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

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

Protein O-mannosylation in the murine brain: Occurrence of Mono-O-Mannosyl glycans and identification of new substrates

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

Known O-mannosylated proteins are located to the cell types identified by the α-O-Man antibody.

<p>A) α-DG as indicated by the IIH6 antibody reactive to matriglycan, and its interacting partner laminin preferentially stained vasculature (arrowhead) and the <i>glia limitans</i>. Purkinje cell layer (asterisks) and granular cell layer (bracket) were stained by the anti-laminin, the IIH6 and α-O-Man antibodies. IIH6 staining was performed following the protocol of Beedle <i>et al</i>. 2012 to detect mouse antigens on mouse tissue [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166119#pone.0166119.ref033" target="_blank">33</a>]. B) Plexin-B2 (Plxnb2) and RPTPζ showed comparable signal distribution as the α-O-Man antibody. Plxnb2 signals were located exclusively to the Purkinje cell layer (asterisks), whereas RPTPζ was primarily located to the granular cell layer in the cerebellum (abbreviated by C). In the cerebral cortex (CC), RPTPζ labeled single cells all of which were also stained for by the α-O-Man antibody. Nuclei were stained by DAPI, sagittal cryosections of WT mouse brains were used. Scale bars = 50 μm.</p

Specificity of the α-O-Man monoclonal antibody.

<p>A) Western blot analysis of recombinant His-tagged proteins [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166119#pone.0166119.ref027" target="_blank">27</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166119#pone.0166119.ref034" target="_blank">34</a>]. The α-O-Man antibody only detected O-mannosylated proteins (hNFASC186 and hDGdel2), whereas non-modified hDG5 was not recognized. B) Immunofluorescent staining with α-O-Man was drastically reduced in the cerebral cortex of POMT2^{f/f;Emx1-Cre+} mice (upper panel). Cerebral cortex layers are indicated on the left (I to VI). In contrast, staining of the Purkinje cell layer was comparable in the cerebellum, where cre is not expressed (lower panels). C) Cryosections of wild-type mouse brain were treated with PNGase F to remove all types of N-glycans. Efficient elimination is demonstrated by <i>Wisteria floribunda</i> agglutinin (WFA) staining, a marker of perineural nets [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166119#pone.0166119.ref038" target="_blank">38</a>] that is reactive to complex type N-glycans [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166119#pone.0166119.ref039" target="_blank">39</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166119#pone.0166119.ref040" target="_blank">40</a>]. No reduction in α-O-Man antibody signal upon N-glycan removal was observed. B,C) Nuclei were counterstained with DAPI, sections were cut sagittally. Scale bars = 50 μm.</p

Localization of O-mannosylated peptides of human RPTPζ.

<p>Schematic model of RPTPζ, illustrating the annotated domains. Plasma membrane (PM) is indicated as a lipid bilayer. Positions of peptides containing extended O-mannosyl glycans (indicated by green circles and -R) and O-linked N-Acetylgalactosamine mucin-type glycans (yellow squares) found by Trinidad and coworkers are shown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166119#pone.0166119.ref029" target="_blank">29</a>]. The position of O-hexosylated peptides identified in this study is indicated as a green circle with a horizontal black bar.</p

Validation of O-mannosylation on RPTPζ.

<p>Samples were dimethyl labeled in the light and medium form, respectively. After treating the light sample with α-mannosidase, both samples were mixed and analyzed by LC-MS/MS. Shown are four extracted ion chromatograms of the m/z values of light and medium labeled peptide LLLPSTATSK in (A) the deglycosylated form (543.8 amu and 547.9 amu) and in (B) the mannosylated form (624.9 amu and 628.9 amu, respectively). The glycopeptide was detected only in the untreated sample (medium labeled), whereas the deglycosylated peptide (light labeled) was only observed after mannosidase treatment. (C) HCD fragment spectrum of precursor mass 543.8 amu and (D) 628.9 amu confirmed the sequence of the deglycosylated and O-mannosylated peptide LLLPSTATSK of RPTPζ.</p

Mono-O-mannosyl glycan staining is pronounced in inhibitory neurons.

<p>Single channel images and merged channels including counterstained nuclei by DAPI (sagittal sections) are shown. A) Co-staining of gephyrin, an inhibitory synapse protein of GABAergic neurons and the α-O-Man antibody on WT murine brain cryosections of the hippocampus (first row) and cerebellum (second row). B) Staining of cryosections from GAD^Cre mice, expressing the cre protein specifically in GABAergic neurons. Cre-labeled GABAergic neurons were positive for α-O-Man antibody staining in the hippocampus (third row) and cerebellum (fourth row). Inlay pictures in A and B, as well as C and D show higher magnification pictures. Asterisks indicate the Purkinje cell layer. C) Peanut agglutinin (PNA), a marker for myelinated axons, did not co-localize with the signal of the α-O-Man antibody in the hippocampus (first row) or the cerebellum (second row). D) Glial marker glial fibrillary acidic protein (GFAP) did not overlap with staining of the α-O-Man antibody. Scale bars = 50 μm.</p