(Received for publication, August 7, 1995; and in revised form, October 2, 1995)
From the
Rabbit polyclonal antibodies against multimeric peptide antigens were found to cross-react to a significant extent with topologically related variants of the parent antigen, where the chirality of each amino acid residue (inverso derivatives), or the peptide sequence orientation (retro derivatives), was inverted or where both modifications were simultaneously introduced (retro-inverso derivatives). All peptide variants displayed similar recognition properties for antibodies and similar dose-dependent inhibitory effects on the interaction between immobilized parent antigen and corresponding antibodies. Importance of peptide side chain topology on antigenicity was evaluated analyzing the recognition properties of two sequence-simplified parent peptide variants, one lacking of the side chains in the sequence odd position and the other in even position. These two variants, prepared introducing glycine residues alternatively in the parent peptide sequence, were found to cross-react to a significant extent with the original antibody raised against the parent peptide. Analysis of molecular models of peptide enantiomeric variants in the elongated all-trans configuration suggested that the topological equivalence of alternating side chains could lead to the formation of similar recognition surfaces, thus mimicking the parent peptide antigenic structure.
Peptides biological activity is most often dependent upon well defined primary and secondary structure elements, even if in many cases peptide topology can be altered, by amino acid chirality inversion (inverso modification) (1, 2, 3) or by peptide reversal of amino acid sequence (retro modification)(4) , without altering the peptide functional activity(1, 2, 3, 4) . Retro-inverso modification of biologically active peptides is the most common peptidomimetic approach used to enhance peptide stability against the action of proteolytic enzymes and thus prolong their half-life in vivo(5, 6) . In addition, this type of modification, leaving unaltered the topology of the peptide side chain and interconverting carbonyl and amide groups from their position, constitutes a useful approach to elucidate the role of peptide backbone polarity in molecular recognition events(7) . End group modified retro-inverso peptides are isomers of linear peptides in which the direction of the sequence is reversed, the chirality of each amino acid residue is inverted, and the end groups are modified to obtain close complementarity and full topological relationship to the parent peptide. Retro-inverso peptide have been found to retain recognition properties (8) or biological activity as well as the parent peptide in many cases(9, 10, 11) , once their end groups were suitably modified.
While the effect of peptide chirality and backbone modification has been extensively investigated in terms of biological reactivity, a limited amount of work has been carried out in order to assess the importance of peptide topochemistry on antibody recognition. Earlier studies suggested the lack of cross-antigenicity between enantiomeric peptides(12, 13, 14) , as well as between enantiomeric proteins, such as rubredoxin and its all-D analogue(15) . More recently the stereochemical requirements for peptide antigenicity have been investigated with renewed interest, since preliminary studies in a limited number of cases suggested the existence of antibody cross-recognition with topologically related variants of the parent peptide. In the first study, cross-recognition between the COOH-terminal fragment 130-135 of histone H3 (IRGERA) and the corresponding all-D analogue was experimentally observed with antibodies of the IgG3 isotype raised in mice against the parent peptide(16) . In addition, it was shown than antibodies raised against the all-D peptide variant were able to recognize the all-L parent peptide equally well. In both cases, the corresponding antibodies recognized also the parent protein histone H3. On the other hand, anti-parent peptide antibodies of the IgG1, IgG2a, and IgG2b isotypes failed to bind the all-D peptide. The analysis of peptide topology importance on antigenicity has been extended in a further study, evaluating the effect of backbone polarity reversal (retro derivative), or amino acid chirality and backbone inversion (retro-inverso derivative), on antibody recognition(17) . Antibodies to the peptide analogues were produced in BALB/c mice and showed a strong correlation between cross-recognition properties and peptide topology, since mouse antibodies of the IgG1, IgG2a, and IgG2b isotypes against the parent peptide recognized only the peptide retro-inverso derivative, while antibodies of the same isotype against the inverso (all-D) peptide recognized only the corresponding retro-peptide and vice versa. These observations were supported by other studies on the cross-antigenicity between a cyclic peptide analogous to the third complementarity-determining region (CDR3) in immunoglobulin and the corresponding retro-inverso isomer(18) . Retro-inverso peptides are strongly topologically correlated with the parent peptide, since the resulting side chain disposition is similar to the parent peptide, but carbonyl and amide groups are interconverted from their positions(6, 7) . Similarly, inverso peptide are strongly correlated with the retro derivative of the parent peptide for the same reason, while no apparent structural relationship can be envisioned between retro and retro-inverso peptides, as well as between the inverso and the all-L parent peptide, since in both cases the peptides display an enantiomeric relationship. The stringent side chains topological equivalence between linear all-L peptides and the corresponding retro-inverso derivatives, or between retro and inverso peptides, could provide a sufficient explanation for the observed cross-antigenicity, while cross-recognition between parent antigen and its retro or inverso variants apparently lack of a rational mechanistic explanation.
