To overcome this problem, the invasion-associated men-ingococcal OpaJ129 and OpaB128 proteins, both of which bind to CEACAM1, were produced in this study in E.. Plasmids pCR2.1 containin
Trang 1Conformational analysis of opacity proteins from Neisseria
meningitidis
Marien I de Jonge1,2, Martine P Bos3, Hendrik J Hamstra1, Wim Jiskoot4, Peter van Ulsen4,
Jan Tommassen4, Loek van Alphen1and Peter van der Ley1
1
Laboratory of Vaccine Research, National Institute of Public Health and the Environment, RIVM Bilthoven, the Netherlands;
2
Department of Medical Microbiology, University of Amsterdam/AMC, Amsterdam, the Netherlands;3Department of Molecular Microbiology and4Department of Pharmaceutics, Utrecht University, Utrecht, the Netherlands
Opacity-associated (Opa) proteins are outer membrane
proteins which play a critical role in the adhesion of
patho-genic Neisseria spp to epithelial and endothelial cells and
polymorphonuclear neutrophils The adherence is mainly
mediated by the CD66-epitope-containing members of the
carcinoembryonic-antigen family of human cell-adhesion
molecules (CEACAM) For the analysis of the specific
in-teractions of individual Opa proteins with their receptors,
pure protein is needed in its native conformation In this
study, we describe the isolation and structural analysis of
opacity proteins OpaJ129 and OpaB128 derived from
Neisseria meningitidisstrain H44/76 When the Opa proteins
were produced with the phoE signal sequence in Escherichia
coli, they were localized at the cell surface and the
recom-binant bacteria were found to specifically interact with
CEACAM1 For refolding and purification, the proteins
were overproduced without their signal sequences in E coli,
resulting in its cytoplasmic accumulation in the form of inclusion bodies After solubilization of the inclusion bodies
in urea, the proteins could be folded efficiently in vitro, under alkaline conditions by dilution in ethanolamine and the detergent n-dodecyl-N,N-dimethyl-1-ammonio-3-propane-sulfonate (SB12) The structure of the refolded and purified proteins, determined by circular dichroism, indicated a high content of b-sheet conformation, which is consistent with previously proposed topology models for Opa proteins A clear difference was found between the binding of refolded
vs denatured OpaJ protein to the N-A1 domain of CEA-CAM1 Almost no binding was found with the denatured Opa protein, showing that the Opa–receptor interaction is conformation-dependent
Keywords: Opa protein; Neisseria meningitidis; CEACAM receptor; in vitro folding; conformation
The pathogenic bacteria Neisseria meningitidis and Neisseria
gonorrhoeae express a family of genes encoding outer
membrane proteins that are structurally related but highly
polymorphic These proteins were originally identified as
colony-opacity-associated (Opa) proteins [1] Opa proteins
appear to play a critical role in the intimate adhesion of the
bacteria to epithelial and endothelial cells and to
polymor-phonuclear neutrophils [2,3] The majority of Opa proteins
bind to carcinoembryonic antigen cell-adhesion molecules
(CEACAM, formerly called CD66) [4] CEACAM proteins
are expressed on various epithelial and endothelial cells as
well as on some lymphoid and myeloid cells [5] A minority of
the Opa proteins target heparan sulfate proteoglycans
(HSPG) [6,7] Recently, several Opa proteins were found
that did not bind to any of these human receptors, suggesting
that these Opa proteins have additional functions or that they recognize additional receptors [8] Opa-receptor-mediated adhesion can lead to invasion of the bacteria into the different cell types expressing CEACAM proteins [8,9] Opa expres-sion was found in mucosal as well as disease isolates, 87.5%
of meningococcal strains tested bind to CEACAM1 [10] Although the binding specificity of the variable Opa proteins to the conserved human receptors has been studied extensively [11], not much is known about the binding sites present in the Opa proteins A two-dimensional topology model has been proposed, in which the Opa proteins form eight-stranded b-barrels, exposing four loops at the cell surface [12] The variability of the Opa proteins is mainly concentrated in surface-exposed loops two and three An intriguing question is how the binding function of the Opa proteins can be conserved despite the hypervariability observed
Pure protein is needed for detailed structure–function relationship studies addressing this question Due to phase-variable expression of the Opa proteins in Neisseria spp [13], it is difficult to purify individual Opa proteins without contamination of other Opa proteins from neisserial cells
To overcome this problem, the invasion-associated men-ingococcal OpaJ129 and OpaB128 proteins, both of which bind to CEACAM1, were produced in this study in E coli
We developed a method to produce pure, folded and functional Opa protein The Opa proteins were isolated in the form of inclusion bodies and subsequently folded in vitro
Correspondence to M de Jonge, Laboratory of Vaccine Research,
National Institute of Public Health and the Environment,
RIVM Bilthoven, the Netherlands.
