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*
Department of Molecular Genetics and Microbiology and
New York State Department of Health, State University of New York, Stony Brook, NY 11794
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously reported on an IgG1
murine mAb, CB2, to OspB that had
complement-independent bactericidal properties (15, 16, 17).
Another complement-independent bactericidal Ab has also been reported
(18). These Abs do not kill B. burgdorferi by
agglutination, since their Fab also have bactericidal activity
(15, 17, 18). Other complement-independent bactericidal
Abs have been identified to epitopes in OspA, OspB, and p39
(19, 20, 21, 22), but the action of their Fab was not studied.
Collectively, it appears that Borrelia are extremely
susceptible to the direct action of Abs.
The epitope for CB2 was mapped to a hydrophilic region in the carboxyl terminus of OspB with a unique requirement for lysine at position 253 for binding, with identical results obtained for H6831, another mAb to OspB (16, 18). These Abs were so effective in their bactericidal action that they have been used to select for mutants. Mutants selected by the bactericidal action of these Abs are still the only ones known for this species (15, 16, 18, 23, 24, 25, 26).
Additional studies showed that the formation of an Ag (OspB)-Ab (CB2) complex led to the lysis of the outer membrane as the end result of the bactericidal activity (17). A polar bleb composed of a Fab-CB2 OspB complex preceded the formation of a spheroplast. The spheroplasts contained both OspA and OspB and were a terminally irreversible stage in the bactericidal process induced by Fab-CB2 (17). Outer membrane destabilization by Fab-CB2, but not cell wall or cytoplasmic membrane alterations, was also demonstrated experimentally (17).
Direct bactericidal action of Abs may be an overlooked, but fundamental, mechanism of host defense, certainly as applied to infections with Borrelia and possibly to infections with other bacteria. As an initial approach to understanding the mechanism of action of CB2, we have considered two possibilities: 1) CB2 functions as a catalytic Ab and cleaves the OspB directly; and 2) CB2 binds the OspB and causes a conformational change in the OspB. By either mechanism, lysis would then ensue as a result of the altered OspB. The results of our study suggest that CB2 is not a catalytic Ab; however, CB2 does effect a change in OspB that can be measured by the reduced susceptibility of this OspB to cleavage by proteases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DNA encoding for OspB was obtained by PCR from genomic DNA
extracted from B. burgdorferi B31 (1) and
B. burgdorferi BEP4 (27, 28). A 5'
oligonucleotide containing a unique NdeI site
(5'-GCACACTTCATATGAAAGGTGCTGAGTCAATTGG-3') that primes 55
nucleotides into the open reading frame from the N-terminus, thus
deleting the consensus sequence required by signal peptidase II, and a
second primer containing an XhoI site
(5'-CTAGCTCGAGTTATTTTAAAGCGTTTTT-3') that was complementary to the
coding strand of the 3' end of the gene were used to amplify the DNA.
All PCR reactions were performed in 100-µl volumes in a GeneAmp PCR
System 9600 (Perkin-Elmer, Norwalk, CT) and contained 200 µM of each
of the four dNTPs, 200 nM each of both primers, 2–5 mM
MgCl2, 1x PCR buffer (10 mM Tris-HCl, pH 8.3,
and 50 mM KCl), 1 ng of genomic DNA, and 0.5–1 U of AmpliTaq DNA
polymerase (Perkin-Elmer). The amplification profile consisted of
denaturation at 94°C for 30 s, annealing at 40°C for 30
s, and extension at 72°C for 2 min for a total of 30 cycles. The PCR
products were purified by use of the Wizard PCR Preps DNA Purification
System (Promega, Madison, WI). The ends of the amplimers were cut with
NdeI and XhoI and directionally ligated into the
expression vector (pET28a+ containing a His tag
leader sequence, Novagen, Madison, WI) cut with the same restriction
enzymes. Competent cells (Subcloning Efficiency Escherichia
coli DH5
, Life Technologies, Gaithersburg, MD) were transformed
with the ligation mixture according to the manufacturer’s protocol.
Transformants were plated on LB agar plates (1% BactoTryptone (Difco,
Detroit, MI), 0.5% Bacto-Yeast Extract (Difco), 1% NaCl, and 1.5%
agar) containing 50 µg/ml kanamycin and incubated overnight at
37°C. Appropriate constructs were identified by restriction analysis
and agarose gel electrophoresis using 1% agarose and 1x TAE (40 mM
Tris acetate and 2 mM EDTA). The nucleotide sequence of the clones was
determined with the ABI BigDye terminator cycle sequencing system (PE
Applied Biosystems, Foster City, CA) at the Center for the Analysis and
Synthesis of Macromolecules (State University of New York, Stony
Brook, NY).
