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.     

2E8 Binds to the High Affinity I-domain in a Metal Ion-dependent Manner: A SECOND GENERATION MONOCLONAL ANTIBODY SELECTIVELY TARGETING ACTIVATED LFA-1*

The activation of leukocyte function-associated antigen-1 (LFA-1) plays a critical role in regulating immune responses. The metal ion-dependent adhesion site on the I-domain of LFA-1 α_L subunit is the key recognition site for ligand binding. Upon activation, conformation changes in the I-domain can lead LFA-1 from the low affinity state to the high affinity (HA) state. Using the purified HA I-domain locked by disulfide bonds for immunization, we developed an mAb, 2E8, that specifically binds to cells expressing the HA LFA-1. The surface plasmon resonance analysis has shown that 2E8 only binds to the HA I-domain and that the dissociation constant (K_D) for HA I-domain is 197 nm. The binding of 2E8 to the HA I-domain is metal ion-dependent, and the affinity decreased as Mn²⁺ was replaced sequentially by Mg²⁺ and Ca²⁺. Surface plasmon resonance analysis demonstrates that 2E8 inhibits the interaction of HA I-domain and ICAM-1. Furthermore, we found that 2E8 can detect activated LFA-1 on both JY and Jurkat cells using flow cytometry and parallel plate adhesion assay. In addition, 2E8 inhibits JY cell adhesion to human umbilical vein endothelial cells and homotypic aggregation. 2E8 treatment reduces the proliferation of both human CD4⁺ and CD8⁺ T cells upon OKT3 stimulation without the impairment of their cytolytic function. Taken together, these data demonstrate that 2E8 is specific for the high affinity form of LFA-1 and that 2E8 inhibits LFA-1/ICAM-1 interactions. As a novel activation-specific monoclonal antibody, 2E8 is a potentially useful reagent for blocking high affinity LFA-1 and modulating T cell activation in research and therapeutics.     

Comparative normal mode analysis of LFA-1 integrin I-domains

The conformational dynamics of the Inserted domain (I-domain) from the lymphocyte function-associated antigen-1 (LFA-1) was investigated by normal mode analysis of multiple structures of the low, intermediate, and high affinity states. LFA-1 is an integrin expressed on leukocytes and is of critical importance in adhesion reactions, like antigen-specific responses, homing, and diapedesis. The main ligand binding site of LFA-1 is the I-domain, which recognizes intercellular adhesion molecules (ICAMs), members of the immunoglobulin superfamily. From experimental crystal structures, a large-scale conformational change of, among others, the α7 helix of the I-domain has been observed leading to the proposal that these structural changes are linked to the conformational regulation of LFA-1. The results from the present calculations show that structural changes of the α7 helix consistent with those observed in the crystal structures are significantly sampled by the low frequency modes. This was found to be particularly true for the low affinity state of the I-domain, indicating that low frequency motions favor the conformational transition implicated in activation. However, beyond the simple downward shift of the helix implied by the crystal structures, the calculations further show that there is a noticeable swinging-out motion of the helix. The consequences of this motion are discussed in the context of integrin activation and inhibition. Moreover, significant changes in the atomic-level dynamics and in long-range correlated motions of the I-domain were found to occur upon binding of the natural ligand ICAM. These changes were more local upon binding of an allosteric inhibitor. The present study opens the question of how changes in dynamics may contribute to the long-range transmission of signal upon ICAM binding by the LFA-1 I-domain.     

Analysis of Cell-Free Human α1 Integrin with a Monoclonal Antibody to the I-Domain: Detection in Ocular Fluid and Function as an Adhesion Substrate

The α1β1 integrin, an inserted (I) domain containing collagen receptor, is expressed in the cell surface membrane of normal and malignant cells, and may play a role in their migration through tissues or in metastatic spread. Here we report that a functional anti-human α1β1 integrin monoclonal antibody (mAb) (1B3.1) directly and specifically binds plastic bound recombinant human α1 I-domain protein containing the collagen binding site. Detection was diminished by acidification of the I-domain protein but was enhanced by increasing concentrations of Mg²⁺ cation. Furthermore, we detected binding of the mAb to proteins from the ocular fluids of 6 patients, with the highest concentration, corresponding to 22.1 ng/ml of I-domain, found in a sample from the eye of a patient with metastatic lung adenocarcinoma. Interestingly, we found that both SKNSH neuroblastoma cells and virally transformed human T cells adhered specifically to plastic wells coated with either immobilized collagen IV oral I-domain. MAb 1B3.1 inhibited adhesion to collagen IV but not to immobilized I-domain. These results suggest a novel function for cell free α1 I-domain as a substrate for cellular adhesion, which may have relevance in tumor spread in vivo.     

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.     

Moieties of Complement iC3b Recognized by the I-domain of Integrin αXβ2

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, αMβ2 and αXβ2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective β2-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.