Role of Capsid Structure and Membrane Protein Processing in Determining the Size and Copy Number of Peptides Displayed on the Major Coat Protein of Filamentous Bacteriophage

Malik, Pratap; Terry, Tamsin D.; Gowda, Lalitha R.; Langara, Amagoia; Petukhov, S. A.; Symmons, Martyn F.; Welsh, Liam; Marvin, D.A.; Perham, Richard N.

doi:10.1006/jmbi.1996.0378

Cited by 129 publications

(97 citation statements)

References 43 publications

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“…11 Thus, variation in the level of expression of particular peptides may occur; this is estimated to be between 10 and 30% of the 2700 copies of pVIII display peptides. 22,23 In the present study, the highly reactive phagotopes reached a similar level of reactivity at high concentrations of CII-C1 (Fig. 1a), suggesting that a comparable level of peptide was in fact displayed on each of these phagotopes.…”

Section: Discussionsupporting

confidence: 68%

Phagotopes derived by antibody screening of phage‐displayed random peptide libraries vary in immunoreactivity: Studies using an exemplary monoclonal antibody, CII‐C1, to type II collagen

1999

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A phage-displayed random peptide library is created when random oligonucleotides are inserted at the 5′ end of one of the genes for the coat proteins of filamentous bacteriophage, gIII or gVIII, thus allowing for expression of foreign peptides as pIII or pVIII fusion proteins on the phage surface. Accordingly, screening of phage-displayed random peptide libraries with antisera to a given antigen can reveal mimotopes of B cell epitopes of that antigen. A phage that displays a particular peptide selected by an antibody is known as a phagotope. Currently efforts are being made to optimize both experimental protocols and the interpretation of data from phagedisplayed library screening. [1][2][3][4] Although phagotopes selected by a given antiserum should react in immunoassays with that selecting antiserum, such phagotopes usually exhibit a diverse array of peptide sequences and their immunoreactivity may vary. Furthermore, this reactivity also can vary according to the assay format used. 5 There is a need to investigate the possible sources of variation in immunoreactivity with the selecting antibody of phagotopes derived from random peptide phage-displayed libraries.We have previously isolated phagotopes by screening phage-displayed random peptide libraries with a monoclonal antibody (mAb), CII-C1, to type II collagen. 6-8 Both the antibody and the antigen are very well characterized. The conformational epitope for CII-C1 on native type II collagen, C1, has been deduced by use of a series of chimeric collagens in which overlapping fragments of type II collagen were inserted into type X collagen; the minimum primary sequence required for CII-CI reactivity was ARGLT. 8 In addition, the complementarity determining regions (CDR) of CII-C1 have been sequenced. 9,10 Our results from screening phage-displayed random peptide libraries with CII-C1 have been consistent with knowledge of the CII-C1 epitope, in that most of the selected phagotopes have at least one basic and one hydrophobic residue within the expressed peptide. 7 The most frequently isolated phagotope expressed the peptide RRLPFGSQM and many, but not all, expressed peptides with variations on both the motifs RRL and FGxQ. We have proposed that the glutamine, which is present in most of the phage-displayed peptides, but does not occur in the C1 epitope, may bind to the CDR3 of CII-C1. Because the reactivity of CII-C1 with the conformational C1 epitope on Immunology and Cell Biology (1999) 77, 483-490 Research ArticlePhagotopes derived by antibody screening of phage-displayed random peptide libraries vary in immunoreactivity: Studies using an exemplary monoclonal antibody, CII-C1, to type II collagen JANET M DAVIES, MERRILL J ROWLEY and IAN R MACKAY Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, AustraliaSummary Antibody screening of phage-displayed random peptide libraries to identify mimotopes of conformational epitopes is promising. However, because interpretations can be difficult, an exemplary system has been used in...

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Section: Discussionsupporting

confidence: 68%

Phagotopes derived by antibody screening of phage‐displayed random peptide libraries vary in immunoreactivity: Studies using an exemplary monoclonal antibody, CII‐C1, to type II collagen

1999

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“…It is an asymmetric particle about 6 nm in diameter and 800-2,000 nm long, comprising a sheath of about 2,700 copies of the major coat protein gpVIII [22]. The recombinant pVIII usually is present in about 100-200 copies per virion if the fusion proteins are less than 100 amino acid residues in size [23]. In the present study, about 1.6Â10 -8 M [(5Â10 13 pfu/l) Â200/(6.02Â10 23 )] of the recombinant pVIII, corresponding to 0.75 lg/ml soluble OVA, was used.…”

Section: Discussionmentioning

confidence: 99%

Cross-presentation of phage particle antigen in MHC class II and endoplasmic reticulum marker-positive compartments

Wan

Zhou

et al. 2005

Eur. J. Immunol.

