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Feb 2020

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Quantitative Characterization of the Amount and Length of (1,3)-β-D-glucan for Functional and Mechanistic Analysis of Fungal (1,3)-β-D-glucan Synthase
通过(1,3)-β-d-葡聚糖量和长度的定量表征对真菌(1,3)-β-d-葡聚糖合酶进行功能和机理分析   

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Abstract

(1,3)-β-d-Glucan synthase (GS) is an essential enzyme for fungal cell wall biosynthesis that catalyzes the synthesis of (1,3)-β-d-glucan, a major and vital component of the cell wall. GS is a proven target of antifungal antibiotics including FDA-approved echinocandin derivatives; however, the function and mechanism of GS remain largely uncharacterized due to the absence of informative activity assays. Previously, a radioactive assay and reducing end modification have been used to characterize GS activity. The radioactive assay determines only the total amount of glucan formed through glucose incorporation and does not report the length of the polymers produced. The glucan length has been characterized by reducing end modification, but this method is unsuitable for mechanistic studies due to the very high detection limit of millimolar amounts and the labor intensiveness of the technique. Consequently, fundamental aspects of GS catalysis, such as the polymer length specificity, remain ambiguous. We have developed a size exclusion chromatography (SEC)-based method that allows detailed functional and mechanistic characterization of GS. The approach harnesses the pH-dependent solubility of (1,3)-β-d-glucan, where (1,3)-β-d-glucan forms water-soluble random coils under basic pH conditions, and can be analyzed by SEC using pulsed amperometric detection (PAD) and radioactivity counting (RC). This approach allows quantitative characterization of the total amount and length of glucan produced by GS with minimal workup and a d-glucose (Glc) detection limit of ~100 pmol. Consequently, this approach was successfully used for the kinetic characterization of GS, providing the first detailed mechanistic insight into GS catalysis. Due to its sensitivity, the assay is applicable to the characterization of GS from any fungi and can be adapted to study other polysaccharide synthases.

Keywords: (1,3)-β-D-Glucan synthase ((1,3)-β-D-葡聚糖合成酶), (1,3)-β-D-Glucan ((1,3)-β-D-葡聚糖), Product entrapment (产品滞留), Pulsed amperometric detection (脉冲安培检测), Fungal cell wall (真菌细胞壁)

Background

Characterization of polysaccharides is fundamental to our understanding of various biological processes, such as cell wall biosynthesis in bacteria, fungi, and plants, biofilm formation by microbes, and the formation of extracellular matrix in humans. Although the characterization of short and soluble oligosaccharides can be performed using a variety of methods including thin-layer chromatography, high-performance liquid chromatography, and mass spectrometry, significant technical challenges remain with respect to the characterization of long, biologically relevant polymers. As a result, molecular details regarding the size and mechanism of biosynthesis of large polysaccharides remain mostly unknown.


Over the past two decades, many methods have been developed to study charged and water-soluble polysaccharides. For example, hyaluronan has been characterized using paper chromatography (Tlapak-Simmons et al., 2005) or electrophoresis (Krupa et al., 2007), and bacterial peptidoglycan has been characterized by electrophoresis (Barrett et al., 2007); however, few methods are available for the characterization of charge-neutral and water-insoluble polysaccharides. Among such polysaccharides, (1,3)-β-d-glucan is an essential structural component of the fungal cell wall (Munro, 2013), and its biosynthetic enzyme, (1,3)-β-d-glucan synthase (GS), is a proven target of FDA-approved antifungal antibiotics (Douglas, 2001). Thus, characterizing the mechanism of catalysis and inhibition of GS is imperative to understand fungal cell wall biosynthesis and the mechanism of action and resistance against GS-targeted antifungal drugs. However, despite GS activity having been known since the 1980s (Shematek et al., 1980), detailed mechanistic characterization has not been possible due to the absence of appropriate methods to quantitatively evaluate the amount and length of (1,3)-β-d-glucan. Consequently, many fundamental aspects of this enzyme, such as the product length specificity, remain ambiguous.


GS has been characterized using radioactive assays (Shematek et al., 1980) that quantitate the total amount of water-insoluble (1,3)-β-d-glucan by determining the amount of incorporated d-glucose (Glc). While this method determines the overall activity of GS, it does not reveal the length of the polymer products. The length of GS products has been characterized using reducing end modification (Shematek et al., 1980), where the reducing end of (1,3)-β-d-glucan is reduced to sorbitol and the polymer hydrolyzed to monosaccharides. Subsequently, the length is determined based on the ratio between Glc and sorbitol. This analysis suggests that the average length of (1,3)-β-d-glucan produced by GS in a crude membrane preparation is 60–80 mer (Shematek et al., 1980); however, this approach requires a large amount of (1,3)-β-d-glucan, usually in millimolar quantities. Moreover, the method can underestimate the length by cleavage via a peeling reaction or other mechanisms during the workup or purification (Chhetri et al., 2020); therefore, there currently exists no suitable method for the detailed mechanistic characterization of GS.


More recently, size exclusion chromatography (SEC) has been used to characterize the length of water-insoluble polysaccharides; however, the chromatography conditions and detection methods frequently limit its use in detailed mechanistic investigations. For example, fungal cell wall glucan and chitin were analyzed by SEC after carboxymethyl derivatization with monochloroacetic acid using radioactivity as the detection method (Cabib and Duran, 2005; Cabib, 2009). This chemical derivatization solubilizes otherwise water-insoluble glucan and chitin and allows SEC analysis using an aqueous solvent. However, since the derivatization is not quantitative, the absolute length of the polymers was not determined, and only the relative lengths of glucan- and chitin-containing isolated cell walls were determined (Cabib et al., 2012).


SEC has also been applied to characterize bacterial cellulose synthase. In this case, cellulose was solubilized in dimethylacetamide containing 8% LiCl (w/v) and analyzed by gel permeation chromatography coupled to multi-angle light scattering (GPC-MALS) (McManus et al., 2018). This approach avoids the pitfalls of chemical derivatization and is potentially applicable to many other polysaccharides. However, the length was determined only under the steady-state of enzyme catalysis, and elongation of the cellulose polymer was not detectable, likely due to the limited sensitivity of refractive index detection.


Here, we report the protocol used for the determination of the amount and length of (1,3)-β-d-glucan using SEC in aqueous sodium hydroxide with pulsed amperometric detection (PAD) and radioactivity counting (RC). This protocol overcomes the aforementioned limitations: PAD and RC allow the characterization of the length and distribution of glucan polymers at sensitivities appropriate for the mechanistic characterization of GS, and the use of aqueous sodium hydroxide as a solvent allows solubilization of the otherwise water-insoluble (1,3)-β-d-glucan. The detection limit of this approach (~100 pmol) is greater than four orders of magnitude lower than reducing end modification previously reported for (1,3)-β-d-glucan characterization. One important limitation of this assay is that it requires reasonably pure GS. So far, the method does not work with crude membrane fractions due to the presence of proteins that perturb the migration of glucan through the SEC column. Therefore, in this protocol, we describe both the preparation of partially purified GS using the product entrapment method and the SEC assay. The product entrapment yields GS with 20–30% purity on SDS-PAGE and a specific activity of ~1,000 nmol/min/mg. While our characterization suggests that the impurities in this preparation does not affect the apparent function of GS (either glucan length or kinetics) (Chhetri et al., 2020), it is critical to remove proteins from the glucan samples by washing with SDS. This protocol has been used to study the mechanism of GS catalysis and successfully detected (1,3)-β-d-glucan elongation between ~1,000 and ~8,000 mer for the first time in the over 40-year-long history of GS (Chhetri et al., 2020). The facile chain length determination was also coupled to blocked substrate analogs to unambiguously determine the direction of polymerization, one of the key but challenging mechanistic questions in polysaccharide biosynthesis (Chhetri et al., 2020). These applications demonstrate the significance of the SEC-based GS activity assays. Similar approaches could be adapted to study the activities of other polysaccharide synthases such as glycogen and hyaluronan synthases.


Materials and Reagents

  1. 50 ml Falcon tubes (VWR, catalog number: 89039-656)

  2. 70 ml polycarbonate bottle assembly, 38 × 102 mm (Beckman Coulter, catalog number: 355622)

  3. 3.5 ml open-top thick-wall polycarbonate ultracentrifuge tubes, 13 × 51 mm (Beckman Coulter, catalog number: 349622)

  4. Acclaim SEC-1000 column 7 µm 4.6 × 300 mm (Thermo Fisher, catalog number: 079724)

  5. Acclaim SEC-1000 Guard Column 7 µm 4.6 × 33 mm (Thermo Fisher, catalog number: 082739)

  6. Trans-Blot Turbo RTA Transfer Kit LF PVDF (Bio-Rad, catalog number: 1704274)

  7. MultiScreenHTS FC Filter Plate, 1.2/0.65 µm (Millipore Sigma, catalog number: MSFCN6B5)

  8. MultiScreenHTS Vacuum Manifold (Millipore Sigma, catalog number: MSVMHTS00)

  9. 6” wood handle cotton swab (VWR, catalog number: 89031-270)

  10. Peptic digest of animal tissue (Meat peptone; Criterion, catalog number: C7482)

  11. Saccharomyces cerevisiae BY4741 (ATCC, catalog number: 201388)

  12. Yeast extract (Criterion, catalog number: C7342)

  13. d-Glucose (VWR, catalog number: BDH9230)

  14. Agar (Acros, catalog number: 443570010)

  15. 0.5 mm glass beads (Scientific Industries, catalog number: SI-BG05)

    Note: Prior to use, the glass beads should be cleaned with bleach, soaked overnight, and subsequently washed with deionized water until the pH of the water wash is neutral based on pH testing strips, usually after 10 washes. Finally, the beads should be washed twice with isopropanol and dried overnight.

