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Rat α1 macroglobulin inhibits hemagglutination by influenza C virus
183
Virus Research, 2 (1985) 183-192 Elsevier
VRR 00175
Rat cylmacroglobulin inhibits hemagglutination influenza C virus G. Herrler
I**, R. Geyer
2, H.-P.
Miiller
3, S. Stirm
2 and H.-D.
Klenk
by
’
’ Institut ftir Virologie, 2 Biochemisches Institut, and ’ Institut ftirBakteriologie und Immunologic, Justus - Liebig - Universitijt Giessen, D - 6300 Giessen, F. R. G. (Accepted
5 December
1984)
Purified cu,-macroglobulin (RMG) isolated from rat plasma was found to be a potent inhibitor of hemagglutination by influenza C virus. Neuraminidase treatment of purified RMG reduced its inhibitory activity by more than 80% indicating that sialic acid is required for maximal HI-activity. The inhibitory activity of RMG was shown to be sensitive to the receptor-destroying activity (RDA) of influenza C virus. Methylation analysis of the glycopeptides of RMG indicated the presence of only one major type of oligosaccharide which is a complex N-linked oligosaccharide with a biantennary structure. Comparison of the glycopeptides before and after neuraminidase treatment revealed that the oligosaccharides are terminated by sialic acid residues attached to galactose residues at position C-6. Methylation analysis was also performed on RMG which had lost its inhibitory activity upon incubation with RDA of influenza C virus. No difference between the glycopeptides of native and inactive RMG could be detected. Galactose was found to be substituted at position C-6 in both samples, indicating that also the oligosaccharides of inactive RMG are terminated by sialic acid. The implications of these results are discussed. influenza C virus, cy,-macroglobulin, glutination inhibitor
rat plasma,
neuraminic
acid, sialic acid, hemag-
Introduction Neuraminic acid bound to glycoproteins or glycolipids is known to be an essential component of the cellular receptor for influenza A and B viruses as well as for
* To whom reprint 0168-1702/85/$03.30
requests
should be addressed.
0 1985 Elsevier Science Publishers
B.V. (Biomedical
Division)
184 paramyxoviruses. These viruses contain a neuraminidase which enables them to destroy their own receptor. The cellular receptor for influenza C virus has been suggested to be different from neuraminic acid, mainly for two reasons: (i) A receptor-destroying activity (RDA) has been reported to be associated with influenza C virus (Hirst, 1950), but no evidence for the presence of a neuraminidase could be obtained (Kendal, 1975). (ii) The cellular receptors for influenza C virus have been reported to be resistant to the action of neuraminidases from both viral and bacterial origin (Hirst, 1950; Kendal, 1975). Recently, we found, however, that under appropriate conditions neuraminidases from both Clostridium perfringens and Vibrio cholerae (VC) are able to destroy the cellular receptor for influenza C virus on chicken red blood cells (Herrler et al., 1984). Rat serum which is known to be a potent inhibitor of hemagglutination by influenza C virus (Styk, 1955), also loses its activity upon incubation with bacterial neuraminidases (Herrler et al., 1984). These results indicate that neuraminic acid may be involved in binding of influenza C virus to cells. Elucidation of the cellular receptor for influenza C virus is hampered by the lack of a well-defined compound which is able to inhibit binding of influenza C virus to cells. Therefore we attempted to purify the hemagglutination-inhibitor present in rat serum. In the present communication we show that a,-macroglobulin (RMG) isolated from rat plasma is the major inhibitory compound. We determined how the inhibitory activity of RMG is affected by incubation with VC-neuraminidase and by treatment with RDA associated with influenza C virus, respectively. A carbohydrate analysis of the glycopeptides of RMG is presented.
Materials and Methods Virus
Strain Johannesburg (JHB)/1/66 of influenza C virus was grown in embryonated eggs as described previously (Herrler, et al., 1979). Purification
of RMG
In rat plasma two kinds of macroglobulin with similar physicochemical properties have been demonstrated. While cY,-macroglobulin is present in normal plasma, a,-macroglobulin is detectable in significant concentrations only after injuries or during inflammation. Rat a,-macroglobulin (RMG) was purified following the protocol for the purification of human cY,-macroglobulin (van Leuven et al., 1980) which is the only macroglobulin present in human plasma and analogous to RMG. Briefly, the purification steps consisted of: (i) treatment with BaCl, and BaSO,; (ii) precipitation between 4 and 8% of polyethyleneglycol 6000; (iii) molecular sieve chromatography on Ultrogel ACA 22 (LKB, Sweden); and (iv) affinity chromatography on Fractogel TSK AF-Blue (Merck, Darmstadt, F.R.G.) instead of Blue-Sepharose. Assay for RMG
RMG was assayed by its ability
to bind trypsin.
Trypsin
bound
to macroglobulin
185 is still able to cleave small synthetic substrates but is not accessible to soybean trypsin inhibitor. The method described by Ganrot (1966) was modified for use in microtiter plates. N-benzoyl-L-arginyl-4-nitroanilide (Merck, Darmstadt, F.R.G.) was used as chromogenic substrate. Cleavage of the substrate by trypsin-amidase (TA) activity was monitored by measurement of the optical density at 405 nm. Assay for hemagglutination
inhibition (HI) activity
Samples to be analysed for HI activity were diluted with PBS in serial twofold steps. To 50 ~1 of each dilution an equal volume of diluted allantoic fluid containing 4 HA-units of influenza C virus as well as 50 ~1 of a 1% suspension of chicken erythrocytes were added. After incubation at 4°C for 60 min the HI-titers of the samples were determined as the reciprocal values of the dilution causing 50% hemagglutination inhibition. Neuraminidase
treatment of RMG
Purified RMG containing 40 pg or absence of 20 sample contained to neuraminidase. Incubation
was dissolved in 0.02 M CaCl,/O.l M MES, pH 6.5. Samples of RMG in a final volume of 70 ~1 were incubated in the presence mU of VC-neuraminidase (Behringwerke, Marburg, F.R.G.). One lop3 M, 2,3-dehydro-2-deoxynueraminic acid (DDN) in addition After 90 min at 37’C, the HI-titer of each sample was determined.
