Aug 25, 2006 - Robert S. HodgesS, Robin J. Heaton, and J. M. Robert Parker. From the Department of Biochemistry and the Medical Research Council of ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 263, No. 24, Issue of August 25, pp. 11768-11775,1988 Printed in U.S. A.
Antigen-Antibody Interaction SYNTHETICPEPTIDES DEFINE LINEAR ANTIGENIC DETERMINANTS RECOGNIZED BY MONOCLONAL ANTIBODIES DIRECTED TO THE CYTOPLASMIC CARBOXYL TERMINUS OF RHODOPSIN* (Received for publication, January 25, 1988)
Robert S. HodgesS, Robin J. Heaton, and J. M. Robert Parker From the Department of Biochemistry and the Medical Research Council of Canada Group in Protein Structure andFunction, University of Alberta, Edmonton, Alberta T6G 2H7,Canada
Laurie Molday and RobertS. Molday From the Department of Biochemistry, The University of British Columbia, Vancouver, British Columbia V6T 1 W5
The specificities of four monoclonal antibodies rho nikov et al., 1982; Hargrave et al., 1983), a model of rhodopsin 1D4, lCS,3A6, and 3D6 prepared by immunization of structure has been proposed in which the polypeptide chain rod outer segments containing rhodopsin have been spans the membrane lipid bilayer seven times. Labeling and defined using synthetic peptides. All of these antibodies proteolysis studies (Molday and MacKenzie, 1983; Clark and interact within the 18 residues at theCOOH terminus Molday, 1979) have indicated that the COOH terminus is of rhodopsin and recognize linear antigenic determi- exposed on the cytoplasmic side of the disc membrane whereas nants of 4-11 residues. Twenty-seven synthetic peptide analogs of varying lengths of native sequence or the NH2 terminus is exposed on the lumen or interdisc surface. containing single amino acid substitutions at each po- Four monoclonal antibodies to bovine rhodopsin have been sition of the COOH-terminal18 residues have provided shown to bind to the COOH-terminal region (MacKenzie et some insight into the mechanism of antigen-antibody al., 1984). These monoclonal antibodies would be ideal canbinding. Our results clearly demonstratethat antibod- didates to study the interaction of antibody and antigensince ies can be highlyspecific at key positions as shown by the known antigenic region is small (18 residues or less) and the loss of binding on single amino acid substitutions synthetic peptides are able to compete with native protein for in the bindingsite. In contrast singleamino acid sub- binding to anti-protein antibodies raised to rhodopsin-constitutions at other positions in the binding site only taining disc segments. Although it is generally thought that affect affinity for some antibodies. Ionic interactions monoclonal antibodies recognize discontinuous epitopes (van candominate immunogenic determinants. Immuno- Regenmortel, 1986; Benjamin et al., 1984), we report that genic determinants are not restricted to highly charged monoclonal antibodies to the COOH terminus of intact rhoof a protein andmay dopsin-containing discs recognize residues confined to small hydrophilic regionson the surface be dominated by hydrophobic interactions. Although linear epitopes ranging from 4 to 11 residues. In a previous certain side chains can dominate the interactionof the study of antigen-antibody interaction, we reported that a antigen with antibody, our results are in agreement with the interpretation that the free energies of all the NH2-terminal acetylated residue was critical for the binding contact points are additive and a certain free energy of antibodies to EDP208 pilin protein, and this binding was must be present to achieve binding. Antibodies with restricted to a NH2-terminal pentapeptide (Worobec et al., different specificities directed to the same region of 1985). Similarly the NH,-terminal acetylated amino acid was the protein antigen can be produced in an immune found to be essential for the binding of synthetic peptides to response. Peptide antigens representing regions of a anti-cytochrome c antibodies (Paterson, 1985). In this report protein antigen bind best to the anti-protein antibody we describe results of antigen-antibody interactions at the when the sequence is shortened to contain only those COOH terminus of rhodopsin, the importance of the free residues binding to thespecificity site in theantibody. COOH-terminal a-carboxyl group, the additive binding effect Cross-reactivity between protein antigens can be ex- of hydrophobic and hydrophilic residues, and thatmonoclonal plained by conservation of the critical residues in the antibodies with a variety of specificities, both ionic and hycombining site. drophobic, can recognize small linear determinants. MATERIALS AND METHODS
Rhodopsin is the major membrane glycoprotein in rod outer segment disc membranes of vertebrate retinalrod photoreceptor cells. On the basis of protein sequence analysis (Ovchin-
* This work was supported by the Medical Research Council of Canada (R. S. H. and R. S. M.), equipment grants from the Alberta Heritage Foundation for Medical Research (to R. S. H.), and National Institutes of Health Grant EY-02422 (to R. S. M.). The costs of Publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. $ To whom correspondence should be addressed.
