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The Journal of Immunology, 2001, 167: 5129-5135.
Copyright © 2001 by The American Association of Immunologists

Structural Analysis of Mutants of High-Affinity and Low-Affinity p-Azophenylarsonate-Specific Antibodies Generated by Alanine Scanning of Heavy Chain Complementarity-Determining Region 21

Behnaz Parhami-Seren2,*, Malini Viswanathan{dagger}, Roland K. Strong{ddagger} and Michael N. Margolies{dagger}

* Department of Biochemistry, College of Medicine, University of Vermont, Burlington, VT 05405; {dagger} Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129; and {ddagger} Fred Hutchinson Cancer Research Center, Seattle, WA 98104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Alanine scanning was used to determine the affinity contributions of 10 side chain amino acids (residues at position 50–60 inclusive) of H chain complementarity-determining region 2 (HCDR2) of the somatically mutated high-affinity anti-p-azophenylarsonate Ab, 36–71. Each mutated H chain gene was expressed in the context of mutated (36–71L) and the unmutated (36–65L) L chains to also assess the contribution of L chain mutations to affinity. Combined data from fluorescence quenching, direct binding, inhibition, and capture assays indicated that mutating H:Tyr50 and H:Tyr57 to Ala in the 36–71 H chain results in significant loss of binding with both mutated (36–71L) or unmutated (36–65L) L chain, although the decrease was more pronounced when unmutated L chain was used. All other HCDR2 mutations in 36–71 had minimal effect on Ab affinity when expressed with 36–71 L chain. However, in the context of unmutated L chain, of H:Gly54 to Ala resulted in significant loss of binding, while Abs containing Asn52 to Ala, Pro53 to Ala, or Ile58 to Ala mutation exhibited 4.3- to 7.1-fold reduced affinities. When alanine scanning was performed instead on certain HCDR2 residues of the germline-encoded (unmutated) 36–65 Ab and expressed with unmutated L chain as Fab in bacteria, these mutants exhibited affinities similar to or slightly higher than the wild-type 36–65. These findings indicate an important role of certain HCDR2 side chain residues on Ab affinity and the constraints imposed by L chain mutations in maintaining Ag binding.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Somatic mutation among p-azophenylarsonate (Ars)3 Abs in certain inbred mouse strains is a major source of Ab structural diversity (1, 2), which requires antigenic challenge for its induction. However, combinatorial and gene junctional diversity occurs during the Ag-independent (early) stages of differentiation (3).

The two A/J anti-Ars mAbs, 36–65 and 36–71 (4), share a dominant idiotype through the use of the same combination of VH, DH, JH, V{kappa}, and J{kappa} genes (1, 2, 5, 6, 7, 8). However, the affinity of 36–71 is 100- to 200-fold higher than 36–65 (9, 10). Two mechanisms have been shown to contribute to the higher affinity of 36–71 Ab: D gene junctional rearrangement and accumulation and selection of favorable somatic mutations in the H chain (11).

It was shown by Sharon et al. (9, 11) that when two contiguous residues in H chain complementarity-determining region (HCDR2) of the germline-encoded 36–65 Ab were mutated from Thr58 to Ile and Lys59 to Thr (similar to 36–71) (Fig. 1Go), Ab affinity increased 8-fold. The affinity of an Ab with an additional mutation at the DH-JH junction (Tyr107 to Lys in 36–65 HCDR3) (Fig. 1Go) was 240-fold higher than 36–65 wild type (wt) (9). It was also shown that, taken together, the 11 L chain mutations in 36–71 Ab do not affect Ab affinity, as expression of 36–71 H chain with either 36–71L or 36–65L resulted in Abs with indistinguishable affinity, despite the fact that several L chain mutations in 36–71 are in CDRs (Fig. 1Go) (11). Thus, high-affinity binding in 36–71 was associated with mutations in the H chain. However, in the crystal structure of several Abs, including 36–71 (12, 13), the involvement of L chain CDR residues in the VH-VL contact is significant, ranging from 26 to 57% of all atomic interactions (14). In addition, 28% of LCDR3 contacts are with HCDR2 (14).



