Name: concanavalin a-based sedimentation assay to measure substrate binding of glucan phosphatases
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Description: Watch this Scientific Journal Video about Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases at JoVE.com

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 amylo...

<ol>
	<li>Meekins, D. A., Vander Kooi, C. W., Gentry, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+mechanisms+of+plant+glucan+phosphatases+in+starch+metabolism.">Structural mechanisms of plant glucan phosphatases in starch metabolism.</a> The FEBS Journal. 283 (13), 2427-2447 (2016).</li><li>Gentry, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phosphatase+laforin+crosses+evolutionary+boundaries+and+links+carbohydrate+metabolism+to+neuronal+disease.">The phosphatase laforin crosses evolutionary boundaries and links carbohydrate metabolism to neuronal disease.</a> The Journal of Cell Biology. 178 (3), 477-488 (2007).</li><li>Worby, C. A., Gentry, M. S., Dixon, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laforin,+a+dual+specificity+phosphatase+that+dephosphorylates+complex+carbohydrates.">Laforin, a dual specificity phosphatase that dephosphorylates complex carbohydrates.</a> The Journal of Biological Chemistry. 281 (41), 30412-30418 (2006).</li><li>Gentry, M. S., Pace, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+the+glucan+phosphatase+laforin+is+linked+to+rates+of+molecular+evolution+and+the+glucan+metabolism+of+the+organism.">Conservation of the glucan phosphatase laforin is linked to rates of molecular evolution and the glucan metabolism of the organism.</a> BMC Evolutionary Biology. 9, 138 (2009).</li><li>Niittyla, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similar+protein+phosphatases+control+starch+metabolism+in+plants+and+glycogen+metabolism+in+mammals.">Similar protein phosphatases control starch metabolism in plants and glycogen metabolism in mammals.</a> The Journal of Biological Chemistry. 281 (17), 11815-11818 (2006).</li><li>Kotting, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STARCH-EXCESS4+is+a+laforin-like+Phosphoglucan+phosphatase+required+for+starch+degradation+in+Arabidopsis+thaliana.">STARCH-EXCESS4 is a laforin-like Phosphoglucan phosphatase required for starch degradation in Arabidopsis thaliana.</a> The Plant Cell. 21 (1), 334-346 (2009).</li><li>Comparot-Moss, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+putative+phosphatase,+LSF1,+is+required+for+normal+starch+turnover+in+Arabidopsis+leaves.">A putative phosphatase, LSF1, is required for normal starch turnover in Arabidopsis leaves.</a> Plant Physiology. 152 (2), 685-697 (2010).</li><li>Zeeman, S. 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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 bindi...

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.

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			<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&#160;</td>
			<td>1703930</td>
			<td></td>
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			<td>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>208291</td>
			<td></td>
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			<td>C-Digit blot scanner</td>
			<td>LICOR</td>
			<td>3600-00</td>
			<td>Blot scanner</td>
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			<td>Complete protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>11836170001</td>
			<td></td>
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		<tr>
			<td>Concanavalin A-sepharose beads</td>
			<td>Sigma-Aldrich</td>
			<td>C9017</td>
			<td>This product contains&#160; in 0.1 M acetate buffer, pH 6, containing 1 M NaCl, 1 mM CaCl2, 1 mM MnCl2, and 1 mM MgCl2 in 20% ethanol&#160;</td>
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			<td>Centrifuge</td>
			<td>Eppendorf&#160;</td>
			<td>5425R</td>
			<td></td>
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			<td>Glycine</td>
			<td>Fisher Scientific</td>
			<td>BP381-5</td>
			<td></td>
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			<td>GraphPad Prism 8.0 software</td>
			<td>GraphPad&#160;</td>
			<td>Version 8.0</td>
			<td>Data analysis software&#160;</td>
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			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H8651</td>
			<td></td>
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			<td>Image Studio</td>
			<td>LICOR</td>
			<td>3600-501</td>
			<td>Acquisition Software</td>
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			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td></td>
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			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A452SK-4</td>
			<td></td>
		</tr>
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			<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>
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			<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>
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			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>MP1TWEEN201</td>
			<td></td>
		</tr>
		<tr>
			<td>Westernsure premium chemiluminescence substrate&#160;</td>
			<td>LI-COR</td>
			<td>&#160;926-95000</td>
			<td></td>
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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 frac...

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 ...

