Multiple Enzyme Approach for the Characterization of Glycan Modifications on the C‑Terminus of the Intestinal MUC2Mucin

The polymeric mucin MUC2 constitutes the main structural component of the mucus that covers the colon epithelium. The protein’s central mucin domain is highly O-glycosylated and binds water to provide lubrication and prevent dehydration, binds bacteria, and separates the bacteria from the epithelial cells. Glycosylation outside the mucin domain is suggested to be important for proper protein folding and protection against intestinal proteases. However, glycosylation of these regions of the MUC2 has not been extensively studied. A purified 250 kDa recombinant protein containing the last 981 amino acids of human MUC2 was produced in CHO-K1 cells. The protein was analyzed before and after PNGase F treatment, followed by in-gel digestion with trypsin, chymotrypsin, subtilisin, or Asp-N. Peptides were analyzed by nLC/MS/MS using a combination of CID, ETD, and HCD fragmentation. The multiple enzyme approach increased peptide coverage from 36% when only using trypsin, to 86%. Seventeen of the 18 <i>N</i>-glycan consensus sites were identified as glycosylated. Fifty-six <i>N</i>-glycopeptides covering 10 <i>N</i>-glycan sites, and 14 <i>O</i>-glycopeptides were sequenced and characterized. The presented method of protein digestion can be used to gain better insights into the density and complexity of glycosylation of complex glycoproteins such as mucins

Multiple Enzyme Approach for the Characterization of Glycan Modifications on the C‑Terminus of the Intestinal MUC2Mucin

The polymeric mucin MUC2 constitutes the main structural component of the mucus that covers the colon epithelium. The protein’s central mucin domain is highly O-glycosylated and binds water to provide lubrication and prevent dehydration, binds bacteria, and separates the bacteria from the epithelial cells. Glycosylation outside the mucin domain is suggested to be important for proper protein folding and protection against intestinal proteases. However, glycosylation of these regions of the MUC2 has not been extensively studied. A purified 250 kDa recombinant protein containing the last 981 amino acids of human MUC2 was produced in CHO-K1 cells. The protein was analyzed before and after PNGase F treatment, followed by in-gel digestion with trypsin, chymotrypsin, subtilisin, or Asp-N. Peptides were analyzed by nLC/MS/MS using a combination of CID, ETD, and HCD fragmentation. The multiple enzyme approach increased peptide coverage from 36% when only using trypsin, to 86%. Seventeen of the 18 <i>N</i>-glycan consensus sites were identified as glycosylated. Fifty-six <i>N</i>-glycopeptides covering 10 <i>N</i>-glycan sites, and 14 <i>O</i>-glycopeptides were sequenced and characterized. The presented method of protein digestion can be used to gain better insights into the density and complexity of glycosylation of complex glycoproteins such as mucins

Multiple Enzyme Approach for the Characterization of Glycan Modifications on the C‑Terminus of the Intestinal MUC2Mucin

The polymeric mucin MUC2 constitutes the main structural component of the mucus that covers the colon epithelium. The protein’s central mucin domain is highly O-glycosylated and binds water to provide lubrication and prevent dehydration, binds bacteria, and separates the bacteria from the epithelial cells. Glycosylation outside the mucin domain is suggested to be important for proper protein folding and protection against intestinal proteases. However, glycosylation of these regions of the MUC2 has not been extensively studied. A purified 250 kDa recombinant protein containing the last 981 amino acids of human MUC2 was produced in CHO-K1 cells. The protein was analyzed before and after PNGase F treatment, followed by in-gel digestion with trypsin, chymotrypsin, subtilisin, or Asp-N. Peptides were analyzed by nLC/MS/MS using a combination of CID, ETD, and HCD fragmentation. The multiple enzyme approach increased peptide coverage from 36% when only using trypsin, to 86%. Seventeen of the 18 <i>N</i>-glycan consensus sites were identified as glycosylated. Fifty-six <i>N</i>-glycopeptides covering 10 <i>N</i>-glycan sites, and 14 <i>O</i>-glycopeptides were sequenced and characterized. The presented method of protein digestion can be used to gain better insights into the density and complexity of glycosylation of complex glycoproteins such as mucins