Fax: + 31 030 2744429, Tel.: + 31 030 2743999,
E-mail: Marien.de.Jonge@RIVM.nl
Abbreviations: CEACAM, carcinoembryonic-antigen cell adhesion
molecule; HSPG, heparan sulfate proteoglycans; IPTG, isopropyl
thio-b- D -galactoside; OMC, outer membrane complex; Opa proteins,
opacity associated proteins; SB12,
n-dodecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate.
(Received 12 July 2002, accepted 5 September 2002)
Trang 2The structure of refolded Opa protein was studied by
circular dichroism (CD) spectroscopy The spectra are
indicative of a high content of b-strands, consistent with the
previously proposed structural models Refolded Opa
protein was shown to be functional by specific binding to
the N-A1 part of CEACAM1
M A T E R I A L S A N D M E T H O D S
Construction of the expression systems
The genes encoding OpaJ129 and OpaB128 were isolated
from H44/76 using Taq polymerase (Amersham,
Piscata-way, NJ, USA) and general opa primers (5¢-CTTCT
CTTCTCTTCCGCAGC-3¢ and 5¢-TCGGTATCGGGG
AATCAGAA-3¢), cloned into plasmid pCR2.1 (Topo TA
cloning kit, Invitrogen, Carlsbad, CA, USA) and
subse-quently sequenced using M13-forward and M13-reverse
primers (Invitrogen) Plasmids pCR2.1 containing opaJ129
and opaB128 were used to amplify the DNA sequences
encoding the mature OpaB128 and OpaJ129 proteins with
Taq polymerase The primers used (5¢-AGCGCCCA
TGGCAAGTGAAG-3¢ and 5¢-GGCATCGGGATCCG
GGAATCAG-3¢) were based on the DNA sequence of
opaB128 and opaJ129 of N meningitidis strains H44/76
(unpublished) and 190/87 (GenBank accession no
AF016285) [12] The primers contained base substitutions
(underlined) to introduce NcoI and BamHI cleavage sites,
respectively The PCR product was cloned in plasmid
pCR2.1 The NcoI–BamHI fragment was isolated from the
resulting plasmid and ligated into the NcoI–BamHI digested
expression vector pET11d (New England Biolabs, Inc.,
Beverly, MA, USA) downstream of the inducible T7
promoter In the resulting construct, the codon for the first
amino acid residue of the mature Opa protein was situated
directly downstream of the ATG start codon The sequences
of the inserts were checked by DNA sequencing, using the
DNA sequencing kit and the ABI Prism 310 genetic
analyser, according to the instructions of the manufacturer
(ABI Prism, Perkin Elmer Applied Biosystems, Warrington,
UK)
Plasmids pET11d-opaB128 and pET11d-opaJ129 were
used to transform the E coli strain BL21 (DE3) containing
a chromosomal copy of the T7 RNA polymerase gene
under control of the lac promotor [14] Plasmid-containing
derivatives of this strain were grown at 37C in
Luria-Bertani (LB) medium (BIO 101, Inc., Carlsbad, CA,
USA) supplemented with 100 lgÆmL)1ampicillin (Sigma,
St Louis, MO, USA)
The OpaB128 and OpaJ129 expression at the cell surface
of E coli strain CE1265 was realized using the expression
vector pMR05, containing the complete phoE gene [15]
PCR amplifications were performed on pCR2.1 containing
either opaB128 or opaJ129 using Taq polymerase and
mutagenic primers (5¢-ATAGATCTCGGGGAATCAG
AAGCG-3¢ and 5¢-CTTCTCTTCTCTTCTGCAGC-3¢)
to generate a PstI site between the signal sequence and the
mature portion and a BglII site behind the stopcodon of
opaB128 and opaJ129 The PstI–BglII fragments of
opaB128 and opaJ129 were used to replace a PstI–BglII
fragment of the phoE gene in pMR05, resulting in an
in-frame fusion of opa to the signal peptide of phoE and
expression from the phoE promotor The resulting plasmids
were used for transformation of strain CE1265, which expresses the pho regulon constitutively due to a phoR mutation [16] Expression of OpaB128 and OpaJ129 was determined by assaying the binding of monoclonal anti-bodies MN20E12.70 (M de Jonge, G Vidarson, H H van Dijken, P Hoogerhout, L van Alphen, J Dankert & P van der Ley, unpublished results) and 15-1-P5.5 [18] in a colony blotting experiment [19] The bla-opaB fusion construction, which resulted in E coli surface expression of gonococcal OpaB, is described by Belland et al [3] The surface expression was confirmed with immunofluorescence Cells were washed with NaCl/Piand after blocking overnight in NaCl/Pi with 3% BSA, incubated with 15-1-P5.5 [18] (diluted 1 : 100) for 1 h, followed by an incubation with Alexa-conjugated goat anti-(mouse IgG) (Molecular Probes Inc., Eugene, OR, USA) (diluted 1 : 300) for 1 h After washing three times, cells were again fixated in NaCl/Piwith 2% formaldehyde (Merck, Darmstadt, Germany) The construction of the recombinant N-domains of the different CEACAM proteins is described by Bos et al [20]
