(Received for publication, December 26, 1995; and in revised form, March 11, 1996)
From the
We previously isolated nucleic acid-binding antibody fragments (Fab) from bacteriophage display libraries representing the immunoglobulin repertoire of autoimmune mice to expedite the analysis of antibody-DNA recognition. In the present study, the binding properties of one such anti-DNA Fab, high affinity single-stranded (ss) DNA-binding Fab (DNA-1), were defined using equilibrium gel filtration and fluorescence titration. Results demonstrated that DNA-1 had a marked preference for oligo(dT) (100 nM dissociation constant) and required oligo(dT) >5 nucleotides in length. A detailed analysis of the involvement of the individual heavy chain (H) complementarity-determining regions (CDR) ensued using previously constructed HCDR transplantation mutants between DNA-1 and low affinity ssDNA-binding Fab (D5), a Fab that binds poorly to DNA (Calcutt, M. J. Komissarov, A. A., Marchbank, M. T., and Deutscher, S. L.(1996) Gene (Amst.) 168, 9-14). Circular dichroism studies indicated that the wild type and mutant Fab studied were of similar overall secondary structure and may contain similar combining site shapes. The conversion of D5 to a high affinity oligo(dT)-binding Fab occurred only in the presence of DNA-1 HCDR3. Results with site-specific mutants in HCDR1 further suggested a role of residue 33 in interaction with nucleic acid. The results of these studies are compared with previously published data on DNA-antibody recognition.
The presence of circulating antibodies that recognize DNA or RNA
is diagnostic of certain autoimmune diseases including systemic lupus
erythematosus(1, 2) . Anti-DNA antibodies are also
produced in autoimmune mice and have facilitated the study of systemic
lupus erythematosus and nucleic acid-protein interactions. The well
developed hybridoma technology for monoclonal antibody production has
made possible the production of numerous murine anti-DNA antibodies.
Consequently, there is substantial amino acid sequence information on
murine anti-DNA antibodies(3, 4, 5) ,
although significantly less is known about the exact mechanism of
binding and interactions between autoantibodies and DNA. Murine
monoclonal anti-DNA antibodies exhibit a high frequency of basic and
aromatic amino acid residues in the hypervariable or
complementarity-determining regions (CDR) ()contained in the
antigen binding fragment (Fab)(6, 7) . Mutagenesis
studies (8, 9) and three-dimensional structure
analyses of two Fab-oligo(dT) complexes (10, 11) have
substantiated a direct role of these residues in nucleic acid
interaction.
In general, the heavy chain (H) is thought to contribute more than the light chain to antigen binding, especially through HCDR3(12) . HCDR transplantation studies of an anti-fluorescein single chain antibody (SCA) 4-4-20 and an anti-ssDNA SCA BV04-01 have analyzed the roles of individual HCDR in combining site formation(13) . Data from the transplantation studies indicated that hybrids containing HCDR3 or HCDR1 of SCA BV04-01 bound oligo(dT). No binding occurred, however, when both HCDR1 and -3 of SCA BV04-01 were present in the context of HCDR2 of SCA BV04-01(13, 14) . While studies have demonstrated the importance of HCDR3 in binding nucleic acid(15, 16) , the roles of HCDR1 and -2 in this interaction are less clear.
We previously isolated ssDNA-binding Fab from combinatorial bacteriophage display libraries derived from the immunoglobulin repertoire of an autoimmune MRL/lpr mouse(17) . Immunological methods were used to characterize two of these Fab, in particular, a high affinity ssDNA-binding Fab (DNA-1) and a Fab (D5) that bound DNA poorly. DNA-1 and D5 were identical in genetic background and were very similar in amino acid sequence, suggesting the Fab may contain similar combining sites. All possible combinations of HCDR transplantation mutants (TM) were previously created between DNA-1 and D5 in order to ascertain the key domains involved in high affinity binding. Results of immunoprecipitation analyses with the HCDR TM suggested HCDR3 was most important in ssDNA binding(18) .
