Human apolipoprotein A-I binds amyloid-beta and prevents A beta-induced neurotoxicity

Aggregates of the amyloid-P peptide (A beta) play a central role in the pathogenesis of Alzheimer`s disease (AD). Identification of proteins that physiologically bind A beta and modulate its aggregation and neurotoxicity could lead to the development of novel disease-modifying approaches in AD. By screening a phage display peptide library for high affinity ligands of aggregated A beta(1-42), We isolated a peptide homologous to a highly conserved amino acid sequence present in the N-terminus of apolipoprotein A-I (apoA-I). We show that purified human apoA-I and A beta form non-covalent complexes and that interaction with apoA-I affects the morphology of amyloid aggregates formed by A beta. Significantly, A beta/apoA-I complexes were also detected in cerebrospinal fluid from AD patients. Interestingly, apoA-I and apoA-I-containing reconstituted high density lipoprotein particles protect hippocampal neuronal cultures from A beta-induced oxidative stress and neurodegeneration. These results suggest that human apoA-I modulates A beta aggregation and A beta-induced neuronal damage and that the A beta-binding domain in apoA-I may constitute a novel framework for the design of inhibitors of A beta toxicity. (C) 2009 Published by Elsevier Ltd.

Identification of peptide ligands to the chemokine receptor CCR5 and their maturation by gene shuffling.

The determination of protein-protein interactions and their role in diverse pathophysiological processes is a promising approach to the identification of molecules of therapeutic potential. This paper describes the identification of peptidic CCR5 receptor ligands as potential drug leads against HIV-1 infection using in vitro evolution based on phage display. A phage-displayed peptide library was used to select for anti-CCR5 peptide. Further in vitro evolution of the peptide by exon shuffling was performed to identify peptides with optimized characteristics for CCR5 receptor. This peptide inhibited HIV coreceptor activity in a cell fusion assay with an IC50 of 5 microM. It did not exhibit either agonistic or antagonistic activity on CCR5 in the concentration range used. To our knowledge, this is a first report that describes the identification of peptide ligands specific to the CCR5 receptor from a phage-displayed library and the maturation of the selected peptide sequence by gene shuffling.

New human kallikrein 14: enzymatic characterization and inhibitors development.

