Moieties of Complement iC3b Recognized by the I-domain of Integrin αXß2.

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMß2 and αXß2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective ß2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α'- chain (α'NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α'NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.

Characterization of αX I-Domain Binding to Receptors for Advanced Glycation End Products (RAGE).

The ß2 integrins are cell surface transmembrane proteins regulating leukocyte functions, such as adhesion and migration. Two members of ß2 integrin, αMß2 and αXß2, share the leukocyte distribution profile and integrin αXß2 is involved in antigen presentation in dendritic cells and transendothelial migration of monocytes and macrophages to atherosclerotic lesions. Receptor for advanced glycation end products (RAGE), a member of cell adhesion molecules, plays an important role in chronic inflammation and atherosclerosis. Although RAGE and αXß2 play an important role in inflammatory response and the pathogenesis of atherosclerosis, the nature of their interaction and structure involved in the binding remain poorly defined. In this study, using I-domain as a ligand binding motif of αXß2, we characterize the binding nature and the interacting moieties of αX I-domain and RAGE. Their binding requires divalent cations (Mg2+ and Mn2+) and shows an affinity on the sub-micro molar level: the dissociation constant of αX I-domains binding to RAGE being 0.49 µM. Furthermore, the αX I-domains recognize the V-domain, but not the C1 and C2-domains of RAGE. The acidic amino acid substitutions on the ligand binding site of αX I-domain significantly reduce the I-domain binding activity to soluble RAGE and the alanine substitutions of basic amino acids on the flat surface of the V-domain prevent the V-domain binding to αX I-domain. In conclusion, the main mechanism of αX I-domain binding to RAGE is a charge interaction, in which the acidic moieties of αX I-domains, including E244, and D249, recognize the basic residues on the RAGE V-domain encompassing K39, K43, K44, R104, and K107.

Characterization of the residues of αX I-domain and ICAM-1 mediating their interactions.

Integrin αXß2 performs a significant role in leukocyte functions including phagocytosis and migration, and binds to a variety of ligands, including fibrinogen, iC3b, and ICAM-1. A particular domain of the α subunit of the integrin - the αX I-domain - is a ligand binding site, and the interaction of the αX I-domain and ICAM-1 on the endothelium is an important step in leukocyte extravasation. In order to elucidate the structural aspects of this interaction, we defined the moieties of the αX and ICAM-1 relevant to their interaction in this study. It was determined that the ICAM-1 binding sites of the αX I-domain were located in the α3α4, ßDα5, and ßFα7 loops at the top surface of the I-domain. The residues Q(202), K(242), K(243), E(298) and D(299) on these loops were crucial for the recognition of ICAM-1. Among these residues, K(242) and K(243) on the ßDα5 loop were found to be the most salient, thereby suggesting an ionic interaction between these proteins. Domain 3 of ICAM-1 was identified as a primary binding site for the αX I-domain. Two regions of domain 3 (D(229)QRLNPTV and E(254)DEGTQRL) perform critical functions in the binding of the αX I-domain. Especially, the residue E(254)DEG, is most important with regard to the αX I-domain.

Direct Thy-1/alphaVbeta3 integrin interaction mediates neuron to astrocyte communication.

Thy-1 is an abundant neuronal glycoprotein of poorly defined function. We recently provided evidence indicating that Thy-1 clusters a beta3-containing integrin in astrocytes to induce tyrosine phosphorylation, RhoA activation and the formation of focal adhesions and stress fibers. To date, the alpha subunit partner of beta3 integrin in DI TNC1 astrocytes is unknown. Similarly, the ability of neuronal, membrane-bound Thy-1 to trigger astrocyte signaling via integrin engagement remains speculation. Here, evidence that alphav forms an alphavbeta3 heterodimer in DI TNC1 astrocytes was obtained. In neuron-astrocyte association assays, the presence of either anti-alphav or anti-beta3 integrin antibodies reduced cell-cell interaction demonstrating the requirement of both integrin subunits for this association. Moreover, anti-Thy-1 antibodies blocked stimulation of astrocytes by neurons but not the binding of these two cell types. Thus, neuron-astrocyte association involved binding between molecular components in addition to the Thy-1-integrin; however, the signaling events leading to focal adhesion formation in astrocytes depended exclusively on the latter interaction. Additionally, wild-type (RLD) but not mutated (RLE) Thy-1 was shown to directly interact with alphavbeta3 integrin by Surface Plasmon Resonance analysis. This interaction was promoted by divalent cations and was species-independent. Together, these results demonstrate that the alphavbeta3 integrin heterodimer interacts directly with Thy-1 present on neuronal cells to stimulate astrocytes.

Identification of critical residues for plasminogen binding by the alphaX I-domain of the beta2 integrin, alphaXbeta2.