Binding of His-tagged CEACAM fragments
to bacterial cells The binding of His-tagged CEACAM fragments to bacter-ial cells was measured as described previously [20] The expression of the N-terminal domains of the CEACAM proteins was regulated by the inducible T7 promoter The Opa-expressing bacteria (3· 108) in 200 lL Hepes buffer (10 mMHepes, pH 7.4, 145 mMNaCl, 5 mMKCl, 0.5 mM MgCl2 and 1 mM CaCl2) were incubated with 10 lL of cleared bacterial cell lysate containing the His-tagged N-terminal domains of either CEACAM1 or CEACAM8, for 20 min at 37C Bacteria were collected by centrifuga-tion (5 min at 2000 g) and washed with 1 mL of Hepes buffer The pellet was resuspended, and processed for SDS/ PAGE and Western blotting, with monoclonal antibody 4B12 (1 : 5000) [21] for the detection of OpaJ129 and anti-His6 monoclonal Ig (1 : 10 000) (Amersham Pharmacia Biotech GmbH, Freiburg, Germany) for the detection of the N-terminal domains of the CEACAM proteins Production and purification of inclusion bodies Cultures of the E coli strain BL21 (DE3) containing either pET11d-opaB128 or pET11d-opaJ129, grown overnight at
37C, were diluted 1 : 10 into fresh LB medium supple-mented with 0.5% glucose (Fluka, Buchs, Switzerland) and
100 lgÆmL)1 ampicillin When the culture reached an optical density of 660 nm (D660) of 0.6, isopropyl
thio-b-D-galactoside (IPTG) (Boehringer Mannheim, Germany) was added to a final concentration of 1 mM After 3 h of incubation at 37C, the cells were harvested by centrifuga-tion at 4500 r.p.m for 15 min at 4C (Centrikon T324, Rotor A6.9, Kontron Instruments, Milan, Italy) The pellet was washed with 10 mMTris/HCl (pH 8) and centrifuged at
4500 r.p.m for 15 min at 4C in the same rotor After resuspension in the same buffer, cells were disrupted using a French Press (SLM-Aminco) at 9000 p.s.i three times The inclusion bodies were collected by a low-speed centrifuga-tion step at 2800 g for 10 min at 4C (Megafuge 1.0 R, Hereaus sepatech, Germany) The pellet was resuspended in
8 urea, 50 m glycine (pH 8.0) Ultracentrifugation at
Trang 3100 000 g for 2.5 h at 4C was used to remove residual
membrane fragments and the supernatant was stored at
4C The protein concentration was determined with the
Pierce protein assay (Pierce, Rockford, IL, USA) using BSA
as a standard Proteins were separated by SDS/PAGE
with 0.4 mM thioglycolic acid (Sigma-Aldrich, Steinheim,
Germany), included in the separating gel After blotting on
poly(vinylidene difluoride) (Millipore, Bedford, MA, USA)
membranes and staining of the blots with Coomassie
Brilliant Blue, protein bands were cut from the membranes
and used for N-terminal sequencing
Semi-native-polyacrylamide gel electrophoresis
To determine the heat-modifiability of wild type OpaB128
and OpaJ129, outer membrane complexes (OMCs) were
isolated from N meningtidis strain H44/76 according to the
protocol described by Davies et al [22] The expression of
wild-type OpaJ129 was determined by Western blotting
using monoclonal antibody 15-1-P5.5 [18] The expression
of wild-type OpaB128 was determined by Western blotting
using monoclonal antibody MN20E12.70 (M de Jonge, G
Vidarson, H H van Dijken, P Hoogerhout, L van Alphen,
J Dankert & P van der Ley, unpublished results)
Semi-native polyacrylamide gel electrophoresis was
per-formed by using SDS-free 11% polyacrylamide gels
Loading buffer containing either 0.1% or 2.0% SDS
(Fluka, Buchs, Switzerland) was added to the samples
which were subsequently incubated at room temperature
and 100C, respectively After electrophoresis, protein
bands were visualized with Coomassie Brilliant Blue (Fluka,
Buchs, Switzerland)
Refolding and purification
To find the optimal folding conditions, buffers with
different NaCl concentrations (100–300 mM) and final urea
concentrations (125 mM)1M) were tested at different pH
values ranging from pH 7.2–12.0 Furthermore, different
protein dilutions (1 : 20 to 1 : 200) and n-dodecyl-N,
N-dimethyl-1-ammonio-3-propanesulfonate (SB-12, Fluka,
Buchs, Switzerland) concentrations were tested All
refold-ing experiments were performed overnight at 4C