of RMG with influenza C virus
Influenza C virus was pelleted from 5 ml of allantoic fluid by centrifugation in a SW55 TiA rotor at 35000 rpm for 30 min. Virus obtained in the sediment was resuspended in 200 ~1 of PBS lacking Ca*+- and Mg*+-ions. The HA titer of this suspension was determined to be 213 HA units/ml. RMG (1 mg) dissolved in 1.5 ml of phosphate buffer (0.05 M, pH 7.4) was kept on ice and mixed with 50 ~1 of the concentrated influenza C virus preparation. At various times aliquots of 300 ~1 were taken and incubated at 37°C. At the end of the incubation time samples were put on ice and diluted to a volume of 0.7 ml with ice-cold PBS. Virions were sedimented by centrifugation as described above and the HI-activity of the supernatants was determined. SDS-polyacrylamide
gel electrophoresis
Electrophoretic analysis of RMG was performed using 5-15s gradient gels and the Tris-glycine buffer systems described by Laemmli (1970). Samples were prepared for electrophoresis by mixing with an equal volume of sample buffer at room temperature. Reducing agents were omitted. Preparation
of glycopeptides
of RMG
RMG dissolved in phosphate buffer was dialysed against a buffer containing 0.02 M CaCl,/O.l M Tris-HCl, pH 7.8. Glycopeptides were prepared by digestion of 1 mg of RMG in 500 ~1 of Tris-buffer with 1 mg of pronase E (Serva, Heidelberg, F.R.G.) which had been predigested for 2 h at 37°C. After incubation at 37°C for 24 h the digested sample was desalted on a Biogel P2 column (0.5 cm X 100 cm). The
186
glycopeptides were eluted with distilled water and collected from the void volume fractions as monitored by measurement of the optical density at 280 nm. Methylation analysis Isolated glycopeptides were permethylated according to Hakomori (1964). The partially methylated alditol acetates obtained after hydrolysis etc. were analyzed by capillary gas chromatography-mass fragmentography as detailed previously (Jansson et al., 1976; Geyer et al., 1983).
Results Purification of rat a,-macroglobulin It has been shown previously that upon molecular sieve chromatography the HI activity of rat serum elutes faster than the IgG fraction (O’Callaghan et al., 1980). This finding in connection with the high HI titers of up to 5 X lo4 HI-units/ml of rat serum (Styk, 1955) indicated that a major high molecular weight component, such as RMGz is responsible for the inhibitory activity. Therefore, we analysed whether RMG is an HI inhibitor.
RMG
60
100
120 Fraction
I
number
Fig. 1. Molecular sieve chromatography of the polyethylene gfycol concentration from Fractions of 2.5 ml were collected and 0), and trypsin-amidase activity (OFig. 2. SDS-polyacrylamide
160
rat plasma proteins. Plasma proteins precipitated by raising 4% to 8% were applied on a column of Ultrogel ACA 22. analyzed for protein content (A280, -), HI-activity (TA, l 0).
gel electrophoresis
of purified
RMG.
187 Following protocols for the isolation of a,-macroglobulin from human plasma, RMG was purified from rat plasma. RMG is precipitated from plasma by raising the concentration of PEG from 4% to 8%. This precipitate contained 95% of the HI-activity. Further purification was achieved by molecular sieve chromatography. As shown in Fig. 1, measurement of the optical density at 280 nm revealed two protein peaks. The smaller of both peaks (fractions 95-108) contained all the RMG as determined by its ability to bind trypsin (see OD 405 nm). The majority of the HI-activity was also found in these fractions. A minor peak of the HI-activity was observed at fractions 115-125. After a final chromatography on Fractogel TSK AF-Blue, purified RMG was obtained in the run through. Analysis of purified RMG by SDS-polyacrylamide gel electrophoresis is shown in Fig. 2. Only one major band is present which represents RMG as indicated by comigration with human a,-macroglobulin (not shown). A faint band is visible at the top of the gel. Whether this protein is a contaminant or an aggregate of RMG is not clear. For purified RMG a HI-activity of about 2” HI-units/mg p rotein was determined. Because of the high HI activity of this preparation it is unlikely that a minor contaminant is responsible for the inhibitory activity. Effect of neuraminidase on the HI activity of RMG Recently we have shown that the HI activity of rat serum is sensitive to the action of bacterial neuraminidases (Herrler et al., 1984). Having a purified inhibitor available we wanted to know whether neuraminic acid is also essential for HI activity of RMG. Purified RMG was incubated at 37°C in the presence or absence of VC-neuraminidase. After 90 min the HI-titer of the samples was determined. The result is shown in Table 1. Incubation with VC-neuraminidase reduced the HI activity by more than 80%. In the presence of the neuraminidase inhibitor DDN no reduction was observed. The results from Table 1 indicate that sialic acid is essential for optimal HI activity of RMG. Effect of RDA of influenza C virus on the HI activity of RMG A receptor-destroying activity has been shown to be associated with influenza C virus. We analysed whether or not this activity is also effective using RMG as substrate. Purified RMG was mixed with a concentrated preparation of influenza C virus and incubated at 37°C for various times. After incubation virions were removed by ultracentrifugation. The supernatant was assayed for HI activity. As shown in Fig. 3 with increasing incubation time at 37°C the HI activity of the supernatant decreased reaching 6% after 60 min. This result indicates that the RDA TABLE
1
Effect of VC-neuraminidase
on the HI activity
of purified
RMG
Enzyme present
HI-units/ml
None VC-neuraminidase VC-neuraminidase
4800 900 4800
+ lo- 3 M DDN
188
I
0
10
60
30 mcubation
time
(mm)
Fig. 3. Effect of RDA associated with influenza C virus on the HI activity of purified RMG. RMG was incubated with a concentrated preparation of influenza C virions for various times at 37°C. The residual HI activity was determined as described under Materials and Methods.
