Role of N-linked Oligosaccharide Chains in the Processing and Antigenicity of Measles Virus...

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Journal of General Virology (1994), 75, 1043 1052. Printed in Great Britain 1043

Role of N-linked oligosaccharide chains in the processing and antigenicity of measles virus haemagglutinin protein

Aizhong Hu,* Roberto Cattaneo , t Stefan Schwartz and Erling Norrby

Department of Virology, School of Medicine, Karolinska Institute, c/o SMI, S-105 21 Stockholm, Sweden

The effects of N-linked oligosaccharides on the haemagglutinin (H) protein of measles virus (MV) were assessed with respect to the processing and antigenicity of the molecule. The functional glycosylation sites on the H protein were determined by eliminating each of the five potential positions, Asn-168, Asn-187, Asn-200, Asn-215 and Asn-238, for N-linked glycosylation by oligonucleotide-directed mutagenesis on a eDNA clone. Expression of the mutant H proteins in BHK-21 cells by a recombinant vaccinia virus encoding T7 polymerase indicated that the first four sites were used in the H glycoprotein for the addition of N-linked oligosaccharide chains. Heterogeneity of oligosaccharide processing was demonstrated. One of the four glycosylation sites had a different carbohydrate structure from those of the other three glycosylation sites and this varied glycosylation was responsible for the appearance of two forms of the H protein. The functional glycosylation sites were

systematically removed in various combinations from the H protein to form a panel of mutants in which the role of carbohydrate chains, singly or in different combinations, could be evaluated. Investigations of these glycosylation mutants indicated that (i) two of the four individual carbohydrate side-chains have a large influence on the antigenicity of the molecule; (ii) individual carbohydrate side-chains have little effect on the folding and oligomerization of the molecule, and are not sufficient or necessary alone to facilitate the transport of the molecule to the plasma membrane; (iii) at least two carbohydrate side-chains are required for the H protein to move along the exocytic pathway to the plasma membrane and various combinations of oligosaccharide side-chains, irrespective of the carbohydrate localizations, influence equally the processing of the molecule.

Introduction

Asparagine-linked (N-linked) glycosylation is one of the most common post-translational modifications of proteins in the exocytic pathway of eukaryotic cells. The addition of N-linked oligosaccharides starts in the endoplasmic reticulum (ER) when the target sequence Asn-X-Ser/Thr (where X is any amino acid except possibly proline and aspartic acid) is exposed in the lumen of the ER (Kornfeld & Kornfeld, 1985). Pro- cessing of these carbohydrate moieties begins immedi- ately following addition of the oligosaccharide in the ER and continues as the glycoproteins pass through the Golgi complex, thus producing two major classes of side- chains: high mannose chains containing only the sugar residues added in the ER and complex chains resulting from further trimming and addition of carbohydrate chains carried out in the Golgi complex (Kornfeld &

t Present address: Institut fiir Molekularbiologie I, Universit~it Ziirich, H6nggenberg, CH-8093 Zfirich, Switzerland.

Kornfeld, 1985). Several approaches have been used to study the functional role of N-linked oligosaccharide side-chains in cells. These include the use of agents that disrupt the addition or modification of glycoproteins, use of glycan-processing-deficient cell lines, and oligonucleotide-directed mutagenesis of cDNAs encoding the proteins to either remove or add sites for carbohydrates. A variety of functions for N-linked oligosaccharides have been suggested (Olden et al., 1982; Rademacher et al., 1988). Oligosaccharides on glycoproteins play a role in the initiation and maintenance of folding into the biologically active conformation, in maintenance of protein stability and solubility, in protection of the polypeptide backbone from proteolytic degradation, in the targeting of glycoproteins to various subcellular compartments and to the cell surface, in promotion of specific adhesion of cells during devel- opment and in influencing the antigenicity and immuno- genicity of glycoproteins.

The haemagglutinin (H) glycoprotein of measles virus (MV) is an integral component of the virion envelope. The H protein plays an essential role in the initiation of