Rod Outer Segment Membranes-Bovine rod outer segments were purified by sucrose-density centrifugation under dim red light as previously described (Wong and Molday, 1986). Rod outer segment disc membranes used as a source of rhodopsin were obtained by hypotonic lysis of rod outer segments followed by flotation on 5% Ficoll according to themethod of Smith et al. (1975). Protein concentration was determined by the Lowry method using bovine serum albumin as a standard (Lowry et al., 1951). Antibodies-Antibovine rhodopsin monoclonal antibodies (rho 1D4, 3.46, 1C5 and rho 3D6) were derived from culture fluids of hybridoma cell lines (MacKenzie et al., 1984; Hicks and Molday, 1986). Goat antimouse immunoglobulin antibody used in indirect solidphase radioimmune assays was iodinated with NalZ5Iby the chlor-
11768
11769
Antigen-Antibody Interaction amine-T method (Hunter and Greenwood, 1962) and had a specific activity of 1-2 X 10‘ dpm/pg. Solid-phase Radioimmune Assays-For radioimmune assays, 96well flexvinyl microtiter plates were coated with rhodopsin by drying down 25 plof Triton X-100-solubilizedrod outer segment membranes. The plates were rinsed in phosphate-buffered saline (0.01 M sodium phosphate, 0.15 M NaC1) containing 1%bovine serum albumin prior to use. Competition assays were carried out by incubating 25 p1 of serial dilutions of the peptide antigen with 25 plof hybridoma culture fluid a t a dilution which gave 80-90% antibody saturation as measured in standard solid-phase radioimmune assays (MacKenzie and Molday, 1982). The concentration of all peptides were determined by amino acid analysis of stock solutions, which has an error of +-5%,or by weight which when compared to amino acid analysis had an accuracy of &lo%. After 30-60 min at 25 “C, 25 p1 from each well was transferred to therhodopsin-coated microtiter plates. The microtiter plates were incubated for 30 min, washed in phosphate-buffered saline, and treated with 25 11 of ‘261-labeledgoat antimouse immunoglobulin antibody (10 pg/ml; 1-2 X 10’ dpm/pg). After 30-60 min, the plates were again washed in phosphate-buffered saline, cut, and counted in a Beckman 8000 gamma counter. Variations in the 160 values (peptide concentration required to obtain half-maximum inhibition of antibody binding to rhodopsin) were observed for different stock hybridoma cell culture fluids and rod outer segment preparations. In repetitive assays, however, 160 values for a given peptide agreed within -C50%. Variations in hybridoma supernatant are due to the quantity of monoclonal antibody secreted per ml of fluid and the dilution used in the competition assay. Differences in 160values were used to get around this difference. When different hybridoma cell culture fluids and rod outer segment preparations were used the native peptide sequence was always used as a control to ensure the relative accuracy of the peptide analog results. A variation in 160 values between different peptide analogs of 2-fold or greater is considered significant. Peptide Synthesis-Unless otherwise stated,all chemicals used were reagent-grade. Diisopropylethylamine, dichloromethane (DCM),’ trifluoroacetic acid, and dimethylformamide were obtained from General Intermediates of Canada. Diisopropylethylamine, DCM, and trifluoroacetic acid were redistilled before use. HPLCgrade acetonitrile was obtained from either Fischer Scientific, or Caledon Laboratories. Double-distilled water was purified by passage througha Milli-Q water purification system. t-Butyloxycarbonylamino acids were obtained from Institut Armand Frappier (Laval, Quebec), Protein Research Foundation (Osaka, Japan), andBachem Fine Chemicals. Co-poly (styrene, 1%divinylbenzene) chloromethyl resin (0.9 mmol Cl/g resin) was obtained from Pierce Chemical Co. Co-poly (styrene, 1%divinylbenzene) benzhydrylamine-HC1 resin (0.9 mmolNHz/g resin)was obtained from Institut Armand Frappier. Twenty-seven analogs were synthesized using the general procedure for solid-phase peptide synthesis described by Merrifield (Stewart and Young,1984; Erickson and Merrifield, 1976) on either a Beckman 990 Peptide Synthesizer, or an Applied Biosystems 430A Peptide Synthesizer. All peptides with a free carboxyl terminus were initiated by esterification of the cesium salt of the COOH-terminal amino acid to co-poly (styrene, 1%divinylbenzene) chloromethyl resin (Stewart and Young, 1984). Substitutions of approximately 0.9 mmol of amino acid/g resin were obtained. For peptides with an amide COOH terminus, co-poly (styrene, 1%divinylbenzene) benzhydrylamine-HC1was neutralized with 5% diisopropylethylamine for 1 h and theCOOH-terminal aminoacid was coupled to theresin with dicyclohexylcarbodiimide for 1 h (1:l equivalent, protected amino acid/dicyclohexylcarbodiimide). Side-chain protecting groups for t-butyloxycarbonyl-aminoacids used were: Lys(2-chlorobenzyloxycarbonyl), Asp(0-benzyl), Glu(0benzyl), Ser(benzyl), and Thr(benzy1). Removal of the t-butyloxycarbony1 protecting group at each cycle was done in 50% trifluoroacetic acid/DCM for 20 min. This was followed by neutralization in 5% diisopropylethylamine. All amino acids were coupled using DCCactivated couplings (1:l equivalent, protected amino acid/DCC) in DCM, except Thr which was coupled as a symmetric anhydride (2:l equivalent, protected amino acid/DCC) in DCM, and Gln and Asn, which were coupled in dimethylformamide as N-hydroxybenzotriazole active esters. Double couplings of 1 h each were performed at each step of the synthesis. The completed peptides were deprotected as The abbreviations used are: DCM, dichloromethane; DCC, dicyclohexylcarbodiimide; HPLC, high performance liquid chromatography.
