(Received for publication, April 7, 1995; and in revised form, May 25, 1995)
From the
Infection of cells by influenza C virus is known to be initiated
by virus attachment to cell surface glycoconjugates containing N-acetyl-9-O-acetylneuraminic acid. Using an in
vitro virus binding assay, we have detected this carbohydrate on
several glycoproteins of Madin-Darby canine kidney cells (type I), a
polarized epithelial cell line permissive for infection with influenza
C virus. Among these proteins, only one was found to be present to a
significant extent on the cell surface. This protein, gp40, was
characterized as an O-glycosylated (mucin-type) integral
membrane protein of 40 kDa, which was predominantly localized on the
apical plasma membrane of filter-grown cells. It is a major cell
surface sialoglycoprotein in this cell line and was shown to be subject
to constitutive and rapid endocytosis. Thus, this glycoprotein can
mediate not only the binding of influenza C virus to the cell surface,
but also its delivery to endosomes, where penetration occurs by
membrane fusion. Other highly sialylated cell surface glycoproteins
were also detected but did not mediate influenza C virus binding to a
significant extent, indicating that only gp40 contains
9-O-acetylated sialic acids. Enveloped viruses enter their host cells by attachment to
receptor molecules located in the plasma membrane followed by a fusion
process between the viral envelope and the cell membrane, either
directly at the cell surface or within endosomes after uptake of the
viral receptor-virus complex by endocytosis. As an increasing number of
virus receptors has been identified in recent years, it turned out that
in many instances viruses selectively recognize specific membrane
proteins for attachment to the cell surface, which is considered to be
a major determinant for their host range and tissue
tropism(1) . On the other hand, it has been known for many
years that sialic acid is the receptor determinant for influenza
viruses and several other viruses. Since this acidic sugar is
conjugated to many cellular glycoproteins and glycolipids, influenza
viruses in theory have the choice between multiple receptors. However,
sialic acids and sialylated oligosaccharides are very diverse in
structure, and it has been shown for influenza A and B viruses that
pronounced preferences exist with regard to the sialylated
oligosaccharide sequences recognized(2) . Another type of
specificity has been observed with influenza C virus. In contrast to
influenza A and B viruses, for which N-acetylneuraminic acid
(Neu5Ac) Madin-Darby canine kidney (MDCK) cells are
polarized epithelial cells which are widely used in cell biology for
the study of protein transport and cell polarity(10) . A
subline of these cells (MDCK I), characterized by a high trans-epithelial electrical resistance, is susceptible to
influenza C virus infection. In the present study, we analyzed the
glycoproteins of MDCK I cells for their ability to mediate influenza C
virus binding as well as for their distribution between the cell
surface and intracellular compartments. We describe a cellular
glycoprotein which is the major surface protein recognized by influenza
C virus and which is subject to rapid endocytosis. This glycoprotein
has therefore the characteristics expected from an influenza C virus
receptor.
Figure 1:
Binding of influenza C virus to MDCK I
glycoproteins. Glycoproteins were precipitated from MDCK I cell lysates
by the immobilized lectins indicated on top of the lanes. The
glycoproteins were separated by SDS-PAGE, transferred to
nitrocellulose, and probed with influenza C virus. Bound virus was
detected using a chromogenic esterase substrate, which was cleaved by
the viral O-acetylesterase.
Because of its
ability to precipitate a great number of glycoproteins which are
recognized by influenza C virus, we used WGA to further study the
specificity of the viral interaction with cellular proteins. Fig. 2A shows that influenza C virus binding to these
glycoproteins was abolished when the blots were pretreated with
sialidase (lane c) or with sodium hydroxide (lane d).
The latter treatment hydrolyzes ester linkages such as the
9-O-acetyl ester of sialic acids. As a further control, we
also tested glycoproteins from influenza C virus-infected cells. The
viral glycoprotein is synthesized at the rough endoplasmatic reticulum
and is transported to the plasma membrane via the constitutive
secretory pathway. On the way to the cell surface, it passes the Golgi
compartment where the cellular O-acetyltransferase is thought
to be located(23, 24) . Therefore, HEF has the chance
to de-O-acetylate both intracellular and surface
sialoglycoproteins with the consequence that influenza C virus cannot
bind to them in the ligand blot assay (lane b). For detection
of sialoglycoproteins, a parallel blot was subjected to mild
metaperiodate oxidation. The oxidized sialic acids were labeled with
digoxigenin and detected by anti-digoxigenin Fab fragments conjugated
to alkaline phosphatase (Fig. 2B, lane a).
De-O-acetylation of sialic acids either by acetylesterase
activity during influenza C virus infection (lane b) or by
alkaline treatment of the blots (lane d) did not impair the
reaction, whereas sialidase treatment abolished the detection of
glycoproteins (lane c). These findings demonstrate that
influenza C virus binds to MDCK I glycoproteins via Neu5,9Ac
Figure 2:
Analysis of the influenza C virus binding
specificity. MDCK I cells were used either as non-infected cells (lanes a, c, and d) or 20 h after infection
with influenza C virus (lanes b). Glycoproteins were
precipitated from cell lysates by immobilized WGA, electrophoresed on
polyacrylamide gels, and blotted to nitrocellulose membranes. The
nitrocellulose was cut into strips that were incubated with PBS (lanes a and b), sialidase (lanes c), or 0.1 M NaOH (lanes d) as described under
``Experimental Procedures.'' The nitrocellulose strips were
probed with influenza C virus (A) or subjected to mild
metaperiodate oxidation (B). Oxidized sialic acid residues
were labeled with digoxigenin and detected using anti-digoxigenin Fab
fragments conjugated to alkaline
phosphatase.
