Distinct mutations in the glycogen debranching enzyme found in glycogen storage disease type III lead to impairment in diverse cellular functions

Abstract

Glycogen storage disease type III (GSDIII) is a metabolic disorder characterized by a deficiency in the glycogen debranching enzyme, amylo-1,6-glucosidase,4-alpha-glucanotransferase (AGL). Patients with GSDIII commonly exhibit hypoglycemia, along with variable organ dysfunction of the liver, muscle or heart tissues. The AGL protein binds to glycogen through its C-terminal region, and possesses two separate domains for the transferase and glucosidase activities. Most causative mutations are nonsense, and how they affect the enzyme is not well understood. Here we investigated four rare missense mutations to determine the molecular basis of how they affect AGL function leading to GSDIII. The L620P mutant primarily abolishes transferase activity while the R1147G variant impairs glucosidase function. Interestingly, mutations in the carbohydrate-binding domain (CBD; G1448R and Y1445ins) are more severe in nature, leading to significant loss of all enzymatic activities and carbohydrate binding ability, as well as enhancing targeting for proteasomal degradation. This region (Y1445-G1448R) displays virtual identity across human and bacterial species, suggesting an important role that has been conserved throughout evolution. Our results clearly indicate that inactivation of either enzymatic activity is sufficient to cause GSDIII disease and suggest that the CBD of AGL plays a major role to coordinate its functions and regulation by the ubiquitin-proteasome system.

Mutation Analysis in Glycogen Storage Disease Type III Patients in the Netherlands: Novel Genotype-Phenotype Relationships and Five Novel Mutations in the AGL Gene

“…1). This makes a pathogenic effect of these mutations on GDE function very probable (Cheng et al 2009). As the c.3911delA, p.Asn1304fs causes a frameshift with the new reading frame ending in a stop codon at position 10, this is considered to be a pathogenic mutation as well.…”

“…However, pathogenic missense mutations have been described. Cheng et al showed that missense mutations located in the active sites produce a GSD III phenotype in which there is total or partial abolishment of GDE activity depending on the location of the mutation (Cheng et al 2009). …”

Molecular features of 23 patients with glycogen storage disease type III in Turkey: a novel mutation p.R1147G associated with isolated glucosidase deficiency, along with 9 AGL mutations

“…23 Moreover, we expressed p.R1147G mutant and normal AGL proteins in COS cells transiently, and measured both glucosidase and transferase activities. 7 The p.R1147G exhibited negligible glucosidase activity, but retained 40% of transferase activity compared with wild AGL. p.R1147G might alter the tertiary conformation of the enzyme and slightly reduce the transferase activity.…”

“…In vitro functional analysis of p.R1147G, which was identified in patient 1, has been reported elsewhere. 7 A gene coding human glycogen debranching enzyme (gene symbol: AGL) has been shown to be 85 kb in length and composed of 35 exons, encoding a 7.0-kb mRNA. 8 Molecular analyses of GSD IIIa and IIIb have been performed in several ethnic populations and over 60 different AGL mutations have been reported to date (Human Gene Mutation Database; http://www.hgmd.cf.ac.uk/ac/index.php).…”

Molecular diagnosis of glycogen storage disease and disorders with overlapping clinical symptoms by massive parallel sequencing

“…[13][14][15] Missense mutations are scarce. 13,16,17 No mutation was prevalent, but c.348_373del26 was present in three mutant alleles (21%). Loss-of-function mutations in PHKA2 made up nearly 71% of the total; only 50% of the mutations deposited for this gene in the Human Gene Mutation Database belong to this category.…”