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. Author manuscript; available in PMC: 2012 Jul 17.
Published in final edited form as: Biochim Biophys Acta. 2001 Oct 31;1521(1-3):135–140. doi: 10.1016/s0167-4781(01)00253-6

Cloning and sequence analysis of cDNAs encoding the heavy and light chain variable regions of an Ab2beta anti-idiotypic monoclonal antibody possessing an internal image of cocaine

Mitchell Ho 1,1, Mariangela Segre 1,*
PMCID: PMC3398996  NIHMSID: NIHMS388615  PMID: 11690646

Abstract

We report here the cloning and sequence analysis of cDNAs encoding the variable regions of an Ab2beta anti-idiotypic monoclonal antibody (K1-4c, gamma1kappa) that mimics the configuration of cocaine. The Ab2beta specifically binds to the human dopamine transporter as shown by confocal immunofluorescence microscopy. The sequence of the heavy chain complementarity-determining region 3 of K1-4c is strikingly similar to that of a monoclonal antibody (F11.2.32) specific for HIV-1 protease. Three or four amino acids in the epitope recognized by the anti-HIV-1 protease antibody are also present in the third extracellular loop of the dopamine transporter. This epitope is within the conserved region of the known transporters for dopamine, norepinephrine and serotonin in Homo sapiens, Rattus norvegicus, Caenorhabditis elegans and Drosophila melanogaster.

Keywords: Anti-idiotypic monoclonal antibody, Dopamine transporter, Cocaine, Nucleotide sequence

Cocaine addiction remains a major public health problem in industrialized countries. Up until now, efforts to find effective therapies for cocaine addiction have been unsuccessful. The addictive properties of cocaine are thought to result from inhibition of dopamine re-uptake by the dopamine transporter (DAT) in and around the synapses. By binding to the DAT, cocaine inhibits dopamine uptake and thus prolongs signaling at key brain synapses. Hydropathicity analysis of the DAT reveals a topology of 12 putative transmembrane domains (TMs) with intracellularly oriented amino and carboxyl termini and a large N-glycosylated extracellular loop between TMs 3 and 4 [1,2]. Despite intensive study, the precise molecular mechanism of dopamine transport and of its inhibition by cocaine remains unclear. Recent studies of the DAT demonstrated the existence of differential sites for dopamine translocation and cocaine binding [2–4]. However, the precise binding site(s) of cocaine on the DAT has not yet been determined.

We have generated anti-idiotypic monoclonal antibodies (Ab2β), possessing the internal image of cocaine in their antigen-combining site, that elicited a specific anti-cocaine antibody response when injected into mice [5]. Several of these Ab2β inhibited dopamine uptake at levels ranging from 40 to 90% of the inhibition given by cocaine itself when tested on our neuroblastoma cells stably expressing the human DAT (hDAT) [6,7]. The different levels of inhibition seen with the various Ab2β antibodies may reflect the different ways in which they mimic the cocaine molecule. In view of this, we propose that the Ab2β might be used as a powerful therapeutic tool against cocaine addiction since they compete with cocaine for its binding site but do not interfere with dopamine uptake as much as cocaine does [6]. To rule out the possibility of steric interference by the antibody molecule bound to the hDAT with the dopamine uptake process, we sought to clone the cDNA encoding the heavy (H) and light (L) chain variable (V) regions of our selected Ab2β and generate peptides derived from their antigen-combining sites.

The concept of internal image represents an essential element of the network theory formulated by Niels Jerne [8]. The idiotypic network is the inescapable consequence of the dual nature of the antibody molecule, which recognizes the antigen with its antigen-combining site (paratope) and is in turn recognized by other antibody molecules by virtue of its idiotypic determinants (idiotopes). When injected into an animal, an antibody generated in response to an antigen (Ab1) induces different sets of anti-idiotypic antibodies specific for its many idiotopes. One set of anti-idiotypic antibodies (Ab2β) recognizes the Ab1 paratope or a discrete portion of its antigen-combining site, and it is regarded as the “internal image” of the antigen. This Ab2βdue to its configuration which mimics the molecular configuration of the antigen, is capable of inducing Ab1-like Ab3 antibodies specific for the antigen, and of inhibiting the binding of the antigen to its specific antibody or Ab1. This antigenic mimicry does not necessarily depend on amino-acid sequence homology but involves similar binding interactions. In fact, antibodies can mimic molecules which are not proteins [9]. Erlanger’s group developed an anti-idiotypic antibody mimicking taxol, a non-protein molecule with a molecular weight (MW) of 853.9 Daltons (Da) [10]. We developed anti-idiotypic antibodies which mimic the configuration of cocaine (MW: 339.8 Da).

