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Apr 2019

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In vitro Measurement of CMP-Sialic Acid Transporter Activity in Reconstituted Proteoliposomes
重组蛋白脂质体中CMP-唾液酸转运体活性的体外检测   

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Abstract

Nucleotide-sugar transporters (NSTs) facilitate eukaryotic cellular glycosylation by transporting nucleotide-sugar conjugates into the Golgi lumen and endoplasmic reticulum for use by glycosyltransferases, while also transferring nucleotide monophosphate byproducts to the cytoplasm. Mutations in this family of proteins can cause a number of significant cellular pathologies, and wild type members can act as virulence factors for many parasites and fungi. Here, we describe an in vitro assay to measure the transport activity of the CMP-sialic acid transporter (CST), one of seven NSTs found in mammals. While in vitro transport assays have been previously described for CST, these studies failed to account for the fact that 1) commercially available stocks of CMP-sialic acid (CMP-Sia) are composed of ~10% of the higher-affinity CMP and 2) CMP-Sia is hydrolyzed into CMP and sialic acid in aqueous solutions. Herein we describe a method for treating CMP-Sia with a nonselective phosphatase, Antarctic phosphatase, to convert all free CMP to cytidine. This allows us to accurately measure substrate affinities and transport kinetics for purified CST reconstituted into proteoliposomes.

Keywords: Protein purification (蛋白纯化), Transport assay (转运体检测), Nucleotide-sugar transporters (核苷糖转运子), SLC35 (SLC35), CMP (CMP), CMP-Sia (CMP-Sia), Sialic acid (唾液酸), Lipid vesicle reconstitution (脂质囊泡重组)

Background

Once synthesized in the cytoplasm or nucleus, nucleotide-coupled sugars are transported into the lumen of the endoplasmic reticulum (ER) or Golgi apparatus by nucleotide-sugar transporters (NSTs) (Aoki et al., 2003). Within these subcellular compartments, glycosyltransferases utilize the sugar moieties to glycosylate lipids and proteins, producing nucleotide monophosphates (NMPs) as a byproduct (Capasso and Hirschberg, 1984; Milla and Hirschberg, 1989; Waldman and Rudnick, 1990; Tiralongo et al., 2006). Many glycosyltransferases are inhibited by NMPs, and the lumenal concentration of the latter must be kept low in order to allow for proper glycan synthesis (Hirschberg et al., 1998). To facilitate this, NSTs act as antiporters by transporting their corresponding NMP back to the cytoplasm.

By regulating the concentration of nucleotide sugars in the Golgi and ER lumens, NSTs have a direct impact on glycosylation–the most common form of protein and lipid modification. Mutations that impair NST function can therefore impair proper protein folding, stability, and functionality, with many adverse physiological effects (Dwek et al., 2002; Moremen et al., 2012; Ohtsubo and Marth, 2006; Stanley, 2011). There are a number of debilitating genetic diseases arising from mutations in the solute carrier 35 (SLC35) gene family, from which NSTs are derived (Jaeken and Matthijs, 2007; Song, 2013). Additionally, because NSTs are virulence factors for pathogens, they are potential targets for antiparasitic and antifungal drugs (Descoteaux et al., 1995; Ma et al., 1997; Hong et al., 2000; Engel et al., 2009; Caffaro et al., 2013; Liu et al., 2013). Studies have also shown that blocking NSTs can inhibit tumor metastasis, as altered cell surface protein glycosylation profiles are often a feature of cancerous cells (Caffaro and Hirschberg, 2006; Ohtsubo and Marth, 2006; Esko and Bertozzi, 2009; Song, 2013; Hadley et al., 2014; Stowell et al., 2015; Wang et al., 2016).

Given the importance of NSTs, it is necessary to not only understand how they transport their physiological substrates, but also how mutations and potential inhibitors affect their transport activity. Functional characterization of these transporters via transport assays and other means is essential in understanding genetic pathologies and is a key component in drug development. Herein we describe an in vitro method for measuring the uptake of CMP-sialic acid (CMP-Sia) into proteoliposomes reconstituted with the CMP-Sia transporter (CST), one of seven known NSTs found in humans. While developing this method, we realized that commercial stocks of CMP-Sia contain approximately 10% CMP (Ahuja and Whorton, 2019). This observation, coupled with the known fact that CMP-Sia is hydrolyzed in aqueous solution to CMP and sialic acid (Beau et al., 1984; Horenstein and Bruner, 1996), presented a problem for structural and functional characterization of CST because the affinity of CMP towards CST is approximately 100 times higher than that of CMP-Sia (Ahuja and Whorton, 2019). For transport assays, this abundance of CMP would lead to errors in determining the affinity and transport kinetics of CMP-Sia–an issue that, to our knowledge, had not been addressed in the literature.

While methods have been described to purify CMP from CMP-Sia (Beau et al., 1984), we ultimately decided to not pursue these since significant amounts of CMP would still be generated through CMP-Sia hydrolysis during long-duration experiments (e.g., multi-day crystallization trials). In addition, although several CMP-Sia derivatives have been described which are resistant to hydrolysis (Burkart et al., 2000; Kajihara et al., 2011; Watts and Withers, 2004), we decided not to employ these since they may have different affinities and transport kinetics than unmodified CMP-Sia, and because they are not commercially available and would thus require custom synthesis. We therefore developed a method of using a nonselective nucleotide phosphatase, Antarctic phosphatase (AnP), to convert all CMP in CMP-Sia solutions to cytidine, which does not have a measurable affinity for CST and would not affect the outcome of functional characterization studies. This approach has allowed us to determine reliable affinity and transport rate constants for CMP-Sia transport by CST. We anticipate that this approach would also be useful for studies of other aspects of glycosylation machinery that require CMP-free solutions of CMP-Sia. Although CMP-Sia is the only nucleotide sugar known to rapidly hydrolyze in aqueous solutions, since AnP is nonselective, it may also be useful for the study of other NSTs if commercial stocks of their nucleotide-sugar substrates also contain high levels of a higher-affinity NMP.

