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Keywords:

  • inflammatory bowel disease;
  • IBD;
  • oligosaccharide;
  • glycan;
  • ulcerative colitis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  7. Supporting Information

Background:

The MUC2 mucin organizes the two mucus layers in the colon. This mucin carries a large number of O-glycans that are assumed to be attachment sites for the commensal flora found in the outer mucus layer.

Methods:

Single biopsies from the sigmoid colon of controls (25) and patients with inactive (13) or active (15) ulcerative colitis (UC) were collected during routine colonoscopy. The insoluble MUC2 mucin was prepared and separated by gel electrophoresis, its relative amount estimated, its O-glycans released, and glycans analyzed by novel sensitive glycomics chromatography / mass spectrometry providing information on glycan structures and relative abundances. The glycosylation pattern was related to the degree of mucosal inflammation and clinical severity of the disease.

Results:

The relative abundance of MUC2 showed high individual variability. Two major glycan profiles were found; a normal pattern in the control and inactive UC patients and an aberrant profile in patients with active colitis with an increase in a subset of the smaller glycans and a decrease of several complex glycans. The magnitude of this phenomenon was significantly related to both the degree of inflammation in the biopsies and also to some extent the severity of disease course. The aberrant profile was further shown to be reversible upon remission.

Conclusions:

In the majority of the active UC patients MUC2 mucin has an altered glycan profile as compared to inactive UC and control patients. Patients with strong alterations in the glycan pattern tended to have a more severe disease course. (Inflamm Bowel Dis 2011)

Ulcerative colitis (UC) has an incidence of more than 40 per 100,000 individuals in Westernized countries.1 The main symptoms of patients with an active inflammation are rectal bleeding and diarrhea. Longstanding UC also leads to an increased risk for colonic cancer. There is a general agreement that the tissue damage in UC patients is mediated by an overactive mucosal response to the colon bacterial flora, but the molecular mechanisms behind the disease remain unknown.1

The mucus layer covering the colonocytes can be up to 800 μm thick as measured in rat.2 It is mainly made up of the gel-forming mucin MUC2, one of a family of four human gel-forming mucins.3 The colon mucus barrier consists of two layers, one easily removable outer loose layer and an inner mucus layer firmly attached to the epithelial cells, with Muc2 being a major constituent of both.4 The inner mucus layer forms a physical barrier that the colonic bacteria are unable to penetrate under healthy conditions.4 However, this inner mucus layer can be penetrated as exemplified by the colon parasite Entamoeba histolytica that cleaves the MUC2 mucin and disrupts this layer.5

The MUC2 mucin is produced by the colonic goblet cells and generates large net-like polymers by its ability to form disulfide-bond stabilized C-terminal end-to-end dimers and N-terminal trimer complexes.6, 7 During biosynthesis, MUC2 is O-glycosylated in the Golgi apparatus by the attachment of GalNAc residues to the hydroxyl group of serine and threonine of the protein backbone. Together with proline, the O-glycans are found in the two central mucin domains of MUC2. The carbohydrate part of MUC2 can constitute 80% of the total mucin mass and a fully glycosylated MUC2 monomer has a mass of ≈2.5 MDa. The secreted MUC2 mucin gel is thus an enormous net-like polymer. The stored and secreted MUC2 mucin is insoluble in 6M guanidinium chloride,8 a property related to the presence of nonreducible bonds.9, 10

The mucin O-glycans are largely concentrated in the mucin domains, resembling bottle brushes with the protein core at its center and the oligosaccharides as its bristles. Important functions of the glycans are to cover the protein backbone and thus protect the mucin from proteolytic enzymes, while at the same time they bind water to generate a gel. Glycans further act as attachment sites for bacteria at the same time as they are an important food source.11–13 There are several studies of glycosylation in UC patients showing alterations.14–17 Among the reported alterations, the increased expression of the short O-linked antigen sialyl-Tn (STn) is of special interest as it is implicated in colon cancer.18

We recently described the MUC2 O-glycans in the sigmoid colon of 25 healthy human individuals.19 More than 100 structures were identified and the majority had a high degree of sialylation and sulfation. Surprisingly, the glycosylation pattern in this group was relatively uniform. The analysis was performed by recently improved sensitive methods for small-scale isolation of MUC2 mucin and analysis of its O-glycosylation in colonic biopsies. We have now used this approach on a group of UC patients with variable degrees of disease activity. A majority of the UC patients with active disease had shifted their glycosylation pattern towards smaller and less complex glycans.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  7. Supporting Information

