Biomedical subjects

Katsumi Maenaka

Publications and source records attributed to Katsumi Maenaka.

At least 19 recordsLinked to original sources

PubMed2007

Cutting Edge: Allele-specific and peptide-dependent interactions between KIR3DL1 and HLA-A and HLA-B.

Although it is clear that KIR3DL1 recognizes Bw4(+) HLA-B, the role of Bw4(+) HLA-A allotypes as KIR3DL1 ligands is controversial. We therefore examined the binding of tetrameric HLA-A and -B complexes, including HLA*2402, a common Bw4(+) HLA-A allotype, to KIR3DL1*001, *005, *007, and *1502 allotypes. Only Bw4(+) tetramers bound KIR3DL1. Three of four HLA-A*2402 tetramers bound one or more KIR3DL1 allotypes and all four KIR3DL1 allotypes bound to one or more HLA-A*2402 tetramers, but with different binding specificities. Only KIR3DL1*005 bound both HLA-A*2402 and HLA-B*5703 tetramers. HLA-A*2402-expressing target cells were resistant to lysis by NK cells expressing KIR3DL1*001 or *005. This study shows that HLA-A*2402 is a ligand for KIR3DL1 and demonstrates how the binding of KIR3DL1 to Bw4(+) ligands depends upon the bound peptide as well as HLA and KIR3DL1 polymorphism.

Alleles↗

PubMed2006

Detection of weak ligand interactions of leukocyte Ig-like receptor B1 by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) can directly and quickly detect the translational diffusion of individual fluorescence-labeled molecules in solutions. Although FCS analyses for protein-protein interactions have been performed, the very weak interactions generally observed in cell-cell recognition of the immune system have not been examined in detail. Here, we report the FCS analysis for low-affinity and fast-kinetic binding (K(d) greater than muM range) of the human inhibitory immune cell surface receptor, leukocyte immunoglobulin-like receptor B1 (LILRB1), to its ligands, MHC (major histocompatibility complex) class I molecules (MHCIs) by using the single-molecule FCS detection system which requires only a small amount of sample. Since the random labeling technique for LILRB1 disturbed the MHCI binding, we performed site-specific labeling of LILRB1 by introducing a cysteine residue at the C-terminus, which could be covalently attached with the fluorescence reagent, Alexa647. This technique can be applied to other type I membrane receptors. The low-affinity binding of LILRB1-Alexa647 to MHCIs (HLA-Cw4, and -G1) was detected by FCS, even though non-labeled MHCIs were only twice as big as the labeled LILRB1. Their dissociation constants (7.5 muM (HLA-Cw4) and 5.7 muM (HLA-G1)) could be determined and were consistent with surface plasmon resonance (SPR) data. These results indicate that the single-molecule FCS detection system is capable of analyzing the binding characteristics of immune cell surface receptors even in difficult cases such as (1) small amount of protein samples, (2) small difference in molecular weight and (3) weak affinity. Therefore, it is a powerful tool for characterization and high throughput inhibitor screening of a wide variety of cell-cell recognition receptors involved in immunologically relevant events.

Antigens, CD↗

PubMed2006

Structural basis for recognition of the nonclassical MHC molecule HLA-G by the leukocyte Ig-like receptor B2 (LILRB2/LIR2/ILT4/CD85d).

HLA-G is a nonclassical MHC class I (MHCI) molecule that can suppress a wide range of immune responses in the maternal-fetal interface. The human inhibitory immune receptors leukocyte Ig-like receptor (LILR) B1 [also called LIR1, Ig-like transcript 2 (ILT2), or CD85j] and LILRB2 (LIR2/ILT4/CD85d) preferentially recognize HLA-G. HLA-G inherently exhibits various forms, including beta(2)-microglobulin (beta(2)m)-free and disulfide-linked dimer forms. Notably, LILRB1 cannot recognize the beta(2)m-free form of HLA-G or HLA-B27, but LILRB2 can recognize the beta(2)m-free form of HLA-B27. To date, the structural basis for HLA-G/LILR recognition remains to be examined. Here, we report the 2.5-A resolution crystal structure of the LILRB2/HLA-G complex. LILRB2 exhibits an overlapping but distinct MHCI recognition mode compared with LILRB1 and dominantly recognizes the hydrophobic site of the HLA-G alpha3 domain. NMR binding studies also confirmed these LILR recognition differences on both conformed (heavy chain/peptide/beta(2)m) and free forms of beta(2)m. Binding studies using beta(2)m-free MHCIs revealed differential beta(2)m-dependent LILR-binding specificities. These results suggest that subtle structural differences between LILRB family members cause the distinct binding specificities to various forms of HLA-G and other MHCIs, which may in turn regulate immune suppression.

