EPI-peptide designer: a tool for designing peptide ligand libraries based on epitope-paratope interactions.

MOTIVATION: Antibodies are an important class of biological drugs, but with limitations, such as inadequate pharmacokinetics, adverse immunogenicity and high production costs. Synthetic peptides for the desired target represent an important alternative to antibodies. However, no computational tool exists to guide the design of these peptides. RESULTS: To identify the interacting residues in a given antibody-antigen (Ab-Ag) interface we used Interface Interacting Residue (I2R), a selection method based on computed molecular interactions. The aggregation of all the molecular interactions between epitope and paratope residues allowed us to transform the 3D Ab-Ag complex structures into interface graphs. Based on these data and the probability of molecular interaction we developed EPI-Peptide Designer tool that uses predicted paratope residues for an epitope of interest to generate targeted peptide ligand libraries. EPI-Peptide Designer successfully predicted 301 peptides able to bind to LiD1 target protein (65% of the experimentally tested peptides), an enrichment of 22% compared to randomly generated peptides. This tool should enable the development of a new generation of synthetic interacting peptides that could be very useful in the biosensor, diagnostic and therapeutic fields. AVAILABILITY AND IMPLEMENTATION: All software developed in this work are available at http://www.biocomp.icb.ufmg.br/biocomp/ CONTACT: liza@icb.ufmg.br SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Protein-Protein and Peptide-Protein Interactions of NudE-Like 1 (Ndel1): A Protein Involved in Schizophrenia.

Schizophrenia (SCZ) is a devastating chronic mental disease determined by genetic and environmental factors, which susceptibility may involve an impaired neural migration during the neurodevelopmental process. Several candidate risk genes potentially associated with SCZ were related to the formation of protein complexes that ultimately mediate alterations in the neuroplasticity. The most studied SCZ risk gene is the Disrupted-in-Schizophrenia 1 (DISC1) gene, which functions seem to depend on the binding with cytoskeleton proteins, as the Nuclear-distribution gene E homolog like-1 (Ndel1) protein among others. Interestingly, Ndel1 is the only binding partner of DISC1 proteins with oligopeptidase activity, besides playing roles in multiple processes, including cytoskeletal organization, cell signaling, neuron migration, and neurite outgrowth. It is still not clear if the protein-protein interaction between Ndel1 and DISC1 is enough to explain all cellular functions attributed to these proteins, but there are several lines of evidence suggesting the importance of the catalytic activity of Ndel1 for the neurite outgrowth and neuron migration during embryogenesis. Recent works of the group have demonstrated the modulation of Ndel1 activity by DISC1, which is hypothetically impaired in SCZ patients. In fact, more recently, we also showed a lower Ndel1 activity in the plasma of SCZ patients compared to control health subjects, but the physiopathological significance of this feature is still unknown. Here we discuss Ndel1 ligands involved in protein-protein complex formations related to neurodevelopmental diseases, as (1) lissencephaly or Miller-Dieker Syndrome (MDS), which is characterized by the typical craniofacial features and abnormal smooth cerebral surface, and as (2) SCZ, since they both seem to be determined by defects in neuronal migration. Although impaired lissencephaly protein Lis1 complex formation with Ndel1 is the leading cause of lissencephaly, this binding does not affect Ndel1 oligopeptidase activity. On the other hand, although MDS and SCZ may be both determined by an abnormal neuronal migration, DISC1 complex formation with Ndel1 was shown to inhibit Ndel1 activity. Also differently of MDS, SCZ needs inputs from environmental factors, while lissencephaly is not likely dependent or affected by the environment. Several other proteins and peptide ligands were described for Ndel1, Lis1 and DISC1, thanks to the employment of biochemical, immunochemical, and biological (using cells or living animals) assays, including heterologous expression and also simply by purification from nature of these proteins in the complex form. Effects of the post-translational modifications of these proteins are also discussed here. Taken together, the data presented here show in essence how protein-protein and proteinpeptide interactions can underlie fundamental processes as cell division, maturation and migration, necessary for adequate formation of a complex structured tissue as the brain. A special attention was given to Ndel1 as this protein binds to either proteins or peptides, besides having proteolytic activity. Moreover, Ndel1 seems to be the key protein underlying two seemingly unrelated diseases with highly complex etiology, as lissencephaly and SCZ.

Generation and molecular characterization of a monoclonal antibody reactive with conserved epitope in sphingomyelinases D from Loxosceles spider venoms.

We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29-36kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism.

Biochemical and immunological characteristics of Peruvian Loxosceles laeta spider venom: neutralization of its toxic effects by anti-loxoscelic antivenoms.