I
I
I
I
20
10
ELUTION TIME ( m i d FIG. 1. Representative reversed-phase chromatographic elution profiles on analytical columns of the crude synthetic peptides. Panel A, peptide 5, Fig. 1 [Ac(Ala2’)R(1’-12’)-OH]; Column, SynChropak RP-8, 4.1 mm inner diameter X 150 mm, 6.5 pm, 300 A, (2-8. Panel B , peptide 1, Fig. 1 [AcR(1’-18’)-OH]; Column, Pharmacia PEP RPC, 5 mm inner diameter x 50 mm, 5 pm, 100 A, C-18. The solvent system used for both columns was a linear AB gradient where A = 0.05% trifluoroacetic acid/HzO and B = 0.05% trifluoroacetic acid/acetonitrile. Gradient rate was 1%B/min with flow rates of 1 ml/min. The crude peptides were purified as outlined under “Materials and Methods.” described previously and acetylated in asolution of toluene, pyridine, and acetic anhydride (3:3:1) for 1 h. The programs used for attachment of each aminoacid using the Beckman 990 synthesizer were the same as previously described (Worobec et al., 1985; Parker and Hodges, 1985). Coupling procedures for the Applied Biosystems synthesizer have been described (Kent andLewis, 1985). Peptides werecleaved from the resin with HF (20 ml/g resin) containing anisole (2 ml/g resin), and ethanedithiol(20drops/g resin) a t 4 “Cfor 45 min. The solvents were removed under reduced pressure a t 4 “Cfor at least 3h. The resin was washed with ether, and extracted with 3 X 10 ml trifluoroacetic acid. The trifluoroacetic acid was evaporated and thepeptide redissolved in water and lyophilized. Purification by HPLC-The crude peptides were purified using reversed-phase chromatography on either a SynChropak RP-P C-18, 300 A, 6.5pm column (250 X IO mm inner diameter) or a Rainin Dynamax Macro C-18, 300 A, 1 2 pm column (250 X 21.4 mm inner diameter) with a linear AB gradient where A = 0.05% trifluoroacetic acid/HzO and B = 0.05% trifluoroacetic acid/acetonitrile. The gradientrate was 1%B/min with flow rates of 2 ml/min for the SynChropak column and 10 ml/min for the Rainin column. Representative HPLC elution profiles of the 12-residue and 18-residue crude synthetic peptides before purification are shown in Fig. 1. RESULTS
Four antibovine rhodopsin monoclonal antibodies, rho 1D4, 3A6, 1C5, and 3D6, were previously shownto bind to bovine rhodopsin in the COOH-terminal 18 residues, 1’-18’ where 1’ is the COOH-terminalresidue(MacKenzie et al., 1984; Hicks and Molday, 1986). To localize further the antibody-
Antigen-Antibody Interaction
11770
binding sites and examine the importance of each residue in this 18-residue peptide to antibody binding, the 27 peptides shown in Fig. 2 were synthesized, purified, and examined in a solid-phase competitive inhibition assay. To demonstrate the effect of each amino acid side chain on antibody binding, the following single amino acid substitutions of each side chain were carried out; when the amino acid side chain in the native sequence was alanine, aglycine residue was substituted; all larger side chains were substituted by alanine except for the acidic residues aspartic and glutamic acid (positions 8 ', 17', and 18', Fig. 2) which were substituted by the uncharged isosteric residues, asparagine and glutamine, respectively (peptides 11, 20, and 21, Fig. 2). These Asn and Gln substitutions would examine the importance of ionic interactions in antigen-antibody binding while leaving all other interactions with that residue intact (van der Waals, hydrophobic or hydrogen bonding). The two peptide chain-lengths of 1'-12' or 1'-18' were selected based upon previous results where competitive inhibition studies indicated that antibody rho 3A6 required a length 1'-12' and larger whereas antibody rho 1C5 required peptide 1'-18' (MacKenzie et al., 1984). Antibody rho 1D4 binding was not inhibitedby peptides 2'-13' or 3'-18', indicating that theCOOH-terminal alanine residue of rhodopsin was required. Competition studies using COOHterminal peptides l"4' to 1'-18' were equally effective inhibitors of antibody rho 3D6 binding to immobilizedbovine
PEPTIDE NUMBER
I
PIPTIOE NAME
rhodopsin (Hicksand Molday,1986). Therefore, synthetic analogs of lengths l ' - l Z ' and 1'-18' seemed appropriate to test antigen-antibody binding of these four monoclonal antibodies. Based upon the results with these analogs, the minimum lengths for maximum antibody binding would then be synthesized. All peptides representing internal regions of the rhodopsin sequence were synthesized as the Ne-acetylated and COOHterminal amides to prevent the introduction of charged groups near the antibody-binding site. This is of particular importance, as the minimum peptide chain length is synthesized which maximizes antibody binding. Talbot andHodges (1981) clearly demonstrated the importance of not introducing N" or C" charged groups into a binding site peptide which originates from an internal sequence of protein molecule. These workers determined the minimal inhibitory peptide of the troponin I inhibitory sequence is 12 residues and that the N"amino groups or C"-carboxyl group significantly affected the ability of this peptide to inhibit the actomyosin ATPase. This precaution of N"-acetylation and C"-amides should always becarried out since these groups are isosteric with the peptide bonds at either end of the peptide. Although, in this study the 150 values for Ne-acetylated 1'-12' or 1'-18' were indistinguishable from the nonacetylated 1'-12' or 1'-18' peptides when tested with rho 1D4 and rho 3A6, this precaution was observed for all shorterpeptides.
SEOUENCE 1 8 ' 1 7 ' 1 6 ' 1 5 ' 1 4 1 3 ' 1 2 ' 1 1 ' 10' 9' 8'
7'
6'
5'
4' 3'
2'
1'
Rc-Rrp-Glu-Rla-Ser-Tr-Thr-Val-Ser-Lyr-Thr-Glu-Thr-Ser-Gln-UaI-Rla-Pro-Rla-OH
2 3
4
5
W
6
RC"
7
Rc-Ual
8
FIG. 2. Amino acid sequence of synthetic peptide analogs of the reCOOH-terminal AcR(l"18') gion of bovine rhodopsin. The numbering of the sequence begins from the COOH-terminal end of the protein with the COOH-terminal residue denoted 1'. Nomenclature example, Ac(Ala13')R(1'18')-OH, synthetic NHz-terminal-acetylated rhodopsin fragment, residues 1'18' with a COOH-terminal carboxyl group and alanine substituted at position 13'. Amide denotes a COOH-terminal amide. The circled residues denote the position of the amino acid substitution.