RESUME DESTINE A UN LARGE PUBLIC En biologie, si une découverte permet de répondre à quelques questions, en général elle en engendre beaucoup d'autres. C'est ce qui s'est produit récemment dans le monde des kallicréines. De la famille des protéases, protéines ayant la faculté de couper plus ou moins spécifiquement d'autres protéines pour exercer un rôle biologique, la famille des kallicréines humaines n'était composée que de 3 membres lors du siècle dernier. Parmi eux, une kallicréine mondialement utilisée pour détecter le cancer de la prostate, le PSA. En 2000, un chercheur de l'hôpital universitaire Mont Sinaï à Toronto, le Professeur Eleftherios Diamandis, a découvert la présence de 12 nouveaux gènes appartenant à cette famille, situés sur le même chromosome que les 3 premières kallicréines. Cette découverte majeure a placé les spécialistes des kallicréines face à une montagne d'interrogations car les fonctions de ces nouvelles protéases étaient totalement inconnues. La kallicréine humaine 14 (hK14) présente un intérêt particulier, car elle se retrouve associée à différents cancers, notamment les carcinomes ovariens et mammaires. Cette association ne répond cependant pas à la fonction de cette protéase. L'objectif de ce travail de thèse était donc de découvrir, dans un premier temps, la spécificité de cette nouvelle kallicréine, c'est-à-dire le type de coupure qu'elle engendre au niveau des protéines qu'elle cible. Utilisant une technologie de pointe qui exploite la propriété des bactériophages à se répliquer dans les bactéries à l'infini, des dizaines de millions de combinaisons protéiques aléatoires ont été présentées à hK14, qui a pu sélectionner celles qui lui étaient favorables pour la coupure. Cette technique qualitative porte le nom de Phage Display Substrate. Une fois la sélection réalisée, il fallait transférer ces séquences coupées ou substrats dans un système permettant de donner une valeur quantitative à l'efficacité de coupure. Pour cela nous avons développé une technologie qui permet d'évaluer cette efficacité en utilisant des protéines fluorescentes de méduse, modifiées génétiquement, dont l'excitation de la première (CFP : cyan fluorescent protein) par la lumière à une certaine longue d'onde permet le transfert d'énergie à la seconde (YFP : yellow fluorescent protein), via un substrat qui les lie. Pour que ce transfert d'énergie se produise, il faut que les deux protéines fluorescentes soient proches, comme c'est le cas lorsqu'elles sont liées par un substrat. La coupure de ce lien provoque un changement de transfert d'énergie qui est quantifiable en utilisant un spectrofluoromètre. Cette technologie permet donc de suivre la réaction d'hydrolyse (coupure) des protéases. Afin de poursuivre certaines expériences permettant de mieux comprendre la fonction biologique d'hK14 ainsi que son éventuelle implication dans le cancer, nous avons développé des inhibiteurs spécifiques d'hK14. Les séquences qui on été le plus efficacement coupées par hK14 ont été utilisées pour transformer deux types d'inhibiteurs classiques, qui circulent dans notre sang, en inhibiteurs d'hK14 hautement efficaces et spécifiques. Selon les résultats obtenus in vitro, ils pourront être évalués in vivo en tant que traitement potentiel contre le cancer. RESUME Les protéases sont des enzymes impliquées dans des processus physiologiques mais aussi parfois pathologiques. La famille des kallicréines tissulaires humaines représente le plus grand groupe de protéases humaines, dont plusieurs pourraient participer au développement de certaines maladies. D'autre part, ces protéases sont apparues comme des marqueurs de pathogénicité potentiels, notamment dans les cas de cancers hormono-dépendants. La kallicréine humaine 14 a été récemment découverte et son implication dans quelques maladies, particulièrement dans le cas de tumeurs, semble probable. En effet, son expression génique est augmentée au niveau des tissus cancéreux de la prostate et du sein et son expression protéique s'est révélée plus élevée dans le sérum de patientes atteintes d'un cancer du sein ou des ovaires. Cependant, comme c'est le cas pour la plupart des kallicréines, sa fonction est encore inconnue. Afin de mieux connaître son rôle biologique et/ou pathologique, nous avons décidé de caractériser son activité enzymatique. Nous avons tout d'abord mis au point un système de substrats entièrement biologique permettant d'étudier in vitro l'activité des protéases. Ce système est basé sur le phénomène de FRET, à savoir le transfert d'énergie de résonance fluorescente qui intervient entre deux molécules fluorescentes voisines si le spectre d'émission de la protéine donneuse chevauche le spectre d'excitation de la protéine receveuse. Nous avons fusionné de manière covalente une protéine fluorescente bleue (CFP) et une jaune (YFP) en les liant avec diverses séquences. Par clivage de la séquence de liaison, une perte du transfert d'énergie peut être mesurée par un spectrofluoromètre. Cette technologie représente un moyen facile de suivre la réaction d'hydrolyse des protéases. Les conditions optimales de production de ces substrats CFP-YFP ont été déterminées, de même que les paramètres pouvant éventuellement influencer le FRET. Ce système possède une grande résistance à la protéolyse non spécifique et est applicable à un grand nombre de protéase. Contrairement aux substrats fluorogéniques, il permet d'étudier