This manuscript describes the general biochemical properties and immunological characteristics of Peruvian spider Loxosceles laeta venom (PLlv), which is responsible for the largest number of accidents involving venomous animals in Peru. In this work, we observed that the venom of this spider is more lethal to mice when compared with L. laeta venom from Brazil (BLlv). The LD50 of PLlv was 1.213 mg/kg when the venom was intradermally injected. The venom displayed sphingomyelinase activity and produced dermonecrotic, hemorrhagic and edema effects in rabbits. 2-D SDS-PAGE separation of the soluble venoms resulted in a protein profile ranging from 20 to 205 kDa. Anti-PLlv and anti-BLlv sera produced in rabbits and assayed by ELISA showed that rabbit antibodies cross-reacted with PLlv and BLlv and also with other Brazilian Loxosceles venoms. Western blotting analysis showed that bands corresponding to 25-35 kDa are the proteins best recognized in every Loxosceles spp venoms analyzed. The immunized rabbits displayed protective effect after challenge with PLlv and BLlv. In vitro assays with horse anti-loxoscelic antivenoms produced in Brazil and Peru demonstrated that these commercial antivenoms were efficient to inhibit the sphingomyelinase activity of PLlv and BLlv.

Generation and characterization of a recombinant chimeric protein (rCpLi) consisting of B-cell epitopes of a dermonecrotic protein from Loxosceles intermedia spider venom.

A chimeric protein was constructed expressing three epitopes of LiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. This species is responsible for a large number of accidents involving spiders in Brazil. We demonstrated that the chimeric protein (rCpLi) generated is atoxic and that antibodies previously developed in rabbits against synthetic epitopes reactive with rCpLi in ELISA and immunoblot assays. The antibody response in rabbits against the rCpLi was evaluated by ELISA and we have detected an antibody response in all immunized animals. Overlapping peptides covering the amino acid sequence of the rCpLi were synthesized on a cellulose membrane, and their recognition by rabbit anti-rCpLi serum assessed. Three different antigenic regions were identified. The percentage of inhibition of the dermonecrotic, hemorrhagic and edematogenic activities caused by the recombinant protein LiD1r in naïve rabbits was assessed by pre-incubation with anti-rCpLi antibodies. Anti-rCpLi induced good dermonecrotic and hemorrhagic protection. The levels of protection were similar to the antiboides anti-LiD1r. In summary, we have developed a polyepitope recombinant chimeric protein capable of inducing multiple responses of neutralizing antibodies in a rabbit model. This engineered protein may be a promising candidate for therapeutic serum development or vaccination.

Protection against the toxic effects of Loxosceles intermedia spider venom elicited by mimotope peptides.

The venom of Loxosceles intermedia (Li) spiders is responsible for cutaneous lesions and other clinical manifestations. We previously reported that the monoclonal antibody LimAb7 can neutralize the dermonecrotic activity of crude Li venom. In this study, we observed that this antibody recognizes several proteins from the venom dermonecrotic fraction (DNF), including LiD1. Identifying the epitope of such a neutralizing antibody could help designing immunogens for producing therapeutic sera or vaccination approaches. To this aim, two sets of 25- and 15-mer overlapping peptides that cover the complete amino acid sequence of LiD1 were synthesized using the SPOT technique. None of them was recognized by LimAb7, suggesting that the epitope is discontinuous. Then, the screening of four peptide phage-display libraries yielded four possible epitope mimics that, however, did not show any obvious similarity with the LiD1 sequence. These mimotopes, together with a 3D model of LiD1, were used to predict with the MIMOP bioinformatic tool the putative epitope region (residues C197, Y224, W225, T226, D228, K229, R230, T232 and Y248 of LiD1) recognized by LimAb7. This analysis and the results of alanine-scanning experiments highlighted a few residues (such as W225 and D228) that are found in the active site of different SMases D and that may be important for LiD1 enzymatic activity. Finally, the only mimotope NCNKNDHLFACW that interacts with LimAb7 by SPOT and its analog NSNKNDHLFASW were used as immunogens in rabbits. The resulting antibodies could neutralize some of the biological effects induced by crude Li venom, demonstrating a mimotope-induced protection against L. intermedia venom.

A protective immune response against lethal, dermonecrotic and hemorrhagic effects of Loxosceles intermedia venom elicited by a 27-residue peptide.

Antibodies raised against recombinant Loxosceles intermedia dermonecrotic protein isoform 1 (rLiD1) display neutralizing capacity for the L. intermedia whole venom. We previously found that an immunodominant continuous B-cell epitope, recognized by these antibodies corresponds to a region of the protein known to be involved in the active site. In this study, we extend previous work by preparing a 27-residue synthetic replica of this epitope ((25)NLGANSIETDVSFDDNANPEYTYHGIP(51)) and using it as an immunogen in mice and rabbits. The immunization process induced antibodies that protected mice from a lethal dose of L. intermedia crude venom and rabbits against the dermonecrotic effects of rLiD1. An Ala scan of the epitope indicated that 4 residues, E44, Y45, T46 and Y47, are essential (over 70% decrease in binding upon replacement with alanine) for antibody recognition. The possible mechanisms of neutralization are discussed in light of these findings.

Design of antibody-reactive peptides from discontinuous parts of scorpion toxins.

