Differences in the post-translational mo
Biotechnol.Appl.Biochem.(2000)32,27–33(Printed in Great
Britain)
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NingXia Fang,Ian H.Frazer and Germain J.P.Fernando 1
Centre for Immunology and Cancer Research ,University of Queensland Department of Medicine ,Princess Alexandra Hospital ,Brisbane ,Qld .4102,Australia
Virus-like particles (VLPs)are being currently investi-gated in vaccines against viral infections in humans.There are different recombinant-protein-expression systems available for obtaining the necessary VLP preparation for vaccination.However,the differences in post-translational modi?cations of the recombinant proteins obtained and their differences in ef?cacy in eliciting an anti-viral response in vaccines are not well established.In this study we have compared the post-translational modi?cations of human papillomavirus type-6b major capsid protein L1(HPV 6bL1)expressed using recombinant baculovirus (rBV)in Sf9(Spodoptera frugiperda )insect cells,with the protein expressed us-ing recombinant vaccinia virus (rVV)in CV-1kidney epithelial cells.Two-dimensional gel electrophoresis of biosynthetically labelled rBV-expressed HPV 6bL1showed several post-translationally modi?edvariants of the protein,whereas rVV-expressed HPV 6bL1showed only a few variants.Phosphorylations were detected at threonine and serine residues for the L1expressed from rBV compared with phosphorylation at serine residues only for the L1expressed from rVV.HPV 6bL1expressed using rBV incorporated [3H]mannose and [3H]galactose,whereas HPV 6bL1expressed using rVV incorporated only [3H]galactose.We conclude that post-translational modi?cation of recombinant HPV 6bL1can differ according to the system used for its expression.Since recombinant L1protein is a potential human-vaccine candidate,the implication of the obser-ved differences in post-translational modi?cations on immunogenicity of L1VLPs warrants investigation.
Introduction
Human papillomaviruses (HPVs)are responsible for a range of human disease,including common genital warts (con-dylomata acuminata),squamous laryngeal papillomas and cervical cancer,and vaccination against these infections has been proposed [1].Since HPV virions cannot be propagated easily in vitro ,nor obtained in reasonable quantities from lesions in vivo ,recombinant DNA techniques have been
developed to assemble synthetic papillomavirus virions in vitro using several vectors,including vaccinia virus [2],Semliki Forest virus [3],baculovirus [4]and yeast [5].Virus-like particles (VLPs)produced from the various recombinant systems appear to have similar morphology to the naturally occurring virion [6].In recent years,capsid-based vaccines against papillomavirus have been shown to induce protec-tion against the virus in animal models [7,8].Others have reported induction of viral-neutralizing antibodies against HPV 11[9],HPV 33[10],bovine papillomavirus [11]and cotton-tail-rabbit papillomavirus [7,8].
A range of eukaryotic expression systems are available for expression of recombinant proteins.The primary sequence of the expressed protein is generally independent of the expression system.However,different expression systems can result in differing post-translational modi?-cations of the recombinant protein [12,13].These different modi?cations might affect protein folding or immunogenicity,and may be of importance for vaccine design.
Previously we determined the post-translational modi-?cation of HPV type-6b major capsid protein L1(HPV 6bL1)when expressed in a vaccinia-virus expression system [14].Here we have compared the differences in post-translational modi?cations of the HPV 6bL1capsid protein expressed from a baculovirus protein-expression system with that expressed from the vaccinia-virus protein expression sys-tem.By speci?c labelling of the HPV 6bL1protein,two-dimensional (2D)electrophoresis,immunoprecipitation and endoglycosidase digestion,we have determined the dif-ferences in the phosphorylation,myristoylation,palmitoy-Key words:glycosylation,phosphorylation,vaccine,virus-like particles,warts.Abbreviations used:HPV,human papillomavirus;HPV 6bL1,HPV type-6b major capsid protein L1;HPV 16,HPV type 16;rBV,recombinant baculovirus;rVV,recombinant vaccinia virus;wt,wild-type virus;VLP,virus-like particle;rBV HPV 6bL1,HPV 6bL1protein produced using a baculovirus expression system;rVV HPV 6bL1,HPV 6bL1protein
produced using a vaccinia-virus expression system;2D,two-dimensional;FBS,foetal bovine serum;pfu,plaque-forming units;mAb,monoclonal antibody;HRP,horseradish peroxidase;IEF,isoelectric focusing;TLE,thin-layer electrophoresis;PNGase F,peptide N-glycosidase F;SV40,simian virus 40.1
To whom correspondence should be addressed (e-mail gfernando !https://www.360docs.net/doc/4e12435163.html,.au).