les acides aminés se trouvant des deux côtés du site de clivage. Ce système étant entièrement biologique, il est le reflet des interactions protéine-protéine et représente un outil biologique facile, bon marché et rapide pour caractériser les protéases. Dans un premier temps, hK14 a été mise en présence d' une banque de haute diversité de pentapeptides aléatoires présentée à la surface de phages afin d'identifier des substrats spécifiques. Ensuite, le système CFP-YFP a été employé pour trier les peptides sélectionnés afin d'identifier les séquences de substrats les plus sensibles et spécifiques pour hK14. Nous avons montré, qu'en plus de sa prévisible activité de type trypsine, hK14 possède aussi une très surprenante activité de type chymotrypsine. Les séquences les plus sensibles ont été choisies pour cribler la banque de donnée Swissprot, permettant ainsi l'identification de 6 substrats protéiques humains potentiels pour hK14. Trois d'entre eux, la laminine α-5, le collagène IV et la matriline-4, qui sont des composants de la matrice extracellulaire, ont démontré une grande susceptibilité à l'hydrolyse par hK14. De plus, la séparation éléctrophorétique a montré que la dégradation de la laminine α-5 et de la matriline-4 par hK14 devait se produire aux sites identifiés par la technologie du phage display. Pour terminer, nous avons transformé, par mutagenèse dirigée, deux serpines (inhibiteurs de protéases de type sérine) connues, AAT et ACT (alpha anti-trypsine et alpha anti-chymotrypsine), qui inhibent un vaste éventail d'enzymes humaines en inhibiteurs d'hK14 hautement efficaces et spécifiques. Ces inhibiteurs pourront être utilisés d'une part pour poursuivre certaines expériences permettant de mieux comprendre l'implication d'hK14 dans des voies physiologiques ou dans le cancer et d'autre part pour les évaluer in vivo en tant que traitement potentiel contre le cancer. SUMMARY Proteases consist of enzymes involved in physiological events, but also, in case of dysregulation, in pathogenicity. The human tissue kallikrein family represents the largest human protease cluster and includes several members that either could participate in the course of certain diseases or emerged as potential biological markers, especially in hormone dependent cancers. The human kallikrein 14 has been recently discovered and suggested implications in some disorders, particularly in tumors since its gene expression is up-regulated in prostate and breast cancer tissues and its protein expression increased in the serum of patients with breast and ovarian cancers. However, like most kallikreins, its function remains unknown. To better understand hK14 biological and/or pathological role, we decided to characterize its enzymatic activity. First of all, we developped a biological system suitable for in vitro study of protease activity. This system is based on the so-called FRET phenomenon, that is the Fluorescence Resonance Energy Transfer that occurs between two nearby fluorescent proteins if the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. We fused covalently a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) with diverses sequences. Upon cleavage of the linker sequence by protease, the loss of energy transfer can be measured by a spectrofluorometer allowing an easy following of hydrolysis reaction. The optimal conditions to produce in bacterial system these CFP-YFP substrates were determined as well as the parameters that could eventually influence the FRET. This system demonstrated a high degree of resistance to non-specific proteolysis and applicability to various conditions corresponding to a great number of existing proteases. Other avantages are the possibility to study the amino acids located both sides of the cleavage site as well as the interest to work in a full biological system reflecting protein-protein interaction. A phage substrate library with exhaustive diversity was used prior to CFP-substrate-YFP system to isolate specific human kallikrein 14 substrates. After that the CFP-YFP system was used to sort peptides and identify highly sensitive and specific substrate sequences for hK14. We showed that besides its predictable trypsin-like activity, hK14 also possesses a surprising chymotrypsin-like activity. The screening of the Swissprot database was achieved with the most sensitive sequences and allowed the identification of 6 potential human protein substrates for hK14. Three of them, laminin α-5, collagen IV and matrilin-4, which are components of the extracellular matrix were incubated with hK14, by which they were efficiently hydrolyzed. Moreover, electrophoretic separation revealed that degradation of laminin α-5 and matrilin-4 by hK14 generated fragments with identical molecular size than the predicted N-terminal fragments that would result from hK14 specific cleavage, proving the value of phage display substrate to identify potential substrates. Finally, with site-directed mutagenesis, we transformed two well-known serpins (serine protease inhibitors), AAT and ACT (alpha anti-trypsin and alpha anti-chymotrypsin), which inhibit a vast spectrum of human enzymes into highly efficient and specific hK14 inhibitors. These inhibitors will be used to pursue experiments that could help understand hK14 implication in physiological pathways as well as in cancer biology and also to perform their in vivo evalution as potential cancer treatment.