Materials and Reagents

  1. 50 ml polypropylene conical tubes (Falcon, catalog number: 352098 )
  2. High-g-rated microfuge tubes (Beckman Coulter, catalog number: 357448 )
  3. 15 ml 50k MWCO Centrifugal Filter (Millipore, catalog number: UFC905024 )
  4. 4 ml 50k MWCO Centrifugal Filter (Millipore, catalog number: UFC805024 )
  5. 0.4 μm extruder filter (Whatman, catalog number: 800282 )
  6. 0.22 μm mixed cellulose ester membrane (Millipore, catalog number: GSTF02500 )
  7. 7 ml glass scintillation vials (Fisher Scientific, catalog number: 03-340-4A )
  8. SEC Column (GE Life Sciences Superdex 200 Increase 10/30 GL; catalog number: 28-9909-44 )
  9. 1 cm diameter glass column (Kimble Flex-Column, catalog number: 420401 )
  10. Bio-Beads SM-2 adsorbent media (Bio-Rad, catalog number: 152-8920 )
  11. cDNA for the Slc35a1 gene; mouse CMP-sialic acid transporter; mCST (Biobasic, Uniprot: Q61420)
  12. EasySelect Pichia Expression Kit (Thermo Fisher, catalog number: K174001 )
  13. Pichia pastoris strain SMD1168H (Thermo Fisher, catalog number: C18400 )
  14. PreScission protease
    Note: We express and purify this ourselves, but it can also be obtained commercially, e.g., GE Healthcare, catalog number: 27084301 .
  15. Cytidine 5’-monophospho-N-acetylneuraminic acid; CMP-sialic acid; CMP-Sia (Carbosynth, catalog number: MC04391 )
  16. [3H]CMP-sialic acid (American Radiolabeled Chemicals, catalog number: ART 0147-50 μCi )
  17. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HEPES (Fisher Scientific, catalog number: BP310-1 )
  18. Sodium chloride; NaCl (Fisher Scientific, catalog number: S271-3 )
  19. Potassium chloride; KCl (Fisher Scientific, catalog number: P217-3 )
  20. Magnesium chloride; MgCl2 (Fisher Scientific, catalog number: M33-500 )
  21. Zinc chloride; ZnCl2 (Acros Organics, catalog number: 380130050 )
  22. Benzamidine hydrochloride hydrate (Acros Organics, catalog number: 105240250 )
  23. Imidazole (Fisher Scientific, catalog number: O3196-500 )
  24. Deoxyribonuclease I; DNase I grade II, from bovine pancreas (Sigma-Aldrich, catalog number: 10104159001 )
  25. Pepstatin A (Fisher Scientific, catalog number: BP2671-25 )
  26. Leupeptin hemisulfate (Fisher Scientific, catalog number: BP2662-100 )
  27. Aprotinin from bovine lung (Fisher Scientific, catalog number: BP2503-10 )
  28. Phenylmethanesulfonyl fluoride (Acros Organics, catalog number: 215740010 )
  29. N-dodecyl-β-D-maltopyranoside solgrade; DDM (Anatrace, catalog number: D310S )
  30. N-dodecyl-β-D-maltopyranoside anagrade; DDM (Anatrace, catalog number: D310 )
  31. 20% DDM (solgrade) solution made in water; stored at -20 °C
  32. Methanol (Fisher Scientific, catalog number: A452-SK4 )
  33. 1,3-Bis[tris(hydroxymethyl)amino]propane; Bis-tris-propane-HCl (Acros Organics, catalog number: 202640250 )
  34. Sodium hydroxide; NaOH (Fisher Scientific, catalog number: S318-1 )
  35. TALON metal affinity resin (Takara, catalog number: 635504 )
  36. DL-1,4-Dithiothreitol; DTT (Acros Organics, catalog number: 16568-0250 )
  37. Ethylenediamine tetraacetic acid, disodium salt; EDTA (Fisher Scientific, catalog number: S311-500 )
  38. Yeast polar lipid extract; YPL (Avanti, catalog number: 190001C )
  39. Pentane (Fisher Scientific, catalog number: P399-1 )
  40. Antarctic phosphatase (New England Biolabs, catalog number: M0289S )
  41. Ultima Gold scintillation cocktail (PerkinElmer, catalog number: 6013329 )
  42. Argon gas, at least grade 4.8
  43. Ultrapure water
  44. Liquid nitrogen
  45. Lysis Buffer (see Recipes)
  46. Buffer A (see Recipes)
  47. Buffer B (see Recipes)
  48. Buffer C (see Recipes) 
  49. AnP Buffer (see Recipes)

Equipment

  1. Pipettes
  2. -80 °C freezer
  3. Miller (Retsch, model: MM400 )
  4. Centrifuge
  5. 50 ml Grinding Jars with 25 mm Grinding Balls (Retsch, catalog numbers: 01.462.0216 and 05.368.0105 , respectively)
  6. Glass Hamilton syringe (Hamilton, catalog number: 81365 )
  7. 12-well filter vacuum manifold (Millipore, model: 1225 , catalog number: XX2702550)
  8. Sonicator (Avanti, model: G112SP1T_B )
  9. Mini extruder (Avanti, catalog number: 610000 )
  10. Liquid Scintillation Counter, e.g., Beckman LS 6000IC

Software

  1. Prism 6 (GraphPad)

Procedure

  1. CST Protein Expression and Purification
    1. Generate Pichia pastoris stable cell lines expressing CST according to manufacturer instructions (EasySelect Pichia Expression Kit, Thermo Fisher).
      Notes:
      1. Briefly, this entails first sub-cloning CST cDNA into the pPICZ vector. This vector is linearized and then introduced into Pichia pastoris (strain SMD1168H) cells by electroporation. Successful transformants are selected on zeocin-containing growth media.
      2. While the bulk of our structural and biochemical characterization of CST has been with the mouse protein, we have observed that the human protein has near-identical properties. Therefore, the methods described here should be applicable to the study of either type of CST.
    2. Express CST in P. pastoris according to manufacturer instructions. We typically induce expression in methanol-containing media for 20-24 h at 25 °C. We routinely grow 6 x 1 L cultures, yielding approximately 10 g cell paste per liter of culture.
    3. Harvest cells by centrifugation at 4,000 x g for 15 min. Discard supernatant and freeze cell pellets with liquid nitrogen.
    4. Lyse frozen cells in a mixer mill. We typically lyse 10 g of frozen cell paste per 50 ml milling chamber for 5 cycles of 3 min at 25 Hz. Chambers are cooled in liquid nitrogen between cycles.
    5. Resuspend milled cells in 2.5 ml of lysis buffer (Recipe 1) per gram of cells and incubate for 1.5 h at 4 °C.
    6. Centrifuge lysate at 35,000 x g for 35 min at 4 °C.
    7. Collect supernatants, adjust pH to 7.2 using 5 M NaOH, and then add imidazole to 5 mM.
    8. Equilibrate TALON resin in Buffer A (Recipe 2). Use 0.175 ml of packed resin per gram of cells.
    9. Add pH-adjusted supernatant to TALON resin and incubate at 4 °C for 2 h under gentle rotation.
    10. Wash resin in batch with 5 column volumes of Buffer A containing 5 mM imidazole. Pellet at 1,250 x g for 5 min and resuspend in 1 column volume of Buffer A.
    11. Load resin onto a 1 cm diameter glass column, and wash using a peristaltic pump at approximately 1 ml/min with 5 column volumes of Buffer A containing 20 mM imidazole, followed by 2 column volumes of Buffer A containing 40 mM imidazole.
    12. Elute protein with at least 5 column volumes of Buffer A containing 300 mM imidazole.
    13. Pool eluted protein and add 1 mM DTT and 1 mM EDTA.
    14. Add PreScission protease at a mass ratio of 1:20 protease:total protein, and dialyze overnight at 4 °C against Buffer A. Dialyze against a sufficient volume to reduce the imidazole concentration to 5 mM or lower. For example, dialyze against a volume that is at least 60 times the sample volume.
    15. Run cleaved protein over a 1 ml TALON column equilibrated in Buffer A. Collect the initial flow-through. Wash the column twice with one column volume buffer A. Pool the flow-through and washes.
      Note: This step is necessary to remove contaminants that are not resolved from CST during size exclusion chromatography (next step).
    16. Concentrate the pooled sample to at least 500 μl in a 50k MWCO concentrator and run on a 10/300 Superdex 200 Increase gel filtration column equilibrated in Buffer B (Recipe 3).
    17. Combine peak fractions for incorporating into proteoliposomes.

