This study was supported by the National Science Foundation award MCB-2012074. The authors thank Dr. Craig W. Vander Kooi of the University of Florida Department of Biochemistry and Molecular Biology for valuable discussions and support. The authors also thank Dr. Matthew S. Gentry of the University of Florida Department of Biochemistry and Molecular Biology for his support. We would like to thank Dr. Sara Lagalwar, chair of the Skidmore College Neuroscience program, for allowing us to use the LICOR C-digit blot scanner for western blot imaging.&#160;

1. Preparation of ConA-Sepharose beads
<ol>
	<li>Make 250 mL of a binding buffer containing 67 mM HEPES (pH 7.5), 10 mM MgCl2, and 0.2 mM CaCl2. Adjust the pH using 1 M NaOH solution.</li>
	<li>Pipette 250 &#956;L of ConA-Sepharose bead suspension into a 1.5 mL microcentrifuge tube. Centrifuge the contents at 10,000 x g for 30 s at 4 &#176;C. Discard the supernatant. 
	NOTE: 250 &#181;L of ConA-Sepharose beads in a 1.5 mL microcentrifuge tube is needed for each amylopectin concentration used for the assay.</li>
	<li>Add 750 &#181;L of the binding buffer to each tube containing 250 &#181;L of ConA-Sepharose beads. Centrifuge the tubes at 10,000 x g for 1 min at 4 &#176;C. Remove the supernatant. Repeat this step 2x to ensure that the beads are appropriately washed and equilibrated with the binding buffer.</li>
</ol>
2. Preparation of amylopectin solutions
<ol>
	<li>Make a stock solution of 10 mg/mL potato amylopectin. Amylopectin is water-insoluble and can be solubilized by heat. To solubilize, add 0.1 g of potato amylopectin to 10 mL of distilled water. Heat the suspension in a water bath at 80 &#176;C for 1 h or until the solution is no longer cloudy.</li>
	<li>Allow the solution to return to room temperature (RT), with repeated vortexing to avoid clumping.</li>
	<li>Alcohol-alkaline treatment is an alternative method to solubilize amylopectin substrates. To solubilize using this method, follow the steps below.
	<ol>
		<li>Suspend 0.5 g of amylopectin substrate in 5 mL of 20% ethanol and 5 mL of 2 M NaOH. Stir the contents vigorously for 15-20 min at RT.</li>
		<li>Next, add 10 mL of water, and adjust the pH of the solution to 6.5 by adding 2 M HCl. Bring up the volume of the resulting solution to 50 mL with distilled water to make a 10 mg/mL amylopectin solution.</li>
	</ol>
	</li>
	<li>Dilute the 10 mg/mL solubilized amylopectin solution to make a series of 2 mL of diluted amylopectin solutions. For example, perform half dilutions of 10 mg/mL to prepare a series of amylopectin concentrations (5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.625 mg/mL, 0.3125 mg/mL, 0.156 mg/mL, 0.078 mg/mL, 0.039 mg/mL, 0.019 mg/mL, and 0 mg/mL).</li>
</ol>
3. Preparation of ConA-Sepharose: amylopectin beads
<ol>
	<li>Add 250 &#181;L of each diluted amylopectin solution to 1.5 mL microcentrifuge tubes containing 250 &#181;L of ConA-Sepharose beads pre-equilibrated in binding buffer. Mix the contents well. Label the tubes with the corresponding amylopectin concentration.</li>
	<li>Incubate the contents on a rotating wheel at 4 &#176;C for 30 min. 
	NOTE: There is no change in the ConA-Sepharose:amylopectin bound complex over time after 20 min. The 30 min incubation time was chosen by varying incubation times from 10 min to 1 h to ensure equilibrium was reached.</li>