  16. Liquid nitrogen (Airgas, catalog number: NI 240LT22)

  17. Ethylenediaminetetraacetic acid, proteomics grade (EDTA; VWR, catalog number: M101)

  18. Sodium chloride (EMD Millipore, catalog number: SX0420-5)

  19. Phenylmethylsulfonyl fluoride (PMSF; Acros Organics, catalog number: 215740100)

  20. Tris base (Sigma, catalog number: T6066)

  21. β-Mercaptoethanol (VWR, catalog number: M131)

  22. Glycerol (EMD Millipore, catalog number: GX0185-6)

  23. Pierce 660-nm Assay (Thermo Scientific, catalog number: 1861426)

  24. EZQ Protein Quantitation Kit (Thermo Scientific, catalog number: R33200)

  25. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; VWR, catalog number: 0465)

  26. Cholesteryl hemisuccinate Tris Salt (CHS; Anatrace, catalog number: CH210)

  27. Dithiothreitol (DTT; VWR, catalog number: 97061-338)

  28. Guanosine 5′-[γ-thio]triphosphate tetralithium salt (GTPγS; Sigma, catalog number: G8634)

  29. 4–20% Mini-PROTEAN TGX Stain-Free Protein Gels, 10 well, 50 µl (BioRad, catalog number: 4568094)

  30. UDP-[U-14C]-d-Glucose (UDP-[14C]Glc; American Radioactive Chemicals, catalog number: ARC0154)

  31. UDP-d-Glucose disodium salt (UDP-Glc; Carbosynth, catalog number: MU08960)

  32. Sodium dodecyl sulfate (SDS; Sigma, catalog number: 75746)

  33. Ethanol 190 proof (Koptec, catalog number: V1101)

  34. Trichloroacetic acid (TCA; EMD Millipore, catalog number: TX1045)

  35. 50% (w/w) sodium hydroxide solution (NaOH; Fisher Chemical, catalog number: SS254-500)

  36. ASTM Type I water (Ricca, catalog number: 9150-5)

  37. Isopropanol (Fisher Chemical, catalog number: A416P).

  38. Pullulan standard (Showa Denko K.K., catalog number: P-82)

  39. Primary antibodies:

    1. Anti-Fks1p (a gift from J.-P Latgé, Institut Pasteur) (Beauvais et al., 2001)

    2. Anti-Rho1p (a gift from Y. Ohya, University of Tokyo) (Qadota et al., 1996)

  40. Goat anti-rabbit IgG-HRP secondary antibody (Southern Biotech, catalog number: 4030-05)

  41. Radiance HRP substrate for CCD imaging (Azure Biosystems, catalog number: AC2101)

  42. Dionex ED electrochemical detector disposable electrodes, gold on PTFE (Thermo Fisher, catalog number: 066480)

  43. 40% sterile glucose solution (see Recipes)

  44. Yeast Peptone Dextrose (YPD) media (see Recipes)

  45. 0.5 M EDTA stock, pH 8.0 (see Recipes)

  46. Breaking Buffer (see Recipes)

  47. Membrane Buffer (see Recipes)

  48. KF Mix (see Recipes)

  49. 2× Assay Buffer (see Recipes)

Equipment

  1. 500 ml baffled flasks (Chemglass, catalog number: CLS-2044-05)

  2. 2.8 L baffled flasks (Chemglass, catalog number: CLS-2022)

  3. -80°C freezer (VWR symphony Ultra-Low Temperature Freezer, model: DW-86L638H)

  4. -20°C freezer (VWR, catalog number: 82027-388)

  5. Tweezers (VWR, catalog number: 82027-388)

  6. Magnetic stir bar (VWR, catalog number: 58948-98)

  7. 7 ml Dounce tissue grinder with type A and B pestles (Kimble, catalog number: D9063)

  8. Sample pestle with a tube, 1.5 ml (Research Products International, catalog number: 199226)

  9. Bead beater (Biospec, model: BeadBeater)

  10. Type 45-Ti rotor (Beckman Coulter, model: 339160)

  11. Beckman L7-55 Ultra-speed Centrifuge (Beckman, model: L7-55)

  12. TLA 100.3 Fixed-Angle Rotor (Beckman Coulter, model: 349490)

  13. Beckman Optima TL-100 Ultracentrifuge (Beckman, model: Optima TL-100)

  14. Stir plate (Labnet Accuplate Analog Magnetic Stirrer, model: D0310)

  15. BioVortexer (Biospec, model: 1083)

  16. Water bath sonicator (NEY, model: ULTRAsonik 28H)

  17. ICS-5000+ DC Chromatography/Detector System (Thermo Fisher, model: ICS-5000+)

  18. Scintillation counter (Beckman Coulter, model: LS 6500)

  19. Fraction collector (Pharmacia Biotech, model: FRAC-100)

Software

  1. Microsoft Excel (Microsoft, https://www.microsoft.com/en-us/microsoft-365/excel)

  2. Chromeleon 7.2 Thermo Scientific Dionex Chromeleon Chromatography Data System (Thermo Fisher Scientific, https://www.thermofisher.com/order/catalog/product/CHROMELEON7#/CHROMELEON7)

Procedure

  1. Saccharomyces cerevisiae culture

    1. Streak S. cerevisiae from glycerol stocks onto fresh YPD agar plates (2% agar) and incubate the plates at 30°C for 2–3 days. Plates usually last ~2 weeks.

    2. Prepare 2 × 200 ml YPD media in 500 ml baffled flasks and 12 × 1.5 L YPD media in 2.8 L baffled flasks.

    3. Pick a single colony from the plate and inoculate 2 ml YPD media in a culture tube. Incubate cultures with 200 rpm shaking at 30°C for ~8 h. This is usually performed early in the morning of the day before the large-scale culture.

    4. At the end of the day, inoculate 2 × 200 ml YPD media in 500 ml baffled flasks with 1 ml pre-culture started in the morning, and incubate with 200 rpm shaking overnight at 30°C. Cultures should reach OD600 of 10–12.

    5. The next morning, add an aliquot (30 ml) of the overnight pre-culture to each of 12 flasks containing 1.5 L YPD media, resulting in an initial OD600 of 0.2–0.3. Incubate the cultures with 200 rpm shaking at 30°C until the OD600 reaches 0.8–1.2; typically, this takes ~6 h from inoculation.

    6. Harvest cells by centrifugation at 5,000 × g, 8°C for 25 min.

    7. Wash the pellets once with 10 mM EDTA buffer (pH 8.0) (~30 ml per 20 g cell paste).

    8. Determine the wet cell weight and store pellets in a -80°C freezer until use. Typically, 50–60 g wet cell paste is obtained.


  2. GS preparation

    1. Membrane fraction preparation

      1. Thaw the frozen cell pellets on ice.

      2. Resuspend the cell pellets in 150 ml ice-cold breaking buffer.

      3. Transfer the suspension to the bead beater chamber with ~150 ml ice-cold, clean 0.5 mm glass beads. Top up the chamber with buffer to minimize the available air space once the chamber is assembled, and ensure that the chamber is well covered by the ice-water. Lyse cells by bead beating with 5 × 1 min pulses, with a 10-min rest between each pulse.

      4. Centrifuge at 1,500 × g for 15 min at 4°C to clear the lysate; separate the clarified lysate.

      5. Resuspend the insoluble material in breaking buffer (100 ml) and repeat Steps B1c and B1d to ensure breaking > 95% of cells.

      6. Combine the clarified lysates from the two bead beating cycles and transfer to 70-ml ultracentrifuge tubes.

      7. Centrifuge the lysate at 100,000 × g for 1 h at 4°C. A brown pellet should be visible at the bottom of the tubes. This pellet consists mostly of membrane proteins, lipids, and any other molecules such as glycans tightly associated with them.

      8. Resuspend the pellets in ~80 ml membrane buffer using a 7-ml Dounce homogenizer and the loose pestle (labeled by the manufacturer as “A”) for ~10 strokes, or until no more large pellet chunks remain, and then the tight pestle (labeled by the manufacturer as “B”) for an additional ~10 strokes to ensure thorough resuspension of the membrane pellet.

      9. Determine the protein concentration using the 660 nm Assay Kit and BSA standards according to the manufacturer’s protocol. To ensure appropriate reading, the resuspended membrane fraction is typically diluted 50–200-fold.

      10. Adjust the final volume of the resuspended membrane fractions with membrane buffer to a final concentration of ~5 mg/ml protein. Typically, an 18 L culture yields ~100 ml resuspended membrane fraction.

      11. Flash-freeze 20 ml aliquots in 50 ml Falcon tubes in liquid nitrogen, and store in a -80°C freezer until ready to proceed to the next step (Figure 2, lane 1).

        Note: We call this resuspension a “membrane fraction,” which can be stored with minimal loss of activity if freeze–thawing is minimized.

    2. Detergent solubilization

      1. Thaw aliquots of the membrane fraction on ice and transfer to an appropriately sized, chilled Erlenmeyer flask containing a stir bar and place in an ice-water bath. Due to the ability to store the purified GS, we suggest using the entire prepared membrane fraction to obtain a 2.5-ml batch of purified GS.

      2. While stirring the thawed membrane fractions, add GTPγS (27.5 μM final concentration), DTT (6.88 mM), and NaCl (192 mM).

      3. Add a 10% (w/v) CHAPS/2% (w/v) CHS solution dropwise to achieve final concentrations of 0.688% CHAPS and 0.138% CHS.

      4. Allow the suspension to stir for 30 min in an ice-water bath using a stir bar and stir plate. It is important to mix the solution gently to prevent inactivation of GS; thus, set the stirring such that the surface of the solution forms a small vortex without any bubbles. This step can also be performed using a rotator.

      5. Transfer the solution to 70-ml centrifuge tubes and balance.

      6. Centrifuge at 100,000 × g for 30 min at 4°C.

      7. Retain the supernatant as the “detergent-solubilized fraction” (Figure 2, lane 2). If this solution is not immediately used for the product entrapment purification, it can be flash-frozen and stored at -80°C with minimal loss of activity.

    3. Product entrapment purification (see Figure 1 for a flow chart of the procedure)



      Figure 1. Flow chart of the product entrapment purification of GS. Steps in the protocol and the corresponding SDS-PAGE lanes in Figure 2 are described in parentheses. Key fractions containing GS are bolded.


      1. Add a 0.125× volume of KF mix and UDP-Glc (4 mM final concentration) to the detergent-solubilized fraction. Incubate at 30°C for 20–30 min. Periodically invert the tube to ensure sufficient mixing.

      2. Chill the reaction on ice for 15 min. Insoluble glucan should become visible in the solution.

      3. Collect the insoluble glucan and bound proteins by centrifugation at 5,000 × g for 5 min at 4°C (see Figure 3 for an image of the pellet). Save an aliquot of the supernatant (product entrapment unbound fraction; Figure 2, lane 3) for protein concentration determination (Step B3o) and SDS-PAGE analysis (Step B3p).

      4. Wash the resulting glucan/protein pellet by resuspension in 1.5 ml wash buffer and homogenization using the 7-ml homogenizer and the tight pestle “B”.

      5. Collect the glucan/protein pellet by centrifugation at 5,000 × g for 5 min at 4°C. Save the supernatant for later characterization.

      6. Repeat Steps B3d and B3e three more times.

      7. Resuspend the pellet in 1.5 ml wash buffer as described in Step B3d, transfer the resuspension to a 3.5-ml ultracentrifuge tube, and collect the pellet by ultracentrifugation at 400,000 × g, 4°C for 10 min. Combine the supernatant from Steps B3e and B3g and store as the “product entrapment pellet wash fraction” (Figure 2, lane 4) for later characterization.

      8. Release GS from the insoluble glucan by resuspending the pellet in 0.5 ml extraction buffer, followed by homogenization for ~10 s using the hand-held electronic homogenizer (BioVortexer).

