A role for AGL ubiquitination in the glycogen storage disorders of Lafora and Cori's disease

Abstract

Cori's disease is a glycogen storage disorder characterized by a deficiency in the glycogen debranching enzyme, amylo-1,6-glucosidase,4-alpha-glucanotransferase (AGL). Here, we demonstrate that the G1448R genetic variant of AGL is unable to bind to glycogen and displays decreased stability that is rescued by proteasomal inhibition. AGL G1448R is more highly ubiquitinated than its wild-type counterpart and forms aggresomes upon proteasome impairment. Furthermore, the E3 ubiquitin ligase Malin interacts with and promotes the ubiquitination of AGL. Malin is known to be mutated in Lafora disease, an autosomal recessive disorder clinically characterized by the accumulation of polyglucosan bodies resembling poorly branched glycogen. Transfection studies in HepG2 cells demonstrate that AGL is cytoplasmic whereas Malin is predominately nuclear. However, after depletion of glycogen stores for 4 h, approximately 90% of transfected cells exhibit partial nuclear staining for AGL. Furthermore, stimulation of cells with agents that elevate cAMP increases Malin levels and Malin/AGL complex formation. Refeeding mice for 2 h after an overnight fast causes a reduction in hepatic AGL levels by 48%. Taken together, these results indicate that binding to glycogen crucially regulates the stability of AGL and, further, that its ubiquitination may play an important role in the pathophysiology of both Lafora and Cori's disease.

Figure 1.

Disrupting the CBD of AGL leads to decreased protein stability. (A) Schematic of wild-type and mutant AGL proteins. AGL possesses transferase, glucosidase, and CBDs. Truncations and a G1448R mutant were made. (B) AGL mutants are unable to bind amylose resin. The indicated constructs were transfected into COS cells, and lysates were subjected to a pull-down assay using amylose resin beads. (C) AGL mutants do not fractionate into the glycogen-enriched fraction. Transfected COS cells were subjected to differential centrifugation, and the proportion of AGL proteins in each fraction was determined by immunoblotting. (D) AGL carbohydrate-binding mutants exhibit decreased protein stability. Cells were transfected with Flag-tagged wild-type, ΔCBD, or G1448R AGL. The next day, cells were treated with cycloheximide and labeled with ³⁵S methionine for 2 h. Cell lysates were immunoprecipitated with anti-Flag beads and subjected to SDS-PAGE. Gels were transferred to nitrocellulose membranes and processed for autoradiography. (E) Proteasomal inhibition increases the levels of mutant AGL proteins. Transfected cells were treated with 100 μg/mL cycloheximide with or without 10 μM MG-132 for 6 h. Cell lysates were then analyzed by immunoblotting with the indicated antibodies. (F) Mutant AGL proteins exhibit increased ubiquitination. Cells were transfected as in B, in addition with HA-UB. Cells were lysed in denaturing immunoprecipitation buffer, and lysates were immunoprecipitated with anti-Flag antibodies. Immunoprecipitates were immunoblotted with the indicated antibodies.

Figure 2.

The AGL (G1448R) mutant forms aggresomes. (A) Disrupting the CBD of AGL induces inclusion body formation upon proteasomal impairment. Flag-tagged wild-type (WT), ΔCBD, or G1448R AGL proteins were immunostained with Flag antibodies (green), and nuclei were visualized with propidium iodide (PI, red). Localization of AGL in COS cells growing in culture in the absence (left panels) or presence (right panels) of proteasome inhibitor. Cells were transfected with AGL constructs for 12 h, and then 10 μM MG-132 was included during the media change and cells were allowed to continue for an additional 12 h before immunofluorescence analysis. (B–E) The AGL (G1448R)-containing inclusions possess aggresome-like properties. Flag-tagged AGL G1448R was transfected in COS cells, and 12 h later, 10 μM MG-132 was included during the media change. Cells were allowed to grow for 12 more hours before immunofluorescence analysis with the indicated antibodies. Costaining reveals that Flag-AGL G1448R inclusions (green) colocalize with HA-tagged ubiquitin (red) (B), the molecular chaperone HSP90 (red) (C), and the centrosomal marker γ-tubulin (red) (D). (E) Costaining with α-tubulin (red) reveals the G1448R inclusion is “caged” by a microtubule network. (F) Disruption of the microtubule network by 1 μM nocodazole for 2 h results in loss of G1448R inclusion formation.

Figure 3.

