O-Linked GlcNAc in serotype-2 adenovirus fibre

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<ul><li><p>Eur. J. Biochem. 184,205-211 (1989) FEBS 1989 </p><p>@Linked GlcNAc in serotype-2 adenovirus fibre Marie-Laure CAILLET-BOUDIN ', Gerard STRECKER' and Jean-Claude MICHALSKI * </p><p>' Laboratoire de Chimie Biologique et Unite de Recherche Associte du Centre National de la Recherche Scientifique no. 217, Unite de Virologie Moltculaire de I'lnstitut National de la Sante et de la Recherche Medicale (Unite 233), Lille </p><p>Universite des Sciences et Tcchniques de Lille Flandres-Artois </p><p>(Received March 29/May 26, 1989) - EJB 89 0380 </p><p>Serotype-2 adenovirus fibre is shown to possess an 0-linked GlcNAc residue and to have affinity for wheat germ agglutinin. The cytoplasmic and nuclear fibres are both glycosylated. Glycosylation seems to take place in the cytoplasm since most of the [ ''C]ClcN-labelled fibre is found in this compartment, little label being associated with the microsomes. Glycosylation of the fibre was not affected by inhibitors of N- and 0-glycosylation. </p><p>A variation in fibre glycosylation is observed among adenovirus. Among the serotypes tested, only serotype- 5 adenovirus (another subgroup C virus) also incorporated [14C]GlcN into its fibre, but did not possess affinity for wheat-germ agglutinin. </p><p>The GlcNAc is located in the N-terminal two-thirds of the fibre and more probably in the N-terminal one- third. The free or penton-base-associated fibres are similarly glycosylated. These results suggest that glycosylation is not involved in viral adsorption and in assembly with the capsid penton base. Thus, glycosylation might be a characteristic feature of subgroup C viruses. </p><p>Adenovirus is a well-characterized double-stranded DNA virus encapsulated in a icosahedral capsid. The latter is composed of at least nine structural proteins. Ishibashi and Maize1 [l] have shown that one adenovirus type-2 early pro- tein and one late protein were glycosylated, i.e. proteins synthesized respectively before or after DNA viral replication. The glycosylated early-protein gene is mapped in the early region E3 [2]. This protein has an apparent molecular mass of 19 kDa. It is synthesized and glycosylated in the endoplasmic reticulum, processed from a precursor and kept in the endo- plasmic reticulum [ 3 - 51. Its glycan is linked to the protein by a classical GlcNAc-Asn linkage [6]. </p><p>The glycosylated late protein of the adenovirus was ident- ified as being the fibre [l]. This protein is anchored in the penton base at the capsid vertices by its shaft part, the knob being needed for viral adsorption onto the cell receptors (reviewed in [7]). ['4C]GlcNH, was incorporated into the fibre polypeptide. The sugar-protein linkage is alkali sensitive [l]. </p><p>GlcNAc residues occur both in N-glycosidic-type and 0- glycosidic-type glycoproteins. In the N-glycans, GlcNAc is involved in the N-glycosidic bond with the amide nitrogen of asparagine. In 0-linked glycans, GlcNAc residues most commonly occur in oligosaccharides linked to polypeptide by a GalNAc residue (reviewed in [8, 91). Recently, evidence for an 0-linked GlcNAc was reported [lo, 1 I]. Since then several proteins with such an 0-linked GlcNac were described, such as the proteins of the nuclear pore complex [12- 141, a human erythrocyte cytoplasmic protein [15], nuclear proteins [16, 171 and the cytomegalovirus 149-kDa protein [ 181. </p><p>In this paper, we studied adenovirus type-2 fibre and ident- ified 0-linked GlcNAc residues. Glycosylation probably takes </p><p>Corrrspondence to M. L. Caillet-Boudin, Unite de Virologie Moleculaire de I'INSERM [U. 2331, 2 place de Verdun, F-59045 Lille Ccdex, France </p><p>Ahhreviations. WGA, Wheat-germ agglutinin; Con A, concana- valin A; GalNAc, N-acetyl-P-D-galactosamine. </p><p>place in the