Multiple Enzyme Approach for the Characterization of Glycan Modifications on the C‑Terminus of the Intestinal MUC2Mucin

The polymeric mucin MUC2 constitutes the main structural component of the mucus that covers the colon epithelium. The protein’s central mucin domain is highly O-glycosylated and binds water to provide lubrication and prevent dehydration, binds bacteria, and separates the bacteria from the epithelial cells. Glycosylation outside the mucin domain is suggested to be important for proper protein folding and protection against intestinal proteases. However, glycosylation of these regions of the MUC2 has not been extensively studied. A purified 250 kDa recombinant protein containing the last 981 amino acids of human MUC2 was produced in CHO-K1 cells. The protein was analyzed before and after PNGase F treatment, followed by in-gel digestion with trypsin, chymotrypsin, subtilisin, or Asp-N. Peptides were analyzed by nLC/MS/MS using a combination of CID, ETD, and HCD fragmentation. The multiple enzyme approach increased peptide coverage from 36% when only using trypsin, to 86%. Seventeen of the 18 <i>N</i>-glycan consensus sites were identified as glycosylated. Fifty-six <i>N</i>-glycopeptides covering 10 <i>N</i>-glycan sites, and 14 <i>O</i>-glycopeptides were sequenced and characterized. The presented method of protein digestion can be used to gain better insights into the density and complexity of glycosylation of complex glycoproteins such as mucins

Local accumulation of neutrophils in the colon of Muc2^-/- mice.

<p>LP cells from proximal, middle or distal colon sections of Muc2^-/- and Muc2^+/- littermates were analyzed by flow cytometry. (A) Viable neutrophils were identified as 7AAD^-MHCII^-CD11b⁺Ly6G⁺ cells. The numbers represent the percent of cells in the indicated gate. (B) The frequency of neutrophils among viable LP cells for all mice examined is shown. The dashed line indicates the “cut off” value of 0.36%[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130750#pone.0130750.ref023" target="_blank">23</a>]). (C) The frequency of CD11b⁺MHC^-/low cells among viable LP cells of all mice examined is depicted. Each symbol represents an individual mouse and the horizontal line indicates the mean. Results are from 12 independent experiments with a total of 14–22 mice per group. Statistical significance was assessed using the Kruskal-Wallis test followed by Dunn’s multiple comparison test.</p

Altered frequency of iMP populations in the colon of Muc2^-/- mice.

<p>LP cells from proximal, middle and distal colon of Muc2^+/- controls, Muc2^-/-, and colitic Muc2^-/- mice were analyzed by flow cytometry. (A) A representative dot plot of a Muc2^+/- mouse showing gating of viable NK1.1^-TCR^-CD19^-CD11c⁺MHC-II⁺ cells is depicted in the left two dot plots and further analysis of gated CD11c⁺MHC-II⁺ cells for expression of CD103 and CD11b for the indicated genotype of mouse is shown to the right. B-D depict the frequency of CD103⁺CD11b^- "P1" DCs (B), CD103⁺CD11b⁺ "P2" DCs (C), and CD103^-CD11b⁺ "P3" iMPs (D) among viable LP cells in the indicated gates. E-G show the absolute number of viable CD103⁺CD11b^- "P1" DCs (E), CD103⁺CD11b⁺ “P2” DCs (F) and CD103^-CD11b⁺ “P3” iMPs (G). Each symbol represents an individual mouse. For B-G results from the proximal (left column), middle (center column) and distal (right column) colon are shown. The mean of each group is indicated by the horizontal line. Segregating Muc2^-/- mice into colitic and non-colitic is performed according to neutrophil influx in the respective colon segment. Data are from 12 independent experiments that analyzed 14–22 mice per group. Statistical significance between groups was assessed using the Kruskal-Wallis test followed by Dunn’s multiple comparison test.</p

MUC2 positive cells on domes of mouse, rat and human Peyer's patches.

