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Comparison of the carbohydrate composition of α₂-macroglobulin from controls and patients with cystic… Park, Carol Maylene 1984

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COMPARISON OF THE CARBOHYDRATE COMPOSITION OF a 2-MACR0GL0BULIN FROM CONTROLS AND PATIENTS WITH CYSTIC FIBROSIS By CAROL MAYLENE PARK B.Sc, The University of British Columbia, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Medical Genetics) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1984 ©Carol Maylene Park, 1984 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 M £ D i £ ^ L - 6 E ? O e T l c S The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date C C T O B E f i 12- , l ? ^ 1 ^ DE-6 (3/81) ABSTRACT The putative involvement of c^-macroglobulin (c^M) i n the pathogenesis of C y s t i c F i b r o s i s (CF) has long been a subject of controversy. Previous reports have indicated that there are a l t e r a t i o n s i n the carbohydrate moieties of O^M. The objective of the present study was to compare the carbohydrate composition of o^M i s o l a t e d from the plasma of patients with CF and from the plasma of age- and sex-matched normal c o n t r o l s . The g a s - l i q u i d chromatographic procedure of Lehnhardt and Winzler was investigated using both o^ M and c^-acid glycoprotein and modified to obtain the required s e n s i t i v i t y , p r e c i s i o n and accuracy for these analyses. Investigation of the hydrolysis of both sugar mixtures and glycoproteins showed that s i g n i f i c a n t losses of the released sugars occurred with extended hydrolysis and that use of the recommended hydrolysis times led to underestimation of the absolute carbohydrate composition. Analysis of the rates of degradation of mixtures of free sugars indicated that not a l l are degraded at the same rate. This implies that estimates of glycoprotein composition based on molar r a t i o s are also i n error. Therefore, hydrolysis times were i n d i v i d u a l l y determined for the two glycoproteins studied; those chosen were a compromise between maximal " release and minimal destruction of the various carbohydrate components. In contrast to the hydrolysis time recommended by Lehnhardt and Winzler (40 hr for c^-acid glycoprotein), the h y d r o l y s i s times selected for o^-acid glycoprotein and a 2 M > w e r e 30 hr and 35 hr, r e s p e c t i v e l y . - i i i -The analysis of o^M from s i x CF patients and t h e i r age- and sex-matched normal controls using the modified g a s - l i q u i d chromatographic procedure indicated that there were no s i g n i f i c a n t differences between the carbohydrate compositions of the glycoproteins. The carbohydrate composition of CF a^M expressed as mean + S.D. pmole carbohydrate per 100 mg protein was: fucose, 0.70 +_ 0.12; mannose, 14.07 _+ 1.31;. galactose, 6.72 +_ 0.65; glucosamine, 15.38 + 1.59; s i a l i c a cid, 5.52 +_ 0.33 and that of normal c o n t r o l o^M was: fucose, 0.69 +_ 0.11; mannose, 14.42 _+ 1.21; galactose, 6.91 +_ 0.52; glucosamine, 16.13 _+ 1.77; s i a l i c a cid, 5.58 +_ 0.31. Therefore, contrary to previous reports, t h i s t h e s i s demonstrates that there i s no differ e n c e i n the carbohydrate composition of a 9M i n CF. - iv -TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF ABBREVIATIONS ix ACKNOWLEDGEMENTS x i i INTRODUCTION I. Cystic Fibrosis: Clinical and Pathological Aspects 1 II. a2~Macroglobulin A. a^ M as a Glycoprotein 4 B. a^M as a Protease Modulator 10 C. a^M in Cystic Fibrosis 10 III. Analysis of the Carbohydrate Composition of Glycoproteins 13 MATERIALS AND METHODS I. Materials 18 II. Subjects 19 III. Methods A. General Methods _ 19 B. Isolation of a^ M from Plasma 1. Isolation Procedure 20 2. Radial Immunodiffusion 20 3. Quantitation of Protein 21 - V -PAGE C. Assessment of Pur i t y of ct M Isolates 1. Immunoelectrophoresis 21 2. SDS-PAGE 22 D. Carbohydrate Analysis of a 2 M 1. Preparation of A l d i t o l Acetate Standards for Gas-Liquid Chromatography ( g l c ) 2. Quantitation of Neutral Sugars and Hexosamines by glc 3. Quantitation of S i a l i c Acid by Colorimetric Assay RESULTS I. I s o l a t i o n of o ^ M from CF and Normal Control Plasma A. Plasma 012M Concentrations 29 B. Column Chromatography of CF and Control Plasma 29 C. Problems Caused by T r i s Buffer 29 I I . Assessment of Pur i t y of 0.2^ Isolates A. Immunoelectrophoresis 36 B. SDS-PAGE 41 I I I . Quantitation of Neutral Sugars and Hexosamines by g l c A. Glc of A l d i t o l Acetates 41 B. Determination of Molar Response Factors and Relative Retention Times of the A l d i t o l Acetate 41 C. Materials I n t e r f e r i n g with the Quantitation of Neutral Sugars and Hexosamines by g l c 46 22 23 28 - v i -PAGE D. Assessment of the Recovery of Sugars by the glc Procedures 53 E. Time Required for Hydrolysis 58 F. Effect of Sodium Bicarbonate on the Quantitation of Neutral and Amino Sugars by glc 68 G. Precision of the Analytical Methods 73 H. Proportionality of Response with Amount of Glycoprotein 73 IV. Analysis of the Carbohydrate Composition of o^ M from CF Patients and Normal Controls. 78 DISCUSSION 83 APPENDIX A 93 APPENDIX B 94 APPENDIX C 96 APPENDIX D 97 APPENDIX E 98 APPENDIX F 99 BIBIOGRAPHY 100 - v i i -LIST OF TABLES TABLE PAGE I Plasma ct2M levels in CF patients and normal controls as measured by radial immunodiffusion 30 II Determination of the sialic acid and protein content ai-acid glycoprotein (ajAG) in water and in 25 mM sodium bicarbonate 37 III Means + S.D. of the relative retention times and molar response factors for the alditol acetates 47 IV Mean recoveries of the components of unhydrolyzed mixtures of monosaccharides 54 V Response factors of the alditol acetates prepared from mixtures of unhydrolyzed monosaccarides 56 VI Comparison of the rates of loss of free sugars during acid hydrolysis 69 VII Carbohydrate composition of aj-acid glycoprotein (ajAG) in water and in 25 mM sodium bicarbonate 71 VIII Precision of the analytical method on aj-acid glycoprotein 74 IX Carbohydrate composition of o^ M from CF patients and normal controls 79 X Carbohydrate composition of a2M from CF patients and normal controls expressed as mean mole % calculated from the data in Table IX 81 XI Comparison of literature values for the carbohydrate comparison composition of normal 0:2^ with values obtained in this study 88 - v i i i -LIST OF FIGURES PAGE 1 Structural features of oligosaccharide units of glycoproteins 6 2 A. Oligosaccharide precursor of simple and complex 8 carbohydrate moieties B. Dolichol phosphate 3 Outline of the procedure for the estimation of neutral and amino sugars from glycoproteins by glc 24 4 Cibacron Blue Sepharose chromatography of normal human plasma 32 5 Immunoadsorption chromatography of CBS pool 34 6 Immunoelectrophoresis of o^ M isolates 39 7 Densitometric scans of control and CF o^ M analyzed on 5% polyacrylamide tube gels in the presence of SDS 42 8 Gas-liquid chromatography of the alditol acetates on GP 3% SP-2340 on 100/120 mesh Supelcoport 44 9 Analysis of the neutral sugars of aj_-acid glycoprotein by glc. Contaminants in drying agents 49 10 Analysis of the neutral sugars of aj_-acid glycoprotein by glc. Contaminants in methanol 51 11 Release of sugars from normal 012M by acid hydrolysis 60 12 Release of sugars from normal aj_-acid glycoprotein by acid hydrolysis 62 13 Release of galactose from lactose by acid hydrolysis 64 14 Loss of free sugars during acid hydrolysis 66 15 Proportionality of the quantity of carbohydrate found to quantity of glycoprotein ( ai-acid glycoprotein) hydrolyzed for 35 hr by the standard method 76 - ix -LIST OF ABBREVIATIONS o^AG otj-acid glycoprotein CL^M c^-macroglobulin Asn asparagine BSA bovine serum albumin CBS Cibacron Blue Sepharose CF c y s t i c f i b r o s i s cm centimeter Con A concanavalin A cone. concentrated C.V. c o e f f i c i e n t of v a r i a t i o n Fuc fucose g gram Gal galactose gl c g a s - l i q u i d chromatography GlcNAc N-acetylglucosamine hr hour ID i n t e r n a l diameter Ig immmunoglobulin L l i t e r M molar ma milliampere Man mannose - x -MeCl2 methylene chloride MeOH methanol mg milligram min minute ml milliliter mM millimolar MRF molar response factor mv millivolt MW molecular weight ul microliter umol micromole n number in sample nmol nanomole nm nanometer P statistical probability PAGE polyacrylamide gel electrophoresis psi pounds per square inch r correlation coefficient RID radial immunodiffusion S.D. standard deviation SDS sodium dodecyl sulfate Ser serine Thr Threonine TMS trimethylsilyl - x i -T r i s tris-hydroxymethylaminomethylmethane v v o l t WGA wheat germ a g g l u t i n i n w/w weight per weight w/v . . : weight per volume y year < less than < l e s s than or equal to °C degrees Celsius a standard deviation - x i i -ACKNOWLEDGEMENTS I am indebted to my research supervisors, Dr. D.A. Applegarth and Dr. P.E. Reid, for their enthusiastic support of this project and for providing constant encouragement and guidance. Thanks are also extended to the other members of my supervisory committee, Dr. C.J. Eaves and Dr. W.R. McMaster, for their helpful advice and criticism. The invaluable assistance of Dr. L.T.K. Wong and Dr. A.G.F. Davidson in obtaining blood samples from CF patients is gratefully acknowledged. My thanks also to the volunteers who donated blood samples for this study. In addition, I would like to thank: Ian Mcdonald for his kind assistance in obtaining blood samples and isolating o^ M and his constant interest in this work, Charles Ramey for offering many patient explanations and expert advice on technical matters, Dr. M.A. Bridges for sharing his expertise on c^ M, and Johan Janzen for his useful discussions and help with data analysis and execution of the figures. I am also grateful to Anne Bishop, Pat Bernoe and a l l those who assisted in the expert preparation of this manuscript. Finally, I wish to thank the Canadian Cystic Fibrosis Foundation for generous financial support and the University of British Columbia for the award of a Summer University Graduate Fellowship. - x i i i -"Learning i s acquired by reading books; but the much more necessary learning, the knowledge of the world, i s only to be acquired by reading men, and studying a l l the various e d i t i o n s of them." Lord C h e s t e r f i e l d : Letters to His Son, March 16, 1752. - 1 -INTRODUCTION I. Cystic F i b r o s i s : C l i n i c a l and Pathological Aspects With an estimated incidence of 1 i n 2000 l i v e b i r t h s , C y s t i c F i b r o s i s (CF) i s the most common l i f e - t h r e a t e n i n g i n h e r i t e d disorder of Caucasian populations (Wood et a l . , 1976). Pancreatic i n s u f f i c i e n c y , elevated sweat sodium chl o r i d e concentrations and chronic pulmonary disease are the major c l i n i c a l findings i n CF (Di Sant'Agnese and Davis, 1976). Genetic studies suggest an autosomal mode of inheritance for the CF t r a i t ( s ) (Thompson, 1980) and heterozygote advantage has been proposed as a mechanism for the maintenance of CF genes at a high l e v e l i n the Caucasian population (Dankset a l . , 1965; Hirschhorn, 1973). Cystic f i b r o s i s a f f e c t s the body's exocrine glands. The abnormally v i s c i d mucous secretions c h a r a c t e r i s t i c of t h i s disorder cause obstruction of a number of ducts and passageways including the respiratory t r a c t , small i n t e s t i n e , mucus-secreting s a l i v a r y glands, pancreas, b i l i a r y t r a c t and g e n i t a l t r a c t (Wood et a l . , 1976). Obstruction and r e s u l t i n g c y s t i c d i l a t i o n of the pancreatic ducts leads to f i b r o s i s of the pancreas hence the o r