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Degradation of rat colonic epithelial glycoproteins by cell free extracts of rat faeces Poon, Hiron Hay-Lun 1982

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DEGRADATION OF RAT COLONIC EPITHELIAL GLYCOPROTEINS  BY CELL FREE EXTRACTS OF RAT FAECES by HIRON HAY-LUN POON B. S c , The U n i v e r s i t y of B r i t i s h Columbia, 1980 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept t h i s t h e s i s as conforming to the required standard The U n i v e r s i t y of B r i t i s h Columbia October 1982 0 Hiron Hay-Lun Poon, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) - i i -ABSTRACT The luminal surface of the gastrointestinal t r a c t i s coated with a thick, mucilaginous e p i t h e l i a l glycoprotein containing mucus whose functions are believed to include both l u b r i c a t i o n of the faecal stream and protection of the underlying mucosal e p i t h e l i a l c e l l s . Chemical and histochemical investigations have shown that the colonic e p i t h e l i a l glycoproteins of man and rat contain terminal non-reducing s i a l i c acid residues, the great majority of which have 0-acetyl substituents located at position C4 and/or on the polyhydroxy side chain. Although the b i o l o g i c a l role of such s i a l i c acids i s unknown the observations that (a) 4-0-acetyl s i a l i c acids are resistant to b a c t e r i a l neuraminidases; (b) s i a l i c acids protect some glycoproteins against proteolysis; and (c) most faecal, b a c t e r i a l , enzymes are exo-qlycosidases have led to the suggestion that O-acetylated s i a l i c acids may contribute to the " i n t e g r i t y of the mucus b a r r i e r " by retarding the degradation of the mucus glycoproteins. In the present study, the degradation of the p u r i f i e d colonic e p i t h e l i a l glycoproteins of Wistar rat colon, bovine submandibular mucin, human seromucoid and rat seromucoid, and the hydrolysis of a r t i f i c i a l p-nitrophenyl substrates by the enzymes present i n fresh faeces from Wistar rats i s reported. S t e r i l e , c e l l free, extracts of Wistar rat faeces i n a "minimal media" at pH7.0 contain ( i ) exo-glycosidases capable of hydrolyzing the a-linked L-fucose and the 3 -linked D-galactose, N-acetyl-D-glucosamine and probably - i i i -the N-acetyl-D-galactosamine residues of rat colonic epithelial glycoproteins; ( i i ) neuraminidase(s) capable of removing s i a l i c acids both with or without side chain substituents from bovine submandibular mucin and rat colonic epithelial glycoproteins; and ( i i i ) an esterase which removes 0-acetyl substituents from the side chain of s i a l i c acid residues. Studies of the removal of s i a l i c acids from bovine submandibular mucin and rat colonic epithelial glycoproteins indicated that: (i) the rate of removal of O-acetylated s i a l i c acids was apparently dependent upon the 4-0-acetylated s i a l i c acid content of the substrate; ( i i ) s i a l i c acids were removed more rapidly from de-O-acetylated glycoproteins; and ( i i i ) the faecal enzymes remove a greater proportion of the s i a l i c acid of both the de-O-acetylated and native glycoproteins than was removed with Vibrio cholera neuraminidase. This suggests that the removal of s i a l i c acids i s mediated by the esterase induced de-O-acetylation of 4-0-acetyl s i a l i c acids, together with the exo-glycosidase catalyzed cleavage of carbohydrate residues which sterically inhibit neuraminidase activity. However, the possibility of a neuraminidase capable of cleaving 4-0-acetyl s i a l i c acid residues has not been eliminated. Enzymes were also detected which (i) hydrolyzed p-nitrophenyl glycosides and p-nitrophenyl acetate; and ( i i ) destroyed N-acetyl neuraminic acid. The results of the study demonstrated that the degradation of colonic epithelial glycoproteins by the faecal enzyme extracts from Wistar rat apparently involved the combined actions of glycosidases, neuraminidase, and an esterase. This suggested that changes in either the proportion and/or the activity of the various faecal enzymes could lead to the breakdown of the - i v otherwise normal mucus barrier. Therefore, small changes i n the b a c t e r i a l f l o r a i n disease such as ulcerative c o l i t i s could be of considerable significance. - V -TABLE OF CONTENTS 11 ix xi xiv xv ABSTRACT TABLE OF CONTENTS V LIST OF TABLES LIST OF FIGURES LIST OF APPENDICES ACKNOWLEDGEMENTS 1 ABBREVIATIONS xvi INTRODUCTION 1 I. Objective 1 II. Large Intestinal Glycoproteins 1 (a) Definition of glycoproteins 1 (b) Chemistry of the large intestinal glycoproteins 2 (c) Characteristics and functions of intestinal mucins 5 (d) Degradation of large intestinal mucins 6 III. Alterations of Large Intestinal Mucins in Inflammatory Bowel Disease 8 (a) Ulcerative colitis 8 (1) Definition 8 (2) Changes in epithelial glycoproteins associated with ulcerative colitis 8 (i) Histochemical studies 8 (ii) Chemical studies 9 - v i (b) Carrageenan-induced c o l i t i s i n animals 12 (1) General background 12 (2) Changes i n e p i t h e l i a l glycoproteins associated with carrageenan c o l i t i s 13 IV. Theories of Pathogenesis of Ulcerative C o l i t i s 13 (a) Psychsomatic disease 14 (b) Familial 14 (c) Immunological disorders 15 (d) Mucolytic enzymes , 16 (e) Infection 17 V. The Role of Gut Flora i n Carrageenan-Induced C o l i t i s 19 VI. Rationale for the Present Investigation 20 MATERIALS AND METHODS 21 (I) MATERIALS 21 (II) METHODS 21 A. Preparation of Substrates 22 1. Preparation of Glycoproteins 22 (a) Preparation of human and rat seromucoid 22 (b) Preparation of bovine submandibular glycoproteins (BS4) 22 (c) Preparation of C 4 BSM 23 (d) Preparation of rat colonic e p i t h e l i a l glycoproteins 23 (i) Isolation of colonic e p i t h e l i a l c e l l s 23 „ - v i i -(ii) Extraction of colonic epithelial glycoproteins 26 ( i i i ) Purification of colonic epithelial glycoproteins 26 (e) Preparation of saponified glycoproteins 31 2. Preparation of p nitrophenyl substrates 31 B. Preparation of Rat Faecal Enzymes 31 (II) Analytical Methods 36 1. Colorimetric Analyses 36 (a) Estimation of free and ketosidically bound sialic acids 36 (b) Estimation of the percentage of sialic acids substituted at positions C 7 and/or C Q 37 (c) Estimation of the percentage of sialic acids substituted at position C^ 38 (d) Determination of enzymatic activities towards p-nitrophenyl substrates 39 (e) Determination of units of activities of neuraminidase and NANA-degrading enzyme AO 2. Qualitative Identification of Sugars by Paper Chromatography 40 3. Quantitative Analysis of Sugars by Gas-Liquid Chromatography 41 4. Bacteriological Studies 41 (III) GENERAL EXPERIMENTAL DESIGN 42 RESULTS 48 (I) PRELIMINARY EXPERIMENTS 48 (a) Preparation of glycoprotein substrates 48 - v i i i -(b) Effects of minimal media on the colorimetric analysis of s i a l i c acids 59 (c) Effects of heating at b o i l i n g water bath on s i a l i c acids 62 (d) Determination of a c t i v i t i e s of glycosidases and esterase 62 (i ) Glycosidases 62 ( i i ) Esterase 62 (e) Determination of a c t i v i t i e s of neuraminidase and NANA-degrading enzyme 65 ( i ) Neuraminidase 65 ( i i ) NANA-degrading enzyme 65 (f) Qualitative i d e n t i f i c a t i o n of sugars by paper chromatography 65 (II) KINETIC STUDIES 69 (a) Neuraminidase 69 (b) De-O-Acetylase 83 ( i ) Percentage of Cy+Cg substituted s i a l i c acids 83 ( i i ) Percentage of C^ substituted s i a l i c acids 100 (c) NANA-Degrading Enzyme 112 (d) Glycosidases 112 (e) Bacteriological Studies 116 DISCUSSION 127 FUTURE STUDIES 146 REFERENCES 148 APPENDICES 157 - i x -LIST OF TABLES TABLE PAGE 1 Chemical composition and characteristics of e p i t h e l i a l glyco-proteins isolated from the upper and lower halves of the Wistar rat colons 4 2 Histochemical alterations of the colonic mucosubstance i n ulcerative c o l i t i s 10 3 Chemical changes of large i n t e s t i n a l glycoproteins i n ulcerative c o l i t i s 11 4 Types of substrates used i n the study of degradation of glyco-proteins by rat faecal enzymes 32 5 Mo l a r i t i e s of p-nitrophenyl substrates used 33 6 Percentage of s i a l i c acids with 0-acetyl substituents at positions C4 and C7+C8 i n the glycoproteins used as substrates for the study of rat faecal enzymes 60 7 Effects of minimal media on the colorimetric analysis of free and bound s i a l i c acids 61 8 Qualitative i d e n t i f i c a t i o n of sugars by paper chromatography 68 9 Percentage of s i a l i c acids released by digestion of the saponified and non-saponified glycoproteins from the upper and lower halves of Wistar rat colon with c e l l free f i l t r a t e (rat enzymes) of Wistar rat faeces 71 10 Percentage of s i a l i c acids released by digestion of human sero-mucoid and saponified and non-saponified samples of bovine submandibular mucin and rat seromucoid by rat faecal enzymes 72 11 S t a t i s t i c a l analysis of the s u s c e p t i b i l i t y of each glycoprotein to rat faecal neuraminidase 74 12 Percentage of s i a l i c acid residues removed from glycoprotein substrates by incubationg with v i b r i o cholera neuraminidase and by rat faecal enzymes 84 - X -TABLE PAGE 13 Percentage of sialic acids remaining substituted at positions C4 and C7+C8 of the rat colonic epithelial glycoproteins after digestion with rat faecal enzymes 85 14 Percentage of sialic acids remaining substituted at positions C4 and C7+C8 of the bovine submandibular mucin (BSM), C^B3A and rat seromucoid, after digestion with rat faecal enzymes 87 15 The percentage of C4 and side chain de-0-acetylation from sialic acids of various glycoprotein substrates 92 16 Statistical analysis of the rates of C4 and C7+C8 de-O-acetylation from different glycoprotein substrates 94 17 Distribution of the various populations of free and glycosidically bound sialic acids which are either side chain substituted or which have no side chain substituents 96 18 Distribution of the various populations of free and glycosidically bound sialic acids which are either side chain substituted or ' which have no side chain substituents 98 19 Mean specific activities of glycosidases detected in the rat faecal enzyme extracts 115 20 Hydrolysis of sugars by rat faecal enzymes from the rat upper colonic epithelial glycoprotein 117 21 Hydrolysis of sugars by rat faecal enzymes from the rat lower colonic epithelial glycoprotein 118 - x i -LIST GF FIGURES FIGURE PAGE 1 S i a l i c acid (N-acetyl neuraminic acid) 3 2 Flow diagram of the method for the i s o l a t i o n of intact rat colonic e p i t h e l i a l c e l l s 3 Flow diagram of the method for the extraction of rat colonic e p i t h e l i a l glycoproteins 4 Flow diagram of the method for the p u r i f i c a t i o n of rat colonic 6 Analysis of the degradation of glycoproteins by rat faecal enzymes 43 7 Flow diagram of the method for the preparation of samples for gas-liquid chromatographic analysis of hexosamines and neutral sugars 46 8 Fractionation of the crude rat upper colonic e p i t h e l i a l glyco-protein on Agarose A15M gel 49 9 DEAE cellulose ion-exchange column chromatography of the s i a l i c a c id-rich fractions (SA) obtained from Agarose A15M gel of crude rat upper colonic e p i t h e l i a l glycoprotein 51 10 Fractionation of the crude rat lower colonic e p i t h e l i a l glyco-protein on Agarose A15M gel 53 11 DEAE cell u l o s e ion-exchange column chromatography of the s i a l i c a c i d - r i c h fractions (SA) obtained from Agarose A15M gel of crude rat lower colonic e p i t h e l i a l glycoprotein 55 12 Electrophoretic patterns of crude and pu r i f i e d Wistar rat colonic e p i t h e l i a l glycoproteins 57 13 Hydrolysis of p-nitrophenyl acetate by minimal media and rat faecal enzyme extracts 63 14 Separation and i d e n t i f i c a t i o n of sugars present i n the faecal enzyme digest by paper chromatography 66 e p i t h e l i a l glycoproteins 29 5 Preparation of rat faecal enzymes 34 - x i i -FIGURE PAGE 15 Degradation of saponified and non-saponified rat upper glyco-protein by rat faecal enzymes 75 16 Degradation of saponified and non-saponified rat lower glyco-protein by rat faecal enzymes 77 17 Degradation of saponified and non-saponified bovine submandibular mucin by rat faecal enzymes 79 18 Hydrolysis of s i a l i c acid residues from saponified and non-saponified rat seromucoid and non-saponified human seromucoid by rat faecal neuraminidase 81 19 Removal of 0-acetyl groups from positions C7+C8 of s i a l i c acid residues of BSM and rat colonic e p i t h e l i a l glycoproteins by rat faecal enzymes 89 20 Digestion of rat upper e p i t h e l i a l glycoprotein by rat faecal enzymes 101 21 Digestion of rat lower e p i t h e l i a l glycoprotein by rat faecal enzymes 103 22 Digestion of bovine submandibular mucin by rat faecal enzymes 105 23 Removal of 0-acetyl groups from position C4 of s i a l i c acid residues of BSM and rat colonic e p i t h e l i a l glycoproteins by rat faecal enzymes 107 24 Removal of s i a l i c acid residues from saponified and non-saponified C4BSM by rat faecal enzymes 110 25 Degradation of N-acetyl neuraminic acid by NANA-degrading enzyme present i n the rat faecal enzyme extracts 113 26 Hydrolysis of s i a l i c acid, fucose, galactose, and N-acetyl glucosamine from the saponified rat upper glycoprotein by rat faecal enzymes 119 27 Hydrolysis of s i a l i c acid, fucose, galactose, and N-acetyl glucosamine from the non-saponified rat upper glycoprotein by rat faecal enzymes 121 - x i i i -FIGURE PAGE 28 Hydrolysis of s i a l i c a cid, fucose, galactose, and N-acetyl glucosamine from the saponified rat lower glycoprotein by rat faecal enzymes 123 29 Hydrolysis of s i a l i c acid, fucose, galactose, and N-acetyl glucosamine from the non-saponified rat upper glycoprotein by rat faecal enzymes 125 30 Schematic of the possible d i s t r i b u t i o n of sugar residues on the oligosaccharide chains of Wistar rat colonic e p i t h e l i a l glyco-proteins 135 31 Diagrammatic representation of the scheme proposed for the hydrolysis and degradation of side chain and C4 substituted s i a l i c acids from rat colonic e p i t h e l i a l glycoproteins 138 32 Diagrammatic i l l u s t r a t i o n of the possible role of the e p i t h e l i a l glycoprotein degrading enzyme system i n the pathogenesis of ulcerative c o l i t i s 142 - xiv -LIST OF APPENDICES APPENDIX PAGE Al Chemical composition of Purina Formulab Chow #5008 157 A2 Chemical composition of minimal media 158 A3 Chemical composition of 0.01% phosphate-buffered saline (PBS-EDTA) solution 159 A4 Equation E l for the calculation of the percentage of side chain and C4 de-0-acetylation from glycoprotein substrates 160 A5 Derivation of equations for the estimation of the concentrations of both free and g l y c o s i d i c a l l y bound s i a l i c acids with or without side chain substituents 161 - XV -ACKNOWLEDGMENTS My thanks and gratitude to my supervisor, Dr. P h i l i p E. Reid, who has made t h i s work possible through his advice, guidance, and continuous support. Thanks are also extended to members of my research committee, Dr. J. Anderson, Dr. M. Bernstein, the l a t e Professor C.F.A. Cu l l i n g , Dr. H. Freeman, and Dr. 3. Frohlich, for t h e i r support and the many valuable suggestions. I would l i k e to thank Mrs. Janet Reid for proof-reading the manuscript. Special thanks to Mr. Charles Ramey for part i c i p a t i n g i n many valuable discussions and for providing technical help. I am also indebted to Dr. Ann G. Skidmore, Division of Medical Microbiology, Acute Care Hospital, University of B r i t i s h Columbia, for performing the bacteriological studies. I would also l i k e to thank the Department of Pathology for allowing me to use the WANG word processor i n the preparation of t h i s thesis. This research project was supported by an operating grant from the Medical Research Council of Canada. I also wish to thank the Canadian Foundation for I l e i t i s and C o l i t i s for the award of the f i r s t Alexander Finkelstein Summer Research Scholarship and the University of B r i t i s h Columbia for the award of a Graduate Summer Scholarship. - xvi -ABBREVIATIONS Rat Upper Glycoprotein = Epithelial glycoprotein isolated from the upper half of Wistar rat colon Rat Lower Glycoprotein = Epithelial glycoprotein isolated from the lower half of Wistar rat colon BSM = Bovine submandibular mucin BSM = BSM digested exhaustively with Vibrio cholera neuraminidase C Q Sialic acids = Free and/or bound sialic acids which do not contain any 0-acetyl substituent Sialic acids = Sialic acids which are 0-acetyl substituted at position C^ . C7+Cg Sialic acids = Free and/or