Sulphation and secretion of the predominant secretory human colonic mucin MUC2 in ulcerative colitis

Abstract

Background: Decreased synthesis of the predominant secretory human colonic mucin (MUC2) occurs during active ulcerative colitis.

Aims: To study possible alterations in mucin sulphation and mucin secretion, which could be the cause of decreased mucosal protection in ulcerative colitis.

Methods: Colonic biopsy specimens from patients with active ulcerative colitis, ulcerative colitis in remission, and controls were metabolically labelled with [35S]-amino acids or [35S]-sulphate, chase incubated and analysed by SDS-PAGE, followed by quantitation of mature [35S]-labelled MUC2. For quantitation of total MUC2, which includes non-radiolabelled and radiolabelled MUC2, dot blotting was performed, using a MUC2 monoclonal antibody.

Results: Between patient groups, no significant differences were found in [35S]-sulphate content of secreted MUC2 or in the secreted percentage of either [35S]-amino acid labelled MUC2 or total MUC2. During active ulcerative colitis, secretion of [35S]-sulphate labelled MUC2 was significantly increased twofold, whereas [35S]-sulphate incorporation into MUC2 was significantly reduced to half.

Conclusions: During active ulcerative colitis, less MUC2 is secreted, because MUC2 synthesis is decreased while the secreted percentage of MUC2 is unaltered. Furthermore, sulphate content of secreted MUC2 is unaltered by a specific compensatory mechanism, because sulphated MUC2 is preferentially secreted while sulphate incorporation into MUC2 is reduced.

Figure 1

PAS staining of mature MUC2. A representative example of analyses of duplicate biopsy specimens of one control patient. After pulse labelling with either [³⁵S]-amino acids or [³⁵S]-sulphate and four hour chase incubations, the homogenates of the tissue (t) and the media (m) were analysed on reducing 4% SDS-PAGE and stained with PAS. The right hand arrow indicates the position of mature MUC2. The left hand arrow denotes the border between the running and stacking gel. Molecular mass markers of 600, 300, and 205 kDa are indicated on the left.

Figure 2

Identification of mature MUC2 in colonic homogenates. Representative examples of analyses of the biopsy specimens of each patient group are shown: controls (specimens 1 and 2), active ulcerative colitis (UC) (specimens 3 and 4), or ulcerative colitis in remission (specimens 5 and 6). Specimens were pulse labelled with either (A) [³⁵S]-amino acids (two duplicate specimens per patient) or (B) [³⁵S]-sulphate (two duplicate specimens per patient) and chase incubated for four hours, followed by analysis of the homogenates of the tissue (lanes t) and of the media (lanes m) on 4% reducing SDS-PAGE. The radioactive signal of the mature MUC2 band, indicated by the right hand arrows, was quantified using a PhosphorImager, followed by exposure to x ray film (shown here). The left hand arrows denote the border between the running and stacking gel. Molecular mass markers of 600, 300, and 205 kDa are indicated on the left.

Figure 3

Secretion of total MUC2. All ³⁵S-labelled homogenates were dot blotted and incubated with an anti-MUC2 monoclonal antibody and ¹²⁵I-labelled protein A. The elicited ¹²⁵I signal was then quantified and the secreted percentage of total MUC2 was calculated. Average values were then calculated per patient from the quadruplicate values of the biopsy specimens, and the mean values per patient group were subsequently calculated. Each point represents the average value of one patient. Mean (SEM) value per patient group: controls (29.5 (1.41)%), active ulcerative colitis (UC) (28.5 (2.21)%) and ulcerative colitis in remission (29.4 (2.9)1%). Horizontal bars denote the mean of each patient group.

Figure 4

Secretion of [³⁵S]-amino acid labelled MUC2. [³⁵S]-amino acid labelled homogenates were analysed on reducing SDS-PAGE, and the MUC2 band was quantified. The secreted percentage of MUC2 was calculated per biopsy specimen. Average values were calculated per patient from the duplicate values of the specimens, and the mean values per patient group were determined. Each point represents the average value of one patient. Mean (SEM) values per patient group: controls (22.17 (2.55)%), active ulcerative colitis (UC) (23.08 (2.55)%) and ulcerative colitis in remission (23.47 (4.16)%). Horizontal bars denote the mean of each patient group.

Figure 5

Secretion of [³⁵S]-sulphate labelled MUC2. [³⁵S]-sulphate labelled homogenates were analysed on reducing SDS-PAGE, and the MUC2 band was quantified. The secreted percentage of MUC2 was calculated per biopsy specimen. Average values were calculated per patient from the duplicate values of the specimens, and the mean values per patient group were determined. Each point represents the average value of one patient. Mean (SEM) values per patient group: controls (16.23 (2.27)%), active ulcerative colitis (UC) (38.63 (4.3)%) and ulcerative colitis in remission (19.97 (2.27)%). Horizontal bars denote the mean of each patient group.

Figure 6

[³⁵S]-sulphate incorporation into MUC2. To determine [³⁵S]-sulphate incorporation into MUC2, the sum of the amounts of [³⁵S]-sulphate labelled MUC2 in tissue and media homogenates was expressed relative to the sum of the amounts of [³⁵S]-amino acid labelled MUC2 in tissue and media homogenates. Each point represents one patient. Mean (SEM) values per patient group: controls (57.35 (7.1)), active ulcerative colitis (UC) (31.04 (6.43)) and ulcerative colitis in remission (56.63 (9.28)). Horizontal bars denote the mean of each patient group.

Figure 7

[³⁵S]-sulphate content of secreted MUC2. To determine [³⁵S]-sulphate content of secreted MUC2, the amount of [³⁵S]-sulphate labelled MUC2 in the medium was expressed relative to the amount of [³⁵S]-amino acid labelled MUC2 in the medium. Each point represents one patient. Mean (SEM) values per patient group: controls (50.23 (10.33)), active ulcerative colitis (UC) (46.14 (12.59)) and ulcerative colitis in remission (55.91 (12.5)). Horizontal bars denote the mean of each patient group.