      9. Incubate the resuspension overnight at 4°C.

      10. Centrifuge the resuspension at 400,000 × g, 4 °C for 10 min and separate the supernatant and pellet.

      11. Resuspend the pellet from Step B3j in 0.5 ml extraction buffer, followed by homogenization for ~10 s using the hand-held electronic homogenizer.

      12. Incubate the suspension for 10 min on ice, centrifuge the resuspension at 400,000 × g (100,000 rpm using the TLA100.3 rotor), 4°C for 10 min, and separate the supernatant and pellet.

      13. Repeat Steps B3k and B3l three more times.

      14. Combine the supernatants from Steps B3j and B3l to obtain ~2.5 ml “product entrapment purified GS” (Figure 2, lane 6).

      15. Resuspend the pellet in 0.5 ml extraction buffer as described in Step B3k, and adjust the volume to 1.0 ml with extraction buffer to obtain the “product entrapment residual glucan bound fraction” (Figure 2, lane 5).



        Figure 2. Characterization of GS purification. SDS-PAGE (A) and Western blots using the anti-Fks1p (B) or anti-Rho1p (C) antibody. Each lane was loaded with 5.3 μg protein. Lane: 1, membrane fraction (see Procedure section B1k); 2, detergent-solubilized fraction (see Procedure section B2g); 3, product entrapment unbound fraction (see Procedure section B3c); 4, product entrapment pellet wash (see Procedure section B3g); 5, product entrapment residual glucan bound fraction (see Procedure section B3n); 6, product entrapment purified GS (see Procedure section B3m). The red arrow indicates the band corresponding to Fks1p, and the green arrow indicates the band corresponding to Rho1p. Reprinted with permission from Chhetri et al. (2020).



        Figure 3. Image of a glucan pellet formed after product entrapment Step B3c. The pellet was prepared from 30–40 ml product entrapment reaction (Step B3a) in a 50-ml centrifuge tube.


      16. Determine the protein concentration at each step of the purification using the EZQ Assay Kit and BSA as the standard. Typically, this protocol yields 0.73 ± 0.12 mg GS from 18 L yeast culture. Knowledge of the protein concentration is critical to performing the GS activity assay and determining the specific activity of GS.

      17. Analyze 1–10 µg protein from each purification step using SDS-PAGE with a 4–20% stain-free gel (Bio-Rad). Determine the purity of GS according to densitometry of the Fks1p band relative to the other bands (see Figure 2A). We typically obtain GS at 20–30% purity. Western blotting can also be used to ensure the copurification of Fks1p and Rho1p, two known components of GS.

      18. Flash-freeze and store aliquots of the purified GS at -80°C.


  3. Typical enzyme activity assays

    1. Prepare stock and working solutions of UDP-[14C]Glc

      1. To prepare a stock solution of radioactive UDP-Glc, dilute 500 µl UDP-[14C]Glc in 70% EtOH to a total volume of 3.5 ml with diH2O by washing the vial with at least 3 × 500 µl volumes of diH2O. Divide the solution into 4 tubes and freeze at -20°C. Once frozen, lyophilize the tubes overnight to dryness. Prepare a 50 mM solution of non-radioactive UDP-Glc and add 3 µmol (60 µl) to each tube, resulting in UDP-Glc solutions of ~10,000 cpm/nmol. Typically, we prepare a stock solution with high radioactivity and dilute based on the purpose of the experiment.

      2. Dilute 1 µl resulting solutions 1,000× with water.

      3. Determine the concentration of the diluted UDP-[14C]Glc prepared in Step C1b using the absorbance at 261 nm and the molar extinction coefficient (ϵ) of uridine at 261 nm (10.1 mM/cm).

      4. To determine the radioactivity of the diluted UDP-[14C]Glc used in Step C1b, transfer 0, 2, 4, 6, 8, 10, 20, 40, 60, 80, 100, and 200 µl diluted sample to scintillation vials, add 3 ml scintillation fluid, and count the radioactivity on a liquid scintillation counter (LSC).

      5. Plot the amount (nmol) of UDP-[14C]Glc according to the radioactivity (cpm) of each sample. The specific radioactivity can be calculated by the slope of a linear fit (cpm/nmol).

      6. To prepare a working solution of UDP-[14C]Glc, dilute the stock solution with non-radioactive UDP-Glc and determine the specific radioactivity by following the Steps C1b–C1e. In our experience, UDP-[14C]Glc with ~800 cpm/nmol specific radioactivity allows detection of glucan formation from 5 µl reaction solution with as short as a 30-s incubation.

    2. Thaw aliquots of stored GS on ice. We typically perform activity assays with a 30–50 µg/ml final concentration of protein in a 30-µl scale.

    3. Prepare a 2× assay buffer as described in the Recipe section.

    4. Incubate the 2× assay buffer and GS in a 30°C water bath for 15 min.

    5. Prepare filter plates for quenching and filtering the insoluble glucan. Add 200 µl 10% w/v TCA to each filter plate well. Each well will be used to quench each timepoint.

    6. Initiate reactions by adding 1 volume of GS to 1 volume of 2× assay buffer. Mix the solution by pipetting up and down as well as closing the caps of the tubes and gently flicking and briefly vortexing before returning to the water bath. Note that this is important for reproducible results as the GS is stored in 30% glycerol, which requires thorough mixing to yield a homogeneous solution.

    7. Incubate the reaction at 30°C.

    8. At each time point, transfer a 5–20 µl aliquot of the reaction to the filter plate wells containing 200 µl 10% w/v TCA (prepared in Step C5). Mix the solution by pipetting up and down.

    9. After quenching all the time points, apply a vacuum using the vacuum manifold to filter the quenched solution. Wash the filter with 3 × 200 µl 10% TCA, followed by 2 × 200 µl EtOH to remove unincorporated UDP-[14C]Glc.

    10. The filters can be allowed to dry overnight or can be analyzed immediately. Remove the plastic support from the bottom of the plate and position the well over an empty scintillation vial. Using a cotton swab, push the filter through the PVDF membrane into the scintillation vial. Break the swab handle so that the vial cap can be closed. If the PVDF membrane remains attached to the plate, transfer to the scintillation vial using a pair of tweezers.

    11. Add 3 ml scintillation fluid, tightly close the vial, and vortex and shake to ensure that the filter is submerged.

    12. Determine the radioactivity of each vial. We set the scintillation counter to a preset time of 3 min, or a % error of 5.0, whichever was shorter.


  4. Quantitative detection of GS product by SEC-PAD analysis

    1. Thaw the frozen GS solution on ice.

    2. Prepare a 2× assay buffer.

    3. Preincubate both the GS and the 2× assay buffer for 15 min in a 30°C water bath.

    4. Prepare the filter plates for quenching and filtering the insoluble glucan. Add 100 µl or an appropriate volume of 10% w/v TCA to each well of the plate. See Step D7.

    5. Initiate the reaction by mixing equal volumes of GS and 2× assay buffer, and mix well by pipetting, flicking, and brief vortexing before returning to the water bath. Note that this is important for reproducible results as the GS is stored in 30% glycerol, which can result in poor mixing and irreproducible results. See Step C6.

    6. Incubate the reaction at 30°C.

    7. At defined time points, remove two aliquots and quench separately.

      1. Quench the first aliquot by mixing 5 µl reaction mixture with 100 µl 10% TCA in one of the filter plate wells. This quenched solution will be used to determine the total amount of glucan.

      2. Quench the second aliquot of reaction mixture (25–200 µl) with 5–10 equivalents of 2% (w/v) SDS solution in a separate well. This quenched solution will be used for length determination by SEC. Do not use acid quenching for length determination since acid-denatured membrane proteins are difficult to remove and interfere with the SEC analysis.

        Note: As the filter plate wells only fit ~250 µl volume, for larger aliquots, reactions need to be quenched in larger tubes and transferred to a single well for workup.

    8. Wash the well(s) from Step D7a with 3 × 200 µl 10% TCA, followed by 3 × 200 µl EtOH.

    9. Wash the well(s) from Step D7b with 5 × 200 µl 2% SDS, 5 × 200 µl H2O, and 5 × 200 µl EtOH. The wash with SDS is critical for removing proteins that interact with glucan since they interfere with the SEC analysis.

    10. Dry the filters in the wells overnight under a vacuum.

    11. Transfer the filters from Step D8 to liquid scintillation vials for LSC to determine the total amount of glucose incorporated in the reaction at each timepoint. See Step C10.

    12. Transfer the filters form Step D9 to a 1.5-ml screw top vial and add 200 µl 1 M NaOH.

      Note: Use freshly diluted NaOH from a 50% w/w solution.

    13. Briefly centrifuge to ensure that the filter is well submerged in the solution, and then sonicate in an ice-water bath for 10 min.

      Note: Longer sonication or sonication at higher temperatures can result in more significant degradation of the glucan polymers and affect the final length calculation and elution profile of the glucan product.

    14. Briefly centrifuge the mixture again and transfer 100 µl to a freash 1.5-ml vial.

    15. Using a wide-bore pipet tip (or regular pipet tip with the extremity cut off to increase the diameter), break apart the filter and resuspend in the solution.

    16. Briefly centrifuge the mixture, remove an additional ~80 µl from the tube, and combine with the solution in Step D14.

    17. Centrifuge the combined solution from Step D16 at 16,000 × g for 10 min at 4°C to remove any particulates.

    18. Transfer 150 µl supernatant to an HPLC vial insert for SEC analysis.

    19. Transfer a 5-µl aliquot of the supernatant to a scintillation vial and analyze by LSC to determine the glucan recovery yield. We typically recover 60–90% of the radioactivity.

    20. Analyze 25 µl each sample by SEC-PAD-RC using the ICS-5000+ DC Chromatography/Detector System equipped with an Acclaim SEC-1000 column at 30°C. Chromatography is performed by isocratic elution with 10 mM NaOH at a flow rate of 0.3 ml/min and monitored by PAD (Gold, Carbo, Quad waveform).

    21. Using a fraction collector, fractionate the elution every 0.5 min during 5–15 min of the chromatography, transfer 120 µl fraction to scintillation vials, and analyze by LSC to determine the radioactive glucose in each fraction.

    22. To determine a standard curve for calibration of the SEC, inject pullulan standards P-82 (Showa Denko K.K.) and analyze under the same conditions as described in Step D20.

    23. Export the chromatography data from the Chameleon software into Excel to calculate the properties of the samples, as detailed in section Data analysis B.

Data analysis

  1. SEC standard curve

    1. Determine the retention time of the pullulan standards at the peak maximum.

    2. Using the peak maximum molecular weight (Mp) for the pullulan standards provided by the manufacturer, plot the log (Mp) of the standard vs. retention time of the pullulan standards to generate a linear standard curve (see Figure 4).