This concanavalin A-based in vitro sedimentation assay is designed to measure binding affinity of glucan phosphatases against different STA substrates. The information we gain from this method is fundamental to our understanding of glucan phosphatase family of enzymes. Carbohydrate protein interactions are relatively weak, therefore current methods cannot detect glucan phosphatase and glucan interactions.Our lectin based cosedimentation assay can detect binding affinity in the millimolar range. The characterization of SEX4 mutants as well as other members of the glucan phosphatase family of enzymes demonstrates the ability of this assay to quantify protein carbohydrate interactions. One of the most challenging parts of the assay is to optimize the concentration range of the substrate.We recommend starting with a broader range to get an idea and then narrowing down for a specific range. To begin, pipette 250 microliters of ConA-sepharose beads suspension into a 1.5 milliliter microcentrifuge tube. Centrifuge the contents at 10, 000 G for 30 seconds at four degrees Celsius.After discarding the supernatant, add 750 microliters of the binding buffer to each tube containing 250 microliters of ConA-sepharose beads. Centrifuge the tubes at 10, 000 G for one minute at four degrees Celsius and remove the supernatant. Next, prepare twofold dilutions of the solubilized amylopectin solution to make a series of two milliliters diluted amylopectin solutions.To prepare the ConA-sepharose amylopectin beads, add 250 microliters of each diluted amylopectin solution to the tubes containing 250 microliters of ConA-sepharose beads preequilibrated and binding buffer. Mix the contents well and label the tubes with the corresponding amylopectin concentration. Incubate the tube contents on a rotating wheel at four degrees Celsius for 30 minutes and centrifuge the tubes at 10, 000 G for one minute.Mix 250 microliters of ConA-sepharose amylopectin beads with 100 microliters of the binding buffer containing 10 micrograms of starch excess four, or SEX4 protein, 10 millimolar of dithiothreitol, and 10 micromolar of protease inhibitor cocktail. Incubate the contents at four degrees Celsius for 45 minutes with gentle rotation. Then centrifuge the tubes at 10, 000 G for one minute and carefully pipette 50 microliters of the supernatant into a new 1.5 milliliter microcentrifuge tube.Add 20 microliters of 4X SDS page dye and 10 microliters of distilled water to each tube containing 50 microliters of the collected supernatant fractions and label them as S.Similarly, add 20 microliters of 4X SDS page dye and 80 microliters of distilled water into the tubes containing washed ConA-sepharose amylopectin SEX4 beads. After adding dye and water, heat all the samples at 95 degrees Celsius for 10 minutes. Once the ConA-sepharose amylopectin SEX4 beads are centrifuged at 10, 000 G for one minute, save the supernatant and label it as P.Load 40 microliters of the unbound protein samples labeled as S into 4 to 12%precast polyacrylamide gel wells from the lowest substrate concentration to the highest, reserving the first lane for a protein molecular weight marker.Use a second gel to load the glucan bound protein samples labeled P.Add freshly prepared SDS page running buffer to both chambers of the apparatus and run the gel at 150 volts for 35 minutes or until the dye front reaches the bottom of the gel. Then take the gel cassette from the apparatus and remove the spacers and glass plates to separate the gel for western blot analysis. Make one liter of transfer buffer containing 5.8 grams of tris base, 2.9 grams of glycine, 0.37 grams of SDS, and 200 milliliters of methanol.To transfer the size separated proteins from the polyacrylamide gel to a nitrocellulose membrane, assemble the sponges, filter papers, gel, and nitrocellulose membrane according to Western transfer protocol and run at 70 volts for one hour. After running the gel, incubate the nitrocellulose membrane in a protein solution of 1 to 5%bovine serum albumin or milk protein in 50 milliliters of TBST buffer for one hour to prevent non-specific protein binding. Wash the membrane three times using TBST buffer to remove any unbound blocking solution.Next, incubate the membrane for one hour with the diluted horseradish peroxidase linked antibody specific for histidine tagged protein. Then wash the membrane three times in TBST buffer to remove any unbound antibodies. For digital imaging, make a solution of equal parts of chemiluminescent substrate solutions, 750 microliters each in a 1.5 milliliter tube, and incubate the membrane for at least five minutes in the solution.Place the membrane protein side down on the blot scanner and run the acquisition software to quantify the protein in both the the pellet and supernatant fractions. To acquire the data, start 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.In the saturating binding experiment, plot the percentage of protein bound versus amylopectin concentration and fit the data using data analysis software to calculate the dissociation constant, Kd.The binding capacity of SEX4 to ConA-sepharose amylopectin beads was analyzed using SDS page. The presence of SEX4 protein in the pellet fraction and bovine serum albumin in the supernatant fraction was noted. W278A, an known SEX4 mutant with significantly reduced glucan binding ability, appeared in the supernatant fraction, indicating the utility and specificity of this assay.Western blotting analysis revealed a significant increase in the detection of SEX4 and the method is specific for detecting glucan phosphates with an end terminal histidine tag. The cosedimentation assay was tested at three different SEX4 concentrations. While two micrograms of SEX4 is sufficient to visualize through chemiluminescence detection, using five or 10 micrograms of SEX4 allows for more accurate detection.Also, increased SEX4 binding with increased amylopectin concentration was noted. The binding affinity of amylopectin and SEX4 wild type protein at different concentrations was determined and the percent protein bound data to the specific one site binding model gave a Kd of approximately 1.03 milligrams per milliliter. The applicability of this method to measure the binding of laforin, LSF2, corn SEX4, and potato SEX4 against potato amylopectin was assessed.The results demonstrated that the tested proteins from agronomically important crops are also differentially bound to amylopectin. It is crucial to remember the normalization of all quantitative measurements in the pellet and supernatant fractions to the total protein loaded. To ensure all amylopectin is bound to the beads, save the supernatant fractions to perform the deep glucose assay later.Glucan phosphatase is employed distinct mechanisms to bind, locate, and dephosphorylate carbohydrates. This method allows for the exploration of glucan phosphatase's binding to different structural domains within large polymeric complexes for position specific dephosphorylation.

Research

JoVE Journal

Biochemistry

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 ...

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

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

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 ...