From the
N-Linked glycosylation usually occurs at the sequon,
Asn-X-Ser/Thr. In this sequon, the side chain of the hydroxy
amino acid (Ser or Thr) may play a direct catalytic role in the
enzymatic transfer of core oligosaccharides to the Asn residue. Using
recombinant variants of rabies virus glycoprotein (RGP), we examined
the influence of the hydroxy amino acid on core glycosylation
efficiency. A variant of RGP containing a single Asn-X-Ser
sequon at Asn
One of the most common types of protein modification is N-linked glycosylation, in which oligosaccharides are added to
specific Asn residues(1, 2) . N-Linked
glycosylation plays a critical role in the expression of most cell
surface and secreted proteins in eukaryotes by promoting their delivery
to the cell surface or extracellular space. N-Linked
glycosylation may also be required for protein stability, antigenicity,
or biological function(3, 4) . Often, these effects
depend on the number and position of the oligosaccharide chains added
to a protein(3, 5) . This is determined during core
glycosylation, in which the presynthesized core oligosaccharide,
Glc
Using this
available information, many investigators have used site-directed
mutagenesis to introduce novel Asn-X-Ser/Thr sequons into
proteins to study the role of glycosylation in protein
transport(15, 16, 17, 18) ,
stability(17, 19) , proteolytic processing(16) ,
and biological activity(20, 21, 22) . Other
studies have used the glycosylation of novel sequons to mark the
transfer of defined regions of proteins into the rough endoplasmic
reticulum lumen to determine protein topology(23, 24) .
Although the introduction of novel glycosylation sites into proteins
has great potential for modulating the expression and function of
recombinant proteins, many sequons introduced into proteins by
site-directed mutagenesis are not glycosylated (15, 21, 22) or are glycosylated inefficiently (20) for reasons which are not understood. In some studies, as
many as three of four novel sequons engineered into a protein fail to
be glycosylated at all(17) . Further characterization of the
protein signals which regulate N-linked glycosylation at
specific sequons is essential for understanding natural glycoprotein
synthesis and for facilitating recombinant protein design.
A number
of studies have compared the amino acid sequences of glycoproteins in
order to identify protein signals which influence core glycosylation.
Those studies suggest that the amino acid at the hydroxy position of a
sequon (i.e. Ser or Thr) may influence the likelihood of
glycosylation at that site, since Asn-X-Thr sequons are
glycosylated two to three times more often than Asn-X-Ser
sequons(6, 9) . Studies using peptides as
oligosaccharide acceptors in membrane preparations also demonstrate a
greater acceptor activity of peptides containing an Asn-X-Thr
sequon than peptides containing an Asn-X-Ser
sequon(25) .
In this study, we examine the impact of the
hydroxy amino acid on the core glycosylation efficiency of rabies virus
glycoprotein (RGP).
Previous studies have suggested that the
presence or absence of a membrane anchor can influence the core
glycosylation of sequons in some proteins(20) . We have shown
previously that the core glycosylation efficiency of sequons in RGP is
not affected by removal of the membrane anchor(26) . We extended
those studies by examining whether the influence of the hydroxy amino
acid on the core glycosylation of sequon 1 was also observed when the
membrane anchor of RGP was removed. To examine this, we generated
additional variants of RGP which contain stop codons at position 434,
immediately upstream of the membrane anchor (Fig. 1A,
arrow). We found that substitution of Thr for Ser at sequon 1
up-regulated core glycosylation at Asn
A variety of factors have been shown to influence the core
glycosylation of sequons in glycoproteins. These include changes in the
amino acid sequence near an Asn residue(36) , deletion or
addition of other sequons in the protein(37, 38) , and
alterations in the position of a sequon relative to the NH
This study examines the
influence of the hydroxy amino acid in an Asn-X-Ser/Thr sequon
on the regulation of core glycosylation efficiency. Previous studies
have found that Asn-X-Thr sequons are more likely to be
glycosylated than Asn-X-Ser sequons in
proteins(6, 9) . However, those studies do not consider
the effect of the hydroxy amino acid on the efficiency of core
glycosylation of sequons which are glycosylated. Other studies using
presynthesized peptides as oligosaccharide acceptors demonstrate that
