Roberts, B., Antonopoulos, A., Haslam, SM, Dicker, AJ, Mcneilly, TN, Johnston, SL, Dell, A., Knox

16 Pages·2013·1.49 MB·English

Roberts, B., Antonopoulos, A., Haslam, S.M., Dicker, A.J., Mcneilly, T.N.,
Johnston, S.L., Dell, A., Knox, D.P., and Britton, C. (2013) Novel
expression of Haemonchus contortus vaccine candidate aminopeptidase
H11 using the free-living nematode Caenorhabditis elegans. Veterinary
Research, 44 (111). ISSN 0928-4249

Copyright © 2013 The Authors.

http://eprints.gla.ac.uk/89670/

Deposited on: 20 January 2014

Enlighten – Research publications by members of the University of Glasgow
http://eprints.gla.ac.uk
Robertsetal.VeterinaryResearch2013,44:111
http://www.veterinaryresearch.org/content/44/1/111 VETERINARY RESEARCH
RESEARCH Open Access
Novel expression of Haemonchus contortus
vaccine candidate aminopeptidase H11 using the
free-living nematode Caenorhabditis elegans
Brett Roberts1, Aristotelis Antonopoulos2, Stuart M Haslam2, Alison J Dicker3, Tom N McNeilly3,
Stephanie L Johnston1, Anne Dell2, David P Knox3 and Collette Britton1*
Abstract
With the problem of parasitic nematodedrugresistance increasing, vaccine development offers an alternative
sustainable control approach. For some parasitic nematodes, native extracts enriched for specific proteins are highly
protective. However, recombinantforms of theseproteins have failedto replicate this protection. This is thought to
be due to differences inglycosylation and/or conformation between native and recombinantproteins.We have
exploited the free-living nematode Caenorhabditis elegans to examine its suitability as an alternative system for
recombinant expression of parasitic nematode vaccine candidates. We focussed on Haemonchus contortus
aminopeptidase H11glycoprotein,whichisenrichedinagutmembranefractioncapableofinducingsignificant
protectionagainstthisimportantovinegastrointestinalnematode.WeshowthatH.contortusH11expressedin
C.elegansisenzymaticallyactiveandMALDImassspectrometryidentifiessimilardi-andtri-fucosylatedstructures
tothoseon native H11, with fucose at the 3- and/or 6-positions of the proximal GlcNAc. Some glycan structural
differences were observed, such as lack of LDNF. Serum antibody to native H11 binds to C. elegans recombinant
H11 and most of the antibody to rH11 or native H11 is directed to glycan moieties. Despite these similarities, no
reduction in worm burden or faecal egg count was observed following immunisation of sheep with C. elegans-
expressed recombinant H11 protein. The findings suggest that the di- and tri-fucosylated N-glycans expressed on
rH11 do not contribute to the protective effect of H11 and that additional components present in native
H11-enriched extract are likely required for enhancing the antibody response necessary for protection.
Introduction areurgentlyneeded.Significantprotectionagainstlivestock
Parasiticnematode infectionsoflivestockareresponsible nematode infections has been achieved following vaccin-
forsignificanteconomiclossesandwelfare concerns glo- ation with native protein extracts, demonstrating that vac-
bally. Control currently relies on the use of anthelmintic cination is feasible [5-7]. However, from a production
drugs, however the widespread problem of parasite an- and cost perspective, the long term aim is to develop
thelmintic resistance means that this approach is becom- molecularly defined vaccines amenable to commercial
ing unsustainable [1,2]. The recent introduction of a new development, without the need for native parasite ma-
class of anthelmintic, the aminoacetonitrile derivatives terial and the associated ethical and safety concerns.
(ADDs; monepantel), offers an alternative [3]. However Currently there is no molecularly defined vaccine avail-
resistance to this new drug class has already been re- ableforanyparasiticnematode.
ported in nematodes of sheep and goats in less than A significant amount of data is available from vaccine
2yearsofuse[4].Alternative,sustainablecontrolmeasures, trialsagainsttheblood-feedinggastrointestinal(GI)nema-
such as vaccination and improved pasture management, tode of sheep and goats, Haemonchus contortus. Native
proteins extracted from the adult parasite gut or from
*Correspondence:[email protected] excretory-secretory (ES) products are capable of indu-
1InstituteofInfection,ImmunityandInflammation,CollegeofMedical,
cing high levels of protection (up to 90% reduction in
VeterinaryandLifeSciences,UniversityofGlasgow,BearsdenRoad,Glasgow
G611QH,UK faecal egg counts (FEC) and 75% reduction in worm
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Robertsetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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burden)[7].Protectivegutfractionsincludeagalactose- activity and antibody recognition of native and recom-
binding glycoprotein complex termed H-gal-GP enriched binant protein. Our findings have important relevance
formetalloandasparticproteases,athiol-bindingfraction to expression of other nematode vaccine candidates and
enriched for cysteine proteases, and a Concanavalin A requirementsforprotectiveimmunity.