Figure 3:
Cellular distribution of
9-O-acetylated sialoglycoproteins in MDCK I cells. Confluent
MDCK I monolayers were surface biotinylated at 4 °C. A,
the monolayers were either incubated with MES puffer (lanes a and c) or treated with C. perfringens sialidase (300 milliunits/ml in MES puffer) for 60 min at 37
°C (lanes b and d). After cell lysis,
glycoproteins were precipitated either by immobilized WGA (lanes a and b) or by immobilized streptavidin (lanes c and d). After SDS-PAGE and transfer to nitrocellulose,
the precipitated proteins were probed with influenza C virus. The
position of gp40, the major cell surface glycoprotein recognized by
influenza C virus is indicated.
For detection of
major cell surface sialoglycoproteins irrespective of O-acetylation, Western blots of biotin-labeled and
streptavidin-precipitated cell surface proteins were subjected to mild
metaperiodate oxidation followed by labeling of the oxidized sialic
acids with digoxigenin. The reaction with an antibody directed to
digoxigenin revealed the presence of several cell surface
sialoglycoproteins including gp40 (Fig. 4A, lane
a). Only the latter was recognized to a significant extent when
the Western blots were probed with influenza C virus (lane b).
De-O-acetylation of sialic acids by NaOH pretreatment
abolished the virus binding but did not alter the antibody reaction
(not shown; see also Fig. 2B for comparison). The
lectin from Arachis hypogea (PNA) exhibits a stringent
specificity for the sequence Gal
Figure 4:
Detection of major MDCK I cell surface
sialoglycoproteins. Confluent MDCK I monolayers were surface
biotinylated, and the labeled proteins were precipitated from cell
lysates by immobilized streptavidin. A, the precipitated
proteins were separated by SDS-PAGE, blotted to nitrocellulose, and
either subjected to mild metaperiodate oxidation with subsequent
labeling and detection of sialic acid residues via an enzyme-linked
immunoassay (lane a) or probed with influenza C virus (lane b). B, Precipitated proteins were either
incubated with acetate buffer (lanes a and c) or
treated with V. cholerae sialidase (lanes b and d) as described under ``Experimental
Procedures.'' Western blots of the precipitates were either probed
with PNA (lanes a and b) or influenza C virus (lanes c and d) as described under
``Experimental Procedures.'' The position of gp40 is
indicated.
Figure 7:
Effect of glycosidases on gp40. Confluent
MDCK I monolayers were surface biotinylated and extracted with the
detergent Triton X-114. After temperature-induced phase separation, the
labeled proteins were precipitated from the detergent phase with
immobilized streptavidin. The precipitates were treated with buffer (lanes a), V. cholerae sialidase (lanes
b), N-glycosidase F (lanes c), sialidase + N-glycosidase F (lanes d), O-glycosidase (lanes e), sialidase + O-glycosidase (lanes
f) as described under ``Experimental Procedures.'' The
digested samples were separated by SDS-PAGE and transferred to
nitrocellulose. A, the blots were probed with influenza C
virus. B, biotinylated proteins were visualized by
strepavidin-horseradish peroxidase. The arrows mark the
undigested and digested forms of gp40. The 35 kDa band in lanes
a, c, and e is not related to gp40 because its
molecular mass was reduced after sialidase treatment and it was not
recognized by influenza C virus.
Figure 5:
Distribution of gp40 between the apical
and basolateral plasma membrane of MDCK I cells. Filter-grown MDCK I
monolayers were surface labeled by addition of sulfo-NHS-biotin to
either the apical (lanes a and c) or basolateral (lanes b and d) compartment of the filter chamber.
The biotinylated proteins were recovered from cell lysates by
streptavidin precipitation, run on SDS-PAGE, and blotted to
nitrocellulose. Biotinylated proteins were detected with
streptavidin-horseradish peroxidase (lanes a and b).
After incubation of the blots with influenza C virus, bound virions
were visualized by esterase activity (lanes c and d).
The position of gp40 is indicated.
Figure 6:
Analysis of the membrane association of
9-O-acetylated sialoglycoproteins. Confluent MDCK I monolayers
were surface biotinylated and extracted with the detergent Triton
X-114. After temperature-induced phase separation the resulting
detergent phases (lanes a and c) and aqueous phases (lanes b and d) were either subjected to
precipitation with immobilized WGA (lanes a and b) or
immobilized streptavidin (lanes c and d). The
precipitates were run on 8% polyacrylamide gels, transferred to
nitrocellulose, and probed with influenza C virus. The position of gp40
is indicated.
The O-sialoglycoprotease from Pasteurella haemolytica has been shown to specifically cleave
mucin-type sialoglycoproteins(31, 32) . As shown in Fig. 8, treatment of biotinylated MDCK I cells with this enzyme
resulted in a drastically reduced binding of influenza C virus to gp40
(compare lane c with d). Parallel detection of the
biotin groups by streptavidin-horseradish peroxidase shows that only
gp40 but not other cell surface proteins had been proteolytically
cleaved (lanes a and b, longer exposure time; lanes a` and b`, shorter exposure time). Two bands in
the range of 22 kDa became visible following O-sialoglycoprotease treatment (lane b) and are
thought to represent proteolytic fragments of gp40. Part of gp40
escaped digestion by O-sialoglycoprotease possibly due to
internalization (see below). Taken together, these results indicate
that gp40 has characteristics typical for mucin-type glycoproteins.
Figure 8:
Effect of P. haemolytica
O-sialoglycoprotease on gp40. Surface biotinylated, confluent MDCK
I monolayers were treated with either PBS+ (lanes a and c) or O-sialoglycoprotease in PBS+ (lanes b and d) for 60 min at 37 °C. The biotinylated proteins
were precipitated from the cell lysates with immobilized streptavidin.
Western blots of the precipitates were probed with either
streptavidin-horseradish peroxidase (lanes a and b)
or influenza C virus (lanes c and d). The exposure
times for chemoluminescent detection were 30 s for lanes a and b and 15 s for lanes a` and b`.