peptides containing Asn-X-Thr sequons are glycosylated more
efficiently than peptides containing Asn-X-Ser sequons and
that Asn-X-Cys sequons in peptides can serve as weak
oligosaccharide acceptors(25) . This effect of the hydroxy amino
acid on core glycosylation has been explained by a catalytic model, in
which the hydroxy amino acid actively participates in oligosaccharide
transfer through a series of hydrogen bond transfer reactions involving
oligosaccharyltransferase, the hydroxy amino acid side chain, and the
We do not detect
glycosylation of RGP(1-)C39 in the cell-free system. This is not
surprising, since Asn-X-Cys sequons are rarely glycosylated (9) and are relatively poor oligosaccharide acceptors (25). The
inability to isolate RGP(1-)C39 from cell lysates following
immunoprecipitation may refect inappropriate folding, destabilization,
or aggregation of the protein; this could result from a lack of
glycosylation. An unglycosylated RGP variant which lacks all three
sequons is also not detectable in stably transfected cells under these
conditions(14) . It is also possible that the presence of Cys at
position 39 blocks glycosylation indirectly by altering disulfide
bonding in RGP; altered disulfide bonding could also cause misfolding,
destabilization, or aggregation of the protein(45) . Two other
Cys residues are present at the amino terminus of RGP, at positions 24
and 35(32) ; those residues are predicted to be present in the
endoplasmic reticulum lumen when Asn
Our findings suggest that the amino acid at the hydroxy position of
a sequon should be considered in the design of novel sequons in
recombinant proteins. In particular, our findings suggest that
Asn-X-Thr is generally a more efficient sequon than
Asn-X-Ser and should be used whenever possible to maximize the
likelihood of efficient glycosylation. Studies in which novel sequons
are introduced into recombinant proteins by genetic engineering often
do not provide information about the hydroxy amino acid at the
glycosylation sites. However, in a series of studies which provide this
information, we find that 92% () of novel Asn-X-Thr sequons
were glycosylated, in contrast to only 57% () of novel
Asn-X-Ser
sequons(15, 20, 21, 22) .
By altering
the hydroxy amino acid at sequon 1 in RGP, we have generated novel
forms of RGP which are fully glycosylated at all three sequons. This
demonstrates that efficient core glycosylation at sequon 1 does not
impair the efficient core glycosylation of sequons 2 and 3. This lack
of interaction between sequons which we observe in RGP has also been
observed in ovalbumin(46) . In other proteins, altering the
glycosylation at one sequon in a protein does appear to impact on the
glycosylation of other sequons(37, 38) . Therefore,
effects of sequon interaction must be determined in individual proteins
on a case by case basis.
The novel forms of RGP generated in this
study may provide further information about the role of site-specific
glycosylation of RGP in rabies virus infection. Previous analysis of
RGP isolated directly from viral particles of the ERA strain (used in
the present study) and the challenge virus standard strain had not
detected glycosylation at sequon 1(47, 48) . In
contrast, our studies clearly demonstrate a potential for this site to
be glycosylated at low levels in both cell-free systems and transfected
cells. The failure to detect RGP glycosylated at all three sequons in
viral particles may reflect a relationship between glycosylation at
Asn
We thank Peter Curtis of The Wistar Institute for his
critical evaluation of the manuscript and Jean Smith of The Centers for
Disease Control for generously providing the monoclonal antibodies used
in this study.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
was modified by site-directed mutagenesis to
change the sequon to either Asn-X-Cys or Asn-X-Thr.
The impact of these changes on core glycosylation efficiency was
assessed by expressing the variants in a cell-free
transcription/translation/glycosylation system and in transfected
tissue culture cells. Substitution of Cys at position 39 blocks
glycosylation, whereas substitution of Thr dramatically increases core
glycosylation efficiency of Asn
in both membrane-anchored
and secreted forms of RGP. The substitution of Thr for Ser also
dramatically enhances the level of expression and cell surface delivery
of RGP when the sequon at Asn
is the only sequon in the
protein. Novel forms of membrane-anchored and secreted RGP which are
fully glycosylated at all three sequons were also generated by
substitution of Thr at position 39.