bound sialic acids which are side chain substituted with 0-acetyl groups NANA = N-acetyl neuraminic acid PRT = Periodate resorcinol assay PRS = Periodate resorcinol assay without the periodate oxidation step TBA = Thiobarbiturate assay for free sialic acid PRBA = Periodate resorcinol assay for bound sialic acid - xvi i -"Making many books there is no end; and much study is a weariness of the flesh. Let us hear the conclusion of the whole matter: Fear God, and keep His commandments; for this is the whole duty of man. " Ecclesiastes 12:12-13 - xv i i i -- 1 -INTRODUCTION I. Objective The purpose of the investigation described i n t h i s thesis was to study the ki n e t i c s of the degradation of colonic e p i t h e l i a l glycoproteins, isolated from the male Wistar r a t , by c e l l - f r e e extracts of Wistar rat faeces. The significance of t h i s study with respect to the possible role of gastrointest-i n a l bacteria i n the pathogenesis of ulcerative c o l i t i s w i l l be discussed. I I . Large I n t e s t i n a l Glycoproteins (a) D e f i n i t i o n of glycoproteins Glycoproteins are glycoconjugates which have carbohydrates covalently linked to proteins (1). The proportion of carbohydrate i n different glycoproteins ranges from 2-3% to up to 90% of the t o t a l weight (2). Glycoproteins d i f f e r from proteoglycans, another subclass of glycoconjugates, i n t h e i r carbohydrate side chains (3). The carbohydrates i n glycoproteins consist of many highly branched oligosaccharide chains without repeating units while proteoglycans contain long chain carbohydrates with repeating disaccharide units (3). The two substances also occur i n different tissues. Glycoproteins are present i n body f l u i d , seromucous secretions as well as on the surface of c e l l membranes whereas proteoglycans occur mainly i n sk e l e t a l and other supportive tissues (4). - 2 -(b) Chemistry of the large i n t e s t i n a l glycoproteins The chemistry of glycoproteins has been reviewed extensively by Gottschalk (1), Sharon (3), and Lennarz (4), Clamp (5). The type of glycoprotein that w i l l be discussed i n t h i s thesis i s the mucous type produced and secreted by the large 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 . Although the i n t e s t i n a l glycoproteins have no unique amino acid composit-ion, they c h a r a c t e r i s t i c a l l y contain large quantities of serine, threonine, and proline and only small quantities of arginine, l y s i n e , and aromatic amino acids. The carbohydrate prosthetic groups share certain monosaccharides i n common, fucose, galactose, N-acetyl glucosamine, N-acetyl galactosamine and s i a l i c acid (6). Mannose i s reportedly to be absent i n most i n t e s t i n a l glycoproteins (7). The s i a l i c acids, the N-acetyl or N-glycolyl derivative of the parent neuraminic acid, are located terminally at the non-reducing end of the carbohydrate prosthetic groups of many i n t e s t i n a l glycoproteins (1,3,6). I t has been shown that fucose and other sugars may occupy the terminal position of certain oligosaccharide chains (7-8). N-Acetyl neuraminic acid (NANA, s i a l i c acid) i s a 9-carbon ketulosonic acid (figure 1). In i n t e s t i n a l mucins, s i a l i c acids may or may not be substituted with 0-acetyl groups at position C^ and/or at various positions on the polyhydroxy side chain ( i . e . Cj, C g, and/or C^) (9-10). NANA with an a x i a l negatively charged carboxylic acid group i s believed to play an important role i n establishing cross-linkages between the glycoprotein molecules and hence i n the promotion of molecular r i g i d i t y (11-12). The glycoproteins produced by the upper and lower halves of the Wistar rat - 3 -Figure 1: Sialic acid (N-acetyl neuraminic acid). O-Acetyl groups may or may not present at positions C4, C7, CQ, and/or C9 of the sialic acid. - 3 - ^  Figure 1; S i a l i c acid (N-acetyl neuraminic acid). 0-Acetyl groups may or may not present at positions C 4 , C 7 , C 8 , and/or C9 of the s i a l i c acid. H 1 rv •0 •OP. •OK 9' CH,OH D I U 7H—OH 8|—OR 9' CH20H 6 I 0 7 j—OH 8 I— 9 ' CH,CR C - = unsubstituted s i a l i c acids - 4 -Table 1: Chemical composition and characteristics of epithelial glyco-proteins isolated from the upper and lower halves of the Wistar rat colons. MOLAR RATIO* Rat Upper Rat Lower Fucose 1.00 1.00 Galactose 2.00 2.40 Glucosamine 0.97 0.80 Galactosamine 1.55 1.32 Sialic acid 1.17 1.20 PERCENT DRY WEIGHT* Neutral sugars 21.53 20.98 Hexosamines 22.81 16.58 Sialic acid 14.89 13.07 Proteins 13.69 14.42 Note: *Unpublished data (13). - 5 -colons have different chemical characteristics (Table 1) (9,13). The percentage composition of the sugars present i n the two glycoproteins are s l i g h t l y different. The e p i t h e l i a l glycoprotein of the upper colon has more side chain (C7+Cg) and substituted s i a l i c acids than that of the rat lower colon. Since Vibrio cholera neuraminidase cannot hydrolyze substituted s i a l i c acids from i n t a c t glycoproteins (10,14), the glycoprotein from the upper colon i s more neuraminidase resistant than that from the rat lower colon. The possible protective role of the side chain 0-acetyl groups to neuraminidase digestion has been investigated by Schauer and Failland (15). They found that i n bovine submandibular mucin the side chain substituted s i a l i c acids were susceptible to Vibrio cholera neuraminidase but the unsubstituted s i a l i c acids were degraded faster. (c) Characteristics and functions of i n t e s t i n a l mucins The i n t e s t i n a l mucins are glycoproteins which are produced and secreted by specialized types of e p i t h e l i a l c e l l s called goblet c e l l s which are located throughout most of the length of the gas t r o i n t e s t i n a l t r a c t . Once the glycoproteins are secreted by the goblet c e l l s , they are incorporated into the mucus layer, a complex sheet of proteins, glycoproteins, c e l l s , bacteria, food debris, ions and water, covering and protecting the underlying epithelium (5). The functions of the ga s t r o i n t e s t i n a l mucins are not t o t a l l y understood but they are assumed to serve as ( i ) a lubricant for the mucous membrane (2,12,16); and ( i i ) a barr i e r against (a) physical invasion by bacteria and - 6 -(b) enzymatic digestion of the underlying tissues by the digestive j u i c e (1,17-20). The r e l a t i v e resistance to proteolysis of the mucins i s believed to be due to s i a l i c acid residues (1,19-22). I t has been speculated that the colonic mucins are partly protected against b a c t e r i a l degradation by the presence of neuraminidase resistant 0-acetyl s i a l i c acids (12,23-25). Thus, the i n t e g r i t y of the carbohydrate moiety may be essential for the survival of the glycoproteins, which i n turn protects the underlying epithelium. (d) Degradation of large i n t e s t i n a l mucins The ga s t r o i n t e s t i n a l t r a c t s of man and animals are populated by a large number of bacteria. These bacteria are i n close contact with both the faecal materials and the i n t e s t i n a l mucosa. The establishment of a normal gut f l o r a i s quite important for the well-being of the host. Not only do the established colonies l i m i t the growth of some pathogens, they also i n d i r e c t l y provide some of the nutrients for the e p i t h e l i a l c e l l s (26-27). Since the gut f l o r a and the host's i n t e s t i n a l mucosa can be said to exist i n a "symbiotic" state, i t i s r a t i o n a l for one to assume that these bacteria w i l l u t i l i z e both the faecal materials and the i n t e s t i n a l mucins as the i r sources of n u t r i t i o n . Lindstedt et al.(28) found that, as compared to conventional rats, germ free rats had an increased amount of proteins and hexosamines i n the caecal contents. The e p i t h e l i a l layer of the caecal wall i n the germ free rats were also found to be "thicker" than that from the conventional rats. Hoskins et al.(29) found that the mucous constituents i n - 7 -the stools of germ-free and conventional rats were different. Germ-free rats excreted more proteins (1.6x), hexoses (9.6x), hexosamines (7.4x), and s i a l i c acids (90x) than conventional animals. When the germ-free animals were "innoculated" with the normal f l o r a , the faecal weight and composition became the same as that of the conventional rats. Hoskins et al.(30-32) also demonstrated that the gastrointestinal glycoproteins possess ABO blood group a c t i v i t y and that the i n t e s t i n a l micro-organisms, not the host's tissues, produce faecal blood group destroying enzymes. Prizont e_t a l . (33) have i d e n t i f i e d various b a c t e r i a l glycosidases i n rat caecal contents. These glycosidases, blood group destroying enzymes, and mucin-destroying enzymes could be detected i n both the faecal contents of rats and human beings (33-38). Furthermore, these enzymes were believed to be produced by a small percentages (approximately 1%) of the t o t a l faecal b a c t e r i a l population (36-38). The evidence that has been gathered to-date undoubtedly indicates that the enteric micro-flora produce the enzymes necessary for degrading the carbohydrates of macromolecules i n the gut lumen. The significance of these findings, although not f u l l y understood at present, may provide us with a means of determining whether or not there i s an association between ba c t e r i a l enzymes and i n t e s t i n a l disorders such as inflammatory bowel disease. - 8 -I I I . Alterations of Large I n t e s t i n a l Mucins i n Inflammatory Bowel Disease (a) Ulcerative c o l i t i s (1) D e f i n i t i o n Ulcerative c o l i t i s (UC) i s a chronic inflammatory disease of unknown etiology (39) primarily affecting the colon. Ulceration usually starts from the d i s t a l part of the colon and progresses to the ascending colon. Histopathologically, ulceration occurs mainly at the mucosal layer and only i n acute stages w i l l i t extend beyond the muscularis mucosa. UC i s characterized by remissions and exacerbations. Patients suffering from the disease have an increased chance of developing colonic carcinoma (40-43). Ulcerative c o l i t i s can be acquired at any age. The age d i s t r i b u t i o n of the patients with t h i s disease, however, has a bimodal pattern with the largest number of people affected between 15-20 and 55-60 (44). This pattern can be observed among both sexes. A recent study by Garland et_ al_. (45) on the incidence of UC i n 15 areas of the United States showed that the bimodal age d i s t r i b u t i o n i n white males (20-29 & 70-79) diffe r e d from that i n females (30-39 & 70-79). The disease i s more common amongst whites, urban dwellers, and certain ethnic groups such as Jews (46). The significance of the age, sex, ethnic and geographic d i s t r i b u t i o n of patients with UC, although i t may have important e t i o l o g i c a l implications, i s not yet f u l l y understood. (2) Changes i n e p i t h e l i a l glycoproteins associated with ulcerative c o l i t i s ( i ) Histochemical studies Changes i n the histochemical pattern of the colonic glycoproteins have - 9 -been demonstrated i n association with ulcerative c o l i t i s (table 2) (47-55). F i l i p e et a l . (49,56) found that i n severe cases of ulcerative c o l i t i s , there was a diminution of sulphated and acid non-sulphated mucosubstances. There was also a s i g n i f i c a n t reduction i n the quantity of mucin i n the goblet c e l l s . Culling et a l . (54-55,57) have shown that i n ulcerative c o l i t i s there i s , as compared to normal, a reduction i n the degree of side chain substitution of the s i a l i c acids of the e p i t h e l i a l glycoproteins. However, the significance of these changes remains unknown, ( i i ) Chemical studies Table 3 summarizes the results of the chemical studies of colonic e p i t h e l i a l glycoproteins i n ulcerative c o l i t i s (57-62). Teague et al.(59) observed that fractionation of colonic mucus, isolated from mucosal biopsies obtained from individuals with i n t e s t i n a l disorders and from normal ind i v i d u a l s , with Sephedax G50 and Sepharose 2B yielded an included and an excluded fraction. Both fractions contained fucose, galactose, mannose, N-acetyl glucosamine, N-acetyl galactosamine, and s i a l i c acid. There was more mannose i n the included f r a c t i o n , and the amount of mannose i n t h i s fraction was s i g n i f i c a n t l y increased i n ulcerative c o l i t i s . Fraser et_ a l . (60) studied the glycoproteins isolated from mucosal scrapings of both normal colons and colons from ulcerative c o l i t i s patients, the mucous glycoproteins were found to contain increased quantities of mannose and s i g n i f i c a n t l y smaller quantities of the amino acids threonine and serine. In organ culture experiments, McDermott et al.(61) demonstrated an increase i n synthesis and secretion of glycoproteins i n r e c t a l e p i t h e l i a l c e l l s isolated Table 2: Histochemical a l t e r a t ion of the colonic mucosubstance i n ulcerative c o l i t i s , (reproduced from 70) I n v e e t i g a t o r ( s ) S e v e r i t y K e u t r a l Mucins Observations Sialomucins Sulphomucins Remarks. HardsVy et a l . (4r7 ) mucus substance e l t h i tv decreased or absent Creco et a l . (48) m i l d aevere present (goblet c e l l of sigmoid) absent present absent decreased absent F i l i p e (49) absent trace decreased c o r r e l a t e w i t h the s e v e r i t y of the disease Cad (50) moderate severe moderately increased decreased increased decreased Lev (51) e a r l y stage moderate reduced increased reduced reduced adjacent to u l c e r a t i o n F i l i p e & Dawson aoderate reduction aoderate reduction c o r -(52) r e l a t e with the s e v e r i t y of the inflammatory response i n lamina p r o p r i a H e l l a t r o a 6 Fisher mucosubstance e i t h e r moderately decreased or p a r t i a l l y preserved C u l l i n g et a K reduction i n the 0-acetyl substituted s i a l i c a c i d most evident at the u l c e r (54,55) margins Table 3: Large i n t e s t i n a l glycoproteins and u lcerative c o l i t i s : chemical investigations, (reproduced from 70) I n v e s t i g a t o r ( s ) Region of Methodology and Obaervations Remarks large i n t e s t i n e Kemarxs Sorgel & I n g e l -f i n g e r (58) rec t a l i I I l g ? t l o n Y l t h Vpertonic phosphate solution} (a) nitrogen contents 50Z higher i n u l c e r a t i v e c o l i t i s ; (b) immunological studies -u l c e r a t i v e c o l i t i s glycoproteins d e f i c i e n t i n a and 0 g l o b u l i n s Teague et a l . (59) colon scrapings - mucus biopsies - fractionated w i t h Sephadex G50 and with Sepharose 2B; two f r a c t i o n s (a) included (b) excluded} There was more mannose i n the included f r a c t i o n and the amount of mannose was s i g n i f i c a n t l y increased i n u l c e r a t i v e c o l i t i s F r a s e r et a l . (60) colon mucosal scrapings; (a) increased mannose, (b) s i g n i f i c a n t l y smaller q u a n t i t i e s of threonine and serine, therefore u l c e r a t i v e colitis associated with changes In glycoprotein composition Involving a fewer number of carbohydrate prosthetic groups MacDermott et a l . ( 61 ) r e c t a l e p i t h e l i a l c e l l s organ c u l t u r e , u l c e r a t i v e c o l i t i s - epithelial cells - e x h i b i t an increased glycoprotein synthesis and secretion ' Reid «t a l . (6£> formaline f i x e d colon ti a s u e specimens from u l c e r a t i v e c o l i t i s patients - i s o l a t e d glycoproteins} (a) s i a l i c acids - s i g n i f i c a n t l y reduced 0-acetyl s u b s t i t u t e (at C4 and at polyhydroxy side chain)} (b) a l t e r a t i o n s with f u c o s e / s i a l i c acid, gaiactoae/fueose and glucosaoine/galactosamine molar r a t i o s - 12 -from UC patients. Reid et al.