	<li>Centrifuge the tubes at 10,000 x g for 1 min. Collect the supernatant in a newly labeled 1.5 mL microcentrifuge tube. Save these supernatant fractions to perform D-glucose assay12 (acid hydrolysis of amylopectin followed by UV determination of glucose via enzymatic assay). This step is necessary to ensure all amylopectin is bound to the beads.</li>
	<li>Add 750 &#181;L of binding buffer to the ConA-Sepharose:amylopectin beads. Centrifuge the tubes at 10,000 x g for 1 min. Discard the supernatant to remove any unbound amylopectin molecules.</li>
	<li>Repeat step 3.4 to ensure sufficient washing. Each tube now contains ConA-Sepharose beads bound to varying amounts of amylopectin substrates.</li>
</ol>
4. Incubating SEX4 with ConA-Sepharose:amylopectin beads
<ol>
	<li>Mix 250 &#181;L of ConA-Sepharose:amylopectin beads with 100 &#181;L of the binding buffer which includes 10 &#181;g of SEX4 protein, 10 mM dithiothreitol (DTT), and 10 &#181;M protease inhibitor cocktail (PIC). Note that the total volume in each tube is 350 &#181;L. 
	NOTE: A protease inhibitor cocktail is added as a precautionary step to avoid any unnecessary SEX4 degradation. This is an optional step. In this assay, the recombinant protein Arabidopsis thaliana SEX4 (AtSEX4) is used. The purified protein contains an N-terminal histidine tag necessary for detecting the protein via chemiluminescence. Detailed information on glucan phosphatase purifications is described in previous publications14,20,24.</li>
	<li>Incubate the protein and ConA-Sepharose:amylopectin bead suspension at 4 &#176;C for 45 min with gentle rotation. 
	NOTE: The 45 min incubation time is chosen to ensure equilibrium is reached for the complex.</li>
	<li>Centrifuge the tubes at 10,000 x g for 1 min. Pipette 50 &#181;L of the supernatant carefully using a gel loading tip into a new 1.5 mL microcentrifuge tube. Add 20 &#181;L of 4x SDS-PAGE dye and 10 &#181;L of water to each tube containing 50 &#181;L of the collected supernatant fractions. Heat the samples at 95 &#176;C for 10 min. Save these samples for running the SDS-PAGE gels. Ensure 10 new tubes labeled &#34;supernatant (S)&#34; have the corresponding substrate concentrations.</li>
	<li>Add 750 &#181;L of the binding buffer to the ConA-Sepharose:amylopectin: SEX4 beads to remove any unbound protein from the beads. Centrifuge the tubes at 10,000 x g for 1 min. Repeat this step one more time to ensure proper washing. Discard the supernatant.</li>
	<li>Add 20 &#181;L of 4x SDS-PAGE dye and 80 &#181;L of distilled water into the tubes containing washed ConA-Sepharose:amylopectin:SEX4 beads. Heat the samples at 95 &#176;C for 10 min and centrifuge at 10,000 x g for 1 min.</li>
	<li>Discard the pellet and save the supernatant for running the SDS-PAGE gels. Pipette 80 &#181;L of the supernatant into new tubes and label them as &#34;pellet (P)&#34;.</li>
</ol>
5. Running SDS-PAGE gels
<ol>
	<li>Load 40 &#181;L of the unbound protein samples (made in step 2.3, labeled S) into 4%-12% precast polyacrylamide gel wells from the lowest substrate concentration to the highest, but keep the first lane free to load the protein molecular weight marker. Use a second gel to load 10 bound protein samples made in step 2.5 (labeled as P).</li>