associated with influenza C virus is able to destroy the HI activity of RMG which therefore should be an useful substrate for the elucidation of the receptor-destroying activity. Methylation analysis of the glycopeptides of RMG The neuraminidase sensitivity of the HI activity of RMG suggests that the carbohydrate moiety of RMG is important for the inhibitory activity. Therefore we analyzed the structure of the oligosaccharides of RMG. The glycopeptides obtained TABLE
2
Methylation Peracetate
analysis of a
of glycopeptides
from RMG
Peak ratio b obtained from glycopeptides of RMG after treatment with: Influenza
2,3,4,6-ManOH 3,4,6-ManOH 3,6-ManOH 2,4-ManOH
0.25
’
2,3,4,6-GalOH 2,4,6-GalOH 2,3,4-GalOH 3,4,6-GlcN(Me)AcOH 3,6_GlcN(Me)AcOH
&O trace 1,05 0.1 0.3 2.0 0.15 3.5
C virus
VC-neuraminidase
0,15 120
0.25 2,0
trace
trace
l,O
1s)
0.15 0.4 1.8 0.1 3.15
2.05 0.2 0.15 0.15 3.3
a 3,4,6-ManOH, 3,4,6-tri-0-methylmannitol; 3,4,6-GlcN(Me)AcOH, 2-deoxy-2-(N-methyl)acetamido3,4,6-tri-0-methylglucitol, etc. b Peak ratios are based on 3,4,6-ManOH peracetate integrals = 2.0 (underscored). ’ Corrected for losses during hydrolysis (Conchie et al., 1982). Glycopeptides from native RMG, from RMG which had lost its HI-activity upon incubation with influenza C virus, and VC-neuraminidase-treated glycopeptides from native RMG were subjected to methylation analysis as described under Materials and Methods.
189 SA-2%6-Gal-lk-GlcNAc-l-L02-Mm-1
SA-2
*6-Gal-l
~L-Glct4Ac-l&2-Mm-l
\
a
OL /6
;Man-l~L-GlcNAc-l~I-GkNAc
Fig. 4. Proposed structure of the major oligosaccharide present on RMG. The structure is based on results obtained from methylation analyses of glycopeptides of RMG. The anomeric configuration of the linkage between individual sugars has not been determined, but has been inferred from other biantennary oligosaccharides (Montreuil, 1982; Vliegenthart et al., 1983).
from RMG after digestion with pronase were permethylated and analyzed by combined gas chromatography and mass spectrometry (GC/MS). The results presented in Table 2 are compatible with a biantennary oligosaccharide the structure of which is shown in Fig. 4. No evidence for the occurrence of fucose or of bisecting glucosamine was obtained. The absence of tri- or tetraantennary oligosaccharides is indicated by failure to detect 3,4_dimethylmannose and only trace amounts of 3,6dimethylmannose, respectively. Galactose residues were found to be substituted predominantly at position C-6 as evidenced by the occurrence of 2,3,4-trimethylgalactose residues. Only a minor fraction of galactose residues was substituted at position C-3. Treatment of the glycopeptides with VC-neuraminidase caused a decrease of the value of 2,3,4-trimethylgalactose by more than 90% and a concomitant increase of 2,3,4,6tetramethylgalactose. This result indicates that both branches of the biantennary oligosaccharide of RMG are terminated by sialic acid residues attached to galactose residues predominantly at position C-4. Glycopeptides of RMG, which had lost its HI activity due to the RDA of influenza C virus, were analyzed in the same way. As can be seen in Table 2, no major difference is detectable. A slight reduction of the value for 3,6-dimethyl-Nacetylglucosamine is probably within the range of experimental error, because no concomitant reduction of the value for 2,3,4-trimethylgalactose was observed. Galactose residues of RMG treated with influenza C virus were found to be substituted at position C-6, as in the case of native RMG, suggesting that sialic acid residues are not lost during the destruction of the HI-activity of RMG by influenza C virus. In conclusion, the destruction of the inhibitory activity of RMG by RDA of influenza C virus is not correlated with a change of the oligosaccharide structure detectable by this kind of analysis.
