Antiidiotypic Antibody Recognizes an Amiloride Binding Domain within the alpha  Subunit of the Epithelial Na+ Channel*

Thomas Kieber-EmmonsDagger , Chaomei Lin§parallel , Mary H. Foster§, and Thomas R. Kleyman§**Dagger Dagger

From the Departments of § Medicine, Dagger  Pathology, and ** Physiology, University of Pennsylvania, and the  Veterans Affairs Medical Center, Philadelphia, Pennsylvania 19104

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We previously raised an antibody (RA6.3) by an antiidiotypic approach which was designed to be directed against an amiloride binding domain on the epithelial Na+ channel (ENaC). This antibody mimicked amiloride in that it inhibited transepithelial Na+ transport across A6 cell monolayers. RA6.3 recognized a 72-kDa polypeptide in A6 epithelia treated with tunicamycin, consistent with the size of nonglycosylated Xenopus laevis alpha ENaC. RA6.3 specifically recognized an amiloride binding domain within the alpha -subunit of mouse and bovine ENaC. The deduced amino acid sequence of RA6.3 was used to generate a three-dimensional model structure of the antibody. The combining site of RA6.3 was epitope mapped using a novel computer-based strategy. Organic residues that potentially interact with the RA6.3 combining site were identified by data base screening using the program LUDI. Selected residues docked to the antibody in a manner corresponding to the ordered linear array of amino acid residues within an amiloride binding domain on the alpha -subunit of ENaC. A synthetic peptide spanning this domain inhibited the binding of RA6.3 to alpha ENaC. This analysis provided a novel approach to develop models of antibody-antigen interaction as well as a molecular perspective of RA6.3 binding to an amiloride binding domain within alpha ENaC.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Epithelial Na+ channels (ENaCs)1 are expressed in a variety of tissues, including the distal nephron of the kidney, airway and alveolar epithelia in the lung, surface cells in the distal colon, urinary bladder epithelia, skin, and ducts within salivary and sweat glands (1). These transporters facilitate the movement of Na+ across the apical (or luminal) plasma membrane and have a critical role in extracellular fluid volume homeostasis, control of blood pressure, fetal lung maturation, and maintenance of airway fluids (1). ENaCs consist of at least three homologous subunits, termed alpha -, beta -, and gamma ENaC, and are thought to form a tetrameric complex consisting of 2alpha -, 1beta -, and 1gamma -subunits (2-5), although one group has suggested a subunit stoichiometry of 3alpha -, 3beta -, and 3gamma -subunits (6). cDNAs encoding these Na+ channel subunits have been isolated and characterized from a variety of species. Each ENaC subunit has two predicted membrane-spanning domains. The amino- and carboxyl-terminal regions of the ENaCs are cytoplasmic, and each subunit has a large ectodomain (7-9). Domains of largely hydrophobic residues are located immediately following the putative first membrane-spanning domains and immediately preceding the second membrane-spanning domains of alpha -, beta - and gamma rENaC (8, 10, 11). The hydrophobic domain immediately preceding the second membrane-spanning region of ENaC (referred to as the H2 domain) may insert in the membrane and form part of the channel pore (3, 7, 12).

The diuretic drug amiloride is a prototypic inhibitor of epithelial Na+ channels. Several sites have been identified within the epithelial Na+ channel which participate in amiloride binding. Mutagenesis studies suggest that residues preceding the second membrane-spanning domain (i.e. H2 domain) of alpha -, beta -, and gamma ENaC and residues within the second membrane-spanning (M2) domain of alpha ENaC participate in amiloride binding. These data provide strong evidence that amiloride interacts with residues within or in close proximity to the channel pore (3, 12, 13). We recently identified a 6-amino acid residue tract within the extracellular domain of the alpha -subunit of ENaC which participates in amiloride binding (14). The identification of this 6-residue tract was based on defining the interaction of an anti-amiloride antibody with amiloride and consequent sequence homology between the anti-amiloride antibody and a residue tract within the extracellular domain of the alpha ENaC (14-16).

Antibodies raised against antihormone (or antiligand) antibodies have been shown to bind the hormone's (or ligand's) physiologic receptor (17). These antiidiotypic antibodies, in effect, function as antireceptor antibodies. We previously generated a monoclonal antiidiotypic antibody (RA6.3) that mimics the effect of amiloride by inhibiting Na+ transport across A6 cell monolayers (18). The antibody also inhibits the amiloride-sensitive mechanoelectric channel of outer hair cells in adult cochlea.2 The antibody was used to immunoprecipitate putative Na+ channels from A6 cells and to immunolocalize putative Na+ channels in rabbit kidney and in a Xenopus epithelial (A6) cell line. However, the interaction of RA6.3 with cloned ENaC subunits has not been demonstrated.

