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1.
Int J Biol Macromol ; 271(Pt 2): 132588, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38788878

ABSTRACT

Interface residues at sites of protein-protein interaction (PPI) are the focus for affinity optimisation. However, protein hydrophobic cores (HCs) play critical roles and shape the protein surface. We hypothesise that manipulating protein HCs can enhance PPI interaction affinities. A cell stress molecule, major histocompatibility complex class I chain-related protein A (MICA), binds to the natural killer group 2D (NKG2D) homodimer to form three molecule interactions. MICA was used as a study subject to support our hypothesis. We redesigned MICA HCs by directed mutagenesis and isolated high-affinity variants through a newly designed partial-denature panning (PDP) method. A few mutations in MICA HCs increased the NKG2D-MICA interaction affinity by 325-5613-fold. Crystal structures of the NKG2D-MICA variant complexes indicated that mutagenesis of MICA HCs stabilised helical elements for decreasing intermolecular interactive free energy (ΔG) of the NKG2D-MICA heterotrimer. The repacking of MICA HC mutants maintained overall surface residues and the authentic binding specificity of MICA. In conclusion, this study provides a new method for MICA redesign and affinity optimisation through HC manipulation without mutating PPI interface residues. Our study introduces a novel approach to protein manipulation, potentially expanding the toolkit for protein affinity optimisation.


Subject(s)
Histocompatibility Antigens Class I , Hydrophobic and Hydrophilic Interactions , Protein Binding , Histocompatibility Antigens Class I/metabolism , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/genetics , Humans , NK Cell Lectin-Like Receptor Subfamily K/metabolism , NK Cell Lectin-Like Receptor Subfamily K/chemistry , NK Cell Lectin-Like Receptor Subfamily K/genetics , Models, Molecular , Mutation
2.
Chemistry ; 30(30): e202400660, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38527187

ABSTRACT

C-type lectins are a large superfamily of proteins involved in a multitude of biological processes. In particular, their involvement in immunity and homeostasis has rendered them attractive targets for diverse therapeutic interventions. They share a characteristic C-type lectin-like domain whose adaptability enables them to bind a broad spectrum of ligands beyond the originally defined canonical Ca2+-dependent carbohydrate binding. Together with variable domain architecture and high-level conformational plasticity, this enables C-type lectins to meet diverse functional demands. Secondary sites provide another layer of regulation and are often intricately linked to functional diversity. Located remote from the canonical primary binding site, secondary sites can accommodate ligands with other physicochemical properties and alter protein dynamics, thus enhancing selectivity and enabling fine-tuning of the biological response. In this review, we outline the structural determinants allowing C-type lectins to perform a large variety of tasks and to accommodate the ligands associated with it. Using the six well-characterized Ca2+-dependent and Ca2+-independent C-type lectin receptors DC-SIGN, langerin, MGL, dectin-1, CLEC-2 and NKG2D as examples, we focus on the characteristics of non-canonical interactions and secondary sites and their potential use in drug discovery endeavors.


Subject(s)
Lectins, C-Type , Lectins, C-Type/chemistry , Lectins, C-Type/metabolism , Humans , Ligands , Binding Sites , Calcium/metabolism , Calcium/chemistry , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/metabolism , Protein Binding , Mannose-Binding Lectins/chemistry , Mannose-Binding Lectins/metabolism , Mannose-Binding Lectin/chemistry , Mannose-Binding Lectin/metabolism , NK Cell Lectin-Like Receptor Subfamily K/chemistry , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Antigens, CD/chemistry , Antigens, CD/metabolism
3.
EMBO J ; 41(2): e107739, 2022 12 17.
Article in English | MEDLINE | ID: mdl-34913508

ABSTRACT

Stimulatory immune receptor NKG2D binds diverse ligands to elicit differential anti-tumor and anti-virus immune responses. Two conflicting degeneracy recognition models based on static crystal structures and in-solution binding affinities have been considered for almost two decades. Whether and how NKG2D recognizes and discriminates diverse ligands still remain unclear. Using live-cell-based single-molecule biomechanical assay, we characterized the in situ binding kinetics of NKG2D interacting with different ligands in the absence or presence of mechanical force. We found that mechanical force application selectively prolonged NKG2D interaction lifetimes with the ligands MICA and MICB, but not with ULBPs, and that force-strengthened binding is much more pronounced for MICA than for other ligands. We also integrated steered molecular dynamics simulations and mutagenesis to reveal force-induced rotational conformational changes of MICA, involving formation of additional hydrogen bonds on its binding interface with NKG2D, impeding MICA dissociation under force. We further provided a kinetic triggering model to reveal that force-dependent affinity determines NKG2D ligand discrimination and its downstream NK cell activation. Together, our results demonstrate that NKG2D has a discrimination power to recognize different ligands, which depends on selective mechanical force-induced ligand conformational changes.