      Figure 4. Calibration curve of SEC-PAD using commercial pullulan as a standard. A. Representative chromatograms of the commercial pullulan standards P-800, P-100, and P-10. Numbers in parentheses represent the Mp of the standards. B. A calibration curve with pullulan standards compared with the GS product. Modified with permission from Chhetri et al. (2020).


  2. Analysis of SEC-PAD data

    1. Export the SEC-PAD data for the GS assays in text format and plot the chromatogram (Figure 5A).

    2. Calculate the average retention time (ti, Figure 5B) between every two consecutive data points in the SEC-PAD chromatogram. For our experiments, this resulted in 2,400 slices (PAD data collected every 1/120th s).

    3. Using the calibration curve determined in Step A2, calculate the molecular weight (Mi) at the average retention time (ti) for each slice (Figure 5B).

    4. Calculate the average PAD signal (Pi) for each slice by taking the average of the two PAD data points in each slice (Figure 5B).

    5. Determine the area of each slice (Ai) using the following equation: Ai = Mi × Pi.

    6. To determine the total area of the peak of interest (A, Figure 5C), calculate the sum of Ai for all the slices in the peak: A = ∑Ai.

    7. To determine the weight fraction (Wi) of each slice i of the peak of interest, divide Ai for each slice by the total area of the peak (A).

    8. To determine the weight average molecular weight (Mw), calculate the sum of Mi × Wi for all the slices in the peak: Mw = ∑(Mi × Wi).

    9. To determine the number average molecular weight (Mn), divide 1 by the sum of Wi/Mi for all the slices in the peak: Mn = 1/∑(Wi/Mi).

    10. Calculate the polydispersity index (PDI) for the sample: PDI = Mw/Mn.

    11. The Mp, Mw, and Mn can be converted to the peak maximum degree of polymerization (Xp), weight average degree of polymerization (Xw), and number average degree of polymerization (Xn) by dividing each by the mass of polymerized glucose (C6H10O5, 162.14).



      Figure 5. Determination of glucan molecular weight parameters from an SEC-PAD chromatogram. A. Chromatogram plotted with a retention time as the x-axis. B. Magnified view of the chromatogram in A with molecular weight as the x-axis. Representative slice i is highlighted. (C) Chromatogram plotted with molecular weight as the x-axis. The peak area used for the determination of the polymer length parameters is highlighted.


  3. Analysis of radioactivity data

    1. Enter the LSC readout for the fractions in cpm in MS Excel. Adjust for the delay in the retention times of RC relative to that of PAD caused by the tubing between the PAD and the fraction collector.

    2. Using the specific radioactivity of UDP-[14C]Glc, convert the cpm into nmol to determine the amount of Glc incorporated into (1,3)-β-d-glucan. Remember to adjust for the difference in the quenched volumes, dilutions, and yield recovery during the workup for each data point.


  4. Preparation of the overlay of SEC-PAD and radioactivity chromatograms

    Overlay of the PAD and RC chromatograms helps visual assessment of the data. For the overlay, we established a quantitative approach as described below.

    1. Plot the PAD and RC chromatograms as described in Sections B and C (Figure 6A).

    2. Calculate the difference (Di) between the PAD value and radioactivity at each data point of the RC chromatogram.

    3. Calculate the sum of the square of Di, ∑(Di)2 for the peak of interest.

    4. Determine the scaling factor for the PAD chromatogram that minimizes ∑(Di)2. For example, a scaling factor of 5 was determined for Figure 6A since 5× magnification of the PAD chromatogram minimized ∑(Di)2.

    5. Using the scaling factor, adjust the y-axis scale in the PAD chromatogram (Figure 6B).



      Figure 6. Preparation of the overlay of SEC-PAD and RC chromatograms using least-squares function approximation. A. Original unmodified PAD (solid line) and RC (diamonds) chromatograms. Di represents the difference between the PAD and RC values at data point i. B. Scale-adjusted overlay of the PAD (solid line) and RC (diamonds) chromatograms. The y-axis of the PAD chromatogram was adjusted by minimizing the ∑(Di)2 in (A).

Notes

  1. When preparing the 2× assay buffer, remember to account for the 2-fold dilution of the UDP-Glc upon mixing with GS.

  2. Store the 50% (w/w) NaOH solution with minimal exposure to CO2 to minimize contamination with sodium carbonate and bicarbonate that interfere with PAD analysis.

  3. Make the SEC-PAD-RC eluents with degassed ASTM Type I water to minimize ion contaminants that can affect the background signal of the PAD response.

Recipes

  1. 40% sterile glucose solution

    400 g (40% w/v) d-glucose

    Adjust to 1 L with diH2O

    Filter-sterilize

  2. Yeast Peptone Dextrose (YPD) media

    20 g peptic digest of animal tissue (meat peptone)

    10 g yeast extract

    For solid media, add 20 g agar

    Adjust to 950 ml with diH2O

    Autoclave

    Add 50 ml (2% w/v) 40% sterile glucose solution (filter sterilized)

  3. 0.5 M EDTA stock, pH 8.0

    186.12 g EDTA disodium dihydrate salt

    Adjust to 800 ml with diH2O

    Cool solution to 4°C in an ice-water bath

    Adjust pH to 8.0 with NaOH

    Adjust volume to 1,000 ml with diH2O

  4. Breaking Buffer

    29.22 g (0.5 M) sodium chloride

    20 ml (10 mM) 0.5 M EDTA stock, pH 8.0

    Adjust volume to 1,000 ml with diH2O

    Add 10 ml (1 mM) 100× PMSF stock solution in 2-propanol (storable at -20°C) immediately prior to use

  5. Membrane Buffer

    50 mM Tris, pH 8.0

    10 mM EDTA, pH 8.0

    333.3 ml (33% v/v) glycerol

    Adjust volume to 1,000 ml with diH2O

    Add 70.92 μl (1 mM) β-mercaptoethanol and cool to 4°C prior to use

  6. KF Mix

    200 mM Tris stock, pH 8.0

    4 mM EDTA stock, pH 8.0

    871.5 mg (0.3 M) KF

    Adjust volume to 50 ml with diH2O

  7. 10% CHAPS, 2% CHS stock

    5 g (10% w/v final) CHAPS

    Dissolve in 40 ml diH2O with sonication

    Add 1 g (2% w/v final) CHS Tris salt

    Note: CHS Tris salt was found to be more soluble than CHS.

    Sonicate ~1 h then rock overnight at RT

    Adjust volume to 50 ml with diH2O

    Store at -20°C

  8. Extraction Buffer

    48 ml membrane buffer

    2 ml 10% CHAPS (0.4% final) and 2% CHS stock (0.08% final)

    1 mM DTT

    Add a stock solution of GTPγS at 4 μM final concentration

  9. Wash Buffer

    50 ml extraction buffer

    5 mM UDP-Glc

  10. 2× Assay Buffer

    10 mM (or any appropriate concentration) UDP-[U-14C]Glc (with specific activity of ∼800 cpm/nmol, or adjusted as necessary for experiments)

    150 mM Tris, pH 7.5

    1.5 mM EDTA

    1.5% (w/v) BSA

    50 mM KF

    0.04 mM GTPγS

  11. 10% TCA

    100 g TCA

    Add diH2O up to 1 L

  12. 2% SDS

    2 g (2% w/v) SDS

    Add diH2O up to 100 ml

Acknowledgments

This work was funded in part by Duke University Medical Center and the National Institute of General Medical Sciences Grant R01 GM115729 (to K.Y.). A.L. was supported by the Tri-Institutional Molecular Mycology and Pathogenesis Training Program from the National Institute of Allergy and Infectious Diseases (T32AI052080). The authors thank Dr. Yoshikazu Ohya at the University of Tokyo for the anti-Rho1p antibody and Dr. Jean-Paul Latge at the Institut Pasteur for the anti-Fks1p antibody.

  This protocol is a detailed version of the protocol used in a recent publication by the authors (Chhetri et al., 2020).

Competing interests

The authors declare no conflicts of interest.

References

  1. Barrett, D., Wang, T. S., Yuan, Y., Zhang, Y., Kahne, D. and Walker, S. (2007). Analysis of glycan polymers produced by peptidoglycan glycosyltransferases. J Biol Chem 282(44): 31964-31971.
  2. Beauvais, A., Bruneau, J. M., Mol, P. C., Buitrago, M. J., Legrand, R. and Latge, J. P. (2001). Glucan synthase complex of Aspergillus fumigatus. J Bacteriol 183(7): 2273-2279.
  3. Cabib, E. (2009). Two novel techniques for determination of polysaccharide cross-links show that Crh1p and Crh2p attach chitin to both beta(1-6)- and beta(1-3)glucan in the Saccharomyces cerevisiae cell wall. Eukaryot Cell 8(11): 1626-1636.
  4. Cabib, E. and Duran, A. (2005). Synthase III-dependent chitin is bound to different acceptors depending on location on the cell wall of budding yeast. J Biol Chem 280(10): 9170-9179.
  5. Cabib, E., Blanco, N. and Arroyo, J. (2012). Presence of a large beta(1-3)glucan linked to chitin at the Saccharomyces cerevisiae mother-bud neck suggests involvement in localized growth control. Eukaryot Cell 11(4): 388-400.
  6. Chhetri, A., Loksztejn, A., Nguyen, H., Pianalto, K. M., Kim, M. J., Hong, J., Alspaugh, J. A. and Yokoyama, K. (2020). Length Specificity and Polymerization Mechanism of (1,3)-beta-d-Glucan Synthase in Fungal Cell Wall Biosynthesis. Biochemistry 59(5): 682-693.
  7. Douglas, C. M. (2001). Fungal beta(1,3)-D-glucan synthesis. Med Mycol 39 (Suppl 1) 55-66.
  8. Krupa, J. C., Shaya, D., Chi, L., Linhardt, R. J., Cygler, M., Withers, S. G. and Mort, J. S. (2007). Quantitative continuous assay for hyaluronan synthase. Anal Biochem 361(2): 218-225.
  9. McManus, J. B., Yang, H., Wilson, L., Kubicki, J. D. and Tien, M. (2018). Initiation, Elongation, and Termination of Bacterial Cellulose Synthesis. ACS Omega 3(3): 2690-2698.
  10. Munro, C. A. (2013). Chitin and glucan, the yin and yang of the fungal cell wall, implications for antifungal drug discovery and therapy. Adv Appl Microbiol 83: 145-172.
  11. Qadota, H., Python, C. P., Inoue, S. B., Arisawa, M., Anraku, Y., Zheng, Y., Watanabe, T., Levin, D. E. and Ohya, Y. (1996). Identification of yeast Rho1p GTPase as a regulatory subunit of 1,3-beta-glucan synthase. Science 272(5259): 279-281.
  12. Shematek, E. M., Braatz, J. A. and Cabib, E. (1980). Biosynthesis of the yeast cell wall. I. Preparation and properties of beta-(1 leads to 3)glucan synthetase. J Biol Chem 255(3): 888-894.
  13. Tlapak-Simmons, V. L., Baron, C. A., Gotschall, R., Haque, D., Canfield, W. M. and Weigel, P. H. (2005). Hyaluronan biosynthesis by class I streptococcal hyaluronan synthases occurs at the reducing end. J Biol Chem 280(13): 13012-13018.


