Molecular mechanism of the recognition of bacterially cleaved immunoglobulin by the immune regulatory receptor LILRA2

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. 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. Leukocyte immunoglobulin (Ig)-like receptors (LILRs or LIRs), also 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 (1Kuroki K. Furukawa A. Maenaka K. Molecular recognition of paired receptors in the immune system.Front. Microbiol. 2012; 3 (23293633): 42910.3389/fmicb.2012.00429Crossref PubMed Scopus (41) Google Scholar). LILRBs (LILR subfamily B) are considered immune checkpoint molecules, like PD-1 and CTLA-4 (2Sharma P. Allison J.P. Immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential.Cell. 2015; 161 (25860605): 205-21410.1016/j.cell.2015.03.030Abstract Full Text Full Text PDF PubMed Scopus (1517) Google Scholar, 3Kang X. Kim J. Deng M. John S. Chen H. Wu G. Phan H. Zhang C.C. 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Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G.Proc. Natl. Acad. Sci. U S A. 2003; 100 (12853576): 8856-886110.1073/pnas.1431057100Crossref PubMed Scopus (439) Google Scholar, 5Chapman T.L. Heikeman A.P. Bjorkman P.J. The inhibitory receptor LIR-1 uses a common binding interaction to recognize class I MHC molecules and the viral homolog UL18.Immunity. 1999; 11 (10591185): 603-61310.1016/S1074-7613(00)80135-1Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 6Borges L. Hsu M.L. Fanger N. Kubin M. Cosman D. A family of human lymphoid and myeloid Ig-like receptors, some of which bind to MHC class I molecules.J. Immunol. 1997; 159 (9548455): 5192-5196PubMed Google Scholar, 7Cosman D. Fanger N. Borges L. Kubin M. Chin W. Peterson L. Hsu M.L. 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Recently, Hirayasu et al. reported that LILRA2 ligands are cleaved Igs whose VH domain is cleaved by proteases secreted by bacteria, such as Legionella pneumophila, Haemophilus influenzae, Streptococcus pneumoniae, and mycoplasma (14Hirayasu K. Saito F. Suenaga T. Shida K. Arase N. Oikawa K. Yamaoka T. Murota H. Chibana H. Nakagawa I. Kubori T. Nagai H. Nakamaru Y. Katayama I. Colonna M. et al.Microbially cleaved immunoglobulins are sensed by the innate immune receptor LILRA2.Nat. Microbiol. 2016; 1 (27572839): 1605410.1038/nmicrobiol.2016.54Crossref PubMed Scopus (26) Google Scholar). The cleaved Igs were found in L. pneumophilia-infected mice and pus fluid from S. pneumoniae-infected patients (14Hirayasu K. Saito F. Suenaga T. Shida K. Arase N. Oikawa K. Yamaoka T. Murota H. Chibana H. Nakagawa I. Kubori T. Nagai H. Nakamaru Y. Katayama I. Colonna M. et al.Microbially cleaved immunoglobulins are sensed by the innate immune receptor LILRA2.Nat. Microbiol. 2016; 1 (27572839): 1605410.1038/nmicrobiol.2016.54Crossref PubMed Scopus (26) Google Scholar). Moreover, Mycoplasma Ig binding protein has been reported to recruit Mycoplasma Ig protease to cleave the heavy chain of Ig (15Arfi Y. Minder L. Di Primo C. Le Roy A. Ebel C. Coquet L. Claverol S. Vashee S. Jores J. Blanchard A. Sirand-Pugnet P. MIB-MIP is a mycoplasma system that captures and cleaves immunoglobulin G.Proc. Natl. Acad. Sci. U S A. 2016; 113 (27114507): 5406-541110.1073/pnas.1600546113Crossref PubMed Scopus (48) Google Scholar). Therefore, the cleavage of Igs is considered a highly conserved immune evasion mechanism for bacteria (14Hirayasu K. Saito F. Suenaga T. Shida K. Arase N. Oikawa K. Yamaoka T. Murota H. Chibana H. Nakagawa I. Kubori T. Nagai H. Nakamaru Y. Katayama I. Colonna M. et al.Microbially cleaved immunoglobulins are sensed by the innate immune receptor LILRA2.Nat. Microbiol. 