(Received for publication, September 14, 1994; and in revised form, November 2, 1994)
From the
Sialic acids are essential components of the cell surface receptors of many microorganisms including viruses. A synthetic, N-substituted D-mannosamine derivative has been shown to act as precursor for structurally altered sialic acid incorporated into glycoconjugates in vivo (Kayser, H., Zeitler, R., Kannicht, C., Grunow, D., Nuck, R., and Reutter, W.(1992) J. Biol. Chem. 267, 16934-16938).
In this study we have analyzed the potential of three different sialic acid precursor analogues to modulate sialic acid-dependent virus receptor function on different cells. We show that treatment with these D-mannosamine derivatives can result in the structural modification of about 50% of total cellular sialic acid content. Treatment interfered drastically and specifically with sialic acid-dependent infection of two distinct primate polyoma viruses. Both inhibition (over 95%) and enhancement (up to 7-fold) of virus binding and infection were observed depending on the N-acyl substitution at the C-5 position of sialic acid. These effects were attributed to the synthesis of metabolically modified, sialylated virus receptors, carrying elongated N-acyl groups, with altered binding affinities for virus particles.
Thus, the principle of biosynthetic modification of sialic acid by application of appropriate sialic acid precursors to tissue culture or in vivo offers new means to specifically influence sialic acid-dependent ligand-receptor interactions and could be a potent tool to further clarify the biological functions of sialic acid, in particular its N-acyl side chain.
Sialic acid ()is the most abundant terminal sugar
moiety on the surface of eukaryotic cells. As a component of
oligosaccharides on glucoconjugates, sialic acids are crucial for many
biological processes (for review see (1) and (2) )
including functional cell surface receptors for viruses such as
influenza viruses, reoviruses, paramyxoviruses, coronaviruses,
encephalomyocarditis virus, and polyoma
viruses(3, 4, 5, 6, 7, 8, 9, 10) .
The most abundant sialic acid, N-acetylneuraminic acid(11) , is synthesized in vivo from N-acetylated D-mannosamine or D-glucosamine as precursors (12) . These compounds are finally converted to CMP-activated N-acetylneuraminic acid, which is transferred in the Golgi apparatus onto oligosaccharide chains of glycoconjugates(12, 13) . It has recently been demonstrated that mammalian cells can take up synthetic N-substituted D-glucosamine and D-mannosamine derivatives and metabolize them in the sialic acid pathway (see Fig. 1)(14, 15) . As a consequence, structurally altered sialic acids with substituted N-acyl side chains were incorporated into various glycoconjugates(15, 16) .
Figure 1: Metabolism of N-propanoyl-D-mannosamine. This chemically synthesized amino sugar analogue follows the metabolic route of N-acetyl-D-mannosamine. It serves as a model for other N-acyl derivatives. For experimental details see Refs. 15 and 16). PEP, phosphoenol pyruvate.
In cells carrying
modified sialic acids, we investigated virus-receptor interactions of
three different members of the Polyomavirus genus. Polyoma
viruses are small DNA tumor viruses with a 45-nm diameter, nonenveloped
icosahedral particle consisting of the three nonglycosylated structural
viral proteins VP1, VP2, and VP3. The B-lymphotropic papovavirus ()(LPV) (17) and the human polyoma virus BK (BKV) (18) both use distinct sialylated receptors for
infection(10, 19) , whereas infection by the highly
related simian virus 40 (SV40) is sialidase-resistant(20) .
Here we describe that biosynthetically generated N-substituted
sialic acids can interfere with sialic acid-dependent biological
functions in a dominant fashion and specific for the introduced N-acyl substitution.
Further purification and
final quantification were achieved by reversed phase HPLC with
fluorescent labeled compounds. The derivation was performed according
to a method by Reuter and Schauer(24) . Labeled neuraminic
acids were chromatographed using a reversed phase C18 column
(Lichrosorb C18 5 µm, 250 4 mm (inner diameter), Knauer,
Berlin, Germany) with a fluorescence detector. Eluent A contained
distilled water while eluent B contained acetonitrile/methanol (60:40,
v/v). The flow rate was 1 ml/min, and all separations were carried out
using a gradient that first ran for 20 min in the isocratic mode with
10% B; then was raised to 25% B in 25 min and finally to 50% B in
another 15 min.