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  		FIGURE 1. Amino acid sequence differences in H and L chain of 36–71 and 36–65 mAbs and mutant Abs. Sequential numbering is used. H chain amino acid residues of 36–71: Pro53, Gly54, Asn55, Gly56, Tyr57, Ile58, Thr59, Tyr60, Asn77, Glu100, and Lys107 correspond to Pro52a, Gly53, Asn54, Gly55, Tyr56, Ile57, Thr58, Tyr59, Asn76, Glu96, and Lys100c, respectively, using Kabat numbering (36 ) and as reported by Sharon et al. (9 ). Only the amino acid positions at which 36–65 and 36–71 H and L chains differ are shown.

 
One way to determine the role of HCDR2 residues in Ab affinity and in L chain interaction is to perform alanine scanning in which single Ala mutations, which eliminate the side chain beyond the {beta} carbon, are introduced to replace amino acids in HCDR2 of mutated 36–71 and unmutated 36–65 VH. The mutated H chains were expressed with either mutated or unmutated L chain to assess the role of each residue on affinity and interaction with L chain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

The production of the murine A/J hybridoma cell lines 36–65 and 36–71 ({gamma}1, {kappa}), and 36–65 and 36–71 transfectomas (IgG2b ({gamma}2b), {kappa}) and their H chain loss variants 36–65L and 36–71L were previously described (4, 11, 15). {gamma}2b 36–65 and {gamma}2b 36–71 cell lines are transfectomas that are the result of the transfection of the respective VH regions linked to {gamma}2b C region genes into their respective L chain-producing cell lines.

Synthesis and purification of hapten and hapten conjugates

Ars-N-acetyl-L-Tyr (Ars-Tyr) was prepared by a modification of the method described by Tabachnick and Sobotka (16), as described earlier (10).

Alanine-scanning mutagenesis and expression of mutant Abs in eukaryotic cells

Mutations were introduced into the cloned 36–71 VDJ gene in M13, as described (11). Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). The mutant genes were sequenced to confirm each mutation by the dideoxy sequencing method (sequenase kit; United States Biochemical, Cleveland, OH). Each mutated H chain gene was then subcloned in an expression vector constructed by Sompuram and Sharon (17). Mutant genes were transfected into L chain-producing hybridoma cell lines 36–65L and 36–71L by electroporation. Mutant Abs were selected using solid-phase ELISA, as reported (10, 18).

Alanine-scanning mutagenesis of 36–65 VH in pComb3 and Fab expression in bacteria

Cloning, expression, and characterization of 36–65 Fab (36–65 H chain (VH plus CH1) and L chain (V{kappa} plus C{kappa}) in the pComb3 vector (19)) were reported previously (20). This construct contained an engineered XhoI restriction site at amino acid position 42 of the 36–65 VH gene. Ala mutations were introduced in HCDR2 at positions 52, 54, 57, 58, and 59 separately using the mega primer method (21). Briefly, PCR fragments of 110 bp were generated using an antisense mutagenic primer and a sense primer that hybridized to amino acid positions 32–36 of the 36–65 VH gene. The PCR fragments (mega primer) generated by this method contained an XhoI site and were then used with a second antisense primer that hybridized to the N terminus of gene III to include the SpeI site in the PCR fragments. These PCR fragments (573 bp) that contained mutated V genes flanked by XhoI and SpeI sites were digested with the two restriction enzymes and were cloned into the XhoI-SpeI-digested pComb3 expression vector containing the complementary 36–65 H chain sequences and the 36–65 L chain V gene.

The mutated 36–65 VH genes in pComb-3 were electroporated separately into Escherichia coli XL-1 Blue cells. Nucleotide sequences of the selected phagemid clones were determined to confirm each mutation. Single colonies were grown overnight, and soluble Fab-gene III fusion proteins were purified as described below.