Subject(s)
NK Cell Lectin-Like Receptor Subfamily K/chemistry , Binding Sites , Cells, Cultured , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/metabolism , Humans , K562 Cells , Ligands , Mechanical Phenomena , Molecular Dynamics Simulation , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Protein Binding , Single Molecule Imaging
4.
Front Immunol ; 9: 1820, 2018.
Article in English | MEDLINE | ID: mdl-30166984

ABSTRACT

NKG2D is a major regulator of the activity of cytotoxic cells and interacts with eight different ligands (NKG2DL) from two families of MIC and ULBP proteins. The selective forces that drove evolution of NKG2DL are uncertain, but are likely to have been dominated by infectious disease and cancer. Of interest, NKG2DL are some of the most polymorphic genes outside the MHC locus and the study of these is uncovering a range of novel observations regarding the structure and function of NKG2DL. Polymorphism is present within all NKG2DL members and varies markedly within different populations. Allelic variation influences functional responses through three major mechanisms. First, it may drive differential levels of protein expression, modulate subcellular trafficking, or regulate release of soluble isoforms. In addition, it may alter the affinity of interaction with NKG2D or modulate cytotoxic activity from the target cell. In particular, ligands with high affinity for NKG2D are associated with down regulation of this protein on the effector cell, effectively limiting cytotoxic activity in a negative-feedback circuit. Given these observations, it is not surprising that NKG2DL alleles are associated with relative risk for development of several clinical disorders and the critical role of the NKG2D:NKG2DL interaction is demonstrated in many murine models. Increased understanding of the biophysical and functional consequences of this polymorphism is likely to provide insights into novel immunotherapeutic approaches.


Subject(s)
Disease Susceptibility , NK Cell Lectin-Like Receptor Subfamily K/genetics , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Polymorphism, Genetic , Animals , Clonal Evolution/drug effects , Clonal Selection, Antigen-Mediated , Cytotoxicity, Immunologic , Humans , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Ligands , NK Cell Lectin-Like Receptor Subfamily K/chemistry , Protein Binding , Signal Transduction , Structure-Activity Relationship
5.
Front Immunol ; 9: 926, 2018.
Article in English | MEDLINE | ID: mdl-29765374

ABSTRACT

Natural killer (NK) cells are immune innate effectors playing a pivotal role in the immunosurveillance of multiple myeloma (MM) since they are able to directly recognize and kill MM cells. In this regard, among activating receptors expressed by NK cells, NKG2D represents an important receptor for the recognition of MM cells, being its ligands expressed by tumor cells, and being able to trigger NK cell cytotoxicity. The MHC class I-related molecule A (MICA) is one of the NKG2D ligands; it is encoded by highly polymorphic genes and exists as membrane-bound and soluble isoforms. Soluble MICA (sMICA) is overexpressed in the serum of MM patients, and its levels correlate with tumor progression. Interestingly, a methionine (Met) to valine (Val) substitution at position 129 of the α2 heavy chain domain classifies the MICA alleles into strong (MICA-129Met) and weak (MICA-129Val) binders to NKG2D receptor. We addressed whether the genetic polymorphisms in the MICA-129 alleles could affect MICA release during MM progression. The frequencies of Val/Val, Val/Met, and Met/Met MICA-129 genotypes in a cohort of 137 MM patients were 36, 43, and 22%, respectively. Interestingly, patients characterized by a Val/Val genotype exhibited the highest levels of sMICA in the sera. In addition, analysis of the frequencies of MICA-129 genotypes among different MM disease states revealed that Val/Val patients had a significant higher frequency of relapse. Interestingly, NKG2D was downmodulated in NK cells derived from MICA-129Met/Met MM patients. Results obtained by structural modeling analysis suggested that the Met to Val dimorphism could affect the capacity of MICA to form an optimal template for NKG2D recognition. In conclusion, our findings indicate that the MICA-129Val/Val variant is associated with significantly higher levels of sMICA and the progression of MM, strongly suggesting that the usage of soluble MICA as prognostic marker has to be definitely combined with the patient MICA genotype.


Subject(s)
Alleles , Genetic Predisposition to Disease , Histocompatibility Antigens Class I/blood , Histocompatibility Antigens Class I/genetics , Multiple Myeloma/blood , Multiple Myeloma/genetics , Polymorphism, Genetic , Aged , Aged, 80 and over , Amino Acid Substitution , Disease Progression , Enzyme-Linked Immunosorbent Assay , Female , Gene Expression Regulation, Neoplastic , Genetic Association Studies , Genotype , Histocompatibility Antigens Class I/chemistry , Humans , Immunophenotyping , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Male , Middle Aged , Models, Molecular , Molecular Typing , Multiple Myeloma/pathology , NK Cell Lectin-Like Receptor Subfamily K/chemistry , NK Cell Lectin-Like Receptor Subfamily K/genetics , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Protein Binding , Protein Conformation , Structure-Activity Relationship
6.
Sci Signal ; 10(481)2017 May 30.
Article in English | MEDLINE | ID: mdl-28559451

