Study of the oligosaccharide units from mucus glycoproteins of meconium from normal infants and from cases of cystic fibrosis with meconium ileus

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Clinical Science (7) 57,44545 Study of the oligosaccharide units from mucus glycoproteins of meconium from normal infants and from cases of cystic fibrosis with meconium ileus J. R. CLAMP AND M.

1 Clinical Science (7) 57,44545 Study of the oligosaccharide units from mucus glycoproteins of meconium from normal infants and from cases of cystic fibrosis with meconium ileus J. R. CLAMP AND M.GOUGH Clinical Research Laboratories. Department of Medicine, Medical School, Bristol, UX. (Received January 7; accepted 7 July 7) Summary. The mucus glycoproteins in meconium from normal infants and from infants having cystic fibrosis with meconium ileus have been studied.. Whereas normal meconium contained about 56 proteinbound carbohydrate, the meconium from cystic fibrosis contained only about 6.. Glycopolypeptides were prepared from the mucus glycoproteins. The oligosaccharide units from this material were released and fractionated. The fractions ranged widely in size and composition. 4. The fractions from cystic fibrosis specimens had a significantly higher content of fucose than those from normal specimens. Key words: cystic fibrosis, glycoproteins, meconium, mucus. Introduction The first stools passed by a newborn infant are called meconium. This is a sterile, viscid material consisting largely of mucus glycoprotein (Fraser & Clamp, 75), but containing in addition bile pigments, inorganic material, protein etc. (Harries, 78). The mucus glycoproteins possess bloodgroup activities (Buchanan & Rapoport, 5; Fraser & Clamp, 75) that are characteristic of the infant and do not have a maternal component. Correspondence: Dr J. R. Clamp, Clinical Research Laboratories, Department of Medicine, Medical School, Bristol BS8 ltd, U.K. Meconium therefore is a convenient and plentiful source of sterile material for the study of the mucus glycoproteins of the foetus and the newborn. In addition, knowledge of normal meconium enables comparisons to be made with meconium from cases of cystic fibrosis presenting as meconium ileus. Methods Specimens of meconium were stored at OOC until required. The bloodgroup activity of each specimen was determined (Boorman & Dodd, 7) in doubling dilutions, and the meconium was pooled accordingly to give five samples, designated A, B, AB, H and N, where N represents meconium devoid of A, B or H bloodgroup activity. All patients with cystic fibrosis had been operated on for intestinal obstruction and were found to have meconium ileus. Patient CF has a sibling with cystic fibrosis. The sodium content of sweat was 4 mmol/l for patient CF, mmolh for patient CF and 5 mmolh for patient CF. The diagnosis of cystic fibrosis was supported by the subsequent history, namely recurrent respiratory infections and evidence of pancreatic insufficiency. The samples were dialysed exhaustively against distilled water and freezedried (dialysed meconium). Each sample was subjected to proteolytic digestion by the method of Fraser & Clamp (75) and then exhaustively dialysed against distilled water and freezedried (egested meconium). The 445

