Identification of PSA peptide mimotopes using phage display peptide library

Prostate cancer (PCa) is one of the most common types of cancer in men in the United States and is the second leading cause of cancer related death in men. Clinically, secreted prostate specific antigen (PSA) has gained recognition because of its proteolytic activity being directly linked to PCa cell proliferation leading to disease initiation and progression. Using phage display technology, we identified four distinct cyclical peptides. These peptides apart from differences in their amino acid sequence, elicited minimal cross reactive antibody responses against each other. One of the four peptides analyzed produced an antibody response that recognizes the PSA protein. We demonstrate that the synthetic PSA peptide mimics identified in our study are immunologically active and produce neutralizing activity and this has relevance and utility for prostate cancer disease progression.     

Production and characterization of peptide mimotopes of phenolic glycolipid-I of Mycobacterium leprae

Phenolic glycolipid-I (PGL-I), a Mycobacterium leprae-specific antigen, has been widely used for the serodiagnosis of leprosy and has been implicated in the pathogenesis of leprosy. In an effort to produce an alternate antigen of PGL-I, the mimotope peptides of PGL-I, W(T/R)LGPY(V/M), were obtained using a monoclonal antibody, III603.8, specific to PGL-I by a phage library. The biotin-labeled predominant mimotope peptide of PGLP1, WTLGPYV, bound to III603.8 in a dose-dependent manner in an immunoassay. However, PGLP1 did not bind to anti-PGL-I antibodies in the serum samples from leprosy patients that were reactive to PGL-I. Although the mimotope peptide of WTLGPYV was not effective as an alternate antigen of PGL-I for the serodiagnosis of leprosy, but it would be of interest to know how the mimotope peptides mimic the role of PGL-I antigen in the pathogenesis of M. leprae infection.     

Identification of peptide mimotopes of gp96 using single-chain antibody library

Heat shock proteins such as gp96 are immunogenic and are widely used as vaccines in immunotherapy of cancers. The present study focuses on the use of peptide mimotopes as immunotherapeutic vaccines for prostate cancer. To this end, we developed a 15-mer gp96 peptide mimotope specifically reactive to MAT-LyLu gp96–peptide complex using combinatorial single-chain antibody and peptide phage display library. The immunogenicity of the synthesized gp96 mimotope was analyzed initially in normal BALB/c mice in combination with various adjuvants such as complete Freund’s adjuvant (CFA), aluminum salts (ALUM), granulocyte-macrophage colony-stimulating factor (GM-CSF), and liposome, of which CFA served as a positive control. The antibody response was determined and found that the gp96 mimotope with ALUM showed a significant increase in antibody titer, followed by GM-CSF and liposomes. Further, the T cell (CD4⁺ and CD8⁺) populations from splenocytes, as well as IgG isotypes, interleukin-4, and interleukin-5 of gp96 mimotope with ALUM-immunized animals, were analyzed. The results suggest that the gp96 mimotope may elicit a potent and effective antitumor antibody response. Further, the study identifies ALUM and GM-CSF as adjuvant options to drive an appropriate protective immune response as these adjuvants have prior use in humans.     

Identification of peptide mimotopes of Trypanosoma brucei gambiense variant surface glycoproteins

Background

The current antibody detection tests for the diagnosis of gambiense human African trypanosomiasis (HAT) are based on native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. These native VSGs are difficult to produce, and contain non-specific epitopes that may cause cross-reactions. We aimed to identify mimotopic peptides for epitopes of T.b. gambiense VSGs that, when produced synthetically, can replace the native proteins in antibody detection tests.

Methodology/Principal Findings

PhD.-12 and PhD.-C7C phage display peptide libraries were screened with mouse monoclonal antibodies against the predominant VSGs LiTat 1.3 and LiTat 1.5 of T.b. gambiense. Thirty seven different peptide sequences corresponding to a linear LiTat 1.5 VSG epitope and 17 sequences corresponding to a discontinuous LiTat 1.3 VSG epitope were identified. Seventeen of 22 synthetic peptides inhibited the binding of their homologous monoclonal to VSG LiTat 1.5 or LiTat 1.3. Binding of these monoclonal antibodies to respectively six and three synthetic mimotopic peptides of LiTat 1.5 and LiTat 1.3 was significantly inhibited by HAT sera (p<0.05).

Conclusions/Significance

We successfully identified peptides that mimic epitopes on the native trypanosomal VSGs LiTat 1.5 and LiTat 1.3. These mimotopes might have potential for the diagnosis of human African trypanosomiasis but require further evaluation and testing with a large panel of HAT positive and negative sera.     