Antigens, CD↗

PubMed2006

MHC class I-like MILL molecules are beta2-microglobulin-associated, GPI-anchored glycoproteins that do not require TAP for cell surface expression.

MILL (MHC class I-like located near the leukocyte receptor complex) is a family of MHC class I-like molecules encoded outside the MHC, which displays the highest sequence similarity to human MICA/B molecules among known class I molecules. In the present study, we show that the two members of the mouse MILL family, MILL1 and MILL2, are GPI-anchored glycoproteins associated with beta2-microglobulin (beta2m) and that cell surface expression of MILL1 or MILL2 does not require functional TAP molecules. MILL1 and MILL2 molecules expressed in bacteria could be refolded in the presence of beta2m, without adding any peptides. Hence, neither MILL1 nor MILL2 is likely to be involved in the presentation of peptides. Immunohistochemical analysis revealed that MILL1 is expressed in a subpopulation of thymic medullary epithelial cells and a restricted region of inner root sheaths in hair follicles. The present study provides additional evidence that MILL is a class I family distinct from MICA/B.

Animals↗

PubMed2006

Crystal structure of the human monocyte-activating receptor, "Group 2" leukocyte Ig-like receptor A5 (LILRA5/LIR9/ILT11).

Human leukocyte Ig-like receptor B1 (LILRB1) and B2 (LILRB2) belong to "Group 1" receptors and recognize a broad range of major histocompatibility complex class I molecules (MHCIs). In contrast, "Group 2" receptors show low similarity with LILRB1/B2, and their ligands remain to be identified. To date, the structural and functional characteristics of Group 2 LILRs are poorly understood. Here we report the crystal structure of the extracellular domain of LILRA5, which is an activating Group 2 LILR expressed on monocytes and neutrophils. Unexpectedly, the structure showed large changes in structural conformation and charge distribution in the region corresponding to the MHCI binding site of LILRB1/B2, which are also distinct from killer cell Ig-like receptors and Fc alpha receptors. These changes probably confer the structural hindrance for the MHCI binding, and their key amino acid substitutions are well conserved in Group 2 LILRs. Consistently, the surface plasmon resonance and flow cytometric analyses demonstrated that LILRA5 exhibited no affinities to all tested MHCIs. These results raised the possibility that LILRA5 as well as Group 2 LILRs do not play a role in any MHCI recognition but could possibly bind to non-MHCI ligand(s) on the target cells to provide a novel immune regulation mechanism.

Amino Acid Sequence↗

PubMed2006

Efficient leukocyte Ig-like receptor signaling and crystal structure of disulfide-linked HLA-G dimer.

HLA-G is a nonclassical major histocompatibility complex class I (MHCI) molecule, which is expressed in trophoblasts and confers immunological tolerance in the maternal-fetal interface by binding to leukocyte Ig-like receptors (LILRs, also called as LIR/ILT/CD85) and CD8. HLA-G is expressed in disulfide-linked dimer form both in solution and at the cell surface. Interestingly, MHCI dimer formations have been involved in pathogenesis and T cell activation. The structure and receptor binding characteristics of MHCI dimers have never been evaluated. Here we performed binding studies showing that the HLA-G dimer exhibited higher overall affinity to LILRB1/2 than the monomer by significant avidity effects. Furthermore, the cell reporter assay demonstrated that the dimer formation remarkably enhanced the LILRB1-mediated signaling at the cellular level. We further determined the crystal structure of the wild-type dimer of HLA-G with the intermolecular Cys(42)-Cys(42) disulfide bond. This dimer structure showed the oblique configuration to expose two LILR/CD8-binding sites upward from the membrane easily accessible for receptors, providing plausible 1:2 (HLA-G dimer:receptors) complex models. These results indicated that the HLA-G dimer conferred increased avidity in a proper structural orientation to induce efficient LILR signaling, resulting in the dominant immunosuppressive effects. Moreover, structural and functional implications for other MHCI dimers observed in activated T cells and the pathogenic allele, HLA-B27, are discussed.

