Biochemical Society Transactions (1 992) 20 95s Loss of sulphate in human during ulcerative colitis
colonic
mucinr
CORFIBLD, C. DO AMARAL CORFIELD, WAGNER, *B.F. WARREN, R.A. MOUNTFORD, +D.C.C. BARTOLO h J.R. C W .
a position well in excess of the marker (IgM).
990
KDa
A.P. S.A.
University Departments of Medicine, *Pathology and +Surgery, Bristol Royal Infirmary, Bristol, BS2 8HW. UK. The mucus glycoproteins synthesized by the colorectal mucosa are normally rich in carbohydrate 0-sulphate esters and sialic ac i ds giving these molecules a characteristic high charge density in addition to their high molecular weight [l]. Ulcerative colitis is an inflamnatory disease characterized histochemically by a reduction in goblet cell mucin staining for sulphate 121. No biochemical data has been presented to support or refute these histochemical data. We have compared the mucin synthesis and secretion in a series of patients with ulcerative colitis to that in histologically normal colon. Rectal tissue was placed in organ culture under standard condition8 [3] and incubated with 92 kBq sodium [ 5S]sulphate and 370 kBq [ HI-glucosamine. The viability of the cultured tissue was evaluated by [ 3H]-thymi dine incorporation over 24h and was found to be linear. The histology of tissue cultured for 24h was examined histologically and found to show no morpho 1 ogi ca 1 or other detectable abnormalities. After 24 h incubation at 37OC the secreted (medium) and cellular soluble fraction obtained after homogenization in lml phosphate buffered saline pH 7.0 containing protease inhibitors [3] were c llected. The rate of incorporation of [%]-glucosamine over 24h was linear for both secreted and cellular soluble fractions. The mucin rich excluded Vo fraction was prepared from these two soluble fractions by chromatography on columns (30 x lcm) of Sepharose CL 4B. The nature of the excluded Vo peak was examined to assess the proportion of non-mucus glycoprotein contaminants present. Samples were analysed by centrifugation in CsCl density gradients containing 4M-guanidine hydrochloride [41. Single peaks with a density of 1.41-1.45 were observed for all samples analysed. Digestion with pronase resulted in a limited reduction of molecular weight as fudged by elution of the product as a single included peak on Sepharose CL 4B. Incubation with hyaluronidase or chondroitinase ABC had no effect on the elution profile. Alkaline beta elimination r ulted in the comp ete removal of the [%]-sulphate and [ Hlglucosamine label to give an included peak on Sepharose CL 4B which was completely excluded when fractionated on BioGel P6 (100 x lcm) in O.1M-pyridine acetate pH 5.0. The two isotopes gave concomitant elution profiles on these two gel filtration media. SDS-PAGE was carried out on 3% gels 151 followed by autoradiography to assess the presence of contaminants and high molecular weight mucins. Low molecular weight contamination was negligible in secreted and cellular soluble fractions and normal samples analysed showed a smear migrating at
I
I
by Sepharose CL 4B is expressed as the ratio and dpmlug DNA for cellular and secreted fractions. INCORPORATION + SD ----__---
Cellular
ULCERATIVE COLITIS (n=7)
NORMAL (n=9)
P*
[35SII I3HI
0.31+0.23
1.93+0.79
colonic
mucinr
CORFIBLD, C. DO AMARAL CORFIELD, WAGNER, *B.F. WARREN, R.A. MOUNTFORD, +D.C.C. BARTOLO h J.R. C W .
a position well in excess of the marker (IgM).
990
KDa
A.P. S.A.
University Departments of Medicine, *Pathology and +Surgery, Bristol Royal Infirmary, Bristol, BS2 8HW. UK. The mucus glycoproteins synthesized by the colorectal mucosa are normally rich in carbohydrate 0-sulphate esters and sialic ac i ds giving these molecules a characteristic high charge density in addition to their high molecular weight [l]. Ulcerative colitis is an inflamnatory disease characterized histochemically by a reduction in goblet cell mucin staining for sulphate 121. No biochemical data has been presented to support or refute these histochemical data. We have compared the mucin synthesis and secretion in a series of patients with ulcerative colitis to that in histologically normal colon. Rectal tissue was placed in organ culture under standard condition8 [3] and incubated with 92 kBq sodium [ 5S]sulphate and 370 kBq [ HI-glucosamine. The viability of the cultured tissue was evaluated by [ 3H]-thymi dine incorporation over 24h and was found to be linear. The histology of tissue cultured for 24h was examined histologically and found to show no morpho 1 ogi ca 1 or other detectable abnormalities. After 24 h incubation at 37OC the secreted (medium) and cellular soluble fraction obtained after homogenization in lml phosphate buffered saline pH 7.0 containing protease inhibitors [3] were c llected. The rate of incorporation of [%]-glucosamine over 24h was linear for both secreted and cellular soluble fractions. The mucin rich excluded Vo fraction was prepared from these two soluble fractions by chromatography on columns (30 x lcm) of Sepharose CL 4B. The nature of the excluded Vo peak was examined to assess the proportion of non-mucus glycoprotein contaminants present. Samples were analysed by centrifugation in CsCl density gradients containing 4M-guanidine hydrochloride [41. Single peaks with a density of 1.41-1.45 were observed for all samples analysed. Digestion with pronase resulted in a limited reduction of molecular weight as fudged by elution of the product as a single included peak on Sepharose CL 4B. Incubation with hyaluronidase or chondroitinase ABC had no effect on the elution profile. Alkaline beta elimination r ulted in the comp ete removal of the [%]-sulphate and [ Hlglucosamine label to give an included peak on Sepharose CL 4B which was completely excluded when fractionated on BioGel P6 (100 x lcm) in O.1M-pyridine acetate pH 5.0. The two isotopes gave concomitant elution profiles on these two gel filtration media. SDS-PAGE was carried out on 3% gels 151 followed by autoradiography to assess the presence of contaminants and high molecular weight mucins. Low molecular weight contamination was negligible in secreted and cellular soluble fractions and normal samples analysed showed a smear migrating at
I
I
by Sepharose CL 4B is expressed as the ratio and dpmlug DNA for cellular and secreted fractions. INCORPORATION + SD ----__---
Cellular
ULCERATIVE COLITIS (n=7)
NORMAL (n=9)
P*
[35SII I3HI
0.31+0.23
1.93+0.79