In the optimal folding procedure, Opa (10 mgÆmL)1)
dissolved in 8M urea and 50 mM glycine (pH 8.0) was
diluted 100-fold in refolding buffer containing 328 mM
ethanolamine (pH 12), 0.5% SB12 (i.e approximately
5· critical micelle concentration) Prior to use, SB12 was
purified by passing a solution of the detergent in methanol/
chloroform (1 : 1, v/v) over an Al2O3column, to remove all
acidic impurities present in the commercial preparation [23]
After incubation overnight at 4C, the refolding mix was
neutralized with HCl to pH 7.5 and 10 mMTris was added
to buffer the solution Subsequently, to remove
ethanol-amine the solution was washed in a concentrator (Schleicher
and Schuell, Dassel, Germany) with 10 mMTris/HCl, 0.5%
SB12 (pH 7.5) (buffer A)
An SP-Sepharose-HP column (volume 15 mL)
(Amer-sham Pharmacia Biotech Europe GmbH, Freiburg,
Ger-many) was equilibrated with buffer A, loaded with
approximately 10 mg refolded OpaJ129 and washed twice
with buffer A with pH 7.5 and pH 8.5 The proteins were
eluted with a linear gradient of NaCl from 0–1 in
120 mL To check folding and purification, SDS/PAGE was performed under seminative and denaturing conditions The folded and purified proteins were stored at)20 C Circular dichroism spectroscopy
Circular dichroism (CD) spectra were recorded at 25C with
a dual-beam DSM 1000 CD spectrophotometer (On-Line Instrument Systems, Bogart, GA, USA) The subtractive double-grating monochromator was equipped with a fixed disk, holographic gratings, and 1.24-mm slits For far-UV and near-UV measurements, gratings with 2400 lines per mm (blaze wavelength 230 nm) and 600 lines per mm (blaze wavelength 300 nm), respectively, were used Far-UV spec-tra were recorded from 250 to 200 nm (cell-path length 0.5 mm) For near-UV measurements (320–250 nm), cells with a path length of 1 cm were used The Opa protein concentration was 0.54 mgÆmL)1 The results depicted represent the average of at least six repeated scans (step resolution 1 nm, 1 s each step), from which the correspond-ing buffer spectrum was subtracted The measured CD signals were converted to molar ellipticity [h], based on a mean residual weight of 112 (OpaB128) or 111.5 (OpaJ129) For the comparison between folded and denatured protein, folded protein in buffer A was incubated for
20 min at 100C with 1.85% SDS
Immunodotblotting Opa proteins were diluted to 1 lg per 100 lL in 10 mM Tris/HCl pH 7.5, 0.2% SB-12 (native) or in 2% SDS and incubated at 100C (denatured) and spotted on to nitro-cellulose (1 lg per spot) Filters were blocked with NaCl/Pi/ T/P (NaCl/Pi, 0.01% Tween-20, 0.5% Protifar) and subse-quently incubated for 1 h with 1 mL of receptor sample
in 10 mL NaCl/Pi/T/P The blots were incubated with
1 : 10.000 diluted anti-His Ig (Amersham), followed by
1 : 10.000 dilution of peroxidase-conjugated goat anti-mouse Ig (BioSource, Camarillo, CA, USA) and ECL detection (Pierce)
OpaDprotein is a purified Opa preparation from meningococcal membranes [21] (generously donated by
M Achtman, Max-Planck Institute, Berlin, Germany) This preparation was successfully used previously to detect CEACAM binding in a dot-blot assay [10]
The N- and A1 domains of CEACAM1 were amplified from CEACAM cDNA (gift from M Kuroki, Fukuoka University, Fukuoka, Japan) with primer pair 5¢-ATCATA TGCAGCTCACTACTGAATCCATGCC-3¢ and 5¢-AT CGGGATCCCTAACTCACTGGGT TCTGTATTTC-3¢ and cloned into pET15a (Invitrogen) using NdeI and BamHI sites included in the primers This results in an N-terminal 6·His-tag addition to the CEACAM N-domain The two domains with the His-tag were subcloned into pET26b (Invitrogen) using NcoI and BamHI restriction, resulting in plasmid pVB1 This vector adds the pelB signal sequence to the CEACAM domains, which allows secretion of the protein into the periplasm BL21 cells containing pVB1 were grown in LB containing 50 lgÆmL)1kanamycin to a D600of 0.6 Cells were induced with 0.2 mM IPTG and grown overnight at room temperature The induced cell pellet was resuspended in 200 mMTris/HCl pH 8.0, 0.5 mMEDTA, 0.5 sucrose Lysozyme was added to 60 lgÆmL)1 and
Trang 4the suspension was diluted 2· with 0.5 mM EDTA and
incubated for 10 min at room temperature Cells were
collected by centrifugation for 2 min at 8000 g (Eppendorf
centrifuge) and the supernatant was collected as the
