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INTRODUCTION |
Relapsing fever and Lyme disease are bacterial infectious diseases
caused by spirochetes of the genus Borrelia. In contrast to
other spirochetes that affect humans, such as the syphilis agent
Treponema pallidum, their transmission to vertebrate hosts depends on arthropod vectors (1). Throughout their vector-host life
cycle, Borrelia cells display abundant lipoproteins on their surfaces. The genomic sequence of the Lyme disease spirochete Borrelia burgdorferi revealed over 130 putative lipoprotein
genes on its chromosome and plasmids (2, 3). With the exception of 3 matrix-binding proteins (4, 5), their specific functions have not been determined.
Included in this class of lipoproteins with unknown function is the
tick-associated Outer Surface Protein A
(OspA)1 (6), which is in
current use as a Lyme disease vaccine for humans (7). OspA is anchored
to the outer membrane via an N-terminal triacyl-modified cysteine (8,
9) and is intrinsically resistant to proteases such as trypsin, despite
its surface accessibility and high lysine content (10-12). The OspA
crystal structure revealed 21 antiparallel
-strands and one
C-terminal
-helix, which fold into globular N- and C-terminal
domains connected by an unusual, freestanding nonglobular
-sheet
(13-15).
Less is known about the structure of the major outer membrane
lipoproteins of relapsing fever Borreliae, which are
immunodominant and determine serotype (16). Collectively called
variable membrane proteins, they have been divided into two families:
the variable large proteins (Vlps) of 36-40 kDa and the variable small
proteins (Vsps) of 20-23 kDa (17). Vlp and Vsp proteins appear unique to the genus Borrelia. They have been described in the
relapsing fever species Borrelia turicatae (17-20),
Borrelia hermsii (21), Borrelia recurrentis (22),
and Borrelia crocidurae (23). B. burgdorferi
OspC, a major outer surface protein in early Lyme disease (24), is
phylogenetically related to the Vsps (17), which has led to the term
Vsp-OspC family.
While their signal peptides necessary for proper translocation and
processing are conserved, the mature Vlp and Vsp-OspC lipoproteins are
highly polymorphic. OspC amino acid sequences of different Lyme disease
Borreliae can vary as much as 25% (25, 26). There is
evidence that this variation is maintained, even within a local population, by frequency-dependent balancing selection
(27). Vlp and Vsp proteins diverge even more, with 40 to 80% amino
acid identities among them (17, 28). In contrast to the single plasmid-encoded ospC of B. burgdorferi (29),
several archival copies of B. hermsii vsp and vlp
genes are maintained on linear plasmids and sequentially expressed from
a promoter site after gene conversions or DNA rearrangements (21, 30,
31). The resulting multiphasic antigenic variation of Vsps and Vlps
allows the spirochete to repeatedly evade the host's immune response, which leads to recurrent spirochetemia and the characteristic febrile
episodes (16). This strategy is analogous to that for variant surface
glycoproteins (VSG) of African trypanosomes, the PfEMP1 proteins of the
malarial parasite Plasmodium falciparum, and the PilE/PilS
and Opa surface proteins of Neisseria species (32, 33).
Experimental relapsing fever in mice has demonstrated a role for Vsp
proteins in differential tissue localization as well as for avoidance
of the immune response. In a clonal population of B. turicatae, expression of VspB was associated with high densities of spirochetes in the blood, while expression of VspA led to early invasion and persistent infection of the central nervous system (17-19, 34, 35). VspA may help to evacuate the bacterium to an
immunopriviledged niche, while VspB may facilitate efficient transmission to the next feeding tick. Tissue culture assays yielded clues about pathogenesis mechanisms. B. turicatae expressing
VspA penetrated human umbilical vein epithelial cell monolayers more readily than those expressing VspB (18). On the other hand, VspB
increased binding of the spirochete to mammalian endothelial and glial
cells (36), predominantly by the direct interaction of VspB with host
cell surface glycosaminoglycans (37).
Structural data for Vsp-OspC proteins are limited. NMR studies of
B. burgdorferi strain B31 OspC have suggested that the core of OspC consists of four
-helices, while the N- and C-terminal sequences are highly flexible (38). OspC dimers were observed both with
recombinant protein in solution (38) as well as with cell
surface-exposed native lipoprotein (11). However, the effect of
sequence variation on Vsp-OspC protein structure, and thus the
structural basis of their established functions, remained unknown. To
address this, we determined structural features of VspA and three other
members of the Vsp-OspC protein family by biochemical and biophysical
methods. A comparison indicates that Vsp-OspC proteins share a highly
-helical, compact fold, which includes a dimerization domain and
confers protease resistance to a large central portion of the Vsps. For
high resolution x-ray structure determination, we obtained two forms of
VspA crystals.