	<li>Add freshly prepared 1x SDS-PAGE running buffer to both chambers of the apparatus. Run the gel at 150 V for 35 min or until the dye front reaches the bottom of the gel.</li>
	<li>Remove the run gel from the apparatus and remove the spacers and glass plates. Use the separated gel to run a western blot analysis.</li>
</ol>
6. Western blotting for chemiluminescence detection14,15
NOTE: This method can be easily modified/adapted depending on the western blotting equipment that users have in their labs.
<ol>
	<li>Make 1 L of transfer buffer containing 5.8 g of Tris base, 2.9 g of glycine, 0.37 g of SDS, and 200 mL of methanol.</li>
	<li>Transfer the size-separated proteins from the polyacrylamide gel to a nitrocellulose membrane. Briefly assemble the sponges, filter papers, gel, and nitrocellulose membrane according to western transfer protocol14,15. Run at 70 V for 1 h.</li>
	<li>To prevent nonspecific protein binding, incubatethe nitrocellulose membrane containing the protein solution of 1%-5% bovine serum albumin (BSA) or milk protein in 50 mL of TBST buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 0.1% Tween 20) for 1 h. Wash the membrane 3x using TBST buffer to remove any unbound blocking solution.</li>
	<li>Incubate the membrane with a horseradish peroxidase (HRP)-linked antibody specific for His-tagged protein for 1 h. Wash the membrane 3x in TBST buffer to remove any unbound antibodies. Use a 1:2,000 dilution of antibody to TBST for optimal reproducibility and sensitivity.</li>
	<li>The HRP enzyme-linked antibody binds specifically to the histidine tag of the SEX4 protein, which yields a band in the presence of chemiluminescence reagents. For digital imaging, make a solution of equal parts of chemiluminescent substrate solutions (750 &#181;L each) in a 1.5 mL tube. Incubate the membrane for at least 5 min in the solution.</li>
	<li>Place the membrane protein side down on the blot scanner and run the acquisition software to quantify the protein in both the pellet and supernatant fractions.</li>
</ol>
7. Data analysis
<ol>
	<li>Perform the quantitative signal measurements using the acquisition software with the blot scanner. Normalize all quantitative measurements in the supernatant and pellet fractions to the total protein loaded. 
	NOTE: The software allows to quantify the intensity of each protein band in the supernatant and pellet fractions.</li>
	<li>In the saturating binding experiment, plot the percentage of protein-bound versus amylopectin concentration. Fit the data to Y = Bmax x X/(KD + X), using data analysis software to calculate KD. 
	&#8203;NOTE: Bmax is the maximum specific binding, the Y-axis is the percentage of protein bound, the and X-axis is the amylopectin concentration.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64700/64700fig01.jpg" /> 
Figure 1: Overview of the ConA-Sepharose sedimentation assay workflow. (A)&#160;Preparation of ConA-Sepharose beads. (B) Incubation with amylopectin substrate. (C) Incubation with SEX4 protein. (D) Separation of bound and unbound protein fractions through centrifugation. (E) Separation of protein through SDS-PAGE. (F) Western-blot analysis. (G) Chemiluminescence detection of His-tagged SEX4 protein. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64700/64700fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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</ol>

