The antioxidant potential of various wheat crusts correlates with AGE content independently of acrylamide

Epidemiological studies have indicated that the consumption of whole-grain products is associated with a reduced risk of cardiovascular diseases, type II diabetes, and cancer. In the case of bread, high amounts of antioxidants and advanced glycation end products (AGEs) are formed during baking by the Maillard reaction in the bread crust; however, the formation of potentially harmful compounds such as acrylamide also occurs. This study investigated the antioxidant responses of different soluble extracts from whole-grain wheat bread crust extracts (WBCEs) in the context of the asparagine, AGE, and acrylamide content. For that, we analyzed nine bread wheat cultivars grown at three different locations in Germany (Hohenheim, Eckartsweier, and Oberer Lindenhof). We determined the asparagine content in the flour of the 27 wheat cultivars and the acrylamide content in the crust, and measured the antioxidant potential using the induced expression of the antioxidant genes GCLM and HMOX1 in HeLa cells. Our study uncovered, for the first time, that the wheat crust’s antioxidant potential correlates with the AGE content, but not with the acrylamide content. Mass spectrometric analyses of WBCEs for identifying AGE-modified proteins relevant to the antioxidant potential were unsuccessful. However, we did identify the wheat cultivars with a high antioxidant potential while forming less acrylamide, such as Glaucus and Lear. Our findings indicate that the security of BCEs with antioxidative and cardioprotective potential can be improved by choosing the right wheat variety

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

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

Mono-O-mannosyl glycans localize to distinct cell types throughout the murine brain.

<p>Sagittal section of WT murine brain counterstained with DAPI for nuclei labeling. A) Broad staining was achieved as shown in the overview (scale bar = 100 μm) and at higher magnifications in the cerebral cortex (1), the cerebellum (2), and the hippocampus (3). In more detail, staining in the cerebellum included single cells of the molecular layer, cells of the granular cell layer and cells of the Purkinje cell layer (indicated by asterisks). In the hippocampus, cells of the <i>cornu ammonis</i> region were labeled including their neuronal projections as indicated by arrowheads (picture 3 shows cells of the CA2 field). Individual cells of regions II to V of the cerebral cortex were stained by the α-O-Man antibody. Co-localization of mono-O-mannosyl glycans with neuronal cell marker (NeuN/Fox3) in the hippocampus (B) or with Purkinje cell marker (Calbindin) in the cerebellum (C) showing single channel signal and merged channels. NeuN-labeled hippocampal neurons of the <i>cornu ammonis</i> 2 (CA2) region were stained by the α-O-Man antibody. Purkinje cell localization of mono-O-mannosyl glycans was demonstrated by co-localization with Calbindin. Scale bar = 50 μm.</p

Schematic workflow for the enrichment of peptides bearing O-linked mannose.

<p>For the detailed protocol please refer to the Materials and Methods section. In brief, murine brains were extracted and pulverized before protein extraction. Protein suspensions were digested with trypsin and N-glycans removed by PNGase F treatment. Tryptic peptides were thereafter subjected to ConA LWAC to enrich for mono-O-mannosylated peptides. Elution fractions were either analyzed directly (a), or after dimethyl labeling (medium (M), light (L)) and α-mannosidase treatment (b) by LC-MS/MS and HCD fragmentation according to previously described protocols [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166119#pone.0166119.ref031" target="_blank">31</a>].</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

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

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

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