Structural insights into the de novo biosynthesis of glycogen

CARRIZO, MARÍA E.

Workshop; Tercer Encuentro y Primer Workshop de la Red Argentina de Tecnología Enzimática; 2021

Red Argentina de Tecnología Enzimática

Glycogenin is the autoglucosyltransferase that initiates the polymerization of glucose to prime the de novo biosynthesis of glycogen. Its three-dimensional structure and studies by size exclusion chromatography indicated that the enzyme exists in solution mainly as a dimer. Based on kinetic studies it was proposed that autoglucosylation might occur by intradimer intersubunit glucosylation. However, we demonstrated that the protein could also exist as a monomer at lower concentrations and that this species is able to autoglucosylate by an intramolecular mechanism. According to this result, we then evaluated whether an intrasubunit glucosylation mechanism might also be involved in dimer autoglucosylation. Through the analysis of the activity of heterodimers formed by mutants unable to transfer or receive glucose, we conclude that both, intrasubunit and intersubunit reaction mechanisms are necessary for the dimeric enzyme to acquire maximum autoglucosylation.The requirement of glycogenin to initiate de novo polysaccharide biosynthesis became more evident with the report of a metabolic disorder called Glycogen Storage Disease (GSD) type XV, caused by mutations in glycogenin-1 gene, one of the human enzyme isoforms. Patients with GSD type XV do not store glycogen, although in most cases they accumulate an abnormally structured polysaccharide with a lower degree of branching named polyglucosan. To date, less than 30 patients with GSD type XV have been reported worldwide, who have a late onset of the disease and frequently exhibit progressive muscle weakness, and in some cases cardiomyopathy. The first described glycogenin mutation associated with GSD type XV was Thr82Met. Since Thr82 is not involved in the active site of glycogenin, it was interesting to analyze how its mutation causes the loss of the enzyme activity. For this purpose, we introduced the mutation into the well-studied rabbit muscle glycogenin and solved its crystal structure. The structure and the functional studies carried out in Thr82Ser and Thr82Val mutants revealed the critical role of a single hydrogen bond to Asp162 which is lost when Thr is replaced by Met.After glycogenin generates the oligosaccharide primer, glycogen synthase and branching enzyme catalyze the elongation and branching of the polysaccharide. This process is subjected to a complex regulatory mechanism at the level of glycogen synthase. In contrast, the regulation of glycogenin has not been extensively studied. In the search for possible regulators of this enzyme, a new protein was found which was named GNIP (Glycogenin Interacting Protein). Autoglucosylation of glycogenin was stimulated by GNIP2, a truncated form of GNIP, showing an almost 4-fold increase in Vmax with no change in Km for UDP-glucose. Recent studies demonstrate that GNIP, now also called TRIM7, is an E3 ubiquitin ligase that belongs to the TRIM family of proteins and is involved in the development of tumors, atherosclerosis and in the pathogenesis of the infection caused by certain viruses, including Zika virus. Therefore, TRIM7 is gaining increasing interest due to its potential as a therapeutic target. In this regard, its C-terminal domain, named B30.2, may be particularly important since specificity determinants of many TRIM proteins lie in this domain. In order to contribute to the understanding of TRIM7 mechanisms of action, we have solved the crystal structure of its B30.2 domain. Based on this structure and sequence conservation analysis, we prepared several mutants that allowed us to define the region of the protein involved in the interaction with glycogenin. Our data provide useful information that can be used to target TRIM7 B30.2 domain for the development of potential therapeutic agents.