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J. gen. ViroL (I977), 37, 625-628
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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).
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626
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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
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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
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628
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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~"
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(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.
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