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

Summary

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.

Abstract

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. As a proof of concept, the dissociation constant (K_D) 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.

Introduction

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 protists^2,3,4. Plants contain three known glucan phosphatases: Starch Excess4 (SEX4), Like Sex Four1 (LSF1), and Like Sex Four2 (LSF2)^5,6,7. Plants that l....

Protocol

1. Preparation of ConA-Sepharose beads

1. 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.
2. Pipette 250 μL 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 °C. Discard the supernatant.
   NOTE: 250 µL of ConA-Sepharose beads in a 1.5 mL microcentrifuge tube is needed for each amylopectin concentration used for the assay.
3. Add 750 µL of the binding buffer to each tube containing 250 µL of ConA-Sepharose beads. ....

Results

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
6x-His Tag monoclonal antibody (HIS.H8), HRP	Therm Fisher Scientific	MA1-21315-HRP
Biorad gel electrophoresis and Western blot kit	Biorad	1703930
Calcium chloride	Sigma-Aldrich	208291
C-Digit blot scanner	LICOR	3600-00	Blot scanner
Complete protease inhibitor cocktail	Sigma-Aldrich	11836170001
Concanavalin A-sepharose beads	Sigma-Aldrich	C9017	This product contains  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
Centrifuge	Eppendorf	5425R
Glycine	Fisher Scientific	BP381-5
GraphPad Prism 8.0 software	GraphPad	Version 8.0	Data analysis software
HEPES	Sigma-Aldrich	H8651
Image Studio	LICOR	3600-501	Acquisition Software
Magnesium chloride	Sigma-Aldrich	M2670
Methanol	Fisher Scientific	A452SK-4
Sodium dodecyl sulfate	Fisher Scientific	PI28312
Potato amylopectin	Sigma-Aldrich	A8515
Precast SDSPAGE Gels	Genscript	M00653S
Tris base	Fisher Scientific	BP154-1
Tween 20	Fisher Scientific	MP1TWEEN201
Westernsure premium chemiluminescence substrate	LI-COR	926-95000