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EXPERIMENTAL PROCEDURES |
Expression and Purification of Recombinant Histidine-tagged
Fusion Proteins--
Non-lipidated, soluble fusion proteins with
N-terminal histidine tags (hisVspA, hisVspB, hisVsp26, and hisOspC)
were obtained as described for hisVspA (17). Briefly, gene segments
lacking the N-terminal signal sequence and lipidation site were
amplified by polymerase chain reaction using Taq
thermostable DNA polymerase (Roche Molecular Biochemicals). Forward and
reverse primers for hisVspB, hisVsp26, and hisOspC are listed in Table
I. The polymerase chain reaction products
were ligated into pET15b (Novagen) and cloned in Escherichia
coli BL21(DE3) (Novagen). Recombinant proteins were expressed
after induction with isopropyl-
-D-thiogalactopyranoside and subsequently purified by nickel affinity chromatography as described (17, 19). All four proteins eluted >95% pure at an
imidazole concentration of 300 mM. Recombinant,
non-lipidated OspA was provided by R. Huebner (Aventis, Swiftwater,
PA).
Expression and Purification of N-terminal Truncated Core
Proteins--
To obtain core proteins S34VspA and S34VspB,
vspA and vspB sequences starting with the
Ser34 codon and ending with the translational stop codon
were amplified from plasmid clones (see above and Refs. 17 and 19) by
polymerase chain reaction using Pwo polymerase (Roche
Molecular Biochemicals). The forward primer for both sequences was
S34vspAB-fwd. Reverse primers were S34vspA-rev and S34vspB-rev,
respectively (see Table I). The polymerase chain reaction products were
digested with NdeI and BamHI and ligated into
pET29b (Novagen). Recombinant plasmids pET29::S34vspA and
pET29b::S34vspB were obtained after transformation of
E. coli Inv
F' (Invitrogen). E. coli BL21(DE3) (Novagen) transformants containing the plasmids were grown overnight from single colonies at 30 °C in LB containing 30 µg of
kanamycin/ml. A 1:50 dilution of the overnight cultures was grown at
37 °C in Terrific broth (39) with kanamycin. Expression was induced
with isopropyl-
-D-thiogalactopyranoside at a final
concentration of 1 mM when the culture OD595
reached 0.9. Incubation continued overnight at 30 °C. Cells were
harvested by centrifugation at 8,000 × g for 10 min at
4 °C and resuspended in 0.2 × volume of anion exchange column
buffers: (i) 20 mM Tris hydrochloride, 0.02% NaN3, pH 9.0 (AIEX-A), for S34VspA and (ii) 20 mM ethanolamine hydrochloride, 0.02% NaN3, pH
10.0 (AIEX-B), for S34VspB. Cleared lysates were obtained by
ultrasonication, centrifugation at 15,000 × g for 10 min and passing through a 0.45-µm pore size filter (Nalgene).
Cleared lysates were passed over a HiPrep 16/10 Q XL anion exchange
column (Pharmacia) in AIEX-A or -B column buffers. Under the respective
conditions both S34VspA and S34VspB bound quantitatively to the matrix.
The proteins were eluted in a linear gradient of AIEX-A or -B column
buffers containing 0 to 200 mM NaCl. S34VspA and S34VspB
eluted at salt concentrations of 80 and 130 mM,
respectively. Peak fractions containing the recombinant proteins were
pooled, concentrated using Centricon Plus-20 centrifugal concentrators (Amicon), and dialyzed overnight against deionized H2O at
4 °C. Protein aliquots were flash-frozen on dry ice and stored at
80 °C.
Proteolytic and Cross-linking Assays--
Recombinant purified
proteins were treated separately with four proteases. The two serine
proteases, trypsin and plasmin (Roche Molecular Biochemicals), were
used at 10 to 400 µg/ml or 1 mg/ml, respectively, in 100 mM Tris-HCl, pH 8.5, while lysosomal cysteine protease
cathepsin B and aspartyl protease cathepsin D (Calbiochem) were used at
concentrations of 1.5 units/ml in 250 mM Na citrate, 1 mM EDTA, 2 mM dithiothreitol, pH 5.5. Reactions
were incubated at 37 °C for 1 h and then stopped with Roche
Complete protease inhibitor mixture supplemented with 1 µM pepstatin.