Cloning of 36–71 VH gene in pComb-3 and its expression in bacteria

The 36–71 VH gene from M13 (11) was amplified using a sense primer that contained a MunI restriction site and hybridized to the nucleotide sequences at the N terminus encoding aa 4–9 of the 36–71 H chain gene. The antisense primer contained a BseRI site and hybridized to sequences encoding aa 115–120 in JH2. The PCR fragment digested with MunI-BseRI was subcloned in MunI-SpeI-digested pComb-3 containing the complementary N-terminal amino acids, JH2-CH1 and the 36–65 L chain (20). Thus, the 36–71 VH gene was expressed with the 36–65 L chain. The nucleotide sequences of the full-length cloned VH gene were determined and found to be identical to the previously reported sequence of 36–71wt (9, 11). Recombinant 36–71H/65L Fab was purified and characterized as described below. Previously, we were unable to obtain the expression of this construct (22). Attempts to express the 36–71 VH gene with the 36–71 VL gene in bacteria were unsuccessful.

Ab purification

Transfectoma Abs or bacterial Fabs were purified from 1 L of culture medium by affinity chromatography on Ars-bovine {gamma}-globulin (BGG) coupled to Sepharose. Nonbinders were purified on goat anti-mouse Fab coupled to Sepharose (Zymed Laboratories, San Francisco, CA). Abs and Fabs were concentrated using Centriprep (30,000 or 10,000 m.w. cutoff for Abs and Fabs, respectively) (Amicon, Beverly, MA) and subjected to gel filtration using Ultrogel ACA34 columns (LKB Instruments, Bromma, Sweden) to separate monomers from aggregates (10).

Direct binding ELISA

Direct binding solid-phase ELISA was used to compare the binding pattern of site-directed mutant Abs, as described (10, 18). Wells of microtiter plates were coated by adding 25 µl of 5 µg/ml in PBS of Ars-BGG. Twenty-five microliters of affinity-purified Ab (0.075–20 µg/ml, 2-fold dilution) were added to each well. Binding was detected using HRP anti-mouse Ig.

Affinity determinations

Competition ELISA was used to determine the relative affinity of each Ab for Ars-Tyr (10). Inhibition of binding of Abs (concentration equivalent to 30–40% binding to Ars-BGG) was determined in the absence or presence of free Ars-Tyr (0.2–500 µM, 2-fold dilutions). Binding was detected using HRP anti-mouse Ig. Percent inhibition was calculated from the following formula: (OD450 in the presence of 1% BSA - OD450 in the presence of Ars-Tyr)/OD450 in the presence of 1% BSA) x 100. Relative affinity (IC50) is the µM concentration of Ars-Tyr that inhibits 50% of the binding of Abs to immobilized Ars-BGG.

Binding constants for Ars-Tyr were determined by fluorescence quenching using a Hitachi F-4500 spectrophotometer (Hitachi Instruments, San Jose, CA), as described (23). Control titration was conducted using an Ab that uses the same combination of VH and VL gene segments as 36–65 and 36–71 Abs, but due to a deleterious mutation in HCDR3, does not bind Ars-Tyr (Parhami-Seren, unpublished results). Ka was calculated using a curve-fitting program (11).

Capture assays

Capture assays were used to confirm Ab/Fab binding to Ars-BGG. Affinity-purified Abs/Fabs (100 µl, 10 µg/ml in PBS) were immobilized in the wells of microtiter plates overnight. Binding of 125I-labeled Ars-BGG (125I-Ars-BGG; 100 µl in 1% BSA/PBS, 40,000 cpm/4 ng), 125I-labeled goat anti-mouse {gamma}2b (for Abs), or 125I-labeled anti-mouse Fab (for bacterial Fabs) was measured after 2 h in a gamma counter (10).