Previous data obtained using our Ab2β in dopamine uptake and inhibition assays strongly suggest the binding of Ab2β to the hDAT [7]. Here we confirm by confocal immunofluorescence microscopy (Fig.1) the direct binding of one Ab2β (K1-4c, D6) to mouse neuroblastoma N1E-115 cells stably expressing the hDAT. The 6C6 cell line used in this study was obtained by single cell subcloning [11] of the hDAT-transfected N1E-115 cell line generated in our laboratory [7]. Using a competitive inhibition enzyme-linked immunosorbent assay (ELISA), we ascertained that the binding of K1-4c to the hDAT was inhibited by soluble cocaine or cocaine-bovine albumin (BSA) as well as by the Ab1 anti-cocaine antibody (K1-4) used to generate the Ab2βK1-4c); it was not inhibited by BSA or by an irrelevant antibody (anti-OVA) (Ho and Segre, unpublished data). These results demonstrated that the antigen-combining site of the Ab2β bound to the DAT at the site of cocaine binding. The affinity constant (Ka = 7.0 × 106 M−1) of K1-4c for the hDAT was determined by Scatchard plot [12] following the protocol described by Bator and Reading [13] with the only modification of using papain-digested K1-4c Fab fragments containing only one antigen-combining site instead of the whole antibody molecule. We then cloned the cDNA fragments which encode the variable regions of heavy (VH) and light (VL) chains of K1-4c and determined their nucleotide sequences. We compared the deduced amino acid sequences with those of germline V segments and with the most homologous monoclonal antibodies known to date.

Fig. 1.

Fig. 1

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Confocal immunofluorescence microscopy of Ab2β binding to hDAT-expressing neuronal cells. Cells from the mouse neuroblastoma N1E-115 cell line, stably expressing hDAT, (Clone 6C6) were fixed with 1% glutaraldehyde (A) and incubated with a cocaine anti-idiotypic (Ab2β) monoclonal antibody (K1-4c) (B) or an irrelevant monoclonal antibody (anti-OVA) as a control (C). The cells were then incubated with PE (phycoerythrin R)-conjugated anti-mouse IgG, diluted 1:100. Confocal images were generated on an Olympus FluoView 300 confocal laser scanning system with an Olympus BX50 microscope. Bar, 50 μm.

A total mRNA fraction was isolated from 1 × 106 K1-4c hybridoma cells using the Quick Prep Micro mRNA Purification Kit (Amersham Pharmacia Biotech). Complementary DNA was produced from mRNA using First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech) and random hexanucleotide primers. Genes coding for the variable domains were amplified from cDNA by polymerase chain reaction (PCR) using a thermal cycler (MJ Research). MVH1-5’ (5’-CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC-3’) and MVH2-3’ (5’-GGAAAGCTTATAGACAGATGGGGGTGTCGTTTTGGC-3’) were used as primers for VH corresponding to the mouse heavy chain framework 1 (FR1) region and γ1 constant region according to Kabat database (January 3, 2001 Dataset, http://immuno.bme.nwu.edu/) [14,15] and Wang et al. [16]. Similarly, MVL1-5’ (5’-GGGAATTCGAYATTGTGMTSACMCARWCTMCA-3’) and MVL2-3’ (5’-GGTAAGCTTGGATACAGTTGGTGCAGCATC-3’) were used as primers for VL corresponding to the mouse light chain FR1 and constant region. Restriction sites, which are underlined, are EcoRI and HindIII. Standard abbreviations have been used for mixed sites: K, G or T; M, A or C; N, A, C, G or T; S, C or G; R, A or G; V, A, C or G; W, A or T; Y, C or T. The cycling program started with 94°C for 10 min and continued with 30 cycles of the following thermocycling conditions: 94°C for 1 min, 45°C for 1 min and 72°C for 2 min per cycle (Taq DNA polymerase, Life Technologies). The amplified gene fragments were purified on 1% (w/v) agarose and converted to blunt and phosphorylated forms. Following a brief heat activation step, DNA fragments approximately 400bp containing VH and VL genes were cloned into the EcoRV sites of pETBlue-2 (Novagen) to yield pET.K1-4c.VH.1 and pET.K1-4c.VL.1, respectively. The K1-4c VH and VL inserts were sequenced using a 5’-primer (pETBlue2Up, 5’-GTCACGACGTTGTAAAACGACGGCC-3’) by the High Throughput Sequencing and Genotyping Unit at the Center for Comparative and Functional Genomics at the University of Illinois. Sequence analyses were done using the Biology Workbench 3.2 utility (http://biology.ncsa.uiuc.edu/), BLAST 2.0 Server (http://www.ncbi.nlm.nih.gov:80/BLAST/), the Wisconsin Package (Genetics Computer Group, Madison, WI) and Protein Explorer (http://www.umass.edu/microbio/chime/explorer/). The nucleotide sequences of the VH and VL and their deduced amino acid sequences are shown in Figs. 2 and 4. Comparison of the nucleotide sequences of the K1-4c heavy and light chain-specific cDNAs with the immunoglobulin genes listed in the Genbank database showed that the sequences have not been reported previously. The sequences of the VH and VL of Ab2β K1-4c (D6) have been assigned the GenBank accession numbers AY028960 and AY028961, respectively.

