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2E8 binds to the high affinity I-domain in a metal ion-dependent manner: a second generation monoclonal antibody selectively targeting activated LFA-1.

ABSTRACT: 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 ?(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 Mn(2+) was replaced sequentially by Mg(2+) and Ca(2+). 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.

SUBMITTER: Carreno R

PROVIDER: S-EPMC2963386 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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2E8 binds to the high affinity I-domain in a metal ion-dependent manner: a second generation monoclonal antibody selectively targeting activated LFA-1.

Carreño Roberto R Brown Wells S WS Li Dan D Hernandez Jessica A JA Wang Yang Y Kim Tae Kon TK Craft John W JW Komanduri Krishna V KV Radvanyi Laszlo G LG Hwu Patrick P Molldrem Jeffrey J JJ Legge Glen B GB McIntyre Bradley W BW Ma Qing Q

The Journal of biological chemistry 20100819 43

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 t ...[more]

PMID: 20724473

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Project description:The marine toxin goniodomin A (GDA) is a polycyclic macrolide containing a spiroacetal and three cyclic ethers as part of the macrocycle backbone. GDA is produced by three species of the Alexandrium genus of dinoflagellates, blooms of which are associated with "red tides", which are widely dispersed and can cause significant harm to marine life. The toxicity of GDA has been attributed to stabilization of the filamentous form of the actin group of structural proteins, but the structural basis for its binding is not known. Japanese workers, capitalizing on the assumed rigidity of the heavily substituted macrolide ring, assigned the relative configuration and conformation by relying on NMR coupling constants and NOEs; the absolute configuration was assigned by degradation to a fragment that was compared with synthetic material. We have confirmed the absolute structure and broad features of the conformation by X-ray crystallography but have found GDA to complex with alkali metal ions in spite of two of the heterocyclic rings facing outward. Such an arrangement would have been expected to impair the ability of GDA to form a crown-ether-type multidentate complex. GDA shows preference for K+, Rb+, and Cs+ over Li+ and Na+ in determinations of relative affinities by TLC on metal-ion-impregnated silica gel plates and by electrospray mass spectrometry. NMR studies employing the K+ complex of GDA, formed from potassium tetrakis[pentafluorophenyl]borate (KBArF20), reveal a major alteration of the conformation of the macrolide ring. These observations argue against the prior assumption of rigidity of the ring. Alterations in chemical shifts, coupling constants, and NOEs indicate the involvement of most of the molecule other than ring F. Molecular mechanics simulations suggest K+ forms a heptacoordinate complex involving OA, OB, OC, OD, OE, and the C-26 and C-27 hydroxy groups. We speculate that complexation of K+ with GDA electrostatically stabilizes the complex of GDA with filamentous actin in marine animals due to the protein being negatively charged at physiological pH. GDA may also cause potassium leakage through cell membranes. This study provides insight into the structural features and chemistry of GDA that may be responsible for significant ecological damage associated with the GDA-producing algal blooms.

Dataset Information

2E8 binds to the high affinity I-domain in a metal ion-dependent manner: a second generation monoclonal antibody selectively targeting activated LFA-1.

Publications

2E8 binds to the high affinity I-domain in a metal ion-dependent manner: a second generation monoclonal antibody selectively targeting activated LFA-1.

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