i g i n a l name for t h i s disorder, c y s t i c f i b r o s i s of the pancreas (Anderson, 1938). Another consequence of t h i s obstruction i s the deficiency of enzymes required for digestion which leads to malabsorption, steatorrhea and malnutrition (Park and Grand, 1981). Inspissation of mucus i n the b i l e ducts may lead to b i l i a r y c i r r h o s i s (Park and Grand, 1981) while obstruction of the ducts of the genitourinary t r a c t s r e s u l t s i n reduced f e r t i l i t y and often s t e r i l i t y , e s p e c i a l l y i n male patients - 2 -(Wood et a l . , 1976). Chronic pulmonary i n f e c t i o n as a r e s u l t of i n s p i s s a t i o n of mucus secretions i n the lung airways and subsequent c o l o n i z a t i o n by Staphylococcus aureus and P'seudomonas aeruginosa i s one of the major complications of CF (Talamo et a l . , 1983). Death, often as a r e s u l t of progressive lung damage, usually occurs before or i n early adulthood. Although the serous glands are morphologically and p h y s i o l o g i c a l l y normal, there i s an elevation i n the sodium and chloride content of eccrine sweat. The detection of elevated sweat chlo r i d e by the."sweat t e s t " i s used i n the diagnosis of CF (Davis and Di Sant'Agnese, 1980). Despite advances i n the c l i n i c a l management of CF patients i n recent years, the basic biochemical defect i n CF remains unknown. Research i n t o the nature of the basic defect has proceeded i n many d i f f e r e n t d i r e c t i o n s . One hypothesis maintains that abnormalities i n glycoprotein metabolism give r i s e to glycoproteins with a l t e r e d physicochemical properties which are thought to contribute to the pulmonary and g a s t r o i n t e s t i n a l pathology i n CF patients. However, many of the studies on mucus glycoproteins, glycosidases and glycosyltransferases i n CF have yielded contradictory r e s u l t s and no consistent f i n d i n g has emerged i n support of t h i s hypothesis (Alhadeff, 1978). Since the exocrine glands are associated with autonomic nerve f i b e r s , dysfunction of the autonomic nervous system i s another possible explanation for the observed exocrinopathy i n CF. While autonomic drugs such as isoproterenol and pil o c a r p i n e induce changes i n the mucous glands of animals s i m i l a r to those found i n CF, studies - 3 -by other i n v e s t i g a t o r s have f a i l e d to demonstrate any u l t r a s t r u c t u r a l or histochemical abnormalities i n the autonomic nerve f i b e r s of CF patients (Di Sant'Agnese and Davis, 1976). The presence of pathogenic polypeptide factors ("CF factors") i n the serum, s a l i v a and sweat of patients with CF has been reported. Spock et a l . (1967) detected a factor i n CF serum which caused c i l i a r y dyskinesis i n rabbit tracheal explants. A second type of factor present i n the sweat and s a l i v a of CF patients was shown to i n h i b i t sodium reabsorption i n rat parotid and human sweat glands (Mangos and McSherry, 1967; Mangos et a l . , 1967; Mangos, 1973). CF factors are reported to be c a t i o n i c polypeptides r i c h i n arginine and l y s i n e residues (Mangos and McSherry, 1968; Bowman et a l . , 1969) and some inv e s t i g a t o r s have postulated that these small peptides are responsible for the defective mucociliary transport i n the r e s p i r a t o r y t r a c t and for the elevation of sodium chloride i n the sweat of CF patients (Wilson, 1979). These factors are not s p e c i f i c to CF, however, (Conover et a l . , 1973; Caplin and Haynes, 1973; Schmoyer et a l . , 1972) and t h e i r nature and mechanism of action remains unknown. One area which has been extensively investigated i n the past few years i s the involvement of proteases. Following reports of a deficiency i n arginine esterase a c t i v i t y i n CF serum, several workers studied the i n t e r a c t i o n of serum proteases with the protease modulator, o^-macroglobulin ( a2 M)« T n e involvement of a 2 M i° C f r i s discussed on p. i o . - 4 -II. a^-Macroglobulin o^-Macroglobulin (o^M) i s a high molecular weight glycoprotein (MW = 725,000) (H a l l and Roberts, 1978) found i n high concentrations i n human plasma (265 mg/100 ml) (James et a l . , 1966). The major s i t e of synthesis of o^M i s the l i v e r b u t . f i b r o b l a s t s and lymphocytes may also produce t h i s glycoprotein (Starkey and Barrett, 1977). A. a^M as a Glycoprotein In common with a great majority of plasma glycoproteins o^M i s believed to have oligosaccharide units N - g l y c o s i d i c a l l y linked to asparagine residues i n the protein core (Sottrup-Jensen et a l . , 1983). A common s t r u c t u r a l feature of the oligosaccharide units i s the inner core branched pentasaccharide shown i n F i g . 1A. In various glycoproteins the peripheral mannose residues of t h i s core region are substituted by ei t h e r a d d i t i o n a l mannose residues r e s u l t i n g i n the "simple" high mannose type of oligosaccharide unit ( F i g . IB) or by two to four units to N-acetyllactosamine (Gal 3 1,4 GlcNAC) bearing terminal s i a l i c acid residues to form the "complex" N-acetyllactosamine type of unit ( F i g . IC). Oligosaccharide units belonging to t h i s l a t t e r group may be further c l a s s i f i e d as b i - , t r i -and tetraantennary structures ( F i g . IC). Small amounts of fucose may be present i n complex type units (Montreuil, 1980). The biosynthesis of both simple and complex types of oligosaccharide units occurs v i a a l i p i d - l i n k e d intermediate. As shown i n F i g . 2A, the oligosaccharide precursor consists of three glucose, nine mannose and two N-acetylglucosamine residues l i n k e d by a - 5 -pyrophosphate bridge to the polyisoprenoid l i p i d , d o l i c h o l ( F i g . 2B). The oligosaccharide i s transferred en bloc to an asparginyl residue i n the sequence Asn - X - Ser/Thr (where X = one of a variety of amino acids) on the nascent, ribosome-bound polypeptide chain. An oligosaccharide transferase i n the rough endoplasmic reticulum i s believed to be responsible for t h i s process (Struck and Lennarz, 1980). The next step i n the sequence involves processing of the protein-bound oligosaccharide i n the Golgi apparatus. This begins with the removal of three glucose and zero to six of the nine mannose residues by s p e c i f i c glycosidases (Sharon and L i s , 1982). Removal of zero to four of these mannose residues r e s u l t s i n a high-mannose type of structure ( F i g . IB). The heptasaccharide, Man^GlcNAc2 r e s u l t i n g from the removal of four mannose residues may then undergo a s e r i e s of elongation reactions mediated by s p e c i f i c glycosyltransferases located i n the Golgi apparatus leading to the formation of complex type structures ( F i g . IC). The biosynthetic pathway for these complex oligosaccharides has been extensively reviewed by Schachter (1984). I t has been demonstrated that the carbohydrate moieties of plasma glycoproteins serve as recognition markers and play a r o l e i n c o n t r o l l i n g t h e i r l i f e t i m e i n the c i r c u l a t o r y system. Morell et a l . , (1971), using animal models, showed that removal of s i a l i c a c i d residues from various plasma glycoproteins i n c l u d i n g a 2M, enhances t h e i r rate of clearance from c i r c u l a t i o n and that the uptake of asialoglycoproteins depends on the recognition by the hepatocytes of galactose residues exposed a f t e r removal of s i a l i c acid (Ashwell and Morell, 1974). - 6 -F i g . 1 S t r u c t u r a l features of oligosaccharide units of glycoproteins (Montreuil, 1980) SA, s i a l i c acid; Gal, galactose; GlcNAc, N-acetylglucosamine; Man, mannose; Fuc, fucose; Asn, asparagine A. CORE STRUCTURE Man, : Man GlcNAc GlcNAc Asn Man' B. SIMPLE HIGH-MANNOSE TYPE Man Man, "Man, Man Man • ,Man GlcNAc GlcNAc Man Man • Man' C. COMPLEX N-ACETYLLACTOSAMINE TYPE a) BIANTENNARY SA Gal GlcNAc Man . SA Gal- GlcNAc Man-^ b) TRIANTENNARY SA Gal GlcNAc Man. SA Gal G l c N A c - ^ SA Gal GlcNAC Man' C) TETRAANTENNARY SA Gal GlcNAc Man, SA Gal GlcNAc SA Gal GlcNAc > SA Gal GlcNAc •Man' •Man GlcNAc-Man GlcNAc • Man GlcNAc • - 8 -Fig. 2 A. Oligosaccharide precursor of simple and complex carbohydrate moieties. B. Dolichol phosphate - 9 -A. Man Man^ Man / \ Man Man-' Man GlcNAc GlcNAc-P- P-Dolichol Glc Glc Glc Man Man Man' B. 0 II OL CH-, ' 0-P-0CH2-CH2-CH-CH2-(CH2-CH=C-CH2)17-CH2-CH=C-CH3 - 10 -B. g^ M as a Protease Modulator A unique feature of o^ M is its property of acting as a "molecular trap" for several proteases. Once the protease is enclosed within the o^ M molecule, its activity towards high molecular substrates is inhibited while activity towards low molecular weight substrates is retained; in this way c^ M is able to modulate proteolytic activity. a^M-protease complexes are rapidly cleared from the circulation by the reticuloendothelial system (Barrett, 1981). A hypothesis for the mechanism of "trapping" of proteases by OjM was formulated by Barrett and Starkey and can be summarized as follows (Barrett, 1981): g^ M consists of four identical subunits each bearing a region which is susceptible to limited proteolysis (now known as the "bait" region). Cleavage of this region in one or more of the subunits leads to a conformational change in thea^M molecule such that i t physically encloses the protease. Only molecules of MW < 10,000 are able to diffuse through the trap thus the protease retains its activity towards small molecular weight substrates while interaction with larger substrates and inhibitors is sterically hindered. C. a^M in Cystic Fibrosis A proposed role forg 2M in the pathogenesis of CF arose from the observation that the body fluids of CF patients are deficient in arginine-esterase activity (Rao and Nadler, 1972; Rao et al., 1972; Rao and Nadler, 1974; Chan et al., 1977). When CF and control plasma was analyzed for arginine esterases using immunoelectrophoretic techniques, - 11 -Shapira et al. (1976) found that the fraction of arginine esterase activity usually associated in complex witho^M was missing. In order to examine the possibility that CF a2M may have failed to complex arginine esterases, these investigators measured the trypsin binding capacity of CF a2M and found i t to be significantly depressed. This suggested that the deficiency of a2M-complexed arginine esterase activity might be a reflection of a molecular defect in CF a2M rather than a deficiency of protease molecules. Further studies with a 2 M isolated from CF and control plasma indicated that there were differences in: i) the molar binding ratios of proteases to c*2M (Shapira et al, 1976; Shapira et al, 1977a), i i ) the kinetic interaction of purified a2M-bovine trypsin complexes with the low molecular substrate benzoyl arginine ethyl ester (Shapira et al., 1977a), i i i ) the stability of the complexes (Shapira et al., 1977b), and iv) the gel electrophoretic behaviour of the complexes in the presence of SDS. It was therefore hypothesized that there is a molecular defect in CF a2M which impairs its ability to regulate proteolytic enzyme activity leading to a deficiency of arginine esterase-type activity in CF plasma and exocrine glands. This would result in the accumulation of the lysine- and arginine-rich CF factors giving rise to disturbed mucociliary transport and increased excretion of sodium chloride in sweat (Wilson, 1979). - 12 -Several studies suggested that the a l t e r e d f u n c t i o n a l properties of CF a 2M might be secondary to abnormalities i n i t s carbohydrate composition. CF a 2M was shown by Ben-Yoseph et a l . (1979) to have a 40% decreased s i a l i c acid content when compared with