Regulation of Glycogen Synthase Kinase-3ß by Phosphorylation and O-ß-Linked N-Acetylglucosaminylation: Implications on Tau Protein Phosphorylation.

Glycogen synthase kinase 3 (GSK3) plays a pivotal role in signaling pathways involved in insulin metabolism and the pathogenesis of neurodegenerative disorders. In particular, the GSK3ß isoform is implicated in Alzheimer's disease (AD) as one of the key kinases involved in the hyperphosphorylation of tau protein, one of the neuropathological hallmarks of AD. As a constitutively active serine/threonine kinase, GSK3 is inactivated by Akt/PKB-mediated phosphorylation of Ser9 in the N-terminal disordered domain, and for most of its substrates, requires priming (prephosphorylation) by another kinase that targets the substrate to a phosphate-specific pocket near the active site. GSK3 has also been shown to be post-translationally modified by O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation), with still unknown functions. Here, we have found that binding of Akt inhibits GSK3ß kinase activity on both primed and unprimed tau substrates. Akt-mediated Ser9 phosphorylation restores the GSK3ß kinase activity only on primed tau, thereby selectively inactivating GSK3ß toward unprimed tau protein. Additionally, we have shown that GSK3ß is highly O-GlcNAcylated at multiple sites within the kinase domain and the disordered N- and C-terminal domains, including Ser9. In contrast to Akt-mediated regulation, neither the O-GlcNAc transferase nor O-GlcNAcylation significantly alters GSK3ß kinase activity, but high O-GlcNAc levels reduce Ser9 phosphorylation by Akt. Reciprocally, Akt phosphorylation downregulates the overall O-GlcNAcylation of GSK3ß, indicating a crosstalk between both post-translational modifications. Our results indicate that specific O-GlcNAc profiles may be involved in the phosphorylation-dependent Akt-mediated regulation of GSK3ß kinase activity.

Phosphorylation of Tau Protein by CDK2/cyclin A and GSK3ß Recombinant Kinases: Analysis of Phosphorylation Patterns by Nuclear Magnetic Resonance Spectroscopy.

Posttranslational modifications (PTMs) of proteins can be investigated by Nuclear Magnetic Resonance (NMR) spectroscopy as a powerful analytical tool to define modification sites, their relative stoichiometry, and crosstalk between modifications. As a Structural Biology method, NMR provides important additional information on changes in protein conformation and dynamics upon modification as well as a mapping of binding sites upon biomolecular interactions. Indeed, PTMs not only mediate functional modulation in protein-protein interactions, but can also induce diverse structural responses with different biological outcomes. Here we present protocols that have been developed for the production and phosphorylation of the neuronal tau protein. Under its aggregated form, tau is a hallmark of Alzheimer's disease and other neurodegenerative diseases named tauopathies involving tau dysfunction and/or mutations. As a common feature shared by various tauopathies, tau aggregates are found into a form displaying an increased, abnormal phosphorylation, also referred to hyperphosphorylation. We have used NMR to investigate the phosphorylation patterns of tau induced by several kinases or cell extracts, how phosphorylation affects the local and overall conformation of tau, its interactions with partners (proteins, DNA, small-molecules, etc.) including tubulin and microtubules, and its capacity to form insoluble fibrillar aggregates. We present here detailed protocols for in vitro phosphorylation of tau by the recombinant kinases CDK2/cyclin A and GSK3ß, the production of the recombinant kinases thereof, as well as the analytical characterization of phosphorylated tau by NMR spectroscopy.

Minimal epitope for Mannitou IgM on paucimannose-carrying glycoproteins.

Paucimannosidic glycans are restricted to the core structure [Man1-3GlcNAc2Fuc0-1] of N-glycans and are rarely found in mammalian tissues. Yet, especially [Man2-3GlcNAc2Fuc1] have been found significantly upregulated in tumors, including in colorectal and liver cancer. Mannitou IgM is a murine monoclonal antibody that was previously shown to recognize Man3GlcNAc2 with an almost exclusive selectivity. Here, we have sought the definition of the minimal glycan epitope of Mannitou IgM, initiated by screening on a newly designed paucimannosidic glycan microarray; among the best binders were Man3GlcNAc2 and its α1,6 core-fucosylated variant, Man3GlcNAc2Fuc1. Unexpectedly and in contrast to earlier findings, Man5GlcNAc2-type structures bind equally well and a large tolerance was observed for substitutions on the α1,6 arm. It was confirmed that any substitution on the single α1,3-linked mannose completely abolishes binding. Surface plasmon resonance for kinetic measurements of Mannitou IgM binding, either directly on the glycans or as presented on omega-1 and kappa-5 soluble egg antigens from the helminth parasite Schistosoma mansoni, showed submicromolar affinities. To characterize the epitope in greater and atomic detail, saturation transfer difference nuclear magnetic resonance spectroscopy was performed with the Mannitou antigen-binding fragment. The STD-NMR data demonstrated the strongest interactions with the aliphatic protons H1 and H2 of the α1-3-linked mannose and weaker imprints on its H3, H4 and H5 protons. In conclusion, Mannitou IgM binding requires a nonsubstituted α1,3-linked mannose branch of paucimannose also on proteins, making it a highly specific tool for the distinction of concurrent human tumor-associated carbohydrate antigens.

Binding of Gold(III) Porphyrin by the Pro-metastatic Regulatory Protein Human Galectin-3.

Gold(III) porphyrin presents an attractive alternative to the use of, for example, cisplatin in chemotherapy. However, approaches that allow to selectively target cancer cells are highly sought. Many plant and mammalian lectins have been shown to bind oligosaccharide sequences of the aberrant glycosylation pattern found on cancerous tumors. For example human galectin-3, of the galectin family specific for ß-galactoside, is overexpressed in the extracellular matrix of tumorigenous and metastatic tissues. We searched for non-carbohydrate ligands for galectin-3 that can guide a cytotoxic drug to the cancer cells by maintaining its affinity for tumor associated carbohydrate antigens. Previous findings showed that zinc tetrasulfonatophenylporphyrin can bind galectin-3 with sub-micromolar affinity without disturbing lactose binding. Gold(III) porphyrin is not only cytotoxic to cancer cells, it knows also a potential application as photosensitiser in photodynamic therapy. We investigated the binding of gold(III) porphyrin to galectin-3 using different biophysical interaction techniques and demonstrated a low micromolar affinity of human galectin-3 for the cytotoxic compound. Co-crystallization attempts in order to understand the binding mode of gold porphyrin to galectin-3 failed, but molecular docking emphasized a highly populated secondary binding site that does not hinder lactose or Thomsen Friendenreich disaccharide binding. This suggests that gold(III) porphyrin might significantly enhance its concentration and delivery to cancer cells by binding to human galectin-3 that keeps its orientation towards tumor associated carbohydrate antigens.

Heptylmannose-functionalized cellulose for the binding and specific detection of pathogenic E. coli.

We developed a chemical method to covalently functionalize cellulose nanofibers and cellulose paper with mannoside ligands displaying a strong affinity for the FimH adhesin from pathogenic E. coli strains. Mannose-grafted cellulose proved efficient to selectively bind FimH lectin and discriminate pathogenic E. coli strains from non-pathogenic ones. These modified papers are valuable tools for diagnosing infections promoted by E. coli, such as cystitis or inflammatory bowel diseases, and the concept may be applicable to other life-threatening pathogens.