key: cord-0007255-x2uga2gl
authors: Geyer, Hildegard; Will, Christiane; Feldmann, Heinz; Klenk, Hans-Dieter; Geyer, Rudolf
title: Carbohydrate structure of Marburg virus glycoprotein
date: 1992-08-03
journal: Glycobiology
DOI: 10.1093/glycob/2.4.299
sha: 9b07b113eda5b0b704c57b13ccc0baf6498f7979
doc_id: 7255
cord_uid: x2uga2gl

Marburg virus was propagated in E6 cells, a cloned cell line of Vero cells, in the presence of [6-(3)H]glucosamine Radiolabelled viral glycoprotein was digested with trypsin, and oligosaccharides were liberated by sequential treatment with endo-β-N-acetylglucosaminidase H, peptide-N(4)-(N-acetyl-β-glucosaminyl)asparagine amidase F and O-glycosidase, by β-elimination, and by alkaline hydrolysis. After fractionation by HPLC and gel filtration, glycans were characterized chromatographically, by digestion with exoglycosidases and, in part, by methylation analysis and liquid secondary ion mass spectrometry. The oligosaccharide structures thus established include oligomannosidic and hybrid-type N-glycans, as well as neutral fucosylated bitri- and tetraantennary species, most of which carry an additional bisecting N-acetylglucosamine. In addition, high amounts of neutral mucin-type O-glycans with type-1 and type-2 core structures were detected. None of the glycans present in this viral glycoprotein carried sialic acid residues.

Marburg virus, as well as the morphologically related Ebola virus, are the prototype members of a new virus family, called Filoviridae (Kiley etal, 1982) , which constitutes the third family of non-segmented negative-strand RNA viruses within the newiy proposed order Mononegavirales besides the Paramyxoviridae and the Rhabdoviridae (ICTV, 1990) . Recently, a third member of this virus family, which is antigenically closely related to Ebola virus, was isolated from cynomolgus monkeys (Macaca Jascicularis) imported to the USA from the Philippines (Centres for Disease Control, 1989; Jahrling et al., 1990) . This agent is called Reston virus.

Marburg virus and Ebola virus are extremely pathogenic for humans, causing a severe haemorrhagic disease with mortality rates of 35% for Marburg virus (Martini and Siegert, 1971 ) and up to 88% for Ebola virus (subtype Zaire) (Johnson etal., 1977) . Filovirus infections were unknown until 1967, when Marburg virus was first isolated in outbreaks among laboratory workers in Germany and Yugoslavia. The source of these simultaneous outbreaks of viral haemorrhagic disease has been attributed to virus-infected African green monkeys (Cercopithecus aethiops) imported from Uganda (Martini and Siegert, 1971) .

For metabolic labelling, Marburg virus was propagated in E6 cells, a cloned line of Vero cells. The radioactive nutrients were added 5 days post-infection for 24 h. Virus particles were isolated from the supernatant, purified by gradient centrifugation and sedimentation steps, and the radiolabelled viral proteins were analysed by SDS gel electrophoresis followed by fluorography ( Figure 1A ). In vivo labelling with [ 35 S]methionine marked all viral structural proteins ( Figure 1A , lane 1), whereas only the glycoprotein was radiolabelled when D-[6-3 H]glucosamine was used ( Figure 1, lane 2) . Starting from tissue culture supernatant of -2.5 x 10 8 infected cells, metabolically labelled with 2 mCi D-[6-3 H]glucosamine, viral glycoprotein comprising 2 x 10 6 c.p.m. was obtained. For determination of the radioactive sugar constituents, aliquots were hydrolysed and the monosaccharides released were identified by co-chromatography with monosaccharide standards in high-pH anion-exchange (HPAE)-HPLC. The results demonstrated that the [ 3 H]glucosamine-labelled viral glycoprotein contained radiolabelled N-acetylglucosamine, N-acetylgalactosamine and galactose in a ratio of -9.8:1.35:1, indicating that small amounts of [6-3 H]glucosamine have been metabolized (Kiley et al., 1988) . Owing to the experimental conditions, the viral polypeptide VP24 is not visible. (B) Reactivity of sialic aad-specific MAA agglutinin to filovirus glycoproteins. Viral proteins were subjected to SDS gel electrophoresis before (-) and after (+) treatment with sialidase from V.cholerae, blotted onto nitrocellulose, incubated with digoxigeninconjugated MAA and visualized using an anti-digoxigenin antibody. Lane 1, Marburg virus; lane 2, Ebola virus; lane 3, Reston virus; lane 4, fetuin (control). The beginning of the running gel is marked by an asterisk.

into A/-acetylgalactosamine and galactose during metabolic labelling of the glycoprotein. Radiolabelled sialic acid residues were not detected (data not shown).

The affinity of carbohydrate-specific lectins for Marburg virus glycoprotein has already been described (Feldmann et al., 1991) . In order to analyse for the presence of sialic acid substituents, the same assay was performed using Maackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin (SNA). MAA recognizes sialic acid linked (a2-3) to galactose in N-and 0-glycans, whereas SNA recognizes sialic acid linked (a2-6) to galactose. For comparison, the respective glycoproteins of two related filoviruses (Ebola and Reston virus), propagated in the same cell line, were also tested. In contrast to SNA, which did not react with any of the three glycoproteins (data not shown), MAA bound to Ebola and Reston virus, but not to Marburg virus glycoprotein ( Figure IB) . Binding of the lectin was abolished after treatment of the glycoproteins with sialidase, demonstrating the carbohydrate-specific reaction of MAA. Thus, Ebola and Reston virus glycoproteins obviously comprise (a2-3)-linked sialic acid, whereas Marburg virus glycoprotein seems to lack sialic acid substituents.