The authors declare no conflicts of interest.

This study demonstrates the successful development of a novel in vitro sedimentation assay that allows determination of the binding affinity of glucan-glucan phosphatase interactions. The assay design takes advantage of the specific binding of lectin ConA to glucans via the hydroxyl residues of glucose to indirectly capture solubilized carbohydrate substrates onto Sepharose beads. This allows the separation of bound and unbound protein fractions via centrifugation and determination of the binding affinity of solubilized glucan substrates and glucan phosphatases. All of the glucan phosphatases tested were found to specifically bind to amylopectin, with no detectable binding to ConA-Sepharose beads in the absence of amylopectin. 
The experiment was performed to measure the binding affinity with a small, fixed amount of SEX4 protein and a range of amylopectin concentrations attached to ConA-Sepharose beads. An excess of ConA-Sepharose beads was used to ensure the highest amylopectin concentration binding to beads. The complete binding of amylopectin was tested via a glucose assay. The protocol presented in the manuscript utilizes quantitative sedimentation to separate bound and unbound protein fractions, as well as gel electrophoresis and western analysis to measure the amount of SEX4 in the supernatant compared with the amount bound to the ConA-Sepharose:amylopectin beads. Quantifying the pellet fraction requires minimal washing to not disturb the binding equilibrium. To avoid any underestimation of pellet fraction, rather than examining the pellet, measuring the concentration of free SEX4 in the supernatant and calculating the difference of the bound SEX4:amylopectin complex is sufficient.
The in vitro sedimentation assay described here determined the differential binding affinity of SEX4 wild-type protein. Interestingly, the determined binding affinity of SEX4 wild-type protein using the developed in vitro sedimentation assay falls within the higher range of the reported value determined with a gel shift assay25. The binding assay should be sensitive to lower substrate concentrations and straightforward, whereas the gel shift assay requires making individual native gels in the presence of varying amounts of glucan substrates. This time-consuming method limits the number of concentrations that can be tested, and a limited substrate concentration range can be tested. Instead, sample preparation is fast and reliable with our in vitro sedimentation assay. Comparing SEX4 wild-type protein and SEX4 C198S mutant reveals a tenfold binding affinity difference. In this regard, this assay is sensitive to determine the binding of SEX4 mutant proteins quantitatively and qualitatively. 
Protein-carbohydrate interactions are vital for many important biological processes, including catalysis, cell signaling, and recognition. They are also gaining increasing attention due to their important roles in the pharmaceutical and food industries. X-ray crystallography is widely used to obtain protein structures at the atomic level. However, the structural heterogeneity and flexibility of carbohydrate substrates make it harder to obtain carbohydrate-bound protein structures. Recently cryo-EM and nuclear magnetic resonance (NMR) spectroscopy have also been employed to determine the structures of carbohydrate-binding proteins26. Despite the many advancements in high-resolution structural techniques, it is challenging to obtain structures of carbohydrate-protein complexes. As a result, only a limited number of protein-carbohydrate complex structures have been solved experimentally. Furthermore, crystal structures only provide a snapshot of a protein at a certain conformation, and not the dynamic nature of substrate binding. Alternatively, several biophysical techniques, including isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR), are used to measure protein-carbohydrate interactions27. However, these methods are also not without limitations. One main challenge in determining protein-carbohydrate interactions is their relatively weak interactions compared to other protein-ligand interactions. The interactions within the range of high micromolar to low micromolar range significantly reduces the usability of ITC and SPR. Indirect methods, such as gel shift and pull-down assays, have gained significant attention for determining interactions that are difficult to assess by other methods25. Therefore, this binding assay is a widely applicable approach for sensitive measurements of protein and soluble carbohydrate interactions. 
Glycogen and starch are major carbohydrate storage molecules made of branched polymers of glucose. The branching pattern, flexibility, and the presence of different microdomains within the starch and glycogen require interacting proteins to adopt unique substrate binding mechanisms. Much understanding of substrate recognition mechanisms of SEX4 has come from X-ray crystallographic studies coupled with structure-guided mutagenesis. To dephosphorylate starch, SEX4 must interact with significantly different microdomains of the starch granule to locate phosphates for position-specific dephosphorylation. Experimentally, it remains a challenge to determine the binding affinities of glucan phosphatases, given the weak interactions between the protein and the glucans and the structural flexibility and heterogeneity of carbohydrates. This manuscript presents a method to determine binding affinities of glucan phosphatase-carbohydrate interactions using a Concanavalin A (ConA)-based in vitro sedimentation assay. Glucan phosphatases employ distinct mechanisms to bind, locate, and dephosphorylate carbohydrates. Previously, ITC has been employed to measure the binding affinity of laforin and linear oligosaccharide chains. However, no study has revealed the binding affinities of glucan phosphatases with their physiological substrates via direct techniques such as ITC and SPR. To better understand the utility of this assay for determining substrate binding of glucan phosphatases, the binding of laforin, LSF2, and SEX4 against amylopectin was assessed. Future studies can be done to experimentally determine the binding affinity of all glucan phosphatases and their physiologically relevant glucan substrates. One of the critical steps of the method is to determine the optimum incubation times and substrate concentration range for the assay. Each glucan phosphatase binds differently with the substrate, and the method is optimized for SEX4. It is important to carry out initial optimization experiments to figure out these parameters. One other limitation is the use of histidine tagged protein for the assay. However, if antibodies are present for the study protein, the method can be easily optimized for any glucan-interacting protein.

