title: Novel method for detection of glycogen in cells.
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 g lutathione S-transferase, a cM c eptitope and the tbd1 BM (GYSC) for use as a glycogen-binding probe, which can be detected with secondary antibodies against glutathione S-transferase 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Skurat
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: Dyann M
authors.initials: DM
authors.lastname: Segvich
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: Anna A
authors.initials: AA
authors.lastname: DePaoli-Roach
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Roach
mesh_terms: D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay; D005947:Glucose / Q000378:metabolism*; D005982:Glutathione Transferase / Q000737:chemistry; D006003:Glycogen / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006008:Glycogen Storage Disease / Q000175:diagnosis* / Q000378:metabolism; D006801:Humans; D020192:Lafora Disease / Q000175:diagnosis* / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D051379:Mice; D009124:Muscle Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D005947:Glucose; D005982:Glutathione Transferase; D006003:Glycogen; D008565:Membrane Proteins; D009124:Muscle Proteins; D011994:Recombinant Proteins; C116148:STBD1 protein, human
keywords: CBM20; Lafora disease; Pompe disease; glycogen; immunofluorescence