Figure 9:
Kinetics of influenza C virus
internalization. Influenza C virus was allowed to bind to MDCK I
monolayers (10
Figure 10:
Endocytosis of surface-labeled gp40. A, confluent MDCK I monolayers were labeled at 4 °C with
sulfo-NHS-biotin and incubated for 45 min at the indicated
temperatures. After cell surface digestion with proteinase K at 4
°C, protease-resistant biotinylated proteins were precipitated from
cell lysates with immobilized streptavidin, electrophoresed on
polyacrylamide gels, transferred to nitrocellulose, and probed with
influenza C virus. Control cells (first lane) were kept at 4
°C and were not digested with protease. B, time course of
gp40 internalization. The endocytosis assays was performed at 37 °C
for the time intervals indicated and quantitated by densitometric
scanning of the Western blots.
A great number of viruses including influenza viruses,
paramyxoviruses, coronaviruses, polyomaviruses, rotaviruses, and
reoviruses are known to require sialic acid as a crucial part of
cellular receptors for attachment to cell surfaces(1) . For
some influenza viruses, the type of sialic acid and the structure of
the oligosaccharide backbone required for optimal binding has been
elucidated(2, 4, 36) . On the other hand, the
glycoconjugates on the surface of permissive cell lines that mediate
virus attachment and virus infection have not been identified. We have
previously shown that MDCK cells (type I) are highly susceptible to
infection by influenza C virus but are rendered resistant to infection
by pretreatment with sialidase(37) . The susceptibility to
infection is entirely restored after resialylation of the cells with
Neu5,9Ac In
this study, we demonstrated the direct binding of influenza C virus to
MDCK I glycoproteins by probing Western blots with intact virions.
Lectin precipitation resulted in specific enrichment of cellular
glycoproteins that facilitated the detection of virus binding. Among
the various glycoproteins recognized by influenza C virus one
glycoprotein was remarkable for two properties not shared by the
others. It was the major protein expressed at the cell surface, and it
showed the characteristics of an integral membrane protein. Because of
its apparent molecular mass of 40 kDa, this glycoprotein has been
designated gp40. It is present at both plasma membrane domains of
filter grown cells with a stronger apical expression. Our finding that
gp40 is subject to rapid endocytosis is most important because it shows
that gp40 is able to exhibit a dual receptor function. It can bring
about the binding of influenza C virus to the cell surface and mediate
its delivery to the endosomal compartment, which provides the acidic
milieu required for the fusion of the viral envelope with the cellular
membrane. A critical point in this context is the kinetics of
internalization because infection of a cell by influenza viruses will
only be successful, if virus internalization proceeds more rapidly than
the elution of the virus from the receptor caused by the
receptor-destroying enzyme. Endocytosis of gp40 has been shown to be a
very rapid process with a kinetics similar to that of virus
internalization. The rapid endocytosis of various known plasma membrane
receptors, for example those for transferrin, low density lipoprotein,
and asialoglycoproteins, is based on a common
mechanism(40, 41, 42, 43) : adaptor
proteins (adaptins) recognize a short sequence in the cytoplasmic tail
of these receptors and induce the formation of a clathrin coat. In this
way, the receptors are preferentially concentrated in coated pits,
which readily invaginate and pinch off the membrane, while other plasma
membrane proteins lacking an endocytosis signal are excluded from the
clathrin-coated pits and will internalize only
slowly(44, 45) . Further studies have to show if this
mechanism is also responsible for the rapid endocytosis of gp40. A
surprising finding of our work was that influenza C virus
preferentially binds to a single cell surface glycoprotein. The reason
for this might be that gp40 is expressed in larger amounts or that it
is sialylated to a larger extent than are other cell surface
glycoproteins. We have found that gp40 is a major sialoglycoprotein of
the MDCK I plasma membrane. However, we detected also other prominent
sialoglycoproteins that did not mediate virus binding. Thus, the
quantitative aspect alone is not sufficient to explain the selectivity
of virus binding. In spite of its sialylation, removal of sialic acids
did not result in a shift to a lower molecular weight. This unusual
behavior is typically seen in heavily O-glycosylated
sialoglycoproteins(46) . In agreement with these observations,
gp40 was found to be susceptible to P. haemolyticaO-sialoglycoprotease, a neutral metalloprotease that has
been shown to specifically cleave mucin-type sialoglycoproteins like
glycophorin, sialophorin (CD43), or CD34(31, 32) . In
addition, the lectins M. amurensis-agglutinin and SNA
that recognize sialic acids on N-glycans but not on O-glycans(13, 47) , failed to bind to gp40.
Other characteristics of mucin-type sialoglycoproteins, like the
predominance of O-glycosylation compared to N-glycosylation and the weak reactivity with protein stains
were also found with gp40 indicating that this protein is a mucin-like
glycoprotein. Secreted mucins, e.g. ovomucin, have been known
for a long time as potent inhibitors of hemagglutination by influenza
viruses(48, 49) . The macromolecular structure of
these compounds is crucial since the tryptic glycopeptides of mucins or
small sialyloligosaccharides had no inhibitory effect(49) . The
binding of influenza viruses to erythrocytes is mediated by
glycophorin, another mucin-type glycoprotein. In contrast to most
soluble mucins which have molecular masses of often several hundred
kDa, glycophorin resembles gp40 in being a small integral glycoprotein
of about 31 kDa. Typical for mucins is the high level of O-glycosylation at clusters of serine- and threonine-rich
sequences(50) . The clustering of the O-linked
carbohydrates may be favorable for a polyvalent interaction and thus
enhance the binding avidity of influenza C virus. Interestingly, the
preferential binding of simian rotaviruses to O-linked
sialoglycoconjugates and mucins is also thought to be due to clustering
of sialic acids(51) . O-Acetylated sialic acids
have been detected in quite diverse types of glycoconjugates including
gangliosides, mucins, serum glycoproteins, and membrane
proteins(52) . For example, rat serum glycoproteins and bovine
submandibular mucin, which both have a high content of
Neu5,9Ac We have previously shown that the
lack of appropriate receptors on the cell surface is a major reason for
the restricted cell tropism of influenza C virus(37) . However,
it is not understood whether this phenomenon is due to a general low
level of 9-O-acetylation in these cells or due to lack of an
appropriate cell surface molecule capable of mediating both efficient
binding and internalization of influenza C virus. To our knowledge,
gp40 is the first defined physiological receptor glycoprotein of an
influenza virus. Correlating cell type-specific expression of gp40 with
the ability of cells to support influenza C virus infection should help
to evaluate the role of gp40 and of O-acetyltransferase
activity in the cell tropism of this virus. It would be interesting to
see if recombinant expression of gp40 in a non-permissive cell line
will be sufficient to render these cells permissive. As various
coronaviruses have been shown to use 9-O-acetylated sialic
acids for attachment to cell surfaces (56, 57) , gp40
may be a promising receptor candidate also for these viruses.