Man
GlcNAc
, is transferred from a
lipid precursor to an Asn residue by the enzyme
oligosaccharyltransferase(1, 2, 6) . Despite the
importance of N-linked glycosylation, little is known about
the protein signals which control the efficiency of oligosaccharide
addition at specific Asn residues. The tripeptide sequence (or sequon)
asparagine-X-serine or asparagine-X-threonine
(Asn-X-Ser/Thr) is generally required for N-linked
glycosylation(7) . In this sequon, X can be any amino
acid, although proline (Pro) is rarely found at this
position(8, 9) . Only rare examples of N-linked
glycosylation at other sequences (e.g. Asn-X-Cys and
Asn-Gly-Gly-Thr) have been described(9) . However, although the
Asn-X-Ser/Thr sequon is generally required for N-linked glycosylation, many such sequons are glycosylated
inefficiently(10, 11, 12, 13, 14) ,
and many sequons are not glycosylated at all (9).
(
)Our previous studies
demonstrate that the sequon at Asn
in RGP is inefficiently
glycosylated in both cell-free and whole cell
systems(14, 26) . In the present study, we use
site-directed mutagenesis to replace the Ser at position 39 with either
Thr or Cys and test directly the effect of these mutations on core
glycosylation of Asn
. We find that the hydroxy amino acid
can have a dramatic effect on the efficiency of core glycosylation and
in doing so can influence protein expression.
Site-directed Mutagenesis and Plasmid
Construction
Site-directed mutagenesis was performed using the
polymerase chain reaction-based method, splicing by overlap extension
(27) as described previously(14) . The Ser at position 39 of
RGP(1-) was replaced with either Thr or Cys to generate plasmids
RGP(1-)T39 and RGP(1-)C39, respectively. For this mutagenesis, the
codon TCA (Ser) was changed to ACA (Thr) or to TGT (Cys).
In both cases, a PvuII site near sequon 1 was deleted by a
single nucleotide substitution to facilitate screening of recombinant
plasmids; this latter mutation did not alter the amino acid sequence.
The amplified products containing the desired mutations were digested
with HindIII and XhoI to generate a 422-base pair
fragment, which was ligated into the HindIII and XhoI
sites of RGP(1-), replacing the corresponding sequence (14). The
resulting plasmids were isolated following transformation into Escherichia coli HB101 and were screened by restriction enzyme
analysis. The full HindIII-XhoI insert in each
plasmid was analyzed by DNA sequencing to confirm the presence of the
desired mutations and the absence of unwanted base changes introduced
during polymerase chain reaction amplification. Additional plasmids
containing Thr at position 39 were generated by subcloning the HindIII-XhoI fragment of RGP(1-)T39 into the
corresponding position of RGP(WT), RGP(1-)T434,
(
)and RGP(WT)T434 to generate plasmids RGP(WT)T39,
RGP(1-)T434T39, and RGP(WT)T434T39, respectively.
In Vitro Transcription and Expression in the Cell-free
System
RNA encoding RGP variants was generated by in vitro transcription with T7 RNA polymerase as described previously (14). In vitro translation was performed using a rabbit reticulocyte
lysate system supplemented with [S]methionine
and dog pancreas microsomes, and translation products were analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography as
described(14, 26) .
Transfection of Plasmids into CHO Cells
Plasmids
were expressed in the CHO cell line, Lec 1(28) , as described
previously (14). Plasmids encoding RGP(1-)C39 and RGP(1-)T39 were
co-transfected with the selectable plasmid, pSV2Neo(29) , into
Lec 1 cells using a polybrene method(30) . G418-resistant
colonies were amplified and screened for expression of the encoded
proteins by indirect immunofluorescence of fixed cells using the mouse
monoclonal anti-rabies antibody 80-6(31) . Colonies expressing
the encoded proteins were subcloned by a single round of limiting
dilution. Transient transfection of plasmids into Lec 1 cells was
performed using a DEAE-dextran method as described
previously(14) .
Metabolic Labeling and Preparation of Cell
Lysates
For metabolic labeling, 80-90% confluent 100-mm
dishes of cells expressing each RGP variant were washed twice with
Hank's balanced salt solution. Cells were preincubated at 37
°C for 2 h in complete medium (14) containing 50 µg/ml
castanospermine (Genzyme Corp., Cambridge, MA), washed three times with
Hank's balanced salt solution, and then incubated at 37 °C
for 4 h with 5 ml of methionine-free -minimal essential medium
containing 50 µCi of [
S]methionine and 50
µg/ml castanospermine. Cell lysates were prepared essentially as
described(14) , except that cells were resuspended and lysed in
200 µl of lysis buffer containing 50 mM Tris, pH 8, 150
mM NaCl, 1% Triton X-100, and 200 µg/ml
phenylmethylsulfonyl fluoride.