(62) have shown that as compared to normal the e p i t h e l i a l glycoproteins p u r i f i e d from the formalin fixed specimens of the affected colons from ulcerative c o l i t i s patients contained s i a l i c acids with s i g n i f i c a n t l y less 0-acetyl substituents (C^ and Cy+Cg). In addition, the molar r a t i o s of fucose to s i a l i c acids, galactose to fucose and glucosamine to galactosamine were different from e p i t h e l i a l glycoproteins obtained from the resection margins ( h i s t o l o g i c a l l y normal) of cases of colonic carcinomas. (b) Carrageenan-induced c o l i t i s i n animals (1) General background 5 Native carrageenans (1-8x10 MW) can be extracted from many species of red seaweeds (63). They are sulphated polysaccharides composed of D-galactose and 3,6 anhydro-O-galactose. Degraded carrageenan (30,000 MW) can be prepared by mild acid hydrolysis of the native molecules (63). Marcus and Watt (64-67) and others (68-69) have demonstrated that both the native and degraded forms of carrageenan cause large bowel ulceration i n various types of laboratory animals. The carrageenan induced ulceration i n rabbits and guinea pigs (64, 66-70) i s well documented but the effect of the molecule on rats and mice i s controversial (61,68-69,71). The v a l i d i t y of carrageenan c o l i t i s as an experimental model for human ulcerative c o l i t i s has been discussed elsewhere (71-73). This experimental disease has been employed as a model system for the study of large bowel ulceration (63-73). - 13 -(2) Changes i n e p i t h e l i a l glycoproteins associated with carrageenan c o l i t i s The chemical, histochemical and h i s t o l o g i c a l changes associated with the degraded carrageenan-induced large bowel ulceration i n rabbits have been studied extensively by Al-Suhail et al.(70,74-75). His results indicate that degraded carrageenan-induced large bowel ulceration of rabbits was accompanied by a reduction i n the percentages of C^ and side chain substituted s i a l i c acids of the e p i t h e l i a l glycoproteins. This change preceded both the mucosal ulceration and the presence of a s i g n i f i c a n t inflammatory response. The reduction of neuraminidase resistant s i a l i c acids i n carrageenan-c o l i t i s presumably renders the glycoproteins more prone to b a c t e r i a l degradation. This would lead to the breakdown of the mucous barrier and consequently to the direct exposure of the l i n i n g epithelium to the b a c t e r i a l f l o r a and t h e i r metabolic products which could lead to tissue injury and subsequently an inflammatory response. Ulceration could result from the "over-reaction" of the host's tissues and/or from the dir e c t actions of the ba c t e r i a l f l o r a (e.g. production of toxins). IV. Theories of Pathogenesis of Ulcerative C o l i t i s : Ulcerative c o l i t i s was recognized over a century ago but the etiology of the disease remains unknown (76-79). The p r i n c i p a l hypotheses which have been advanced to explain the development of c o l i t i s can be c l a s s i f i e d as follows: - 14 -(a) Psychosomatic disease This hypothesis was f i r s t proposed by Murray i n 1930 (80). The argument for t h i s theory was that UC patients often had different behaviour patterns from normal individuals. They were apt to be i n f a n t i l e , dependent, passive, egocentric, hesitant, and uncertain. I t was c h a r a c t e r i s t i c of them that they "bottled-up" t h e i r feelings. This theory was quite popular several decades ago but i t has not gained a general acceptance from c l i n i c i a n s . There are undoubtedly changes i n the attitude of l i f e of patients during severe attacks of c o l i t i s . I t i s not known, however, whether these changes i n behaviour are the r e s u l t s of the disease or vice versa. To-date there i s l i t t l e evidence that psychogenic factors are responsible for the i n i t i a t i o n of ulcerative c o l i t i s . However, the evidence seems to suggest that emotional factors may be important i n maintaining or prolonging an existing attack of c o l i t i s , or even that they may participate i n the exacerbation of the disease. (b) Familial The concept of an i n d i v i d u a l predispostion to ulcerative c o l i t i s was suggested by Kirsner and Palmer (81). The f a m i l i a l incidence i s estimated to be about 20 percent, first-degree r e l a t i v e s rather that second-degree r e l a t i v e s being more vulnerable. - 15 -Ulcerative c o l i t i s has also been reported to have association with other genetic disorders, p a r t i c u l a r l y ankylosing spondylitis (AA). The observation that certain races of people are more prone to contract the disease i s also noteworthy (A6). There i s no consistent association between s p e c i f i c HLA antigens and ulcerative c o l i t i s (82). The existence of certain HLA l o c i which are "high r i s k " for UC remains controversial (AO,79,83-85). (c) Immunological disorders The possible role of a fundamental immune mechanism i n the pathogenesis of ulcerative c o l i t i s was f i r s t discussed by Kirsner and Palmer (81). Occasional association of UC with immunological disorders such as i r i t i s , erythema nodosum, pyoderma gangrenosum, autoimmune hemolytic anaemia, etc. (AA,78-79,86), are consistent with the idea that some form of immunological disorder i s present i n certain UC patients. At one time ulcerative c o l i t i s was thought to be caused by tissue hypersensitivity or a l l e r g i c reactions to certain food antigens. Although the increased histamine levels noted i n r e c t a l mucosa of some patients with UC are compatible with an a l l e r g i c reaction (87), attempts to treat UC patients with disodium cromoglycate, an agent used i n the treatment of asthma to s t a b i l i z e mast c e l l s , were unsuccessful (88). Further, there are also no deficiencies i n the major complement components or t h e i r regulators i n UC (89-90). Immunodeficiency i s also not associated with patients with UC although the disease occur i n occasional patients with immunoglobulin deficiencies (91-96). - 16 -Circulating antigen-antibody complexes are present in some UC patients (97-99). Their manifestation does not correlate, however, with the severity or location, of the disease. Although such complexes are often linked to systemic complications (79), the antigenic components of the complexes are unknown. The origin of these antigens may be important. Kirsner has pointed out that "gut-derived immune complexes could contribute to the intestinal inflammation in ulcerative c o l i t i s by activating the complement sequence or by ini t i a t i n g lymphocytoxicity against intestinal c e l l s " (79). Serum from patients with UC may contain "anticolon antibodies" (100-103). These antibodies react in vitro with mucus-secreting colonic epithelial c e l l s , allogeneic foetal colonic tissues, and colonic epithelium from germ-free rats (79,104). It i s known that E. c o l i 014 present in human gut flora contains antigenic determinants which cross-react with colonic epithelium (104). It i s not known, however, whether or not intestinal mucosal damage i s a result or a consequence of the "anti-colon antibodies". The evidence to-date does not establish a disorder of immunologic mechanism as the primary cause of ulcerative c o l i t s . The impaired immune responses noted occasionally in certain patients appear to be the consequence, not the cause, of the disease. The manifestation of these "defects" may contribute, however, to complications of the disease. (d) Mucolytic enzymes Meyer et_ al.(105) f i r s t postulated that ulcerative c o l i t i s resulted from the enzymatic destruction of the colonic surface mucus layer rendering the - 17 -colon more s u s c e p t i b l e to a t t a c k by b a c t e r i a l and/or other agents. Lysozyme was thought to be the key enzyme re s p o n s i b l e f o r the d e s t r u c t i o n of the mucus l a y e r . However, various enzyme i n h i b i t o r s administered to UC p a t i e n t s f a i l e d to improve the c l i n i c a l course o f the disease. In 1950 Glass et al.(106) showed that lysozyme was incapable of d i g e s t i n g human mucus and the theory was subsequently abandoned. (e) I n f e c t i o n B a c t e r i a have been i m p l i c a t e d i n the pathogenesis of u l c e r a t i v e c o l i t i s f o r s e v e r a l decades. Many of the c l i n i c a l and morphological features of u l c e r a t i v e c o l i t i s are c o n s i s t e n t with an inflammatory r e a c t i o n caused by b a c t e r i a l i n f e c t i o n (76). M i c r o b i a l i n f e c t i o n may exacerbate UC or p r e c i p i t a t e recurrences. Although a d m i n i s t r a t i o n of a n t i m i c r o b i a l agents b r i n g s about c l i n i c a l improvement i n some cases of c o l i t i s (107), they do not cure the disease (44). The i n i t i a l c l a i m by Bargen (108) t h a t a diplococcus was r e s p o n s i b l e f o r u l c e r a t i v e c o l i t i s was challenged and subsequently disproved by many i n v e s t i g a t o r s (109-110). Other micro-organisms a l s o i m p l i c a t e d i n the pathogenesis of UC i n c l u d e Entamoeba h i s t o l y t i c a (111), Bacteriodes necrophorum (112), and Mycobacteria (113-114). However, these organisms could not be found i n the s t o o l s of a l l UC p a t i e n t s and f u r t h e r were found to be present i n seemingly healthy s u b j e c t s . To-date, no one s i n g l e species of b a c t e r i a has been i s o l a t e d and demonstrated as the primary e t i o l o g i c a l agent i n u l c e r a t i v e c o l i t s . Under normal c o n d i t i o n s the i n t e s t i n a l mucosa i s an e f f e c t i v e b a r r i e r to - 18 -micro-organisms. It is logical to assume that the disruption of the integrity of the mucosa, as is seen in ulcerative colitis, could be induced by infection. Because the pathologic changes caused by Shigella infection closely mimic those of ulcerative colitis in the acute period (115), the idea that UC is caused by bacterial infection is becoming more and more attractive. If bacterial infection were the primary etiologic agent in ulcerative colits, one would expect to see a qualitative and/or a quantitative change in the composition of the gut flora. Seneca et_ al.(116) determined the total bacterial count and the coliform count in faeces from patients with UC and normal subjects and obtained higher counts in UC patients. Cooke (117), however, could find no significant difference between the viable bacterial count of 20 specimens from faeces of UC patients and normal subjects. In two independent studies by Gorbach ejt al. (118) and Jacomina et 8^.(119) i t was found that the gut flora of UC patients were quite unstable; there were substantial increase in the number of coliform but l i t t l e or no change in the bacteriodes population. Although i t may appear that the gut flora in ulcerative colitis patients is different from that of the normal individuals, such changes could well be the result of the disease rather than the cause. It is known, for example, that diarrhea may cause changes in the micro-flora, regardless of the primary etiology (120). It is s t i l l premature at this stage to assume that alteration in the gut flora is the primary etiologic agent in the pathogenesis of ulcerative colitis. Early attempts to isolate a pathogenic virus in ulcerative colitis have yielded largely negative results (121-122). Recently, Gitnick et al.(123) - 19 -have been able to i s o l a t e a new vir u s , with a mean p a r t i c l e diameter of 60 nm, from ulcerative c o l i t i s patients. This result suggested an association between viruses and UC but did not establish an e t i o l o g i c relationship. Despite a l l the negative r e s u l t s , attempts are s t i l l being made to try to develop new techniques for the i s o l a t i o n of a possible v i r a l e t i o l o g i c a l agent. V. The Role of Out Flora i n Carrageenan-induced c o l i t i s There have been few studies of the gut f l o r a i n degraded carrageenan-induced c o l i t i s . Van Der Waaij e_t al.(124) showed that selective elimination of aerobic gram negative i n t e s t i n a l f l o r a i n guinea pigs fed 2% degraded carrageenan resulted i n granuloma formation with no ulceration. Onderdonk e_t al.(125) found that the caecal contents of guinea pigs given carrageenan contained increased numbers of coliforms and gram negative anaerobic bacteria. In addition, i t was found that selective depletion of obligate anaerobes by treating the carrageenan-fed guinea pigs concomittantly with clindamycin (0.1 mg/ml) or metronidazole (2 mg/ml) resulted i n zero and 8%, respectively, caecal ulceration. It was noted that germ-free animals given carrageenan did not develop t y p i c a l lesions. These findings suggest that bacteria are an important factor i n the development of carrageenan-induced c o l i t i s i n guinea pigs. - 20 -VI. Rationale for the Present Investigation Since colonic e p i t h e l i a l mucins are degraded by the i n t e s t i n a l f l o r a , i t seems l i k e l y that the synthesis and degradation of the colonic e p i t h e l i a l glycoproteins exist i n a steady state. The O-acetylated s i a l i c acids on the glycoproteins protect the macromolecules from degradation by b a c t e r i a l neuraminidase, hence presumably protecting them from proteolysis and possibly glycosidases. There are changes i n the chemistry and histochemistry of the colonic e p i t h e l i a l mucins i n both human ulcerative c o l i t i s and carrageenan induced c o l i t i s i n animals such that the glycoproteins contain less O-acetylated s i a l i c acids; hence, the mucins are more susceptible to digestion by b a c t e r i a l neuraminidase. Bacteria of the large bowel are important i n the production of caecal ulceration i n carrageenan-induced c o l i t i s . I t has, therefore, been suggested (70,74-75) that ulcerative c o l i t i s may be a consequence of a change i n the steady state caused by either (a) a change i n the synthesis of mucosal glycoproteins and/or; (b) a q u a l i t a t i v e and/or quantitative change i n the composition of the b a c t e r i a l f l o r a . Investigation of the degradation of colonic e p i t h e l i a l glycoproteins by faecal enzymes may test t h i s hypothesis. The objective of t h i s thesis was to study, i n a systematic fashion, the k i n e t i c s of the degradation of colonic e p i t h e l i a l glycoproteins by c e l l - f r e e extracts of faeces. Wistar rat colonic e p i t h e l i a l glycoproteins were used as an experimental model and rat faeces were used as the sole source of enzymes. - 21 -MATERIALS AND METHODS (I) MATERIALS Young male Wistar rats weighing approximately 150 grams were purchased from the Animal Care Unit of the University of British Columbia. The animals were kept in the Department Animal Room and fed with Purina Formulab Chow code #5008 (appendix Al) and water ad libitum. All rats were housed in separate cages for a minimum of 7 days before being used in the experiments. Vibrio cholera Neuraminidase (E.C.3.2.1.18, 1.0 I.U./ml) was purchased from Behringwerke, bovine submandibular mucin (BSM) from Boehringer Mannheim Corp., and N-acetyl neuraminic acid (A grade) from California Biochemicals. Out-dated human blood was obtained from the Red Cross of Canada. Para-nitrophenyl glycosides and acetate were purchased from the Sigma Chemical Company. All chemicals were of reagent grade or better and were purchased either from J.T. Baker Chemical Co., or from Fisher Scientific Co. Dialysis tubing (size #27) was obtained from VWR Scientific Inc. (II) METHODS Colorimetric analysis was performed using a Beckman Model 25 spectrophotometer (working volume 81 ul, path length 1 cm). All calculations were done with a Hewlett Packard (HP) HP33E programmable calculator and a Radio Shack's TRS-80 level II Basic computer. Statistical analysis was done with a HP41CV programmable calculator. - 22 -A. PREPARATION OF SUBSTRATES 1. Preparation of Glycoproteins (a) Preparation of human and rat seromucoid (126) Out-dated human blood or fresh rat blood was centrifuged at 1,000 xg for 30 minutes to separate the serum from the c e l l u l a r components. The serum was cooled to 0°C and an equal volume of ice-cold 1.2M_ perchloric acid was added drop by drop with constant s t i r r i n g to the serum. The precipitate was removed by centrifugation at 1,200 xg and 4°C for 20 minutes and the supernatant was neutralized to pH 7.0 by the addition of concentrated ammonium hydroxide. The solution was then exhaustively dialyzed against running d i s t i l l e d water. The dialyzed sample was freeze-dried and redissolved i n d i s t i l l e d water ( s i a l i c acid concentrations: human seromucoid 254 ug/ml, rat seromucoid 145 ug/ml). (b) Preparation of bovine submandibular mucins (B3A) Bovine submandibular mucin (500 mg) was dissolved i n d i s t i l l e d water (5 mg/ml) and washed with portions of d i s t i l l e d water ( t o t a l volume 1 l i t r e ) i n an Amicon u l t r a f i l t r a t i o n c e l l (model 202) equipped with a Diaflo >M50 f i l t e r (exclusion l i m i t 50,000 M.W.). The retentate was lyo p h i l i z e d and re-dissolved i n d i s t i l l e d water to a f i n a l concentration of 1 mg/ml. - 23 -(c) Preparation of C4BSM (127) Five hundred milligrams of bovine submandibular mucin were incubated with 2.0 ml of Vibrio cholera neuraminidase (1 I.U./ml) i