Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain1
Author for correspondence: José Melero. Fax +34 91 509 7919. e-mail jmelero{at}isciii.es
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Abstract |
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Introduction |
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The G protein (as with other paramyxovirus attachment proteins) is a type II glycoprotein with a single hydrophobic region near the N-terminal end that serves as both signal peptide and membrane anchor. However, the G protein has a number of genetic, chemical and structural characteristics that are unique among paramyxovirus attachment proteins and, indeed, among the receptor-binding proteins of any other virus. The protein precursor, 292299 amino acids in length (depending on the virus strain) (Sullender et al., 1991 ; Martínez et al., 1999
) migrates in SDSPAGE as a 32 kDa polypeptide, whereas the mature G protein migrates as a wide band of 8090 kDa. Much of this apparent size difference is due to extensive glycosylation of the polypeptide backbone during maturation which increases the protein mass but also affects its interaction with SDS. The majority of the potential glycosylation sites are clustered in two regions of the G protein ectodomain, with amino acid compositions similar to that of mucins produced by epithelial cells (Wertz et al., 1985
). The mucin-like regions are highly variable among HRSV isolates (Cane et al., 1991
; Sullender et al., 1991
) and they flank a central conserved segment of the G protein ectodomain including a cluster of four cysteines that has been proposed as the receptor binding site (Johnson et al., 1987
). A soluble/secreted form of the G protein (Gs) (Hendrick et al., 1988
) is generated by initiation of translation at an alternative in-frame AUG codon located in the middle of the hydrophobic transmembrane region (Roberts et al., 1994
). Gs shares structural characteristics with the membrane-anchor form of G, such as extensive glycosylation and reactivity with all the anti-G monoclonal antibodies (MAbs) identified so far. Gs has been associated with pulmonary eosinophilia following HRSV challenge of BALB/c mice sensitized to the attachment protein (Bembridge et al., 1998
).
HRSV isolates are classified into two antigenic groups by their reactivity with MAbs (Anderson et al., 1985 ; Mufson et al., 1985
). The G protein shows the highest degree of genetic and antigenic variation among HRSV gene products of viruses from both antigenic groups (Johnson et al., 1987
). Within each group, the G protein also shows the highest degree of antigenic and genetic diversity (García et al., 1994
; Cane & Pringle, 1995
). Three types of epitopes, recognized by MAbs, have been identified in the G protein: (i) conserved epitopes, shared by all human isolates, (ii) group-specific epitopes, shared by viruses of the same antigenic group and (iii) strain-specific or variable epitopes, present only in a subset of viruses of the same antigenic group (Martínez et al., 1997
). Epitopes of the first two types are located in the central conserved segment of the G molecule whereas the strain-specific epitopes are restricted almost exclusively to the variable C-terminal third (reviewed in Melero et al., 1997
). Interestingly, all the epitopes identified so far in the G protein do not seem to require the native protein conformation for antibody binding (i.e. all anti-G MAbs react with the G protein band in Western blots).
Given the extensive glycosylation of the mature G protein, it is not surprising that carbohydrates influence both virus infectivity and antigenicity of the G glycoprotein (Lambert, 1988 ; Palomo et al., 1991
). Moreover, cell-type specific glycosylation of G influences its electrophoretic mobility and its reactivity with sugar-specific reagents and MAbs directed against strain-specific epitopes (García-Beato et al., 1996
). In contrast, the conserved and group-specific epitopes of G are unaffected by host-specific glycosylation, in agreement with their location in the central conserved segment of the G ectodomain which is essentially devoid of potential glycosylation sites.
In order to gain further insights into the structure of the G glycoprotein, we have purified Gs from supernatants of HRSV-infected cells and have subjected it to different protease treatments. The results obtained indicate that the G protein C-terminal third is partially resistant to protease digestion and that it is glycosylated in a cell-dependent manner that influences the expression of certain epitopes located in this region of the molecule.
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Methods |
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Antibodies.
MAbs raised against the G proteins of either Long or Mon/3/88 viruses have been described (García-Barreno et al., 1989 ; Martínez et al., 1997
). They were used either individually or as a pool of antibodies that included the following: 021/1G, 021/19G, 63G, 68G and 78G. Rabbit hyperimmune anti-HRSV serum raised against purified Long virus has been described previously (Palomo et al., 1991
).
Purification of Gs.
Either HEp-2 or M6 cells growing in 100 mm Petri dishes were infected with the viruses indicated in the figure legends, as previously described (García-Beato et al., 1996 ). One of the dishes was labelled with 100 µCi [3H]glucosamine, from 24 h post-infection until harvesting, and radioactivity was monitored during the purification process. When cytopathic effect was maximal (48 h post-infection), cell supernatants were harvested, pooled and cleared of cell debris by centrifugation at 3000 g for 5 min. Then, the supernatants were ultracentrifuged at 65000 g for 3 h at 4 °C to remove virus particles and precipitated with ammonium sulfate at 65% saturation. The pellet was dissolved in buffer A (20 mM TrisHCl pH 7·5, 0·5 M NaCl) and dialysed against a large excess of the same buffer.