Identification of mimotopes of the Japanese encephalitis virus envelope protein using phage-displayed combinatorial peptide library

The phage-displayed combinatorial peptide library is a revolutionary method for discovering epitopes, in particular conformational epitopes. In this study, we characterized a Japanese encephalitis virus (JEV) conformational epitope by biopanning of phage-displayed random peptide libraries with a JEV envelope (E) protein-specific monoclonal antibody (mAb) 2H2. Eleven identified phage clones with high affinity to mAb 2H2 were identified using direct and inhibitory binding ELISA. Sequence alignment, structure modeling and mutational analysis revealed that the identified mimotopes for mAb 2H2 possess a conserved motif X(1)(D/E)(Y/T/S)X(2), fitting into a region at the domain III lateral surface of the E protein. The results of our study could provide useful information on the development of effective mimotope-based vaccines and diagnostic kits for the JEV infection.     

Construction and characterization of an HCV-derived multi-epitope peptide antigen containing B-cell HVR1 mimotopes and T-cell conserved epitopes

Hepatitis C(HCV) genome is highly variable,particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene.The variability of HCV genome has been a major obstacle for de-veloping HCV vaccines.Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes,we synthesized an minigene of HCV-derived multi-epitope peptide an-tigen(CMEP) ,which contains 9 B-cell HVR1 mimotopes in E2,2 conserved CTL epitopes in C,1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3.This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP.The immunogenic properties of CEMP were characterized by HCV infected patients' sera,and found that the reactivity frequency reached 75%.The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%.Meanwhile,we constructed an HCV DNA vaccine candidate,plasmid pVAX1.0-st-CMEP carrying the recombinant gene(st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene.Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody,which was of the same cross reactivity as the fusion protein GST-CMEP.Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes,and would be of the value as a candidate for the development of HCV vaccines.     

Construction and characterization of an HCV-derived multi-epitope peptide antigen containing B-cell HVR1 mimotopes and T-cell conserved epitopes

Hepatitis C (HCV) genome is highly variable, particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene. The variability of HCV genome has been a major obstacle for developing HCV vaccines. Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes, we synthesized an minigene of HCV-derived multi-epitope peptide antigen (CMEP), which contains 9 B-cell HVR1 mimotopes in E2, 2 conserved CTL epitopes in C, 1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3. This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP. The immunogenic properties of CEMP were characterized by HCV infected patients’ sera, and found that the reactivity frequency reached 75%. The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%. Meanwhile, we constructed an HCV DNA vaccine candidate, plasmid pVAX1.0-st-CMEP carrying the recombinant gene (st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene. Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody, which was of the same cross reactivity as the fusion protein GST-CMEP. Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes, and would be of the value as a candidate for the development of HCV vaccines.     

Identification of mimotopes with diagnostic potential for Trypanosoma brucei gambiense variant surface glycoproteins using human antibody fractions

Background

At present, screening of the population at risk for gambiense human African trypanosomiasis (HAT) is based on detection of antibodies against native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. Drawbacks of these native VSGs include culture of infective T.b. gambiense trypanosomes in laboratory rodents, necessary for production, and the exposure of non-specific epitopes that may cause cross-reactions. We therefore aimed at identifying peptides that mimic epitopes, hence called “mimotopes,” specific to T.b. gambiense VSGs and that may replace the native proteins in antibody detection tests.

Methodology/Principal Findings

A Ph.D.-12 peptide phage display library was screened with polyclonal antibodies from patient sera, previously affinity purified on VSG LiTat 1.3 or LiTat 1.5. The peptide sequences were derived from the DNA sequence of the selected phages and synthesised as biotinylated peptides. Respectively, eighteen and twenty different mimotopes were identified for VSG LiTat 1.3 and LiTat 1.5, of which six and five were retained for assessment of their diagnostic performance. Based on alignment of the peptide sequences on the original protein sequence of VSG LiTat 1.3 and 1.5, three additional peptides were synthesised. We evaluated the diagnostic performance of the synthetic peptides in indirect ELISA with 102 sera from HAT patients and 102 endemic negative controls. All mimotopes had areas under the curve (AUCs) of ≥0.85, indicating their diagnostic potential. One peptide corresponding to the VSG LiTat 1.3 protein sequence also had an AUC of ≥0.85, while the peptide based on the sequence of VSG LiTat 1.5 had an AUC of only 0.79.

Conclusions/Significance

We delivered the proof of principle that mimotopes for T.b. gambiense VSGs, with diagnostic potential, can be selected by phage display using polyclonal human antibodies.     

Identification and characterization of murine IRAK-M

Interleukin-1 receptor-associated-kinases (IRAKs) are signal transduction mediators of the Toll/IL-1 receptor family, which comprise several transmembrane proteins involved in host defense mechanisms. Today four different human IRAKs (hu-IRAK-1, hu-IRAK-2, hu-IRAK-M, hu-IRAK-4) and two murine IRAKs (mouse pelle like kinase (mPLK) and mu-IRAK-4) have been described. Here we report the identification and characterization of murine IRAK-M (mu-IRAK-M), a mouse homologue to human IRAK-M (hu-IRAK-M). These IRAK-M molecules show 71% sequence identity, a comparable cellular expression, and functional similarities with respect to signal transduction capacity and kinase activity, suggesting functional homology in signalling in human and mouse cells.     