Patient Data and Collection of Colonic Biopsies

Study subjects were recruited among patients undergoing routine colonoscopy at Sahlgrens' University Hospital, Gothenburg, Sweden. Biopsies from the sigmoid colon were collected from 53 patients and all ABO blood groups were represented among these. Ethical permission for the study was obtained from the Ethical Committee of the Sahlgren University Hospital and all patients gave written informed consent. In all, 28 UC patients were included in the study and their disease activity was assessed from medical records, colonoscopy findings, and histopathological evaluation. Fifteen patients with UC had active extensive colitis (UCa) and the remaining 13 patients with UC had low activity or were in remission (UCi). Three of the patients with UC (patients 395 = 579, 435 = 627, 386 = 580) revisited the endoscopy unit ≈2 years after the first analysis. Biopsies were also assessed from 25 control subjects (Ctrl) who underwent colonoscopy for investigation of anemia, rectal bleeding, or polyp surveillance. The control subjects showed normal colonic mucosa both macroscopically and microscopically without any signs of inflammation. No biased selection of patients was made and the small differences in number of patients were due to technical reasons and had no relation to the patient or disease. The biopsies were directly placed in liquid nitrogen and stored at −80°C.

At least three biopsies for morphological analysis were taken from the studied segment. The biopsies were fixed, formalin-embedded, and evaluated by a trained pathologist according to normal hospital procedures. The inflammatory activity, as stated in the pathology report, was evaluated blindly by an experienced gastroenterologist who did not have access to glycan expression or clinical data and graded according to Sandborn's histological activity scale (0–4).20 Sandborn score 0–1 is regarded as inactive disease with no presence of neutrophils, but level 1 has hypercellular lamina propria. Score 2–4 is regarded as active disease with infiltration of neutrophils, cryptitis, and crypt abscesses of increasing degree. Disease course (range 0–6) was assessed on the basis of the following factors (1 point/factor): continuous systemic treatment with 5-aminosalicylate (5-ASA); >1 exacerbation/year; occurrence of a systemic inflammatory response as reflected by elevated C-reactive protein (CRP); requirement of azathioprine treatment; requirement of tumor necrosis factor alpha (TNF-α) inhibitor treatment; consideration for surgery.

Mucin Preparation and Analysis

One or two sigmoid colonic biopsies (≈5–10 mg) were homogenized in an Eppendorf tube in 200 μL 6M guanidinium chloride, 5 mM EDTA, 0.01 M NaH2PO4 with protease inhibitors.19 The guanidinium chloride insoluble MUC2 mucin was recovered semipurified in the pellet.10 The reduced (dithiotreitol) and alkylated (4-vinylpyridine or iodoactamide) mucins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).19 The composite gels contain agarose (0.5%–1% gradient), polyacrylamide (0%–6%), and glycerol (0%–10%).21 The electrophoresis was developed in borate/Tris buffer (192 mM boric acid, 1 mM EDTA, 0.1% SDS, pH adjusted to 7.6 with Tris base) on ice at 12 mA/gel for 16 hours. For proteomics, the gel was stained with Alcian blue. For glycomics, the gel was blotted at 40W for 5 hours onto Immobilon (PSQ) membrane in 25 mM Tris, 192 mM glycine, 0.04% SDS, 20% methanol on ice, and the bands were visualized with Alcian blue.

For quantification of the MUC2 (including trace amounts of MUC5B) monomer, 10% of the sample was analyzed by SDS-PAGE. Two patient samples were used as standards on all gels. The proteins were stained with Sypro Ruby (Invitrogen, La Jolla, CA) and the gels were scanned by 2D-2920 Master Imager (GE Healthcare, Milwaukee, WI) with 65,000 pixels as maximum intensity. Evaluation was performed with ImageJ software (NIH, Bethesda, MD).