Expression of anti-Z-DNA single chain antibody variable fragment on the filamentous phage surface

We describe the expression of an anti-Z-DNA single chain variable region antibody fragment (scFv) on a filamentous phage surface. Four vectors for phage display were constructed. Two of them are able to display multiple copies of the antibody fragment, and the others can be used to make monovalent libraries. The vectors use different promoter/leader sequences to direct the expression of the fused proteins. All were able to promote the assembly of fusion virion particles. In this paper we also show the affinity selection (biopanning) of those phage-antibodies based on the capacity of their products to recognize the antigen. We used biotinylated Z-DNA and the selection was performed in a solution phase fashion. The data presented here indicate that these vectors can be further used to construct anti-nucleic acid antibody fragment libraries that can be used to study the basis of nucleic acid-protein interaction and its role in autoimmunity mechanisms.

Produção dos fragmentos de anticorpos recombinantes scFv-N e scFv-S1 e suas aplicações na detecção e diferenciação do Vírus da Bronquite Infecciosa

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Serrumab: A human monoclonal antibody that counters the biochemical and immunological effects of Tityus serrulatus venom

In Brazil, the species Tityus serrulatus is responsible for the most severe cases of scorpion envenomation. There is currently a need for new scorpion anti-venoms that are more effective and less harmful. This study attempted to produce human monoclonal antibodies capable of inhibiting the activity of T. serrulatus venom (TsV), using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Four rounds of phage antibody selection were performed, and the round with the highest phage antibody titer was chosen for the production of monoclonal phage antibodies and for further analysis. The scFv 2A, designated serrumab, was selected for the production and purification of soluble antibody fragments. In a murine peritoneal macrophage cell line (J774.1), in vitro assays of the cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, and IL-10 were performed. In male BALB/c mice, in vivo assays of plasma urea, creatinine, aspartate transaminase, and glucose were performed, as well as of neutrophil recruitment and leukocyte counts. It was found that serrumab inhibited the TsV-induced increases in the production of IL-6, TNF alpha, and IL-10 in J774.1 cells. The in vivo inhibition assay showed that serrumab also prevented TsV-induced increases in the plasma levels of urea, creatinine, aspartate transaminase, and glucose, as well as preventing the TsV-induced increase in neutrophil recruitment. The results indicate that the human monoclonal antibody serrumab is a candidate for inclusion in a mixture of specific antibodies to the various toxins present in TsV. Therefore, serrumab shows promise for use in the production of new anti-venom.

Targeting the Blood-brain Barrier with a Non-canonical Iron-mimicry Mechanism

Treatment of central nervous system (CNS) diseases is limited by the blood-brain barrier (BBB), a selective vascular interface restricting passage of most molecules from blood into brain. Specific transport systems have evolved allowing circulating polar molecules to cross the BBB and gain access to the brain parenchyma. However, to date, few ligands exploiting such systems have proven clinically viable in the setting of CNS diseases. We reasoned that combinatorial phage-display screenings in vivo would yield peptides capable of crossing the BBB and allow for the development of ligand-directed targeting strategies of the brain. Here we show the identification of a peptide mediating systemic targeting to the normal brain and to an orthotopic human glioma model. We demonstrate that this peptide functionally mimics iron through an allosteric mechanism and that a non-canonical association of (i) transferrin, (ii) the iron-mimic ligand motif, and (iii) transferrin receptor mediates binding and transport of particles across the BBB. We also show that in orthotopic human glioma xenografts, a combination of transferrin receptor over-expression plus extended vascular permeability and ligand retention result in remarkable brain tumor targeting. Moreover, such tumor targeting attributes enables Herpes simplex virus thymidine kinase-mediated gene therapy of intracranial tumors for molecular genetic imaging and suicide gene delivery with ganciclovir. Finally, we expand our data by analyzing a large panel of primary CNS tumors through comprehensive tissue microarrays. Together, our approach and results provide a translational avenue for the detection and treatment of brain tumors.