In
this study we have investigated the cross-recognition properties of
polyclonal antibodies raised in rabbits against a 15-residue peptide
(P15) able to bind interleukin 2 and at the same time to inhibit its
interaction with the p55 interleukin 2 receptor subunit(19) .
The peptide antigen was produced in a tetrameric form
(MAP()-P15) starting from a tetradentate lysine core for
direct immunization(20) , and cross-antigenicity with a series
of topochemically related peptides, such as inverso, retro, and
retro-inverso variants was evaluated in direct binding and competition
experiments by ELISA. To further examine the role of side chain
topology of enantiomeric antigens on cross-antigenicity, two other
variants of the parent peptide were prepared, where the parent peptide
amino acid sequence was changed in order to display only side chains in
alternating position (1, 3, 5, 7, . . . or 2, 4, 6, 8, . . . ) of the
parent peptide original sequence, by replacing corresponding residues
with glycine residues. Recognition properties of sequence-simplified
variants were then evaluated by direct and competitive ELISA
experiments.
For competition experiments, various concentrations of inhibitor (MAP, conjugated peptide, or linear peptide) were incubated for 3 h at 37 °C with anti-MAP antibodies. The mixture was then added to MAP or BSA-conjugated peptide-coated wells.
After 1 h of incubation the plates were washed five times with PBS. For anti-MAP antibody detection, wells were filled with 100 µl of a horseradish peroxidase labeled goat anti-rabbit (IgG) immunoglobulin solution diluted 1:1000 with PBS-B. The plates were then left to stand for 1 h at room temperature in a humid covered box, washed with PBS 5 times, an then filled with chromogenic substrate solution consisting of 1 mg/ml o-phenylenediamine in 0.1 M sodium citrate buffer, pH 5.0, containing 5 mM hydrogen peroxide. The absorbance at 450 nm was determined with a model 2250 EIA Reader (Bio-Rad).
Figure 1: Sequences of the linear peptide P15 and its tetrameric form MAP-P15 used for immunization and the corresponding retro, retro-inverso, inverso variants, and sequence-simplified variants (+G)P15 and (-G)P15. Lower-case notation is used for amino acids in the D configuration.
Figure 2:
Inhibition of the binding between
anti-MAP-P15 antibody and immobilized MAP-P15 by BSA-conjugated linear
peptide P15 (), retro P15 (
), inverso P15 (
),
retro-inverso P15 (
), and scrambled P15 (
). Data are
representative of five different
experiments.
Figure 3: Topological relationships between enantiomeric antigens. A, superimposed molecular models of P15 fragment 10-15 (ADLDAR) and its inverso derivative in the elongated configuration, showing the alignment of residues in position 11 (Asp), 13 (Asp), and 15 (Arg). B, structures superimposed in order to align residues in position 10 (Ala), 12 (Leu), and 14 (Ala).
Figure 4: Topological relationships between parent peptide and sequence-simplified variants. Planar (A) and axial views (B) of the molecular models of P15 fragment 10-15 (ADLDAR) and the corresponding sequence-simplified variants (+G)P15 and (-G)P15 in the elongated all-trans configuration.
Figure 5:
Cross-recognition properties of
sequence-simplified peptide variants. A, binding of
affinity-purified anti-MAP-P15 antibody to immobilized P15 (),
(+G)P15 (
), (-G)P15 (
), and to scrambled P15
(
) conjugated to BSA, adsorbed to microtiter plates at
concentration 5 µg/well. B, inhibition of the binding
between anti-MAP-P15 antibody and immobilized MAP-P15 by P15 (
),
(+G)P15 (
), (-G)P15 (
), and scrambled P15
(
) conjugated to BSA. Data are representative of five different
experiments.
Experimental evidences of antigenic cross-recognition between
linear antigens and the corresponding retro-inverso isomers have been
reported only recently in at least three different cases. In the first
study, monospecific rabbit antibodies were used as conformational
probes to demonstrate surface similarities between a cyclic peptide
analogous to the third complementarity-determining region (CDR3) in
immunoglobulin and the corresponding retro-inverso isomer(18) .
