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Published online before print August 1, 2006, 10.1110/ps.062236806
Protein Science (2006), 15:2141-2148. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Effects of mutation at the D-JH junction on affinity, specificity, and idiotypy of anti-progesterone antibody DB3

Mingyue He1, Maureen Hamon1, Hong Liu1, Adam L. Corper2 and Michael J. Taussig1

1 Technology Research Group, The Babraham Institute, Cambridge CB2 4AT, United Kingdom
2 Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA

(RECEIVED March 22, 2006; FINAL REVISION June 2, 2006; ACCEPTED June 21, 2006)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
The crystal structures of the Fab' fragment of the anti-progesterone monoclonal antibody DB3 and its complexes with steroid haptens have shown that the D-JH junctional residue TrpH100 is a key contributor to binding site interactions with ligands. The indole group of TrpH100 also undergoes a significant conformational change between the bound and unliganded states, effectively opening and closing the combining site pocket. In order to explore the effect of substitutions at this position on steroid recognition, we have carried out mutagenesis on a construct encoding a three-domain single-chain fragment (VH/K) of DB3 expressed in Escherichia coli. TrpH100 was replaced by 13 different amino acids or deleted, and the functional and antigenic properties of the mutated fragments were analyzed. Most substitutions, including small, hydrophobic, hydrophilic, neutral, and negatively charged side chains, were reduced or abolished binding to free progesterone, although binding to progesterone-BSA was partially retained. The reduction in antigen binding was paralleled by alteration of the idiotype associated with the DB3 combining site. In contrast, the replacement of TrpH100 by Arg produced a mutant that retained wild-type antibody affinity and idiotype, but with altered specificity. Significant changes in this mutant included increased relative affinities of 104-fold for progesterone-3-carboxymethyloxime and 10-fold for aetiocholanolone. Our results demonstrate an essential role for the junctional residue H100 in determining steroid-binding specificity and combining site idiotype and show that these properties can be changed by a single amino acid substitution at this position.

Keywords: anti-progesterone; single-chain antibody fragment; idiotype; site-directed mutagenesis; antibody engineering


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
DB3 is one of a panel of mouse monoclonal antibodies (mAbs) raised against progesterone-11{alpha}-hemisuccinyl-BSA (Wright et al. 1982; Stura et al. 1987; Ellis et al. 1988). DB3 has a high affinity for progesterone (Ka = 109 M–1), but cross-reacts with some structurally different progesterone conjugates and derivatives (Fig. 1). X-ray crystallographic structures of the Fab' fragment, both unliganded and complexed with diverse steroids, indicate a key role for the heavy-chain variable region (VH) residue TrpH100, which is encoded at the D-JH junction and is part of the H-CDR3 loop (Fig. 2A) (Arevalo et al. 1993a, b, 1994). In the Fab'–progesterone complex, the D-ring and C20-21 methylketone group of the steroid are buried in a hydrophobic cavity formed mainly by the aromatic side chains of residues TrpH50, TyrH97, TrpH100, and PheH100b, while the steroid A-ring is close to the surface and bound between TrpH100 and ValL94. As well as making critical contacts to the ligand, the indole side chain of TrpH100 undergoes a significant conformational change of 5–6 Å between the bound and unliganded states of the combining site, effectively opening and closing the pocket, which may serve to stabilize the hydrophobic, unliganded steroid-binding area. DNA sequencing analysis of 12 anti-progesterone mAbs (including DB3) revealed that the structurally identified contact residues are almost always conserved, with the same VH and VL genes and similar CDR3 loops being expressed repeatedly; TrpH100 is present in 10 anti-progesterone mAbs and substituted by Tyr in two (Table 1).


Figure 1
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  		Figure 1. Structures of steroid ligands used in this study.

 


Figure 2
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  		Figure 2. Structure of the DB3 combining site. (A) Crystal structure of the complex between DB3 and progesterone (Arevalo et al. 1993a) showing some of the contact residues. Light and the heavy chains are in blue and purple, respectively; steroid oxygen atoms in red; steroid A-ring asterisked. (B) Model of site with TrpH100 replaced by arginine. (C) Superposition of A and B.