  2. Proteoliposome Reconstitution
    1. To incorporate purified CST into lipid vesicles, the lipids must first be transformed into small unilamellar vesicles (SUVs). Since the lipid stocks are supplied dissolved in chloroform, the first step in this process involves evaporating away the chloroform before the lipids can be resuspended in an aqueous buffer.
    2. A typical reconstitution uses 40 mg of lipids (yeast polar lipid extract). To aliquot this amount, use a glass Hamilton syringe with a Teflon plunger to measure 1.6 ml of a 25 mg/ml solution of lipids dissolved in chloroform. Place the lipid solution into a glass test tube and evaporate the chloroform using a gentle stream of argon gas. Then, re-dissolve the dried lipids with ~2 ml pentane and then evaporate the pentane using a gentle stream of argon. Repeat this pentane wash once more and then place the dried lipids in a vacuum desiccator overnight to remove any residual solvent.
    3. Re-suspend the dried lipids in Buffer C (Recipe 4) at 11.1 mg/ml and sonicate for 2-3 h in a bath sonicator. Use a ring stand and clamp to suspend the glass test tube in the bath such that the lipid suspension is completely submerged. Run the sonicator in 5-10 min intervals to prevent overheating (above ~40 °C). During this time, the solution should change from opaque to translucent indicating the formation of SUVs.
    4. Add DDM to a final concentration of 5 mM, using a 20% stock solution, to partially solubilize the SUVs. Vortex gently and then incubate for 10-15 min at room temperature.
    5. Sonicate again for up to 1 h, in 5-10 min intervals, until the solution becomes semitransparent.
    6. Add 200 μg of purified CST from the previous section. The protein should be sufficiently concentrated using a 50k MWCO concentrator such that the addition of protein to the lipids does not dilute the lipids below 10 mg/ml. For protein-free vesicle controls, substitute CST for an equivalent volume of Buffer B. For filling the vesicles with a substrate (e.g., 300 μM CMP), add the desired substrate at this step.
      Note: This protein:lipid mass ratio of 1:200 was empirically determined to give the best signal to noise in our assay. Other proteins or lipid compositions may require different protein:lipid ratios.
    7. Bring the final lipid concentration to 10 mg/ml by diluting with Buffer C, if necessary, then incubate for 1 h at 4 °C.
    8. To form proteoliposomes, remove DDM by adding Bio-Beads to 100 mg/ml. Incubate for 2 h at 4 °C under gentle rotation, then briefly pellet Bio-Beads and transfer supernatant to fresh Bio-Beads at 100 mg/ml. Incubate again for 2 h at 4 °C under gentle rotation, then transfer to a third fresh batch of Bio-Beads at 100 mg/ml. Incubate this overnight at 4 °C under gentle rotation. The finished proteoliposomes should be noticeably less translucent at the end of this incubation.
      Notes:
      1. Prepare Bio-Beads by adding 30 ml methanol to ~2 g of Bio-Beads in a 50 ml Falcon tube. Rotate beads for 15 min, then remove methanol by applying a vacuum to a small hole created in the bottom of the Falcon tube using a needle. Wash the beads four times in 50 ml water, removing water by applying a vacuum. Wash the beads once in 10 ml of Buffer C, and then store under Buffer C until needed.
      2. To obtain the desired amount of beads, briefly drain the washed beads by vacuum and weigh out the desired amount using a balance.
    9. Aliquot the liposomes and flash freeze in liquid nitrogen. Store at -80 °C.
      Notes:
      1. The liposomes can be stored at -80 °C for at least several months.
      2. We can typically incorporate about 60% of the starting amount of protein into the vesicles.
      3. To determine the efficiency of protein incorporation, solubilize a small amount of vesicles using DDM, and run on a gel filtration column. Then compare the peak height to an equivalent protein sample that was not incorporated into vesicles.

  3. CMP-Sia Ligand Purification
    Notes:
    1. Commercial stocks of CMP-Sia contain approximately 10% CMP and will further hydrolyze to form free CMP and Sia over time. For transport assays, all free CMP must first be converted to cytidine–which has a very low affinity for CST–using Antarctic phosphatase (AnP).
    2. We screened a number of commercially-available phosphatases. Most were able to effectively convert CMP to cytidine, but we ultimately selected AnP because it retains high enzymatic activity in acidic pHs, which is the pH range where most of our crystallization hits were obtained.
    3. The hydrolysis of CMP-Sia is temperature and pH dependent (faster at higher temperature and/or lower pH), so keep AnP-treated CMP-Sia solutions in neutral pH buffers and on ice during use and store at -80 °C.
    4. We determined that the rate of hydrolysis of CMP-Sia in the transport assay buffer (Buffer C) is 0.005%/min at room temperature. This should be a factor to consider when designing and interpreting data from transport assay experiments. However, given that most of our transport experiments used relatively low concentrations of CMP-Sia and short incubation times, we did not consider the small amount of CMP generated to be significant.
    1. The Antarctic phosphatase (AnP) stock from NEB is 5,000 U/ml. This is not concentrated enough to adequately treat a concentrated (100 mM) stock of CMP-Sia; therefore, the AnP must be concentrated. To do this, first dilute the AnP to 100 U/ml in AnP Buffer (Recipe 5) (to reduce glycerol concentration), then concentrate to 10,000 U/ml with a 10k MWCO concentrator.
    2. Dissolve CMP-Sia powder into the 10,000 U/ml AnP solution to a final concentration of 100 mM. Add ZnCl2 to a final concentration of 0.2 mM and MgCl2 to a final concentration of 2 mM.
    3. Incubate this mixture for 8 h at room temperature.
    4. Remove AnP by filtering the mixture through a 3k MWCO concentrator, which retains the 70 kDa AnP.
      Note: We confirmed that we do not observe any AnP activity after this step.
    5. If desired, confirm the elimination of CMP form the CMP-Sia stock by HPLC analysis.
    6. Aliquot and freeze at -80 °C. Aliquots can be stored for at least several months.