In order to gain a better understanding of DNA-1-oligo(dT) interactions, we undertook a thorough examination of the equilibrium binding properties of highly purified DNA-1, D5, HCDR TM, and mutant Fab containing site-specific amino acid substitutions. Fluorescence spectroscopy, as well as equilibrium gel filtration, was used to characterize the binding of Fab to oligo(dT). The results demonstrated that DNA-1 had a marked preference for oligo(dT) of at least 15 nucleotides and bound with a 1:1 stoichiometry. The HCDR transplantation studies showed that HCDR3 of DNA-1 plays a critical role in oligo(dT) binding and residues in HCDR1 may also affect this interaction.
VSX, a derivative of the plasmid pBC containing a
chloramphenicol-resistance gene (Stratagene) and lacking the VspI and SacI restriction enzyme sites, was used as
an intermediate vector for mutagenesis and sequencing procedures.
Site-directed mutagenesis was performed using splice overlap
PCR(20) . Typically, two fragments of DNA-1 or D5 H DNA in VSX
were amplified in two separate reactions using standard PCR conditions,
resulting in fragments overlapping by at least 10 nucleotides.
Full-length Fd (V + C
) products were
generated by annealing and extension of the two overlapping fragments
and were amplified by 25 cycles of PCR following the addition of
flanking T3 and T7 primers. The final products were purified, cut with XhoI (Boehringer Mannheim) and VspI (Life
Technologies, Inc.) restriction enzymes, and ligated into similarly cut
DNA-1- or D5-VSX plasmid. Having verified the desired mutation had been
introduced, an XhoI-BamHI fragment containing the
V
region was subcloned into XhoI-BamHI
cut DNA-1-pComb3 or D5-pComb3, thus replacing the wild type sequence.
Using this two-step procedure, only a 140-nucleotide fragment derived
from PCR was present in the final construct, thus reducing the amount
of DNA sequencing necessary to check for mutations caused by Taq DNA polymerase (Perkin-Elmer) errors. The correct mutation was
verified by DNA sequencing using a Sequenase 2.0 kit (U. S. Biochemical
Corp.). The relevant H amino acid sequences of DNA-1, D5, TM, as well
as site-specific mutants are shown in Fig. 1.
Figure 1:
Comparison of DNA-1 and D5 heavy
chain variable domains and design of mutants. A, comparison of
DNA-1 and D5 V. For the FR, only those residues that differ
between the two Fabs are shown. For CDR, all residues are shown. The bold amino acid residues represent the residues that were
changed as a result of site-directed mutagenesis. Numbers of the
residues for site-specific mutagenesis are indicated below the
sequences. The numbering system of Kabat et al.(33) was used to indicate the lengths of the FR and CDR. B, transplantation and site-specific mutants. The 1 or 5 in each box represents the origin of each CDR either
from DNA-1 or D5, respectively. For the last three Fab the name given
represents the original residue, its number, and the new residue
following site-specific mutagenesis. All Fab contained DNA-1 light
chain.
Figure 2: Poly(dT)-induced tryptophan fluorescence quenching of DNA-1. DNA-1 (200 nM; solid line) in TBS was excited at 292 nm, and fluorescence emission spectrum from 310 to 400 nm was collected. Saturating levels of poly(dT) (400 nM) were subsequently added, and quenched spectrum (broken line) was obtained. Spectra were corrected for Raman scattering.
The percentage of functional DNA-1 in the
preparations was determined by binding to oligo(dT)-Sepharose, in order
to accurately determine subsequent K values. All
of the Fab reversibly bound to this resin and eluted with 500 mM sodium phosphate, pH 7.0, suggesting that approximately 100% of
the purified Fab was functional.
Figure 3:
Fluorescence titrations of DNA-1 with
oligonucleotides of different length. The fractions of free DNA-1 were
calculated and displayed as a function of total concentration of
oligonucleotides. Titration curves for (dT) (
),
(dT)
(
), (dT)
(
), and
(dT)
(
) are shown. Calculated K
values are included in Table 2.
Figure 4:
Stoichiometry determination by
fluorescence titration and equilibrium gel filtration. A,
stoichiometric titration of 250 nM DNA-1 with increasing
amounts of (dT) (
) or (dT)
(
).