0001-2204 © 1994 SGM


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1044 A. Hu and others

infection by binding to a specific receptor on susceptible cells. An acute infection is followed by lifelong immunity in which efficient neutralizing antibodies against the H protein play a pivotal role (Norrby & Oxman, 1990). H glycoprotein is an oligomeric protein that is thought to be a homodimer or oligomer (Hardwick & Hussell, 1978). From studies of the intracellular processing and antigenic maturation of the H protein in virus-infected cells, we have shown that the H protein, like paramyxo- virus haemagglutinin-neuraminidase (Mottet et al., 1986; Ng et al., 1989; Vidal et al., 1989; Waxham et al., 1986), oligomerizes relatively slowly (tl/2 about 30 min) (A. Hu, J. K6vamees & E. Norrby, unpublished results) compared with some other viral glycoproteins, such as vesicular stomatitis virus G and influenza virus haemagglutinin (tl/2 6 to 10 min) (Gething et al., 1986; Copeland et al., 1986; Kreis & Lodish, 1986). N-linked glycosylation is possibly involved in the formation of discontinuous epitopes since monoclonal antibodies (MAbs) that react with such epitopes fail to recognize the H protein from MV-infected cells treated with tunicamycin (TM) and neutralization escape mutants selected by one MAb inactivate an N-linked glycosylation site on the MV H protein (Hu et al., 1993; A. Hu, J. K6vamees & E. Norrby, unpublished results).

In this report, we analysed the role of N-linked glycosylation on the processing and antigenicity of MV H glycoprotein from the Edmonton strain. Oligonucleotide-directed mutagenesis was used to gen- erate a panel of MV H glycosylation mutants which were subsequently analysed by transient expression in BHK- 21 cells. Our data showed that four of the five potential sites were used for N-linked carbohydrate additions in the H glycoprotein. Based on the functional sites, a panel of H glycosylation mutants were constructed and this allowed us to evaluate the effect of carbohydrate chains, individually or in various combinations, on the processing and antigenicity of MV H glycoprotein.

Single-stranded DNA was prepared according to a standard protocol (Sambrook et al., 1989). Oligonucleotide-directed site-specific mutagenesis was performed using a kit according to the instructions of the supplier (Bio-Rad). To obtain mutant cDNA lacking a particular single natural consensus site, we performed oligonucleotide-directed mutagenesis using a single oligonucleofide designed to produce the required change. The combinations of mutation sites were generated by performing successive site-directed mutagenesis procedures on previously mutagenized templates. Confirmation of successful mutagenesis was obtained by dideoxynucleotide chain termination sequencing of alkali-denatured plasmid DNA according to the protocol of the supplier (Pharmacia). The four sites used for N-linked glycosylation (see Results) are designated gl, g2, g3 and g4, corresponding to Asn found at H protein residues 168, 187, 200 and 215, respectively (Alkhatib & Briedis, 1986). A total of 13 N-linked glycosylation mutants were generated, each designated Hg(n), where n is a set of numbers defining the N-linked glycosylation sites that are used: e.g., a mutant lacking the gl site is Hg234 and the non-glycosylated mutant is Hg0 (see Fig. 1).

Transfection. BHK-21 cells were passaged the day before transfection and grown to about 70 % confluence in MEM supplemented with 10 % FCS in 25 cm 2 tissue culture plates, The medium was removed and the cells were washed three times with serum-free MEM. The cells were then infected with the recombinant vaccinia virus vTF7-3 at a multiplicity of infection of about 15 p.f.u./cell and incubated at 37 °C for 45 rain. The virus inoculum was then removed and replaced with 1 ml of fresh Opti-MEM (Gibco BRL). The cells were transfected with 4 lag of plasmid DNA using 15 lag lipofectin as instructed by the supplier (Gibco BRL).

Metabolic labelling and pulse-chase analysis. At 5 h post-transfection, the medium was replaced with MEM containing 1/20 of the normal content of methionine and the ceils were incubated at 37 °C for 30 min. The cells were then labelled with 100 laCi/ml of [35S]methionine for 30 min and the medium was removed at the end of pulse radiolabelling. The monolayer was washed three times with cold PBS, overlaid with MEM containing 0.5% FCS and incubated for 2 h. The labelled cells were harvested in 1 ml radioimmunoprecipitation assay (RIPA) buffer (2% Triton X-100, 0-15M-NaC1, 0-6M-KCI, 0-5M-MgCla, 5mM- EDTA, I mM-PMSF, 1% aprotinin, 0.01 M-Tri~HCI pH 7.8). Nuclei and cell debris were removed by centrifugation at 15000g in a microfuge and the supernatants were kept for immunoprecipitation.

RIPA. This technique has been described in detail previously (Sheshberadaran et al., 1983). Lysates (100 lal) from the transfected

Methods Cells and virus. BHK-21 cells were grown in Eagle's minimum

essential medium (MEM) supplemented with 10 % inactivated fetal calf serum (FCS). Propagation and purification of the recombinant vaccinia virus encoding T7 polymerase (vTF7-3) (Fuerst et al., 1986) grown in BHK-21 cells was carried out essentially as described previously (Mackett et al., 1985).