cytoplasm and was not affected by either N- or 0-glycosylation inhibitors. Fibre glycosylation was shown to vary among different adenovirus serotypes and GlcNAc might be a characteristic feature of subgroup-C adenoviruses. The role of this 0-linked GlcNAc is discussed. </p><p>MATERIALS AND METHODS </p><p>Virus and cells To purify viruses, human adenovirus types 2 , 3, 4, 5 , 7 , 9 </p><p>and wild type were grown on KB cells maintained in suspen- sion culture at 4 x l o 5 cells/ml in Eagle's minimum essential medium supplemented with 5% horse serum. Cells were in- fected at a multiplicity of infection of 10 - 20 plaque-forming units/cell. </p><p>To test glycosylation inhibitors and to prepare micro- somes, adenovirus type-2 infection was performed on HeLa cell monolayers in Dulbecco's modified Eagle's medium. </p><p>PuriJicution of adenovirus type-2jibre and penfon Soluble fibre and penton were purified from adenovirus- </p><p>type-2-infected KB cells by a four-step procedure, namely fluorocarbon extraction, ammonium sulfate precipitation, DEAE-Sephadex and hydroxyapatite chromatography [19]. </p><p>Labelling conditions </p><p>To label glycoproteins, D-U-['~C]G~CN (300 Ci/mol, Com- missariat a 1'Energie Atomique, Saclay, France) was added in a normal culture medium while ~ - [ 2 - ~ H ] M a n (16 Ci/mmol, New England Nuclear, USA) was added after 20-fold concen- tration of the cells in glucose-depleted medium. </p><p>To label proteins, met (600 - 700 Ci/mmol, Amer- sham, UK), ~- [3 ,4-~H~]Val (35 Ci/mmol, France) or [I4C]- </p></li><li><p>206 </p><p>formic acid (sodium salt; 60 Ci/mol, Commissariat a 1'Energie Atomique, Saclay, France) were added in appropriate amino- acid-depleted medium. </p><p>To purify the fibre from labelled infected cells, radioac- tivity was added at 1 pCi for labelled amino acids and at 0.125 pCi/ml for ['4C]GlcN. To obtain a highly labelled cell extract, radioactivity was used at 15 pCi/mmol for a shorter time (1 h or 6 h). </p><p>Analysis of sugar composition The fiber was washed by three precipitation rounds in 50% </p><p>ethanol/water (by vol.), was then lyophilized and submitted to acidic methanolysis (methanol/0.5 M HCl, 80"C, 24 h). Monosaccharides were analyzed by GLC as trifluoroacetate derivatives according to Zanetta et al. 1201. </p><p>Acid hydrolysis of [ ''C]GlcN-labelled fiber was performed in 4 M trifluoroacetic acid at 100C for 24 h. The hydrolysate was dried by evaporation under reduced pressure, resuspended in water and analyzed by thin-layer chro- matography (Silica gel 60, Merck, Darmstadt, FRG) with n- butanol/acetic acid/water (2: 1 : 1 ; by vol.) as a solvent. Galactosamine and glucosamine (chloride salts, Sigma) were used as standards. For determination of radioactivity, the plate was scraped every 0.5 cm, the silica was resuspended in water (500 pl) and 4.5 ml scintillation cocktail was added. </p><p>Release qf oligosaccharides from the j h r hy reductive ulkaline treatment or hydrazinolysis </p><p>[14C]GlcN-radiolabelled fiber was treated with 2 mlO.1 M NaOH, 1 M KBH, at 37C for 24 h. The reaction was stopped by adding Dowex 50x8 (20 - 50 mesh, H + form). Borate was eliminated as methylborate by repeated evaporation under reduced pressure with methanol. </p><p>The reaction mixture was chromatographed on a Bio-Gel P-4 column (200-400 mesh; 1 x 150 cm), in 50 mM acetic acid. Radioactive material was pooled and further analyzed by descending paper chromatography on Whatman 3 paper with rz-butanollacetic acid/water (4: 1 : 5 ; by vol.) as solvent. Glucosaminitol was identified after de-N-acetylation with 6 M HCI (IOOT, 4 h) by ion-exchange chromatography on a column of AG 50 WX8 (Hi form; Bio-Rad; 0.5 x 20 cm), equilibrated and eluted with 0.3 M HCI 1211. </p><p>Hydrazinolysis was according to [22]. </p><p>Electrophoretic techniques </p><p>Analytical SDSIPAGE. Polypeptide