 


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  		Table 1. H-CDR3 sequences of 12 mouse monoclonal anti-progesterone antibodies

 
The DB3 combining site has been expressed in Escherichia coli as a three-domain, single-chain fragment (VH/K) in which the VH domain is linked to the complete {kappa} light chain, and which possesses similar binding characteristics to the parent antibody (He et al. 1995a). The functional expression of DB3 VH/K provides an efficient system for investigating the role of key contact residues in determining antibody specificity and affinity using site-directed mutagenesis. Here we report the effects of mutation at TrpH100 on DB3 affinity and ligand specificity. While most substitutions are deleterious both to binding and idiotype, replacement of Trp by Arg produced a progesterone-binding mutant with the same affinity as DB3, but altered specificity properties.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
Sequence conservation in anti-progesterone antibodies
Table 1 shows the H-CDR3 sequences in the VH domains among a panel of mouse anti-progesterone mAbs. It is evident that the steroid contact residues TyrH97, TrpH100, and PheH100b are highly conserved, or conservatively substituted, while the intervening residues, which are not directly involved in the ligand binding, show considerable variability.

E. coli expression of mutants
The mutations generated at heavy chain residue H100 were Tyr, Phe, Leu, Ile, Ala, Cys, Ser, Asp, Glu, Arg, Asn, Gln, Pro, Gly, and a deletion. Western blotting showed that the mutant proteins were secreted into the periplasm of E. coli at similar levels (Fig. 3) and ran as monomers under nonreducing conditions on SDS-PAGE (data not shown). The periplasmic extracts were used directly to analyze the properties of mutated VH/K fragments.


Figure 3
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  		Figure 3. Western blot of wild type and mutants of DB3 VH/K. Samples were run on 10% SDS-PAGE and electrotransferred to an Immobilon-P membrane (PVDF); the blot was probed with HRP-coupled sheep anti-mouse {kappa}.

 
Binding properties of mutants
Progesterone-BSA binding
Binding of VH/K mutants to progesterone-BSA in ELISA showed activity comparable to that of wild-type DB3 VH/K for the Tyr, Phe, Cys, and Arg mutants; the others were all weaker, with four- to eightfold reduction of the wild-type DB3 titer (Fig. 4A).


Figure 4
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  		Figure 4. Antigen-binding and idiotypic properties of periplasmic extracts of E. coli expressing VH/K mutants. (A) Binding activity by ELISA on progesterone-BSA coated wells; (B) Competitive inhibition of binding by free progesterone (100 ng/mL) in ELISA; (C) Competitive inhibition by rabbit polyclonal anti-DB3-idiotype (10 µg/mL). Results are means of duplicates. (d) Deletion; (Co.) negative control.

 
Inhibition by free progesterone
Binding of VH/K mutants to progesterone-BSA was performed in the presence of free progesterone as the competitive inhibitor. Whereas wild-type DB3 VH/K was 80% inhibited by 100 ng/mL of free progesterone, a similar inhibition was only obtained with the TrpH100Arg mutant (Fig. 4B). There was weak inhibition of the TrpH100Tyr mutant (10%), but none for the other mutants, indicating their inability to recognize free progesterone.

Inhibition by anti-idiotypic antibodies
The idiotypes of wild-type DB3 VH/K and mutants were compared by competitive ELISA using purified rabbit polyclonal anti-DB3-idiotype antibodies as inhibitors. With the anti-idiotype antibodies at 10 µg/mL, binding of wild-type DB3 and the TrpH100Arg mutant were both blocked by about 65% and the TrpH100Tyr mutant by about 50% (Fig. 4C). All other mutations at H100 led to major loss of the DB3 idiotype; titration showed a consistent reduction in relative affinity for anti-idiotype of about 100-fold.