Discussion While a variety of compounds, including human and equine a,-macroglobulins, are known to inhibit hemagglutination caused by influenza A and B viruses (for a review see Gottschalk et al., 1972) only rat serum has been reported to be able to prevent influenza C virus from agglutinating chicken erythrocytes (Styk, 1955). We have purified a,-macroglobulin (RMG) from rat plasma and shown that this protein is a potent inhibitor of hemagglutination by influenza C virus, accounting for the
190 majority of the HI activity found in rat plasma. Furthermore, we have found that the inhibitory activity of RMG is sensitive to the receptor-destroying activity (RDA) of influenza C virus. Both findings suggest that there are some structural similarities between RMG and the cellular receptor for influenza C virus on chicken erythrocytes. Due to this structural similarity RMG not only serves as a substrate of the RDA of influenza C virus but it also competes with the cellular receptor for the viral hemagglutinin thus preventing influenza C virus from agglutinating chicken red blood cells. A characteristic feature of RMG, which may be required for the HI activity is the large size of this glycoprotein (about 7.4 X lo5 daltons). Glycopeptides obtained after pronase digestion of RMG are devoid of inhibitory activity (not shown). The importance of the molecular size of a glycoprotein for its ability to function as a hemagglutination inhibitor has been noted earlier for other influenza viruses (for review see Gottschalk et al., 1972). For instance, ovine submaxillary mucin loses its HI activity upon digestion with trypsin (Gottschalk and Fazekas de St. Groth, 1960). On the other hand, cr,-acid glycoprotein, which is a poor inhibitor of influenza A viruses, becomes a potent inhibitor after increasing its size either by crosslinking of individual molecules or by aggregation following heat treatment (Morawiecki and Lisowska, 1965; Whitehead, 1965). The HI activity of RMG has been found to be sensitive to the action of VC-neuraminidase. This result indicates that the sialic acid portion of RMG is important for the inhibitory activity. Methylation analysis of the glycopeptides of RMG revealed the presence of only one type of carbohydrate which is a complex N-linked oligosaccharide with a biantennary structure. Oligosaccharides of this type were reported to be present on both human and rabbit serum transferrin (Spik et al., 1975; Leger et al., 1978). Human as well as rabbit sera are unable to inhibit hemagglutination by influenza C virus (Styk, 1955). Therefore, it is reasonable to assume that human and rabbit serum transferrins are not inhibitors of influenza C virus, either. At present it is unknown whether these transferrins are inactive because of their smaller size (about 7 x lo4 daltons) compared to RMG or because of some additional structural requirements present on RMG but absent on transferrins. Our previous results suggested that sialic acid is involved in binding of influenza C virus to cells. We have proposed that the role of sialic acid as receptor for influenza C virus may depend on one or more of the following: (1) the nature of the adjacent sugar(s); (2) the type of linkage between neuraminic acid and the adjacent sugar residue; (3) modification of neuraminic acid, e.g. O-acetylation (Herrler et al., 1984). The results of the methylation analyses indicate that a modification of the neuraminic acid may be an important determinant for the HI activity. It remains to be seen whether the structure of the oligosaccharide core or the linkage of neuraminic acid to the core are important, too. On the basis of previous observations we have also speculated that the RDA of influenza C virus may be either an esterase that deacetylates neuraminic acid or an endoglycosidase removing a neuraminic acid-containing oligosaccharide, or a neuraminidase with an unusual substrate specificity. In the light of the data presented here an endoglycosidase can be ruled out because RDA of influenza C virus leaves
191 the core of the RMG-oligosaccharides intact. A neuraminidase activity would be compatible with our results only if it removes the terminal residue from an oligomeric form of neuraminic acid (a-NeuAc-(2-8)-aNeuAc). However, oligomeric neuraminic acid has not been found on plasma glycoproteins of the rat (Finne et al., 1977). Whether RDA of influenza C virus is an esterase, is the subject of current investigations.
Acknowledgements
The technical assistance of W. Mink and S. Kuhnhardt is gratefully acknowledged. We thank R. Rott for helpful discussions and Mrs. A. Becker for typing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 47, Virologie).
References Conchie, J., Hay, A.J. and Lomax, J.A. (1982) A comparison of some hydrolytic and gas chromatographic procedures used in methylation analysis of the carbohydrate units of glycopeptides. Carbohydr. Res. 103, 129-132. Finne, J., Krusius, T., Rauvala, H. and Hemminki, K. (1977) The disialosyl group of glycoproteins. Occurrence in different tissues and cellular membranes. Eur. J. Biochem. 77, 319-323. Ganrot, P.O. (1966) Determination of alpha-2-macroglobulin as trypsin-protein esterase. Clin. Chim. Acta 14,493-501. Geyer, R., Geyer, H., Kiihnhardt, S., Mink, W. and Stirm, S. (1983) Methylation analysis of complex carbohydrates in small amounts: Capillary gas chromatography-mass fragmentography of methylalditol acetates obtained from N-glycosidically linked glycoprotein oligosaccharides. Anal. B&hem. 133, 197-207. Gottschalk, A. and Fazekas de St. Groth, S. (1960) Studies on mucoproteins. III. The accessibility to trypsin of the susceptible bonds in ovine submaxillary gland mucoprotein. B&him. Biophys. Acta 43, 513-519. Gottschalk, A., Belyavin, G. and Biddle, F. (1972) Glycoproteins as influenza virus hemagglutinin inhibitors and as cellular virus receptors. In: Glycoproteins. Their Composition, Structure and Function (A. Gottschalk, ed.), pp. 1082-1096. Elsevier, Amsterdam. Hakomori, S. (1964) Rapid permethylation of glycolipid and polysaccharide catalyzed by methylsulfinylcarbanion in dimethyl sulfoxide. J. B&hem. 55, 205-211. Herrler, G., Compans,R.W. and Meier-Ewert, H. (1979) A precursor glycoprotein in influenza C virus. Virology 99, 49-56. Herrler, G., Rott, R. and Klenk, H.-D. (1985) Neuraminic acid is involved in the binding of influenza C virus to erythrocytes. Virology, in press. Hirst, G.K. (1950) The relationship of a new strain of virus to those of the mumps-NDV-influenza group. J. Exp. Med. 91,177-185. Jansson, P.E., Kenne, L., Liedgren, H., Lindberg, B. and Lbnngren, J. (1976) A practical guide to the methylation analysis of carbohydrates. Chem. Commun. Univ. Stockh. 8, l-75. Kendal, A.P. (1975) A comparison of ‘influenza c’ with prototype myxoviruses: Receptor-destroying activity (neuraminidase) and structural polypeptides. Virology 65, 87-99. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685.