n 0.05M sodium acetate buffer (containing 0.1% calcium chloride and 0.02% sodium azide), pH5.5, for 72 hours at 37°C. The digest was u l t r a - f i l t e r e d using an XM50 f i l t e r and washed successively with portions of d i s t i l l e d water (2 l i t r e s f i n a l volume). The retentate, containing the BSM, was assayed for neuraminidase a c t i v i t y and stored frozen at -20°C. (d) Preparation of rat colonic e p i t h e l i a l glycoproteins Rat colonic e p i t h e l i a l glycoproteins were prepared from homogenates of rat colonic e p i t h e l i a l c e l l s as follows (9,128): ( i ) I s o l a tion of colonic e p i t h e l i a l c e l l s (figure 2) Young male Wistar rats (150-200 grams) were k i l l e d by ether anaesthesia. The colons were resected, inverted, i n f l a t e d with ice-cold 0.9% saline and ligated at both ends with surgical thread. The junction between the upper and lower halves of the colon was also ligated and the colon was then cut into two pieces; each of which were then placed i n separate containers. E p i t h e l i a l c e l l s were obtained by alternately shaking the colons, i n 0.01% PBS-EDTA solution (appendix A2) for 5 minutes and then incubating i n the same solution at 37°C for 5-10 minutes. This process was repeated three times with fresh portions of the buffer. The pooled c e l l suspension was centrifuged at 900 xg and 4°C for 10 minutes and the pe l l e t containing the e p i t h e l i a l c e l l s was washed twice with ice-cold PBS (without EDTA) solution. - 24 -Figure 2: Flow diagram of the method for the i s o l a t i o n of intact rat colonic e p i t h e l i a l c e l l s - 25 -MALE WISTAR RAT 1/ 2/ killed by ether colon resected RAT COLON 1/ everted 2/ inflated with 0.9% saline 3/ divided in "upper" and "lower" halves 4/ shaken with 0.01% PBS-EDTA (x3) centrifuged at 900 Xg, 4°C, 10 minutes CELL SUSPENSION I SUPERNATANT PELLET washed X2 with ice-cold PBS ISOLATED EPITHELIAL CELLS - 26 -( i i ) Extraction of colonic e p i t h e l i a l glycoproteins (figure 3) E p i t h e l i a l c e l l s (1 volume) were suspended i n 1M NaCl (10-15 volumes) and sonicated at 0°C with a Braunsonic 1510 sonicator f i t t e d with a macroprobe at a setting of 300 watts for 8 x 30 second periods . The homogenate was then centrifuged at 4°C and 1,000 xg for 15 minutes. The resultant supernatant was re-centrifuged at 105,000 xg and 4°C for 1 hour. The supernatant obtained was then concentrated to a smaller volume i n an Amicon u l t r a - f i l t r a t i o n c e l l using an M 50 f i l t e r to y i e l d the crude glycoprotein extract. ( i i i ) P u r i f i c a t i o n of colonic e p i t h e l i a l glycoproteins (figure 4) Crude e p i t h e l i a l glycoprotein extracts were applied to a column of Biogel A15M (100-200 mesh) agarose packed i n a Pharmacia SR25 column equiped with A-16 (Pharmacia) adaptors. Samples were eluted with 1M_ NaCl and each f r a c t i o n (10 ml) was scanned at 280 nm for an approximate estimation of proteins and assayed for s i a l i c acids with the periodate-resorcinol assay (KOH/PRT) (see page 37). The s i a l i c a cid-rich fractions were concentrated, dialyzed against 0.02M_ pyridine-HC1 buffer (pH5.5) over night, and then applied to a column (27 x 3.4 cm) packed with DE22 (Whatman) DEAE cel l u l o s e . The column was eluted with a l i n e a r gradient (0-2M) of NaCl i n 0.02M pyridine-HC1, pH5.5 and the fractions were monitored for s i a l i c acid. The s i a l i c a c i d - r i c h fractions were concentrated, dialyzed against d i s t i l l e d water and l y o p h i l i z e d to y i e l d the p u r i f i e d rat colonic e p i t h e l i a l glycoproteins. The purity of the rat colonic glycoproteins was tested by cellulose acetate electrophoresis. This was carried out on 6" x 1" s t r i p s of Sepraphore - 27 -Figure 3: Flow diagram of the method for the extraction of rat colonic e p i t h e l i a l glycoproteins - 28 -ISOLATED EPITHELIAL CELLS 1/ suspended in JM_ NaCl 2/ sonated 8x30 seconds, 300 watts, 0°C 3/ centrifuged at l,000Xg, 15 minutes, 4°C 4/ centrifuged at 105,000Xg, 1 hour, 4°C SUPERNATANT CONTAINING EPITHELIAL GLYCOPROTEINS PELLET Discarded - 29 -Figure 4: Flow diagram of the method for the p u r i f i c a t i o n of rat colonic e p i t h e l i a l glycoproteins - 30 -CRUDE EPITHELIAL GLYCOPROTEINS 1/ gel chromatography Biogel A15M eluted with JM NaCl 2/ fractions monitored at 280 3/ assayed for sialic acid SIALIC ACID FRACTIONS PROTEIN RICH FRACTIONS 1/ concentrated and dialyzed against 0.02M_ pyridine-HCl buffer, pH5.5 2/ ion-exchange chromatography on DEAE Cellulose eluted ^ with a linear gradient (0-2M_)"bf NaCl in 0.02M pyridine-HCl PROTEIN-RICH FRACTIONS SIALIC ACID-RICH FRACTIONS 1/ concentrated 2/ dialyzed against distilled water 3/ lyophilized RAT COLONIC EPITHELIAL GLYCOPROTEINS - 31 -I I I (Gslman Instrument Co.) using T r i s - b a r b i t a l buffer (high resolution buffer, Gelman), pH8.8, for 30 minutes at 300 volts. Strips were stained with Alcian Blue (pH3.0) (129). Cellulose acetate electrophoresis was also performed on the crude glycoproteins. Electrophoretic mobilities were measured r e l a t i v e to heparin. (e) Preparation of saponified glycoproteins Saponified (KOH) substrates were prepared by adding 1 volume of IN KOH to 9 volumes of substrate. After 30 minutes at room temperature 1 volume of IN H^ SO^  was added to neutralized the samples. Non-saponified substrates were prepared by adding 2 volumes of^O.SM K 2S0^ to 9 volumes of the glycoproteins. Table 4 shows the various types of substrates used i n the studies. 2. Preparation of para-nitrophenyl substrates Para-nitrophenyl glycosides and acetate were dissolved i n d i s t i l l e d water (table 5) and stored at 0°C. B. Preparation of Rat Faecal Enzymes Fresh faecal p e l l e t s from male Wistar rats were collected immediately after defaecation and suspended i n minimal media (appendix A3) i n a 20% W/V r a t i o (figure 5). The suspension was homogenized by vortexing with glass beads and the debris was removed by f i l t r a t i o n through glass-wool. The f i l t r a t e was - 32 -Table 4: Types of substrates used in the study of degradation of glycoproteins by rat faecal enzymes. SUBSTRATE SAPONIFIED NON-SAPONIFIED Rat Upper X X Rat Lower X X Bovine Sub-mandibular X X Mucins (BSM) C 4 BSM X X Rat Seromucoid X X Human Sero- X mucoid N-Acetyl neuraminic X acid Distilled H20 used as control I - 33 -Table 5: Molarit ies of para-nitrophenyl substrates used. Para-Nitro phenyl Substrates CONCENTRATION (M) Acetate 0.01 a-L-fucoside 0.01 g-L-fucoside 0.01 a-0-galactopyrano -side 0.01 £4) -galactopyrano-side 0.01 B-D-galactopyrano-side (ortho-NP) 0.01 a-O-glucoside 0.01 8-0 glucoside 0.01 N-Acetyl-3-D-galactosaminide 0.002* N -Acetyl -a -D-glucosaminide 0.0025* N-Acetyl-3-D-glucosaminide 0.004* * Substrates were insoluble in d i s t i l l e d water above this concentration - 34 -Figure 5: Preparation of rat faecal enzymes - 35 -20% W/V FAECAL HCMOGENATE (IN MINIMAL MEDIA) 1/ centrifuge at 10,000 xg, 10 min 2/ centrifuge at 105,000 xg, 60 min., 15°C PELLET SUPERNATANT DISCARD 1/ millipore filter, 0.45u 2/ 0.02% NaN3 added "ENZWIE" - 36 -centrifuged at 1,000 xg for 10 minutes and the supernatant recentrifuged at 105,000 xg for 1 hour at 15 °C. The ultra-supernatant was then filtered through a 0.45u millipore fi l t e r (millipore) and sodium azide (final concentration 0.02%) was added to prevent bacterial growth. Boiled faecal enzyme extract was prepared by heating a portion of the extract at 100°C for 30-45 minutes. Both the boiled and untreated enzyme extracts were used immediately. (Ill) ANALYTICAL METHODS 1. Colorimetric Analyses (a) Estimation of free and ketosidically bound sialic acids This was performed according to the method of Culling et al. (130) Aliquots of samples (30 ul) containing free and/or bound sialic acids were oxidized with 0.025M_ sodium periodate (NalO^) (15 ul) in 0.067M phosphate buffer, pH7.0, and after 20 minutes at room temperature the reaction was stopped by the addition of 2% sodium arsenite in 0.5N HC1 (12 ul). (i) Free sialic acid was estimated as follows (TBA): The oxidized samples (47 ul) were treated with thiobarbituric acid reagent (120 ul) prepared according to the procedure of Aminoff (131). After heating at 100°C for 8 minutes, the samples were cooled, centrifuged to spin down the condensate and then n-butanol/HCl solution (95:5 v/v) (300 ul) was added. The samples were mixed, re-centrifuged and the upper layer read at 549nm. - 37 -Standards were prepared from serial dilutions of known quantities of N-acetyl neuraminic acid dissolved in distilled water or in solution consisting of 5 parts of water and 3 parts of the minimal media, (ii) Bound sialic acid was estimated as follows (PRBA): The oxidized samples (47 ul) were mixed with resorcinol reagent (80 ul) prepared according to the procedure of Jourdian et al.(132) After heating at 100°C for 8 minutes, the samples were cooled, centrifuged and then t-butanol (80 ul) was added. The resultant mixture was heated at 37°C for 3 minutes. Samples were then mixed, cooled to room temperature, and read at 630 nm. Standards were prepared from at least 4 concentrations of glycosidically bound sialic acid (human seromucoid). (b) Estimation of the percentage of sialic acids substituted at positions C7  and/or C8. This was carried out according to the method of Reid et_ al. (14) (i) Saponified samples (KOH/PRT): Saponified samples (33 ul) were oxidized with 0.0^M_ NalO^ (7 ul) at 0°C for 35 minutes. Resorcinol reagent (80 ul) was then added and after 3-5 minutes the mixture was heated at 100°C for 8 minutes. The samples were cooled, centrifuged and t-butanol (80 ul) was added and the mixture was heated at 37°C for 3 minutes. The samples were then re-mixed and read at 630 nm. (ii) Non-saponified samples ( P R T ) : Non-saponified samples were treated as above. ( i i i ) The PRS estimation of sialic acid was performed as described in (i) - 38 -except that the samples were treated with pre-mixed solution of resorcinol reagent and 0.04M NaI04 (87 ul). The values obtained for KOH/PRT, PRT, and PRS were then substituted into the following equation: %C7C8 = /KOH/PRT - PRT\x 100% \K0H/PRT - PRS/ (c) Estimation of the percentage of sialic acids substituted at position CA This was procedure was carried out according to the method of Reid et al.(14) Saponified samples were prepared by adding 1 volume of IN KOH (7 ul) to 9 volumes of samples (63 ul). After 30 minuses at room temperature 1 volume of IN H2S0^ (7 ul) was added to neutralize the samples. Non-saponified samples were prepared by adding 2 volumes of 0.5M KjSO^ (14 ul) to 9 volumes of the samples (63 ul). To both the saponified and non-saponified samples (77 ul) were added 0.5M sodium acetate buffer (containing 0.1% calcium chloride and 0.02% sodium azide) (9 ul, pH5.5) and 0.2 I.U. of Vibrio cholera neuraminidase (9 ul). The solutions were incubated at 37°C for 24 hours, centrifuged and a further aliquot of 0.2 I.U. of neuraminidase (9 ul) was added, the samples were incubated for another 24 hours. The digestion was stopped by saponifying the samples, at room temperature for 30 minutes, with IN KOH (12 ul), they were then neutralized with an aliquot of 1JN H2S0^ (12 ul). All the samples were then assayed for free and ketosidically bound sialic acids by the methods previously described. The percentage of 0-acetyl group substituted at position - 39 -C, of sialic acid was calculated from the equation given below: % = 1 -j% sialic acid released from non-KOH glycoproteins \ X 100% dns / sialic acid released from KOH glycoprotei(d) Determination of enzymatic activities towards p-nitrophenyl substrates The procedure employed was a slight modification of the method of Huggins et al. (133) The analysis was carried out as follows: minimal media (600 ul, pH7.0) was mixed with p-nitrophenyl (PNP) substrates (200 ul). Rat faecal enzymes (200 ul) were added to the buffered substrate, mixed rapidly and incubated at 37°C. At time intervals, samples (100 ul) were mixed with 0.1 NaOH (500 ul) and read at 400nm. Para-nitrophenol was used as a standard (12.6 - 62.7 nmoles/ml). The standards (100 ul) were added to 0.1N NaOH (500 ul) as well as to the minimal media (500 ul) and read at 400nm. The standard curve was obtained by plotting the difference in absorbance between the NaOH and minimal media standards against the concentration of p-nitrophenol. When PNP-acetate was used, the procedure was modified by using 2 extra controls. One tube contained only the substrate (200 ul) and the minimal media (800 ul) whereas the second tube contained the media (600 ul), the enzyme (200 ul), and distilled water (200 ul). Aliquots of samples (100 ul) were removed at intervals and mixed with 500 ul of minimal media (pH7.0) and read at 400nm. One unit of activity was defined as the amount of rat faecal enzymes - 40 -required to release 1 micromole (umole) of para-nitrophenol per minute at 37°C and pH7.0 and the specific activity was expressed as milliunit per milligram wet weight faeces. (e) Determination of activities of neuraminidase and NANA-degrading enzyme Human seromucoid and N-acetyl neuraminic acid were used as substrates for the determination of the activities of neuraminidase and NANA-degrading enzyme, respectively. Substrates (human seromucoid 254 ug sialic acid/ml, N-acetyl neuraminic acid 100 ug sialic acid/ml) (125 ul) were incubated with rat faecal enzymes (25 ul) and minimal media (50 ul) at 37°C. Human seromucoid was incubated for a period of 15 minutes whereas N-acetyl neuraminic acid was incubated for 30 minutes. Distilled water plus enzymes was used as the control for "background colour". One unit of activity was defined as the amount of enzyme required to release or degrade 1 micromole (umole) of N-acetyl neuraminic acid per minute at 37°C and pH7.0 and the specific activity was expressed as mU/milligram wet weight faeces. 2. Qualitative Identification of Sugars by Paper Chromatography Paper chromatography was performed by the descending method on sheets (20 x 45 cm) of Whatman #1 fi l t e r paper. Chromatograms were developed for 19 hours at room temperature in ethyl acetate, pyridine, water (8:2:1 v/v) and sugars were detected by an alkaline silver nitrate procedure (134). Sugars were identified by comparison with standards chromatographed on the same -41 -chromatogram and the mobilities were expressed relative to that of fucose. 3. Quantitative Analysis of Sugars by Gas-Liquid Chromatography (Glc) Gas-liquid chromatography was carried out on a Hewlett Packard (model No. 7610A) high efficiency dual column gas liquid chromatograph fitted with dual hydrogen flame ionization detectors and on-column injection. Nitrogen was used as carrier gas. Neutral sugars and hexosamines were estimated by the glc of the corresponding polyhydric alcohol acetates (PHA) using a U-shaped glass column (183 cm x 6 mm i.d.) containing 3% SP2340 on gas chrome Q 100-200 mesh. The chromatograph was operated isothermally at 205°C for the analysis of neutral sugars and at 240°C for the analysis of hexosamines. Peak areas were measured with a Hewlett Packard (model No. 3370B) electronic integrator. Inositol was used as the internal standard for glc analysis of hexosamines and arabinitol as the internal standard for the neutral sugars. 