Following digestion with trypsin (see Scheme 1), glycopeptides obtained were treated with endo-/3-A/-acetylglucosaminidase H (endo H) and the oligosaccharides released (4.27% of total radioactivity) were separated from residual glycopeptides by Bio-Gel P-4 chromatography. After reduction, the oligosaccharide alditols obtained were separated by HPAE-HPLC, yielding four fractions (H1-H4) which represented 0.6, 1.7, 1.3 and 0.6% of total radioactivity ( Figure 2A) .

Endo H-resistant glycopeptides were treated with peptide-A rt -(A'-acetyl-^-glucosaminyl)asparagine amidase F (PNGase F) and subjected to reversed-phase HPLC (RP-HPLQ. Two glycan fractions (Fl and F2) comprising 3.23 and 40.9% of total radioactivity were thus isolated (see Scheme 1). The remaining 51.6% of radioactivity still co-eluted with peptides (data not shown). No changes in the elution profile were observed when treatment with PNGase F was repeated once. The minor glycan fraction Fl was not analysed further, whereas fraction F2 glycans were reduced and subjected to anionexchange HPLC. The result revealed that the vast majority of radioactivity (94.2%) eluted at the position of neutral sugar chains ( Figure 3A ). Treatment of the trace amounts of charged material with sialidase from Vibrio cholerae did not change the elution position of these compounds, excluding the presence of sialylated oligosaccharides. Neutral species were freed from the charged material by preparative anion-exchange HPLC and separated by HPAE-HPLC, yielding four main subtractions (F21-24, see Figure 2B ) which comprised 4, 17, 3.5 and 6.4% of total radioactivity, respectively.

Endo H-and PNGase F-resistant glycopeptides were treated with C-glycosidase and subjected to gel filtration. About 2.4% of total radioactivity was liberated and eluted at ~ 3 glucose units, i.e. the position of a Gal-GalNAc disaccharide (fraction Ol in Scheme 1). An aliquot of the remaining glycopeptide fraction was then sequentially treated with sialidase and O-glycosidase without any further release of radioactivity. Following reductive /3-elimination and RP-HPLC, however, 32 % of total radioactivity was recovered in the oligosaccharide fraction, whereas 17% still remained bound to peptides. The reduced Oglycans obtained were analysed for the presence of negatively charged components by anion-exchange HPLC.

The results demonstrated that this glycan fraction contained exclusively neutral oligosaccharides (see Figure 3B ). O-Glycans were fractionated by HPLC on a LiChrosorb-NH 2 column, yielding four major subfractions (02-05) which comprised 2.8, 1.3, 24.6 and 2.1% of total radioactivity ( Figure 4 ). Glycans still bound to peptides (cf. Scheme 1) were liberated by alkaline hydrolysis and separated from remaining peptides by RP-HPLC. Oligosaccharides obtained were fractionated by HPAE-HPLC, leading to an oligosaccharide pattern which was almost identical to that of F2 glycans (data not shown). Starting from the glycoprotein preparation described above, -25-500 pmol of mostly individual glycans were thus obtained.

Oligosaccharide alditol fractions H2 and H3, F21-F24 and O2-O4 (cf. Figures 2 and 4 , and Scheme 1) were subjected to methylation analysis (see, for example, Figure 5 ). The results are summarized in Table I . Owing to the small amounts of material, fractions were only qualitatively characterized. The presence or absence of the corresponding alditol derivatives, however, was clearly significant in all cases. In addition, major fractions F22 and 04 were studied by liquid secondary ion mass spectrometry (LSIMS) ( Figure 6 ).

For determination of the anomeric configurations of monosaccharide constituents, as well as for enzymic sequencing of the major glycan fractions, oligosaccharide alditols H2, H3, F21-F24 and O1-O5 were degraded by successive treatment with the exoglycosidases listed in Table n . Untreated glycans and reaction products were analysed by Bio-Gel P-4 chromatography and their sizes were determined by co-chromatography with glucose oligomers (Yamashita etai, 1982; Kobata etal, 1987 ; see, for example, Figures 7 and 8) . For chromatographic characterization, the main oligosaccharide fractions were further subjected to LiChrosorb-Diol-(endo H-sensitive glycans) or LiChrosorb-NH 2 -HPLC (PNGase F-released species) and their chromatographic behaviour was compared to that of standard oligosaccharides (data not shown).

In conjunction with the chromatographic data obtained in HPAE-, LiChrosorb-Diol-and LiChrosorb-NH 2 -HPLC, analytical results permit the structural conclusions summarized in Table HI . Assuming that all JV-acetylglucosamine and yV-acetylgalactosamine residues are equally labelled, and disregarding the fact that galactose residues are labelled to a small extent too, the relative amount of each glycan was roughly estimated from the distribution of 3 H radioactivity in the different structures. Given values include corresponding glycans obtained after alkaline hydrolysis (see Scheme 1).

Endo H-sensitive glycans (~9 mol%) comprise highmannose and hybrid-type glycans. Methylation analysis (cf. Table I ) of fraction H2 (3.3 mol%) revealed the presence of terminal galactose (2,3,4,6-GalOH) and 4-substituted A'-acetylglucosamine (3,6-GlcN[Me]AcOH), in addition to terminal (2,3,4,6-ManOH), 2-substituted (3,4,6-ManOH) and 3,6-disubstituted (2,4-ManOH) mannose residues and 4-substituted yv-acetylglucosaminitol (l,3,5,6-GlcN[Me]AcOH). In gel filtration, two radioactive peaks could be detected eluting at 10.4 Oligosacchaiide alditols obtained after treatment of tryptic glycopeptides with endo H (A) or PNGase F-(B) and reduction (see Scheme 1) were separated by HPAE-HPLC on a CarboPac PA1 column (4 x 250 mm) using a gradient of sodium acetate in 0.1 M NaOH as indicated. Fractions (0.35 ml) were collected at 1 ml/min and monitored for radioactivity by liquid scintillation counting. Fractions subjected to structural analysis were pooled as indicated by horizontal brackets. Numbers (3-7) with arrows are elution volumes of isomaltosyl oligosaccharide alditols with 3-7 glucose units. and 8.6 glucose units (Table II) . Sequential treatment with /3-galactosidase, a-mannosidase and /3-JV-acetylhexosaminidase resulted in the removal of one or zero galactose, three or six mannose and one or zero GlcNAc residues, respectively. Oligosaccharide alditols obtained after treatment of tryptic peptides with PNGase F and reduction (A) or reductive /S-elimination (B; see Scheme 1) were analysed by anion-exchange HPLC using a Carbopak PA 1 column (4 x 250 mm) and a linear gradient of sodium acetate in bidisnUed water as indicated. Fractions (0.5 ml) were collected at 1 ml/min and monitored for radioactivity. N and numbers (1-4) with arrows are elution volumes of neutral glycans and sialylated oligosaccharide standards with 1-4 sialic acid residues.