In the second study, poly- or monoclonal antibodies raised against the
COOH-terminal hexapeptide of histone H3 by injecting BALB/c mice with
peptides covalently coupled to small unilamellar liposomes containing
monophosphoryl lipid A, exhibited a clear cross-reactivity with the
retro-inverso derivative(17, 22) . Similarly,
antibodies raised against the retro derivative cross-reacted with the
inverso derivative, but not with the parent linear antigen or the
retro-inverso form, at least for antibodies of the IgG1, IgG2a, and
IgG2b isotypes. Only antibodies of the IgG3 isotype displayed a
generalized cross-reactivity, as in our case, with the parent peptide
and the retro, retro-inverso, and inverso derivatives. In the third
very recent study, similar cross-reactivity was found between the major
antigenic site of foot-and-mouth disease virus and its end
group-modified retro-inverso isomer(23) . Cumulatively these
results ruled out the importance of the peptide backbone as the common
structural recognition element providing cross-reactivity between
linear antigens and the corresponding topologically related variants,
since retro-inverso peptides, while structurally similar to the linear
parent peptide in the side chain disposition, have totally inverted
carbonyl and amide groups in the backbone. The hypothesis of the
limited role of peptide backbone on antibody recognition was further
supported from data deriving from a different study, where it has been
shown that pseudopeptide analogues of the COOH-terminal hexapeptide of
histone H3 obtained by systematically replacing, in each analogue, one
peptide bond at a time by a reduced peptide bond Y
(CH-NH), maintained recognition properties for poly-
or monoclonal antibodies raised against the original parent
peptide(24) .
In our study, antigenic cross-recognition was found not only between the parent peptide and its retro-inverso derivative, or between the inverso and the retro derivatives, but with the complete set of topologically related peptides, to the same extent and with similar characteristics. These observations are in agreement with data on the cross-recognition properties of IgG3 mouse monoclonal antibodies against the COOH-terminal histone H3 hexapeptide with the all-D derivative(16, 17) . While structural similarities between retro-inverso and parent peptide, or between inverso and retro derivatives of the parent peptide, are clearly evident from a simple visual inspection of the peptide molecular models, and could constitute the simplest reason to explain cross-reactivity, no structural relatedness can be apparently detected between the parent peptide and its inverso or retro derivative. Inverso peptides are mirror images of the linear parent peptide, and consequently also the side chains are oriented in a nonsuperimposable way. The same is true for the retro and retro-inverso derivatives. But if enantiomeric peptides are aligned in the trans configuration, side chains in alternating residues (1, 3, 5, . . . or alternatively 2, 4, 6, . . . ) can be superimposed, thus leading to topologically equivalent molecular surfaces formed by alternating residues. The epitope recognized by the antibody in this case is formed not by a linear array of residues, but by alternating residues. This suggest that a peptide antigen can display two recognition surfaces, one formed by residues in odd position, the other by residues in even position, and that probably both surfaces can take part in antibody recognition events, even independently. The cross-recognition dependence on the alternating linear array of side chains has been confirmed by experiments with sequence-simplified variants of the parent peptide, which represent a molecular dissection of the parent peptide side chains topology. Peptide variants, where only parent peptide side chains in alternating position were left, still retained a certain degree of cross-antigenicity. These findings have considerable significance for a more detailed understanding of antigen-antibody interaction and could constitute an interesting route to design sequence-simplified peptides able to cross-react with antibodies of predetermined specificity. In addition, the observed cross-reactivity between peptides displaying complementary side chains could suggest the possibility of incorporating a 2-fold antibody specificity on peptide antigens, by generating chimeric peptides characterized by side chains in alternating position belonging respectively to two different antigens. The structural model proposed in our study to explain cross-recognition between enantiomeric antigens is based on the assumption that peptide binding occur in an extended and flexible conformation. A growing number of linear peptide antigens are seen to adopt an extended conformation upon binding to the corresponding antibodies(25, 26, 27) . Similarly, extended conformations dominates T-cell epitope-major histocompatibility complex of class I or II complexes(28, 29, 30) .
Our study also points out that, at least in the case systems analyzed, topologically related variants of the parent antigen do not need end group modification to display cross-antigenicity.
The
generality of the phenomenon of antigenic cross-reactivity has also
been confirmed with two other polyclonal antibodies raised against a
multimeric 13 residue peptide (GFRKYLHFRRHLL) and a 14-residue peptide
(RKFLAGLRARRLKF). In both cases the same type of antigenic
cross-recognition was observed with the complete series of
topochemically related peptides. ()
Inverso- and retro-inverso peptides are particularly stable to proteolytic treatments since their inverted chiral configuration is not easily recognized by enzymes, making these derivatives useful for their prolonged half-life in vitro and in vivo. The stability and cross-antigenicity of retro-inverso and inverso peptides could provide a novel and interesting route for the treatment of various diseases associated to the immune system(31) .