简介

[摘要] (1,3)-β- d葡聚糖合成酶(GS)是一个必不可少的真菌细胞壁的生物合成,其催化(1,3)-β-合成酶d葡聚糖,一个重要的和关键的成分细胞壁。GS是包括FDA批准的棘皮菌素衍生物在内的抗真菌抗生素的可靠靶点;ħ H但是,功能和GS的机制在很大程度上仍然未表征由于缺少信息活性测定的小号 。以前,放射性分析和还原末端修饰已用于表征GS活性。放射性测定仅确定通过葡萄糖掺入形成的葡聚糖的总量,并且没有报告所产生的聚合物的长度。葡聚糖长度的特点是还原端修饰,但第是方法不适用于机理研究由于毫摩尔的非常高的检测限AR量和技术的劳动密集。因此,GS催化的基本方面,例如聚合物长度特异性,仍然是模棱两可的。我们已经开发了一种基于尺寸排阻色谱(SEC)的方法,该方法可以对GS进行详细的功能和机理表征。该方法利用了(1,3)-β- d-葡聚糖的pH依赖性溶解度,其中(1,3)-β- d-葡聚糖在碱性pH条件下形成水溶性无规卷曲,可以通过SEC分析使用脉冲安培检测(PAD)和放射性计数(RC)。这种方法允许通过GS以最小的后处理和产生的葡聚糖的总量和长度的定量表征一个d -葡萄糖(GLC)检测限的〜100pmol的。因此,成功使用这种方法的GS的运动特性,provid荷兰国际集团首次详细机械洞察GS催化作用。因其敏感性,该试验是适用于所述characteriz的通货膨胀从任何真菌GS和可以适于研究其他多糖合酶。


[背景]多糖的表征是我们的各种生物过程的理解基本,如细菌,真菌细胞壁生物合成,和植物,由微生物生物膜的形成,和细胞外基质的在人类中的形成。尽管可以使用多种方法(包括薄层色谱法,高效液相色谱法和质谱法)对短而可溶性的寡糖进行表征,但是在表征长的,生物学相关的聚合物方面仍然存在重大的技术挑战。结果,关于大多糖的生物合成的大小和机理的分子细节仍然是未知的。

在过去的二十年中,已经开发出许多方法来研究带电荷的和水溶性的多糖。例如,乙酰透明质酸已通过纸层析(Tlapak-Simmons等,2005 )或电泳(Krupa等,2007 )进行了表征,细菌肽聚糖的电泳已得到了表征(Barrett等,2007 )。^ h H但是,一些方法可用于的电中性和水不溶性多糖的表征。在这些多糖中,(1,3)-β- d-葡聚糖是真菌细胞壁的重要结构成分(Munro,2013 ),其生物合成酶是(1,3)-β- d-葡聚糖合酶(GS) ),是FDA的一个行之有效的目标-批准抗真菌抗生素(道格拉斯,2001年)。因此,表征的催化和抑制机制GS是势在必行理解真菌细胞壁生物合成和作用和抗GS-目标的机制编抗真菌药。然而,尽管GS活性公顷咏自80年代以来,已知(Shematek等人,1980 ),详述机械表征一直不可能由于缺乏适当的方法进行定量评价的数量和长度(1,3)-β - ð葡聚糖。因此,这种酶的许多基本方面,例如产物长度特异性,仍然是模棱两可的。

GS已被使用放射性测定法,其特征在于(Shematek等人,1980年)该孔定量TAT电子商务不溶于水的总量(1,3)-β- d葡聚糖通过确定掺入的量d -葡萄糖(GLC)。尽管该方法确定了GS的总体活性,但并未揭示聚合物产物的长度。GS产品的长度已被使用还原端修饰,其特征在于(Shematek等人,1980 ),其中还原性的(1,3)端-β- d葡聚糖我和s还原为山梨醇的聚合物水解成单糖。接着,长度我根据GLC和山梨糖醇之间的比率S确定。该分析表明小号其的平均长度(1,3)-β- d葡聚糖通过GS在粗膜标本制作我第60 - 80聚体(Shematek等人,1980 ); ħ H但是,这种方法需要大量的(1,3)-β- d葡聚糖,通常以毫摩尔AR quantit IES 。此外,c中的方法的低估的长度通过切割通过后处理或纯化过程中的剥离或反应的其它机制(刹帝利等人,2020 ); 吨herefore,当前存在对GS的详细机械特性没有合适的方法。

最近,尺寸排阻色谱法(SEC)已被用来表征水不溶性多糖的长度。^ h H但是,色谱条件和检测方法往往限制其U SE的详细机理研究。例如,使用放射线作为检测方法,用一氯乙酸将氧甲基甲基化后,通过SEC对真菌细胞壁葡聚糖和几丁质进行了分析(Cabib和Duran,2005; Cabib,2009 )。这种化学衍生作用可溶解否则不溶于水的葡聚糖和几丁质,并允许使用水性溶剂进行SEC分析。然而,由于衍生是不定量的,聚合物的绝对长度没有测定,和葡聚糖仅相对长度-和壳多糖的含测定分离的细胞壁(Cabib等人,2012 )。

SEC也已用于表征细菌纤维素合酶。在这种情况下,纤维素在溶解了二甲基乙酰胺的含8%的LiCl(W / V)和由耦合凝胶渗透色谱法分析,以多角度光散射(GPC-MALS) (麦克马纳斯等人,2018 )。这种方法避免了化学衍生化的陷阱,并可能适用于许多其他多糖。然而,该长度仅酶催化的稳态下测定和伸长的纤维素聚合物是不可检测的,这可能是由于有限的灵敏度折射率检测。

在这里,我们报告用于在氢氧化钠水溶液中使用SEC的脉冲安培检测(PAD)和放射性计数(RC)来确定(1,3)-β- d-葡聚糖的量和长度的方案。该协议克服上述的限制:PAD和RC允许适当的在灵敏度的长度和葡聚糖聚合物的分布的表征的GS的机械特性,并且使用氢氧化钠水溶液作为溶剂允许的增溶的否则不溶于水的( 1,3)-β- d-葡聚糖。这种方法(约100皮摩尔)的检测极限是更大的数量级,比四个数量低于减少先前对(1,3)报道端改性-β- d葡聚糖characterizati上。该测定法的一个重要限制是它需要相当纯的GS。迄今为止,该方法不与粗膜部分工作小号由于扰乱的葡聚糖迁移蛋白的存在通过SEC柱。因此,在该协议中,我们描述了使用产物包埋法和SEC测定法制备部分纯化的GS的方法。产物截留后得到的GS在SDS-PAGE上的纯度为20 %至30%,比活度约为1,000 nmol / min / mg。虽然我们的表征表明小号,在这种制剂中的杂质不影响GS(无论是葡聚糖长度或动力学)的表观功能(刹帝利等人,2020 ),它我的批判通过洗涤用SDS以除去从葡聚糖样品的蛋白质。该协议已经被用于研究GS催化机制和成功地检测到(1,3)-β- d 〜1000之间葡聚糖伸长和〜8000聚体在超过40年的在第一时间- GS的很长的历史(Chhetri等人,2020年)。简便的链长测定也与封闭的底物类似物偶联以明确确定聚合方向,这是多糖生物合成中关键但具有挑战性的机械问题之一(Chhetri等人,2020 )。这些应用证明了基于SEC的GS活性测定的重要性。可以采用类似的方法来研究其他多糖合酶(如糖原和透明质酸合酶)的活性。

关键字:(1,3)-β-D-葡聚糖合成酶, (1,3)-β-D-葡聚糖, 产品滞留, 脉冲安培检测, 真菌细胞壁

材料和试剂

1. 50毫升隼吨ubes (VWR,目录号:89039-656)       

2. 70毫升聚碳酸酯瓶组件,38 × 102毫米(贝克曼库尔特,目录号:355622)       

3. 3.5毫升ö笔顶厚壁聚碳酸酯超速离心管小号,13 × 51毫米(Beckman Coulter公司,目录号:349622)       

4. Acclaim SEC-1000色谱柱7 µm 4.6×300 mm(Thermo Fisher ,目录号:079724)     

5. Acclaim SEC-1000保护柱7 µm 4.6×33 mm(Thermo Fisher ,目录号:082739)     

6. Trans-Blot Turbo RTA转移套件LF PVDF(Bio-Rad,目录号:1704274)       

7. MultiScreen HTS FC滤板,1.2 / 0.65 µm(Millipore Sigma,目录号:MSFCN6B5)     

8. MultiScreen HTS真空歧管(Millipore Sigma,目录号:MSVMHTS00)     

9. 6”瓦特OOD ħ andle Ç奥多小号WAB(VWR,目录号:89031-270)     

10.动物组织的消化性消化物(M食用蛋白ept;标准,目录号:C7482)   

11.酿酒酵母BY4741(ATCC,目录号:201388)   

12.酵母提取物(标准,目录号:C7342)   

13. d-葡萄糖(VWR,目录号:BDH9230)   

14.琼脂(阿克罗斯(Acros),目录号:443570010)   

15. 0.5毫米克玻璃珠(科学工业,目录号:SI-BG05)   

注意:使用前,玻璃珠应先用漂白剂清洗,浸泡一整夜,然后再用去离子水洗涤,直到根据pH试纸测得的水洗液的pH为中性(通常是在洗涤10次后)。最后,将珠应该两次洗涤我sopropanol并干燥过夜。


16.液氮(空气,目录号:NI 240LT22)   

17.乙二胺四乙酸,蛋白质组学等级(EDTA; VWR,目录号:M101)   

18.氯化钠(EMD密理博,目录号:SX0420-5)   

19.苯甲基磺酰氟(PMSF; Acros Organics,目录号:215740100)   

20. Tris base(Sigma,目录号:T6066 )   

21. β巯基乙醇(VWR,目录号:M131)   

22.甘油(EMD密理博,目录号:GX0185-6)   

23. Pierce 660-nm检测(Thermo Scientific,目录号:1861426)   

24. EZQ P rotein Q uantitation ķ它物(Thermo Scientific,目录号:R33200)   

25. 3-[(3-氯氨基丙基)二甲基铵] -1-丙烷磺酸盐(CHAPS; VWR,目录号:0465)   

26.胆固醇半琥珀酸酯三盐(CHS; Anatrace,目录号:CH210)   

27.二硫苏糖醇(DTT; VWR,目录号:97061-338)   