2016; 1 (27572839): 1605410.1038/nmicrobiol.2016.54Crossref PubMed Scopus (26) Google Scholar). In contrast, Hirayasu et al. also found that LILRA2 ingeniously bound to the cleaved Igs and stimulated the immune system to inhibit bacterial growth. For example, the growth of L. pneumophila was inhibited by stimulating primary monocytes with LILRA2 (14Hirayasu K. Saito F. Suenaga T. Shida K. Arase N. Oikawa K. Yamaoka T. Murota H. Chibana H. Nakagawa I. Kubori T. Nagai H. Nakamaru Y. Katayama I. Colonna M. et al.Microbially cleaved immunoglobulins are sensed by the innate immune receptor LILRA2.Nat. Microbiol. 2016; 1 (27572839): 1605410.1038/nmicrobiol.2016.54Crossref PubMed Scopus (26) Google Scholar). This is a novel immune activation mechanism for sensing immune evasion by bacteria; however, the detailed molecular recognition mechanism remains unknown. Here, we report the biophysical characteristics of the interaction between LILRA2 and N-truncated Ig. We purified WT LILRA2 and N-truncated Ig and determined the binding affinity, kinetics, and thermodynamic characteristics of the Ig interaction using surface plasmon resonance Furthermore, we identified the N-truncated Ig binding site on LILRA2 using mutagenesis We also N-truncated Ig and Ig by force microscopy These results molecular insights into cleaved Ig recognition by LILRA2 as as strategies for regulation of immune The for the expression of proteins in this are in A and and The domains 1 and 2 of the extracellular region of human LILRA2 receptor were expressed in as and in by a (4Shiroishi M. Tsumoto K. Amano K. Shirakihara Y. Colonna M. Braud V.M. Allan D.S. Makadzange A. Rowland-Jones S. Willcox B. Jones E.Y. van der Merwe P.A. Kumagai I. Maenaka K. Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G.Proc. Natl. Acad. Sci. U S A. 2003; 100 (12853576): 8856-886110.1073/pnas.1431057100Crossref PubMed Scopus (439) Google Scholar). The was purified by and as a on analysis using analysis indicated that the protein was highly purified and for The domains 1 to of the extracellular region of LILRA2 was to a and expressed in cells. The protein was purified by The that also as a and an of the LILRA2 a Y. F. F. B. and of the myeloid activating receptor immunoglobulin-like transcript 1 2007; PubMed Scopus Google Scholar, Y. F. F. H. L. Y. P. of myeloid cell activating receptor leukocyte Ig-like receptor domain molecular for to MHC PubMed Scopus Google Scholar). However, the of the WT LILRA2 as a LILRA2 with the region of LILRA2 with was expressed by cells as a secreted as (14Hirayasu K. Saito F. Suenaga T. Shida K. Arase N. Oikawa K. Yamaoka T. Murota H. Chibana H. Nakagawa I. Kubori T. Nagai H. Nakamaru Y. Katayama I. Colonna M. et al.Microbially cleaved immunoglobulins are sensed by the innate immune receptor LILRA2.Nat. Microbiol. 2016; 1 (27572839): 1605410.1038/nmicrobiol.2016.54Crossref PubMed Scopus (26) Google Scholar). The protein was purified from the by protein affinity by Ig of heavy chain and of and N-truncated Ig to of the heavy chain of and of the were also expressed and purified by the as the The of the protein was by analysis analysis was to the molecular recognition mechanism of LILRA2 by N-truncated using the purified proteins protein was a N-truncated Ig or Ig a was immobilized by the coupling binding of LILRA2 to N-truncated Ig was but not to the Ig not show specific binding to N-truncated Ig by a sequence of to This was with which indicated that not bind to N-truncated using the system with on cells (14Hirayasu K. Saito F. Suenaga T. Shida K. Arase N. Oikawa K. Yamaoka T. Murota H. Chibana H. Nakagawa I. Kubori T. Nagai H. Nakamaru Y. Katayama I. Colonna M. et al.Microbially cleaved immunoglobulins are sensed by the innate immune receptor LILRA2.Nat. Microbiol. 