Name: concanavalin a-based sedimentation assay to measure substrate binding of glucan phosphatases
Uploaded: 2022-12-23T14:50:00
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. C., Northrop, F., Smith, A. M., Rees, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+starch-accumulating+mutant+of+Arabidopsis+thaliana+deficient+in+a+chloroplastic+starch-hydrolysing+enzyme.">A starch-accumulating mutant of Arabidopsis thaliana deficient in a chloroplastic starch-hydrolysing enzyme.</a> The Plant Journal: For Cell and Molecular Biology. 15 (3), 357-365 (1998).</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=Identification+of+a+novel+enzyme+required+for+starch+metabolism+in+Arabidopsis+leaves.+The+phosphoglucan,+water+dikinase.">Identification of a novel enzyme required for starch metabolism in Arabidopsis leaves. The phosphoglucan, water dikinase.</a> Plant Physiology. 137 (1), 242-252 (2005).</li><li>Tagliabracci, V. 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=Laforin+is+a+glycogen+phosphatase,+deficiency+of+which+leads+to+elevated+phosphorylation+of+glycogen+in+vivo.">Laforin is a glycogen phosphatase, deficiency of which leads to elevated phosphorylation of glycogen in vivo.</a> Proceedings of the National Academy of Sciences. 104 (49), 19262-19266 (2007).</li><li>Gentry, M. S., Guinovart, J. J., Minassian, B. A., Roach, P. J., Serratosa, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lafora+disease+offers+a+unique+window+into+neuronal+glycogen+metabolism.">Lafora disease offers a unique window into neuronal glycogen metabolism.</a> The Journal of Biological Chemistry. 293 (19), 7117-7125 (2018).</li><li>Brewer, M. 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A., 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=Structure+of+the+Arabidopsis+glucan+phosphatase+like+sex+four2+reveals+a+unique+mechanism+for+starch+dephosphorylation.">Structure of the Arabidopsis glucan phosphatase like sex four2 reveals a unique mechanism for starch dephosphorylation.</a> The Plant Cell. 25 (6), 2302-2314 (2013).</li><li>Smith, A. M., Zeeman, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Starch:+A+flexible,+adaptable+carbon+store+coupled+to+plant+growth.">Starch: A flexible, adaptable carbon store coupled to plant growth.</a> Annual Review of Plant Biology. 71, 217-245 (2020).</li><li>Jane, J., Kasemuwan, T., Chen, J. F., Juliano, B. 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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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		<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&#160;</td>
			<td>1703930</td>
			<td></td>
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			<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>
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		<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&#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>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf&#160;</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&#160;</td>
			<td>Version 8.0</td>
			<td>Data analysis software&#160;</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&#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 ...

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