School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX and 2 Celltech Therapeutics Ltd, 216 Bath Road, Slough, Berkshire SL1 4EN, UK
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: conserved binding site motif/polyamines/recombinant anti-polyamine antibodies
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
At present, high-performance liquid chromatography (HPLC) is routinely used for polyamine analysis (Das, 1992). The need to derivatize samples prior to analysis by HPLC has limited the use of polyamines as prognostic indicators. An alternative method that is quick, sensitive and requires minimal sample handling is immunoassay, which exploits the specificity of monoclonal antibodies (mabs). Several anti-polyamine monoclonal antibodies have been described (Garthwaite et al., 1993
; Delcros et al., 1995
; Fujiwara and Masuyama, 1995
; Catcheside et al., 1996
; Johnston et al., 1997
), including JSJ-1, JAC-1 and IAG-1, which were raised and characterized in this laboratory. All exhibit some level of cross reactivity with other polyamine group members, which has limited their usefulness in clinical analysis.
The binding site of an antibody, responsible for antigen binding and recognition, is comprised of six polypeptide segments (also called CDRs), three of which come from the heavy chain and three from the light chain N-terminal variable (v) domains. The CDRs, particularly CDR3 of the heavy chain, are stretches of high sequence variability, which are separated by relatively invariant framework sequences (Davis and Padlan, 1990). If we could determine key residues involved in polyamine binding, we could engineer a second generation of anti-polyamine antibodies with prescribed specificities and affinities.
Recently, the crystal structure of PotD, the primary receptor of the polyamine transport system in Escherichia coli, has been published (Sugiyama et al., 1996). It showed the polyamine-binding site to be a cleft lying at the interface between domains. Acidic residues in the cleft recognized the positively charged amino groups of spermidine while aromatic side chains anchored the methylene backbone. Loss of polyamine binding by our antibodies in solutions of high ionic strength (Johnston et al., 1997
) suggests that the polyamineantibody interaction also involves acidic residues.
In this we paper report the cloning, sequence analysis and mutagenesis of the variable regions of the three anti-polyamine antibodies JSJ-1, JAC-1 and IAG-1. From analysis of molecular models generated from the amino acid sequence of our antibodies we have identified a polyamine-binding motif. This is present in all the anti-polyamine antibodies we have studied to date.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
All chemicals were obtained from Sigma UK (Poole, Dorset, UK). Restriction enzymes were obtained from New England Biolabs (Hitchin, Hertfordshire, UK), Taq polymerase from Perkin-Elmer (Warrington, UK) and secondary antibodies from Amersham (Little Chalfont, Buckinghamshire, UK). Oligomers were prepared by Oswel DNA Service (Southampton, UK). Plasmid kits from Qiagen (Crawley, West Sussex, UK) were used.
Hybridoma production
Balb/c mice were immunized with spermineovalbumin or spermidine conjugated to ovalbumin via a glutarate linker, prepared as described previously by reaction with 1-ethyl-3-(3-diethylaminopropyl)carbodiimide (EDC) (Goodfriend et al., 1964). Hybridomas were produced by immortalization of splenocytes by fusion with P3.X63.Ag8.653 myeloma cells (Kohler and Milstein, 1975
). Sera and hybridoma supernatant were analysed for spermine- or spermidine-specific antibodies using spermine- or spermidine-specific direct binding enzyme-linked immunoassays (ELISAs) as described previously (Garthwaite et al., 1993
; Catcheside et al., 1996
). Antibody cross reactivities were determined by competitive ELISAs in which free polyamine and bound polyamine conjugate complex compete for antibody (Garthwaite et al., 1993
; Catcheside et al., 1996
).
Antibody variable region cloning
The variable regions of the three anti-polyamine antibody heavy and light chains were cloned from their respective hybridoma cells by RT-PCR using degenerate forward primers to the immunoglobulin leader sequences and reverse primers to the framework FR4 regions (Johnston and Athwal, 1998).
The primers introduced the enzyme restriction sites BstB1/SplI and HindIII/ApaI into the light and heavy variable domains, respectively, for cloning into the mammalian expression vectors. The heavy and light chains were cloned into separate vectors pJJcH (heavy) and pJJcL (light) (see Figure 1).
|
Transfection of expression vectors into CHO cells
Transfection of CHO cells with expression vectors containing heavy and light chains were carried out according to Lopata et al. (1984). The day prior to transfection, 7090% confluent CHO cells were diluted to 5x105 cells/ml and left to grow at 37°C overnight. The next day the medium (DMEM) was removed and replaced with an equal volume of fresh medium and left for 3 h at 37°C. Calcium phosphate precipitate was prepared by adding 925 µl of TE, 100 µl of light chain vector (50 µg of DNA), 100 µl of heavy chain vector (50 µg of DNA) and 125 µl of 2.5 M calcium chloride to 1250 µl of HEPES-buffered saline. The precipitate was immediately added to the medium on the cells and left for 3 h at 37°C. The medium was removed and the cells were shocked for 1 min with 10 ml of 15% glycerol in phosphate-buffered saline (PBS). They were then washed with 10 ml of PBS and incubated for 4872 h in growth medium containing 10 mM sodium butyrate. The medium was removed and centrifuged to remove dead cells.