ABSTRACT

NKG2D (natural killer group 2, member D) is an activating receptor found on the surface of immune cells, including natural killer (NK) cells, which regulates innate and adaptive immunity through recognition of the stress-induced ligands ULBP1 (UL16 binding protein 1) to ULBP6 and MICA/B. Similar to class I human leukocyte antigen (HLA), these NKG2D ligands have a major histocompatibility complex-like fold and exhibit pronounced polymorphism, which influences human disease susceptibility. However, whereas class I HLA polymorphisms occur predominantly in the α1α2 groove and affect antigen binding, the effects of most NKG2D ligand polymorphisms are unclear. We studied the molecular and functional consequences of the two major alleles of ULBP6, the most polymorphic ULBP gene, which are associated with autoimmunity and relapse after stem cell transplantation. Surface plasmon resonance and crystallography studies revealed that the arginine-to-leucine polymorphism within ULBP0602 affected the NKG2D-ULBP6 interaction by generating an energetic hotspot. This resulted in an NKG2D-ULBP0602 affinity of 15.5 nM, which is 10- to 1000-fold greater than the affinities of other ULBP-NKG2D interactions and limited NKG2D-mediated activation. In addition, soluble ULBP0602 exhibited high-affinity competitive binding for NKG2D and partially suppressed NKG2D-mediated activation of NK cells by other NKG2D ligands. These effects resulted in a decrease in a range of NKG2D-mediated effector functions. Our results reveal that ULBP polymorphisms affect the strength of human lymphocyte responses to cellular stress signals and may offer opportunities for therapeutic intervention.


Subject(s)
Hematologic Neoplasms/pathology , Killer Cells, Natural/pathology , Membrane Proteins/metabolism , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Polymorphism, Genetic , T-Lymphocytes/pathology , Binding, Competitive , Cell Membrane/metabolism , Cells, Cultured , Hematologic Neoplasms/genetics , Hematologic Neoplasms/immunology , Hematologic Neoplasms/metabolism , Histocompatibility Antigens Class I/immunology , Humans , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Ligands , Membrane Proteins/chemistry , Membrane Proteins/genetics , NK Cell Lectin-Like Receptor Subfamily K/chemistry , NK Cell Lectin-Like Receptor Subfamily K/genetics , Protein Conformation , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
7.
Mol Immunol ; 66(2): 418-27, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25983110

ABSTRACT

Malignant cells expressing NKG2D ligands on their cell surface can be directly sensed and killed by NKG2D-bearing lymphocytes. To ensure this immune recognition, accumulating evidence suggests that NKG2D ligands are trafficed via alternative pathways to the cell surface. We have previously shown that the NKG2D ligand ULBP2 traffics over an invariant chain (Ii)-dependent pathway to the cell surface. This study set out to elucidate how Ii regulates ULBP2 cell-surface transport: We discovered conserved tryptophan (Trp) residues in the primary protein sequence of ULBP1-6 but not in the related MICA/B. Substitution of Trp to alanine resulted in cell-surface inhibition of ULBP2 in different cancer cell lines. Moreover, the mutated ULBP2 constructs were retained and not degraded inside the cell, indicating a crucial role of this conserved Trp-motif in trafficking. Finally, overexpression of Ii increased surface expression of wt ULBP2 while Trp-mutants could not be expressed, proposing that this Trp-motif is required for an Ii-dependent cell-surface transport of ULBP2. Aberrant soluble ULBP2 is immunosuppressive. Thus, targeting a distinct protein module on the ULBP2 sequence could counteract this abnormal expression of ULBP2.


Subject(s)
Antigens, Differentiation, B-Lymphocyte/genetics , Gene Expression Regulation, Neoplastic , Histocompatibility Antigens Class II/genetics , Intercellular Signaling Peptides and Proteins/genetics , NK Cell Lectin-Like Receptor Subfamily K/genetics , Tryptophan/genetics , Alanine/chemistry , Alanine/genetics , Alanine/immunology , Amino Acid Sequence , Amino Acid Substitution , Antigens, Differentiation, B-Lymphocyte/chemistry , Antigens, Differentiation, B-Lymphocyte/immunology , Binding Sites , Cell Line, Tumor , GPI-Linked Proteins/chemistry , GPI-Linked Proteins/genetics , GPI-Linked Proteins/immunology , HEK293 Cells , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class II/chemistry , Histocompatibility Antigens Class II/immunology , Humans , Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/immunology , Jurkat Cells , Models, Molecular , Molecular Sequence Data , NK Cell Lectin-Like Receptor Subfamily K/chemistry , NK Cell Lectin-Like Receptor Subfamily K/immunology , Protein Binding , Protein Interaction Domains and Motifs , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Transport , Sequence Alignment , Signal Transduction , Tryptophan/chemistry , Tryptophan/immunology
8.
Protein Pept Lett ; 22(5): 460-9, 2015.
Article in English | MEDLINE | ID: mdl-25925769