Alcian blue-stained bands from SDS-PAGE were excised, trypsinized, and the peptides identified using a LTQ-ICR(FT) mass spectrometer.22

Identification and Quantification of Mucin O-glycans

The sigmoid colon mucins were separated by SDS-PAGE and the bands blotted to an Immobilon membrane.19 The bands were visualized with Alcian blue and the oligosaccharides from the MUC2 monomer band were released by reductive β-elimination.19, 21 The glycans were resuspended in dd-H2O (15 μL) prior to analysis of 1 μL on a Hypercarb column (180 μm id) coupled to an LTQ mass spectrometer (Thermo, Pittsburgh, PA).19 All patients were analyzed both by total ion chromatograms and by analyzing the individual mass spectra of all components for each patient. After this, the 26 most abundant glycans were semiquantified with the software DeCyder MS (GE Healthcare).19 The majority of these glycans exist as isomers and for these the intensities were summarized for quantification. For statistical analysis, the nonparametric Mann–Whitney test was used for evaluating group differences. For correlation of glycan levels with degree of inflammation and disease course, regression analysis followed by an F-test was used. A P-level of < 0.05 was regarded as statistically significant. Statistical calculations were performed in SPSS 16.0 (Chicago, IL).

Ethical Considerations

Ethical permission for the study was obtained from the Ethical Committee of Sahlgren University Hospital. All patients were informed in writing and orally by a specially trained nurse. All patients were informed that participation in this study was voluntary and that did not affect their treatment. All patients gave written informed consent. The biopsies were kept coded and patient information kept anonymous.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  7. Supporting Information

Patients undergoing endoscopy for reasons other than suspected inflammatory bowel disease (IBD) (e.g., anemia of unknown origin) and without macro- or microscopic inflammation were used as control subjects (Ctrl) (n = 25). Patients having an active colitis, UCa (n = 15), or an inactive colitis UCi (n = 13) were classified according to Sandborn's histological activity scale.20 Patient details are given in Supplementary Tables S1–S2.

MUC2 Mucin Amounts in Sigmoid Colon

The guanidinium chloride insoluble mucins were isolated from the sigmoid colon. The reduced and alkylated insoluble fraction was separated by SDS-PAGE. Typically, three high molecular mass protein bands were visualized by a protein stain (Fig. 1A). These bands were identified as MUC2 mucin using proteomics with matching peptides predominantly in its less glycosylated N- and C-terminal parts (not shown). The fastest migrating band (MUC2 band 1) has a mass of ≈2.5 MDa and is the MUC2 monomer. The slower migrating bands are the MUC2 nonreducible dimer (MUC2 band 2) and oligomers (MUC2 band 3).9, 10 The MUC5B mucin was also identified in trace amounts in the monomeric MUC2 band. MUC5B has previously been observed in small quantities in a subset of goblet cells at the bottom of the colonic crypts.23 MUC5AC has previously been observed in UC by immunohistochemistry,24 but could not be identified here.

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Figure 1. Separation and semiquantification of MUC2 mucin. (A) Single sigmoid colon biopsies of UC and control patients were homogenized and the insoluble MUC2 mucin was extracted in guanidinium chloride and separated by composite SDS-PAGE and further visualized with Sypro Ruby protein stain. (B) The relative amount of MUC2 in one biopsy was estimated from the intensity of the stained MUC2 band 1. Mean values of the relative amounts are given with ±1 SD for the three patient groups: Ctrl (n = 21), UCi (n = 13), and UCa (n = 7). No significant differences were observed.

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The MUC2 amounts were further semiquantified in 41 patients (21 controls, 13 UCi, and 7 UCa) by scanning the Sypro Ruby-stained monomer band. Duplicate or triplicate quantifications of the MUC2 band of the same biopsy or multiple biopsies from one patient analyzed on different gels gave similar results (not shown). The biopsy surface area was best correlated to the amount of mucosa in contrast to the weight that reflected more the depth of the biopsy. No correlation to number of goblet cells was made, as the amount of mucus is due to number of goblet cells and to the number and filling of their mucin granulae, where the latter parameters are difficult to estimate. A large variability between individuals in the MUC2 mucin amounts was shown and no significant differences between patient groups could be demonstrated (Fig. 1B). Two patients with active UC (patients 434 and 580 = 386) had no sigmoid MUC2 expression as stained with Alcian blue, and these patients also showed a corresponding decrease in mucus production in the crypts according to histology.