Immunoprecipitation
Cell lysates were precleared
by incubation with protein A beads shaking for 16 h at 4 °C.
Radiolabeled RGP-related proteins were immunoprecipitated from
precleared cell lysates by adding polyclonal anti-rabies antiserum at a
1:100 dilution and shaking for 1 h at 4 °C. Immune complexes were
isolated by adding protein A beads and shaking for 1 h at 4 °C.
Protein A beads were then washed, and immunoprecipitated proteins were
eluted into sample buffer and analyzed by SDS-polyacrylamide gel
electrophoresis as described previously(14) .
Immunofluorescence
Cell surface expression of
RGP-related proteins was examined by indirect immunofluorescence of
live cells as described previously(14) . The secondary antibody
used for staining was affinity-purified, fluorescein-conjugated, goat
anti-mouse Ig (Organon Teknika Corp., Durham, NC) which was diluted
1:50 in -minimal essential medium and precleared by centrifugation
at 12,000
g for 30 min. Intracellular expression of
RGP proteins was examined by indirect immunofluorescence of cells
following fixation and permeabilization. Cells grown on glass
coverslips were rinsed twice with phosphate buffered saline (PBS),
fixed, and permeabilized with methanol:acetone (1:1) for 5 min at
-20 °C, and air-dried. Indirect immunofluorescent staining of
fixed cells was performed at room temperature. Cells were preincubated
for 1 h in PBS containing 5% goat serum (GS). Culture supernatant
containing the primary mouse monoclonal anti-RGP antibody 80-6 was
precleared by centrifugation at 12,000
g for 30 min,
diluted with an equal volume of PBS, and supplemented with GS at a
final concentration of 5%. Cells were incubated with the primary
antibody preparation for 45 min and then washed three times for 10 min
each in PBS containing 5% GS and 1% Triton X-100. The secondary goat
anti-mouse antibody described above was diluted to a final
concentration of 1:100 in PBS containing 5% GS. Cells were incubated
with the secondary antibody preparation for 45 min, washed three times
as described above, rinsed in PBS, mounted in PBS:glycerol (1:1), and
examined by epifluorescence and phase contrast microscopy. The
monoclonal antibodies used for staining of live cells included 509-6
and 523-11 from The Wistar Institute, Philadelphia, PA, and 80-6, 36-6,
111-6, and 105-2, which were generously provided by J. Smith from the
Centers for Disease Control, Atlanta, GA.
Generation of RGP Variants with Amino Acid
Substitutions at the Hydroxy Position
In the studies described
below, RGP is used as a model glycoprotein to study the influence of
the hydroxy amino acid on core glycosylation efficiency. RGP from the
ERA strain is a 505-amino acid type 1 transmembrane protein with three
Asn-X-Ser/Thr sequons for N-linked glycosylation at
Asn (sequon 1), Asn
(sequon 2), and
Asn
(sequon 3) (Fig. 1A, RGP(WT); Refs. 32
and 33). The cDNA encoding this protein was previously subcloned into
the pSG5 vector to generate a plasmid encoding RGP(WT) (34). We
previously generated a variant of RGP(WT) with a single sequon at
Asn
by replacing the Thr in sequons 2 and 3 with Ala (Fig. 1B, RGP(1-); Ref. 14). For this study, additional
variants were generated from RGP(1-), in which the Ser in sequon 1 was
replaced with Thr or Cys by site-directed mutagenesis (Fig. 1B, RGP(1-)T39 and RGP(1-)C39,
respectively).
Figure 1:
Structure of RGP(WT) and RGP variants.
The structures of RGP(WT) and RGP variants are shown. Dark bars indicate the presence of an Asn-X-Ser/Thr sequon at
Asn (sequon 1), Asn
(sequon 2), or
Asn
(sequon 3). Open bars represent the deletion
of the corresponding sequon by site-directed mutagenesis in which the
Ser or Thr residue in the sequon was replaced with Ala. The
transmembrane domain is indicated by light stippling, and the
cytoplasmic tail is indicated by dark stippling. The amino
acid sequence at sequon 1 is indicated for each protein. An arrow indicates the position of the stop codon in truncated RGP mutants
(position 434). Panel A, RGP(WT) and RGP(WT)T39 each contain
three sequons for N-linked glycosylation. B, RGP(1-)
and RGP(1-)T39 each contain a single sequon at Asn
;
RGP(1-)C39 contains the sequence Asn-X-Cys at
Asn
, which can also serve as an oligosaccharide addition
site.