For in situ protease treatment, B. turicatae Oz1
serotype A cells were grown in BSK II medium and harvested as described
(40). Intact Borrelia cells were treated in situ
with trypsin (Roche Molecular Biochemicals) as described (41).
Cross-linking of surface-exposed proteins was performed using
formaldehyde as described for B. burgdorferi OspC (11).
Protein Gel Electrophoresis and Immunoblot
Analysis--
Proteins were separated on 12% polyacrylamide-SDS gels
and visualized by Coomassie Blue staining. For immunoblots, proteins were electrophoretically transferred to nitrocellulose membranes (Immobilon-NC, Millipore) as described (42). Membranes were rinsed in
20 mM Tris, 500 mM NaCl, pH 7.5 (TBS), and
either air-dried or processed directly. 5% Dry milk in TBS with 0.05%
Tween 20 was used for membrane blocking and subsequent incubations for 1 h each. TBS with 0.05% Tween 20 alone was used for intervening washes. Rabbit antisera were used at 1:500 dilutions, and mouse monoclonal antibody hybridoma supernatants at a 1:10 dilution. Alkaline
phosphatase-conjugated Protein A/G (ImmunoPure Protein A/G CIP
conjugate, Pierce) at a 1:5000 dilution was used as the second ligand,
and a stabilized alkaline phosphatase substrate solution (1-Step
NBT/BCIP, Pierce) was used for colorimetric detection.
N-terminal Protein Sequence Analysis and Mass
Spectrometry--
N-terminal protein sequences of recombinant proteins
were determined on a Model 477A protein sequencer (Applied Biosystems) from protein samples previously purified by gel electrophoresis and
transfer to a polyvinylidene difluoride membrane (43). Molecular masses
of purified recombinant proteins were determined by matrix-assisted laser desorption ionization-time of flight on a Voyager biospectrometry workstation (Perseptive Biosystems) using sinapinic acid as matrix.
Circular Dichroism Spectroscopy and Secondary Structure
Prediction--
Circular dichroism (CD) spectra were obtained using a
Jasco J-720 spectropolarimeter. Proteins were diluted to a
concentration of 20 µM in deionized H2O.
Samples were placed in 0.1-mm path length cells at 25 °C and spectra
were acquired using a scan speed of 20 nm/min, response time of 1 s, bandwidth of 1.0 nm, and step resolution of 0.5 nm. 20 Acquisitions
between 260 and 180 nm wavelength were co-added. Spectral data were
deconvoluted using the DICROPROT program suite (44) and secondary
structure ratios were determined by the least squares fit method using
the Fasman algorithm. Predictions of secondary structure were obtained
using software available on the protein sequence analysis server at the
Biomolecular Engineering Research Center of Boston University
(45).
Gel Filtration Liquid Chromatography--
For size
determination, proteins were separated on a HiPrep 16/60 Sephacryl
S-200 high-resolution column (Amersham Pharmacia Biotech). The column
was first equilibrated with column running buffer (20 mM
Tris-HCl, pH 8.0, 200 mM NaCl, 0.2% NaN3) and
then calibrated separately with blue dextran 2000 (for void volume determination), albumin (67 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), and ribonuclease A (13.7 kDa) (Amersham Pharmacia Biotech). Approximately 10 mg of concentrated recombinant proteins previously purified by anion exchange chromatography were loaded in 1 ml of column running buffer. For each protein, the partition constant
Kav was calculated from the peak elution volume
using the following formula: Kav = (Ve 2
Vo)/(Vt
Vo), where Ve = elution volume,
Vo = column void volume, and Vt = total bed volume.