Appropriate constructs were introduced into E. coli BL21(DE3) (29), and the recombinant protein was overexpressed and purified (Novagen). BL21(DE3) transformants were streaked on LB agar plates with 50 µg/ml kanamycin. Overnight starter cultures from individual isolates were inoculated into a larger volume of LB with 50 µg/ml kanamycin and incubated at 37°C. Cultures were induced by adding 1 mM isopropyl-ß-D-thiogalactoside when an OD600 of 0.5 was attained, and the expression was continued for 3 h. The cells were harvested by centrifugation, and the truncated OspB (tOspB) was purified from the soluble fraction of the lysate under nondenaturing conditions on a nickel column that bound the His tag sequence. The tOspB was eluted from the nickel column in 0.02 M Tris-HCl (pH 7.9) buffer containing 60 mM imidazole and 0.5 M NaCl (wash buffer). The purified tOspB was concentrated on a Centriprep-10 concentrator (Amicon, Beverly, MA). The purity and identity of the recombinant proteins were determined by Tricine SDS-PAGE and Western blot analysis using mAb CB2 to detect B31-tOspB and mAb H4610 to detect B31- and BEP4-tOspBs. The concentrations of the recombinant proteins were determined using the MicroBCA protein assay (Pierce, Rockford, IL). The physical state of the purified tOspBs was determined by size-exclusion chromatography and native PAGE.
Monoclonal Abs
mAb CB2 (mouse IgG1) to strain B31 OspB (15) was
obtained from hybridoma cells grown in tissue culture under serum-free
conditions using RPMI 1640 (Life Technologies, Grand Island, NY)
supplemented with 1 mM sodium pyruvate, 100 U/ml penicillin, 100
µg/ml streptomycin, and 1% Nutridoma NS (Roche, Indianapolis, IN).
The mAb was purified from filtered culture supernatant by affinity
chromatography on protein G-Sepharose 4FF using fast protein liquid
chromatography (Pharmacia, Piscataway, NJ). The purified mAb was
dialyzed against 0.02 M sodium phosphate (pH 7) buffer,
filter-sterilized (0.45-µm pore size filter), and stored at 4°C.
mAb MOPC21 (mouse IgG1) was obtained from Sigma (St. Louis, MO) and
further purified on protein G-Sepharose as detailed above. mAb H4610,
directed against OspB, was a gift from Tom Schwan (Rocky Mountain
Laboratory, Hamilton, MT). The protein concentration was determined
from the 280 nm absorption, assuming 1 mg/ml mouse IgG has an
A280 of 1.35.
Limited proteolysis
Recombinant tOsp was subjected to limited proteolysis in the presence and the absence of mAb. Typically, tOspB (0.2 mg/ml) was preincubated with CB2 (1 mg/ml) for 1 h to allow time for Ab to bind, then enzyme was added and allowed to act for the desired amount of time. The digestion was terminated by adding Tricine SDS-PAGE sample buffer (1 part 3x sample buffer to 2 parts incubation mixture) and heating for 3 min in a boiling water bath. Controls consisted of incubations without enzyme and incubations without tOspB. The tOspB (8 mg/ml) was in 0.02 M Tris-HCl (pH 7.9) buffer with 60 mM imidazole and 0.5 M NaCl. The CB2 (2.73 mg/ml) was in 0.02 M sodium phosphate (pH 7) buffer. The enzymes used were trypsin (modified to prevent autolysis and unmodified), endoproteinase Arg-C, endoproteinase Asp-N, and endoproteinase Glu-C (sequencing grade proteases, Roche). Incubation conditions for each were as follows. For trypsin, 1 µl of B31-tOspB was mixed with 14 µl of CB2, 3 µl of sterile water, and 20 µl of 0.1 M Tris-HCl (pH 8) buffer with 8 mM CaCl2. After 1 h at 34°C, 2 µl of modified trypsin (100 µg/ml in 1 mM HCl) or unmodified trypsin (5 µg/ml in 1 mM HCl) was added, and the incubation was continued for 1 h at 34°C. For endoproteinase