Incubation with a-l,2-specific mannosidase from Aspergillus oryzae and subsequent gel filtration demonstrated that the glycan originally eluting at 8.6 glucose units lost two mannose residues, whereas the elution position of species eluting at 10.4 glucose units was not changed (data not shown). Thus, it can be concluded that fraction H2 contained the two glycans depicted in Table HI. Fraction H3 (3.7 mol%) contained solely Man 8 GlcNAcOH species. This can be concluded from methylation data (presence of 2,3,4,6-ManOH, 3,4,6-ManOH, 2,4-ManOH and 1,3,5,6-GlcN[Me]AcOH and absence of other derivatives; see Table I ) as well as from results obtained after degradation with a-mannosidase (see Table II ) and a-l,2-specific mannosidase (data not shown). The isomeric structure of this species is proposed on the basis of its co-chromatography with the corresponding Man g GlcNAcOH standard in LiChrosorb-Diol-HPLC (data not shown), which can be separated from other isomers under the conditions used (Geyer et al., 1984a) .

Minor fractions HI and H4 (~1 mol% each) contained hybrid-type species and traces of MangGlcNAcOH, as evidenced by LiChrosorb-Diol-HPLC, gel filtration and digestion with a-mannosidase and a-l,2-mannosidase (data not shown). Owing to the small amounts of material, these fractions were not analysed further. -elimination were fractionated by HPLC on a LiChrosorb-NH 2 column (5 /un, 4.6 X 250 mm) using a gradient of acetonitrile in bidisOlled water as indicated. RadiolabeUed glycans were monitored by a continuous-flow radioactivity detector. Fractions (0.5 ml) were collected at 1 ml/min and pooled as indicated by horizontal brackets. The elution position of fraction 01 disaccharide is shown by an arrow.

Marburg virus glycoprotein. Partially methylated alditol acetates obtained after methylation, hydrolysis, reduction and peracetylation were analysed by capillary gas chromatography-mass fragmentography using a fused-silica, bonded-phase DB-1 column (0.25 mm i.d., 60 m length). Detection was achieved by chemical ionization with ammonia and selective monitoring of the ions (M+NHJ* (hexitol acetates) or (M+H) + (aminohexitol acetates). Alditol derivatives were assigned as follows: (1) 2,3,4-FucOH;

(2) 2,3,4,6-GalOH; (3) 3,6-ManOH; (4) 3,4-ManOH; (5) 2-ManOH; (6) 3,4,6-GlcN(Me)AcOH; (7) l,3,5-GlcN(Me)AcOH; (8) 3,6-GlcN(Me)AcOH; (9) 1,4,5-GalN(Me)AcOH (for an explanation of this short-hand notation see the legend to Table I )-The peak designated (a) represents 2,3,6-GlcOH, probably derived from cellulose contaminants. Subtle variations in the retention times of (2) and (6) arise from slight changes in the temperature gradients used.

Fraction F21 still comprised a mixture of complex-type glycans eluting at 15.2 (major component; 2.2 mol%) and 17.8 glucose units (minor fraction; 0.9 mol%) in gel filtration (Table  H) . For exoglycosidase studies, the two constituents were separated prior to digestion with /3-galactosidase. Analysis of the reaction products revealed the release of two and three galactose residues, respectively. Subsequent treatment with /3-N-acetylhexosaminidase using standard amounts of enzyme (see Materials and methods) resulted in the removal of zero or one N-acetylglucosamine residue. Only small amounts of the fragment eluting at 14.1 glucose units could be converted to a product co-eluting at 8.2 glucose units with authentic standard Man 3 GlcNAc(Fuc)GlcNAcOH (cf . Table II) . Using a 10-fold higher concentration of the enzyme, however, both components yielded the fucosylated core fragment eluting at 8.2 glucose units. In agreement with data reported by Yamashita et al. (1983a) it may, therefore, be assumed that the majority of fraction F21 glycans carry a bisecting N-acetylglucosamine residue (GlcNAc-9) at Man-3. This assumption is corroborated by methylation data revealing the presence of terminal N-acetylglucosamine and 3,4,6-trisubstituted mannose in addition to terminal fucose, terminal galactose, 2-substituted as well as traces of 2,4-and 3,6-disubstituted mannose, 4-substituted N-acetylglucosamine and 4,6-disubstituted N-acetylglucosaminitol residues (see Table I ). Taken together, the data obtained indicate the presence of fucosylated biantennary species carrying a bisecting N-acetylglucosamine residue (major component), as well as triantennary glycans (minor components), about half of which are similarly substituted by GlcNAc-9. Obviously, the latter species represented a contamination by F22 glycans (see below).