We now provide evidence that RA6.3 recognizes the alpha -subunit of ENaC and is directed against a region within the extracellular domain of alpha ENaC containing the 6-residue amiloride binding domain we identified previously (14-16). Analysis of the topology of the antigen combining site of RA6.3 provided an opportunity to examine potential interactions of RA6.3 with an amiloride binding domain within alpha ENaC. We report the primary sequence of the variable regions of the light (L) and heavy (H) chains of RA6.3. A molecular structure of RA6.3 was created by homology modeling, and an epitope map of potential amino acid side chains that could interact with the RA6.3 combining site (15, 19, 20) was generated using a structure-based computer screening approach. This epitope map confirmed the potential interaction of an alpha ENaC amiloride binding domain with RA6.3. The theoretical epitope mapping approach may have role in examining protein-protein interactions in other systems.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Electrophoresis reagents were purchased from Bio-Rad and Tran35S-label from ICN (Costa Mesa, CA). All other reagents were purchased from Sigma and were reagent grade unless indicated otherwise.

Cell Culture-- A6 cells were derived from Xenopus laevis kidney. A subclone of this cell line (2F3) was a gift from Drs. J. P. Kraehenbuhl and B. C. Rossier (University of Lausanne). A6 cells between passages 92 and 96 were seeded on 4.7-cm2 polycarbonate filters from Costar (Cambridge, MA) at 106 cells/cm2 and maintained for 7-15 days in a humidified 28 °C incubator with 5% CO2 in a medium containing Leibovitz's medium L-15 (7 parts) and Coon's medium F-12 (3 parts), modified for amphibian cells by adjusting the final HCO3- concentration to 25 mM and the final osmolality of 230 mosmol/kg H2O, and supplemented with 5% (v/v) fetal calf serum (21). A6 cell monolayers were transferred to a modified Ussing chamber and bathed in a Ringer's saline containing [mM] 100 NaCl, 4 KCl, 2.5 NaHCO3, 1 K2HPO4, 1 CaCl2, 11 glucose, and buffered with HEPES. Electrical measurements were performed with a modified Ussing chamber and a DVC-1000 voltage clamp (World Precision Instruments) as described previously (21, 22). The amiloride-sensitive component of the short circuit current was determined by adding 10-5 M amiloride to the luminal solution. Data are reported as mean short circuit current ± S.E.

Metabolic Labeling and Immunoprecipitation-- Cells were incubated overnight (16 h) with A6 medium supplemented with 300 nM aldosterone without tunicamycin, or with either 0.5 or 5 µg/ml tunicamycin. Cells were washed three times with methionine, cysteine, and serum-free A6 medium supplemented with 300 nM aldosterone and 0, 0.5, or 5 µg/ml tunicamycin and then incubated for 15 min with 1 mCi/ml [35S]methionine/[35S]cysteine added to the basolateral surface as described previously (23, 24). Cells were then washed once and incubated for 1 h at 28 °C in A6 medium supplemented with 2.5 mM methionine, 2.5 mM cysteine, 300 nM aldosterone, and 0, 0.5, or 5 µg/ml tunicamycin. Immunoprecipitations were performed as described previously (18) with a 1:250 dilution of RA6.3 (raised in ascites) or control ascites (Sigma). The immunoprecipitated proteins were eluted into a SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (3% (w/v) SDS, 15% (w/v) sucrose and 92.5 mM Tris-HCl, pH 6.9). Samples were incubated with 10 mM dithiothreitol and boiled for 1 min prior to SDS-PAGE, which was performed as described previously (18).

In Vitro Translation, Immunoprecipitation, and Immunoblot-- Mouse and bovine alpha ENaC were transcribed and translated in vitro using the TNTTM-coupled reticulocyte lysate system (Promega, Madison, WI) according to the manufacturer's instructions. 20 µl of in vitro translated alpha mENaC was diluted to 0.5 ml of a buffer containing 0.4% (w/v) sodium deoxycholate, 1% (v/v) Nonidet P-40, 50 mM EGTA, 10 mM Tris-HCl, pH 7.4, containing a protease inhibitors mixture (10 µM phenylmethylsulfonyl fluoride, 1 µM antipain, 1 µM leupeptin, 1 µM pepstatin A). Solubilized proteins were preabsorbed for 1 h at 4 °C with 50 µl of protein G-agarose, centrifuged to pellet the protein A-agarose, and then incubated for 16 h with RA6.3 (1:500 dilution) in the absence or presence of an excess of the peptide DAVREWYRFHYINILSRLSD (20 µg) at 4 °C. Protein G-agarose (50 µl) was then added, and after a 1-h incubation at 4 °C, the agarose beads were washed three times with a buffer containing 150 mM NaCl, 5 mM EGTA, 1% (v/v) Triton X-100, 50 mM Tris, pH 7.4, and protease inhibitors and twice with a buffer containing 0.1% (w/v) SDS, 2 mM EGTA, 10 mM Tris-HCl, pH 7.4, and protease inhibitors. Precipitated samples were eluted into SDS-PAGE sample buffer (3% (w/v) SDS, 15% (w/v) sucrose, and 92.5 mM Tris-HCl, pH 6.9). Samples were incubated with 10 mM dithiothreitol for 20 min and subjected to 7.5% SDS-PAGE and autoradiography.