Oligonucleotide-directed mutagenesis. Plasmid pert1 containing the full-length eDNA encoding the MV H protein has been described previously (Eschle, 1988). Five 25-mer oligonucleotides with the desired mismatched nucleotide in the middle of sequences synthesized to be complementary to H cDNA sequences encoding the five N-linked glycosylation consensus sites (Asn-X Ser/Thr) were utilized to substitute codons for serine residues in place of those encoding asparagiue residues in the five consensus sequences present in MV H protein (Table 1).

Table 1. Nucleotide substitutions and corresponding amino acid alterations in mutants with changes in the potential N-linked glycosylation consensus sequences

Glycosylation Amino acid Wild-type Mutant site residue sequence sequence

g 1 168 A s ~ S e ~ T h r Ser-Ser-Thr AAC TCA-ACT AGC-TCA-ACT

g2 187 Asn-Cys-Ser Ser-Cy~Ser AAC-TGC-TCA AGC-TGC-TCA

g3 200 Asn-Met-Ser Ser-Me~Ser A A C ~ T G TCG AGC-ATG-TCG

g4 215 Asn Val Ser Ser-Val-Ser AAT-GTG-TCA AGT-GTG TCA

g -- 238 Asn-Leu-Ser Ser-Leu-Ser AAT-CTG-AGC AGT-CTG-AGC


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Measles virus haemagglutinin N-linked glycans 1045

cells were mixed with 2 gl MAb and the final volume was adjusted to 500 ~tl using RIPA buffer. The mixture of antigen and antibody was adsorbed onto Protein A bound to Sepharose CL-4B (Pharmacia), washed three times with RIPA washing buffer (RIPA buffer without protease inhibitors) and once with 0.15M-NaC1, 0.01 M-Tri~HC1 pH 8-0. The purified immune complexes were dried, mixed with protein sample buffer [3 % SDS, 3 % 2-mercaptoethanol (2-ME), 10 % glycerol and 0'1% EDTA], boiled for 3mix, and finally fractionated by SDS-PAGE. For the analysis of non-reduced proteins, 2-ME was omitted from the sample buffer. The gels were analysed by fluoro- graphy. Quantification of signals on gels was done by laser scanning densitometry.

Drug treatment. Endoglycosidase H (Endo H) digestion was performed as described previously (Hu et al., 1993). For tunicamycin (TM) treatment, the drug (final concentration of 2 gg/ml) was added at 4 h post-transfection and remained present throughout the transfection.

Indirect immunofluorescence. This technique has been described previously (Norrby et at., 1982). BHK-21 cells were grown to 70% confluence on microscope coverslips before transfection. Five hours post-transfection, the cells were washed with PBS and fixed with 3 % paraformaldehyde in PBS for 30 mix. After washing three times with PBS, the cells were permeabilized with 0.1% Triton X-100 in PBS for 30 rain. Following three further washes with PBS, cells were treated with primary antibody at room temperature for 30 min. Cells were then rinsed with PBS and incubated with goat anti-mouse immunoglobulin (IgG) conjugated with fluorescein isothiocyanate (FITC; Cappel Laboratories) for 30 rain. Cells were washed again with PBS before mounting of the coverslips on glass slides for photography.

Results

Determination of functionaI N-linked glycosylation sites in the H glycoprotein and construction of a panel of glycosylation mutants

The predicted amino acid sequence indicates that the H protein from the Edmonston strain contains five potential N-linked glycosylation sites (Alkhatib & Briedis, 1986). Partial Endo H digestion shows that four of these are used (Cattaneo & Rose, 1993). However, it remains to be determined which sites are actually used. To address this question, we constructed a panel of H site-directed mutants in which the consensus sequences for N-linked glycosylation were altered singly and these mutants were expressed transiently in BHK-21 cells. Oligonucleotide- directed mutagenesis was employed to introduce a one- nucleotide alteration in the codon for asparagine, resulting in a single amino acid substitution at each potential glycosylation site. The addition of N-linked carbohydrates was blocked by altering the Asn-X-Ser/ Thr at asparagine residues 168, 187, 200, 215 and 238 to Ser-X-Ser/Thr. The asparagine-encoding codons at each site were replaced with serine-encoding codons, since an asparagine to serine alteration is conservative due to the similarity in bulk and polar nature of the side- chains. Five single-site mutants were generated. The derivation of the nomenclature for each mutant is described in Methods (see also Fig. 1).

The H glycosylation mutants were expressed using recombinant vaccinia virus encoding the T7 RNA polymerase. The expression of the mutant proteins was analysed by metabolic labelling, RIPA and SDS-PAGE.