By the same line of evidence, fraction F22 (10.9 mol%) could be similarly deduced to comprise glycans with bisecting A'-acetylglucosamine (see Figure 7A -D and Table H ). Since F22 species represented a major glycan fraction of Marburg virus glycoprotein, these oligosaccharides could be further studied by LSIMS. As shown in Figure 6A -C, the analysis revealed a quasi-molecular ion ([M+H] + ) at m/z 2932 consistent with a sugar composition of Hex^HexNAc5dHexHex-NAcOH where Hex is hexose, HexNAc is A/-acetylhexosamine, HexNAcOH is jV-acetylhexosaminitol and dHex is deoxyhexose. In addition, characteristic fragment ions were observed at m/z 228 (HexNAc + minus methanol), 260 (HexNAc + ), 432 (HexHexNAc + minus methanol), 464 (HexHexNAc + ) and 450/2465 (fission of the chitobiose unit). LSIMS further revealed a significant amount of undermethylated products, which are commonly observed when glycans with bisecting JV-acetylglucosamine residues are subjected to permethylation (R.Geyer, unpublished observation). Methylation analysis revealed the presence of terminal fucose, terminal galactose, 2-substituted, 2,4-disubstituted and 3,4,6-trisubstituted mannose, terminal and 4-substituted A^-acetylglucosamine and 4,6-disubstituted /V-acetylglucosaminitol. Thus, fraction F22 species can be assumed to comprise the triantennary structure proposed in Table HI. Fraction F23 glycans (2.3 mol%) were identified as fucosylated tetraantennary species. Sequential digestion with /3-galactosidase and jS-A^-acetylhexosaminidase resulted in the liberation of four galactose and four N-acetylglucosamine residues, respectively. As shown below, the resulting fragment eluting at 8.2 glucose units again represented the fucosylated pentasaccharide core structure. Methylation analysis revealed the presence of 2,4-, 2,6-and 3,6-disubstituted mannose residues, and the absence of 3,4,6-trisubstituted mannose and terminal N-acetylglucosamine, demonstrating that this fraction represented tetraantennary glycans without bisecting N-acetylglucosamine.

Methylation data of fraction F24 (3.5 mol%) revealed the presence of terminal fucose and terminal galactose, 2,4-and 2,6-disubstituted and 3,4,6-trisubstituted mannose, terminal and 4-substituted Af-acetylglucosamine, and 4,6-disubstituted N-acetylglucosaminitol (see Figure 5A and Table I ). This is consistent with data from exoglycosidase studies which demonstrated the presence of four lactosamine antennae and bisecting Af-acetylglucosamine. Thus, fraction F24 can be concluded to represent the fucosylated tetraantennary species depicted in Table m .

Core fragments (8.2 glucose units in gel filtration) obtained from fractions F21-F24 were pooled and sequentially degraded with a-mannosidase, /3-mannosidase, /3-A'-acetylhexosaminidase and a-fucosidase. As shown in Table II , two a-linked and one /3-linked mannose, one A^-acetylglucosamine and one fucose residue could be released, leaving radioactive Af-acetylglucosaminitol (cf. Figure 7E-H) .

O-Glycans constitute more than half of oligosaccharides bound to Marburg virus glycoprotein. Fractions 01 and O2 (7.2 and 8.2 mol%) both originate from the disaccharide Gal/33GalNAc, as evidenced by its sensitivity to 0-glycosidase (only Ol) and /3-galactosidase from bovine testes, and resistance to the corresponding /3-1,4-specific enzyme from Diplococcus pneumoniae (Distler and Jourdian, 1973; Paulson et al., 1978) . Consequently, methylation analysis of fraction O2 species revealed the presence of terminal galactose and 3-substituted //-acetylgalactosaminitol (cf. Table I ). The fact that part of these glycans (i.e. O2 species) were not liberated by 0-glycosidase may be ascribed to steric hindrance.

Sequential degradation of fraction 03 glycans (1.9 mol%) with /3-A'-acetylhexosaminidase and /3-galactosidase (from bovine testes) resulted in the liberation of one HexNAc and one galactose residue, producing a radioactive fragment which coeluted with HexNAcOH at 2.5 glucose units. The HexNAc residue liberated was collected and identified as N-acetylglucosamine by HPAE-HPLC (data not shown). Methylation analysis revealed the presence of terminal galactose, terminal N-acetylglucosamine and 3,6-disubstituted N-acetylgalactosaminitol (see Figure 5B and Table I ), demonstrating that these glycans represented a type 2 core (Carraway and Hull, 1989) with the structure Gal/33[GlcNAc/S6]GalNAcOH. It is noteworthy that terminal galactosyl residues could not be liberated by treatment with high amounts of /3-galactosidase from bovine testes prior to digestion with /3-Af-acetymexosarninidase (cf . Table H) . Obviously, this enzyme is sterically hindered by the simultaneous presence of 7V-acetylglucosamine residues linked to C-6 of Af-acetylgalactosaminitol.

Fraction O4 glycans comprise -36.2 mol% of the total carbohydrate constituents of Marburg virus glycoprotein. LSIMS analysis revealed a quasimolecular ion ([M + H] + ) at m/z 961.6 consistent with a sugar composition of Hex 2 Hex-NAcHexNAcOH ( Figure 6D ). Sequential digestion with /3-galactosidase from D.pneumoniae and /3-galactosidase from bovine testes demonstrated the release of only one /34-linked galactose. Subsequent treatment with /3-yV-acetylhexosaminidase and /3-galactosidase liberated one /3-linked A'-acetylglucosamine and again one /3-linked galactose residue, leaving a radioactive fragment co-eluting with HexNAcOH (cf. Table  IT ). In contrast, methylation analysis revealed only the presence of terminal galactose, 4-substituted N-acetylglucosamine and 3,6-disubstituted A^-acetylgalactosaminitol, ruling out a linear molecule with a type-1 core. As already discussed in the case of fraction O3 glycans, the terminal galactose residue linked to C-3 of N-acetylgalactosaminitol obviously can not be removed by treatment of the tetrasaccharide with /3-galactosidase from bovine testes due to steric hindrance. Thus, it can be concluded that fraction O4 glycans comprised the branched tetrasaccharide depicted in Table IH .

