Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains xi, 190 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

A origem das doenças autoimunes é multifatorial, sendo que envolve condições ambientais e predisposição genética, dificultando sua identificação. Muitos pesquisadores têm estudado a associação entre agentes infecciosos e autoimunidade, a qual pode ser disparada pelo processo conhecido por mimetismo molecular. Neste caso, respostas imunes cruzadas envolvendo antígenos próprios têm sido documentadas. O presente projeto tem como objetivo a busca in silico por associações entre epítopos de microrganismos e autoantígenos humanos. Iniciaram-se as análises pela identificação de semelhanças de sequências de aminoácidos entre epítopos de microrganismos e autoantígenos humanos por meio do alinhamento local de sequências efetuado pelo programa BLASTP. As sequências de epítopos dos microrganismos e autoantígenos humanos foram previamente adquiridas nos bancos de dados Immune Epitope Database and Analysis Resource (IEDB) e no Genbank, respectivamente. Foram também realizadas modelagens de estruturas proteicas para o antígeno e o autoantígeno que obtiveram melhores valores de alinhamento, com base no valor do E-value, por meio dos programas Modeller e Rosetta. Por fim, a predição de epítopos foi executada, pelo uso dos softwares NetMHC e NetMHCII, para avaliar a possibilidade de epítopos de microrganismos e de autoantígenos humanos se associarem aos mesmos alelos de HLA. Como resultado, foram encontradas similaridades tanto de sequências proteicas quanto de afinidade a 4 tipos de alelos de HLA entre um epítopo do antígeno LSA-1 de Plasmodium falciparum e o autoantígeno de miosina, o que sugere uma associação entre eles, atingindo o objetivo deste trabalho. / The origin of autoimmune diseases is multifactorial. It involves environmental conditions and genetic predisposition that difficulties its identification. Several researchers have studied the association between infectious agents and autoimmunity, which can be initiated by a process named molecular mimicry. In this case, cross immune responses involving self antigens have been documented. This project aims to search in silico for associations between microorganisms epitopes and human autoantigens. The first step was the identification of similarities in amino acid sequences between microorganisms epitopes and human autoantigens by use of sequence local alignment performed by the program blastp. The sequences of the microorganisms epitope and the human autoantigens had been previously acquired in the Immune Epitope Database and Analysis Resource (IEDB) and Genbank, respectively. The modeling of protein structures for the antigen and autoantigen was also carried out to show the best alignment values, based on the E-value, using the programs Modeller and Rosetta. Finally, the prediction of epitopes was performed by use of NetMHC and NetMHCII softwares to evaluate the possibility of microorganisms epitopes and human autoantigens join the same HLA alleles. Similarities of protein sequences was found for both. It was possible to observe affinity of 4 HLA alleles between an epitope from LSA-1 Plasmodium falciparum antigen and the myosin, suggesting an association between them, reaching the goal of this work.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.

