Probing the Basis of Antibody Reactivity with a Panel of Constrained Peptide Libraries Displayed by Filamentous Phage

Bonnycastle, Lori L.; Mehroke, Jarnail S.; Rashed, M. A.; Gong, Xiaobo; Scott, Jamie K.

doi:10.1006/jmbi.1996.0284

Cited by 153 publications

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“…Panning and Screening of Phage-displayed Peptides-Four phagedisplayed libraries (LX-8/f88.4, X 15 /f88.4, X 8 CX 8 /f88.4, and X 30 /f88.4) in which peptides were displayed at the N terminus of the pVIII coat protein on the surface of the filamentous bacteriophage M13, were obtained from Dr. J. Scott (Simon Fraser University, Burnaby, Canada) (45). Phages specific for aAb T13 were selected by biopanning as described previously (46).…”

Section: Methodsmentioning

confidence: 99%

Localization of the Discontinuous Immunodominant Region Recognized by Human Anti-thyroperoxidase Autoantibodies in Autoimmune Thyroid Diseases

Bresson¹,

Cérutti²,

Devauchelle³

et al. 2003

Journal of Biological Chemistry

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The discontinuous immunodominant region (IDR) recognized by autoantibodies directed against the thyroperoxidase (TPO) molecule, a major autoantigen in autoimmune thyroid diseases, has not yet been completely localized. By using peptide phage-displayed technology, we identified three critical motifs, LXPEXD, QSYP, and EX(E/D)PPV, within selected mimotopes which interacted with the human recombinant anti-TPO autoantibody (aAb) T13, derived from an antibody phage-displayed library obtained from thyroid-infiltrating TPO-selected B cells of Graves' disease patients. Mimotope sequence alignment on the TPO molecule, together with the binding analysis of the T13 aAb on TPO mutants expressed by Chinese hamster ovary cells, demonstrated that regions 353-363, 377-386, and 713-720 from the myeloperoxidase-like domain and region 766 -775 from the complement control protein-like domain are a part of the IDR recognized by the recombinant aAb T13. Furthermore, we demonstrated that these regions were involved in the binding to TPO of sera containing TPO-specific autoantibodies from patients suffering from Hashimoto's and Graves' autoimmune diseases. Identification of the IDR could lead to improved diagnosis of thyroid autoimmune diseases by engineering "mini-TPO" as a target autoantigen or designing therapeutic peptides able to block undesired autoimmune responses.Human thyroid peroxidase (TPO), 1 described previously as the "thyroid microsomal antigen" (1), is a membrane-bound enzyme expressed at the apical pole of thyrocytes (2). TPO generates the functional form of thyroglobulin by iodination and coupling of tyrosine residues (3). During autoimmune thyroid diseases (AITD), TPO represents a major target for the immune system (4, 5), leading to high titer TPO-specific autoantibodies (aAbs) in the sera of patients suffering from Hashimoto's thyroiditis and Graves' disease. Besides their role as efficient and early diagnostic markers of AITD, TPO-specific aAbs also act as effector molecules either through modulating antigen presentation to T cells or by mediating thyroid destruction after complement activation or antibody-dependent cell cytotoxicity (6 -12). Alignment studies and structural homologies have shown that TPO is formed by three distinct domains: a myeloperoxidase (MPO)-like, a complement control protein (CCP)-like, and an EGF-like domain, from the N-to the Cterminal extremities (13). Although the structure of each domain has been elucidated in part by three-dimensional modeling (13-16), the full three-dimensional structure of TPO remains unknown, even though low resolution crystals have been obtained (17,18). The flexibility observed for the hinge regions probably make difficult the exact positioning of each domain in relation to the others (15).These observations denote a highly complex structure of TPO, thus explaining the reason why TPO aAbs from patients' sera preferentially recognize discontinuous epitopes on . Different approaches have been used to determine the epitopic regions recognized by anti-TPO aAbs from ...

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Section: Methodsmentioning

confidence: 99%

Localization of the Discontinuous Immunodominant Region Recognized by Human Anti-thyroperoxidase Autoantibodies in Autoimmune Thyroid Diseases

Bresson¹,

Cérutti²,

Devauchelle³

et al. 2003

Journal of Biological Chemistry

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“…The phage-displayed peptide libraries are as described previously (29). Human MAb 2G12 Fab was produced as before (11).…”

Section: Methodsmentioning

confidence: 99%

“…Phage were produced in E. coli K91 cells, following Bonnycastle et al (29). Electrocompetent, F − E. coli MC1061 cells were used for library construction, and strain CJ236 was used to amplify the phage used as a source of single-stranded viral DNA for site-directed mutagenesis.…”

Section: Bacterial Strains and Dna Constructsmentioning

confidence: 99%

“…The 2G12.1 peptide sublibrary was constructed using the f88-4 phage vector (30); singlestranded, covalently closed circular DNA was used as template, following the procedure described in (29), and a degenerate oligonucleotide was synthesized using the two-column, "divide-couple-recombine method," as described by Haaparanta and Huse (31). In the resulting library, the amino acids at each position were either from the 2G12.1 peptide or a random residue encoded by a degenerate NNK codon (in which n=A, G, C, or T and K=G or T).…”

Section: Bacterial Strains and Dna Constructsmentioning

confidence: 99%

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A peptide inhibitor of HIV‐1 neutralizing antibody 2G12 is not a structural mimic of the natural carbohydrate epitope on gp120

Menéndez

Calarese

Stanfield³

et al. 2008

FASEB j.