normal control a 2M. This decrease i n s i a l i c a c i d content would be expected to s i g n i f i c a n t l y a l t e r the electrophoretic mobility of t h e a 2 M molecule. Two-dimensional gel electrophoretic analyses, however, did not reveal any s i g n i f i c a n t d i f f e r e n c e s between preparations from controls and CF patients (Comings et a l . , 1980). Studies comparing the binding of CF and normal a 2M to various l e c t i n s indicate possible d i f f e r e n c e s i n the carbohydrate composition of the two species may e x i s t . The binding of CF a 2M to the l e c t i n s concanavalin A (Con A) and wheat germ ag g l u t i n i n (WGA) was lower than normal (Ben-Yoseph et £l., 1979) and these authors suggest that t h i s finding r e f l e c t s a decrease i n the number or the a c c e s s i b i l i t y of D-mannose, N-acetyl-D-glucosamine and possibly s i a l i c acid residues i n CF a 2M. However Shapira and Menendez (1980) showed that, as compared to normal, CF a 2M displayed s i g n i f i c a n t l y increased Con A binding. More recently, Ben-Yoseph et a l . (1981) investigated the incorporation of s i a l i c a c i d into asialoglycoproteins by the s i a l y t r a n s f e r a s e s found i n the plasma and f i b r o b l a s t s of normal controls and CF patients. Desialylated preparations of f e t u i n , c o n t r o l and CF a 2M were used as acceptors. S i a l y l a t i o n was reduced when CF a 2M was employed as the s i a l i c acid acceptor but not when f e t u i n or c o n t r o l a 9M was used; s i a l y l a t i o n was also independent of the source - 13 -(CF or normal) of the s i a l y l t r a n s f e r a s e . These data provide a d d i t i o n a l evidence for a carbohydrate a l t e r a t i o n i n CF o^M. In contrast to the studies discussed above, other i n v e s t i g a t o r s have shown that CF and normal a 2M a r e i d e n t i c a l with regard to gel electrophoretic patterns, subunit molecular weight and t r y p s i n cleavage products (Burdon et a l . , 1980; Parsons and Romeo, 1980). Following a c a r e f u l i n v e s t i g a t i o n , Bridges et a l . , (1982b) concluded that there were no differences between CF and normal c o n t r o l a^M with repect to: i ) molar protease binding, i i ) i n t e r a c t i o n of bovine c a t i o n i c t rypsin - a 2M complexes with benzoyl arginine ethyl ester, i i i ) s t a b i l i t y of a 2M-trypsin complexes, and i v ) subunit structure. While i t i s doubtful that functional differences between CF and normal a 2M e x i s t , i t i s evident that the issue of carbohydrate a l t e r a t i o n s i n CF a 2M warrants further i n v e s t i g a t i o n . This thesis therefore addresses the question: . Is there an a l t e r a t i o n i n the carbohydrate composition of plasma a 2-macroglobulin i n c y s t i c f i b r o s i s ? III. Analysis of the Carbohydrate Composition of Glycoproteins The carbohydrate content of a 2M i s low (8-10%) (Barrett, 1981) and only l i m i t e d amounts are a v a i l a b l e from p e d i a t r i c patients. I t was therefore necessary to s e l e c t a highly s e n s i t i v e , precise and accurate method for the analysis of the carbohydrate composition o f a 2 M from CF patients and c o n t r o l s . As has been extensively discussed by Dutton - 14 -(1973) and Clamp et a l . , (1972), g a s - l i q u i d chromatography (glc) i s a technique which f u l f i l l s these requirements and has been used extensively for the quantitation of the carbohydrate components of glycoproteins. I t affords greater s e n s i t i v i t y and s p e c i f i c i t y than c o l o r i m e t r i c methods and the separation, i d e n t i f i c a t i o n and quantitation of submicrogram amounts of carbohydrate can be achieved i n a s i n g l e procedure. Three main steps are involved i n the g l c analysis of the carbohydrate moieties of glycoproteins: the glycoprotein sample i s f i r s t hydrolyzed, a s u i t a b l e d e r i v a t i v e of the released monosaccharides i s prepared and these compounds are then analyzed by g l c . A variety of carbohydrate d e r i v a t i v e s may be employed, of which the t r i m e t h y l s i l y l ether (TMS) and a l d i t o l acetate d e r i v a t i v e s have been most commonly used. A disadvantage of the TMS d e r i v a t i z a t i o n procedure i s that several glycosides can be formed per monosaccharide • r e s u l t i n g i n a complex chromatogram. I d e n t i f i c a t i o n and quantitation of the sugar components may therefore be d i f f i c u l t (Sweeley et a l . , 1963; Clamp et a l . , 1967). A more s a t i s f a c t o r y method employs the a l d i t o l acetate d e r i v a t i v e s . Reduction of the monosaccharides to t h e i r a l d i t o l s p r i o r to d e r i v a t i z a t i o n eliminates the problem of anomerization and a s i n g l e acetylated d e r i v a t i v e i s formed for each sugar (Sawardeker et a l . , 1965). However, reduction of the ketone group of s i a l i c acids would be expected to y i e l d two compounds and thus i t i s simpler to quantitate s i a l i c a c i d by an alternate method. Of the c o l o r i m e t r i c assays a v a i l a b l e for the estimation of s i a l i c acids those most commonly used are the methods developed by Warren (1959), Aminoff (1961) and Jourdian et a l . (1971). - 15 -A variety of g l c procedures have been developed for the estimation of neutral and amino sugars i n glycoproteins as t h e i r a l d i t o l acetates. The procedure for the quantitation of the neutral sugars i n glycoproteins described by Lehnhardt and Winzler (1968) involves hydrolysis of the glycoprotein sample i n the presence of Dowex 50 r e s i n (H+ form), addition of an i n t e r n a l standard, reduction of the released sugars with sodium borohydride, a c e t y l a t i o n of the r e s u l t i n g alcohols with a c e t i c anhydride and pyridine, and glc of the r e s u l t i n g a l d i t o l acetates. Hydrolysis i n the presence of a cation exchange r e s i n (Kim et a l . , 1967) has the advantage that amino acids are bound to the r e s i n and hence do not i n t e r a c t with and destroy the neutral sugars. Another advantage of the method i s that i t employs an i n t e r n a l standard. Addition of an i n t e r n a l standard allows not only i d e n t i f i c a t i o n of the various components but also accounts for any losses of the sugars during subsequent steps i n the procedure. Simultaneous estimation of neutral and amino sugars i n glycoproteins can be c a r r i e d out by the method of Niedermeier (1971); t h i s procedure does not, however, u t i l i z e r e s i n hydrolysis and temperature programming of the chromatograph i s required i n order to obtain adequate separation of the components. A l t e r n a t i v e l y , by including a nitrous acid deamination step, i t i s possible to convert the hexosamines to t h e i r corresponding 2,5-anhydro sugars and analyze the derived p o l y o l acetates together with those of the neutral sugars (Niedermeier, 1971; Porter, 1975). - 16 -Reid et a l . (unpubl.), using the procedure of Lehnhardt and Winzler, were able to quantitate the neutral and amino sugars present i n a s i n g l e hydrolysate. Resin-bound hexosamines were eluted with hydrochloric acid and the derived acetates were analyzed by g l c . This modified method r e t a i n s the advantages of the o r i g i n a l r e s i n hydrolysis method as i t employs an i n t e r n a l standard and the hydrolysis conditions are i d e n t i c a l to those of Lehnhardt and Winzler (1968) and much milder than those used by Niedermeier (1971). A further advantage i s that excellent separation of the a l d i t o l acetate d e r i v a t i v e s can be obtained under isothermal conditions. Therefore, t h i s method was chosen for the analysis of the neutral and amino sugar content o f a ^ M . In addition, the method of Jourdian et a_l. (1971) was selected for the quantitation of s i a l i c a cid, as i t i s a s e n s i t i v e and e a s i l y performed method which does not require h y d r o l y t i c or enzymatic release of s i a l i c acid from the glycoprotein. As none of these methods had previously been applied t oc^M, i t was necessary to f i r s t evaluate t h e i r s e n s i t i v i t y , p r e c i s i o n and accuracy and, i f required, modify them i n order to obtain the best possible a n a l y t i c a l conditions for the study. - 17 -The objectives of this thesis were therefore to: A. evaluate and, i f necessary, modify the analytical procedures selected to allow quantitation of the small amounts of carbohydrate present in the a2M samples available from pediatric patients. B. use these methods to determine whether or not the carbohydrate composition of o^ M isolated from CF patients differs from that of normal controls. - 18 -MATERIALS AND METHODS I. Materials The chemicals and materials used in the isolation ofa^M were obtained from the following sources: Blue Sepharose CL-6B, CNBr-activated Sepharose AB (Pharmacia); NOR-Partigen o^-macroglobulin radial immunodiffusion plates, protein standard serum (human) for NOR-partigen, rabbit antisera against human haptoglobin a2M, IgA, IgG, IgM, transferrin, albumin, a-^-antitrypsin, ceruloplasmin and whole serum (Behring/Hoechst Canada); soybean trypsin inhibitor No. T-9003, polybrene, bovine serum albumin No. A-7638 (Sigma); Aquasil (Pierce); Tris barbital-sodium barbital high resolution buffer pH 8.8 No. 51104, human control serum for electrophoresis (Gelman); electrophoresis-grade acrylamide, N,N-methylene bisacrylamide, N,N,N',N'-tetramethylethylenediamine and ammonium persulfate (Bio-Rad); YM 30 ultrafiltration membranes (Amicon). For the chemical analyses of o^ M isolates and model glycoproteins, the chemicals and supplies listed below were used: D-Mannose, D-galactose, L-arabinose, N-acetyl-D-glucosamine, D-glucosamine hydrochloride (Calbiochem A grade); D-galactitol, D-mannitol (Eastman); a-lactose monohydrate (Fisher); L-fucose, D-mannosamine hydrochloride (Sigma); GP 3% SP-2340 on 100/120 mesh Supelcoport, Thermogreen LB-1 septa (Supelco); dimethyldichlorosilane, silicone rubber septa, - 19 -R e a c t i - v i a l s (Pierce); A.C.S. Spectranalyzed methanol, A.C.S. c e r t i f i e d pyridine and a c e t i c anhydride (Fisher); anion exchange r e s i n AG 1-X8 200-400 mesh [ C l ~ form] and cation exchange r e s i n AG 50W-X8 200-400 mesh [H + form] (Bio-Rad); gas chromatographic syringes (Hamilton). The carbohydrates l i s t e d above were used without further p u r i f i c a t i o n with the exception of lactose which was r e c r y s t a l l i z e d once according to the method of Hudson (1902). A l l other chemicals employed were of reagent grade or better. A preparation of human seromucoid (0.6M p e r c h l o r i c acid soluble f r a c t i o n of human serum) of known s i a l i c a c i d content was generously supplied by Dr. P.E. Reid, Dept. of Pathology, U.B.C. A g i f t of pure lyophilized-.o^-acid glycoprotein from Drs. G. Strecker and B. Fournet, Laboratoire de chimie biologique, Universite des sciences et techniques de L i l l e , France i s g r a t e f u l l y acknowledged. II . Subjects Non-fasting blood samples were obtained from six CF patients and six age- and sex-matched normal controls. Control subjects were apparently healthy i n d i v i d u a l s with no family h i s t o r y of CF. I I I . Methods A. General Methods A Buchi Rotavap rotary evaporator with a 37°C water bath was - 20 -used for a l l evaporations at reduced pressure. Melting points were determined with a Drechsel melting point apparatus. A Beckman Model 25 spectrophotometer was used for a l l spectrophotometric measurements and densitometric analysis of tube gels was performed with a Helena Auto-Scan densitometer. B. I s o l a t i o n of g^M from Plasma 1) I s o l a t i o n Procedure a^-Macroglobulin was i s o l a t e d from 5 ml plasma samples by dye a f f i n i t y chromatography on Cibacron Blue Sepharose followed by immunoadsorption chromatography dire c t e d against the major protein contaminants. Procedures for blood c o l l e c t i o n , harvesting plasma and column chromatography were those described by Bridges et a l . (1982a). Following immunoadsorption chromatography, f r a c t i o n s containing g^M were pooled, the protein content was determined by e x t i n c t i o n at 280 nm (Dunn and Spiro, 1967) and samples were concentrated to approximately 2 mg/ml on an u l t r a f i l t r a t i o n apparatus f i t t e d with YM 30 membranes (MW retention l i m i t = 30,000). The concentrate was then subjected to exchange d i a l y s i s against 5 volumes of 25 mM sodium bicarbonate approximately pH 8.3. After determination of the protein content of the g 2M i s o l a t e s by c o l o r i m e t r i c assay (see below p.21), aliquots of known volume were dispensed as required for the various analyses and stored at -20°C for up to 6 months. 