periplasmic fraction containing the Opa receptor
R E S U L T S
Binding of Opa proteins to CEACAM1
N meningitidisstrain H44/76 can make four different Opa
proteins, some of which appear to be associated with the
ability to invade human nasopharyngeal cells [24] Among
those, major differences in the hypervariable regions were
found between OpaB128 and OpaJ129; the other two Opa
proteins have sequences closely resembling either one (M de
Jonge, G Vidarson, H H van Dijken, P Hoogerhout, L
van Alphen, J Dankert & P van der Ley, unpublished
results) The majority of Opa proteins bind to CEACAM
proteins, reviewed by Billker et al [11] The binding of
OpaB128 and OpaJ129 to CEACAM1 and CEACAM8
was determined, using E coli cells expressing this Opa
protein at the cell surface For this purpose we cloned the
fragment of opaB128 and opaJ129 encoding the mature
domain of the protein downstream of the promoter and the
signal sequence-encoding part of the phosphate-limitation
inducible phoE gene of E coli The resulting plasmids were
used to transform E coli strain CE1265, which expresses the
pho regulon constitutively due to a phoR mutation [16]
Surface expression of OpaB 128 and OpaJ129 was
con-firmed by immunoblotting outer membrane complexes
(OMCs) (Fig 1) with monoclonal antibodies MN20E12.70
and 15-1-P5.5 We determined binding of soluble his-tagged
N-terminal domains of CEACAM1 or CEACAM8 to
recombinant OpaB128 or OpaJ129 expressed at the cell
surface with an anti-His monoclonal antibody, as previously
described by Bos et al [20] The E coli bacteria expressing
either OpaB128 or OpaJ129 at the cell-surface were
incubated with N-domains of the two different CEACAM
proteins (Fig 2, lanes 5–8) Surface expressed recombinant
gonococcal OpaB protein was included in these experiments
as a positive control (Fig 2, lanes 1 and 2) After
incubation, the bacterial cells were collected by
centrifuga-tion and the proteins were separated by SDS/PAGE After
blotting to nitrocellulose filters, the presence of CEACAM
in the bacterial cell pellets was evaluated with an anti-His Ig
Like the bacteria expressing the gonococcal OpaB protein
(lane 1 and 2) the bacteria expressing OpaB128 or OpaJ129
bound to CEACAM1 (lane 5 and 7) while no binding was found with CEACAM8 (lanes 6 and 8)
Expression system for Opa proteins
To obtain large quantities of OpaB128 and OpaJ129 protein, part of the opa sequence encoding the mature Opa protein without the signal sequence was cloned into pET11d under the control of the inducible T7 promoter The recombinant genes were expressed in the E coli strain BL21 (DE3) upon addition of IPTG The Opa proteins accumulated in the cytoplasm as inclusion bodies, which could be separated from the other cell components by centrifugation After dissolving these inclusion bodies in 8M urea followed by an ultracentrifugation step to remove residual membrane fragments, the Opa protein in the supernatant was approximately 90% pure as determined
by SDS/PAGE (Fig 3A) N-terminal amino acid sequen-cing of the purified proteins revealed the sequence ASEDG,
Fig 1 Western blots showing the heat-modifiability of OpaB128 and OpaJ129 expressed in N meningitidis and E coli OMCs of H44/76 expres-sing either OpaB128 or OpaJ129 and OMCs of E coli strain CE1265 containing the phoE-opa fusion plasmid pMR05-opaB128 or pMR05-opaJ129 were separated by seminative-PAGE and analysed by Western blotting, using either the OpaB128 or OpaJ129 specific monoclonal antibody (MN20E12.70 or 15-1-P5.5, respectively) Samples were treated in sample buffer containing 0.1% SDS at room temperature (RT) or 2.0% SDS at
100 C, prior to electrophoresis.
Fig 2 Binding of His-tagged N-domains of CEACAM1 and 8 by OpaJ129- and OpaB128-expressing E coli cells The binding of the N-terminal fragments of CEACAM1 and CEACAM8 by MS11-OpaB-expressing E coli (lanes 1 and 2), by E coli not expressing an Opa protein (lanes 3 and 4), binding of OpaJ129- and OpaB128-expressing E coli to CEACAM1 (lane 5 and 7, respectively) and to CEACAM 8 (lane 6 and 8, respectively) was studied The bacteria were incubated with cleared lysates of E coli containing the N-domains of the CEACAM proteins and were processed for immunoblotting Bound N-domain was detected with anti-His Ig Opa protein expres-sion of the variants was evaluated with mAb 4B12.