Arg-C, 1 µl of B31-tOspB was mixed with 14 µl of CB2 and 20 µl of 0.1 M Tris-HCl (pH 8) buffer with 8 mM CaCl2. After 1 h at 34°C, 4 µl of activation solution (50 mM DTT, 5 mM EDTA) and 2 µl of Arg-C (100 µg/ml in 50 mM Tris-HCl (pH 8), 10 mM CaCl2, and 5 mM EDTA) were added, and the incubation was continued for 1 h at 34°C. For endoproteinase Asp-N, 1 µl of B31-tOspB was mixed with 14 µl of CB2 and 20 µl of 0.1 M sodium phosphate (pH 8) buffer. After 1 h at 34°C, 5 µl of Asp-N (40 µg/ml in 10 mM Tris-HCl, pH 7.5) was added, and the incubation was continued for 1 h at 34°C. For endoproteinase Glu-C, 1 µl of B31-tOspB was mixed with 14 µl of CB2, 1.4 µl of sterile water, 1.6 µl of 100 mM CaCl2, and 20 µl of 50 mM ammonium carbonate (pH 7.8) buffer. After 1 h at room temperature, 2 µl of Glu-C (100 µg/ml in sterile water) was added, and the incubation was continued for 4 h at room temperature. For the incubations containing no mAb, 14 µl of 0.02 M sodium phosphate (pH 7) buffer was added in place of the Ab.
Time-course studies
Time-course studies were performed with trypsin (modified enzyme) and Arg-C. For the trypsin study, 7 µl of B31-tOspB (8 mg/ml) was mixed with 21 µl of sterile water, 140 µl of 0.1 M Tris-HCl (pH 8) buffer with 8 mM CaCl2, and either 98 µl of CB2 (2.73 mg/ml) or 98 µl of 0.02 M sodium phosphate (pH 7) buffer. After 1 h at 34°C, 38 µl was removed from each and mixed with 2 µl of 1 mM HCl to serve as the zero point (no enzyme control), and then 12 µl of modified trypsin (100 µg/ml in 1 mM HCl) was added to each of the remaining 228-µl aliquots. These incubations were continued at 34°C, and 40-µl aliquots were removed after 5, 15, 30, 60, and 100 min. Immediately after sampling, the 40-µl aliquots were mixed with 3x SDS-PAGE sample buffer (20 µl) and heated for 3 min at 100°C. For the Arg-C study, 8 µl of B31-tOspB was mixed with 160 µl of 0.1 M Tris-HCl (pH 8) buffer with 8 mM CaCl2 and either 112 µl of CB2 (2.73 mg/ml) or 112 µl of 0.02 M sodium phosphate (pH 7) buffer. After 1 h at 34°C, 32 µl of activation solution (50 mM DTT and 5 mM EDTA) was added to each incubation. To serve as the zero time point, 39 µl was removed from each and mixed with 20 µl of 3x SDS-PAGE sample buffer. After heating for 3 min at 100°C, 2 µl of Arg-C (100 µg/ml in 50 mM Tris-HCl (pH 8), 10 mM CaCl2, and 5 mM EDTA) was added to each, and the tubes were reheated for 1 min at 100°C to ensure inactivation of the enzyme. A 14-µl aliquot of the same Arg-C stock was added to each of the remaining 273 µl, and these incubations were continued at 34°C. Aliquots (41 µl) were removed after 1, 2, 4, 8, 24, and 48 h; each was immediately mixed with 20 µl of 3x SDS-PAGE sample buffer and heated for 3 min at 100°C.
Dose-response studies
Dose-response studies were conducted to determine the effect of the dose of CB2 on tryptic digestion of B31- or BEP4-tOspBs. Aliquots (0, 0.9, 1.8, 3.5, 7, 14, and 28 µl) of a stock solution of CB2 (2.73 mg/ml) were taken, and each was diluted to a final volume of 28 µl with 0.02 M sodium phosphate (pH 7) buffer. To each was added 40 µl of 0.1 M Tris-HCl (pH 8) buffer with 8 mM CaCl2 and either 2 µl of B31-tOspB (8 mg/ml) and 6 µl of sterile water (B31 experiment) or 4 µl of BEP4-tOspB (4 mg/ml) and 4 µl of sterile water (BEP4 experiment). These were incubated for 1 h at 34°C to allow time for Ab to bind. Trypsin (4 µl of 0.1 mg/ml modified enzyme in 1 mM HCl) was added, and the incubations were continued for 1 h at 34°C. To stop digestion, 40 µl of 3x SDS-PAGE sample buffer was added to each tube, and the tubes were heated for 3 min at 100°C.