192 Leger, D., Tordera, V., Spitz, G., Dorland, L., Haverkamp, J. and Vliegenthart. J.F.G. (1978) Structure determination of the single glycan of rabbit serotransferrin by methylation analysis and 360 MHz ‘H NMR spectroscopy. FEBS Lett. 93, 225-260. Montreuil, J. (1982) Glycoproteins. Compr. Biochem. 19B, (Part II), l-188. Morawiecki, A. and Lisowska, E. (1965) Polymerized orosomucoid an inhibitor of influenza virus hemagglutination. B&hem. Biophys. Res. Commun. 18, 606-610. O’Callaghan, R.J., Gohd, R.S. and Labat, D.D. (1980) Human antibody to influenza C virus: its age related distribution and distinction from receptor analogs. Infect, Immun. 30, 500-505. Spik. G., Bayard, B., Fournet, B., Strecker, G., Bouquelet, S. and Montreuil, J. (1975) Studies on glycoconjugates. LXIV. Complete structure of two carbohydrate units of human serotransferrin. FEBS Lett. 50, 296-299. Styk, B. (1955) Non-specific inhibitors in normal rat serum for the influenza C virus. Folia Biol. 1, 207-212. Van Leuven, F., Cassiman, J.J. and Van Den Berghe, H. (1980) Primary amines inhibit recycling of a2 M receptors in fibroblasts. Cell 20, 37-43. Vliegenthart, J.F.G., Dorland, L. and Van Halbeek, H. (1983) High-resolution ‘H-nuclear magnetic resonance spectroscopy as a tool in the structural analysis of carbohydrates related to glycoproteins. Adv. Carbohydr. Chem. Biochem. 41, 209-374. Whitehead, P.H. (1965) Viral inhibition by polymerized orosomucoid. Biochem. J. 95, 8p. (Manuscript
received
7 November
1984)
Virus Research, 2 (1985) 183-192 Elsevier
VRR 00175
Rat cylmacroglobulin inhibits hemagglutination influenza C virus G. Herrler
I**, R. Geyer
2, H.-P.
Miiller
3, S. Stirm
2 and H.-D.
Klenk
by
’
’ Institut ftir Virologie, 2 Biochemisches Institut, and ’ Institut ftirBakteriologie und Immunologic, Justus - Liebig - Universitijt Giessen, D - 6300 Giessen, F. R. G. (Accepted
5 December
1984)
Purified cu,-macroglobulin (RMG) isolated from rat plasma was found to be a potent inhibitor of hemagglutination by influenza C virus. Neuraminidase treatment of purified RMG reduced its inhibitory activity by more than 80% indicating that sialic acid is required for maximal HI-activity. The inhibitory activity of RMG was shown to be sensitive to the receptor-destroying activity (RDA) of influenza C virus. Methylation analysis of the glycopeptides of RMG indicated the presence of only one major type of oligosaccharide which is a complex N-linked oligosaccharide with a biantennary structure. Comparison of the glycopeptides before and after neuraminidase treatment revealed that the oligosaccharides are terminated by sialic acid residues attached to galactose residues at position C-6. Methylation analysis was also performed on RMG which had lost its inhibitory activity upon incubation with RDA of influenza C virus. No difference between the glycopeptides of native and inactive RMG could be detected. Galactose was found to be substituted at position C-6 in both samples, indicating that also the oligosaccharides of inactive RMG are terminated by sialic acid. The implications of these results are discussed. influenza C virus, cy,-macroglobulin, glutination inhibitor
rat plasma,
neuraminic
acid, sialic acid, hemag-
Introduction Neuraminic acid bound to glycoproteins or glycolipids is known to be an essential component of the cellular receptor for influenza A and B viruses as well as for
* To whom reprint 0168-1702/85/$03.30
requests
should be addressed.
0 1985 Elsevier Science Publishers
B.V. (Biomedical
Division)
184 paramyxoviruses. These viruses contain a neuraminidase which enables them to destroy their own receptor. The cellular receptor for influenza C virus has been suggested to be different from neuraminic acid, mainly for two reasons: (i) A receptor-destroying activity (RDA) has been reported to be associated with influenza C virus (Hirst, 1950), but no evidence for the presence of a neuraminidase could be obtained (Kendal, 1975). (ii) The cellular receptors for influenza C virus have been reported to be resistant to the action of neuraminidases from both viral and bacterial origin (Hirst, 1950; Kendal, 1975). Recently, we found, however, that under appropriate conditions neuraminidases from both Clostridium perfringens and Vibrio cholerae (VC) are able to destroy the cellular receptor for influenza C virus on chicken red blood cells (Herrler et al., 1984). Rat serum which is known to be a potent inhibitor of hemagglutination by influenza C virus (Styk, 1955), also loses its activity upon incubation with bacterial neuraminidases (Herrler et al., 1984). These results indicate that neuraminic acid may be involved in binding of influenza C virus to cells. Elucidation of the cellular receptor for influenza C virus is hampered by the lack of a well-defined compound which is able to inhibit binding of influenza C virus to cells. Therefore we attempted to purify the hemagglutination-inhibitor present in rat serum. In the present communication we show that a,-macroglobulin (RMG) isolated from rat plasma is the major inhibitory compound. We determined how the inhibitory activity of RMG is affected by incubation with VC-neuraminidase and by treatment with RDA associated with influenza C virus, respectively. A carbohydrate analysis of the glycopeptides of RMG is presented.
Materials and Methods Virus
Strain Johannesburg (JHB)/1/66 of influenza C virus was grown in embryonated eggs as described previously (Herrler, et al., 1979). Purification
of RMG
In rat plasma two kinds of macroglobulin with similar physicochemical properties have been demonstrated. While cY,-macroglobulin is present in normal plasma, a,-macroglobulin is detectable in significant concentrations only after injuries or during inflammation. Rat a,-macroglobulin (RMG) was purified following the protocol for the purification of human cY,-macroglobulin (van Leuven et al., 1980) which is the only macroglobulin present in human plasma and analogous to RMG. Briefly, the purification steps consisted of: (i) treatment with BaCl, and BaSO,; (ii) precipitation between 4 and 8% of polyethyleneglycol 6000; (iii) molecular sieve chromatography on Ultrogel ACA 22 (LKB, Sweden); and (iv) affinity chromatography on Fractogel TSK AF-Blue (Merck, Darmstadt, F.R.G.) instead of Blue-Sepharose. Assay for RMG
RMG was assayed by its ability
to bind trypsin.