Quantitative Profiling of MUC2 Mucin O-glycosylation

To analyze the O-glycosylation of the MUC2 mucin, the insoluble mucins were blotted to PVDF membranes, stained with Alcian blue, and the O-glycans released by β-elimination from the MUC2 monomer band (band 1, Fig. 1A). The oligosaccharides were separated on a Hypercarb column and the ions detected with negative ion mode mass spectrometry (MS) and MSn. Figure 2 shows examples of liquid chromatography traces from the separation of glycans released from one control patient and one patient with UCa. The control patient has a characteristic “control” glycan profile with relatively small amounts of the shorter glycans and the presence of larger, late eluting peaks representing more complex glycans. This profile is characteristic for control patients, in which we previously have identified more than 100 different glycans and could show a surprisingly uniform glycan repertoire and quantitative distribution among 25 control patients.19 However, the chromatographic trace of the UCa patient shows a substantially different peak pattern (Fig. 2). The intensities of the shorter glycans are increased and the larger, more complex glycans are absent or decreased in amounts.

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Figure 2. O-glycan profiles of MUC2 mucin as revealed by the total ion intensities from nanoLC/MS of a control (patient 441) and a UCa (patient 436) patient. The numbers indicated refer to the individual glycans and their corresponding mass as previously described.19

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To further investigate the differences in the profiles of the patient groups, the relative O-glycan amounts on the insoluble MUC2 of sigmoid colon was semiquantified and the relative expression of the 26 most abundant glycans was calculated by integration of the individual peak areas in the chromatograms. The relative mean amounts are shown in Figure 3A and the relative amounts of six key glycans in each individual patient are presented in Figure 3B. The glycans are labeled with their corresponding molecular mass and their relative amounts were based on the combined intensities of their singly and doubly charged ions. When oligosaccharide isomers were present, their intensities were also combined in the quantification (except for 716a and b). The relative glycan amounts in each patient are presented in Supplementary Table S3.

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Figure 3. The relative amounts of different O-glycans on the MUC2 mucin in biopsies from sigmoid colon. (A) The relative mean amounts (±1 SD) of the 26 most abundant glycans from the semiquantification of the molecular ion intensities in sigmoid colon of control patients (blue, n = 25), UCi (black, n = 11), and UCa (red, n = 14) patients. (B) Relative amounts of six abundant O-glycans in all 50 analyzed patients, controls in blue, inactive UC in black, and active UC in red. The relative amount of the glycans 513, 716a, 716b, 1104, 1315, and 1372, expressed as percentage of the total amount of the 26 glycans used for quantification, on the y-axis. The individual patients each represent one column on the x-axis. Control (n = 25), UCi (n = 11), and UCa (n = 14). The difference between UCa and Ctrl is significant (P < 0.05) for 513 (P < 0.0001), 716a (P = 0.021), 1104 (P < 0.0001), and 1372 (P = 0.001). The difference between UCa and UCi is significant for 513 (P = 0.002), 716 a (P = 0.037), 1104 (P = 0.001), and 1372 (P = 0.017). No statistical significance could be proven between the UCi and Ctrl groups. Filled arrows indicate major alterations in the UCa patients.

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The most abundant glycans in the control patients were the two isomers of the trisaccharide 716 (relative mean amounts of 16% and 13%), together with substantial amounts of the 1104 (15%) and 1372 (13%) glycans (Fig. 3A). This pattern, typical for control patients, was also found in all the UCi group patients except for one, and in 6 out of 14 of the UCa patients. In contrast, the majority of the UCa patients (8 out of 14) had another pattern with increased amounts of the 716a isomer (relative amount of 22%) and decreased levels of the 1104 (5%) and 1372 (5%) glycans. The most compelling difference was the increased amount of the STn antigen (the 513 glycan), with relative mean amounts of 18% as compared to 2% in the control patients (Fig. 3A).