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MAb 2G12 neutralizes HIV-1 by binding with high affinity to a cluster of high-mannose oligosaccharides on the envelope glycoprotein, gp120. Screening of phage-displayed peptide libraries with 2G12 identified peptides that bind specifically, with K d s ranging from 0.4 to 200 μM. The crystal structure of a 21-mer peptide ligand in complex with 2G12 Fab was determined at 2.8 Å resolution. Comparison of this structure with previous structures of 2G12-carbohydrate complexes revealed striking differences in the mechanism of 2G12 binding to peptide vs. carbohydrate. The peptide occupies a site different from, but adjacent to, the primary carbohydrate-binding site on 2G12, and makes only slightly fewer contacts to the Fab than Man 9 GlcNAc 2 (51 vs. 56, respectively). However, only two antibody contacts with the peptide are hydrogen bonds in contrast to six with Man 9 GlcNAc 2 , and only three of the antibody residues that interact with Man 9 GlcNAc 2 also contact the peptide. Thus, this mechanism of peptide binding to 2G12 does not support structural mimicry of the native carbohydrate epitope on gp120, since it neither replicates the oligosaccharide footprint on the antibody nor most of the contact residues. Moreover, 2G12.1 peptide is not an immunogenic mimic of the 2G12 epitope, since antisera produced against it did not bind gp120.-Menendez, A., Calarese, D. A., Stanfield, R. L., Chow, K. C., Scanlan, C. N., Kunert, R., Katinger, H., Burton, D. R., Wilson, I. A., Scott, J. K. A peptide inhibitor of HIV-1 neutralizing antibody 2G12 is not a structural mimic of the natural carbohydrate epitope on gp120. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptHuman monoclonal antibody (MAb) 2G12 efficiently neutralizes a broad range of HIV-1 primary isolates in vitro (1-3) and protects from in vivo viral challenge in macaques in combination with other antibodies (4-6). MAb 2G12 binds with high affinity to a unique, conserved epitope on the HIV-1 envelope that is formed by a cluster of N-linked, high-mannose glycan groups on the "silent" face of gp120 (7-10). Crystal structures of 2G12 Fab alone, and in complex with oligosaccharides Man 9 GlcNAc 2 and Manα1-2Man, revealed that two Fab monomers assemble into an interlocked, domain-swapped dimer, that forms an extensive, multivalent binding surface (11). Four Man 9 GlcNAc 2 moieties were observed bound to the Fab dimer in the crystal structure, occupying the two canonical antigen-binding sites, and two novel ones located at the assembled interface of the two interlocked V H domains. Since the identification of its epitope, MAb 2G12 has received significant attention as a template for HIV-1 vaccine development. Considerable effort has been directed at characterizing its oligosaccharide-binding profile (12)(13)(14)(15), and the generation of novel antigens in the form of synthetic oligomannoses (13,16,17) or short, synthetic glycopeptides (18-20). In an attempt to generate immunogens that elicit 2G12-like antibodies, we chose to isolate peptides t...

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“…The wells were washed six times and bound Ab was detected by a 45 min incubation at room temperature with goat anti-mouse IgG (Fc specific):HRP conjugate (Pierce), diluted 1/1500 in TBS containing 0.1% Tween 20. HRP was detected with 35 μl 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS; Sigma) solution containing H 2 O 2 [51]. The OD 405-490 of each reaction was measured by an EL 312e ELISA plate reader (Bio-Tek).…”

Section: Elisa and Determination Of Serum Titersmentioning

confidence: 99%

Filamentous phage as an immunogenic carrier to elicit focused antibody responses against a synthetic peptide

Houten

Zwick

Menéndez

et al. 2006

Vaccine

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Filamentous bacteriophage are widely used as immunogenic carriers for "phage-displayed" recombinant peptides. Here we report that they are an effective immunogenic carrier for synthetic peptides. The f1.K phage was engineered to have an additional Lys residue near the N-terminus of the major coat protein, pVIII, so as to enhance access to chemical cross-linking agents. The dimeric synthetic peptide, B2

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Probing the Basis of Antibody Reactivity with a Panel of Constrained Peptide Libraries Displayed by Filamentous Phage

Cited by 153 publications

References 0 publications

Localization of the Discontinuous Immunodominant Region Recognized by Human Anti-thyroperoxidase Autoantibodies in Autoimmune Thyroid Diseases

Localization of the Discontinuous Immunodominant Region Recognized by Human Anti-thyroperoxidase Autoantibodies in Autoimmune Thyroid Diseases

A peptide inhibitor of HIV‐1 neutralizing antibody 2G12 is not a structural mimic of the natural carbohydrate epitope on gp120

Filamentous phage as an immunogenic carrier to elicit focused antibody responses against a synthetic peptide

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