Crystallographic refinement

The structure of the 36–71 Fab, 6FAB reported previously (12), was further refined against the original diffraction data set through cycles of rebuilding (using x fit) and positional and B factor refinement in X-PLOR (24, 25). The final R factor was 19.6% on all F > 2 {varsigma} in the 6- to 1.90-Å range. The R free was not calculated, as this was not a standard practice (26). Final model geometry is tight (rmsd bond lengths, 0.013 Å; rmsd bond angles, 1.911°). A total of 112 water molecules was included in the final model vs 70 in 6FAB. During re-refinement, aside from adjustments of several lysine and arginine side chains and the rotamer utilization of several residues, the conformation of several side chains was significantly altered, particularly Asp41 and Glu154 in the L chain, and Asn55, Tyr57, Tyr102, and Arg221 in the H chain. The register of residues 52–57 in the L chain was corrected. An additional residue was built onto the C terminus of the L chain, and the first and last residues of the H chain were deleted. The only alteration that potentially affects the interaction with hapten is H chain Tyr57. The updated coordinates have been deposited as Brookhaven Protein Data Bank file 1JFQ.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Alanine-scanning mutagenesis of 36–71 H chain expressed with 36–71 L chain; impact on affinity

To assess the contribution of each amino acid in the HCDR2 loop to Ab affinity, amino acid residues at positions 50–60, inclusive, of Ab 36–71, were mutated to Ala (Fig. 1Go). We included only residues 50–60 of HCDR2 based on the loop structure of 36–71, as deduced by analysis of the crystal structure of 36–71 Fab (12). Each H chain mutant gene shown in Fig. 1Go was subcloned in a {gamma}2b-gene-containing expression vector and introduced into 36–71 L chain-producing cells.

In direct binding assays of affinity-purified Abs immobilized on Ars-BGG, all 36–71 VH gene mutants that were expressed in the context of 36–71 L chain (36–71H/36–71L) bound Ars-BGG, except for the Ab with a Tyr50Ala.4 However, this Ab bound to anti-{gamma}2b-coated wells (data not shown), indicating that it was expressed and secreted (data not shown).

In fluorescence-quenching assays, the majority of the 36–71H/36–71L mutant Abs showed slight decrease (1.1- to 2-fold) in their affinities (Table IGo). As previously shown (11), mutation of Tyr to Ala at position 50 resulted in complete loss of binding of the Ab to Ars-Tyr, consistent with results of direct binding to Ars-BGG. Substitution of Ala for Tyr at position 57 resulted in a 5.8-fold decrease in affinity.


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  		Table I. Affinity (Ka) of 36–71 H chain mutants1

 
Impact of 36–65 L chain on affinity of 36–71 H chain Ala-scanning mutants

In direct binding ELISA to Ars-BGG, the binding pattern of 36–71 VH gene mutants expressed with an unmutated 36–65 L chain (36–71H/36–65L) was heterogeneous compared with the 36–71H/36–71L mutants. All Abs bound anti-{gamma}2b-coated wells to a similar degree, indicating that the heterogeneous binding to Ars-BGG is not due to significant differences in Ab concentration (data not shown). The mutant Abs Tyr50Ala, Pro53Ala, and Gly54Ala mutant Abs exhibited significant decreases in their binding to Ars-BGG. Table IGo shows the intrinsic affinity of each Ab for Ars-Tyr measured by fluorescence quenching. Affinities for 36–71H/36–65L Abs containing Tyr50Ala, Gly54Ala, or Tyr57Ala mutants were too low to measure; three other mutant Abs, Asn52Ala, Pro53Ala, and Ile58Ala, showed between 4.3- and 7.2-fold decrease in their affinity. The latter three H chain gene mutants did not show significant decreases in their affinities when expressed instead with 36–71 L chain (Table IGo).

Relative affinities (IC50) of 36–71H/36–71L and 36–71H/36–65L HCDR2 mutants

Competition ELISA was used to determine the relative affinity of each Ab for Ars-Tyr. In most cases, the results were consistent with the fluorescence-quenching assays (Table IGo). All 36–71H/36–71L mutant Abs exhibited relative affinities equal to or slightly lower than that of 36–71wt Ab (data not shown), with the exception of Tyr57Ala (Fig. 2Go). In inhibition assays, the IC50 of Tyr57Ala was 15-fold lower than that of 36–71wt Ab. The relative affinity of 36–65wt was 112-fold less than 36–71wt, consistent with fluorescence-quenching data (Table IGo). Because H chain Tyr50Ala/36–71L did not bind Ars-BGG, the IC50 could not be measured.