binding fraction enriched for aminopeptidase H11. How-
ever, attempts to mimic the protective effects of these Materials and methods
nativeextractsusingrecombinantformsoftheenriched IdentificationofgenomiclocationofH.contortusH11genes
proteases expressed in bacteria, yeast or insect cells have The available H. contortus genome data (strain MHco3
proved unsuccessful [6,8]. Protection studies against the (ISE))[23,24]wassearchedbytBLASTnusingaminoacid
cattle GI nematode Ostertagia ostertagi have similarly sequences of all available H11 isoforms. This identified
demonstrated significant reductions in egg output using a number of overlapping scaffolds encoding known
an ES fraction highly enriched for two activation- H11 sequences and identified a novel sequence, named
associated secreted proteins (ASP-1 and ASP-2) [9]. H11-5 [GenBank Accession number KF381362]. Tandem
However,vaccinationwithbaculovirus-expressedASP-1 arrangement of H11 genes was indicated by scaffold
protein failed to induce any protection [10]. There has sequence analysis and confirmed experimentally by PCR
been much speculation as to why recombinant parasitic on genomic DNA, extracted by standard method from
nematode proteins fail to induce protective immunity. H. contortus adult worms ((MHco3(ISE) strain), using
Possible explanations include incorrect folding, lack of DNAprimersdesignedtothe5′and3′endsofeachH11
glycosylationofbacterially-expressedproteins,inappropri- gene coding sequence. All primer sequences are available
ate glycosylation of yeast or insect-expressed proteins, onrequest.
induction of lower avidity antibodies or, alternatively,
that the dominant proteins identified in protective native RNAextractionfromH.contortusandsemi-quantitative
fractionsarenotsolelyresponsibleforprotection[8]. RT-PCR
Gene rescue studieshavepreviously demonstrated that Total RNA wasextracted from H. contortusadult worms
parasiteproteinscan beexpressedinabiologically active using an RNAeasy kit (Qiagen) after grinding the worms
forminthefree-livingnematodeC.elegans[11-14].How- in liquid nitrogen. Reverse Transcription was performed
ever, expression of parasite proteins with complex post- using an AffinityScript cDNA Synthesis kit (Stratagene)
translationalmodificationshasnotbeentestedinC.elegans. as per manufacturer’s instructions. Semi-quantitative
InthisstudywefocusonH.contortusH11aminopeptidase, RT-PCR was carried out as described previously [25],
due to the high level of protection achieved with native using constitutively expressed superoxide dismutase gene
gut extracts enriched for H11 and the unusual glycosyl- (Hc-sod-1) as a reference [26]. Relative levels of each H11
ation identified on the native protein. Previous mass geneweredeterminedusingImageJ[27].
spectrometric analysis identified unusual fucosylated
modifications on native H11, including core α1-3 and GenerationofH.contortusH11expressionconstructs
α1-6 fucosylation [15]. The former is not found on An expression cassette containing 1.76 kb of promoter
mammalian glycans and has been shown to be highly sequence of C. elegans cathepsin L protease gene cpl-1
antigenic when present on plant and insect glycopro- and 500 bp of Ce-cpl-1 3′ UTR was generated in the
teins [16]. The core α1-3 Fuc epitope is recognised by TOPO2.1vector(Invitrogen)bystandardcloningmethods
IgE antibody from H. contortus infected sheep and is as previously described [12]. The cDNA region encoding
speculated to contribute to the induction of a Th2 re- the putative signal peptide sequence (first 29 amino acids)
sponse [17]. Core α1-3 and α1-6 fucosylation structures of the H. contortus Hmcp-6 gene (accession number
have also been identified on C. elegans glycoproteins GQ223792) was inserted between the cloned Ce-cpl-1
(though it should be noted that these are often addition- promoter and 3′ UTR regions. The H. contortus H11
ally substituted with galactose residues) [18-20] and have coding sequence for each isoform (approximately 2.9 kb)
been shown to induce Th2 type immune responses in was inserted into the expression cassette between the
mice,similartoparasiteglycans[21].α1-3andα1-6fuco- Hmcp-6 signal peptide-encoding region and Ce-cpl-1
syltransferases have been characterised from C. elegans 3′UTR using Nru I and Xho I restriction sites at these