(
)is the primary receptor determinant,
influenza C virus exclusively binds to a modified form of this sialic
acid which is acetylated at position C-9
(Neu5,9Ac
)(3, 4) . The receptor binding
(hemagglutinating) activity of influenza C virus is a function of the
single viral spike glycoprotein HEF, which is also responsible for the
receptor destroying and the fusion activities of this
virus(5) . The receptor destroying activity has been identified
as an acetylesterase which catalyzes the de-O-acetylation of
Neu5,9Ac
(3, 6) . The inactivation of the
receptor determinant may be important for facilitating the elution of
progeny virus from infected cells, for preventing self-aggregation of
virions, and/or for inactivating competitive inhibitors. Recent data
suggest that the acetylesterase may also be involved in virus
entry(7) . However, the role of the receptor-destroying enzyme
in this process is not understood because the fusion activity of HEF,
which is known to be essential for virus penetration(8) , is
not dependent on the inactivation of the receptors (9) . Fusion
is believed to be due to a hydrophobic amino acid sequence, which for
its activation requires the proteolytic cleavage of the glycoprotein
into the subunits HEF
and HEF
, and the exposure
to low pH. Because of the dependence on acidic pH, it is assumed that
influenza C virus, like influenza A and B viruses, is internalized by
cells via receptor-mediated endocytosis. Acidification of the endosomal
compartment by a vacuolar ATPase is believed to induce a conformational
change in the viral glycoprotein which results in the fusion of the
endosomal and the viral membrane. As receptor binding and receptor
inactivation are competitive activities, influenza C virus may bind to
certain glycoconjugates of the plasma membrane and, if these molecules
are not internalized in time, will elute from them without infecting
the cell. Thus, aside from proper glycosylation, a further important
criterion for a functional influenza C virus receptor is its rapid
internalization.
Materials
The following reagents were obtained
from Boehringer Mannheim GmbH (Mannheim, Germany): proteinase K, N-glycosidase F from Flavobacterium meningosepticum, O-glycosidase from Diplococcus pneumoniae,
2,3-dehydro-2-desoxy-N-acetylneuraminic acid,
digoxigenin-3-O-succinyl--amidocaproic acid hydrazide
hydrochloride, polyclonal sheep anti-digoxigenin Fab fragments
conjugated to alkaline phosphatase, nitro blue tetrazolium chloride
(NBT), 5-bromo-4-chloro-3-indolyl phosphate 4-toluidine salt (BCIP).
The following reagents were purchased from Sigma (Deisenhofen,
Germany): wheat germ agglutinin (WGA)-agarose beads, Jacalin agarose
beads, concanavalin A-agarose beads,
-naphthyl acetate,
4-chloro-2-methylbenzenediazonium salt (Fast Red TR salt), sialidase
from Clostridium perfringens (Type X). Sambucus nigra agglutinin (SNA)-agarose beads were from Medac GmbH (Hamburg,
Germany). Lens culinaris agglutinin (LCA)-agarose beads were
obtained from Pharmacia LKB (Freiburg, Germany).
Sulfo-succinimido-biotin (sulfo-NHS-biotin) and streptavidin-agarose
beads were supplied by Pierce Chemical Co. ECL detection reagent and
prestained protein molecular weight markers were purchased from
Amersham Buchler GmbH (Braunschweig, Germany). Vibrio cholerae sialidase was from Behring AG (Marburg, Germany). PI-specific
phospholipase C from Bacillus thuringiensis was obtained from
Oxford Glycosystems (Oxford, United Kingdom). Pasteurella
haemolyticaO-sialoglycoprotease was from Accurate
Chemical & Scientific Co. (Westbury, NY). Stock solutions of BCIP
(50 mg/ml in dimethylformamide), NBT (75 mg/ml in 70%
dimethylformamide), and
-naphthyl acetate (10 mg/ml in acetone)
were stored at -20 °C.
Virus
Influenza C virus (strain Johannesburg/1/66)
was grown in the allantoic cavity of 8-day-old embryonated chicken
eggs. After 3 days at 33 °C, the allantoic fluid was harvested and
clarified by low speed centrifugation (1000 g, 15 min, 4
°C). Aliquots were stored frozen at -80 °C.
Hemagglutination activity of viruses was determined in microtiter
plates. Serial 2-fold virus dilutions of 50 µl each were prepared
in phosphate-buffered saline (PBS) and 50 µl of a 0.5% suspension
of chicken erythrocytes were added to each dilution. After 60 min at 4
°C, the hemagglutination activity (HA-units/ml) was determined as
the reciprocal value of the highest dilution causing complete
agglutination.
Cell Culture
MDCK cells (type I) were grown in
Eagle`s minimal essential medium supplemented with 10% fetal calf
serum. For studies of cell polarity, 0.4-µm pore size polycarbonate
Transwell filters (Costar, Cambridge, MA) were used. On the fourth day
of culture, the polarization of the monolayer was assessed by measuring
the electrical resistance between the apical and the basolateral
compartments of the filter chamber using a Millicell-ERS instrument
(Millipore). Only filter cultures with an electrical resistance of at
least 2000 cm
were used for experiments.