For virus infection cells were washed once with cold PBS and were then incubated either with LPV (BJA-B cells, 4 °C, 3 h) or with BKV or SV40 (Vero cells, 4 °C, various times). After removing unbound virus from the cultures by three washing cycles with medium, infected cells were cultured at 37 °C. In BJA-B cells without prior treatment by N-acylmannosamines the applied LPV dose yielded 15-20% immunofluorescence-positive cells (see below) 48 h after infection. For BKV the infectious dose was chosen to yield 1.5-2.5% infected Vero cells in order to allow an exact determination of the increase in infection in ManNProp- and ManNBut-treated cells (see Fig. 3, a and c).
Figure 3:
Biosynthesis of N-substituted
neuraminic acid derivatives in host cell lines treated with N-substituted D-mannosamines. Three representative
electron impact mass spectrograms of extracted N-acylneuraminic acids are shown: a, N-acetylneuraminic acid
(5-acetamido-3,5-dideoxy-D-glycero--D-galactopyranosyl-2-onic
acid) from ManNAc-pretreated (control) Vero cells; b, N-propanoylneuraminic acid
(3,5-dideoxy-5[propanoyl-amido]-D-glycero-
-D-galactopyranosyl-2-onic
acid) from ManNProp-pretreated Vero cells; and c, N-butanoylneuraminic acid
(5[butanoyl-amido]-3,5-dideoxy-D-glycero-
-D-galactopyranosyl-2-onic
acid) from ManNBut-pretreated BJA-B cells. m/e defines the
ionic mass/ionic charge of fragments generated from neuraminic acids
during mass spectroscopy. Fragment peaks characteristic for the
individual N-acylderivatives (15) are indicated. Note
the m/e increase of 14 for each additional methylene group in
corresponding fragments. At m/e 285 a fragment peak constant
for all N-acylneuraminic acids occurs. The chair conformations
of the main peak fragment of N-acetylneuraminic acid (m/e 356) (a), N-propanoylneuraminic acid (m/e 370) (b), and N-butanoylneuraminic acid (m/e 384) (c) are shown to indicate the elongation of the N-acyl group.
Figure 4: Reduction or enhancement of host cell susceptibility to LPV or BKV infection by pretreatment with sialic acid precursor analogues. a, the chair conformation of the applied N-substituted D-mannosamines is shown with R indicating the modified N-acyl group. b, host cell lines BJA-B and Vero were cultured for 48 h in the presence of the sialic acid precursor analogues ManNProp, ManNBut, or ManNPent or as control an equivalent volume of PBS and subsequently infected with LPV or BKV, respectively. Virus-infected cells were identified by immunofluorescence staining for LPV and BKV capsid proteins 48 h after infection (this time allows the completion of only one viral replication cycle). Similar numbers of cells are present in microphotographs of each panel as determined by nuclear counterstaining (not shown).
To demonstrate the biosynthesis of structurally modified sialic acids and their incorporation into glycoconjugates after pretreatment of BJA-B and Vero cells by either ManNProp, ManNBut, or ManNPent, extracted sialic acids were separated by HPLC (Fig. 2) and identified by gas chromatography-mass spectrometry (Fig. 3), as has been shown previously for serum glycoproteins of ManNProp-treated rats(15) . The resulting sialic acid derivatives carry the identical N-acyl group substitution as the applied sialic acid precursor analogue, thus resulting in an elongation of the physiological N-acetyl group at position C-5 of sialic acid by one, two, or three methylene groups, respectively (see chair conformations in Fig. 4a). The amount of modified sialic acids incorporated in pretreated cells relative to the amount of physiological N-acetylneuraminic acid was quantified after HPLC separation of the extracted sialic acids (Fig. 2). About 50% of physiological neuraminic acid was replaced by the N-acyl-modified neuraminic acid in both of the cell lines tested after treatment with each of the three D-mannosamine derivatives (Table 1).
Figure 2: Detection of biosynthetically modified sialic acids from Vero cells with two different HPLC methods. a-d, anion exchange chromatography of purified neuraminic acids with pulsed amperometric detection. Glucose 6-phosphate (Glc-6-P) was used as an internal standard. See ``Experimental Procedures'' for details. e and f, reversed phase chromatography of fluorescence labeled neuraminic acids. N-glycolylneuraminic acid (NGNA) was used as an internal standard. a and e, reference chromatograms. The chromatograms of cells treated with ManNAc (b), ManNProp (c), ManNBut (d), and ManNPent (f) are shown. NANA, N-acetylneuraminic acid; NPropNA, N-propanoylneuraminic acid; NButNA, N-butanoylneuraminic acid; NPentNA, N-pentanoylneuraminic acid; PAD, pulsed amperometric detection.
