Triacylglycerol mimetics regulate membrane interactions of glycogen branching enzyme: implications for therapy

Abstract

Adult polyglucosan body disease (APBD) is a neurological disorder characterized by adult-onset neurogenic bladder, spasticity, weakness, and sensory loss. The disease is caused by aberrant glycogen branching enzyme (GBE) (GBE1Y329S) yielding less branched, globular, and soluble glycogen, which tends to aggregate. We explore here whether, despite being a soluble enzyme, GBE1 activity is regulated by protein-membrane interactions. Because soluble proteins can contact a wide variety of cell membranes, we investigated the interactions of purified WT and GBE1Y329S proteins with different types of model membranes (liposomes). Interestingly, both triheptanoin and some triacylglycerol mimetics (TGMs) we have designed (TGM0 and TGM5) markedly enhance GBE1Y329S activity, possibly enough for reversing APBD symptoms. We show that the GBE1Y329S mutation exposes a hydrophobic amino acid stretch, which can either stabilize and enhance or alternatively, reduce the enzyme activity via alteration of protein-membrane interactions. Additionally, we found that WT, but not Y329S, GBE1 activity is modulated by Ca²⁺ and phosphatidylserine, probably associated with GBE1-mediated regulation of energy consumption and storage. The thermal stabilization and increase in GBE1Y329S activity induced by TGM5 and its omega-3 oil structure suggest that this molecule has a considerable therapeutic potential for treating APBD.

Keywords: adult polyglucosan body disease; diseases; drug therapy; membrane lipid therapy; metabolic disease; protein-membrane interactions; triglycerides; triheptanoin.

Fig. 1.

Human GBE1 and GBE1Y329S amino acid sequences. The one-letter code amino acid sequence for the WT GBE1 used in the present study. A C-terminal 6xHis tag was added for purification and immunodetection purposes. In red, the Y-to-S alteration found in APBD patients is shown.

Fig. 2.

WT and GBE1Y329S protein purification. Proteins were purified by affinity chromatography on Ni-NTA columns, fractionated by SDS-PAGE, and stained with Coomassie blue (see the Materials and Methods for more details). GBE1Y329S was eluted at higher imidazole concentrations than WT GBE1, which anticipates relevant structural alterations. M, Mr standards.

Fig. 3.

WT GBE1 and GBE1Y329S binding to model membranes. Model membranes of PC, PC:PE (2:3 molar ratio), PC:PS (3:2), PC:PE:PS (3:4:3), PC:PE:Cho:SM (1:1:1:1), and PC:PE:PS:Cho:SM (2.3:2:2.6:2.3:0.8) were used. The bars indicate the proportion (mean ± SEM) of WT and GBE1Y329S protein bound to the model membranes resembling different membrane types or membrane microdomains. Representative immunoblots of each binding experiment are shown in the same order as in the graph. The data represent the mean ± SEM of from three to four independent experiments. *P < 0.05; **P < 0.01.

Fig. 4.

Binding of WT GBE1 and GBE1Y329S to PC membranes with different triacylglycerol compositions. A: Binding of WT GBE1 and GBE1Y329S to PC membranes with increasing concentrations of TH and TGM0 (5, 10, and 20 mol%). B: Binding of WT GBE1 and GBE1Y329S to PC membranes in the presence or absence of various TGMs (20 mol%). The bars show the mean proportion of each GBE1 protein bound to model membranes (bound GBE1 protein relative to the total GBE1 protein) and representative immunoblots of each binding experiment are also shown. In all cases the data represent the mean ± SEM of from three to seven independent experiments. **P < 0.01. #Indicates a significant difference in the binding of GBE1Y329S with respect to WT GBE1 (P < 0.05).

Fig. 5.

Effect of TGMs on GBE1 activity. A: Purified recombinant WT GBE1 (GBE) and the Y329S mutant (GBE YS) protein at the concentrations indicated were assayed for GBE1 activity (12) at different concentrations and times (left panel) and the correlation between these effects and those produced in patient’s cells were compared (right panel). B, D: Activity of GBE1Y329S (solid bars) in the presence or absence of different PC membranes and TGMs. Activity was calculated relative to the activity of WT GBE1 in the presence of PC membranes alone [GBE1 (C): 100%]. C, E: Correlation between GBE1Y329S activity and binding to lipid membranes after 30 min (panel C) or 60 min of the reaction (E). The data shown are the proportion of WT GBE1 binding or its activity in PC bilayers (100%), and they represent the mean ± SEM of from three to seven independent experiments. *P < 0.05.

Fig. 6.

Effect of TH, TGM0, and TGM5 on the thermal stability of WT GBE1 and GBE1Y329S. Changes in T_m (melting temperature that defines the thermal stability) of WT GBE1 (A–D) and GBE1Y329S (E–H) proteins were determined by DSF. These representative graphs show the absolute (dotted line) and relative (continuous line) fluorescence and the increase in T_m (bar graphs) induced by PC membranes in the presence or absence of TH, TGM0, or TGM5 (20 mol%).

Fig. 7.

Effects of TH, TGM0, and TGM5 on the thermal stability of GBE1Y329S. Upper panel: The differences in T_m between WT GBE1 and mutated GBE1 (without lipids, dotted lines) and the differences in T_m between WT and GBE1Y329S treated with TH, TGM0, and TGM5. Left lower panel: DSF profiles of GBE1Y329S (black points) and GBE1Y329S in presence of 5, 10, and 25 μM of TGM0 (small, medium, and big blue points, respectively) and of 5, 10, and 25 μM of TGM5 (red points of different sizes as above). Right lower panel: DSF profiles of GBE1Y329S (black points) and GBE1Y329S in presence of 5, 10, and 25 μM of TH (small, medium, and big green points, respectively).

Fig. 8.

Structure and effects of TH, and of TGM0, TGM 1, and TGM 5 on GBE1Y329S activity in APBD patient’s PBMCs. Upper panel: APBD PBMCs were incubated overnight in the presence or absence (APBD) of 300 μM of the triacylglycerides indicated. After harvesting the PBMCs, GBE1Y329S activity was determined as described [mean ± SEM; *P < 0.05 (12)]. Lower panel: Structure of TGM0 and TGM5. Right panel: DSC experiments showing the effect of TGM0 and TGM5 on the lipid structure of POPE membranes. The first peak corresponds to the solid-to-liquid phase transition and the second peak accounts for the lamellar-to-hexagonal (H_II) phase transition.

Fig. 9.

Effect of calcium and anionic phospholipids on GBE1 activity and binding to model lipid membranes. Binding of WT GBE1 (A) (open bars) and GBE1Y329S (C) (solid bars) to PC, PC:PS (3:2, mol:mol) or PC:PIP₂ (19:1, mol:mol) membranes in the presence (red) or absence (black) of Ca²⁺. The data represent the mean ± SEM values of from three to four independent experiments. **P < 0.01, effect of Ca²⁺; #P < 0.05, effect of PS. Correlation between GBE1 (both WT and GBE1Y329S) activity and membrane binding in the presence of Ca²⁺ (red) for 30 min (B) or 60 min (D). GBE1 activity and binding were expressed relative to the values for the WT protein in the presence PC (100%, black).

Fig. 10.

WT and GBE1Y329S structure. A: Kyte-Doolittle hydrophobicity plots of WT and GBE1Y329S showing changes between the proteins. B: Surface probability plots showing the relevant changes between the WT and mutant proteins. The boxes, red lines, and asterisks show the region around Y/S329 in detail. See Table 3 for hydrophobicity and surface probability values (19 amino acid windows). C: GBE1 structure showing the Y329 amino acid (orange) and the C terminus (yellow).