ABSTRACT

Major Histocompatibility Complex class I-related chain molecules A (MICA) and receptor Natural killer group 2 member D (NKG2D) are important membrane proteins with immunosurveillance properties which could serve as therapeutic targets for immunotherapy. However, expression of MICA and NKG2D in E. coli often leads to the formation of inclusion bodies. Here, we present simple, inexpensive and convenient protocol for the solubilization and refolding of inclusion bodies of MICA and NKG2D expressed in E. coli. The inclusion bodies were firstly dissolved in strong chaotropic reagent (8M urea) and subsequently purified by immobilized-metal affinity column. The denatured MICA/NKG2D was refolded by gradually removing both denaturant (8M urea) and imidazole via dialysis in dialysis buffer of pH 7.4. The appropriate pH of the dialysis buffer was selected based on the theoretical isoelectric points of MICA and NKG2D which were 5.0 and 5.2 respectively. The folded MICA and NKG2D demonstrated the capacity to bind to recombinant NKG2D and MICA respectively by ELISA, Western blot and Surface Plasmon Resonance (SPR) assays. Additionally, the folded MICA and NKG2D demonstrated significant binding to NKG2D-positive Human leukemic cell line U937 and MICA-positive Human pancreatic carcinoma, epithelial-like cell line (PANC-1) respectively, suggesting successful refolding. Successful refolding was further confirmed by Circular Dichroism spectroscopy (CD). We have successfully dissolved, refolded and characterized inclusion bodies of MICA/NKG2D expressed in E. coli using simple, inexpensive and convenient protocol which can be carried out in laboratories under-resourced.


Subject(s)
Histocompatibility Antigens Class I/chemistry , Inclusion Bodies/chemistry , NK Cell Lectin-Like Receptor Subfamily K/chemistry , Protein Refolding , Cell Line, Tumor , Cloning, Molecular , Escherichia coli/chemistry , Escherichia coli/genetics , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/isolation & purification , Histocompatibility Antigens Class I/metabolism , Humans , Inclusion Bodies/genetics , NK Cell Lectin-Like Receptor Subfamily K/genetics , NK Cell Lectin-Like Receptor Subfamily K/isolation & purification , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Protein Binding , Solubility
9.
Nanotechnology ; 25(47): 475101, 2014 Nov 28.
Article in English | MEDLINE | ID: mdl-25371538

ABSTRACT

Iron oxide nanoparticles (IONPs) hold great potential for cancer therapy. Actively targeting IONPs to tumor cells can further increase therapeutic efficacy and decrease off-target side effects. To target tumor cells, a natural killer (NK) cell activating receptor, NKG2D, was utilized to develop pan-tumor targeting IONPs. NKG2D ligands are expressed on many tumor types and its ligands are not found on most normal tissues under steady state conditions. The data showed that mouse and human fragment crystallizable (Fc)-fusion NKG2D (Fc-NKG2D) coated IONPs (NKG2D/NPs) can target multiple NKG2D ligand positive tumor types in vitro in a dose dependent manner by magnetic cell sorting. Tumor targeting effect was robust even under a very low tumor cell to normal cell ratio and targeting efficiency correlated with NKG2D ligand expression level on tumor cells. Furthermore, the magnetic separation platform utilized to test NKG2D/NP specificity has the potential to be developed into high throughput screening strategies to identify ideal fusion proteins or antibodies for targeting IONPs. In conclusion, NKG2D/NPs can be used to target multiple tumor types and magnetic separation platform can facilitate the proof-of-concept phase of tumor targeting IONP development.


Subject(s)
Magnetite Nanoparticles/chemistry , NK Cell Lectin-Like Receptor Subfamily K/chemistry , Neoplasms/drug therapy , Animals , Humans , Magnetite Nanoparticles/therapeutic use , Mice , NK Cell Lectin-Like Receptor Subfamily K/therapeutic use
10.
PLoS One ; 9(9): e108925, 2014.
Article in English | MEDLINE | ID: mdl-25268476

ABSTRACT

Tumor-derived exosomes, which are nanometer-sized extracellular vesicles of endosomal origin, have emerged as promoters of tumor immune evasion but their role in prostate cancer (PC) progression is poorly understood. In this study, we investigated the ability of prostate tumor-derived exosomes to downregulate NKG2D expression on natural killer (NK) and CD8+ T cells. NKG2D is an activating cytotoxicity receptor whose aberrant loss in cancer plays an important role in immune suppression. Using flow cytometry, we found that exosomes produced by human PC cells express ligands for NKG2D on their surface. The NKG2D ligand-expressing prostate tumor-derived exosomes selectively induced downregulation of NKG2D on NK and CD8+ T cells in a dose-dependent manner, leading to impaired cytotoxic function in vitro. Consistent with these findings, patients with castration-resistant PC (CRPC) showed a significant decrease in surface NKG2D expression on circulating NK and CD8+ T cells compared to healthy individuals. Tumor-derived exosomes are likely involved in this NKG2D downregulation, since incubation of healthy lymphocytes with exosomes isolated from serum or plasma of CRPC patients triggered downregulation of NKG2D expression in effector lymphocytes. These data suggest prostate tumor-derived exosomes as down-regulators of the NKG2D-mediated cytotoxic response in PC patients, thus promoting immune suppression and tumor escape.


