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 O-Man 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.

Keywords: O-glycosylation; Orbitrap; POMGnT1; glycoproteomics; mass spectrometry.

Fig. 1.

Mining the O-Man glycoproteome by ZFN gene targeting of POMGNT1. (A) Knockout of POMGNT1 and COSMC abrogates elongation of O-Man (Left) and O-GalNAc (Right) type glycosylation, respectively, resulting in truncated homogenous O-glycan structures limited to O-Man and O-GalNAc (in some cells NeuAcα2–3GalNAc). Note that POMGNT2-mediated elongation of O-Man will not be affected by this gene targeting strategy. (B) Double knockout of POMGNT1 and COSMC in MDA-MB-231 cells allows for enrichment of O-Man glycoproteins from the cell culture supernatant using either a short Con A mannose-binding lectin column (captures both O-Man and N-glycoproteins) or for O-GalNAc glycoprotein enrichment on a short VVA αGalNAc-binding lectin column. Enriched eluates from these columns or total cell lysates can then be treated with proteases and PNGase F to remove N-glycans before glycopeptides are isolated by LWAC with a long Con A column and sequenced by nanoflow liquid chromatography-mass spectrometry (nLC-MS/MS).

Fig. 2.

Schematic representation of the identified O-Man and O-GalNAc glycosylation sites in the mucin-like domain of human α-dystroglycan. Glycopeptides covered by the nLC-MS/MS analysis are underlined.

Fig. 3.

Cadherins are the major class of O-mannosylated proteins. (A) Schematic drawing of extracellular domains of classical type 1 and type 2 cadherins. White circles illustrate potential O-Man glycosites predicted based on sequence conservation of Ser/Thr residues in alignments. Green-white circles show glycosites that were ambiguously identified, i.e., a glycopeptide was identified, but localization of the glycan was not identifiable by ETD. The O-GalNAc site identified by the O-GalNAc SimpleCell strategy is shown as a yellow square (21). Predicted O-GalNAc sites based on sequence identity are depicted as white squares. The O-Man sites are located either on or at the border of β-strand B and G. (B) Evolutionary conservation of predicted O-Man glycosites in type 1 and 2 cadherins. Representative analysis of CADH1 and CAD11 are shown with prediction of highly conserved and distinct patterns of distribution of glycosites from human to zebra fish in type 1 and type 2 cadherins. O-Man glycosites identified are depicted as green circles. These also include glycosites found in the crystal structures of mouse CADH1 and CADH2 (31).