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  		FIGURE 2. Inhibition of binding of Abs to immobilized Ars-BGG by free Ars-Tyr. Abs (concentration equivalent to 30–40% binding to Ars-BGG) were incubated in the absence or presence of indicated concentrations of Ars-Tyr. Ab binding was determined using HRP anti-mouse Ig. Each point is a mean of duplicate determinations.

 
The relative affinity of 36–71H/36–65L mutant Abs, Asn52Ala, Pro53Ala, and Ile58Ala, was also measured in inhibition assays (data not shown). A decrease of 7.5- to 14-fold was observed for these Abs, consistent with the results of fluorescence quenching (Table IGo).

In general, expression of 36–71 H chain mutants with the 36–65 L chain resulted in more significant decreases in Ab affinity as compared with Abs in which 36–71 VH mutants were coexpressed with the homologous L chain (36–71).

Results of binding of 36–71H/36–71L and 36–71H/36–65L mutants in capture assays

Fluorescence quenching measures Ab affinity for free hapten Ars-Tyr, and the solid-phase inhibition assay measures the relative affinity for hapten-protein conjugate. While an affinity for Abs H chain 36–71 Tyr50Ala and Gly54Ala in 36–65L could not be measured by both fluorescence quenching (Table IGo) and competition ELISA, 36–71 H chain Tyr57Ala/36–65L had no measurable affinity in fluorescence quenching, but bound to Ars-BGG in direct binding assays, presumably via avidity effects in the solid-phase assay (data not shown). We used capture assays to test the ability of immobilized Abs to bind to soluble 125I-Ars-BGG. All 36–71H/36–71L mutant Abs could capture between 30 and 43% of the labeled probe, except for Tyr50Ala/36–71L, which showed 6% capturing ability indistinguishable from controls (data not shown). All the Abs in this group, including Tyr50Ala, bound 75% of 125I-labeled anti-{gamma}2b, indicating that the Abs were immobilized in the wells (data not shown).

In the group of 36–71H/36–65L mutant Abs (Fig. 3Go), more heterogeneity in binding was observed as compared with 36–71H/36–71L Abs. H chain Gly54Ala and Tyr57Ala mutants captured 1 and 5% of the labeled Ars-BGG, respectively, while Tyr50Ala and Pro53Ala captured 11–16% of the probe. All other Abs, including 36–65wt and 36–71wt, bound between 30 and 45% of 125I-Ars-BGG (Fig. 3Go). All Abs bound 75% of the 125I-labeled anti-{gamma}2b probe, indicating that Abs were immobilized (data not shown).



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  		FIGURE 3. Direct binding of 125I-Ars-BGG to immobilized affinity-purified transfectoma Abs. Transfectoma Abs (36–71H/36–65L) were immobilized in the wells of microtiter plates. Binding of 125I-Ars-BGG (40,000 cpm, 4 ng) to Abs was determined. Each point is a mean of three determinations.

 
Characterization of 36–65 HCDR2 alanine-scanning mutants coexpressed with 36–65 L chain in E. coli

To further examine the contribution of L chain to observed affinity variations, in 36–71H/36–65L Abs, Ala scanning was performed on selected residues in the 36–65 HCDR2. The mutated VH genes were expressed in bacteria with the homologous L chain (36–65L) as Fab. The mutations Asn52Ala, Gly54Ala, Tyr57Ala, Thr58Ala, and Lys59Ala did not affect direct binding to Ars-BGG of the 36–65 H chain-based mutant Fabs, as compared with 36–65wt (data not shown). Results of fluorescence quenching of these sets of mutant Fabs are shown in Table IIGo. All 36–65H/36–65L mutants exhibited affinities similar to or slightly higher (Lys59Ala) than that of 36–65wt. Fluorescence quenching for 36–65 Tyr57Ala/36–65L could not be measured, although 36–65 Tyr57Ala/36–65L Ab exhibited a direct binding pattern and relative affinity by competition ELISA comparable with 36–65wt Fab (data not shown).