2016; 1 (27572839): 1605410.1038/nmicrobiol.2016.54Crossref PubMed Scopus (26) Google Scholar). We to μm) or to 15 μm) to N-truncated and Igs immobilized on the to the by binding analysis The of the interaction between or and N-truncated Ig were determined to and These that not of LILRA2 a in the N-truncated Ig and μm) was an N-truncated Ig immobilized to the by using a binding The and were and The from the was from the and analysis provide of the of typically have or antigen binding binding expressed on the cell surface due to an avidity The N-truncated Ig has receptor binding also the avidity on LILRA2 expressed on the cell this avidity N-truncated Ig was the immobilized a the opposite Furthermore, to the in of binding or binding and N-truncated Ig were by The of the of and N-truncated Ig for that of and N-truncated Ig an which was not for protein analysis revealed that this and N-truncated Ig These indicated that N-truncated Ig has LILRA2 binding and a significant avidity for LILRA2 We the of N-truncated Ig by differential scanning calorimetry analysis to thermodynamic analysis of the N-truncated Ig with of and the of analysis of the Ig was also with of and to the of and as by et al. A of Fab a of 2007; PubMed Scopus Google Scholar). the N-truncated Ig was purified by protein affinity the of the N-truncated Ig has a to that of Ig. Therefore, the of in N-truncated Ig was to the of the of N-truncated Ig. The other of in N-truncated Ig was to the of the Fab of N-truncated Ig. The of N-truncated that of the were that of that Fab truncation the of N-truncated the of the Fab We Hoff analysis using the determined by to the thermodynamic of this The binding entropy and were determined by using the Hoff as in the was found to and was that this interaction is with a entropy and was as and force microscopy was to the structural of N-truncated Ig. of Igs in and show that Ig and The Fab and are is 3 = with reported T. H. T. T. M. T. the with force 167 PubMed Scopus Google Scholar). Moreover, that the between Fab and or of Ig are highly the are the Ig is and has a structural and are for the other N-truncated Ig and exhibited and with highly to Ig is = These results that N truncation does not cause significant changes in and that the of Fab is of that of the region and and and and We LILRA2 on a of the primary sequence of LILRA2 and 1 LILRs to the N-truncated Ig binding site on LILRA2 in LILRA2 with conserved in other 1 LILRs were identified to and We also on that are not conserved 1 LILRs and the and of were to that of the WT protein analysis of LILRA2 that domain 1 and and domain 2 and bound to the N-truncated Ig with to that of the However, domain 2 not bind to the N-truncated Ig and and These results that the region on LILRA2 domain 2 is in N-truncated Ig We an protein to this of the were to that of the WT protein analysis that the protein does not bind to the N-truncated Ig LILRA2 and were to the N-truncated Ig binding region on the of the LILR family and by the analysis that LILRA2 and bound to N-truncated Ig but LILRA2 and not of on the that are on the surface of domain 2 on the mutagenesis study, we a of the Ig of binding of LILRA2 for N-truncated of are as are as from or from of are as are as from or from in a In this study, we analysis using to the of the interaction between LILRA2 and N-truncated Ig. LILRA2 binds to N-truncated Ig with a fast and with of that LILRA2 rapidly N-truncated Ig in The weak affinity of Ig interaction (Kd of μm) is typically in which recognition A avidity was in N-truncated Ig was an LILRA2 immobilized In the of the of and N-truncated Ig an which the and N-truncated that of and