Sequential enzymic digestion of fraction 05 glycans (2.1 mol%) revealed the presence of two different hexasaccharides (see Figure 8 and Table H ). Liberation of one or two terminal Galj34 residues by treatment with /3-galactosidase from D.pneumoniae ( Figure 8A ) and one or two /3-linked N-acetylglucosaminyl residues by incubation with /3-A/-acetylhexosaminidase ( Figure 8B ) resulted in a tetra-and a disaccharide fragment eluting at 6.5 and 3.5 glucose units, respectively ( Figure 8C ), which could be further degraded to HexNAcOH ( Figure 8D-F) . Although methylation data were not available .5/2.5 3.5 C 2.5 /3-Galactosidase b |3-Galactosidase' i /3-/V-Acetylhexosaminidase /S-Galactosidase" 1 /S-A'-Acetylhexosaminidase /S-GalactosidaseN eutral oligosaccharide alditol fractions (H2, H3 and F21-F24 in Figure 2 ; O2-O5 in Figure 4 ; 01 see Scheme 1) isolated from Marburg virus glycoprotein, metabolically labelled with [ 3 H]glucosamine, were sequentially digested with the enzymes indicated and the products were analysed by gel filtration using a Bio-Gel P-4 column. The results are expressed as the number of glucose units (Yamashita et al., 1982 (Yamashita et al., , 1983b Kobata et al., 1987) of the fragments obtained. 'Fractions contained at least two different oligosaccharide species. b /3-Galactosidase from D.pneumoniae. 'After reduction^ the corresponding oligosaccharide alditol elutes at 3.5 glucose units. d /9-Galactosidase from bovine testes. "Liberated HexNAc eluted at ~2 glucose units and was identified as GlcNAc.

in this case, it may be assumed that 05 glycans represent 04 species elongated by an additional lactosamine unit at either galactose. The linkage positions of the corresponding N-acetylglucosamine residues, however, were not determined. Obviously, the galactose linked to C-3 of N-acetylgalactosaminitol is again resistant to jS-galactosidase from bovine testes as long as N-acetylgalactosaminitol is further substituted at C-6.

This study describes the first detailed carbohydrate structure analysis of the envelope glycoprotein of a filovirus. Marburg virus, a highly pathogenic member of this family, was propagated in E6 cells which were cultivated in the presence of [6-3 H]glucosamine. Starting from the supernatant (100 ml) of five tissue culture flasks only, radiolabelled virus particles were harvested and purified by gradient centrifugation. Since only the viral glycoprotein was metabolically labelled by this procedure (cf. Figure 1) , the protein preparation, obtained after disintegration of the virions with detergents, could be subjected to carbohydrate analysis directly without further purification of the glycoprotein.

Oligosaccharide constituents were sequentially released from tryptic glycopeptides by enzymatic as well as chemical cleavage, and fractionated by different HPLC methods. Oligosac-charides obtained were studied by sequential degradation with exoglycosidases, methylation analysis and, in part, by LSIMS. The results reveal that Marburg virus glycoprotein is heterogeneously glycosylated in E6 cells. About 9 mol% of total glycans were of the high-mannose or hybrid type, whereas 19 mol% comprised fucosylated bi-, tri-and tetraantennary complex-type species, most of which carried a bisecting /V-acetylglucosamine residue. The majority of oligosaccharides (~55 mol%) were found to represent O-linked carbohydrate chains with 2-6 sugar residues. Owing to the small amounts of starting material, the structures of the remaining glycans could not be elucidated.

The results obtained are in agreement with previous data (Feldmann et al., 1991) demonstrating the presence of highmannose and complex-type N-glycans, as well as (9-glycosidically linked Gal/?3GalNAc disaccharides by endoglycosidase treatment of the total glycoprotein and by lectin studies. Furthermore, in that study the reaction of peanut agglutinin with this glycoprotein was not extinguished by treatment with 0-glycosidase. Similar results were now obtained in the case of tryptic peptides. Only about half the amount of disaccharide present could be released by repeated enzyme treatment, whereas the remaining half had to be liberated by reductive |3-elimination. Possibly due to steric hindrance, part of the linkages between N-acetylgalactosamine and the corresponding serine (or threonine) residue seem to be inaccessible to this Fig. 7 . Sequential degradation of oligosaccharide aiditol fraction F22 from Marburg virus glycoprotein with exoglycosidases. Fraction F22 (A) was successively digested with 0-galactosidase (B) and 0-W-acetylhexosaminidase (C, standard conditions; D, 10-fold amount). The core fragment thus obtained was combined with core fragments of fractions F21, F23 and F24, and degraded by a-mannosidase (E), 0-mannosidase (F), 0-W-acetylhexosamiriidase (G) and a-fucosidase (H). Starting material and reaction products were chromatographed on a calibrated Bio-Gel P-4 column (-400 mesh; 16 x 600 mm) at 55°C using 0.02% aqueous sodium azide as eluant. Fractions (0.4 ml) were collected at 0.2 ml/min and monitored for radioactivity. Brackets indicate fractions pooled for further degradation. Column calibration with glucose oligomers (2-20 glucose residues) is shown by arrows. enzyme. The exact positions of O-glycosylation sites, however, remain to be elucidated. Nucleotide sequencing of the corresponding gene revealed that Marburg virus glycoprotein contains several potential N-glycosylation sites, of which the a-amino groups of asparagine residues are connected with the carboxyl group of lysine (data not shown). Thus, tryptic digestion leads, in part, to glycopeptides having glycosylated asparagine residues at their N-termini. Since PNGase F does not release asparaginelinked glycans from such glycopeptides (Tarentino et al., 1985) , the PNGase F-resistant glycopeptides obtained in this study obviously represent this type of molecule.

Anion-exchange HPLC of fraction F2 revealed the presence of trace amounts of charged material, the chromatographic properties of which, however, were not influenced by treatment with sialidase, indicating that this material did not contain sialic acids. Since Marburg virus glycoprotein can be radiolabelled with neither [ 35 S]sulphate nor [ 32 P]orthophosphate (data not shown), it may be concluded that this charged material represented small GlcNAc-peptides produced by endo H treatment which were not separated off by RP-HPLC.