Cultivation of BJA-B cells in the presence of the sialic acid precursor analogues ManNProp, ManNBut, or ManNPent for 2 days reduced their LPV binding capacity by 75, 86, and 85%, respectively (Fig. 5c). Similarly, the susceptibility of these pretreated cells to LPV infection was impaired by up to 97% in comparison with cells pretreated with control saline buffer (Fig. 4b, Fig. 5, a and b). 50% inhibition of LPV infection was reached at a concentration in tissue culture of approximately 0.4 mM for ManNProp and 0.8 mM for ManNBut (Fig. 5a).
Figure 5: Reduction of LPV infection (a and b) and binding (c) in host cells containing N-substituted sialic acids. a, BJA-B cells were cultured in the presence of different concentrations of ManNProp, ManNBut, the physiological sialic acid precursor ManNAc (Sigma, Germany), or PBS (control) for 48 h and subsequently infected with LPV. 48 h after infection the concentration of viral antigen in cell extracts was quantified by LPV VP1-specific enzyme-linked immunosorbent assay(10) . The values shown represent the arithmetic means from triplicate samples (± S.D.) relative to the concentration of viral antigen present in control cells. b, after ManNProp pretreatment (10 mM) of 6-48 h, cells were infected with LPV and analyzed as described above. For each time point, LPV infection in ManNProp-treated cells is given relative to LPV infection in control cells (set as 100%). c, the LPV binding capacity of BJA-B cells pretreated with different N-acyl-D-mannosamines (5 mM, 48 h) or PBS (control) was tested in a nonradioactive, indirect virus binding assay where a constant amount of virus is incubated with increasing cell numbers(10) . Values represent the amount of virus bound relative to the total virus offered for binding and are arithmetic means (±S.D.) from triplicate samples. The cell numbers required to bind 37.5% of the administered LPV were used to determine LPV binding capacities of pretreated cells relative to control cells.
D-Mannosamine has been reported to inhibit the biosynthesis of glycosylphosphatidylinositol anchors in mammalian cells(28) . In order to determine whether the observed inhibition of LPV infection was specific for the modified N-acyl group and not an effect of the D-mannosamine residue, cells were also pretreated with the physiological sialic acid precursor ManNAc, differing from ManNProp by only one methylene group. Yet, neither LPV binding (Fig. 5c) nor infection (Fig. 5a) was significantly affected by this D-mannosamine derivative.
In order to examine whether pretreatment with ManNProp, ManNBut, or ManNPent, respectively, also had an effect on other steps of the virus replication cycle apart from virus binding, purified LPV DNA was transfected by the DEAE-dextran method. This experiment yielded similar amounts of virus for all cells, irrespective of their pretreatment (not shown), indicating that the impaired susceptibility to infection involves events prior to virus gene expression and DNA replication. Taken together, these experiments strongly suggest that already the first step of LPV infection, i.e. virus attachment, was impaired by the presence of structurally altered sialic acids in membrane glycoconjugates and by this mechanism hindered LPV infection.
Figure 6: Sialic acid-dependent BKV infection (a and c) but not sialic acid-independent SV40 infection (b) is affected in Vero cells precultivated in the presence of N-substituted D-mannosamines (5 mM, 48 h). BKV (a) or SV40 (b) (same symbols as in a) was allowed to attach at 4 °C for 6 min to 14 h to pretreated Vero cells. Unbound virus was washed away after the attachment phase, and then cells were incubated at 37 °C for 48 h before the percentage of virus-infected cells was determined by immunofluorescence. c, the increased BKV susceptibility of ManNProp- and ManNBut-pretreated Vero cells is sensitive to sialidase treatment. Values given are the mean of two independent experiments.
It has been shown previously that N-propanoylneuraminic acid, incorporated into glycoconjugates in vivo, can be enzymatically or chemically cleaved(15) . In ManNProp- and ManNBut-pretreated Vero cells V. cholerae sialidase reduced the elevated levels of BKV infection by 88 and 82%, respectively (Fig. 6c), demonstrating the essential role of terminal sialic acids on the cell surface for the enhanced susceptibility to BKV infection.
