Systemic lupus erythematosus (SLE) is a chronic multiorgan autoimmune disorder characterized by abundant immune complex (IC) deposition, with nephritis being a major cause of morbidity and mortality. Yet, IC deposition alone is not sufficient for disease development suggesting that additional factors dictate the propensity for developing target organ injury. Genome-wide association studies have identified polymorphisms in the leukocyte integrin Mac-1 (CD11b/CD18, ITGAM) that associate with lupus nephritis. Although Mac-1 promotes inflammation by triggering leukocyte recruitment and cytotoxic functions, there is emerging evidence that it may also serve protective roles under certain conditions. We demonstrate that Mac-1 deficiency in the context of the uniquely human FcgRIIA a receptor that binds IgG-IC, promotes susceptibility to lupus nephritis in two independent animal models. Analysis of renal tissue and intravital microscopy revealed that Mac-1 modulates neutrophil recruitment by FcgRIIA. The SLE-associated variant of Mac-1 rs1143679 (R77H), results in reduced Mac-1 functions, but the underlying mechanism remains undefined. CD18 integrin mediated adhesion is a multistep process that begins with affinity changes for ligand via transmission of allosteric signals. Moreover, mechanical forces (e.g. shear flow) paradoxically increase the lifetime of integrin-ligand bonds, referred to as "catch-bonds". Here, we show that expression of Mac-1 R77H on neutrophils, and blocking antibodies to the extracellular b-propeller domain in which it resides, markedly impairs Mac-1 adhesion to ligand under shear flow. R77H expressing cells exhibit a shift in equilibrium towards a bent conformation, a lower affinity and on- and off- rate for ligand and an inability to form catch-bonds. Additional mutants and activating antibodies reveal that R77H prevents allosteric signal transmission to the aI-domain required for productive ligand binding.

abstract: Integrins are a family of αβ heterodimeric transmembrane receptors. As an important class of adhesion receptors, integrins mediate cell adhesion, migration, and transformation through bidirectional signaling across the plasma membrane. Among the 24 different types of integrins, which are notorious for their capacity to recognize multiple ligands, the leukocyte integrin αMβ2 (Mac-1) is the most promiscuous member. In contrast to other integrins, Mac1 is unique with respect to its preference for cationic ligands. In this thesis, a new Mac-1 cationic ligand named pleiotrophin (PTN) is uncovered. PTN is an important cytokine and growth factor. Its activities in mitogenesis and angiogenesis have been extensively researched, but its function on immune cells was not widely explored. In this research, the cell biology and biochemical evidences show that PTN can regulate various Mac-1-expressing cells functions through the activation of the extracellular signal regulated kinases. Direct interactions between PTN and the αM I-domain, the major ligand-binding domain of Mac-1, has been shown using biolayer interferometry analyses and confirmed by solution NMR spectroscopy. The binding epitopes and the binding mechanism of PTN and αM I-domain interaction were further revealed by peptide array analysis and microscale thermophoresis. The data suggested that PTN’s thrombospondin type-1 repeat (TSR) domains and αM I-domain metal-ion-dependent adhesion site (MIDAS) are the major binding sites. In addition, this interaction followed a novel metal-ion independent binding mechanism which has not been found in other integrins. After a series of characterizations of αM I-domain using both experimental and computational methods, it showed that activated αM I-domain is significantly more dynamic than inactive αM I-domain, and the dynamics seem to modulate the effect of Mg2+ on its interactions with cationic ligands. To further explore the PTN induced Mac-1 structure rearrangement, intact Mac-1 was studied by negative stain electron microscopy. The results showed that the Mac-1 exhibited a very heterogeneous conformation distribution in detergents. In contrast, the Mac-1 adopted predominantly the bent conformation in phospholipid nanodisc condition. This Mac-1 nanodisc model provides a new platform for studying intact Mac-1 activation mechanism in a more physiologically relevant manner in the future. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