The beta2 integrins on leukocytes play important roles in cell adhesion, migration and phagocytosis. One of the beta2 integrins, alphaXbeta2 (CD11c/CD18), is known to bind ligands such as fibrinogen, Thy-1 and iC3b, but its function is not well characterized. To understand its biological roles, we attempted to identify novel ligands. The functional moiety of alphaXbeta2, the alphaX I-domain, was found to bind plasminogen, the zymogen of plasmin, with moderate affinity (1.92 X 10-(6) M) in the presence of Mg(2+) or Mn(2+). The betaD-alpha5 loop of the alphaX I-domain proved to be responsible for binding, and lysine residues (Lys(242), Lys(243)) in the loop were the most important for recognizing plasminogen. An excess amount of the lysine analog, 6-aminohexanoic acid, inhibited alphaX I-domain binding to plasminogen, indicating that binding is lysine-dependent. The results of this study indicate that leukocytes regulate plasminogen activation, and consequently plasmin activities, through an interaction with alphaXbeta2 integrin.

Critical residues of alphaX I-domain recognizing fibrinogen central domain.

Integrin alphaXbeta2 (CD11c/CD18), which binds several ligands such as fibrinogen and iC3b, has important roles in leukocyte functions including phagocytosis and migration. Establishment of structure and functional relationship in alphaX I-domain, which is a ligand-binding moiety, is important in understanding leukocyte biology and integrin function. Previously we showed that two loops (alpha3-alpha4, betaD-alpha5) around a ligand-binding face of alphaX I-domain are important for the binding of the fibrinogen molecule. In this study, we took the further step of identifying critical residues in these loops and in a supportive loop (betaF-alpha7) for fibrinogen fragment E, the central domain of fibrinogen. The residues S(199) and Q(202) in the alpha3-alpha4 loop and K(243), Y(250) in the betaD-alpha5 loop are critical for the ligand. The residues K(242), D(249), K(251), and D(252) are important but less critical for fibrinogen fragment E. The involvement of the residues in the 3-dimensional model of the I-domain suggests that several amino acid sequences in fibrinogen fragment E are responsible for alphaX I-domain. Sequence comparisons with alphaM I-domain reveal that most of the critical residues shown in alphaX I-domain are also conserved in alphaM and may have important roles in fibrinogen central domain recognition in alphaM I-domain as well.

Characterization of alphaX I-domain binding to Thy-1.

The beta2 integrins are found exclusively in leukocytes and they are composed of a common beta chain, CD18, and one of four unique alpha chains, CD11a (alphaL subunit), CD11b (alphaM subunit), CD11c (alphaX subunit), or CD11d (alphaD subunit). alphaX-beta2 which binds several ligands including fibrinogen and iC3b is expressed in monocytes/macrophages and dendritic cells playing an important role in the host defense. Despite the unique characteristics on expression and regulation, alphaX-beta2 is less functionally characterized than other beta2 integrins. To understand the biological function of alphaX-beta2 more, we tested the possibility that alphaX-beta2 binds Thy-1, a membrane protein involved in cell adhesion and signaling regulation in neurons and T cells. Here we report that a ligand binding moiety of alphaX-beta2, the I-domain, bound Thy-1 in a specific and divalent cation-dependent manner. The dissociation constant (K(D)) of alphaX I-domain binding to Thy-1 was 1.16muM and the affinity of the binding was roughly 2-fold higher than that of alphaM I-domain. Amino acid substitutions on the betaD-alpha5 of alphaX I-domain (D249, KE243/244) showed low affinities for Thy-1 while other point mutations on alpha3-alpha4 and betaE-alpha6 loops of I-domain did not, suggesting that Thy-1 recognizes the portion of a betaD-alpha5 loop, possibly alpha5 helix. Taken together, these results indicate that alphaX-beta2 specifically interacts with Thy-1. Additionally, kinetic analysis reveals a moderate affinity interaction in the presence of divalent cations. Given the reported role of Thy-1 in the regulation of T cell homeostasis and proliferation, it is tempting to speculate that alphaX-beta2 may be involved in Thy-1 function.

Loops within the CD11c I domain critical for specific recognition of fibrinogen.

The I domains of CD11 are responsible for the binding of ligands and have a unique structure with 6-7 alpha helices and 6 beta sheets with interconnecting loops. To determine loops recognizing fibrinogen in CD11c I domain, five oligopeptides corresponding to CD11c loops were used to prevent fibrinogen binding to isolated CD11c I domain. The results of the inhibition experiment indicated that all of the loops except the one between E-beta sheet and 6-alpha helix were involved in the binding to fibrinogen. The peptide beta D alpha 5 and alpha 3 alpha 4 showed higher inhibitory activity than others, and the combination of four peptides blocked fibrinogen binding to the I domain completely. These peptides (beta A alpha 1, alpha 3 alpha 4, beta D alpha 5 and beta F alpha 7) could block THP-1 cell binding to fibrinogen coated surface as well. Alanine substitution of amino acids on the I domain such as Y249A and Q201A (which are on the loops beta D-alpha 5 and alpha 3-alpha 4 respectively) abolished fibrinogen binding, while mutation on the loop beta E-alpha 6 (Q273A) had no effect on fibrinogen binding. Taken together, the results from this study suggest that the loops on the top of CD11c I domain such as loop beta A-alpha 1, alpha 3-alpha 4, beta D-alpha 5 and beta F-alpha 7 are involved in fibrinogen binding, and two loops (alpha 3-alpha 4 and beta D-alpha 5) are more important than others for the recognition of fibrinogen.