2) Radial Immunodiffusion The a„M content of 5 y l a l i q u o t s of plasma and p u r i f i e d g 9M - 21 -was measured by si n g l e r a d i a l immunodiffusion using NOR-Partigen a^-macroglobulin immunodiffusion plates and the appropriate protein standard serum. Immunodiffusion and evaluation of the plates was c a r r i e d out according to the manufacturer's d i r e c t i o n s . 3 ) Quantitation of Protein The protein content of theo^M i s o l a t e s was determined according to the method of Lowry et a l . (1951). The procedure for the analysis of proteins i n so l u t i o n was miniaturized to give a f i n a l assay volume of 650 y l . Results are expressed as mg Lowry protein using bovine serum albumin as standard. IV. Assessment of Pur i t y of a^M Isolates The purity o f a 2 M i s o l a t e s was assessed by Immunoelectrophoresis with p o l y s p e c i f i c and monospecific antisera and by SDS-PAGE. 1) Immunoelectrophoresis Immunoelectrophoresis was performed by a micro-method on glass microscope s l i d e s coated with 1% (w/v) agar i n T r i s barbital-sodium b a r b i t a l buffer pH 8.8 e s s e n t i a l l y as described by Bridges (1981). Samples containing 10 ug protein were applied to the lower wells cut i n the agar and human c o n t r o l serum (2y 1) was applied to the upper wells. Electrophoresis was performed at 250 v for 2 hr at room temperature. Following electrophoresis, a l o n g i t u d i n a l trough was cut between the two wells i n each gel and p o l y s p e c i f i c or monospecific antiserum (25y1) was added. Immunodiffusion was c a r r i e d out i n a humid chamber at room temperature for 18 hr. The gels were then - 22 -soaked i n 0.9% s a l i n e for 2 hr, rinsed with d i s t i l l e d water and a i r - d r i e d overnight. Staining was accomplished by immersing the gels i n 0.25% (w/v) Coomassie blue R-250 i n 45% (v/v) aqueous methanol containing 9% a c e t i c acid for 1 hr; gels were subsequently destained i n a s o l u t i o n of 30% (v/v) aqueous methanol containing 5% a c e t i c a c i d . A l l a 2M i s o l a t e s were tested with rabbit antisera against human serum,.o^M, IgG, IgA, IgM, a ^ - a n t i t r y p s i n , ceruloplasmin, t r a n s f e r r i n , albumin and haptoglobin. Previous work by Bridges et a l . (1982a) had confirmed that these were the contaminants found i n the » 2 M obtained by the above i s o l a t i o n procedure. 2) SDS-PAGE Polyacrylamide tube?gels (5% t o t a l acrylamide, 2.6% of t h i s as methylene bisacrylamide) were prepared according to the method of Weber and Osborn (1972). Samples (10 y l ) containing approximately 20 yg protein were mixed with 90 y l of sample buffer (0.01 M sodium phosphate pH 7.2, 1% SDS, 10% g l y c e r o l , 0.002% bromophenol blue) and electrophoresis was c a r r i e d out at room temperature at 3 ma per tube for 30 min. The current was increased to 5 ma per tube for the remainder of the run (5-6 h r ) . Proteins were v i s u a l i z e d by s t a i n i n g with 0.25% (w/v) Coomassie blue R-250 i n 45% (v/v) aqueous methanol containing 9% a c e t i c a c i d . The gels were then destained i n a s o l u t i o n of 5% (v/v) aqueous methanol containing 7.5% a c e t i c acid for 2-3 days. The gels were evaluated by densitometry at 595 nm. V. Carbohydrate Analysis of a 2M 1) Preparation of A l d i t o l Acetate Standards for Gas-liquid  Chromatography (glc) - 23 -The a l d i t o l acetates of D-galactose, L-fucose, D-mannose, and L-arabinose, were synthesized and r e c r y s t a l l i z e d to constant melting point. The melting points of these compounds were as follows: a r a b i n i t o l pentaacetate, 72-74°C; f u c i t o l pentaacetate, 119-120°C; mannitol hexaacetate, 119-121°C; and g a l a c t i t o l hexaacetate, 158-160°C. The acetates of D-glucosamine and D-mannosamine were obtained as syrups. (This procedure i s described i n d e t a i l i n Appendix B.) 2) Quantitation of Neutral Sugars and Hexosamines by glc The methodology described below and outlined i n F i g . 3 was developed from procedures described by Lehnhardt and Winzler (1968) and Niedermeier and Tomana (1974). A l l glassware was washed with chromic/sulfuric acid cleaning s o l u t i o n (Fisher) at 80°C, rinsed with d i s t i l l e d water and oven-dried overnight at 100°C to ensure removal of any contaminating material. Glycoprotein samples (180 u1) containing a minimum of 20-50 y g t o t a l carbohydrate were pipetted and accurately weighed into 0.3 ml R e a c t i - v i a l s and equal volumes of a 40% (w/v) suspension of AG 50W-X8 (H+) 200-400 mesh i n 0.02 M HC1 were added (see Appendix A for r e s i n preparation.) After s e a l i n g the v i a l s with s i l i c o n e rubber septa, hydrolysis was c a r r i e d out i n an oven at 100°C for an appropriate period of time. (The s e l e c t i o n of s u i t a b l e hydrolysis times forc^M and other glycoproteins i s discussed i n the Results and Discussion sections. F o r a 9 M , a hydrolysis time of 35 hr was - 24 -Fig. 3 Outline of the procedure for the estimation of neutral and amino sugars from glycoproteins by glc - 25 -ONCOPROTEIN SAMPLE 1. hydrolyze with AG 50W (H+) 20% (w/w) i n 0.01 M HC1, 100°C, cool 2. i n j e c t i n t e r n a l standards(arabinose and mannosamine) 3. t r a n s f e r to 1 ml p l a s t i c pipette t i p plugged with glass wool and drain i n t o column of AG KHCO5) A. elute both resins with H2O and 50% aq. MeOH 6. dry then reduce with 0.1 M NaBH^ at room temperature, A hr 7. dry then d i s t i l l with MeOH/HCl and MeOH 8. acetylate with a c e t i c anhydride and pyr i d i n e , 100°C, 2 x 15 min 9. centrifuge, dry supernatant 10. d i s s o l v e residue i n MeCl2 5. elute AG 50W with j 0.3 M HC1 AMINO SUGARS NEUTRAL SUGARS V 11. analyze by g l c at 2A0°C 1 12. analyze by g l c at 200°C - 26 -used.) A d e s c r i p t i o n of the apparatus used to mix and heat samples during hydrolysis i s included as Appendix C. Following hydrolysis, the samples were cooled to room temperature and al i q u o t s of both arabinose (25 u l , 0.03 ymol) and mannosamine HC1 (50 u l , 0.06 umol) i n d i s t i l l e d water were injec t e d through each septum with a Hamilton syringe. Each sample was mixed thoroughly (to allow binding of mannosamine to the resin) and the contents of the v i a l were then transferred to a 1 ml p l a s t i c disposable pipette t i p plugged with glass wool which was stacked on top of a second pipette t i p containing a bed (300 y l ) of AG 1-X8 (HC0~) 200-400 mesh r e s i n (Appendix A) on a glass wool plug support. The eluate from t h i s s e r i e s of "columns" was c o l l e c t e d i n a 15 ml Pyrex c o n i c a l centrifuge tube f i t t e d with a female $ 13 ground glass j o i n t . Portions (6 x 300 y l ) of d i s t i l l e d water were used to rinse the v i a l and these washings were transferred to the columns. The columns were then eluted with 50% (v/v) aqueous methanol ( 2 x 1 ml) and allowed to drain. The neutral sugars were contained i n the eluates plus washings. The two columns were separated and that containing the AG 50W r e s i n was eluted with 0.3 M HC1 ( 2 x 1 ml). The a c i d i c eluate containing the amino sugars was c o l l e c t e d i n a separate centrifuge tube. Neutral and a c i d i c eluates were evaporated to dryness under reduced pressure at 37°C. Reduction was c a r r i e d out by adding 0.1 M sodium borohydride (1 ml) to each dried sample. After 4 hr at room temperature, g l a c i a l a c e t i c acid (60 y l ) was added to each sample to terminate the reaction - 27 -and a l l f r a c t i o n s were subsequently evaporated to dryness. Excess borate was removed as v o l a t i l e trimethyl borate by d i s t i l l i n g with portions of 0.1% (v/v) HC1 i n methanol (A x 1 ml) followed by methanol (2 x 1 ml). The dry residue was t r i t u r a t e d with a mixture of pyridine (200 y l ) and a c e t i c anhydride (200 y l ) , and then the centrifuge tubes were f i t t e d with glass stoppers and heated for 15 min i n an oven at 100°C. The samples were mixed well, reheated at 100°C for 15 min, cooled and then centrifuged to p e l l e t the i n s o l u b l e inorganic s a l t s . The supernatants were transferred to 3 ml glass c o n i c a l centrifuge tubes using glass Pasteur pipettes and dried i n vacuo over fresh sodium hydroxide and s u l f u r i c a c i d for a minimum of 6 hr. Samples were removed from the dessicator and stored at room temperature u n t i l analyzed (within one week). Derivatives are reported to be stable for up to 3 months (Lehnhardt and Winzler, 1968). The residue from each sample was dissolved i n methylene c h l o r i d e (10 y l ) and an aliquot (3 y l ) was analyzed on a Hewlett Packard 7610A gas chromatograph f i t t e d with a flame i o n i z a t i o n detector and on-column i n j e c t i o n . Chromatography was c a r r i e d out isothermally at 200°C (for neutral sugars) or 240°C (for hexosamines) on 183 cm x 2 mm ID s i l i c o n i z e d glass columns packed with GP 3% SP-2340 on 100/120 mesh Supelcoport using nitrogen as the c a r r i e r gas. Peak areas were recorded with a 3370B e l e c t r o n i c integrator. D e t a i l s of column preparation and g l c operating conditions are given i n Appendices D,E and F, r e s p e c t i v e l y . - 28 -3) Quantitation of S i a l i c Acid by Colorimetric Assay Total s i a l i c a c i d was estimated by the method of Jourdian et a l . (1971) with the following modifications (Reid et a l . , unpubl.) Samples (27 y l ) containing 0.2-4.0 yg s i a l i c ' a c i d were saponified by treatment with 1 M potassium hydroxide (3 y l ) for 30 min at room temperature then neutralized with 0.5 M s u l f u r i c acid (3 y l ) . After addition of 0.04 M sodium metaperiodate (7 yl)', the samples were mixed well and placed i n an i c e bath for 35 min. Resorcinol reagent (80 y l ) was added, the samples were mixed well and, a f t e r standing 5 min, heated i n a b o i l i n g water bath for 8 min. After cooling to room temperature, t-butanol (80 y l ) was added and the samples were reheated at 37°C for 5 min and read at 630 nm. Human seromucoid was employed as a standard and was used without s a p o n i f i c a t i o n . - 29 -RESULTS I. I s o l a t i o n ofg^M from CF and Control Plasma A. Plasma g^M Concentrations As shown i n Table I, the plasma a^M l e v e l s of the CF patients were not s i g n i f i c a n t l y d i f f e r e n t from those of the c o n t r o l s . As previously observed (Ganrot and Schersten, 1967; T u n s t a l l et a l . , 1975; Wagner et a l . , 1982), there was a general decrease i n plasma g 2M concentration with increasing age. The values obtained were si m i l a r to those reported for t h i s age group by Wagner et a l . (1982). B. Column Chromatography of CF and Control Plasma F i g . A shows the t y p i c a l e l u t i o n p r o f i l e obtained when plasma was subjected to Cibacron Blue Sepharose column chromatography. The f i r s t peak (CBS pool 1, F i g . A) contained 80 percent of the a^M applied. The f r a c t i o n s c o n s t i t u t i n g t h i s peak were pooled and further fractionated using an immunoadsorption column to remove the major plasma protein contaminants. As shown i n F i g . 