Binding properties of the TrpH100Arg mutant
The affinity (Ka) of the DB3 TrpH100Arg mutant for progesterone, determined by equilibrium dialysis, was 0.9 x 109 M–1, the same as that of wild-type DB3 VH/K. Competitive inhibition ELISA using free progesterone as the inhibitor also confirmed the affinity (Fig. 5). Binding specificity of the mutant was tested in the presence of different free steroids and progesterone derivatives as inhibitors (Figs. 5, 6), measuring the IC50 for each (Table 2). In comparison with wild-type DB3 VH/K, a number of changes in specificity were observed. The relative affinity for aetiocholanolone was increased 10-fold, whereas that for 5beta-dihydroprogesterone was reduced by a similar amount (Fig. 5). The most striking change was in binding of progesterone-3-CMO (Fig. 6), where there was an increased relative affinity of more than 104-fold, as indicated by the decrease in IC50 from 10 µM to 0.5 nM. Binding to 5{alpha}-dihydroprogesterone and testosterone was unaltered.


Figure 5
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  		Figure 5. Steroid specificity of DB3 wild-type VH/K and TrpH100Arg mutant. Binding to progesterone-BSA of fixed amounts of wild-type DB3 VH/K (A) and the TrpH100Arg mutant (B) was carried out in the presence of free progesterone, aetiocholanolone, 5{alpha}-dihydroprogesterone, 5beta-dihydroprogesterone, or testosterone as inhibitors. Results are means of duplicates.

 


Figure 6
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  		Figure 6. Binding specificity of wild-type DB3 VH/K and the TrpH100Arg mutant for progesterone derivatives. Binding to progesterone-BSA of wild-type DB3 VH/K (A) and the TrpH100Arg mutant (B) was carried out in the presence of free progesterone-3-CMO (C3-CMO), progesterone-6-HMS (C6-HMS), progesterone-11{alpha}-HMS (C11{alpha}-HMS), or progesterone-21-HMS (C21-HMS). Results are means of duplicates.

 


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  		Table 2. Relative affinities (as IC50) of free steroids for wild-type DB3 and TrpH100Arg VH/K antibody fragments

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
Here we describe the effects of mutation of the contact residue TrpH100 on the combining site of the anti-progesterone antibody DB3. TrpH100 is the N-terminal (5') residue of the rearranged JH1 segment, i.e., the D-JH junctional residue. Despite the genetic potential for variability at this position, through use of different JH segments and alternative rearrangement positions, TrpH100 is conserved in 10 of 12 anti-progesterone mAbs sequenced and conservatively replaced as Tyr in two others (Table 1). The importance of the D-JH junctional residue in determining antibody affinity, either alone or combination with VH-D junctional residues, has also been found in anti-p-azophenylarsonate antibodies (Parhami-Seren and Margolies 1996). X-ray crystallographic analysis of DB3 has shown that TrpH100 makes a major contribution to steroid binding through several van der Waals contacts with the steroid A ring in the progesterone–DB3 complex (Fig. 2A) (Arevalo et al. 1993a), and to the B ring, D ring, and methyl groups in the aeticholanolone–DB3 complex, where the orientation of steroid is rotated by ~180° along the long axis (Arevalo et al. 1994). Interestingly, in the unliganded DB3 site, the indole side chain of TrpH100 is rotated to occupy partly the binding pocket and block ligand access. A dynamic equilibrium between the open and closed forms of the site is required to allow the steroid to bind. The role of TrpH100 in DB3 has been analyzed computationally by alanine scanning, confirming the large contribution to the ligand-binding free energy (Peräkylä and Nordman 2001).