Cell Surface Labeling
MDCK I cells were grown to
confluence in 94-mm Petri dishes (about 7 10
cells), rinsed with ice-cold phosphate-buffered saline, pH 7.4,
containing 1 mM Ca
and 1 mM Mg
(PBS+), and cooled in this buffer for 10
min at 4 °C. Cell surface proteins were labeled by incubating the
cells with sulfo-NHS-biotin (0.5 mg/ml in PBS+, 3 ml/dish) for 30
min at 4 °C with gentle agitation. The monolayers were washed once
with ice-cold PBS+ containing 0.1 M glycine and incubated
in the same buffer for 15 min at 4 °C. For selective labeling of
the apical or basolateral surface of filter-grown cells, the
biotinylation reagents were added either to the apical (1.5 ml) or
basolateral (2.5 ml) compartment of the filter chamber, while the
opposite compartment was incubated with PBS+/0.1 M glycine.
Streptavidin and Lectin Precipitation
Cell
monolayers derivatized with biotin were scraped from the Petri dishes
into ice-cold PBS, pH 7.4, using a rubber policeman. The cells were
pelleted by centrifugation (600 g, 10 min, 4 °C),
resuspended in 500 µl of 20 mM Tris-HCl, pH 7.5, and lysed
by addition of 500 µl of 2
concentrated RIPA-buffer (2%
Triton X-100, 2% deoxycholate, 0.2% SDS, 400 mM NaCl, 40
mM Tris-HCl, pH 7.5, 20 mM iodoacetamide, 2
mM PMSF, 100 units/ml aprotinin). Following incubation on ice
for 60 min, insoluble material was removed by centrifugation (105,000
g, 60 min, 4 °C). The cell lysate (1 ml) received
100 µl of a 50% slurry of streptavidin agarose (prewashed three
times with RIPA buffer) and was incubated overnight at 4 °C while
rotating head over tail. The streptavidin agarose was pelleted by
centrifugation (14,000
g, 2 min, 4 °C), washed
three times with RIPA buffer and once with 20 mM Tris-HCl, pH
6.8. Precipitated proteins were eluted by heating the
streptavidin-agarose in 50 µl of 2
concentrated reducing
SDS sample buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 10%
glycerol, 0.02% bromphenol blue, 200 mM dithiothreitol) at 95
°C for 10 min with constant agitation. Isolation of cellular
glycoproteins by immobilized lectins was performed in principle like
the streptavidin precipitation differing only in the lysis buffer used
(final concentration: 1% Triton X-100, 200 mM NaCl, 20 mM Tris-HCl, pH 7.5, 2 µg/ml leupeptin, 2 µg/ml pepstatin).
In addition, divalent cations Ca
,
Mg
, and Mn
(1 mM each)
were added to the lysates prior to overnight incubation with
immobilized lectins at 4 °C. Triton lysis buffer without divalent
cations was used for washing the beads.
In Vitro Virus Binding Assay
Cell surface proteins
isolated by streptavidin precipitation or cellular glycoproteins
isolated by lectin precipitation were separated by 10% SDS-PAGE under
reducing conditions (11) and transferred to nitrocellulose
membranes by electroblotting(12) . To prevent loss of O-acetyl groups, electroblotting was modified by lowering the
pH values of the two buffers at the anode side from 10.4 to 9.0 (facing
the anode) and 7.4 (facing the nitrocellulose), respectively.
Nonspecific binding sites were blocked by incubating the nitrocellulose
with PBS containing 1% BSA for 60 min at room temperature. The blots
were washed with PBS for 5 min. After removal of the buffer, some drops
of virus suspension with a hemagglutinating activity of at least 512
HAU/ml were added and spread over the whole blot by covering it with a
piece of parafilm. Virus was allowed to bind for 60 min at 4 °C.
The nitrocellulose was washed three times with ice-cold PBS containing
0.05% Tween 20, 5 min each, and incubated with a filtered solution of
PBS containing 1 mM -naphthyl acetate and 0.1%
(mass/volume) 4-chloro-2-methylbenzenediazonium salt. The esterase
reaction was allowed to take place at room temperature and was stopped
by rinsing the blots with water. For alkaline hydrolysis of O-acetyl esters, the nitrocellulose membranes were incubated
directly after electroblotting with 0.1 M NaOH for 30 min at 4
°C and washed three times with PBS, 5 min each. For detection of
glycoproteins the blots were probed with different digoxigenin-labeled
lectins as described previously(13) .
Detection of Sialic Acid and Biotin Residues
For
selective oxidation of sialic acids, proteins immobilized on
nitrocellulose were incubated in the dark for 20 min at 0 °C with 1
mM sodium metaperiodate in 100 mM acetate buffer, pH
5.5. The membranes were washed as above, and oxidized sialic acids were
labeled by incubation with digoxigenin-succinyl--amidocaproic acid
(1:5000) in 100 mM acetate buffer, pH 5.5, for 60 min at room
temperature. The blots were rinsed twice in PBS and washed three times,
10 min each with PBS before incubation with 1% BSA in PBS overnight at
4 °C. After washing the membrane as above, digoxigenin-labeled
sialoglycoproteins were detected by incubating the blots with
anti-digoxigenin Fab fragments conjugated to alkaline phosphatase
(1:1000 in PBS) for 60 min at room temperature. The blots were washed
and immersed at room temperature in a solution of 37.5 µl of BCIP
and 50 µl of NBT diluted in 10 ml of 100 mM Tris-HCl, pH
9.5, 50 mM MgCl
, 100 mM NaCl. The
reaction was stopped by rinsing the blots with water. For detection of
biotinylated proteins, blots were blocked with 1% BSA in PBS for 60 min
at room temperature or overnight at 4 °C. After washing the
nitrocellulose three times, 5 min each with PBS, some drops of
streptavidin-horseradish peroxidase (1:1000) in PBS were added, the
blots covered with a piece of parafilm, and the
streptavidin-horseradish peroxidase allowed to bind for 60 min at 4
°C. The blots were rinsed twice with PBS containing 0.1% Tween 20
and washed with this buffer three times for 10 min each. A
chemoluminescent substrate (ECL detection reagent, Amersham,
Braunschweig) was used for detection of streptavidin-horseradish
peroxidase by short exposure to an autoradiography film.