Trypsin
bound
to macroglobulin
185 is still able to cleave small synthetic substrates but is not accessible to soybean trypsin inhibitor. The method described by Ganrot (1966) was modified for use in microtiter plates. N-benzoyl-L-arginyl-4-nitroanilide (Merck, Darmstadt, F.R.G.) was used as chromogenic substrate. Cleavage of the substrate by trypsin-amidase (TA) activity was monitored by measurement of the optical density at 405 nm. Assay for hemagglutination
inhibition (HI) activity
Samples to be analysed for HI activity were diluted with PBS in serial twofold steps. To 50 ~1 of each dilution an equal volume of diluted allantoic fluid containing 4 HA-units of influenza C virus as well as 50 ~1 of a 1% suspension of chicken erythrocytes were added. After incubation at 4°C for 60 min the HI-titers of the samples were determined as the reciprocal values of the dilution causing 50% hemagglutination inhibition. Neuraminidase
treatment of RMG
Purified RMG containing 40 pg or absence of 20 sample contained to neuraminidase. Incubation
was dissolved in 0.02 M CaCl,/O.l M MES, pH 6.5. Samples of RMG in a final volume of 70 ~1 were incubated in the presence mU of VC-neuraminidase (Behringwerke, Marburg, F.R.G.). One lop3 M, 2,3-dehydro-2-deoxynueraminic acid (DDN) in addition After 90 min at 37’C, the HI-titer of each sample was determined.
of RMG with influenza C virus
Influenza C virus was pelleted from 5 ml of allantoic fluid by centrifugation in a SW55 TiA rotor at 35000 rpm for 30 min. Virus obtained in the sediment was resuspended in 200 ~1 of PBS lacking Ca*+- and Mg*+-ions. The HA titer of this suspension was determined to be 213 HA units/ml. RMG (1 mg) dissolved in 1.5 ml of phosphate buffer (0.05 M, pH 7.4) was kept on ice and mixed with 50 ~1 of the concentrated influenza C virus preparation. At various times aliquots of 300 ~1 were taken and incubated at 37°C. At the end of the incubation time samples were put on ice and diluted to a volume of 0.7 ml with ice-cold PBS. Virions were sedimented by centrifugation as described above and the HI-activity of the supernatants was determined. SDS-polyacrylamide
gel electrophoresis
Electrophoretic analysis of RMG was performed using 5-15s gradient gels and the Tris-glycine buffer systems described by Laemmli (1970). Samples were prepared for electrophoresis by mixing with an equal volume of sample buffer at room temperature. Reducing agents were omitted. Preparation
of glycopeptides
of RMG
RMG dissolved in phosphate buffer was dialysed against a buffer containing 0.02 M CaCl,/O.l M Tris-HCl, pH 7.8. Glycopeptides were prepared by digestion of 1 mg of RMG in 500 ~1 of Tris-buffer with 1 mg of pronase E (Serva, Heidelberg, F.R.G.) which had been predigested for 2 h at 37°C. After incubation at 37°C for 24 h the digested sample was desalted on a Biogel P2 column (0.5 cm X 100 cm). The
186
glycopeptides were eluted with distilled water and collected from the void volume fractions as monitored by measurement of the optical density at 280 nm. Methylation analysis Isolated glycopeptides were permethylated according to Hakomori (1964). The partially methylated alditol acetates obtained after hydrolysis etc. were analyzed by capillary gas chromatography-mass fragmentography as detailed previously (Jansson et al., 1976; Geyer et al., 1983).
Results Purification of rat a,-macroglobulin It has been shown previously that upon molecular sieve chromatography the HI activity of rat serum elutes faster than the IgG fraction (O’Callaghan et al., 1980). This finding in connection with the high HI titers of up to 5 X lo4 HI-units/ml of rat serum (Styk, 1955) indicated that a major high molecular weight component, such as RMGz is responsible for the inhibitory activity. Therefore, we analysed whether RMG is an HI inhibitor.
RMG
60
100
120 Fraction
I
number
Fig. 1. Molecular sieve chromatography of the polyethylene gfycol concentration from Fractions of 2.5 ml were collected and 0), and trypsin-amidase activity (OFig. 2. SDS-polyacrylamide
160
rat plasma proteins. Plasma proteins precipitated by raising 4% to 8% were applied on a column of Ultrogel ACA 22. analyzed for protein content (A280, -), HI-activity (TA, l 0).
gel electrophoresis
of purified
RMG.