These findings indicate that TrpH100 is a crucially important residue in DB3. To examine its role in more detail, we performed site-directed mutagenesis of H100 on a three-domain DB3 VH/K fragment, which is functionally expressed in the periplasm of E. coli (He et al. 1995a). Previous studies have shown that DB3 VH/K extracted from E. coli periplasm retains closely similar properties, with regard to affinity and specificity, to those of the intact antibody (He et al. 1995a,b). While wild-type DB3 VH/K bound well to both free progesterone and progesterone–BSA, most H100 mutants had no ability to bind free progesterone, but partially retained binding to progesterone-BSA (Fig. 4A). The distinction between recognition of free and carrier-conjugated progesterone was apparent even after substitution by other aromatic residues (Tyr, Phe). In both of the latter, progesterone-BSA binding was similar to that of wild-type DB3 VH/K in OD and titer, but inhibition by free progesterone was weak for TrpH100Tyr and undetectable for TrpH100Phe (Fig. 4B). This suggests that even those conservative substitutions alter the combining site sufficiently to prevent binding of free steroid, but the likely additional interactions conferred by the HMS linker and the putative BSA epitope enable binding to progesterone-BSA to occur. With the exception of TrpH100Arg, this pattern of dependence on the presence of the carrier for binding was repeated with most of the other mutants. Compared with wild-type DB3 VH/K, they generally showed only a four- to eightfold reduction in binding titer for progesterone–BSA, but no detectable binding of free progesterone by competitive inhibition ELISA or radioimmunoassay. A ranking of the activity of the mutants in terms of their binding titers against progesterone-BSA was as follows: Arg > (Tyr,Phe,Cys) > (Pro,Asn,Gln,Ser,Leu,Ile) > (Gly,Ala,Glu,Asp,del).

It is noteworthy that in two naturally occurring mAbs (11/32, 11/64) the presence of TyrH100 (Table 1) is associated with increased affinity for free progesterone. In both, the sequence difference results from use of the JH4 segment instead of JH1. In addition, they also have an altered contact at the base of the pocket, namely LeuH100b in place of PheH100b (apparently a result of somatic mutation), and changes in VL CDR3 (Leu instead of Pro at position 96, the VL-JL junction). These other changes may modify the effect of replacing Trp by Tyr at H100.

Most H100 mutations also led to alteration in the idiotype associated with the DB3 combining site, as shown by a reduced ability to inhibit progesterone-BSA binding with rabbit anti-DB3-idiotype antibodies (Fig. 4C). The idiotype is mostly specific to DB3, although the rabbit reagent shows a cross-reaction of up to 10% with other anti-progesterone antibodies encoded by the same VH and VL genes (Taussig et al. 1986). The decreased idiotypic reactivity of the mutants was approximately in line with that of progesterone-BSA binding, with no reduction for TrpH100Arg, 10% reactivity for TrpH100Tyr, and less for the other mutants. In contrast to the reduced binding activities of the other mutants, TrpH100Arg retained both the ability to bind free progesterone and expression of the native DB3 idiotype. This suggests that TrpH100 itself is not part of the idiotypic determinant, but rather that alterations which abolish binding of free steroid change the conformation of the CDR loops in such a way as to decrease idiotypic recognition.

Nevertheless, the TrpH100Arg mutant does show some significant changes in specificity of steroid binding from that of DB3 VH/K (Table 2). Particularly striking is an increased binding of progesterone-3-CMO of ~104-fold compared with wild-type DB3 VH/K. The preferential binding to progesterone-3-CMO was also demonstrated by phage display of the TrpH100Arg mutant, where enrichment was much better when panning on progesterone-3-CMO-BSA than on progesterone-11{alpha}-BSA (Chappel et al. 1998). The effect of the Arg substitution is to make progesterone-3-CMO the ligand with the highest affinity for the mutated DB3 site.

These results indicate that ArgH100 is capable of maintaining the conformation of the combining site and interacting with progesterone as effectively as Trp, but that fine specificity is altered by the mutation. Although chemically not a conservative replacement, functional substitution of Trp by Arg could be accounted for by their similarly large surface areas, the long hydrophobic Arg side chain which enables it to make extensive van der Waals interactions and the pseudo-aromatic properties of the guanidinium group (Richardson and Richardson 1989; Mian et al. 1991). Modeling shows that the long side chain of Arg at H100 could make van der Waals interaction with the steroid skeleton with good shape complementarity and that Arg could also maintain interactions with other parts of the DB3 V-regions (Fig. 2B,C). For example, in the DB3 Fab'–steroid complexes, TrpH100 is stacked between the steroid and TyrL32. For TrpH100Arg, {pi}–{pi} interaction between the guanidinium and TyrL32 could also occur, which may contribute to the stability of the mutant. The improved binding of progesterone-3-CMO could imply an interaction between the Arg residue and the CMO group, and indeed when modeled on the structure of DB3, the guanidinium group of Arg and the CMO group are in close proximity. Since the latter contains four potential hydrogen-bond acceptors and the guanidinium has five potential hydrogen bond donors (Borders et al. 1994), the increased affinity may result from the multiple H-bonding. The improved binding of aetiocholanolone is less explicable, being too small to make modeling meaningful.