Tricine SDS-PAGE and Western blots
Tricine SDS-PAGE was conducted on 10% gels according to the method of Schägger and von Jagow (30). Minislab gels (8 x 9 x 0.1 cm) were cast with a 10-well comb, and samples (10 µl/well) were applied and run for 3 h and 40 min at 25 mA/gel. The gels were fixed in 50% methanol-12% acetic acid, stained with 0.1% Coomassie brilliant blue R-250 in 50% methanol-12% acetic acid, and destained in 50% methanol-12% acetic acid. The stained gels were scanned on a model GS700 imaging densitometer (Bio-Rad, Hercules, CA) to determine band densities (expressed as OD x mm). Prestained protein standards (BRL, Gaithersburg, MD) were run on each gel to allow estimation of m.w.s.
Proteins were electroblotted onto nitrocellulose (31), blocked in PBS containing 2% casein, incubated for 1 h each in primary Ab (2 µg/ml CB2 or 1/50 dilution of H4610 hybridoma culture supernatant) and secondary Ab (1/1000 dilution of a 0.2 mg/ml stock of alkaline phosphatase conjugated to goat anti-mouse IgG (Fisher, Pittsburgh, PA)), and reacted with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (Kirkegaard & Perry, Gaithersburg, MD).
N-terminal sequences
Sequence analysis was conducted on bands electroblotted in CAPS (pH 11) transfer buffer onto Immobilon PSQ polyvinylidene difluoride membrane (Millipore, Bedford, MA) and visualized with Coomassie brilliant blue R-250 (32). N-terminal sequences were determined on a model 477A protein sequencer (Applied Biosystems) operated at the Tufts University Core Facility, Tufts Medical Center (Boston, MA).
Size-exclusion chromatography
Native, undenatured tOspB from strain B31 was subjected to size-exclusion chromatography (AKTA chromatography system, Pharmacia, Piscataway, NJ) to determine its physical state before CB2 binding. Samples of the B31-tOspB (100 µl of an 8 mg/ml stock) and standard proteins were applied to a 1.0-cm x 30-cm column of Superdex 200 (Pharmacia) and eluted at a flow rate of 0.25 ml/min with 0.05 M Tris-HCl (pH 8) buffer containing 4 mM CaCl2 and 12.5 mM NaCl at 4°C. The standards used were thyroglobulin (670 kDa), catalase (232 kDa), BSA (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12 kDa). A linear relation between molecular size and elution volume was obtained by plotting log Mr vs [Ve - Vo]/[Vt -Vo], where Ve is the elution volume as determined from the peak absorbance at 280 nm, Vo is the void volume (Ve of blue dextran), and Vt is the total volume (Ve of the salts as determined from the peak conductivity reading).
Native PAGE
Native PAGE was conducted using the histidine/MOPS buffer system of Thomas and Hodes (33). This method allows separation of positively charged proteins in a discontinuous system at near-neutral pH. Minislab gels (8 x 9 x 0.1 cm) were cast with 4% stacking gel and 7.5, 10, or 12.5% separating gel. Ammonium persulfate and TEMED were used to initiate polymerization in separating gel (each 0.05%) and in stacking gel (each 0.08%). Glycerol (0.13 g/ml) was added to the separating gel before gelation. CaCl2 (4 mM) was added to both separating and stacking gels and to the electrophoresis buffer. Samples of the purified tOspB in 0.05 M Tris-HCl (pH 8) buffer with 4 mM CaCl2 and 12.5 mM NaCl were mixed with sample buffer (30% glycerol and 0.2 M MOPS-KOH, pH 8) in the ratio of 2 parts sample to 1 part sample buffer, loaded onto the gel (10 µl/well), and run for 3 h at 8 mA/gel with tap water cooling. Methylene blue (a tracking dye) was added to some of the samples to allow calculation of Rf values. Bands were visualized by staining with Coomassie brilliant blue R-250. Ferguson plots were constructed by plotting log Rf vs percent acrylamide (34).
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Results |
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As a first step to understanding how CB2 causes bacteriolysis of B. burgdorferi, we chose to study the initial interaction between CB2 and its Ag, OspB. Previous studies had shown that CB2 binds to OspB in the region of lysine 253, near the C terminus (16). Here, we sought to determine whether binding of CB2 to its epitope affected the tertiary structure of the OspB so as to make it more or less susceptible to cleavage by proteases.