187 Following protocols for the isolation of a,-macroglobulin from human plasma, RMG was purified from rat plasma. RMG is precipitated from plasma by raising the concentration of PEG from 4% to 8%. This precipitate contained 95% of the HI-activity. Further purification was achieved by molecular sieve chromatography. As shown in Fig. 1, measurement of the optical density at 280 nm revealed two protein peaks. The smaller of both peaks (fractions 95-108) contained all the RMG as determined by its ability to bind trypsin (see OD 405 nm). The majority of the HI-activity was also found in these fractions. A minor peak of the HI-activity was observed at fractions 115-125. After a final chromatography on Fractogel TSK AF-Blue, purified RMG was obtained in the run through. Analysis of purified RMG by SDS-polyacrylamide gel electrophoresis is shown in Fig. 2. Only one major band is present which represents RMG as indicated by comigration with human a,-macroglobulin (not shown). A faint band is visible at the top of the gel. Whether this protein is a contaminant or an aggregate of RMG is not clear. For purified RMG a HI-activity of about 2” HI-units/mg p rotein was determined. Because of the high HI activity of this preparation it is unlikely that a minor contaminant is responsible for the inhibitory activity. Effect of neuraminidase on the HI activity of RMG Recently we have shown that the HI activity of rat serum is sensitive to the action of bacterial neuraminidases (Herrler et al., 1984). Having a purified inhibitor available we wanted to know whether neuraminic acid is also essential for HI activity of RMG. Purified RMG was incubated at 37°C in the presence or absence of VC-neuraminidase. After 90 min the HI-titer of the samples was determined. The result is shown in Table 1. Incubation with VC-neuraminidase reduced the HI activity by more than 80%. In the presence of the neuraminidase inhibitor DDN no reduction was observed. The results from Table 1 indicate that sialic acid is essential for optimal HI activity of RMG. Effect of RDA of influenza C virus on the HI activity of RMG A receptor-destroying activity has been shown to be associated with influenza C virus. We analysed whether or not this activity is also effective using RMG as substrate. Purified RMG was mixed with a concentrated preparation of influenza C virus and incubated at 37°C for various times. After incubation virions were removed by ultracentrifugation. The supernatant was assayed for HI activity. As shown in Fig. 3 with increasing incubation time at 37°C the HI activity of the supernatant decreased reaching 6% after 60 min. This result indicates that the RDA TABLE
1
Effect of VC-neuraminidase
on the HI activity
of purified
RMG
Enzyme present
HI-units/ml
None VC-neuraminidase VC-neuraminidase
4800 900 4800
+ lo- 3 M DDN
188
I
0
10
60
30 mcubation
time
(mm)
Fig. 3. Effect of RDA associated with influenza C virus on the HI activity of purified RMG. RMG was incubated with a concentrated preparation of influenza C virions for various times at 37°C. The residual HI activity was determined as described under Materials and Methods.
associated with influenza C virus is able to destroy the HI activity of RMG which therefore should be an useful substrate for the elucidation of the receptor-destroying activity. Methylation analysis of the glycopeptides of RMG The neuraminidase sensitivity of the HI activity of RMG suggests that the carbohydrate moiety of RMG is important for the inhibitory activity. Therefore we analyzed the structure of the oligosaccharides of RMG. The glycopeptides obtained TABLE
2
Methylation Peracetate
analysis of a
of glycopeptides
from RMG
Peak ratio b obtained from glycopeptides of RMG after treatment with: Influenza
2,3,4,6-ManOH 3,4,6-ManOH 3,6-ManOH 2,4-ManOH
0.25
’
2,3,4,6-GalOH 2,4,6-GalOH 2,3,4-GalOH 3,4,6-GlcN(Me)AcOH 3,6_GlcN(Me)AcOH
&O trace 1,05 0.1 0.3 2.0 0.15 3.5
C virus
VC-neuraminidase
0,15 120
0.25 2,0
trace
trace
l,O
1s)
0.15 0.4 1.8 0.1 3.15
2.05 0.2 0.15 0.15 3.3
a 3,4,6-ManOH, 3,4,6-tri-0-methylmannitol; 3,4,6-GlcN(Me)AcOH, 2-deoxy-2-(N-methyl)acetamido3,4,6-tri-0-methylglucitol, etc. b Peak ratios are based on 3,4,6-ManOH peracetate integrals = 2.0 (underscored). ’ Corrected for losses during hydrolysis (Conchie et al., 1982). Glycopeptides from native RMG, from RMG which had lost its HI-activity upon incubation with influenza C virus, and VC-neuraminidase-treated glycopeptides from native RMG were subjected to methylation analysis as described under Materials and Methods.
189 SA-2%6-Gal-lk-GlcNAc-l-L02-Mm-1
SA-2
*6-Gal-l
~L-Glct4Ac-l&2-Mm-l
\
a
OL /6
;Man-l~L-GlcNAc-l~I-GkNAc
Fig. 4. Proposed structure of the major oligosaccharide present on RMG. The structure is based on results obtained from methylation analyses of glycopeptides of RMG. The anomeric configuration of the linkage between individual sugars has not been determined, but has been inferred from other biantennary oligosaccharides (Montreuil, 1982; Vliegenthart et al., 1983).
from RMG after digestion with pronase were permethylated and analyzed by combined gas chromatography and mass spectrometry (GC/MS). The results presented in Table 2 are compatible with a biantennary oligosaccharide the structure of which is shown in Fig. 4. No evidence for the occurrence of fucose or of bisecting glucosamine was obtained. The absence of tri- or tetraantennary oligosaccharides is indicated by failure to detect 3,4_dimethylmannose and only trace amounts of 3,6dimethylmannose, respectively. Galactose residues were found to be substituted predominantly at position C-6 as evidenced by the occurrence of 2,3,4-trimethylgalactose residues. Only a minor fraction of galactose residues was substituted at position C-3. Treatment of the glycopeptides with VC-neuraminidase caused a decrease of the value of 2,3,4-trimethylgalactose by more than 90% and a concomitant increase of 2,3,4,6tetramethylgalactose. This result indicates that both branches of the biantennary oligosaccharide of RMG are terminated by sialic acid residues attached to galactose residues predominantly at position C-4. Glycopeptides of RMG, which had lost its HI activity due to the RDA of influenza C virus, were analyzed in the same way. As can be seen in Table 2, no major difference is detectable. A slight reduction of the value for 3,6-dimethyl-Nacetylglucosamine is probably within the range of experimental error, because no concomitant reduction of the value for 2,3,4-trimethylgalactose was observed. Galactose residues of RMG treated with influenza C virus were found to be substituted at position C-6, as in the case of native RMG, suggesting that sialic acid residues are not lost during the destruction of the HI-activity of RMG by influenza C virus. In conclusion, the destruction of the inhibitory activity of RMG by RDA of influenza C virus is not correlated with a change of the oligosaccharide structure detectable by this kind of analysis.