5, the a^M was obtained as a s i n g l e peak. No differences were observed i n the behaviour of CF and c o n t r o l plasma during column chromatography. C. Problems Caused by T r i s Buffer The procedure for the i s o l a t i o n of a 2 M (Bridges et a l . , 1982a) employs 50 mM Tris-HCl pH 7.5 as a buffer. The s i a l i c acid content of glycoprotein samples i n T r i s could not be determined by the modified periodate-resorcinol assay as T r i s suppressed the formation of the chromagen i n t h i s assay. The problem could be solved by carrying out the periodate oxidation i n 0.125 N r^SO^. At t h i s pH T r i s i s protonated and presumably does not undergo periodate oxidation. T r i s - 30 -Table I Plasma a 2M l e v e l s i n CF patients and normal controls as measured by r a d i a l immunodiffusion (p.20). - 31 -Age (yr) Sex Plasma Normal a 9M (mq/100 ml) CFl 7 M 406 375 10 F • 404 343 12 M 406 438 13 M 511 544 14 F 358 300 18 M 331 253 i D i fferences between normal and C F plasma o^M concentrations were not s i g n i f i c a n t when evaluated by paired t t e s t . - 32 -F i g . 4 Cibacron Blue Sepharose chromatography of normal human plasma. Five ml of harvested plasma Csee p.20 ) was applied to a s i l i c o n i z e d column (1.5 x 90 cm) containing Cibacron Blue Sepharose CL-6B. The column was eluted with 50 mM T r i s HC1, pH 8.0 containing 3 mM sodium azide at a rate of 1.5-2.0 ml/hr. Two ml f r a c t i o n s were c o l l e c t e d and monitored for absorbance at 280 nm. A l l steps were c a r r i e d out at 4°C. The f r a c t i o n s c o n s t i s t u t i n g the f i r s t peak were then monitored for ct2M and plasma protein contaminants by RID before being pooled (CBS pool). F R A C T I O N N U M B E R - 34 -F i g . 5 Immunoadsorption chromatography of CBS pool. The CBS pool ( F i g . 4) was applied to a s i l i c o n i z e d column (1.6 x 28 cm) containing an immunoadsorbent prepared by binding antibodies against contaminating protein species to Sepharose 4B (Bridges et a l . , 1982a). a 2M was eluted with 50 mM T r i s HC1, pH 7.5 containing 250 mM sodium ch l o r i d e and 3 mM sodium azide at a rate of 15 ml/hr. Two ml f r a c t i o n s were c o l l e c t e d and monitored for absorbance at 280 nm. A l l steps were c a r r i e d out at 4°C. - 35 -0.6 h O oo CM < 0.4 0.2 h 10 20 F R A C T I O N 30 NUMBER 40 - 36 -also i n t e r f e r e d with the quantitation of neutral and amino sugars by glc (see Discussion section) and since i t i s known to i n t e r f e r e i n the Lowry protein assay (Rej and Richards, 1974; Peterson, 1979), exchange d i a l y s i s against 25 mM sodium bicarbonate was c a r r i e d out, p r i o r to concentration of the material obtained a f t e r immunoadsorption chromatography, i n order to remove T r i s buffer. Sodium bicarbonate was an appropriate substitute for T r i s as the pH of a 25 mM s o l u t i o n (pH = 8.3) exceeds the i s o e l e c t r i c point of c*2M (pi = 5.0 - 5.5) thus a 2M remains i n s o l u t i o n (Starkey and Barrett, 1977). Furthermore, as shown i n Table II analysis of a model glycoprotein, a-j-acid glycoprotein demonstrated that 25 mM sodium bicarbonate does not i n t e r f e r e with e i t h e r the Lowry protein or periodate-resorcinol assays. Quantitation of neutral and amino sugars by g l c could also be c a r r i e d out i n the presence of sodium bicarbonate as discussed below (p. 63 ). II Assessment of Purity of a 2M Isolates Two methods were used to assess the purity of t h e a 2 M i s o l a t e s : Immunoelectrophoresis and SDS-PAGE. A. Immunoelectrophoresis Immunoelectrophoresis of a 2M i s o l a t e s against antiserum to whole human serum yielded a s i n g l e p r e c i p i t i n band ( F i g . 6). Repetition of the analysis using rabbit antiserum to human a 2M p o s i t i v e l y i d e n t i f i e d the material i n the arc a s « 2 M . None of the p o t e n t i a l plasma protein contaminants were detected i n the f i n a l a 2M preparations; approximately 5 percent (w/w) contamination would have been detected (Bridges et a l . , 1982a). - 37 -Table II Determination of the s i a l i c a c i d and protein content of aj_-acid glycoprotein (aj_AG) i n water and i n 25 mM sodium bicarbonate (n = 4). - 38 -a,AG i n water a,AG i n 25 mM NaHCO Protein 0.81 + 0.02 0.80 + 0.0A (mg/ml) S i a l i c acid 29.A +1.8 28.2 + 0.9 1 (ymol/100 mg protein) Differences between the r e s u l t s of analyses performed i n water and i n sodium bicarbonate were not s i g n i f i c a n t when evaluated by the t s t a t i s t i c for the comparison of two means. - 39 -F i g . 6 Immunoelectrophoresis of o^M i s o l a t e s The upper well of each s l i d e contained human serum (2 y l ) ; o^M i s o l a t e d from normal plasma (10 yg) was added to the lower well. A. D i f f u s i o n against rabbit antiserum to whole human serum B. D i f f u s i o n against rabbit antiserum to human a ?M - 40 -A B + + - 41 -B. SDS-PAGE A s i n g l e high molecular weight band was observed when c^M (10 yg) was analyzed by SDS-PAGE. Densitometric analysis of the SDS-PAGE tube gels showed that a l l CF and normal preparations were free of detectable contaminants ( F i g . 7 ) . I l l . Quantitation of Neutral Sugars and Hexosamines by g l c A. Glc of A l d i t o l Acetates Solutions i n methylene c h l o r i d e of mixtures of the a l d i t o l acetates of fucose, arabinose, mannose and galactose (10 ymol/ml) and of the a l d i t o l acetates of mannosamine and glucosamine (lOvmol/ml) yielded well-resolved symmetrical peaks when a l i q u o t s (3 y l ) were analyzed by g l c . Excellent r e s u l t s were obtained under isothermal conditions. Pig. 8 shows t y p i c a l chromatograms of the a l d i t o l acetates of neutral and amino sugars. B. Determination of Molar Response Factors and R e l a t i v e  Retention Times of the A l d i t o l Acetates The detector responses and r e l a t i v e retention times of the a l d i t o l acetates of neutral and amino sugars were c a l c u l a t e d r e l a t i v e to a r a b i n i t o l and mannosaminitol acetates, r e s p e c t i v e l y . The r e s u l t s of the g l c analysis of three mixtures containing equimolar amounts of e i t h e r the a l d i t o l acetates of fucose, arabinose, mannose and galactose (see Appendix B for synthesis) or the a l d i t o l acetates of mannosamine and glucosamine (synthesis i s o u t l i n e d i n Appendix B) prepared i n methylene c h l o r i d e (10 ymol/ml of each sugar) and analyzed i n t r i p l i c a t e were used to c a l c u l a t e the r e l a t i v e molar response fa c t o r s from the following equation. - 42 -F i g . 7 Densitometric scans of c o n t r o l (A) and CF (B) a 2M (20 ug) analyzed on 5% polyacrylamide tube gels i n the presence of SDS - 44 -Fig. 8 Gas-liquid chromatography of the alditol acetates on GP 3% SP-2340 on 100/120 mesh Supelcoport A. neutral monosaccharides at 200°C B. hexosamines at 240°C 1 = fucose; 2 = arabinose; 3 = mannose; 4 = galactose; 5 = glucosamine; 6 = mannosamine - 4 6 -MRFu = peak area X moles standard peak area standard moles u where MRFu = molar response factor of u and standard = a r a b i n i t o l pentaacetate ( f o r neutral sugars) or mannosaminitol hexacetate (for amino sugars) The r e s u l t s obtained are shown i n Table I I I . C. Materials I n t e r f e r i n g with the Quantitation of Neutral Sugars  and Hexosamines by glc Due to the l i m i t e d amounts of a.^ a v a i l a b l e for the analyses and i t s r e l a t i v e l y low carbohydrate content, i t was necessary to operate the gas chromatograph at l e v e l s of high s e n s i t i v i t y . Under these circumstances, contaminants which have not previously been reported to i n t e r f e r e with the quantitaton of the a l d i t o l acetate d e r i v a t i v e s of the carbohydrate exponents were detected i n s i g n i f i c a n t amounts. Material which co-eluted with f u c i t o l acetate ( F i g . 9) presented a major problem and was eliminated by ensuring that the acetates were dried i n vacuo over fresh drying agents. Additional contaminants which i n t e r f e r e d with the quantitation of the a l d i t o l acetates arose from the use of reagent grade methanol and could be eliminated by using spectro A.C.S. grade methanol ( F i g . 10). In addition, i t was imperative to wash glassware with a chromic/sulfuric acid mixture i n order to remove traces of detergent which also gave r i s e to i n t e r f e r i n g peaks. - 47 -Table III Means _+ S.D. of the r e l a t i v e retention times and molar response factors for the a l d i t o l acetates The values shown represent the average of three r e p l i c a t e preparations analyzed i n t r i p l i c a t e . The retention time f o r a r a b i n i t o l pentacetate was 8 min while mannosaminitol hexacetate had a retention time of 2 2 min under the operating conditions described (see Appendix F) . The retention times varied between runs depending upon the exact column conditions employed. I t should be noted that with the column packing used the absolute retention times decreased with the age of the column. - 48 -Parent Relative Molar response monosaccharide retention factor Neutral sugars Fucose Arabinose Mannose Galactose Amino sugars Glucosamine Mannosamine 0.63 + 0.01 1.00 2.28 + 0.04 2.62 + 0.05 0.88 + 0.00 1.00 1.14 + 0.03 1.00 1.13 + 0.03 1.15 + 0.03 0.85 + 0.02 1.00 - 49 -F i g . 9 Analysis of the neutral sugars of M A preparation of a M i n water was analyzed according to the procedure ou t l i n e on p. 23 . A. Acetylated sugars were drie d i n vacuo over NaOH/conc. H2SO4 which had previously been used to dry a c e t y l a t i o n mixtures. B. Acetylation mixture was dried i n vacuo over fresh NaOH/conc. H2SO4. 1 = fucose; 2 = arabinose ( i n t e r n a l standard); 3 = mannose; 4 = galactose D E T E C T O R R E S P O N S E - 51 -F i g . 10 Analysis of the neutral sugars of <^ M by g l c . a, M i n water was analyzed according to the standard procedure. A. Reagent grade methanol was used to remove excess borate. B. Spectranalyzed ACS grade methanol was employed. 