28.鸟苷5 ' -[γ-硫代]三磷酸四锂盐(GTPγS ;Sigma,目录号:G8634)   

29. 4 – 20%Mini-PROTEAN TGX无污蛋白凝胶,10孔,50 µl(BioRad,目录号:4568094)   

30. UDP- [U- 14 C] - d -葡萄糖(UDP- [ 14 C] GLC;美国放射性化学,目录号:ARC0154)   

31. UDP- d-葡萄糖二钠盐(UDP-Glc;Carbosynth,目录号:MU08960)   

32.十二烷基硫酸钠(SDS; Sigma,目录号:75746)   

33.乙醇190证明(Koptec,目录号:V1101)   

34.三氯乙酸(TCA; EMD密理博,目录号:TX1045)   

35. 50%(w / w)的氢氧化钠溶液(NaOH; Fisher Chemical,目录号:SS254-500)   

36. ASTM I类水(里卡,目录号:9150-5)   

37.异丙醇(Fisher Chemical,目录号:A416P)。   

38. Pullulan标准(昭和电工株式会社,目录号:P-82)   

39.一抗:   

Anti-Fks1p(J.-PLatgé的礼物,巴斯德研究所的礼物)(Beauvais等人,2001年)
Anti-Rho1p(东京大学Y. Ohya的礼物)(Qadota等人,1996年)
40.山羊抗兔IgG-HRP二抗(Southern Biotech,目录号:4030-05) 

41.用于CCD成像的辐射HRP基板(Azure Biosystems,目录号:AC2101) 

42. Dionex ED电化学检测器一次性电极,PTFE上的金(Thermo Fisher,目录号:066480) 

43. 40%无菌葡萄糖溶液(请参阅食谱) 

44.酵母蛋白ept右旋糖(YPD)培养基(请参阅食谱) 

45. 0.5 M EDTA储备液,pH 8.0(请参见配方) 

46.破损缓冲区(请参阅食谱) 

47.膜缓冲液(请参阅食谱) 

48. KF M ix(请参阅食谱) 

49. 2 ×分析缓冲液(请参见配方) 



设备


500毫升带挡板的烧瓶(化学玻璃,目录号:CLS-2044-05)
2.8升带挡板的烧瓶(化学玻璃,目录号:CLS-2022)
-80°C冰柜(VWR交响乐超低温冰柜,型号:DW-86L638H)
-20°C冷冻室(VWR,目录号:82027-388 )
镊子(VWR,目录号:82027-388)
电磁搅拌棒(VWR,目录号:58948-98)
7毫升Dounce tissu e研磨器,带有A型和B型杵(Kimble,目录号:D9063)
1.5毫升带管样品杵(国际研究产品,目录号:199226)
搅珠器(Biospec ,型号:BeadBeater)
45-Ti型转子(Beckman Coulter,型号339160 )
贝克曼L7-55超速离心机(贝克曼,型号:L7-55)
TLA 100.3定角转子(贝克曼库尔特(Beckman Coulter),型号349490)
贝克曼Optima TL-100超速离心机(贝克曼,型号:Optima TL-100)
搅拌板(Labnet Accuplate模拟电磁搅拌器,型号:D0310)
BioVortexer(Biospec,型号:1083)
水浴超声波仪(NEY,型号:ULTRAsonik 28H)
ICS-5000 + DC色谱/检测器系统(Thermo Fisher ,型号:ICS-5000 +)
闪烁计数器(贝克曼库尔特,型号:LS 6500)
馏分收集器(Pharmacia Biotech,型号:FRAC-100)


软件


Microsoft Excel(Microsoft,https://www.microsoft.com/zh-cn/microsoft-365/excel)
Chromeleon 7.2 Thermo Scientific Dionex Chromeleon色谱数据系统(Thermo Fisher Scientific,https://www.thermofisher.com/order/catalog/product/CHROMELEON7#/CHROMELEON7)
程序


A.酿酒酵母培养     

1.条纹酿酒酵母从甘油储存到新鲜YPD琼脂平板(2%琼脂)上并在30℃下孵育板2 - 3天。平板通常持续约2周。     

2.在500毫升带挡板的烧瓶中准备2 × 200毫升YPD培养基,在2.8升带挡板的烧瓶中准备12 × 1.5升YPD培养基。     

3.从平板上挑一个菌落,并在培养管中接种2 ml YPD培养基。在30°C下以200 rpm摇动孵育培养物约8小时。这通常是在大规模文化之前的清晨执行。     

4.一天结束时,将2 × 200 ml YPD培养基接种在500 ml带挡板的烧瓶中,并从早上开始进行1 ml预培养,然后在30°C下以200 rpm摇动孵育过夜。文化应达到OD 600 (10 – 12)。     

5. Ť他第二天早上,添加了过夜预培养物的等分试样(30ml)中的每一个含有1.5L的YPD培养基12个培养瓶中,导致在初始OD 600为0.2 - 0.3。孵育以200rpm的速度在30℃下振荡培养直至培养物的OD 600个达到0.8 - 1.2 ; 吨ypically,这需要〜6小时从接种。     

6.通过在5,000 × g ,8°C下离心25分钟收获细胞。     

7.洗的粒料一次用10mM EDTA缓冲液(pH值8.0 )(〜30米升每20克细胞糊)。     

8.确定湿细胞重量和存储粒料一个-80℃冰箱中直至使用。通常,可获得50 – 60 g的湿细胞糊。     



B. GS准备     

1.膜级分的制备     

一种。在冰上解冻冷冻的细胞沉淀。     

b。Resu将细胞沉淀物置于150毫升冰冷破碎缓冲液中。     

C。用约150 ml冰冷,干净的0.5 mm玻璃珠将悬浮液转移到搅拌器中。顶部向上腔室用缓冲液以最小化可用空气空间一旦该腔室被组装时,并且确保该室被很好地覆盖由所述冰-水中。溶解细胞,珠磨,用5 × 1分钟的脉冲,具有一10 -每个脉冲之间分钟的休息。     

d。在4°C下以1,500 × g离心15分钟以清除裂解液;分离澄清的裂解液。     

e。重悬不溶性物质在破碎缓冲液(100毫升)和重复小号TEPS B1 c和B1 d,以确保破坏>细胞的95%。     

F。从TW合并澄清的裂解物ø珠磨周期,并转移到70 -毫升超速离心管。       

G。将裂解物在100,000 × g下于4°C离心1小时。在试管底部应该可以看到一个棕色的沉淀物。该颗粒包括主要膜蛋白,脂质,和任何其它分子,例如与它们紧密结合聚糖。     

H。使用7 ml Dounce匀浆器和松杵(由制造商标记为“ A”)将小丸重悬于〜80 ml膜缓冲液中约10次,或直到不再剩下大块小丸,然后将紧小杵(由制造商标记为“ B”)另外的约10个冲程,以确保彻底重悬膜沉淀。     

一世。确定使用的660nm测定蛋白质浓度ķ它和BSA标准根据制造商的方案。为了确保适当的阅读,再悬浮的膜级分我š通常稀释50 - 200 -倍。       

j。调节与膜缓冲液再悬浮的膜级分的最终体积至约5毫克/孔的最终浓度毫升蛋白质。通常,18 L培养物产生约100 ml重悬的膜级分。       

k。闪光灯-冻结20 ml的等分50 ml的的Falco Ñ吨ubes在液氮中,并储存在一个-80℃冷冻机中直至准备进行到下一个步骤(图2,泳道1)。     

注:我们把这种悬浮“膜部分,”它可以冻结,如果存储与活性损失最小的-解冻被最小化。


2.洗涤剂增溶     

一种。在冰和转印膜部分的到解冻等分试样的适当尺寸,冷却三角烧瓶含有搅拌棒和地点在冰-水浴中。由于存储纯化GS的能力,我们建议我们荷兰国际集团整个制备的膜部分来获得2.5 -毫升批次纯化GS的。     

b。在搅拌融化的膜级分的同时,添加GTPγS(27.5μM最终浓度),DTT(6.88 mM)和NaCl(192 mM)。     

C。滴加10%(w / v)CHAPS / 2%(w / v)CHS溶液,以达到0.688%CHAPS和0.138%CHS的最终浓度。     

d。使用搅拌棒和搅拌板在冰水浴中将悬浮液搅拌30分钟。重要的是轻轻混合溶液以防止GS失活; 因此,进行搅拌,使溶液表面形成一个小的旋涡,没有任何气泡。此步骤也可以使用旋转器执行。     

e。将该溶液转移至70 -毫升离心管中并平衡。     

F。在4°C下以100,000 × g离心30分钟。       

G。保留上清液为“洗涤剂增溶级分” (图2,泳道2)。如果此溶液不能立即用于产物包封纯化,则可以将其快速冷冻并在-80°C下保存,而活性损失最小。     

3.产品截留纯化(该过程的流程图请参见图1)     







的图1流程图的GS的产物截留纯化。在协议的步骤和所述图2对应的SDS-PAGE车道parenthes描述ë秒。包含GS的关键部分以粗体显示。


一种。甲DD一个0.125 × KF的体积混合和UDP-GLC(4mM的最终浓度)吨Ô洗涤剂solubiliz编分数。在30°C孵育20 – 30分钟。定期倒转试管以确保充分混合。     

b。将反应在冰上冷却15分钟。不溶性葡聚糖应在溶液中可见。     

C。通过在4°C下以5,000 × g离心5分钟收集不溶的葡聚糖和结合的蛋白质(有关沉淀的n图像,请参见图3 )。保留上清液的等分试样(产物截留的未结合部分;图2,泳道3),用于蛋白浓度测定(步骤B 3o)和SDS-PAGE分析(步骤B3 p)。       

d。通过resuspen洗涤所得的葡聚糖/蛋白质沉淀锡永于1.5ml洗涤缓冲液和homogeniz通货膨胀使用的7毫升匀化器和紧杵“B”。     

e。通过在4°C下以5,000 × g离心5分钟收集葡聚糖/蛋白质沉淀。保存上清液,以备以后表征。     

F。再重复三遍步骤B3d和B3e 。       

G。如步骤B3d所述,将沉淀物重悬于1.5 ml洗涤缓冲液中,将重悬液转移至3.5 ml超速离心管中,并在400,000 × g ,4°C下超速离心10分钟以收集沉淀物。结合从上清液小号TEPS B3 e和B3克和存储作为所述“产品截留沉淀洗涤级分”(图2,泳道4),用于以后的表征。     