Alternatively, in vitro translated alpha bENaC (5 µl) was subjected to 7.5% SDS-PAGE, and proteins were transferred to nitrocellulose or polyvinylidene difluoride membranes as described previously (23, 24). Western blot analysis was performed using an alkaline phosphatase-linked chemiluminescent detection system (Western Light Plus) according to the manufacturer's instructions. In brief, nitrocellulose membranes were incubated in a blocking buffer consisting of 68 mM NaCl, 75 mM NaPO4, pH 7.4 (PBS), supplemented with 0.2% (w/v) purified casein, 0.1% (v/v) Tween 20, for 1 h at room temperature. Membranes were then incubated with blocking buffer containing either the antibody RA6.3 (1:500 dilution) in the absence or presence of the peptide DAVREWYRFHYINILSRLSD. Membranes were subsequently washed twice with PBS supplemented with 0.1% (v/v) Tween 20 and then incubated with a biotinylated anti-mouse IgG antibody (1:20,000 dilution in blocking buffer) at room temperature for 30 min. Membranes were washed twice in PBS and 0.1% Tween 20, incubated with a streptavidin-alkaline phosphatase conjugate (1:20,000 dilution in blocking buffer) at room temperature for 30 min, and washed again three times with PBS and Tween. The membranes were washed in assay buffer (0.1 M diethanolamine, 1 mM MgCl2, pH 10), incubated for 5 min in a substrate solution (0.25 mM CSPD in assay buffer) at room temperature, and then exposed to film.

Oligonucleotide Synthesis-- The mouse Cgamma primer was the 15-mer oligodeoxynucleotide 5'-GGCCAGTGGATAGAC-3'. The mouse Cgamma probe was 5'-TGGGG(CG)TGTTGTTTT-3'. The mouse Ckappa primer was 5'-CTGCTCACTGGATGGTGGGA-3', and the Ckappa probe was 5'-AGATGGATACAGTTGGT-3'. The anchor primer for polymerase chain reaction was a 33-mer oligodeoxynucleotide 5'-CACTGCAGAAGCTTGGATCCCCCCCCCCCCCCC-3' containing PstI, HindIII, and BamHI restriction sites. Probes were 5'-end radiolabeled with T4 polynucleotide kinase and [gamma -32P]ATP as described previously (25) and purified over NENSORB-20 cartridges (NEN Life Science Products).

Cloning and Sequencing of the Variable Region and H Chain of RA6.3-- The nucleotide sequence of the H region of RA6.3 was obtained using methods described previously (15). Briefly, total RNA was extracted from RA6.3 hybridoma cells cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum by the guanidinium/CsCl method as described previously. Reverse transcription was performed using RNA as a template with Moloney mouse leukemia virus reverse transcriptase and Cgamma oligonucleotide primer. Escherichia coli DNA polymerase I (26-28) was then used to synthesize double-stranded cDNA. T4 DNA polymerase was used to generate blunt ended cDNA, which was ligated into SmaI-digested and dephosphorylated M13. E. coli DH5-alpha F'IQ were transformed with recombinant phagemids and clones screened with the Cgamma (H chain) oligonucleotide probe (25). Positive plaques were purified and sequenced by incorporation of dideoxynucleotides using the Sequenase version 2.0 DNA sequencing kit. Nucleotide sequences were confirmed by sequencing a second independent clone.

Cloning and Sequencing of the Variable Region L Chain of RA6.3-- The method of random extension of cDNA ends was used to obtain the L chain variable region cDNA. Reverse transcription was performed using RNA from RA6.3 hybridoma cell as a template with Moloney mouse leukemia virus reverse transcriptase and Ckappa oligonucleotide primer at 37 °C for 1 h. An oligo(dG) tail was added to the cDNA with terminal deoxynucleotide transferase. A polymerase chain reaction was performed using poly(dC) and the Ckappa 3'-primer to amplify oligo(dG)-tailed cDNA. Samples were initially denatured at 92 °C for 8 min followed by 35 cycles of 92 °C for 1 min, 58 °C for 2 min, and 72 °C for 3 min and a final extension for 7 min at 72 °C. Products of the correct predicted size were cloned into a pCR vector (Invitrogen) and sequenced using the Ckappa oligonucleotide as a sequencing primer, as described above.

