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The partial characterization of the epithelial glycoprotein from normal and diseased colons Tsang, Wai-Chiu 1975

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THE PARTIAL CHARACTERIZATION OF THE EPITHELIAL GLYCOPROTEIN FROM NORMAL AND DISEASED COLONS by WAI-CHIU TSANG B.Sc. U n i v e r s i t y of B r i t i s h Columbia A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department o f PATHOLOGY We ac c e p t t h i s t h e s i s as conforming t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH A p r i l , 1975. COLUMBIA In present ing th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ive rs i t y of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f r ee ly a v a i l a b l e for reference and study. I fur ther agree that permission for extensive copying of th is thes is for s c h o l a r l y purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or p u b l i c a t i o n of th is thes is fo r f i n a n c i a l gain sha l l not be allowed without my wr i t ten permiss ion . Department of PATHOLOGY The Un ivers i ty of B r i t i s h Columbia Vancouver 8, Canada Date February, 1975 i i ABSTRACT T h i s study r e p o r t s the i s o l a t i o n , f r a c t i o n a t i o n and chemical c h a r a c t e r i z a t i o n of a p p a r e n t l y homogenous and undegraded e p i t h e l i a l g l y c o p r o t e i n s from normal human co l o n s and from p a t i e n t s w i t h u l c e r a t i v e c o l i t i s , Crohn's d i s e a s e and ischaemic c o l i t i s of the c o l o n . C o l o n i c e p i t h e l i a l c e l l s were removed from each specimen by shaking w i t h EDTA and the g l y c o -p r o t e i n s were e x t r a c t e d ' f r o m the sonated c e l l s w i t h 1M NaCl. Combined agarose g e l and DEAE c e l l u l o s e chromatography of the pooled crude e x t r a c t from each normal and d i s e a s e d sample y i e l d e d two a p p a r e n t l y homogeneous f r a c t i o n s A and B. Agarose g e l e l e c t r o p h o r e s i s r e v e a l e d t h a t the m o b i l i t y o f the second DEAE f r a c t i o n (B) was about the same as t h a t o f h e p a r i n and twice as f a s t as t h a t o f f r a c t i o n A. A p p l i c a t i o n o f gas-l i q u i d chromatography, UV-spectroscopy, agarose g e l e l e c t r o -p h o r e s i s a t pH 3.0, c o l o r i m e t r i c hexose a n a l y s i s , phosphorus and Indole assays suggested t h a t f r a c t i o n B was DNA. F r a c t i o n A g l y c o p r o t e i n s appeared t o be homogeneous by DEAE chromatography, agarose g e l and c e l l u l o s e a c e t a t e e l e c t r o -phoreses a t pH 8.8. Chemical analyses of these g l y c o p r o t e i n s r e v e a l e d the presence of fucose, g a l a c t o s e , hexosamine, s i a l i c a c i d , p r o t e i n and l i t t l e or no phosphorus. The molar r a t i o o f the carbohydrate components of each sample was s i g n i f i c a n t l y i i i d i f f e r e n t from t h a t of the normal, and i t v a r i e d among the d i f f e r e n t d i s e a s e s . In each of the g l y c o p r o t e i n s s t u d i e d , 4 d i f f e r e n t types o f s i a l i c a c i d s were found i n d i f f e r e n t q u a n t i t i e s and they were: ( 1 ) u n s u b s t i t u t e d or C9 s u b s t i t u t e d s i a l i c a c i d s , ( i i ) s i a l i c a c i d s s u b s t i t u t e d at C4 and or a t C9, ( i i i ) s i a l i c a c i d s s u b s t i t u t e d a t C7 and or C8, (iv) s i a l i c a c i d s s u b s t i t u t e d at C4 and a t C7 and or C8. Gas-l i q u i d chromatography r e v e a l e d t h a t these s u b s t i t u e n t s were a l k a l i n e - l a b i l e O - a c e t y l groups. The s i g n i f i c a n c e o f the above d i f f e r e n c e s were d i s c u s s e d . u-pc I 1. INTRODUCTION 1 1. L i t e r a t u r e Review. A. Chemistry of the i n t e s t i n a l g l y c o -p r o t e i n s . B. The involvement of g l y c o p r o t e i n s i n u l c e r a t i v e c o l i t i s . C. The involvement of g l y c o p r o t e i n s i n Crohn's d i s e a s e . 2. The Pre s e n t I n v e s t i g a t i o n I I . MATERIALS AND METHODS 17 1. P r e p a r a t i o n of Samples f o r GLC and C o l o r -i m e t r i c a n a l y s e s . 2. Gas L i q u i d Chromatographic A n a l y s e s , A. N e u t r a l sugars, B. O - a c e t y l groups. 3. C o l o r i m e t r i c A n a l y s e s . 4. E l e c t r o p h o r e s i s S t u d i e s . 5. P r e l i m i n a r y Experiments on the I s o l a t i o n of Rat C o l o n i c E p i t h e l i a l G l y c o p r o t e i n s . A. I s o l a t i o n o f c o l o n i c e p i t h e l i a l c e l l s . B. E x t r a c t i o n of c o l o n i c g l y c o p r o t e i n s . C. F r a c t i o n a t i o n o f c o l o n i c g l y c o p r o t e i n s . 6. I s o l a t i o n o f Human C o l o n i c E p i t h e l i a l G l y c o p r o t e i n s . A. Source of specimens. B. I s o l a t i o n o f mucosal c e l l s from human c o l o n . C. E x t r a c t i o n and f r a c t i o n a t i o n o f c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s . 7. S a p o n i f i c a t i o n S t u d i e s . 8. D i g e s t i o n S t u d i e s w i t h Neuraminidase. 9. Immunodiffusion S t u d i e s . 10. P r e p a r a t i o n o f A n t i s e r a . V page I I I . RESULTS AND DISCUSSION 33 1. I s o l a t i o n o f E p i t h e l i a l C e l l s . 2. E x t r a c t i o n o f C o l o n i c E p i t h e l i a l G l y c o p r o t e i n s . 3. F r a c t i o n a t i o n o f C o l o n i c E p i t h e l i a l G l y c o p r o t e i n s . A. P r e l i m i n a r y model experiments w i t h r a t g l y c o p r o t e i n s . B. Experiments on human c o l o n i c e p i t h e l i a l c e l l s . 4. Blood Contamination. 5. Analyses o f F r a c t i o n s B. 6. Analyses of F r a c t i o n s A. 7. S a p o n i f i c a t i o n S t u d i e s . 8. D i g e s t i o n Study w i t h V i b r i o C h o l e r a Neuraminidase. 9. GLC an a l y s e s o f O - a c e t y l groups. 10. General D i s c u s s i o n , 11. F u r t h e r Work Proposed. IV. BIBLIOGRAPHY 68 APPENDIX 72 v i LIST OF TABLES Page. 1. Percentage r e c o v e r y o f hexose and s i a l i c a c i d 35 a f t e r c o n c e n t r a t i o n of the 105,000g supernatant of i n t e s t i n a l e p i t h e l i a l g l y c o p r o t e i n by the v a r i o u s p r o c e d u r e s . 2. E l e c t r o p h o r e t i c m o b i l i t i e s o f the samples ob- 41 t a i n e d from the v a r i o u s stages i n the f r a c t i o n -a t i o n o f c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s ex-t r a c t e d from normal and d i s e a s e d human c o l o n s . 3. Chemical a n a l y s e s o f F r a c t i o n s B. 44 4. Molar r a t i o s of sugars i n c o l o n i c e p i t h e l i a l 45 g l y c o p r o t e i n s ( F r a c t i o n s A ) . 5. Chemical a n a l y s e s of c o l o n i c e p i t h e l i a l g l y c o - 46 p r o t e i n s ( F r a c t i o n s A ) . 6. Percentage s i d e c h a i n s u b s t i t u t i o n i n the s i a l i c 51 a c i d s o f c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s . 7. S u b s t i t u t i o n p a t t e r n s o f s i a l i c a c i d s i n c o l o n i c 55 e p i t h e l i a l g l y c o p r o t e i n s . 8. Molar r a t i o s o f sugars found i n c o l o n i c e p i t h e l i a l 61 g l y c o p r o t e i n s expressed as percen t a g e s . v i i LIST OF FIGURES page 1. Flow diagram of the method used for the i s o l a t i o n 20 of i n t a c t ra t co lon ic e p i t h e l i a l c e l l s . 2. Flow diagram of the method for the ex t rac t ion of 22 g lycoprote in from ra t c o l o n i c e p i t h e l i a l c e l l s . 3. Flow diagram of the method for the f r a c t i o n a t i o n 23 or r a t co lon ic e p i t h e l i a l g lycoprote ins . 4. Flow diagram of the procedure for the d ige s t ion 28 studies of co lon ic e p i t h e l i a l g lycoprote ins of r a t and human using v i b r i o cholera neuraminidase. 5. Photomicrographs of sect ions from untreated and 31 EDTA-treated human co lon . 6. Ce l lu lo se acetate e lec t rophores i s of ra t c o l o n i c 36 e p i t h e l i a l g lycoprote ins at pH 8.8. Procedure used to monitor the f r a c t i o n a t i o n procedure. 7. Agarose A15M column chromatography of the 105,000g . 38 supernatant extracted from the i s o l a t e d c o l o n i c e p i t h e l i a l c e l l s of a human sample resected for carcinoma of the co lon . 8. DEAE c e l l u l o s e ion-exchange column chromatography 40 of the carbohydrate-r ich f r a c t i o n A l obtained from agarose A15M column chromatography of human co lon ic c e l l sap obtained from a sample resected for carcinoma of the c o l o n . 9. I l l u s t r a t i o n of a t y p i c a l agarose ge l e l e c t r o - 42 phoresis pat tern of the various f rac t ions i n the f r a c t i o n a t i o n of the 105,000g supernatant of a human sample. 10. G a s - l i q u i d chromatograph of the t r i m e t h y l s i l y l 47 ethers of the neutra l sugars of co lon ic e p i t h e l i a l g lycoprote in extracted from the h i s t o l o g i c a l l y normal part of s u r g i c a l carcinoma specimens obtained from pat ients undergoing colectomy. 11. I l l u s t r a t i o n of a t y p i c a l s apon i f i ca t ion time curve 49. of human co lon ic e p i t h e l i a l g l y c o p r o t e i n . ACKNOWLEDGEMENTS The author wishes to thank h i s s u p e r v i s o r Dr. P. Reid f o r h i s guidance and encouragement over the d u r a t i o n of t h i s work. In a d d i t i o n , thanks are extended to members o f h i s committee, Mr. C.F.C. C u l l i n g , Dr.G. G. S. Dutton, Dr. S. French and Dr. P. Va s s a r . The author i s indebted to Mr. C. Ramey f o r h i s a s s i s t a n c e w i t h some o f the chemical analyses and i n p r o o f - r e a d i n g t h i s t h e s i s , to the s t a f f s o f the h i s t o l o g y l a b o r a t o r y i n the Department o f Pathology f o r the p r e p a r a t i o n of h i s t o l o g i c a l s e c t i o n s , and to o t h e r members o f the F a c u l t y and s t a f f o f the Department of Pathology f o r t h e i r c o n s t a n t c o - o p e r a t i o n . F i n a n c i a l support was p r o v i d e d by the M e d i c a l Research C o u n c i l o f Canada i n the form o f a s t u d e n t s h i p . X INTRODUCTION The r e s e a r c h d e s c r i b e d i n t h i s t h e s i s concerned the i s o l a t i o n and c h a r a c t e r i z a t i o n of e p i t h e l i a l g l y c o -p r o t e i n s from normal and d i s e a s e d human c o l o n s . Chemical and h i s t o c h e m i c a l evidence suggested t h a t g l y c o p r o t e i n s 1 2 p l a y a r o l e i n i n t e s t i n a l d i s e a s e s ' and i t was f e l t t h a t such a study c o u l d c o n t r i b u t e to a b e t t e r u n d e r s t a n d i n g of the e t i o l o g y and pathogenesis o f c o l o n i c d i s e a s e s p a r t i -c u l a r l y u l c e r a t i v e c o l i t i s and Crohn's d i s e a s e . The chemistry of g l y c o p r o t e i n s has been extensive^-3 l y reviewed by G o t t s c h a l k . G l y c o p r o t e i n s are b e s t d e f i n e d as "conjugated p r o t e i n s c o n t a i n i n g as p r o s t h e t i c group (s) one or more h e t e r o s a c c h a r i d e ( s ) , the l a t t e r are u s u a l l y branched, w i t h a r e l a t i v e l y low number o f sugar r e s i d u e s , l a c k i n g a s e r i a l l y r e p e a t i n g u n i t and bound c o v a l e n t l y t o 4 the p o l y p e p t i d e c h a i n . A major p o r t i o n o f the carbohydrate i n animal t i s s u e s i s found i n conjugated form w i t h p r o t e i n s . G l y c o p r o t e i n s are a d i v e r s e group of compounds s i n c e the c o v a l e n t a s s o c i a t i o n o f carbohydrate w i t h p r o t e i n occurs i n a , wide range of p r o t e i n t ypes. While they have no unique amino a c i d composition, they do c o n t a i n a c h a r a c t e r i s t i c group of sugars t h a t i n c l u d e D - g a l a c t o s e , D-mannose, D-glucose, L - f u c o s e , D-xylose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine and v a r i o u s d e r i v a t i v e s o f neuraminic a c i d (the s i a l i c a c i d s ) . The carbohydrate content of these g l y c o p r o t e i n s may vary from 2 l e s s than one p e r c e n t to more than e i g h t p e r c e n t o f the weight o f the molecule, and as many as seven sugar types may be p r e s e n t i n any gi v e n p r o t e i n . " There i s c o n s i d e r a b l e c o n f u s i o n i n the t e r m i n o l o g y used t o d e s c r i b e macromolecules whose composition i s i n acco r d w i t h t h i s d e f i n i t i o n . Terms t h a t have been employed i n c l u d e : mucin, mucoprotein, mucopolysaccharide, mucosubstance, s u l -phomucin, fucomucin, s i a l o m u c i n , carboxymucin, a c i d muco-p o l y s a c c h a r i d e , g lycosaminoglycan and g l y c o p r o t e i n . In t h i s t h e s i s , o n l y two terms w i l l be used t o d e s c r i b e these types of macromolecules - (a) g l y c o p r o t e i n and Cb) g l y c o s a m i n o g l y c a n . Any macromolecules which c o n t a i n s one or more o f the f o l l o w i n g carbohydrate polymers: h y a l u r o n i c a c i d , h e p a r i n s u l p h a t e , k e r a -tan s u l p h a t e , dermatan sulphate and c h r o n d r o i t i