A naturally occurring Arg residue at the D-JH junction occurs in two Diels-Alderase catalytic antibodies 39-A11 and 1E9. Interestingly, the H and L chains of 39-A11, 1E9, and DB3 are all derived from the highly restricted VGAM3.8 and Vk5.1 gene families (Romesberg et al. 1998; Xu et al. 1999). Structural analysis and comparison show that they share some of the same antigen contact residues (AsnH35, TrpH47, TrpH50, and ProL96) and superposition of either the H-CDR3 and L-CDR3 loops or the binding pockets of the three antibodies suggests that ArgH100 in 39-A11 and 1E9 may play a similar structural role to that of TrpH100 in DB3.

As in DB3, the crystal structure of the high-affinity anti-digoxin antibody 26–10 shows that TrpH100 is again a major contact residue for the steroid moiety (Jeffrey et al. 1993). Substitution of Trp with Arg at H100 by PCR mutagenesis generated an ArgH100 mutant that retained full antigen-binding activity (Burks et al. 1997). Phage-based selection from a randomized H-CDR3 library of 26–10 using digoxin analogs led in many cases to substitution of TrpH100 by Arg, with improved binding to the analog. It was also found that ArgH100 was a permissible and frequently replaced residue among the phage-selected mutants, whereas Tyr or Phe were not (Short et al. 2001). Interestingly, site-directed replacement of TrpH100 by Arg in 26–10 retained binding for digoxin, though with a sixfold reduced affinity, and altered the specificity of the mutant toward improved binding to digoxin analogs substituted at the C16 position (Krykbaev et al. 2001). The ArgH100 mutant was also used as the template for further phage display of mutations in CDR1 contact residues, leading to selection of restored wild-type specificity in a double mutant (Short et al. 2002).

In conclusion, our results confirm the importance of the D-JH junctional residue in determining affinity, specificity, and idiotypy in the anti-progesterone antibody system. They reinforce the results of sequence comparisons that indicate a high degree of conservation at this position and confirm the X-ray crystallographic analysis of DB3 showing the critical contribution of TrpH100 in the antigen binding. This study also reveals a role of TrpH100 in maintaining the idiotypy of the combining site, which may reflect CDR conformation. In our screen, the only amino acid that could functionally replace Trp was Arg. The high sensitivity of combining site properties to more conservative substitutions (Tyr, Phe) is surprising, while the effects of Arg substitution are consistent with the characteristics of this amino acid. These observations support others showing that single substitution in critical locations of the combining site can radically alter the antigen recognition properties of an antibody (Kussie et al. 1994).


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
Steroids and conjugates
The following were obtained from Steraloids, Inc.: progesterone, progesterone-3-carboxymethyloxime (CMO), progesterone-11{alpha}-hemisuccinate (HMS), progesterone-6-HMS, progesterone-21-HMS, 5{alpha}-dihydroprogesterone, 5beta-dihydroprogesterone, aetiocholanolone, and testosterone. Progesterone-11{alpha}-HMS-BSA (progesterone-BSA) was from Sigma.

Anti-idiotype antibodies
Rabbit polyclonal anti-DB3-idiotype was purified by removal of anti-immunoglobulin on a column of normal mouse immunoglobulin followed by absorption and elution from a DB3 column (Taussig et al. 1986).

Enzymes
Restriction enzymes (Northumbria Biological Labs), Taq polymerase (Boehringer Mannheim), and T4 DNA ligase (Northumbria Biological Labs) were used according to the suppliers’ recommendations.