Model Building and Energy Refinement-- Antibody modeling was performed as described previously (15, 29-31). For modeling of the H chain complementarity determining region (CDR) 3 region in particular, we utilized both a knowledge-based approach to search for suitable structures that fit a geometry to the base residues of the CDR3 domain (32-34) and a conformational search procedure (35-38). The latter involved use of the program AbM (Oxford Molecular Inc.). CDRs and the framework regions of identified templates were mutated to those of the respective antibody H and L chains using Insight II (Biosym Technologies) or automatically assigned by AbM. The program Discover (version 2.95, Biosym Technologies) was used for conformational calculations with the supplied consistent valence force field parameters. The respective modeled structure was energy optimized to convergence. Molecular dynamics at 300 K and 600 K was used to alleviate any further close contacts within the antibodies (15, 29-31).

LUDI Search-- In the exploration of the potential epitope recognized by RA6.3, we made use of the program LUDI (39) (Ligand Design, Biosym Technologies) to identify amino acid residues that potentially interact with the RA6.3 combining site as described previously (20). A LUDI search was performed using standard default values and fragment library supplied with the program to identify organic fragments that potentially interact with residues within the RA6.3 binding site. Amino acid residues with side chains similar in structure to these organic fragments were identified. In evaluating these fragments, we compared amino acid residues identified by LUDI relative to the VRDWYRFH sequence such that the fragments could occupy nonredundant sites, be spatially far enough to accommodate the peptide backbone, and follow the appropriate ordered sequence of amino acid residues. In effect, one wants to "stitch" the fragments together to form a peptide that fits within the RA6.3 combining site. To identify possible fragments that are similar to the VRDWYRFH sequence, the radius of interaction, which defines the size of spheres in which LUDI is to fit appropriate fragments, was set as incrementing radii from 5 to 14 Å. The VRDWYRFH peptide was built using Insight II and positioned relative to the docked LUDI fragments. The peptide backbone and side chain torsional angles were rotated until the side chains of the peptide were approximate to the corresponding LUDI fragments. The peptide-RA6.3 complex was subjected to energy optimization and molecular dynamics simulations to relieve steric overlap.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RA6.3 Recognizes alpha ENaC-- RA6.3 is a monoclonal antibody raised by an antiidiotypic approach designed to be directed against an amiloride binding site within the epithelial Na+ channel (18). We demonstrated previously that RA6.3 immunoprecipitates a complex of polypeptides expressed at the apical plasma membrane of A6 epithelia. Cells were treated with tunicamycin to inhibit N-linked glycosylation in order to identify non-N-linked glycosylated polypeptide(s) recognized by RA6.3.

A6 cells were incubated overnight (16 h) in the presence (0.5 or 5 µg/ml) or absence of tunicamycin. Treatment of A6 cells with tunicamycin for 16 h led to a reduction in short circuit current from 12.4 µA × cm-2 (0 tunicamycin) to 0.7 µA × cm-2 (0.5 µg/ml tunicamycin) and 0.5 µA × cm-2 (5 µg/ml tunicamycin). The A6 monolayers were metabolically labeled in the absence or presence (0.5 or 5 µg/ml) of tunicamycin, detergent solubilized, and subjected to immunoprecipitation with RA6.3. The immunoprecipitate was analyzed by SDS-PAGE and autoradiography. In the absence of tunicamycin, RA6.3 immunoprecipitated a complex of polypeptides from metabolically labeled A6 cells, as we have observed previous (18, 40) (Fig. 1A, left panel). However, in the presence of 0.5 or 5 µg/ml tunicamycin, RA6.3 immunoprecipitated a 72-kDa polypeptide from A6 cells, consistent with the predicted size of nonglycosylated alpha xENaC (Fig. 1A, left panel) (41). This 72-kDa polypeptide was not immunoprecipitated when control ascites was used for immunoprecipitation (Fig. 1B). Interestingly, an additional polypeptide was immunoprecipitated with an apparent molecular mass of 35-45 kDa and is in agreement with the mass of a novel short chain alcohol dehydrogenase we cloned previously from an A6 cell expression library based on its binding to RA6.3 (42). Treatment of A6 epithelia with tunicamycin altered the profile of total labeled cellular proteins (Fig. 1A, right panel).