Transmembrane anchor gl g2 g3 g4

Y Y Y Y I 168 187 200 215 238

NH 2 COOH

Hg234 Y Y Y

Hg134 Y Y Y

g124 Y Y Y

Hgl23-- Y Y Y

Hgl2

Hg34

Hgl4

Y Y

Y

Y

%- t %_

%__

Hg24

Hgl

Y Y

Y

%-

%-

r g2 5"

Hg3 Y / ~ -

Hg4 x(, / / ~

Fig. 1. Schematic diagram of MV H gene products. The H wild-type (H) protein (617 amino acids) is shown on the top of the figure. Symbols: Y, functional N-linked glycosylation sites; v, unused glycosylation site. The designation of the carbohydrate chains (gl, g2, g3 and g4) are indicated above each site. H glycosylation mutants are represented with the designated name shown at the left of each diagram. The residue nmnbers indicate the Asn residue in the H amino acid sequence. The sites are numbered sequentially from the N to C terminus of the protein. Figures are not drawn to scale.


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(a) -TM +TM

eq ~ eq

(b) -TM +TM TM +TM

¢ 'q ~ c 'q

(c) TM +TM

% % % ~ % % % % ~ %

Fig. 2. Expression of H protein glycosylation mutants. The transfected cells were metabolically labelled, at 5 h post-transfection, with [35S]methionine for 3 h in the absence ( - T M ) or presence (+TM) of TM (2 gg/ml). Equal amounts of cell lysates containing labelled proteins were immunoprecipitated with MAb 1-29 and analysed by SDS-PAGE. (a) Single-site mutants. (b) Double-site mutants. (c) Triple- and quadruple-site mutants. Only the relevant section of the autoradiogram is shown.

Wild-type H expressed two forms of the glycoprotein of 214,. 77K and 79K which represented heterogeneity of glycosylation modifications at different sites in BHK-21 cells (see below). The glycosylation-site mutant polypeptides Hg234, Hg134, Hg124 and Hg123 (asparagine to serine alteration at positions 168, 187, 200 and 215, respectively) showed an increased electrophoretic mobility compared with wild-type H protein (Fig. 2a, -TM) . Synthesis of these mutant polypeptides in the presence of TM (Fig. 2a, +TM) indicated that the differences in mobility resulted from differences in carbohydrate addition, since all the mutants showed the same mobility as unglycosylated wild-type H protein. This provides evidence that sites gl, g2, g3 and g4 are used for N-linked glycosylation. Deletion of the carbohydrate attachment site at position 238 did not cause an alteration in mobility on SDS-PAGE compared with wild-type H protein (Fig. 2 a, H g - ) , suggesting that this site is not glycosylated. The mutant H g - will not be

(a) H Hg234 Hg134 Hg124 Hg123

(b) Hgl2 Hg34 Hg24 Hgl4

,,~ ,,b ~b ,3

(c) Hgl Hg2 Hg3 Hg4 Hg0

Fig. 3. Immunoreactivity of glycosylation mutant proteins with a panel of MAbs. Equal amount of cell lysates containing labelled proteins were immunoprecipitated with different MAbs as illustrated and the polypeptides were analysed by SDS-PAGE. (a) Single-site mutants. (b) Double-site mutants. (c) Triple- and quadruple-site mutants. Only the relevant section of the autoradiogram is shown.

described further in this report, but it was examined in further assays and found to be indistinguishable in every way from wild-type H, which suggests that at least at this site the substitution from asparagine to serine does not have a deleterious effect on the integrity of the H protein.

With the knowledge that four sites on the H glycoprotein are used for N-linked glycosylation, further mutants in which two, three or all four sites were mutated were generated as described in Methods and are schematically illustrated in Fig. 1. Expression of the double-, triple- and quadruple-site glycosylation mutant polypeptides showed alterations in electrophoretic mobility compared with wild-type H protein (Fig. 2 b and c, - T M ) , which are in agreement with the deletion of two, three or all four carbohydrate chains. When the mutant polypeptides were synthesized in the presence of TM (Fig. 2 b and c, + TM), they all showed the same mobility as non-glycosylated wild-type H protein, indicating that the mobility differences seen in the absence of TM are caused by differences in glycosylation.