Rls-OH
9 10
Rc-Ual
Rls-OH
II
flr-Ual
Rla-OH
12
Rc-UaI
Rh-OH
13 14 15 16
17 I8 19 20 Rla-OH
21 22
~c-Rsp-6lu-Rla-Ser-~r-Thr-Ual-Ser-Lyr-Tr-6lu-amide
23
Rc-Thr-Thr-Val-Ser-Lys-Thr-Glu-amide
24
Ac-Val-Ser-Lyr-Rr-Glu-amide
25
Ac-Thr-Glu-Thr-Ser-GIn-Ual-Ala-Pro-RIa-OH
26
Rc-Thr-Ser-Gln-Val-Ala-Pro-Ala-OH
27
Ac-Ual-Ala-Pro-Rla-OH
Antigen-Antibody Interaction Importance of the COOH-terminal a-Carboxyl Group to Antibody Binding-Synthetic peptides 2 and 3, AcR(1'-12')-OH and AcR(1'-12')-amide, respectively, were compared in the solid-phase competitive inhibition assay. The results shown in Table I indicate that theCOOH-terminal a-carboxyl group is essential for antibody binding to both rho 1D4 and rho 3D6. The Iso value increased from 0.9 to 158 p~ when comparing peptide 2 and 3, respectively, for antibody rho 1D4 binding and peptide 3 showed no binding to antibody rho 3D6 (Table I).In contrast antibody rho 3A6 bound equally well to peptides 2 and 3 indicating that the a-carboxyl group of the COOH-terminal alanine residue is not involved in binding to this antibody. Importance of Each Amino Acid Side C h i n to Antibody Binding-Systematic replacement of each residue in the COOH-terminal 18-residue sequence was made in either the 12- or 18-residue peptide. The substitutions for each of the first 12 positions of the COOH-terminal sequence were made in analogs of the 1'-12' sequence and are shown in Fig. 2, peptides 4-15. The substitutions for each of the positions 1318were made in analogs of the 1'-18' sequence and areshown in Fig. 2, peptides 16-21. All four antibodies used in this study bound to the 18-residue peptide containing the COOH-terminal residue of rhodopsin. In addition, three antibodies, rho 1D4,3A6, and 3D6 bound to the12-residue peptide containing the COOH-terminal residue of rhodopsin. Monoclonal Antibody rho 104 Binding Studies-As shown in Fig. 3 and Table I, the region 1'-9' is important for peptide binding to monoclonal antibody rho 1D4. These results suggest that side chains of residues l', 10'-18' make only weak or insignificant contributions to antibody rho 1D4 binding. Substantial decreases in antibody binding, as reflected by the 150 values in Table I, were seen with peptides 5, 7, 9, 11, and 12, where proline 2', valine 4', serine 6', and threonine 9' were replaced with alanine and glutamic acid 8' was replaced
11771
with glutamine (Iw values of2000, 63, 125, 10, and 6.3 pM, respectively, when compared to peptide 2 of0.9 pM representing a7-2220-fold decrease). The most important residues interacting with antibody rho 1D4 werealanine 3', glutamine 5', and threonine7' where no detectable binding was observed when these positions were substituted by glycine 3', alanine 5', and 7', respectively. The results of the competitive inhibition assays for monoclonal rho 1D4 antibody binding are summarized graphically in Fig. 3 where the solid bars indicate the importance of each residue to antibody binding. The results shown in Fig. 3 suggested that the minimum peptide sequence required for maximum antibody rho 1D4 binding was residues 1'-9'. This conclusion was verified by the data shown in Table I1 where binding increased approximately 6-fold whencomparing peptide 25 (1'-9') with peptide 2 (1'-12'). It appears that the 1'-9' peptide reproducibly serves as a more effective antigen than the 1'-12' peptide. Interestingly, peptide 26 (1'-7') which contained the residues demonstrated to be critical for binding (Fig, 3) showed no detectable binding. It appears as if the loss of binding energy from residues 8' and 9', although small, was substantial enough to prevent binding from a peptide containing residues 1'-7'. MacKenzie et al. (1984) showed that peptide 1'4' bound to antibody rho 1D4. Another possibility is the loss of hydrogen bonds to thepeptide bonds on removing residues 8' and 9'. Monoclonal Antibody rho 3A6 Binding Studies-As shown in Fig. 3 and TableI, the side chains in the region 8'-12' are important for peptide binding to monoclonal antibody rho 3A6. These resultssuggest that theside chains of residues 1'7' and 13'"' make only weak or insignificant contributions to antibody rho 3A6 binding. Substantial decreases in antibody binding, as reflected by the IsOvalues in Table I were seen for peptides 12 and 15 where threonine 9' and valine 12' were replaced with alanine (I50values of 1000 and 350 p ~
TABLE I Effect of single amino acid substitutions on peptidebinding to anti-rhodopsinmonoclonal antibodies Monoclonal antibody Peptide no.
Peptide region
Group altered from -+ to
1D4 1C5
2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 16 17 18 19 20 21
l"12' l"12' l"12' l"12' l"12' l"12' l"12' 1'-12' 1'-12' l"12' l"12' l"12' l"12' l"12'
None
COOH + amide 1'Ala + Gly 2'Pro + Ala 3'Ala + Gly 4'Val+ Ala 5'Gln + Ala 6'Ser + Ala 7'Thr + Ala 8'Glu + Gln 9'Thr + Ala 1O'Lys + Ala 11'Ser + Ala 12'Val- Ala
3A6
3D6 ~
IW"
0.9 158 2.5 2000 63 125 6.3 10.0 1.0 0.5 0.3
LogIMl-LogI"50b
154
0 2.3 0.5 3.4 1.8 2.2
28 28 25 35 35 56 24 35 31 1000 350
-
0.9 1.1 0.1 -0.3 -0.5
LogI~O-LogI"50
0 0 -0.05 0.09 0.09 0.30 -0.07 0.09 0.04 -
1.55 1.09
0.8 0 4 0 1'"' None 2.5 l"18' 13'Thr + Ala 1.3 0.21 10 0.4 50 '"'1 14'Thr + Ala 1.2 0.18 101.5 0.4 1.2 0.18 4 0 4.2 l"l8' 15'Ser + Ala 1.2 0.18 4 0 28 l"l8' 16'Ala + Gly 1.2 0.18 10 l"l8' 17'Glu + Gln 0.4 8.0 l"l8' 18'Asp + Asn0.24 1.4 10 0.4 6.3 Iw, competitor concentration (PM)required for 50% inhibition in the competitive radioimmune assay. b160", is the I W value for either the N"-acetylated 1'-12' peptide [AcR(1'-12')-OH, peptide 21 or the Noacetylated 1'-18' peptide [AcR(1'-18')-OH, peptide 11 with each monoclonal antibody. The value LogIW-LogIso" is used for graphicalcomparisons of the effect of single amino acid substitutions. The dash indicates no measureable binding to the monoclonal antibody.
-
,
11772
Antigen-Antibody Interaction
A
WW
1
BB
3.4
0 Ov) U
FIG. 3. Panel A , effects of single amino acid substitutions inthe synthetic peptide AcR(l'-18')-OH or AcR(1'12')-OH representing the bovine rhodopsin COOH-terminal sequence on binding to anti-rhodopsin monoclonal antibodies. The hatched bars denote the results of competition assays with monoclonal rho 3A6 and the solid bars with monoclonal rho 1D4. See Fig. 2 for the amino acid sequence of all synthetic analogs, Table I, and textfor details. Panel B, carboxyl-terminal sequence of bovine rhodopsin showing the location of the linear antigenic determinants for the various anti-rhodopsin monoclonal antibodies on the basis of synthetic peptide inhibition studies. Solid line represents segment which givesmaximum antibody binding; dashed line represents smallest observed segment which exhibits antibody binding.
