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

Abstract

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

Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast

“…Analysis of the identified proteins revealed that a large proportion (n = 83, 29%) were known or predicted nuclear and/or cytosolic proteins without recognizable signal peptides. This finding was in striking contrast to our previous analysis of the O-Man glycoproteome of human cells, where all identified glycoproteins were known or predicted to traffic the secretory pathway (16). We therefore also inspected all identified glycoproteins with signal peptides for the specific localization of O-glycosites with respect to known or predicted membrane orientation and found sites predicted to be located in the cytosolic compartment (Dataset S1).…”

“…An additional 4 U PNGase F was added and incubated at 37°C for 4 h. The digests were acidified with 12 μL TFA, incubated at 37°C for 20 min, cleared by centrifugation at 10,000 × g for 10 min, and purified by Sep-Pak C18 (Waters) columns. The LWAC protocol for isolation of O-Man glycopeptides was as previously described (16). Sep-Pak-purified peptides were concentrated by evaporation, and the reduced solution was diluted with an equal volume of 2 × ConA buffer A (40 mM Tris·HCl, pH 7.4, 300 mM NaCl, 2 mM CaCl 2 /MgCl 2 / MnCl 2 /ZnCl 2 , 1 M urea) before loading in a 2.8-m-long ConA lectin agarose column.…”

“…nLC-MS/MS and Data Analysis. Mass spectrometric analyses were performed essentially as previously described (16). Samples were analyzed on a setup composed of an EASY-nLC 1000 (Thermo Fisher Scientific) interfaced via a nanoSpray Flex ion source to an LTQ-Orbitrap Velos Pro hybrid spectrometer (Thermo Fisher Scientific).…”

“…We previously used the same strategy to characterize the human O-Man glycoproteome and found no evidence of O-mannosylation of nucleocytoplasmic proteins (16). Because yeast is also the only known eukaryote to lack the OGT/OGA enzymes responsible for O-GlcNAcylation, it is likely that nucleocytoplas- sensor in yeast.…”

“…Our knowledge of the proteins undergoing O-Man glycosylation in yeast and the specific sites of glycosylation is limited (11,12), but recently we developed a glycoproteomic strategy to probe the O-Man glycoproteome of human cells using genetic engineering to simplify the O-glycan structures, the so-called "SimpleCell" strategy, in combination with Concanavalin A (ConA) lectin chromatography for enrichment of glycopeptides and mass spectrometric sequencing (16). This resulted in identification of a large number of O-Man glycoproteins and O-Man glycosites, demonstrating, for example, that cadherins and protocadherins are major carriers of O-Man glycans.…”