Abstract

Glycogen branching enzyme 1 (GBE1) plays an essential role in glycogen biosynthesis by generating α-1,6-glucosidic branches from α-1,4-linked glucose chains, to increase solubility of the glycogen polymer. Mutations in the GBE1 gene lead to the heterogeneous early-onset glycogen storage disorder type IV (GSDIV) or the late-onset adult polyglucosan body disease (APBD). To better understand this essential enzyme, we crystallized human GBE1 in the apo form, and in complex with a tetra- or hepta-saccharide. The GBE1 structure reveals a conserved amylase core that houses the active centre for the branching reaction and harbours almost all GSDIV and APBD mutations. A non-catalytic binding cleft, proximal to the site of the common APBD mutation p.Y329S, was found to bind the tetra- and hepta-saccharides and may represent a higher-affinity site employed to anchor the complex glycogen substrate for the branching reaction. Expression of recombinant GBE1-p.Y329S resulted in drastically reduced protein yield and solubility compared with wild type, suggesting this disease allele causes protein misfolding and may be amenable to small molecule stabilization. To explore this, we generated a structural model of GBE1-p.Y329S and designed peptides ab initio to stabilize the mutation. As proof-of-principle, we evaluated treatment of one tetra-peptide, Leu-Thr-Lys-Glu, in APBD patient cells. We demonstrate intracellular transport of this peptide, its binding and stabilization of GBE1-p.Y329S, and 2-fold increased mutant enzymatic activity compared with untreated patient cells. Together, our data provide the rationale and starting point for the screening of small molecule chaperones, which could become novel therapies for this disease.

Highlights

  • Glycogen is a compact polymer of α-1,4-linked glucose units regularly branched with α-1,6-glucosidic bonds, serving as the main carbohydrate store and energy reserve across many phyla [1]

  • Using the molecular replacement method with the Oryza sativa starch branching enzyme I (SBE1; PDB: 3AMK; 54% identity to human GBE1 (hGBE1)) as search model, we have determined the structure of hGBE1trunc in the apo form, and in complex with the tetra-saccharide acarbose or hepta-saccharide maltoheptaose, to the resolution range of 2.7–2.8 Å (Supplementary Material, Table S1)

  • A structural overlay of hGBE1 with reported branching enzyme structures from O. sativa SBE1 [17] (PDB: 3AMK, Cα-RMSD: 1.4 Å, sequence identity: 54%) and M. tuberculosis GBE [19] (3K1D, 2.1 Å, 28%) (Fig. 1C) highlights the conserved catalytic core housing the active site within a canonical6 barrel [16]

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Summary

Introduction

Glycogen is a compact polymer of α-1,4-linked glucose units regularly branched with α-1,6-glucosidic bonds, serving as the main carbohydrate store and energy reserve across many phyla [1]. Published at: Froese, D Sean; Michaeli, Amit; McCorvie, Thomas J; Krojer, Tobias; Sasi, Meitav; Melaev, Esther; Goldblum, Amiram; Zatsepin, Maria; Lossos, Alexander; Álvarez, Rafael; Escribá, Pablo V; Minassian, Berge A; von Delft, Frank; Kakhlon, Or; Yue, Wyatt W (2015).

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