©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Hydroxy Amino Acid in an Asn-X-Ser/Thr Sequon Can Influence N-Linked Core Glycosylation Efficiency and the Level of Expression of a Cell Surface Glycoprotein (*)

Lakshmi Kasturi (1), James R. Eshleman (1), William H. Wunner (2), Susan H. Shakin-Eshleman (1)(§)

From the (1)Institute of Pathology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106 and (2)The Wistar Institute, Philadelphia, Pennsylvania 19106

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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, GlcManGlcNAc, 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).

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).()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.


MATERIALS AND METHODS

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.


RESULTS

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.

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 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 with Thr in RGP(WT) Produces a Novel form of RGP Fully Glycosylated at all Three Sequons

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.


DISCUSSION

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 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) .

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 -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.

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 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).

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 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.


FOOTNOTES

*
This work was supported in part by a Research Initiation Grant from the Ohio Regents Challenge Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institute of Pathology, Case Western Reserve University, School of Medicine, 2085 Adelbert Rd., Cleveland, OH 44106. Tel.: 216-844-8006; Fax: 216-844-1810.

The abbreviations used are: RGP, rabies virus glycoprotein; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; GS, goat serum; WT, wild type.

To avoid any confusion it should be noted that throughout this paper T434 indicates truncation of the protein at residue 434, whereas T39 represents threonine at position 39.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Hubbard, S. C., and Ivatt, R. J.(1981) Annu. Rev. Biochem.50, 555-583 [CrossRef][Medline] [Order article via Infotrieve]
  2. Kornfeld, R., and Kornfeld, S.(1985) Annu. Rev. Biochem.54, 631-664 [CrossRef][Medline] [Order article via Infotrieve]
  3. Varki, A.(1993) Glycobiology3, 97-130 [Abstract]
  4. Opdenakker, G., Rudd, P. M., Ponting, C. P., and Dwek, R. A.(1993) FASEB J.7, 1330-1337 [Abstract/Free Full Text]
  5. Shakin-Eshleman, S. H., and Spitalnik, S. L.(1993) Trends Glycosci. Glycotechnol.5, 355-368
  6. Kaplan, H. A., Welply, J. K., and Lennarz, W. J.(1987) Biochim. Biophys. Acta906, 161-173 [Medline] [Order article via Infotrieve]
  7. Marshall, R. D.(1974) Biochem. Soc. Symp.40, 17-26 [Medline] [Order article via Infotrieve]
  8. Mononen, I., and Karjalainen, E.(1984) Biochim. Biophys. Acta788, 364-367
  9. Gavel, Y., and von Heijne, G.(1990) Protein Eng.3, 433-442 [Abstract]
  10. Plummer, T. H., and Hirs, C. H. W.(1964) J. Biol. Chem.239, 2530-2538 [Free Full Text]
  11. Pohl, G., Kallstrom, M., Bergsdorf, N., Wallen, P., and Jornvall, H. (1984) Biochemistry23, 3701-3707 [Medline] [Order article via Infotrieve]
  12. Thim, L., Bjoern, S., Christensen, M., Nicolaisen, E. M., Lund-Hansen, T., Pedersen, A. H., and Hedner, U.(1988) Biochemistry27, 7785-7793 [Medline] [Order article via Infotrieve]
  13. Curling, E. M. A., Hayter, P. M., Baines, A. J., Bull, A. T., Gull, K., Strange, P. G., and Jenkins, N.(1990) Biochem. J.272, 333-337 [Medline] [Order article via Infotrieve]
  14. Shakin-Eshleman, S. H., Remaley, A. T., Eshleman, J. R., Wunner, W. H., and Spitalnik, S. L.(1992) J. Biol. Chem.267, 10690-10698 [Abstract/Free Full Text]
  15. Machamer, C. E., and Rose, J. K.(1988) J. Biol. Chem.263, 5948-5954 [Abstract/Free Full Text]
  16. Ladenheim, R. G., Seidah, N. G., and Rougeon, F.(1991) Eur. J. Biochem.198, 535-540 [Abstract]
  17. Nakamura, S., Takasaki, H., Kobayashi, K., and Kato, A.(1993) J. Biol. Chem.268, 12706-12712 [Abstract/Free Full Text]
  18. Roberts, P. C., Garten, W., and Klenk, H.-D.(1993) J. Virol.67, 3048-3060 [Abstract]