A origem das doenças autoimunes é multifatorial, sendo que envolve condições ambientais e predisposição genética, dificultando sua identificação. Muitos pesquisadores têm estudado a associação entre agentes infecciosos e autoimunidade, a qual pode ser disparada pelo processo conhecido por mimetismo molecular. Neste caso, respostas imunes cruzadas envolvendo antígenos próprios têm sido documentadas. O presente projeto tem como objetivo a busca in silico por associações entre epítopos de microrganismos e autoantígenos humanos. Iniciaram-se as análises pela identificação de semelhanças de sequências de aminoácidos entre epítopos de microrganismos e autoantígenos humanos por meio do alinhamento local de sequências efetuado pelo programa BLASTP. As sequências de epítopos dos microrganismos e autoantígenos humanos foram previamente adquiridas nos bancos de dados Immune Epitope Database and Analysis Resource (IEDB) e no Genbank, respectivamente. Foram também realizadas modelagens de estruturas proteicas para o antígeno e o autoantígeno que obtiveram melhores valores de alinhamento, com base no valor do E-value, por meio dos programas Modeller e Rosetta. Por fim, a predição de epítopos foi executada, pelo uso dos softwares NetMHC e NetMHCII, para avaliar a possibilidade de epítopos de microrganismos e de autoantígenos humanos se associarem aos mesmos alelos de HLA. Como resultado, foram encontradas similaridades tanto de sequências proteicas quanto de afinidade a 4 tipos de alelos de HLA entre um epítopo do antígeno LSA-1 de Plasmodium falciparum e o autoantígeno de miosina, o que sugere uma associação entre eles, atingindo o objetivo deste trabalho. / The origin of autoimmune diseases is multifactorial. It involves environmental conditions and genetic predisposition that difficulties its identification. Several researchers have studied the association between infectious agents and autoimmunity, which can be initiated by a process named molecular mimicry. In this case, cross immune responses involving self antigens have been documented. This project aims to search in silico for associations between microorganisms epitopes and human autoantigens. The first step was the identification of similarities in amino acid sequences between microorganisms epitopes and human autoantigens by use of sequence local alignment performed by the program blastp. The sequences of the microorganisms epitope and the human autoantigens had been previously acquired in the Immune Epitope Database and Analysis Resource (IEDB) and Genbank, respectively. The modeling of protein structures for the antigen and autoantigen was also carried out to show the best alignment values, based on the E-value, using the programs Modeller and Rosetta. Finally, the prediction of epitopes was performed by use of NetMHC and NetMHCII softwares to evaluate the possibility of microorganisms epitopes and human autoantigens join the same HLA alleles. Similarities of protein sequences was found for both. It was possible to observe affinity of 4 HLA alleles between an epitope from LSA-1 Plasmodium falciparum antigen and the myosin, suggesting an association between them, reaching the goal of this work.

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Pacientes com doença reumática cardíaca (DRC) desenvolvem lesões valvares mediadas por linfócitos T CD4+, capazes de reconhecer cruzadamente proteínas cardíacas e estreptocócicas pelo mecanismo de mimetismo molecular. Neste trabalho empregamos uma biblioteca peptídica de Phage Display para identificar auto-antígenos cardíacos capazes de serem reconhecidos por duas linhagens intralesionais de linfócitos T e um clone derivado de uma das linhagens isolados de pacientes com DRC. A análise dos peptídeos dos fagos em banco de dados de proteínas revelou novos epitopos da miosina cardíaca, laminina, vimentina e outras proteínas coiled-coil, provavelmente involvidos no processo auto-imune da DRC. Outras moléculas inflamatórias como citocinas, integrinas e fatores de crescimento também foram identificadas / Rheumatic heart disease (RHD) patients develop valvar lesions with CD4+ T lymphocytes infiltrating the heart. Molecular mimicry between streptococcal and cardiac proteins recognized by these T cells may explain these auto-aggressive lesions. In the present work we used a Phage Display peptide library to identify cardiac antigens which could be recognized by two heart infiltrate T cell lines and by a T cell clone derived from one of the lines which were isolated from RHD patients. Using the protein data bank to analyse the phage peptides, we observed that many sequences showed homology with cardiac myosin, laminin, vimentin and other coiled-coil proteins, suggesting the involvement of these proteins in the autoimmune process of RHD. Other inflammatory molecules such as cytokines and integrins were also identified