<p>Fluorescent staining of Muc2 reveals mucin containing cells in the FAE of a mouse (A), rat (B) and human (C) ileal PP. Bars = 50 µm. Inset in panel C shows the MUC2 positive cells at higher magnification (bar = 10 µm). Muc2 staining is green, nuclei are blue and FAE is indicated by dashed lines. (D) MUC2 positive cells and nuclei in FAE were counted in sections from 5 mice, 5 rats and 5 humans. Values are presented as median (25^th and 75^th percentile). The percentage of goblet cells was larger in human FAE compared to mouse FAE (<i>P</i><0.001, ***) and rat domes (<i>P</i><0.05, *).</p

Transmission electron micrographs of mouse, rat and human Peyer's patches show secreting goblet cells.

<p>(A) Secreting goblet cell in mouse FAE. (B) M cell in mouse FAE. (C) Secreting goblet cell in a rat FAE. (D) Two M cells next to each other in a rat FAE. (E) Secreting goblet cell in human FAE. (F) Mucus on top of a human FAE, mucus border indicated by black arrow and mucus marked by black star. Bars = 2 µm.</p

CD103⁺CD11b⁺ Dendritic Cells Induce T_h17 T Cells in Muc2-Deficient Mice with Extensively Spread Colitis

<div><p>Mucus alterations are a feature of ulcerative colitis (UC) and can drive inflammation by compromising the mucosal barrier to luminal bacteria. The exact pathogenesis of UC remains unclear, but CD4⁺ T cells reacting to commensal antigens appear to contribute to pathology. Given the unique capacity of dendritic cells (DCs) to activate naive T cells, colon DCs may activate pathogenic T cells and contribute to disease. Using Muc2^-/- mice, which lack a functional mucus barrier and develop spontaneous colitis, we show that colitic animals have reduced colon CD103⁺CD11b^- DCs and increased CD103^-CD11b⁺ phagocytes. Moreover, changes in colonic DC subsets and distinct cytokine patterns distinguish mice with distally localized colitis from mice with colitis spread proximally. Specifically, mice with proximally spread, but not distally contained, colitis have increased IL-1β, IL-6, IL-17, TNFα, and IFNγ combined with decreased IL-10 in the distal colon. These individuals also have increased numbers of CD103⁺CD11b⁺ DCs in the distal colon. CD103⁺CD11b⁺ DCs isolated from colitic but not noncolitic mice induced robust differentiation of Th17 cells but not Th1 cells <i>ex vivo</i>. In contrast, CD103^-CD11b⁺ DCs from colitic Muc2^-/- mice induced Th17 as well as Th1 differentiation. Thus, the local environment influences the capacity of intestinal DC subsets to induce T cell proliferation and differentiation, with CD103⁺CD11b⁺ DCs inducing IL-17-producing T cells being a key feature of extensively spread colitis.</p></div

Cytokine profiles in the distal colon of colitic mice correlate with increased CD103⁺CD11b⁺ P2 DCs in the same colon segment.

<p>LP cells of the other half of the same distal colon segment of the same mice used for cytokine analyses in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130750#pone.0130750.g003" target="_blank">Fig 3</a> were analyzed by flow cytometry. Cells were gated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130750#pone.0130750.g002" target="_blank">Fig 2A</a>. (A-C) The frequency of DCs belonging to subsets P1 (A), P2 (B) or P3 (C) among viable MHCII⁺CD11c^hi cells is shown. (D-E) The absolute number of DCs belonging to subsets P1 (D), P2 (E) or P3 (F) among 10⁶ viable LP cells is depicted. Each symbol represents an individual mouse and the solid line indicates the mean of each group. Statistical significance between groups was assessed using the Kruskal-Wallis test followed by Dunn’s multiple comparison test.</p