Glucan phosphatases are members of a functionally diverse subfamily of dual specificity phosphatases (DSPs) within the protein tyrosine phosphatase (PTP) superfamily1. They have been found in most life forms, including widely divergent photosynthetic organisms, humans, vertebrates, and some invertebrates and protists2,3,4. Plants contain three known glucan phosphatases: Starch Excess4 (SEX4), Like Sex Four1 (LSF1), and Like Sex Four2 (LSF2)5,6,7. Plants that lack glucan phosphatases display decreased rates of transitory starch degradation and accumulation of starch in the leaves8,9. Laforin is the founding member of the glucan phosphatase family that dephosphorylates glycogen in vertebrates and humans3,10. The mutations of laforin result in neurodegenerative Lafora disease, a fatal autosomal recessive form of epilepsy11. Glucan phosphatases are necessary for glycogen and starch metabolism and have emerged as important enzymes for modulating starch content in plants and treating neurodegenerative Lafora disease12,13. Recent X-ray crystallography studies on glucan phosphatases with model glucan substrates have shed light on substrate binding and the catalytic mechanism of glucan dephosphorylation14,15,16,17. However, the current understanding of how glucan phosphatases bind to their physiological substrates is incomplete.
Starch is an insoluble polymer of glucose made of 80%-90% amylopectin and 10%-20% amylose18. The substrates for plant glucan phosphatases are phosphorylated carbohydrate molecules, such as glycogen and starch granules. The phosphorylated glucosyl residues are present at a 1:600 phosphate:glucosyl residue ratio. Interestingly, the phosphates are present only on the amylopectin molecules19. The main plant glucan phosphatase SEX4 acts on the starch granule to dephosphorylate amylopectin molecules. The X-ray crystal structure of SEX4 combined with structure-guided mutagenesis studies has demonstrated the unique substrate specificities of SEX4 for different positions within a glucan structure15. We recently showed that the biologically relevant activity of SEX4 can only be observed when acting on its solubilized amylopectin substrates20. However, understanding glucan-SEX4 interactions has proven to be difficult due to the structural complexity of the substrate, broader binding specificities, and low binding affinities between the protein and its substrates. These issues have hindered the ability to utilize methods commonly used in protein-ligand interactions, such as isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) spectroscopy, and enzyme-linked immunosorbent assay (ELISA)-based assays.
Interestingly, much of our understanding of carbohydrate-protein interactions have come from studying lectins. Concanavalin A (ConA) is a legume lectin family of proteins originally extracted from the jack bean. ConA binds carbohydrates with high specificity, which is advantageous for its use in drug targeting and delivery applications. The binding of ConA to a variety of substrates containing nonreducing &#945;-D-mannosyl and &#945;-D-glucosyl has been extensively studied19,20. Commercially available ConA-bound Sepharose beads are commonly used to purify glycoproteins and glycolipids21. ConA binds to these glucans via C3, C4, and C6 hydroxyl groups of the glucose residues. ConA-Sepharose beads have also been successfully used to measure the binding of glycogen-protein and starch-protein interactions22,23. In this study, we used ConA-Sepharose beads to develop a binding assay to measure the binding specificities of glucan phosphatase-amylopectin interactions.
Previously, a ConA-based sedimentation assay was employed to assess glucan phosphatase substrate binding ability14,20,24. In this study, the same strategy was used to develop a novel method to determine the binding affinity of glucan-glucan phosphatase and carbohydrate interactions. This method also has an advantage for investigating various solubilized carbohydrate-protein interactions.

<table><tbody><tr><td>6x-His Tag monoclonal antibody (HIS.H8), HRP</td><td>Therm Fisher Scientific</td><td>MA1-21315-HRP</td><td></td></tr><tr><td>Biorad gel electrophoresis and Western blot kit</td><td>Biorad&amp;nbsp;</td><td>1703930</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma-Aldrich</td><td>208291</td><td></td></tr><tr><td>C-Digit blot scanner</td><td>LICOR</td><td>3600-00</td><td>Blot scanner</td></tr><tr><td>Complete protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>11836170001</td><td></td></tr><tr><td>Concanavalin A-sepharose beads</td><td>Sigma-Aldrich</td><td>C9017</td><td>This product contains&amp;nbsp; in 0.1 M acetate buffer, pH 6, containing 1 M NaCl, 1 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 1 mM MnCl&lt;sub&gt;2&lt;/sub&gt;, and 1 mM MgCl&lt;sub&gt;2&lt;/sub&gt; in 20% ethanol&amp;nbsp;</td></tr><tr><td>Centrifuge</td><td>Eppendorf&amp;nbsp;</td><td>5425R</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP381-5</td><td></td></tr><tr><td>GraphPad Prism 8.0 software</td><td>GraphPad&amp;nbsp;</td><td>Version 8.0</td><td>Data analysis software&amp;nbsp;</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H8651</td><td></td></tr><tr><td>Image Studio</td><td>LICOR</td><td>3600-501</td><td>Acquisition Software</td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A452SK-4</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Fisher Scientific</td><td>PI28312</td><td></td></tr><tr><td>Potato amylopectin</td><td>Sigma-Aldrich</td><td>A8515</td><td></td></tr><tr><td>Precast SDSPAGE Gels</td><td>Genscript</td><td>M00653S</td><td></td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP154-1</td><td></td></tr><tr><td>Tween 20</td><td>Fisher Scientific</td><td>MP1TWEEN201</td><td></td></tr><tr><td>Westernsure premium chemiluminescence substrate&amp;nbsp;</td><td>LI-COR</td><td>&amp;nbsp;926-95000</td><td></td></tr></tbody></table>

concanavalin a-based sedimentation assay to measure substrate binding of glucan phosphatases