Crystallization of S34VspA and preliminary diffraction
analysis--
Recombinant purified S34VspA was dialyzed against
deionized H2O, then diluted to 8 mg/ml. Crystals suitable
for structure determination by x-ray diffraction methods were obtained
using standard vapor diffusion techniques. Aggregated thin plates were produced in conditions numbers 6 and 22 during initial screening with
Hampton Research sparse matrix kit I. These conditions share precipitant and buffer conditions of 30% PEG 4000 and 0.1 M Tris/HCl, pH 8.5, but differ in salt additive of 0.2 M MgCl2 or 0.2 M
NaCH2COOH, respectively. The precipitant, salt, and pH were
varied systematically about these two conditions to optimize crystal
size and quality (see "Results"). S34VspA crystals were
characterized using a conventional rotating anode x-ray source and
native diffraction data were collected at beamline X12C of the
Brookhaven National Laboratory National Synchrotron Light Source on a
CCD detector. Diffraction images were processed with DENZO/SCALEPACK
(46).
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RESULTS |
Vsp-OspC Protein Purification--
While pathogenesis-associated
functions of Borrelia Vsp proteins, e.g. in
antigenic variation and tissue tropism, have been established, the
structural basis for these functions remain unknown. One question is
how the observed primary protein sequence polymorphisms affect
secondary, tertiary, and quarternary protein structure. Beginning to
address this by biochemical and biophysical methods, we first expressed
VspA of B. turicatae in E. coli. We chose this protein to focus on because of its association with nervous system invasion. Two related proteins, VspB of the same strain of B. turicatae and Vsp26 of B. hermsii, as well as the more
distantly related OspC of B. burgdorferi, were also
expressed. Their conserved signal peptides and overall amino acid
identities of 40 to 70% are representative of the entire Vsp-OspC
protein family (Fig. 1). The proteins
were purified in an N-terminal histidine-tagged form by standard nickel
affinity chromatography. In these constructs (hisVspA, hisVspB,
hisVsp26, and hisOspC), the Vsp-OspC sequences start with residue
Asn20 (Fig. 1), i.e. they lack the signal
peptide and acylation site at Cys19. The recombinant
proteins therefore were soluble, but otherwise mimic the full-length
processed form.

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Fig. 1.
Amino acid sequence alignment of VspA, VspB,
Vsp26, and OspC. The protein sequence alignment was
performed using CLUSTAL X (71). Color coding of conserved residues is
according to Gonnet Pam250 matrix groups. Asterisks (*) in
the alignment header indicate fully conserved amino acid residues,
colons (:) indicate conservation of "strong" Gonnet
Pam250 matrix group residues, and periods (.) conservation of
"weaker" group residues. The shaded area of the
alignment header indicates the protease resistant core determined for
VspA, VspB, and Vsp26 (see also Fig. 2). An L denotes the
lipid-modified cysteine (C19). Shaded bars and black
frames in the alignment footer indicate predicted Vsp and
NMR-determined OspC -helices ( 1 to 4) (38), respectively (see
also Fig. 5B). Amino acid residues are numbered to the
right of the alignments according to the full-length,
unprocessed protein sequences.
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Protease Resistance of Vsp Core Proteins--
Previous studies
showed that B. burgdorferi OspA and OspC are highly
resistant to trypsin in situ, i.e. when expressed
in a native, lipidated form on the surface of the bacteria (10, 11) and
that protease resistance is an intrinsic property of recombinant OspA
(12). To determine whether VspA and the related proteins are also
protease resistant, we incubated recombinant hisVspA, hisVspB,
hisVsp26, and hisOspC with trypsin at concentrations ranging from 12.5 µg to 400 µg/ml for 1 h at 37 °C. Recombinant, non-lipidated OspA and casein (Sigma) were used as controls (Fig. 2).

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Fig. 2.
Protease-resistant cores of recombinant Vsp
proteins. Recombinant proteins were incubated in the appropriate
reaction buffers either without proteases ( ), 100 µg/ml trypsin
(t), or 1.5 units/ml of cathepsin B (cB) and
cathepsin D (cD). Proteins were separated on a 12% SDS-PAGE
gel. Approximately 2 mg of protein/lane was loaded and visualized using
a Coomassie stain (Pierce). The sizes of protein size markers (Life
Technologies, Inc.) are indicated to the left.
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As expected, casein was digested to completion, while recombinant OspA
was unaffected by trypsin. In contrast to the study of in
situ OspC (11), several independently purified recombinant hisOspC
preparations did not exhibit comparable trypsin resistance in
vitro. This difference in results suggests that OspC is either stabilized by the lipid-mediated attachment to the cell surface, or
that the recombinant hisOspC obtained from our construct is destabilized by the additional, non-native sequence at its N terminus.