Triton X-114 Phase Separation and Enzyme
Treatments
Cell surface biotinylated MDCK I cells (7
10
cells) were extracted with the detergent Triton X-114
followed by temperature-induced phase separation of the
extracts(14) . Reextracted aqueous and detergent phases were
diluted to 1 ml with Tris-buffered saline (TBS) and subjected to WGA
precipitation or streptavidin precipitation. Precipitates were probed
with influenza C virus in a ligand blot assay (see above).
Phosphatidylinositol-specific phospholipase C treatment of detergent
phases with subsequent temperature-induced phase separation was
performed as described(14) . For treatment with glycosidases,
the biotin-labeled proteins were precipitated from the detergent phase
with immobilized streptavidin and were digested with one of the
following glycosidases while still bound to the streptavidin-agarose:
100 milliunits of sialidase from V. cholerae (50
mM acetate buffer, pH 5.5, 154 mM NaCl, 9 mM CaCl
), 2 units of N-glycosidase F (50 mM phosphate buffer, pH 7.0, 1% Triton X-100, 1 mM
2,3-dehydro-2-deoxy-N-acetylneuraminic acid), 5.7 milliunits
of O-glycosidase (cf. buffer for N-glycosidase F). Glycosidases (diluted in 100 µl of
buffer) were added to the streptavidin-agarose and incubated in a
thermomixer 5436 (Eppendorf) at 37 °C with agitation for 60 min
(sialidase) or overnight (N-glycosidase F, O-glycosidase). Of note, all glycosidase treatments were
performed in the presence of leupeptin (1 µg/ml), pepstatin (1
µg/ml), and PMSF (1 mM). The streptavidin-agarose was
washed and the biotinylated proteins eluted as described above (see the
previous section on streptavidin and lectin precipitation). The eluted
proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose,
and probed with influenza C virus or streptavidin-horseradish
peroxidase (see previous sections). Polyacrylamide gels were silver
stained according to the methods of Merril et al.(15, 16) using two commercially available kits
(Silver Stain Kit, Silver Stain Plus Kit, Bio-Rad,
Mnchen). Desialylation of glycoproteins
transferred to nitrocellulose was performed after blocking the membrane
with 1% BSA in PBS (60 min, room temperature) by adding some drops of V. cholerae sialidase (1 units/ml, buffer as above)
to the blot and covering it with a piece of parafilm. Sialidase was
allowed to act for 2 h at 37 °C in a humid atmosphere followed by
three wash steps with PBS, 5 min each. For P. haemolyticaO-sialoglycoprotease treatment, biotinylated MDCK I
monolayers (4
10
cells) were incubated for 60 min
at 37 °C with 1 ml of PBS+ in the presence or absence of the
enzyme (25 µl). After washing the cells three times with ice-cold
PBS+, they were lysed and subjected to streptavidin precipitation
(see above). The precipitated cell surface proteins were analyzed by
the in vitro virus binding assay and streptavidin-horseradish
peroxidase binding (see above).
Endocytosis Assay
Surface-labeled cells were
incubated with PBS+ at 37 °C. At different times, cells were
rinsed with ice-cold PBS, and cell surface proteins were digested with
1 mg/ml proteinase K in PBS containing 5 mM EDTA at 4 °C
for 45 min. The digestion was stopped by addition of 2 mM PMSF. Protease-resistant, biotinylated proteins were precipitated
from cell lysates with immobilized streptavidin and internalized
influenza C virus receptors were detected by the in vitro virus binding assay described above. Quantitation was performed by
densitometric scanning of the blots.Kinetics of Virus Internalization
MDCK I cell
monolayers (1-2 10
cells) were washed twice
with ice-cold PBS+ and influenza C virus (about 5 HAU/ml in
PBS+) was allowed to bind to the monolayers for 60 min at 4
°C. Virus not adsorbed to the cells was washed away with ice-cold
PBS+. The monolayers were incubated with medium at 37 °C for
different time periods after which the cells were rinsed with ice-cold
PBS+. Virus that had not been internalized was neutralized at the
cell surface by a polyclonal antiserum (1:100 in PBS+) for 60 min
at 4 °C. The monolayers were washed three times with ice-cold
PBS+ and incubated with 2 ml of medium without fetal calf serum at
33 °C. After 24 and 48 h, 100 µl of medium was removed and
virus released in the supernatant titrated by a hemagglutination assay
(see above).
The Receptor Determinant Neu5,9Ac
We analyzed the
glycoproteins of MDCK I cells for their ability to mediate influenza C
virus attachment. Glycoproteins were precipitated from cell lysates
using immobilized lectins of different specificity. ConA preferentially
interacts with N-linked oligosaccharides of the high mannose
and hybrid type, weakly with bi-antennary complex structures, and not
with tri- and tetra-antennary complex
oligosaccharides(17, 18) . Recognition by lentil
lectin (LCA) requires both the presence of two Is
Present on Several MDCK I Glycoproteins
-linked mannose
residues and the presence of fucose attached to the Asn-linked GlcNAc.
Exposure of terminal GlcNAc residues enhances the binding(19) .
Jacalin recognizes O-glycans of the sequence
Gal
1-3GalNAc-Ser/Thr. This sequence with an additional
sialic acid residue
2,3-linked to Gal is also
recognized(20) . WGA shows affinity for GlcNAc and sialic acid
residues(21) , and SNA specifically binds to sialic acids
2,6-linked to Gal(22) . The precipitated glycoproteins
were separated by SDS-PAGE, transferred to nitrocellulose membranes,
and probed with influenza C virus. The viral acetylesterase activity
was used to directly visualize receptor-bound virions with a
chromogenic esterase substrate. As shown in Fig. 1, influenza C
virus recognized a different number of glycoproteins depending on the
lectin used for precipitation. A strong binding was mediated by a
cluster of glycoproteins in the 38-55 kDa range precipitated by
both WGA and Jacalin. Glycoproteins precipitated by the other lectins
were only poorly recognized by influenza C virus with the exception of
a 50-55-kDa glycoprotein precipitated by LCA.
residues.
