Specific Binding of a Synthetic Peptide Derived from an Antibody Complementarity Determining Region to Phosphatidylserine

Sets of short (12 residues) cellulose-bound synthetic overlapping peptides derived from the sequences of the variable regions of the heavy and light chains of three different antibodies (an anti-thyroglobulin antibody, the HyHEL-5 anti-lysozyme antibody, and an anti-angiotensin II antibody) were used to systematically assess the antigen binding capacity of peptides from the antibody paratope outside their natural molecular context. Peptides enclosing one or several of the complementarity determining region (CDR) residues had antigen binding activity, although the most active peptides were not necessarily those bearing the greatest number of CDR residues. Several residues from the framework region, preceding or following the CDR, were found to play a role in binding. Affinity constants from 4.1 ؋ 10 ؊7 to 6.7 ؋ 10 ؊8 M ؊1 for the soluble form of 9 lysozymebinding dodecapeptides were measured by BIAcore analysis. Alanine scanning of lysozyme-binding hexapeptides from the HyHEL-5 sequence identified 38 residues important for binding, of which 22 corresponded to residues that had been shown by x-ray crystallography to be at the interface between HyHEL-5 and lysozyme. Our results could be of interest for the rational identification of biologically active peptides derived from antibody sequences and in providing an experimental basis for mutagenesis of the antibody paratope.Antibody molecules bind antigens with high affinity and specificity by synergistically using multiple noncovalent forces. The combining site (paratope), whose shape is complementary to the epitope on the antigen, is made up of the hypervariable regions, also called complementarity determining regions (CDRs) 1 (1). It is commonly accepted that there are three CDRs in the light chain (L1, L2, and L3) and in the heavy chain (H1, H2, and H3). These CDRs fold into turn structures that are stabilized by the ␤-sheet framework of the variable domains. The interface between antibodies and antigens has been precisely described by x-ray crystallographic studies, and several complexes between Fab fragments of monoclonal antibodies and peptide or protein antigens have been recently described (for reviews see Refs. 2-4). The structures of antibody-antigen complexes indicate that at least four of the CDRs, and in some cases all six CDRs, contribute to antigen binding (5). Residues in the framework have rarely been reported to participate in this interaction (6, 7).Antibody-peptide or antibody-protein complexes are excellent model systems to study the physicochemical requirements for molecular recognition. Unfortunately, it is a difficult task to obtain crystals suitable for the structural elucidation of antibody fragments in complex with proteins or peptides. Therefore, other approaches to obtain information about the key residues involved in the interaction would be very useful, in particular for paratope mutagenesis. Some workers have demonstrated that synthetic peptides derived from the amino acid sequences of CDRs bind antigens with specificities similar to t...

Section: Discussionsupporting

confidence: 74%

Systematic Exploration of the Antigen Binding Activity of Synthetic Peptides Isolated from the Variable Regions of Immunoglobulins

Laune

Molina

Ferrières

et al. 1997

“…Antibody variable domains comprise a framework of ␤-sheets surmounted by antigen-binding loops. We can postulate that critical residues, identified in the Spot assay and confirmed by preliminary Alascan analysis (data not shown), located in the ␤-sheet framework closely underlying the CDRs, probably do not participate in direct interaction with CD4, but could induce a binding conformational state mimicking some of (36). These six critical residues from the framework regions of the ST40 antibody possess one or several of these characteristics, in agreement with previous results obtained in our laboratory on the interactions of mAb HyHEL-5/lysozyme (40) and mAb Tg10/thyroglobulin and mAb 4D8/ angiotensin II.…”

Section: Discussionmentioning

confidence: 75%

“…The diversity of paratopes is mainly generated by the sequences of the CDRs found in V H and V L , which are exposed hypervariable loop structures. Antigen binding by peptide sequences from selected CDRs of mAbs has been demonstrated to have specificities similar to those of the original antibody molecule (31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Our previous results showed that the systematic exploration of the antigen binding capacity of short peptides derived from an antibody sequence leads to the identification of numerous paratope-derived peptides (PDPs) that display significant affinity for the antigen (40).…”

Section: Entry Into Cells (5-7) Cd4 Is a Member Of The Immunoglobulimentioning

confidence: 99%

Synthetic Peptides Derived from the Variable Regions of an Anti-CD4 Monoclonal Antibody Bind to CD4 and Inhibit HIV-1 Promoter Activation in Virus-infected Cells