[22], indicating that glycosylation pathways and the positions. The H11 coding regions were amplified using
resulting glycan modifications are similar in free-living PfuUltra II Fusion HS DNA Polymerase (Stratagene)
andparasiticnematodes.C.elegansmaythereforepresent and H. contortus adult worm cDNA as template. The
a suitable system for expression and analysis of parasite 5′ primers were designed to amplify downstream of the
glycans as important immunogens. Here we express encoded N-terminal transmembrane domain (35 amino
recombinant H. contortus H11 protein in C. elegans acids) of each H11 gene. The 3′ primers contained
and characterise the glycosylation pattern, enzymatic Afe I sites to allow for the insertion of a 10 amino
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acid C-terminal His tag-encoding sequence. A C. elegans Proteinidentificationbymassspectrometry
synthetic intron (SI) was inserted into available blunt A sample of C. elegans-expressed recombinant H11-4
end restriction sites within the H11 genes to improve (rH11-4) protein purified by cobalt chelation chromatog-
transgene expression [28]. A diagrammatic representa- raphywassubjectedtoSDS-PAGE,stainedwithCoomassie
tion of the final H11 expression construct is shown in blueandthemajorbandexcised.Standardmassspectrom-
Additional file 1. The integrity of all constructs was veri- etry procedure was performed with in-gel trypsin digest,
fiedbyautomatedDNAsequencing(EurofinsMWG). extraction of tryptic peptides, fractionation by RP-HPLC
and online nano-electropsray tandem mass spectrometry
(MS). Data was searched against the National Center for
C.eleganstransgenesis Biotechnology Information (NCBI) database using the
Each H11 gene expression construct was microinjected Mascot search engine (Proteomics Services, University
into the gonads of C. elegans adult hermaphrodite worms of Glasgow). Native H11 protein was extracted from
of strain DR96 (unc-76(e911)) as simple free arrays at a H. contortus adult worms (day 28) by solubilisation of a
final concentration of 10 μg/mL, together with the rescue PBS homogenate pellet in 1% thesit and purified using
marker plasmid p76-16B [29] at a final concentration of Concanavalin A (Con A) sepharose (Sigma). Protein ex-
10μg/mLand1kbladderDNA(100μg/mL)[30].Trans- tract was separated on a 4-15% SDS-PAGE gel, stained
genicanimalswereselectedonthebasisofreversionfrom with Coomassie blue and the major band of approxi-
anuncoordinated(unc)towild-typephenotype. mately 110 kDa excised and subject to MS as described
above(ProteomicsUnit,MoredunResearchInstitute).
CultureofC.eleganstransgenicwormsandprotein SDS-PAGEandWesternblotting
purification Protein samples were boiled for 5 min in an equal
C. elegans worms transformed with the H11 expression volume of 2×SDS-PAGE loading buffer containing 5%
constructs were grown on 9 cm diameter peptone rich β-mercaptoethanol and separated by 4-15% gradient
plates seeded with N22 bacteria [31] for approximately SDS-PAGE. Proteins were visualised with Coomassie
seven days. Worms were washed from the plates in blue or western blotting carried out by transferring the
0.1 M NaClandseparatedfrombacteriabycentrifugation proteins onto PVDF membrane (Amersham). Blots
at3000rpmona60%(w/v)sucrosegradient.Wormswere were probed with a variety of antibodies: mouse anti-
collected from the upper phase and washed three times in HIS (C-terminal) (Invitrogen; 1 in 4000 dilution), sheep
ice cold 0.1 M NaCl by centrifugation at 3000 rpm. The antiserum to native H11-enriched gut membrane extract
worm pellet was resuspended in an equal volume of 1× (1in 2000 dilution),Con A-horseradish peroxidase (HRP;
lysis buffer (50 mM NaPO , 300 mM NaCl, 10 mM imid- Sigma L6397; 0.5 μg/mL), mouse IgA TEPC-15 antibody
4
azole) and stored at −70 °C. After thawing on ice, the vol- (Sigma M1421, 1 in 2500 dilution) and sheep antiserum
ume was made up to 10 mL with 1× lysis buffer. Worms raised to C. elegans-expressed recombinant H11 (1 in
were sonicated for 10 cycles of 10 s on/20 s off on ice, 2000 dilution). HRP-conjugated secondary antibodies were
followed by homogenisation in a standard glass homogen- routinely used at 1 in 10 000 dilution and binding de-
iser. The worm extract was centrifuged at 13 000 rpm for tected using the Enhanced Chemiluminscent (ECL)
3 min and the cleared lysate collected and stored on ice. system (GE Healthcare).