The triple-site glycosylation mutant proteins showed a single protein band, indicating that the individual oligosaccharide structure is relatively homologous. The mutant Hg4 displayed a slightly lower mobility than the mutant Hgl, Hg2 or Hg3 (Fig. 2c, - T M ) . When the mutant polypeptides were produced in the presence of TM (Fig. 2 c, + TM), the same electrophoretic mobility


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Measles virus haemagglutinin N-linked gIycans 1047

Table 2. MAb reactivity, Endo H sensitivity and cell surface expression of wild-type and mutant proteins

MAb reactivity* Endo H Cell surface

Protein 16-DE6 1-44 1-41 sensitivityt expression:~

H + + + + + + + + + + + + R Yes Hg234 + + + + + + + + + + + R Yes Hg134 + + + + + + + + + + + R Yes Hg124 + + + + + + + + + + R Yes Hg123 + + + + + + + + + + + R Yes Hgl2 + + + + + + + R Yes Hg34 + + + + + + + + + + + R Yes Hg24 + + + + + + + + + + R Yes Hgl4 + + + + + + + R Yes Hgl - + + - S No Hg2 - + + - S No Hg3 + + + + + S No Hg4 + + + + + S No HgO - + - N A N o

* Where there were two protein bands, the combination of the two signals was calculated. The data are presented as the amount of protein immunoprecipitated by MAbs relative to that precipitated by MAb 1-29: + + + + , 70 to 100%; + + + , 40 to 70%; ++ , 20 to 40%, +, 5 to 20 %; - , < 5 %. Data are from three independent experiments.

tAbbreviations: S, carbohydrate chains were sensitive to Endo H digestion; R, some carbohydrates were found to be resistant to Endo H digestion; NA, not applicable. Data are from three independent experiments.

Cell surface expression was analysed by indirect immunofluorescence with MAb 1-29. Data are from three independent experiments.

was observed, suggesting that the oligosaccharide at glycosylation site g4 had a different structure f rom those at the rest of the glycosylation sites. This heterogeneity in glycosylation processing was further reflected in the mutants in which glycosylation site g4 was involved. All the mutants containing this site (Hgl4, Hg24, Hg34, Hg234, Hg134 and Hg124) showed some effects on the H protein migration characteristics. The combination of g2 and g4 showed the most pronounced synergistic effect. This effect was further observed in the mutant Hg124 which showed different migration properties compared with the other single-site glycosylation mutants (Fig. 2 a, - T M ) . The mutant Hg123 lacking the glycosylation site g4 showed a relatively homogeneous protein band (Fig. 2 a, - T M ) , which again supported the assumption that heterogeneity in glycosylation was mainly dependent on that of site g4.

Immunoreactivity of the glycosylation mutant proteins with a panel of MAbs

The contribution of the individual carbohydrate chains to the folding and antigenicity of MV H protein was determined using a panel of H protein-specific MAbs which recognize continuous (MAb 1-29) or discontinuous (MAbs 16-DE6, 1-41 and 1-44) epitopes (A. Hu, J. K6va- mees & E. Norrby, unpublished results). The transfected

cells were pulse-labelled for 30 min and incubated in the chase medium for 2 h for the processing and maturat ion of the protein. Cells were lysed, the labelled glycosylation mutant proteins were immunoprecipitated with individual MAbs and the polypeptides were analysed by SDS-PAGE. The autoradiograms were quantitatively interpreted by densitometry. To measure the immuno- logical reactivity of the glycosylation mutant proteins, we compared the amount of the H protein immunoprecipitated by the MAbs reacting with discontinuous epitopes with that precipitated by the MAb recognizing a continuous epitope. The results are shown in Fig. 3 and summarized in Table 2. All mutants, including the unglycosylated mutant Hg0, were found to react with MAb 1-29 as well as MAb 1-44, but the reactivity with the latter was weaker. Removal of any single glycosylation site at positions gl , g2, g3 or g4 had no effect on the ability of the H protein to react with the MAbs specific for conformational epitopes (Fig. 3 a), indicating that no single carbohydrate chain appears to be critical for the formation of such epitopes. When the double-site mutants were examined, mutants Hg34 and Hg24 were found to retain nearly full M A b reactivity, whereas mutants Hg l2 and Hg l4 showed a markedly reduced reactivity (Fig. 3b). Further analysis of the triple-site mutants which each contain a single functional glycosylation site showed that the presence of the gl or g2 carbohydrate chains alone did not cause retention of the MAb reactivity (Fig. 3 c). However, the mutants Hg3 and Hg4, which respectively have the g3 and g4 carbohydrate chains alone, showed approximately 20% of MAb reactivity. These results suggest that these carbohydrate chains have a large influence on the conformational epitopes of the H protein.