1 = fucuse; 2 = arabinose ( i n t e r n a l standard) 2 = mannose; A = galactose D E T E C T O R R E S P O N S E D E T E C T O R R E S P O N S E - 53 -D. Assessment of the Recovery of Sugars Assayed by the g l c  Procedure In order to determine the recoveries of monosaccharides assayed by the g l c procedure, aliquots (180 y l ) each containing accurately weighed amounts of L-fucose, D-mannose, D-galactose and N-acetyl-D-glucosamine (0.5 - 80 nmol) plus the appropriate i n t e r n a l standard were mixed with AG 50W-X8 [H +] re s i n 40% (w/w) i n 0.02 M HC1 (180 y l ) and subjected to the standard procedure (p. 23 ) omitting the hydrolysis step. The quantity of each sugar was calculated from the following equation: moles sugar = Peak area of sugar X Moles standard Peak area of standard MRF* As shown i n Table IV, the amounts of neutral and amino sugars recovered (expressed as percent) were s t a t i s t i c a l l y d i f f e r e n t from the amounts of the sugars added. However, there was no s i g n i f i c a n t difference between the recoveries of fucose, mannose and galactose. In view of t h i s , response factors were calculated from an analysis of an unhydrolyzed mixture of sugars of known composition. Other i n v e s t i g a t o r s have used a s i m i l a r method and express t h e i r data as molar response fa c t o r s . As shown i n Table V, when the data were *MRF cal c u l a t e d from a weighed quantity of a l d i t o l acetate (see p.41). - 54 -Table IV Mean recoveries of the components of unhydrolyzed mixtures of monosaccharides. - 55 -Monosaccharide % recovery''' (mean + S.D.) 2 Neutral sugars Fucose 91 + 11 (18) Mannose 9 3 + 1 2 (18) Galactose 8 9 + 7 (18) Amino sugars Glucosamine 7 9 + 8 (15) The values for a l l sugars were s i g n i f i c a n t l y d i f f e r e n t from 100% when analysed by t s t a t i s t i c for comparison of two means The values for neutral sugars did not d i f f e r from one another using the t s t a t i s t i c for the comparison of two means - 56 -Table V Response factors of the a l d i t o l acetates prepared from mixtures of unhydrolyzed monosaccharides. This Porter Niedermeier Metz Neidermeier Shaw Lehnhardt study (1975) & Tomana et al. (1971) & Moss & Winzler (1974) (1971) (1969) (1968) Neutral sugars Fucose 1.018 1.05 1.10 1.07 0.99 1.089 Arabinose 1.000 1.00 1.00 1.00 1.00 1.000 Galactose 1.048 1.21 1.15 1.14 1.09 1.211 Mannose 1.014 1.33 1.15 1.25 1.18 1.182 Amino sugars Glucosamine 0.688 1.24 Mannosamine 1.000 1.00 - 58 -expressed i n t h i s way, they f e l l within the s c a t t e r of values reported by these i n v e s t i g a t o r s although the value obtained f o r glucosamine d i f f e r e d r a d i c a l l y from that reported by Niedermeier and Tomana (1974). E. Time Required for Hydrolysis As ct^M had not previously been analyzed using the r e s i n hydrolysis method, i t was considered necessary to e s t a b l i s h the optimal time for h y d r o l y s i s . This was determined by hydrolyzing a l i q u o t s of normal c^M (180 y l ) containing 300-400 ug protein i n the presence of AG 50W-X8 (H +) ion exchange r e s i n i n 0.01 M HC1 at 100°C for the following times: 3,6,10,20,30,40,50,60 and 70 hr; two separate a l i q u o t s were hydrolyzed at each time i n t e r v a l . In add i t i o n , the hyd r o l y s i s of the following models was also investigated over a s i m i l a r time period: c ^ - a c i d glycoprotein (90 ug), a mixture of neutral and amino sugars i n approximately the same t o t a l amounts as expected i n 300-400 ug c*2M (fucose, 2 nmol; mannose 40 nmol; galactose 20 nmol; glucosamine 40 nmol) and lactose (100 nmol) i n the presence and absence of bovine serum albumin (100 yg). On hydrolysis of a2M, fucose and galactose were released most ra p i d l y as shown i n F i g . 11; the maximum amounts of these sugars were obtained a f t e r 6 hr of h y d r o l y s i s . Mannose and glucosamine were released at somewhat slower rates with maximum amounts of mannose and glucosamine detected a f t e r approximately 20 hr. As shown, there was considerable degradation of the released sugars with extended hydrolysis times. On the basis of t h i s study, the h y d r o l y s i s time selected f o r a 0M was 35 hr; t h i s ensured maximal release of mannose and - 59 -glucosamine while minimizing the acid degradation of the more ra p i d l y released galactose and fucose. The release of sugars from a^-acid glycoprotein ( F i g . 12) followed a s i m i l a r pattern to that found with o^M. Fucose was released most r a p i d l y (maximal release at 6 hr), followed by release of galactose and glucosamine (10 hr) and mannose (20 h r ) . The r e l a t i v e rates of release of the various sugars are s i m i l a r to those observed by Lehnhardt and Winzler (1968). A hydrolysis time of 30 hr was selected for a^-acid glycoprotein rather than the AO hr reported previously (Lehnhardt and Winzler, 1968). Figs. 11 and 12 demonstrate that, as shown previously (Lehnhardt and Winzler, 1968; Porter, 1975), sugars were released from glycoproteins at d i f f e r e n t rates. However, i n contrast to these reports, l o s s of the released sugars was observed with extended hydrolysis times. This was apparently not due to i n t e r a c t i o n of the free sugars with amino acids as studies on hydrolysis of lactose demonstrated that the release and subsequent loss of galactose was not affected by the presence of bovine serum albumin ( F i g . 13). Presumably, the observed l o s s of the released sugars was a consequence of acid degradation since i t was also observed when mixtures of free sugars were hydrolyzed ( F i g . 1A). Analysis of the data obtained from the hydrolysis of the free sugars ( F i g . 1A) indicated that there were s i g n i f i c a n t d ifferences i n the rates of degradation of the various sugars. The data shown were re-analyzed by expressing them as the slopes of the regression l i n e s - 60 -Fig. 11 Release of sugars from normal o^ M by acid hydrolysis at 100°C in 0.01 M HC1 in the presence of 20% (w/v) AG 50W-X8 [H+] 200-400 mesh resin (-Q-) , glucosamine; (-•-) , galactose ; (-•-) , mannose; , fucose. p.mol C A R B O H Y D R A T E 1 0 0 mg P R O T E I N - T9 -- 62 -F i g . 12 Release of sugars from a j - a c i d glycoprotein by aid hydroloysis at 100°C i n 0.01 M HC1 i n the presence of 20% (w/v) AG 50W-X 8 [H+] 200-400 mesh r e s i n (- Q-) , glucosamine; (-•-) , mannose; (-O-) , galactose ; (-•-) , fucose. p.mol C A R B O H Y D R A T E 100 mg P R O T E I N ro A o> oo o o o o o o o - e9 -- 64 -F i g . 13 Release of galactose from lactose by acid hydrolysis at 100°C i n 0.01 M HC1 i n the presence of 20% (w/v) AG 50W-X 8 [H +] 200-400 mesh r e s i n . Lactose (10 ymol) was hydrolyzed i n the presence and absence of bovine serum albumin (100 yg). > galactose released from lactose (-• -) > galactose released from lactose i n the presence of bovine serum albumin. - 65 -- 66 -F i g . 14 Loss of free sugars during acid hydrolysis at 100°C i n 0.01 M HC1 i n the presence of 20% (w/v) AG 50W-X 8 [H+] 200-400 mesh r e s i n . A mixture containing glucosamine, mannose, galactose and fucose was analyzed according to the standard procedure. (-o-) , glucosamine; (-•-) , mannose; (-o-) , galactose ; (-•-) , fucose. - 67 -31VHuAH0aaVD \omr\ - 68 -(Mendenhall et a l . , 1981). This analysis (Table VI) indicated that fucose was degraded at a s i g n i f i c a n t l y d i f f e r e n t rate to mannose (P< 0.001), galactose (P<.001) and glucosamine (P<0.001). In addition, mannose was degraded at a d i f f e r e n t rate to galactose. (P<0.01). F. E f f e c t of Sodium Bicarbonate on the Quantitation of Neutral  and Amino Sugars by g l c . In order to determine whether or not sodium bicarbonate i n t e r f e r e d with the glc procedure, preparations of a^-acid glycoprotein (a^AG) i n water and i n 25 mM sodium bicarbonate were hydrolyzed for 35 hr and c a r r i e d through the standard procedure as a-^-acid glycoprotein was used as a model for a 2M. The hydrolysis time used for t h i s and subsequent studies was that selected for a 2M. There was no s i g n i f i c a n t difference between the r e s u l t s obtained from analyses performed i n sodium bicarbonate and those obtained when the analyses were c a r r i e d out i n water (Table VII). - 69 -Table VI Comparison of the rates of l o s s of free sugars during acid hydrolysis. The data shown i n F i g . 14 were expressed as a percentage of the amount of each monosaccharide detected at zero time. The regression l i n e s through these data points were determined and the slopes were compared using a t s t a t i s t i c for two means (Mendenhall et a l . , 1981). - 70 -Monosaccharide Slope S.D. Fucose 1 -0.91 0.09 2 Mannose -0.67 0.03 Galactose -0.60- 0.05 Glucosamine -0.62 0.07 The slope estimated from the fucose data was s i g n i f i c a n t l y d i f f e r e n t from those obtained from the mannose, galactose and glucosamine data (P<0.001). The slope estimated from the mannose data was s i g n i f i c a n t l y d i f f e r e n t from those obtained using fucose and galactose data (P< 0.001). - 71 -Table VII Carbohydrate composition of a ^ - a c i d glycoprotein (aj_AG) i n water and i n 25 mM sodium bicarbonate. A s o l u t i o n of ajAG (1.64 mg/ml i n water) was d i l u t e d 1:1 with e i t h e r water or 50 mM NaHC03 and the s i a l i c acid and protein content of each was analyzed i n quadruplicate by the Lowry and periodate r e s o r c i n o l assays. - 72 -umol/100 mg protein a-^ AG in water a^ AG in 25 mM sodium (n=3) bicarbonate (n=3) Fucose Mannose Galactose Glucosamine t statistic for the comparison of two means. Results for samples analyzed in sodium bicarbonate were not significantly different from those for samples analyzed in water. 8.8 + 0.5 AA.8 + 1.8 56.3 + 3.2 102.8 + 2.8 8.5 + O.A AA.l + 1.21 56.3 + 2.71 103.2 + 3.81 - 73 -G. Pr e c i s i o n of the A n a l y t i c a l Methods The p r e c i s i o n of an a n a l y t i c a l method as defined by Reed and Henry (1974) i s "the v a r i a t i o n of r e s u l t s obtained by a method when the same sample i s run repeatedly" and i s expressed as a c o e f f i c i e n t of v a r i a t i o n , C.V.: C.V. = o/x x 100% where a = standard deviation x = mean The p r e c i s i o n of the g l c method (n=6) and the periodate r e s o r c i n o l assay (n=9) was determined by analysis of a preparation of a^-acid glycoprotein i n water. The c o e f f i c i e n t s of v a r i a t i o n shown i n Table VIII f e l l within the range of values (C.V. = 2-10%) reported by other i n v e s t i g a t o r s (Niedermeier, 1971; Niedermeier and Tomana, 1974). H. P r o p o r t i o n a l i t y of Response with Amount of Glycoprotein In order to demonstrate that the amounts of carbohydrate estimated by the g l c procedure were proportional to the amount of glycoprotein analyzed, 90,180 and 270 yg of ctj-acid glycoprotein were hydrolyzed for 35 hr according the outlined g l c procedure (p23). The amounts of fucose, mannose, galactose and glucosamine released r e l a t i v e to the amount of a^-acid glycoprotein hydrolyzed are shown i n F i g . 15. These r e s u l t s demonstrate that there i s a l i n e a r r e l a t i o n s h i p between the amount of carbohydrate estimated by the glc method and the amount of glycoprotein hydrolyzed. - 74 -Table VIII P r e c i s i o n of the a n a l y t i c a l method on a j - a c i d glycoprotein Following hydrolysis for 35 hr, the neutral and amino sugar content of a preparation of a^-acid glycoprotein i n water was estimated according to the g l c procedure described on p. 23 (n=6). The s i a l i c a c i d content of the unhydrolyzed preparation was determined by periodate r e s o r c i n o l assay (n=9). The c o e f f i c i e n t s of v a r i a t i o n (C.V.) were calculated from the equation given i n the text. - 75 -Monosaccharide Mean Concentration o C.V. (umol/100 mg protein) (%) Fucose 8.63 0.43 5.0 Mannose 44.43 1.43 3.2 Galactose 56.30 2.64 4.7 Glucosamine 102.99 2.99 2.9 S i a l i c Acid 27.63 1.39 4.8 - 76 -F i g . 