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  		Table II. Affinity (Ka) of 36–65 H chain mutants1

 
To confirm the binding of the H chain 36–65 Tyr57Ala/36–65L mutant Fab, a capture assay was used. This mutant Fab could capture 125I-Ars-BGG to the same extent as 36–65wt (data not shown). Two different assays (fluorescence-quenching and capture assay) indicated that when residues Tyr50, Gly54, and Tyr57, in 36–71 H chain, were mutated to Ala and coexpressed with 36–65 L chain, all exhibited low binding affinity for hapten. In the context of the 36–71 L chain, however, only the 36–71 H chain Tyr50Ala Ab lost its ability to bind to Ars, and Tyr57Ala showed 6-fold reduced affinity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We probed the role of side chain residues in the HCDR2 loop of an anti-Ars Ab on affinity and interaction with the L chain using Ala-scanning mutagenesis. The high-affinity binding of 36–71 can be accounted for by two mutations in HCDR2 (Thr58Ile and Lys59Thr) and one difference at the H chain junctional amino acid (Tyr107Lys) compared with the germline-encoded Ab 36–65 (9, 11, 27), although the respective Abs differ by many other mutations (Fig. 1Go). A high rate of recurrent mutations at H chain positions 58 and 59 was observed among in vivo elicited secondary immune response anti-Ars Abs (9). Mutations at two other HCDR2 positions, 52 and 55, are also associated with small affinity increases (B. Parhami-Seren, unpublished data). Combined mutations at positions 52, 55, 58, and 59 of Ab 36–65 increased Ab affinity up to 5-fold, also indicating synergistic interactions among HCDR2 residues in enhancing affinity (28).

Because mutations in the HCDR2 loop are associated with increased affinity among canonical anti-Ars Abs, analysis of side chain residue identity is important for understanding the modulation of Ab affinity. In addition, because the L chain of 36–71 exhibits 11 somatic mutations as compared with the germline-encoded 36–65 L chain (Fig. 1Go), the effect of mutated and unmutated L chains on the affinity of HCDR2 site-directed mutant Abs was measured. Residues in L and H chains are commonly involved in a number of interactions across the domains (14). Analysis of these contacts in the crystal structure of 36–71 shows that residues in L chain CDR3 interact with both the H chain framework region 2 and HCDR2 (12).

The high-affinity Ab 36–71 was used for alanine scanning because its crystal structure was determined at 1.9 Å resolution (12), and a model of the interaction of Ars with the combining site was proposed (13).

The Tyr residue at position 50 was proposed to be a contact residue to phenyl-Ars (12, 17), and thus mutation in this position was expected to abolish Ab binding. This was demonstrated experimentally for both Abs 36–65 and 36–71 (10, 17). Mutation at H chain position 50 affected Ab affinity regardless of the identity of the L chain (Table IGo). In the 36–71 crystal structure, the phenyl ring of Tyr50 stacks against the phenyl ring of Ars. Moreover, the hydroxyl of Tyr50 makes a strong hydrogen bond with a water molecule that is in the binding cavity. The substitution of Ala for Tyr at this position would thus energetically be unfavorable to binding. Although the three-dimensional structure for 36–65 Ab is not available, previous experimental data employing mutagenesis of putative contact residues indicated that the overall binding site geometry is conserved among both 36–71 and 36–65 (10, 17).