Thewormpelletwasresuspendedin3mLof1×lysisbuffer
and re-sonicated and homogenised. After centrifugation, Site-directedmutagenesisofH.contortusH11-4
the lysates were pooled, added to 300 μL of HisPur Cobalt The three putative N-glycosylation sites of H11-4 (identi-
Resin (ThermoScientific) and mixed gently on a roller at fied as described in [32]) were altered by site-directed
4 °C for 2 h. The solution was loaded onto a 1 mL poly- mutagenesis using the QuikChange Lightning Multi
propylene column (Qiagen) and the resin washed with Site-Directed Mutagenesis Kit (Stratagene) according to
4mLlysisbuffer,followedby4mLoflysisbuffercontain- themanufacturer’sinstructions.Threegenespecificprimers
ing 0.04 M imidazole. Bound protein was eluted with six were used in which the codon for asparagine was altered
aliquots of 150 μL of 1× lysis buffer containing 0.125 M to that for glutamine. DNA sequencing confirmed alter-
imidazole.Elutedproteinwasdialysedagainst1×PBSusing ationofallthreecodons(EurofinsMWG).
Slide-a-Lyzer cassettes (Thermo Scientific) and the protein
concentrationestimatedusingtheCoomassieProteinAssay MildperiodatetreatmentofH11proteins
Reagent (Thermo Scientific). Protein was analysed by PVDF membrane containing native H11-enriched extract
4-15% gradient SDS polyacrylamide gel electrophoresis orrH11-4proteinwaswashedwith30%isopropanol,10%
(SDS-PAGE; Bio-Rad minisystem) followed by Coomassie acetic acid for 5 min, before being rinsed in 50 mM so-
staining(0.25%). dium acetate pH 4.5, 50 mM NaCl buffer. The following
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incubations were all performed at 4 °C in the dark. The incubation at room temperature) was determined at
blotwasincubated in the abovebuffercontaining10mM 405 nm using a Beckman DU530 spectrophotometer.
sodium metaperiodate for 1 h, rinsed 3 times in water To test inhibitor sensitivity, assays were carried out as
before being incubated in 20 mM Tris, pH 7.2, 150 mM above, but with pre-incubation for 10 min in amasta-
NaCl, 200 mM glycerol for 20 min. After rinsing 3 times tin or bestatin (both at 10 μM final concentration)
in 20 mM Tris, pH 7.2, 150 mM NaCl, the standard prior to the addition of pNA substrate.
immunoblottingprotocolwasfollowed[33].
Vaccinetrial
Glycananalysis Six month old indoor housed, worm-free Suffolk-cross
A sample of rH11-4 protein was processed as previously lambs were allocated into groups of seven, balanced for
described [34]. Briefly, rH11-4 protein was subjected to sexandweight.Onegroupreceivedthreeimmunisations
reduction in 4 M guanidine HCl (Pierce) containing (sub-cutaneously) of C. elegans-expressed rH11-1 and
2 mg/mL dithiothreitol, carboxymethylation and trypsin rH11-4 protein (a combination of 10 μg of each protein)
digestion, and the digested glycoprotein was purified by or rH11-4 and rH11-5 co-expressed in C. elegans (20 μg
C -Sep-Pak (Waters Corp., Hertfordshire, UK). N-linked total protein) at three weekly intervals and were chal-
18
glycans were released by peptide:N-glycosidase F (EC lenged with 5000 infective L3 stage H. contortus on the
3.5.1.52, Roche Applied Science) and peptide:N-glycosi- day of the final immunisation (day 42). Vax Saponin
dase A (EC 3.5.1.52, Roche Applied Science) digestions. (Guiness Chemical Products Ltd) was used as adjuvant
N-linked glycans were then permethylated using the at a final concentration of 1 mg/mL and the final vac-
sodium hydroxide procedure, and finally, the permethy- cine volume was adjusted to 1 mL with 1× TBS. Blood
lated N-linked glycans were purified by C -Sep-Pak. All was sampled at weekly intervals and FEC monitored
18
permethylatedsamples weredissolved in10μL of metha- threetimesweeklyfrom day14afterchallengeuntilnec-
nol,and1μLofdissolvedsamplewaspremixedwith1μL ropsy at day 35 when worm numbers were counted, as
of matrix (20 mg/mL 2,5-dihydroxybenzoic acid in 70% previously described [36]. Control groups were injected
(v/v) aqueous methanol). The mixture was then spotted with 1×TBS and adjuvant only or with 40 μg of native
onto a target plate (2×0.5 μL), and dried under vacuum. gut membrane extract enriched for H11 plus adjuvant,
MSdatawereacquiredusingaVoyager-DESTRMALDI- following the method of [37]. All experimental proce-
TOF (Applied Biosystems, Darmstadt, Germany). MS/MS dures were approved by the Moredun Research Institute
data were acquired using a 4800 MALDI-TOF/TOF Experiments and Ethics committee and carried out in
(Applied Biosystems) mass spectrometer. The collision accordance with the Animals (Scientific Procedures) Act
energy was set to either 1 or 2 kV, and argon was used of1986.