Subject(s)
CD8-Positive T-Lymphocytes/metabolism , Exosomes/metabolism , Killer Cells, Natural/metabolism , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Prostatic Neoplasms/pathology , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , CD8-Positive T-Lymphocytes/immunology , Castration , Cell Line , Down-Regulation/drug effects , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/metabolism , Humans , Immune Evasion/drug effects , Intracellular Signaling Peptides and Proteins/immunology , Intracellular Signaling Peptides and Proteins/metabolism , K562 Cells , Killer Cells, Natural/immunology , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/immunology , Male , NK Cell Lectin-Like Receptor Subfamily K/chemistry , Prostatic Neoplasms/metabolism
11.
Semin Immunopathol ; 36(6): 641-50, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25141793

ABSTRACT

Natural killer (NK) cells play an important role in virus control during infection. Many viruses have developed mechanisms for subversion of NK cell responses. Murine cytomegalovirus (MCMV) is exceptionally successful in avoiding NK cell control. Here, we summarize the major MCMV evasion mechanisms targeting NK cell functions and their role in viral pathogenesis. The mechanisms by which NK cells regulate CD8(+) T cell response, particularly with respect to the role of NK cell receptors recognizing viral antigens, are discussed. In addition, we discuss the role of NK cell receptors in generation and maintenance of memory NK cells. Final part of this review illustrates how the NK cell response and its viral regulation can be exploited in designing recombinant viral vectors able to induce robust and protective CD8(+) T cell response.


Subject(s)
Herpesviridae Infections/immunology , Killer Cells, Natural/immunology , Muromegalovirus/immunology , Animals , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Herpesviridae Infections/metabolism , Herpesviridae Infections/prevention & control , Herpesviridae Infections/virology , Host-Pathogen Interactions/immunology , Immunologic Memory , Immunomodulation , Killer Cells, Natural/metabolism , Ligands , Lymphocyte Activation , Mice , Muromegalovirus/pathogenicity , NK Cell Lectin-Like Receptor Subfamily A/metabolism , NK Cell Lectin-Like Receptor Subfamily K/chemistry , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Natural Cytotoxicity Triggering Receptor 1/chemistry , Natural Cytotoxicity Triggering Receptor 1/metabolism , Protein Binding , Receptors, Natural Killer Cell/metabolism , Vaccines, Attenuated/immunology , Viral Vaccines/immunology
12.
Int Immunol ; 26(11): 597-606, 2014 Nov.
Article in English | MEDLINE | ID: mdl-24860119

ABSTRACT

The natural killer group 2 membrane D (NKG2D) receptor is an NK-activating receptor that plays an important role in host defense against tumors and viral infections. Although the marmoset is an important and reliable animal model, especially for the study of human-specific viral infections, functional characterization of NKG2D on marmoset NK cells has not previously been conducted. In the present study, we investigated a subpopulation of marmoset NK cells that express NKG2D and exhibit cytolytic potential. On the basis of their CD16 and CD56 expression patterns, marmoset NK cells can be classified into three subpopulations: CD16(+) CD56(-), CD16(-) CD56(+) and CD16(-) CD56(-) cells. NKG2D expression on marmoset CD16(+) CD56(-) and CD16(-) CD56(+) splenocytes was confirmed using an NKG2D ligand composed of an MHC class I chain-related molecule A (MICA)-Fc fusion protein. When marmoset splenocytes were cultured with IL-2 for 4 days, NKG2D expression was retained on CD16(+) CD56(-) and CD16(-) CD56(+). In addition, CD16(+) CD56(+) cells within the marmoset NK population appeared which expressed NKG2D after IL-2 stimulation. IL-2-activated marmoset NK cells showed strong cytolytic activity against K562 target cells and target cells stably expressing MICA. Further, the cytolytic activity of marmoset splenocytes was significantly reduced after addition of MICA-Fc fusion protein. Thus, NKG2D functions as an activating receptor on marmoset NK cells that possesses cytotoxic potential, and phenotypic profiles of marmoset NK cell subpopulations are similar to those seen in humans.


Subject(s)
Callithrix/immunology , Callithrix/metabolism , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Lymphocyte Activation , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/pharmacology , Callithrix/genetics , Cell Line , Cell Membrane/metabolism , Cloning, Molecular , Cross Reactions/immunology , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Killer Cells, Natural/drug effects , Lymphocytes/immunology , Lymphocytes/metabolism , Molecular Sequence Data , NK Cell Lectin-Like Receptor Subfamily K/antagonists & inhibitors , NK Cell Lectin-Like Receptor Subfamily K/chemistry , NK Cell Lectin-Like Receptor Subfamily K/genetics , Phenotype , Recombinant Fusion Proteins/pharmacology , Sequence Alignment , Spleen/cytology , Spleen/immunology
13.
J Virol ; 87(2): 840-50, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23115291

ABSTRACT

The NKG2D receptor is expressed on the surface of NK, T, and macrophage lineage cells and plays an important role in antiviral and antitumor immunity. To evade NKG2D recognition, herpesviruses block the expression of NKG2D ligands on the surface of infected cells using a diverse repertoire of sabotage methods. Cowpox and monkeypox viruses have taken an alternate approach by encoding a soluble NKG2D ligand, the orthopoxvirus major histocompatibility complex (MHC) class I-like protein (OMCP), which can block NKG2D-mediated cytotoxicity. This approach has the advantage of targeting a single conserved receptor instead of numerous host ligands that exhibit significant sequence diversity. Here, we show that OMCP binds the NKG2D homodimer as a monomer and competitively blocks host ligand engagement. We have also determined the 2.25-Å-resolution crystal structure of OMCP from the cowpox virus Brighton Red strain, revealing a truncated MHC class I-like platform domain consisting of a beta sheet flanked with two antiparallel alpha helices. OMCP is generally similar in structure to known host NKG2D ligands but has notable variations in regions typically used to engage NKG2D. Additionally, the determinants responsible for the 14-fold-higher affinity of OMCP for human than for murine NKG2D were mapped to a single loop in the NKG2D ligand-binding pocket.