Antibody purification and quantitation
Antibodies were purified using a batch method from culture supernatant by protein A and quantitated against a known standard in an assembly assay (Whittle et al., 1987).
Antibody specificity was determined in polyamine-specific direct and competitive ELISAs (Garthwaite et al., 1993; Catcheside et al., 1996
; Johnston et al., 1997
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
At Royal Holloway three anti-polyamine monoclonal antibodies, IAG-1 (Garthwaite et al., 1993), JAC-1 (Catcheside et al., 1996
) and JSJ-1 (Johnston et al., 1997
), have been produced and characterized in direct and competitive binding ELISAs. Their properties are listed in Table I
. All three antibodies are specific for a single polyamine species (IAG-1, spermine; JAC-1 and JSJ-1, spermidine) but they exhibit limited cross reactivity with other polyamine family members.
|
The variable regions of the heavy and light chains of all three antibodies were cloned from their respective hybridoma cells by RT-PCR as described in Materials and methods. The heavy chains were cloned into pJJcH, producing JSJ-1H, IAG-1H and JAC-1H. The light chains were cloned into pJJcL yielding JSJ-1L, IAG-1L and JAC-1L. The heavy and light chain vectors were transformed into chemically competent E.coli pmosBlue cells and plasmid DNA was prepared from three insert positive colonies. The DNA from all three colonies was sequenced and a consensus DNA sequence was established for each antibody heavy and light chain. The resultant sequences were translated and are shown in Figure 2.
|
Before detailed analysis of the rescued variable regions was undertaken, it was necessary to establish that the recombinant chimeric antibodies had unaltered polyamine-binding properties.
Chimeric whole antibodies (murine variable and human constant domains) were produced by co-transfection of the appropriate heavy and light chain vector pairs into CHO cells (Whittle et al., 1987). Three days post-transfection recombinant antibodies were purified from culture supernatants using protein A (Lopata et al., 1984
) and quantitated in an assembly assay against a known standard. All three antibodies were produced in similar amounts (IAG-1 23.5, JAC-1 21.3 and JSJ-1 25.1 µg/ml).
The authenticity of the recombinant antibodies was confirmed by comparison with the murine equivalent in direct binding assays. The binding profiles of the parental murine and recombinant chimeric antibodies are indistinguishable, as shown in Figure 3.
|
Comparison of the heavy and light chain sequences of the three anti-polyamine antibodies (Figure 2) showed (a) no conserved `polyamine specific' heavy chain sequences and (b) two light chains of remarkable homology. The light chains of JSJ-1 and IAG-1 are 93% homologous at the amino acid level. Only three non-conservative amino acid substitutions distinguish these two light chains. Two of these, a tyrosine to histidine and tyrosine to serine, occur in CDR1 while the third, a change from phenylalanine to valine, occurs in CDR2. Another anti-polyamine antibody sequenced in this laboratory shares this light chain homology (the three light chains are 92% homologous). This fourth antibody has the IAG-1 CDR1 and the JSJ-1 CDR2. It is unable to distinguish between spermine and spermidine in binding assays. Conservation of light chain sequence may be important for function or it may reflect a near germ line light chain. Germ line analysis of the JSJ-1, IAG-1 and the fourth antibody light chains revealed that they are all VK1 subgroupII with J segment Jk2. Their sequences are sufficiently divergent from that of the germ line to suggest a functional role for this light chain in polyamine binding. The JAC-1 light chain is significantly different from the others (VK4/5 subgroup IV, Jk2). It has a high serine content (27.5%, compared with 17.1% for JSJ-1 and 18.2% for IAG-1).