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lation and glycosylation of HPV 6bL1produced by these two systems.Materials and methods
Cells and antibodies
Spodoptera frugiperda 9(Sf9)insect cells were grown at 27m C in Sf 900II medium (Gibco BRL,Gaithersburg,MD,U.S.A.)supplemented with 10%foetal bovine serum (FBS).CV-1cells were maintained at 37m C in Dulbecco’s modi?ed Eagle’s medium (DMEM;ICN)supplemented with 10%FBS,2mM L -glutamine,100units \ml penicillin and 0.01%strep-tomycin.
All antibodies used have been described previously [14].
Recombinant HPV 6bL1virus
Construction of recombinant HPV 6bL1baculovirus (rBV)and recombinant HPV 6bL1vaccinia virus (rVV)was described in [14,15].
Induction of recombinant HPV 6bL1protein
Sf9insect cells were infected with 10plaque-forming units (pfu)\cell of rBV HPV 6bL1virus in Sf 900II medium supplemented with 10%FBS and cultured at 27m C for 72h.CV-1cells was infected with 1pfu \cell of rVV HPV 6bL1virus and grown in DMEM supplemented with 2.5%FBS,at 37m C,for 48h.
Puri?cation of recombinant L1VLPs
The preparation of VLPs has been described [14].Brie?y,the L1-infected cell pellet was suspended in PBS.After homo-genization and sonication the sample was subjected to ultracentrifugation with a 20%sucrose cushion and then with CsCl (1.3g \ml).The particulate VLPs band was collected.The VLP’s purity was tested by SDS \PAGE and Western blotting.The VLP’s morphology was con?rmed by electron microscopy.
Metabolic labelling of recombinant HPV 6bL1with [32P]phosphate
Sf9cells were infected with rBV HPV 6bL1virus at 20pfu \cell for 20h and starved for 0.5h in phosphate-free Grace’s medium.The starved cells were labelled with 0.1mCi \ml [32P]phosphate (370MBq \ml,10mCi \ml;Amersham Life Science,Little Chalfont,Bucks.,U.K.).Post-infection (44h),the cells were harvested and then lysed in RIPA buffer (50mM Tris,pH 8.0\150mM NaCl \1%Noni-det P40\0.5%deoxycholate \0.1%SDS \4μg \ml pepstatin A \5μg \ml leupeptin \1mM PMSF \1mM EDTA)supple-mented with 1mM sodium vanadate \50mM NaF.CV-1cells were infected with rVV HPV 6bL1virus and labelled
as described in [14].rBV HPV type 16(HPV 16)E7protein [16]was used as a control.
Metabolic labelling of recombinant HPV 6bL1with
[3H]myristic acid and [3H]palmitic acid
Sf9cells were infected as above at 10pfu \cell of the virus.The infected cells were labelled as described in [14].
Metabolic labelling of the L1protein with [3H]monosaccharides
HPV 6bL1-infected Sf9cells were starved for 15min in glucose-free medium supplemented with 2.5%dialysed FBS and labelled with 50μCi \ml D -[2-3H]mannose (37MBq \ml,16.5Ci \mmol;Amersham),or with 50μCi \ml D -[6-3H]-galactose (37MBq \ml,31.0Ci \mmol;Amersham).Post-infection (2days),the cells were harvested and lysed in RIPA buffer.rVV HPV 6bL1-infected CV-1cells were metabolically labelled as described previously [14].HPV 16L1protein from rVV [17]was used as a control.
