temperature, 2 passes.) 5 mL of 10%:1% (w:w) stock of OGNG/CHS was added per litre of cell culture and the volume was adjusted by the addition of EXB to a final volume of 50 mL/L of cell culture. The sample was rotated in the cold room for 1h, then unlysed cells and cell debris was removed by centrifugation at 35,000g for 45 min at 4oC.
Column 1: Co2+ talon resin (0.5 ml volume in a gravity-flow column):
Wash Buffer: 50 mM HEPES (pH 7.5), 5mM MgCl2, 10 mM imidazole (pH 8.0), 200 mM NaCl , 2 mM TCEP, 5 % Glycerol, 0.18 % OGNG, 0.018 % CHS, 0.0036 % cardiolipin
Elution Buffer: 50 mM HEPES (pH 7.5), 5 mM MgCl2, 250 mM imidazole (pH 8.0) , 200 mM NaCl, 2 mM TCEP, 5% Glycerol, 0.18 % OGNG, 0.018 % CHS, 0.0036 % cardiolipin
SEC Buffer: 20 mM HEPES (pH 7.5) , 5mM MgCl2, 200 mM NaCl, 2 mM TCEP, 0.12 % OGNG, 0.012 % CHS, 0.0024 % cardiolipin
The detergent-extracted membrane protein from each litre of cells was combined with 1ml of pre-equilibrated slurry of 50% Co2+ talon resin (previously washed 2 times with H2O and 4 times with EXB). The sample was rotated in the cold room for 1h then poured onto an econo column. The residual talon resin was washed with 5ml wash buffer and pipetted onto the econo column. The talon resin was washed with 30x talon resin volume of WB and the protein eluted with 2x Talon resin volume of elution buffer in 1ml fractions. The peak fractions were combined and the imidazole removed using two PD10 columns per purification (pre-equilibrated with SEC buffer). Protein was eluted from the PD10 columns using SEC buffer.
TEV protease cleavage and reverse purification
TEV protease was added at a ratio of 10:1 (DPAGT1:TEV protease (w:w)). Samples were incubated on ice in the cold room overnight. 2 mL of a 50 % slurry of Talon resin (pre-equilibrated as above) were added and the sample was rotated in the cold room for an hour, poured into an econo column and the flowthrough collected and centrifuged at 21,500 rpm in a Beckman JA25.5 rotor for 5 min at 4°C. The supernatant was removed and kept.
Column 2: Superdex 200 Size Exclusion Chromatography
The protein sample was concentrated in a 30 kDa PES concentrator (pre-equilibrated with SEC buffer) and concentrated, with mixing every 5 mins, to a final volume of 500 μL. After centrifugation at 20,000 g for 10 min the sample was subjected to size exclusion chromatography on a Superdex 200 column in SEC buffer. DPAGT1 protein was then concentrated with a Sartorius 2ml PES 50 kDa concentrator pre-equilibrated with SEC buffer (without detergent), at 3,220 g, with mixing every 5 mins. The protein was centrifuged at 20,000 g for 15 mins, then flash frozen in liquid nitrogen. The final concentration was ~30 mg/mL.
Assay protocols
Thermostability using the tryptophan fluorescence as readout
Samples with a volume of 40 μL were prepared containing 0.5 mg/mL protein and 50 μM compound or 5% DMSO in SEC buffer and a glass capillary was dipped into each sample, with the capillary held horizontally to ensure that it was full of the sample. The capillaries were placed on the capillary holder on the Prometheus. Triplicates of each sample were prepared. A discovery scan was performed to ensure that the values were roughly the same for all the samples. A melting curve from 20˚C to 95˚C at 5˚C/min was performed. The minimum of the first derivative of the 330/350 nm ratio was used to determine the Tm1/2.
Activity assays using radiolabelled 14C UDP-GlcNAc
2 μL of 2 μM protein in SEC buffer supplemented with 5mM extra MgCl2 and 1% OGNG/CHS/cardiolipin and dolichyl monophosphate was combined with 2 μL of UDP-N-acetyl [1-14C] D-glucosamine in the same buffer and incubated at 37 °C for 21 min. The reaction was terminated by the addition of 6 μl of 100% methanol and immediately transferred onto ice. 1 μL of sample is spotted onto a silica coated TLC plate in triplicate and run with a mobile phase consisting of choloform, methanol, and water at a 65:25:4 relative ratios. After running the TLC plate was thoroughly dried, wrapped in cling film, incubated with a phosphor imaging substrate for 4 days and phosphor was then imaged. The pixel density of the spot corresponding to the hydrophobic product is divided by combined pixel density of the product and the substrate and multiplied by the known concentration of substrate added to ascertain the concentration of product formed.