  4. Transport Assay
    1. Thaw the lipid vesicles from Section B and extrude 10 times through a 0.4 μm Whatman Nuclepore filter using an Avanti mini extruder.
    2. Remove external CMP by pelleting the vesicles via ultracentrifugation at 194,800 x g for 60 min at 4 °C, using high-g rated 1.5 ml Beckman tubes. 45 μl vesicles are needed per assay point.
    3. Discard supernatant and rinse pellet twice with 500 μl of Buffer C. Then resuspend the pellet in Buffer C. The final volume should be 0.444 times the starting volume; however, it is helpful to initially only add a small volume (~50 μl) of Buffer C to make it easier to completely homogenize the vesicles before bringing the sample up to the final volume. Store on ice until needed.
    4. For each assay point, initiate transport by taking 20 μl of the resuspended vesicles and adding 30 μl of Buffer C with the desired concentration of AnP-treated CMP-Sia containing 30-50 nM [3H]CMP-Sia (20 Ci/mmol).
      Note: We have not evaluated the purity of the [3H]CMP-Sia that we use for transport assays, but we do not anticipate the presence of any CMP impurity to be a significant problem for the following reasons: 1) the tritium is on the Sia moiety so we don’t have to worry about detecting [3H]CMP transport, and free Sia has a non-detectable affinity for CST and is most likely not transported; 2) the [3H]CMP-Sia is only included as a tracer and is typically diluted at least 100x with cold CMP-Sia for the assay; therefore, the relative concentration of any CMP will be very low.
    5. Incubate the mixture for the desired time and temperature depending on the experiment. We typically incubate at 23 °C for convenience and to minimize CMP-Sia hydrolysis. At this temperature, the vesicles appear to reach equilibrium by 10 min, with the rate of uptake being linear up until 30 s.
    6. Stop transport by adding 0.6 ml of ice-cold Buffer C and store on ice.
    7. To separate the vesicles from the external solution, filter the mixture through a 0.22 μm mixed cellulose ester membrane. To reduce background counts (non-specific adherence of [3H]CMP-Sia to the filters), it helps to first pre-wash the filters with 2 ml ice-cold Buffer C before applying the vesicles. After the vesicles have filtered through, wash the filters three times with 2 ml of ice-cold Buffer C. We typically use a 12-well filter vacuum manifold (MilliporeSigma) that accommodates 25 mm diameter filters.
    8. Place filters in 7 ml scintillation vials, add 5 ml scintillation cocktail, and count in a liquid scintillation counter.

Data analysis

  1. To determine background counts, use values from protein-containing vesicles mixed with the hot substrate and then immediately quenched by adding 0.6 ml ice-cold Buffer C. Alternatively, protein-free vesicles that undergo the same experimental conditions as protein-containing vesicles can be used. We have found that these approaches yield similar results.
  2. Subtract the background counts from the total counts for each sample to determine specific counts and convert to mol/min for substrate transport.
  3. Fit data to a Michaelis-Menten model to determine Km and Vmax (see Figure 1 for example data).


    Figure 1. Functional properties of mouse CST. A. Time course of 30 μM CMP-Sia uptake for purified mouse CST (mCST) reconstituted into lipid vesicles with (black) or without (red) 300 μM CMP inside the vesicles. (Inset), the first minute of CMP-Sia uptake for mCST reconstituted into CMP-filled vesicles is shown to illustrate the linear relationship between uptake and time for up to 30 s, which we used to determine the initial velocity of transport for a given concentration of CMP-Sia. B. The initial velocity of CMP-Sia uptake for mCST reconstituted into CMP-filled vesicles is plotted as a function of substrate concentration to determine the Km and Vmax for transport. In all panels, the symbols show the mean ± standard error of the mean (SEM) for n = 2 (A) or n = 4 (B). Adapted from Ahuja and Whorton, 2019.

Recipes

  1. Lysis Buffer
    50 mM HEPES, pH 7.5
    150 mM NaCl
    0.01 mg/ml deoxyribonuclease I
    0.7 μg/ml pepstatin
    1 μg/ml leupeptin
    1 μg/ml aprotinin
    1 mM benzamidine
    0.5 mM phenylmethylsulfonyl fluoride
    2% (w/v) DDM (solgrade)
  2. Buffer A
    50 mM HEPES, pH 7.5
    150 mM NaCl
    0.1% (w/v) DDM (solgrade)
  3. Buffer B (Gel Filtration Buffer)
    25 mM HEPES, pH 7.5
    150 mM NaCl
    0.1% (w/v) DDM (anagrade)
    5 mM DTT
    1 mM EDTA
  4. Buffer C (Reconstitution Buffer)
    20 mM HEPES, pH 7.5
    0.1 M KCl
  5. AnP Buffer
    50 mM Bis-Tris-Propane-HCl, pH 6.0
    0.1 mM ZnCl2
    1 mM MgCl2

Acknowledgments

This work was supported by NIH grant R01GM130909, and by Oregon Health and Science University. This protocol has been expanded from previous work (Ahuja and Whorton, 2019).

Competing interests

The authors declare no competing interests.