0"
2.4
-1
I
5:
M
1.4
m
0 -1
0.4
Native Sequence
AC
18'
17' 1 6 ' 15' 1 4 13'
12' 1 1 ' 1 0 '
9'
7'
8'
6'
4
5'
3'
2'
I'
OH
Ac-D-E-A-S-T-T-V-S-K-T-E-T-S-Q-V-A-P-A-OH
Substitution
. . . . .A .A .A . A .A . A . A . Q . A . A . A . A . G . A . G . amide
NH; N Q G
6
I
3A6 """
104 " " " "
18'
12'
9'
-
" "
8'
1' -Asp-Glu-Ala-Ser-Thr-Thr-Val-Ser-Lys-Thr-Glu-Thr-Ser-Gln-Val-Ala-Pro-Ala-COOH
I TABLE I1 Effects of chain length on peptide binding to anti-rhodopsin monoclonal antibodies Monoclonal antibody
Peptide no.
Peptide region or ROS"
I,* PM
l"12' 1D4
3A6 8"18' 8"12' 1C5
2 25 26 24 2 22 23 24 1 2 22 23 24
1'-9' l"7' 8"12' ROS l"12' 8"14' ROS l"18' l"12' 8'-18' 8"14' 8"12' ROS
6.3 1.3 0.02 59' 1.38 3.58 19.60 0.08 0.40 -
0.21 263 >loo0 0.03 l"12'3D6 2 140 l"4' 27 140 ROS 0.06 ROS denotes rhodopsin outer segment disc membranes. The dash (-) indicates no measurable binding to themonoclonal antibody. The IW values determined vary slightly with the preparation of ROS, and themonoclonal antibody containing hybridoma cell culture ~ fluid. For example the I50 value for peptide 1'-12' was 28 / r (Table I) and 59 ~ I M(Table 11). For this reason all peptides are compared to the native sequences 1'-12' or 1'-18' in each assay.
respectively, when compared to peptide 2 of 28 pM representing a 36- and 13-fold decrease). The most important residues in interacting with antibody rho 3A6 were glutamic acid 8',
"""-
" " " " "
1C5
I
I
306
I
lysine lo', and serine 11' where no detectable binding was observed, when these positions were substituted by glutamine 8', alanine lo', and ll',respectively. The importance of serine 11' andthreonine 9' also has been suggested in previous studies indicating that kinase-catalyzed phosphorylation of rhodopsin inhibits the binding of antibody rho 3A6 to rhodopsin (Molday and MacKenzie, 1985). The results of the competitive inhibition assays for monoclonal rho 3A6 antibody binding are summarized graphically in Fig. 3 where the hatched bars indicate the importance of each residue to antibody binding. The results shown in Fig. 3 suggestedthat minimum peptide sequence required for maximum antibody rho 3A6 binding is residues 8'-12'. This conclusion wasverified by the data shown in Table I1 and Fig. 4. Fig. 4 is representative of the data obtained by solid-phase radioimmune competitive inhibition assays displayed in Tables I and 11. Binding increased approximately 3-fold when peptide 24 (8'-12') is compared with peptide 2 (1'-12'). However, as the8'-12' sequence was extended to 8'-14' (peptide 23) and 8"'' (peptide 22) binding increased by 5- and 14-fold, respectively, when compared to peptide 24 (8'-12') (Table 11). Interestingly, peptide 1 (1'18') binds 7 times better than peptide 2 (1'-12') (Table I) while peptide 22 (8'"') binds 43 times better than peptide 2 (1'-12') (Table 11). These results suggest that, although extension of the 1'-12' peptide to 1'"' increases the binding affinity to monoclonal rho 3A6, the removal of unnecessary residues in the 1'-7' region results in a further substantial increase in binding affinity. Monoclonal Antibody rho1C5 Binding Studies-In a similar fashion to the monoclonal binding studies with rho 3A6, the binding region for antibody rho 1C5 was localized to the 8'18' region (Fig. 3B, Table 11) and the importance of residues
Interaction Antigen-Antibody
8
3
0
6
X
0
a
a
4
2n 2
0
-3
-2
-1
0
2
1
Log Antigon Concentration ( I “
FIG.4. Competitive inhibition of monoclonal antibody rho 3 A 6 binding to rhodopsin by synthetic peptide analogs of the COOH-terminal region of rhodopsin. The closed circles denote peptides 2, AcR(1’-12’)-OH; the open triangles, peptide 24, AcR(8’12’)amide; the open squares,peptide 23, AcR(8’-14’) amide,and the closed triangles, peptide 22, AcR(8’-18’) amide. 1‘ Rod
0 0 0
Ala
-
COOH
Cone
@- Ser -@-Val -@-Pro - Ala
-
COOH
Thr - Ser - G l n - Val
-
Ala - Pro
-
11773
respectively, in peptide 2 (1’-12’). The contributions of all other side chains in the COOH-terminal sequence were insignificant to antibody rho 3D6 binding. These results suggest that the minimum sequence required for maximal antibody binding is 1’-4’. This conclusion is in agreement with the results of Hicks and Molday (1986) who showedthat peptides of lengths 1’-4’ to 1’-18’ were equally effective inhibitors of rho 3D6 antibody binding to immobilized bovine rhodopsin. No inhibition was observed with the 1‘-2’ peptide or with peptide 2’-13’. Our results clearly show that removal of either the ionized carboxyl group by substitution with an isosteric amide group or removal of the @-carbon of alanine 1’ by replacement with glycine completely removed peptide binding to antibody rho 3D6. A very interesting observation can explain the cross-reactivity between rho 3D6 and opsin from cone cells (Hicks and Molday, 1986). The sequence of the COOH terminus of rod (Hargrave and Fong, 1977) and green and red cone protein (Nathans et al., 1986) is shown in Fig. 5. Absolute identity in sequence is observed for the residues suggested in this study to be important for rho 3D6 binding, that is, the side chains of 4‘, 2‘, and 1’ and the a-COOH groups of 1’.The sequence differences occur at positions 3’ and 5’ which were shown to be unimportant for peptide binding to the antibody rho 3D6. Obviously increase in the size of the side chain at position 3’ from alanine in rhodopsin to serine in opsin is not able to interfere with antibody binding, suggesting that this side chain may not be in the antibody-binding site. DISCUSSION