Discussion While a variety of compounds, including human and equine a,-macroglobulins, are known to inhibit hemagglutination caused by influenza A and B viruses (for a review see Gottschalk et al., 1972) only rat serum has been reported to be able to prevent influenza C virus from agglutinating chicken erythrocytes (Styk, 1955). We have purified a,-macroglobulin (RMG) from rat plasma and shown that this protein is a potent inhibitor of hemagglutination by influenza C virus, accounting for the
190 majority of the HI activity found in rat plasma. Furthermore, we have found that the inhibitory activity of RMG is sensitive to the receptor-destroying activity (RDA) of influenza C virus. Both findings suggest that there are some structural similarities between RMG and the cellular receptor for influenza C virus on chicken erythrocytes. Due to this structural similarity RMG not only serves as a substrate of the RDA of influenza C virus but it also competes with the cellular receptor for the viral hemagglutinin thus preventing influenza C virus from agglutinating chicken red blood cells. A characteristic feature of RMG, which may be required for the HI activity is the large size of this glycoprotein (about 7.4 X lo5 daltons). Glycopeptides obtained after pronase digestion of RMG are devoid of inhibitory activity (not shown). The importance of the molecular size of a glycoprotein for its ability to function as a hemagglutination inhibitor has been noted earlier for other influenza viruses (for review see Gottschalk et al., 1972). For instance, ovine submaxillary mucin loses its HI activity upon digestion with trypsin (Gottschalk and Fazekas de St. Groth, 1960). On the other hand, cr,-acid glycoprotein, which is a poor inhibitor of influenza A viruses, becomes a potent inhibitor after increasing its size either by crosslinking of individual molecules or by aggregation following heat treatment (Morawiecki and Lisowska, 1965; Whitehead, 1965). The HI activity of RMG has been found to be sensitive to the action of VC-neuraminidase. This result indicates that the sialic acid portion of RMG is important for the inhibitory activity. Methylation analysis of the glycopeptides of RMG revealed the presence of only one type of carbohydrate which is a complex N-linked oligosaccharide with a biantennary structure. Oligosaccharides of this type were reported to be present on both human and rabbit serum transferrin (Spik et al., 1975; Leger et al., 1978). Human as well as rabbit sera are unable to inhibit hemagglutination by influenza C virus (Styk, 1955). Therefore, it is reasonable to assume that human and rabbit serum transferrins are not inhibitors of influenza C virus, either. At present it is unknown whether these transferrins are inactive because of their smaller size (about 7 x lo4 daltons) compared to RMG or because of some additional structural requirements present on RMG but absent on transferrins. Our previous results suggested that sialic acid is involved in binding of influenza C virus to cells. We have proposed that the role of sialic acid as receptor for influenza C virus may depend on one or more of the following: (1) the nature of the adjacent sugar(s); (2) the type of linkage between neuraminic acid and the adjacent sugar residue; (3) modification of neuraminic acid, e.g. O-acetylation (Herrler et al., 1984). The results of the methylation analyses indicate that a modification of the neuraminic acid may be an important determinant for the HI activity. It remains to be seen whether the structure of the oligosaccharide core or the linkage of neuraminic acid to the core are important, too. On the basis of previous observations we have also speculated that the RDA of influenza C virus may be either an esterase that deacetylates neuraminic acid or an endoglycosidase removing a neuraminic acid-containing oligosaccharide, or a neuraminidase with an unusual substrate specificity. In the light of the data presented here an endoglycosidase can be ruled out because RDA of influenza C virus leaves
191 the core of the RMG-oligosaccharides intact. A neuraminidase activity would be compatible with our results only if it removes the terminal residue from an oligomeric form of neuraminic acid (a-NeuAc-(2-8)-aNeuAc). However, oligomeric neuraminic acid has not been found on plasma glycoproteins of the rat (Finne et al., 1977). Whether RDA of influenza C virus is an esterase, is the subject of current investigations.
Acknowledgements
The technical assistance of W. Mink and S. Kuhnhardt is gratefully acknowledged. We thank R. Rott for helpful discussions and Mrs. A. Becker for typing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 47, Virologie).
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192 Leger, D., Tordera, V., Spitz, G., Dorland, L., Haverkamp, J. and Vliegenthart. J.F.G. (1978) Structure determination of the single glycan of rabbit serotransferrin by methylation analysis and 360 MHz ‘H NMR spectroscopy. FEBS Lett. 93, 225-260. Montreuil, J. (1982) Glycoproteins. Compr. Biochem. 19B, (Part II), l-188. Morawiecki, A. and Lisowska, E. (1965) Polymerized orosomucoid an inhibitor of influenza virus hemagglutination. B&hem. Biophys. Res. Commun. 18, 606-610. O’Callaghan, R.J., Gohd, R.S. and Labat, D.D. (1980) Human antibody to influenza C virus: its age related distribution and distinction from receptor analogs. Infect, Immun. 30, 500-505. Spik. G., Bayard, B., Fournet, B., Strecker, G., Bouquelet, S. and Montreuil, J. (1975) Studies on glycoconjugates. LXIV. Complete structure of two carbohydrate units of human serotransferrin. FEBS Lett. 50, 296-299. Styk, B. (1955) Non-specific inhibitors in normal rat serum for the influenza C virus. Folia Biol. 1, 207-212. Van Leuven, F., Cassiman, J.J. and Van Den Berghe, H. (1980) Primary amines inhibit recycling of a2 M receptors in fibroblasts. Cell 20, 37-43. Vliegenthart, J.F.G., Dorland, L. and Van Halbeek, H. (1983) High-resolution ‘H-nuclear magnetic resonance spectroscopy as a tool in the structural analysis of carbohydrates related to glycoproteins. Adv. Carbohydr. Chem. Biochem. 41, 209-374. Whitehead, P.H. (1965) Viral inhibition by polymerized orosomucoid. Biochem. J. 95, 8p. (Manuscript
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