4. Bacteriological Studies The cell free extract of Wistar rat faeces was tested for sterility by performing (a) the Gram's stain method for bacteria, and (b) 24 hours aerobic culture and 2 days anaerobic culture on blood agar plates. Bacteriological cultures were performed by Dr. Ann G. Skidmore, Department of Microbiology, Acute Care Hospital, University of British Columbia. -ta-in. GENERAL EXPERIMENTAL DESIGN A typical experiment was performed as illustrated in figure 6 and as described below: Fresh and boiled rat faecal enzyme extracts were prepared as described previously. Freshly prepared faecal enzyme extracts were assayed for neuraminidase, esterase, glycosidases, and NANA-degrading enzyme activity. Samples were also taken for anaerobic and aerobic cultures to test for bacteriological sterility. Boiled enzymes were used as controls. The remaining portion, of the enzyme extracts (3 parts) was incubated with the glycoprotein substrate (5 parts, 0.8 mg/ml water) at 37°C. Aliquots (1600 ul) were removed from the digest at various time intervals, heated in a boiling water bath for 30 minutes, cooled and stored at -20°C. The samples were thawed and aliquots were assayed for free and bound sialic acids and for the percentage of sialic acids substituted at Cy+Cg. The remainder of the samples were used in the ultra-filtration procedure below. k A known quantity of the incubate (1312 ul) was ultra-filtered through an Amicon CF50A cone (exclusion limit 50,000 M.W.). Aliquots of the filtrate (162 ul) were then analyzed for the percentage of sialic acids substituted at C7+Cg. The retentate (macromolecular fraction) was washed with three portions of distilled water (1000 ul) and the pooled filtrates were stored at -20°C for later use. The retentate was reconstituted with distilled water (5 X 200 ul) and assayed for the percentages of C^  and Cy+Cg substituted sialic acids. An identical control experiment was conducted concomitantly - 43 -Figure 6: Analysis of the degradation of glycoproteins by rat faecal enzymes - 44 -FAECAL ENZWES* 1/ Incubate with substrates at 37°C 2/ Aliquots taken out at time intervals 3/ Heat in boiling water bath for 30 minutes INCUBATES ULTRAFILTRATION CF50A X RETENTATE FILTRATE reconstituted in distilled water Estimate 1/ % sialic acid C4 2/ % sialic acid C7+C8 SUGAR ANALYSES -Determination of activities of: 1/ Neuraminidase 2/ Esterase 3/ Glycosidases-4/ NANA-degrading enzyme -Estimate 1/ Free sialic acid 2/ Bound sialic acid 3/ % sialic acid C7+C8 sialic acid Cy+Cg de-ionized *Boiled enzymes were used as control - 45 -using boiled enzyme. The pooled filtrates were thawed and de-ionized by passage through beds of Dowex-50 [H+] and Dowex-1 [HCO^ ] ion exchange resins (figure 7). The neutral eluates were concentrated to dryness and analyzed for neutral sugars by the gas-liquid chromatography (glc) of the derived polyhydric alcohol acetates. Elution of the Dowex-50 [H+] resins with 0.3N HC1 yielded hexosamines which were also analyzed by the glc of the derived polyhydric alcohol acetates. A typical kinetic experiment, excluding the glc analysis of sugars, could be accomplished in 2 to 3 weeks. - 46 -Figure 7: Flow diagram of the method for the preparation of samples for the gas-liquid chromatographic analysis of hexosamines and neutral sugars. - 47 -FILTRATE 1/ known quantity of inositol (internal standard) added 2/ washed 3X1000 ul H20 DOWEX 50 [H+] eluted with 3X1000 ul 0.3N HC1 DOWEX 1 [HCO3] HEXOSAMINES NEUTRAL SUGARS - 48 -RESULTS (I) PRELIMINARY EXPERIMENTS Preliminary experiments were used to establish the optimal conditions for each assay system employed. The effect of minimal media on each type of colorimetric analysis was investigated. Paper chromatography was used to determine whether the observed glycosidase activities directed against ar t i f i c i a l p-nitrophenyl substrates were also directed against the natural substrates, namely, the rat colonic epithelial glycoproteins. The objective of the kinetic experiment was to study the kinetics of the degradation of Wistar rat colonic epithelial glycoproteins and other types of macromolecular substrates (bovine submandibular mucin, human and rat seromucoid). (a) Preparation of glycoprotein substrates Purified rat colonic epithelial glycoproteins were prepared, as described by Reid et al.(9), by a combination of the Agarose A15 M gel (figures 8 and 10) and DEAE cellulose (figures 9 and 11) chromatography of the crude glycoproteins obtained from the epithelial cells of the upper and lower halves of Wistar rat colon. As shown in figure 12 these glycoproteins migrated as single bands on cellulose acetate electrophoresis. C^ BSM was prepared by digestion of the crude commercial glycoprotein with Vibrio cholera neuraminidase while the seromucoids were obtained from the - 49 -Figure 8: Fractionation of the crude rat upper colonic epithelial. glycoprotein on Agarose A15M gel. Fractions were eluted with 1M NaCl solution. Protein (-) was monitored at 0.D.280 and sialic acid (-Owas estimated by the periodate-resorcinol assay (PRT). The sialic acid-rich fractions were concentrated and re-fractionated by ion-exchange chromatography. 200 300 400 500 ELUTION VOLUME (ML) - 51 -Figure 9: DEAE Cellulose ion-exchange column chromatography of the sialic acid-rich fraction (SA) obtained from Agarose A15M chromato-graphy of crude rat upper colonic epithelial glycoprotein. Fractions were eluted with 0.02M_ pyridine-HCl buffer, pH5.5, containing an increasing NaCl gradient (0-2M_, — ) . Protein (••) was monitored at 0.D.280 and sialic acid (-) estimated by the PRT assay. The sialic acid-rich peak, containing the "purified" rat upper colonic epithelial glycoprotein, was eluted at a concentration of 0.2^ NaCl. n ELUTION VOLUME (ML) - 53 -Figure 10: Fractionation of the crude rat lower colonic epithelial glycoprotein from Agarose A15M gel. Fractions were eluted with ]M NaCl solution. Protein (-) was monitored at 0.D.280 and sialic acid (••) was estimated by the periodate-resorcinol assay (PRT). The sialic acid-rich fractions were concentrated and re-fractionated by ion-exchange chromatography. - 55 -Figure 11: DEAE Cellulose ion-exchange column chromatography of the sialic acid-rich fraction (SA) obtained from Agarose A15M chromato-graphy of crude rat lower colonic epithelial glycoprotein. Fractions were eluted with 0.02M_ pyridine-HCl buffer, pH5.5, containing an increasing NaCl gradient (0-2M_, — ) . Protein (••) was monitored at 0.D.280 and sialic acid (-) estimated by the PRT assay. The sialic acid-rich peak, containing the "purified" rat lower colonic epithelial glycoprotein, was eluted at a concentration of 0.2M_ NaCl. - 9 S -- 57 -Figure 12: Electrophoretic patterns of crude and purified Wistar rat colonic epithelial glycoproteins. Cellular acetate electrophoresis was carried out on strips (6" x 1") of Sepraphore III (Gelman Instrument Co.) using Tris barbital sodium barbital buffer (high resolution buffer, Gelmen), pH8.8 at 300 volts for 40 minutes with a current of 2-3 mA/strip. Strips were stained with Alcian Blue at pH3.0 (129). I. heparin; II. crude rat upper glyco-protein in 0.02M pyridine-HCl buffer, pH5.5; III. purified rat upper glycoprotein; IV. crude rat lower glycoprotein in 0.02M pyridine-HCl buffer, pH5.5; V. purified rat lower glycoprotein; S. application line. - 59 -0.6M_ perchloric acid soluble fraction of human and rat sera. The 0-acetyl substitution pattern of the sialic acids of these glycoproteins is shown in Table 6. As reported previously (9) the glycoprotein from the upper half of rat colon contained a significantly greater percentage of sialic acids substituted at C7+CQ and at but the actual values obtained differed somewhat from those reported previously (9). Digestion of BSM with V.c. neuraminidase resulted in a large increase in the percentage of Cy+Cg and substituted sialic acids but did not, as had been expected, yield a glycoprotein in which a l l the sialic acids had substituents. As expected, human seromucoid contained no 0-acetyl sialic acids but surprisingly these were detected in the corresponding fraction from rat serum. As far as I am aware this has not been reported previously. (b) Effects of minimal media on the colorimetric analysis of sialic acids Table 7 shows the effect of minimal media on the analytical procedures, the PRT, the PRBA, and the TBA, used to analyze sialic acids. As will be seen minimal media had no effect upon either the PRT or PRBA assays but in the TBA assay the extinction coefficient decreased as the proportion of minimal media was increased. Since the oxidation conditions used in the TBA and PRBA assays were identical i t would appear that minimal media inhibits the formation of the chromogen. The mechanism of this inhibition is unknown. It was necessary, however, to analyze free sialic acid using standards made up in the same proportion of minimal media as that present in the enzyme digests. - 60 -Table 6: Percentages of sialic acids with 0-acetyl substituents at (i) position C4 and (ii) positions C7+C8 in the glyco-proteins used as substrates for the study of rat faecal enzymes. SUBSTRATE %C4 %C7+C8 Rat Upper 64 + 5 (21)* 64 + 4 (53) Rat Lower 53 + 5 (21) 45 + 5 (58) BSM 26 + 5 (20) 53 + 5 (48) C4 BSM 74 + 4 (3) 80 + 6 (7) Rat Sero- ' 26 + 4 (3) 57 + 5 (9) mucoid Human Sero- 0 (5) 0 (5) mucoid * Mean + SD (n) - 61 -Table 7: Effects of Minimal Media on the colorimetric analysis of free sialic acid (N-acetyl neuraminic acid) with the TBA, and PRT methods, and of bound sialic acid (human seromucoid) with the PRBA method. Extinction Coefficient* Minimal Media/Water TBA PRBA PRT 0/5 48,700 - 20,900 23,300 1/5 45,000 19,300 2/5 40,000 20,100 3/5 36,800 21,000 23,300 ^Extinction coefficient is defined as the O.D. of 1 micromole (umole) of free or bound sialic acid in 1 millilitre (ml) of water in a cell of path length of 1 centimeter (cm). 6 - 62 -(c) Effects of boiling on free sialic acids Heating samples of sialic acids in minimal media at 100°C for 30 minutes had no effect on (i) the estimation of sialic acid by the TBA procedure and (ii) the determination of sialic acids substituted at Cy+Cg. It was, therefore, feasible to employ heating at 100°C to denature the faecal enzymes. (d) Determination of activities of glycosidases and esterase (i) Glycosidases: Preliminary experiments showed that with a l l the p-nitrophenyl glycosides used the rate of hydrolysis with rat faecal enzymes remained linear for at least 60 minutes of incubation at 37°C. Units of enzyme activity (glycosidases) were therefore calculated from the amount of p-nitrophenol released within the first 15 minutes at 37°C. An enzyme blank was used to substract the "colour" contributed by the faecal enzymes. (ii) Esterase: The hydrolysis of PNP-acetate was also shown to be linear over the first 15 minutes. This substrate, however, was completely hydrolyzed by 0.1N NaOH or 0.1N Na2C0^ and therefore the reaction could not be stopped by the addition of alkali. Furthermore, PNP-acetate was also hydrolyzed to a small extent by minimal media (figure 13). It was necessary therefore to include a minimal media blank and to read the colour of the samples immediately. The "net" amount of p-nitrophenol released was calculated as described in the material and method section (see p.39). - 63 -Figure 13: Hydrolysis of p-nitrophenyl acetate by minimal media (+) and rat faecal enzyme extracts (•) at 37°C, pH7.0. - 65 -(e) Determination of activities of neuraminidase and NANA-degrading enzyme (i) Neuraminidase: The linear range of the hydrolysis of the sialic acid of human seromucoid by the rat faecal enzyme neuraminidase was found to be 90 minutes. Fifteen minutes were selected for the calculation of the units of neuraminidase activity. (ii) NANA-degrading enzyme: The amount of free sialic acid which disappeared from the incubate, as monitored by the TBA assay, was determined over a period of 50 minutes. The rate of disappearance of N-acetyl neuraminic acid was linear up to 30 minutes and this time was chosen for the calculation of the units of activity of the NANA-degrading enzyme. (f) Qualitative identification of sugars by paper chromatography The epithelial glycoprotein from the lower half of rat colon was incubated with rat faecal enzymes for 24 hours at 37°C and the digest examined by paper chromatography; a similar incubation with boiled enzyme extract was used as control. The results of these studies, shown in figure 14 and table 8, indicated that digestion of the glycoprotein with rat faecal enzymes caused the release of fucose, galactose, and possibly N-acetyl hexosamine. It was clear therefore that the rat faecal enzymes contained glycosidases capable of acting upon macromolecular substrates. - 66 -Figure 14: Separation and identification of sugars present in the faecal enzyme digest by paper chromatography. The paper chromatogram was developed for 19 hours at room temperature with a solvent system which consisted of ethyl acetate, pyridine, and distilled water (8:2:1, v/v/v). Sugars were detected by an alkaline silver nitrate procedure (134). Sl-3 = standards consisted of galactosamine, glucosamine, galactose, glucose, N-acetyl galactosamine, mannose, N-acetyl glucosamine, and/or fucose (in the order of increasing mobility). ALO = rat lower glycoprotein (RL) digested with rat faecal enzymes for 0 hour, i.e. 0 time control. AL24 = RL digested with rat faecal enzymes for 24 hours BLO = RL digested with boiled rat faecal enzymes for 0 hour BL24 = RL digested with boiled rat faecal enzymes for 24 hours - 6 7 -- 68 -Table 8: Qualitative identification of sugars by paper chromatography. Paper chromatograms were developed for 19 hours at room temperature with a solvent system which consisted of ethyl acetate, pyridine, and distilled water (8:2:1, v/v/v). Abbreviations used are as in figure 14. "^^s*ample Rfucose > V v > >\^ SI ALO AL24 S2 BLO BL24 S3 Galactosamine 0.09 0. 09 0.10 Glucosamine 0.11 Galactose 0.47 0. 42 0.44 X X 0.47 Glucose 0.58 0 53 0.56 x X 0.57 N-Acetyl 0 67 0.69 0.70 x X Galactosamine Mannose 0.72 0.74 x X 0.74 N-Acetyl 0 80 0.78 0.79 Glucosamine Fucose 1.00 1 00 1.00 X X 1.00 Others 1 .27 1.30 X X 1 .45 1.48 X X Note: Rfucose = Distance of unknown from start line over distance of fucose to start line. (II) KINETIC STUDIES - 69 -The results of the studies of the kinetics of the degradation of the glycoproteins are summarized in tables 9-10 and illustrated in figures 15-18. Since analysis of the data obtained indicated that (i) the relative proportions of the enzymes varied between individual preparations of the enzyme extract and (ii) degradation of the glycoproteins involved the combined action of more than one enzyme, i t proved impossible to express the results in terms of the average value for the four batches of enzyme extract used. Results are therefore reported for each of the individual experiments (Table 9-10). (a) Neuraminidase Studies of the neuraminidase(s) present in the rat faecal enzyme extracts (tables 9-11 and illustrated in figures 15-18 for experiment #2) showed that, regardless of either the substrate or the batch of enzyme used, sialic acids were removed significantly faster from the de-O-acetylated (saponified) glycoproteins than from the glycoproteins containing 0-acetyl sialic acids. As will be seen the rate of hydrolysis of the mucin glycoproteins was apparently a function of the percentage of sialic acids with substituents at Cy+Cg and/or at C^ . Thus the sialic acids of BSM (%C^  26+5; %C7+Cg 53 +_ 5) were removed faster than those of the glycoprotein of the lower half of rat colon (%C. 53 + ^ ; %C-,+C0 45 + 5); these in turn were removed faster than those of the glycoprotein from the upper half of rat colon (%C4 64 + 4; %C7+Cg 64+5). It was of interest that the - 70 -Table 9: Percentage of sialic acids released by digestion of the saponified and non-saponified glycoproteins from the upper and lower halves of Wistar rat colon with cell free filtrate (rat enzyme) of Wistar rat faeces. a) Values were based on assays for the quantity of glycosidically bound sialic acids present in the digests. b) Units (specific activities) are expressed as milliunits/mg wet weight faeces where 1 milliunit = 1 umole sialic acid released per minute at 37°C pH 7.0 from human seromucoid. RU = Epithelial glycoprotein from the upper half of rat colon KRU = Saponified epithelial glycoprotein from the upper half of rat colon RL = Epithelial glycoprotein from the lower half of rat colon KRU = Saponified epithelial glycoprotein from the lower half of rat colon - 71 -Sialic Acid Released % a Expt # 1 2 3 N'daseb units 13.1 5.3 1.7 Substrate Time (hrs) RU KRU RL KRL RU KRU RL KRL RU KRU RL KRL 0 0 0 0 0 0 0 0 0 0 0 0 0 .5 11 12 3 16 1.0 2 10 9 21 18 19 9 31 6 11 10 13 2.0 23 30 24 49 3.0 29 33 4.0 34 42 46 62 23 31 34 34 6.0 34 44 8.0 42 55 64 75 12.0 50 60 71 67 44 54 64 64 24.0 71 77 85 88 64 80 86 85 36.0 73 77 84 85 48.0 76 68 86 -93.0 94 91 90 94 - 72 -Table 10: Percentage of sialic acids released by digestion of (i) human seromucoid and (ii) saponified and non-saponified samples of bovine submandibular gland mucin and rat seromucoid by cell free filtrates of (rat enzyme) of Wistar rat faeces. Values and units were calculated as in Table 9. BSM = Bovine submandibular mucin KBS = Saponified bovine submandibular mucin RNS = Rat seromucoid KRNS = Saponified rat seromucoid HNS = Human Seromucoid C4BSM = Bovine submandibular gland exhaustively digested with \/.c_. neuraminidase C4KBSM = Saponified bovine submandibular gland exhaustively digested with V.c. neuraminidase - 73 -Sialic Acid Released % a Expt # 1 2 3 N'daseb units 5.3 1.7 2.7 Substrate Time (hrs) BSM KBSM BSM KBSM BSM KBSM RNS KRNS HNS C 4 BSM C 4 KBSM 0 0 0 0 0 0 0 0 0 0 0 0 0.5 8 17 - - 2 3 4 23 15 0 17 1 23 33 18 42 17 21 22 46 45 15 28 2 38 52 - - 23 31 44 58 46 26 46 4 62 74 64 78 47 50 69 76 58 37 56 8 85 90 - 69 69 76 78 73 56 62 12 86 88 24 93 95 - 88 84 90 87 83 81 77 36 93 93 65 93 90 92 84 86 86 87 93 97 96 - 74 -Table 11: Statistical analysis of the susceptibility to neuraminidase of each glycoprotein present in the rat faecal enzyme extracts. Comparisons were made between the