The thick line represents the average between the points. B, elution profiles for binding of DNA-1 with
P-labeled (dT)
. Solutions of DNA-1 (1
(
), 0.1 (
), and 0.01 µM (
)) were
preincubated 30 min at 4 °C with the same concentration of
P-labeled oligonucleotide in TBS plus BSA (0.2 mg/ml).
Solutions (150 µl) were applied to columns, equilibrated with the
same buffer, and eluted. Fractions (100 µl) were collected and
monitored for radioactivity. Data obtained from a series of experiments
were used for the generation of binding isotherms. The binding
parameters were calculated using a single binding site curve-fitting
procedure (Table 2). DNA-1 recovery (greater than 90%) and
elution volumes, for both free DNA-1 and oligonucleotides, were
determined in independent experiments.
The DNA-1-oligonucleotide complex was examined for ionic strength sensitivity using fluorescence titrations. The initial fluorescence signal was almost completely recovered with NaCl concentrations greater than 1 M, establishing that the changes in DNA-1 fluorescence observed upon the addition of oligo(dT) were the result of a reversible interaction (data not shown). The dissociation of the complex at high salt concentrations is also indicative that aggregation or precipitation had not occurred. The same effect probably led to elution of DNA-1 from oligo(dT)-Sepharose (mentioned previously).
Figure 5:
CD spectra of DNA-1, D5, and HCDR TM.
Spectra of proteins (0.2-0.8 mg/ml, 50 mM phosphate
buffer, pH 7.0) were obtained in the range of 205-240 nm at room
temperature and corrected for contributions of buffer components.
-, DNA-1; -
, D5; -, 1 1
5; - -, 5 5 1; - - -, 5 1 5;
, 5 1 1; -
-, 1 5 1; -
-
, 1 5 5.
Positively charged amino acid residues might also be
involved in the interaction with oligonucleotide at the Fab combining
site(8, 9) . D5 contains a Lys at position 31, in
place of a Ser at this position in DNA-1. In addition, the FR2
difference of an Arg at position 40 in D5, versus a Lys in
DNA-1, may also be of importance. Both mutations, K31S and R40K, were
created in TM 551 and DNA-1, respectively (Fig. 1). The mutant
Fab were purified and analyzed by fluorescence titration. Neither
substitution significantly altered the K or
maximal quenching values, suggesting differences at position 31 or 40
are not key for nucleic acid interaction (data not shown). These
results indicate that Trp-33 is solely responsible for the observed
increased quenching and K
difference.
We previously described the isolation and preliminary characterization of DNA-1, the first anti-nucleic acid Fab obtained from a combinatorial bacteriophage Fab display library generated from autoimmune sources(17) . In the present study, both fluorescence quenching and equilibrium gel filtration techniques were employed to evaluate the binding parameters of DNA-1 and TM under solution equilibrium conditions.
Fluorescence titrations were
performed with polynucleotides of different base composition to examine
the specificity of DNA-1-ligand interactions. The results demonstrated
that DNA-1 preferred poly(dT). A similar selectivity has also been
observed for the ssDNA binding proteins Pf3 (27) and
g5p(28) , and it was proposed that the affinity of these
interactions was inversely related to nucleotide stacking in the free
homopolymer. Thus, the K values of DNA-1 were less
for poly(dT) and poly(U) presumably because they have the most
unstacked structure.
Binding, as monitored by fluorescence quenching
titration, indicated the optimal ligand for DNA-1 Fab interaction was
approximately 15 nucleotides, although smaller oligo(dT) could bind
with reduced affinity. DNA-1 bound to oligo(dT) (n
15) with an affinity of 100-150 nM and a
stoichiometry of 1 mol of oligonucleotide per mol of Fab. As expected,
there was a significant decrease in the affinity of DNA-1 for oligo(dT)
in the presence of high salt concentrations. Thus, formation of the
DNA-1-oligonucleotide complex was reversible and not a result of
aggregation. The K
values and stoichiometry of the
complex obtained by fluorescence measurements were verified by
equilibrium gel filtration experiments. These results were similar to
those reported for anti-ssDNA Fab BV04-01, which had been shown
to contain a binding site size of 6-8 nucleotides and a K
of 450 nM for
(dT)
(29) . X-ray crystallographic data obtained for
a complex of BV04-01 with (dT)
led to the conclusion
that the antigen combining site was a shallow cleft(11) . DNA-1
and BV04-01, while varying in CDR amino acid sequence, had
similar binding properties for ligands of the same repetitive
structure, perhaps reflecting similarly shaped combining sites.