The fact that Marburg virus glycoprotein glycans completely lack sialic acid residues is corroborated by lectin studies demonstrating that neither SNA nor MAA recognizing Structures were deduced from the analytical results described. The molar ratios were roughly estimated from the distribution of 3 H radioactivity incorporated into /V-acetylglucosamine and Af-acetylgalactosamine residues. In the structure of F24, the designation of sugar residues in Af-acetyllactosaminic glycans (Vliegenthart a al., 1983) is indicated.

(a2-6)-and (a2-3)-linked sialic acids, respectively, reacted with this glycoprotein. In contrast, the presence, of (a2-3)linked sialic acid residues could be clearly shown in the case of the glycoproteins from the morphologically and genetically related Ebola and Reston viruses propagated in the same cell line. Thus, the sialylation capacity of E6 cells is not generally abolished by filovirus infection. It might be possible, however, that the expression of sialylated glycoproteins is selectively affected by Marburg virus, but further analysis, e.g., of cellular glycoproteins before and after Marburg virus infection, are required to clarify this point. Alternatively, one might assume that Marburg virus glycoprotein has intrinsic sialidase activity similar to corresponding envelope glycoproteins of para-and orthomyxoviruses. Up to now, however, such enzymatic activity could not be detected in mature virus particles. Possibly, the glycoprotein does not reach the intracellular compartments involved in the sialylation of glycoconjugates.

Only a limited number of viral glycoproteins have been reported to carry O-glycans. While glycoprotein El of murine coronavirus carries exclusively CMinked sugar chains (Niemann etai, 1984) , herpes simplex virus glycoproteins (Oloffson etai, 1981; Dall'Olio etai, 1985; Serafini-Cessi et al., 1988) , vaccinia virus haemagglutinin (Shida and Dales, 1981) , G protein of respiratory syncytial virus (Gruber and Levine, 1985) , glycoprotein 65 encoded by Friend spleen focus-forming virus (Gliniak and Kabat, 1989; Pinter and Honnen, 1989) and glycoprotein 71 of Friend murine leukaemia virus (Geyer etai., 1990) comprise both O-and N-linked oligosacharides, similar to Marburg virus glycoprotein. Since carbohydrate side chains can influence the antigenicity and immunogenicity of viral glycoproteins either by masking or by stabilization of polypeptide epitopes [for review, see Klenk (1990) ], the heterogeneous glycosylation pattern of Marburg virus glycoprotein might be an important parameter determining the chances of success of the human immune response during infection. In addition, the substitution by large amounts of O-and /V-glycans could influence the maintenance of the biologically active conformation, and protect the peptide chain from proteolytic attack.

The origin and structures of oligosaccharide standards used for column calibration were given in detail previously (Pfeiffer etai., 1989 (Pfeiffer etai., , 1990 ). a-l,2-Specific mannosidase from A.oryzae was generously provided by Dr H.Yamaguchi (University of Osaka, Osaka, Japan).

The Musoke strain of Marburg virus isolated in 1980 in Kenya (Smith et al., 1982) has been used. The virus was propagated in E6 cells, a cloned line of Vero cells (ATCC CRL 1586), as follows. E6 cells were infected with Marburg virus, strain Musoke, at a multiplicity of infection of 10~2 plaque-forming units/cell. Following an adsorption period of 30 min at 37°C, cells were incubated in Dulbecco's medium containing 2% fetal calf serum. In vivo radioactive labelling of viral proteins was performed 5 days post-infection. The maintenance medium was removed and the cell monolayers were washed twice with glucose-deficient Dulbecco's medium containing 1.8 g fructose/1 (for labelling with [ 3 H]glucosamine) or methionine-deficient medium (for labelling with ["SJmethionine). Cells were incubated in deficiency medium for 2 h. The incubation was continued for 24 h at 37 °C in the specific nutrient-deficient Dulbecco's medium to which radioactive nutrients were added as follows: 20 jiCi/ml of D-[6-3 H]glucosamine or 20 fiCi/ml of ["Slmethionine (Amersham Buchler, Braunschweig, FRG). Isolation and purification of the virus were as described previously (Feldmann etai., 1991; MQhlberger etai., 1992) . Briefly, culture fluid was clarified by centrifijgation at 6000 g (Minifuge RF, Heraeus, FRG) for 15 min at 4°C and virus particles were isolated by centrifugation in a SW28 rotor through a 20% sucrose cushion in TNE buffer (0.01 M Tris HC1, pH 7.4; 0.15 M NaCl; 2 mM EDTA) at 113 000# for 2 h at 4°C. The pellet was resuspended in TNE buffer and the virus particles were purified by gradient centrifugation in a SW28 rotor using a gradient of 0-40% (w/v) potassium tartrate, 30-0% glycerol in TNE buffer at 160 000 g for 16 h at 4°C. The virus band was isolated, diluted 1 in TNE buffer, pelleted by centrifugation in a SW41 rotor at 210 000 g for 30 min at 4"C and resuspended in TNE buffer. The virus was disintegrated by adding SDS up to a final concentration of 1%. For carbohydrate structure analysis the glycoprotein, metabolically labelled with [6-3 H]glucosamine, was precipitated with acetone in order to remove salts and detergents, and redissolved in buffer for proteolytic digestion.

Electrophoresis of proteins was carried out as described by Laemmli (1970) . Metabolically labelled virion proteins were visualized by fluorography as follows. Gels were soaked for 30 min in 30% methanol and 10% glacial acetic acid for protein fixation, treated for 30 min with ENLJGHTNING™ autoradiography enhancer (NEN, Bad Homburg, FRG), dried and exposed to an X-ray film (Kodak). For calibration, rainbow [ 14 C]methylated protein molecular mass markers (Amersham) were used.