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates.&#160;As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

One of the key features of the glucan phosphatase family of proteins is their ability to bind to glucan substrates. First, the binding capacity of SEX4 to ConA-Sepharose:amylopectin beads was analyzed using SDS-PAGE (Figure 2A). Bovine serum albumin (BSA) served as a negative control to detect any nonspecific binding of proteins to the ConA-Sepharose:amylopectin beads. The SDS-PAGE analysis of proteins showed the presence of SEX4 protein in the pellet fraction and BSA in the supernatant fraction. W278A, a known SEX4 mutant with significantly reduced glucan binding ability, was also included in the assay. The W278A mutant appeared in the supernatant fraction, indicating the utility and specificity of this assay for detecting the glucan binding ability of SEX4 proteins. While SDS-PAGE visualized the SEX4 proteins, there was concern about the sensitivity and use of small glucan phosphatases; For instance, the ConA lectin protein could potentially interfere with detecting small proteins such as LSF2. To test whether this method could detect only the glucan phosphatases, a western blot was performed using an antibody specific to the N-terminal histidine tag. Indeed, there was a significant increase in the detection of SEX4, and the method is specific for detecting glucan phosphatases with an N-terminal histidine tag (Figure 2B). Quantitative measurement of ligand-protein interaction experiments requires performing essential controls for establishing the appropriate ligand and protein concentrations. Therefore, the co-sedimentation assay was tested next at three different SEX4 concentrations (Figure 2C-E). While 2 &#181;g of SEX4 is sufficient to visualize via chemiluminescence detection, using 5 or 10 &#181;g of SEX4 allows for a more accurate detection of the partial binding. The SEX4 binding against varying amylopectin concentrations (0.5 mg/mL, 1.0 mg/mL, and 5 mg/mL) was also tested. These results also indicate increased SEX4 binding with increased amylopectin concentration, with saturated binding at 5 mg/mL of amylopectin.
To determine the binding affinity of amylopectin and SEX4 wild-type protein, 10 &#181;g of SEX4 was incubated with amylopectin (up to 5 mg/mL), and binding at each concentration was determined (Figure 3A). The fitting of percent protein-bound data to the specific, one-site binding model gave a KD of 1.03 &#177; 0.23 mg/mL (Figure 3B). There is no available crystal structure of SEX4 wild-type protein; instead, the crystal structure of catalytically inactive SEX4 C198S mutant reveals the binding interface of SEX4, made of DSP and carbohydrate-binding molecule (CBM) domains. The binding affinity of SEX4 C198S and amylopectin interaction was determined to have a KD of 0.11 &#177; 0.05 mg/mL for C198S mutant proteins; a ~10 times increased the binding affinity compared to SEX4 wild-type protein. While the exact mechanism of the increased binding of C198S is not understood, it is intriguing to see how a single-point mutation of SEX4 results in a significant increase in glucan binding ability.
Next, the applicability of this method to measure the binding of laforin, LSF2, corn (Zea mays) SEX4, and potato (Solanum tuberosum) SEX4 against potato amylopectin was assessed. Using the protocol presented here, the results demonstrated that laforin, LSF2, and SEX4 from agronomically important crops also differentially bind to amylopectin, suggesting different binding mechanisms for each protein (Figure 4A,B). Collectively, these findings revealed a successful method development for the determination of substrate binding of glucan phosphatase with amylopectin substrates.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64700/64700fig02.jpg" /> 
Figure 2: Visualization of pellet (P) and supernatant (S) SEX4 fractions and optimization of the assay parameters. (A)&#160;Visualization of 5 &#181;g of SEX4 protein (34 kDa) in the pellet and supernatant fractions using SDS-PAGE and Coomassie staining. The protein bands at 25 kDa and below are for the ConA protein attached to Sepharose beads. (B)&#160;Visualization of SEX4 proteins in the pellet and supernatant fractions via western analysis. 5 &#181;g of SEX4 protein was detected via an antibody developed for the histidine tag. (C-E)&#160;The western analysis was done using varying amounts of SEX4 protein (2 &#181;g, 5 &#181;g, and 10 &#181;g) and amylopectin concentrations (0.5 mg/mL, 1.0 mg/mL, and 5.0 mg/mL). A volume of 250 &#181;L of beads and an incubation time of 30 min were used for all experiments to ensure equilibrium binding. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64700/64700fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64700/64700fig03.jpg" /> 
Figure 3: Quantitative in vitro sedimentation assay. (A)&#160;Representative data of supernatant depletion and the gradual increase of pellet signal of SEX4 wild-type protein as the amylopectin concentration increased from 0 mg/mL to 5 mg/mL. (B) The percentage of SEX4 wild-type protein bound against varying amounts of amylopectin immobilized onto ConA-Sepharose beads. The beads were pelleted at 10,000 x g for 1 min, and both the supernatant and pellet proteins were separated by SDS-PAGE, detected by western analysis, and quantified to measure the percent bound. Data represent the average &#177; SD of three replicates. (C) Representative data of&#160;supernatant depletion and the gradual increase of pellet signal of SEX4 C198S protein as amylopectin concentration increased from 0 mg/mL to 5 mg/mL. (D) The percentage of SEX4 C198S protein bound against varying amounts of amylopectin immobilized onto ConA-Sepharose beads. The beads were pelleted at 10,000 x g for 1 min, and both the supernatant and pellet proteins were separated by SDS-PAGE, detected by western analysis, and quantified to measure the percent bound. Data represent the average &#177; SD of three replicates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64700/64700fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64700/64700fig04.jpg" /> 
Figure 4: Substrate binding of SEX4 orthologs, laforin, LSF2 against solubilized&#160; amylopectin. (A) Representative binding of each protein with 5 mg/mL of amylopectin immobilized in ConA-Sepharose beads. (B) The percentage of bound protein was calculated using the normalized chemiluminescence signals obtained for three independent experiments. Data represent the mean &#177; SD of three replicates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64700/64700fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases at JoVE.com