The Amm VIII protein was previously isolated from the venom of the scorpion Androctonus mauretanicus mauretanicus. Despite 87% identity with AaH II, the most toxic alpha-type scorpion toxin, Amm VIII is not toxic to mice. However, antisera against Amm VIII protect mice from AaH II lethal action. Here, we report that the Amm VIII protein elicits antibodies that only recognize discontinuous-type epitopes since we could not observe any antibody binding to overlapping 12-mer peptides covering the whole Amm VIII sequence. By using a new bioinformatic tool, 24 peptides mimicking discontinuous regions of Amm VIII were designed in silico, then prepared by Spot synthesis. Seven of these discontinuous-continuous peptides were recognized by anti-Amm VIII antibodies. Analysis of the 3D location of the segments that compose the antigenically reactive discontinuous-continuous peptides, allowed us to group those antigenic segments into three regions of Amm VIII, putatively corresponding to discontinuous antigenic regions of alpha-type scorpion toxins. Anti-Amm VIII antibodies were also found to cross-react towards several of the discontinuous-continuous peptides designed from the AaH II structure, pointing to a possible involvement of the corresponding discontinuous epitopes in the capacity displayed by anti-Amm VIII antibodies to neutralize AaH II. Altogether, our results show that it is possible to design antibody-reactive peptides from discontinuous parts of scorpion toxins. The position of the reactive segments in the structural context of scorpion toxins highlights the antigenic properties of the Amm VIII anatoxin and concurs to explain the capacity of anti-Amm VIII antibodies to neutralize the potent AaH II toxin.

Immunodiagnosis of human neurocysticercosis using a synthetic peptide selected by phage-display.

The usefulness of a synthetic peptide in the serodiagnosis of Taenia solium human neurocysticercosis (NC) has been evaluated. Phage-displayed peptides were screened with human antibodies to scolex protein antigen from cysticercus cellulosae (SPACc). One clone was found to interact specifically with anti-SPACc IgGs. The corresponding synthetic peptide was found to be recognized in ELISA by NC patient's sera. The study was carried out with sera from 28 confirmed NC patients, 13 control sera and 73 sera from patients suffering from other infectious diseases. A 93% sensibility and a 94.3% specificity was achieved. Figures of 89% and 31.4% of sensibility and specificity were obtained in a SPACc-based ELISA. Immunoblotting of SPACc with anti-peptide antibodies revealed a single band of approximately 45 kDa in 1D and four 45 kDa isoforms in 2D-gel electrophoresis. A strong and specific immunostaining in the fibers beneath the suckers, at the base of the rostellum, and in the tissue surrounding the scolex of cysticerci was observed by immunomicroscopy. Our results show that a peptide-based immunodiagnostic of neurocisticercosis can be envisioned.

Functional characterization and epitope analysis of a recombinant dermonecrotic protein from Loxosceles intermedia spider.

In the present study the recombinant form (recLiD1) of a dermonecrotic protein present in the Brazilian brown spider Loxosceles intermedia venom was expressed in Escherichia coli cells and purified by reversed-phase HPLC using a C8 Vydac column. About 25.8mg of purified recLiD1 was produced from a litre of bacterial culture. SDS/PAGE and immunoblot analysis of the recombinant protein revealed an apparent molecular weight of 32-35kDa. The later result was confirmed by mass spectrometry (32,758Da). recLiD1 displayed dermonecrotic and platelet aggregation activities which were qualitatively similar to that displayed by the crude L. intermedia venom. However, very low sphingomyelinase D enzymatic activity and complement-dependent haemolytic activities were observed. recLiD1 immunized BALB/c mice developed an antibody response. Anti-recLiD1 antibodies recognized L. intermedia venom in an indirect enzyme-linked immunosorbent assay (ELISA) and cross-reacted with crude venoms from L. intermedia, L. gaucho and L. laeta. An in vivo protection assay carried out 5 weeks after the end of the immunization protocol showed that 75% of the vaccinated mice could resist the challenge by 2.5LD(50) of L. intermedia venom. To characterize epitopes associated with protective antibodies, we prepare sets of immobilized synthetic 15 mer overlapping peptides covering the complete amino acid sequences of the recLiD1. Antibodies revealed one antigenic region in the N-terminal part of the toxin. The amino acid sequence of this epitope was found in several dermonecrotic proteins and some of its residues have been implicated with the active site of the toxin.

[Patient at hemorrhagic risk. Prevention and local control]. / Il paziente a rischio emorragico. Profilassi e accorgimenti locali.

The Authors make a short analysis of the main alterations of the coagulative system and of the anticoagulant medicines, used in patients with normal hemostasis, but suffering from cardiovascular disease. It is underlined the importance of a close collaboration between the odontologist and the hematologist or the general practitioner, to be able to make a correct pre-operating prophylaxis, if necessary. All the local procedures that the odontologist must prepare to prevent or at least stop the possible hemorrhagic troubles are finally analysed.