H。通过将沉淀物重悬于0.5 ml提取缓冲液中,从不溶性葡聚糖中释放GS,然后使用手持式电子均质器(BioVortexer)均质化〜10 s。     

一世。将重悬液在4°C下孵育过夜。       

j。在400,000 × g ,4°C下将重悬液离心10分钟,然后分离上清液和沉淀。       

k。将S tep B3 j中的沉淀重悬于0.5 ml提取缓冲液中,然后使用手持式电子均质器均质化〜10 s。     

l。孵育悬浮液10分钟,在冰上,在400000离心再悬浮×克(使用TLA100.3转子100000转),4℃下10分钟,并分离上清液和沉淀。       

米 重复小号TEPS B3 k和B3升三多次。   

。合并上清液从小号TEPS B3 j和B3升,得到〜2.5毫升“产品截留纯化GS”(图2,泳道6)。     

o。重悬沉淀在0.5ml抽提缓冲液如描述在小号TEP B3 k,以及用提取缓冲液调节音量至1.0ml得到的“产品截留的残余葡聚糖结合级分”(图2,泳道5)。     







图2. GS纯化的表征。SDS-PAGE(A)和Western印迹使用的抗Fks1p(B)或抗Rho1p(C)抗体。每个车道都装有5.3 μ g蛋白质。泳道:1,膜级分(见“程序” B1k);2,洗涤剂增溶级分(见程序B2g部分);3,产品截留的未结合部分(见程序B3c部分);4,将产品截留的沉淀物洗净(见步骤B3g部分);5,产物截留残留葡聚糖的结合级分(见步骤B3n部分);6,产品截留纯化的GS(参见步骤B3m)。红色箭头指示对应于Fks1p带,和绿色箭头表示对应于Rho1p的波段。授权转载来自刹帝利ê t铝,。,(2020年)。






图3.图片产品包封步骤B3C后形成的葡聚糖颗粒。将丸粒从30制备- 40米升产品截留反应(步骤B3A)是一个50 -米升离心管中。


p。在使用EZQ纯化的每个步骤确定蛋白浓度甲SSAY ķ它和BSA作为所述标准。通常,此方案可从18 L酵母培养物中产生0.73±0.12 mg GS。ķ的nowledge的蛋白质浓度是关键执行荷兰国际集团的GS活性分析和determin荷兰国际集团GS的比活性。     

q。分析1 -从用SDS-PAGE用4每个纯化步骤10微克蛋白质- 20%,不含染色凝胶(Bio-Rad公司)。确定GS的纯度根据相对于Fks1p频带到另一个频带(光密度小号EE图2A)。我们通常以20 – 30%的纯度获得GS 。蛋白质印迹法也可以用于确保GS的两个已知成分Fks1p和Rho1p的共纯化。     

河 闪光灯-冻结并存储在纯化GS的liquots - 80℃。     



C.典型的酶活性测定     

1.准备UDP- [ 14 C] Glc的库存和工作解决方案     

一种。要制备放射性UDP-Glc储备液,请用至少3 × 500 µl体积的diH洗涤小瓶,然后用diH 2 O将500 µl UDP- [ 14 C] Glc稀释在70%乙醇中,使其总体积为3.5 ml 。2 O.除以溶液进入4个试管,并冷冻在-20℃。冷冻后,将试管冻干过夜。准备一个50 mM的非放射性UDP-Glc溶液,并向每个试管中加入3 µmol(60 µl ),得到的UDP-Glc溶液约为10,000 cpm / nmol。通常,我们根据实验目的制备具有高放射性并稀释的储备溶液。     

b。稀1微升所得溶液1 ,000 ×水。     

C。使用261 nm处的吸光度和261 nm(10.1 mM / cm)尿苷的摩尔消光系数(ε),确定在S tep C1 b中制备的稀释的UDP- [ 14 C] Glc的浓度。     

d。为了确定稀UDP-的放射性[ 14 C]在GLC使用小号TEP C1 B,调0,2,4,6,8,10,20,40,60,80,100,和200微升稀释的样品到闪烁小瓶,加入3毫升闪烁液,和计数的上液体闪烁计数器(LSC)的放射性。     

e。根据每个样品的放射性(cpm)绘制UDP- [ 14 C] Glc的量(nmol)。可以通过线性拟合的斜率(cpm / nmol)来计算比放射性。     

F。要准备UDP- [ 14 C] Glc的工作溶液,请用非放射性UDP-Glc稀释储备溶液,并按照S teps C1 b – C1 e确定比放射性。根据我们的经验,UDP- [ 14 C]与GLC〜800 CPM /纳摩尔比放射性允许小号检测从5葡聚糖形成微升的反应溶液与短至一个30-S温育。       

2.在冰上解冻储存的GS的等分试样。我们通常用执行活性测定一个30 -在30蛋白的50μg/ ml的终浓度-微升规模。     

3.如“配方”部分所述,准备2 ×化验缓冲液。     

4.在30 °C水浴中将2 ×分析缓冲液和GS孵育15分钟。     

5.准备用于淬灭和过滤不溶葡聚糖的滤板。向每个滤板孔中加入200 µl 10%w / v TCA。每个孔将用于淬灭每个时间点。     

6.通过将1体积的GS加到1体积的2 ×分析缓冲液中来启动反应。通过上下吹打以及关闭试管盖混合溶液,然后轻轻摇动并短暂涡旋,然后返回水浴。请注意,这对于可重现的结果很重要,因为GS储存在30%的甘油中,这需要彻底混合以产生均匀的溶液。     

7.在30°C下孵育反应。     

8.在每个时间点,转印5 -反应的20微升等分试样到含有200μl的10%w / v的TCA(中制备的过滤器板的孔小号TEP Ç 5)。上下吹打混合溶液。     

9.淬灭所有时间点后,使用真空歧管施加真空以过滤淬灭的溶液。用3 × 200 µl 10%TCA洗涤过滤器,然后用2 × 200 µl EtOH洗涤以除去未结合的UDP- [ 14 C] Glc。     

10.可以让过滤器干燥过夜,或者可以立即进行分析。从板的底部移去塑料支撑,然后将孔放在空的闪烁瓶上。用棉签将过滤器穿过PVDF膜推入闪烁瓶中。打破棉签手柄,以便可以关闭小瓶盖。如果PVDF膜仍然附着在板上,请用镊子将其转移到闪烁瓶中。 

11.加入3 ml的闪烁液,紧密地封闭的小瓶中,并涡旋并摇动以确保该过滤器被浸没。 

12.确定每个样品瓶的放射性。我们将闪烁计数器设置为3分钟的预设时间,或将%误差设置为5.0,以较短者为准。 



D.通过SEC-PAD分析定量检测GS产品     

1.解冻的冰冻结GS的解决方案。     

2.准备一个2 ×化验缓冲液。     

3.预温育两者GS和2 ×在30℃水浴中15分钟,测定缓冲液。     

4.准备的过滤板进行淬火并过滤不溶性葡聚糖。在平板的每个孔中加入100 µl或适当体积的10%w / v TCA 。小号EE小号TEP d 7。     

5.启动所述通过混合GS等体积和2反应×测定缓冲液中,并通过移液拌匀,轻弹,并简短涡旋返回到水浴之前。请注意,这对于可重现的结果很重要,因为GS储存在30%的甘油中,这可能导致混合不良和不可重现的结果。请参阅步骤C6。     

6.在30°C下孵育反应。     

7.在定义的时间点,取出两等份并分别淬灭。     

一种。通过将5 µl反应混合物与100 µl 10%TCA混合,在一个滤板孔中淬灭第一等分试样。该淬灭的溶液将用于确定葡聚糖的总量。     

b。淬灭反应的第二等份混合物(25 - 5 200微升)- 10个当量的2%(W / V)SDS中的单独的孔溶液中。该淬灭的溶液将用于通过SEC确定长度。不要使用酸淬灭来确定长度,因为酸变性的膜蛋白很难去除并且不会干扰SEC分析。     

注意:由于滤板孔仅适合〜250 µl体积,对于较大的等分试样,需要在更大的试管中将反应淬灭,然后转移到单个孔中进行后处理。


8.用3 × 200 µl 10%TCA,然后用3 × 200 µl EtOH洗涤S tep D 7a中的孔。     

9.从洗井(一个或多个)小号TEP d 7b中,用5 × 200μl的2%SDS,5 × 200微升水中2 ö ,和5 × 200μl的EtOH中。用SDS洗涤是至关重要为祛瘀荷兰国际集团的蛋白质即与葡聚糖相互作用,因为它们与SEC分析干涉。     

10.干下过夜孔中的过滤器一真空。 

11.将过滤器从S tep D 8转移到用于LSC的液体闪烁瓶中,以确定在每个时间点反应中掺入的葡萄糖总量。请参阅步骤C10。 

12.传输滤波器形成小号TEP d 9到1.5 -毫升螺旋盖小瓶中,并添加200微升的1M NaOH。 

注意:使用50%w / w溶液中的新鲜稀释的NaOH。


13.短暂离心以确保该过滤器在该溶液中浸没井,然后在冰声处理-水浴中10分钟。 

注意:较长的超声处理或在较高温度下进行超声处理可导致葡聚糖聚合物的降解更为显着,并影响葡聚糖产品的最终长度计算和洗脱曲线。


14.简言之离心机再次e是混合物和100微升转移至freash 1.5 -毫升小瓶中。 

15.使用宽-孔吸管尖(或常规吸管尖与末端切断,以增加直径),掰开在溶液中的过滤器和再悬浮。 

16.简短地离心混合物,再从管中移出约80 µl ,并在S tep D 14中与溶液合并。 

17.在4°C下以16,000 × g的离心力将来自S tep D 16的混合溶液离心10分钟,以去除任何颗粒。 

18.将150 µl上清液转移到HP LC样品瓶插入物中以进行SEC分析。 

19.转移5 -微升的等份上清液到闪烁小瓶中,并且分析由LSC确定葡聚糖回收率。通常,我们可以回收60 %至90%的放射性。 

20.使用装有Acclaim SEC-1000色谱柱的ICS-5000 + DC色谱/检测器系统,在30°C下通过SEC-PAD-RC分析25 µl的每个样品。色谱法是通过用10 mM NaOH以0.3 ml / min的流速等度洗脱进行的,并通过PAD进行监测(金,Carbo,Quad波形)。 

21.使用级分收集器,分馏EL ution每0.5分钟期间5 - 15分钟的色谱法,转移120 μl的分数至闪烁瓶中,并通过LSC分析,以确定在每个级分放射性葡萄糖。 

22.要确定的标准曲线为calibrat的离子的SEC,注入支链淀粉标准P-82(昭和电工株式会社制),并在相同条件下分析如所描述小号TEP d 20。 

23.将色谱数据从Chameleon软件导出到Excel中以计算样品的属性,如数据a分析B中所述。 



数据分析


答:美国证券交易委员会的标准曲线     

1.确定支链淀粉标准品在最大峰处的保留时间。     

2. ü唱最大峰值分子量(Mp)由制造商提供的支链淀粉的标准,绘制日志的的支链淀粉标准的标准相对于保留时间(MP),以产生线性标准曲线(小号Ë E图4 )。     







图4.使用商业支链淀粉作为标准品的SEC-PAD的校准曲线。一。商业支链淀粉标准品P-800,P-100和P-10的代表性色谱图。括号中的数字表示标准的Mp。乙。与支链淀粉的标准曲线的标准相比,与该GS产品。经Chhetri等人的许可进行了修改。,(2020年)。