Although the processed VspA, VspB, and Vsp26 proteins have 27 to 30 predicted trypsin sites distributed over the whole length of their
protein sequences, the three proteins were merely truncated 5 to 8 kDa
by trypsin digestion (Fig. 2). Similar results were obtained with
plasmin, which has trypsin-like activity. Yet, to reach an equivalent
cleavage efficiency, plasmin concentrations had to be increased to 1 mg/ml, which corresponds to about 10 times the physiological
concentration (47). This indicated that most of the sites were not
accessible to the protease, thus yielding a protease-resistant core. No
further truncations were observed with trypsin concentrations of up to
10 mg/ml, and VspA maintained its protease-resistant core even after
being boiled for 10 min (not shown). Protein sequencing of the
trypsin-treated VspA, VspB, and Vsp26 protein cores revealed the
N-terminal amino acids in all three cases to be SDGTV. This sequence
can be found starting at a conserved serine at position 34 (Ser34) (Fig. 1).
To purify the protease-resistant cores and for further structural
analysis of VspA (see below), we constructed N-terminal truncated
versions of VspA and VspB starting at Ser34 (S34VspA and
S34VspB). A methionine codon was added to initiate translation.
S34VspA and S34VspB proteins were purified to >95% purity from crude
E. coli lysates in a single-step anion-exchange chromatography step. Trypsin truncated S34VspA and S34VspB by approximately 1 kDa, revealing a succeptible site at the C terminus as
well (Fig. 2). The location of this site, i.e. the C
terminus of the core protein, was derived from matrix-assisted laser
desorption ionization-time of flight mass spectrometry data. The mass
of undigested S34VspA was determined as a control, yielding a major peak of 18,507 Da. It corresponds to a protein starting with
Ser34 and ending with Asn214 with a predicted
size of 18,475 Da. As confirmed by N-terminal sequencing, the f-Met was
removed, probably by the E. coli methionyl aminopeptidase
(48). For trypsin-treated S34VspA, the mass of the major peak was
17,739 Da, which corresponds to a predicted 17,700 Da protein starting
with Ser34 and ending with Lys206. The mass
difference of 768 Da would also match the removal of 8 C-terminal amino
acids, a predicted 792 Da.
To assess the resistance to other physiologically relevant proteases,
we treated the proteins with two lysosomal proteases. Cathepsin B is a
broad specificity cysteine endopeptidase with preference for R-R bonds,
but also has peptidyl dipeptidase activity, liberating C-terminal
dipeptides. Cathepsin D is an aspartic endopeptidase with pepsin A-like
activity, cleaving F-V, Q-A, A-L, L-Y, Y-L, G-F, F-F, and F-Y bonds
(49). Incubation conditions were derived from the ones used to study
antigen presentation of ovalbumin (50). Casein was digested by
both proteases, while OspA appeared unaffected. Neither VspA, VspB,
Vsp26, nor OspC was cleaved by cathepsin D, although they contain
1 to 4 predicted endopeptidase sites (Fig. 1).
Cathepsin B treatment resulted in several distinct protein bands for
all Vsp proteins. Interestingly, the smallest bands for VspA and VspB,
presumably the proteolytic end products, were similar if not identical
in size to the ones obtained with trypsin. We therefore performed mass
spectrometry on cathepsin B-digested S34VspA. The mass of the final
proteolytic product, 17,733 Da, was almost identical to
trypsin-digested VspA. This suggests that cathepsin B removes 8 amino
acids from the C terminus of S34VspA, which would be consistent with
its peptidyl dipeptidase activity. Together, these data indicate that
Vsp proteins, although differing in primary amino acid sequence, have a
conserved core that is resistant to proteases with different specificities.
Vsp-OspC Protein Dimerization In Solution--
Huang et
al. (38) have recently shown that a truncated recombinant form of
B. burgdorferi OspC forms a 37-kDa dimer in solution. To see
if this is a common structural feature of the Vsp-OspC protein family,
we determined the size of the recombinant proteins by gel filtration.
Purified recombinant proteins were loaded on a high-resolution
Sephacryl column, which had been calibrated with protein size
standards. The apparent molecular masses were calculated from
the partition coefficients to be as follows: hisVspA, 43.5 kDa;
hisVspB, 42.5 kDa; hisVsp26, 49.2 kDa; hisOspC, 49.2 kDa; S34VspA, 38.5 kDa; S34VspB, 40.5 kDa (see Fig. 3). The
predicted monomer sizes for the recombinant proteins range from 18.6 kDa (S34VspA) to 23 kDa (hisVsp26). This shows that VspA, like the other relapsing fever Vsp proteins and the related OspC, forms dimers
in solution.