2 446 J. R. Clamp and M. Gough dried material (4 mg) was dissolved in 5 ml of sodium chloride solution ( 5 mmol/l) and applied to a column (8 cm x.5 cm) of Sephadex G5 and eluted with sodium chloride solution. The effluent was monitored for the presence of hexose (Dubois, Gilles, Hamilton, Rebers & Smith, 56) and the hexosecontaining peaks were pooled according to the profile to give three fractions designated 5, 5 and 5. Each fraction was dialysed against distilled water and freezedried. This procedure was repeated on the remaining digested meconium until the whole sample had been processed in this way. The fraction 5 was now subjected to p elimination with reduction, a modification (Fraser, 74) of the Carlson (68) technique being used. A portion of 5 ( mg) was dissolved in ml of potassium hydroxide solution (5 mmol/l) containing sodium borohydride (4.5 g) and the solution kept at 45OC for days. At the end of this time the ph was adjusted to 5. with acetic acid and the solution freezedried. The dried material was extracted with water (8 ml), centrifuged (8 OOO g for min at 5OC) and the supernatant solution freezedried. This dried material was extracted with water ( ml), centrifuged and freezedried as before. The dried material was again extracted with water ( ml), centrifuged, freezeconcentrated and applied to a column (6 cm x.4 cm) of Sephadex G, which was then eluted with water. The hexosecontaining fractions were identified as previously described, pooled and freezedried. The dried material was dissolved in water ( ml) and applied to a column (5 cm x.4 cm) of Sephadex G5 and eluted with water. The effluent was monitored for hexose as before and the fractions were pooled according to the profile and freezedried. The last peak to emerge from the Sephadex G5 column was poorly resolved and this fraction was separated on a column of Sephadex G5 under identical conditions. The carbohydrate content of glycoproteins, glycopolypeptides and oligosaccharides was determined by gas chromatography with the method of Clamp (77). Results In common with most secretions containing mucus, meconium possesses bloodgroup activity. A, B and H activities are of particular interest because these are associated with mucus glycoproteins and are determined by the presence of certain monosaccharides in nonreducing endgroup positions. The normal meconium specimens were therefore pooled to give five different samples, namely: those with blood group A (A); those with blood group B (B); those with both A and B (AB); those devoid of A and B but possessing H activity (H) and those devoid of A, B and H activities (N). Meconium contains a significant amount of lowmolecularweight material, chiefly inorganic salts and bile components (Harries, 78), and these were removed in the first dialysis step. meconium at this stage contained close to 5% carbohydrate (Table l), which is consistent with previous reports (Fraser & Clamp, 75; Harries, 78) on the content of mucus glycoproteins in meconium. There is little difference between the various normal meconium results, but the cystic fibrosis samples are markedly different in containing only about % of total carbohydrate. The major contaminants at this stage are proteins, particularly plasma albumin and other glycoprotein materials such as globulins and TABLE. Carbohydrate composition of dialysed meconium Mean values? SD are shown. A B AB H N A No. of specimens Carbohydrate content Fucose Mannose Galactose N Acetylglucosamine N Acety lgalactosamine Sialic acid Total carbohydrate (%) 5 48 f 6 f 884 f f 7 86 f 4 f 4 48 f 6 4f 87 f 6 65 f 5 5 f 7 f f 7 65 f 7 5 f f 4 8 f 8 4 f k 8 56 f 7 8 f 7 56 f 87 4 f 6 48 f f 8 8 f 8 75 f 84 7f 78 f f 4 f 8 4f 8 f 6 6 f 5 5 f