Analysis of Core Glycosylation of RGP Variants in a
Cell-free System
To analyze the influence of the hydroxy amino
acid on core glycosylation efficiency, we expressed RGP variants in a
cell-free transcription/translation/glycosylation system. RNA was
generated from plasmids by in vitro transcription with T7 RNA
polymerase and was translated in a rabbit reticulocyte lysate system
supplemented with [S]methionine and canine
pancreas microsomes(14) . We have shown previously that this
system removes the amino-terminal signal sequence from RGP and adds N-linked core oligosaccharides(14) . Translation
products were separated by gel electrophoresis and detected by
autoradiography (Fig. 2).
Figure 2:
Core
glycosylation of RGP variants in the cell-free system. RNA encoding
selected RGP variants was expressed in the cell-free system with
{S] methionine in the presence (+) or
absence (-) of pancreatic microsomes (PM), and the
translation products were analyzed by polyacrylamide gel
electrophoresis and autoradiography. Variants included RGP(1-) (lanes A and B), RGP(1-)T39 (lane C),
RGP(WT) (lane D), and RGP(WT)T39 (lane E). The
migration positions of proteins containing zero, one, two, and three
core oligosaccharides are shown on the right.
Expression of RGP(1-) in the
cell-free system in the absence of microsomes yields a major product
which represents the unglycosylated protein with the amino-terminal
signal sequence attached (Fig. 2, lane A; Ref. 14). In
the presence of microsomes, expression of RGP(1-) yields two products (Fig. 2, lane B), which we have shown previously
represent the unglycosylated protein with the signal sequence removed (lower band) and the protein glycosylated with a single core
oligosaccharide (upper band; Refs. 14 and 26). Expression of
RGP(1-)C39, which contains the sequence Asn-X-Cys at
Asn, yields only the unglycosylated product (data not
shown), indicating that sequon 1 is not glycosylated when cysteine is
present at the hydroxy position; this result was expected, since
Asn-X-Cys sequences are rarely glycosylated. Remarkably,
expression of RGP(1-)T39 yields predominantly the glycosylated product (Fig. 2, lane C); this demonstrates that the single,
conservative amino acid substitution of Thr for Ser at the hydroxy
position of a sequon can dramatically up-regulate the efficiency of
oligosaccharide addition.
in a variant
truncated at position 434, analogous to the effect seen in the
full-length, membrane-anchored protein (data not shown).
Analysis of Core Glycosylation of RGP Variants in
Transfected Cells
We next examined the influence of the hydroxy
amino acid on the core glycosylation efficiency of sequon 1 in
transfected tissue culture cells. To do this, plasmids encoding
RGP(1-)C39 and RGP(1-)T39 were stably transfected into Lec 1 CHO
cells. Transfected cells were screened by indirect immunofluorescence
for expression of RGP proteins, and cell lines expressing each protein
were subcloned by limiting dilution. To analyze core glycosylation of
the RGP variants in the transfected cells, we blocked the processing of
core oligosaccharides which normally occurs in the rough endoplasmic
reticulum and Golgi apparatus. This processing is partially blocked in
the Lec 1 cell line due to a deficiency in GlcNAc-T1
transferase(28) . To completely block processing, cells were
incubated before and during metabolic labeling with castanospermine, an
inhibitor of glucosidases I and II(35) . We have shown that
castanospermine blocks the processing of core oligosaccharides on RGP
under these conditions(14) . The cell lines expressing
RGP(1-)T39 and RGP(1-)C39 were analyzed in parallel with previously
isolated, stably transfected Lec 1 cells expressing RGP(1-) and
RGP(-3)(14) , and with untransfected Lec 1 cells as a control.