The schematic structures of some of the more abundant oligosaccharides and their biosynthetic pathways are presented in a flowchart in Figure 4. The accumulation of the 513 precursor glycan in the UCa group was statistically significant (P < 0.05) as compared to both the control group (P < 0.0001) and the UCi group (P = 0.002). The glycan 878 is the precursor for the 1104, 1315, and 1372 compounds and was found in similar amounts in all patients (not shown). The patients (8/14 UCa and 1/11 UCi) with an accumulation of smaller glycans had subsequently reduced levels of the blood group Lewis-type glycan 1104 and the Sda/Cad-type glycan 1372, but interestingly not of the 1315 glycan formed from the same precursor. The reduction of the 1104 and 1372 glycans in the UCa group also showed statistical significance (P < 0.05) as compared to the control group (P < 0.0001 and P = 0.001) and the UCi group (P = 0.001 and P = 0.017).

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Figure 4. Schematic graph showing a plausible biosynthetic pathway for a selection of seven key oligosaccharides identified. The symbols are explained in the figure. The dotted arrows indicate the effect of one or several glycosyltransferases. S/T is an abbreviation for the amino acids serine or threonine and Gal for galactose, GalNAc for N-acetylgalactosamine, GlcNAc for N-acetylglucosamine, Fuc for fucose, and NeuAc for sialic acid. Filled arrows indicate major alterations in the UCa patients.

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Correlation of MUC2 Mucin O-glycosylation Patterns with Degree of Inflammatory Activity and Disease Course

A disturbed glycosylation pattern has been reported previously in active UC,14–17, 25 but the implications of this phenomenon have not been assessed. One possibility is that a severe inflammation has more pronounced effects on glycosylation, i.e., that it is a consequence of the inflammation per se. Another possibility is that some patients have a built-in vulnerability in their glycosylation machinery, and therefore develop a more severe disease course. To tentatively test these two hypotheses, we correlated the relative amounts of the glycans 513 and 1104 with 1) the degree of inflammatory activity in the biopsies and 2) disease course (Fig. 5).

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Figure 5. Correlation of the relative amounts of the glycans 513 and 1104 with the inflammatory disease activity (Sandborn's inflammatory score) and disease course indices. The inflammatory disease activity was graded according to Sandborn's histological activity scale (0–4) on the basis of the presence of neutrophils, cryptitis, and crypt abscesses and are given in Supplementary Table S2. The disease course was scored as a sum of the six factors: >1 exacerbation/year, systemic inflammatory response, 5-ASA, azathioprine or TNF-α inhibitor treatment regimens and consideration for surgery as given in Supplementary Table S4. The relation between the relative amounts of the 513 glycan and the inflammatory disease activity was statistically significant (P = 0.007), as was the relation to disease course (P = 0.006). For the 1104 glycan, the relation to the inflammatory disease activity was statistically significant (P = 0.0005), while the relation to disease course was not statistically significant (P = 0.302).

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The degree of inflammatory activity was scored according to Sandborn's histological activity scale (Supplementary Table S2) and the disease course was scored as a sum of six different factors (Supplementary S4). Figure 5 shows a significant relation (P < 0.05) between the amount of 513 and both the degree of inflammatory activity and disease course (P = 0.007 and P = 0.006). The 1104 glycan showed an opposite trend, but only the relation to inflammatory disease activity was statistically significant (P = 0.0005).

Since UC patients in remission had a similar glycan pattern as controls, while most of the UC patients with active disease had a different pattern, it could be possible that the glycosylation pattern is related to inflammatory activity rather than the disease per se. When we analyzed the MUC2 O-glycans of one patient (patient 435) with active UC in 2006, this patient showed the active disease glycan profile. However, when an additional colonoscopy was made in 2008 this patient was in remission (patient 435 = 627) and showed that the glycan pattern was normalized to the control pattern (Fig. 6). This change in glycan pattern was not observed when a patient in remission 2006 (patient 395) was reanalyzed in 2008 when still in remission (patient 395 = 579).

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Figure 6. The O-glycans of sigmoid biopsies from two patients (patients 435 and 395) was analyzed in 2006 when patient 435 had an active disease and patient 395 was in remission. In 2008 additional biopsies were collected when both patients were in remission. The histogram shows relative amounts of five key MUC2 glycans (513, 716a, 716b, 1104, and 1372) for the patients at the two timepoints.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  7. Supporting Information

In the present study the relative amounts and glycosylation patterns of MUC2 from the sigmoid colon of patients with both inactive and active UC was compared with that in control subjects. As the glycosylation varies along the intestine, only sigmoid colon was studied where all biopsies were taken from the same area with a similar-looking mucosa. Major differences in glycosylation patterns between controls and active disease patients were observed and were significantly related to both the degree of inflammation and disease course.

