Synapsin-1 and tau reciprocal O-GlcNAcylation and phosphorylation sites in mouse brain synaptosomes

O-linked N-acetylglucosamine (O-GlcNAc) represents a key regulatory post-translational modification (PTM) that is reversible and often reciprocal with phosphorylation of serine and threonine at the same or nearby residues. Although recent technical advances in O-GlcNAc site-mapping methods combined with mass spectrometry (MS) techniques have facilitated study of the fundamental roles of O-GlcNAcylation in cellular processes, an efficient technique for examining the dynamic, reciprocal relationships between O-GlcNAcylation and phosphorylation is needed to provide greater insights into the regulatory functions of O-GlcNAcylation. Here, we describe a strategy for selectively identifying both O-GlcNAc- and phospho-modified sites. This strategy involves metal affinity separation of O-GlcNAcylated and phosphorylated peptides, beta-elimination of O-GlcNAcyl or phosphoryl functional groups from the separated peptides followed by dithiothreitol (DTT) conjugation (BEMAD), affinity purification of DTT-conjugated peptides using thiol affinity chromatography, and identification of formerly O-GlcNAcylated or phosphorylated peptides by MS. The combined metal affinity separation and BEMAD approach allows selective enrichment of O-GlcNAcylated peptides over phosphorylated counterparts. Using this approach with mouse brain synaptosomes, we identified the serine residue at 605 of the synapsin-1 peptide, 603QASQAGPGPR612, and the serine residue at 692 of the tau peptide, 688SPVVSGDTSPR698, which were found to be potential reciprocal O-GlcNAcylation and phosphorylation sites. These results demonstrate that our strategy enables mapping of the reciprocal site occupancy of O-GlcNAcylation and phosphorylation of proteins, which permits the assessment of cross-talk between these two PTMs and their regulatory roles.

Transient downregulation of protein O-N-acetylglucosaminylation by treatment of high-dose nicotinamide in human cells

Nicotinamide at millimolar concentrations affects cell survival in various conditions, and is being utilized therapeutically in many human diseases. However, the effect of an acute treatment of nicotinamide at such high dose on gene expression and cellular metabolism has rarely been determined previously. In this study, we found that levels of O-N-acetylglucosamin(O- GlcNAc)ylated proteins including Sp1 acutely decreased upon treatment of 10 mM nicotinamide. Concomitantly, Sp1 protein level decreased rapidly through accelerated proteasome-mediated proteolysis. Cotreatment of glucosamine or 2-deoxyglucose, which inhibits protein deGlcNAcylation, effectively blocked the decrease induced by nicotinamide. Interestingly, the decline in the levels of Sp1 and protein O- GlcNAcylation was only transient lasting for two days post treatment, and this pattern matched closely the rapid fluctuation of the cellular [NAD(+)]. Our results suggest a possible link between cellular nicotinamide metabolism and protein O-GlcNAcylation, and an existence of cellular [NAD(+)] homeostasis.

Localization of sugar-binding proteins in Trypanosoma cruzi using gold-labeled neoglycoproteins

Neoglicoproteínas marcadas com ouro coloidal foram utilizadas para localizaçäo de sítios de ligaçäo de carboidratos em Trypanosoma cruzi. Sítios de ligaçäo para N-acetil-D-glicosamina e D-galactose foram observadas na superfície de cerca de 80 e 5 a 10% das formas tripomastigotas, respectivamente. Sítios de ligaçäo para D-manose näo foram observados na superfície de tripomastigotas. Neoglicoproteínas que reconhecem N-acetil-D-glicosamina, D-galactose e D-manose näo se ligam á superficie das formas epimastigota e amastigota. Marcaçäo de núcleo e cinetoplasto foi observada com a neoglicoproteína que reconhece N-acetil-D-glicosamina. Os resultados obtidos säo discutidos levando em consideraçäo o papel que proteínas que se ligam a carboidratos podem desempenhar no processo de interaçäo entre T. cruzi e a célula hospedeira