Equal amounts of purified MAbs 021/1G and 021/21G (Martínez et al., 1997 ) were covalently bound to CNBr-activated Sepharose as specified in the manufacturers instructions. The cell supernatants, processed as indicated above, were passed through the immunoaffinity column which was then washed extensively with buffer A. The retained material was eluted with a buffer containing 0·1 M glycine pH 2·5, 0·2 M NaCl and 0·2% octylglycoside and neutralized immediately after elution with saturated Tris. Elution was followed by radioactivity counts and the presence of Gs in the fractions was evaluated by ELISA. Positive fractions were pooled together and ELISA using specific MAbs was used to rule out the presence of other HRSV proteins (particularly F protein).
Protease digestion and Western blot.
Different amounts of Gs protein, purified as indicated before, were subjected to SDSPAGE, electrotransferred to Hybond PVDF membranes and blotted with the pool of anti-G MAbs as previously described (García-Beato et al., 1996 ), except that the chemoluminescence substrate ECL (Amersham) was used. The lowest amount of Gs that generated a visible band in the blot was incubated with proteases as indicated in the figure legends. After digestion, an excess of SDSPAGE sample buffer was added and, after boiling, the samples were resolved by electrophoresis and visualized by Western blot. Cell extracts of infected cells were made up in buffer containing 10 mM TrisHCl pH 7·6, 140 mM NaCl, 5 mM EDTA, 1% Triton X-100 and 1% sodium deoxycholate and analysed by SDSPAGE and Western blot in a similar manner.
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Results |
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Fig. 3(B) shows a diagram of the G protein primary structure and the location of the epitopes recognized by MAbs that were tested for reactivity with the 50 and 35 kDa Gs fragments. The location of epitopes in the G protein sequence is based mainly on the identification of amino acid changes in escape mutants selected with anti-G MAbs (reviewed in Melero et al., 1997
). From the results obtained, it is concluded that the large fragment (50 kDa) spans from epitope 63G (amino acids 204209) to the C-terminal end of the G polypeptide. The 35 kDa fragment, however, starts at the same position but ends after epitopes 021/9G and 021/16G (amino acid 244). Thus, the entire C-terminal third of the G protein shows partial resistance to protease digestion and, within that region, the segment spanning amino acids 204244 is particularly resistance to protease degradation. It should be stressed that our methodology did not allow precise delineation of the fragment limits since only some of the amino acids determining most epitopes are known and the number of epitopes tested did not cover the entire G protein sequence. Resistance to protease digestion could not be related to absence of potential cutting sites, which were distributed more or less uniformly throughout the G protein sequence (data not shown).
Comparison of protease-resistant fragments of Gs obtained from HEp-2 and M6 cells
We have shown previously that the G protein glycosylation pattern is determined by the host-cell machinery, as detected by reactivity with lectins and carbohydrate-specific antibodies (García-Beato et al., 1996 ). For instance, HRSV infection of M6 cells a highly differentiated mucin-producing epithelial cell line derived from colon carcinoma generated a G protein band of lower electrophoretic mobility than HEp-2 cells. In addition, the reactivity of either M6- or HEp-2-derived G proteins with sugar-specific reagents was different. Since the C-terminal end of the G protein has multiple potential glycosylation sites, we decided to explore the host-cell influence upon G protein sensitivity to protease digestion.
Gs (Long strain form) was purified from the supernatant of either HRSV-infected HEp-2 or M6 cells and subjected to partial digestion with S. aureus V8 protease (Fig. 4). Whereas similar 35 kDa fragments were identified after digestion of the Gs obtained from either cell line, there was a substantial size difference in the large fragment (50 kDa for Gs from HEp-2 and 70 kDa from M6 cells). Reactivity of the Gs fragments with diagnostic MAbs indicated that the M6-derived 35 and 70 kDa fragments were equivalent to the 35 and 50 kDa fragments derived from HEp-2 cells, respectively (not shown). Thus, the size difference between the 50 and 70 kDa fragments was attributed to host-cell-specific glycosylations (see later under Discussion).
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Host cell influence on expression of epitope 63G
Epitope 63G was mapped previously in the G protein primary structure by the reactivity of antibody 63G with certain frame-shift mutants of the HRSV G glycoprotein (García-Barreno et al., 1990 ) and with a set of synthetic peptides (García-Barreno et al., 1992
). Amino acids 204209 were found to be essential for antibody binding.