N-truncated Ig for protein These that LILRA2 binds to N-truncated Ig by avidity The avidity is in other molecules, such as and LILRB1 and -B2 Bjorkman P.J. into immune responses from the of human and with 2003; PubMed Scopus Google Scholar, C.L. C. Wu C. Bjorkman P.J. binding of to suggests a mechanism for activation of 2003; PubMed Scopus Google Scholar, H. L. J. of receptor binding by that human Full Text Full Text PDF PubMed Scopus Google Scholar, G. Y. growth binding to the extracellular domain of the receptor on a surface using a system that avidity significantly to affinity binding.J. 2003; PubMed Scopus Google Scholar, M. K. T. L. I. Arase H. Tsumoto K. Kumagai I. D. Maenaka K. leukocyte Ig-like receptor signaling and of Full Text Full Text PDF PubMed Scopus Google Scholar). We that the which by cross-linking the binding of N-truncated immune activation through LILRA2. the LILRA2 expression is also an the characteristics of the LILRA2 cells with bacteria secreted not the cleaved heavy chain but also the heavy chain (14Hirayasu K. Saito F. Suenaga T. Shida K. Arase N. Oikawa K. Yamaoka T. Murota H. Chibana H. Nakagawa I. Kubori T. Nagai H. Nakamaru Y. Katayama I. Colonna M. et al.Microbially cleaved immunoglobulins are sensed by the innate immune receptor LILRA2.Nat. Microbiol. 2016; 1 (27572839): 1605410.1038/nmicrobiol.2016.54Crossref PubMed Scopus (26) Google Scholar). A with cleaved heavy chain and heavy chain in (15Arfi Y. Minder L. Di Primo C. Le Roy A. Ebel C. Coquet L. Claverol S. Vashee S. Jores J. Blanchard A. Sirand-Pugnet P. MIB-MIP is a mycoplasma system that captures and cleaves immunoglobulin G.Proc. Natl. Acad. Sci. U S A. 2016; 113 (27114507): 5406-541110.1073/pnas.1600546113Crossref PubMed Scopus (48) Google Scholar). binding analysis in indicated that bind to LILRA2 but are rapidly In contrast, the that cleaved heavy have binding affinity and immune cells through LILRA2. Therefore, LILRA2 Igs that have cleaved Fab is to bind to with affinity due to avidity and to the immune LILRA2 specifically binds to the N-truncated Ig but not to the that LILRA2 the and the of cleaved VH of N-truncated Ig. analysis the instability of the the domain with the VH domain of the Immunol. PubMed Scopus Google Scholar). Therefore, instability is considered to from the of the and of the cleaved VH of N-truncated Ig. Ig interaction was by binding with small entropy we that LILRA2 binding to N-truncated Ig the of of the N-truncated Ig and results in binding with entropy to that of receptor binding van der Merwe P.A. binding to a recognition 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, D.C. that T cell are 1999; 11 Full Text Full Text PDF PubMed Scopus Google Scholar). However, the entropy was with a of which is that of the interaction and the of binding and 1997; PubMed Scopus Google Scholar). In we determined the which changes a in the S. A. the between binding and 2016; PubMed Scopus Google Scholar, L. C. J. The of recognition 1999; PubMed Scopus Google Scholar). For example, which structural changes for a P. M. R. J. M.L. of the HIV binding Natl. Acad. Sci. U S A. PubMed Scopus Google Scholar). The of Ig was with the of cell surface such as and a in protein The interaction fast and with and small entropy loss, using the hydrophobic site composed of and of LILRA2. These results a hydrophobic of The of N-truncated and Igs revealed that of not but also These from the of the proteins toward as for the R. P.A. R. and recognition of on 2016; PubMed Google Scholar). We also that for N-truncated and Igs are highly due to the Therefore, the and of N-truncated and Ig were to that N truncation does not and and and and We the of a of and N-truncated