percentages of sialic acid liberated by the same enzyme preparation at period betwsen 1/2 and 24 hours (Tables 9 and 10). Statistical comparisons were made with the paired t-test method and the level of significance was set as a value =0.05. Rat Upper Rat Lower BSA KOH Non KOH KOH Non KOH KOH Non KOH Rat KOH Upper Non KOH -7.36 (17)* 0.01>a >0.001 . Rat Lower KOH 5.70 (13) a<0.001 — ton KOH 3.63 (13) 0.01 >a > 0.001 -3.03 (11) 0.02> a> 0.01 KOH — — — — ton KOH 4.30 (10) 0.01 >a >0.001 5.89 (10) 0.001 >a -3.77 (14) 0.01>a >0.001 *t (df), a . - 75 -Figure 15: Degradation of saponified (•••CJ---) and non-saponified (—+—) rat upper epithelial glycoprotein by rat faecal enzymes. The results were expressed as the percentage of sialic acids removed from the glycoproteins by the neuraminidase(s) present in the rat faecal enzyme extracts. PERCENT SIALIC ACID RELEASED i—» o cn - 9L -- 77 -Figure 16: Degradation of saponified (••••*••) and non-saponified (—+—) rat lower epithelial glycoprotein by rat faecal enzymes. The results were expressed as the percentage of sialic acids removed from the glycoproteins by the neuraminidase(s) present in the rat faecal enzyme extracts. PERCENT S I A L I C ACID RELEASED cn - QL -- 79 -Figure 17; Degradation of saponified (•••O***) and non-saponified (—+—) bovine submandibular mucin by rat faecal enzymes. The results were expressed as the percentage of sialic acids removed from the glycoproteins by the neuraminidase(s) present in the rat faecal enzyme extracts. \ PERCENT SIALIC ACID. RELEASED - 0 8 -- 81 -Figure 18: Hydrolysis of sialic acid residues from saponified (•••<>•;•) and non-saponified (—+—) rat seromucoid and non-saponified human seromucoid (— D — ) by neuraminidase(s) present in the rat faecal enzyme extracts. PERCENT SIALIC ACID RELEASED - 28 -- 83 -de-O-acetylated sialic acids of the rat lower glycoprotein were removed faster than those of the upper glycoprotein. This implies that there is some stereochemical differences between the two glycoproteins such that the sialic acids of the lower glycoprotein are more accessible to the neuraminidase. Table 12 shows that rat faecal enzymes removed a very much greater proportion of the sialic acids of the glycoproteins with 0-acetyl substituted sialic acids than did digestion with Vibrio cholera neuraminidase. In contrast, upon digestion of a glycoprotein without 0-acetyl sialic acids (human seromucoid) both the enzymes released the same proportion of the sialic acid. In view of (a) the data discussed above and (b) the fact that Vibrio  cholera neuraminidase is known to (i) be inactive against -0-acetyl sialic acids (14) and (ii) hydrolyze side chain substituted sialic acids more slowly than their unsubstituted counter parts (15), i t seemed propable that either the rat faecal enzymes contained a "de-O-acetylase" and/or that the rat faecal extracts contained a neuraminidase capable of removing C^-O-acetyl sialic acids. Experiments designed to test these possibilities are described below: (b) De-O-acetylase (esterase) (i) Percentage of C7+C8 substituted sialic acids As shown in tables 13-14 and illustrated in figure 19 for experiment #2, there was a reduction in side chain substitution upon incubation of glycoproteins with the rat faecal enzymes. This reduction was observed with a l l the glycoprotein substrates studied. Although each substrate had a different percentage of side chain substituted sialic acids the rate of - 84 -Table 12: Percentage of sialic acid residues removed from glycoprotein substrates by incubating with Vibrio cholera neuraminidase for 48 hours and by rat faecal enzymes for 24-48 hours. Substrate % Sialic Acid Removed + SD (n) Vibrio Cholera Neuraminidase Rat Faecal Enzymes Bovine Sub-mandibular mucin (BSM) 64+7 (22) 91+3 (3) Rat Upper 20 + 2 (17) 78 + 15 (4) Rat Lower 34+4 (17) 85+1 (3) Human Sero-mucoid 84+8 (6)* 85+2 (2) * Vibrio cholera neuraminidase released 100% of sialic acids when incubated for a further 24 hours - 85 -Table 13: Percentage of sialic acid remaining substituted at positions C7+C8 and C4 of the glycoproteins from the upper and lower halves of Wistar rat colon, after digestion with cell free filtrate (rat enzymes) of Wistar rat faeces. D = % C7+C8 detected in the F = % C7+C8 detected in the C4 = % C4 substituted sialic enzyme digest filtrate (free sialic acids) acid of the total glycoprotein 1 E»Dt # 1 2 —' * 3 M'dase 1 3 . 1 5 . 3 1-7 Esterase 27 .9 71 .9 6 . 1 >Eubstrate - J SIALIC ACID SUBST ITUTED AT T l m e V ( h r s ) \ RU R L B U RL • • nu RL c 1 C 7 6 8 (D) : 7 C 8 ..IF) C 1 L 7 U 8 C 7 C 8 (F) (D) ° 7 C 8 (F) C 1 (D) '7^8 (F) C 1 C 7 C 8 C 7 C 8 0 62 0 66 17 0 13 58 0 69 12 0 18 59 0 69 _DJ. 17 0 1 52 0 . 5 60 19 10 50 22 35 1 . 0 60 13 13 13 37 35 61 56 52 36 31 22 59 11 62 31 51 32 2 . 0 55 37 50 31 17 16 3 - 0 50 18 -1 . 0 53 29 30 11 28 11 26 13 37 9 38 15 6 . 0 39 25 23 8 . 0 10 28 27 0 19 5 1 2 . 0 30 18 15 21 28 20 27 30 22 15 16 2 24 . 0 23 5 7 32 - 23 13 9 10 20 3 3 8 . 0 16 31 3 1 0 3 1 18 .0. 22 19 1 21 -93.0 0 0 7 0 8 0 - 87 -Table 14: Percentage of sialic acid of the bovine submandibular mucin (BSM), C4BSM and rat seromucoid, which remained substituted at positions C7+C8 and C4, after digestion with cell-free filtrate of Wistar rat faeces. D F C 4 = % C7+C8 detected in the enzyme digest = % C7+C8 detected in the filtrate (free sialic acid) = % C4 substituted sialic acid of the total glycoprotein - 89 -Figure 19: Removal of 0-acetyl groups from positions CJ+CQ of sialic acid residues of BSM (•••••••), rat upper ( + ), and rat lower (•••$•••) glycoproteins by rat faecal enzymes. Insert: enlarged scale of the first 40 hours of digestion. PERCENT C7 + C8 SIALIC ACIDS .- 06 -- 91 - • de-0-acetylation could be normalized by expressing the results in terms of the percentage of side chain de-0-acetylation as indicated by equation 1 (El appendix A4). The rate of side chain de-0-acetylation calculated from equation El indicated that side chain 0-acetyl groups were removed most slowly from the sialic acids of the rat upper glycoprotein (table 15). The rate of removal from the rat lower glycoprotein and BSM was similar and faster than from the upper glycoprotein. The rates of de-0-acetylation from these three glycoproteins were compared and analyzed statistically using the paired t'-test (table 16). Analysis of the filtrates demonstrated the presence of side chain substituted sialic acids, indicating that the rat neuraminidase was capable of removing such sialic acids. The possibility that these side chain sialic acids appeared in the filtrates as the result of an enzyme, oligo-saccharidase, cleaving the prosthetic group at some other points of the oligosaccharide chain was eliminated because no glycosidically bound sialic acids could be detected in the filtrates. Since (i) side chain substituted sialic acids could be assayed in the digests, the filtrates and the retentates, and (ii) the quantities of the free and bound sialic acids were also measurable in these fractions, i t was possible using equations E2-E5 (see appendix A5) to calculate the various populations of free and glycosidically bound sialic acids which were either side chain substituted or which had no side chain substituents. Tables 17-18 show the distribution of the four categories of sialic acids during the course - 92 -Table 15: The percentages of C4 and side chain (C7+C8) de-O-acetylation from sialic acids of various glycoprotein substrates. RU = rat upper colonic epithelial glycoprotein RL = rat lower colonic epithelial glycoprotein BSM = bovine submandibular mucin CB = C4BSM RNS = rat seromucoid X C4 de-O-acetylation % C7C8 de-O-acetylation Exp't 1 2 3 4 1 2 3 4 Substrate RU RL RU RL BSM RU RL BSM BSM CB RNS RU RL RU RL BSM RU RL BSM BSM CB RNS Time (hrs) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.5 28 33 18 11 1 0 0 12 3 8 12 1 35 19 24 55 29 9 38 8 38 0 75 4 8 0 15 18 1 27 21 7 8 29 2 27 66 38 72 11 85 5 27 18 14 9 31 3 19 4 28 77 100 46 72 69 53 100 9 74 56 81 35 44 26 47 6 65 38 i 8 61 89 100 63 81 99 31 100 54 56 47 56 12 78 94 67 96 100 51 54 55 68 71 24 89 87 95 100 94 100 100 63 31 61 100 77 80 78 69 36 95 98 100 73 100 87 48 98 65 49 65 91 67 75 93 95 89 100 100 100 100 100 - 94 -Table 16: Statistical analysis of the rates of de-0-acetylation of C4 and C7+C.8 substituents from different glycoprotein substrates. Comparisons were made between the percentages of C4 and C7+C8 de-O-acetylated (calculated from equation El, p.160 and Table 15) by the same enzyme pre-paration from the different glycoprotein substrates at period between 1/2 and 24 hours. Statistical comparisons were made with the paired t-test method and the level of significance was set at a value = 0.05. - 95 -Table 16a Rat Upper Rat Lower BSM C7C8 C A C7C8 C A C 7 C 8 RU C 7 C 8 t=-5. 9 1 ( 1 1 ) * 0.001 > a t=-2 . 26 (9 ) 0.05 > a > 0.02 t=2.A8(7) 0.02 > a > 0.01 C A t= A . 2 6(8) 0.01> a > 0.001 t=2.A3(6) 0.05 > a > 0.02 RL C7C8 t=-2 . 89 (9 ) 0.02 > a > 0.01 t=l.A2(6) NS C A t=0.78(7) NS BSM C7C8 t= A . 7 6(8) 0.01 > a > 0.001 C A Table 16b C 4B^I BSM C7C8 C A C 7C 8 CA CA 3SM C 7C 8 t=-2.61(5) 0.05 > a > 0.02 t=l.Al(5) NS C A t=l.02(A) NS 3SM c 7 c 8 t=A.76(15) 0.001 > a C A * t ( d f ) , a . - 96 -Table 17: Distribution of the various populations of free and glyco-sidically bound sialic acids which are either side chain substituted (CJCQ) or which have no side chain substituents ( C Q ) . Values are given in micrograms (ug) sialic acids. GLYCOPROTI SIN RAT UPPER RAT LOWER BSM FREE SIALIC ACID BOUND SIALIC ACIC FREE SIALIC ACID BOUND SIALIC ACID FREE SIALIC ACID BOUND SIALIC ACID TIME (HRS Co c 7 c 8 c 0 C7C8 C 0 C7C8 c 0 C7C8 c 0 C7C8 CO C7C8 0 7 . 7 0 38.7 82.0 3.1 2 . 9 56.7 38.5 7.2 0 3 7 . 2 13.7 0 . 5 1 . 9 1 . 9 10 . 7 67.2 6.1 1 . 9 51.9 3 7 . 1 7 . 7 0 39.2 35.0 1 1.6 6.0 15.8 5 3 . 6 10.7 5.1 7 3 . 6 12 . 9 9 . 9 6 . 2 32.0 3 0 . 5 O i 2 11.8 7.0 3 7 . 6 51.9 23.1 1 . 8 31.6 11.1 22 . 3 9-5 30.1 19.7 EH cu 1 20. 3 8.3 23.1 56.2 36.2 11.1 38 . 7 12.1 11.8 11.1 11 . 5 16.6 X w e 32.1 12.3 2 9 . 8 39 .9 5 7 . 7 13-5 25.9 7 . 9 7 0 . 8 16.0 •8.1 1 . 3 21 71.2 10.5 22.1 21 . 5 75.0 1 8 . 7 8 . 9 1 . 5 77 . 9 5.1 5.1 0 . 6 0 0 . 9 0 30 . 3 58.0 0 0 10 . 7 5 0 . 9 0.6 0 2 9 . 5 33-9 1 5.1 3.8 30.7 52.0 1.9 5 . 6 5 2 . 3 2 9 . 6 12 . 3 8 . 3 25.5 26.2 ' m f-i 15.5 1 1 . 3 29 . 6 38.1 23.1 11.1 13.1 17.0 2 3 . 8 22.6 11.1 8.7 Cu X UJ 12 36.2 15.7 27.2 22.6 6 0 . 8 11 .9 .27.8 5 . 3 12.1 20 . 5 9.0 0 36 56.2 25.5 18,1 5.1 90 .3 2.1 11.9 2.9 58.1 7.0 1.2 0 93 0 0 5.1 0 91.0 7.8 8.9 0 2.7 0 1.7 0 - 98 -Table 18; Distribution of the various populations of free and glyco-sidically bound sialic acids which are either side chain substituted (C7C.8) or which have no side chain substituents ( C Q ) . Values are given in micrograms (ug) sialic acids. GLYCOPROTEIN C,BSH BiiM KAT SEkGMUCOID TIME (HKS) FREE SIALIC ACID C 0 C 7 C 8 BOUND SIALIC ACID C 0 c 7 c B FREE SIALIC ACID c 0 c ? c b BOUND SIALIC ACID C 0 c 7 c 8 FREE SIALIC ACID C 0 c 7 c e BOUND SIALIC ACID Co c 7 c 8 0 0.5 1 2 0 B 20 65 0 0 0 0.1 2.6 9.9 15.7 0 0 0 1.0 3.0 2.2 1.2 GLYCOPROTEIN 0.5 7.2 6.0 0.7 10.2 6.1 1.7 26.1 23.2 20.1 17.B 5.1 7.5 0.1 RAT UPPER 0 0.9 3.3 10.0 20 .0 39.9 72.1 37.3 0 0 0.8 5.3 6.8 8 .7 5.0 0 38.3 35.2 28.1 31.1 21.9 13.6 7.5 6.0 00.3 05.7 00.1 33.0 21.9 12.0 2.6 0 RAT LOWER 0 0.9 3.6 B.5 12.6 17.5 17.5 17.2 0 0.5 1.6 3.9 0.9 0.6 3.1 3.1 FREE SIALIC ACID BOUND SIALIC ACID FREE SIALIC ACID BOUND SIALIC ACID TIME (HRS) c 0 c 7 c 8 Co C 7 C 8 CO c 7 c 8 Co C 7 C 8 0 0 0 39.0 60.7 0 0 53.7 OS . l 1 6.0 0.6 01.0 56.9 12 .0 7.0 06.1 32.2 3 11.1 10.3 30.3 00.0 * 6 20.2 6.8 35.0 30.9 r 12 07.3 10.5 28.2 21.6 39. 3 15.5 20.0 8.1 X " 20 50.0 3.0 18.2 10.7 08 50.6 11.6 20.0 0 10.1 12.5 9.8 9.3 3.9 0.9 2.1 1.6 10.2 7.0 6.0 2.1 2.0 0 0 0 • - 100 -of the enzyme digestion. All batches of enzyme preparations yielded similar results and therefore only one set of data is illustrated in figures 20-22. As shown in figures 20-22, the three glycoproteins studied showed a continuous reduction in the amount of bound sialic acids, both with and without side chain substitution. The quantity of the free C-,+C0 substituted sialic acids increased initially and then decreased again as the incubation period was extended. With the rat lower glycoprotein the amount of unsubstituted (C^) sialic acids continued to increase and until i t reached a plateau. With the rat upper glycoprotein and BSvi, the population of C Q sialic acids increased to a maximum at 36 hours and subsequently decreased to zero at 93 hours. It was of interest that the i n i t i a l rates of the appearance of the free C Q and C7+Cg sialic acids were approximately the same with a l l the glycoproteins studied. This suggested that the neuraminidase that hydrolyzed the sialic acids could act on the sialic acid residues regardless of their side chain substitutions, (ii) Percentage of C4 substituted sialic acids Tables 13-14 and figure 23 show that in addition to the decrease in side chain substituted sialic acids there was also a decrease in the substituted sialic acids. Equation El was used to calculate and compare the rates of C4 de-0-acetylation from the three substrates. This calculation depended upon the assumption that the neuraminidase did not cleave substituted sialic acids. If the neuraminidase could remove substituted sialic acids, the calculated, rate would be an over estimation of the actual value because the - 101 -Figure 20: Digestion of rat upper epithelial glycoprotein by rat faecal enzyme extracts showing the distribution of C Q (— + —) and C7+C8 substituted (•••£•••) free and Cg ( ) and C7+C8 substituted (•••••••) bound sialic acids. MICROGRAMS (UG) S I A L I C ACID - ZOl -- 103 -Figure 21: Digestion of rat lower epithelial glycoprotein by rat faecal enzyme extracts showing the distribution of C Q (—•< + —) and C7+C8 substituted (•••<>•••) free and C Q ( ) and C 7 + C Q substituted (•••••••) bound sialic acids. MICROGRAMS (UG) SIALIC ACID - KIT -- 105 -Figure 22: Digestion of bovine submandibular mucin (BSM) by rat faecal enzyme extracts showing the distribution of C Q (•••<0"**) and Cy+Cg substituted ( i ) free and C Q ( ) and Cy+Cg substituted (•••••••) bound sialic acids. - 107 -Figure 23: Removal of 0-acetyl groups from position C4 of sialic acid residues of BSM ( • ), rat upper (— + — ) , and rat lower (•••••••) glycoproteins by rat faecal enzymes. - 108 -- 109 -percentage of substituted sialic acids (numerator of equation El) would be under estimated. As will be seen in table 16, there was no apparent difference between the rates of de-0-acetylation of the rat lower glycoprotein and BSM. 0-acetyl groups were removed most slowly from the rat upper glycoprotein. The difference in the rates of de-0-acetylation observed between the rat upper and lower glycoproteins suggested that sialic acid residues on the two glycoproteins were not equally accessible to the rat faecal enzymes. Further analysis of the data showed that in a l l three cases, the rate of removal of C4 0-acetyl groups was faster than the removal of the side chain 0-acetyl groups (table 16). However, this did not eliminate the possibility that substituted sialic acids could be removed by the neuraminidase. Since the validity of equation El used for the calculation of the rate of de-0-acetylation depended on the assumption that the substituted sialic acids were not removed by the rat faecal neuraminidase, i t was important to determine whether the enzyme could act on substituted sialic acids. To study this possibility, bovine submandibular mucin (BSM) was exhaustively digested with V.c. neuraminidase and used as a substrate (C^ BSM) for the rat faecal enzymes (figure 24). As will be seen the de-O-acetylated C^ BSM was degraded at a much faster rate than the C^ BSM. The presence of a lag period of 1/2 hour in the hydrolysis of sialic acids suggested that the substituent must be removed before the neuraminidase could act on the sialic acid. Further, as calculations indicated that the removal of C A 0-acetyl groups from the BSM and C^ BSM were the same, i t - 110 -Figure 24: Removal of sialic acid residues from saponified (•••<>•••) and non-saponified ( + ) bovine submandibular mucin which had been digested exhaustively with V.C_. neuraminidase (C4BSM). - I l l -Q3SV313H. QI3V 3I1VIS lN33H3d - 112 -would appear that de-0-acetylation at is the rate limiting step in the removal of the sialic acids. (c) NANA-degrading enzyme: It was observed that the total amount (free and bound) of sialic acids present in the digests decreased during the course of incubation with the rat enzymes. Incubation of the faecal enzymes with N-acetyl neuraminic acid confirmed the presence of the NANA-degrading enzyme (figure 25). The results were unlikely to be the consequence of (a) bacterial contamination because both aerobic and anaerobic cultures failed to grow any bacteria; and (b) spontaneous "auto-degradation" of the sialic acids because the effect was not observed with the boiled control extract. This result implied that an enzyme was present in the rat faecal enzymes which converted the sialic acids into a form which was not TBA reactive. It seems possible that this enzyme degrades the carbon chain of sialic acid, i.e. i t is a pyruvate lyase. (d) Glycosidases: Table 19 shows that the rat faecal enzyme extracts contained a number of glycosidases capable of hydrolyzing artif i c i a l p-nitrophenyl glycoside substrates. Variations in the activities of these enzymes occured in the different preparations of rat faecal enzymes but no activity was demonstrated in the boiled control preparations. However, the existence of glycosidases active against p-nitrophenyl substrates does not prove that such enzymes are - 113 -Figure 25: Degradation of N-acetyl neuraminic acid by NANA-degrading enzyme present in the rat faecal enzyme extracts. - frit -- 115 -Table 19: Mean s p e c i f i c a c t i v i t i e s o f g l y c o s i d a s e s and o t h e r enyzmes d e t e c t e d i n the r a t f a e c a l enzyme e x t r a c t s . The a c t i v i t i e s o f these enzymes were d i r e c t e d a g a i n s t a r t i f i c i a l p - n i t r o p h e n y l s u b s t r a t e s . One u n i t o f a c t i v i t y was d e f i n e d as the amount o f enzymes r e q u i r e d t o r e l e a s e 1 micromole o f p a r a - n i t r o p h e n o l p er minute a t 37°C and pH7.0 and the s p e c i f i c a c t i v i t y was expressed as m i l l i u n i t per m i l l i g r a m wet weight faeces. Enzyme Su b s t r a t e Mean S p e c i f i c A c t i v i t y + SD (N) Range oi -L-Fucosidase PNP-a-L-Fucoside 1 1 + 6 (10) 3 - 1 8 a -D-Galact- PNp _a_D - G a l a c t o s i d e 282 + 127 (10) 111 - 471 o s i d a s e k D - G a l a c t - PNP-B-O-Galactoside 188 + 64 (10) 89 - 266 o s i d a s e °4)-Glucosidase • PNP-a4D-Glucoside 57 + 26 (10) . 