Neurofascin 186 Is O-Mannosylated within and Outside of the Mucin Domain

Pacharra, Sandra; Hanisch, Franz‐Georg; Breloy, Isabelle

doi:10.1021/pr200996y

Cited by 39 publications

(28 citation statements)

References 25 publications

(42 reference statements)

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“…α-DG contains both O-Man and O-GalNAc glycans in this region (22)(23)(24), and some sites are potentially occupied by either type of glycosylation (25). This dual-occupancy was also proposed in neurofascin 186 (13). We reasoned that we could identify O-Man sites and the interplay between the two types of O-glycosylation on α-DG and other proteins in MDA-MB-231 with both types of glycosylation simplified, making both O-Man and O-GalNAc glycopeptides easily identifiable.…”

Section: Resultsmentioning

confidence: 59%

“…Using this strategy O-Man glycopeptides from α-DG and a total of 51 unique O-Man glycoproteins were identified, comprising a total of 235 O-Man glycosites (Table 1 and Dataset S1). We did not identify any of the individual proteins more recently suggested to carry O-Man glycans (10)(11)(12)(13)(14), which could be due to a number of reasons including experimental limitations as well as lack of expression of these proteins in MDA-MB-231 cells. Identification of O-Man Glycosites on α-DG.…”

Section: Resultsmentioning

confidence: 87%

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Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins

Vester-Christensen¹,

Halim²,

Joshi³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

145

187

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The metazoan O-mannose (O-Man) glycoproteome is largely unknown. It has been shown that up to 30% of brain O-glycans are of the O-Man type, but essentially only alpha-dystroglycan (α-DG) of the dystrophin-glycoprotein complex is well characterized as an O-Man glycoprotein. Defects in O-Man glycosylation underlie congenital muscular dystrophies and considerable efforts have been devoted to explore this O-glycoproteome without much success. Here, we used our SimpleCell strategy using nuclease-mediated gene editing of a human cell line (MDA-MB-231) to reduce the structural heterogeneity of O-Man glycans and to probe the OMan glycoproteome. In this breast cancer cell line we found that O-Man glycosylation is primarily found on cadherins and plexins on β-strands in extracellular cadherin and Ig-like, plexin and transcription factor domains. The positions and evolutionary conservation of O-Man glycans in cadherins suggest that they play important functional roles for this large group of cell adhesion glycoproteins, which can now be addressed. The developed O-Man SimpleCell strategy is applicable to most types of cell lines and enables proteome-wide discovery of O-Man protein glycosylation.POMGnT1 | O-glycosylation | Orbitrap | mass spectrometry | glycoproteomics

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Section: Resultsmentioning

confidence: 59%

Section: Resultsmentioning

confidence: 87%

Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins

Vester-Christensen¹,

Halim²,

Joshi³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

145

187

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show abstract

“…Although this type of glycosylation is most frequently discussed in connection with alpha-dystroglycan, other proteins such as CD24, neurofascin, and receptor tyrosine phosphatase beta have also been shown to contain O-mannosyl structures (63)(64)(65). However, the exact site of such modifications has not been identified for these proteins.…”

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

confidence: 99%

N- and O-Glycosylation in the Murine Synaptosome

Trinidad

Schoepfer²,

Burlingame

et al. 2013

Molecular & Cellular Proteomics

159

230

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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

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“…However, recent glycomics efforts (7,9) and the identification of additional O-mannosylated proteins (8)(9)(10)(11)32) indicated that O-mannosyl glycans contribute to more cellular processes than initially expected. Given the crucial role of cadherin-based cell-cell contacts in tissue morphogenesis and maintenance (37), our findings shed light on the molecular basis of some aberrations observed in CMDs.…”

Section: O-mannosyl Glycans Directly Affect Cadherin-mediated Cell Admentioning

confidence: 99%

Protein O-mannosylation is crucial for E-cadherin–mediated cell adhesion

Lommel¹,

Winterhalter

Willer³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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In recent years protein O-mannosylation has become a focus of attention as a pathomechanism underlying severe congenital muscular dystrophies associated with neuronal migration defects. A key feature of these disorders is the lack of O-mannosyl glycans on α-dystroglycan, resulting in abnormal basement membrane formation. Additional functions of O-mannosylation are still largely unknown. Here, we identify the essential cell-cell adhesion glycoprotein epithelial (E)-cadherin as an O-mannosylated protein and establish a functional link between O-mannosyl glycans and cadherin-mediated cell-cell adhesion. By genetically and pharmacologically blocking protein O-mannosyltransferases, we found that this posttranslational modification is essential for preimplantation development of the mouse embryo. O-mannosylation-deficient embryos failed to proceed from the morula to the blastocyst stage because of defects in the molecular architecture of cell-cell contact sites, including the adherens and tight junctions. Using mass spectrometry, we demonstrate that O-mannosyl glycans are present on E-cadherin, the major cell-adhesion molecule of blastomeres, and present evidence that this modification is generally conserved in cadherins. Further, the use of newly raised antibodies specific for an O-mannosyl-conjugated epitope revealed that these glycans are present on early mouse embryos. Finally, our cell-aggregation assays demonstrated that O-mannosyl glycans are crucial for cadherin-based cell adhesion. Our results redefine the significance of O-mannosylation in humans and other mammals, showing the immense impact of cadherins on normal as well as pathogenic cell behavior.POMT2 | mouse preimplantation development | protein-glycosylation | O-glycans | POMT1

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Neurofascin 186 Is O-Mannosylated within and Outside of the Mucin Domain

Cited by 39 publications

References 25 publications

Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins

Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins

N- and O-Glycosylation in the Murine Synaptosome

Protein O-mannosylation is crucial for E-cadherin–mediated cell adhesion

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