Trang 5exactly corresponding to the predicted N-terminus of the
mature Opa This indicated that the N-terminal methionine
encoded by the ATG initiation codon was efficiently
removed in vivo by the methionine endopeptidase [25]
Folding experiments with Opa proteins
Correct folding of recombinant Opa protein was evaluated
using the property that native Opa proteins migrate faster
in semi-native PAGE than heat-denatured forms [1] To
check whether this heat-modifiability character also applies
to OpaB128 and OpaJ129, OMCs were isolated from
N meningitidis strain H44/76 expressing them, as
deter-mined by Western blotting using monoclonal antibodies
MN20E12.70 (M de Jonge, G Vidarson, H H van Dijken,
P Hoogerhout, L van Alphen, J Dankert & P van der Ley,
unpublished results) or 15-1-P5.5 [18], respectively
Semi-native PAGE, followed by Western blotting, confirmed that
both OpaB128 and OpaJ129 migrated with an apparent
molecular mass of 23 kDa, whereas completely unfolded
OpaB128 or OpaJ129 migrates as a protein of 27 kDa
(Fig 1) The heat-modifiability of wild type OpaB128 and
OpaJ129 was taken as marker for correct folding of the
proteins purified from the inclusion bodies The correct
folding of OpaB128 and OpaJ129 expressed at the surface of
E coli strain CE1265 was confirmed in the same assay
(Fig 1)
We diluted the urea-solubilized protein solution
(10 mgÆmL)1) 100-fold in various buffers with different
pHs, all containing 0.5% SB12 (w/v) and incubated the
samples overnight at 4C When the pH of the refolding
buffer was below 10, no or hardly any refolding was
observed However, in 328 mM ethanolamine buffer with
pH 10.5 (i.e just above the calculated pI of OpaJ129 and
OpaB128, 10.3 and 10.4, respectively) almost 50% of
OpaJ129 and > 50% of OpaB proved to be refolded
according to semi-native PAGE analysis (data not shown)
To increase the folding efficiency, several buffering substances and final protein concentrations were tested
at different pH values, and different NaCl and urea concentrations Although inclusion of 200 mM NaCl in the refolding buffer improved refolding considerably this condition was not applied further, because salt interferes with the subsequent protein purification by ion-exchange chromatography The variation in protein and urea con-centrations had almost no effect (data not shown) How-ever, at pHs further above the calculated pI of OpaB128 and OpaJ129 the folding appeared very efficient (Fig 3A) Summarizing, efficient refolding was achieved by a 100-fold dilution of 10 mgÆmL)1Opa protein solubilized in 8Murea
in folding buffer containing 328 mM ethanolamine and 0.5% (w/v) SB12 The optimal pH for efficient refolding was
11 for OpaB128 and 12 for OpaJ129 More than 95% of the protein proved to adopt a folded state under these conditions, as shown by semi-native PAGE (Fig 3) Purification of folded Opa proteins
To remove unfolded protein and other contaminants, the
in vitro folded OpaJ129 proteins were purified by ion-exchange chromatography We observed that during the purification by anion-exchange chromatography (Q-Seph-arose HP at pH 12) a substantial proportion of the refolded OpaJ129 protein eluted in the denatured state Probably, the protein was unstable in the alkaline conditions applied during purification Therefore, the pH was reduced from 12
to 7.5 after refolding This procedure did not affect the folding state of either OpaB128 or OpaJ129 as determined
by seminative PAGE (data not shown) The neutralized protein solution was applied to a cation-exchange column (SP-Sepharose HP at pH 7.5) Protein was eluted from the column with a linear salt gradient, resulting in the elution of either folded OpaB128 or folded OpaJ129 as a single peak Apparently, due to a difference in affinity, the folded protein
Fig 3 Semi-native PAGE analysis of in vitro folding of OpaB128 (A) and OpaJ129 (B) (A) Semi-native-PAGE analysis of in vitro folding of unpurified OpaB128 (Coomassie stained) Lane 1 isolated inclusion bodies Lane 2 in vitro folded protein Lane 3 denatured protein Lane 4 and 5,
in vitro folded and denatured OpaB after additional purification Lane 6, molecular mass marker Samples 2 and 4 were incubated at room temperature in loading buffer containing 0.1% SDS, samples 1, 3 and 5 were incubated at 100 C in loading buffer containing 2.0% SDS prior to electrophoresis (B) Semi-native PAGE analysis of in vitro folding of unpurified OpaJ129 (Coomassie stained) Lane 1, isolated inclusion bodies Lane 2, in vitro folded protein Lane 3, denatured protein Samples 1 and 3 were incubated as samples 1, 3 and 5 (Fig 3A) and sample 2 was treated
as sample 2 and 4 (Fig 3A) (C) Coomassie stained polyacrylamide gel showing in vitro folded OpaJ129, after additional purification Purified protein samples 1 and 2 were treated as samples 2 and 3 (Fig 3A), respectively.