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Fig. 1.   RA6.3 recognizes a 72-kDa polypeptide in tunicamycin-treated A6 epithelia. A6 monolayers were incubated overnight with or without tunicamycin, metabolically labeled with [35S]methionine/[35S]cysteine for 15 min, and then chased for 1 h with an excess of unlabeled methionine/cysteine. The cells were detergent solubilized, subjected to immunoprecipitation with RA6.3, and labeled polypeptides were analyzed by a 5-13% gradient SDS-PAGE and autoradiography (panel A, left). Alternatively, cell lysate was subjected directly to SDS-PAGE and autoradiography to examine the profile of total metabolically labeled proteins (panel A, right). The concentrations of tunicamycin used (0, 0.5, or 5 µg/ml) are indicated at the top of the figure. A complex of polypeptides was recovered after immunoprecipitation with RA6.3 from metabolically labeled A6 cells that were not exposed to tunicamycin. A 72- and a 35-45-kDa polypeptide were recovered from cells treated with 0.5 or 5 µg/ml tunicamycin. The 72-kDa polypeptide is consistent with the predicted size of nonglycosylated Xenopus alpha ENaC. A change in the profile of total labeled A6 cell proteins was detected in cells treated with tunicamycin compared with control cells not exposed to tunicamycin (panel A, right). The migration of molecular mass standards is indicated to the left of the figure. The 72-kDa polypeptide was not recovered if nonimmune ascites (panel B, lane 2) was substituted for RA6.3 (panel B, lane 1) for immunoprecipitation.

To provide additional evidence that RA6.3 recognizes the alpha -subunit of ENaC, in vitro transcription/translation of mouse alpha ENaC was performed in the presence of [35S]methionine/[35S]cysteine. The translated product was analyzed by SDS-PAGE and autoradiography (Fig. 2A). A 72-kDa polypeptide was observed for mouse alpha ENaC, in agreement with its predicted molecular mass. Alternatively, the translation product was product subjected to immunoprecipitation with RA6.3 (1:500 dilution) in the absence or presence of the peptide DAVREWYRFHYINILSRLSD, corresponding to residues 273-292 of rat alpha ENaC, residues 273-292 within mouse alpha ENaC (with the exception that residue 291 in the mouse sequence is Pro), and residues 226-245 within bovine alpha ENaC (with the exception that residues 243-245 in the bovine sequence are RRQ). RA6.3 specifically immunoprecipitated in vitro translated mouse alpha ENaC (Fig. 2A). We also observed that RA6.3 specifically recognized in vitro translated bovine alpha ENaC (Fig. 2B).


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Fig. 2.   RA6.3 recognizes in vitro translated alpha ENaC. Panel A, mouse alpha ENaC cDNA was in vitro transcribed and translated in the presence of [35S]methionine/[35S]cysteine and subjected to 7.5% SDS-PAGE (lane 1). Alternatively, alpha mENaC was subjected to immunoprecipitation with RA6.3 in the absence (lane 2) or presence (lane 3) of the peptide DAVREWYRFHYINILSRLSD, which contains an amiloride binding domain (WYRFHY) that we have identified previously (14). RA6.3 specifically recognized in vitro translated alpha mENaC. This figure is representative of two separate experiments. Migration of molecular mass standards is indicated to the left of panel B. Bovine alpha ENaC cDNA was in vitro transcribed and translated in the presence of [35S]methionine/[35S]cysteine as described under "Experimental Procedures," subjected to 7.5% SDS-PAGE, transferred to nitrocellulose, and probed with RA6.3 in the absence (lane 1) or presence (lane 3) of a peptide corresponding to residues 273-292 of alpha rENaC and containing the tract WYRFHY. As a control, the blot was probed with mouse ascites (lane 2). A 75-kDa polypeptide was observed (arrow). RA6.3 specifically recognized in vitro translated alpha bENaC. This figure is representative of three separate experiments. Migration of molecular mass standards is indicated to the left of the figure.

Cloning and Sequencing of the L and H Chain cDNA of RA6.3-- RA6.3 H chain variable region cDNA was synthesized and introduced into M13. Individual subclones were isolated on the basis of hybridization to a mouse Cgamma probe. Sequence analysis of the clones revealed that the H chain is a member of the VH-36-60 gene family, subgroup I(A). RA6.3 L chain variable region cDNA was obtained by random extension of cDNA ends. The L chain is encoded by a member of the Vkappa 23 subgroup. The H chain and L chain cDNAs and deduced amino acid sequences are illustrated in Fig. 3.


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Fig. 3.   cDNA and deduced amino acid sequences of monoclonal antibody RA6.3 H chain and L chain variable regions. CDRs are underlined.