Folding and oligomerization of the glycosylation mutant proteins

The folding state of the glycosylation mutant proteins was investigated using non-reducing gels. BHK-21 cells were transfected with plasmids containing H glycosylation mutant cDNAs, labelled with [35S]methionine for 30 rain and incubated in the chase medium for 2 h. Cells were lysed and the H proteins were immunoprecipi- tared with MAb 1-29 which recognizes a continuous epitope on the H protein, and the immunoprecipitates were separated by S D S - P A G E under reducing (Fig. 2 and Fig. 3) and non-reducing conditions (Fig. 4). Wild- type and mutant H proteins migrated differently in the presence and absence of 2-ME. In the absence of 2-ME, little monomer form of the H protein was observed and most of the molecules displayed the migration pattern of dimers or of larger aggregates. The single- and double- site mutant H proteins were all folding-competent,


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(a) ., ~ , = ~ , = ,* -* = = = = % % ~ V~ % % " =

Aggregate ]

Dimer -

Monomer --

(b)

Aggregate I

Dimer -

- - 2 0 0 K

-- 92-5K

- 69K

- 2 0 0 K

Fig. 4. Analysis of H glycosylation mutants under non-reducing conditions. (a) Equal amounts of cell lysates were immunoprecipitated with MAb 1-29. The samples were analysed by SDS-PAGE under non-reducing conditions. Positions of the monomer, dimer, and aggregate of the glycosylation mutants are indicated on the left. The positions of M r markers are indicated on the right. (b) Same gel as (a), but only the top section of the film which was deliberately overexposed is shown. Positions of the dimer and aggregate of the glycosylation mutants are indicated on the left.

ee5 e¢~ ¢',1 ¢',1 ¢',1 ~ ~l" '~"

Monomer- : -80K

Hg0 - i~, - 69K

Fig. 5. Processing of wild-type and glycosylation mutant H proteins. Cell lysates were prepared and were immunoprecipitated with MAb 1-29. The immune complexes were digested with Endo H overnight and the polypeptides were analysed by SDS-PAGE. The positions of unglycosylated H protein (Hg0) and the monomer form of the H protein are indicated on the left. The positions of M r markers are shown on the right.

a l though the quant i ty o f folded proteins was reduced compared with the wild-type protein. When three glycosylation sites were inactivated, properly folded protein could be seen only after overexposure o f the film (Fig. 4b), indicating that these mutan t H proteins were unstable a n d / o r susceptible to proteolytic degradat ion. N o dimer form of the mutan t protein could be observed when the mutan t Hg0 protein was analysed under non- reducing condit ions and this mu tan t protein was aggreg- ated on the top o f the gel, suggesting that the mutan t protein was totally defective in folding and most o f the molecules existed in the form of aggregates. A protein band of Mr approximately 200K was observed in all

lanes; this p robab ly represented a complex between the H protein and a cellular protein. Aggregates at the top o f the gel were observed a m o n g all mutants . The aggregate materials were greatly reduced in amoun t under reducing condit ions (data not shown), which is consistent with the assumption that they are disulphide-linked. There was a large difference in the ratio o f the amoun t o f the dimers to that o f the aggregates between the mutan ts having two or more than two glycosylation sites and the mutants with a single glycosylation site or wi thout any such site.

Intracellular transport o f the glycosylation mutant proteins

Pulse-chase analysis has been used to examine the matura t ion o f the H protein in MV-infected cells (A. Hu, J. K6vamees & E. Nor rby , unpublished results). To extend these studies and determine what effect removal o f a specific N-linked glycosylation site had on the matura t ion o f the glycoprotein, the processing of the mutan t proteins was analysed. There is evidence that p roper folding and ol igomerizat ion are prerequisites for t ranspor t o f integral membrane proteins f rom the E R (Copeland et al., 1986, 1988; Gething et al., 1986; Kreis & Lodish, 1986). To determine whether the glycosylat ion mu tan t proteins acquire resistance to Endo H digestion,


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Measles virus haemagglutinin N-linked glycans 1049

a marker of the modification of carbohydrate chains from simple to complex form in the medial Golgi complex, transfected cells were labelled with [35S]methionine for 30 min, followed by incubation in chase medium for 2 h to allow processing and maturation of the mutant molecules. H proteins were immunoprecipitated with MAb 1-29, digested with Endo H and analysed by SDS-PAGE. The results are shown in Fig. 5 and summarized in Table 2. The Endo H-resistant form of the H protein was occasionally observed in the triple- site glycosylation mutants only after overexposure of the film and hence these mutant proteins were considered to be the molecules retained in the ER, although the occurrence of a small portion of the molecules in the Golgi could not be excluded. The results of most of the mutant H proteins showed a good correlation between the acquisition of reactivity to the MAbs detecting conformational epitopes, representing folding and oligomerization, and the conversion of some H carbohydrate chains to the Endo H-resistant forms (Table 2), indicating intracellular transport from the ER to the medial Golgi complex.