n 4 and 6 s u l p h a t e w i l l be termed glycosaminoglycans; a l l o t h e r macromolecules w i l l be d e s i g n a t e d g l y c o p r o t e i n s . A c c o r d i n g t o t h i s c l a s s i f i c a t i o n g lycosaminoglycans (with the e x c e p t i o n o f k e r a t a n sulphate) c o n t a i n u r o n i c a c i d as a major carbohydrate c o n s t i t u e n t w h i l e g l y c o p r o t e i n s n o r m a l l y c o n t a i n s i a l i c a c i d . Glycosaminoglycans are mainly found i n c o n n e c t i v e t i s s u e s w h i l e the g l y c o p r o t e i n s d i s c u s s e d here are assumed t o be of e p i t h e l i a l o r i g i n . U l c e r a t i v e c o l i t i s i s a d i s e a s e o f unknown e t i o l o g y . I t i s a d i f f u s e , n o n - s p e c i f i c inflammatory c o n d i t i o n o f the 3. mucous membrane of the l a r g e i n t e s t i n e . The inflammation i s of an exudative and v a s c u l a r type and g e n e r a l l y u n p r o d u c t i v e of g r a n u l a t i o n t i s s u e or f i b r o s i s . Although t h i s d i s e a s e can occur f o r the f i r s t time a t any age, the peak age of i n c i d e n c e f o r e i t h e r sex on f i r s t attendance at h o s p i t a l i s i n the t h i r d decade. I t has been e s t i m a t e d t h a t the r i s k of cancer i n p a t i e n t s w i t h u l c e r a t i v e c o l i t i s i s between f i v e and ten times g r e a t e r than the r i s k i n the g e n e r a l p o p u l a t i o n ^'^. Changes i n the e p i t h e l i a l g l y c o p r o t e i n s have been demonstrated both h i s t o c h e m i c a l l y and c h e m i c a l l y i n t h i s d i s e a s e ^ , but whether these changes are a cause or a consequence o f the d i s e a s e i s unknown. Chemistry o f the I n t e s t i n a l G l y c o p r o t e i n s V i s c o u s s e c r e t i o n s of the e p i t h e l i a l c e l l s are found throughout the g a s t r o i n t e s t i n a l tract"'"''". These s e c r e t i o n s e x i s t as h i g h l y hydrated g e l s the apparent f u n c t i o n s of which are to l u b r i c a t e the mucous membrane, t o p r o t e c t i t from mechanical and 12 chemical damage and from a t t a c k by enzymes and b a c t e r i a C h e m i c a l l y they are g l y c o p r o t e i n s . I t i s s p e c u l a t e d t h a t the carbohydrate moiety forms a p h y s i c a l b a r r i e r which prevents pro-t e o l y s i s o f the p r o t e i n core (3). Thus, the i n t e g r i t y of the carbohydrate moiety may be e s s e n t i a l f o r the s u r v i v a l o f the g l y c o p r o t e i n , which i n t u r n imparts p r o t e c t i o n t o the mucosal e p i t h e l i a l c e l l s . 4 There have been s u r p r i s i n g l y few s t u d i e s of i n t e s t i n a l g l y c o p r o t e i n s . The procedures employed i n the i s o l a t i o n and f r a c t i o n a t i o n o f these g l y c o p r o t e i n s have been reviewed by 3 JL 3 14 1 2 G o t t s c h a l k , Marshal , Horowitz , and S p i r o ' . Three types of s t a r t i n g m a t e r i a l have been employed f o r the e x t r a c t i o n o f 11 15-22 these g l y c o p r o t e i n s : (a) mucosal s c r a p i n g s ' , (b) minced 2 3 2 4 2 5 2 6 co l o n ' and (c) r e c t a l i r r i g a t e s ' . The use of such s t a r t i n g m a t e r i a l s make i t u n l i k e l y t h a t compounds of p u r e l y e p i t h e l i a l o r i g i n were i s o l a t e d s i n c e s c r a p i n g s and minced organs may c o n t a i n c o n n e c t i v e t i s s u e contaminants such as the 15 16 20 22 glycosaminoglycans found i n some s t u d i e s ' ' , w h i l e i r r i g a t e s c o u l d be contaminated w i t h the products of f a e c a l stream b a c t e r i a and enzymatic d e g r a d a t i o n o f g l y c o p r o t e i n s . In many cases, n a t i v e g l y c o p r o t e i n s were not o b t a i n e d because p r o t e o l y s i s was used i n the i s o l a t i o n procedure. Thus, i t i s d i f f i c u l t i f not i m p o s s i b l e t o r e l a t e any of the s t r u c t u r a l data o b t a i n e d t o a s p e c i f i c g l y c o p r o t e i n . Chemical analyses o f the i n t e s t i n a l g l y c o p r o t e i n s * i • 9,11,15-17,23 . , - . . of s e v e r a l s p e c i e s r e v e a l e d the presence of p r o t e i n , s i a l i c a c i d , f u c o s e , hexosamine and g a l a c t o s e . Only i n the case o f sheep c o l o n i c mucin ^was an attempt made to perform a d e t a i l e d s t r u c t u r a l a n a l y s i s . A n a l y t i c a l data o f l i m i t e d scope was r e p o r t e d i n the o t h e r s t u d i e s . Thus, very l i t t l e i s known about the chemical and macromolecular s t r u c t u r e o f the i n t e s t i n a l g l y c o p r o t e i n s o f man and animals. 3 Animal G l y c o p r o t e i n S t u d i e s 15 Werner s t u d i e d the composition o f washed mucosal s c r a p i n g s from g a s t r o - i n t e s t i n a l t i s s u e s o f the p i g . G l y c o -p r o t e i n s were shown to be a major component of the v a r i o u s mucins. On the b a s i s of the a n a l y t i c a l d a t a , Werner p o s t u -l a t e d the e x i s t e n c e of two d i f f e r e n t i n t e s t i n a l g l y c o p r o t e i n s , one r i c h i n f u c o s e , and the o t h e r r i c h i n s i a l i c a c i d . Inoue and Yosizawa"^ examined the g l y c o p e p t i d e s o b t a i n e d by the d i g e s t i o n of p i g c o l o n i c mucosal s c r a p i n g s w i t h pronase. A l l the f r a c t i o n s o b t a i n e d were a c i d i c and c o n t a i n e d O-sulphate e s t e r . They c o u l d be s e p a r a t e d i n t o two groups: g l y c o p e p t i d e s and g l y c o s a m i n o g l y c a n s . The g l y c o -5 p e p t i d e s -(molecular weight 2-3 X 10 ) c o n t a i n e d g a l a c t o s e , f u c o s e , glucosamine, galactosamine, s i a l i c a c i d and s u l p h a t e . The glycosaminoglycan f r a c t i o n s , p r e s e n t i n much s m a l l e r amounts than the g l y c o p e p t i d e s , c o n t a i n e d h e p a r i n s u l p h a t e , h e p a r i n and c h o n d r o i t i n s u l p h a t e . I t was assumed t h a t the glycosaminoglycans r e p r e s e n t e d c o n n e c t i v e t i s s u e contaminants. 17 Nemoto and Yosizawa o b t a i n e d s i m i l a r mixed f r a c t i o n s from pronase d i g e s t s of mucosal s c r a p i n g s o f r a b b i t c o l o n and s m a l l i n t e s t i n e . Glycosaminoglycans have a l s o been found i n mucosal s c r a p i n g s of dog c o l o n 6 Kent e t a l have c a r r i e d out d e t a i l e d s t u d i e s on the water s o l u b l e g l y c o p r o t e i n s o b t a i n e d from s c r a p i n g s o f sheep c o l o n i c mucosa. Four macromolecular f r a c t i o n s were found, w i t h s e d i m e n t a t i o n c o e f f i c i e n t s of 9.58, 6.1, 3.25 and 2.15 r e s p e c t i v e l y . The major f r a c t i o n (9.58S) was a s i a l i c a c i d - c o n t a i n i n g g l y c o p r o t e i n w i t h a m o l e c u l a r weight o f about 2 X 10^. I n c u b a t i o n of t h i s f r a c t i o n w i t h papain r e s u l t e d i n a r a p i d r e d u c t i o n i n v i s c o s i t y without'any l o s s of d i a l y s a b l e , c arbohydrate, and the p r o d u c t i o n o f a p a r t i a l l y degraded 5 g l y c o p r o t e i n of m o l e c u l a r weight of 1 X 10 . The second f r a -c t i o n (6.IS) was s u b s t a n t i a l l y f r e e of s i a l i c a c i d , and c o u l d be degraded by papain t o a 1.7S f r a c t i o n (molecular weight 2 X 4 10 ). The remaining two f r a c t i o n s (3.2S and 2.15S) c o n t a i n e d 2 7 28 m ainly n u c l e o p r o t e i n s . I n subsequent s t u d i e s ' an i s o t o p e l a b e l l e d 9.58S g l y c o p r o t e i n was i s o l a t e d from s c r a p i n g s o f sheep c o l o n i c mucosal t i s s u e which had been i n c u b a t e d i n Krebs-Ringer b u f f e r f o r 4 hours i n the presence of oxygen and such m e t a b o l i t e s as D-(1- 1 4C) and D-(2- 1 4C) g l u c o s e , L - (U- 1 4C) 35 2-t h r e o n m e and SO^ . N e i t h e r the 6. IS g l y c o p r o t e i n nor the two n u c l e o p r p t e i n f r a c t i o n s were l a b e l l e d under these c o n d i t i o n s . Chemical a n a l y s i s of the 9.58S g l y c o p r o t e i n showed t h a t i t c o n t a i n e d 25% p r o t e i n , 72% carbohydrate and 4.6% e s t e r s u l p h a t e . The s i a l i c a c i d r e s i d u e s of the f r a c t i o n , r e p r e s e n t i n g 13.7% of the g l y c o p r o t e i n , were made up o f 6 3% N - g l y c o l l y l n e u r a m i n i c a c i d and 37% N - a c e t y l n e u r a m i n i c a c i d . The ease w i t h which these r e s i d u e s c o u l d be h y d r o l y z e d by d i l u t e a c i d s or neuraminidase i m p l i e d t h a t they o c c u p i e d t e r m i n a l p o s i t i o n s i n a "branched 7. o l i g o s a c c h a r i d e s t r u c t u r e v . S e q u e n t i a l "Smith d e g r a d a t i o n " 35 of the S - l a b e l l e d 9.58S g l y c o p r o t e i n i n d i c a t e d t h a t t h i s g l y c o p r o t e i n had a p e p t i d e "backbone" t o which an a r r a y of o l i g o s a c c h a r i d e s ( s i a l i c a c i d - g a l a c t o s e - a m i n o sugar o r fucose galactose-amino sugar) were a t t a c h e d , p r o b a b l y through B-aspartamido l i n k a g e s . Some of the amino sugar r e s i d u e s ad-j a c e n t t o the p e p t i d e c h a i n were s u l p h a t e d . A g l y c o p r o t e i n c o n t a i n i n g s u l p h a t e and s i a l i c a c i d was e x t r a c t e d from mucosal s c r a p i n g s o f r a t s m a l l i n t e s t i n e 19 w i t h i s o t o n i c s a l i n e . Chromatography of t h i s s a l t - s o l u b l e g l y c o p r o t e i n on a DEAE c e l l u l o s e column gave th r e e f r a c t i o n s . The major f r a c t i o n was c a r b o h y d r a t e - r i c h and c o n t a i n e d s u l -phate. On u l t r a c e n t r i f u g a t i o n and d i s c g e l e l e c t r o p h o r e s i s t h i s f r a c t i o n was r e s o l v e d i n t o two components, which c o u l d be p a r t i a l l y s e p a r a t e d by Sepharose-4B chromatography. Both s u b f r a c t i o n s had the same chemic a l composition as the parent m i x ture. Galactosamine, glucosamine, f u c o s e , g a l a c t o s e and s i a l i c a c i d were p r e s e n t i n a l l f r a c t i o n s . The amount of sul p h a t e d e t e c t e d was 2.4%, 40% o f which was a c i d l a b i l e . Both s i a l i c a c i d and fucose were r a p i d l y r e l e a s e d by a c i d h y d r o l y s i s s u g g e s t i n g t h a t they o c c u p i e d t e r m i n a l p o s i t i o n s . Threonine, s e r i n e and p r o l i n e made up 69% o f the t o t a l amino a c i d r e s i d u e s . The r e s u l t s o b t a i n e d from a l k a l i n e b o r o h y d r i d e treatment o f the g l y c o p r o t e i n s t r o n g l y suggested t h a t galactosamine r e s i d u e s were l i n k e d t o the p e p t i d e through O - g l y c o s i d i c l i n k a g e s i n v o l v i n g s e r i n e and t h r e o n i n e r e s i d u e s . F o r s t n e r e t a l ^ u i s o l a t e d a h i g h - m o l e c u l a r -weight g l y c o p r o t e i n (HMG) from mucosal s c r a p i n g s of r a t s m a l l i n t e s t i n e . A n t i b o d i e s a g a i n s t HMG were prepared i n r a b b i t s and the f l u o r e s c e i n - l a b e l l e d a n t i s e r a so o b t a i n e d was found to s t a i n the s u p r a n u c l e a r mucous v e s i c l e s of g o b l e t c e l l s i n the body of the stomach and mucus-producing c e l l s i n the s u b l i n g u a l g l a n d . I t was concluded t h a t HMG was a major component of r a t s m a l l i n t e s t i n e g o b l e t - c e l l 21 mucus. In a l a t e r study , HMG was found to be a n e g a t i v e l y charged macromolecule w i t h a m o l e c u l a r weight of about 2 X 10 and c o n t a i n e d 12% p r o t e i n , 23% hexose, 22.4% hexosamine, 10% s i a l i c a c i d , 6.6% fucose and l e s s than 1% s u l p h a t e . One major and two minor components were i d e n t i f i e d by acrylamide d i s c g e l e l e c t r o p h o r e s i s and by a n a l y t i c a l u l t r a c e n t r i f u g a t i o n . Human G l y c o p r o t e i n S t u d i e s 25 R o e l f e t a l , have examined human r e c t a l i r r i g a t e s . Gas chromatographic a n a l y s i s of h y d r o l y s a t e s o f the i r r i g a t e s r e v e a l e d the presence of f u c o s e , mannosamine, galactosamine, glucosamine, N - a c e t y l - n e u r a m i n i c a c i d and N - a c e t y l - g a l a c t o s a m i n e . The presence o f mannosamine was thought to r e p r e s e n t the e p i -m e r i z a t i o n o f glucosamine as a consequence of h e a t i n g w i t h p y r i d i n e . y Two r e c t a l mucous plugs , obtained from c h i l d r e n with c y s t i c f i b r o s i s of the pancreas and r e l a t i v e l y uncon-taminated by mater ia l s such as faeces and b lood , were 2 6 examined by Johansen . The samples were inso lub le i n a wide v a r i e t y of solvents i n c l u d i n g water, 3,6, and 9 M urea, formamide, saturated calcium c h l o r i d e , ethanol (9:1 v / v ) , 0.1 M cys te ine , s a l t so lut ions and mineral ac ids . They d i s so lved slowly i n 0.1 M sodium hydroxide. Both samples contained ga lactose , fucose, s i a l i c ac id and N-acetylglucosamine. One sample contained traces of mannose. Uronic ac id was not found i n e i t h e r sample. S i a l i c ac id appeared to occupy a terminal p o s i t i o n , as i t could be removed from the o l i g o -saccharide chain by v i b r i o cholera neuraminidase. Both g lycoprote ins were found to be r e s i s t a n t to t r y p s i n , chrymotrypsin, pepsin and lysozyme, but could be dispersed with papain, 23 Sky-Peck et a l have i s o l a t e d glycopeptides and glycosaminoglycans from p r o t e o l y t i c digests of whole human co lon . Human colons were minced and the crude homogenate was incubated with papain at 6 0 ° C for 24 hours. Treatment of the incubate with 1% ce ty lpyr id in ium c h l o r i d e , (CPC), to pre-c i p i t a t e the glycosaminoglycans present , y i e lded a CPC complex and a supernatant. Treatment of the mater ia l obtained from the CPC complex with t e s t i c u l a r hyaluronidase followed by f r a c t i o n -at ion of the digest by a combination of B ioge l P30 and DEAE 1( c e l l u l o s e chromatography y i e lded a major hexose-r ich f r a c t i o n free of uronic a c i d . This material ' appeared to be homogenous by u l t r a c e n t r i f u g a t i o n and free boundary e lec t rophores i s at pH 8.4 and at pH 4.6, Chemical analys i s revealed the presence of hexose, s i a l i c a c i d , hexosamine and fucose. The supernatant from the CPC p r e c i p i t a t i o n contained hexose. On f r a c t i o n a t i o n on DEAE c e l l u l o s e chromatography i t y i e lded three d i s t i n c t f r a c t i o n s . Galactose, mannose, hexosamine, fucose and s i a l i c ac id were present i n each of these f r a c t i o n s . Hexuronic ac id could not be detected. In two of the f rac t ions hexosamine was present only as glucosamine while the t h i r d contained only galactosamine. 