  19. Omura, H., Otsu, M., Yoshimori, T., Tashiro, Y., and Kikuchi, M. (1992) Eur. J. Biochem.210, 591-599 [Abstract]
  20. Guan, J., Machamer, C. E., and Rose, J. K.(1985) Cell42, 489-496 [Medline] [Order article via Infotrieve]
  21. Gallagher, P., Henneberry, J., Wilson, I., Sambrook, J., and Gething, M.-J.(1988) J. Cell Biol.107, 2059-2073 [Abstract]
  22. Wright, A., Tao, M., Kabat, E. A., and Morrison, S. L.(1991) EMBO J.10, 2717-2723 [Abstract]
  23. Chavez, R. A., and Hall, Z. W.(1991) J. Biol. Chem.266, 15532-15538 [Abstract/Free Full Text]
  24. Olender, E. H., and Simoni, R. D.(1992) J. Biol. Chem.267, 4223-4235 [Abstract/Free Full Text]
  25. Bause, E., and Legler, G.(1981) Biochem. J.195, 639-644 [Medline] [Order article via Infotrieve]
  26. Shakin-Eshleman, S. H., Wunner, W. H., and Spitalnik, S. L.(1993) Biochemistry32, 9465-9472 [Medline] [Order article via Infotrieve]
  27. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59 [CrossRef][Medline] [Order article via Infotrieve]
  28. Stanley, P.(1984) Annu. Rev. Genet.18, 525-552 [CrossRef][Medline] [Order article via Infotrieve]
  29. Southern, P. J., and Berg, P.(1982) J. Mol. Appl. Genet.1, 327-341 [Medline] [Order article via Infotrieve]
  30. Sambrook, J., Fritsch, E. F., and Maniatis, T.(1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., p. 16.47, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  31. Smith, J. S., Sumner, J. W., and Roumillat, L. F.(1984) J. Clin. Microbiol.19, 267-272 [Medline] [Order article via Infotrieve]
  32. Anilionis, A., Wunner, W. H., and Curtis, P. J.(1981) Nature294, 275-278 [Medline] [Order article via Infotrieve]
  33. Wunner, W. H., Larson, J. K., Dietzschold, B., and Smith, C. L. (1988) Rev. Infect. Dis.10, Suppl. 4, S771-S784
  34. Burger, S. R., Remaley, A. T., Danley, J. M., Moore, J., Muschel, R. J., Wunner, W. H., and Spitalnik, S. L.(1991) J. Gen. Virol.72, 359-367 [Abstract]
  35. Elbein, A. D.(1987) Annu. Rev. Biochem.56, 497-534 [CrossRef][Medline] [Order article via Infotrieve]
  36. Hasegawa, M., Orita, T., Kojima, T., Tomonoh, K., Hirata, Y., and Ochi, N.(1992) Eur. J. Biochem.210, 9-12 [Abstract]
  37. Grinnell, B. W., Walls, J. D., and Gerlitz, B.(1991) J. Biol. Chem.266, 9778-9785 [Abstract/Free Full Text]
  38. Rau, S., Geyer, R., and Friedrich, R. W.(1993) J. Gen. Virol.74, 699-703 [Abstract]
  39. Livi, G. P., Lillquist, J. S., Miles, L. M., Ferrara, A., Sathe, G. M., Simon, P. L., Meyers, C. A., Gorman, J. A., and Young, P. R.(1991) J. Biol. Chem.266, 15348-15355 [Abstract/Free Full Text]
  40. Kane, S.(1993) J. Biol. Chem.268, 11456-11362 [Abstract/Free Full Text]
  41. Nilsson, I., and von Heijne, G.(1993) J. Biol. Chem.268, 5798-5801 [Abstract/Free Full Text]
  42. Bulleid, N. J., Bassel-Duby, R. S., Freedman, R. B., Sambrook, J. F., and Gething, M. J.(1992) Biochem. J.286, 275-280 [Medline] [Order article via Infotrieve]
  43. Morimoto, K., Kawai, A., and Mifune, K.(1992) J. Gen. Virol.73, 335-345 [Abstract]
  44. Bause, E., Warmuth, R., and Wesemann, M.(1989) in Tenth International Symposium on Glycoconjugates (Sharon, N., Lis, H., Duksin, D., and Kahane, I., eds) September 10-15, Jerusalem, Israel
  45. Doms, R. W., Lamb, R. A., Rose, J. K., and Helenius, A.(1993) Virology193, 545-562 [CrossRef][Medline] [Order article via Infotrieve]
  46. Sheares, B. T.(1988) J. Biol. Chem.263, 12778-12782 [Abstract/Free Full Text]
  47. Dietzschold, B.(1977) J. Virol.23, 286-293 [Medline] [Order article via Infotrieve]
  48. Wunner, W. H., Dietzschold, B., Smith, C. L., Lafon, M., and Golub, E. (1985) Virology140, 1-12 [Medline] [Order article via Infotrieve]
  49. Wunner, W. H., Dietzschold, B., Curtis, P. J., and Wiktor, T. J.(1983) J. Gen. Virol.64, 1649-1656 [Medline] [Order article via Infotrieve]
  50. Yelverton, E., Norton, S., Obijeski, J. F., and Goeddel, D. V.(1983) Science219, 614-619 [Medline] [Order article via Infotrieve]
  51. Benmansour, A., Brahimi, M., Tuffereau, C., Coulon, P., Lafay, F., and Flamand, A.(1992) Virology187, 33-45 [Medline] [Order article via Infotrieve]
  52. Fodor, I., Grabko, V. I., Khozinski, V. V., and Selimov, M. A.(1994) Arch. Virol.135, 451-459 [Medline] [Order article via Infotrieve]
  53. Otvos, L., Urge, L., Xiang, Z. Q., Krivulka, G. R., Nagy, L., Szendrei, G. I., and Ertl, H. C. J.(1994) Biochim. Biophys. Acta1224, 68-76 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.