B. SEC-PAD数据分析     

1.以文本格式导出用于GS测定的SEC-PAD数据,并绘制色谱图(图5A)。     

2.计算SEC-PAD色谱图中每两个连续数据点之间的平均保留时间(t i ,图5B)。对于我们的实验中,这导致了2 ,400片(PAD数据收集每1/120个或多个)。     

3.使用校准曲线d etermined在小号TEP A2,计算分子量(M我在平均滞留时间)(吨我)针对每个切片(图5B)。     

4.通过取每个切片中两个PAD数据点的平均值来计算每个切片的平均PAD信号(P i )(图5B)。     

5.使用以下公式确定每个切片的面积(A i ):A i = M i × P i 。     

6.要确定感兴趣峰的总面积(A,图5C),请计算峰中所有切片的A i之和:A = ∑ A i 。     

7.要确定感兴趣峰的每个切片i的重量分数(W i ),请将每个切片的A i除以峰的总面积(A)。     

8.为了确定重均分子量(Mw),计算M的总和我× w ^我对所有在峰值切片:MW = Σ (M我× w ^我)。     

9.要确定数均分子量(Mn),请用1除以峰中所有切片的W i / M i之和:Mn = 1 / ∑ (W i / M i )。     

10.计算样品的多分散指数(PDI):PDI = Mw / Mn。 

11.在MP,Mw和锰可以通过将每个被转化成聚合的最大峰度(XP),聚合(XW)的重均程度,以及数均聚合度(XN)的由聚合的葡萄糖的质量(C 6 H ^ 10 ø 5 ,162.14)。 







图5.从SEC-PAD色谱图中确定葡聚糖分子量参数。一。色谱图以保留时间为x轴绘制。乙。分子量为x轴的A中色谱图的放大图。代表切片i突出显示。(C)以分子量为x轴绘制的色谱图。突出显示了用于确定聚合物长度参数的峰面积。


C.分析[R adioactivity数据     

1.输入LSC读出用于在MS Excel以cpm的级分。调整RC相对于PAD的保留时间的延迟,该延迟是由PAD和馏分收集器之间的管路引起的。     

2.使用UDP- [ 14 C] Glc的比放射性,将cpm转换为nmol,以确定掺入(1,3)-β - d-葡聚糖的Glc的量。切记在每个数据点的处理过程中针对淬灭体积,稀释度和产率回收率之间的差异进行调整。     



D. SEC-PAD的覆盖的制备和[R adioactivity色谱     

PAD和RC色谱图的叠加有助于对数据进行视觉评估。对于覆盖层,我们建立了一种定量方法,如下所述。


1.按照B节和C节(图6A)所述绘制PAD和RC色谱图。     

2.计算RC色谱图每个数据点的PAD值和放射性之间的差(Di)。     

3.计算感兴趣峰的Di,∑ (Di)2的平方和。     

4.确定的缩放因子为最小化的PAD色谱Σ (Di)和2 。例如,5的缩放因子被确定为图6A中,因为5 ×放大率的PAD色谱最小化的Σ (Di)和2 。     

5.使用比例因子,在PAD色谱图中调整y轴比例(图6B)。     







图6.使用最小二乘函数近似法制备SEC-PAD和RC色谱图的叠加图。一。未经修改的原始PAD(实线)和RC(钻石)色谱图。D i代表数据点i处PAD和RC值之间的差。乙。PAD(实线)和RC(钻石)色谱图的比例调整重叠图。通过最小化(A)中的∑ (D i )2来调整PAD色谱图的y轴。


笔记


制备2 ×检测缓冲液时,请记住将UDP-Glc与GS混合稀释2倍。
储存50%(w / w)的NaOH溶液,并使CO 2的暴露量最小,以最大程度地减少碳酸钠和碳酸氢钠对PAD分析的污染。
用脱气的ASTM I型水制备SEC-PAD-RC洗脱液,以最大程度地减少可能影响PAD响应本底信号的离子污染。


菜谱


40%无菌葡萄糖溶液
400 g(40%w / v)d - g葡萄糖


用diH 2 O调节至1 L


过滤-消毒


是的蛋白P右旋糖(YPD)培养基
2 0克p动物组织的eptic消化(肉蛋白胨)                                       

10克ÿ东提取


对于固体培养基,添加20克一个GAR


用diH 2 O调节至950 ml


高压釜


加入50 ml (2%w / v)40%无菌葡萄糖溶液(过滤器灭菌)


0.5 M EDTA储备液,pH 8.0
186.12 g EDTA二水合二钠盐


用diH 2 O调节至800 ml


在冰水浴中将溶液冷却至4 °C


用NaOH调节pH值到8.0


调整体积至1 ,000个ml的与DIH 2 ö


破坏缓冲区
29.22克(0.5M)小号裂果Ç hloride


20 ml (10 mM)0.5 M EDTA储备液,pH 8.0


调整体积至1 ,000个ml的与DIH 2 ö


立即在使用前添加10 ml (1 mM)100 × PMSF的2-丙醇储备溶液(可在-20°C储存)。


膜缓冲液
50 mM Tris ,pH 8.0


10 mM EDTA ,pH 8.0


333.3 ml的(33%V / V)克lycerol


调整体积至1 ,000个ml的与DIH 2 ö


添加70.92 μ升(1毫摩尔)β巯基乙醇并冷却至4之前℃至使用


KF中号IX
200 mM Tris储备液,pH 8.0


4 mM EDTA储备液,pH 8.0


871.5毫克(0.3百万)KF


用diH 2 O将体积调节至50 ml


10%CHAPS,2%CHS期权
5克(最终含量的10%w / v)


超声处理溶于40 ml diH 2 O


加入1 g(最终2%w / v)CHS Tris盐


注意:发现CHS Tris盐比CHS更可溶。


超声处理约1小时,然后在室温摇动过夜


用diH 2 O将体积调节至50 ml


储存在-20°C


提取缓冲液
48 ml膜缓冲液


2 ml 10%CHAPS (最终含量0.4%)和2%CHS储备液(最终含量0.08%)


1毫米DTT


加入终浓度为4μM的GTPγS储备溶液


洗涤缓冲液
50 ml的È xtraction缓冲


5毫米UDP-Glc


2 ×检测缓冲液
10 mM(或任何合适的浓度)UDP- [U- 14 C] Glc(比活度约为800 cpm / nmol,或根据实验需要进行调整)


150 mM Tris ,pH 7.5


1.5毫米EDTA


1.5%(w / v)BSA


50毫米KF


0.04毫米GTPγS


10%三氯乙酸
100克TCA


加入diH 2 O至1 L


2%SDS
2克(2%w / v)SDS


加入diH 2 O至100 ml


致谢


这项工作WA Š资助的杜克大学医学中心和部分在普通医学科学格兰特R01 GM115729研究所(至KY)。美国国家过敏和传染病研究所(T32AI052080)的三机构分子真菌学和发病机制培训计划为AL提供了支持。作者感谢东京大学的Yoshikazu Ohya博士的抗Rho1p抗体和巴斯德研究所的Jean-PaulLatgé博士的抗Fks1p抗体。


该协议是在所使用的协议的详细版本一个最近的出版物由作者(刹帝利等人,2020 )。


利益争夺


作者宣称没有利益冲突。


参考


Barrett,D.,Wang,TS,Yuan,Y.,Zhang,Y.,Kahne,D. and Walker,S.(2007年)。分析由肽聚糖糖基转移酶产生的聚糖聚合物。生物化学杂志282(44):31964-31971。
博韦A.,布鲁诺JM,摩尔M.PC,布伊特拉戈MJ,勒格朗R. 烟曲霉的葡聚糖合酶复合物。细菌学杂志183(7):2273-2279。
Cabib,E。(2009)。两种测定多糖交联的新技术表明,Crh1p和Crh2p将几丁质连接到酿酒酵母细胞壁中的β(1-6)-和β(1-3)葡聚糖上。真核细胞8(11):1626-1636。
Cabib,E.和Duran,A.(2005)。依赖于合酶III的几丁质结合到不同受体上,这取决于发芽酵母在细胞壁上的位置。生物化学杂志280(10):9170-9179。
E. Cabib,N。Blanco和J. Arroyo(2012)。在酿酒酵母母芽颈处存在与甲壳质连接的大β(1-3)葡聚糖,提示参与了局部生长控制。真核细胞11(4):388-400。
Chhetri,A.,Loksztejn,A.,Nguyen,H.,Pianalto,KM,Kim,MJ,Hong,J.,Alspaugh,JA和Yokoyama,K.(2020)。(1,3)-β-d-葡聚糖合酶在真菌细胞壁生物合成中的长度特异性和聚合机理。生物化学59(5):682-693。
道格拉斯,CM(2001)。真菌β(1,3)-D-葡聚糖合成。Med Mycol 39(Suppl 1)55-66。
Krupa,JC,Shaya,D.,Chi,L.,Linhardt,RJ,Cygler,M.,Withers,SG和Mort,JS(2007)。透明质酸合酶的定量连续测定。Anal Biochem 361(2):218-225。
McManus,JB,Yang,H.,Wilson,L.,Kubicki,JD和Tien,M.(2018)。细菌纤维素合成的起始,延伸和终止。ACS欧米茄3(3):2690-2698。
加州芒罗(2013)。几丁质和葡聚糖是真菌细胞壁的阴阳,它们对抗真菌药物的发现和治疗具有重要意义。微生物学进展83:145-172。
Qadota,H.,Python,CP,井上SB,Arisawa,M.,Anraku,Y.,Zheng,Y.,Watanabe,T.,Levin,DE和Ohya,Y.(1996)。鉴定酵母Rho1p GTPase作为1,3-β-葡聚糖合酶的调节亚基。科学272(5259):279-281。
EM的Shematek,JA的Braatz和E的Cabib(1980)。酵母细胞壁的生物合成。I.β-(1)的制备和性质导致3)葡聚糖合成酶。生物化学杂志255(3):888-894。
Tlapak-Simmons,VL,Baron,CA,Gotschall,R.,Haque,D.,Canfield,WM和Weigel,PH(2005)。由I类链球菌透明质酸合酶进行的透明质酸生物合成发生在还原端。生物化学杂志280(13):13012-13018。
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引用:Chhetri, A., Loksztejn, A. and Yokoyama, K. (2021). Quantitative Characterization of the Amount and Length of (1,3)-β-D-glucan for Functional and Mechanistic Analysis of Fungal (1,3)-β-D-glucan Synthase. Bio-protocol 11(8): e3995. DOI: 10.21769/BioProtoc.3995.
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