Thisstudy was carried outas part of this laboratory’s investigation of the specificity of antibodies produced to a protein immunogen. The results of the present study suggest that linear immunogenic determinants on native proteinsmay sequence differences. not only be abundant but are generally small in length and in the 13‘-18’ region determined (Table I). Both antibodies vary from 4-11 amino acid residues (Fig. 3B). These results rho 3A6 and rho 1C5 show maximal binding to the peptide also suggest that linear immunogenic determinants on the containing the residues 8’-18’. Antibody rho 1C5 binds 2-fold surface of native proteins can be considerably smaller than more tightly to peptide 22 (8’-18’) than peptide 1 (1’-18’) the minimum of 7 residues suggested by Lerner et al. (1981). (Table 11). These results again demonstrate that shortening The linear determinant for antibody rho 3D6 is the smallest the chain length to remove residues unimportant to antibody native protein immunogenic determinant yet to be reported binding (1’-7’ region) results in an increase in binding affinity (contained within 4 residues and involves only three of the for the peptide. Although antibodies rho 1C5 and rho 3A6 four side chains). Recently, Worobec et al. (1985) reported a bind to the 8’-18’ region, different residues are involved in linear immunogenic determinant at the NH2 terminus of a binding. For example, alanine 16’ has no effect on antibody pilin protein (contained within 5 residues). In fact this deterrho 3A6 binding but is of major importance to antibody rho minant was the immunodominant region of the pilin protein 5-residue peptide was capable of 1C5 binding when replaced by glycineresidue at 16’ (I5ovalues (11,500 daltons),anda of 28 p~ for peptide 19 [Ac-(Gly)l6’R(1’-18’)-OH] compared titrating 80% of the polyclonal antibodies produced to the to 2.5 p~ (11-folddecrease) for peptide 1containing the native native protein. Similarly, only three of five side chains were sequence 1’-18’, Table I). Similarly, threonine 13’ substituted of major importance for antibody binding. Interestingly, these by alanine in the 1’-18’ peptide results in a 20-fold decrease two small determinants involved the NH2 or COOH terminus in antibody rho 1C5 binding compared to a 2.5-fold decrease of the protein. The 4-residue determinant on rhodopsin for for antibody rho 3A6 (compare peptides 1 and 16, Table I). binding antibody rho 3D6 involvesthe ionized a-COOH group Threonine 14’ is unimportant for antibody rho 1C5 binding, at the COOH terminus of the protein and the 5-residue but a 2.5-fold decrease in binding is observed for rho 3A6, determinant for EDP208 pilus-specific antibodies involved when peptide 17, Ac(Alal4’)R(1’-18’)-OH is compared to the Ne-acetyl group at theNH2 terminus. This study has allowed us to accurately delineate the funcpeptide 1. Monoclonal Antibody rho 306 Binding Studies-As shown tional groups involved in the antigen-antibody interactionfor in Fig. 3B, Tables I and 11, the region 1’-4’ is important for three anti-rhodopsin monoclonal antibodies, rho 1D4, 3A6, peptide binding to monoclonal antibody rho 3D6. The effect and 3D6. The following general observations can be made: Ionic Interactions-In this report binding through ionic of single amino acid substitutions on peptide binding to antirhodopsin monoclonal antibody rho 3D6 is shown in Table I. interactions has been shown to be very important in antigenThe most important side chains in antibody interaction are antibody interactions at the COOH terminus of a protein. alanine l’, proline 2‘, and valine 4’ where no detectable The binding of monoclonal antibodies rho 1D4 and rho 3D6 binding was observed when these positions were substituted to synthetic peptides was dramatically reduced or prevented by glycine at position 1‘ and alanine at positions 2‘ and 4’, when the free a-carboxyl group of the COOH-terminal Ala is
FIG.5. The amino acid sequence of the COOH-terminal region of bovine rhodopsin from rod cells and bovine opsin from human red and green cone cells. The circled residuesdenote
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Interaction
Antigen-Antibody
replaced by an amide group. Landsteiner and Van der Scheer (1934) were the first to suggest that thedistal part of a hapten is immunodominant. Using dipeptides as haptens, these researchers demonstrated that the COOH-terminal amino acid provided the major binding energy in antigen-antibody interaction. Schechter et al. (1971) showed that the free carboxyl terminus is critical in the binding of antipolyalanyl peptide antibodies. This binding was abolished if peptides containing COOH-terminal amides were used. Similar results have been reported by Gras-Masse et al. (1985) in raising antipeptide antibodies to streptococcal M protein peptides. Ionic interactions can also dominate immunogenic determinants at sites other than the COOH terminus. This was observed for antibody rho 3A6 where the peptide antigen does not bind when glutamic acid 8’ or lysine 10’ are replaced by glutamine and alanine,respectively. Getzoff et al. (1987) have described a recognition, induced-fit, and binding model for antibody binding to myohemerythrin where the critical ionic recognition step is followedby interaction with previously buried residues, leading to binding. Hydrophobic Interactions-Immunogenic determinants are not restricted to highly charged hydrophilic regions on the surface of a protein. For example, when any one of the 3 residues (alanine 3‘, glutamine 5’, and threonine 7’) of the peptide antigen for rho 1D4 is replaced by glycine at 3’ or alanine at 5’ and 7’, binding of peptide antigen to antibody is totally prevented(Fig. 3).Of the 9 residues involved in this determinant (1’-9’) only the a-COOH group and the side chain of glutamic acid 8’ would be charged. The loss of the charged groups on Glu-8’ results in only a 7-fold decrease in antibody binding whereas replacement of the COOH-terminal carboxyl group by an amide group in the peptide antigen causes a 175-fold decrease. Similarly, the 3 side chains involved in antigen binding to antibody rho 3D6 are all hydrophobic (Val-4’, Pro-2’, and Ala-l‘, Table I) with the only hydrophilic residue being the charged a-carboxyl group to