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Fig. 3.
Dimerization of Vsp-OspC proteins in
solution. Recombinant proteins previously purified by anion
exchange chromatograpy were separated by gel filtration liquid
chromatography. Albumin, chymotrypsinogen A, ovalbumin, and
ribonuclease A (Amersham Pharmacia Biotech) served as size standards.
Kav partition coefficient values were calculated
based on the peak elution volumes and plotted against the log of the
proteins' predicted molecular weights (MW, in kDa). The
diagonal line represents a logarithmic regression derived
from the protein standard values.
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VspA Dimerization and Protease Resistance In Situ--
In a next
step, we investigated whether the dimerization and protease resistance
of Vsp proteins could also be observed in situ,
i.e. on the surface of intact Borrelia cells.
Dimerization has recently been shown for in situ B. burgdorferi OspC (11). We applied the same approach to VspA
expressed by B. turicatae Oz1 serotype A. Cross-linking of
closely associated surface proteins by formaldehyde revealed that VspA
dimers form also in situ (Fig. 4A).

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Fig. 4.
VspA dimerization and protease resistance
in situ. Intact B. turicatae serotype
A (B.t. A) cells expressing VspA were incubated
with either (A) 1% formaldehyde (form.) or
(B) 200 µg/ml trypsin (tryp.). Total cellular
proteins were separated on on a 12% SDS-polyacrylamide electrophoresis
gel, transferred to nitrocellulose membranes, and probed with anti-VspA
polyclonal rabbit antisera. For the trypsin protease assay, both the
pelleted, cell-associated proteins (p) and the reaction
supernatant (s) were loaded in equivalent volumes. S34VspA
protein was also included in the reactions as a size control. Sizes of
protein markers in kDa are indicated at the left of each
panel. In panel A, labeled arrows to the
right indicate the sizes of VspA monomers and dimers derived
from the size standards. In panel B, L, indicates lipidated,
NL, non-lipidated forms of VspA.
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We next assessed whether the native VspA protein dimers also exhibited
the protease-resistant properties observed with the recombinant
protein. Accessibility of the C-terminal trypsin sites would lead to a
truncated, yet cell-associated protein. If the N-terminal sites in both
dimer subunits were accessible as well, the protein parts containing
the two lipid anchors would be cleaved and the cores would be released
into the supernatant. We therefore incubated intact B. turicatae cells with trypsin and then analyzed the pelleted
cell-associated proteins and solubilized proteins in the supernantant.
VspA was detected using an anti-VspA polyclonal antibody (Fig.
4B). In untreated B. turicatae cell pellets, VspA was found in its full-length lipidated form running at about 22 kDa. No
protein was detectable in the supernatant. In the cell-associated fraction of trypsin-treated B. turicatae cells however, two
additional forms of VspA approximately 20 and 18 kDa in size were
detected, with the 20-kDa band being the most prominent. In the
supernatant, only the 18-kDa band was detected. The 20-kDa band likely
represents the C-terminal truncated lipidated protein, while the 18-kDa
band corresponds in size to the VspA core. This indicates that both the
N- and C-terminal domains were accessible to proteases such as trypsin.
It also suggests that the VspA core can still be anchored to the
surface via association with a lipidated dimer subunit, and that
release into the surrounding environment occurs only after cleavage of
both membrane anchor-containing peptides.
Conserved Vsp-OspC Secondary Structure--
We used CD
spectroscopy as a first approach to compare the secondary structures of
the recombinant Vsps and OspC (Fig.
5A). The obtained mean residue
ellipticity values were plotted against the wavelength. Peaks at 192 nm
and two troughs at 208 and 222 nm are indicative of predominantly
-helical secondary structures. The CD spectra of VspA, VspB, Vsp26,
and OspC overlap extensively, thus the high helix content appears
conserved among the Vsp-OspC protein family. Deconvolution of spectral
data lead to estimates of secondary structure ratios. For
histidine-tagged proteins, the mean values for
-helical,
-sheet,
and coiled secondary structure were 71, 2, and 27%, respectively. For
the N-terminal truncated proteins, the coiled structure content
decreased to 16%, with
-helix and
-sheet ratios of 80 and
4%.