Structure determination procedures
All data were collected at Diamond Light Source (beamlines I24, I04-1 and I04) to resolutions between 3.2-3.6 Å. Data were processed, reduced and scaled using XDS and AIMLESS.
The crystals belong to space group P6522 and contain a single DPAGT1 monomer in the asymmetric unit with 70 % solvent. Initial phase estimates were obtained using molecular replacement (MR). Briefly, an initial search model was built automatically using the PHYRE server based on the coordinates of the bacterial homolog MraY (23 % sequence identity; PDB: 4J72). However, this simple homology model failed to produce any meaningful MR solutions when used in isolation in PHASER. The PHYRE model was then subjected to model pre-refinement using the procedures implemented in MR-ROSETTA in PHENIX and the resultant five best-scoring output models were trimmed at their termini and the TM9/TM10 cytoplasmic loop region and superposed for use as an ensemble search model in PHASER. A marginal but consistent solution was obtained that exhibited sensible crystal packing in space group P6522 but both the initial maps and model refinement were inconclusive. The model positioned using PHASER was converted to a poly-alanine trace and recycled into MR-ROSETTA, using model_already_placed=True option. The resultant MR-ROSETTA output model had an R/Rfree of 42/46 and the electron density maps showed new features not present in the input coordinates that indicated that the structure had been successfully phased.
Using the MR-ROSETTA solution as a starting point, the remaining regions of the DPAGT1 structure could be built manually using COOT and the wildtype 3.6 Å native data. However, the novel 54 amino acid cytoplasmic insertion domain between TM9 and TM10 was poorly ordered and proved difficult to trace. This region was primarily traced using the electron density maps for a UDP-GlcNAc complex as substrate binding results in partial stabilisation of the TM9/10 insertion domain. Sequence assignment was aided by using the sulphur anomalous signal from a dataset collected at a wavelength of 1.7 Å. Anomalous difference maps combined with anomalous substructure completion using PHASER-EP clearly revealed the location of 18 of the expected 22 sulphur positions and helped to confirm the sequence register. Additional experimental phasing information was provided by a Pr3+ derivative. The resultant model for the entire chain was then refined against both the apo V264G (3.2 Å) and V264G tunicamycin (3.4 Å). The representative final model comprises the entire polypeptide chain between residues Leu7 and Gln400 apart from a flexible loop connecting TM2 and TM3 and part of the poorly-ordered lumenal hairpin (152-161).
- Belaya, K., Finlayson, S., Slater, C. R., Cossins, J., Liu, W. W., Maxwell, S., McGowan, S. J., Maslau, S., Twigg, S. R., Walls, T. J., Pascual Pascual, S. I., Palace, J., and Beeson, D. (2012) Mutations in DPAGT1 cause a limb-girdle congenital myasthenic syndrome with tubular aggregates. Am J Hum Genet 91, 193-201
- Wu, X., Rush, J. S., Karaoglu, D., Krasnewich, D., Lubinsky, M. S., Waechter, C. J., Gilmore, R., and Freeze, H. H. (2003) Deficiency of UDP-GlcNAc:Dolichol Phosphate N-Acetylglucosamine-1 Phosphate Transferase (DPAGT1) causes a novel congenital disorder of Glycosylation Type Ij. Hum Mutat 22, 144-150
- Wurde, A. E., Reunert, J., Rust, S., Hertzberg, C., Haverkamper, S., Nurnberg, G., Nurnberg, P., Lehle, L., Rossi, R., and Marquardt, T. (2012) Congenital disorder of glycosylation type Ij (CDG-Ij, DPAGT1-CDG): extending the clinical and molecular spectrum of a rare disease. Mol Genet Metab 105, 634-641
- Iqbal, Z., Shahzad, M., Vissers, L. E., van Scherpenzeel, M., Gilissen, C., Razzaq, A., Zahoor, M. Y., Khan, S. N., Kleefstra, T., Veltman, J. A., de Brouwer, A. P., Lefeber, D. J., van Bokhoven, H., and Riazuddin, S. (2013) A compound heterozygous mutation in DPAGT1 results in a congenital disorder of glycosylation with a relatively mild phenotype. Eur J Hum Genet 21, 844-849
- Thiel, C., and Korner, C. (2011) Mouse models for congenital disorders of glycosylation. J Inherit Metab Dis 34, 879-889
- Varelas, X., Bouchie, M. P., and Kukuruzinska, M. A. (2014) Protein N-glycosylation in oral cancer: dysregulated cellular networks among DPAGT1, E-cadherin adhesion and canonical Wnt signaling. Glycobiology 24, 579-591
- Chung, B. C., Zhao, J., Gillespie, R. A., Kwon, D. Y., Guan, Z., Hong, J., Zhou, P., and Lee, S. Y. (2013) Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis. Science 341, 1012-1016
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