3 Mucus glycoproteins of meconium TNILE. Carbohydrate composition of digested meconium Mean dues f SD are shown. 447 A B AB H N A Carbohydrate content (nmolrmg) Fucose 47 f 6 44 f 7 78 f 4 46 f f 5 f 5 Mannose f 4of 8 8 f8 f +5 88f76 Galactose 8 f 8 88 f 86 5 f 47 f 76 f 7 f 8 N Acetylgl~co~d~ 7 f 6 78 f f 6 8 f 7 8f f 47 NAwtykalactw.de 48 f 6 f 7 f 8 47 f 7 f 5 44 f 5 Sialic acid 76 f 4 4 f 74?r 74 f 8 5 f 54 f 4 Recovery (%) cellular components. These protein components of meconium are susceptible to proteolysis and are largely removed during the digestion and dialysis procedures. Mucus glycoproteins, on the other hand, are relatively resistant to proteolysis and remain as highmolecularweight glycopolypeptides (Gibbons, 78). The analysis of digested meconium (Table ) shows that there has been a considerable enrichment of the carbohydrate content, particularly of the cystic fibrosis samples, which show about a sixfold increase in total carbohydrate. Mucus glycopolypptides do not contain mannose (Allen, 78; Gibbons, 78) and the presence of this monosaccharide in all the digested meconium samples (Table ) indicates some contamination by other carbohydratecontaining material. This can be removed, however, by gelpermeation chromatography with Sephadex G 5. The first peak to emerge (5) was devoid of mannose (Table ) and this material was used for subsequent studies. The oligosaccharide units in mucus glycoproteins are glycosidically linked and these linkages to protein are alkalilabile. The reaction is carried out in the presence of excess reducing agent, which stabilizes the liberated oligosaccharide unit from alkaline degradation by reducing the linkage Nacetylgalactosamine to the corresponding glycitol, Nacetylgalactosami. The reduction step has additional advantages in identifying the linkage monosaccharide, establishing the equivalent weight of the oligosaccharide unit and monitoring any nonspecific cleavage by the appearance of other glycitols. The conditions for cleavage with reduction of mucus glycoproteins were established by Carlson (68). These conditions as modified by Fraser (74) release over % of the oligosaccharide units from mucus glycoproteins of meconium. The overall recovery of carbohydrate from the glycoprotein to the final isolation of oligosaccharide units was not significantly Werent between normal and cystic fibrosis, beiig 7% and 5% respectively. These overall recoveries are reasonable in view of the cumulative losses during the various extraction and chromatographic procedures. Table 4 gives the molar yield of the various oligosaccharide fractions for each meconium sample. On average, about 7% of the oligosaccharides contain from five to nine residues, although there are slight differences between the various samples. The range of oligosaccharides is very wide, from just the linkage monosaccharide to units containing residues. There does not appear to be any significant difference between normal and cystic fibrosis meconium in the size distribution of the oligosaccharide units. Table 5 compares the compositions of the oligosaccharide fractions from cystic fibrosis material with those from normal material. The comparisons are made on the basis of similar contents of galactose and Nacetylglucosamine. Apart from those units that could be so compared, a number of oligosaccharides were found in the cystic fibrosis material that were not found in normal material. For example, a unit was found containing eleven residues of galactose and nine of Nacetylglucosamine which had nine residues of fucose. Only one of the oligosaccharide fractions was present in all the normal meconium samples. This fraction contained one residue of fucose, three residues of galactose, two of Nacetylglucosamine and one residue of Nacetylneuraminic acid, together with the linkage Nacetylgalactosamine. No fraction from A meconium contained a signiticant amount of Nacetylgalactosamine apart from the linkage residue. Obviously this mono

$~ ~ ~ 448 J. R. Clamp and M. Gough TABLE. Carbohydrate composition of meconium fractions Mean values f SD are shown.$

4 ~ ~ ~ 448 J. R. Clamp and M. Gough TABLE. Carbohydrate composition of meconium fractions Mean values f SD are shown. A B AB H N A Fraction I 5 carbohydrate content Fucose 7 f 48 Mannose Galactose 7 f 75 N Acaylglucosamine 85 f 5 N Acetylgalactosamine 57 f 65 Sialic acid 46 f Yield (%) 5 45 f 44 f f f 4 77 f f 5 8 f 67 8 f f 4 7 f f 4 6 f 8 7 f 66 5 f 5 4? 4 5 f 7 4 f 7 5 f 55 4 f 4 56 f f 68 f 4 88 f f 77 f Fraction 5 carbohydrate content Fucose 6 f 5 Mannose f Galactose 77 f 8 N Acetylglucosamine 66 f 7 N Acetylgalactosamine 7 f 4 Sialic acid f 6 Yield (%) 6 58 f f 5 5 f f 5 55 k 8 f f 87 f f f 47 5 f 4 f 7 58 f 6 f f 48 5 f 54 f 5 47 f f 46 5 f 8 6 f 45 f 64 6 f 6 6 f 5 88 f 6 4 f f 5 8 Fraction 5 carbohydrate content Fucose 7 f 6 Mannose 456 f 4 Galactose 8 f 86 N Acetylglucosamine 77 f N Acetylgalactosamine 86 f 75 Sialic acid f 47 Yield (%) f f 6 74 f f f f f I 7 f f f 8 87 f f 6 7 f 8 5 f f 78 f 5 74 f 8 5 f f 7 f f 5 4 f 7 _C Total recovery (%) (7) (8) (7) (77) saccharide must be present in nonreducing terminal positions in order to confer A activity, but the number of units possessing such terminal residues must be very small in any particular fraction. Discussion When dialysed meconium is analysed for content of proteinbound carbohydrate, a striking difference is found between normal and cystic fibrosis material (Table ). Whereas normal meconium contains about 5% carbohydrate, that from cystic fibrosis contains only %. The lower content of mucus glycoproteins and correspondingly larger amounts of albumin in cystic fibrosis meconium may play a part in altering the physicochemical properties of meconium in meconium ileus (List, Findlay, Forstner 6t Forstner, 78). As might be expected, the carbohydrate contents are not significantly different after exhaustive proteolytic digestion had removed most of the contaminating protein (Table ). The analyses of the mucus glycopolypeptides (5, Table ) show interesting differences, particularly in the fucose content. Thus N has only half the fucose content of the others and this is accompanied by a higher content of the core monosaccharides, Nacetylglucosamine and galactose. The lower content of fucose in N is consistent with the known distribution of terminal residues in nonsecretors. No oligosaccharides from N contained more than two residues of fucose and indeed more than threequarters of the N oligosaccharides contained only one residue or no fucose at all. The cystic fibrosis mucus glycopolypeptides ( 5, Table ) had greater amounts of fucose and lesser amounts of Nacetylneuraminic acid (sialic acid) than normal. The reciprocal relationship between these two terminal monosaccharides was first noticed by Dische (6). The greater amounts of fucose were reflected in the oligo