The work embodied in this thesis pertains to an attempt to understand better, the molecular basis of protein-carbohydrate recognition. For this purpose a systematic study was undertaken, not only of the energetics of lectin-sugar interactions, which serve as molecular recognition prototype of protein-carbohydrate interactions, but also of the complex effects of solvent water molecules surrounding both the species in solution state. The systems chosen for investigation include the specific recognition of sugars by lectins from diverse families, leguminosae and moraceae. The following salient aspects of the molecular recognition process constitute the focus of this thesis: • Effect of site specifically modified, deoxy-, fluorodeoxy-, or methoxy- substituted D-galactopyranoside binding to lectins. Isothermal titration calorimetric (ITC) investigations of the binding of these sugars to a model lectin permitted the correct prediction of the architecture of the primary binding site in the absence of x-ray crystal or NMR structure of the combining site (Ref. 7). The study provided the only unambiguous means of a site specific mapping of the hydrogen-bond donor- acceptor relationship of the monosaccharide within the primary combining site of the lectin. • Novel features of lectin-sugar recognition. Molecular interactions and forces contributing to the stabilization of the saccharides in the primary combining site of lectins. Binding of site specifically modified fluoro- substituted D- galactopyranosides to WBA I led to the demonstration of the involvement of C- F««»H-0 hydrogen bonds in stabilizing the saccharide within the combining site of lectin (Ref. 7). Implication of the novel C-H«**O hydrogen bonds at the specificity determining C-4 position in enabling the methoxy- substituted D- galactopyranoside to be stabilized within the primary binding site of galactose specific lectins WBA I and jacalin. • Development of a novel coupled osmotic-thermodynamic approach for investigating the role of water molecules in determining the specificity of lectin- sugar interactions. The results obtained led to the first direct demonstration of a differential uptake of water molecules accompanying the specific process of recognition of sugars by lectins (Ref 2) • On the origin of enthalpy-entropy compensation, a ubiquitous phenomenon accompanying the thermodynamics of several ligand binding reactions in aqueous solutions in general and the molecular recognition involving all known lectin-sugar interactions, in particular. The results provide the first unequivocal solution state proof of water reorganization as the source of enthalpy-entropy compensation (Ref 3). A new diagnostic test of a true osmotic effect in molecular recognition phenomena was proposed (Ref. 2) and validated (Ref. 3). As an introduction, Chapter 1 is a comprehensive review of literature that touches upon the diverse properties of lectins and our present understanding of their multifarious roles and applications, which has led to their christening, perhaps appropriately, as molecules that mediate the 'social' functions of cells and tissues. Although a challenge it is still, to decipher the "glycocode", it is apparent that the fundamental basis of the recognition function of lectin-sugar interactions is the initial specific binding of the saccharide molecule by the globular proteinaceous lectin molecule. It is imperative, therefore, that an incisive investigation of the origin of specificity of the binding reaction as well as the solvent effects influencing both the interacting species be undertaken for a better understanding of the complete molecular recognition process. Towards this end is introduced in Chapter 1 our present understanding of the results on lectin-sugar interactions from two complementary approaches viz structural, including X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, as well as thermodvnamic ones, which have provided important information on the architecture of the combining sites, the dynamic modes of saccharide recognition and forces involved therein. Despite a detailed knowledge available from such methods, a structure-energetics correlation has persisted as a current challenge of the field. Towards achieving this goal, studies on the energetics of the recognition of sugars by lectins were undertaken, with an aim to better understand the origin of specificity of lectin-sugar interactions. This thesis attempts to provide new insights on some of the possible lacunae precluding structure-energetics correlation and suggests ways to overcome them. Chapter 2 deals with ITC investigation of the effect of deoxy-, fluorodeoxy-, and methoxy- substitutions on the binding of monosaccharides to the primary combining site of the lectin WBA I isolated from the mature seeds of the leguminosae family member Psophocarpus tetragonolobus as well as the moraceae lectin jacalin. These studies provide valuable information on the hydrogen-bond donor-acceptor relationships within the combining site of the lectins wherein the sugar molecule is liganded with the amino-acid residues of the lectin. This study is relevant for understanding