Limited proteolysis has been employed to study the effect of ligand binding on protein conformation (35, 36, 37). However, ligands are typically low m.w. components that would not be expected to act as substrates of proteases, while CB2 could act as a potential substrate. Thus, for this approach to work, enzyme conditions needed to be found wherein OspB acted as a substrate while CB2 did not.
Truncated OspB (tOspB) that lacked N-terminal lipidation was employed in these studies to avoid the use of detergents. Recombinant tOspB prepared from strain B31 consisted of an N-terminal His tag followed by the OspB sequence of strain B31, beginning at residue 4 of the mature protein (predicted molecular mass, 32.2 kDa). This tOspB gave a single band on Tricine SDS-PAGE (apparent molecular mass, 33.8 kDa) and reacted on Western blots with CB2 and H4610, both specific for OspB (16, 17). Size-exclusion chromatography of the undenatured protein gave an apparent molecular mass of 45.5 kDa, indicating that the tOspB existed in the form of monomers. Overnight incubation of this tOspB with or without CB2 produced no breakdown products as determined by Tricine SDS-PAGE, indicating that the tOspB was not autolytic and that the CB2 was not catalytic against the tOspB, at least under the chosen reaction conditions.
Four proteases were tried as potential probes of tOspB structure.
Incubation conditions were found for each of these proteases wherein
the tOspB was cleaved (Fig. 1
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A–D, lane 3) while the mAb was not (Fig. 1, A–D, lane 6). Two of the proteases, trypsin and
Arg-C, produced a different pattern of cleavage, dependent on whether
the mAb was present in the incubation (Fig. 1, A and B,
lanes 3 and 4). The remaining two proteases,
Asp-N and Glu-C, produced the same pattern of cleavage regardless of
the absence or the presence of the mAb (Fig. 1, C and
D, lanes 3 and 4).
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Tryptic cleavage of B31-tOspB in the absence (Fig. 2A) or the presence (Fig. 2B) of CB2 produced three bands with
Mr of 32.2 kDa (band 2), 28.8 kDa
(band 3), and 28.1 kDa (band 4). In the absence of CB2 (Fig. 2A), the rate of cleavage of tOspB was approximately twice
that in the presence of CB2 (Fig. 2B). Band 2 disappeared
from the reaction without CB2 at approximately twice the rate as from
the reaction with CB2. Band 4 appeared in the reaction without CB2 at
approximately twice the rate as in the reaction with CB2. The amount of
band 3 present in the reaction without CB2 after 30 min (Fig. 2A) was about the same as the amount of band 3 present in
the reaction with CB2 after 15 min (Fig. 2B).
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), with two major products of 31.9 kDa
(band 2) and 28.8 kDa (band 3). Similar to the tryptic digestion, CB2
slowed the rate of cleavage of the tOspB by Arg-C. Near complete
cleavage of band 2 required 2 h in the absence of CB2 (Fig. 3A) and 8 h in the presence of CB2 (Fig. 3B). At 24 h, band 3 was present in the reaction with
CB2, but was essentially gone from the reaction without CB2. At 48
h, band 3 was present in the reaction with CB2 at approximately 50% of
the maximum level (data not shown).
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The sites cleaved in the B31-tOspB by trypsin and Arg-C in the
presence or the absence of CB2 were determined by N-terminal sequence
analysis of the electroblotted bands (Table I). For trypsin and Arg-C, the sites
cleaved were the same regardless of whether CB2 was present. In the
case of trypsin, bands 2 and 3 each gave two N-terminal sequences,
indicating that there were two cleavage products present in each band
(Table I). Therefore, trypsin cleaved the tOspB at five sites (Fig. 4: sites 2a, 2b, 3a, 3b, and 4), while
Arg-C cleaved the tOspB at 2 sites (Fig. 4, sites 2 and 3). Trypsin
site 2a and Arg-C site 2 were in the His tag sequence. The remaining
sites were within the body of the OspB, confined to a short stretch
near the N-terminus (Fig. 4).
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From the sequence data it was clear that the sites cleaved by trypsin and Arg-C were the same regardless of whether CB2 was present in the reaction. However, the time-course studies indicated that when CB2 was present in the reaction, the rate of cleavage was decreased. To rule out the possibility that this was a nonspecific effect of the mAb, we tested an irrelevant mAb of identical isotype (MOPC21, IgG1) and determined that it had no effect on the pattern or rate of cleavage of the tOspB by trypsin (data not shown).