Structural Properties of RA6.3-- Sequence comparisons with known immunoglobulin crystal structures in the Brookhaven Protein Data Base provided templates for the variable regions of RA6.3 (Fig. 4). The primary structure of the L chain of the anti-lysozyme antibody (3HFM) in complex with lysozyme displayed 78% identity with the RA6.3 L chain through the CDR3 region (Fig. 4). For the H chain of RA6.3, we identified the anti-dinitrophenyl antibody (1BAF) also in complex with its ligand, as a template displaying 83% homology with RA6.3 up to CDR3. The H chain of 3HFM displays 81% homology with RA6.3 (Fig. 4), with a 1-residue deletion in the CDR1 region of 3HFM. Superpositioning these two templates up to CDR3 and accounting for the 1-residue deletion in 3HFM results in a root mean square deviation of 0.99 Å, indicating that these two templates are similar within their CDR1 and CDR2 domains and framework regions. These two templates for the RA6.3 L and H chain were also identified by the AbM program.


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Fig. 4.   Sequence comparisons of RA6.3 H chain and L chain with immunoglobulins of known crystal structure. Numbering of amino acid residues is according to Kabat et al. (68).

To model the CDR3 region of the RA6.3 H chain, we first examined CDR3 folding patterns of several immunoglobulins whose structures have been determined by crystallography. Based upon the sequence similarities between 1BAF with RA6.3, we utilized the 1BAF template and searched for loops in the Brookhaven Protein Data Bank using the program Insight II. Two potential structures found within antibodies were identified which differ in conformation but retain the same CDR3 length as RA6.3. These structures correspond to the lambda  dimer MCG (2MCG) and the antibody YST9.1 (1MAM) (Fig. 4). Both loop structures were spliced into the 1BAF template. A dynamics run was performed to relieve any short contacts in the modeled structures. Minimization of the average conformation from 100 structures for each model, derived from a molecular dynamics production run, resulted in average structures that differed in energy by 20 kcal, with the MCG spliced loop being the energetically preferred structure. As expected, the major difference in the structures of these two models was in the conformation of the CDR3 domain of the H chain. The low energy average conformation generated by the AbM program was similar to the MCG-derived conformer type. Its CDR2 region was different from the starting conformation of the 1BAF template (root mean square 1.2 Å). This structure was 6 kcal more stable than the MCG spliced structure. Consequently, both model structures (generated by the knowledge-based approach and by the AbM program) were used for screening of the LUDI fragments.

Peptide Placement within the RA6.3 Combining Site-- A point within the middle of the RA6.3 combining site was used to initiate the LUDI search. 101 small organic molecules were identified based on their ability to bind to the combining site within the RA6.3 structure generated by the knowledge-based approach, and 128 small organic molecules were identified based on binding to the AbM-generated structure. Most of the organic molecules that bound to the two RA6.3 structures were redundant for the same set of potential hydrogen bond donors or acceptors. Fig. 5, A and B, illustrates the placement of small organic fragments within the RA6.3 structure generated by the knowledge-based approach. Organic structures were observed to interact with both H and L chain amino acids within the various CDRs of RA6.3. A lipophilic group representative of a valine side chain was bounded by Leu-96 L chain (CDR3L) and Tyr-50 H chain (CDR2H) of RA6.3. A Trp-like residue was bounded by Tyr-50 L chain (CDR2L), and Ser-91 L chain (CDR3L), Asn-92 L chain (CDR3L) of RA6.3. A guanidinium head group formed hydrogen bonds with Glu-53 L chain within CDR2L of RA6.3. A Phe-like residue was bounded by Tyr-34 H chain and Tyr-50 H chain. A His-like group also interacted with Tyr-34 H chain and Tyr-50 H chain.


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Fig. 5.   Docking peptide fragments to the RA6.3 model: LUDI search. A variety of nonoverlapping molecular species were identified to fit within the RA6.3 combining site (panels A and B). Fragments identified by LUDI were compared with the side chains of amino acid residues within the sequence VRDWYRFH. Fragments were identified which occupied nonredundant sites and were spatially close enough to accommodate the peptide backbone (panel C). We have found that the best match involves the VRDWYRFH sequence such that the corresponding Val (position 1), Trp (position 4), Arg (position 6), and Phe (position 7) occupied the relative positions shown in panels C and D. The interaction of the peptide fragment with RA6.3 results in a favorable interaction such that the functional groups of the peptide are spatially similar to the organic fragments identified by the LUDI search for the structure generated by the knowledge-based approach. Panel D is a representative model of the VRDWYRFH peptide bound to RA6.3.