the origin of specificity of monosaccharide binding within the primary combining site of the lectins. It has recently become apparent that there is a predisposition in three-dimensional space, of the donor-acceptor pairs within the sugar binding site of the lectins. Hence there appears to be a stereochemical basis of distinguishing the recognition of the donor group vis-a-vis that of the acceptor group and that their spatial disposition determines the specificity of the saccharide recognition. Unambiguous assignment of which of the groups within the hydrogen bonded pairs is a donor and which one is the acceptor assumes greater importance. The ITC measurements of the binding of deoxy-, flurodeoxy-and methoxy-derivatives of D-galactopyranoside (oc-D-Gal) to the basic lectin from winged bean Psophocarpus tetragonolobus, WBA I revealed that each of the ligands bind to WBA I with the same stoichiometry of one per subunit (29 kDa) of WBA I. The binding enthalpies for various derivatives were essentially independent of temperature and showed complementary changes with respect to binding entropies. Replacement of the hydroxyl group by fluorine or hydrogen on C3 and C4 of the galactopyranoside eliminated their binding to the lectin, consistent with C3-OH and C4-OH acting as hydrogen bond donors. The affinity for C2 derivatives of galactose decreased in the order: GalNAc>2MeOGal>2FGal=Gal>2HGal which suggests that both polar and non-polar residues surround the C2 locus of galactose, consistent with the observed high affinity of WBA I towards GalNAc, where the acetamido group at C2 position is probably stabilized by both non-polar interactions with the methyl-group and polar interactions with the carbonyl group. The binding of C6 derivatives followed the order: Gal>6FGal>D-Fuc»6MeOGal=L-Ara indicating the presence of favourable polar interactions with a hydrogen bond donor in the vicinity. Based on these results the hydrogen bond donor-acceptor relationship of the complexation of methyl-a-D-galactopyranoside with the primary combining site of WBA I was proposed (Ref. /), which was subsequently validated by the crystal structure of methyl-a-D-galactopyranoside complexed with WBA I. This chapter also describes the results from ITC studies on the binding of monosaccharides and disaccharides to the lectin jacalin isolated from the mature seeds of the moraceae family member Artocarpus integrifolia. The novel observation about the existence of C-F*«*H-0 and C-H**»O hydrogen bonds in lectin-sugar interactions is also discussed in this chapter. Chapter 3 is a description of the detailed investigation on the role of water molecules in influencing the energetics of lectin-sugar recognition. A novel coupled osmotic-thermodynamic approach was developed to dissect the role of water molecules in determining the recognition of the sugars by lectins. For this purpose, the model system of mannotriose-concanavalin A was used because atomic level structural information on these complexes were available. The work described in this chapter, is the first solution state evidence for the role of water molecules in the specific interaction of carbohydrates with a legume lectin, concanavalin A (Con A) (Ref. 2). Sugar binding to Con A was accompanied by linear changes in the logarithm of binding constants as a function of neutral osmolyte strength, and were described by well defined negative slopes characteristic for each sugar. As these changes were independent of the chemical nature of the osmolyte used, the results were rationalized in terms of a true osmotic effect. It was demonstrated that the specific recognition of the branched trimannoside (3,6-di-0-(a-D-mannopyranosyl)~a-D-mannopyranoside), the individual dimannosidic arms (3-<9-(a-D-mannopyranosyl)-a-D-mannopyranoside, and 6-0-(a-D-marmopyranosyl)-a-D-mannopyranoside) and the monomeric unit D-mannopyranoside by Con A was accompanied by differential uptake of water molecules; 1,3 and 5 respectively. We also observed a conservation of the compensatory behaviour of binding enthalpies and entropies in the presence as well as absence of osmolytes. This provided the first definitive evidence that water-reorganization plays a direct role in effecting the phenomenon of enthalpy-entropy compensation in protein-ligand interactions in general and lectin-sugar interactions in particular, and that the specificity of lectin-sugar recognition is characterized by a differential uptake of water molecules. Chapter 3 also describes the first experimental identification of the origin of enthalpy-entropy compensation (EEC), a ubiquitous phenomenon accompanying the thermodynamics of multifarious biomolecular recognition processes. By coupling direct microcalorimetry with osmotic stress technique, an experimental handle was devised to test the hypothesis that solvent reorganization could be the source of EEC. The results provided an unequivocal demonstration that an osmotic change in water activity alone, at the same temperature and pH, is