Novel method for detection of glycogen in cells

Glycobiology, 2017, vol. 27, no. 5, 416–424 doi: 10.1093/glycob/cwx005 Advance Access Publication Date: 15 February 2017 Original Article Cell Biology Novel method for detection of glycogen in cells Alexander V Skurat1, Dyann M Segvich, Anna A DePaoli-Roach, and Peter J Roach Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA To whom correspondence should be addressed: Tel: +1-317-278-1764; Fax: +1-317-274-4686; e-mail: 1 Received 4 August 2016; Revised 4 November 2016; Editorial decision 4 January 2017; Accepted 9 January 2017 Abstract Glycogen, a branched polymer of glucose, functions as an energy reserve in many living organisms. Abnormalities in glycogen metabolism, usually excessive accumulation, can be caused genetically, most often through mutation of the enzymes directly involved in synthesis and degradation of the polymer leading to a variety of glycogen storage diseases (GSDs). Microscopic visualization of glycogen deposits in cells and tissues is important for the study of normal glycogen metabolism as well as diagnosis of GSDs. Here, we describe a method for the detection of glycogen using a renewable, recombinant protein which contains the carbohydrate-binding module (CBM) from starch-binding domain containing protein 1 (Stbd1). We generated a fusion protein containing glutathione S-transferase, a cMyc eptitope and the Stbd1CBM (GYSC) for use as a glycogen-binding probe, which can be detected with secondary antibodies against glutathione Stransferase or cMyc. By enzyme-linked immunosorbent assay, we demonstrate that GYSC binds glycogen and two other polymers of glucose, amylopectin and amylose. Immunofluorescence staining of cultured cells indicate a GYSC-specific signal that is co-localized with signals obtained with anti-glycogen or anti-glycogen synthase antibodies. GYSC-positive staining inside of lysosomes is observed in individual muscle fibers isolated from mice deficient in lysosomal enzyme acid alpha-glucosidase, a well-characterized model of GSD II (Pompe disease). Co-localized GYSC and glycogen signals are also found in muscle fibers isolated from mice deficient in malin, a model for Lafora disease. These data indicate that GYSC is a novel probe that can be used to study glycogen metabolism under normal and pathological conditions. Key words: CBM20, glycogen, immunofluorescence, Lafora disease, Pompe disease Introduction Glycogen is a branched polymer of glucose and is the primary carbohydrate storage form in animals (Agius 2008; Roach et al. 2012). Glycogen synthesis and degradation are tightly controlled by complex regulatory mechanisms (Agius 2008; Roach et al. 2012; Adeva-Andany et al. 2016). Disturbances in this regulation or in the metabolic enzymes themselves can result in aberrant glycogen stores, resulting in a glycogenosis or glycogen storage disease (GSD) (Chen and Burchell 1995; DiMauro and Lamperti 2001; DiMauro and Spiegel 2011; Oldfors and DiMauro 2013). These are monogenic, congenital disorders in which a genetic defect results in abnormal amounts and/or structure of glycogen. The first examples involved mutations linked directly to glycogen metabolizing enzymes, such as GSD II (Pompe disease) in which the lysosomal α-glucosidase is defective (Reuser et al. 1995; Hirschhorn and Reuser 2000; Raben et al. 2002). Histochemical staining of glycogen is therefore important diagnostically, the most commonly used method being periodic acid Schiff (PAS) staining that visualizes molecules with a high percentage of carbohydrate content (McManus 1946; Hotchkiss 1948). One disadvantage of PAS staining for the detection of glycogen in tissues is lack of specificity. Besides glycogen, PAS staining detects other carbohydrate containing molecules including glycoproteins, © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: 416 417 Detection of glycogen in cells Results Design, expression and purification of a glycogenbinding probe Previous studies had demonstrated that Stbd1 binds to glycogen in vitro and co-localized with glycogen in mammalian cells (Jiang et al. 2010). To generate a probe capable of detecting glycogen, we designed a bacterial expression plasmid that would encode a fusion protein containing glutathione S-transferase (GST), a cMyc-tag and a carbohydrate-binding module corresponding to residues 261–358 of human Stbd1 (Figure 1 A). The resulting protein, named GYSC, was expressed in Escherichia coli strain BL21, purified by absorption on glutathione-agarose beads, eluted with glutathione and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Purified fractions contained a predominant band with Mr ~ 39 kDa (Figure 1B). Carbohydrate-binding specificities of GYSC and anti-glycogen antibody In order to characterize the carbohydrate-specificity of GYSC, we used glycogen and two other glycogen-like polymers, amylopectin and amylose, which differ from glycogen in their degree of branching. Mammalian glycogen is branched on average at 1 in ~13 glucose residues, amylopectin 1 in 20–25 and amylose is scarcely branched at all (Roach et al. 2012; Roach and Zeeman 2016). Binding analysis was performed by enzyme-linked immunosorbent assay (ELISA). GYSC bound to all three polymers of glucose with slightly higher affinity to amylopectin and lower affinity to amylose, as compared to glycogen (Figure 2 A). Half maximal effective concentrations (EC50) of glycogen, amylopectin and amylose for GYSC binding were 0.18, 0.07 and 0.57 μg/mL, respectively. We also analyzed binding of the Baba antibody (Baba 1993) to the same Fig. 1. Scheme for experimental procedures with GYSC and analysis of purified GYSC by SDS-PAGE. In (A), a fusion protein GYSC containing the CBM20 domain from human Stbd1 with attached two epitope tags, cMyc (myc) and GST is shown. The CBM20 domain serves for binding of GYSC to glycogen, whereas GST-tag is used for detection of glycogen-bound GYSC by either ELISA or immunofluorescent microscopy. In both methods, the GYSC–glycogen complex is incubated first with rabbit anti-GST antibody and then with anti-rabbit secondary antibody conjugated with HRP (ELISA) or fluorophore (Texas Red is shown as an example). In (B), Coomassie blue stained SDS-PAGE gel of purified GYSC is shown. Numbers on the left indicate migration of molecular mass markers (in kDa). GYSC, a fusion protein composed of glutathione S-transferase, a cMyc eptitope and the Stbd1CBM; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; CBM, carbohydrate-binding module; GST, glutathione S-transferase; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase.This figure is available in black and white in print and in color at Glycobiology online. polysaccharides. As expected, this antibody bound glycogen (EC50 = 0.92 μg/mL) but it did not recognize amylopectin or amylose (Figure 2B), consistent with a (...truncated)