Site-directed mutagenesis
Mutations at position H100 (Kabat numbering) were introduced into the DB3 VH/K construct by PCR using the megaprimer method (Sarker and Sommer 1990). A mutagenic oligonucleotide and two flanking oligonucleotides, containing restriction enzyme sites for XhoI and SacI, respectively, were used in sequential PCR. Initially, the SacI 3' primer (5'-CATCTGGAGCTCGGCCAGTGGATAGACAGATGG-3') and the mutagenic oligonucleotide (5'-TACGTCAACNNC/GTACTTCGAT-3') were used to generate a PCR fragment, which was then used as a primer for the second round of PCR with the XhoI 5' primer (5'-AGGTCCAGCTCGAGCAGTCTGG-3'). N is a mixture of the four nucleotides. The underlined sequences are sites for restriction digestion by SacI (GAGCTC) and XhoI (CTCGAG). The resulting PCR product was inserted into the DB3 VH/K construct as an XhoI–SacI fragment. Individual clones and mutations were identified by DNA sequencing.

Expression and analysis of mutants
E. coli expression of mutants of DB3 VH/K, preparation of soluble periplasmic extracts, and protein characterization were as described (He et al. 1995a). The expression level of VH/K fragments was analyzed by Western blotting, developed with horseradish peroxidase (HRP)-coupled anti-mouse {kappa} antibody (The Binding Site Co.), and ECL (Amersham), followed by densitometry of stained bands using a Joyce Loebel Chromoscan 3. Concentrations of VH/K mutants were adjusted to equivalence, by densitometry, for comparison of binding assays.

Progesterone-BSA binding ELISA
Ninety-six-well polystyrene microtiter plates were coated with progesterone-BSA, 3 µg/mL in phosphate-buffered saline (PBS) overnight at 4°C and blocked with 10% fetal calf serum for 2 h. Samples were titrated in the wells and incubated for 1–2 h at 4°C. After washing the plates with PBS containing 0.05% Tween 20 (pH 7.5), binding was detected with sheep anti-mouse {kappa} light chain conjugated to horseradish peroxidase (HRP) (The Binding Site Co.). Color was developed by addition of tetramethyl benzidine substrate (Sigma) and H2O2 in acetate buffer (pH6.0); optical density (OD) was read at 450 nm.

Competitive inhibition ELISA immunoassay
Fixed concentrations of native and mutated DB3 VH/K samples were mixed with either 100 ng/mL of free steroids, 10 µg/mL of rabbit anti-DB3 idiotype, or serial dilutions of these reagents from 10 pg/mL to 10 µg/mL. The mixtures were incubated on progesterone-BSA coated plates and binding developed as in the ELISA assay. Percent inhibition was calculated as 1 – (OD450 antibody with inhibitor)/(OD450 antibody without inhibitor) x 100%.

Equilibrium dialysis
An EMD 1/4 equilibrium microvolume dialyzer (Hoefer Scientific Instruments) fitted with a 6000–8000 Da cut-off membrane was used according to the supplier's instructions. Samples (200 µL) at constant concentration were loaded on one side of the membrane and 3H-progesterone (3.7–4.8 TBq/mM) (Amersham Life Sciences), with activity from 2000 to 300,000 dpm/200 mL of PBS gelatin, on the other. The dialyzer was rotated for 16 h at ambient temperature. 3H-progesterone on both sides of the membrane was determined by scintillation counting using Ultimagold liquid scintillation cocktail and a Tri-Carb2500TR liquid scintillation spectrometer. Bound and free progesterone were calculated and association constants determined from a Langmuir plot.


   Footnotes
 
Reprint requests to: Dr. Mingyue He or Dr. Michael J. Taussig, Technology Research Group, The Babraham Institute, Cambridge CB2 4AT, UK; e-mail: mingyue.he{at}bbsrc.ac.uk or mike.taussig{at}bbsrc.ac.uk; fax: +44-1223-496045.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062236806.

Abbreviations: Progesterone-BSA, progesterone-conjugated bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; CDR, complementarity determining region; H, antibody heavy chain.


   Acknowledgments
 
Research at the Babraham Institute is supported by the Biotechnology and Biological Sciences Research Council, UK.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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P. Verdino, C. Aldag, D. Hilvert, and I. A. Wilson
Closely related antibody receptors exploit fundamentally different strategies for steroid recognition
PNAS, August 19, 2008; 105(33): 11725 - 11730.
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