Subject(s)
Cowpox virus/chemistry , NK Cell Lectin-Like Receptor Subfamily K/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , Animals , Cowpox virus/pathogenicity , Crystallography, X-Ray , Humans , Mice , Models, Molecular , Molecular Sequence Data , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Protein Binding , Protein Structure, Quaternary , Sequence Homology, Amino Acid , Viral Proteins/metabolism
14.
Proc Natl Acad Sci U S A ; 109(51): E3578-87, 2012 Dec 18.
Article in English | MEDLINE | ID: mdl-23169621

ABSTRACT

Natural killer (NK) cells are activated by engagement of the NKG2D receptor with ligands on target cells stressed by infection or tumorigenesis. Several human and rodent cytomegalovirus (CMV) immunoevasins down-regulate surface expression of NKG2D ligands. The mouse CMV MHC class I (MHC-I)-like m152/gp40 glycoprotein down-regulates retinoic acid early inducible-1 (RAE1) NKG2D ligands as well as host MHC-I. Here we describe the crystal structure of an m152/RAE1γ complex and confirm the intermolecular contacts by mutagenesis. m152 interacts in a pincer-like manner with two sites on the α1 and α2 helices of RAE1 reminiscent of the NKG2D interaction with RAE1. This structure of an MHC-I-like immunoevasin/MHC-I-like ligand complex explains the binding specificity of m152 for RAE1 and allows modeling of the interaction of m152 with classical MHC-I and of related viral immunoevasins.


Subject(s)
Major Histocompatibility Complex/immunology , Membrane Glycoproteins/metabolism , Muromegalovirus/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , Viral Proteins/metabolism , Animals , Drosophila/metabolism , Escherichia coli/metabolism , Gene Expression Regulation, Viral , HEK293 Cells , Humans , Immune System , Killer Cells, Natural/metabolism , Ligands , Mice , Molecular Conformation , Mutation , NK Cell Lectin-Like Receptor Subfamily K/chemistry , Protein Conformation , Protein Isoforms , Protein Structure, Secondary , Surface Plasmon Resonance , X-Ray Diffraction
15.
J Immunol Methods ; 366(1-2): 52-9, 2011 Mar 07.
Article in English | MEDLINE | ID: mdl-21281644

ABSTRACT

NK cell function is regulated by the integration of signals from activating and inhibitory receptors. We developed an assay to study the effect of co-crosslinking NK cell receptors in pair-wise combinations without the need to purify NK cells. Monoclonal antibodies recognising inhibitory and activating receptors were coated to flat bottomed tissue culture plates and degranulation was measured within unfractionated, freshly isolated resting or cytokine activated peripheral blood mononuclear cells by flow cytometric analysis of CD107a expression. Measured degranulation responses were NK cell specific, since no expression of CD107a was induced in gated T cells. We detected enhancement of degranulation in response to combinations of antibodies against activating NK cell receptors, including CD16, NKG2D, NKp30 and NKp46 compared to each antibody when combined with an isotype matched control antibody. Co-crosslinking of NKG2A resulted in the inhibition of degranulation measured in response to anti-NKp30 or anti-NKp46 alone in both resting or cytokine pre-activated NK cells, but had no effect on CD16 or NKG2D mediated responses. Interferon gamma production was assayed by intracellular cytokine staining and in cell culture supernatants after receptor crosslinking. No IFN-γ could be detected from resting NK cells after receptor crosslinking whereas the pattern of IFN-γ production in cytokine pre-activated NK cells reflected that observed for degranulation. We conclude that this assay is suitable for the analysis of the impact of NK cell receptor co-crosslinking on multiple NK cell functions and has the potential for application to pathologic conditions where limited numbers of cells are available for study.


Subject(s)
Immunoassay/methods , Killer Cells, Natural/immunology , Receptors, Natural Killer Cell/immunology , Antibodies, Monoclonal , Cell Degranulation/immunology , Cell Separation , Cross-Linking Reagents , Cytokines/metabolism , Flow Cytometry , Humans , In Vitro Techniques , Interferon-gamma/biosynthesis , Lymphocyte Activation , Lysosomal-Associated Membrane Protein 1/metabolism , NK Cell Lectin-Like Receptor Subfamily C/chemistry , NK Cell Lectin-Like Receptor Subfamily C/immunology , NK Cell Lectin-Like Receptor Subfamily K/chemistry , NK Cell Lectin-Like Receptor Subfamily K/immunology , Natural Cytotoxicity Triggering Receptor 1/chemistry , Natural Cytotoxicity Triggering Receptor 1/immunology , Receptors, Natural Killer Cell/chemistry
16.
Biol Pharm Bull ; 34(1): 8-12, 2011.
Article in English | MEDLINE | ID: mdl-21212510