The splenic autoimmune response to ADAMTS13 in thrombotic thrombocytopenic purpura contains recurrent antigen-binding CDR3 motifs.

Acquired thrombotic thrombocytopenic purpura (TTP) is the consequence of a severe ADAMTS13 deficiency resulting from autoantibodies inhibiting ADAMTS13 or accelerating its clearance. Despite the success of plasma exchange the risk of relapse is high. From 2 patients (A and B), splenectomized for recurrent episodes of acquired TTP, the splenic B-cell response against ADAMTS13 was characterized through generation of human monoclonal anti-ADAMTS13 autoantibodies (mAbs) by cloning an immunoglobulin G (IgG)4κ- and IgG4λ-Fab library using phage display technology and by Epstein-Barr virus transformation of switched memory B cells (CD19+/CD27+/IgG+). Sequence analysis of the anti-ADAMTS13 IgGs of both patients revealed that the VH gene use was limited in our patients to VH1-3 (55%), VH1-69 (17%), VH3-30 (7%), and VH4-28 (21%) and contained 8 unique and thus far not reported heavy-chain complementarity determining region 3 motifs, of which 4 were shared by the 2 patients. The discovery of several highly similar anti-ADAMTS13 autoantibodies in 2 unrelated TTP patients suggests that the autoimmune response is antigen driven, because the probability that such similar immunoglobulin rearrangements happen by chance is very low (< 10(-9)).

A high-affinity natural autoantibody from human cord blood defines a physiologically relevant epitope on the FcepsilonRIalpha.

Natural Abs represent the indigenous immune repertoire and are thus present at birth and persist throughout life. Previously, human autoantibodies to the alpha domain of the high-affinity IgE receptor (FcepsilonRIalpha) have been isolated from Ab libraries derived from normal donors and patients with chronic urticaria. To investigate whether these anti-FcepsilonRIalpha Abs are present in the germline repertoire, we constructed a phage Fab display library from human cord blood, which represents the naive immune repertoire before exposure to exogenous Ags. All isolated clones specific to the FcepsilonRIalpha had the same sequence. This single IgM Ab, named CBMalpha8, was strictly in germline configuration and had high affinity and functional in vitro anaphylactogenic activity. Inhibition experiments indicated an overlapping epitope on the FcepsilonRIalpha recognized by both CBMalpha8 and the previously isolated anti-FcepsilonRIalpha Abs from autoimmune and healthy donors. This common epitope on FcepsilonRIalpha coincides with the binding site for IgE. Affinity measurements demonstrated the presence of Abs showing CBMalpha8-like specificity, but with a significantly lower affinity in i.v. Ig, a therapeutic multidonor IgG preparation. We propose a hypothesis of escape mutants, whereby the resulting lower affinity IgG anti-FcepsilonRIalpha Abs are rendered less likely to compete with IgE for binding to FcepsilonRIalpha.

Identification and characterization of a molecular marker in epithelial ovarian cancer

Carcinomas that arise from the ovarian surface epithelium represent a great challenge in gynecologic oncology. Although the prognosis of ovarian cancer is influenced by many factors capable of predicting clinical outcome, including tumor stage, pathological grade, and amount of residual disease following primary surgery, the biological aspects of ovarian cancer are not completely understood, thus implying that there may be other predictive indicators that could be used independently or in conjunction with these factors to provide a clearer clinical picture. The identification of additional markers with biological relevance is desirable. To identify disease-associated peptides, a phage display random peptide library was used to screen immunoglobulins derived from a patient with ovarian cancer. One peptide was markedly enriched following three rounds of affinity selection. The presence of autoantibodies against the peptide was examined in a panel of ovarian cancer patients. Stage IV patients exhibited a high percentage of positive reactivity (59%). This was in contrast to stage III patients, who only displayed 7% positive reactivity. Antibodies against the peptide were affinity purified, and heat-shock protein 90 (Hsp90) was identified as the corresponding autoantigen. The expression profile of the identified antigen was determined. Hsp90 was expressed in all sections examined regardless of degree of anaplasia. This thesis shows that utilizing the humoral response to ovarian cancer can be used to identify a tumor antigen in ovarian cancer. The data show that certain antigens may be expressed in ovarian tumors independent of the disease stage or grade, whereas circulating antibodies against such epitopes are only found in a subset of patients. ^

