Dissection of \u03b14\u03b27 integrin regulation by Rap1 using novel conformation-specific monoclonal anti-\u03b27 antibodies

Tsuyoshi Sato,Sayaka Ishihara,Koko Katagiri,Ryoya Marui,Junichi Takagi

doi:10.1038/s41598-020-70111-0

Tsuyoshi Sato, Sayaka Ishihara + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-70111-0

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Abstract

Integrin activation is associated with conformational regulation. In this study, we developed a system to evaluate conformational changes in α4β7 integrin. We first inserted the PA tag into the plexin-semaphorin-integrin (PSI) domain of β7 chain, which reacted with an anti-PA tag antibody (NZ-1) in an Mn2+-dependent manner. The small GTPase Rap1 deficiency, as well as chemokine stimulation and the introduction of the active form of Rap1, Rap1V12, enhanced the binding of NZ-1 to the PA-tagged mutant integrin, and increased the binding affinity to mucosal addressing cell adhesion molecule-1 (MAdCAM-1). Furthermore, we generated two kinds of hybridomas producing monoclonal antibodies (mAbs) that recognized Mn2+-dependent epitopes of β7. Both epitopes were exposed to bind to mAbs on the cells by the introduction of Rap1V12. Although one epitope in the PSI domain of β7 was exposed on Rap1-deficienct cells, the other epitope in the hybrid domain of β7 was not. These data indicate that the conversion of Rap1-GDP to GTP exerts two distinct effects stepwise on the conformation of α4β7. The induction of colitis by Rap1-deficient CD4+ effector/memory T cells suggests that the removal of constraining effect by Rap1-GDP on α4β7 is sufficient for homing of these pathogenic T cells into colon lamina propria (LP).

Highlights

Integrin activation is associated with conformational regulation
To probe the conformational state of α4β7 using the PA-tagNZ-1 system (Fig. 1a), a proB cell line (BAF cells), in which the β7 chain was knocked-out using CRISPR/ Cas9-mediated genome editing, and β7 chains cDNA which were inserted PA tag into 4 locations (PAins 1: 23/24, PAins 2: 29/30, PAins 3: 427/428, PAins 4: 431/432) were introduced into BAF cells (Fig. 1b,c). These insertion mutants of β7-expressing cells were stained with NZ-1 or FIB504 in the presence of 1 mM Ca2+/Mg2+ or 0.5 mM Mn2+, and analyzed by flow cytometry
In our previous p aper[10], we demonstrated that T cell-specific Rap1-deficient mice developed severe colitis with infiltration of C D4+ TEM cells into colon lamina propria (LP) and that adoptive transfer of these cells into normal mice induced colitis

Summary

Introduction

Integrin activation is associated with conformational regulation. In this study, we developed a system to evaluate conformational changes in α4β7 integrin. We generated two kinds of hybridomas producing monoclonal antibodies (mAbs) that recognized Mn2+-dependent epitopes of β7 Both epitopes were exposed to bind to mAbs on the cells by the introduction of Rap1V12. One epitope in the PSI domain of β7 was exposed on Rap1-deficienct cells, the other epitope in the hybrid domain of β7 was not These data indicate that the conversion of Rap1-GDP to GTP exerts two distinct effects stepwise on the conformation of α4β7. Rap[1] deficiency leads to lymphopenia and the generation of pathogenic T EM cells in lymph nodes It facilitates homing of TEM cells into the colon, which exacerbates spontaneous T-cell-dependent colitis and tubular a denomas[10]. Previous studies showed that integrin extracellular domains existed in distinct global conformational states that differed

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