Intracellular localization of the glycosylation mutant H proteins

The subcellular localization of the glycosylation mutant proteins was examined by indirect immunofluorescence. Immunostaining was performed on fixed intact or fixed and permeabilized transfected BHK-21 cells using MAb 1-29 and FITC-conjugated goat anti-mouse IgG. An example representing wild-type, single-, double-, triple- and quadruple-site mutants is shown in Fig. 6. Cells expressing wild-type H protein exhibited internal staining throughout the cytoplasmic reticulum as well as in the juxtanuclear region (Fig. 6a), and cell surface staining was readily detectable (Fig. 6b). In contrast, the unglycosylated H protein showed no surface staining (Fig. 6j), and the internal staining pattern showed that this protein was limited to reticular perinuclear structures (Fig. 6 i), indicating the presence of unfolded molecules in the ER. The results of the indirect immunofluorescence analysis for the entire panel of mutants are presented in Table 2. We found that the H glycosylation mutants (Hg234, Hg134, Hg124, Hg123, Hgl2, Hg34, Hg24 and Hg 14) that had molecules recognizable by MAbs reacting with conformational epitopes and that acquired some Endo H-resistant carbohydrate chains could be detected on the cell surface. The mutants (Hg3 and Hg4) that had molecules recognizable by the MAbs reacting with conformational epitopes and that did not acquire Endo H-resistant forms of carbohydrate chains did not express a detectable level of H protein at the cell surface. This should not be surprising since conformational antigenic

Internal Surface

Fig. 6. Indirect immunofluorescence of wild-type and mutant H proteins. Transfected BHK-2I cells on coverslips were fixed in 3% paraformaldehyde in PBS for cell surface fluorescence (b, d, f, h and j) or fixed then permeabilized in 0.1% Triton X-100 (a, c, e, g and 0. H proteins were stained with MAb 1-29, followed by FITC-conjugated goat anti-mouse IgG. (a, b) wild-type; (c, d) Hg234; (e,f) Hgl2; (g, h) Hgl ; (i,j) Hg0.

epitope formation of the MV H protein occurs in the ER (A. Hu, J. K6vamees & E. Norrby, unpublished results) and the locally folded conformation-competent protein does not necessarily indicate a transport-competent molecule.

Discussion Accumulated data have shown that not all the N-linked glycosylation target sequences Asn-X-Ser/Thr are actually used (Kornfeld & Kornfeld, 1985). The amino acid sequence of the MV H glycoprotein contains five potential sites for the addition of N-linked oligosaccharide chains. Deletion and analysis of the individual sites indicate that four sites are used (asparagine 168, 187, 200 and 215) and that asparagine residue 238 is not utilized. The reason for this remains unclear. It is generally considered that a polypeptide influences its own glycosylation by controlling accessibility of oligo- saccharyltransferase. Inspection of the amino acid


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1050 A. H u and others

sequence of MV H protein reveals that this potential N- linked glycan acceptor site is flanked by three positively charged amino acids, residues Lys-236, Lys-242 and Arg- 243 (Alkhatib & Briedis, 1986). The ability of the tripeptide signal to achieve a favourable conformation by interacting with neighbouring amino acids may have a significant effect on the realization, rate and extent of glycosylation (Kornfeld & Kornfeld, 1985). This positively charged fragment may convey an unfavourable element to the conformation of the target sequence and hence the acceptor site at asparagine 238 may not be accessible to the glycosylation enzyme.

Carbohydrate modifications at individual glycosylation sites were relatively homogeneous. However, heterogeneity of oligosaccharide structure was observed at the glycosylation site g4, which was responsible for the appearance of two populations of the H glycoprotein in BHK-21 cells. The heterogeneity may be a result of altered carbohydrate processing, since pulse-chase analysis showed that the 79K form of the H protein could be seen in the processed samples, but not in the pulse- labelled samples (data not shown). Heterogeneity of glycosylation is also observed when the same plasmid (pert1) is used for transfection in HeLa cells (Cattaneo & Rose, 1993). The heterogeneity in glycosylation is therefore unlikely to be host cell-dependent, but is an intrinsic property dictated by the primary amino acid sequence of the H protein. Two types of H protein caused by altered glycosylation processing have also been observed in virus-infected ceils (Graves, 1981; Ogura et al., 1991). Thus, heterogeneity in H protein glycosylation probably also exists in virus lyric infections. This type of observation has been made previously, e.g. in studies of herpesvirus glycoproteins (Sodora et al., 1989; Tikoo et al., 1993) and of the cellular glycoprotein Thy-1 (Rademacher et al., 1988). However, the signifi- cance of glycosylation heterogeneity remains unclear.