22 Korhonen and Makela removed human co lon ic mucosa at the l e v e l of the lamina muscularis mucosae. The t i s sue obtained was d r i e d at 6 0 ° C for 24 hours , homogenized, and then heated for 20 minutes i n a b o i l i n g water bath . Pronase d i -gest ion of the heated homogenate was c a r r i e d out at 6 0 ° for 48 hours and the glycopeptides obtained were extracted with t r i -ch loroace t i c a c i d . E lec t rophores i s of the extract revealed the presence of four components, three of which contained s u l -phate. A l l these glycopeptides contained p r o t e i n , hexose, fucose, hexosamine, s i a l i c ac id and uronic a c i d . The hexuronic ac id was thought to be present i n the c o l o n i c mucosa as a g lycoprote in component and not as a connective t i s sue contaminant. Kawaski e t a l ^ e x t r a c t e d g l y c o p r o t e i n s from human c o l o n w i t h e t h y l e n e g l y c o l . The major g l y c o p r o t e i n o b t a i n e d c o n t a i n e d as much as 80% carbohydrate, composed of glucosamine, galactosamine, g a l a c t o s e , f u c o s e , s i a l i c a c i d and e s t e r s u l p h a t e . Threonine, s e r i n e and p r o l i n e were the predominant amino a c i d s . Immunofluorescent s t u d i e s , u s i n g a n t i s e r a a g a i n s t these g l y c o p r o t e i n s were s a i d t o show t h a t they were a n t i -g e n i c a l l y s i m i l a r t o g l y c o p r o t e i n s i n " i n t e s t i n a l i s e d g a s t r i c mucosa" but were u n r e a c t i v e towards normal g a s t r i c g l a n d s . The Involvement of G l y c o p r o t e i n s i n U l c e r a t i v e C o l i t i s Changes i n the p a t t e r n s of e p i t h e l i a l g l y c o p r o t e i n s a s s o c i a t e d w i t h u l c e r a t i v e c o l i t i s have been demonstrated by both chem i c a l and h i s t o c h e m i c a l methods. A d e t a i l e d knowledge of the normal and p a t h o l o g i c a l d i s t r i b u t i o n o f these d i f f e r e n t types o f g l y c o p r o t e i n s i s an important p r e r e q u i s i t e f o r any attempt t o e l u c i d a t e t h e i r b i o l o g i c a l s i g n i f i c a n c e . Although h i s t o c h e m i c a l methods can be presumed t o show the presence and l o c a t i o n o f some of these g l y c o p r o t e i n s i n a t i s s u e , the ex a c t c h e m i c a l nature o f such g l y c o p r o t e i n s cannot be determined 23 by h i s t o c h e m i s t r y H i s t o c h e m i c a l S t u d i e s The normal p a t t e r n o f the e p i t h e l i a l g l y c o p r o t e i n s i n the c o l o n and rectum of man, as r e v e a l e d by h i s t o c h e m i c a l methods, has been d e s c r i b e d by s e v e r a l i n v e s t i g a t o r s ' There i s g e n e r a l agreement t h a t both s u l p h a t e d and s i a l i c a c i d c o n t a i n i n g g l y c o p r o t e i n s are p r e s e n t i n the c o l o n i c mucosa of man but t h e r e i s some d i f f e r e n c e i n o p i n i o n r e g a r d i n g the d i s t r i b u t i o n o f these m a t e r i a l s between the s u r f a c e e p i t h e l i u m 3 6 and the upper and lower h a l v e s o f the c r y p t s . Changes i n the p a t t e r n of the e p i t h e l i a l g l y c o p r o t e i n s are a s s o c i a t e d 7 8 w i t h u l c e r a t i v e c o l i t i s ' , but th e r e i s no g e n e r a l agreement as t o the nature o f these changes o t h e r than t h a t t h e r e i s a decrease i n the q u a n t i t y o f g l y c o p r o t e i n p r e s e n t . Indeed i n one study the d i s t r i b u t i o n of the c o l o n i c g l y c o p r o t e i n s v a r i e d from case t o case. Chemical I n v e s t i g a t i o n s 9 S o e r g e l e t a l o b t a i n e d r e c t a l mucus from normal s u b j e c t s and from p a t i e n t s w i t h u l c e r a t i v e c o l i t i s by i r r i g a t i o n of the rectum w i t h h y p e r t o n i c phosphate s o l u t i o n . Chemical a n a l y s e s r e v e a l e d the presence o f f u c o s e , g a l a c t o s e , hexosamine and s i a l i c a c i d i n m a t e r i a l i s o l a t e d from both normal and d i s e a s e d c o l o n s . The carbohydrate composition appeared t o remain c o n s t a n t , but the n i t r o g e n content was 50 per cent h i g h e r i n the samples i s o l a t e d from d i s e a s e d t i s s u e than from normal t i s s u e . Immunologic s t u d i e s (double d i f f u s i o n ) showed t h a t g l y c o p r o t e i n s from normal human r e c t a l mucosa contained a variable number of serum proteins, par t i -cularly c< and fl g lobulin. These serum proteins were absent in mucus from ulcerative c o l i t i s patients. The authors postulated that these globulins might form an integral part of rectal mucus and that highly viscous °<^ - glycoproteins might contribute to the physical and chemical properties of the mucus. Teague et a l 1 ^ , in a study using mucosal biopsies and mucosal scrapings, reported the presence of two glyco-proteins in colonic mucus. Both contained fucose, galactose, N-acetyl-glucosamine, N-acetyl-galactosamine and s i a l i c acid. One however, contained mannose, whereas in the other mannose was a minor component. The amount of the mannose-rich component was s ignif icantly increased in ulcerative c o l i t i s . The poss ib i l i ty that ulcerative c o l i t i s i s an 5 37-42 auto-immune disorder has been extensively investigated ' 3 8 Broberger and Perlmann found that the sera of 90% of children and 40% of the adults with ulcerative c o l i t i s con-tained an antibody that haemagglutinated with sheep red blood ce l l s pre-coated with a phenol extract of s ter i le human colon. Fluorescein labelled gamma globulin from such patients was ab-sorbed preferential ly onto the ep i the l i a l ce l l s of the crypts of the c o l o n . A b s o r p t i o n was thought t o be s p e c i f i c f o r s e r a from u l c e r a t i v e c o l i t i s p a t i e n t s i n t h a t the a n t i g e n i n v o l v e d was immunologically u n r e l a t e d t o the ABH bl o o d group substances. F u r t h e r , f l u o r e s c e n t antibody l a b e l l i n g showed t h a t s e r a from some p a t i e n t s w i t h u l c e r a t i v e c o l i t i s r e a c t e d 40 w i t h the cytoplasm of t h e i r own c o l o n i c e p i t h e l i a l c e l l s On chemical a n a l y s i s , the phenol e x t r a c t o f s t e r i l e human c o l o n was found t o c o n t a i n g a l a c t o s e , g l u c o s e , mannose, fucose, x y l o s e , hexosamine and t r a c e s of 3,6-dideoxyhexose, s i a l i c a c i d and an a c i d h y d r o l y z a b l e l i p i d . Thus, i f u l c e r a -t i v e c o l i t i s i s an autoimmune d i s e a s e , i t i s p o s s i b l e t h a t g l y c o p r o t e i n s might p l a y a c e n t r a l r o l e i n the a n t i g e n i c 44 s t i m u l u s Chemical changes i n c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s have been a s s o c i a t e d w i t h u l c e r a t i v e c o l i t i s . Although these changes may be n o n - s p e c i f i c ^ ' ^ ' ^ , a d e t a i l e d knowledge of c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s i n normal s u b j e c t s and those w i t h u l c e r a t i v e c o l i t i s c o u l d c o n t r i b u t e t o a b e t t e r understand-i n g o f the e t i o l o g y and pathogenesis o f the d i s e a s e and may a i d i n i t s d i a g n o s i s . From the p r e s e n t l i t e r a t u r e , i t appears t h a t o n l y crude g l y c o p r o t e i n e x t r a c t s have been i n v e s t i g a t e d , and as was p o i n t e d out above, they are pro b a b l y contaminated w i t h g a s t r o i n t e s t i n a l s e c r e t i o n s and the product of b a c t e r i a l d e g r a d a t i o n . 15. The Involvement of G l y c o p r o t e i n s i n Crohn's Disease Crohn's d i s e a s e i s a c h r o n i c inflammation o f the s m a l l and l a r g e i n t e s t i n e . D i f f u s e Crohn's d i s e a s e o f the l a r g e i n t e s t i n e i s o f t e n misdiagnosed c l i n i c a l l y , r a d i o -. . 5 l o g i c a l l y and h i s t o p a t h o l o g i c a l l y as u l c e r a t i v e c o l i t i s . In a chemical a n a l y s i s o f the i n t e s t i n a l w a l l i n 47 a case o f Crohn's d i s e a s e o f the ileum, Seppala e t a l r e p o r t e d t h a t the glycosaminoglycan content was h i g h e r i n the d i s e a s e d than i n the h e a l t h y t i s s u e , whereas the contents of t o t a l c o l l a g e n , n e u t r a l - s a l t - s o l u b l e and a c i d - s o l u b l e c o l l a g e n were the same i n the d i s e a s e and the h e a l t h y p a r t s of the i n t e s t i n e . On e l e c t r o p h o r e s i s , the glycosaminoglycans i s o l a t e d from the di s e a s e d i n t e s t i n e always showed the presence of an e x t r a , slow-moving, f r a c t i o n which was s i m i l a r t o t h a t found i n g l y -cosaminoglycans i s o l a t e d from g r a n u l a t i o n t i s s u e o f r a t s . T h i s f r a c t i o n was thought t o be g l y c o p r o t e i n i n n a t u r e , but i t was not f u r t h e r i n v e s t i g a t e d . To t h i s degree t h e r e f o r e , chemical changes i n g l y c o -p r o t e i n s have been i m p l i c a t e d i n Crohn's d i s e a s e . No o t h e r study has been c a r r i e d out t o c o n f i r m t h i s f i n d i n g . D e s p i t e e x t e n s i v e i n v e s t i g a t i o n s t o r e v e a l the c l i n i c a l , r a d i o l o g i c a l and p a t h o l o g i c a l d i f f e r e n c e s between u l c e r a t i v e c o l i t i s and Crohn's d i s e a s e , i t i s sometimes d i f f i c u l t t o make a d i f f e r e n t -5 i a l d i a g n o s i s . I t i s hoped t h a t a d e t a i l e d knowledge of the chemical d i f f e r e n c e s , should they e x i s t i n the c o l o n i c g l y c o p r o t e i n s i n these d i s e a s e e n t i t i e s , might a i d i n the d i f f e r e n t i a l d i a g n o s i s o f these two d i s e a s e s . The P r e s e n t I n v e s t i g a t i o n In t h i s t h e s i s , the author d e s c r i b e s the i s o l a t i o n f r a c t i o n a t i o n and c h a r a c t e r i z a t i o n of human c o l o n i c g l y c o -p r o t e i n s from p u r e l y e p i t h e l i a l s o u r c e s . E p i t h e l i a l g l y c o -p r o t e i n s were o b t a i n e d from both normal and d i s e a s e d human co l o n s p a r t i c u l a r l y those r e s e c t e d f o r u l c e r a t i v e c o l i t i s and Crohn's d i s e a s e . In p r e l i m i n a r y s t u d i e s , r a t c o l o n was used as a model f o r experiments w i t h human t i s s u e . I t was found t h a t procedures s u i t a b l e f o r r a t c o l o n i c t i s s u e s c o u l d be success f u l l y a p p l i e d t o a study of human c o l o n i c t i s s u e . MATERIALS AND METHODS I. Preparat ion of samples for GLC and Co lor imet r i c A n a l y s i s . In genera l , samples for the various analyses were drawn from a stock aqueous so lu t ion of the g lycoprote in of accurately known concentrat ion (usually 2 mg g lycoprote in per m l ) . I I . Gas L i q u i d Chromatographic A n a l y s i s . Gas l i q u i d chromatography (GLC) was c a r r i e d out on a Hewlett Packard model No. 7 610A high e f f i c i e n c y dual columan gas chromatography f i t t e d with dual hydrogen flame i o n i z a t i o n detectors and "on column i n j e c t i o n " . Helium was used as c a r r i e r gas. The fo l lowing columns were used: (a) 8' x 1/4" o .d.copper columns conta in ing 10% S . E . 