O-Linked N-Acetylglucosaminylation Is Involved in the Ca2+ Activation Properties of Rat Skeletal Muscle

Abstract

O-Linked N-acetylglucosaminylation termed O-GlcNAc is a dynamic cytosolic and nuclear glycosylation that is dependent both on glucose flow through the hexosamine biosynthesis pathway and on phosphorylation because of the existence of a balance between phosphorylation and O-GlcNAc. This glycosylation is a ubiquitous post-translational modification, which probably plays an important role in many aspects of protein functions. We have previously reported that, in skeletal muscle, proteins of the glycolytic pathway, energetic metabolism, and contractile proteins were O-GlcNAc-modified and that O-GlcNAc variations could control the muscle protein homeostasis and be implicated in the regulation of muscular atrophy.In this paper, we report O-N-acetylglucosaminylation of a number of key contractile proteins (i.e. myosin heavy and light chains and actin), which suggests that this glycosylation could be involved in skeletal muscle contraction. Moreover, our results showed that incubation of skeletal muscle skinned fibers in N-acetyl-D-glucosamine, in a concentration solution known to inhibit O-GlcNAc-dependent interactions, induced a decrease in calcium sensitivity and affinity of muscular fibers, whereas the cooperativity of the thin filament proteins was not modified. Thus, our results suggest that O-GlcNAc is involved in contractile protein interactions and could thereby modulate muscle contraction. O-Linked N-acetylglucosaminylation, termed O-GlcNAc,5 is a regulatory post-translational modification that occurs in nuclear and cytosolic proteins, corresponding to the addition of a unique monosaccharide, N-acetyl-D-glucosamine, to a serine and threonine hydroxyl group by a ␤-linkage (1). Because of the existence of the UDP-GlcNAc-peptide-␤-GlcNAc transferase, which transfers the monosaccharide into proteins (2, 3), and the N-acetyl-␤-D-glucosaminidase, which removes it (4), OGlcNAc is more similar to phosphorylation than classical glycosylation. The half-life of the monosaccharide is shorter than the half-life of the protein backbone (5), indicating that the O-GlcNAc cycle could rapidly respond to cellular signals (6). All of the known O-GlcNAc proteins described to date are also phosphoproteins (7) (for review, see Ref. 8). Modifications by O-GlcNAc or phosphorylation could occur on the same site (9) or at neighboring sites (10); this competition between O-GlcNAc and phosphorylation is called the "Yin-Yang" process.O-GlcNAc has been described to play a role in various cell functions: in nuclear transport (11-13); in protein degradation, with a reversible inhibition of the proteasome itself (14) and a protection of the modified protein against proteasomal degradation (9, 15, 16); and in regulation of protein expression, with the regulation of transcription (10, 17-19) and translation (20). The involvement of O-GlcNAc in protein-protein interactions has been described in different biological systems. Indeed, many proteins playing a key role in organization and assembly of cytoskeleton are O-GlcNAc-modified, including cytok...

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