Localization of AGL, Laforin, and Malin under glycogenolytic conditions. (A) Malin is a predominately nuclear protein. COS cells were transfected with Malin-myc for 12 h (low expression) or 24 h (high expression) and processed for immunofluorescence analysis. Cells were stained with anti-myc antibodies (for Malin) and propidium iodide (PI). (B) Localization of Malin-myc in HepG2 cells. (C) AGL and Laforin exhibit partial nuclear staining upon treatment with glycogenolytic agents. HepG2 cells were transfected with the indicated constructs, treated with 50 μM FSK/500 μM IBMX in glucose-free media or left in regular media for 2 h, and then processed for immunofluorescence analysis. (D) Quantification of AGL nuclear staining with different treatments. Cells were scored as positive when nuclear staining was at least as strong as cytoplasmic staining. Error bars represent SEM. (E) Corresponding glycogen levels during the treatments. Levels were normalized to untreated cells. Error bars represent SEM. (F) Inhibition of glycogen phosphorylase prevents nuclear staining of AGL. Cells expressing HA-AGL were preincubated with or without 1 μM phosphorylase inhibitor for 1 h, before treatment with the glycogenolytic agents. New inhibitor was added when medium was changed. Cells were scored as in D. (G) Corresponding glycogen levels during the treatments. Levels were normalized to untreated cells. Error bars, SEM.

Figure 4.

Agents that elevate cAMP increase Malin levels and Malin/AGL complexes. (A) Malin interacts with AGL. 293T cells were grown to 50%–75% confluence in 6-cm dishes prior to transfection with Fugene 6. Flag-AGL was transfected with vector, wild type, or C26S Malin-myc. Cell lysates were immunoprecipitated with anti-myc antibodies and probed for the presence of Flag-AGL. (B) Overexpression of Malin promotes AGL ubiquitination. Cells were transfected as in A, in addition with HA-UB. Cells were lysed in denaturing buffer, and lysates were immunoprecipitated with anti-Flag antibodies. Immunoprecipitates were immunoblotted with the indicated antibodies. (C,D) Increased Malin/AGL coimmunoprecipitation during treatment with FSK but not glucose deprivation alone. Flag-AGL and C26S Malin-myc were cotransfected into 293T cells for 16 h, followed by treatment with the indicated reagents. Lysates were subjected to immunoprecipitations with anti-myc antibodies and analyzed for the presence of Flag-AGL. (E) FSK, but not glucose deprivation, causes an increase in Malin levels. Malin-myc was transfected into 293T cells and treated with the indicated reagents. Lysates were analyzed with the indicated antibodies.

Figure 5.

Regulation of AGL levels in liver and primary hepatocytes during fasting–refeeding. (A) AGL levels in mouse liver decrease during the refeeding after an overnight fast. Three-month-old female C57Bl/6 mice were either allowed to eat freely (Ad. lib.), fasted for 24 h, or refed for 2 h after fasting. Liver lysates were obtained and processed for immunoblotting with the indicated antibodies. Each lane represents one mouse. (B) AGL levels during the time course of refeeding. Fasted mice were allowed to be refed for the indicated times, and then liver lysates were processed as above. (C) Quantification of AGL levels. Three mice were analyzed in each case. Error bars represent SEM. (D) Liver glycogen levels during fasting and refeeding. Glycogen levels are expressed as micromolar glucose released from amyloglucosidase digestion normalized to the weight of the liver. Three mice were analyzed in each case. Error bars represent SEM. (E) AGL levels in primary mouse hepatocytes during a starvation/refeeding cycle. Starvation was induced by glucose/serum deprivation and addition of 100 nM glucagon for either 1 or 7 h. After starvation for 1 h, refeeding was initiated by replacing the media with regular growth medium supplemented with 100 nM insulin. Lysates were then obtained and processed for immunoblotting with the indicated antibodies.

Figure 6.

AGL colocalizes with polyglucosans formed by overexpression of glycogen synthase. (A) Expression of myc-tagged glycogen synthase (myc-GS) causes the formation of inclusions that stain positive for periodic acid Schiff (PAS). HepG2 cells were transfected with myc-GS for 16 h, fixed in formalin, and processed for immunofluorescence microscopy using anti-myc antibodies (green) or PAS stain. Black arrows point to positive PAS stain. (B) Expression of myc-GS causes wild type but not the ΔCBD mutant of AGL to aggregate around the PAS-stain-positive inclusions. HepG2 cells were transfected for 16 h with myc-GS together with either HA-tagged wild-type AGL (HA-AGL) or HA-AGL ΔCBD. Cells were fixed in formalin and processed for immunofluorescence microscopy using anti-HA (green) and anti-myc (red) antibodies. White arrows indicate colocalization of HA-AGL and myc-GS.

Figure 7.

Regulation of AGL during glycogenolysis. AGL binds to glycogen and is involved in the debranching process. Signals that induce glycogenolysis trigger the release of AGL and allow it to enter the nucleus. There, Malin promotes its ubiquitination. Signals that elevate cAMP levels may further enhance this process by increasing the levels of Malin. Subsequent degradation of AGL probably involves an additional signal that might be related to the down-regulation of cAMP/PKA signaling. In Lafora disease, loss of Malin function could potentially lead to increased AGL levels and/or activity. Conversely, in Cori’s disease, the G1448R mutant is unable to bind glycogen and undergoes rapid proteasomal-mediated degradation.