A galectina-1 (Gal-1) é uma lectina homodimérica multifuncional capaz de reconhecer e se ligar a beta-galactosídeos por meio de um domínio denominado carbohydrate recognition domain (CRD). A Gal-1 humana (Gal-1h) e a Gal-1 de camundongo (Gal-1c) mantêm 88,15% de homologia e, apesar de não existirem mutações em aminoácidos-chave do CRD, há substituições próximas a esses resíduos. Considerando as implicações dessas diferenças em estrutura e função, e que é comum a utilização de modelos murinos para estudar a função Gal-1, o presente trabalho objetiva analisar comparativamente a Gal-1c e a Gal-1h por meio de ensaios de cristalização e determinação estrutural da Gal-1c, além da avaliação comparativa da atividade lectínica da Gal-1h e da Gal-1c por glycan array e hemaglutinação. Também foi avaliada a capacidade de ambas as Gal-1 em induzir a exposição de fosfatidilserina (FS) em neutrófilos ativados provenientes de medula de camundongos normais ou deficientes de ?-2 integrina (Mac-1), de modo a investigar se a interação Gal-1/Mac-1 estaria envolvida nesse processo. Preparações homogêneas e ativas de Gal-1c e Gal-1h foram utilizadas nos ensaios. Os cristais de Gal-1c foram obtidos em 20% de polietilenoglicol 3350 e 0,2 M de fluoreto de amônio. Os dados de difração de raios X foram coletados e processados, obtendo-se uma estrutura com resolução de 2,4 Å. Observou-se que substituições de aminoácidos entre a Gal-1c e a Gal-1h estão localizadas em regiões expostas ao solvente, próximas do CRD e distantes da interface de dimerização. A análise comparativa entre Gal-1c e Gal-1h mostrou que estas substituições conferem a Gal-1c um caráter mais polar, com consequente aumento da distribuição de volume molecular. Nos ensaios de hemaglutinação, pode-se observar que é necessária uma concentração 2 vezes maior de Gal-1c para aglutinar eritrócitos humanos, de carneiro e de coelho na mesma proporção que a Gal-1h. Por meio do glycan array, pode-se determinar o perfil de ligação a glicanas de ambas as Gal-1. As duas Gal-1 apresentam afinidade por glicanas ramificadas contendo galactose terminal, e a Gal-1h apresentou maior intensidade de ligação às glicanas quando comparada à Gal-1c. Preparações de Gal-1c e Gal-1h induzem níveis semelhantes de exposição de FS na superfície de neutrófilos deficientes ou não de Mac-1, sugerindo que a interação Gal-1/Mac-1 não esteja envolvida no processo de exposição de FS na superfície de neutrófilos ativados. Assim, a diferença sequencial entre a Gal-1c e a Gal-1h é capaz de gerar diferenças estruturais consideráveis que implicam no reconhecimento diferencial de glicanas, o que, entretanto, não se reflete na capacidade de indução de FS na superfície de neutrófilos ativados. Além disso, a interação Gal-1/Mac-1 parece não participar desse processo, o que pode indicar que o papel da Gal-1 no turnover de neutrófilos, via reconhecimento fagocítico, seja um processo complexo e independente dessa interação. / Galectin-1 (Gal-1) is a homodimeric and multifunctional lectin that recognizes and binds to beta-galactoside by a carbohydrate recognition domain (CRD). Human Gal-1 (hGal-1) and mouse Gal-1 (mGal-1) are 88.15% identical, and although there are no mutations in key amino acids within the CRD, there are differences in the amino acids sequence near the CRD. Given the potential of these differences to alter overall structure and function, and the common utilization of murine models to study Gal-1 function, we sought to directly compare key biochemical features of hGal and mGal-1. Thus, we performed crystallization and structure determination assays of mGal-1, and determined the carbohydrate binding specificy of mGal-1 and hGal-1 using a glycan array and using hemagglutination assay. We also evaluated the ability of both Gal-1 to induce exposure of phosphatidylserine (PS) in activated neutrophils from the bone marrow of normal or ?-2 integrin (Mac-1) deficient mice, in order to investigate the involvement of Gal-1/Mac-1 interaction in this process. To accomplish this, homogeneous and active preparations of hGal-1 and mGal-1 were used in the study. mGal-1 crystals were obtained in 20% polyethylene glycol 3350 and 0.2 M ammonium fluoride. Data from X-ray diffraction were collected and processed, yielding a structure with a final resolution of 2.4 Å. The amino acid substitutions found between mGal-1 and hGaI-1 are detected on the solvent-exposed surfaces where the CRDs are located and not on the proteins dimerization surfaces. A comparative structural analysis between mGal-1 and hGal-1 shows that these amino acid substitutions confer to mGal-1 a greater number of ionizable residues, polar character, appearance of the acid regions clustered, and a slight increase of volume distribution. In hemagglutination assays, twice the concentration of mGal-1 was required to cause equivalent agglutination of human, sheep or rabbit erythrocytes as hGal-1. Glycan array analysis demonstrated that both galectins have affinity for branched glycans containing terminal galactose residues. However, hGal-1 appeared to display higher levels of binding that mGal-1. Preparations of mGal-1 and hGal-1 induced similar levels of PS exposure on normal or Mac-1 deficient neutrophils, suggesting that the interaction Gal-1/Mac-1 is not involved in this process. Thus, hGal-1 and mGal-1 appear to possess considerable differences in glycan recognition that likely reflects subtle difference in amino acid sequence. Furthermore, the interaction Gal-1/Mac-1 do not appear to participate in this PS exposure process, which suggest that other Gal-1 receptors are likely important in this process.

