Novel method for detection of glycogen in cells

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)416-424
Number of pages9
JournalGlycobiology
Volume27
Issue number5
DOIs
StatePublished - May 1 2017

Fingerprint

Glycogen
Glycogen Storage Disease Type II
Glycogen Storage Disease
Glucans
Metabolism
Lafora Disease
Staining and Labeling
Amylopectin
Muscle
Muscles
Glycogen Synthase
Amylose
Enzymes
alpha-Glucosidases
Antibodies
Lysosomes
Glutathione Transferase
Recombinant Proteins
Immunosorbents
Starch

Keywords

  • CBM20
  • glycogen
  • immunofluorescence
  • Lafora disease
  • Pompe disease

ASJC Scopus subject areas

  • Biochemistry

Cite this

Novel method for detection of glycogen in cells. / Skurat, Alexander; Segvich, Dyann M.; De Paoli-Roach, Anna; Roach, Peter.

In: Glycobiology, Vol. 27, No. 5, 01.05.2017, p. 416-424.

Research output: Contribution to journalArticle

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