Identification and characterization of sORF-encoded polypeptides

Abstract

Molecular biology, genomics and proteomics methods have been utilized to reveal a non-annotated class of endogenous polypeptides (small proteins and peptides) encoded by short open reading frames (sORFs), or small open reading frames (smORFs). We refer to these polypeptides as s(m)ORF-encoded polypeptides or SEPs. The early SEPs were identified via genetic screens, and many of the RNAs that contain s(m)ORFs were originally considered to be non-coding; however, elegant work in bacteria and flies demonstrated that these s(m)ORFs code for functional polypeptides as small as 11-amino acids in length. The discovery of these initial SEPs led to search for these molecules using methods such as ribosome profiling and proteomics, which have revealed the existence of many SEPs, including novel human SEPs. Unlike screens, omics methods do not necessarily link a SEP to a cellular or biological function, but functional genomic and proteomic strategies have demonstrated that at least some of these newly discovered SEPs have biochemical and cellular functions. Here, we provide an overview of these results and discuss the future directions in this emerging field.     

Identification and characterization of bacterial cutinase

Cutinase, which exists in both fungi and bacteria, catalyzes the cleavage of the ester bonds of cutin. Fungal cutinases have been extensively studied, however, reports on bacterial cutinases have been limited due to the lack of knowledge concerning the identity of their open reading frames. In the present study, the cutinase from Thermobifida fusca was induced by cutin and purified to homogeneity by following p-nitrophenyl butyrate hydrolyzing activity. Peptide mass fingerprinting analysis of the wild-type enzyme matched two proteins, Tfu_0883 and Tfu_0882, which are 93% identical in sequence. Both proteins were cloned and overexpressed in their mature form. Recombinant Tfu_0883 and Tfu_0882 display very similar enzymatic properties and were confirmed to be cutinases by their capability to hydrolyze the ester bonds of cutin. Comparative characterization of Fusarium solani pisi and T. fusca cutinases indicated that they have similar substrate specificity and catalytic properties except that the T. fusca enzymes are thermally more stable. Homology modeling revealed that T. fusca cutinases adopt an alpha/beta-hydrolase fold that exhibits both similarities and variations from the fungal cutinase structure. A serine hydrolase catalytic mechanism involving a Ser(170)-His(248)-Asp(216) (Tfu_0883 numbering) catalytic triad was supported by active site-directed inhibition studies and mutational analyses. This is the first report of cutinase encoding genes from bacterial sources.     

Identification and characterization of Kingella kingae

Kingella kingae is a rarely isolated opportunistic pathogen in the family Neisseriaceae. Thirteen strains of this organism, including six strains isolated in Denmark, were characterized. They formed a homogeneous group of small, non-motile, fastidious Gram-negative rods which were beta-haemolytic, oxidase positive, catalase negative and saccharolytic. Acid was produced for glucose and maltose. Growth was most pronounced aerobically but corroding colonies increased considerably in size during anaerobic incubation.     

Identification and characterization of taxilin isoforms

The syntaxin family is implicated in intracellular vesicle traffic. We have recently identified taxilin, a novel syntaxin-binding protein, which has a long coiled-coil region in its C-terminal half. A database search has revealed the presence of two other molecules having a long coiled-coil region homologous to that of taxilin in mammals. Then, we here attempted to isolate and characterize the two molecules. Both the two molecules stoichiometrically interacted with several syntaxin family members. Then, we renamed original taxilin alpha-taxilin and named the two molecules beta- and gamma-taxilins, respectively. Beta-taxilin was a human homologue of chicken MDP77. Gamma-taxilin was an uncharacterized protein and Northern blot analysis revealed that gamma-taxilin was ubiquitously expressed. Beta- and gamma-taxilins preferentially interacted with syntaxin-1a and -4, respectively. The taxilin family members mutually interacted with the syntaxin family members. These results indicate that there is the taxilin family composed of at least three members in mammals.     

Therapeutic potential of yeast killer toxin-like antibodies and mimotopes

This review focuses on the potential of yeast killer toxin (KT)-like antibodies (KTAbs), that mimic a wide-spectrum KT through interaction with specific cell wall receptors (KTR) and their molecular derivatives (killer mimotopes), as putative new tools for transdisease anti-infective therapy. KTAbs are produced during the course of experimental and natural infections caused by KTR-bearing micro-organisms. They have been produced by idiotypic vaccination with a KT-neutralizing mAb, also in their monoclonal and recombinant formats. KTAbs and KTAbs-derived mimotopes may exert a strong therapeutic activity against mucosal and systemic infections caused by eukaryotic and prokaryotic pathogenic agents, thus representing new potential wide-spectrum antibiotics.     