ABSTRACT

Lectin-like receptors natural killer group 2D (NKG2D) and CD94 on natural killer (NK) cells bind to α2,3-NeuAc-containing N-glycans and heparin/heparan sulfate (HS). Using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rGST-NKG2Dlec) and CD94 (rGST-CD94lec), we evaluated their binding affinities (K(d)) to high sialyl Lewis X (sLeX)-expressing transferrin secreted by HepG2 cells (HepTf) and heparin-conjugated bovine serum albumin (Heparin-BSA), using quartz crystal microbalance (QCM) and enzyme immunoassay (EIA) microplate methods. K(d) values obtained by linear reciprocal plots revealed good coincidence between the two methods. K(d) values of rGST-NKG2Dlec obtained by QCM and EIA, respectively, were 1.19 and 1.11 µM for heparin-BSA >0.30 and 0.20 µM for HepTf, while those of rGST-CD94lec were 1.31 and 1.45 µM for HepTf >0.37 and 0.36 µM for heparin-BSA. These results suggested that these glycans can interact with NKG2D and CD94 to modulate NK cell-dependent cytotoxicity.


Subject(s)
Heparin/chemistry , NK Cell Lectin-Like Receptor Subfamily D/metabolism , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Oligosaccharides/chemistry , Polysaccharides/chemistry , Cell Line , Gene Expression Regulation/physiology , Hep G2 Cells , Humans , Immunoenzyme Techniques , NK Cell Lectin-Like Receptor Subfamily D/chemistry , NK Cell Lectin-Like Receptor Subfamily D/genetics , NK Cell Lectin-Like Receptor Subfamily K/chemistry , NK Cell Lectin-Like Receptor Subfamily K/genetics , Protein Binding , Sialyl Lewis X Antigen
17.
Mol Immunol ; 48(4): 516-23, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21074271

ABSTRACT

The homodimeric, activating natural killer cell receptor NKG2D interacts with multiple monomeric ligands polyspecifically, yet without central conformational flexibility. Crystal structures of multiple NKG2D-ligand interactions have identified the NKG2D tyrosine pair Tyr 152 and Tyr 199 as forming multiple specific but diverse interactions with MICA and related proteins. Here we systematically altered each tyrosine to tryptophan, phenylalanine, isoleucine, leucine, valine, serine, and alanine to measure the effect of mutation on affinity and thermodynamics for binding a range of similar ligands: MICA, the higher-affinity ligand MICB, and MICdesign, a high-affinity version of MICA that shares all NKG2D contact residues with MICA. Affinity and residue size were related: tryptophan could often substitute for tyrosine without loss of affinity; loss of the tyrosine hydroxyl through mutation to phenylalanine was tolerated more at position 152 than 199; and the smallest residues coincide with lowest affinities in general. NKG2D mutant van't Hoff binding thermodynamics generally show that substitution of other residues for tyrosine causes a moderate positive or flat van't Hoff slope consistent with moderate loss of binding enthalpy. One set of NKG2D mutations caused MICA to adopt a positive van't Hoff slope corresponding to absorption of heat, and another set caused MICB to adopt a negative slope of greater heat release than wild-type. MICdesign shared one example of the first set with MICA and one of the second set with MICB. When the NKG2D mutation affinities were arranged according to change in nonpolar surface area and compared to results from specific antibody-antigen and protein-peptide interactions, it was found that hydrophobic surface loss in NKG2D reduced binding affinity less than reported in the other contexts. The hydrophobic effect at the center of the NKG2D binding appears more similar to that at the periphery of an antibody-antigen binding site than at its center. Therefore the polyspecific NKG2D binding site is more tolerant of structural alteration in general than either an antibody-antigen or protein-peptide binding site, and this tolerance may adapt NKG2D to a broad range of protein surfaces with micromolar affinity.


Subject(s)
Mutation/genetics , NK Cell Lectin-Like Receptor Subfamily K/chemistry , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Tyrosine/genetics , Tyrosine/metabolism , Crystallography, X-Ray , Histocompatibility Antigens Class I/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Ligands , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Binding , Solvents , Thermodynamics
18.
Nat Immunol ; 11(11): 1023-9, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20890284

ABSTRACT

Many receptors that activate cells of the immune system are multisubunit membrane protein complexes in which ligand recognition and signaling functions are contributed by separate protein modules. Receptors and signaling subunits assemble through contacts among basic and acidic residues in their transmembrane domains to form the functional complexes. Here we report the nuclear magnetic resonance (NMR) structure of the membrane-embedded, heterotrimeric assembly formed by association of the DAP12 signaling module with the natural killer (NK) cell-activating receptor NKG2C. The main intramembrane contact site is formed by a complex electrostatic network involving five hydrophilic transmembrane residues. Functional mutagenesis demonstrated that similar polar intramembrane motifs are also important for assembly of the NK cell-activating NKG2D-DAP10 complex and the T cell antigen receptor (TCR)-invariant signaling protein CD3 complex. This structural motif therefore lies at the core of the molecular organization of many activating immunoreceptors.


