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Effect of O-Sulphate Groups in Lactose and N-Acetylneuraminyl-Lactose on Their Enzymic Hydrolysis by Nasi MIAN, Caroline E

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Effect of O-Sulphate Groups in Lactose and N-Acetylneuraminyl-Lactose on Their Enzymic Hydrolysis by Nasi MIAN, Caroline E Biochem. J. (1979) 181, 387-399 387 Printed in Great Britain Effect of O-Sulphate Groups in Lactose and N-Acetylneuraminyl-lactose on their Enzymic Hydrolysis By Nasi MIAN, Caroline E. ANDERSON and Paul W. KENT Glycoprotein Research Unit, Science Laboratories, University of Durham, Durham DH1 3LE, U.K. (Received 18 December 1978) 1. Lactose 6'-0-sulphate, N-acetylneuraminyl-(a2->3)-D-lactose 6'-0-sulphate, N-acetyl- neuraminyl ?-O-sulphate-(a2-+3)-D-lactose 6'-0-sulphate, N-acetylneuraminyl ?-O-sul- phate-(a2-÷6)-D-lactose and N-acetylneuraminyl-(a2->3)- and -(a2->6)-lactose 6'-O-sul- phate were prepared by chemical sulphation of lactose, N-acetylneuraminyl-lactose and its isomers by using pyridine-SO3 reagent. 2. Significant kinetic differences were observed in the enzymic hydrolysis of the sulphated derivatives compared with unsubstituted sub- strates. 3. In the case of reactions catalysed by rat liver lysosomal and Clostridium perfrin- gens neuraminidases (EC 3.2.1.18), the presence of an 0-sulphate group in the N-acetyl- neuraminyl moiety affected the reaction by decreasing the Km and the Vmax. its presence in the galactosyl moiety affected the reaction by decreasing the Km and increasing the Vmax. and its presence in both N-acetylneuraminyl and galactosyl moieties decreased the Km and the Vmax. of the reaction. 4. Mixed-substrate reaction kinetic data indicated competi- tion between the sulphated and unsubstituted substrates for the same active sites on the neuraminidase molecule. 5. Lactose 6'-O-sulphate neither behaved as a substrate nor acted as an inhibitor with respect to unsubstituted lactose and p-nitrophenyl fi-D-galacto- pyranoside when tested with lactase of suckling rat intestine and Escherichia coli fJ-D- galactosidase (EC 3.2.1.23). 6. Preliminary investigation also indicated that, whereas glucose 6-0-sulphate and glucose 3-0-sulphate were neither substrate nor inhibitor of glucose oxidase (EC 1.1.3.4), galactose 6-0-sulphate was oxidized half as fast as unsub- stituted galactose by galactose dehydrogenase (EC 1.1. 1.48). For a compound to be cleaved by neuraminidase Bearing in mind the resistance of compounds the presence of unsubstituted carboxy groups on the towards neuraminidase action owing to the presence N-acetylneuraminic acid is of great importance of substituents on the N-acetylneuraminic acid mole- (Gottschalk, 1962; Yu & Ledeen, 1969). Bacterial cule and the inhibition of the enzyme activity in the and viral neuraminidases recognize substituents on presence of chemically sulphated sialoglycopeptides, the N-acetylneuraminic acid molecule. The resistance it was considered important to chemically sulphate to hydrolysis by neuraminidase of substances some simple low-molecular-weight substrates and to containing the substituted N-acetylneuraminic acid compare their enzymic hydrolysis reactions with the molecule have been a subject of great interest from unsubstituted compounds. To this end, two enzyme the point of view of specificity of enzyme action systems, neuraminidases (EC 3.2.1.18) from Clostri- (for review see Drzeniek, 1973). In addition, viral dium perfringens and rat liver lysosomes and lactase neuraminidases specifically hydrolyse the different (EC 3.2.1.23) from suckling rat intestine and Escher- types of a-ketosidic linkage between N-acetyl- ichia coli ,B-galactosidase were chosen and chemically neuraminic acid and adjacent carbohydrate mole- sulphated derivatives of N-acetylneuraminyl-lactose cules. The nature of this carbohydrate is of minor and lactose were prepared. In the present paper, we importance for the recognition by these enzymes, report a comparative kinetic study on the action of but large substituents on these carbohydrates make these enzymes on their sulphated and unsubstituted such compounds resistant towards bacterial and substrates and also discuss the biological role of viral neuraminidases owing to steric hindrance 0-sulphate ester groups. (Drzeniek, 1973). However, we have observed that the chemically sulphated sialoglycopeptides inhibited Experimental the hydrolysis of N-acetylneuraminic acid from N-acetylneuraminyl-lactose, bovine submaxillary- Chemical estimations gland mucin and from the native sialoglycopeptides The 2-thiobarbituric acid method of Warren (1959) by steric hindrance (Mian et al., 1979). was used for the estimation of free N-acetylneura- Vol. 181 388 N. MIAN, C. E. ANDERSON AND P. W. KENT minic acid. The resorcinol method of Svennerholm Authentic glucose, galactose and lactose were (1957) was used for the determination of total free eluted with water, whereas N-acetylneuraminic acid and bound N-acetylneuraminic acid. Lactose was was eluted with 0.3 M-formic acid. Galactose 6-0-sul- determined by the anthrone-reagent method des- phate, glucose 6-0-sulphate, glucose 3-0-sulphate cribed by Cook (1976). Glucose was determined by were eluted with 4.5 M-formic acid, whereas