The tendency of a slight decrease in MUC2 amounts in the active UC subjects as compared to the control patients did not reach significance, but has been observed previously.26 However, Tytgat et al did not find any alterations in the MUC2 mRNA levels.27–29 The amounts of MUC2 could potentially be of importance in UC pathogenesis since it might be related to our observation of an inner mucus layer built around the MUC2 network, acting as a barrier to keep the colon bacteria at a distance from the epithelia.4 Lack of Muc2, as in Muc2 null mice,4, 30 or mutations in Muc231 cause spontaneous colitis and later on colon cancer. MUC5B mucin was also observed in small amounts, but no MUC5AC could be identified. MUC5AC has previously been observed in UC by immunohistochemistry.24

No significant differences were observed for the relative MUC2 amounts between the patient groups, but large differences were found in the glycosylation pattern of MUC2. Two major glycan profile patterns were present. One was found among control patients and almost all UC patients in remission. This normal control pattern has been described in detail previously and is surprisingly similar in all studied individuals.19 No glycosylation differences could be related to the ABO or Lewis blood groups,32 and in contrast to earlier suggestions, no highly active Lewis enzyme appears to be present in the distal part of the colon.33 Typical for the control patients is a pattern with heavily sulfated and sialylated glycans as, for example, glycans 1104, 1315, and the Sda/Cad-like glycan 1372.

A different glycan profile was found in a majority of the patients with active UC. This profile was characterized by increased amounts of smaller glycans and lower amounts of some more complex compounds. The precursor of the MUC2 O-glycans is the Tn antigen (GalNAc-S/T), which is also the precursor of the sialyl-Tn antigen (STn, 513 glycan) (Fig. 4). The increased amount of the 513 glycan could be due to an upregulated ST6GalNAc1 transferase, adding the sialic acid to the C-6 position of GalNAcol, or a lower conversion of the Tn glycan into more complex compounds. This is illustrated by a simplified biosynthetic pathway in Figure 4. The patients with an accumulation of smaller glycans had a subsequent decrease in the 1104 and 1372, but not the 1315 glycans. This pattern is difficult to explain by specific alterations in single glycosyltransferases. The amount of the precursor glycan (878) for both 1104 and 1372 was similar in all groups, further suggesting that the profile in active UC patients is more complex and due to change in action, expression, and/or localization of several glycosyltransferases.

There have been several reports describing glycan alterations related to UC. An increased amount of the STn antigen (glycan 513) was demonstrated in UC by Itzkowitz et al25 using immunohistochemistry, a technique with limitations regarding quantitative estimation. Those authors observed an increased amount of STn in patients with active inflammation, but discussed this finding mainly in terms of a risk factor for development of neoplasia. In accordance with this concept, an increased amount of the STn antigen (glycan 513) is frequently observed in different forms of cancer.34 Campbell et al17 further suggested an increased level of the precursor glycan Gal-GalNAcol (TF-antigen, glycan 384) in UC, but we could not observe this increase on MUC2. Additionally, Bodger et al35 have shown in monozygotic twins that both the sibling with IBD and the unaffected twin sibling express the TF-antigen in the superficial epithelium, as compared to only 5 of 29 histologically normal controls. Interestingly, the majority of individuals carrying the TF-antigen also had NF-κB activation in the surface epithelium, indicating a connection between inflammatory responses and glycan alterations. All proteins passing the Golgi compartment will be affected by alteration in the glycosyltransferases present, and by using immunohistochemistry it is not possible to determine which glycoproteins that carry the antigen. Many glycoproteins may have an altered glycosylation in UC, but we have only studied the MUC2 mucin.

An additional observation in the present study, decreased sulfation, has been suggested in studies using sulfate incorporation into mucins, most likely MUC2, in colon explants from UC.14, 16 Others have not observed any alterations in the sulfate content of secreted MUC2, but a preference in secreting the sulfated MUC2.15 However, no structural analysis or thorough quantitative analyses were performed in these studies.