Trang 6was purified from the residual unfolded protein as well as
from other contaminants
Analysis of the Opa protein conformation
by circular dichroism
To test whether the in vitro folded OpaB128 and OpaJ129
had adopted the expected b-sheet conformation, CDspectra
were recorded for folded Opa protein and Opa protein that
was denatured by boiling in 1.85% SDS The far-UV spectra
revealed a clear difference between the secondary structures
of the folded and denatured proteins (Fig 4A) The
charac-teristic feature of the spectrum, recorded for folded OpaB128
was a minimum at 217 nm Characteristic features of the
spectrum, recorded for folded OpaJ129, were a maximum at
approximately 232 nm and a minimum at 215 nm The
minimum negative peaks in this range are indicative for the
content of b-sheet The characteristic features of folded
OpaB128 and OpaJ129 disappeared upon denaturation,
with the minimum shifting to approximately 209 nm and the
maximum disappearing This spectrum suggested the
pres-ence of a considerable proportion of a-helix Apparently,
boiling in SDS induces a non-native structure
Near-UV CDpermits assessment of the differences
between the tertiary structure of folded and denatured
OpaJ129 Figure 4B shows that a less pronounced peak at
approximately 293 nm characterized the near-UV CD
spectrum of folded OpaB128, while folded OpaJ129 was
characterized by two peaks at approximately 265 nm and
293 nm After denaturation, this characteristic feature of the folded OpaJ129 protein changed into a spectrum with a broad positive ellipticity and a maximum at around 270 nm, while the major difference between refolded and denatured OpaB128 was measured between 250 and 265 nm The differences in the spectra between refolded and denatured Opa protein are indicative for a major structural change after denaturation
Functional analysis of purified refolded and denatured Opa protein
In a receptor overlay experiment, equal amounts of refolded and denatured OpaJ and OpaD were applied to nitrocel-lulose and incubated with bacterial lysates containing the CEACAM1-N-A1 domain Binding of CEACAM1-N-A1 was determined by monoclonal anti-His Ig reacting with the His-tagged CEACAM1-N-A1 protein Refolded OpaJ129 bound to CEACAM1-N-A1, consistent with the binding experiments with the OpaJ129-expressing E coli bacteria (Fig 5) The binding of refolded Opa appeared to be conformation-dependent, as no binding was found with denatured OpaJ129
D I S C U S S I O N
The majority of Opa proteins have been shown to speci-fically target members of the CEACAM receptor family [10,26] How this binding function can be conserved
Fig 4 Far-UV (A) and near-UV (B) circular dichroism spectra of refolded and heat-denatured OpaB128 (A) Far-UV circular dichroism spectra of refolded OpaB128 (interrupted line) and heat-denatured OpaB128 in 1.85% SDS containing buffer (solid line) (1) Far-UV circular dichroism spectra of refolded OpaJ129 (interrupted line) and heat-denatured OpaJ129 in 1.85% SDS containing buffer (solid line) (2) (B) Near-UV circular dichroism spectra of refolded OpaB128 (interrupted line) and heat-denatured OpaB128 in 1.85% SDS containing buffer (solid line) (1) Near-UV circular dichroism spectra of refolded OpaJ129 (interrupted line) and heat-denatured OpaJ129 in 1.85% SDS containing buffer (solid line) (2).