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates.

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals ...

concanavalin-based-sedimentation-assay-to-measure-substrate-binding

Nanyang Technological University

Research

JoVE Journal

Biochemistry

1.3K Views.  Skidmore College. This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates. 

Video: Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

Environment

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Biology

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins of several nucleotide-derived secondary messengers (NSMs) have been systematically identified and studied in model organisms. These achievements are mainly due to the development of several new techniques including the capture compound technique and the differential radial capillary action of ligand assay (DRaCALA), which were used to systematically identify target proteins of these small molecules. This paper describes the use of the NSMs, guanosine penta- and tetraphosphates (p)ppGpp, as an example and video demonstration of the DRaCALA technique. Using DRaCALA, 9 out of 20 known and 12 new target proteins of (p)ppGpp were identified in the model organism, Escherichia coli K-12, demonstrating the power of this assay. In principle, DRaCALA could be used for studying small ligands that can be labeled by radioactive isotopes or fluorescent dyes. The critical steps, pros, and cons of DRaCALA are discussed here for further application of this technique.

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Immunology and Infection

Characterization of Cell Wall-Related Enzymes in RWA-Infested Wheat Cultivars

Elucidating &#946;-1,3-Glucanase and Peroxidase Physicochemical Properties of Wheat Cell Wall Defense Mechanism Against Diuraphis noxia Infestation

Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defense responses, including the hypersensitive responses (HR) and induction of pathogenesis-related (PR) proteins, such as &#946;-1,3-glucanase and peroxidase (POD). This study aims to characterize the physicochemical properties of cell wall-associated POD and &#946;-1,3-glucanase and determine their synergism on the cell wall modification during RWASA2-wheat interaction. The susceptible Tugela, moderately resistant Tugela-Dn1, and resistant Tugela-Dn5 cultivars were pregerminated and planted under greenhouse conditions, fertilized 14 days after planting, and irrigated every 3 days. The plants were infested with 20 parthenogenetic individuals of the same RWASA2 clone at the 3-leaf stage, and leaves were harvested at 1 to 14 days post-infestation. The Intercellular wash fluid (IWF) was extracted using vacuum filtration and stored at -20 &#176;C. Leaf residues were crushed into powder and used for cell wall components. POD activity and characterization were determined using 5 mM guaiacol substrate and H2O2, monitoring change in absorbance at 470 nm. &#946;-1,3-glucanase activity, pH, and temperature optimum conditions were demonstrated by measuring the total reducing sugars in the hydrolysate with DNS reagent using &#946;-1,3-glucan and &#946;-1,3-1,4-glucan substrates, measuring the absorbance at 540 nm, and using glucose standard curve. The pH optimum was determined between pH 4 to 9, temperature optimum between 25 and 50 &#176;C, and thermal stability between 30 &#176;C and 70 &#176;C. &#946;-1,3-glucanase substrate specificity was determined at 25 &#176;C and 40 &#176;C using curdlan and barley &#946;-1,3-1,4-glucan substrates. Additionally, the &#946;-1,3-glucanase mode of action was determined using laminaribiose to laminaripentaose. The oligosaccharide hydrolysis product patterns were qualitatively analyzed with thin-layer chromatography (TLC) and quantitatively analyzed with HPLC. The method presented in this study demonstrates a robust approach for infesting wheat with RWA, extracting peroxidase and &#946;-1,3-glucanase from the cell wall region and their comprehensive biochemical characterization.

Author Spotlight: Integrating Biochemical Functions of &#946;-Glucanases and Peroxidase Enzymes in Wheat-RWA Interaction

The present protocol describes procedures used to study and characterize cell wall-related enzymes, mainly &#946;-1,3-glucanase and peroxidase, in wheat plants. Their activity levels increase during wheat-RWA interaction and are involved in the plant defense response through cell wall reinforcement, which deters aphid feeding.

Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defense responses, including the hypersensitive responses (HR) and induction of ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

Measurement of Chitinase Activity in Biological Samples

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body including at baseline. The precise role of chitinase is not known, however it was believed to play important role in digestion and host defense against chitin-containing food and pathogens, respectively. Recent work, including ours, has shown an important role of chitinase and chitinase-like proteins in host immunity and allergic diseases. Importantly, chitinase activities serve as important biomarkers of disease severity in a wide-range of diseases including type 2 inflammatory diseases such as asthma and pulmonary fibrosis. Similarly, patients with genetic disorders like Gaucher disease have significantly elevated chitinase levels, which not only correlate with disease severity but also serve as a reliable biomarker for therapeutic effectiveness. The protocol outlined here describes a simple, quick, and straightforward way to measure chitinase activity in BAL or serum samples of mice and can be widely adapted to human subjects and other model organisms due to the highly conserved nature of the enzymes.

Presented here is a simple method to measure chitinase activity in biological fluids such as bronchoalveolar lavage or serum.

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body ...

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

C. parapsilosis (Cp) is an emerging cause of bloodstream infections in certain populations. The&#160;Candida clade, including Cp, is increasingly developing resistance to the first and the second line of antifungals. Cp is frequently isolated from hands and skin surfaces, as well as the GI tract. Colonization by Candida predisposes individuals to invasive bloodstream infections. To successfully colonize or invade the host, yeast must be able to rapidly adhere to the body surfaces to prevent elimination by host defense mechanisms. Here we describe a method to measure adhesion of Cp to immobilized proteins under physiologic fluid shear, using an end-point adhesion assay in a commercially available multichannel microfluidic device. This method is optimized to improve reproducibility, minimize subjectivity, and allow for the fluorescent quantification of individual isolates. We also demonstrate that some clinical isolates of Cp show increased adhesion when grown in conditions mimicking a mammalian host, whereas a frequently used lab strain, CDC317, is non-adhesive under fluid shear.

Adhesion of Candida parapsilosis to Bovine Serum Albumin under Fluid Shear

Adhesion is an important first step in colonization and pathogenesis for Candida. Here, an in vitro assay is described to measure adhesion of C. parapsilosis isolates to immobilized proteins under fluid shear. A multichannel microfluidics device is used to compare multiple samples in parallel, followed by quantification using fluorescence imaging.

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

Summary

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