For lectin staining, the glycan differentiation kit (Boehnnger, Mannheim) containing digoxigenin-conjugated lectins was used. Viral extracts were subjected to SDS gel electrophoresis before and after treatment with sialidase, blotted onto nitrocellulose, incubated with MAA. or SNA and stained with anti-digoxigenin antibodies conjugated with alkaline phosphatase according to the protocol of the manufacturer, except that the nitrocellulose was incubated overnight at 4°C in the blocking solution.

For the determination of labelled monosaccharide components, a sample (2 x 10 3 c.p.m.) of glycoprotein was hydrolysed in 1 ml of 1 N trifluoroacetic acid at 80°C. After 30 min, an aliquot (500 y\) was taken for the determination of labelled sialic acids, whereas the remaining 500 y\ were further incubated at 80°C for 24 h. Both samples were dried by rotary evaporation, repeatedly redissolved in 1 ml of methanol and dried again. The radiolabelled monosaccharides obtained were identified by HPAE-HPLC (see below).

Marburg virus glycoprotein was digested with trypsin (Serva, Heidelberg, FRG) as described elsewhere (Schlflter et al., 1985) . N-Linked oligosaccharides were released from tryptic glycopeptides by sequential treatment with endo H from Streptomyces griseus and PNGase F from Flavobaaerium meningosepticum (both from Boehringer, Mannheim, FRG), separated from residual (glyco)peptides by gel filtration or RP-HPLC, reduced and desalted as described earlier (Strube etai., 1988; Geyer and Geyer, 1990) . O-Glycans were liberated by incubation with O-glycosidase from D.pneumoniae (Boehringer) in 20 mM sodium phosphate buffer (pH 7.2), 50 mM EDTA, 0.02% sodium azide (50 pkat of enzyme; 100 jil buffer, 24 h, 37°C) and isolated by gel filtration. Remaining O-glycans were released by ^-elimination using 0.8 M sodium borohydride/50 mM sodium hydroxide as described previously (Geyer el al., 1990; Geyer and Geyer, 1992) and separated from peptide material by RP-HPLC. For alkaline hydrolysis of PNGase F-and /S-elimination-resistant glycopeptides, the experimental protocol of Geyer et al. (1984b) was followed. After re-N-and de-0-acetylation, residual peptides were removed by binding to mixed-bed ion-exchange resin (Amberlite AG-MB3; Serva, Heidelberg, FRG) (Strube etai., 1988) .

(i) Desalting of glycopeptides and oligosaccharide alditols, (ii) RP-HPLC for separation of oligosaccharides from (glyco)peptides, (iii) separation of glycans by gel filtration, HPAE-HPLC, HPLC using columns of LiChrosorb-Diol or LiChrosorb-NH 2 (Merck, Darmstadt, FRG) and anion-exchange HPLC at neutral pH using a Carbopak PA1 column, and (iv) chromatographic characterization of oligosaccharide alditol fragments by Bio-Gel P-4 chromatography were carried out as described in detail previously (Schlflter et al., 1985; Strube etai., 1988; Holschbach etai., 1990; Pfeiffer etai., 1990; Wendorf etai., 1991; Geyer and Geyer, 1992) . For fractionation of O-glycans, a LiChrosorb-NH 2 column (250 x 4.6 mm; 5 jtm; Merck, Darmstadt, FRG) equilibrated with 75% aqueous acetonitrile was used and the concentration of acetonitrile was reduced to 50% in 90 min (Lamblin et al., 1984) .

Identification of radiolabelled monosaccharides by HPAE-HPLC was achieved using 125 mM sodium acetate in 100 mM aqueous sodium hydroxide (sialic acids) or 10 mM sodium hydroxide (neutral and amino sugars) as eluents (Hardy et al., 1988) .

Degradation of oligosaccharide alditols with (i) sialidase from V.cholerae (Behringwerke, Marburg, FRG), (ii) /S-A'-acetylhexosaminidase (5 or 50 nkat) and a-mannosidase from jack beans (Sigma, Deisenhofen, FRG), (iii) /3-galactosidase from D.pneumoniae (Boehringer, Mannheim, FRG), (iv) ar-fucosidase from beef kidney (Boehringer), and (v) a-1,2-specific Glycosylation of Marburg virus glvcoprotein mannosidase from A.oryzae was performed as described elsewhere (Geyer et al., 1982 (Geyer et al., , 1984b Strobe et al., 1988; Holschbach et al, 1990) . For treatment with j3-galactosidase from bovine testes (Boehringer) or /3-mannosidase from Helix pomaria (Sigma), oligosaccharide samples were taken up in 50 /d of 100 mM sodhim phosphate/citrate buffer, 0.1 % (w/v) bovine serum albumin (pH 4.4), or 50 mM sodium acetate buffer (pH 4.0) and incubated at 37°C for 24 h with 1 /ikat or 240 nkat of enzyme, respectively.

Oligosaccharide alditols were permethylated (Paz Parente etal., 1985) and hydrolysed. Partially methylated alditol acetates obtained after sodium borohydride reduction and peracetylation were analysed by capillary GLC/MS using the instrumentation and microtechniques described earlier (Geyer et al., 1983) .

LSIMS LSIMS was carried out using a Finnigan MAT 900 mass spectrometer (Finnigan MAT, Bremen, FRG) equipped with an array detector and a caesium gun which was operated at 23 kV with an emission current of 2-3 /iA. Mass spectra of methylated F22 glycans were recorded in positive ion mode at an accelerating potential of 5 kV and a resolution of -4000 (high mass range) or 1000 (low mass range) with a linear magnet scan (30 s/1000 mass unit). Within each mass range, 5 {m/z 160-1000) or 9 (m/z 2000-^tOOO) scans were accumulated. Spectra of methylated O4 glycans were recorded at constant magnetic field solely within the mass range of m/z 920-990 (10 s/scan) using the array detector of the instrument. In this case, 20 scans were accumulated. In all experiments, data were acquired with a DEC 2100 data system and 3-nitrobenzyl alcohol (Aldnch, Steinheim, FRG) was used as matrix.