RGP(-3), which contains a single sequon at Asn, was
shown in previously studies to be fully glycosylated in these
cells(14) . Following metabolic labeling of each cell line with
[
S]methionine, RGPs were immunoprecipitated from
cell lysates using a polyclonal anti-rabies antibody and analyzed by
gel electrophoresis and autoradiography (Fig. 3). These results
demonstrate that RGP(1-) is inefficiently glycosylated in the
transfected cells (Fig. 3, lane C), whereas RGP(-3) is
fully glycosylated (Fig. 3, lane E), consistent with
results from our previous studies(14) . RGP(1-)C39, which
contains an Asn-X-Cys sequon at Asn
, was not
isolated following immunoprecipitation of cell lysates (Fig. 3, lane B). In contrast, we find that the single amino acid
substitution of Thr for Ser at sequon 1 in the variant RGP(1-)T39
dramatically increases the efficiency of core glycosylation in the
transfected cells (Fig. 3, lane D); this result is
consistent with the efficient glycosylation of the Asn-X-Thr
sequon in this variant in the cell-free system.
Figure 3:
Core glycosylation of RGP variants in
transfected CHO cells. The core glycosylation of selected RGP variants
was analyzed in transfected CHO cells. Cell lines expressing
RGP(1-)C39, RGP(1-), RGP(1-)T39, and RGP(-3) were metabolically
labeled with [S]methionine in the presence of
the oligosaccharide processing inhibitor, castanospermine (lanes
B-E, respectively). The radiolabeled RGP variants were
isolated from cell lysates by immunoprecipitation and analyzed by
electrophoresis and autoradiography. Untransfected cells were analyzed
in parallel as a control (lane A). The migration positions of
the unglycosylated protein (0) and the protein glycosylated with a
single core oligosaccharide (1) are indicated on the right.
Analysis of Intracellular and Cell Surface Expression of
RGP Variants in Transfected CHO Cells
We demonstrated previously
that RGP is normally expressed at high levels on the surface of
transfected CHO cells and that cell surface expression requires
glycosylation of at least one sequon in the protein(14) .
Moreover, those studies suggested that the level of cell surface RGP
expression reflects the efficiency of core glycosylation of the protein
when only one sequon is present(14) . To examine further the
role of glycosylation on RGP expression, we compared the cell surface
expression of RGP(1-)C39 and RGP(1-)T39 to that of RGP(1-) and
RGP(WT) in the stably transfected cell lines. This was first assessed
by examining cells following fixation and permeablization (Fig. 4). As shown previously, RGP(1-) is barely detectable in
the transfected cells, whereas RGP(WT) is expressed at high
levels(14) . We find that RGP(1-)C39 is detectable at a low
level similar to that of RGP(1-). In contrast, RGP(1-)T39 is detected
at a level comparable to that of RGP(WT). This demonstrates that the
single amino acid substitution of Thr for Ser in sequon 1 dramatically
increases the expression of RGP(1-).
Figure 4:
Immunofluorescence analysis of cell lines
following fixation and permeabilization. Subclones of CHO cells stably
transfected with plasmids encoding RGP(1-), RGP(1-)C39, RGP(1-)T39,
and RGP(WT) were analyzed by indirect immunofluorescence following
fixation and permeabilization and examined by epifluorescence
microscopy (left) and phase contrast microscopy (right).
To assess the impact of the
hydroxy amino acid on cell surface delivery of RGP, we examined the
transfected cells following indirect immunofluorescent staining of live
cells (Fig. 5). As shown previously, RGP(1-) is detectable at
low levels at the surface of live cells, whereas RGP(WT) is expressed
at high levels(14) . In contrast to RGP(1-), RGP(1-)C39 is not
detected on the cell surface; this finding is consistent with the lack
of glycosylation of this protein as observed in the cell-free system
and with the dependence of RGP on glycosylation for cell surface
transport(14, 34) . In contrast, we find that
RGP(1-)T39 is detected on the cell surface at levels comparable with
that of RGP(WT), demonstrating that the single conservative amino acid
substitution of Thr for Ser at sequon 1 dramatically increases the cell
surface expression of this protein. Similar results were obtained when
CHO cells were examined following transient transfection with the same
plasmids (data not shown). To assess whether RGP(1-)T39 retains
conformational features of RGP(WT), we compared the immunofluorescent
staining of live cells expressing RGP(1-)T39 and RGP(WT) with a panel
of six anti-RGP monoclonal antibodies (see ``Materials and
Methods''). In each case, the staining of RGP(1-)T39 was
comparable with that of RGP(WT) (data not shown).
Figure 5:
Immunofluorescence analysis of live cells.