Fig. 2.

Fig. 2

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Nucleotide and deduced amino acid sequences of the variable region of the heavy chain of K1-4c anti-idiotypic (Ab2β) monoclonal antibody. A dash indicates an exact match between residues; a dot indicates a gap added to the sequence. CDRs 1, 2, 3 and JH4 are shown. The sequence has been assigned GenBank accession number AY028960.

Fig. 4.

Fig. 4

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Nucleotide and deduced amino acid sequences of the variable region of the light chain of K1-4c anti-idiotypic (Ab2β) monoclonal antibody. A dash indicates an exact match between residues; a dot indicates a gap added to the sequence. CDRs 1, 2, 3 and Jκ5 are shown. The sequence has been assigned GenBank accession number AY028961.

The sequence analysis using the Kabat database (January 3, 2001 Dataset, http://immuno.bme.nwu.edu/) [14,15] revealed that the VH of K1-4c belonged to the VH XIV family, subgroup IIID. The complementarity-determining regions (CDR) were assigned using the IMGT database (http://imgt.cines.fr:8104/) [17] . The heavy chain CDR1 (aa 26 – 33), CDR2 (aa 51 – 57) and CDR3 (96 – 103) are located as shown in Fig. 2. Comparison of the VH with mouse germline VH sequences showed that the nucleotide sequence was most homologous to VH283 (GenBank accession number X00163) [18]. A total of 23 nucleotides of VH283 were changed in AY028960 (VH) by possible somatic mutations; 12 amino acid residue changes at aa 3, 5, 31, 33, 50, 52, 56(2), 74, 76, 91, and 96 (2 in CDR1, 3 in CDR2 and 1 in CDR3 partial sequence of VH283). Interestingly, the CDR3 of the VH bears striking homology to a monoclonal antibody (F11.2.32, PDB entry 2HRPH, GenBank accession number 2780903)[19] that recognizes HIV-1 protease (Fig. 2 & 3). It should be noted that the CDR3 of F11.2.32, which represents the antigen-combining site of F11.2.32, has been assigned to include some residues in the diversity (D) and joining (J) gene segments: RSGGIERYDGTYYVMDY [19]. For K1-4c, the corresponding sequence is I-GGITTVWGTYYVMDY. Like F11.2.32 [19], K1-4c recruits the almost identical D (except Trp instead of Asp at aa 104) and the identical long JH4 gene segments (GenBank accession number V00770) [14, 20] possibly to form a large protruding loop for the specificity of the antigen-combining site (Fig. 3). In fact, it has been proposed that in the anti-idiotypic network the D region may contain the internal image of the antigen [21]. Like F11.2.32, the K1-4c antigen-combining site may include several aromatic side chains due to the presence of three tyrosine residues (at aa 58, 107 and 108) and one tryptophane residue at aa 104. As observed in F11.2.32 [19], the hypervariable region of K1-4c may possibly form a hydrophobic cavity. Interestingly, the amino acid sequence Thr-Leu-Pro-Gly of the dopamine transporter located on the third extracellular loop between TMs 5 and 6 and close to TM 5 according to the current topological model [2] is almost identical to the epitope Ser-Leu-Pro-Gly amino acid residues (P37–40) on the 10 amino-acid peptide (PDB entry 2HRPP, GenBank accession number 2780904) recognized by anti-HIV-1 protease F11.2.32 monoclonal antibody. Analysis of chimeric dopamine-norepinephrine transporters demonstrated that the regions from N-terminal through the first five TMs were essential for dopamine translocation, whereas cocaine binding was associated with TMs 6–8 [4]. In that report, Giros et al. postulated that the third extracellular loop between TMs 5 and 6 and the fourth extracellular loop between TMs 7 and 8 were the possible regions of cocaine binding. A recent study [2] of several DAT TM polar mutants revealed that substitution of the polar residue Thr (T285A) in the Thr-Leu-Pro-Gly close to TM5 reduced a cocaine analog CFT [(−)-2-beta-carbomethoxy-3-beta-(4-fluorophenyl)tropane] binding affinity to the DAT by more than 50% and produced a CFT/Dopamine affinity ratio of less than 0.5. It is well documented that the transporters for norepinephrine and serotonin, which have 60 – 70 percent amino acid sequence homology with the DAT, also serve as cocaine recognition sites and their function are also inhibited by cocaine. These transporters and the DAT are encoded by the same gene family. Sequence alignments of the known transporters for dopamine, norepinephrine and serotonin in Homo sapiens (GenBank accession numbers AAG33844, P23975, and X70697, respectively), Rattus norvegicus (GenBank accession numbers I59558, CAA73665, and X63253, respectively), Caenorhabditis elegans (GenBank accession number X19158 for the DAT) and Drosophila melanogaster (GenBank accession number AAF76882 for the DAT and GenBank accession number U04809 for the serotonin transporter) revealed that the amino acid sequence Thr-Leu-Pro-Gly or Ser-Leu-Pro-Gly (for the Drosophila serotonin transporter) was conserved in all of these transporter proteins.