Monnet¹,

Laune²,

Laroche‐Traineau³

et al. 1999

The monoclonal antibody (mAb) ST40, specific for the immunoglobulin complementarity-determining region (CDR) 3-like loop in domain 1 of the CD4 molecule, inhibits human immunodeficiency virus type 1 (HIV-1) promoter activity and viral transcription in HIV-infected cells. To design synthetic peptides from the ST40 paratope that could mimic these biological properties, a set of 220 overlapping 12-mer peptides frameshifted by one residue, corresponding to the deduced ST40 amino acid sequence, was synthesized by the Spot method and tested for binding to recombinant soluble CD4 antigen. Several peptides that included in their sequences amino acids from the CDRs of the antibody and framework residues flanking the CDRs were found to bind soluble CD4. Eleven paratope-derived peptides (termed CM1-CM11) were synthesized in a cyclic and soluble form. All the synthetic peptides showed CD4 binding capacity with affinities ranging from 1.6 to 86.4 nM. Moreover, peptides CM2, CM6, CM7, CM9, and CM11 were able to bind a cyclic peptide corresponding to the CDR3-like loop in domain 1 of CD4 (amino acids 81-92 of CD4). Peptide CM9 from the light chain variable region of mAb ST40 and, to a lesser extent, peptides CM2 and CM11 were able to inhibit HIV-1 promoter long terminal repeat-driven ␤-galactosidase gene expression in the HeLa P4 HIV-1 long terminal repeat ␤-galactosidase indicator cell line infected with HIV-1. The binding of mAb ST40 to CD4 was also efficiently displaced by peptides CM2, CM9, and CM11. Our results indicate that the information gained from a systematic exploration of the antigen binding capacity of synthetic peptides from immunoglobulin variable sequences can lead to the identification of bioactive paratope-derived peptides of potential pharmacological interest.

“…The N-terminal amino acid sequences of the Id8F7-reactive peptide fragments were determined by the amino acid sequencer as described above. The synthetic peptides derived from the consensus sequence of PKC and PS decarboxylase (PSD) were synthesized using an automated peptide synthesizer (Advanced Chemtech, model 396 MPS), and an extra cysteine residue was added to the C-terminal end of the peptides (22). The peptides were purified by reverse-phase HPLC with an ODS 120 column (Tosoh Co.).…”

Section: Methodsmentioning

confidence: 99%

A Novel Phosphatidylserine-binding Peptide Motif Defined by an Anti-idiotypic Monoclonal Antibody

Igarashi¹,

Kaneda²,

Yamaji

et al. 1995

A monoclonal anti-idiotypic antibody, Id8F7, previously shown to bind to a phosphatidylserine (PS)-specific binding site on protein kinase C (PKC) has been used to identify a 12-amino acid consensus sequence shared by PKC and phosphatidylserine decarboxylase (PSD). The 14-amino acid synthetic peptide derived from the corresponding region of PSD (amino acids 351-364 of the enzyme from Chinese hamster ovary cells) bound effectively and specifically to PS, and that derived from rat PKC␥ (amino acids 227-240) bound weakly but specifically to PS. Analysis of binding of Id8F7 to various synthetic peptides revealed that the consensus sequence motif, FXFXLKXXXKXR, is responsible for the interaction with both Id8F7 and PS. The results suggest that the conserved amino acid residues represent a basic structural motif for the specific interaction with PS, and the corresponding regions of PKC and PSD form the PS-specific binding sites of these enzymes.Although it is generally accepted that phospholipids in membranes are essential for the catalytic activity of many membrane-bound enzymes, it is still unclear how phospholipids regulate the activity of the enzymes. Phosphatidylserine (PS) 1 in membranes is known to be an essential cofactor for the activation of protein kinase C (PKC) (1-3) and blood coagulation (4 -6). Recent analyses have shown that it regulates the activity of various enzymes such as c-Raf-1 protein kinase (7), nitric oxide synthase (8), Na ϩ /K ϩ -ATPase (9), Dynamin GTPase (10), and diacylglycerol kinase (11) and acts as a ligand in recognition of apoptotic cells (12,13). PKC is a family of phospholipid-dependent kinases, and its enzymatic activity is allosterically regulated by 1,2-diacyl-sn-glycero-3-phospho-Lserine (PS) (14,15). Analyses of the interaction of conventional PKC (cPKC) with membrane lipids support a two-step model for the activation of cPKC, which includes initial binding to membranes that does not require PS since cPKC binds various acidic phospholipids in a Ca 2ϩ -dependent manner and allosteric activation by specific interaction with PS and diacylglycerol (15). The initial binding of cPKC to membranes is believed to be mediated by the Ca 2ϩ -dependent phospholipid-binding (CaLB) domain (amino acids 186 -233 of PKC␥), a sequence motif that is also found in other cytosolic proteins such as cytosolic phospholipase A 2 , GTPase-activating protein, and synaptotagmin (16,17). However, it has been difficult to validate the presence of the PS-specific binding site on PKC, since the enzyme interacts with multiple phospholipid molecules during activation. Our previous studies showed that the binding to PKC of the monoclonal anti-idiotypic antibody Id8F7 raised against the combining site of the PS-specific antibody is inhibited by PS but not by other phospholipids including the synthetic PS analogue, 1,2-diacyl-sn-glycero-3-phospho-D-serine (18,19). The binding of Id8F7 to PKC is significantly enhanced by the presence of diacylglycerol and is independent of the presence of Ca 2ϩ (19). These observa...