52 on 80 - 100 mesh dia toport S, operated i sothermal ly at 1 9 0 ° C . (b) 6' x 1/4" o . d . copper columns conta ining Chromo-sorb 103, operated i sothermal ly at 1 4 5 ° C . Peak areas were measured with a Hewlett Packard model No. 3370B e l e c t r o n i c in teg ra to r . Analyses were c a r r i e d out by an i n t e r n a l standard method using molar response factors determined i n our 48 laboratory as described by Reid et a l 18. A. N e u t r a l Sugars An a c c u r a t e l y weighed sample (approximately 2 mg) of each f r e e z e - d r i e d m a t e r i a l was h y d r o l y z e d i n a s e a l e d g l a s s tube a t 100°C. f o r 36 to 50 hours a c c o r d i n g t o the procedure 49 of Lehnhardt and W i n z l e r . The f r e e n e u t r a l sugars were analysed as t h e i r t r i m e t h y l s i l y l e t h e r s (TMS-ethers) on column 48 (a) by the method of Reid e t a l u s i n g s o r b i t o l as i n t e r n a l s t a n d a r d . B. 0 - a c e t y l Groups An a c c u r a t e l y weighed sample of each of the l y o p h i -l i z e d g l y c o p r o t e i n s (2 mg) was t r e a t e d w i t h 0.1 M sodium metho-x i d e i n anhydrous methanol (o.5 ml.) a t 4°C. f o r 0.5 h o u r s ^ . The methyl a c e t a t e produced by t r a n s e s t e r i f i c a t i o n was a n a l y s e d on column (b) u s i n g benzene as the i n t e r n a l s t a n d a r d . I I I . C o l o r i m e t r i c Analyses In most assays, v a r i o u s components of the g l y c o p r o t e i n o t h e r than the one under a n a l y s i s were i n c l u d e d as c o n t r o l s . Normally, these were g a l a c t o s e , f u c o s e , N - a c e t y l n e u r a m i n i c a c i d and N - a c e t y l - g l u c o s a m i n e . On o c c a s s i o n , D - r i b o s e , 2-deoxy-D-r i b o s e , c a l f thymus DNA and y e a s t RNA were a l s o used. Hexoses were e s t i m a t e d by the p h e n o l - s u l p h u r i c 51 procedure of Dubois u s i n g a mixture of g a l a c t o s e and fucose (10:3 w/w) as the s t a n d a r d . Fucose was e s t i m a t e d by the 3 52 method of Dische and S h e t t l e s ' . P r o t e i n was measured by 53 the method of Lowry w i t h Hyland c o n t r o l serum ( C a l i f o r n i a , U.S.A.) as standard. Phosphorus was e s t i m a t e d by the p r o c e -54 55 dure of Chen and DNA by the method of C e r i o t t i as m o d i f i e d by S h o r t . ^ . H y d r o l y s a t e s of the g l y c o p r o t e i n s prepared 49 by the method of Lehnhardt and W i n z l e r were an a l y z e d f o r 57 58 hexosamine by m o d i f i c a t i o n of the procedure o f W i n z l e r T o t a l and bound s i a l i c a c i d s were e s t i m a t e d by the p e r i o d a t e 59 r e s o r c i n o l procedures of J o u r d i a n e t a l , and f r e e s i a l i c a c i d s by the method o f Warren^". IV. E l e c t r o p h o r e s i s S t u d i e s C e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s was c a r r i e d out on 6" x 1" s t r i p s of Sepraphore I I I (Gelman Instrument Co.) u s i n g T r i s b a r b i t a l sodium b a r b i t a l b u f f e r (High R e s o l u t i o n B u f f e r , Gelman), pH 8.8, i= 0.1, f o r 30 minutes a t 300 v o l t s . 61 S t r i p s were s t a i n e d w i t h a l c i a n b l u e (pH 3.0). C e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s of r a t g l y c o p r o t e i n samples was a l s o c a r r i e d out i n 0.02 M p y r i d i n e HCl b u f f e r , pH 5.5, and i n 0.1 M p y r i d i n e formate b u f f e r , pH 3.0. Figure 1. Flow diagram of the method for the i s o l a t i o n i n t a c t r a t colonic e p i t h e l i a l c e l l s . 20b WISTAR RAT (150-250 gm.) RAT COLON 1. k i l l e d by ether anesthetia 2. colon resected Small piece for histology Colon Histology Main portion 1. 2. 3. everted i n f l a t e d with 0.9% sa l i n e shaken with 0.01% Na2EDTA in PBS 1 C e l l Suspension Centrifuged 2000 rpm for 10 minutes Supernatant 1. dialyzed (water) 2. freeze-dried 3. stored Isolated E p i t h e l i a 4 C e l l s Agarose ge l e lec t rophores i s was performed with commercial agarose ge l p lates ( A n a l y t i c a l Chemists Inc.) conta ining 1% agarose i n T r i s b a r b i t a l sodium b a r b i t a l bu f fe r , pH 8.8, at a constant voltage of 90 v o l t s for 30 minutes. The plates were stained with a l c i a n b lue , pH 3.0. E l e c t r o p h o r e t i c m o b i l i t i e s were measured r e l a t i v e to hepar in . V. Pre l iminary Experiments on the I s o l a t i o n of Rat Colonic  E p i t h e l i a l Glycoproteins (Figures 1 - 3 ) A. I s o l a t i o n of Colonic E p i t h e l i a l C e l l s I n t e s t i n a l e p i t h e l i a l c e l l s were i s o l a t e d by the 6 2 procedure of C u l l i n g et a l (Figure 1) . Wistar rat s (150-250g) were k i l l e d with ether and the colons were removed. E p i t h e l i a l c e l l s were obtained by shaking the everted, i n f l a t e d colons wi th 0.01% EDTA i n PBS (phosphate-buffered sa l ine) ,. (8 g NaCl , ' Q2.g KC1, 1.15g Na 2 HP0 4 and 0.2 g KH 2 P0 4 i n 1 l i t r e of water) . The colons were a l t e r n a t e l y shaken for 5 minutes and incubated at 3 7 ° C for 5 minutes for a t o t a l per iod of one hour, the shaking medium being changed at 10 and 30 minutes. The pooled c e l l suspension was centr i fuged at 2000 rpm for 10 minutes at 4 ° C . The res idue , which contained e p i t h e l i a l c e l l s , was washed twice with i c e - c o l d PBS and recovered by c e n t r i f u g a t i o n . The pooled supernatants were d i a ly sed against d i s t i l l e d water, freeze dr i ed and stored for future use. Specimens (approximately 1/2 cm) were removed immediately af ter the colons had been everted and at the conclus ion of the f i n a l shaking procedure. These were f ixed i n 10% formolcalcium, pa ra f f in processed and cut at 5 ji. Sections were s ta ined with Hematoxylin and Eos in (H & E) and with p e r i o d i c a c i d / S c h i f f , 6 3 a l c i a n b lue , pH 2.5 . Microscopic examinations of the s ta ined sect ions enabled the degree of c e l l removal to be assessed. 22a. F i g u r e 2. Flow diagram of the method f o r the e x t r a c t i o n of g l y c o p r o t e i n s from r a t c o l o n i c e p i t h e l i a l c e l l s . 22b. EPITHELIAL CELLS 1. Suspended i n 1 M NaCl 2. Sonated 3. Centrifuged at 2000rpm for 10 minutes 4. Supernatant removed and residue resuspended i n 1 M NaCl, steps 2-4 repeated POOLED SUPERNATANT RESIDUE (stored) RESIDUE centrifuged at 20,000 g for 1 hour 1 SUPERNATANT* centrifuged at 105,000 g for 1 hour RESIDUE (stored) SUPERNATANT 1. dialyzed against 1 M NaCl 2. concentrated CONCENTRATED SUPERNATANT 23a. Figure 3. Flow diagram of the method for the f r a c t i o n a t i o n of Rat colonic e p i t h e l i a l glycoproteins. 23b. CONCENTRATED SUPERNATANT Gel chromatography on Biogel A15 M using 1 M NaCl solution as eluant AT. FRACTION A2 FRACTION (Carbohydrate-rich) (Protein-rich) 1. concentrate 2. dialyzed against 0.02 M pyridine-HCl buffer, pH 5.5 3. ion-exchange chromatography on DEAE cellulose using 0.02 M pyridine-HCl buffer, tpH 5.5 as eluant and a NaCl gradient (0-1.5N A FRACTION B FRACTION LEAF 24 OMITTED IN PAGE NUMBERING. B. Ex t rac t ion of Colonic Glycoproteins The procedure for the ex t rac t ion of c o l o n i c g lycoproteins was a modi f ica t ion of a method prev ious ly des-64 cr ibed by Hakkinen for the i s o l a t i o n of ac id mucopoly-saccharide (figure 2) . A suspension of the c o l o n i c e p i -t h e l i a l c e l l s i n ice co ld IM sodium ch lor ide was sonated for periods of 30 seconds with a Biosonik Sonator (Bronswil l S c i e n t i f i c ) at se t t ings of 1 and 80. The c e l l suspension was then centr i fuged at 2000 rpm for 10 minutes and the residue was resuspended i n co ld 1 M sodium ch lor ide so lu t ion and again sonated. This procedure was repeated u n t i l the quanti ty of residue obtained by cent r i fuga t ion was constant. The pooled supernatants were centr i fuged at 20,000 g for 1 hour and the residue was washed with 1 M sodium ch lor ide so lu t ion and recentr i fuged . The combined supernatants obtained were centr i fuged at 105,000 g for 1 hour and the residue was washed i n 1 M sodium ch lor ide and recentr i fuged . The f i n a l pooled supernatants were d ia lyzed against 1 M sodium ch lor ide s o l u t i o n for 24 hours at 4 ° C and concentrated to a convenient volume by means of an Amicon u l t r a f i l t r a t i o n apparatus with an XM 50 membrane, exclus ion l i m i t 50^,000 M.W. . C. F rac t iona t ion of Colonic Glycoproteins The f r a c t i o n a t i o n of the concentrated 105,000 g supernatant i s shown i n f igure 3. The concentrated supernatant was a p p l i e d t o a column of B i o g e l A15M (100-200 mesh) (BIO-RAD L a b o r a t o r i e s ) and e l u t e d w i t h I M NaCl. The carbohydrate-r i c h f r a c t i o n o b t a i n e d was c o n c e n t r a t e d and d i a l y z e d a g a i n s t 0.02 M p y r i d i n e - H C l b u f f e r , pH 5.5 at 4°C f o r 24 hours. The r e t e n t a t e was a p p l i e d t o a column of D E A E - c e l l u l o s e (DE22, Whatman), and was e l u t e d w i t h 0.02 M p y r i d i n e - H C l b u f f e r , pH 5.5, c o n t a i n i n g a convex NaCl g r a d i e n t ( 0 - 1.5 M). The v a r i o u s c a r b o h y d r a t e - r i c h f r a c t i o n s o b t a i n e d were i n d i v i d u a l l y concentrated, d i a l y z e d a g a i n s t d i s t i l l e d water o v e r n i g h t and r e c o v e r e d by l y o p h i l i z a t i o n . In p r e l i m i n a r y experiments, the f r a c t i o n a t i o n procedure was monitored f o r hexoses by the p h e n o l - s u l p h u r i c 51 53 method of Dubois e t a l , f o r p r o t e i n by the Lowry procedure and f o r s i a l i c a c i d by the diphenylamine method of Werner and 6 5 Odin . In l a t e r experiments, the s i a l i c a c i d assay was o m i t t e d . VI I s o l a t i o n of Human C o l o n i c E p i t h e l i a l G l y c o p r o t e i n : A. Source o f Specimens A l l specimens were o b t a i n e d from the pathology department of the Vancouver General H o s p i t a l . They were t r a n s -p o r t e d t o the l a b o r a t o r y i n p l a s t i c bags packed i n i c e . The l e n g t h of time a f t e r surgery and between p r o c e s s i n g was from 1 t o 3 hours. C e l l s were o b t a i n e d from the f o l l o w i n g c o l o n specimens: (a) the h i s t o l o g i c a l l y normal p a r t s of 20 specimens o b t a i n e d from p a t i e n t s of both sexes and o f ages r a n g i n g from 29 to 85,( average 53) and undergoing colectomy f o r carcinoma. The c e l l s from these specimens were pooled and t r e a t e d as one sample. (b) The e n t i r e c o l o n from a 19 year o l d male cadaver which had been s e l e c t e d as kidney donor. (c) S u r g i c a l s p e c i -mens from two cases of u l c e r a t i v e c o l i t i s (two 60 year o l d female p a t i e n t s ) , and one case of i s c h e m i c c o l i t i s (39 year o l d f e male), one case of Crohn's d i s e a s e of the l a r g e bowel and two cases of Crohn's d i s e a s e of the t e r m i n a l i l e u m (36 and 38.year o l d female p a t i e n t s ) . The c e l l s from the two cases of u l c e r a t i v e c o l i t i s were poo l e d as were those from the two cases of Crohn's d i s e a s e of the t e r m i n a l i l e u m . B. I s o l a t i o n of Human C o l o n i c E p i t h e l i a l C e l l s The s u r g i c a l specimens r e c e i v e d from the pathology department a t the Vancouver General H o s p i t a l had a l l been cut l o n g i t u d i n a l l y along the l e n g t h o f the c o l o n . A f t e r the f a t t y t i s s u e s had been removed from the s e r o s a l s u r f a c e , the c o l o n s were s u t u r e d i n t o t h e i r o r i g i n a l t u b u l a r form w i t h the mucosal s u r f a c e on the o u t s i d e . A c y l i n d r i c a l b a l l o o n , was i n s e r t e d i n t o each c o l o n tube and was then i n f l a t e d w i t h i s o t o n i c s a l i n e . F i g u r e 4. Flow diagram of the procedure f o r the d i g e s t i o n s t u d i e s of c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s of r a t and human u s i n g v i b r i o c h o l e r a neuraminidase. Analyses f o r t o t a l (PRT) s i a l i c a c i d s by 59 the p e r i o d a t e r e s o r c i n o l method of J o u r d i a n e t a l were performed on samples of the r e t e n t a t e and the f i l t r a t e which were e i t h e r (1) un t r e a t e d o r (2) t r e a t e d w i t h 0.1 N KOH f o r 1 hour a t room temp-e r a t u r e . Samples (both the KOH t r e a t e d and non-KOH t r e a t e d ) o f the r e t e n t a t e and the f i l t r a t e were f u r t h e r analyzed r e s p e c t i v e l y f o r g l y c o s i d i c a l l y bound s i a l i c a c i d s (PRB) by the p e r i o d a t e 59 r e s o r c i n o l method of J o u r d i a n e t a l and f o r f r e e s i a l i c a c i d s 6 0 (TB) by the t h i o b a r b i t u r i c a c i d method of Warren 283: SAMPLE inc u b a t e d w i t h v i b r i o c h o l e r a a t 37 C f o r 65 hours S u c t i o n f i