Fig. 3.

Fig. 3

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Structure alignment of the VH of K1-4c and of F11.2.32. The three-dimensional structure alignment was done using the MSA3D program in Advanced Protein Explorer. Within the variable region of the heavy chain (H), green indicates exact match between residues; light blue indicates similar match; yellow indicates difference; red indicates mismatch or insufficient sequence data. CDR 1, 2, 3: complementarity-determining region 1, 2, 3; P: peptide shown here in turquoise (PDB entry 2HRPP). Light chain can be seen in dark blue in the background.

The VL of K1-4c belonged to the Vκ IV family, subgroup II. The light chain CDR1 (aa 27 – 36), CDR2 (aa 55 – 57) and CDR3 (94 – 101) are located as shown in Fig. 4. Comparison of the VL with mouse germline Vκ sequences showed that the nucleotide sequence was most homologous to K1A5 (GenBank accession number D00081) [22]. A total of 10 nucleotides of K1A5 appear to have been changed in AY028961 (VL) by possible somatic mutations, resulting in 6 amino acid residue difference at aa 2, 3, 4, 7, 33, and 90. The amino acid sequence of the light chain CDR regions, except for one amino acid (aa 33: Thr instead of Asn) in CDR1, was identical to the sequence of the germline segment K1A5. The J gene segment of VL is identical to mouse germ line Jκ5 gene segment (GeneBank accession number V00777) [23]. Since anti-idiotypic response may depend on selection of non-germ line sequences [24], it is unlikely that the light chain of K1-4c significantly contributes to the specificity of this antibody. It is worth noting that many antibodies of discrete specificities, such as anti-GAT, anti-DNP, anti-flagellin, anti-phosphorylcholine, anti-digoxin, anti-phenyloxazolone, and anti-DNA, used almost the same Vκ subgroup in association with various heavy chains [22]. Random pairing of a given Vκ chain with distinct heavy chains would probably favor an escape from selection in evolution, especially in view of the possible role of the idiotypic network acting as a “built-in” selection pressure [22].

The above observations suggest that the amino acid(s) (Ser/Thr-Leu-Pro-Gly) in the three or four positions close to TM5 of the DAT may contribute to the binding of cocaine. Further studies by point mutation or peptide mapping will be needed to confirm this prediction. We are currently generating the peptides from the antigen-combining region of K1-4c. We will examine whether indeed these peptides inhibit cocaine binding to the DAT without interfering with dopamine uptake. If this is so, these peptides may be used as novel “cocaine antagonist” peptides to study the interaction of cocaine and the dopamine transporter as well as potential therapeutic anti-cocaine addiction agents.

Acknowledgments

We thank Dr. Gary Olsen in Department of Microbiology for helpful advice in the analysis of sequence data, Lou Ann Miller in Center for Microscopic Imaging for assistance with microscopy photographs, and Dr. Elizabeth Greeley for critical reading of the manuscript. This work was supported in part by grant DA10367 from the National Institutes of Health.

Abbreviations

aa

amino acid(s)

bp

base pair(s)

cDNA

DNA complementary to RNA

CDR

complementarity-determining region(s)

DAT

dopamine transporter

hDAT

human dopamine transporter

ELISA

enzyme-linked immunosorbent assay

FR

framework region

H

heavy chain

HIV

human immunodeficiency virus

Ig

immunoglobulin

L

light chain

MW

molecular weight

nt

nucleotide(s)

PCR

polymerase chain reaction

TM

transmembrane domain

V

variable region

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