A galectina-1 (Gal-1) é uma lectina homodimérica multifuncional capaz de reconhecer e se ligar a beta-galactosídeos por meio de um domínio denominado carbohydrate recognition domain (CRD). A Gal-1 humana (Gal-1h) e a Gal-1 de camundongo (Gal-1c) mantêm 88,15% de homologia e, apesar de não existirem mutações em aminoácidos-chave do CRD, há substituições próximas a esses resíduos. Considerando as implicações dessas diferenças em estrutura e função, e que é comum a utilização de modelos murinos para estudar a função Gal-1, o presente trabalho objetiva analisar comparativamente a Gal-1c e a Gal-1h por meio de ensaios de cristalização e determinação estrutural da Gal-1c, além da avaliação comparativa da atividade lectínica da Gal-1h e da Gal-1c por glycan array e hemaglutinação. Também foi avaliada a capacidade de ambas as Gal-1 em induzir a exposição de fosfatidilserina (FS) em neutrófilos ativados provenientes de medula de camundongos normais ou deficientes de ?-2 integrina (Mac-1), de modo a investigar se a interação Gal-1/Mac-1 estaria envolvida nesse processo. Preparações homogêneas e ativas de Gal-1c e Gal-1h foram utilizadas nos ensaios. Os cristais de Gal-1c foram obtidos em 20% de polietilenoglicol 3350 e 0,2 M de fluoreto de amônio. Os dados de difração de raios X foram coletados e processados, obtendo-se uma estrutura com resolução de 2,4 Å. Observou-se que substituições de aminoácidos entre a Gal-1c e a Gal-1h estão localizadas em regiões expostas ao solvente, próximas do CRD e distantes da interface de dimerização. A análise comparativa entre Gal-1c e Gal-1h mostrou que estas substituições conferem a Gal-1c um caráter mais polar, com consequente aumento da distribuição de volume molecular. Nos ensaios de hemaglutinação, pode-se observar que é necessária uma concentração 2 vezes maior de Gal-1c para aglutinar eritrócitos humanos, de carneiro e de coelho na mesma proporção que a Gal-1h. Por meio do glycan array, pode-se determinar o perfil de ligação a glicanas de ambas as Gal-1. As duas Gal-1 apresentam afinidade por glicanas ramificadas contendo galactose terminal, e a Gal-1h apresentou maior intensidade de ligação às glicanas quando comparada à Gal-1c. Preparações de Gal-1c e Gal-1h induzem níveis semelhantes de exposição de FS na superfície de neutrófilos deficientes ou não de Mac-1, sugerindo que a interação Gal-1/Mac-1 não esteja envolvida no processo de exposição de FS na superfície de neutrófilos ativados. Assim, a diferença sequencial entre a Gal-1c e a Gal-1h é capaz de gerar diferenças estruturais consideráveis que implicam no reconhecimento diferencial de glicanas, o que, entretanto, não se reflete na capacidade de indução de FS na superfície de neutrófilos ativados. Além disso, a interação Gal-1/Mac-1 parece não participar desse processo, o que pode indicar que o papel da Gal-1 no turnover de neutrófilos, via reconhecimento fagocítico, seja um processo complexo e independente dessa interação. / Galectin-1 (Gal-1) is a homodimeric and multifunctional lectin that recognizes and binds to beta-galactoside by a carbohydrate recognition domain (CRD). Human Gal-1 (hGal-1) and mouse Gal-1 (mGal-1) are 88.15% identical, and although there are no mutations in key amino acids within the CRD, there are differences in the amino acids sequence near the CRD. Given the potential of these differences to alter overall structure and function, and the common utilization of murine models to study Gal-1 function, we sought to directly compare key biochemical features of hGal and mGal-1. Thus, we performed crystallization and structure determination assays of mGal-1, and determined the carbohydrate binding specificy of mGal-1 and hGal-1 using a glycan array and using hemagglutination assay. We also evaluated the ability of both Gal-1 to induce exposure of phosphatidylserine (PS) in activated neutrophils from the bone marrow of normal or ?-2 integrin (Mac-1) deficient mice, in order to investigate the involvement of Gal-1/Mac-1 interaction in this process. To accomplish this, homogeneous and active preparations of hGal-1 and mGal-1 were used in the study. mGal-1 crystals were obtained in 20% polyethylene glycol 3350 and 0.2 M ammonium fluoride. Data from X-ray diffraction were collected and processed, yielding a structure with a final resolution of 2.4 Å. The amino acid substitutions found between mGal-1 and hGaI-1 are detected on the solvent-exposed surfaces where the CRDs are located and not on the proteins dimerization surfaces. A comparative structural analysis between mGal-1 and hGal-1 shows that these amino acid substitutions confer to mGal-1 a greater number of ionizable residues, polar character, appearance of the acid regions clustered, and a slight increase of volume distribution. In hemagglutination assays, twice the concentration of mGal-1 was required to cause equivalent agglutination of human, sheep or rabbit erythrocytes as hGal-1. Glycan array analysis demonstrated that both galectins have affinity for branched glycans containing terminal galactose residues. However, hGal-1 appeared to display higher levels of binding that mGal-1. Preparations of mGal-1 and hGal-1 induced similar levels of PS exposure on normal or Mac-1 deficient neutrophils, suggesting that the interaction Gal-1/Mac-1 is not involved in this process. Thus, hGal-1 and mGal-1 appear to possess considerable differences in glycan recognition that likely reflects subtle difference in amino acid sequence. Furthermore, the interaction Gal-1/Mac-1 do not appear to participate in this PS exposure process, which suggest that other Gal-1 receptors are likely important in this process.

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