l t e r e d through a d i a l y s i s membrane FILTRATE RETENTATE d i v i d e d i n t o 2 p o r t i o n s and one was t r e a t e d w i t h KOH 1 d i v i d e d i n t o 2 p o r t i o n s and one was t r e a t e d w i t h _j KOH KOH t r e a t e d non-KOH t r e a t e d KOH t r e a t e d non-KOH t r e a t e d 1. TB 1. TB 1. PRT 1. PRT 2. PRT 2. PRT 2. PRB 2. PRB C o l o n i c e p i t e h l i a l c e l l s were removed from each specimen by shaking w i t h 0.02% EDTA i n PBS. The shaking and i n c u b a -t i o n procedures were i d e n t i c a l t o those employed w i t h the r a t c o lons (see f i g u r e 2 ) . H i s t o l o g i c examinations, t o monitor c e l l removal, was performed as d e s c r i b e d on page 21 • C. E x t r a c t i o n and F r a c t i o n a t i o n o f Human C o l o n i c E p i t h e l i a l  G l y c o p r o t e i n s The procedures employed were s i m i l a r t o those d e s c r i b e d w i t h r a t co l o n s (see pages 22 to 24). V I I . S a p o n i f i c a t i o n S t u d i e s A known weight of each f r e e z e d r i e d g l y c o p r o t e i n (o.2 mg/mg) was t r e a t e d w i t h 0.1 N. potassium hydroxide f o r 2 hours at room temperature. A l i q u o t s (0.2 t o 0.5 ml) were withdrawn a t 0, 0.5, 1 and 2 hours and a f t e r n e u t r a l i z a t i o n w i t h the a p p r o p r i a t e q u a n t i t y o f IN s u l p h u r i c a c i d were analyzed f o r s i a l i c a c i d by the p e r i o d a t e r e s o r c i n o l method 59 of J o u r d i a n e t a l V I I I . D i g e s t i o n S t u d i e s These were c a r r i e d out as shown i n f i g u r e 4. An a c c u r a t e l y weighed sample (5 to 12 mg) of each f r e e z e d r i e d g l y c o p r o t e i n was d i s s o l v e d i n 0.05 M sodium a c e t a t e b u f f e r , 3( pH 5.5 ( C o n t a i n i n g 2 X 10 % sodium a z i d e and 1 X 10 % c a l c i u m c h l o r i d e ) and i n c u b a t e d w i t h V i b r i o c h o l e r a neuraminidase (100 u n i t s / ml i n c u b a t e ) (Behringwerke) f o r 24 hours a t 37°C. A f u r t h e r 100 u n i t s of enzyme per ml i n c u b a t e was then added and i n c u b a t i o n was c o n t i n u e d f o r a f u r t h e r 41 hours. The i n c u b a t e was vacuum f i l t e r e d through a d i a l y s i s sac w i t h an e x c l u s i o n l i m i t o f approximately 100,000 MW.. The r e t e n t a t e and d i a l y s a t e was o b t a i n e d were f r e e z e d r i e d and r e d i s s o l v e d i n a known volume of- d i s t i l l e d water. Analyses f o r t o t a l (PRT) s i a l i c 59 a c i d s by the p e r i o d a t e r e s o r c i n o l method of J o u r d i a n e t a l were performed on samples of the r e t e n t a t e and the f i l t r a t e which were e i t h e r (1) u n t r e a t e d or (2) t r e a t e d w i t h 0.1N KOH f o r 1 hour at room temperature. Samples (both the KOH t r e a t e d and non-KOH t r e a t e d ) o f the r e t e n t a t e and the f i l t r a t e were f u r t h e r analyzed r e s p e c t i v e l y f o r g l y c o s i d i c a l l y bound s i a l i c a c i d s 59 (PRB) by the p e r i o d a t e r e s o r c i n o l method of J o u r d i a n e t a l and f o r f r e e s i a l i c a c i d s (TB) by the t h i o b a r b i t u r i c a c i d method o f Warren**^. IX. Immunodiffusion S t u d i e s . These were c a r r i e d out on commercially a v a i l a b l e Ouchterlony p l a t e s (Hyland, C a l i f o r n i a , U.S.A.). Figure 5. Photomicrographs of sections of human colon obtained from specimen resected for carcinoma of the co lon . The t i s sue was taken CA) before and (B) a f ter treatment with 0,02% (0.54 mM) EDTA i n phosphate buffered s a l i n e . In t h i s case, complete removal of e p i t h e l i a l mucus secret ing c e l l s i n (Bl) and (B2) was seen and there was no apparent damage to the basement membrane. Sections (Al) and (Bl) were s ta ined with a l c i a n blue - PAS technique and sections (A2) and (B2) were s ta ined by H & E . 31b. 32. X. P r e p a r a t i o n of A n t i s e r a 6 6 A m o d i f i c a t i o n o f the method of Kopp e t a l was f o l l o w e d i n the p r e p a r a t i o n o f a n t i s e r a a g a i n s t r a t c o l o n i c e p i t h e l i a l g l y c o p r o t e i n . 7.7 mg of f r e e z e d r i e d g l y c o p r o t e i n ( f r a c t i o n A) was suspended i n PBS, pH 7.3, a t a c o n c e n t r a t i o n of 10 mg per ml and was mixed w i t h an equal volume of complete Freunds adjuvant (DIFCO Lab.) by s q u i r t i n g i n t o a shot g l a s s . The mixture was then i n j e c t e d i n t o the f o u r f o o t pads of a 3 l b . female New Zealand r a b b i t . Three weeks l a t e r , a b o o s t e r dose o f the same s t r e n g t h was i n j e c t e d subcutaneously. A f t e r a f u r -t h e r 2 weeks, the r a b b i t was g i v e n a subcutaneous i n j e c t i o n o f a suspension of 6.6 mg o f r a t g l y c o p r o t e i n i n 1.4 ml of a 1:1 mixture of PBS and P e r t u s i s V a c c i n e (Connaught M e d i c a l Research L a b o r a t o r i e s ) . Two weeks a f t e r the l a s t i n j e c t i o n , 5 ml of b l o o d was drawn from the m a r g i n a l v e i n o f the r a b b i t e a r . Serum was o b t a i n e d by c e n t r i f u g i n g the c l o t t e d b l o o d a t 3000 rpm f o r 30 minutes. RESULTS AND DISCUSSION 1. I s o l a t i o n o f E p i t h e l i a l C e l l s Complete c e l l removal was accomplished i n a l l r a t experiments when i n f l a t e d and e v e r t e d r a t c o l o n s were shaken w i t h 0.01% (0.27 mM) EDTA i n PBS. The m a j o r i t y o f the c e l l s were removed d u r i n g the f i r s t 30 minutes o f shaking. The e p i t h e l i a l c e l l suspension was washed twice w i t h PBS and the c e l l s were recovered by c e n t r i f u g a t i o n . The average packed c e l l volume o b t a i n e d was 1.16 + 0.24 ml o f c e l l s per r a t c o l o n . When t h i s i s o l a t i o n procedure was a p p l i e d to specimens of human c o l o n , the use o f 0.02% (0.54 mM) EDTA was necessary to achieve removal o f the e p i t h e l i a l c e l l s . In 40% o f the specimens, complete removal o f c e l l s was accomplished ( f i g u r e 5), w h i l e i n the o t h e r s , from 10 to 90 % o f the c e l l s were removed. The c e l l s a t the base o f the c r y p t s proved to be the most d i f f i c u l t to remove. 2. E x t r a c t i o n o f C o l o n i c E p i t h e l i a l G l y c o p r o t e i n s The i s o l a t e d c e l l s were suspended i n 1 M NaCl, and sonated i n an ice-water b a t h . Normally, complete r u p t u r e o f a l l the c o l o n i c e p i t h e l i a l c e l l s r e q u i r e d 5 to 6 p e r i o d s o f s o n a t i o n of 30 seconds d u r a t i o n each. Sonation was judged to be complete by the occurrence o f a con s t a n t r e s i d u e a f t e r c e n t r i f u g a t i o n a t 2000 rpm f o r 10 minutes. Under the l i g h t microscope, t h i s r e s i d u e appeared t o be composed o f c e l l membrane fragments. The sonate was c e n t r i f u g e d a t 105,000 g and the supernatant o b t a i n e d was u s u a l l y c o n c e n t r a t e d , t o one-tenth o f i t s volume by Amicon u l t r a f i l t r a t i o n membrane ( e x c l u s i o n l i m i t 50,000 M.W.). Recovery of the g l y c o p r o t e i n s was u s u a l l y g r e a t e r than 90% (see page 37 ) . The e f f i c i e n c y o f the s o n a t i o n procedure was com-pared w i t h two ot h e r c u r r e n t l y used homogenization methods. Known volumes o f packed c e l l s were homogenized w i t h (a) a motor-driven P e s t l e OMNI-MIXER, (b) a pr e s s u r e homogenizer and (c) s o n a t i o n . Each homogenate o b t a i n e d was c e n t r i f u g e d s u c c e s s i v e l y a t 2000 rpm, 20,000 g and 105,000 g. The 105,000 supernatant and the 3 r e s i d u e s from each procedure were analyzed f o r p r o t e i n by the 53 Lowry procedure and f o r hexose by the p h e n o l - s u l p h u r i c 51 method o f Dubois e t a l . The r e s u l t s o b t a i n e d i n d i c a t e d t h a t s o n a t i o n gave a more complete e x t r a c t i o n o f the carbohydrate r i c h m a t e r i a l s from the c e l l s , and t h a t t h i s c o u l d be accomplished i n a much s h o r t e r time. Furthermore, s p e c t r o p h o t o m e t r i c e s t i m a -t i o n o f the DNA i n the 105,000 g supernatants i n d i c a t e d t h a t the supernatant from the s o n a t i o n procedure c o n t a i n e d the l e a s t q u a n t i t y o f DNA. TABLE 1. Percentage r e c o v e r y o f hexose and s i a l i c a c i d a f t e r c o n c e n t r a t i o n o f the 105,000 g supernatant of i n t e s t i n a l e p i t h e l i a l g l y c o p r o t e i n by the v a r i o u s procedures Method Hexose r e c o v e r y S i a l i c a c i d (%) r e c o v e r y (%) Sephadex G-25 67.3 5 67 .3 3 Amicon u l t r a -f i l t r a t i o n 83.8 4 85 .8 1 Hollow f i b r e (BIO-RAD) 33.0 1 0 1 Note: (a) The number i n the b r a c k e t r e p r e s e n t s the number o f e x p e r i -ments done t o a r r i v e at the above averages. (b) The degree o f c o n c e n t r a t i o n i n each case was about 8 to 10 times. 51 (c) Hexose was determined by the p h e n o l - s u l p h u r i c procedure of Dubois and s i a l i c a c i d was es t i m a t e d by the diphenylamine method o f Werner 6 5 and Odin (3) The e x c l u s i o n l i m i t o f the membrane used i n Amicon u l t r a - f i l t r a t i o n was 10,000 (PM10). F i g u r e 6. M o n i t o r i n g of the f r a c t i o n a t i o n o f r a t c o l o n i c e p i t h e l i a l g l y c o p r o t e i n s by c e l l u l o s e a c e t a t e e l e c t r o p h o r e s i s at pH 8.8. A l l s t r i p s were s t a i n e d w i t h a l c i a n b l u e at pH 3. I, h e r a p i n standard; I I , 105,000g supernatant; I I I , f r a c t i o n Al.; IV, f r a c t i o n A; V, f r a c t i o n B; VI, f r a c t i o n s A & B combined. 0) 37. During the p r e p a r a t i o n of the g l y c o p r o t e i n s , i t was necessary to c o n c e n t r a t e s o l u t i o n s on at l e a s t t h r e e o c c a s s i o n s . I t was d e s i r a b l e t h e r e f o r e , to choose a concen-t r a t i o n procedure which caused the s m a l l e s t l o s s e s of g l y c o -p r o t e i n . For t h i s reason, t h r e e c u r r e n t l y used methods of c o n c e n t r a t i o n were i n v e s t i g a t e d : namely, (a) Sephadex G-25 ( c o a r s e ) , (b) Amicon u l t r a f i l t r a t i o n and (c) hollow f i b r e c o n c e n t r a t i o n d i a l y s i s . The r e s u l t s of t h i s study, shown i n Table 1, i n d i c a t e d t h a t Amicon u l t r a f i l t r a t i o n l e d t o the s m a l l e s t l o s s of g l y c o p r o t e i n as judged by the r e c o v e r y of s i a l i c a c i d and hexose. By t h i s method, the average r e c o v e r y was more than 85% of the s t a r t i n g m a t e r i a l . A f u r t h e r 5 to 10% of the t o t a l g l y c o p r o t e i n c o u l d be r e c o v e r e d , i n some c a s e s , by r i n s i n g the membrane w i t h 1 M NaCl s o l u t i o n . T h i s procedure which u t i l i z e s membranes w i t h s i z e - l i m i t i n g pores ( u l t r a f i l t r a -t i o n ) , has been c l a i m e d t o be the most s e l e c t i v e method f o r c o n c e n t r a t i n g b i o l o g i c a l m a t e r i a l s and to be the l e a s t i n j u r i o u s 6 7 t o p r o t e i n s o l u t e s . Thus, Amicon u l t r a f i l t r a t i o n was employed i n a l l of the c o n c e n t r a t i o n and most of the d i a l y s i s p r o c e s s e s used i n t h i s work. 3. F r a c t i o n a t i o n of C o l o n i c E p i t h e l i a l G l y c o p r o t e i n s A. P r e l i m i n a r y Model Experiments w i t h Rat G l y c o p r o t e i n s E l e c t r o p h o r e t i c examination of the 105,000 g super-natant a t pH 8.8 i n d i c a t e d the presence of two components Figure 7. Agarose A15M column chromatography of the 105,000 g concentrated supernatant extracted from the i s o l a t e d colonic e p i t h e l i a l c e l l s of a human sample resected for car-cinoma of the colon. Column size 83X 2.5 cm.. Fractions were eluted with 1 M NaCl. Hexose (0) and protein (•) 51 53 were determined by the procedures of Dubois and Lowry respectively. A carbohydrate-rich f r a c t i o n A l and a protein-r i c h f r a c t i o n A2 were obtained. 