A number of studies have indicated that N-linked glycans can influence proper folding, disulphide bond formation and oligomerization of glycoproteins (Gal- lagher et al., 1992; Grigera et al., 1991; Guan et al., 1988; Machamer & Rose, 1988; Machamer et al., 1985; Matzuk & Boime, 1988; Ng et al., 1990; Sodora et al., 1989; Taylor & Wall, 1988; Tikoo et al., 1993; Vidal et al., 1989; Wright et al., 1989). Some studies have suggested that N-linked oligosaccharides affect the conformational integrity of certain proteins, whereas some proteins appear relatively unaffected (Hannink & Donoghue, 1986; Williams & Lamb, 1986). The mutant Hg0 lacking any glycosylation sites was found to produce molecules that formed disulphide-linked aggregates that did not fold properly, did not oligomerize, accumulated in the ER, and were not transported to the cell surface, as was found previously for inappropriately disulphide-

linked aggregates of vesicular stomatitis virus G protein, influenza virus haemagglutinin and simian virus 5 haemagglutinin-neuraminidase (Doms et al., 1988; Machamer & Rose, 1988; Hurtley et al., 1989; Ng et al., 1990). Even MV H proteins with a single side-chain could not efficiently fold or dimerize, and were not efficiently transported to the cell surface.

The neutralization escape mutants selected by MAb 1-44 show a single amino acid alteration, Ser-189 to Pro (Sheshberadaran & Norrby, 1986; Hu et al., 1993). This change inactivates the glycosylation of site g2. In this study, the mutant Hg134 lacking g2 with alteration of Asn-187 to Ser was found to react with MAb 1-44 to the same extent as the wild-type. The observed phenotype of the neutralization escape mutants may therefore result from the change in the amino acid near the glycosylation site rather than the absence of the N-linked oligosaccharide. The glycosylation g2 might thus not be directly involved in this epitope, although a possible indirect influence could not be excluded.

The presence of glycosylation gl, g2 alone, or a total lack of glycosylation yielded a significantly reduced immunoreactivity with the MAbs recognizing discontinuous epitopes, indicating that the g 1 or g2 individually have little effect on the conformation of the discontinuous epitopes. In contrast, the presence of glycosylation g3 or g4 alone allowed moderate immunoreactivity of the H protein to the MAbs specific for the conformational epitopes, suggesting that glycosylation g3 or g4 alone can contribute to the discontinuous epitopes. Interestingly, these two sites are in close proximity to glycosylation site g3 of the corresponding attachment protein of simian virus 5 which has been shown to have an important role in folding, assembly and intracellular transport (Ng et al., 1990).

Mutants containing two oligosaccharide chains were folded and transported to the cell surface and maintained moderate immunoreactivity with MAbs specific for the conformational epitopes, albeit less efficiently than the wild-type protein. The H protein from one case of subacute sclerosing panencephalitis (SSPE) MV displayed the same glycosylation phenotype as the mutant Hgl4. The mutant Hgl4 protein was transport-competent whereas the protein of the SSPE case A is poorly transported to the cell surface (Cattaneo & Rose, 1993), indicating some other mutations besides those causing the inactivation of the N-linked glycosylation are also involved in the process. Alternatively, it may be a result of different expression efficiency in different cell lines. Mutants containing three oligosaccharide chains were found to have biological activity indistinguishable from the wild-type H protein, suggesting that individual carbohydrate side-chains are not necessary for the expression of the structure and function of the H


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Measles virus haemagglutinin N- l inked glycans 1051

molecules. Therefore, the ability of H glycosylation mutant proteins to be transported through the exocytic pathway is not dependent on the presence of any particular carbohydrate chain but on the collective carbohydrate chains that substantially aid in the folding of the H protein to acquire the native structure.

Our sincere thanks go to Drs Yihai Cao, Anita Bergstrrm, Lars Melin and Ralf Pettersson for helping with establishment of the vaccinia virus expression system and Dr Susan Cox for careful reading of the manuscript. We also thank Dr Jan K6vamees for helpful discussion. This work was supported by Swedish Medical Research Council (B93-16X-00116-29A).

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(Received 29 October 1993; Accepted 12 December 1993)

Role of N-linked Oligosaccharide Chains in the Processing and Antigenicity of Measles Virus...

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