This study demonstrates drastic but selective biological effects of sialic acids modified in their N-acyl side chains, which have been synthesized and incorporated in high amounts into cellular glycoconjugates. These modified sialic acids have been biosynthesized in competition with physiological sialic acid in cells cultivated in the presence of N-substituted D-mannosamines, and amounts equal to the physiological N-acetylneuraminic acid can be reached. The N-substituted sialic acids drastically enhance and/or abolish in a dominant fashion host cell susceptibility to sialic acid-dependent virus infections.
The high amount of modified sialic acids with
elongated N-acyl groups synthesized indicates that enzymes and
transport mechanisms in the sialic acid metabolic pathway cannot be
very selective with respect to the N-acyl group of substrate
intermediates. Also, the modified sialic acids and their precursors
apparently are tolerated by the cells, at least in tissue culture. At 5
mMD-mannosamine derivative, when about 50% of total
sialic acids with an N-pentanoyl instead of the physiological N-acetyl side chain was reached, cell viability was not
affected, and cell proliferation was only slightly reduced. Similarly, in vivo treatment of Wistar rats with ManNProp had displayed
no signs of acute toxicity. ()This indicates that, at least
in these established cell lines, either the metabolic and signaling
pathways needed for cellular proliferation and survival in tissue
culture are rather independent of sialic acids or are not very
discriminatory with respect to the N-acyl side chain.
In contrast to this rather inert behavior of central cellular functions, the minor modifications of the sialic acid side chain, i.e. the introduction of uncharged, hydrophobic methylene groups at C-5, had pronounced and specific effects on interactions of host cells with two viruses that are known to depend on cell surface sialic acid for infection. Most probably, the modification of sialic acid forming part of the viral receptor structures was responsible for alteration of the virus infections. The presence of physiological N-acetyl sialic acid and modified N-propanoyl sialic acid in about equal amounts in human B-lymphoma line BJA-B was sufficient to reduce binding of LPV 6-fold and infection over 10-fold. On the other hand, infection by BKV was enhanced 7-fold in monkey epithelium cell line Vero carrying the same modified sialic acid also in about equal amounts as the physiological sialic acid. The kinetic experiment suggests that the increase in infection was mainly due to faster binding, which may reflect an increase either in receptor number or, more probably, in receptor affinity. One could assume that a polyvalent cooperative interaction of the repetitive virus capsid with several cell surface receptor molecules is necessary for particle binding. If so, then even slight receptor affinity changes in either direction could add up to the strong, opposite effects seen here for the two viruses.
The use of these sialic acid precursor analogues allowed the identification of the N-acyl group as a critical determinant of sialic acid-dependent polyoma virus-receptor interaction. This group has previously been implicated in host range determination of a completely different microorganism. Enterotoxigenic Escherichia coli with strain K99 fimbriae, which use sialylated intestinal glycolipids as receptors, specifically recognize N-glycolyl but not N-acetyl substitutions(34) . In addition to viral infections studied here, the principle of biosynthetic structural modification of sialic acid should be applicable to a wide variety of sialic acid-dependent ligand-receptor interactions including other bacteria(35) , parasites(36) , toxins(37) , and lectins(38) .
For the interaction of the influenza A virus hemagglutinin with synthetic sialic acids, N-substitutions were found to reduce (39) or not to alter (40) the binding affinity. Such studies using nuclear magnetic resonance spectroscopy or virus adsorption inhibition assays on erythrocytes could be complemented and extended by assays on virus binding and infection in cultured host cells and animals carrying biosynthetically incorporated N-substituted sialic acids. This should allow a more detailed understanding of the multivalent nature of this virus-receptor interaction.
Synthetic sialic acid derivatives binding with high affinity to influenza A and B virus sialidase (4-amino- and 4-guanidino-2-deoxy-2,3-didehydro-D-N-acetyl sialic acid) (41) or influenza C virus surface glycoprotein (42) can serve as inhibitors of influenza virus infections. The enhanced susceptibility to BKV observed in our study suggests that sialic acid modifications generated in living host cells may also facilitate the in vitro development of soluble high affinity competitors of virus binding as antiviral drugs.
In conclusion, the principle of biosynthetic modification of sialic acid by application of appropriate sialic acid precursors to tissue culture or in vivo offers new means to specifically influence sialic acid-dependent ligand-receptor interactions.
This paper is dedicated to Dr. Dr. Herbert Falk on the occasion of his 70th birthday.