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It has been shown that exogenous antigens can access the MHC class I pathway of professional antigen-processing cells. However, details as to how the MHC class Ipeptide complex forms in the presentation pathway are still poorly understood. Here we used MHC class I-peptide-specific antibodies to investigate the formation and intracellular location of class I-peptide complexes in macrophages. We observed that the formation of class I-peptide complexes occurs within a few hours and lasts for another few hours on the cell surface of macrophages following loading with filamentous phage particles. The class I-peptide complexes in the process were colocalized with MHC class II molecules and endocytic system markers. Moreover, endosomal compartments containing class I-peptide complexes were found within intracellular organelles stained by DiOC6 and calnexin. In addition, the crosspresentation of phage particles was transporter associated with antigen processing (TAP)-dependent and sensitive to proteasome inhibitors and NH 4 Cl. These data suggest that endocytosed phage particles may be processed and cross-presented in organelles positive for phagosome and endoplasmic reticulum (ER) markers via a classical ER MHC class I loading mechanism.

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“…For peptides mimicking discontinuous epitopes or non-protein antigens, not only the linear sequence, but also the conformation of both the peptide and the binding site on the cognate antibody may play a role, to a various extent, in the isolation process, and later in the reactivity of the peptides (39,40). Additionally, the theoretical diversity of peptide libraries is reduced during their preparation steps and the number of peptide copies expressed on the surface of the viral particles may vary, which in turn affects the results of the screening process (41). Therefore, as a rule the initial leading sequences isolated from phage-display libraries are not the most optimal and require further optimization.…”

Section: Discussionmentioning

confidence: 99%

Analysis and optimization of interactions between peptides mimicking the GD2 ganglioside and the monoclonal antibody 14G2a

Horwacik

Kurciński²,

Bzowska³

et al. 2011

Int J Mol Med

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Abstract. Overexpression of the Gd2 ganglioside (Gd2) is a hallmark of neuroblastoma. The antigen is used in neuroblastoma diagnosis and to target newly developed therapies to cancer cells. Peptide mimetics are novel approaches in the design of antigens for vaccine development. We previously reported the isolation of five GD2-mimicking peptides from the LX-8 phage display library with the monoclonal antibody (mAb) 14G2a. The goal of our current study was to analyze and optimize the binding of the peptide mimetics to the mAb 14G2a. Therefore, we performed further experiments and supported them with molecular modeling to investigate structure-activity relationships that are the basis for the observed mimicry of Gd2 by our peptides. Here, we show that the peptides have overlapping binding sites on the mAb, 14G2a and restricted specificity, as they did not crossreact with other ganglioside-specific antibodies tested. In addition we demonstrate that the phage environment was involved in the process of selection of our peptides. The AAEGd sequence taken from the viral major coat protein, p8, and added to the c-termini of the peptides #65, #85 and #94 significantly improved their binding to the mAb, 14G2a. By application of analogs with amino acid substitutions and sequence truncations, we elucidated the structure-activity relationships necessary for the interactions between the 14G2a mAb and the peptide #94 (RCNPNMEPPRCF). We identified amino acids indispensable for the observed Gd2-mimicry by #94 and confirmed a pivotal role of the disulphide bridge between the cysteine residues of #94 for binding to the mAb 14G2a. More importantly, we report five new peptides demonstrating a significant improvement of mAb 14G2a binding. The experimental data were supported and expanded with molecular modeling tools. Taken together, the experimental results and the in silico data allowed us to probe in detail the mechanism of the molecular mimicry of Gd2 by the peptides. Additionally, we significantly optimized binding of the leading peptide sequence #94 to the mAb 14G2a. We can conclude that our findings add to the knowledge on factors governing selections of peptide mimetics from phage-display libraries.

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Role of Capsid Structure and Membrane Protein Processing in Determining the Size and Copy Number of Peptides Displayed on the Major Coat Protein of Filamentous Bacteriophage

Cited by 129 publications

References 43 publications

Phagotopes derived by antibody screening of phage‐displayed random peptide libraries vary in immunoreactivity: Studies using an exemplary monoclonal antibody, CII‐C1, to type II collagen

Phagotopes derived by antibody screening of phage‐displayed random peptide libraries vary in immunoreactivity: Studies using an exemplary monoclonal antibody, CII‐C1, to type II collagen

Cross-presentation of phage particle antigen in MHC class II and endoplasmic reticulum marker-positive compartments

Analysis and optimization of interactions between peptides mimicking the GD2 ganglioside and the monoclonal antibody 14G2a

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