as collision gas. The 4700 calibration standard kit,
Calmix (Applied Biosystems), was used as the external ELISA
calibrant for the MS mode of both instruments, and Microtitre plates were coated overnight at 4 °C with
[Glu1] fibrinopeptide B human (Sigma) was used as 1 μg/mL of a combination of rH11-1 and rH11-4 pro-
an external calibrant for the MS/MS mode of the teins or rH11-4 and rH11-5 co-expressed proteins di-
MALDI-TOF/TOF instrument. luted in 50 mM bicarbonate buffer, pH 9.6. The avidity
The MS and MS/MS data were processed using Data of serum antibody from lambs vaccinated with native
Explorer 4.9 Software (Applied Biosystems). The spectra H11-enriched extract or rH11 protein against the hom-
were subjected to manual assignment and annotation ologous protein was estimated using the thiocyanate
with the aid of the glycobioinformatics tool, GlycoWork- elution method [38], with the addition of 0-5 M KSCN
Bench [35]. The proposed assignments for the selected to all washes. Avidity was estimated as the concentra-
peaks were based on 12C isotopic composition together tion of KSCN which resulted in a 50% reduction in OD
with knowledge of the biosynthetic pathways. The pro- value at a particular serum dilution (1/800). Mild sodium
posed structures were then confirmed by data obtained periodate-treatment (final concentration 5 mM) was
from MS/MS experiments. performed after coating of protein to wells, as previ-
ouslydescribed[39].
Aminopeptidaseactivityassays Antibodyisotypereactivitywasdeterminedusingstand-
Aminopeptidase activity was determined by standard ardELISAmethodwithprimaryantibodyat1/50dilution
assay using L-leucine p-nitroanilide substrate (L-Leu-pNA; and probing with mouse anti-ovine IgG (clone GT-34,
Sigma). Protein sample (500 ng/well) was mixed with Sigma-Aldrich; 1/2500 dilution), IgM (clone VPM13,
100 μL 50 mM NaPO buffer (pH5.5-7.5) and 10 μL Moredun Research Institute; 1/1000 dilution), IgE (clone
4
L-Leu-pNA (0.5 mM final concentration). The initial 2 F1, Moredun Research Institute; 1/100 dilution) or IgA
and final absorbance (after 2 h at 37 °C and overnight (AbDSeroteccloneK842F9;1/1000dilution).Secondary
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antibody binding was detected with anti-mouse IgG-HRP (GAMEN) characteristic of N-aminopeptidases highly
(Sigma-AldrichA2304,1/1000). conserved in all isoforms (Additional file 2). All H11
proteinscontainpredictedN-glycosylationsites,asshown
Statisticalanalysis intheaminoacidsequencealignmentinAdditionalfile1.
Statistical analysis of the FEC and worm burden results Semi-quantitative RT-PCR identified transcript for
was carried out following the guidelines set out in [40] all five isoforms in adult worms, with significantly lower
anddata analysedusingExcel2010. expression in infective L3 stage larvae (Figure 1B). Differ-
ent isoforms showed different patterns of expression,
Results with H11-1 the most abundant isoform in infective
DevelopmentalexpressionofH11genefamily L3 larvae, H11-4 the most abundant isoform in adult
Genes encoding four different isoforms of H. contortus worms and H11-5 only expressed in adult female
H11 were previously identified (H11, H11-1, H11-2 and worms (not shown). Subsequent RNA seq data [23]
H11-4) and in this study we identified a fifth isoform confirmed the male-specific expression of H11-5 and
(H11-5) from the available H. contortus genome data. identified significant enrichment of transcripts encod-
Analysis of overlapping H. contortus scaffolds, in addition ing H11-4, H11 and H11-2 in H. contortus adult gut
to performing PCR on H. contortus genomic DNA, tissue. All five H11 isoforms are present in native H11
showed that all 5 isoforms are tandemly arranged in the protein extract, as indicated by MS peptide sequencing,
genome (Figure 1A). Our findings expand on previous with more peptide matches found to H11-1 than to the
data [41], which showed linkage of H11-4 and H11 other isoforms (Mascott scores ranged from 390–5567).