which is as a by as in considered to and were such were in of or N-truncated Ig the recognition of LILRA2 toward N-truncated Ig. were for the interaction between or and N-truncated Ig In protein not bind to the N-truncated Ig These results that domain 2 is for N-truncated Ig revealed that on and and on the between and of LILRA2 domain 2 to N-truncated Ig of LILRA2 and on the that are in the region of the region The in and on the are on the surface of the protein and are We that the hydrophobic surface composed of and is for the interaction with the and of the cleaved VH of the N-truncated Ig. of N-truncated Igs by LILRA2, with the hydrophobic common N-truncated Therefore, hydrophobic interaction LILRA2 to recognize a of N-truncated Igs Hirayasu et al. found that LILRA2 bound to N-truncated whose VH domain was cleaved the or of the region but not 15 or (14Hirayasu K. Saito F. Suenaga T. Shida K. Arase N. Oikawa K. Yamaoka T. Murota H. Chibana H. Nakagawa I. Kubori T. Nagai H. Nakamaru Y. Katayama I. Colonna M. et al.Microbially cleaved immunoglobulins are sensed by the innate immune receptor LILRA2.Nat. Microbiol. 2016; 1 (27572839): 1605410.1038/nmicrobiol.2016.54Crossref PubMed Scopus (26) Google Scholar). This suggests that the of the region the of the VH domain after cleavage is for In the mutagenesis analysis that for N-truncated Ig are and to the of the domain and the of the domain, after the the other the mutagenesis on LILRA2 suggests that the binding site for N-truncated Ig is a small hydrophobic on the surface of the of the LILRA2 Therefore, the hydrophobic of the LILRA2 domain with the hydrophobic surface of N-truncated the VH domain, the of the domain, the of the LILR or have binding For example, binds to Ig domains of HLA β2 and the domain M. K. L. Tsumoto K. Kumagai I. K. D. Maenaka K. for recognition of the MHC by the leukocyte Ig-like receptor Natl. Acad. Sci. U S A. PubMed Scopus Google Scholar). is to LILRA2 a surface for the The of LILRA2 and is a 1 For example, the residue of LILRA2 is typically This the for LILRA2 not binding to which are common ligands for 1 LILRA2. The N-truncated Ig binding site on LILRA2 is also a novel recognition site 1 LILR family and other immunoglobulin-like receptors, such as which bind to The region between domain 1 and domain 2 of and is reported as the recognition site M. K. L. Tsumoto K. Kumagai I. K. D. Maenaka K. for recognition of the MHC by the leukocyte Ig-like receptor Natl. Acad. Sci. U S A. PubMed Scopus Google Scholar, Bjorkman P.J. of bound to a and viral Immunol. 2003; PubMed Scopus Google Scholar, J. M. cell immunoglobulin-like receptor binds to Natl. Acad. Sci. U S A. PubMed Scopus Google Scholar). LILR have rapidly by F. R. P. of and human leukocyte Ig-like receptor and rapidly Immunol. 2001; 167 PubMed Scopus Google Scholar). of the recognition suggests that LILRA2 to a receptor to by HLA recognition In this study, we report that Ig binding fast and as typically in recognition However, the affinity with immobilized LILRA2 due to the avidity that affinity by the cross-linking of LILRA2 on the cell surface is for Furthermore, analysis the instability of N-truncated Ig with Ig. analysis that the interaction is enthalpy-driven, with entropy and The results that the and of N-truncated Ig were to of Ig. In a mutagenesis revealed that the N-truncated Ig binding site on LILRA2 was the hydrophobic region on domain that LILRA2 binds to the domain and heavy which is The fast kinetics, and entropy by a hydrophobic The of the N-truncated Ig recognition mechanism by LILRA2 in this insights into strategies in regulation of immune Furthermore, the for the of that LILRA2 in to innate N-truncated Ig.