CB2 exhibits dose dependence in its effect on the tryptic cleavage of tOspB
In the previous experiments, the incubation conditions were chosen
such that tOspB and CB2 were present in approximately equal molar
amounts as determined from the protein concentrations (see
Materials and Methods). However, if CB2 were to act on tOspB
through binding at its epitope (Fig. 4
), then one could expect the
maximal effect of the mAb at a ratio of 2 mol of tOspB/mol of mAb
(i.e., at saturation of all IgG binding sites). When we tested a series
of concentrations of CB2 and compared them with a control incubation
containing no Ab, we observed a maximal effect of the mAb at 0.12 mg/ml
of mAb (Fig. 5), corresponding to a ratio
of 8 mol of tOspB/mol of mAb. Because this ratio was calculated using
protein data collected in two different ways
(A280 spectrophotometry and Pierce MicroBCA
protein assay), band densities were determined for the tOspB and CB2
light chain by scanning densitometry, and the ratio of tOspB to mAb was
calculated for each experiment. According to these calculations (not
shown), the maximal effect of the mAb on the tryptic cleavage of tOspB
was observed at 10 mol of tOspB/mol of mAb. Thus, by two different
methods of calculation we have shown that at the point when the mAb was
maximally effective, the amount of CB2 present in the reaction could
bind only a fraction (<25%) of the total tOspB.
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To rule out the possibility that CB2 was acting directly on
trypsin rather than on tOspB, we compared the dose-response curve for
B31-tOspB with that of Bep4-tOspB. OspB from strain BEP4 differs from
OspB from strain B31 in that it contains threonine at position 253
instead of lysine and, thus, does not bind CB2 (16).
Recombinant tOspB from strain Bep4 was prepared in an identical manner
as B31-tOspB and was found to contain the lysine to threonine
substitution (see Materials and Methods). PAGE analysis of
the undenatured proteins indicated that the B31- and BEP4-tOspBs were
identical in size, as a plot of log Rf
vs percent acrylamide gave identical slopes for the two proteins (data
not shown). A comparison of the dose-response curves for the B31-tOspB
(Fig. 6A) and Bep4-tOspB (Fig. 6B) showed that while the mAb had some effect on the tryptic
cleavage of the Bep4-tOspB at the highest concentrations (Fig. 6B, lanes 6 and 7), at 0.12 mg/ml, the
concentration that showed maximal effect with the B31-tOspB (Fig. 6A, lane 4), there was very little effect with
the Bep4-tOspB (Fig. 6B, lane 4). This result
indicated that the mAb did not act directly against the trypsin, as it
would then have affected the tryptic cleavage of both tOspBs equally.
Furthermore, it showed that CB2 needed to bind to tOspB to elicit its
effect on tryptic cleavage.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Catalytic Abs that cleave peptide bonds have been described (38, 39), including human autoantibodies (40, 41, 42) and mouse mAbs (43). No breakdown products were detected when tOspB was incubated with CB2; thus, it appears that CB2 is not a catalytic Ab. However, for several of these catalytic Abs, the active site for catalysis is present only in the light chain (44, 45); thus, it is still possible that other experimental conditions could uncover less obvious catalytic activity of the mAb or its light chain.
Both trypsin and Arg-C produced different patterns of cleavage when CB2
was present in the reaction. Each enzyme cleaved the arginine in the
His tag sequence. Trypsin also cleaved four lysines within the OspB,
while Arg-C cleaved 1 lysine (Fig. 4). Typically, trypsin cleaves after
arginine and lysine, while endoproteinase Arg-C cleaves after arginine
only (32). Under rare circumstances, however, Arg-C has
been shown to cleave after lysine also (46, 47). With
synthetic substrates, Arg-C exhibits a specificity for arginine that is
100-1000 times greater than that for lysine (48). Thus,
the lysine cleaved by Arg-C in OspB is probably more labile (or more
exposed) than the lysines not cleaved by Arg-C (yet cleaved by
trypsin).
With trypsin and Arg-C, CB2 affected the rate of cleavage, but not the identity of the sites cleaved. In both cases, inclusion of CB2 in the reaction slowed the rate of cleavage. Irrelevant mAb did not have this effect, indicating that the effect was specific.