genes, and suggest that the H11 gene family has arisen Based on this data, H11-1, H11-4 and H11-5 were
through recent duplication and divergence. The encoded selected forlarge-scaleproteinexpressioninC.elegans,
proteins share 62-75% amino acid identity, with the followed by glycan and enzymatic characterisation, and
zinc binding domain (HELAH) and exopeptidase motif vaccine testing.
A
scaffold 88.1 scaffold 822.1
(309,898 bp) (109,415 bp)
H11-1 H11-5 H11-4 H11 H11-2
scaffold 186.1
(240,422 bp)
B
0.80
0.70
0.60
n
o
si 0.50
s
e
xpr 0.40
e
of
s 0.30
nit
U
0.20
0.10
0.00
H11 H11-1 H11-2 H11-4 H115
H11 isotype
Figure1GenomicorganisationanddevelopmentalexpressionofH.contortusH11isoforms.(A)H.contortusoverlappinggenomicscaffold
sequencesindicatingtandemarrangementofallfiveH11isoforms.Arrowsindicatethedirectionoftranscriptionfromeachscaffold.(B)Semi-quantitative
RT-PCRofthefiveH.contortusH11genesininfectiveL3larvae(stripedbars)andinadultworms(bluebars).Expressionofeachgeneisshownrelativeto
constitutivelyexpressedcontrolgeneHc-sod-1.MeanoftriplicatePCRsisshown,witherrorbarsshowingthestandarderrorofthemean.
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RecombinantexpressionofH.contortusH11proteins no reactivity. The identity of rH11 was also confirmed by
usingC.elegans MS peptide sequencing. While characterisation focussed
H. contortus H11 isoforms were expressed in transgenic on H11-1, H11-4 and H11-5, all 5 isoforms were able to
C.elegansassolubleproteinsbysubstitutingtheN-terminal be expressed in recombinant form in C. elegans and anti-
transmembrane domain with a short signal peptide se- serum raised to native H11-enriched extract recognised
quence. Previous studies demonstrated that a soluble all rH11 isoforms. For each rH11 protein, the yield was
form of native H11, in which the transmembrane do- approximately 70–100 μg purified protein per mL packed
main was removed, was equally protective compared to volumeofC.eleganstransgenicworms.
full-length H11 [8]. Coomassie staining and Western
blotting using antibody to the C-terminal His tag or EnzymaticactivityofrecombinantH11proteins
antiserum to native H11-enriched extract (nH11) con- expressedinC.elegans
firmed that recombinant H11 protein (rH11) was the Aminopeptidase activity of rH11 proteins expressed in
majorcobalt-bindingproteininaPBS-solubleextractof C. elegans was examined bystandardaminopeptidase en-
C. elegans mixed stage transgenic worms (Figure 2). Pre- zymeassayusing L-leucine p-nitroanilide (pNA)as sub-
bleed serum prior to H11-extract immunisation showed strate. H11 recombinant proteins were enzymatically
active, with rH11-4 and rH11-5 showing greater activity
than H11 and H11-1 isoforms (Figure 3). rH11-4 and
rH11-4
Coomassie Anti-His Anti-nH11 rH11-5 proteins co-expressed and purified from the
same C. elegans transgenic line (rH11-4/5) showed an
increase in aminopeptidase activity relative to singly-
expressed forms, which was consistent in all assays, and
suggestspossibleinteractionand/orstablisationofthese
isoforms (Figure 3A). H11-5 showed greater activity at
115kDa pH 5.5, while H11-4 was more active at pH 7.0, similar
tonative H11-enrichedextract. Aminopeptidaseactivity
of the rH11-4 and rH11-5 co-expressed proteins and
native H11-enriched extract was inhibited to a similar
rH11-1 degree by standard aminopeptidase inhibitors amastatin
Coomassie Anti-His Anti-nH11
and bestatin (60-70% reduction; Figure 3B). The activity
and inhibition observed suggested that the recombinant
H11proteinswerefoldedappropriatelyintoactiveenzymes.