O-Glycosylation pathway for mucin-type glycoproteins

Centres for Disease Control (1989) Ebola virus infections in imported primates-Virginia

Sialylated oligosaccharides (?-glycosidically linked to glycoprotein C from Herpes simplex virus type 1

The purification and properties of 0-galactosidase from bovine testes

Glycosylation and oligomerization of the spike protein of Marburg virus

Glycosylation of the envelope glycoprotein from a polytropic murine retrovirus in two different host cells

Isolation and fractionation of glycoproteinglycans in small amounts

Separation procedure and sugar composition of oligosaccharides in the surface glycoprotein of Friend murine leukemia virus

Methylation analysis of complex carbohydrates in small amounts: Capillary gas chromatography-mass fragmentography of methylalditol acetates obtained from N-glycosidically linked glycoprotein oligosaccharides

Structure of the oligosaccharides sensitive to endo-/3-/v r -acetylglucosaminidase H in the glycoprotein of Friend murine leukemia virus

Major oligosaccharides in the glycoprotein of Friend murine leukemia virus: Structure elucidation by one-and two-dimensional proton nuclear magnetic resonance and methylation analysis

Oligosacchandes at individual glycosylation sites in glycoprotein 71 of Friend murine leukemia virus

Leukemogenic membrane glycoprotein encoded by Friend spleen focus-forming virus: Transport to cell surfaces and shedding are controlled by disulfide-bonded dimerization and by cleavage of a hydrophobic membrane anchor

Respiratory syncytial virus polypeptides. IV. The oligosaccharides of the glycoproteins

Monosaccharide analysis of glycoconjugates by aiuon exchange chromatography with pulsed amperometric detection

Glycosylation of the envelope glycoprotein gpl3O of simian immunodeficiency virus from sooty mangabey

The order Mononegavirales. Paramyxovirus Study Group of the Vertebrate Subcommittee

Preliminary report: isolation of Ebola virus from monkeys imported to USA Lancet

Isolation and partial characterization of a new virus causing acute hemorrhagic fever in Zaire

Filoviridae: a taxonomic home for Marburg and Ebola viruses 7 Intervirology

Physicochemical properties of Marburg virus: evidence for three distinct virus strains and their relationship to Ebola virus

Influence of glycosylation on antigenicity of viral proteins

BioGel P-4 column chromatography of oligosaccharides: Effective size of oligosaccharides expressed in glucose units

Cleavage of structural proteins during the assembly of the head of bacteriophage T4

Further characterization, by a combined high-performance liquid chromatography/1H-NMR approach, of the heterogeneity displayed by the neutral carbohydrate chains of human bronchial mucins

The nucleotide sequence of the L gene of Marburg virus, a filovirus: homologies with paramyxoviruses and rhabdoviruses

The carbohydrates of mouse hepatitis virus (MHV) A59: structures of the O-glycosidically linked oligosacchandes of glycoprotein El

O-glycosidic carbohydratepeptide linkages of Herpes simplex virus glycoproteins

Sialyl-and fucosyltransferases in the biosynthesis of asparaginyl-linked oligosaccharides in glycoproteins

A convenient method for methylation of glycoprotein glycans in small amounts by using lithium methylsulfinyl carbanion

Carbohydrate structure of recombinant human uterine tissue plasminogen activator expressed in mouse epithelial cells

Separation of glycoprotein-JV-glycans by high-pH anion-cxchange chromatography

Biochemical characterization of cellassociated and extracellular products of the Friend spleen focus-forming virus env gene

Sequence analysis of the Marburg virus nucleoprotein gene: Comparison to Ebola virus and other nonsegmented negative-strand RNA viruses

Isolation of glycopeptides containing individual glycosylation sites of Friend murine leukemia virus glycoprotein: Studies of glycosylation by methylation analysis

Comparative study on CMinked oligosacchandes of glycoprotein D of Herpes simplex virus types 1 and 2

Biogenesis of vaccinia: Carbohydrate of the hemagglutinin molecule

Marburgvirus disease in Kenya

Carbohydrate structure of glycoprotein 52 encoded by the polycythemia-inducing strain of Friend spleen focus-forming virus

Jr (1985) Deglycosylation of asparagine-linked glycans by peptide^-glycosidase F

High-resolution, 'H-nuclear magnetic resonance spectroscopy as a tool in the structural analysis of carbohydrates related to glycoproteins

Carbohydrate structure of human pancreatic elastase 1

Analysis of oligosacchandes by gel filtration

Structural studies of the carbohydrate moieties of rat kidney gammaglutaminyltranspeptidase

Structural studies of the sugar chains of hen ovomucoid

We express our gratitude to Werner Mink, Peter Kaese and Siegfried KOhnhardt for methylation, GLC/MS and LSIMS analyses. This project was supported by the Deutsche Forschungsgemeinschaft (Sti 38/21-1; Ge 527/2-1; Sonderforschungsbereich 272, Teilprojekt Z2 and Sonderforschungsbereich 286, Teilprojekt A6). Abbreviations dHex, deoxyhexose; endo H, endo-0-JV-acetylglucosaminidase H; Fuc, L-fucose; GalNAcOH, A'-acetylgalactosaminitol; GlcNAcOH, /V-acetylglucosaminitol; GLC/MS, gas-liquid chromatography/mass spectrometry; Hex, hexose; HexNAc, /V-acetylhexosamine; HexNAcOH, A'-acetylhexosaminitol; HPAE-HPLC, high-pH anion-exchange HPLC; LSIMS, liquid secondary-ion mass spectrometry; MAA, Maackia amurensis agglutinin; PNGase F, peptide-A^/V-acetyl-jS-glucosaminylJasparagine amidase F; RP-HPLC, reversed-phase HPLC; SNA, Sambucus nigra agglutinin.