HCDR
TM were constructed between DNA-1 and D5, in order to examine the
importance of HCDR in ssDNA-binding. These Fab were chosen for HCDR
transplantation because they have different binding properties,
identical genetic background, very similar sequence, and perhaps,
similarly shaped combining sites. The suggestion that DNA-1 and D5
contain similarly shaped combining sites is supported by models of
numerous anti-DNA Fab derived from x-ray crystal studies (30) and from molecular modeling of DNA-1 and D5. ()Immunoprecipitation methods were used previously to
analyze the TM and results indicated numerous CDR combinations,
particularly those including HCDR3 of DNA-1, allowed for immune complex
precipitation. However, the TM did not show a strict specificity for
oligo(dT) in that other nucleic acids including U1RNA were also
recognized(18) . That additional components in the
immunoprecipitation reactions (i.e. anti-Fab antibody) may
have acted to artificially stabilize or destabilize the TM-nucleic acid
complexes remained to be determined.
In the present study, more direct methods of binding analysis, including fluorescence quenching and equilibrium gel filtration, were used to analyze the HCDR TM. Highly purified HCDR TMs were monitored by CD spectroscopy prior to equilibrium binding analyses. Results indicated there were no significant differences in CD spectra, indicative of the conservation of the secondary structure of the Fab. HCDR3 of DNA-1 in the context of all other HCDR combinations of D5 or DNA-1 resulted in the ability of the Fab to bind oligo(dT) with a similar affinity as that of native DNA-1. The TM did not bind well to U1RNA, in contrast to results with immunoprecipitation binding assays (18) . The HCDR1 and 2 of DNA-1 in the context of HCDR3 of D5 or DNA-1 did not drastically affect binding to oligo(dT), suggesting less involvement of these HCDRs in direct interaction. However, the transplantation of HCDR1 from D5 into DNA-1 was accompanied by more than a 50% increase in the maximal value of fluorescence quenching and was due to an additional Trp residue present in HCDR1 of D5. Taken together, the results of the transplantation experiments demonstrated a critical involvement of HCDR3 of DNA-1 in the direct binding of oligo(dT) and, furthermore, suggested the possible involvement of residue 33 of HCDR1 in complex formation.
These data are consistent with previous studies of HCDR3 grafting, which also illuminated the contribution of this region to Z-DNA (15) and dsDNA (16) binding. HCDR transplantation studies with an anti-fluorescein SCA 4-4-20 (pocket-shaped combining site) and the anti-ssDNA SCA BV04-01 (cleft-shaped combining site) resulted in binding with the presence of any two HCDR from the same SCA, tandemly arranged. Transplantation of HCDR1 from SCA 4-4-20 to SCA BV04-01 led to significant changes in CD spectra of the mutants, reflecting possible changes in their secondary structure. These results, and subsequent studies of the stability of SCA 4-4-20 and SCA BV04-01 TM to denaturation(31) , were explained from the point of view that there were drastic differences in the binding site shapes and charge distribution in the combining sites of the constructed TM.
Our results on HCDR transplantation were obtained with TM of similar sequence, secondary structure, and probable combining sites. Therefore, our data may be interpreted in terms of the retention of combining site conformation after CDR transplantation, suggesting the observed differences in oligo(dT) binding were probably due to the contribution of the individual HCDR in ligand interaction. X-ray crystallographic structural investigations are now in progress with liganded DNA-1 and should provide sufficient data to elucidate the combining site structure. A molecular model of DNA-1 has been constructed based on the crystal structure of the mouse monoclonal antibody R19.9, which contains very similar framework sequences and CDR lengths(32) . Analysis of the model indicates that Tyr-97 and Arg-98 (Fig. 1) are at the apex of HCDR3 of DNA-1 and are likely to be most crucial for DNA binding. The importance of the individual amino acids of DNA-1 HCDR3 in ligand interaction, however, awaits future mutagenesis and binding studies.