Subject(s)
Membrane Proteins , Models, Molecular , NK Cell Lectin-Like Receptor Subfamily K/chemistry , Receptors, Immunologic/chemistry , Amino Acid Sequence , Animals , Dimerization , Humans , Killer Cells, Natural/immunology , Magnetic Resonance Spectroscopy , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , NK Cell Lectin-Like Receptor Subfamily K/genetics , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Protein Structure, Tertiary , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Sequence Alignment
19.
J Biol Chem ; 285(22): 16408-15, 2010 May 28.
Article in English | MEDLINE | ID: mdl-20304922

ABSTRACT

NKG2D is an important activating receptor on lymphocytes. In human, it interacts with two groups of ligands: the major histocompatibility complex class I chain-related A/B (MICA/B) family and the UL-16 binding protein (ULBP) family, also known as retinoic acid early transcript (RAET1). MIC proteins are membrane-anchored, but all of the ULBP/RAET1 proteins, except for RAET1E and RAET1G, are glycosylphosphatidylinositol (GPI)-anchored. To address the reason for these differences we studied the association of RAET1G with the membrane. Using epitope-tagged RAET1G protein in conjunction with antibodies to different parts of the molecule and in pulse-chase experiments, we showed that the C terminus of the protein was cleaved soon after protein synthesis. Endoglycosidase H and peptide N-glycosidase treatment and cell surface immunoprecipitation indicated that most of the protein stayed in the endoplasmic reticulum, but some of the cleaved form was modified in the Golgi and transported to the cell surface. We examined the possibility of GPI anchoring of the protein in three ways: (i) Phosphatidylinositol (PI)-specific phospholipase C released the PI-linked form of the protein. (ii) The surface expression pattern of RAET1G decreased in cells defective in GPI anchoring through mutant GPI-amidase. (iii) Site-directed mutagenesis, to disrupt residues predicted to facilitate GPI-anchoring, resulted in diminished surface expression of RAET1G. Thus, a form of RAET1G is GPI-anchored, in line with most other ULBP/RAET1 family proteins. The cytoplasmic tail and transmembrane domains appear to result from gene duplication and frameshift mutation. Together with our previous results, our data suggest that RAET1G is regulated post-translationally to produce a GPI-anchored isoform.


Subject(s)
Carrier Proteins/chemistry , Glycosylphosphatidylinositols/chemistry , Membrane Proteins/chemistry , NK Cell Lectin-Like Receptor Subfamily K/chemistry , Cell Line, Tumor , Cell Membrane/metabolism , Gene Expression Regulation , Golgi Apparatus/metabolism , HeLa Cells , Humans , Killer Cells, Natural/metabolism , Mutation , Protein Isoforms , Protein Processing, Post-Translational , Protein Structure, Tertiary , Type C Phospholipases/metabolism
20.
PLoS Pathog ; 6(1): e1000723, 2010 Jan 15.
Article in English | MEDLINE | ID: mdl-20090832

ABSTRACT

The activating immunoreceptor NKG2D promotes elimination of infected or malignant cells by cytotoxic lymphocytes through engagement of stress-induced MHC class I-related ligands. The human cytomegalovirus (HCMV)-encoded immunoevasin UL16 subverts NKG2D-mediated immune responses by retaining a select group of diverse NKG2D ligands inside the cell. We report here the crystal structure of UL16 in complex with the NKG2D ligand MICB at 1.8 A resolution, revealing the molecular basis for the promiscuous, but highly selective, binding of UL16 to unrelated NKG2D ligands. The immunoglobulin-like UL16 protein utilizes a three-stranded beta-sheet to engage the alpha-helical surface of the MHC class I-like MICB platform domain. Intriguingly, residues at the center of this beta-sheet mimic a central binding motif employed by the structurally unrelated C-type lectin-like NKG2D to facilitate engagement of diverse NKG2D ligands. Using surface plasmon resonance, we find that UL16 binds MICB, ULBP1, and ULBP2 with similar affinities that lie in the nanomolar range (12-66 nM). The ability of UL16 to bind its ligands depends critically on the presence of a glutamine (MICB) or closely related glutamate (ULBP1 and ULBP2) at position 169. An arginine residue at this position however, as found for example in MICA or ULBP3, would cause steric clashes with UL16 residues. The inability of UL16 to bind MICA and ULBP3 can therefore be attributed to single substitutions at key NKG2D ligand locations. This indicates that selective pressure exerted by viral immunoevasins such as UL16 contributed to the diversification of NKG2D ligands.


Subject(s)
Cytomegalovirus/immunology , Histocompatibility Antigens Class I/chemistry , Immune Evasion/immunology , Molecular Mimicry/immunology , NK Cell Lectin-Like Receptor Subfamily K/chemistry , Viral Proteins/chemistry , Amino Acid Sequence , GPI-Linked Proteins , Histocompatibility Antigens Class I/immunology , Humans , Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Ligands , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Molecular Sequence Data , Multiprotein Complexes/chemistry , Multiprotein Complexes/immunology , NK Cell Lectin-Like Receptor Subfamily K/immunology , Protein Binding , Protein Structure, Quaternary , Structure-Activity Relationship , Surface Plasmon Resonance , Viral Proteins/immunology
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