15 P r o p o r t i o n a l i t y of the quantity of carbohydrate found to quantity of glycoprotein ( a j - a c i d glycoprotein) hydrolyzed for 35 hr by the standard method. (-a-) glucosamine,(-o-) galactose-, (-•-) mannose. (-<>-) fucose. - 77 -0.2 Or mg G L Y C O P R O T E I N - 78 -IV. Analysis of the Carbohydrate Composition of a^M from CF  Patients and Normal Controls. The carbohydrate composition of a 2 M i s o l a t e d from six CF patients and s i x age- and sex-matched normal controls i s shown i n Table IX. There was no s i g n i f i c a n t difference i n the neutral sugar, hexosamine, s i a l i c acid or t o t a l carbohydrate content of CF and con t r o l a^A. When monosaccharides were expressed as mole % of t o t a l carbohydrate (Table X), again no s i g n i f i c a n t differences between CF and control ct~M were observed. - 79 -Table IX Carbohydrate composition of a 2M from CF patients and controls. Neutral and amino sugars were estimated by g l c ; samples were analyzed simultaneously i n duplicate on two separate occasions. Estimation of s i a l i c a c i d by periodate r e s o r c i n o l assay was c a r r i e d out i n t r i p l i c a t e twice. The values shown are the mean +_ S.D. of a l l analyses. (For neutral sugars and hexosamines n=A and for s i a l i c acid n=6). pmol carbohydrate/100 mg protein Age/Sex Fucose Hamose Galactose Sialic Acid Total Glucosamine Carbohydrate N CF N CF N CF N CF N CF N CF 7y M 0.72+0.11 0.71*0.13 15.17*1.35 14.41*0.86 6.73*0.39 6.66+0.46 15.87*0.33 15.88*1.04 5.50+0.58 5.70*0.47 43.99 43.36 lOy F 0.61*0.04 0.67*0.10 13.89_»0.79 13.93*1.94 7.11*0.40 7.17*0.52 15.72+1.50 16.00*1.10 5.59*0.19 5.61+0.09 42.92 43.38 12y H 0.73*0.09 0.60*0.09 14.97*0.84 14.10*0.85 6.80*0.53 6.66*^ 0.44 16.49*0.93 15.09*2.08 5.47+0.26 5.61*0.32 44.46 42.06 13y M 0.62*0.09 0.62.0.07 13.83*1.77 15.18.2.02 7.18*0.70 6.68+1.23 17.61+2.17 15.11*1.40 5.66*0.13 5.22*0.15 44.90 42.81 l4y F 0.78*0.08 0.74+0.09 15.15*0.96 13.38*0.71 7.02*0.67 6.65+0.24 17.15*1.90 16.28*1.39 5.49+0.37 5.39_*0.48 45.59 42.44 16y M 0.68*0.07 0.87*0.03 13.49*0.42 13.41.0.63 6.60*0.44 6.51_.0.78 14.14*1.44 14.08*2.02 5.86*0.01 5.66*0.42 40. 77 40.53 Mean 0.69*0.11 0.70*0.12 14.42*1.21 14.07.1.31 6.91*0.52 6.72+0.65 16.13_*1.77 15.38*1.59 5.58+0.31 5.52+0.33 1 No significant differences were observed in the carbohydrate composition of CF and normal *hen data was analysed by paired t-statistlc - 81 -Carbohydrate composition of 012M from CF patients and controls expressed as mean mole % calculated from data i n Table IX. mole % carbohydrate* Fucose Mannose Galactose Glucosamine S i a l i c A c i d N CF N CF N CF N CF N CF 7y M 1.6 1.6 34.5 33.2 15.3 15.4 36.1 36.6 12.5 13.2 lOy F 1.4 1.6 32.4 32.1 16.6 16.5 36.6 36.9 13.0 12.9 12y M 1.6 1.4 33.7 33.5 15.3 15.8 37.1 35.9 12.3 13.4 13y M 1.4 1.4 30.8 35.5 16.0 15.6 39.2 35.3 12.6 12.2 14y F 1.7 1.7 33.2 31.5 15.4 15.7 37.6 38.4 12.1 12.7 18y M 1.6 2.1 33.1 33.1 16.2 16.1 34.7 34.7 14.4 14.0 I No s i g n i f i c a n t d i f f e r e n c e s were observed i n the carbohydrate compos i t ion o f CF and normal ° ( 2 M w n e n data w a s analyzed by pa i r ed t - s t a t i s t i c - 83 -DISCUSSION The aim of t h i s t h e s i s was to compare the carbohydrate composition of CF and co n t r o l o^M. To accomplish t h i s , a method which was highly s e n s i t i v e , precise and accurate was required. The gl c method used, a further development of the procedures used by Lehnhardt & Winzler (1968) and Reid et a l . (unpubl.), met these c r i t e r i a . In order to achieve the required degree of p r e c i s i o n and accuracy i t was found necessary to use highly p u r i f i e d solvents, e s p e c i a l l y methanol, to avoid the accumulation of contaminants which i n t e r f e r e d with quantitation. Arabinose was selected as an i n t e r n a l standard since i t s a l d i t o l acetate d e r i v a t i v e separated well from the a l d i t o l acetate d e r i v a t i v e s of the neutral sugars found i n c^M and i n addition, i t i s not a component of e i t h e r o^M or other plasma glycoproteins. I t was also a more appropriate standard than the a l d i t o l , a r a b i n i t o l , employed by Lehnhardt and Winzler (1968), as i t i s chemically s i m i l a r to the compounds being analyzed. Furthermore, arabinose i s subjected to a l l reactions of the d e r i v a t i z a t i o n procedure including reduction. Arabinose has been s u c c e s s f u l l y u t i l i z e d as an i n t e r n a l standard by Niedermeier (1971). The amino sugars were quantitated r e l a t i v e to the i n t e r n a l standard mannosamine as f i r s t described by Niedermeier and Tomana (1974). The use of a hexosamine as an i n t e r n a l standard (rather than a neutral compound such as i n o s i t o l , for example), accounts more c l o s e l y for any losses incurred during the e l u t i o n of hexosamines from the AG 50W r e s i n . Mannosamine i s a s u i t a b l e i n t e r n a l standard for the - 84 -quantitation of hexosamines i n glycoproteins such as a^M which contain only glucosamine. However, a p p l i c a t i o n of t h i s method to glycoproteins containing galactosamine or galactosamine plus glucosamine would require the use of a column packing capable of reso l v i n g the a l d i t o l acetates of mannosamine and galactosamine, for example, Poly A 103 (Niedermeier, 1971). The excellent separation of the a l d i t o l acetates under isothermal conditions eliminated the need for temperature programming and the balanced dual columns used i n other procedures. The columns proved s u f f i c i e n t l y stable f or 200-300 determinations, thus the complete analysis of normal and CF a 2M could e a s i l y be performed on one column. I t was possible to accurately analyze with a high degree of p r e c i s i o n as l i t t l e as 0.3 mg of o^M which represents 0.03 mg t o t a l carbohydrate. Of the neutral sugar components i t was possible to analyze approximately 0.3 yg of fucose. The method also gave a l i n e a r r e l a t i o n s h i p between the amount of carbohydrate estimated and the amount of glycoprotein hydrolyzed and the p r e c i s i o n of the method as measured by the c o e f f i c i e n t s of v a r i a t i o n for the various sugars was s i m i l a r to that reported by other inv e s t i g a t o r s (Niedermeier, 1971; Niedermeier and Tomana, 1974; Gehrke et a l . , 1979). The c o e f f i c i e n t of v a r i a t i o n obtained for the estimation of s i a l i c acid by periodate r e s o r c i n o l assay (4.8%) was comparable to the c o e f f i c i e n t s of v a r i a t i o n obtained for the other sugars estimated by g l c . Careful i n v e s t i g a t i o n of the hydrolyses of ajAG and revealed that there was s i g n i f i c a n t l o s s of the released sugars with - 85 -extended hydrolysis times. This has not apparently been observed previously. Lehnhardt and Winzler (1968) and Porter (1975) found that the release of sugars reached a plateau and concluded that there was no l o s s of released sugars when hydrolysis was performed for periods of up to 48 hr. Our in v e s t i g a t i o n s indicated that a sui t a b l e h y d r o l y s i s time for a AG was 30 hr while that for a M was 35 hr Lehnhardt and Winzler (1968) and Porter (1975) reported optimum oijAG hydrolysis times of 40 and 48 hr, respectively. Our studies showed however, that when the hydrolysis of a-jAG was extended beyond 40 hr there was a s i g n i f i c a n t reduction i n the quantity of carbohydrate detected. The degradation of released sugars was also demonstrated, using as models, lactose, lactose plus BSA and a mixture of monosaccharides. The hydrolysis of lactose was not affected by the presence of BSA i n d i c a t i n g that the observed loss of free sugars was not a function of the presence of amino acids; presumably, therefore, the sugars are degraded by the ac i d . The data suggests that the acid degradation of the sugars occurs continuously and therefore the degradation of sugars released from glycoproteins would be expected to begin immediately. Hence the maximum quantity of carbohydrate detected i n a glycoprotein hydrolysate w i l l be l e s s than the actual quantity of carbohydrate present i n the glycoprotein. Further, as the free sugars are degraded at d i f f e r e n t rates, i t i s c l e a r that data based upon molar r a t i o s obtained at any sing l e hydrolysis time must also be i n error to some extent. - 86 -A potential method for obtaining values closer the actual values would be to conduct a time course of hydrolysis for each glycoprotein sample. A maximal value for each sugar is obtained at the point at which the rate of release of the sugar equals the rate of its degradation. This does not, however represent the actual amount of carbohydrate present in the glycoprotein. Further, in many cases i t would be difficult to perform such an experiment as large quantities of material would be required. An alternative approach is to conduct one hydrolysis time course study using a sample of the glycoprotein under investigation. (Use of the actual glycoprotein is necessary because as shown in Figs. 11 and 12, glycoproteins may behave differently during hydrolysis. Similarly, free sugars are not a suitable model (Fig. 14) because degradation of sugars which are not glycosidically bound begins immmediately.) Selection of a hydrolysis time which guarantees that complete hydrolysis has occurred is then made on the basis of the data. A correction factor is obtained by estimating the percentage loss of sugar that occurs between the time at which a maximum is reached and the selected hydrolysis time. This is then used to correct the data obtained from other samples of the glycoprotein analyzed at the selected time. For example, analysis of the data in Fig. 11 by this method shows that the potential losses of sugars released from c^M after 35 hr of hydrolysis are as follows: fucose, 23%; galactose, 8%; mannose, 9%; glucosamine, 2%. However, with the exception of fucose, application of such correction factors would alter the values by less than 10%. Furthermore, the values - 87 -would be increased by the same percentage for each of the samples analyzed and thus when comparing the carbohydrate analyses of o^M from CF patients and c o n t r o l s , the i n t e r p r e t a t i o n of the corrected and uncorrected data would be i d e n t i c a l . The values reported i n Table IX are therefore l e f t as the uncorrected values obtained a f t e r 35 hr of h y d r o l y s i s . No attempt was made to compare hydrolysis behaviours of CF and c o n t r o l samples. The assumption made i s that a l l a 2-macroglobulins behave s i m i l a r l y during h y d r o l y s i s . Previous analysis of normal o^M by other i n v e s t i g a t o r s (Table XI) established the presence of fucose, mannose, galactose, glucosamine and s i a l i c a c i d . Colormetric methods were used to quantitate these components. The values for the quantities of the various sugars presented i n t h i s t h e s i s f a l l within the s c a t t e r of the l i t e r a t u r e values (see Table XI) although these values and those of other i n v e s t i g a t o r s are considerably lower than the data reported by Bottiger and Norberg (196A). The value for s i a l i c acid content obtained by H a l l and Roberts (1978) i s s i g n i f i c a n t l y lower than that presented here or reported by others. Calculations based upon t h e i r data give a molar hexose to s i a l i c acid r a t i o of 21:1. Assuming that the oligosaccharide moieties of a^M are of the complex type and that the r a t i o s of mannose to galactose reported here and by Dunn and Spiro (1967) are correct, one may c a l c u l a t e the number of s i a l i c a c i d residues per galactose residue for b i - , t r i - and tetraantennary structures ( F i g . 