C.elegans-expressedrH11proteinisN-glycosylated
115kDa Native H. contortus H11 protein binds to Concanavalin
A lectin (Con A) [42] and previous mass-spectrometric
analysis identifiedN-glycans with highly fucosylatedcore
structures [15]. Interaction with Con A-HRP was exam-
ined by Western blotting, and showed strong binding to
rH11-5
Coomassie Anti-His Anti-nH11 native H11-enriched extract and to recombinant H11-4
(Figure 4A), rH11-1 and rH11-5 proteins. No ConA-HRP
binding was observed with protein expressed from a
rH11-4 gene construct in which the three potential
N-glycosylation sites had been altered by site-directed
mutagenesis (H11-4ΔN-Gly), indicating specific inter-
115kDa
action with N-linked glycans (Figure 4A).
To identify the specific N-linked glycan structures
present on C. elegans-expressed rH11-4 protein, detailed
Figure2ExpressionandpurificationofrecombinantH11-4,
mass-spectrometricanalysiswascarriedoutafterPNGase
H11-1andH11-5expressedinC.elegans.rH11proteinspurified
F and PNGase A enzymatic release. N-glycans were
bycobaltchelationchromatographywereseparatedby4-15%
SDS-PAGEandstainedwithCoomassieblueortransferredtoPVDF analyzed by matrix-assisted laser desorption/ionization–
membraneandprobedwithanti-His(C-terminal) antibodyor time-of-flight (MALDI-TOF) mass spectrometry (MS),
antiserumtonative H11-enrichedextract(Anti-nH11).Antiserum as well as by collisionally activated dissociation (CAD)
fromanimalspriortoH11-extractimmunisationshowednoreactivity
on a MALDI-TOF-TOF instrument [tandem mass
torH11isoforms.
spectrometry (MS/MS)]. Mixtures of N-glycans were
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A
0.8
0.7
m 0.6
n
5
40 0.5
e
nc 0.4
a
b
r 0.3
o
s
b 0.2
A
0.1
0.0
rH11-1 rH11 rH11-4 rH11-5 rH11-4/5 Native H11
extract
B H11 isoform
y 90.0
vit
cti 80.0
A
e 70.0
s
a
ptid 60.0
pe 50.0
o rH11-4/5
n
mi 40.0 native H11 extract
A
of 30.0
n
o 20.0
cti
du 10.0
e
% r 0.0
Amastatin Bestatin
Figure3AminopeptidaseactivityofC.elegans-expressedrecombinantH11proteins.(A)AminopeptidaseactivityofrH11proteinswas
measuredasthechangeinabsorbanceat405nmusingL-leucinep-nitroanilideassubstrate.ActivitywasmeasuredatpH5.5(stripedbars)and
pH7(bluebars)forsinglyexpressedrH11proteins,rH11-4andrH11-5co-expressedinC.elegans(rH11-4/5)andnativeH11-enrichedextract.
500ngtotalproteinwasaddedperassay.Theresultsoftriplicateassaysareshownwitherrorbarsrepresentingthestandarderror.(B)Reduction
inaminopeptidase(AP)activityofrH11-4/5co-expressedproteins(stripedbars)andnativeH11extract(bluebars)wasmeasuredinthepresence
ofamastatinorbestatin,bothat10μMfinalconcentration.Assayswerecarriedoutinduplicateand%reductioncalculatedrelativetopre-incubation
inbufferalone.
permethylated before MS and MS/MS analyses. The structures are shown in more detail in Additional file 3.
spectrum of PNGase F released glycans was domi- The N-glycan profiles are fully consistent with previously
nated by high mannose structures (m/z 1579–2396 publishedC.elegansprofiles[18-20].