5 ~~ Mucus glycoproteins of meconium TABLE 4. Distribution of oligosaccharides in mucus gbmpolypptides from meconium No. of residues in oligosaccharidc unit _ ~ Distribution (mol/loo mol of total oligosaccharides) 44 A B A B H N A TABU 5. Monos~hride compositions of oligosaccharide fractions fmm Vstic fibrosis material compared with those from normal material The results from the two types of material are compared on the basis of similar contents of galactose and Nacetylglucosamine. Oligosaccharide units from normal material with the highest content of fucose have been chosen for comparison. Monosaccharide (ratio to residue of Nacetylgalactosaminitol) Galactose NAcetyl Fucose Sic acid glucosamine

$45 J. R. Clamp and M. Gough saccharides prepared from mucus glycopolypeptides (Table 5). The oligosaccharide fractions from cystic fibrosis usually contain more fucose than their normal counterpart.$

6 45 J. R. Clamp and M. Gough saccharides prepared from mucus glycopolypeptides (Table 5). The oligosaccharide fractions from cystic fibrosis usually contain more fucose than their normal counterpart. Indeed the oligosaccharides from normal meconium never contain more than three residues of fucose, whereas those from cystic fibrosis may contain six to eight residues. It must be emphasized that the observations on mucus glycopolypeptides and oligosaccharides are based on only three cases of cystic fibrosis with meconium ileus. Our confidence in these findings is, hqwever, strengthened by the fact that material from cystic fibrosis has previously been reported to contain a high content of fucose (Dische, di Sant Agnese, Pallavicini & Youlos, 5; Roelfs, Gibbs & Grimn, 67). These observations have been largely ignored, however, in recent discussions on the aetiology of cystic fibrosis. There are a number of reasons for this. Thus it is extremely dimcult to obtain strictly comparable mucuscontaining material from normal subjects and cystic fibrosis patients. Even when such material has been obtained, the analyses have usually been carried out on relatively crude material. As can be seen from Tables, and, the differences do not become manifest until the material has been purified to the mucus glycopolypeptide stage. In addition, until recently, the only techniques available for carbohydrate estimations were colorimetric procedures. Dische himself originated most of these techniques and in his laboratory even small differences in fucose content were significant. The present study overcomes these difficulties. The increased fucose content should have important effects on the properties of mucus. Fucose (6deoxy~galactose) has a hydrophobic group, namely a methyl group, at position C6. An increase in the numbers of these groups would reduce the water associated with the oligosaccharide unit. In addition, neighbouring chains could associate through hydrophobic interactions. Both these effects would increase the gellike properties of mucus and might be important therefore in cystic fibrosis. There are other connections between fucose and cystic fibrosis. For example, an abnormal distribution of mfucosidase has been reported in this disease (Scanlin, Matacic, Pace, Santer & Glick, 78; Scanlin, Matacic & Glick, 7) and a difference has been found in the concanavalin A binding between cystic fibrosis and normal liver a Lfucosidase (Alhadeff & Watkins, 7). It is possible that the oligosaccharide units of mucus glycoproteins after synthesis are subjected to glycosidase action in a similar fashion to Nglycosidically linked units. If, therefore, fucosidase activity was altered in goblet cell membranes, these series of