22 - 97 S-D-Glucosidase PNP-6-O-Glucoside 65 + 26 (10) 29 - 114 N-Acetyl - e-D-Galactosaminidase PNP-N-Acetyl-6 -0-Galactosaminide 6 + 4 (10) 2 - 1 6 N -Acetyl - e-D-Glucosaminidase PNP-N-Acetyl-3-D-Glucosaminide 3 1 + 9 (10) 1 9 - 4 4 Neuraminidase Human seromucoid 9 + 6 (10) 2 - 1 7 E s t e r a s e PNP-Acetate 60 + 2 3 (10) 17 - 28 NANA-degrading N-Acetyl neura-m i n i c a c i d 3 + 2 ( 6 ) 0 - 6 - 116 -capable of hydrolyzing macromolecular substrates. Therefore, to investigate the activity of the enzymes against such substrates the preliminary experiments (see p.64) were expanded by performing gas liquid chromatography analysis for sugars in incubates of glycoproteins with rat faecal enzymes. Tables 20-21 show the quantities of sugars released on incubation of the saponified and intact glycoproteins from the upper and lower halves of rat colon with rat faecal enzymes. These data are illustrated in figures 26-29. Little or no sugar was released from either the saponified or the intact glycoproteins until a significant proportion (10-20%) of the sialic acids had been removed. Furthermore, the rate of release of both galactose and fucose was apparently independent of whether or not the glycoprotein was saponified before incubation with the enzyme extracts. Gas liquid chromatography also indicated the presence of (i) N-acetyl glucosamine; (ii) a peak corresponding to N-acetyl galactosamine and N-acetyl mannosamine (these compounds could be not resolved under the experimental conditions used); and ( i i i ) hexosamines. It was noted that the quantities of neutral sugars detected reached a maximum value at 12-36 hours and then decreased. This was accompanied by the appearance of "new" glc peaks; the quantity of which increased with time. (e) Bacteriological studies: No organisms could be detected in any batch of rat faecal enzyme extract by Gram's stain or by aerobic or anaerobic culture. As expected, similar negative results were obtained after addition of sodium azide (final concentration 0.02% w/v). - 117 -Table 20: Hydrolysis of sugars by rat faecal enzymes from the rat upper colonic epithelial glycoprotein. ug/mg glycoprotein Rat Upper Substrate Sialic Acid Fucose galactose NAc Glu NAc Gal + Time (hrs) Nac Man KOH Ron K N K N K N K RP KOH Exp't 0 0 0 0 0 4.5 0 0 0 0 0 #1 1 25.3 16.8 0 0 4.9 0 1.1 0 1.5 0 3 53.3 34.1 3.9 2.2 13.1 6.6 4.1 1.5 3.2 0 6 78.3 59.1 9.2 4.6 25.6 11.4 6.6 5.6 3.9 4.7 12 86.0 92.3 23.7 8.0 43.6 21.5 16.7 12.5 12.0 8.9 24 104.8 91.8 18.9 11.0 13.0 30.2 1.2 1.0 4.7 9.5 48 98.5 99.4 25.0 14.0 22.3 18.3 4.4 1.9 17.2 9.5 Exp't 0 3.8 1.7 0 0 1.6 0.8 0.7 0 0 0 #3 1 27.3 17.8 0.5 1.4 4.7 2.1 0.7 0 2.7 2.2 4 77.7 51.4 7.1 6.0 11.3 9.2 3.4 2.6 8.1 6.2 12 127.6 101.5 18.6 18.0 51.6 1.2 13.5 10.3 15.4 12.9 36 167.7 159.6 36.1 33.0 18.0 9.2 15.4 1.0 36.6 32.4 93 165.5 195.7 34.5 37.3 0 15.0 2.0 0 36.9 0 - 118 -Table 21: Hydrolysis of sugars by rat faecal enzymes from the rat lower colonic epithelial glycoprotein. Rat Lower Substrate Sialic Acid Fucose galactose NAc Glu NAc Gal + Time (hrs) Nac Man KOH Non K N K N K N K N KOH Exp't 0 0.1 1.3 0 0 1.1 0 0 0 0 0 #1 1 31.7 23.4 0 0 3.0 1.2 0 0 0 0 3 6 12 87.6 101.6 7.5 10.1 71.1 42.6 23.4 16.8 19.3 11.7 24 91.8 110.4 24.3 - 109.6 - 22.8 - 0 -48 - 113.0 9.0 - 54.4 0 - 0 Exp't 0 1.9 0.2 0 0 1.0 0.5 0 0 0 0 #3 1 48.5 20.4 0.9 0.6 2.4 0.7 0.7 0 2.4 1.3 4 99.5 73.9 4.7 3.6 12.9 11.3 2.9 1.7 6.6 4.2 12 147.5 142.0 21.8 19.4 49.3 51.4 14.9 17.4 21.2 21.1 36 181.6 181.1 42.8 45.3 109.7 125.3 43.7 97.4 51.2 45.0 93 176.5 193.1 33.1 57.2 184.1 161.3 2.5 0 72.1 43.4 - 119 -Figure 26: Degradation of saponified rat upper by cell-free filtrate of Wistar rat quantities (ug/mg glycoproteins) of (*••••••), galactose ( ), and hydrolyzed from the glycoprotein by at 37°C. colonic epithelial glycoprotein faeces. The diagram shows the sialic acid ( 1 ), fucose N-acetyl glucosamine (—O—) the rat faecal enzyme extracts - 120 -- 121 -Figure 27: Degradation of non-saponified rat upper colonic epithelial glycoprotein by cell-free filtrate of Wistar rat faeces. The diagram shows the quantities (ug/mg glycoproteins) of sialic acid ( « ), fucose ("•"••••), galactose ( ), and N-acetyl glucosamine (—O—) hydrolyzed from the glycoprotein by the rat faecal enzyme extracts at 37°C. - zzi -- 123 -Figure 28: Degradation of saponified rat lower colonic epithelial glycoprotein by cell-free filtrate of Wistar rat faeces. The diagram shows the quantities (ug/mg glycoproteins) of sialic acid (—HI ), fucose ( • • • O - - ' ) , galactose ( ),and N-acetyl glucosamine (—O—) hydrolyzed from the glycoprotein by the rat faecal enzyme extracts at 370C. - 124 -- 125 -Figure 29: Degradation of non-saponified rat lower colonic epithelial glycoprotein by cell-free filtrate of Wistar rat faeces. The diagram shows the quantities (ug/mg glycoproteins) of sialic acid ( 1 ), fucose (•••••••), galactose ( ), and N-acetyl glucosamine (—Q—) hydrolyzed from the glycoprotein by the rat faecal enzyme extracts at 37°C. AMOUNT OF SUGARS RELEASED (UG/MG GLYCOPROTEINS) n o - 9ZT -- 127 -DISCUSSION Epithelial glycoproteins are a major constituent of the mucin which forms a continuous coating over the gastro-intestinal epithelium and which is assumed to serve both as a lubricant and as a protective barrier for the underlying mucosal epithelial cells (2,12,16). Studies of the colonic epithelial glycoproteins indicate that their carbohydrate prosthetic groups are terminated by sialic acid residues with 0-acetyl substituents located on the polyhydroxy side chain and/or at position C^  (12,14,23-24,126,135-136). The biological function of these sialic acids is unknown. However, because (a) C^ substituted sialic acids are resistant to digestion with bacterial neuraminidases (13); (b) sialic acids have been shown to protect some glycoproteins against proteolysis (137); and (c) bacterial glycosidases are in the main exo-glycosidases (35), i t has been suggested that they serve to protect the glycoprotein against bacterial degradation and therefore help to maintain the integrity of the "mucin barrier". As mucin glycoproteins are degraded by the colonic flora (28-38) i t seems likely that, under normal circumstances, there is a "steady state" in which mucin lost is balanced by mucin synthesis and secretion. Since (a) both human ulcerative colitis and the degraded carrageenan-induced colitis of rabbits are characterized by a reduction in the proportion of the sialic acids of the epithelial glycoproteins with C^ substituents (138-139); and (b) carrageenan colitis can be prevented by the elimination of certain of the large intestinal flora (124-125) i t has been suggested (70,74-75,140) that a pivotal event in - 128 -ulcerative colitis could be a change in the epithelial glycoproteins and/or the bacterial flora such that there is a more rapid removal of the sialic acids. Under such circumstances the steady state would be unbalanced towards degradation, weakening the mucin barrier and permitting attack by constituents of the faecal stream on the underlying mucosal cells. This thesis reports a detailed investigation of the role of sialic acids in the degradation of colonic epithelial glycoprotein by faecal enzymes using as a model system, the Wistar rat. The model was selected because (a) the chemical nature of these glycoproteins is known; (b) such glycoproteins are readily available; and (c) the Wistar rat is cheap, easily maintained, and has the virtue of genetic homogeneity. Further, i t provides a control system for the development of methods for the examination of the degradation of human colonic glycoproteins. Although the degradation of glycoproteins by extracellular enzymes from faecal extracts or cultures of faecal bacteria has been reported by several investigators (28-38), the present study differs in a number of respects. Firstly, the presumed natural substrates, rat colonic epithelial glycoproteins, were studied in addition to the ar t i f i c i a l p-nitrophenyl glycoside substrates, bovine submandibular mucin, human seromucoid and rat seromucoid. The latter is probably a natural substrate for these enzymes because i t might be expected that rat serum protein would be a constituent of faeces. In fact, unpublished studies in our laboratory have demonstrated that there are glycoproteins present in the supernatant from the isolation of epithelial cells which can be - 129 -detected by anti-sera raised against plasma proteins. Secondly, a detailed study was made of the action of the neuraminidase(s) upon O-acetylated and de-O-acetylated sialic acids. Thirdly, the presence of a de-O-acetylase active against free and/or bound O-acetylated sialic acids has been demonstrated for the first time. Fourthly, glycosidases active against the "natural" macromolecular substrate has been confirmed; and fifthly, the presence of a NANA-degrading enzyme was detected. The ease of hydrolysis of the sialic acids was inversely related to the degrees of O-acetylation. Saponified (de-acetylated) glycoproteins were generally degraded faster than the corresponding non-saponified glycoproteins. Epithelial glycoprotein isolated from the upper half of rat colon which contained the most 0-acetyl groups was degraded most slowly. Rat seromucoid and bovine submandibular mucin (BSM) which contained the smallest amount of 0-acetyl substituents were degraded the fastest. Removal of substituents at positions C7/Cg or of sialic acids by the faecal enzymes was indicative of the presence of the de-O-acetylase. This enzyme was basically an esterase which hydrolyzed the ester linkage of the 0-acetyl substituents. It could act on either the free or bound sialic acids. The rates of de-O-acetylation differed for the different glycoproteins in that the rat upper glycoprotein was de-O-acetylated more slowly than the rat lower glycoprotein or BSM. The significance of this is unknown but i t may suggest that some of the substituted sialic acids are more "hidden" than the others. The data indicated that the faecal neuraminidase can hydrolyze sialic - 130 -acids regardless of their side chain substitution. The side chain 0-acetyl groups contribute l i t t l e to the protection of the sialic acids from the neuraminidase. , This is in agreement with with the finding that in BSM the side chain substituted sialic acids are susceptible to Vibrio cholera neuraminidase but the less substituted sialic acids are degraded faster (15). The neuraminidase(s) present in the rat faecal extracts apparently differs from the V.c_. neuraminidase in that the former is active at pH7.0 whereas the optimal pH of the latter is 5.5. Further, in contrast to V.c_. neuraminidase the rat faecal neuraminidase is apparently calcium independent because the minimal media was basically a phosphate buffer which did not contain any calcium ion. Both neuraminidases, however, are unable to hydrolyze C^ substituted sialic acids. Analysis of the data, assuming that a l l acetylated substrates could be hydrolyzed by the rat faecal neuraminidase, shows that the rates of the two de-acetylation processes (i.e. Cy/Cg and C^ ) were different in that the de-O-acetylase hydrolyzed the 0-acetyl group preferentially. If the enzyme recognized only the ester linkage, one would expect the side chain substituents to be removed at the same rate . Therefore, i t is possible that (a) the de-O-acetylase recognizes the CA substituent better than i t does for the side chain 0-acetyl groups; or (b) the side chain 0-acetyl groups are less easily accessible to the enzyme; and/or (c) there was more than one type of esterases acting on the ester linkages. Vibrio cholera neuraminidase hydrolyzed the rat upper and lower colonic epithelial glycoproteins to a lesser extent than the faecal enzyme extracts. - 131 -Hydrolysis with the V.c_. neuraminidase never reached completion because the substituted sialic acids were resistant to this enzyme. In contrast, the cell-free filtrate of the rat faeces removed essentially a l l the sialic acids from the rat colonic epithelial glycoproteins. Apparently this difference was due to the presence of the de-O-acetylase removing the acetyl groups (C^, Cy/Cg) from the sialic acids. Hence, i t seems likely that the de-O-acetylase plays an important role in the rapid removal of sialic acids from glycoproteins or alternatively, the rat faecal neuraminidase is capable of removing 4-0-acetyl sialic acids. If 0-acetyl groups were the only factor that determined the susceptibilty of glycoproteins to digestion by neuraminidase, i t would be logical to expect a l l the saponified substrates to be equally susceptible to neuraminidase digestion. However, saponified rat upper glycoprotein was degraded slower than the saponified rat lower glycoprotein or the BSM. A possible explanation for the differences observed is that either (a) some 0-acetyl linkages were not susceptible to cleavage by alkali; this was highly unlikely because analysis showed that the saponified substrates did not contain 0-acetyl substituted sialic acids; or (b) the structures of these glycoproteins were different from each other, the sialic acids of rat upper glycoprotein being less accessible to neuraminidase than those of the rat lower glycoprotein or BSM. A further difference between the rat faecal neuraminidase and Vibrio  cholera neurmainidase is that the former removes a significantly larger proportion of the sialic acids of the de-O-acetylated rat glycoproteins. A • - I m -possible explanation for this phenomenon is that some of the rat glycoprotein sialic acids are protected from digestion with neuraminidase by the sugars in the immediate vicinity. Kuhn and Wiegandt (141) have, for example, described such a neuraminidase resistant sialic acid residue as a branch point in a monoganglioside. The presence of exo-glycosidases in the rat faecal enzyme extracts could, however, remove the sugars distal to the branch point producing a neuraminidase sensitive sialic acid residue. Data obtained in this study suggested that in addition to the neuraminidase and the de-O-acetylase which act on the sialic acid residues, the rat faecal enzymes contained a third enzyme, termed NANA-degrading enzyme, which degraded free sialic acids. The exact nature of this enzyme is unknown but the data imply that i t breaks down free sialic aicds into components that are not detectable by the colorimetric methods used. One possibility of this enzyme is an acetylneuraminate pyruvate-lyase as described by Schauer et al.