A 40-kDa Glycoprotein Is the Major Cell Surface Protein
Recognized by Influenza C Virus
An important criterion for a
functional virus receptor is its location on the cell surface.
Therefore, we analyzed the distribution of O-acetylated
sialoglycoproteins between the plasma membrane and intracellular
compartments by determining their sensitivity to cell surface digestion
by sialidase at 37 °C (Fig. 3). Comparison with the
untreated control (lane a) indicates that many glycoproteins
were protected from desialylation (lane b). Binding of
influenza C virus to only a few glycoproteins was diminished by the
sialidase treatment, the major band being a protein of 40 kDa. To
confirm this result, we labeled cell surface proteins at 4 °C using
a water-soluble, membrane-impermeable sulfonated N-hydroxysuccinimide ester of biotin. The biotinylated
proteins were specifically precipitated from cell lysates by
immobilized streptavidin. Among the O-acetylated
sialoglycoproteins only the 40-kDa glycoprotein was recovered by this
procedure to a significant extent (lane c). When cell surface
labeling was followed by sialidase treatment, the recognition of this
protein by influenza C virus was totally abolished (lane d).
We repeated the experiment using proteinase K at 4 °C instead of
sialidase at 37 °C to allow effective digestion of cell surface
proteins at a temperature, at which endocytosis is arrested. The result
obtained with this approach (not shown) was the same as that shown in Fig. 3. The most prominent glycoprotein recognized by influenza
C virus was a 40-kDa glycoprotein which mainly resided at the plasma
membrane, whereas many other O-acetylated sialoglycoproteins
were predominantly found in intracellular compartments. The 40-kDa cell
surface sialoglycoprotein was designated gp40.
1-3GalNAc on O-glycans and does not tolerate substitution of Gal or GalNAc
by sialic acid(25) . Fig. 4B shows that PNA did
not react with any cell surface glycoprotein (lane a) unless
they were pretreated with sialidase (lane b). Desialylation
did not markedly change the mobility of gp40 on SDS-PAGE as verified by
staining with streptavidin-horseradish peroxidase (cf.Fig. 7). We found that among several O-glycosylated cell surface proteins only gp40 efficiently
mediated influenza C virus binding (lane c).
Surface Distribution of gp40 on Polarized
Cells
MDCK cells have provided a well characterized in vitro model system for the study of cell polarity(10) .
Polarized cells are characterized by two distinct plasma membrane
domains facing different physiological compartments. The apical and
basolateral membrane are separated by junctional complexes and differ
from each other in their protein and lipid composition. The epithelial
organization found in vivo can be mimicked in culture when the
cells are grown on permeable polycarbonate filters. Using this system,
influenza C virus was able to infect the cells from either membrane
domain, but infection was more efficient when virus was applied to the
apical compartment (Table 1). Progeny virus, titrated by its
hemagglutination activity, was only detected in the apical medium
indicating that maturation and budding of influenza C virus is a polar
process as has been reported(26) . To determine the
distribution of gp40 between the apical and basolateral plasma
membrane, filter-grown cells were selectively biotinylated at either
domain. The labeled proteins were recovered by streptavidin
precipitation, and gp40 was detected by the virus binding assay (Fig. 5). Using this approach, gp40 was labeled at either
domain, but a larger amount was recovered from the apical (lane
c) than from the basolateral (lane d) plasma membrane. As
a control for the equal labeling efficiency on both plasma membrane
domains, the biotinylated apical and basolateral proteins were stained
in parallel with streptavidin-horseradish peroxidase complex (lanes
a and b). The domain-specific distribution of many other
proteins demonstrates the high polarity of filter-grown MDCK I cells.
Gp40 Is an Integral Membrane Protein
Triton X-114
phase separation (27) was used to study the association of gp40
with the plasma membrane. In Fig. 6the binding of influenza C
virus to sialoglycoproteins precipitated from the resulting detergent
and aqueous phases with immobilized WGA is shown. The majority of
proteins recognized by influenza C virus was recovered from the aqueous
phase (lane b) with the exception of a 40-kDa protein which
was enriched in the detergent phase (lane a). Treatment of the
detergent phase with phosphatidylinositol-specific phospholipase C with
subsequent phase separation did not release this glycoprotein into the
aqueous phase (not shown) indicating that it is not attached to the
plasma membrane via a glycosylphosphatidylinositol anchor. The 40-kDa
protein could be biotinylated at the cell surface and precipitated from
the detergent phase with immobilized streptavidin (lane c)
confirming its identity with gp40. Influenza C virus bound also to some
cell surface proteins that were precipitated from the aqueous phase but
to a much lesser extent (lane d). The hydrophobic nature of
gp40 suggests that this cell surface protein contains one or more
membrane-spanning segments.
Gp40 Is a Mucin-type Glycoprotein
We have shown
above that gp40 is a major cell surface sialoglycoprotein of MDCK I
cells, which is bound by Jacalin and PNA, lectins specific for O-glycans. Despite its sialylation, gp40 was not recognized by Maackia amurensis-agglutinin or SNA when Western blots of cell
surface proteins were probed with these two sialic acid-specific
lectins (not shown). To understand this reactivity and to gain a more
detailed insight into the glycosylation of this protein, we analyzed
the susceptibility of gp40 to different exo- and endoglycosidases.