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Fig. 5.
Secondary structure of Vsp-OspC
proteins. A, CD spectra of recombinant Vsp-OspC
proteins. The mean residue ellipticity ( mrd), which
normalizes for the proteins' different Mr, is
plotted against the wavelength. B, secondary structure
predictions of processed protein sequences starting with
Cys19. Amino acid residues are numbered according to the
full-length, unprocessed protein sequences (see also Fig. 1).
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As we currently lack further secondary or tertiary structure
information on the Vsps, we resorted to computer algorithms for further
comparisons. While a comparison of predicted secondary structures
confirmed the experimental data of highly 
helical proteins, they
also revealed further conservation. All studied proteins are calculated
to fold into 4 helices separated by turns (Fig. 5B).
Furthermore, the N and C termini likely form flexible, unordered tails
of approximately 20 and 10 amino acids, respectively. Both these
structural features are in accordance with the NMR-derived data for
OspC (38).
Crystallization of S34VspA--
To date, B. burgdorferi
OspA is the only Borrelia lipoprotein whose crystal
structure has been determined (13-15). Initial attempts to crystallize
the histidine-tagged forms of VspA and VspB were unsuccessful. We
surmised that the predicted flexible, unordered N- and C-terminal
sequences could inhibit crystallization. In our next set of
experiments, we therefore used S34VspA, which lacks most of the
flexible N-terminal tail.
Large single crystals of S34VspA were obtained at either room
temperature or 4 °C using 28% (w/v) polyethylene glycol 4000, 80 mM Tris/HCl, pH 8.5, 15% (v/v) glycerol, and either
100-200 mM nickel chloride (Fig.
6) or magnesium formate (not shown). Nucleation was controlled by reducing the initial concentration of
S34VspA to 6-8 mg/ml. S34VspA/nickel chloride crystals belong to the
monoclinic space group C2, and have unit cell dimensions a = 241.8 Å, b = 69.1 Å,
c = 87.6 Å, and
= 104.9°. S34VspA/magnesium formate crystals belong to space group P21 and have cell
dimensions a = 121.8 Å, b = 68.7 Å,
c = 87.5 Å, and
= 103.4°. The volume of the
asymmetric unit is quite similar in both crystal forms. We anticipate
that four VspA dimers are accommodated with approximately 50% solvent
content.
We have collected two complete diffraction data sets at 100 K. Dataset
I (S34VspA/nickel chloride) has 38,032 reflections merged from 130,123 individual measurements and is 98.9% complete to a resolution of 2.7 Å, with a linear R-merge of 4.2% (14.7% for the highest
resolution shell, 2.8-2.7 Å). Dataset II (S34VspA/magnesium formate)
has 27,615 reflections merged from 84,782 individual measurements and
is 96.2% complete to a resolution of 3.0 Å, with a linear
R-merge of 6.7% (20.9% for the highest resolution shell, 3.11 - 3.0 Å). In both cases the mosaic spread of the crystal was
determined to be 0.95°. We have recently succeeded in obtaining selenomethionine-substituted crystals of S34VspA for structure determination using the multiple anomalous dispersion method (51).
 |
DISCUSSION |
This study has shown that the neurotropism-associated VspA of
B. turicatae shares secondary, tertiary, and quarternary
structural characteristics with other Borrelia Vsp-OspC
family proteins, despite as little as 40% identity in primary
sequence. CD spectroscopy demonstrated conserved, highly
-helical
secondary structures that are predicted to fold into a four-helix
bundle. Supporting this model were the findings that postswitch
mutations in expressed vsp alleles are mainly conservative
(30, 52), and that non-conservative amino acid changes, insertions, and
deletions cluster in regions outside the predicted helices (Fig. 1). We
conclude that localized polymorphisms, e.g. variable loops,
rather than differences in overall domain structures determine specific
Vsp-OspC functions such as antigenic variation and niche selection.
Further validating our model of structural conservation were
experiments showing that the proteins had same size protease-resistant cores and dimerized in a physiologic solution as well as on the bacterial surface. These shared characteristics are likely a
consequence of a common protein fold and conserved residues. Supporting
this, the Vsp cores coincide with the predicted helical domains. The predicted flexible unordered N- and C-terminal tails appear susceptible to proteolysis. In the case of trypsin, the enzyme active site must
obtain access to the target peptide bond (K-X) to accomplish cleavage.