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  19. Ma, D., Russell, D. G., Beverley, S. M. and Turco, S. J. (1997). Golgi GDP-mannose uptake requires Leishmania LPG2. A member of a eukaryotic family of putative nucleotide-sugar transporters. J Biol Chem 272(6): 3799-3805.
  20. Milla, M. E. and Hirschberg, C. B. (1989). Reconstitution of Golgi vesicle CMP-sialic acid and adenosine 3'-phosphate 5'-phosphosulfate transport into proteoliposomes. Proc Natl Acad Sci U S A 86(6): 1786-1790.
  21. Moremen, K. W., Tiemeyer, M. and Nairn, A. V. (2012). Vertebrate protein glycosylation: diversity, synthesis and function. Nat Rev Mol Cell Biol 13(7): 448-462.
  22. Ohtsubo, K. and Marth, J. D. (2006). Glycosylation in cellular mechanisms of health and disease. Cell 126(5): 855-867.
  23. Song, Z. (2013). Roles of the nucleotide sugar transporters (SLC35 family) in health and disease. Mol Aspects Med 34(2-3): 590-600.
  24. Stanley, P. (2011). Golgi glycosylation. Cold Spring Harb Perspect Biol 3(4): a005199.
  25. Stowell, S. R., Ju, T. and Cummings, R. D. (2015). Protein glycosylation in cancer. Annu Rev Pathol 10: 473-510.
  26. Tiralongo, J., Ashikov, A., Routier, F., Eckhardt, M., Bakker, H., Gerardy-Schahn, R. and von Itzstein, M. (2006). Functional expression of the CMP-sialic acid transporter in Escherichia coli and its identification as a simple mobile carrier. Glycobiology 16(1): 73-81.
  27. Waldman, B. C. and Rudnick, G. (1990). UDP-GlcNAc transport across the Golgi membrane: electroneutral exchange for dianionic UMP. Biochemistry 29(1): 44-52.
  28. Wang, L., Liu, Y., Wu, L. and Sun, X. L. (2016). Sialyltransferase inhibition and recent advances. Biochim Biophys Acta 1864(1): 143-153.
  29. Watts, A.G. and Withers, S.G. (2004). The synthesis of some mechanistic probes for sialic acid processing enzymes and the labeling of a sialidase from Trypanosoma rangeli. Can J Chem 82: 1581-1588.

简介

[ 摘要] 核苷酸糖转运蛋白(NST)通过将核苷酸糖结合物转运到高尔基腔和内质网供糖基转移酶使用,同时将核苷酸单磷酸副产物转移到细胞质中,从而促进了真核细胞的糖基化。该蛋白质家族的突变可引起许多重要的细胞病理,野生型成员可充当许多寄生虫和真菌的致病因子。在这里,我们描述了一种体外测定法,以测量CMP-唾液酸转运蛋白(CST)的转运活性,CMP-唾液酸转运蛋白(CST)是在哺乳动物中发现的七个NST之一。虽然在体外 以前已经针对CST进行了转运分析,但这些研究未能说明以下事实:1 )CMP- 唾液酸(CMP-Sia )的商业库存由约10%的高亲和力CMP组成,以及2)CMP- SIA 是水解d 到CMP和在水溶液中的唾液酸。在这里,我们描述了一种用非选择性磷酸酶南极磷酸酶治疗CMP-Sia的方法,以将所有游离CMP转化为胞苷。这使我们能够准确地测量重组为蛋白脂质体的纯化CST的底物亲和力和运输动力学。

[ 背景技术]一旦在细胞质或细胞核合成,核苷酸偶联的糖被输送到内质网的内腔(ER)由核苷酸-糖转运蛋白(NSTS)或高尔基体(青木等人,2003)。在这些亚细胞区室中,糖基转移酶利用糖部分糖基化脂质和蛋白质,产生副产物核苷酸单磷酸酯(NMP)(Capasso和Hirschberg,1984 ;Milla和Hirschberg ,1989; Waldman和Rudnick,1990 ;Tiralongo 等,2006)。 )。NMPs抑制了许多糖基转移酶,为了保持适当的聚糖合成,必须将其内腔浓度保持在较低水平(Hirschberg 等,1998)。为了促进这一点,NST通过将其相应的NMP转运回细胞质而充当反向转运蛋白。

通过调节在高尔基体与ER内腔核苷酸糖的浓度,NSTS对糖基化有直接影响- 蛋白质和脂质修饰的最常见形式。因此,损害NST功能的突变会损害适当的蛋白质折叠,稳定性和功能,并具有许多不利的生理效应(Dwek 等,2002; Moremen 等,2012; Ohtsubo和Marth,2006; Stanley,2011)。由溶质载体35(SLC35)基因家族的突变引起的许多衰弱性遗传疾病均源于NST(Jaeken and Matthijs,2007; Song,2013)。此外,由于NST是病原体的致病因子,因此它们是抗寄生虫药和抗真菌药的潜在靶标(Descoteaux 等,1995; Ma 等,1997 ; Hong 等,2000; Engel 等,2009; Caffaro 等等人,2013; Liu 等人,2013)。研究还表明,阻断NSTs可以抑制肿瘤转移,因为改变的细胞表面蛋白糖基化谱通常是癌细胞的特征(Caffaro和Hirschberg,2006; Ohtsubo和Marth,2006;Esko和Bertozzi,2009; Song,2013;Hadley 等人,2014; Stowell 等人,2015; Wang 等人,2016)。

考虑到NST的重要性,不仅需要了解NST如何转运其生理底物,而且还必须了解突变和潜在抑制剂如何影响其转运活性。通过转运测定和其他手段对这些转运蛋白的功能表征对于理解遗传病理学至关重要,并且是药物开发中的关键组成部分。本文中,我们描述了一种体外方法,用于测量CMP-唾液酸(CMP-Sia)对用CMP-Sia转运蛋白(CST)重构的蛋白脂质体的吸收,该蛋白脂质体是在人类中发现的七个已知NST之一。在开发此方法时,我们意识到CMP-Sia的商业库存包含约10%的CMP(Ahuja和Whorton,2019)。这一观察结果,再加上已知的事实是CMP-Sia的是水解d 在水溶液中CMP和唾液酸(博等人,1984; 霍伦斯坦和布鲁纳,1996 ),提出了一个问题,因为该CST的结构和功能表征CMP对CST的亲和力比CMP-Sia高约100倍(Ahuja和Whorton,2019年)。对于转运分析,CMP的这种丰度将导致确定CMP-Sia 的亲和力和转运动力学方面的错误-据我们所知,这一问题在文献中尚未解决。

虽然已描述了从CMP-Sia中纯化CMP的方法(Beau 等人,1984),但我们最终决定不采用这些方法,因为在长时间的实验中,仍然会通过CMP-Sia水解产生大量的CMP(例如,多天结晶试验)。此外,尽管已描述了几种抗水解的CMP-Sia衍生物(Burkart 等,2000; Kajihara 等,2011; Watts和Withers,2004),但我们决定不使用它们,因为它们可能有不同之处。亲和力和传输动力学比未经修饰的CMP-Sia好,并且因为它们不可商购,因此需要定制合成。因此,我们开发了一种使用非选择性核苷酸磷酸酶南极磷酸酶(AnP)将CMP-Sia溶液中的所有CMP转化为胞苷的方法,该方法对CST没有可测量的亲和力,并且不会影响功能表征研究的结果。这种方法使我们能够确定CST用于CMP-Sia转运的可靠亲和力和转运速率常数。我们预计该方法对于需要CMP-Sia的无CMP解决方案的糖基化机制其他方面的研究也将是有用的。尽管CMP-Sia是已知的唯一在水溶液中能快速水解的核苷酸糖,但由于AnP是非选择性的,如果其核苷酸糖底物的商业库存也含有高水平的高糖基化酶,它也可能用于研究其他NST。亲和力NMP。