Therapeutic potential of yeast killer toxin-like antibodies and mimotopes

This review focuses on the potential of yeast killer toxin (KT)-like antibodies (KTAbs), that mimic a wide-spectrum KT through interaction with specific cell wall receptors (KTR) and their molecular derivatives (killer mimotopes), as putative new tools for transdisease anti-infective therapy. KTAbs are produced during the course of experimental and natural infections caused by KTR-bearing micro-organisms. They have been produced by idiotypic vaccination with a KT-neutralizing mAb, also in their monoclonal and recombinant formats. KTAbs and KTAbs-derived mimotopes may exert a strong therapeutic activity against mucosal and systemic infections caused by eukaryotic and prokaryotic pathogenic agents, thus representing new potential wide-spectrum antibiotics.     

Identification of peptide mimotopes for the fluorescein hapten binding of monoclonal antibody B13‐DE1

Using 6mer and 12mer phage peptide libraries three unique phage clones were identified which specifically bind to a monoclonal anti‐FITC antibody, B13‐DE1. The two 6mer and one 12mer peptide insert sequences are clearly related to each other and contain a high proportion of hydrophobic amino acids. The peptides are bound by the antibody combining site of B13‐DE1 probably in a similar manner to FITC and represent therefore true peptidic mimics of the fluorescein hapten. No reactivity of the peptides could be demonstrated with another monoclonal anti‐fluorescein antibody or with polyclonal anti‐fluorescein antibodies. Immunization of mice with the peptides resulted in the production of antibodies cross‐reacting with all peptides but not with fluorescein. The results show that phage peptide libraries can be used to isolate mimotope peptides which can mimic low molecular weight structures seen by a specific antibody and probably other recognition molecules. Copyright © 1999 John Wiley & Sons, Ltd.     

Identification and characterization of novel NuMA isoforms

The large nuclear mitotic apparatus (NuMA) has been investigated for over 30 years with functions related to the formation and maintenance of mitotic spindle poles during mitosis. However, the existence and functions of NuMA isoforms generated by alternative splicing remains unclear. In the present work, we show that at least seven NuMA isoforms (categorized into long, middle and short groups) generated by alternative splicing from a common NuMA mRNA precursor were discovered in HeLa cells and these isoforms differ mainly at the carboxyl terminus and the coiled-coil domains. Two “hotspot” exons with molecular mass of 3366-nt and 42-nt tend to be spliced during alternative splicing in long and middle groups. Furthermore, full-length coding sequences of long and middle NuMA obtained by using fusion PCR were constructed into GFP-tagged vector to illustrate their cellular localization. Long NuMA mainly localized in the nucleus with absence from nucleoli during interphase and translocated to the spindle poles in mitosis. Middle NuMA displayed the similar cell cycle-dependent distribution pattern as long NuMA. However, expression of NuMA short isoforms revealed a distinct subcellular localization. Short NuMA were present in the cytosol during the whole cycle, without colocalization with mitotic apparatus. These results have allowed us tentatively to explore a new research direction for NuMA’s various functions.     

Identification and characterization of Drosophila melanogaster paramyosin

Paramyosin, a major structural component of thick filaments in invertebrates has been isolated, purified and characterized from whole adult Drosophila melanogaster extracts and a specific polyclonal antibody against it has been prepared. Paramyosin has been identified on the basis of several criteria, including molecular weight, alpha-helicity, species distribution, capability of fiber formation in vitro and sequence. We have used the immunopurified polyclonal antibody to isolate eight clones from a lambda gt11 expression library of Drosophila 1 to 22 h embryo cDNA. The largest clone (pJV9) has been sequenced and encodes the coiled-coil region of D. melanogaster paramyosin that is 47% identical to Caenorhabditis elegans paramyosin. Indirect immunofluorescence in semi-thin sections of adult flies show fluorescence mainly in tubular muscle. Freshly prepared tubular myofibrils decorated with the immunoabsorbed antibody show the A region in the sarcomere as the specific localization of paramyosin. The amount of paramyosin in tubular synchronous muscles of insects appears to be five times higher than in fibrillar insect muscles. There are at least three paramyosin isoforms as shown by isoelectrofocusing separation. The more acidic and less abundant form is phosphorylated as shown by 32P in vivo labeling experiments in adult flies. The developmental pattern of expression of Drosophila paramyosin is presented. This mesoderm-specific protein, immunologically undetectable during gastrulation and early phases of germ band formation, progressively increases during organogenesis to the adult stage. Interestingly, it is also expressed as a major maternal product in the insoluble cytoskeletal fraction of the mature oocyte.