Man5-9GlcNAc2) (Figure 4B). This is fully consistent In addition, MS analysis showed evidence of phos-
with the positive Con A binding experiments. In phorylcholine (PC) modifications on rH11-4 and this
addition pauci-mannose type (m/z 1345–1928 Fuc0- was supported by reactivity of PC TEPC-15 antibody
2Hex3-5GlcNAc2) and minor amounts of complex type (Figure 4D). PNGase F released N-glycans were treated
glycans (m/z 1416–2285 Fuc0-1Hex3-5GlcNAc2-4) were withHF,whichcleavespotentialPCmodifications,prior
identified(Figure4B).ThespectrumofPNGaseAreleased to permethylation and MALDI-MS analysis. The HF
glycans (Figure 4C) in contrast contained only minor treated N-glycans showed an increase in abundance of
amountsof highmannosetype,indicativeofanefficient complex structures terminated with HexNAc residues
initial PNGase F digest. It was instead dominated by (m/z 1416–22693 Fuc0-1Hex3-6GlcNAc2-5), as shown
highly fucosylated pauci-mannose type (m/z 1171–2481 in Additional file 4, thus indicating probable PC substi-
Fuc0-4Hex3-6GlcNAc2). MS/MS analysis of the mo- tution [43]. PC on some helminth proteins has been
lecular ion of m/z 1724 (Fuc2Hex4GlcNAc2) contained showntointerferewithactivationofTcellandBcellre-
structurally informative fragment ions at m/z 1098, 853 sponses [44,45]. We examined whether rH11 proteins
and 648, which confirmed the presence of α1-3 and had any inhibitory effect on activation of helminth-
α1-6 fucosylation, similar to native H11 extract. These naive ovine peripheral blood lymphocytes using Con A
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A D
rH11-4 Nat rH11-4
rH11-4 rH11-4 N-Gly H11 rH11-4 N-Gly
115kDa 115kDa
ConA-HRP TEPC-15
B C
Figure4C.elegans-expressedrH11-4proteinismodifiedbyN-linkedglycosylationandphosphorylcholine.(A)PurifiedrH11-4protein,
rH11-4proteininwhichthethreepotentialN-glycosylationsitesweremutatedbysitedirectedmutagenesis(rH11-4ΔN-Gly)andnativeH11-enriched
extractwereseparatedby4-15%SDS-PAGE,transferredtoPVDFmembraneandprobedwithHRP-labelledConAlectin.(BandC)MALDI-TOFmass
spectrometry(MS)profilesofN-linkedglycansfromrH11-4protein.N-glycansofpurifiedrH11-4werereleasedfromtrypticglycopeptidesbydigestion
withPNGaseF(B)orPNGaseA(C).ProfilesofN-glycansarefromthe50%MeCNfractionfromaC Sep-Pak(MaterialsandMethods).Allmolecular
18
ionsare[M+Na]+.Putativestructuresarebasedoncomposition,tandemMSandbiosyntheticknowledge.Structuresthatshowsugarsoutsideofa
brackethavenotbeenunequivocallydefined.(D)WesternblotofrH11-4andrH11-4ΔN-Glyprobedwithphosphorylcholine(PC)antibodyTEPC-15.
as a polyclonal T cell activator; none was observed burden or worm female:male ratio at necropsy (day 35
as shown in the lymphocyte proliferation assays in post-challenge infection) was observed in lambs vacci-
Additional file 5. natedwiththeC.elegans-expressedrH11proteinscom-
pared to an adjuvant control group (results of trial 2
VaccinetrialsusingC.elegans-expressedH11protein shown in Figures 5A and B and Tables 1 and 2; results
To examine any protection afforded by rH11 protein, of trial 1 were very similar). Lambs vaccinated with
six-montholdlambs(7pergroup)were vaccinatedthree H. contortus native H11-enriched extract showed sig-
times at 3 weekly intervals with rH11 or native H11- nificantreductionsinbothwormburden(93.6%reduction)
enriched extract and challenged at the time of the third and FEC (99.9% reduction) compared to the adjuvant con-
vaccination. In the first trial, protection using rH11-1 trolgroup.
and rH11-4 proteins (10 μg each protein per vaccination)
was examined and a second trial tested any protection AntibodyresponsetorecombinantandnativeH11proteins
afforded by rH11-4 and rH11-5 proteins co-expressed Characterisation of the antibody responses of lambs
in C. elegans (20 μg total protein per vaccination). In immunised with rH11 proteins or native H11-enriched
both trials, no significant difference in FEC, worm extract were examined to identify any quantitative or
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A
B
C D
Figure5Faecaleggoutput,adultwormburdensandELISAantibodyresponsesfollowingvaccinationwithrH11-4/5co-expressedproteins
orwithnativeH11-enrichedextract.Faecaleggcount(FEC)(A)wasmonitoredfromdays14–34post-challengeinfectionandadultwormburdens
(B)countedatnecropsyonday35.ThemeangroupcountfromlambsvaccinatedwithrH11-4/5proteins,nativeH11-enrichedextractorVaxSaponin
adjuvantareshown,witherrorbarsrepresentingthestandarderrorofthemean.(C)ELISAODvaluesofantisera(1/800dilution)reactivitytorH11-4/5
co-expressedproteinsornativeH11extractfollowingimmunisationwiththehomologousantigen.Lambswerevaccinatedondays0,21and42and
challengedwith5000L3onday42,asindicated.(D)Igisotyperesponses(1/50dilution)tonativeH11-enrichedextractmeasuredbyELISA.