1). Calculations of t h i s type i n d i c a t e that i n the best possible case (biantennary structure) which minimizes the number - 88 -Table XI Comparison of l i t e r a t u r e values for the carbohydrate composition of normal «2 M w ^ t h values obtained i n t h i s study. Values i n brackets represent the range. mg/100 mg protein This study Ha l l and Roberts (1978) B o u r i l l o n and Razafimahaleo (1972) Dunn and Spiro (1967b) Bottiger Heimburger Muller-Norberg et a l . Eberhard (1964) TT964) (1956) Fucose 0.12 (0.10-0.14) 0.71 0.24 0.1 Mannose 2.56 (2.41-2.73) 1.86 Galactose 1.23 (1.17-1.29) 1.22 Hexose 2.79 2.97 4.5 3.08 5.6 3.6 4.55 Glucosamine 3.49 (3.11-3.89) 2.47 3.95 3.62 4.2 2.9 2.68 S i a l i c acid 1.72 (1.61-1.81) 0.32 1.83 1.7 2.8 1.8 2.3 Total Carbohydrate 9.12 10.99 8.64 8.40 - 90 -of galactose residues, the galactose to s i a l i c acid r a t i o i s s t i l l 8:1. This implies that such a glycoprotein would be r a p i d l y cleared from the c i r c u l a t i o n (Ashwell and Morell, 1974) and i t therefore seems l i k e l y that the value reported by H a l l and Roberts represents a n a l y t i c a l error or p o s t - i s o l a t i o n a l loss of s i a l i c a c i d . The only data which can be r e a d i l y compared with that obtained by the g l c method used i n t h i s t h e s i s i s that of Dunn and Spiro (1967a,b) who i d e n t i f i e d the hexoses and hexosamines present and were able to estimate by colorimetry the r e l a t i v e proportions of mannose and galactose. While t h e i r values for glucosamine and s i a l i c a c i d content were s i m i l a r to those obtained here i n (Table XI), they obtained l e s s t o t a l hexose and a d i f f e r e n t molar r a t i o of mannose to galactose. These inv e s t i g a t o r s found that maximal release of mannose and galactose during hydrolysis i n 2 N H2S0^ at 100°C occurred a f t e r 4 hr. Degradation of sugars has been shown to occur under l e s s vigorous conditions (1 N H 2S0^ 100°C, Lehnhardt and Winzler, 1968). Therefore the mannose and galactose values reported by Dunn and Spiro (1967b) are l i k e l y underestimates. While the hydrolysis conditions employed i n the present study are somewhat milder, degradation of the released sugars s t i l l occurs and the hydrolysis times selected represent a compromise between maximizing the release of mannose and glucosamine while minimizing the degradation of fucose and galactose. However as discussed i n a previous section, t h i s must also represent an underestimate of the actual carbohydrate content. Analysis of the glycopeptides i s o l a t e d from normal a M suggested - 91 -that there i s considerable microheterogeneity of the oligosaccharide moieties and that the most complete structure i s the tetraantennary type shown i n F i g . 1 (Dunn and Spiro, 1967b). I n s u f f i c i e n t data i s a v a i l a b l e to allow further comments' to be made on the structure of the oligosaccharide moieties of o^M. The l a t t e r would require d e t a i l e d s t r u c t u r a l analyses of p u r i f i e d glycopeptides obtained from o^M. Ben Yoseph et a l . (1979) compared the s i a l i c a c i d content of a^M from three CF patients and three normal controls and found a 40% decrease i n the amount of s i a l i c a c i d present i n CF ct2M. S i g n i f i c a n t differences i n the binding of CF o^M to the l e c t i n s Con A and WGA were also reported (Ben-Yoseph et a l . , 1979; Shapira and Menendez, 1980) although no differences i n the t o t a l hexose content of CF and c o n t r o l a 2M were found (Ben-Yoseph et a l . , 1979). These studies raised the p o s s i b i l i t y of a l t e r a t i o n s i n the carbohydrate composition and/or structure of CF a 2 M -In c o n t r a d i c t i o n , i n the present study, no s i g n i f i c a n t d i f f e r e n c e s were found i n the o v e r a l l carbohydrate content or proportions of the carbohydrate components of o^M i s o l a t e d from six CF patients and t h e i r age- and sex-matched normal c o n t r o l s . Although small dif f e r e n c e s i n carbohydrate composition would have been masked by ei t h e r the inherent a n a l y t i c a l uncertainty of the methods and/or b i o l o g i c a l v a r i a b i l i t y i n the carbohydrate composition of a 2M, only small a n a l y t i c a l and within the population v a r i a t i o n s were observed. In view of the fact that the values for the s i a l i c a c i d content of normal aJA obtained by Ben-Yoseph et a l . , (1979) are i d e n t i c a l to - 92 -those reported herein, i t i s d i f f i c u l t to explain the s i g n i f i c a n t l y -reduced s i a l i c acid content of CF o^M found by these i n v e s t i g a t o r s . Further, Comings et a l . (1980) were unable to detect any differences i n the two-dimensional gel electrophoretic patterns of c o n t r o l and CF o^M; t h i s technique would be expected to reveal any charge differences due to d i f f e r e n c e s i n s i a l i c acid content. Previous studies on the r o l e of i n the pathogenesis of CF have indicated that there are no functional differences i n CF o^M but did not address the question of whether or not there are d i f f e r e n c e s i n i t s carbohydrate composition. In conclusion, the data presented i n t h i s t h e s i s demonstrates that no such differences e x i s t and therefore, i f a 2M i s involved i n the pathogenesis of CF then di f f e r e n c e s i n the o^M molecule must be at a s t r u c t u r a l l e v e l not yet investigated. - 93 -APPENDIX A Preparation of Ion Exchange Resins AG 50W-X8 [H+] resin was prepared as follows. The resin was stirred with excess 1 M NaOH, filtered under vacuum using a glass fiber filter and washed with distilled water until the eluate was neutral. The resin was then stirred with excess 1 M HC1, filtered and washed with distilled water until neutral. The resin was prepared as a 40% (w/v) suspension in 0.02 M HC1. AG 1-X8 [Cl~] resin was converted to its bicarbonate form by the following procedure. The resin was treated with excess 1 M HC1, washed with water, then treated with excess 1 M NaOH and washed with water until neutral as described above. This was followed by addition of excess 1 M NaHCO^  and the resin was washed with water until neutral. A 20% (w/v) suspension of the treated resin was prepared in distilled water. Both resins were prepared immediately before required. - 94 -APPENDIX B Preparation of A l d i t o l Acetate Standards The h e x i t o l acetate d e r i v a t i v e s of D-galactose and D-mannose were prepared from the corresponding h e x i t o l s according to the procedure of Abdel-Akher et a l . (1951) with the following modifications. To the h e x i t o l (10 g) was added pyridine (133 ml) followed by ac e t i c anhydride (78 ml); the mixture was s t i r r e d and, i n the case of mannitol heated gently, i n order to dissolve the h e x i t o l . The ac e t y l a t i o n mixture was l e f t at room temperature overnight a f t e r which i t was poured into a s t i r r e d mixture of i c e and water (1 L ) . The p r e c i p i t a t e was c o l l e c t e d by f i l t r a t i o n and washed with i c e - c o l d water u n t i l the odor of pyridine could no longer be detected. G a l a c t i t o l and mannitol hexaacetates' were r e c r y s t a l l i z e d from absolute ethanol to constant melting point. L - F u c i t o l and L - a r a b i n i t o l pentaacetates were prepared from t h e i r respective parent carbohydrates as follows. To fucose or arabinose (2 g) dissolved i n d i s t i l l e d water (20 ml), was added an equal weight of sodium borohydride i n 20 ml of d i s t i l l e d water. Af t e r 24 hr at room temperature excess sodium borohydride was destroyed by addition of acetone (10 ml) and the reduced product was s t i r r e d for 1 hr with an excess of AG 50W-X2 (H +) ion exchange r e s i n . The r e s i n was removed by f i l t r a t i o n on a Buchner funnel f i t t e d with a glass f i b e r f i l t e r and the f i l t r a t e was evaporated to dryness. Boric acid was removed by d i s t i l l a t i o n with 0.1% HC1 i n methanol (4 x 40 ml) followed by methanol (2 x 40 ml). The f l a s k containing the syrup was stoppered, - 9 5 -placed on i t s side and l e f t at -20 C u n t i l the syrup had frozen. The f l a s k , s t i l l on i t s side, was rotated 180° and a few ml of methanol was added such that the syrup adhering to the side of the f l a s k was suspended above a pool of methanol. C r y s t a l l i z a t i o n of the product was effected by leaving the stoppered f l a s k i n t h i s p o s i t i o n at -20°C overnight. The crude product was c o l l e c t e d by suction on a Buchner funnel and dried i n vacuo before being acetylated according to the procedure outlined above. F u c i t o l and a r a b i n i t o l pentaacetates were r e c r y s t a l l i z e d from absolute ethanol to constant melting point. The po l y o l acetates of D-glucosamine and D-mannosamine could not be obtained i n c r y s t a l l i n e form by the above methods and were consequently prepared by reduction and ac e t y l a t i o n of accurately weighed amounts of the corresponding parent carbohydrates according to the method outlined i n Section V i i . On gas chromatography, a l l of the pol y o l acetates eluted as s i n g l e symmetrical peaks. - 96 -APPENDIX C Apparatus for Mixing and Heating Samples During Hydrolysis R e a c t i - v i a l s were enclosed i n a wire mesh basket and fastened to the bar of a tumbler mixing apparatus i n such a manner as to provide end-over-end mixing of samples; t h i s apparatus was secured to the bottom of a thermostatically c o n t r o l l e d oven set at 100°C. The shaft of the mixing bar extended to the outside through a small hole d r i l l e d i n one side of the oven and connected by f l e x i b l e mechanical drive to a 1/20 HP e l e c t r i c a l motor. - 97 -APPENDIX D Preparation of GP 3% 5P-2340 Columns for Gas-liquid Chromatography An empty U-shaped glass column (183 cm x 2 mm ID) was f i l l e d with 2% (v/v) dimethyldichlorosilane i n toluene using a 30 ml syringe and allowed to stand at room temperature for 15 min. After s i l y l a t i o n , the column was rinsed successively with ten 10 ml portions of toluene and methanol and was a i r - d r i e d for 24 hr. A small plug of glass wool was inserted i n t o one end of the column ~> (detector end). This end of the column was connected to a vacuum l i n e and moderate suction was applied. Column packing was added to the other end of the column v i a a small funnel and even s e t t l i n g of the packing was achieved by v i b r a t i n g the column with the chuck of an e l e c t r i c engraving t o o l . The column was packed to within 5 cm of the open end and then plugged with glass wool. This end was connected to the i n j e c t i o n port of the gas chromatograph. - 98 -APPENDIX E Column Conditioning Procedure The packed column was*fitted with 1/4" Vespel f e r r u l e s , i n s t a l l e d i n the oven of the gas chromatograph and purged at ambient temperature for 30 min with the c a r r i e r gas flowing (N 2 = 25 ml/min, 40 p s i ) . The oven temperature was then programmed from 50°C to 250°C at 2°C/min and maintained at 250°C overnight (approximately 16 h r ) . The detector assembly was disconnected during the conditioning procedure to prevent deposition of contaminants on the c o l l e c t i n g r i n g . - 99 -APPENDIX F Operating Conditions for Gas Chromatography Column temperature: I n j e c t i o n port temperature: Detector temperature: C a r r i e r gas: A i r : Hydrogen: Program mode: Range: 200 C (neutral sugars) 2A0°C (amino sugars) set 25° above column temperature set 25° above column temperature N 2 = 25 ml/min, AO p s i with a u x i l i a r y flow = 35 ml/min 500 ml/min, AO p s i AO ml/min, 20 p s i isothermal 10"5 (signal fed through integrator) Integrator settings Noise suppression: maximum Slope s e n s i t i v i t y up: 0.03 mv/min down: 0.03 mv/min Baseline reset delay: zero Area threshold: 1000 Shoulder co n t r o l front: on rear: 1000 mv - 100 -BIBLIOGRAPHY Abdel-Akher, M., Hamilton, J.K. and Smith, F. (1951) The reduction of sugars with sodium borohydride. J . Am. Chem. Soc. 73, A691-A692. Alhadeff, J.A. 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