observations would be compatible with one another. A further connection would be with the condition of fucosidosis, in which there is a more widespread disturbance of fucosidase activity. It may be significant, therefore, that in fucosidosis there is not only an increased sodium chloride content in sweat but also a tendency to respiratory infections (Durand, Borrone & Della Cella, 6). Acknowledgments We thank the Cystic Fibrosis Research Trust for their generous support. We also thank the staff of Bristol Maternity Hospital for specimens of normal meconium; Miss Helen Noblett, F.R.C.S., Southmead General Hospital, and Dr Brian Webb, F.R.C.P., Taunton and Somerset Hospital, for cystic fibrosis specimens. In addition, we thank Dr E. Atkins, University of Bristol, for helpful discussions. References ALHADEFF, J.A. & Warxms, P. (7) Differential concanavalin A binding of cystic fibrosis and normal liver &Lfucosidase. Biochemical and Biophysical Research Communications, 86,7877. ALLEN, A. (78) Structure of gastrointestinal mucus glycoproteins and the viscous and gelforming properties of mucus. British Medical Bulletin, 4,. BOORMAN, K.E. & DODD, B.E. (7) In: An Introduction to Blood Group Serology, 4th edn, pp. 77. Churchill, London. BUCHANAN, D.J. & RAPOPORT, S. (5) Composition of meconium. Serological study of blood group specific substances found in individual meconiums. Proceedings of the Society for Experimental Biology and Medicine, 77, 4 7. CARLSON, D.M. (68) Structures and immunological propcrties of oligosaccharides isolated from pig submaxillary mucins. Journal of Biological Chemistry, 4,6666. CLAMP, J.R. (77) A gasliquid chromatographic approach to the analysis of carbohydrates. Biochemical Society Transactions, 5,665. DISCHE, Z. (6) Reciprocal relation between fucose and sialic acid in mammalian glycoproteins. Annals of the New York Academy of Sciences, 6,57. DISCHE,.. DI SANT AGNESE, P., PALLAVICINI, c. & YOULOS, J. (5) Composition of mucoprotein fractions from duodenal fluid of patients with cystic fibrosis of the pancreas and from controls. Pediatrics, 4,76. DUBOIS, M., GILLES, K.A., HAMILTON, J.K., ResERs, P.A. & SMITH, F. (56) Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 8, 5&56. DURAND, P., BORRONE, C. & DELLA CELLA, G. (6) Fucosidosis. Journal of Pediatrics, 75,

Mucus glycoproteins of meconium 45 FRASER, D. (74) A Study of the Glycoproteins of Human Mucus, pp. 7. Ph.D. Thesis, University of Bristol. FMSER, D. & CLAW, J.R. (75) The glycoprotein content of meconium.

7 Mucus glycoproteins of meconium 45 FRASER, D. (74) A Study of the Glycoproteins of Human Mucus, pp. 7. Ph.D. Thesis, University of Bristol. FMSER, D. & CLAW, J.R. (75) The glycoprotein content of meconium. Clinica Chimica Acta, 5,7. GIBBONS, R.A. (78) Mucus of the mammalian genital tract. British Medical Bulletin, j4; 48. HARRIES, J.T. (78) Meconium in health and disease. British Medical Bulletin, 4,7578. LIST, S.J., FINDLAY, B.P., FORSTNER, G.G. & FORSTNER, J.F. (78) Enhancement of the viscosity of much by serum albumin. Biochemical Journal, 75, ROELFS, R.E., Gmss, G.E. & G~wm, G.D. (67) The composition of rectal mucus in cystic fibrosis. American Journal OfDiseaseof Children,,44. SCANLIN, T.F., MATACIC, S.S., PACE, M., SANTER, U.V. & GLICK, M.C. (78) Abnormal distribution of cmfucosidase in cystic fibrosis: increased activity in skin fibroblasts. Biochemical and Biophysical Research Communications,, SCANLIN, T.F., MATACK, S.S. & GLICI(, M.C. (7) Abnormal distribution of axfucosidasc in cystic fibrosis. Decrekascd activity in smun Clinica Chimica Acta,,7.

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