Characterization of Staphylococcus aureus adhesins important for respiratory tract infection

Staphylococcus aureus is a leading cause of lower respiratory tract infections in both adult and pediatric populations. In the past two decades, reports have described emergent incidence of severe necrotizing pneumonia in previously healthy individuals, frequently caused by antibiotic resistant strains. Additionally, S. aureus remains the most common cause of ventilator-associated pneumonia, contributing morbidity and mortality in intensive care units. As treatment of infection is made more difficult by the resistance to multiple antibiotics including vancomycin, there is a pressing need for novel strategies to prevent and treat S. aureus infections. Targeting essential mechanisms that promote infection such as adhesion, colonization, invasion, evasion of immune system and signaling may lead to inhibition of pathogenic surge. Staphylococcal adhesins of the MSCRAMM family (microbial surface components recognizing adherent matrix molecules) represent viable targets for such investigations. Understanding the molecular mechanism of binding is the first step toward the development of such therapies. Analysis of bacterial strains isolated from patients with staphylococcal pneumonia show increased expression of protein A, SdrD, SdrC and ClfB, cell surface proteins members of the MSCRAMM family. In this study the interaction of these MSCRAMMs with candidate ligands has been examined. We found that SdrD mediates S. aureus adherence to the lung epithelial cell line A549. Consistently, bacteria expressing SdrD have increased persistence in the lungs of infected mice after bronchoalveolar lavage in comparison with bacteria lacking this protein. Inhibition studies revealed that bacterial attachment can be abolished using neutralizing antibodies against SdrD. Using phage display, neurexin β isoforms were identified as SdrC binding partners. Previous reports postulated that MSCRAMMS bind their ligands by a 'dock, lock and latch' mechanism of interaction. Our data suggested that ClfB, an MSCRAMM responsible for nasal colonization, binds cytokeratin 10 by a 'dock and lock' variant of this model, in which the 'latching' event is not necessary. In summary, we have characterized aspects of molecular interaction between several MSCRAMMS and host components. We hope that continued delineation of these interactions will lead to identification of novel therapeutic targets or preventive strategies against S. aureus infections. ^

Making chemistry selectable by linking it to infectivity

The link between recognition and replication is fundamental to the operation of the immune system. In recent years, modeling this process in a format of phage-display combinatorial libraries has afforded a powerful tool for obtaining valuable antibodies. However, the ability to readily select and isolate rare catalysts would expand the scope of library technology. A technique in which phage infection controlled the link between recognition and replication was applied to show that chemistry is a selectable process. An antibody that operated by covalent catalysis to form an acyl intermediate restored phage infectivity and allowed selection from a library in which the catalyst constituted 1 in 105 members. Three different selection approaches were examined for their convenience and generality. Incorporating these protocols together with well known affinity labels and mechanism-based inactivators should allow the procurement of a wide range of novel catalytic antibodies.

Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold

We demonstrate that the ligand pocket of a lipocalin from Pieris brassicae, the bilin-binding protein (BBP), can be reshaped by combinatorial protein design such that it recognizes fluorescein, an established immunological hapten. For this purpose 16 residues at the center of the binding site, which is formed by four loops on top of an eight-stranded β-barrel, were subjected to random mutagenesis. Fluorescein-binding BBP variants were then selected from the mutant library by bacterial phage display. Three variants were identified that complex fluorescein with high affinity, exhibiting dissociation constants as low as 35.2 nM. Notably, one of these variants effects almost complete quenching of the ligand fluorescence, similarly as an anti-fluorescein antibody. Detailed ligand-binding studies and site-directed mutagenesis experiments indicated (i) that the molecular recognition of fluorescein is specific and (ii) that charged residues at the center of the pocket are responsible for tight complex formation. Sequence comparison of the BBP variants directed against fluorescein with the wild-type protein and with further variants that were selected against several other ligands revealed that all of the randomized amino acid positions are variable. Hence, a lipocalin can be used for generating molecular pockets with a diversity of shapes. We term this class of engineered proteins “anticalins.” Their one-domain scaffold makes them a promising alternative to antibodies to create a stable receptor protein for a ligand of choice.