We examined whether organic molecules identified by LUDI were similar to the structure of the side chains and spatial order of the sequence of residues within the amiloride binding domain VRDWYRFH (Fig. 5C). As mentioned earlier, in effect one wants to stitch the fragments together to form a peptide. We modeled the VRDWYRFH peptide by two methods. First, individual amino acids were oriented with their side chains superimposed on the corresponding molecules identified by LUDI (Fig. 5C). The individual amino acid side chains were restrained, and then the residues were forced to form amide bonds (i.e. form the backbone structure). As expected, such an approach resulted in highly strained conformations. Alternatively, a peptide was built, and the phi , psi  angles rotated until the respective side chains were in close proximity to the molecules identified by LUDI. The VRDWYRFH sequence was modeled such that the corresponding Val (position 1), Trp (position 4), Arg (position 6), and Phe (position 7), approximated positions identified by the related LUDI fragments. The positioned peptide fragment-RA6.3 complex was then energy optimized with a restrained dynamics calculation such that the side chain positions were held approximate to the spatial positions of the LUDI-identified molecules. The complex was again energy optimized to convergence without the imposition of constraints (Fig. 5D). Deviation of the backbone conformation of RA6.3 bound to peptide relative to the unbound RA6.3 structure was found to be only 0.36 Å. This indicates that the placement of the peptide within the antibody combining site did not dramatically alter the overall conformation of the RA6.3 structure.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The generation of the monoclonal antibody RA6.3 was designed such that the antibody was directed against the combining site of anti-amiloride antibodies (such as BA7.1 (43)) and consequently against an amiloride binding domain on the epithelial Na+ channel (18). RA6.3 therefore functions as a molecular mimic of amiloride. Several lines of evidence support the hypothesis that RA6.3 recognized an amiloride binding domain within the alpha -subunit of ENaC. First, RA6.3 recognized a core, non-N-linked glycosylated polypeptide of 72 kDa in Xenopus A6 cells, consistent with the predicted core size of Xenopus alpha ENaC (Fig. 1) (41). Second, a 20-mer peptide containing the putative amiloride binding tract WYRFHY (14) inhibited the binding of RA6.3 to in vitro translated mouse and bovine alpha ENaC (Fig. 2). RA6.3 did not efficiently recognize mouse beta - or gamma ENaC (data not shown).

Previous studies have shown that blocking N-linked glycosylation with tunicamycin inhibits the amiloride-sensitive short circuit current in A6 epithelia and that this is a result of inhibition of functional Na+ channels (44). Our data are consistent with this observation in that tunicamycin both inhibits short circuit current and prevents the assembly of Na+ channels into an oligomeric complex recognized by RA6.3. In contract, N-linked glycosylation is not required for functional expression of alpha ENaC in Xenopus oocytes (9).

Work from several investigators supports the idea that amiloride interacts within the channel pore (45-50). A hydrophobic region (termed H2) preceding the second transmembrane domain of each subunit is thought to contribute to the conduction pore because selected mutations within the region alter cation selectivity or single channel conductance (3, 12, 13). Furthermore, mutations within these H2 regions alter amiloride binding characteristics (3, 12, 13). Selected organic cations other than amiloride, such as 2,4,6-triaminopyrimidine, triamterene, pentamidine, and trimethoprim, also function as epithelial Na+ channel inhibitors, although with Ki values much greater than that of amiloride (51-53). These data suggest that it is the charged guanidine moiety of amiloride and other organic cations which is interacting with the channel pore. Amiloride inhibits the Na+ channel with a submicromolar Ki, and structure-function analyses indicate that the substituted pyrazine ring of amiloride is required for high affinity interactions with the channel and likely has a critical role in stabilizing the binding of amiloride to the channel protein (54, 55).

We propose a model of amiloride binding to the Na+ channel based on the previous studies examining Na+ channel blockers and on the recent description of the tertiary structure of the Streptomyces KcsA K+ channel (56). The Na+ channel pore may be analogous in structure to the KcsA K+ channel that consists of a narrow selectivity filter facing the extracellular space and a wider pore cavity facing the intracellular space (56). Our proposed model of amiloride binding to the Na+ channel has two key elements. First, the charged acylguanidine moiety of amiloride interacts at or near the selectivity filter to block the channel; the carboxyl-terminal portions of the H2 domains of ENaC form the selectivity filter. Second, the substituted pyrazine ring of amiloride interacts with specific regions within ENaC ectodomains to stabilize the amiloride interaction with ENaC. We identified one site within the alpha -subunit ectodomain (residues 278-283) which participates in amiloride binding (14), based on sequence homology with an amiloride binding site on the anti-amiloride antibody BA7.1 (15). We reported that alpha -subunits lacking this 6-residue tract (alpha ENaCDelta 278-283) confer a loss of sensitivity to submicromolar concentrations of amiloride to Na+ channels composed solely of alpha -subunits or to channels composed of alpha -, beta - and gamma -subunits (14). Li et al. (57) have also provided evidence suggesting that the ectodomain of alpha ENaC participates in amiloride binding. In contrast, we reported that alpha -subunit channels formed by the mutant alpha ENaC H282D were insensitive to submicromolar concentrations of amiloride, although alpha H282D,beta ,gamma ENaC was inhibited by amiloride with a Ki similar to wild type alpha , beta ,gamma ENaC (3, 14). Based on these results, we propose that there are several sites within ENaC ectodomains, including residues 278-283 within alpha ENaC, which interact with the substituted pyrazine ring and stabilize the binding of amiloride to the channel.

