Densin‐180: revised membrane topology, domain structure and phosphorylation status

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163

230

We present the first large scale study characterizing both N-and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N-and O-linked carbohydrate structures. Liquid chromatography-MS (LC/ MS) analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N-and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues. Molecular & Cellular

Section: Fig 4 Etd Spectrum Of 3100 Ngat(galglcnacfuc)c(carbamidomementioning

confidence: 92%

N- and O-Glycosylation in the Murine Synaptosome

Trinidad

Schoepfer²,

Burlingame

et al. 2013

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163

230

“…While O-GlcNAcylation occurs almost exclusively on intracellular protein regions, the extracelluar domain of Notch is O-GlcNAcylated (26). Peptides were assigned as ambiguous between O-GalNAcylated or O-GlcNAcylated based upon their annotation in UniProt (downloaded April 2011) as located in extracelluar or luminal regions, without further corrections (27). In the case of transmembrane proteins, UniProt topological information was used when available to determine protein extracellular and cytosolic regions.…”

Section: Methodsmentioning

confidence: 99%

Global Identification and Characterization of Both O-GlcNAcylation and Phosphorylation at the Murine Synapse

Trinidad

Barkan²,

Gulledge³

et al. 2012

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375

416

O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic, reversible monosaccharide modifier of serine and threonine residues on intracellular protein domains. Crosstalk between O-GlcNAcylation and phosphorylation has been hypothesized. Here, we identified over 1750 and 16,500 sites of O-GlcNAcylation and phosphorylation from murine synaptosomes, respectively. In total, 135 (7%) of all O-GlcNAcylation sites were also found to be sites of phosphorylation. Although many proteins were extensively phosphorylated and minimally O-GlcNAcylated, proteins found to be extensively O-GlcNAcylated were almost always phosphorylated to a similar or greater extent, indicating the O-GlcNAcylation system is specifically targeting a subset of the proteome that is also phosphorylated. Both PTMs usually occur on disordered regions of protein structure, within which, the location of O-GlcNAcylation and phosphorylation is virtually random with respect to each other, suggesting that negative crosstalk at the structural level is not a common phenomenon. As a class, protein kinases are found to be more extensively O-GlcNAcylated than proteins in general, indicating the potential for crosstalk of phosphorylation with O

“…Biochemical analyses have revealed that the PSD-enriched samples consist of hundreds of proteins (19 -26). More recently, the abundance of several components in the PSD have been quantified by a variety of techniques (27)(28)(29)(30), and the phosphorylation and glycosylation states of PSD proteins have further been studied (31)(32)(33)(34).…”

Section: Postsynaptic Density (Psd)mentioning

confidence: 99%

A Study of the Spatial Protein Organization of the Postsynaptic Density Isolated from Porcine Cerebral Cortex and Cerebellum

Yun-Hong

Chuang

Chang

et al. 2011

Postsynaptic density (PSD) is a protein supramolecule lying underneath the postsynaptic membrane of excitatory synapses and has been implicated to play important roles in synaptic structure and function in mammalian central nervous system. Here, PSDs were isolated from two distinct regions of porcine brain, cerebral cortex and cerebellum. SDS-PAGE and Western blotting analyses indicated that cerebral and cerebellar PSDs consisted of a similar set of proteins with noticeable differences in the abundance of various proteins between these samples. Subsequently, protein localization in these PSDs was analyzed by using the Nano-Depth-Tagging method. This method involved the use of three synthetic reagents, as agarose beads whose surface was covalently linked with a fluorescent, photoactivable, and cleavable chemical crosslinker by spacers of varied lengths. After its application was verified by using a synthetic complex consisting of four layers of different proteins, the Nano-Depth-Tagging method was used here to yield information concerning the depth distribution of various proteins in the PSD. The results indicated that in both cerebral and cerebellar PSDs, glutamate receptors, actin, and actin binding proteins resided in the peripheral regions within ϳ10 nm deep from the surface and that scaffold proteins, tubulin subunits, microtubule-binding proteins, and membrane cytoskeleton proteins found in mammalian erythrocytes resided in the interiors deeper than 10 nm from the surface in the PSD. Finally, by using the immunoabsorption method, binding partner proteins of two proteins residing in the interiors, PSD-95 and ␣-tubulin, and those of two proteins residing in the peripheral regions, elongation factor-1␣ and calcium, calmodulin-dependent protein kinase II ␣ subunit, of cerebral and cerebellar PSDs were identified. Overall, the results indicate a striking similarity in protein organization between the PSDs isolated from porcine cerebral cortex and cerebellum.