Biotin-labeled cell surface proteins were precipitated with immobilized
streptavidin and subjected to digestion by sialidase and two
endoglycosidases. Gp40 was detected on Western blots either by virus
binding (Fig. 7A) or by streptavidin-horseradish
peroxidase complex (Fig. 7B). Comparison with the
untreated control (lanes a) shows that desialylated gp40 (lanes b) was not recognized by influenza C virus though it
did not show a markedly altered mobility on SDS-PAGE. The
endoglycosidases used were N-glycosidase F, which cleaves all
classes of N-linked carbohydrate chains at the
peptide-carbohydrate linkage(28) , and O-glycosidase,
which exhibits a stringent specificity for the disaccharide core
structure Gal1-3GalNAc cleaving the linkage between GalNAc
and Ser or Thr (29) . These enzymes had no effect both on virus
binding to gp40 and on the electrophoretic mobility (lanes c and e). As O-glycosidase is known not to
tolerate any substitution on Gal or on GalNAc, gp40 was desialylated
prior to incubation with O-glycosidase treatment. After this
treatment, gp40 migrated as two bands of about 35 and 37 kDa (Fig. 7B, lane f). Only the 37 kDa band was
observed when gp40 was treated with O-glycosidase alone,
without addition of a sialidase inhibitor (not shown; the O-glycosidase preparation contained a low sialidase activity,
which could be inhibited by addition of
2,3-dehydro-2-desoxy-N-acetylneuraminic acid). The 37 kDa band
is, therefore, thought to be a partly desialylated form of gp40 that
was not completely digested by the O-glycosidase.
Desialylation of gp40 did not improve its susceptibility to N-glycosidase F (Fig. 7B, lane d).
This result indicates the presence of sialylated O-linked
oligosaccharides of the sequence Gal
1-3GalNAc. Gp40 did not
react with silver stains of the diamine type(16) , but
intensely stained yellow to brown when a silver-staining procedure
adapted from photographic chemical development processes was
used(15) , which is known to stain highly O-glycosylated sialoglycoproteins(30) . After
treatment of gp40 with sialidase and O-glycosidase, the
resulting 35 kDa band was still visualized by this silver-staining
procedure, indicating that it still contained O-glycan
sequences (not shown).
Gp40 Is Subject to Rapid and Constitutive
Endocytosis
Influenza C virus as well as other influenza viruses
require the exposure to low pH to display fusion
activity(33, 34, 35) . As this requirement is
met by the acidic milieu of secondary endosomes, influenza viruses are
thought to enter host cells by receptor-mediated endocytosis. To
understand the dynamics of this process, we first allowed influenza C
virus to bind to MDCK I monolayers for 1 h at 4 °C, a temperature
at which endocytosis is arrested. The cells were then incubated at 37
°C to allow endocytosis to take place. After various times the
process was stopped by putting the cells on ice. Virus which had not
been internalized was inactivated by a neutralizing antiserum.
Internalized virus could escape neutralization and initiate productive
infection. Progeny virus released into the medium was titrated 24 and
48 h post-infection using a hemagglutination assay (Fig. 9).
Some internalization of infectious virus already occurred during the
first 10 min after raising the temperature to 37 °C and increased
as a function of time. We assume that internalization of influenza C
virus is mediated by a receptor which follows a similar endocytosis
kinetics. To test whether gp40 meets this criterion, MDCK I monolayers
were surface labeled with biotin at 4 °C. After an incubation at 33
or 37 °C for 45 min, the cells were rapidly cooled, and gp40 still
exposed at the surface was digested with proteinase K at 4 °C. The
virus binding assay of Fig. 10A shows that gp40, that
had been internalized during the 33 or 37 °C chase and thereby had
acquired resistance to exogenous protease treatment, could be recovered
by streptavidin precipitation, whereas surface-labeled gp40 was
completely digested, when the cells were kept at 4 °C. The kinetics
of gp40 internalization at 37 °C (Fig. 10B) shows
that uptake of gp40 could already be detected 10 min after raising the
temperature and reached a maximum at 40 min. With a longer incubation
time, a decrease of protected gp40 was observed which may be the result
of intracellular degradation or of a recycling process back to the
plasma membrane.
cells) for 60 min at 4 °C. The cells
were incubated at 37 °C for the indicated time periods after which
the cells were rapidly chilled and treated with a neutralizing
antiserum directed against influenza C virus for 60 min at 4 °C.
After 24 h (
) and 48 h (
) at 33 °C, the virus yield
was titrated by measuring the hemagglutination activity of the cell
supernatant.
, whereas other types of sialic acids transferred
are ineffective in this respect (38, 39) . These
results demonstrate that influenza C virus uses Neu5,9Ac
as
a receptor determinant to initiate the infection of MDCK I cells.
, very efficiently inhibit the hemagglutination
activity of influenza C virus(3) . This suggests that the
structural features of the oligosaccharide backbone may only be of
minor importance for the recognition of Neu5,9Ac
by
influenza C virus. This conclusion is supported by the finding that
asialo-erythrocytes which have been resialylated to contain
Neu5,9Ac
in three different defined oligosaccharide
sequences were agglutinated equally well by influenza C
virus(4) . On the other hand, 9-O-acetylation of
sialic acid shows remarkably tissue-specific and developmentally
regulated expression in a variety of systems(53) . In addition,
certain types of sialoglycoconjugates have been found to be
predominantly O-acetylated in a given cell
type(54, 55) . Therefore, it is likely that the sialic
acids of gp40 are selectively O-acetylated rendering this
glycoprotein a preferred ligand for influenza C virus. From the
observation that gp40 was easily oxidized by metaperiodate, we conclude
that only part of its sialic acids are 9-O-acetylated because
this substitution is known to render sialic acid rather resistant to
periodate oxidation(52) . This agrees with the finding that
de-O-acetylation prior to oxidation did not markedly improve
the reaction as would be expected if most sialic acids were
9-O-acetylated. Perhaps 9-O-acetylation is restricted
to certain sialic acid residues of gp40. Interestingly, structural
studies of different O-acetylated gangliosides have shown that
in almost all cases the 9-O-acetyl group is located on a
specific terminal
2,8-linked sialic acid(53) . Future work
has to show what factors are responsible for the preferential O-acetylation of gp40.
, N-acetyl-9-O-acetylneuraminic acid; LCA, Lens culinaris agglutinin; MDCK, Madin-Darby canine kidney;
PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered
saline; PMSF, phenylmethylsulfonylfluoride; PNA, peanut agglutinin;
SNA, Sambucus nigra agglutinin; WGA, wheat germ agglutinin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.