38b which stained with a l c i a n blue at pH 3.0. The m o b i l i t y of the fas ter moving band was s i m i l a r to that of the heparin standard and was approximately twice as fast as that of the slower moving band (see f igure 6 ) . F r a c t i o n a t i o n of t h i s supernatant by g e l chromatography on Bioge l A15M using 1 M NaCl as eluant y ie lded a carbohydrate-r ich f r a c t i o n A l and a carbohydrate-poor but p r o t e i n - r i c h f r a c t i o n A2. An immunodiffusion study revealed that f r a c t i o n A2 reacted with a n t i r a t plasma p r o t e i n prepared i n rabbi t s while f r a c t i o n A l d idnot . Presumably, f r a c t i o n A2 contained plasma prote ins and other low molecular weight substances. F rac t ion A l was concentrated, d i a lyzed against 0.02 M pyr id ine HCl buf fe r , pH 5 .5 , and was then f ract ionated by ion exchange chromatography on DEAE c e l l u l o s e using 0.02 M p y r i d i n e H C l buf fe r , pH 5.5 , conta ining a convex s a l t gradient (0 to 1.5 M NaCl) as e luant . Two carbohydrate-r i c h peaks were obtained which were designated f r ac t ions A and B according to t h e i r order of e l u t i o n . C e l l u l o s e acetate e lec t rophores i s showed that f r a c t i o n B corresponded to the fast moving band in the 105,000 g supernatant while f r a c t i o n A represented the slower moving band (figure 6 ) . This f r a c t i o n a -t i o n procedure was repeated on two new samples of the 105,000 g supernatant of ra t co lon ic e p i t h e l i a l c e l l s , and s i m i l a r r e su l t s were obta ined. 40a. F i g u r e 8, DEAE c e l l u l o s e ion^-exchange column chromatography of the c a r b o h y d r a t e - r i c h f r a c t i o n A l o b t a i n e d from Agarose A15M column chromatography of human c o l o n i c c e l l sap (105,000g supernatant) o b t a i n e d from a sample r e s e c t e d f o r carcinoma of the c o l o n . Column s i z e 83 X 2.5 cm.. F r a c t i o n s were e l u t e d w i t h 0.02 M p y r i d i n e HC1 b u f f e r , pH 5.5, c o n t a i n i n g a convex NaCl g r a d i e n t (0-1.5 M). Two d i s t i n c t peaks were o b t a i n e d ( f r a c t i o n s A and B ) . Hexose was est i m a t e d by the procedure o f D u b o i s ^ 1 and p r o t e i n measured by the method of 53 Lowry . (0) , hexose; (•) , p r o t e i n and (••.•• ) the s a l t g r a d i e n t . 40b. 4: TABLE 2. Agarose ge l e l ec t rophore t i c m o b i l i t i e s of the sample obtained from the various stages i n the f r a c t i o n a t i o n of co lon ic e p i t h e l i a l g lycoprote ins extracted from normal and diseased human colons . SAMPLES Normal (Cancer) Normal Ischemic C o l i t i s Crohn 1s (colon) Crohn 1s (ileum) Ulce ativi C o l i Heparin 1.0 1.0 1.0 1.0 1.0 1.0 105,000g Supernatant 0.29 0.34 0.35 0.22 0.20 0.34 0.79 0.82 0.79 0.89 0.83 0.87 Frac t ion A l 0.29 0.35 0.36 0.19 0.25 0.37 (carbohydrate-rich) 0.74 0.78 0.87 0.91 0.88 0.85 Frac t ion A2 1.0 1.01 1 0.98 2 1.05 1.03 3 1.01 F i r s t DEAE f r a c t i o n 0.35 0.35 0.35 0.20 0.24 0.37 ( f ract ion A) Second DEAE f r a c t i o n 0.82 0.84 4 0.84 0.87 0.87 0.85 ( f rac t ion B) Ca l f thymus DNA cont ro l (streak) 0 to 0.88 0 to 0.89 0 to 0.9 0 to 0.91 0 to 0.58 Note: (1) (2) and (3) ,very f a i n t traces of a l c i a n blue s t a in ing with m o b i l i t i e s of 0.45, 0.41 and 0.3 r e spec t ive ly were found. (4)very f a i n t trace of a l c i a n blue p o s i t i v e mater ia l with m o b i l i t y of 0.34 was observed. Figure 9. I l l u s t r a t i o n of a t y p i c a l agarose gel electrophoresis pattern of the various f r a c t i o n s i n the fractionation of the 105,000g supernatant of a human sample. In t h i s case, the 105,000g supernatant was extracted from the i s o l a t e d colonic e p i t h e l i a l c e l l s of a sample resected for carcinoma of the colon. Heparin was used as the standard. Electrophoresis was performed with commercial agarose plate containing 1% agarose i n T r i s b a r b i t a l sodium b a r b i t a l buffer, pH 8.8, at a constant voltage of 90 v o l t s for 30 minutes. The plates were stained with Alcian blue, pH 3. 1, heparin; 2, 105,000g supernatant; 3, f r a c t i o n A l , 4, f r a c t i o n A2; 5, f r a c t i o n A; 6, f r a c t i o n B; 7, f r a c t i o n s A and B combined. VE direction of migration t n PI II starting point 1 2 3 4 5 6 7 8 B. Experiments on Human Colonic E p i t h e l i a l C e l l s S imi l a r procedures to those developed on the ra t model were appl ied to the ex t rac t ion of g lycoprote ins from human co lon ic e p i t h e l i a l c e l l s . F rac t iona t ion of the 105,000 g supernatant of the 1 M NaCl extract of these c e l l s on B ioge l A15M using 1 M NaCl as eluant y i e lded a carbohydrate-r ich f r a c t i o n A l and a p r o t e i n - r i c h f r a c t i o n A2. (f igure 7) F r a c t i o n A l was d ia lyzed against 0.02 M pyr id ine HC1, pH 5.5 , and was f ract ionated by ion exchange chromatography on DEAE c e l l u l o s e using 0.02 M pyr id ine H C l , pH 5 .5 , conta ining a convex NaCl gradient ( 0 - 1.5 M ) as e luant . Two apparently homogenous f rac t ions ( f ract ions A and B) were obtained from each normal and diseased sample (f igure 8 ) except i n one case of Crohn's disease of the terminal iteum where mul t ip le peaks were observed i n F rac t ion B. The r e su l t s of agarose gel e lec t rophores i s are shown i n table 2. A l l the 105,000 g supernatants were found to show two a l c i a n blue s t a in ing bands (f igure 9 ) . One of these had a m o b i l i t y s i m i l a r to that of heparin while the other migrated at a much slower ra te . F rac t ion A l , obtained by B ioge l A15M f r a c t i o n a t i o n , was found to contain both a l c i a n blue p o s i t i v e bands. DEAE c e l l u l o s e chromatography of A l separated the two bands in to f r ac t ions A and B. F rac t ion B was found to have a m o b i l i t y s i m i l a r to heparin and approximately twice that of F rac t ion A. A l l f r ac t ions B showed s i m i l a r 4 TABLE 3. Chemical Analyses of fra c t i o n s B. (A l l values are expressed i n ug/mg of freeze dried sample). Protein Phosphorus DNA Hexose GLC S i a l i c Hexose acid fucose RAT NORMAL (Cancer) NORMAL ULCERATIVE COLITIS CROHN 1S (Colon) CROHN1S (Ileum) ISCHEMIC COLITIS 84 11.9 22.4 74.0 122.5 67.4 123.5 67.9 76.8 66.8 1080 245 1090 250 16.6 77.9 1180 251 131.8 47.3 920 690 0 1030 235 0 1090 250 0 0 16 15 17 19 16 19 35 CALF THYMUS DNA (CONTROL) 18.3 66.8 1000 210 - 11 RNA (CONTROL) 27.4 76.8 0 340 - 2 Chemical analyses were performed by the following procedures:hexose 51 53 by the method of Dubois , protein by the procedure of Lowry , 54 phosphorus by method of Chen , DNA by the indole procedure of C e r i o t t i ^ as modified by S h o r t ^ and s i a l i c acid by the periodate 59 resor c i n o l procedure of Jourdian . Other than the rat and the two nucleic acid controls, a l l the samples were obtained from human colons. 4 TABLE 4. Molar r a t i o of sugars i n co lon ic e p i t h e l i a l g lyco-proteins ( f rac t ion A) Molar Ratios of Sugar Fucose Galactose Hexosamine S i a l i c ac id RAT 1 3.35 2.2 1.45 NORMAL (Cancer) 1 3 .15 3.7 2.7 NORMAL 1 2.9 2.6 1.7 ULCERATIVE COLITIS 1 2.1 2.1 1.5 CROHN'S (colon) 1 1.35 1.2 0.32 CROHN'S (ileum) 1 5.3 2.1 3.73 ISCHEMIC COLITIS 1 3.4 3.35 3.3 Note: Glycoprote in samples were t reated with Dowex 50H i n 0.01 HCl at 1 0 0 ° C for 36 to 50 hours and the Hydrolysates were analysed for fucose and galactose by GLC and for hexosamine by a modi f i ca t ion ^ o f a procedure of W i n z l e r ^ . S i a l i c ac id values represented the t o t a l s i a l i c ac id obtained from a p e r i o d a t e - r e s o r c i n o l assay of the f u l l y saponif ied g lycoprote in samples. TABLE 5. Chemical analyses of the co lon ic e p i t h e l i a l g lyco-prote ins ( f rac t ion A ) . A l l values are expressed i n ug per mg of freeze dr i ed sample. Pro te in Hexose Phosphorus Fucose RAT 84.3 230 0.2 60.1 NORMAL (Cancer) 58.5 170 0 41.3 NORMAL 65.0 195 0.2 57.6 ULCERATIVE COLITIS 82.8 140 0.2 41.2 CROHN'S (Colon) 67.9 105 0.2 46.2 CROHN'S (Ileum) 116.0 - 0.1 -ISCHEMIC COLITIS 53.7 — 0.1 -Chemical analyses were performed by the fo l lowing procedures: 53 pro te in by the procedure of Lowry , hexose by the phenol-51 su lphur ic procedure of Dubois , phosphorus by the method of Chen ^ 4 and fucose by the procedure of Dische and Shet t le s 4 ' " * (-) ind ica tes that the ana lys i s was not performed. Figure 10. GLC of the t r i m e t h y l s i l y l ethers of the neutral sugars of colonic e p i t h e l i a l glycoprotein. The sample i l l u s t r a t e d i n this t y p i c a l chromatogram was obtained from the h i s t o l o g i c a l l y normal part of surgical carcinoma specimens obtained from patients undergoing colectomy. Only fucose and galactose were detected and each of the two sugars had resolved into three peaks (1) , (2) and (3) . e lec t rophore t i c m o b i l i t i e s . In the case of f r ac t ions A , the two samples obtained from specimens resected for Crohn's disease moved much slower than f rac t ions from other sources. 4. Blood Contamination The c e l l s i s o l a t e d from some human samples were contaminated by b lood . In view of t h i s , the sonation and f r a c t i o n a t i o n procedures described above were c a r r i e d out on samples of whole human b lood . Blood components had a s i m i l a r e luted volume to the included second peak (see above). Thus, none o f . t h e g lycoprote ins examined below appeared to a r i se from contamination with blood p r o t e i n s . 5. Analyses of Fract ions B The re su l t s obtained from an analys i s of f r ac t ions B are shown i n table 3, i n which i s included for comparative purposes s i m i l a r analyses of c a l f thymus DNA and yeast RNA. A l l these f rac t ions absorbed s t rongly at 260nui, f a i l e d to mi-grate on e lec t rophores i s at pH 3.0 and contained no detectable fucose, galactose or hexosamine. Based upon t h e i r high 5 6 phosphorus content, the r e su l t s of the Indole assay and the s i m i l a r i t y of the a n a l y t i c a l r e su l t s to c a l f thymus DNA, f rac t ions B are probably DNA. 48b. The s m a l l q u a n t i t y o f s i a l i c a c i d found i n f r a c t i o n s B u s i n g the p e r i o d a t e - r e s o r c i n a l assay i s p r o b a b l y due to i n t e r f e r e n c e i n t h i s assay by l a r g e q u a n t i t i e s o f DNA The r e s u l t of the DNA t e s t ( i n d o l e ) i n d i c a t e s t h a t i t i s u n l i k e l y t h a t RNA was p r e s e n t i n any of these f r a c t i o n s . The low DNA v a l u e s g i v e n by the sample w i t h Ischaemic c o l i t i s was p r o b a b l y due to the presence of a contaminant. I t i s p robable t h a t t h i s c o n t a m i n a t i o n produced the f a i n t a l c i a n b l u e p o s i t i v e band ( i n agarose g e l e l e c t r o p h o r e s i s ) a s s o c i a t e d w i t h the second DEAE f r a c t i o n of t h i s sample (see t a b l e 2). Of i n t e r e s t i s the Lowry p r o t e i n a n a l y s i s . 12% Lowry p r o t e i n was found i n a l l human samples except f o r the t h r e e o b t a i n e d from c o l o n s r e s e c t e d f o r u l c e r a t i v e c o l i t i s , and Crohn's d i s e a s e , where o n l y 1 t o 2% Lowry p r o t e i n was d e t e c t e d . T h i s low p r o t e i n v a l u e was v e r y s i m i l a r to t h a t found i n the r e f e r e n c e s t a n d a r d , c a l f thymus DNA, the p r o t e i n of which had been removed by e x h a u s t i v e d i a l y s i s . There i s i n s u f f i c i e n t d a t a i n t h i s study t o draw any c o n c l u s i o n on the s i g n i f i c a n c e of these d i f f e r e n c e s i n Lowry p r o t e i n v a l u e s . 6. Analyses of F r a c t i o n s A The r e s u l t s o b t a i n e d from a n a l y s e s o f these f r a c t i o n s are shown i n t a b l e s 4 and 5. A l l the f r a c t i o n s con-t a i n e d f u c o s e , hexose, hexosamine, p r o t e i n and s i a l i c a c i d . Gas chromatographic a n a l y s i s o f the h y d r o l y z a t e s i n d i c a t e d t h a t Figure 1 1. I l l u s t r a t i o n of a t y p i c a l saponi f i ca t ion time curve i n which a sample of human co lon ic e p i t h e l i a l g lycoprote in , obtained from a s u r g i c a l specimen resected for carcinoma of the co lon , was treated with 0.5% KOH. The amount of t o t a l s i a l i c ac id present was estimated at 0.5, 1 and 2 hour 59 by the periodate r e s o r c i n o l method of Jourdian . Saponif icat ions appeared to be completed at 0.5 hours. 300 g a l a c t o s e was the o n l y hexose p r e s e n t ( f i g u r e 10). The molar r a t i o of the sugars i n each sample i s shown i n t a b l e 4. I n -s i g n i f i c a n t q u a n t i t i e s of phosphorus were d e t e c t e d . 7. S a p o n i f i c a t i o n S t u d i e s When u n t r e a t e d and KOH t r e a t e d samples of the same c o l o n i c e p i t h e l i a l g l y c o p r o t e i n were t e s t e d f o r t o t a l s i a l i c a c i d by the p e r i o d a t e r e s o r c i n a l procedure (PRT) o f 59 J o u r d i a n e t a l the amount of s i a l i c a c i d d e t e c t e d was found to be g r e a t e r i n the f u l l y s a p o n i f i e d sample. T h i s i m p l i e d t h a t some s i a l i c a c i d s which were u n d e t e c t a b l e by PRT i n the n a t u r a l s t a t e were rendered d e t e c t a b l e under a l k a l i n e condi?