The heavy chain V domains showed no conserved `polyamine-specific' sequences. However, polyamines are positively charged species at physiological pHs. Ionic interactions between these positively charged species and negatively charged acidic residues in the antibody binding site are likely to be very important in the antibodyantigen interaction. All three of our antibodies contain negatively charged residues in the heavy chain CDRs. We have therefore investigated the effect of increased salt concentration on the binding of the antibodies with their respective antigens. High ionic strength (1 M sodium chloride) is known to disrupt electrostatic interactions. Figure 4 shows that at high salt concentrations binding of the antibody to the free antigen is decreased. For JSJ-1 (B) a small right-hand displacement of the curve is seen and a shift in half-maximum binding from 4.1x106 to 7.9x106 M is observed. For JAC-1 and IAG-1 the displacement of the curve is greater. The shift in half-maximum binding is from 2.2x106 to 1.5x105 M for JAC-1 (A) and from 3.1x107 to 2.9x106 M for IAG-1 (C). This confirms that ionic interactions are involved in the polyamineantibody interaction but that they are more important for polyamine JAC-1 and IAG-1 binding than for the polyamineJSJ-1 interaction.
|
Using the molecular graphics modelling packages AbM (Oxford Molecular), Modeller and InsightII 97 (MSI), homology models of all three of the cloned antibody Fv regions have been generated. All numbering is according to Kabat (Martin, 1996). The antibody framework regions were built by homology from known crystal structures. CDRs which could be assigned into canonical groups were identified automatically and generated. Those that could not were built by the method of Martin et al. (1989). The reconstructed loops were minimized and filtered before a final conformation for each loop was selected. Model building was done in vacuo.
Figure 5 shows contour mapping of the binding sites of the three antibodies with electrostatic charges assigned by Delphi (MSI). The binding sites appear as negatively charged clefts of similar size and orientation in all three antibodies. Closer analysis of the clefts shows that they are closed at one end. This region binds the terminal amino group of the polyamine conjugate complex. The open end of the cleft apparent in all our models accommodates the bulky protein to which the polyamine is conjugated. Only antibodies that can bind the polyamine conjugate complex are detected in our ELISA.
|
|
JAC-1 (Figure 6B).
Two possible spermidine-binding sites can be seen for JAC-1. In the first, the terminal NH3+ of the aminopropyl group of spermidine binds to TyrL34. The secondary amino group forms an ionic interaction with AspH217. The carbon backbone interacts with other aromatic residues including TyrL40 and SerL41. The polyamine is in an extended conformation. In the second, the terminal NH3+ group interacts with AspH143 and AspH141 and the remainder of the molecule interacts with TyrH137 and AspH217. The existence of two spermidine-binding sites of differing affinity is consistent with our experimental data (Johnston et al., 1997
).
JSJ-1 (Figure 6C).
The terminal NH3+ of the aminobutyl group of spermidine interacts with TyrL96, TyrH56 and ThrH50. The secondary amino group interacts with both AspH95 and AspH97, while the aminopropyl amino group binds to SerH52A and the carboxyl group of ArgH31. Spermidine is in an energy minima conformation and fits snugly into the antibody binding cleft.
It is the aminobutyl group, a putrescine moiety, that interacts with the closed end of the antibody binding cleft. We believe that it is this interaction that determines putrescine cross reactivity. Neither IAG-1 nor JAC-1 cross reacts with putrescine (Garthwaite et al., 1993; Catcheside et al., 1996
). In both cases it is the aminopropyl group of their respective polyamines that binds to the closed end of the cleft.
Heavy and light chain shuffling
Simple mutagenesis experiments were undertaken in which the heavy and light chain pairings of the three antibodies were mixed. All possible heavy and light chain combinations, nine in total, were generated. The chimeric whole antibodies were purified as described previously and quantitated in an assembly assay. Three heavylight chain combinations were poorly expressed (Figure 7). Computer models showed that the heavy and light chain CDRs clashed, preventing them from associating (data not shown).
|
|
|
We have proposed a model for polyamineantibody binding in which acidic and polar residues line a negatively charged binding cleft that is partially or completely closed at one end. The specificity of the interaction is determined by the placement of acidic residues in the cleft.
We can define the binding site more specifically for those antibodies that share the JSJ-1IAG-1 light chain. In these antibodies only Tyr96 from the light chain is involved directly in the antibodyantigen interaction. Heavy chain residues H31 and H56 and a D/EXD(D) binding motif from CDR3 are key amino acids for determining antigen-binding affinity.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The heavy and light chain variable regions of all three anti-polyamine antibodies were rescued from the parental hybridoma lines by RTPCR and cloned into mammalian expression vectors. The variable domains were placed upstream of human constant domains. Chimeric whole antibodies (murine variable domains/human constant domains) were produced and purified. The cloned v regions were shown to be authentic by comparison with the murine equivalent in direct and competitive ELISAs.
The DNA sequence of the three anti-polyamine heavy and light chains was translated. The resultant protein sequences showed no common heavy chain sequence motifs. However, two of the three light chains showed considerable sequence homology (93%). Another anti-polyamine antibody has also been shown to have this light chain sequence. The conservation of light chain sequence may be important for function or it may reflect a near germ line light chain. Germ line analysis of the JSJ-1 and IAG-1 light chains revealed that they are both VK1 subgroup II with j segment jk2. However, their sequences are sufficiently divergent from that of the germ line to suggest a functional role in polyamine binding. The simple `mix and match' mutagenesis experiments confirmed the importance of the light chain in conferring antibody specificity and affinity. By simply swapping the light chain from the spermidine-specific JSJ-1 antibody to pair with the spermine-specific IAG-1 heavy chain, a new antibody with greater spermine specificity and affinity was generated.