(142) The activity of this enzyme, isolated from Clostridium perfrinqens, was retarded by 4-0-substituted (80%) and 7-0-substituted (40%) free sialic acids. Glycosidically bound sialic acids, however, could not serve as substrates for the enzyme. Therefore, the possibility that the NANA-degrading enzyme is a pyruvate-lyase and is only active against free sialic acids cannot be eliminated. Many glycosidases have been reported to be present in the faeces and caecal contents of human and rats (28-38). In the present investigation a fucosidase, a galactosidase, and N-acetyl hexosaminidases were detected in the rat faeces using artif i c i a l p-nitrophenyl derivatives and rat colonic - 133 -epithelial glycoproteins. It has not been established whether those enzymes active against the ar t i f i c i a l substrates will also hydrolyze the presumed natural substrates. The rate of hydrolysis of fucose, galactose, and N-acetyl glucosamine from the rat colonic epithelial glycoproteins was the same as from the saponified glycoproteins, suggesting that the 0-acetyl groups on the sialic acid residues did not protect the oligosaccharide chains from hydrolysis by these glycosidases. Differences in the rates of hydrolysis of the sugars of the saponified and the non-saponified glycoproteins would be expected i f sialic acids were the only type of terminal sugar on the carbohydrate chains. N-acetyl glucosamine did not appear in the digests until 4 hours after the digestion had commenced suggesting that this sugar was not located at or near the terminal ends of the oligosaccharide chains. Fucose and galactose, on the contrary, appeared at the same time as the sialic acids but in much smaller quantities. These results imply that these sugar residues may either be located at, or very close to the terminus of the sugar chains, or alternately, that the carbohydrate chains have multiple branches with sialic acid, fucose, and galactose located at the terminal ends of different branches. Since N-acetyl galactosamine and N-acetyl mannosamine appeared together as one single peak on the gas-liquid chromatogram, i t was not possible to determine the exact quantities of each component. N-Acetyl galactosamine is known to be present in rat colonic epithelial glycoproteins (6-7). N-Acetyl mannosamine, however, is not present but i t could be one of the breakdown products of sialic acid i f the NANA-degrading enzyme is a pyruvate lyase. If - 134 -this peak corresponds to N-acetyl galactosamine, the data obtained indicate that the N-acetyl galactosamine was not located near the terminals of the oligosaccharide chains because this sugar also appeared late in the incubates. An alternatve explanation for this is that i f there is a small amount of N-acetyl galactosamine present as the end group and that the putative N-acetyl galactosaminidase has low activity, then i t may require several hours of digestion to release sufficient quantity of sugar to be detected by the method used. The data showed that a small amount of hexosamines without any N-acetyl substituent was present in the hydrolysates of the rat glycoproteins. This small quantity may be the result of (i) the presence in the glycoproteins very small quantities of such hexosamines or alternatively the presence of a de-N-acetylase; (ii) the presence of l i t t l e or no hexosaminidase in the rat faecal enzyme extracts; and/or ( i i i ) the hexosamines being located at sites which well "protected" from the actions of such hexosaminidases. Based on the data obtained from the present investigation i t is possible to devise a simplified model for the structure of the oligosaccharide chains of the rat colonic epithelial glycoprotein. This is illustrated schematically in figure 30. Since sialic acid, fucose, and galactose were hydrolyzed from the glycoprotein quite rapidly, these residues are believed to be located at or near the chain terminals. In fact, sialic acids and fucose are known to be present at the terminal positions of oligosaccharide chains of the rat colonic glycoprotein and they have never been reported to exist as residues in the interior of the chain. Studies by Slomiany e_t al. (7) on the structures of - 135 -Figure 30: Schematic of the possible distribution of sugar residues on the oligosacchaide chains of rat colonic epithelial glycoproteins. Chain #1, 2 and/or 3 may also be present together as substituents on a particular carbohydrate chain. Galactose may also be present in interior positions of the sugar chain. - 136 -1 2 3 Sialic Acid Fucose 1 Galactose Galactose Galactose 1 N-Acetyl Galactosamine and/or N-Acetyl Glucosamine 1 Galactosamine and/or Glucosamine Protein Core of the glycoprotein - 137 -the oligosaccharide chains of Fisher rat colonic mucus glycoprotein have established that the carbohydrate chains are linked 0-glycosidically to the protein core through N-acetyl galactosamine residues. Furthermore, the galactose residue of the carbohydrate chains has been found to exist as (i) the terminal sugar; (ii) substituted with either fucose or with sialic acid, but not by both at the same time; and ( i i i ) one of the interior chain residues. Hence, the putative structure proposed in this study partially agrees with the structure of the oligosaccharide chains of rat mucin glycoproteins elucidated by Slomiany et al.(7) Figure 31 illustrates the proposed scheme for the hydrolysis and degradation of substituted sialic acids from the colonic epithelial glycoprotein. It is assumed that the de-O-acetylase that hydrolyzes the side chain 0-acetyl groups is the same as that for the substituent (enzyme 1). The rat faecal neuraminidase(s) (enzyme 2) will only act on the de-O-acetylated sialic acid; hence, releasing the side chain substituted (C7+Cg) and non-substituted (CQ) sialic acids into the medium. The de-O-acetylase will convert the side chain substituted free sialic acids into the non-substituted form which, subsequently, is hydrolyzed by the NANA-degrading enzyme (enzyme 3). Sialic acids without substituents can be removed by the rat faecal neuraminidase. The present investigation clearly has demonstrated the presence of an emzyme system in rat faeces capable of degrading rat colonic epithelial glycoproteins by means of neuraminidase(s), de-O-acetylase, NANA-degrading enzyme, and several glycosidases. This implies that under normal conditions - 138 -Figure 31: A diagrammatic representation of the scheme proposed for the hydrolysis and degradation of side chain and C4 substituted sialic acids from rat colonic epithelial glycoproteins. G-C7C8 = side chain (C7+C8) substituted bound sialic acids G-CO = non^substituted bound sialic acids C7C8 = side chain (C7C8) substituted free sialic acids CO = non-substituted free sialic acids G-C4 = bound CA substituted sialic acids, 1 = de-O-acetylase 2 = neuraminidase 3 = NANA-degrading enzyme X = unknown products - 139 -- 140 -the mucus glycoproteins are degraded by such enzymes and the intestinal epithelium will compensate for the loss by the synthesis and secretion of more mucins. Although in the present investigation, the origin of the faecal enzymes was not established, varieties of glycosidases and neuraminidases are known to be produced by the gut flora (29-38), I believe that most probably these enzymes are of bacterial origins. Hoskins e_t al. (35-38) have been able to obtain mucin-degrading enzymes from the supernatants of the human faecal innoculae. Further, experiments performed in our laboratory have shown that epithelial glycoproteins are apparently unaffected by lyzosomal enzymes released from the death and lysis of epithelial cells (11). Based upon both clinical and experimental observations Shorter et_ al. (44,78-79) have proposed a working hypothesis for the role of bacteria in the etiology of ulcerative colitis. This hypothesis rests upon the assumption that ulcerative colitis "results from the establishment of a state of hypersensitivity to bacterial antigens normally present in the gastrointestinal tract" (44). Since the faecal flora has close contact with the intestinal epithelium, i t seems likely that "the hypersensitivity state results from (i) bacterial antigens gaining access to the lymphoid tissue of the bowel wall during infancy, perhaps before the normal mucosal block to their uptake is established; or (ii) in adult li f e the mucosa being sensitized •as a result of impairment of the normal mucosal barrier by insults such as bacterial or viral infection, or a temporary depressed immune response" (44). - 141 -The possible role of an epithelial glycoprotein degrading enzyme system in the pathogenesis of ulcerative colitis is illustrated schematically in figure 32. The suggested etiological mechanism is purely speculative. A normal mucosal barrier is maintained when the synthesis and the degradation of the epithelial glycoprotein is in balance. Perturbation of this steady state may occur as a result of (i) malfunctioning in the synthesis of normal epithelial glycoproteins; (ii) deficiency in the protection of the mucosa due to immunological disorders; ( i i i ) an increased activity of the enzymes produced by the gut flora in the degradation of colonic epithelial mucin; (iv) acute or chronic bacterial or viral infection; (v) trauma; and/or (vi) ischemia of the portions of the gastrointestinal tract. Once the normal mucosal barrier is broken, the host's immune system is exposed to (a) enterobacterial antigens, (b) tissue antigens (e.g. intestinal mucin), or (c) other antigens that are normally present in the faecal materials which cross-react with the host's tissues. Exposure of the host's immune system to these "foreign" antigens present in the faecal contents could lead to immunologic inflammation in the bowel wall. In most of the cases there will be temporary irritation of the bowel wall which will then be followed by complete healing of the mucosal barrier. On the contrary, as i t has been suggested by Kirsner and Shorter (79), some individuals who might be "genetically at risk for the development of inflammatory bowel disease" their immune system might become hypersensitized which could lead to the subsequent development of acute tissue inflammatory reactions, hence, ulcerative colitis. - 142 -Figure 32: A diagrammatic illustration of the possible role of the epithelial glycoprotein degrading enzyme system in the pathogenesis of ulcerative colitis. This enzyme system produced by the gut flora may participate in the initiation of the mucosal injury which sub-sequently may not participate further in the perpetuation of the inflammatory reaction. Exposure of the host's immune system to the enterobacterial antigens or own tissue antigens (e.g. glyco-proteins) and modulation of the inflammatory response as pre-disposed by the host's genetical make-up may lead to subsequent development of ulcerative colitis. - 143 -2. 3. 4. Normal mucosal Epithelium break down of the the mucosal barrier as a result of _ J EXTRINSIC FACTORS j actions of the epithelial glycoprotein degrading enzyme system bacterial/viral infection trauma ischaemia . INTRINSIC FACTORS 1. 2. genetically pre-disposed abnormal synthesis and production of epithelial glycoproteins immunodeficiency Sensitization to Enterobacterial and other Antigens Hypersensitivity Reactions and/or Cross Reactivity to Host's Tissue Antigens RECOVERY Inflammatory Reactions modified by Host's Genetic Influences ULCERATIVE COLITIS - 144 -The efficacy of sulfasalazine (SASP) in the treatment of ulcerative colitis has been well documented (143-146). It is known that a proportion of SASP is passed to the colon where the compound is split, presumably by intestinal bacteria, into its components, 5-aminosalicylic acid (5-ASA) and a sulfonamide, sulfapyridine (SP) (147-149). Sulfasalazine's mode of action i s s t i l l unclear (148-149). It may be related to the immunosuppressant or anti-inflammatory properties of the 5-ASA. The effects of sulfapyridine on the faecal flora, however, remain controversial. Some investigators reported an overall increase in Gram positive aerobes and in anaerobic lactobacilli in patients receiving the drug (147, 150). Cooke (151), on the other hand, concluded that there was l i t t l e difference between the faecal flora of UC patients receiving SASP and those not being treated with this drug, in fact, to-date, there is no solid evidence showing that sulfasalazine alters the intestinal microflora of persons with ulcerative colitis; as Gbodman and Oilman (149) have suggested that "any beneficial effect of the drug is probably due to some property other than its antibacterial activity". How does the proposed scheme (figure 32) f i t into the clinically proven effectiveness of sulfasalazine in the treatment of ulcerative colitis? As i t has been suggested earlier that bacteria or bacterial enzymes participate only in the initiation of the disease process, perpetuation of the inflammatory response depends on the host's immune system. Hence, an anti-inflammatory drug rather than an antibiotic would seem to be more effective in the treatment of ulcerative colitis. If microbial infection may exacerbate UC or precipitate recurrences, the sulfonamide component of SASP could be helpful in - 145 -minimizing the incidence of relapses. This "hypothesis" also takes into account the individual variations in immune responsiveness to a given antigen and therefore the genetic aspect of the disease. Kirsner and Palmer (81) first proposed the concept of individual susceptibility to the development of ulcerative colitis because of some heritable predisposition. Chronicity and relapses of the disease may also be a function of the genetic make up of the individual, and the concentration and persistence of the "damaging" antigens. - 146 -Proposed Future Investigation: 1. Enzymes involved in the degradation of the epithelial glycoproteins, particularly the neuraminidase, de-O-acetylase, and the NANA-degrading enzyme, be characterized further. Attempts to separate these enzymes by means of preparative chromatography should be made in order to "visualize" the effects of individual enzymes on the epithelial glycoproteins. 2. The structure of the oligosaccharide chains of the Wistar rat colonic epithelial glycoproteins be elucidated by the 3-elimination method because they may differ from those of the Fisher rat. 3. The analytical methods developed in this study should be applied to the detection of a similar enzyme system in other experimental animals such as guinea pigs and rabbits. Investigate the changes of enzyme activities, i f any, in these animals before and after the induction of carrageenan-colitis. This would be a good animal model to study the possible involvement of bacteria in the pathogenesis of carrageenan- induced colitis. • - 147 -4. 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Compendium of Pharmaceuticals and Specialties, 16th edition. Canadian Pharmaceutical Association, Ontario, 1981. 149. Goodman, L.S., and Gdlman, A., editors. The Pharmacological Basis of Therapeutics, 4th edition. The MacMillan Company, Toronto, 1970. 150. Borbach, S.L., Nahas, L., Plaut, A. G., Weinstein, L., Patterson, J.F., and Levitan, R. Gastroenterology, 54:575 (1968). 151. Cooke, E.M. Out, 10:565 (1969). - 157 -APPENDIX Al Chemical Composition of Purina Formulae- Chow #5008 Protein 23.5% Fat 6.5% Fiber 3.8% TDN 77.0% Ash 6.8% - 158 -Chemical Composition of 0.01% Phosphate-Buffered Saline (PBS-EDTA) Solution NaCl 8.0 gm KC1 0.2 gm Na2HP04 1.15 gm KH2P04 0.2 gm EDTA 0.1 gm in 1 litre of distilled water - 159 -Chemical Composition of Minimal Media, pH7.0 NH4C1 Na2HP04 KH2P04 NaCl MgS047H20 1 gm 7 gm 3 gm 0.5 gm 0.2 gm The final volume is 1 litre. Chemicals must be added in the sequence shown above. - 160 -The percentage of side chain de-0-acetylation from glycoprotein substrates % de-0 -acetylation = 1 - ( % C7C8* at time t \X 100% >% C7C8* at time zero Note: * Same equation is used for the calculation of % de-O-acetylation. An assumption made in this calculation is that the rat faecal neuraminidase cannot hydrolyze substituted sialic acids, i.e. there is no free sub-stituted sialic acid present in the digest. - 161 -A5 Derivation of Equations for the Estimation of the Concentrations of  both Free and Glycosidically Bound Sialic Acids with or without Side  Chain Substituents A = Concentration of Cy+Cg substituted free sialic acids B = Concentration of CQ substituted free sialic acids GA = Concentration of Cy+CQ substituted bound sialic acids GB = Concentration of C Q substituted bound sialic acids Assumptions: 1. TBA on digests determines A + B = X 2. PRBA on digests determines GA + GB = Y 3. Total sialic acids = X + Y = P = A + B + G A + G B 4. % Cy+Cg substituted sialic acids on digests: Z = (GA + A)/P x 100% 5. % Cy+Cg substituted sialic acids on filtrate: M = A/X x 100% 6. % Cy+Cg substituted sialic acids on retentate: N = GA/Y x 100% Since X, Y, P, Z, M, and N can a l l be determined: from 5 A = M x X/ 100 E2 from 1 B = X - A E3 from 6 GA = N x Y/100 from 2 GB = Y - GA E5 

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