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J. gen. ViroL (I977), 37, 625-628 625 Printed in Great Britain The Amino Acid and Carbohydrate Composition of the Neuraminidase of B/Lee/40 Influenza Virus (Accepted 18 July I977) SUMMARY The neuraminidase of B/Lee/4o influenza virus was isolated following detergent dissociation or tryptic digestion of virus particles and the amino acid and carbohydrate compositions of the two preparations are reported. The results indicate that the carbohydrate side-chains of the neuraminidase contain only N-acetylglucosamine, galactose, mannose and fucose, that they are attached by N-acetylglucosamine-asparagine linkages, and that the molecular weights of the neuraminidase subunit and the intact molecule are about 7oooo and 28oooo, respectively. The results also suggest that more than 50 % of the carbohydrate is attached to the membrane-associated 'stalk' of the molecule and that in this 'stalk' region the subunits are linked by disulphide bonds. The membranes of influenza viruses contain two readily distinguishable glycoproteins a haemagglutinin and a neuraminidase. This is a report of the chemical composition of the latter protein, isolated from the B]Lee[4o strain. It has been shown previously that enzymically active neuraminidase molecules can be isolated from viruses of this strain by either detergent disruption of the membrane (Laver, i963) or by trypsinization of intact virus particles (Noll, Aoyagi & Orlando, i962 ) and a comparison of the size and shape of the components isolated by both procedures has been reported (Wrigley et aL 1973). Here, the results of analyses of the amino acid and carbohydrate composition of the enzyme are presented and, again, the properties of the proteins isolated following either detergent treatment or proteolysis of virus particles are compared. The virus was propagated in the allantoic cavity of Io-day-old hens' eggs and purified as described by Skehel & Schild 097I). Neuraminidase was prepared by digesting virus particles (5 mg/ml) in PBS, pH 7"2, with trypsin (I mg/ml) and purified as described by Noll et al. 0962) and Wrigley et al. (I973), or by dissociating virus particles in sodium dodecyl sulphate (2 %) and separating the proteins by electrophoresis on cellulose acetate as described by Laver (r963). The polypeptide composition of the two proteins was determined by polyacrylamide gel electrophoresis and the results are shown in Fig. I. For comparison the polypeptide components of purified virus particles were analysed in parallel. Both neuraminidase preparations contained only one polypeptide of tool. wt. about 70000, in the case of the detergent solubilized protein, and 48000, in the case of the trypsin-released enzyme. Also shown in the figure are the results of analyses of samples dissociated in SDS and urea but not reduced by the addition of fl-mercaptoethanol. It is notable that under these conditions the detergent-released protein has an apparent tool. wt. of about I4OOOOwhereas the trypsin-released protein has an apparent mol. wt. of 45 ooo. Analyses of the two neuraminidase preparations for amino acids and amino sugars were done on a Locarte Mini analyser and details of the elution systems used are given elsewhere (Allen & Neuberger, I973). Their neutral sugar compositions were estimated by gas chromatography after methanolysis and trimethylsilylation (Chambers & Clamp, I97I). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 12:30:39 626 Short communications A B C D HA PD P2 NA' NP~ HA1 HA2 MP Fig. I. The polypeptide components of influenza B virus and the purified neuraminidase preparations. Samples were dissociated at Ioo °C for 2 min in solutions of 8 M-urea and 2 ~oo SDS with or without o'z ~ fl-mercaptoethanol. Following dissociation the polypeptides were separated by electrophoresis at 5 V/cm for 16 h on gels containing I5 ~ acrylamide and the buffers described by Laemmli 0970). The figure shows A and D, the polypeptide components of virus particles; B and E, the polypeptides of SDS-solubilized neuraminidase; and C and F, the polypeptides of trypsinreleased neuraminidase. Samples A, B and C were dissociated in SDS, urea and fl-mercaptoethanol, samples D, E and F in SDS and urea alone. A similar nomenclature to that used for the polypeptides of type A influenza viruses is employed (Skehel, I972): P1 and P2, the two largest polypeptides; NA, neuraminidase; NP, nucleoprotein; HA, unreduced haemagglutinin; HA1 and HA2, the two glycopolypeptidecomponents of the haemagglutinin; MP, matrix protein. Sugar analyses were related quantitatively to the amino acid analyses by adding internal standards (mannitol for sugars and p-fluorophenylalanine for amino acids) to samples from the same stock solution of glycoprotein. The results are given in Table ~. Since the trypsin-released neuraminidase contains a lower proportion of carbohydrate than does the intact molecule the tool. wt. estimate of the former is taken as being more reliable and the calculations of the compositions are based on that value (Wrigley et al. I973). They are also based on an assumption that the trypsin-released enzyme contains all the isoleucine and tyrosine residues of the whole protein. This gives the minimum possible difference in composition and also indicates that if the mol. wt. of 48 ooo is correct for the subunit of the trypsin-released enzyme then the minimum tool. wt. of the intact neuraminidase subunit is 680o0 and that of the intact protein is about 280000. Both preparations of neuraminidase were found to contain only four types of sugar residue- N-acetyi glucosamine, mannose, galactose and f u c o s e - and in this respect are like the other influenza virus glycoprotein, the haemagglutinin (Ward & Dopheide, I976, and unpublished results). Of the amino acids present, only asparagine, serine, threonine Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 12:30:39 Short communications 62 7 Table I. Amino acid and carbohydrate composition of the neuraminidase* SDS-solubilized Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan Total amino acids N-acetyl glucosamine Fucose Mannose Galactose Total carbohydrate Molecular weights 53 43 50 52 28 57 36 27 27 13 25 32 15 r5 2o 27 2I 8 549 17 5 16 II 49 68 ooo Trypsin released 38 28 32 37 2I 44 26 15 I7 Io 25 24 15 II 13 22 I8 5 4oi 8 I I2 3 24 48 ooo Difference 15 I5 18 I5 7 I3 Io 12 Io 3 o 8 o 4 7 5 3 3 I48 9 4 4 8 25 2o ooo * Samples of protein were hydrolysed for 24, 48 and 72 h in constant-boiling HC1 at IIO °C ill vacuo. Values for the quantities of the individual amino acids present were derived from an average of the three hydrolyses, with the exception of threonine and serine, which were extrapolated to zero time and valine and isoleucine for which the values from the 72 h hydrolyses were taken. Halfcystine and methionine values were obtained from the analyses of cysteic acid and methionine sulphone residues resulting from the performic acid oxidation (Hirs, 1967) and subsequent hydrolysis of the protein for 24 h at I Io °C in constantboiling HC1. Tryptophan was determined after hydrolysis of the proteins in 3 N-p-toluene sulphonic acid at Iio°C for 24 h and correction factors were used for its destruction in the presence of carbohydrate (Liu, 1972). Glucosamine was determined on the analyser after hydrolysis of the glycoprotein in 3 N-ptoluene sulphonic acid at Ioo °C for 24 h (Allen & Neuberger, 1973). Neutral sugars were determined by gas chromatography as described by Chambers & Clamp 097I). a n d cysteine are likely to be covalently b o u n d to carbohydrate. However, following solution in a n d dialysis against o'5 N - N a O H for 48 h at 4 °C, n o change in the carbohydrate compositions of the proteins could be detected. Since O-glycosidic linkages involving the hydroxyl groups of serine a n d threonine are k n o w n to be generally alkali-labile by a process of fl-elimination (reviewed by Marshall & Neuberger, I97o ) a n d this p r o b a b l y also applies to S-glycosidic linkages with cysteine, it is unlikely that these a m i n o acids are associated with the carbohydrate chains. This conclusion is reinforced by the absence of N-acetylgalactosamine which is often involved in linkages with serine a n d threonine. It is, therefore, p r o b a b l e that all the carbohydrate of the n e u r a m i n i d a s e is attached through the alkalistable N-acetylglucosamine-asparagine linkage. In different preparations variable a m o u n t s of glucose were f o u n d which are p r o b a b l y due to c o n t a m i n a t i o n of the samples b y celogel or sucrose a n d so this sugar is n o t included in the table of compositions. The possibility c a n n o t , however, be excluded that some of the glucose is covalently b o u n d . Finally, if it is assumed that the differences in composition a n d properties of the two n e u r a m i n i d a s e preparations reflect the c o m p o s i t i o n a n d properties of the m e m b r a n e Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 12:30:39 628 Short communications associated ' s t a l k ' of the intact molecule, two features of this region are worth noting. Firstly, more t h a n 5o ~ of the sugar residues of the intact molecule are associated with the ' s t a l k ' , a finding in agreement with the radiochemical data of Lazdins, Haslam & White 0972), a n d secondly, it appears from the results of the polyacrylamide gel electrophoretic analyses of reduced a n d n o n - r e d u c e d p r e p a r a t i o n s that in the ' s t a l k ' region the subunits are linked by interchain disulphide bonds. W e t h a n k D a v i d Stevens for excellent assistance. Division of Virology National Institute for Medical Research The Ridgeway, Mill Hill London N W 7 I A A ANTONY K. ALLEN* JOHN J. SKEHEL VADIM YUFEROV~" REFERENCES ALLEN, A. K. & NEUBERGER,A. (t973). The purification and properties of the lectin from potato tubers, a hydroxyproline-containingglycoprotein. The Biochemical Journal x35, 3o7-3 I4. CHAMBERS,R. E. & CLAMP,J. R. 0971). An assessment of methanolysis and other factors used in the analysis of carbohydrate-containing materials. The Biochemical Journal I25, Ioo9-IOI8. HIRS, C. ft. W. (I967). Determination of cystine as cysteic acid. Methods in Enzymology xI, 59-62. LAEMMLI,U. K. 097O). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London, ~27, 680-685. LAVER, W. G. (I963). The structure of influenza viruses. 3. Disruption of the virus particle and separation of neuraminidase activity. Virology zo, 251-262. LAZDINS, I., HASLAM, E. A. & WHITE, D. O. (1972). The polypeptides of influenza virus. VI. Composition of the neuraminidase. Virology 49, 758-765. LIU, T.-Y. (I972). Determination of tryptophan. Methods in Enzymologyz5, 44-55. MARSHALL,R. D. &NEUBERGER,A. (I970). Aspects of the structure and metabolism of glycoproteins. Advances in Carbohydrate Chemistry and Biochemistry uS, 407-478. NOLL, H., AOYAGI, T. & ORLANDO, J. (I962). The structural relationship of sialidase to the influenza virus surface. Virology I8, I54-I57. SKEHEL,J. J. (t97Z). Polypeptide synthesis of influenza virus infected cells. Virology 49, 23-36. SKEHEL, J. J. & SCHILD, G. C. (I97I). The polypeptide composition of influenza A viruses. Virology 44, 396-4o8. WARD, C. W. & DOPHEIDE,T. A. A. 0976). Size and chemical composition of influenza virus haemagglutinin chains. FEBS Letters 65, 365-368. WRIGLEY, N. G., SKEHEL,J. J., CHARLWOOD, P. A. & BRAND, C. M. (1973). The size and shape of influenza virus neuraminidase. Virology 5x, 525-529. (Received 3 June 1977) * Present address: Department of Biochemistry, The Charing Cross Hospital Medical School, Hammersmith, London W6. t Present address: Ivanovsky Institute, Ivanovsky Institute of Virology, U.S.S.R. Academy of Medical Sciences, Moscow D-98, U.S.S.R. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 03 May 2017 12:30:39