Protein O-Mannosylation in the Murine Brain: Occurrence of Mono-O-Mannosyl Glycans and Identification of New Substrates

Abstract

Protein O-mannosylation is a post-translational modification essential for correct development of mammals. In humans, deficient O-mannosylation results in severe congenital muscular dystrophies often associated with impaired brain and eye development. Although various O-mannosylated proteins have been identified in the recent years, the distribution of O-mannosyl glycans in the mammalian brain and target proteins are still not well defined. In the present study, rabbit monoclonal antibodies directed against the O-mannosylated peptide YAT(α1-Man)AV were generated. Detailed characterization of clone RKU-1-3-5 revealed that this monoclonal antibody recognizes O-linked mannose also in different peptide and protein contexts. Using this tool, we observed that mono-O-mannosyl glycans occur ubiquitously throughout the murine brain but are especially enriched at inhibitory GABAergic neurons and at the perineural nets. Using a mass spectrometry-based approach, we further identified glycoproteins from the murine brain that bear single O-mannose residues. Among the candidates identified are members of the cadherin and plexin superfamilies and the perineural net protein neurocan. In addition, we identified neurexin 3, a cell adhesion protein involved in synaptic plasticity, and inter-alpha-trypsin inhibitor 5, a protease inhibitor important in stabilizing the extracellular matrix, as new O-mannosylated glycoproteins.

The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins

“…The O-mannosylated and core 2 peptides were synthesized as previously described (39)(40)(41)(42)(43) The O-mannosylated peptides (1 μg each) were added to StcE in a separate reaction under the same conditions. For core 2 synthetic peptides, 40 μL of solutions containing 1 μM peptide was separately dried down via a speedvac.…”

Analysis of Mammalian O-Glycopeptides—We Have Made a Good Start, but There is a Long Way to Go

“…It has been reported that branched O-mannosyl structures displaying HNK-1 epitopes (sulfoglucuronyl "capping") influence cell-cell and cell-matrix interactions in the developing nervous system (66). Recently, protein modification with a single mannose residue has also been reported (67,68).…”

Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins

“…To explore the functions of O-Man glycans on cdhs and pcdhs, we previously used a combinatorial gene-targeting strategy in multiple cell lines and found that the two POMTs are essential for glycosylation of α-DG but not cdhs, pcdhs, and IPT/TIG domaincontaining proteins (13). In contrast to α-DG, we and others (6,10,11,13) also found that O-Man glycans on the latter proteins were not elongated, suggesting that the biosynthesis of these distinct classes of proteins were different. We therefore predicted the existence of a new type of O-Man glycosylation machinery in higher eukaryotes (13).…”

“…Deficiencies in enzymes catalyzing the structurally complex and diverse O-Man glycans on α-DG, including the two human POMT1 and POMT2 genes, underlie a subgroup of congenital muscular dystrophies that have been designated α-dystroglycanopathies because deficient O-Man glycosylation of α-DG disrupts the interaction between the dystrophin glycoprotein complex and the ECM (7)(8)(9). Several studies have also implicated deficiency of POMT2 with E-cdh dysfunction (10)(11)(12), although direct evidence for a role in glycosylation of cdhs and pcdhs is missing. To explore the functions of O-Man glycans on cdhs and pcdhs, we previously used a combinatorial gene-targeting strategy in multiple cell lines and found that the two POMTs are essential for glycosylation of α-DG but not cdhs, pcdhs, and IPT/TIG domaincontaining proteins (13).…”