Glycosylation alterations have also been observed in a number of other diseases. Careful studies of these phenomena suggest that inflammation (and infection) is a mechanism that can trigger such alterations. The Muc2 mucin of the small intestine of both mouse and rat shows an altered glycosylation when infected with an intestinal parasite.36, 37 Mice lacking the Cftr channel as in cystic fibrosis develop bacterial overgrowth in the small intestine and an altered glycosylation.38, 39 The change in glycosylation pattern in the lung mucins of patients with cystic fibrosis has also been linked to inflammation and not to the disease per se.40 In our study there was a significant correlation between glycan expression and both the degree of inflammation and disease course. The latter finding may represent a “sampling error,” as patients having a more severe disease are more likely to be subjected to colonoscopy at a university hospital. The maximal observation time of the patients was ≈2 years, i.e., we do not know if additional patients eventually will be given anti-TNF-α treatment or even be colectomized.

A disturbed glycosylation pattern with a highly abundant 513 glycan and a low abundance of the 1104 glycan may be markers for a more severe disease course, but it is not clear if this is a consequence of the inflammation per se or if it reflects an underlying vulnerability of the mucosa to inflammation. One patient with active UC and the aberrant glycan profile was reexamined after 2 years when in remission. Interestingly, the glycan profile had then returned to the normal control pattern. Another patient who was analyzed at two timepoints of remission showed the normal glycan pattern on both occasions. Patients are thus suggested to be able to alternate between these two glycan patterns and the altered glycan profile is probably due to the level of inflammation. This further suggests that inflammatory response mechanisms are able to alter the biosynthesis of MUC2 O-glycans. Studies in human lung tissue and in pancreatic cancer cell lines have shown that the action of certain glycosyltransferases can be regulated by proinflammatory cytokines.41, 42 Additionally, the MAPK pathway is involved in the transcriptional regulation of the glycosyltransferase C2GnT, a core 2 enzyme active in human airways.43 However, few studies have been performed regarding the influence of inflammatory mechanisms on the glycosylation machinery. Inflammation leads to a strong shift in cell differentiation and it cannot be excluded that the differentiation of goblet cells affects glycosylation. To conclude, the glycan alterations observed in active UC disease are likely not an inherent primary defect, but a consequence of inflammation.

Since the colon harbors such a high number of bacteria that are known to produce numerous glycan degrading enzymes,44 the alterations observed could potentially be due to degradation. However, this is less likely as the increase in the amounts of the STn (glycan 513) cannot be explained by glycan degradation. The lower amounts of certain complex glycans should necessitate very specific degrading enzymes, but no increase in the potential products of such hydrolyses could be observed. Additionally, the biopsies obtained after excessive cleaning of colon for the colonoscopy are virtually devoid of mucus on their surface (not shown). The MUC2 mucin fraction analyzed in this study is insoluble in guanidinium chloride and this fraction is largely derived from the intracellularly stored pool in the goblet cell and partly from the inner germ-free mucus layer and, thus, should not come in close contact with bacteria.4, 8 The outer loose mucus layer inhabiting bacteria is soluble in this chaotropic salt and was removed during preparation. We can thus conclude that the MUC2 mucin glycosylation patterns observed is due to the action of glycosyltransferases in the goblet cell secretory apparatus.

The glycans on mucins are probably important for the selection and maintenance of the intestinal flora. Bacteria have lectins sitting at the outer end of their pili, as recently exemplified by the commensal bacteria Lactobacillus rhamnosus.11, 12 The intestinal flora is relatively species-specific and is selected by the host.45 One of the reasons for this may be the unique glycan repertoire carried by the MUC2 mucin in each species.46, 47 The uniform glycosylation pattern in control patients supports the concept of host glycosylation as an important factor for the selection of the commensal flora also in humans. It should be noticed that several studies have suggested that patients with UC have an altered bacterial flora.48 These observations could be related to the altered MUC2 glycan profile found in patients with active UC.

In conclusion, the MUC2 glycosylation patterns were similar in controls and UC patients in remission. However, in active disease there was a marked shift towards smaller glycans and the magnitude of this shift was significantly correlated with both the degree of inflammation and disease course.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
  7. Supporting Information

Additional supporting information may be found in the online version of this article.

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IBD_21625_sm_SuppTables.xls271KSupporting Information Tables.

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