Conformational mAb as a Tool for Integrin Ligand Discovery

Njus, Ben H.; Chigaev, Alexandre; Waller, Anna; Wlodek, Danuta; Halip, Liliana; Ursu, Oleg; Wang, Wei; Oprea, Tudor I.; Bologa, Cristian; Sklar, Larry A.

doi:10.1089/adt.2009.0203

Cited by 18 publications

(20 citation statements)

References 30 publications

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“…As shown previously, the EC50 for mAb binding is similar to the ligand dissociation constant (K d ) determined using other methods [54]. We observed that the EC50s for LIBS antibody binding for CORM-2 treated samples were larger than for the vehicle treated controls by ~2 fold for resting cells, and ~3 fold for FPR activated cells (Figure 6).…”

Section: Resultssupporting

confidence: 85%

Carbon monoxide down-regulates α4β1 integrin-specific ligand binding and cell adhesion: a possible mechanism for cell mobilization

2014

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BackgroundCarbon monoxide (CO), a byproduct of heme degradation, is attracting growing attention from the scientific community. At physiological concentrations, CO plays a role as a signal messenger that regulates a number of physiological processes. CO releasing molecules are under evaluation in preclinical models for the management of inflammation, sepsis, ischemia/reperfusion injury, and organ transplantation. Because of our discovery that nitric oxide signaling actively down-regulates integrin affinity and cell adhesion, and the similarity between nitric oxide and CO-dependent signaling, we studied the effects of CO on integrin signaling and cell adhesion.ResultsWe used a cell permeable CO releasing molecule (CORM-2) to elevate intracellular CO, and a fluorescent Very Late Antigen-4 (VLA-4, α4β1-integrin)-specific ligand to evaluate the integrin state in real-time on live cells. We show that the binding of the ligand can be rapidly down-modulated in resting cells and after inside-out activation through several Gαi-coupled receptors. Moreover, cell treatment with hemin, a natural source of CO, resulted in comparable VLA-4 ligand dissociation. Inhibition of VLA-4 ligand binding by CO had a dramatic effect on cell-cell interaction in a VLA-4/VCAM-1-dependent cell adhesion system.ConclusionsWe conclude that the CO signaling pathway can rapidly down-modulate binding of the VLA-4 -specific ligand. We propose that CO-regulated integrin deactivation provides a basis for modulation of immune cell adhesion as well as rapid cell mobilization, for example as shown for splenic monocytes in response to surgically induced ischemia of the myocardium.

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Section: Resultssupporting

confidence: 85%

Carbon monoxide down-regulates α4β1 integrin-specific ligand binding and cell adhesion: a possible mechanism for cell mobilization

2014

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“…This approach has been verified for multiple VLA-4 ligands. The EC50s for βl-integrin LIBS mAbs (HUTS-21) binding determined in the mAb-binding experiments were identical to K i s, determined in the competition assay with fluorescent VLA-4 ligand (37). This novel approach can be used for determination of ligand-binding affinities for unlabeled ligands, as well as for previously unknown integrin ligands.…”

Section: Mabs and Integrin Conformationsupporting

confidence: 52%

Overview: Assays for Studying Integrin-Dependent Cell Adhesion

Chigaev

Sklar

2011

Integrin and Cell Adhesion Molecules

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Interaction of the integrin receptors with ligands determines the molecular basis of integrin –dependent cell adhesion. Integrin ligands are typically large proteins with relatively low binding affinities. This makes direct ligand-binding kinetic measurements somewhat difficult. Here we examine several real-time methods, aimed to overcome these experimental limitations and to distinguish the regulation of integrin conformation and affinity. This chapter includes: the use of a small ligand-mimetic probe for studies of inside-out regulation of integrin affinity and unbending, real-time cell aggregation and disaggregation kinetics to probe integrin conformational states and the number of integrin-ligand bonds, as well as the real-time monitoring of ligand -induced epitopes under signaling through G-protein-coupled receptors, and others. Experimental data obtained using these novel methods are summarized in terms of the current model of integrin activation.

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“…Such conformational changes were reported for the α4β1, αLβ2 integrins and have been validated through exposure of the activation-dependent antibody epitopes and x-ray crystallography to visualize resting and extended integrins 10 – 12 …”

Section: Introductionmentioning

confidence: 72%

Evaluating integrin activation with time-resolved flow cytometry

et al. 2018

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Förster resonance energy transfer (FRET) continues to be a useful tool to study movement and interaction between proteins within living cells. When FRET as an optical technique is measured with flow cytometry, conformational changes of proteins can be rapidly measured cell-by-cell for the benefit of screening and profiling. We exploit FRET to study the extent of activation of α4β1 integrin dimers expressed on the surface of leukocytes. The stalk-like transmembrane heterodimers when not active lay bent and upon activation extend outward. Integrin extension is determined by changes in the distance of closest approach between an FRET donor and acceptor, bound at the integrin head and cell membrane, respectively. Time-resolved flow cytometry analysis revealed donor emission increases up to 17%, fluorescence lifetime shifts over 1.0 ns during activation, and FRET efficiencies of 37% and 26% corresponding to the inactive and active integrin state, respectively. Last, a graphical phasor analysis, including population clustering, gating, and formation of an FRET trajectory, added precision to a comparative analysis of populations undergoing FRET, partial donor recovery, and complete donor recovery. This work establishes a quantitative cytometric approach for profiling fluorescence donor decay kinetics during integrin conformational changes on a single-cell level.

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Conformational mAb as a Tool for Integrin Ligand Discovery

Cited by 18 publications

References 30 publications

Carbon monoxide down-regulates α4β1 integrin-specific ligand binding and cell adhesion: a possible mechanism for cell mobilization

Carbon monoxide down-regulates α4β1 integrin-specific ligand binding and cell adhesion: a possible mechanism for cell mobilization

Overview: Assays for Studying Integrin-Dependent Cell Adhesion

Evaluating integrin activation with time-resolved flow cytometry

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