关键字:蛋白纯化, 转运体检测, 核苷糖转运子, SLC35, CMP, CMP-Sia, 唾液酸, 脂质囊泡重组

材料和试剂


 


50毫升p 聚环氧Ç onical 吨ubes中(Falcon ,目录号:352098)
高g级微量离心管(Beckman Coulter,目录号:357448)
15米升50K MWCO离心过滤器(Millipore ,目录号:U ˚F C905024)
4 ml 50k MWCO离心过滤器(Millipore,目录号:UFC805024)
0.4 μ米挤出机滤器(Whatman,目录号:800282)
0.22 μ M混合床柱的纤维素酯膜(Millipore,目录号:GSTF02500)
7毫升玻璃闪烁瓶(Fisher Scientific,目录号:03-340-4A)
SEC色谱柱(GE生命科学Superdex 200增加10/30 GL;目录号:28-9909-44)
直径1厘米的玻璃柱(Kimble Flex-Column,货号:420401)
生物珠SM-2 一dsorbent 米EDIA(生物- Rad公司,目录号:152-8920)
Slc35a1 基因的cDNA ; 小鼠CMP-唾液酸转运蛋白; mCST(生物碱,Uniprot:Q61420)
EasySelect Pichia表达试剂盒(赛默飞世尔(Thermo Fisher),目录号:K174001)
巴斯德毕赤酵母菌株SMD1168H(Thermo Fisher,目录号:C18400)
分裂前蛋白酶
注:W é表达,这洁净自己,但它也可以购得,例如,通用电气医疗集团,目录号:27084301 。


胞苷5'-单磷酸-N-乙酰神经氨酸;CMP-唾液酸;CMP-Sia(Carbosynth,货号:MC04391)
[ 3 H] CMP唾液酸(美国放射性标记的化学品,目录号:ART 0147 -50μCi )
4-(2-羟乙基)-1-哌嗪乙烷磺酸; HEPES(Fisher Scientific,目录号:BP310-1)
氯化钠; NaCl(Fisher Scientific,目录号:S 27 1 -3)
氯化钾; KCl(Fisher Scientific,目录号:P217-3)
氯化镁; MgCl 2 (Fisher Scientific,目录号:M33-500)
氯化锌 ZnCl 2 (Acros Organics,目录号:380130050)
盐酸苄am水合物(Acros Organics,目录号:105240250)
咪唑(Fisher Scientific,目录号:O3196-500)
脱氧核糖核酸酶I;DNase I II级,来自牛胰腺(Sigma-Aldrich,目录号:10104159001)
抑肽酶A(Fisher Scientific,目录号:BP2671-25)
亮丙肽半硫酸盐(Fisher Scientific,目录号:BP2662-100)
牛肺抑肽酶(Fisher Scientific,目录号BP2503-10)
苯甲磺酰氟(Acros Organics,目录号:215740010)
正十二烷基β -D-吡喃麦芽糖苷solgrade; DDM(Anatrace,目录号:D310S)
正十二烷基β -D-吡喃麦芽糖苷anagrade; DDM(Anatrace,目录号:D310)
用水制成的20 %DDM(溶解)溶液;储存在-20°C
甲醇(Fisher Scientific,目录号:A452-SK4)
1,3-双[三(羟甲基)氨基]丙烷; Bis-tris-丙烷-HCl(Acros Organics,目录号:202640250)
氢氧化钠; NaOH(Fisher Scientific,目录号:S318-1)
TALON金属亲和树脂(宝酒,商品目录号:635504)
DL-1,4-二硫苏糖醇; DTT(Acros Organics,目录号:16568-0250)
乙二胺四乙酸二钠盐;EDTA(Fisher Scientific,目录号:S311-500)
酵母极性脂质提取物;YPL(Avanti,目录号:190001C)
戊烷(Fisher Scientific,目录号:P399-1)
南极磷酸酶(新英格兰生物实验室,目录号:M0289S)
Ultima Gold闪烁鸡尾酒(Perkin Elmer,目录号:6013329)
至少4.8级的氩气
超纯水
液氮
裂解缓冲液(请参见食谱)
缓冲区A(请参阅食谱)
缓冲区B(请参阅食谱)
缓冲区C(请参阅食谱)
AnP缓冲区(请参阅食谱)
 


设备


 


P ipettes
-80 °C冷冻室
米勒(Retsch ,型号:MM400)
离心机
50 m l 带有25 mm研磨球的研磨罐(Retsch ,目录号分别为01.462.0216和05.368.0105)
G lass Hamilton注射器(Hamilton ,目录号:81365)
12孔过滤器真空歧管(Millipore ,型号:1225,目录号:XX2702550)
超声仪(Avanti,型号:G112SP1T_B)
小型挤出机(Avanti,目录号:610000)
液体闪烁计数器,例如Beckman LS 6000IC
 


软件


 


棱镜6(GraphPad)
程序


 


CST蛋白表达和纯化
根据生产商的说明(EasySelect Pichia表达试剂盒,Thermo Fisher)生成表达CST的巴斯德毕赤酵母稳定细胞系。
注意小号:


简而言之,这需要先将CST cDNA亚克隆到pPICZ 载体中。将该载体线性化,然后通过电穿孔引入巴斯德毕赤酵母(SMD1168H菌株)细胞。在含zeocin的生长培养基上选择成功的转化体。
虽然我们对CST的大部分结构和生化特性都与小鼠蛋白有关,但我们已经观察到人蛋白具有几乎相同的特性。因此,此处描述的方法应适用于两种CST类型的研究。
根据制造商的说明在巴斯德毕赤酵母中表达CST 。我们通常在25到20-24小时内在含甲醇的培养基中诱导表达 ℃。我们通常生长6 x 1 L培养物,每升培养物产生约10 g细胞糊。
通过以4,000 xg离心15分钟收获细胞。丢弃上清液并用液氮冷冻细胞沉淀。
在搅拌机中裂解冷冻细胞。我们通常在25 Hz的频率下,每50 ml研磨室裂解10 g冷冻细胞糊,进行3分钟的5个循环。循环之间将室在液氮中冷却。
每克细胞在2.5 ml裂解缓冲液(配方1 )中重悬研磨的细胞,并在4 °C 下孵育1.5 h 。
在4 °C下以35,000 xg 离心裂解液35分钟。
收集上清液,使用5 M NaOH将pH调节至7.2,然后将咪唑添加至5 mM。
在缓冲液A中平衡T ALON 树脂(配方2 )。每克细胞使用0.175毫升填充树脂。
向T ALON 树脂中添加pH调节的上清液,并在4 °C 下温和旋转孵育2 h。
娲SH树脂分批用含5mM咪唑的缓冲液A的5个柱体积。以1,250 x g 的速度沉淀5 分钟,然后重悬于1 倍柱体积的缓冲液A中。
将树脂加载到直径为1 cm的玻璃柱上,并使用蠕动泵以大约1 ml / min的速度用5柱体积的含20 mM咪唑的缓冲液A洗涤,然后用2柱体积的含40 mM咪唑的缓冲液A 洗涤。
用至少5 倍柱体积的含有300 mM咪唑的缓冲液A 洗脱的蛋白质。
合并洗脱的蛋白质,并加入1 mM DTT和1 mM EDTA。
以1:20蛋白酶:总蛋白质的质量比添加PreScission蛋白酶,并在4 °C下对缓冲液A 透析过夜。对足够的体积进行透析,以使咪唑浓度降至5 mM或更低。例如,针对至少是样品体积60倍的体积进行透析。
将切割的蛋白质运行在 在缓冲液A中平衡1 m L T ALON 柱。收集初始流通量。用一个色谱柱体积缓冲液A清洗色谱柱两次。合并流通液和洗涤液。
注意:此步骤对于去除在尺寸排阻色谱法(下一步)中未从CST中分离出的污染物是必要的。


浓缩合并样本到至少500 μ 升在50K MWCO集中器和在缓冲液B(平衡的10/300的Superdex 200增加凝胶过滤柱运行配方3 )。
合并峰级分以结合到脂质体中。
 


蛋白脂质体重建
要将纯化的CST掺入脂质囊泡中,必须先将脂质转化为小单层囊泡(SUVs)。由于供应的脂质原料溶解在氯仿中,因此此过程的第一步涉及蒸发掉氯仿,然后将脂质重新悬浮在水性缓冲液中。
典型的复原使用40 mg 脂质(酵母极脂质提取物)。到等分该量,使用玻璃Hamilton注射器用Ť EFLON柱塞测量1.6毫升溶解在氯仿中的脂质的25毫克/毫升溶液。将脂溶液放入玻璃试管中,并用氩气缓慢吹扫氯仿。Ť 母鸡,再溶解干燥的脂质用约2ml 戊烷,然后蒸发戊烷使用的温和的流氩气。再次重复戊烷洗涤一次,然后将干燥的脂质放置在真空干燥器中过夜,以去除残留的溶剂。
重悬的干燥脂质小号在缓冲液C(配方4 在11.1)毫克/毫升并声处理2-3小时在槽式声波处理器。用环架和夹子将玻璃试管悬挂在浴中,使脂质悬浮液完全浸没。每隔5-10分钟运行一次超声仪,以防止过热(约40 °C以上)。在此期间,溶液应从不透明变为半透明,表明SUV 的形成。
使用20 %的储备溶液将DDM加入至终浓度为5 mM ,以部分溶解SUV 。轻轻涡旋,然后在室温下孵育10-15分钟。
以5-10分钟的间隔再次超声处理长达1小时,直到溶液变为半透明。
加入200 μ 从上一节纯化CST的克。所述蛋白质应使用50K MWCO集中器,使得除了蛋白与脂质不稀释低于10的脂质被充分浓缩毫克/米升。对于无蛋白囊泡控制,替换为CST缓冲液B的等效体积对于具有基板(填充囊泡例如,300 μ 中号CMP),在该步骤中添加所希望的衬底。
注意:凭经验确定此蛋白质与脂质的质量比为1:200,以在我们的测定中提供最佳的信噪比。其他蛋白质或脂质组合物可能需要不同的蛋白质:脂质比率。


使最终脂质浓度到10毫克/米升稀释用缓冲液C,在4如果必要的话,然后孵育1个小时℃下。
要形成蛋白脂质体,可通过将Bio-Beads添加至100 mg / ml除去DDM。在4 °C 下轻轻旋转孵育2小时,然后短暂沉淀Bio-Beads,然后将上清液以100 mg / ml的浓度转移至新鲜的Bio-Beads。再次孵育在4 2小时℃的下轻轻旋转,然后在100毫克/米转让给第三新鲜批次生物珠的升。将其在4 °C 下缓慢旋转孵育过夜。孵育结束后,完成的蛋白脂质体应明显不透明。
笔记:


通过在50 ml Falcon管中的约2 g Bio-Beads中添加30 ml甲醇来制备Bio-Beads。旋转珠子15分钟,然后使用针对在Falcon管底部形成的小孔施加真空,以除去甲醇。在50毫升水中洗涤小珠四次,然后抽真空除去水。在10 ml 的缓冲液C中洗一次磁珠,然后在缓冲液C下保存直至需要。 
为了获得所需量的珠子,请在真空中短暂沥干清洗后的珠子,然后用天平称出所需量。
分装脂质体并在液氮中快速冷冻。储存在-80 °C 。
笔记:


脂质体可以在-80 °C下保存至少几个月。
我们通常可以将蛋白质起始量的约60%掺入囊泡中。
为了确定蛋白质掺入的效率,请使用DDM溶解少量的囊泡,然后在凝胶过滤柱上运行。然后将峰高与未掺入囊泡的等效蛋白质样品进行比较。
 


CMP-Sia配体纯化
笔记:


CMP-Sia的商业库存包含约10%的CMP,随着时间的流逝,它们将进一步水解形成游离的CMP和Sia。对于转运测定,必须首先将所有游离CMP转化为胞苷- 对CST的亲和力很低–使用南极磷酸酶(AnP)。
我们筛选了许多商业上可获得的磷酸酶。大多数能够有效地将CMP转化为胞苷,但我们最终选择了AnP,因为它在酸性pH(这是我们获得大多数结晶产物的pH范围)内保留了高酶促活性。
CMP-Sia的水解取决于温度和pH值(在较高的温度和/或较低的pH值下更快),因此在使用过程中,将AnP处理的CMP-Sia溶液保持在中性pH缓冲液和冰中,并在-80 °C下储存。
我们确定,在室温下,在运输测定缓冲液(缓冲液C)中CMP-Sia的水解速率为0.005 %/ min。这是设计和解释转运分析实验数据时要考虑的因素。但是,考虑到我们的大多数运输实验都使用了相对较低的CMP-Sia浓度和较短的孵育时间,因此我们认为少量的CMP并不是很重要。
NEB 的南极磷酸酶(AnP )库存为5,000 U / ml。浓缩程度不足以充分处理CMP-Sia的浓缩(100 mM)储备液;因此,必须集中精力。要做到这一点,首先dilut Ë 的ANP 到100U / ml,在ANP缓冲液(配方5 )(以减少甘油浓度),然后浓缩至10,000 U / ml时,用10K MWCO集中器。
将CMP-Sia粉溶解到10,000 U / ml AnP溶液中至终浓度为100 mM。添加ZnCl 2 至终浓度为0.2 mM,添加MgCl 2 至终浓度为2 mM。  
将该混合物在室温下孵育8小时。
通过3k MWCO浓缩器过滤混合物以除去AnP,该浓缩器保留70 kDa AnP。 
注意:我们确认此步骤后我们没有观察到任何AnP活性。