Trang 7despite the hypervariability of the surface-exposed regions
of the Opa proteins is still an enigma The detailed
identification of the receptor-binding Opa regions would
aid greatly in the development of new vaccines or
antimicrobials specifically targeted at blocking this
essen-tial adhesion process The study of the molecular
inter-actions between the CEACAM receptors and Opa
proteins would be facilitated greatly by the availability
of large quantities of pure Opa proteins This was
achieved in the present study for OpaB128 and OpaJ129,
two representative Opa proteins present in invasive
variants of N meningitidis strain H44/76
Previously, the isolation and purification of Opa proteins
from meningococcal strains has been described [21,27]
However, translation of the constitutively transcribed opa
genes depends on the expression status of the individual opa
loci, which are subject to high-frequency phase variation [28]
Due to this phase variation it is difficult to express and purify
a single individual Opa protein without significant
contam-ination from different Opa proteins expressed from other
loci However, for detailed structure–function analysis pure
protein is needed and by cytoplasmic expression of OpaB128
and OpaJ129 in E coli, we were able to isolate highly pure
protein, not contaminated with other Opa proteins
In order to determine their binding to CEACAM1, we
assessed the binding of surface-expressed OpaB128 and
OpaJ129 in an E coli background to the N-terminus of
CEACAM1 CEACAM8 was taken as a negative control
because it does not function as an Opa receptor as has been
shown for the gonococcal and meningococcal Opa proteins
analyzed to date Both OpaB128 and OpaJ129 bound
specifically to the N-terminal domain of CEACAM1 As
the majority of Opa proteins recognize this receptor,
OpaB128 and OpaJ129 seem to be typical members of this
protein adhesin family [10]
OpaB128 as well as OpaJ129 was expressed
cytoplasmi-cally in E coli in the form of inclusion bodies and
subsequently refolded and purified The characteristic heat
modifiability of Opa proteins was used to monitor their
refolding Similar to other b-barrel outer membrane
proteins such as OmpA (E coli) and P5 (Haemophilus influenzae), this heat-modifiable characteristic of Opa cor-relates with folding into the native structure [29,30] Both OmpA and P5 are integrated into the membrane as eight-stranded b-barrels [31,32] and the same structure has been predicted for Opa proteins [12,33]
The CDmeasurements showed a clear difference between the structure of folded Opa and Opa denatured
by boiling in SDS The far-UV spectrum we recorded for folded Opa resembles that of folded OmpA from E coli [34] and purified P5 from H influenzae [32] The spectra are indicative of a high content of b-strands, consistent with the (proposed) structure of these outer membrane proteins (Fig 4A) The difference between the near-UV CDspectra of folded and denatured Opa supports the conclusion that denatured protein has undergone a major conformational change In the proposed topology model for Opa proteins, 31% of the amino acid chain is predicted to form a transmembrane b-barrel The high content of b-strands reflected in the CDspectra reported here suggests that a significant part of the extracellular loops may also adopt this secondary structure It is thus conceivable that Opa proteins form a more extended b-barrel structure that protrudes from the outer mem-brane into the extracellular space, similar to what was described recently for the OmpT outer membrane prote-ase from E coli [35]
The pH and the salt concentration are the most critical factors in the folding efficiency of Opa It appeared that a
pH above the calculated pI is needed for efficient folding, as has also been found for the OmpA protein from E coli and the PorA protein from N meningitidis [36,37] The present study demonstrates how two different Opa proteins, with approximately 70% homology, can be folded in vitro under similar conditions This method will allow us to establish a collection of different Opa proteins, suitable for studying the interactions with CEACAM receptors
In the receptor binding experiments OpaDwas used as a positive control, as was also done in similar experiments by Virji et al [10] In earlier experiments using only the N-terminus of CEACAM1 we could not find reproducible binding to Opa However, when the N-A1 domain of CEACAM1 was used instead, it became clear that refolded OpaJ129 is functional in receptor binding The binding between Opa and CEACAM1 seemed to be conformation-dependent since almost no binding was found with dena-tured Opa protein
To conclude, with our purification and folding proce-dures, we were able to isolate pure and native OpaB128 and OpaJ129, both adhesins binding to the CEACAM1 recep-tor Conformational analysis of the purified, refolded proteins provided the first experimental evidence for a secondary structure dominated by b-strands, confirming previously proposed topology models Purified and refolded Opa proteins will be used for detailed structural and functional analysis
A C K N O W L E D G E M E N T S
We would like to thank F van der Lecq at the sequencing centre of the Centre for Biomembranes and Lipid Enzymology at Utrecht University for N-terminal protein sequencing We are grateful to
M Kuroki at the Fukuoka University, for the generous gift of Fig 5 Binding of native and denatured OpaD and OpaJ to its receptor
was determined by immunodotblotting.
Trang 8cDNA from CEACAM, to M Achtman at the Max-Planck
Institute in Berlin, for the generous gift of purified OpaDand to
B Kuipers at the RIVM in Bilthoven and W Zollinger at the
Walter Reed Army Institute of Research in Washington for
providing us with monoclonal antibodies We also thank
W van Noppen at the University of Amsterdam/AMC for critically
reading the manuscript.
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