sufficient to result in the conservation of EEC during the molecular recognition of specific ligands by macromolecules belonging to thermodynamically diverse and unrelated systems, a compelling evidence that the primary source of EEC in aqueous solutions is attributable to reorganization of solvent water molecules, thus validating the test for the role of water reorganization as a source of EEC (Ref. 3). This provides the first definitive evidence for the notion that there is a direct involvement of water molecules in originating the EEC effect. Despite the generality of the results it is urged that several systems be subjected to a vigorous application of the coupled osmotic-thermodynamic approach proposed herein before constituting it as a proof. Suffice to say, it is perhaps heartening that at last one has a handle to test the role of water molecules in effecting EEC in the solution state and appreciate the diverse roles played by water molecules in mediating molecular recognition reactions. The proposal presented in Ref 2, that the strong isoequilibrium relationship of enthalpy with entropy during the recognition of saccharides by Con A studied under osmotic stress, be considered as diagnostic of a true osmotic effect was subsequently validated in a thermodynamically diverse and unrelated system of peptide recognition by monoclonal antibody, the results from which are discussed in an Appendix (A) to this thesis (Ref 4). That the stabilities of these lectins are not hampered in the presence of osmolytes was demonstrated using differential scanning calorimetry (DSC) (Ref 2). During the course of these DSC studies, we discovered an unusual feature in an animal galectin. Despite possessing the legume lectin fold, the 14-kDa S- type lectin exhibits multiple oligomeric states that are influenced profoundly by complementary ligands and surprisingly do not dissociate at the denaturation temperature. These results are discussed in an Appendix (B) to this thesis (Ref. 5). The general discussion and conclusions drawn from this work are summarized in chapter 4. Briefly, the following salient conclusions can be drawn from the work presented in this thesis: 1. Unambiguous assignment of hydrogen-bond donor-acceptor relationship at each of the hydroxyl group of the monosaccharide bound to the lectin belonging to different families has been demonstrated (Refs. 1,6). 2. First report of novel hydrogen bonds in lectin-sugar interactions such as C- F«MH-0 (Ref 1) and C-H^*O hydrogen bonds (Ref 6). 3. Unusual structural stabilities in a galectin with a fold similar to that in legume lectins but with starkly different thermodynamic stabilities (Ref 5). 4. We have demonstrated for the first time in solution state, that water molecules are involved in the specific recognition of sugars by concanavalin A (Ref 2). It appears that lectin-sugar recognition reactions are, in general, mediated by a net uptake of water molecules during the binding process (Ref 7). 5. We have provided the first experimental demonstration that reorganization of water molecules is the source of enthalpy-entropy compensation in molecular recognition processes (Ref 3). 6. We provide evidence for another facet in the recognition of antigens by antibodies, viz water release accompanying the binding reaction (Ref 4). The studies reported in this thesis provide the foundation for embarking on a systematic study not only of the origin of specificity of lectin-sugar recognition but also of the complex roles that water molecules play in mediating these molecular recognition processes. These specific binding reactions wherein non-linear thermodynamics predominates and precludes a direct structure-energetics correlation emphasize the need to account for the effect of solvent water molecules in lectin-sugar interactions in particular and, without any overemphasis, in molecular recognition processes in general.

Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease. HTLV-1 Related Endogenous Sequence (HRES-1), a human endogenous retrovirus, produces 2 retroviral-like Gag capsid proteins (p8 and p15) that share significant sequence homology to the U1-subunit of the small ribonucleoprotein complex (U1sn-RNP), an autoantigen of lupus. The central hypothesis is that molecular mimicry between HRES-1 and U1sn-RNP serves as a priming event in SLE via the production of cross-reactive autoantibodies. Anti-HRES-1/U1sn-RNP serological responses in subjects with SLE and comparison populations were characterized. An overlapping peptide set mapping the HRES-1 p8 and p15 proteins was used. SLE subjects produce IgG to several regions of HRES-1. Healthy subjects or those with RA, HIV-1 infection, or HTLV-1-infection produced no significant anti-HRES-1 IgG. Anti-HRES-1 antibodies deposited in the kidneys of patients with SLE glomerulonephritis were identified. Our data suggests that HRES-1 plays a role in SLE by means of a molecular mimicry mechanism with U1sn-RNP.

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