-t i o n s . I t has been r e p o r t e d t h a t the p e r i o d a t e - r e s o r c i n o l procedure f o r the e s t i m a t i o n o f t o t a l ' s i a l i c a c i d depends upon the r e a c t i o n of the r e s o r c i n a l reagent w i t h an aldehyde group, produced by p e r i o d a t e o x i d a t i o n , a t p o s i t i o n C7 of the s i a l i c 59 68 a c i d s i d e c h a i n ' . S u b s t i t u e n t s p r e s e n t a t e i t h e r C7 and or C8 o f the s i d e c h a i n w i l l t h e r e f o r e prevent the clevage o f the bond between C7 and C8 by p e r i o d a t e o x i d a t i o n , l e a v i n g the s i a l i c a c i d u n r e a c t i v e t o PRT. Consequently, the removal of these s u b s t i t u e n t groups c h e m i c a l l y ( f o r example, treatment w i t h KOH) w i l l render the s i a l i c a c i d s r e a c t i v e to PRT. T h i s can be used t o e x p l a i n the d i s c r e p a n c y i n the amount of s i a l i c a c i d d e t e c t e d i n the u n s a p o n i f i e d and f u l l y s a p o n i f i e d samples o f t h e same c o l o n i c e p i t h e l i a l g l y c o p r o t e i n . S u b s t i t u e n t s which are a l k a l i e - l a b i l e must be p r e s e n t at C7 and or C8 of 5. TABLE 6. Percentage of side chain subs t i tu t ion i n the s i a l i c acids of c o l o n i c e p i t h e l i a l g lycopro te ins . Source of Sample Percentage side chain s u b s t i t u t i o n i n s i a l i c acids NORMAL (Cancer) 49.2 NORMAL 25.0 ULCERATIVE COLITIS 55.4 CROHN'S (Colon) 41.0 CROHN'S (Ileum) 53.1 ISCHEMIC COLITIS 61.0 Note: Colonic e p i t h e l i a l g lycoprote ins were saponi f ied i n 0.5%KOH for 120 minutes. Samples of the f u l l y saponi f ied and the unsaponifiec g lycoprote in were tested for t o t a l s i a l i c ac id by the periodate r e s o r c i n o l procedure of Jourdian (59). The percentage of s ide chain subs t i tu t ion i n s i a l i c ac id i s given by the fo l lowing expressioi c PRT PRT Percentage of , , x - . x side chain = 100 X (sap, sample) - (unsap. sample) subs t i tu t ion PRT (sap. sample) where PRT= periodate r e s o r c i n o l estimate of t o t a l s i a l i c a c i d . some of the s i a l i c ac id side chains of t h i s g l y c o p r o t e i n . Furthermore, ana lys i s (GLC) of the methyl acetate produced by treatment of the g lycoprote in with sodium methox ide i n methanol ind ica ted that a l k a l i n e - l a b i l e 0 -ace ty l groups were present i n these g lycoprote ins . In order to obta in a true estimate of the s i a l i c acids present i n the g lycoprote ins under study, i t i s necessary to remove a l l the subst i tuent groups by a l k a l i n e treatment ( saponif icat ion) before the PRT tes t i s app l i ed . To f ind the optimal time for the a l k a l i n e treatment procedure, each co lon ic e p i t h e l i a l was subjected to a s apon i f i c a t ion time study. A t y p i c a l s apon i f i ca t ion time curve i s shown i n f igure 11. For the g lycoprote ins s tud ied , s a p o n i f i c a t i o n was complete between 30 and 60 minutes. As a r e s u l t , 6 0 minutes was chosen as the optimum time for a l l subsequent s apon i f i ca t ion procedure. Thus, for a p a r t i c u l a r g l y c o p r o t e i n , the t o t a l amount of s i a l i c ac id present i s estimated by the PRT value of a f u l l y saponif ied sample. The di f ference i n PRT values of the f u l l y saponi f ied and the unsaponified g lycoprote in i s a measure of the amount of s i a l i c ac id subst i tuted at e i t h e r C7 or C8. Consequently, the percentage of s i a l i c ac id having s ide-chain s u b s t i t u t i o n at C7 or C8 can be c a l c u l a t e d . The re su l t s obtained with the various c o l o n i c g lycoprote ins under study are tabulated i n table 6. As compared with the normals, an increase i n the percentage of s ide chain s u b s t i t u t i o n of s i a l i c acids was found in a l l the diseased colons . However, more normal colons must be analysed.before conclusions can be drawn. 8. Digest ion Study of V i b r i o Cholera Neuraminidase Digest In an independent experiment c a r r i e d out i n our l aboratory , a sample of ra t c o l o n i c e p i t h e l i a l g lyco-pro te in was incubated with v i b r i o cholera neuraminidase for 48 hours. The amount of free s i a l i c a c id released by the enzyme was estimated by the t h i o b a r b i t u r i c ac id procedure of W a r r e n ^ . The r e s u l t s revealed that less than 30% of the t o t a l s i a l i c ac id present i n the g lycoprote in was re leased . F a i l u r e of the enzyme to br ing about t o t a l re lease of the s i a l i c ac id from the g lycoprote in could be a t t r ibu ted to the presence of subst i tuent groups such as 0 -ace ty l groups at C4 of the s i a l i c a c i d , for i t i s genera l ly be l i eved that s i a l i c acids subst i tuted at C4 are r e s i s t a n t to v i b r i o cholera neuraminidase d ige s t ion ^ . I f t h i s were t r u e , then removal of these subst i tuents should increase the sus*-c e p t i b i l i t y of the g lycoprote in to enzyme d i g e s t i o n . Indeed, when a sample of a f u l l y saponi f ied ra t co lon ic e p i t h e l i a l g lycoprote in was t reated with v i b r i o cholera neuraminidase for 53 hours, about 95% of the s i a l i c acids were released as free s i a l i c a c i d . Thus, i t was concluded that s i a l i c acids subst i tuted at C4 were present i n rat c o l o n i c g lycoprote ins and that s apon i f i ca t ion was necessary to br ing about t o t a l l i b e r a t i o n of N-acetylneuraminic ac id by neuraminidase. Substitution of the s i a l i c acids by a l k a l i n e - l a b i l e groups i s not however confined s o l e l y to p o s i t i o n C4 . As was discussed i n the section on s a p o n i f i c a t i o n , the colonic glycoproteins under study have some s i a l i c acids with 0 - a c e t y l groups at p o s i t i o n C7 and or C8 of the polyhydroxy side chain. The digestion study outlined i n figure 4 was designed to permit a p a r t i a l estimation of the percentage sub s t i t u t i o n occurring at each of positions C4, C7 and C8 on the s i a l i c acid residue. After exhaustive digestion of the glycoprotein with v i b r i o cholera neuraminidase, the incubate was vaccuum f i l t e r e d through a membrane with an exclusion l i m i t of approximately 100,000 M.W., S i a l i c acid released by the enzyme would pass through the membrane and be found as free s i a l i c acid i n the f i l t r a t e . As s i a l i c acids substituted at C4 are r e s i s t a n t to v i b r i o cholera neuraminidase digestion, these free s i a l i c acids would not be expected to contain any substitutents at C4. On the other hand, s i a l i c acids found i n the retentate would s t i l l be bound to the glycoprotein and would therefore be substituted at C4. Quantitatively, the amount of free s i a l i c present i n the f i l t r a t e as estimated by the t h i o b a r b i t u r i c acid t e s t of Warren for free s i a l i c acid (TB) would represent the amount of s i a l i c a cid residues free of substitutents at C4. On the other hand, the amount of s i a l i c acid containing substitutents at C4 would be given by the periodate r e s o r c i n o l t e s t (PRB) for bound s i a l i c acid on the retentate. The sum of the free and bound s i a l i c acids TABLE 7. Subs t i tu t ion patterns of s i a l i c acids i n c o l o n i c e p i t h e l i a l g lycopro te ins . % of s u b s t i t u t i o n occurr ing at pos i t ions ^ C 4 + C 7 ° r C 8 NORMAL (Cancer) 34.0 16.3 18.6 . 31.1 NORMAL 58.1 19.8 16 .8 5.3 ULCERATIVE COLITIS 19 .0 12.0 23.4 45 .6 CROHN'S (Colon) 38.3 9.2 22.8 29.7 ISCHEMIC COLITIS 17.2 10.6 21.7 50.4 Note; sample for Crohn's disease of the ileum was not ava i l ab le for t h i s a n a l y s i s . 56 would give a t o t a l amount of s i a l i c acids present. Hence, the percentage of C4 sub s t i t u t i o n occurring i n the s i a l i c acid residue of a glycoprotein can be calc u l a t e d . Saponified and unsaponified samples of both the f i l t r a t e and retentate were analyzed for t o t a l s i a l i c 59 acid by the periodate-resorcinol procedure of Jourdian et a l The degree of su b s t i t u t i o n at C7 or C8 of the f i l t r a t e and the retentate was then calculated (for discussion see previous sections on s a p o n i f i c a t i o n ) . For the f i l t r a t e (containing s i a l i c acids unsubstituted at C4), two types of s i a l i c acids were found. These were estimated as follows: (a) the PRT value fo r the non-KOH treated sample would represent the amount of unsubstituted s i a l i c acids (unsubstituted at C4, C7 and C8) (b) the difference i n PRT values of the KOH treated and non-KOH treated samples would indicate the amount of s i a l i c acids sub-s t i t u t e d at C7 and or C8. In the retentate, two types of s i a l i c acids substituted at C4 were found and estimated as follows: (a) the PRT value of the non-KOH treated retentate sample indicates the amount of s i a l i c acids with no substitutions at C7 or C8. (b) the difference i n PRT values of the KOH treated and non-KOH treated samples would indicate the amount of s i a l i c acids sub-s t i t u t e d at C4, C7 and or C8. 5: With the procedures employed in this study, i t is not possible to differentiate between substitutents at C7 and C8. Neither can the procedures be u t i l i zed to estimate substitutents at C9. The results of this digestion study, performed on human colonic ep i the l i a l glycoproteins obtained from normal and diseased colons, are as shown in table 7. The s i a l i c acid substitution of colonic glycoproteins obtained from diseased colons appears to be different from the controls. Since the results are based on single specimens the results must be interpreted with a large degree of caution. 9. GLC Analysis of 0-acetyl Groups The number of 0-acetyl groups present in the colonic ep i the l ia l glycoprotein was estimated by GLC as the methyl acetate after trans-esterif ication with 0.1 M sodium methoxide in anhydrous methanol. 0-acetyl groups were found in a l l the samples but the results were inconsistent, and i t was not possible to obtain quantitative data. GENERAL DISCUSSION Fract ions A Fract ions A g lycoprote ins appear to be homogeneous by DEAE chromatography, agarose ge l e lec t rophores i s at pH 8.8 and c e l l u l o s e acetate e lec t rophores i s at pH 8.8. Chemical analys i s of these g lycoprote ins revea l the presence of fucose, galactose , hexosamine, s i a l i c a c i d , p ro te in and l i t t l e or no phosphorus. Q u a l i t a t i v e l y , they resemble g lycoprote ins . , . , - . . . 70 . . 11,16,17,24 i s o l a t e d from g a s t r i c ju i ce , co lon ic mucosa ' n • 4. 4.. 11,17,19,21 . . . 9 , i n t e s t i n e mucosa and r e c t a l mucus , and they are apparently uncontaminated with n u c l e i c a c i d . Since the g lyco-prote ins were extracted from washed e p i t h e l i a l c e l l preparat ions they should be'reasonably free of contaminants such as connective t i s sue components and mater ia l s a r i s i n g from the faeca l stream. Fur ther , s ince p r o t e o l y t i c d ige s t ion was avoided, and r e l a t i v e l y mi ld f r a c t i o n a t i o n techniques were employed, i t i s pos s ib le that an undegraded g lycoprote in was obtained. There appears to be no other report i n the l i t e r a t u r e of the i s o l a t i o n and character" i z a t i o n of co lon ic g lycoprote ins from a purely e p i t h e l i a l source. The data presented i n table 4 shows: (a) that there was a d i f ference i n the molar r a t i o of the sugars of the g lyco-pro te in i s o l a t e d from the normal and diseased human co lons , (b) that i n each of the glycoprotein studied, the four d i f f e r e n t types of s i a l i c acids found, (that i s : (i) unsubstituted or C9 substituted s i a l i c acids ( i i ) s i a l i c acids substituted at C4 and or C9, ( i i i ) s i a l i c acids substituted at C7 and or C8, (iv) s i a l i c acids substituted at C4 and at C7 and or C8) were present i n d i f f e r e n t quantities (see table 7). I t i s tempting to conclude that these differences are re l a t e d to changes associated with the disease processes but there i s c l e a r l y i n s u f f i c i e n t data a v a i l a b l e to support such a conclusion. Under normal conditions, v a r i a t i o n s of the chemical compositions of the colonic glycoproteins might occur i n the following manner; (a) along the length of the colon, (b) from the bottom to the top of the crypts of Lieberkuhn. Thus, the differences found i n t h i s study could be attr i b u t e d to the d i f f e r e n t anatomical region of the colon from which the spec i -mens were obtained rather than to the disease process. Also, i n some experiments not a l l the c e l l s were removed, with those at the base of the crypts the most d i f f i c u l t to remove. I t has been reported that colonic goblet c e l l s mature as they 71 migrate from the bottom to the top of the crypts . Failure-to remove the c e l l s i n the lower h a l f of the crypts i n some experiments may therfore have excluded the majority of young c e l l s . In add i t ion to the above, microheterogeneity can occur i n the normal b iosynthes i s of co lon ic e p i t h e l i a l g lyco-pro te in s . Despite extensive i n v e s t i g a t i o n on the b iosynthes i s 3 of i n t e s t i n a l g lycoprote ins , the d e t a i l e d mechanism remains unknown. In genera l , g lycoprote ins are synthesized i n c o l o n i c 3 4 e p i t h e l i a l c e l l s by the fo l lowing mechanism ' . The peptide chain i s synthesized i n the rough endoplasmic rect iculum according the to Orthodox RNA template theory. Sugars are added i n a step-wise sequence as monosaccharide u n i t s . The enzyme attachment of the f i r s t sugar v i a a sugar nucleot ide transferase proceeds while the peptide i s s t i l l attached to the