A β-hairpin structure in a 13-mer peptide that binds α-bungarotoxin with high affinity and neutralizes its toxicity

Snake-venom α-bungarotoxin is a member of the α-neurotoxin family that binds with very high affinity to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. The structure of the complex between α-bungarotoxin and a 13-mer peptide (WRYYESSLEPYPD) that binds the toxin with high affinity, thus inhibiting its interactions with AChR with an IC50 of 2 nM, has been solved by 1H-NMR spectroscopy. The bound peptide folds into a β-hairpin structure created by two antiparallel β-strands, which combine with the already existing triple-stranded β-sheet of the toxin to form a five-stranded intermolecular, antiparallel β-sheet. Peptide residues Y3P, E5P, and L8P have the highest intermolecular contact area, indicating their importance in the binding of α-bungarotoxin; W1P, R2P, and Y4P also contribute significantly to the binding. A large number of characteristic hydrogen bonds and electrostatic and hydrophobic interactions are observed in the complex. The high-affinity peptide exhibits inhibitory potency that is better than any known peptide derived from AChR, and is equal to that of the whole α-subunit of AChR. The high degree of sequence similarity between the peptide and various types of AChRs implies that the binding mode found within the complex might possibly mimic the receptor binding to the toxin. The design of the high-affinity peptide was based on our previous findings: (i) the detection of a lead peptide (MRYYESSLKSYPD) that binds α-bungarotoxin, using a phage-display peptide library, (ii) the information about the three-dimensional structure of α-bungarotoxin/lead-peptide complex, and (iii) the amino acid sequence analysis of different AChRs.

Random phage mimotopes recognized by monoclonal antibodies against the pyruvate dehydrogenase complex-E2 (PDC-E2).

Dihydrolipoamide acetyltransferase, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), is the autoantigen most commonly recognized by autoantibodies in primary biliary cirrhosis (PBC). We identified a peptide mimotope(s) of PDC-E2 by screening a phage-epitope library expressing random dodecapeptides in the pIII coat protein of fd phage using C355.1, a murine monoclonal antibody (mAb) that recognizes a conformation-dependent epitope in the inner lipoyl domain of PDC-E2 and uniquely stains the apical region of bile duct epithelium (BDE) only in patients with PBC. Eight different sequences were identified in 36 phage clones. WMSYPDRTLRTS was present in 29 clones; WESYPFRVGTSL, APKTYVSVSGMV, LTYVSLQGRQGH, LDYVPLKHRHRH, AALWGVKVRHVS, KVLNRIMAGVRH and GNVALVSSRVNA were singly represented. Three common amino acid motifs (W-SYP, TYVS, and VRH) were shared among all peptide sequences. Competitive inhibition of the immunohistochemical staining of PBC BDE was performed by incubating the peptides WMSYPDRTLRTS, WESYPDRTLRTS, APKTYVSVSGMV, and AALWGVKVRHVS with either C355.1 or a second PDC-E2-specific mAb, C150.1. Both mAbs were originally generated to PDC-E2 but map to distinct regions of PDC-E2. Two of the peptides, although selected by reaction with C355.1, strongly inhibited the staining of BDE by C150.1, whereas the peptide APKTYVSVSGMV consistently inhibited the staining of C355.1 on biliary duct epithelium more strongly than the typical mitochondrial staining of hepatocytes. Rabbit sera raised against the peptide WMSYPDRTLRTS stained BDE of livers and isolated bile duct epithelial cells of PBC patients more intensively than controls. The rabbit sera stained all size ducts in normals, but only small/medium-sized ductules in PBC livers. These studies provide evidence that the antigen present in BDE is a molecular mimic of PDC-E2, and not PDC-E2 itself.