analysis was per- formed in a SDS-containing 15% polyacrylamide slab gel (acrylamide/bisacrylamide, 50 : 0.235) overlaid by a 5% spacer gel (acrylamide/bisacrylamide, 50: 1.33) in the discontinuous buffer system described by Laemmli [23]. Samples were de- natured by heating for 2 min at 100 "C in 0.0625 M Tris/HCl, pH 6.8, 4% SDS, 6 M urea, 10% 2-mercaptoethanol, 0.002% bromophenol blue. </p><p>A,lffi'nity electrophoresis. To test adenovirus type-2 native fibre with different lectins, immunoelectrophoresis-like affin- ity electrophoresis was used (as described by Brig-Hansen et al. [24]). Lectins (concanavalin A, wheat-germ agglutinin, Lens culinzaris agglutinin up to 1 mg/ml) were incorporated into the first dimension agarose gel. Anti-(adenovirus type-2 fibre) serum (15 pl/ml) was included in the second dimension agarose gel. </p><p>Immuno-blotting. This was achieved according to Towbin et al. [25]. Western blots were incubated successively with wheat-germ agglutinin (WGA; 30 pg/ml) and avidin-peroxi- dase (25 pg/ml). In the HCI-hydrolyzed-fibre study, biotinylated WGA (Vecor Laboratories, Burlingham, USA; 10 p/ml) was used to intensify the reaction and then streptavidin-peroxidase was added. </p><p>Chemical hydrolysis of the f ibre </p><p>This was as described by Caillet-Boudin et al. [26]. </p><p>Cel1,fractionation </p><p>Rough microsomes were isolated using a modification of the procedure described in 1271. During purification, nuclei and cytoplasm aliquots were stored to test fibre glycosylation in these two cellular compartments. The post-mitochondria1 supernatant was adjusted to 1.8 M sucrose by addition of 2.5 M sucrose in SO mM Tris/HCl, pH 7.4, 50 mM KC1, 5 mM MgClz and laid on 0.5 ml 2 M sucrose. Then, 1 ml 1.55 M sucrose, 7 ml 1.35 M sucrose and 0.5 nil 1 M sucrose were successively laid on. Centrifugation was performed for 15 h at 41 000 rpm in a tft 65 rotor. </p><p>Ainino mid analysis </p><p>Samples of adenovirus fibre corresponding to 0.225 mg protein were hydrolyzed for 24 h with 4 M HCl in sealed tubes under nitrogen and analyzed in a Beckman multichrom amino acid autoanalyser. </p><p>RESULTS </p><p>Giycm characterization </p><p>Purified fibre was subjected to hydrolysis in 4 M HC1 for 24 h at 110C and the hydrolysate was analyzed on an amino acid analyzer. Glucosamine was detected at approximately one residue/polypeptide. Galactosamine was absent. We then made an attempt to label the fibre with ['4C]GlcN and [3H]Man as described in Material and Methods. Label was found only when ['4C]GlcN was used. </p><p>[14C]GlcN was incorporated without metabolic conver- sion because only GlcN was identified by GLC analysis of monosaccharide released after acid methanolysis and by thin- layer chromatography analysis of the monosaccharides re- leased after acid hydrolysis of the ['4C]GlcN-labelled fibre. </p><p>The glycan-protein linkage was alkali labile. Identification of the group liberated by mild alkaline hydrolysis indicated that more than 90% of the radioactivity was present in a fraction comigrating with an acetylglucosaminitol standard. Ion-exchange chromatography after strong acid hydrolysis confirmed the occurrence of glucosaminitol (Fig. 1). N o trace of N-acetylgalactosaminitol was detected after alkaline elimin- ation. This excluded the most common 0-glycosylation species : the 0-GalNAc linkage. Experimental conditions used for the p-elimination reaction excluded the possibility of GlcNAc linked to amino acids such as OH-Lys or OH-Pro, these types of linkages not being hydrolyzed in the alkaline conditions used in this study. </p><p>No GlcNAc was found after the hydrazine treatment. After re-N-acetylation and chromatography on Bio-Gel P-4 all radioactivity was co-eluted as micromolecular material (co-eluting with 3 H 2 0 ) . We assume that, like most 0-linked </p></li><li><p>207 </p><p>A Table 1 Glycosylation