如果需要,请通过HPLC分析确认从CMP-Sia库存中消除了CMP。
分装并在-80 °C 冷冻。等分试样可以存储至少几个月。
 


运输分析
解冻从部分B的脂质囊泡和拉伸10次通过0.4 μ 使用阿凡提微型挤出机米的Whatman的Nuclepore滤波器。
通过使用高g等级的1.5 ml Beckman管在194,800 xg 在4°C下进行60分钟的超速离心来沉淀囊泡,以去除外部CMP 。45个μ 每次测定点需要升囊泡。
弃上清,沉淀冲洗两次,用500 μ 缓冲液C的升。然后重悬在沉淀缓冲液C的最终体积应为起始体积0.444倍; 但是,它是有帮助的最初仅加入小体积(〜50 μ 升缓冲液C),以使其更易于完全使所述样品达到最终体积之前均质化囊泡。存放在冰上,直到需要为止。
对于每个测定点,通过取20发起传输μ 升再悬浮的囊泡中,加入30 μ 升缓冲液C的与ANP处理CMP-Sia的含有30-50纳米[所需浓度3 H] CMP-Sia的(20℃ 我/毫摩尔)。
注意:我们尚未评估用于转运分析的[ 3 H] CMP-Sia 的纯度,但由于以下原因,我们预计任何CMP杂质的存在都不是一个重大问题:1)is是在Sia部分上,因此我们不必担心检测[ 3 H] CMP转运,并且游离Sia对CST具有不可检测的亲和力,很可能不会转运。2)[ 3 H] CMP-Sia仅作为示踪剂使用,通常用冷CMP-Sia稀释至少100倍进行分析;因此,任何CMP的相对浓度都非常低。


根据实验将混合物孵育所需的时间和温度。W¯¯ Ë通常孵育在23 ℃下为方便起见,并尽量减少CMP-Sia的水解。在此温度下,囊泡似乎在10分钟之前达到平衡,直至30 s的摄取速率都是线性的。
停止输送通过添加0.6米升冰上冰冷缓冲液C的和存储。
从外部溶液中分离的囊泡中,f 通过0.22 ILTER混合物μ M混合床柱纤维素酯膜。为减少背景计数([ 3 H] CMP-Sia对过滤器的非特异性粘附),在使用囊泡之前,先用2 ml 冰冷的缓冲液C 预清洗过滤器是有帮助的。囊泡过滤后,用2 ml 冰冷的缓冲液C 清洗过滤器3次。我们通常使用容纳25毫米直径过滤器的12孔过滤器真空歧管(MilliporeSigma)。
将过滤器放入7 ml的闪烁小瓶中,加入5 ml的闪烁鸡尾酒,并在液体闪烁计数器中计数。
 


数据分析


 


要确定背景计数,请使用含蛋白的囊泡与热底物混合后的值,然后立即添加0.6 ml 冰冷的缓冲液C 淬灭。或者,可以使用与含蛋白的囊泡相同的实验条件的无蛋白的囊泡使用。我们发现这些方法产生相似的结果。
从每个样品的总计数中减去背景计数,以确定特定计数,并转换为mol / min用于底物运输。
将数据拟合为一个米氏模型来确定ķ 米和V 最大(小号EE例如图1的数据)。
 


D:\ Reformatting \ 2020-1-6 \ 1902713--1301 Matthew Whorton 784665 \ Figs jpg \图1.jpg


图1 。鼠标CST的功能特性。一。30时间过程μ 中号CMP-Sia的摄取对于纯化小鼠CST(MCST)重构为脂质囊泡与(黑色)或无(红色)300 μ 中号CMP囊泡内。(插图),Ť 他CMP-Sia的摄取为MCST的第一分钟重构为CMP填充的囊泡被示出以说明摄取和时间最长为30秒,这是我们用于确定传输的用于初始速度之间的线性关系给定浓度的CMP-Sia。乙。将重构为CMP的囊泡中的mCST的CMP-Sia吸收初始速度绘制为底物浓度的函数,以确定转运的K m 和V max 。在所有面板中,符号均表示n = 2(A )或n = 4(B )的平均值±平均值的标准误差(SEM )。改编自Ahuja和Whorton,2019。   


 


 


菜谱


 


裂解缓冲液
50 mM HEPES,pH 7.5


150毫米氯化钠


0.01 mg / ml脱氧核糖核酸酶I


胃抑素0.7μg/ ml


1μg/ ml亮肽素


1μg/ ml抑肽酶


1 mM联am


0.5 mM苯基甲基磺酰氟


2%(w / v)DDM(溶剂化)


缓冲液A
50 mM HEPES ,pH 7.5


150毫米氯化钠


0.1%(w / v)DDM(溶剂化)


缓冲液B(凝胶过滤缓冲液)
25 mM HEPES ,pH 7.5


150毫米氯化钠


0.1%(w / v)DDM(降级)


5毫米DTT


1 毫米EDTA


缓冲区C(复原缓冲区)
20 mM HEPES,pH 7.5


0.1 M氯化钾


AnP缓冲液
50 mM Bis-Tris-丙烷-HCl,pH 6.0


0.1 mM氯化锌2


1毫米氯化镁2


 


致谢


 


这项工作得到了美国国立卫生研究院(NIH)资助R01GM130909和俄勒冈州健康与科学大学的支持。该协议已从以前的工作中扩展(Ahuja和Whorton,2019)。


 


利益争夺


 


作者宣称没有利益冲突。


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Copyright Cahill et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Cahill, J., Ahuja, S. and Whorton, M. R. (2020). In vitro Measurement of CMP-Sialic Acid Transporter Activity in Reconstituted Proteoliposomes. Bio-protocol 10(6): e3551. DOI: 10.21769/BioProtoc.3551.
  2. Ahuja, S. and Whorton, M. R. (2019). Structural basis for mammalian nucleotide sugar transport. Elife 8: e45221.
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