The antibody RA6.3 was cloned and sequenced in order to build a structural model of this antibody and to explore potential interactions with alpha ENaC. A computer screening approach (LUDI) was used to identify potential amino acid residues that could theoretically bind to the RA6.3 combining site. Studies using this computerized screening strategy have been reported in the discovery of novel enzyme inhibitors. The use of this approach to design inhibitors of antibody-related protein-protein interactions remains a largely unexplored area with great potential (19, 20). A search for potential amino acid side chain structures that might interact with residues within the RA6.3 antigen binding site was performed using the program LUDI (Fig. 5, A and B). Fragments reflective of amino acid side chains within and flanking the putative amiloride binding domain (i.e. the tract WYRFHY) were observed to interact with the antibody in an ordered linear array, providing a basis to "dock" this peptide sequence to RA6.3 (Fig. 5, C and D).

Current procedures for predicting ligand-antibody interactions are limited, mainly because of the conformational flexibility of ligands and antibodies and the role of solvent in mediating ligand recognition and binding. Consequently, it was not our intent to elucidate the binding mode conformation of peptides that interact with RA6.3, but to validate the potential of the WYRFHY sequence to be recognized by RA6.3. It has been suggested that proteins may actually interact through small critical surface-binding epitopes as in the cases of the human growth hormone (58) and the erythropoietin receptor complexes (59). Such findings suggest that small binding epitopes may be sufficient for the effective blockade of large protein-protein interfaces.

In recent years, several reports on the crystal structures of antibody molecules, with and without bound antigen (for review, see Ref. 60) have provided insight into this process (61), which is described by the lock-and-key and induced fit mechanisms (62). Fabs whose structures have been solved in complex with a peptide have also been solved in their native or unbound form include B1I32 (63), Fab 50.1 (61), Fab 17/9 (64), 8F5 (65), SD6 (66), and 2H1 (67). In these cases, conformational changes in the antibody range from side chain rearrangements and small segmental movements in the CDR loops to a major rearrangement of the H3 loop in Fab 17/9 (64) as well as domain-domain rearrangements. The changes observed in the three-dimensional structure of RA6.3 on binding the 8-mer peptide sequence (VRDWYRFH) are slight. Hence, binding of this peptide to RA6.3 requires that the peptide antigen assume the proper conformation to fit in the antibody combining site, which is largely unchanged in this process. This is most likely the result of modeling RA6.3 from templates that have undergone transitions upon ligand binding. We have shown previously that there are conformational similarities among antibodies that have been solved in complex with ligands (20). In summary, computer screening provides a novel approach to develop models of antibody-antigen interaction and allowed us to generate a molecular perspective of RA6.3 binding to an amiloride binding domain within alpha ENaC.

    ACKNOWLEDGEMENTS

Bovine alpha ENaC was a generous gift from C. M. Fuller and D. J. Benos (University of Alabama at Birmingham). We thank Shaohu Sheng and Farhad Kosari for helpful discussions regarding models of amiloride binding to ENaC.

    FOOTNOTES

* This work was supported in part by a grant from the Department of Veterans Affairs and the National Institutes of Health Grant DK50268. This work was performed during the tenure of an Established Investigatorship Award from the American Heart Association (T. R. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF113618 (H chain) and AF113619 (L chain).

parallel Recipient of a postdoctoral fellowship award from the Cystic Fibrosis Foundation.

Dagger Dagger To whom correspondence should be addressed: Medical Research 151, VA Medical Center, University and Woodland Ave., Philadelphia, PA 19104. E-mail: kleyman{at}mail.med.upenn.edu.

2 C. C. Schulte, J. Meyer, D. N. Furness, C. M. Hackney, T. R. Kleyman, and A. W. Gummer, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: ENaC(s), epithelial Na+ channel(s); L chain, light chain; H chain, heavy chain; PAGE, polyacrylamide gel electrophoresis; CSPD, disodium 3-(4-methoxyspiro(1,2-dioxetane-3,2'-(5'-chloro)tricyclo)3.3.1.13,7)decan)4-yl)phenyl phosphate; CDR, complementarity determining region.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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