With Arg-C, cleavage of the lysine within the body of the OspB took
8 h when CB2 was present in the reaction and only 2 h when
CB2 was not (Fig. 3). The simplest interpretation of this result is
steric hindrance; that is, when the mAb was bound to tOspB it blocked
the enzyme (e.g., Arg-C) from reaching its substrate (in this case,
lysine) and therefore interfered with cleavage. A similar argument
could be made for the trypsin results. Yet, the presence of bound mAb
had no effect on the cleavage of tOspB by either Asp-N or Glu-C (Fig. 1, C and D).
If steric hindrance were to account for the reduced activity of the enzymes against tOspB, then the maximal effect should occur at saturation of all Ab binding sites, that is, 1 mol of mAb to 2 mol of tOspB, assuming that the tOspB was present in monomeric form (see below). With trypsin, the maximal effect was seen at a ratio of 1 mol of mAb to 8 mol of tOspB. Thus, at the point when the mAb was maximally effective, not enough CB2 was present in the reaction to bind all the tOspB. This argues against steric hindrance being the mechanism of action of the mAb.
As noted above, this argument depends on tOspB being present in monomeric form at the outset of the incubation with CB2. From the size-exclusion chromatography, the estimated size of the undenatured B31-tOspB was 45.5 kDa. However, the standards used to calibrate the column were typical globular proteins. OspB, which is 53% homologous to OspA (6), probably exists as a dumbbell-shaped protein (N- and C-terminal globular domains connected by a single-layer ß sheet) as has been shown for OspA (49, 50). Therefore, the size estimated for B31-tOspB using globular proteins as reference represents an overestimate. According to the DNA sequence, monomeric B31-tOspB should have an Mr of 32.2 kDa. Thus, at the outset of the incubation with CB2, B31-tOspB existed as monomers.
A second possibility, that CB2 was inhibiting the activity of trypsin
directly, was also considered. Even though the mAb was not cleaved by
the enzyme (Fig. 1A) and therefore did not appear to serve
as a substrate, it still may have bound trypsin and thus reduced the
effective concentration of the enzyme against tOspB. To address this,
we set up parallel incubations: one containing B31-tOspB and a second
containing BEP4-tOspB. These two tOspBs differed from each
other at 2 aa (Fig. 4); however, both tOspBs produced the
same pattern of cleavage when incubated with trypsin. If CB2 were to
act by removing trypsin from the reaction, then both tOspBs should give
the same result. In fact, the two tOspBs gave different results (Fig. 6). With B31-tOspB, CB2 showed a maximal effect at a ratio of 8 mol of
tOspB to 1 mol of mAb, whereas with BEP4-tOspB, CB2 showed very little
effect at the same molar ratio. From this, we concluded that CB2 was
not acting simply by interfering with the action of the enzyme. Rather,
CB2 was acting through tOspB.
Previous studies had shown that strain BEP4 was not susceptible to CB2-mediated bacteriolysis because its OspB contained threonine at position 253, instead of lysine, and so could not bind the mAb (16). We showed that CB2 did not detect BEP4-tOspB on Western blots, whereas another anti-OspB mAb (H4610), which binds to a different site in OspB, did detect BEP4-tOspB. Taken together, these results suggest that for CB2 to elicit an effect on the tryptic cleavage of B31-tOspB, it must bind tOspB. However, the maximal effect by CB2 was observed at a ratio of 8 mol of tOspB to 1 mol of mAb. At this molar ratio, insufficient amounts of CB2 were present to bind all the tOspB. Therefore, we suggest either 1) the mAb shows turnover; or 2) the mAb propagates its effect through producing a change in tOspB that is then further propagated by the tOspB-tOspB interaction, a form of activity that in some ways resembles that of prions (51, 52, 53). Regardless of the mechanism, these results showed that CB2 effected a change in B31-tOspB that could be measured by decreased susceptibility of tOspB to cleavage by trypsin or Arg-C.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Laura I. Katona, Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, NY 11794-5222. E-mail address:
3 Abbreviations used in this paper: Osp, outer surface protein; tOspB, truncated OspB; B31-tOspB, tOspB from strain B31; BEP4-tOspB, tOspB from strain BEP4; CB2, mAb CB2; H4610, mAb H4610; Arg-C, endoproteinase Arg-C; Asp-N, endoproteinase Asp-N; Glu-C, endoproteinase Glu-C; LB, Luria Bertani.
Received for publication September 3, 1999. Accepted for publication November 17, 1999.
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