This can only be achieved in regions where the main chain atoms are not
involved in formation of stable secondary structural elements (53). In
OspA, the lysines are in antiparallel
-strand sheets or short loops
and therefore protected (15). Similarly, the participation of Vsp-OspC
core lysines in
-helix hydrogen bonding would make these trypsin
sites inaccessible. On the other hand, lysines in the loops may be
shielded from proteolytic attack by dimer subunit interactions. A
comparable mechanism has been observed with two B. burgdorferi surface proteins: OspA closely interacts with an
integral membrane protein, P66, thereby protecting a surface-exposed
loop of P66 from proteolysis (11).
The common, intrinsic property of Vsp-OspC proteins to dimerize appears
mediated by sequences present in the protein cores. This conservation
despite significant divergence between the Vsp and OspC proteins
suggests that some of the invariant core residues participate in this
process. In the absence of further structural information, a stretch of
constant amino acids found at the C-terminal end of the predicted first
-helix (Fig. 1) is currently our strongest candidate for a
dimerization domain.
The effect of Vsp-OspC core features on the proteins' biological
functions could be manifold. Various tick organs have proteases (54,
55), thus protease resistance would be of benefit during the migration
of the spirochetes from the mitgut through the hemolymph to the
salivary glands (1). The resistance to trypsin and plasmin might also
serve the spirochetes in their vertebrate hosts. After host plasminogen
acquisition and activation, surface bound plasmin facilitates the
dissemination of bacteria in the tick and fosters heart and brain
invasion in infected mice (56, 57).
While the lipid moiety of Borrelia lipoproteins such as
B. burgdorferi OspA is a determinant of mitogenicity (58),
the common structural features of Vsp-OspC proteins might modulate host
immune responses as well. An efficient peptide antigen presentation to T cells via the major histocompatibility complex requires the limited
proteolytic processing of exogenous proteins by endosomal and lysosomal
proteases (59). Experiments with specific protease inhibitors and
protease gene knockout mice or the elimination of protease sensitive
sites have shown this (60). Borrelia spirochetes may have
evolved the latter strategy, rendering their major antigens resistant
to proteolytic attack and thus leading to the persistence of Vsp-OspC
core proteins in infected mammals. Furthermore, the multimerization of
the highly abundant Vsp-OspC proteins might produce an array that is
analogous to viral capsid particles (61). Both antigen persistence and
repetitive structure have been associated with a T-independent response
(62, 63). Intriguingly, this type of immune response has been observed
in the clearance of B. turicatae infection (64) and was
important for protective immunity and resolution of Lyme disease in an
animal model (65). The current study has focused on the activity of a
limited selection of proteolytic enzymes in vitro, and
further immunological studies will be needed to comprehensively address
the possible effects of Vsp protease resistance on antigen presentation.
The mechanisms of antigenic variation in African trypanosomes and
relapsing fever Borrelia are strikingly similar (66). A
comparison of the known structural features of these proteins reveals
further convergences. VSGs also share common, highly
-helical folds
and form stable dimer units in solution (67). Both VSGs and Vsp-Vlps
are anchored to the cell's surface via post-translational modifications, the VSGs via a C-terminal glycosylphosphatidylinositol anchor (68), and the Vsp-Vlps, like other Borrelia
lipoproteins (8, 9), via an N-terminal triacyl-modified cysteine.
Interestingly, VSG glycosylphosphatidylinositol anchors are hydrolyzed
by an endogenous trypanosomal phospholipase under stress conditions, which leads to VSG shedding (69). This may expedite the exchange of
coat proteins during an antigenic switch, and at the same time divert
the host immune system from live trypanosomes expressing the same VSG.
A similar mechanism has been recently observed with the oral pathogen
Streptococcus mutans, which expresses an enzyme that
releases the adhesin P1 from its cell wall (70). This activity might
allow the bacterium to shed P1-antibody complexes or to recolonize
after detaching from an adherent surface.
The structure-function analysis of the Vsp and Vlp proteins will likely
yield important clues in our understanding of antigenic variation and
tissue tropism. By integrating the structural information gained from
the VspA crystals obtained here with data from in vitro
immunological, biochemical, and cell biological studies, we will be
able to define the domains interacting with antibodies and other host factors.