While we could not identify polyamine-binding sites from primary sequence, molecular models have enabled us to identify a three-dimensional polyamine-binding motif. This is present in all three of our anti-polyamine antibodies. The polyamine-binding site is comprised primarily of aspartic acid, glutamic acid and tyrosine residues. The specificity of the polyamine interaction is determined by the position of acidic and polar residues along the sides and bottom of the cleft. We have been able to define key residues in antibodies that share the JSJ-1/IAG-1 antibody light chain. These are L96, H31, H56 and an E/DXD(D) motif in CDR3 of the heavy chain.
The polyamine-antibody-binding motif we have described is very similar to that observed in the crystal structure of PotD, the primary receptor of the polyamine transport system in E.coli (Sugiyama et al., 1996). Here also, the positively charged amino groups of spermidine are bound to acidic residues in the binding sitea cleft at the interface between domains. Aromatic residues also contribute to polyamine binding, interacting with the carbon backbone. The conservation of residues involved in polyamine binding in these two independent systems demonstrates mechanistic convergence.
We are now in a position to begin site-directed mutagenesis studies to validate our models. We postulate that in order to obtain increased specificity, the polyamine binding pocket must be closed at both ends and acidic residues placed at appropriate intervals along its length. These must coincide with the positions of the amino groups in the polyamines. However, our anti-polyamine antibodies, as indeed are those of other groups (Garthwaite et al., 1993Delcros et al., 1995
; Fujiwara and Masuyama, 1995
; Catcheside et al., 1996
; Johnston et al., 1997
), are detected by binding to terminally conjugated polyamines in direct or competitive binding ELISAs. Antibodies detected in this way must be open at one end to enable the bulky conjugate to present the polyamine for binding. Conjugation of the polyamine to carrier via secondary amine groups may permit the detection of more specific antibodies. The production of such polyamine conjugates is in progress.
Crystallographic studies are under way to confirm the validity of our models.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Das,I. (1992) Chromatogr. Anal., 21, 57.
Davis,D.R. and Padlan,E.A. (1990) Annu. Rev. Biochem., 59, 439473.[ISI][Medline]
Delcros.J., Clement,S., Thomas,V., Quemener,V. and Moulinoux,J. (1995) J. Immunol. Methods, 185, 191198.[ISI][Medline]
Fujiwara, K. and Masuyama,Y. (1995) Histochem. Cell Biol., 104, 309316.[ISI][Medline]
Garthwaite,I., Stead,A.D. and Rider,C.C. (1993) J.Immunol.Methods, 162, 175178.[ISI][Medline]
Goodfriend,T.L., Levine,L. and Flashman,G.D. (1964) Science, 144, 13441346.[ISI]
Herby,O. (1981) Differentiation, 19, 120.[ISI][Medline]
Johnston,J.S. and Athwal,D.S. (1998) Biochem. Soc.Trans., 26, S38S38.[ISI][Medline]
Johnston,J.S., Beesley,P.W., Rider,C.C., Ahmed,H. and Harris,S. (1997) Hybridoma, 16, 541543.[ISI][Medline]
Kohler,G. and Milstein,C. (1975) Nature, 256, 5253.[ISI][Medline]
Laskowski,R.A. (1995) J. Mol. Graph., 13, 323330.[ISI][Medline]
Laskowski,R.A., Thornton,J.M., Humblet,C. and Singh,J. (1996) J. Mol. Biol., 259, 175201.[ISI][Medline]
Lopata,M.A., Cleveland,D.W. and Sollner-Webb,B. (1984) Nucleic Acids Res., 12, 57075717.[Abstract]
Martin,A.C.R., Cheetham,J.C. and Rees,A.R. (1989) Proc. Natl Acad. Sci. USA, 86, 92689272.[Abstract]
Martin,A.C.R. (1996) Proteins: Struct. Funct. Genet., 25, 130133.[ISI][Medline]
Pegg,A.E. (1986) Biochem. J., 234, 249262.[ISI][Medline]
Sugiyama,S., Vassylyev,D.G., Matsushima,M., Kashiwagi,K., Igarashi,K. and Morikawa,K. (1996) J. Biol. Chem., 271, 95199525.
Whittle,N., Adair,J., Lloyd,C., Jenkins,L., Devine,J., Scholm,J., Raubitschek,A., Colcher,D. and Bodmer,M. (1987) Protein Engng, 1, 499505.[Abstract]
Received August 22, 1998; revised February 10, 1999; accepted February 19, 1999.