STRUCTURAL STABILITY STUDIES OF HUMAN GLYCOGENIN-1 MUTANT ALA16PRO ASSOCIATED WITH GLYCOGEN STORAGE DISEASE XV

MUÑOZ SOSA, CHRISTIAN J.; CURTINO, JUAN A.; CARRIZO, MARÍA E.

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Glycogenin is a glycosyltransferase that catalyzes the transfer of glucose residues from UDP-glucose to itself, creating a linear polymer of approximately 12 glucose units bound by α-1,4-glycosidic linkages. This oligosaccharide chain serves as the primer for the combined action of glycogen synthase and glycogen branching enzyme that will complete the polysaccharide synthesis. In humans, there are two forms of glycogenin, glycogenin-1 and glycogenin-2. Glycogenin-1 (HGN1) is mainly expressed in skeletal muscle and heart and to a lesser extent in lung, kidney, brain, pancreas, and placenta. Instead, glycogenin-2 is primarily expressed in the liver.Glycogen storage disease (GSD) XV is a rare metabolic disorder caused by mutations in the GYG1 gene, which encodes HGN1. To date, eight point mutations have been described in GSD XV patients. One of them was homozygous for an N-terminal missense variant (c.46G>C, p.Ala16Pro) of the protein and exhibited skeletal myopathy with storage of polyglucosan in muscle fibers. The mutation was confirmed at the RNA level but the mutant protein was not detected in the skeletal muscle biopsy of the patient.Since human and rabbit glycogenin amino acid sequences are 93% identical, we have introduced Ala16Pro mutation into rabbit enzyme, the most studied member of the family, and expressed the mutant in E. coli. We have previously described that Ala16Pro mutant was inactive for auto- and transglucosylation and has a diminished substrate binding affinity, probably due to a conformational change. In order to explain the absence of the protein in the patient muscle tissue, in this work we have analyzed its stability and oligomerization state by different in vitro techniques. Here we show that, in contrast to wild type HGN1, which exists as a dimer, the Ala16Pro variant forms soluble high molecular weight oligomers. Besides, our results suggest that the mutant has a less stable conformation, more prone to proteolytic digestion.