residue 1’. Immunogenic determinants may be dominated by hydrophobic interactions as is the case with the rho 3D6 determinant described above. This result agrees with Worobec et al. (1985) where the threemost important functionalgroups to antigen binding were the N“-acetyl methyl group and two leucine side chains at positions 3 and 4 of the 5-residue NH2terminal determinant (replacement of the acetyl group by a formyl group or either leucine residues by a glycine residue resulted in a 8,300-, 6,400-, and 20,000-fold decrease in binding of the peptide antigen to antibody, respectively). Thus, the most stable contact points in the antigen-antibody complex may be hydrophobic. This is reminiscent of observations by Lemieux (1982) who showed that hydrophobic interactions are important in theinteraction of carbohydrates with antibodies and lectins. Also, Atassi (Atassi and Webster, 1983) has reported that antigenic sites are not necessarily hydrophilic and that hydrophobic interactions are very important in binding interactions. In order for hydrophobic determinants to exist, they must be at or near the surface, and this is usually accomplished by a group of nearby charged and noncharged hydrophilic residues which bring this hydrophobic region to the surface. For the antibody rho 3D6 determinant, thisis accomplished by the hydrophilicity of the COOHterminal region 5‘-18‘ where the only hydrophobes are residues valine 12’ and Ala-16’ (Fig. 3). Additive Effect-The observations with antibodies rho 3A6 and rho 1D4 (Fig. 3) suggest that although certain side chains can dominate the interaction of the antigen with antibody, the free energies of all the contact points are additive (Schechter, 1970) and a certain minimum free energy must be present
to achieve binding (Eisen, 1980). For example, whereas peptide 25 (1’-9’) binds most tightly with antibody rho 1D4, peptide 26 (l’”7’) missing 2 residues does not bind (Table 11). These side chains aremuch less important than the a-COOH group of residue 1’ and side chains of residues 2‘-7’ (Fig. 3). Similarly, peptide 22 (8’-18’) binds most tightly with antibody rho 3A6. In this case the loss of residues 15’-18’ results in a decrease binding affinity of 1.7-fold (peptide 23, 8’-14’, Table 11)while the loss of residues 13’-18’ results ina further decrease in binding of 4.0-fold (peptide 24, 8‘-12‘ versus peptide 23,8’-14‘, Table 11). Withthisdeterminant the minimum binding energy still has not been reached with the peptide antigen 8’-12’ which is only 5 residues. These observations strongly suggest a greater specificity with antibody rho 3D6 or rho 1D4 than antibody rho 3A6 and mutations anywhere in theregion of the protein antigen 13’-18‘ may be accommodated with only a small decrease in binding affinity with antibody rho 3A6. Specificity and Affinity-Antibodies with different specificities directed to thesame region of the protein antigen canbe produced in an immune response to a protein antigen. Antibodies rho 1C5 and rho 3A6 bind most tightly to the same peptide antigen 8’”‘ yet their specificities are different. For example the side chain of alanine 16‘ is important for rho 1C5binding but makes no contribution to binding to antibody rho 3A6 (Table I). Similarly, the side chain of alanine 1’ is critical for antibody rho 3D6 binding (this antibody does not bind to peptide 4 with a glycine at position l’, Table I) yet unimportant for rho 1D4 binding (the Iso values are 2.5 and , I, for peptides 4 and 2, respectively). Even 0.9 p ~ Table though both rho 1D4 and rho 3D6 are directed to theCOOHterminal carboxyl group, rho 1D4 is specific for Pro-2’, Ala3’, Gln-5’, and Thr-7’, whereas rho 3D6 is specific for Alal‘,Pro-Z’, and Val-4‘. Interestingly, the combination of binding sites for the four monoclonal antibodies used in thisstudy, each with their own specificity, cover the complete 1‘-18’ sequence which is accessible on rhodopsin. The resultsinthis study clearly demonstrate the high specificity of antibodies. The removal of a single methyl group in the antigenic determinant can totally prevent antibody binding (replacement of alanine 3‘ by glycine in the peptide antigen for antibody rho 1D4(Fig. 3) or replacement of alanine 1’by glycine in the peptide antigen for antibody rho 3D6 (Table I)). Synthetic peptide antigens may contain the entirespecificity site for the protein antigens’ interaction with antibody and yet the protein antigen may bind more strongly to the antibody. This is shown in Table I1 where the antirhodopsin monoclonal antibodies prepared from “native” rhodopsin in disc membranes bound better to native rhodopsin (discs) than the synthetic peptides. The differences ISo values for the most efficient peptide binding and native rhodopsin binding to monoclonals varied from 7-fold weaker in the case of rho 1C5 to 17-, 65-, and 2,330-fold in the case of rho 3A6, rho 1D4, and rho 3D6 (Table 11). Peptide antigens representing regions of a protein antigen bind best to the anti-protein antibody when the sequence is shortened to contain only those residues binding to thespecificity site in the antibody. These results are shown in Table I1 where maximal peptide antigen binding was obtained with peptides 1’-9’ for antibody rho 1D4, 8‘-18’ for antibodies rho 3A6 and rho 1C5 and 1’-4’ for antibody rho 3D6. This can be explained by an increase in peptide flexibility as the peptide is shortened which allows it to fold more easily into the proper orientation for binding in theantibody combining
Interaction
Antigen-Antibody