Immunoprecipitation and Western blotting
The infected cells were harvested by centrifugation and lysed in RIPA buffer.The HPV 6bL1protein was immunopre-cipitated by rabbit anti-6bL1serum.HPV 16E7protein was immunoprecipitated by monoclonal antibody (mAb)6D (see [14]),and HPV 16L1protein was immunoprecipitated by a mixture of anti-16L1mAbs (mAbs 8,15,25and CamVir1).The immuno-complexes were collected with Protein A–Sepharose beads and washed as described in [14].The target proteins were recovered by boiling in reducing sample buffer,subjected to SDS \PAGE [18]and then transferred on to nitrocellulose membranes.The HPV 6bL1protein was detected with mAb 8and anti-mouse Ig–horseradish per-oxidase (HRP)conjugate.The HPV 16L1protein was detected with mAb 8and Protein A–HRP conjugate (Sigma,St.Louis,MO,U.S.A.).HPV 16E7protein was detected with mAb 6D and anti-mouse Ig-HRP conjugate.The Western blot was then developed with chemiluminescence En 3Hance reagent (ECL kit,Amersham).
Detection of 3H signal with ?uorography and β-counting
SDS \PAGE gels were ?xed in methanol \acetic acid \water (45:10:45)and enhanced in Amplify (Amersham).Fluoro-graphy was performed at k 70m C for 2–4weeks.After Western blotting,the blot was probed with antibody,and then sliced and counted in liquid-scintillation cocktail with a β-radiation counter.
2D gel electrophoresis
A slab isoelectric focusing (IEF)gel was cast on Gel Bond PAG ?lm [19],which contained 5%acrylamide gel solution,8M urea,2.5%Pharmalyte 3-10and 2.5%Pharmalyte 5–8
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Post-translational modi?cations of human papillomavirus6bL1
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Figure 22D gel electrophoresis of recombinant HPV 6bL1protein
IEF was performed in an IEF polyacrylamide gel,pH 3–10,containing 8M urea,2.5%Pharmalyte 3–10and 2.5%Pharmalyte 5–8.The 32P-labelled rBV HPV 6bL1-infected Sf9cells and rVV HPV 6bL1-infected CV-1cells were lysed in RIPA buffer.The samples were applied at the cathode side.Focusing was performed at 2000V,20W,25mA and 10m C for 90min.After focusing,the IEF gel strips were equilibrated in reducing sample buffer for 10min and then separated by SDS/PAGE (10%gel).(a ,c )The proteins were transferred on to nitrocellulose membrane and probed with mAb 8and detected with the ECL kit (Amersham).(b ,b h ,d ,d h )Autoradiography was performed by PhosphorImager.rBV HPV 6bL1was shown in (a ),(b )and (b h ).rVV HPV 6bL1was shown in (c ),(d )and (d h ).KD,kDa.
the 6bL1protein from the rBV (Figure 1b,lane 3)is phosphorylated,similar to the rVV (Figure 1b,lane 5).
Phosphorylation pattern of the rBV-produced L1differs from that of the rVV-produced L1
2D electrophoresis showed that rBV HPV 6bL1can be separated into more than 20species (Figure 2a)according to different molecular size and isoelectric point (pI).Around 54–58kDa,L1fractions with three different molecular sizes gave about 15variants,with pI values of around 7.5and 6.5–5.0.The spots of smaller molecular size are presumed to be due to the degradation of L1.There were only about 10variants of L1observed with the rVV system.Autoradio-graphy showed that at least ?ve of the spots from rBV HPV 6bL1(Figure 2b,b h )and one (pH 6.5)from rVV HPV 6bL1
(Figures 2d and 2d h )represent phosphorylated species of L1.These data indicate that the 6bL1protein consists of a group of proteins with different molecular sizes and isoelectric points,depending on the expression systems used,and that more phosphorylated species are seen in rBV HPV 6bL1than in rVV HPV 6bL1.
Recombinant HPV 6bL1produced in the baculovirus expression system is phosphorylated at threonine and serine,whereas when produced in the VV system is only phosphorylated at serine
To compare further the differences in the phosphorylation patterns between rBV HPV 6bL1and rVV HPV 6bL1,the proteins were hydrolysed and the phospho-amino acids were analysed by 2D TLE.HPV 6bL1protein from rBV contained phosphothreonine and phosphoserine,but no
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Post-translational modi?cations of human papillomavirus6bL1
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Figure 5Glycosylation study of HPV 6bL1
rBV HPV 6bL1and rVV HPV 6bL1were metabolically labelled with [3H]mannose (Man)or [3H]galactose (Gal;50μCi/ml each)in glucose-free media.rVV HPV 16L1was used as the control.The L1proteins were immunoprecipitated and analysed by (a )Western blotting and (b )auto-radiography using a PhosphorImager.wt,wild-type virus.KD,kDa;MW,molecular mass.
culture (39.8%).These faster-migrating bands may be from the deglycosylation of 6bL1protein.
