Abstract
Human leukocyte immunoglobulin-like receptors (LILRs) typically regulate immune activation by binding to the human leukocyte antigen class I molecules. LILRA2, a member of the LILR family, was recently reported to bind to other unique ligands, the bacterially degraded Igs (N-truncated Igs), for the activation of immune cells. Therefore, LILRA2 is currently attracting significant attention as a novel innate immune receptor. However, the detailed recognition mechanisms required for this interaction remain unclear. In this study, using several biophysical techniques, we uncovered the molecular mechanism of N-truncated Ig recognition by LILRA2. Surface plasmon resonance analysis disclosed that LILRA2 specifically binds to N-truncated Ig with weak affinity (Kd = 4.8 μm) and fast kinetics. However, immobilized LILRA2 exhibited a significantly enhanced interaction with N-truncated Ig due to avidity effects. This suggests that cell surface-bound LILRA2 rapidly monitors and identifies bi- or multivalent abnormal N-truncated Igs through specific cross-linking to induce immune activation. Van't Hoff analysis revealed that this interaction is enthalpy-driven, with a small entropy loss, and results from differential scanning calorimetry indicated the instability of the putative LILRA2-binding site, the Fab region of the N-truncated Ig. Atomic force microscopy revealed that N truncation does not cause significant structural changes in Ig. Furthermore, mutagenesis analysis identified the hydrophobic region of LILRA2 domain 2 as the N-truncated Ig-binding site, representing a novel ligand-binding site for the LILR family. These results provide detailed insights into the molecular regulation of LILR-mediated immune responses targeting ligands that have been modified by bacteria.
Highlights
Leukocyte immunoglobulin (Ig)-like receptors (LILRs or LIRs), known as Ig-like transcripts (ILTs) or the CD85 family, are expressed on the surface of immune cells, such as monocytes, macrophages, and dendritic cells [1]
LILRA2 D1D4 fused with the Fc region was transiently expressed by HEK293T cells as a secreted protein, as described previously [14]
We report that LILRA2/N-truncated Ig binding shows relatively fast association and dissociation rates, as typically observed in cell-cell recognition mechanisms
Summary
The constructs used for the expression of proteins in this study are summarized in Fig. 1, A and B, and Fig. S1A. We injected LILRA2D1D2 (from 0.16 mM to 40 mM) or LILRA2D1D4 (from 0.12 mM to 15 mM) to N-truncated and full-length Igs immobilized on the chip to determine the dissociation constants (Kd) by equilibrium binding analysis (Fig. 1, E–H). The N-truncated Ig presumably has two potential receptor binding sites and, may exhibit the avidity effect on LILRA2 expressed on the cell surface To examine this avidity effect, Ntruncated Ig was injected onto the LILRA2D1D2 immobilized sensor chip. SDS-PAGE analysis revealed that this peak contained Fc fusion LILRA2D1D4 and N-truncated Ig (Fig. 2C) These data indicated that N-truncated Ig has two LILRA2 binding sites and a significant avidity effect for LILRA2 binding. More LILRA2 mutants (V121E, D127G, H146Q, S147P, and W152S) were designed to further clarify the N-truncated Ig binding region surrounding Trp154, based on the structures of the LILR family members and prepared by the refolding method. Based on the mutagenesis study, we propose a model of the LILRA2/N-truncated Ig complex (Fig. 4D)
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