Except for the Tyr50Ala mutation, the binding of the other 36–71H/36–71L mutant Abs was generally much less sensitive to Ala substitutions (Table IGo). The largest reduction in affinity observed is a 6-fold decrease for the H chain Tyr57Ala mutant. In the refined 36–71 crystal structure5 (Fig. 4Go), the phenyl ring of Tyr57 is at a distance of 4.4 Å from the hapten. It is thus potentially close to the hapten, but the interatomic distances in the case of 36–65 H chain are not known. Substitution of Tyr with Ala would remove the planar aromatic ring close to the hapten. This reduces the affinity, which is in agreement with the results observed. Consistent with this explanation is the significant reduction in the affinity of the 36–71 Tyr57Ala/36–65L Ab. However, the relative affinity of the 36–65 Tyr57Ala/36–65L was comparable with that of 36–65wt Ab in the avidity and capture assays and unmeasurable by fluorescence quenching (data not shown). This indicates a possible difference in the HCDR2 orientation and specific interactions with Ars, between germline (36–65) and mutated (36–71) Abs.



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  		FIGURE 4. Stereo view of the backbone of residues 50–60 in HCDR2 of anti-Ars Ab 36–71. The side chains of Tyr50 and Tyr57 are shown explicitly. The hapten phenyl-Ars is at the left. The apex of the HCDR2 loop is at the top.

 
It was previously shown (11) and also confirmed in the present study that the binding affinity of 36–71 H chain coexpressed with 36–65 L chain is similar to that of 36–71wt Ab (Table IGo). However, certain alanine-scanning mutants of 36–71 H chain that were coexpressed with 36–65 L chain exhibited very different binding affinities compared with 36–71H/36–65L Ab and 36–71wt (Table IGo). The most striking difference was the Gly54Ala mutation. H chain 36–71 Gly54Ala/36–65L had no measurable affinity by fluorescence quenching, inhibition assay, and capture assay (Fig. 3Go), while the affinity of the same mutated VH gene coexpressed with 36–71 L chain was not affected by the same H chain mutation (Table IGo). Similarly, the affinity of 36–65 Gly54Ala/65L Fab was not reduced (Table IIGo). From analysis of the crystal structure of 36–71, we cannot account for the effect of the Gly54Ala mutation. The backbone dihedral angle here is not specific for Gly, and hence could potentially tolerate a substitution. It could be that the packing between the two domains of the heterologous chains is different from that of the homologous chains due to changes in conformational freedom of the HCDR2 loop, and in the former case the flexibility of Gly is required for loop conformation.

It was previously shown that the identity of the L chain has a major effect on Ab affinity depending on the identity of the residue at H chain position 100 (11, 13). For example, when 36–65 H chain was mutated at four positions, Thr58Ile, Lys59Thr, Val100Glu, and Tyr107Lys, and coexpressed with 36–71 L chain, the affinity was 149-fold higher than 36–65wt. However, when the same mutant H chain gene was expressed with 36–65 L chain, the affinity was only 24-fold higher than 36–65wt Ab (11, 13). The results reported in this study indicate that certain mutations in HCDR2 of 36–71 cannot be tolerated in the context of an unmutated L chain.

Mutation of residues at positions 52, 53, 54, 57, and 58 to Ala, in the 36–71H/36–65L Ab, have significant effects on Ab binding. Since the affinity of 36–71H/36–71L and 36–65H/36–65L Abs with Asn52Ala, Gly54Ala, and Thr58Ala was similar to their parent Ab (36–71wt and 36–65wt, respectively) (Table IGo), it appears that the observed differences in affinities between 36–71H/36–71L and 36–71H/36–65L mutant Abs are due to somatic mutations in the L chain. From the crystal structure of 36–71, four of the L:H-contacting residues are mutated in 36–71, namely Phe32, Ile44, and Phe50 from L chain and Lys107 from H chain (Fig. 5Go). This could account for the differences observed in the affinity in 36–71H/36–65L and 36–71H/36–71L mutants.



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  		FIGURE 5. Crystal structure of 36–71 Ab5 (12 ). The L chain backbone is shown in green at the left, and the H chain in gold at the right. The side chain of residues that are mutated in 36–71 as compared with 36–65 is displayed in magenta (L chain) and blue (H chain). The image was produced using the programs MOLSCRIPT and RASTER3D (37 38 ).