Since antibody 63G reacted with the unglycosylated G protein precursor (Palomo et al., 1991 ) and with synthetic peptides, it was thought that epitope 63G was determined by a linear sequence of consecutive amino acids in the G protein primary structure. However, we found that expression of epitope 63G in the mature protein was influenced by cell-type-specific glycosylation of the G molecule (García-Beato et al., 1996
). For instance, as shown in Fig. 6
, antibody 63G reacted with the mature G protein from extracts of infected HEp-2 extracts but did not react with the equivalent protein from M6 cells, although both proteins reacted with a pool of anti-G antibodies. The 70 kDa protease-resistant fragment of Gs from M6 cells did not react with antibody 63G (reflecting the changes observed in the full-length protein) although the equivalent 50 kDa fragment of Gs from HEp-2 cells reacted normally and both fragments reacted with the MAbs pool. Unexpectedly, the 35 kDa fragment from either cell type reacted with antibody 63G to the same extent (Fig. 6
), although this fragment is included in the large protease-resistant fragment. This result indicated that M6-specific modifications of the 70 kDa fragment, located outside the 35 kDa fragment, influenced the expression of epitope 63G, shared by the N-terminal end of both fragments (Fig. 3B
).
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Discussion |
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The dose effect of V8 digestion (Fig. 1) suggested that the large protease-resistant fragment of Gs is an intermediate in the formation of the small fragment, the latter being more resistant to the protease. However, it cannot be formally excluded that both fragments were generated from different subpopulations of Gs. It is also possible that the size differences between the 50 and 70 kDa fragments of Gs derived from HEp-2 and M6 cells, respectively, were determined by differences in the cutting sites of the enzyme in these two proteins. Nevertheless, we favour the idea that the large protease-resistant fragment is an intermediate in the formation of the small fragment and that the 70 kDa fragment is equivalent to the 50 kDa fragment, for the following reasons.
(1) The amount of the small fragment increased with greater amounts of protease at the expense of the large fragment (only shown for the Gs obtained from HEp-2 cells in Fig. 1).
(2) There is no evidence for different subpopulations of Gs. In fact the results in Fig. 6 argue against that possibility. Thus, epitope 63G is not expressed in either the full-length Gs protein or the 70 kDa fragment obtained from M6 cells, yet it is expressed in the 35 kDa fragment derived from those cells. Were the 70 and 35 kDa fragments derived from different Gs subpopulations, epitope 63G should have been detected in the full-length form of its Gs precursor and this was not the case. We interpret the results in Fig. 6
to indicate that epitope 63G is masked in the Gs protein and in the 70 kDa fragment from M6 cells but that further fragmentation unmasks this epitope in the 35 kDa fragment.
(3) The effect of premature termination codons on the sizes of the 50 and 70 kDa fragments lend support to the notion that both fragments shared their C-terminal ends with Gs. The N-terminal end of the 50 kDa fragment was delineated by its reactivity with antibody 63G and its lack of reactivity with antibody 021/18G (see Fig. 3B). Since the 70 kDa fragment did not react with antibody 63G (see above), its N-terminal end could only be defined between the epitopes 021/18G and 68G. However, since the 35 kDa fragment derived from it reacted with 63G, it is likely that the 70 and 50 kDa fragments shared their N termini (or that they are very closely located in the Gs sequence) and that their size differences reflect host-cell-specific glycosylations.
The results in Fig. 7 suggest that O-linked carbohydrates are sufficient to maintain differences in the reactivity of Gs from HEp-2 or M6 cells with antibody 63G. It is feasible that this type of carbohydrate also influences the glycosylation differences between the 50 and 70 kDa fragments, some of which were mapped to a small segment of the G protein ectodomain by using HRSV mutants with premature termination codons (Fig. 5
). It was, however, surprising that no host-derived differences were observed between the small protease-resistant fragments of Gs obtained from either HRSV-infected HEp-2 or M6 cells, despite having multiple O- and N-linked potential glycosylation sites (Fig. 5
). Thus, different segments of the same molecule seem to be differentially modified by host-cell-specific glycosylation machineries. Further refinement of this analysis should identify signals and/or local structures that determine the use of potential glycosylation sites in proteins expressed in different cell types.
We proposed a model of the G protein three-dimensional structure (Melero et al., 1997 ) that accounts for the experimental results presented here. The C-terminal third of G was modelled as a segment folded in two halves to indicate its partial resistance to protease digestion and, within it, the particular resistance to proteases of its N-terminal half. The expression of epitope 63G in Gs and its protease-resistant fragments suggests that certain interactions might exist between the two halves in which the C-terminal third of Gs was subdivided. It is interesting to note that reactivity of certain human convalescent sera with the 35 kDa of Gs obtained from HEp-2 cells was also masked in the 50 kDa fragment (Palomo et al., 1999
), arguing again in favour of interactions between the two halves of that G protein region. These results should be kept in mind when analysing the specificity of anti-G antibodies.
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Acknowledgments |
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References |
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Received 8 September 1999;
accepted 1 December 1999.