Cell surface expression of RGP variants was examined by performing
indirect immunofluorescence on live cultures of CHO cells stably
transfected with plasmids encoding RGP(1-), RGP(1-)C39, RGP(1-)T39,
and RGP(WT). Cells were examined by epifluorescence microscopy (left) and phase contrast microscopy (right).
Substitution of Ser
Our previous studies demonstrate that the full-length,
wild-type protein, RGP(WT), and a secreted variant of this protein,
RGP(WT)T434, are inefficiently glycosylated at sequon 1 and are
efficiently glycosylated at sequons 2 and 3(14, 26) . We
next examined whether introduction of the sequon, Asn-X-Thr,
into RGP(WT) could be used to generate a novel form of RGP with full
glycosylation at all three sequons. To do this, we generated a variant
of RGP(WT) which contained the substitution of Thr for Ser at sequon 1 (Fig. 1A, RGP(WT)T39). Expression of RGP(WT) in the
cell-free system yields a major protein product with two core
oligosaccharides and a minor protein product with three core
oligosaccharides, as described previously (Fig. 2, lane
D; Ref. 14). In contrast, RGP(WT)T39, which contains Thr at the
hydroxy position of sequon 1, is fully glycosylated at all three
sequons (Fig. 2, lane E). This result indicates that
efficient glycosylation at sequon 1 (resulting from the presence of Thr
at position 39) does not interfere with the efficient core
glycosylation at sequons 2 and 3. Similar results were obtained using
secreted variants of these proteins, RGP(WT)T434 and RGP(WT)T434T39
(data not shown). These findings demonstrate that the substitution of
Thr for Ser at sequon 1 can be used to generate novel forms of RGP with
full glycosylation at all three sequons.
with Thr in RGP(WT)
Produces a Novel form of RGP Fully Glycosylated at all Three
Sequons
or COOH terminus of a protein (26, 39, 40) or to the membrane
anchor(41) . Core glycosylation efficiency can also be
influenced by factors which do not affect the primary structure of the
protein. For example, core glycosylation efficiency can be modulated by
alterations in protein folding in cell-free systems(42) , by the
growth conditions of cultured cells (13), or by the type of cells used
for protein expression(43) .
-amide group of asparagine(25) . Studies of peptides
containing Thr derivatives further demonstrate that alterations of the
hydroxy amino acid side chain which impact on local stereochemistry can
interfere with enzymatic transfer of the core
oligosaccharide(44) . The studies in this report provide
additional support for this model by directly demonstrating that
substitution of Thr for Ser at an inefficient Asn-X-Ser sequon
in an intact protein can up-regulate the efficiency of core
glycosylation at that site. This effect is observed using both a
cell-free system and transfected tissue culture cells to examine the
co-translational core glycosylation efficiency of these recombinant
proteins. This amino acid substitution is highly conservative and is
unlikely to induce a major change in protein conformation. In fact, our
immunofluorescence analysis using a series of anti-RGP monoclonal
antibodies reveals that recombinant proteins with this amino acid
substitution are displayed on the surface of transfected cells in a
fashion similar to that of the wild-type protein.
is available for
glycosylation and could interact with the Cys residue at position 39.
Similar interactions have been found to impair glycosylation in other
proteins; in interleukin-6, inefficient glycosylation at Asn
was attributed to a conformational constraint on the sequon due
to disulfide bonding between Cys
and
Cys
(36) . The possibility that Cys
in
RGP impairs glycosylation by disulfide bonding, rather than a direct
effect on core glycosylation, is somewhat less likely, since
substitution of Cys at position 38 did not inhibit, and actually
enhanced core glycosylation at Asn
(data not shown).
and the instability of RGP, trimerization, or
inefficient intracellular transport of RGP in infected cells or the
assembly of RGP into viral particles. This is of interest, since
glycosylation of RGP may influence not only cell surface transport of
the protein(14, 34) , but also its ability to provide
immunogenicity as a viral subunit vaccine against rabies virus
infection(33, 49, 50) . It is interesting to
note that the sequon at Asn
and the surrounding amino acid
sequence is highly conserved among different strains of rabies virus
(51, 52). Moreover, this region of RGP (amino acids 29-41)
appears to serve as an epitope for viral recognition by T-helper
cells(53) . The variants generated in this study will help to
further characterize the role of site-specific glycosylation in the
expression and immunogenicity of RGP and in rabies virus infection.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.