 
The pattern of somatic mutations resulting in amino acid replacements in HCDR2 of in vivo elicited canonical anti-Ars Abs was analyzed. The analysis included amino acid sequences of H chain from 272 in vivo elicited secondary immune response hybridoma Abs (including endogenous, transgenic, and clonally related anti-Ars Abs). These Abs use the same combination of VH and VL genes as 36–65 and 36–71, for which sequence data are available in the region of HCDR2 (references for most VH sequences were listed previously in Refs. 12, 18 and 29, 30, 31, 32, 33 , and unpublished data of L. J. Wysocki, National Jewish Medical and Research Center, Denver, CO). H chain positions 50, 53, 54, and 57 are the most conserved residues (Fig. 6Go). Positions 51, 52, 55, 56, 58, and 59 may be mutated. This pattern of mutation is consistent with the mutagenesis data reported in this work in that the mutations of Tyr50, Pro53, Gly54, and Tyr57 to Ala have significant effects on Ab binding in the context of the 36–65 L chain. The amino acid residues at positions 50, 53, and 54 are replaced in vivo at a low rate (0.6–2%, within the error of sequencing). Among 272 Abs, five Abs showed amino acid substitutions at position 57, of which four were a change from Tyr to Phe, a conservative mutation that results in modest decrease in the affinity (10, 17). H chain residues at positions 51, 52, 55, 56, 58, and 59 were replaced at rates varying from 10 to 64%. The frequency of mutation at H chain position 60 was 3%. Position 60 is located in the base of the HCDR2 loop, as observed in the crystal structure, and would not be directly involved in binding to Ars.



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  		FIGURE 6. Amino acid replacements in HCDR2 of in vivo elicited secondary immune response anti-Ars Abs. HCDR2 amino acid sequences (positions 50–60) of 272 clonally related and unrelated anti-Ars Abs that use the canonical gene segment combination were examined. Percent replacement frequency = (number of observed replacements at each position/total number of Abs sequenced at that position) x 100.

 
The conservation of H chain residues at positions 50, 54, and 57 may indicate that clones carrying mutations at these positions would not be selected by Ag in germinal centers (34, 35). Thus, potentially certain L chain mutations may be deleterious in the context of otherwise favorable H chain mutations; thus, L chain sequence is not neutral, despite the similar binding of 36–71H/36–71L to 36–71H/36–65L.


   Acknowledgments
 
We thank Drs. Jacqueline Sharon and Seshi Sompuram of the Boston University Medical School (Boston, MA) for the eukaryotic expression vector, and Drs. Carlos Barbas III and Dennis Burton of The Scripps Research Institute (La Jolla, CA) for the pComb3 bacterial expression vector. Dr. Lawrence Wysocki of the National Jewish Hospital (Denver, CO) kindly tk;4provided unpublished sequences. We thank Lihua Zhang for excellent technical assistance.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants CA24432 and HL47415 (to M.N.M.) and FIRST Award AI33175 (to B.P.-S.). B.P.-S. was supported in part by National Institutes of Health Grant HL52282. Back

2 Address correspondence and reprint requests to Dr. Behnaz Parhami-Seren, Department of Biochemistry, College of Medicine, University of Vermont, Burlington, VT 05405-0068. E-mail address: bparhami{at}zoo.uvm.edu Back

3 Abbreviations used in this paper: Ars, p-azophenylarsonate; {gamma}2b, IgG2b; Ars-Tyr, Ars-N-acetyl-L-Tyr; BGG, bovine {gamma}-globulin; HCDR, H chain complementarity-determining region; 125I-Ars-BGG, 125I-labeled Ars-BGG; wt, wild type. Back

4 Mutations are denoted by the H chain position preceded by the wt residue in three-letter code, followed by the mutant residue. Back

5 Coordinates for the refined structure were deposited in the Brookhaven Protein Data Bank, Entry 1JFQ. Back

Received for publication April 24, 2001. Accepted for publication August 31, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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