To con?rm that the HPV 6bL1protein is glycosylated,rBV HPV 6bL1-infected Sf9cells were labelled biosyn-thetically with [3H]mannose and [3H]galactose.L1protein was puri?ed by immunoprecipitation and identi?ed by Western blotting (Figure 5a)and ?uorography (Figure 5b).HPV 6bL1from rBV was glycosylated mainly with [3H]mannose (Figure 5b,lane 1)and less with [3H]galactose (Figure 5b,lane 2),compared with rVV HPV 6bL1,which was weakly glycosylated and only incorporated [3H]galactose (Figure 5b,lane 6).HPV 16L1was glycosylated on the heavier fraction (69kDa;Figure 5a,lanes 9and 10),mostly with [3H]mannose (Figure 5b,lane 9),as reported in [14,17].
VLPs formed by rBV HPV 6bL1protein are not glycosylated
We have shown previously that the VLPs formed with rVV HPV 6bL1are not glycosylated [14].To determine whether glycosylated 6bL1protein is incorporated into VLPs when rBV HPV 6bL1was used,the VLPs were puri?ed from the
infected cultures,subjected to endoglycosidase digestions with PNGase F and Endo H,and separated by SDS \PAGE and Western blotting.The gels were incubated with ConA \FITC reagent.Fluorophotography was performed with the UV transilluminator.No differences were observed after PNGase F and Endo H digestions for 6bL1VLPs from both rBV and rVV,suggesting that no N-linked glycosylation could be detected in the 6bL1VLPs by enzyme digestions (results not shown).We conclude that the 6bL1protein from rBV that is incorporated into VLPs is not glycosylated,similar to that observed with the rVV HPV 6bL1protein [14].
Discussion
In the present study we con?rm that the L1protein of HPV is phosphorylated when expressed using rBV [23].L1is more heavily phosphorylated when expressed using baculovirus in Sf9cells than when expressed in CV-1cells using rVV,and is phosphorylated at threonine residues only when expressed in Sf9cells,whereas serine phosphorylation occurs in both expression systems.As no fatty acylation of L1was observed in the current studies,and glycosylated L1was not in-corporated into VLPs in the current or a previous study [17],phosphorylation differences are likely to account for at least some of the immunoreactive 55-kDa L1variants demon-strated in IEF studies on native L1protein [24].Consistent with our ?ndings,phosphorylation of simian virus 40(SV40)T antigen has been demonstrated to differ quantitatively between recombinant protein expressed using rBV in Sf9cells and native protein expressed in SV40-infected monkey cells [13].
The N-glycosylation pathway of baculovirus-infected insect cells differs from that found in higher eukaryotes,and glycoproteins produced in this system typically lack complex biantennary N-linked oligosaccharide side chains containing penultimate galactose and terminal sialic acid residues [12,21,25,26].For example,simple high-mannose glycosyla-tion was observed for rBV-expressed HbsAg (hepatitis B surface antigen),in contrast with the complex-type oligo-saccharides detected on plasma-derived HbsAg [27].We observed in the current study that rBV 6bL1protein is partially glycosylated when produced in Sf9,as described previously for native bovine papillomavirus 1capsid proteins [24]and for rVV-expressed L1for a range of papillomavirus genotypes [14,17,23,28].HPV 16L1expressed using rVV has been shown to contain N-linked high-mannose-type sugars [14,17,24]and could be deglycosylated by Endo H and PNGase F.In contrast,rBV 6bL1protein was,judging by our current results,only partially degraded by PNGase F digestion and was not sensitive to Endo H treatment,suggesting that rBV 6bL1may contain complex N-linked
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Post-translational modi?cations of human papillomavirus6bL1
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