N-acetyl- glucose oxidase reaction by using glucose-test- neuraminic acid 0-sulphate was eluted with 5.OM- combination kit (Boehringer) based on the method formic acid. of Werner et al. (1970). Galactose was estimated by galactose dehydrogenase according to the method Chemical sulphation ofcarbohydrates described by Asp & Dahlqvist (1972). Sulphation of carbohydrates was carried out by The sulphate content of the samples after hydroly- using the pyridine-SO3 reagent (Aldrich Chemical sis in 25 % (v/v) formic acid for 24h at 100°C was Co., Gillingham, Dorset, U.K.) by a modification of determined by the method of Mende & Whitney the method of Lloyd (1960). Carbohydrates were (1978), which involved precipitation of the inorganic dissolved in anhydrous pyridine at 60°C, excess of sulphate on cellulose thin-layer plates with 133BaC12. the pyridine-SO3 was added and the reaction was maintained at 37°C for 12 or 36h with constant Paper chromatography shaking. The reaction mixture was then kept at room temperature for 3 h, cooled to 4°C and then an ice- Descending chromatography on Whatman no. 1 cold suspension of Ba(OH)2 was added with constant paper was performed with solvent system I (ethyl stirring over a period of 10min. After centrifugation acetate/pyridine/water, 10:5:6 by vol.), system II at 5000g at 4°C for 15 min, the sediment was discarded (n-butanol/n-propanol/0.1M-HCl, 1:2:1 by vol.), and solid CO2 was added to the supernatant liquid system III (ethyl acetate/pyridine/acetic acid/water, and centrifugation was repeated. The resulting 5 :5:1 :3 by vol.), and system IV (butan-l-ol/ethanol/ supernatant was concentrated by rotary evaporation water, 3 :1 :1 by vol.) containing 3 % (w/v) cetylpy- at 30°C with repeated additions of water to remove ridinium chloride. The chromatograms were de- excess pyridine and finally concentrated to 4ml. The veloped following a dip in ethanolic NaOH/AgNO3 sulphated products from unreacted carbohydrates (for reducing compounds) or with p-dimethylamino- were separated by repeated ion-exchange chroma- benzaldehyde spray (for N-acetylneuraminic acid). tography on Dowex 1 (X8; formate form). Carbo- hydrates present in the eluted fractions throughout were detected by using anthrone reagent for sulphated Ion-exchange chromatography or unchanged hexoses and resorcinol reagent for The authentic carbohydrate samples were run N-acetylneuraminic acid or its sulphated forms. individually on ion-exchange columns (Dowex 1; X8; Eluted fractions were freed from pyridine/formate by x 1 rotary evaporation at 30°C and concentrated to 1 ml. formate form; 200-400 mesh; 25cm cm) and These were converted salts eluted with a gradual continuous gradient of 0 to finally into potassium by 5.0M-formic acid. Similarly in every preliminary passage through Dowex 5OW (H+ form) followed by run, the sulphation reaction mixture was passed neutralization with KHCO3 (0.1 M). Samples were through an ion-exchange column and eluted with a then concentrated to dryness at room temperature gradual continuous gradient of 0 to 5.0M-pyridine/ in vacuo over P205. formate solution pH4.4 to assess approximately the gradient concentrations that eluted the carbohydrate Identification of 0-sulphate esters in sulphated test-positive materials. In the subsequent ion- substrates exchange chromatography, the columns after being Sulphated sugars used in the present work, i.e. loaded with the reaction mixture were washed with glucose 3-0-sulphate, glucose 6-0-sulphate and water and then eluted with a stepwise gradient galactose 6-0-sulphate (Lloyd, 1960) were all as ranging between minimum and maximum concen- potassium salts. Di- and tri-saccharide sulphates were trations of pyridine/formate required to elute the hydrolysed in 0.1 M-HCI for 90min at 100°C (Ryan carbohydrate material, as assessed from the pre- et al., 1965). Hydrolysis under these conditions liminary runs. This procedure minimized the time yielded monosaccharides and monosaccharide 0-sul- taken for ion-exchange chromatography and im- phates from di- or tri-saccharide derivatives. The proved the recovery of the added material from the hydrolysates werecooled in an ice bath and dried in the columns. The material recovered from these columns presence of NaOH pellets over silica gel in an evacu- was subjected to rechromatography on Dowex ated desiccator. The hydrolysis products were 1 (X8) and was eluted either by using a gradual examined in comparison with the reference authentic continuous gradient or by a narrow-range stepwise compounds by using analytical paper and ion- gradient. exchange chromatography. Samples were loaded on 1979 ENZYMIC DEGRADATION OF O-SULPHATED OLIGOSACCHARIDES 389 Dowex 1 (X8; formate form) columns (25cm x 1 cm) (a) and eluted with water followed by a linear gradient of 4.0 0 to 5.5M-formate. The eluted fractions were freed [I from pyridine/formate and concentrated by rotary evaporation. These were examined chemically, (b) enzymically and by analytical paper chromatography (c) 3.0 _ for the presence of glucose, galactose, N-acetyl- (d) neuraminic acid and their sulphated derivatives. The molar ratios of sulphate to monosaccharides were calculated from their total sulphate contents.
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