in the presence of glycosylation inhibitors Thc ['4C]GlcN and [35S]Met labelling were performed separately </p><p>GlcNAc-ol </p><p>0 </p><p>Distance, c m </p><p>GIcNHz-oI GalNH2-ol r I i ' </p><p>f r t 10 20 LO 60 </p><p>rnl </p><p>Fig. 1. Liberation of N-acetylfilucosaminitol by alkulinelreductive tr-mtment. (A) Paper chromatography. (B) The material migrating like the N-acetyl-glucosaminitol (GlcNAC-ol) by paper chromatography was cluted, hydrolyzed with 6 M HCI for 4 h at 100'C and applied on an AG50 WX8 column in 0.3 hl HCI. Glucosaminitol (GlcNH2-ol) and galiictosaminitol (GalNH2-oI) wcrc added as internal standards </p><p>monosaccharides, GlcNAc was released and largely degraded during hydrazinolysis as suggested by Takasaki et al. [28]. This result together with the observed release of N-acetyl- glucosaminitol after the /?-elimination reaction, confirms the presence of an 0-linked GlcNAc in the adenovirus type-2 fibre. </p><p>The adenovirus tpe-2 fibre shows affinity for WGA, simi- lar to the nuclear-pore protein [12, 141. WGA was able to slow down the fibre electrophoretic migration when incorporated in the agarose gel while neither concanavalin A (Con A) nor Lens culinaris agglutinin affected it. The WGA affinity was also confirmed by Western blotting. GlcNAc (0.1 M) in the incubation buffer inhibited the affinity for by WGA. </p><p>Effect of N- and 0-glycosylujion inhibitors Since the 0-GlcNAc linkage was unusual and only re- </p><p>cently described, specific glycosylation inhibitors were tested. Tunicamycin and 2-deoxy-D-glucose, which are both inhibi- tors of N-linked glycosylations did not affect ['4C]GlcN incor- poration at the usual concentrations (0.5 pg/ml and 10 mM, respectively). At higher concentrations, [14C]GlcN incorpor- ation decreased in the same way as [35S]Met incorporation. Under these conditions, the synthesis of all proteins was de- creased as seen in SDS/polyacrylamide gels (not shown) but the ratio of ["SlMet incorporated to incorporated [14C]GlcN was constant (Table 1). </p><p>Monensin, which inhibits protein transfer from the endo- plasmic reticulum to the Golgi where 0-glycosylation usually </p><p>Inhibitor [Inhibitor] 1 4 ~ ~ 3 5 5 s incorporated </p><p>Tunicamycin 0 PglassaY 0.45 0.1 pg/assay 0.53 0.5 kg/assay 0.47 5 pg/assay 0.48 </p><p>10 pg/assay 0.52 </p><p>2-Deoxy-~-glucose 0 mM 0.057 10 mM 0.042 </p><p>Monensine 0 PM 0.079 1 PM 0.074 5 pM 0.05 </p><p>10 pM 0.04 </p><p>takes place 129, 301, does not inhibit fibre glycosylation (Ta- ble 1). The fibre of all samples had incorporated [14C]GlcNAc as seen by autoradiography of the SDS polyacrylamide gel (not shown). At high concentrations, the same inhibitory ef- fect on protein synthesis was also observed (not shown). </p><p>Intracellulur localization offibre glycosylation </p><p>The fibre is synthesized in the infected cell cytoplasm and quickly transferred to the nucleus [31]. To compare fibre glycosylation in the cytoplasm and nucleus, cellular fraction- ation was performed as described in Material and Methods. The fibres found in the cytoplasm and in the nucleus showed the same affinity for WGA (not shown). </p><p>As N-glycosylation usually takes place in the endoplasmic reticulum, the endoplasmic reticulum of infected cells was prepared by sucrose gradient centrifugation as described in [27]. Viral proteins were labelled by ['4C]GlcN or [35S]Met incorporation for 18-24 h post infection. Most of the [14C]GlcN radioactivity remained in the bottom of the centri- fuge tube and corresponded to the fibre seen in a SDS] polyacrylamide gel. In addition, two small peaks of [14C]GlcN were found in the endoplasmic reticulum region (Fig. 2). With the [35S]Met-labelled extract, we did not see any preferential fibre accumulation through the gradient with regard to the other viral proteins. Most of the glycosylated fibre...</p></li></ul>