polyribosomes or s h o r t l y a f te r t h e i r separat ion . The remainder of the o l igosacchar ide chain i s completed inthe smooth membranes and i n the g o l g i apparatus. The g l y c o s y l transferases are s p e c i f i c for both the acceptor and the donor. The order of the sugar sequence depends on the k i n e t i c sequence of the transferases invo lved . For example, s i a l y -transferases require a high molecular weight acceptor and t h i s explains the terminal p o s i t i o n of s i a l i c ac id i n an o l i g o -saccharide cha in . In sp i te of the non-randomness i n the syn-thes i s of the o l igosacchar ide chain as w e l l as the s p e c i f i c i t y of the t ransferases , t h i s mechanism of synthes i s , unl ike the RNA-directed synthesis of p ro te in s , may introduce microhetero-geneity as a r e s u l t of (a) the production of incomplete o l i g o -saccharide chains and (b) the occurrence of i n c o r r e c t mono-saccharide sequence. The re su l t s i n t h i s work may therefore represent microheterogeneity of one or both of these types . TABLE 8. Molar r a t io s of sugar found i n co lon ic e p i t h e l i a l g lycoprote ins expressed as percentages. FUCOSE GALACTOSE HEXOSAMINE SIALIC ACID NORMAL (Cancer) 9.5 29.9 35.0 25.6 NORMAL 12 .2 35.4 31.7 20.7 ULCERATIVE COLITIS 14.9 31.3 31.3 22.4 CROHN'S (Colon) 25.8 34.8 31.0 8.3 CROHN'S (Ileum) 8.2 43.7 17.3 30.6 ISCHEMIC COLITIS 9.1 30.8 30.3 29.9 Note: Glycoprote in samples were t reated with Dowex 50H T in 0.01 N HCl at 80^C. for 36 to 50 hours and the hydrolysates were analysed for 57 fucose and galactose by GLC and for hexosamine by a modi f i ca t ion 58 of a procedure of Winzler . S i a l i c acids were estimated by the 59 periodate r e s o r c i n o l procedure of Jourdian for t o t a l s i a l i c a c i d . The following discussion i s very tentative therefore, and i t i s r e a l i z e d that the differences discussed may be a r e s u l t of normal v a r i a t i o n s and be e n t i r e l y unrelated to the disease process. Under pathological conditions, changes i n the chemical composition of the colonic e p i t h e l i a l glycoproteins found i n t h i s study might be ascribed to one or both of two general classes of changes (a) a l t e r a t i o n s i n the normal biosynthesis of the glycoproteins at d i f f e r e n t stages of the synthesis and, (b) a l t e r a t i o n s i n the normal degradation of the glycoprotein a f t e r synthesis i s complete. In t h i s study, a general increase i n the proportion of C4 substituted s i a l i c acids was found i n glycoproteins i s o -lated from a l l diseased samples (table 7 ). I t i s now generally believed that s i a l i c acids substituted at C4 are r e s i s t a n t 79 to v i b r i o cholera neuramindase digestion . In a pathologxcal environment, constant removal of non - C 4-substituted s i a l i c acids might occur r e s u l t i n g i n the presence of a high proportion of C 4-substituted s i a l i c acids i n the glycoprotein pool. Thus, these could be c a l l e d " r e s i d u a l " glycoproteins. On the other hand, the increase i n C 4-substituted s i a l i c acids may also, be the r e s u l t of the production of "adaptive" glycoproteins i n the e p i t h e l i a l c e l l s . That i s , the glycoproteins synthesized are capable of surviving the pathological environment. A de t a i l e d k i n e t i c study on the biosynthesis of i n t e s t i n a l glyco-protein i n normal and i n pathological conditions would provide information on whether or not such " r e s i d u a l " or "adaptive" glycoproteins are involved i n these disease processes. When the molar r a t i o s of the sugars of the glycoproteins (fucose, galactose, hexosamine and s i a l i c acid) were expressed as percentage values (table 8) i t was found that the amount of galactose and hexosamines were quite constant i n the glyco-proteins obtained from the d i f f e r e n t diseased tissues of the colon. However, the amount of s i a l i c acids and fucose were found to vary from disease to disease. Of p a r t i c u l a r i n t e r e s t was the low percentage of s i a l i c acids found i n the glyco-protein obtained from Crohn's disease of the large i n t e s t i n e . This low percentage value does not explain the slower mobility of the glycoprotein on agarose gel electrophoresis (table 2), since the glycoprotein obtained from Crohn's disease of the terminal ileum which contained three to four times more s i a l i c a c i d, exhibited a s i m i l a r slow electrophoretic mobility. This low mobility however, was not found i n glycoproteins obtained from u l c e r a t i v e c o l i t i s (table 2). The above f i n d i n g , i f confirmed, might aid i n the d i f f e r e n t i a l diagnosis of u l c e r a t i v e c o l i t i s and Crohn's disease of the colon. It i s i n t e r e s t i n g to note that the percentage of non-substituted s i a l i c acid dropped from 58% i n the normal to about 35% i n glycoproteins from patients with Crohn's disease and from patients with carcinoma and to about 18% i n both u l c e r a t i v e c o l i t i s and ischaemic c o l i t i s . This reduction i s counterbalanced by a corresponding increase i n the percentages of s i a l i c acids subst i tuted at C4 or at C7 and or C8. In genera l , the percentage of s ide chain s u b s t i t u t i o n was elevated i n a l l of the diseases studied and s u p r i s i n g l y inthe t i s sues resected for carcinoma. In view of the high r i s k of m a l i -72 gnant change associated with u l c e r a t i v e c o l i t i s and the increas ing number of reports of carcinoma complicat ing Crohn's disease ^ , t h i s f i n d i n g , i f confirmed, might c o n t r i -bute to an understanding of the e t io logy and pathogenesis of these diseases . Teague et a l ^ r e p o r t e d the presence of two glycoprote ins i n human co lon ic mucosa. One of these, excluded from Sepharose 2B, was mannose f ree ; the other which was of molecular weight, and which contained s i g n i f i c a n t quant i t i e s of mannose. Since mucosal scrapings were the source of g lyco-p r o t e i n , contamination of the preparat ions by serum prote ins i s probable. These would be expected to appear i n the included f r a c t i o n obtained from Sepharose 2B and contr ibute mannose to t h i s f r a c t i o n . I t i s s u r p r i s i n g therefore that serum prote ins could not be detected by immunological methods i n e i t h e r of the Sepharose f r ac t ions obtained by Teague et a l " ^ . Traces of mannose were found to be present i n the g lycoprote ins obtained 2 6 from "mucous plugs" by Johansen , and i n the glycopeptides i s o l a t e d by p r o t e o l y t i c d ige s t ion of minced colon by Sky-Peck t e s t i n a l glycc 11,16,17,19-21,2 23 et al . However, the presence of mannose in intestinal glyco-proteins has not been reported by other workers. The a n a l y t i c a l methods employed i n t h i s i n v e s t i -gation w i l l enable the detection of a quantity of mannose as low as 2% by weight of the galactose present, (that i s , approximately 0.2% of the dry weight of the glycoprotein). Mannose could not be detected i n any of the glycoproteins i s o l a t e d i n t h i s study but i t i s possible that a mannose containing glycoprotein was present i n the low molecular weight 'A2 f r a c t i o n ' obtained from the agarose A15M f r a c t i o n a t i o n . This f r a c t i o n probably corresponds to the included Sepharose 2B f r a c t i o n investigated by Teague et a l 1 ^ and i t i s therefore of i n t e r e s t that the 'A2* f r a c t i o n s tested i n t h i s work contained serum proteins. Further Work Proposed As was mentioned previously (see page 59) / colonic e p i t h e l i a l c e l l s progress through a maturation cycle as they 71 migrate from the bottom to the top of the crypts . In order to assess whether there i s a s i g n i f i c a n t v a r i a t i o n i n the chemical composition of the glycoproteins obtained from colonic c e l l s at d i f f e r e n t phases of t h e i r maturation cyc l e , e p i t h e l i a l c e l l s at d i f f e r e n t l e v e l s of the crypt should be i s o l a t e d . Removal of c e l l s from d i f f e r e n t l e v e l s of the crypts i n rat 73 small i n t e s t i n e had been reported by Weimer . Furthermore, i t i s also necessary to investigate any v a r i a t i o n s i n the glyco-proteins along the whole length of the colon. Once the p o s i t i o n a l v a r i a t i o n s i f any are known, p u r i f i e d g l y c o p r o t e i n s should be o b t a i n e d from normal and d i s e a s e d c o l o n s . Chemical a n a l y s e s o f these should be c a r r i e d out t o t e s t the r e p r o d u c i b i l i t y o f the p r e s e n t d a t a . In a d d i t i o n , the amount o f O - a c e t y l groups and the percentage of each type o f amino a c i d p r e s e n t i n these g l y c o p r o t e i n s should be a s c e r t a i n e d and the s t r u c t u r e o f the o l i g o s a c c h a r i d e u n i t s should be e l u c i d a t e d . The r e s u l t s from these i n v e s t i -g a t i o n s would permit a much b e t t e r comparison o f the g l y c o -p r o t e i n s p r e s e n t i n the normal andin the v a r i o u s p a t h o l o g i c a l c o n d i t i o n s . Once p u r i f i e d g l y c o p r o t e i n s from d e l i n e a t e d areas o f each normal and d i s e a s e d c o l o n i c e p i t h e l i u m have been o b t a i n e d these should be used i n the p r e p a r a t i o n o f a n t i s e r a . Such a n t i s e r a would a l l o w an assessment o f the d i s t r i b u t i o n (or c r o s s r e a c t i v i t y ) o f these g l y c o p r o t e i n s i n the g a s t r o i n t e s t i n a l t r a c t , and i n a d d i t i o n , would f a c i l i t a t e the i n v e s t i g a t i o n o f whether o r not the a n t i g e n i c determinants p r e s e n t i n the g l y c o -p r o t e i n s are d i s e a s e s p e c i f i e d . In view o f the d i f f i c u l t i e s e x p e r i e n c e d i n . o b t a i n i n g human samples, m i n i a t u r i z a t i o n o f the f r a c t i o n a t i o n and c h e m i c a l procedures would enable i n v e s t i g a t i o n s t o be c a r r i e d out on s m a l l samples such as b i o p s y specimens. 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APPENDIX Since the completion of the experimental work reported i n t h i s t h e s i s , an independent study c a r r i e d out i n t h i s laboratory has found that . the estimate of the quant i ty of s i a l i c acids subst i tuted at and or Cg i s i n e r r o r . In the PRT assay, the sample under tes t i s f i r s t t reated with p e r i o d i c a c i d . S i a l i c acids unsubst i tuted at and Cg are suscept ible to periodate ox idat ion r e s u l t i n g i n the formation of carbonyl groups at C^. These carbonyl groups react with the r e s o r c i n o l reagent forming a chromogen, with an absorption maxima at 630 m . . S i a l i c acids subst i tuted at C^ and or C g are r e s i s t an t to periodate oxidat ion and i t was assumed there fore , that they were unreactive towards the r e s o r c i n o l reagent .Recently however, i t has been shown that the r e s o r c i n o l reagent reacts with the and or Cg subst i tuted s i a l i c acids (PRS r e a c t i o n ) . This reac t ion gives a s i g n i f i c a n t O.D. reading at 630 m^ j , thereby r a i s i n g the PRT reading a t t r ibu ted to s i a l i c acids unsubst i tuted at C_ and C 0 Thus, the O.D. reading at 630 m i s no longer a true est imation of C^ unsubst i tuted s i a l i c acids and values reported i n tables 6 and 7. need to be r e c a l c u l a t e d . I t should be noted however, that the PRT values for KOH treated samples need not be corrected as none of the s i a l i c acids contain subst i tutents at C^ and would a l l p a r t i c i p a t e i n the PRT r e a c t i o n . The e x t i n c t i o n c o e f f i c i e n t of the PRS reac t ion i s 0.279 8 times t h a t o f the PRT assay. In r e c a l c u l a t i n g the da t a as r e p o r t e d i n t a b l e s 6 and 7, the f o l l o w i n g equator was employed. (a c t u a l ) P R T ( o b s e r v e d , non-KOH) - 0 - 2 7 9 8 P R T ( K O H ) 0 .7202 The r e c a l c u l a t i n g data are as shown i n t a b l e s A and B. A comparison of the o r i g i n a l and r e v i s e d c a l c u l a t i o n s suggests t h a t the o r i g i n a l i n t e r p r e t a t i o n o f the da t a s t i l l h o l d s t r u e . TABLE A. Percentage side chain s u b s t i t u t i o n i n the s i a l i c acids of colonic e p i t h e l i a l glycoproteins. Source of Sample Percentage side chain s u b s t i t u t i o n i n s i a l i c acids NORMAL (Cancer) 6 5 . 9 NORMAL 3 4 . 9 ULCERATIVE COLITIS 8 0 . 0 CROHN'S (Colon) 4 4 . 0 CROHN'S (Ileum) 7 3 . 4 ISCHEMIC COLITIS 8 4 . 7 TABLE B. Subs t i tu t ion patterns of s i a l i c acids i n c o l o n i c e p i t h e l i a l g lycoprote ins . % of s u b s t i t u t i o n occuring at pos i t ions C C7orC8 C4 C4+C7orC8 NORMAL (Cancer) 27.7 22.6 6.5 43.2 NORMAL 50.4 27.5 14.7 7.4 ULCERATIVE COLITIS 14.3 16.7 5.7 63.3 CROHN'S (Colon) 34.7 12.8 11.3 31.2 ISCHEMIC COLITIS 13.1 14.7 2.1 70.0 

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