site. In contrast, the decrease in binding affinity for peptides compared to thenative protein can be explained by the protein antigen removing the flexible interference and fixing the region in the exact orientation required for binding. This interpretation is supportedby the x-ray diffraction studies of the lysozyme-Fab, protein antigen-antibodycomplex (Amit et al., 1986) where the classical “lock and key” is an adequate simplification to describe this interaction. It should be noted thatthestructure of the complex between antibody and influenza virus neuraminidase show featuresinconsistent with a rigid lock and key model for antigen-antibody interactions and that conformational changes can be induced in the antigen by antibody (Colman et al., 1987). However, both x-ray studies involve discontinuous epitopes where the interface between antigen and antibody involves a minimum of 16 residues on the antigen surface. The native monoclonals described in this report recognize small linear determinants between 4 and 11 residues. It will be interesting to compare the x-ray diffraction results of an antigen-antibody complex involving a linear epitode on the surface of a protein antigen. Cross-reactivity-Cross-reactivity between protein antigens can be explained by conservation of the critical residues in the combining site (see Fig. 3 and Table I for the results of antibody rho 3D6). Many workers have reported using monoclonal antibodies to locate or define antigenic sites. An ideal approach to the problem of formulating a binding mechanism is to study the effect of each residue inthe antigenic site. This is best achieved by the chemical synthesis of antigenic analogs which allow us to study sequences that do not occur naturally. We feel that thestudy presented in thispaper will provide a useful approach for delineating antigenic determinants and examining the molecular basis of antigen-antibody interactions. For example information on the amino acid residues of rhodopsin which are important for monoclonal antibody binding has enabled Oprian et al. (1987) to purify functionally active rhodopsin from COS cells expressing the synthetic gene for bovine rhodopsin using antibody rho 1D4 and a synthetic COOH-terminal peptide in conjunction with immunoaffinity chromatography. The results summarized in Fig. 3 show that antibodies with a variety of specificities, both ionic and hydrophobic, can recognize small linear determinants. REFERENCES Amit, A. G., Mariuzza, R.A., Phillips, S. E. V., and Poljak, R. J. (1986) Science 2 3 3 , 747-753 Atassi, M. Z., and Webster, R. G. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,840-844 Benjamin, D. C., Berzofsky, J. A., East, I. J., Gurd, F. R. N., Hannum, C., Leach, S. J., Margoliash, E., Michael, J. G., Miller, A., Prager, E. M., Reichlin, M., Sercarz, E. E., Smith-Gill, S. J., Todd, P. E., and Wilson, A.C. (1984) Annu. Reu. Immunol. 2 , 67-101 Clark, S. P., and Molday, R. S. (1979) Biochemistry 18,5868-5873 Colman, P. M., Laver, W. G., Varghese, J. N., Baker, A. T., Tulloch,
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P. A., Air, G. M., and Webster, R. G. (1987) Nature 326,358-363 Eisen, H. N. (1980) in Immunology, An Introduction to Molecular and Cellular Principles of the Immune Responses, 2nd Ed., pp. 302-305, Harper and Row, New York Erickson, B. W., and Merrifield, R. B. (1976) in The Proteills (Neurath, H., and Hill, R. L., eds) Third Ed., pp. 257-527, Academic Press, New York Getzoff, E. D., Geysen, H. M., Rodda, S. J., Alexander, H., Tainer, J. A., and Lerner, R. A. (1987) Science 2 3 5 , 1191-1196 Gras-Masse, H., Jolivet, M., Audibert, F., Beachey, E., Chedid, L., and Tartar,A. (1985) in Synthetic Peptides inBiology and Medicine (Alitalo, K., Partanen, P., and Vaheri, A., eds) pp. 105-112, Elsevier Scientific Publishing Co., New York Hargrave, P. A., and Fong, S.-L. (1977) J. Supramol. Struct. 6,559570 Hargrave, P. A., McDowell, J. H., Curtis, D. R., Wang, J. K., Juszczak, E., Fong, S.-L., Mohana Rao, J. K., and Argos, P. (1983) Biophys. Struct. Mech. 9,235-244 Hicks, D., and Molday, R. S. (1986) Exp. Eye. Res. 42,55-71 Hunter, W. M., and Greenwood, F. C. (1962) Nature 194,495-496 Kent, S., and Clark-Lewis, I. (1985) in Synthetic Peptides in Biology and Medicine (Alitalo, K., Partanen, P., and Vaheri, A., eds) pp. 29-57, Elsevier Scientific Publishing Co., New York Landsteiner, K., and Van der Scheer, J. (1934) J. Exp. Med. 59,769780 Lemieux, R. U. (1982) Frontiers in Chemistry, pp. 3-26, Pergamon Press, Oxford Lerner, R. A,, Sutcliff, J. G., and Shinnick, T. M. (1981) Cell 2 3 , 309-310 Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275 MacKenzie, D., and Molday, R. S. (1982) J. Biol. Chem. 2 5 7 , 71007105 MacKenzie, D., Arendt, A., Hargrave, P., McDowell, J. H., Molday, R. S. (1984) Biochemistry 23,6544-6549 Molday, R. S., and MacKenzie, D. (1983) Biochemistry 22,653-660 Molday, R. S., and MacKenzie, D. (1985) Biochemistry 24,776-781 Nathans, J., Thomas, D., and Hogness, D. S. (1986) Science 2 3 2 , 193-202 Oprian, D. D., Molday, R. S., Kaufman, R. J., and Khorana, G . H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,8874-8878 Ovchinnikov, Y.A., Abdulaev, N. G., Feigina, M. Y., Artamonov, I. D., Zolotarev, A. S., Kostina, M. B., Bogachuk, A. S., Miroshnikov, A. I., Martinov, V. I., and Kudelin, A. B. (1982) Bioorg. Khim. 8 , 1011-1014 Parker, J. M. R., and Hodges, R. S. (1985) J. Protein Chem. 3 , 465478 Paterson, Y.(1985) Biochemistry 24,1048-1055 Schechter, I. (1970) Nature 228,639-641 Schechter, I., Clerici, E., and Zazepitzki, E. (1971) Eur. J. Biochen. 18,561-572 Smith, H. E., Stubbs, G. W., and Litman, B. J. (1975) Exp. Eye. Res. 20,211-217 Stewart, J. M., and Young, J. D. (1984) in Solid-Phase Peptide Synthesis 2nd, Ed., Pierce Chemical Co., Rockford, IL Talbot, J. A., and Hodges, R. S. (1981) J. Biol. Chem. 256, 27982802 Van Regenmortel, M. H. V. (1985) in Synthetic Peptides in Biology and Medicine (Alitalo, K., Partanen, P., and Vaheri, A., eds) pp. 67-74, Elsevier Scientific Publishing Co., New York Wong, S., and Molday, R. S. (1986) Biochemistry 25,6294-6300 Worobec, E. A., Paranchych, W., Parker, J. M. R., Taneja, A. K., and Hodges, R. S. (1985) J. Biol. Chem. 2 6 0 , 938-943
