G.P.28 GNE interactions and interactors

Abstract

GNE Myopathy (formerly designated HIBM/DMRV) is a rare neuromuscular recessive disorder caused by missense mutations in GNE, the key enzyme of sialic acid biosynthesis. To date, over 60 different mutations in GNE have been reported to cause the disease worldwide, but a single homozygous missense mutation M712T, is common in the Middle East. The mechanisms leading from GNE mutations to the disease are not yet understood. In an attempt to elucidate the pathophysiological pathway of the disease, we have searched for proteins potentially interacting with GNE. We have previously determined that GNE interacts with α-actinin1, one of the 4 α-actinins isoforms, a family of proteins binding the actin filaments. We studied the interactions of recombinant human GNE protein with other members of the actinin family, in particular α-actinin2 which is located at the Z-disk of the muscle fiber. Using a Surface Plasmon Resonance (SPR)-Biosensor based assay, we found that GNE, in its wild type and M712T mutated forms, also binds α-actinin 2. Furthermore, GNE binds α-actinin1 and α-actinin 2 differently, either at different interaction sites on the GNE protein, or alternatively, by changing the conformational state of GNE. GNE exists in different oligomeric forms, dimers, tetramers and hexamers, which are in a dynamic interplay in the cell. To evaluate the potential effect of common mutations found in GNE myopathy patients, on the oligomeric state of GNE protein, we have investigated GNE–GNE interactions and affinity, and found the highest affinity between wtGNE and wtGNE, the lowest between mutGNE–mutGNE, and intermediate affinity between wt and mutated GNE proteins. These differences could have a direct effect on both the oligomeric state of GNE and on the dynamic interplay between the oligomeric forms of the protein. These studies could contribute new understandings on the mechanism of GNE in health and disease.