Alleles↗

PubMed2005

Entropically driven MHC class I recognition by human inhibitory receptor leukocyte Ig-like receptor B1 (LILRB1/ILT2/CD85j).

The human inhibitory receptor, leukocyte immunoglobulin (Ig)-like receptor B1 (also called Ig-like transcript (ILT) 2, CD85j), is broadly expressed on leukocytes. LILRB1 binds to a wide range of major histocompatibility complex class I molecules (MHCIs) and transduces negative signals that can, for example, prevent killing of MHCI-expressing cells. Here we report the kinetic, thermodynamic, NMR and crystallographic analyses of MHCI recognition by LILRB1. Kinetic studies demonstrated that LILRB1 binds to MHCIs with fast association and dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses showed that LILRB1-MHCI interactions are entropically driven (-TdeltaS = -9.4 approximately -6.6 kcal mol(-1)) with low heat capacity changes (deltaC(p) = -0.22 approximately -0.10 kcal mol(-1) K(-1)). The crystal structures of LILRB1 in the different crystal forms exhibited variation in the elbow angle between the two N-terminal Ig-like domains, indicating interdomain flexibility. Consistently, NMR analysis provided the direct evidence of the conformational changes of LILRB1 upon the MHCI binding. These findings suggest that LILRB1-MHCI interactions, while involving some conformational adjustment, are not accompanied by a very large reduction in conformational flexibility at the binding interface. This mode of binding is distinct from "induced-fit" binding, which is associated with large reductions in conformational flexibility, and would be suitable for rapid engagement of MHCIs to enable fast monitoring of the expression level of MHCIs on target cells.

Antigens, CD↗

PubMed2005

Preparation and crystallization of the disulfide-linked HLA-G dimer.

HLA-G is a non-classical MHC class I, which binds to inhibitory receptors, such as Leukocyte Ig-like receptors, to induce a wide range of tolerogenic immunological effects. HLA-G can be expressed as a disulfide-liked dimer both in solution and at the cell surface. However, the three-dimensional structure of the HLA-G dimer is unknown. Here, we report the crystallization of the disulfide-linked dimer form of HLA-G by adding dithiothreitol (DTT), enabling a 3.2-A data set to be collected. We also show that DTT promotes disulfide bond exchange of refolded HLA-G, whose free cysteine was protected, thus facilitating its dimerization. This technique could also be applied for disulfide-mediated dimer/multimer formation of refolded proteins harbouring free cysteines.

Crystallization↗

PubMed2005

Crystallization and preliminary crystallographic analysis of the N-terminal domain of PriA from Escherichia coli.

PriA, a DEXH-type DNA helicase, binds specifically to the 3' end of DNA through its N-terminal domain, and is a candidate sensor protein that recognizes arrested DNA replication forks in bacteria. We crystallized an N-terminal fragment of PriA in the absence and the presence of oligonucleotides to elucidate the structural basis for the specific recognition of the 3' terminus of DNA.

Adenosine Triphosphatases↗

PubMed2005

Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system.

Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.

Amino Acid Sequence↗

PubMed2005

Expression, crystallization and preliminary diffraction studies of the Pseudomonas putida cytochrome P450cam operon repressor CamR.

The Pseudomonas putida cam repressor (CamR) is a homodimeric protein that binds to the camO DNA operator to inhibit the transcription of the cytochrome P450cam operon camDCAB. CamR has two functional domains: a regulatory domain and a DNA-binding domain. The binding of the inducer D-camphor to the regulatory domain renders the DNA-binding domain unable to bind camO. Native CamR and its selenomethionyl derivative have been overproduced in Escherichia coli and purified. Native CamR was crystallized under the following conditions: (i) 12-14% PEG 4000, 50 mM Na PIPES, 0.1 M KCl, 1% glycerol pH 7.3 at 288 K with and without camphor and (ii) 1.6 M P(i), 50 mM Na PIPES, 2 mM camphor pH 6.7 at 278 K. The selenomethionyl derivative CamR did not crystallize under either of these conditions, but did crystallize using 12.5% PEG MME 550, 25 mM Na PIPES, 2.5 mM MgCl2 pH 7.3 at 298 K. Preliminary X-ray diffraction studies revealed the space group to be orthorhombic (P2(1)2(1)2), with unit-cell parameters a = 48.0, b = 73.3, c = 105.7 A. Native and selenomethionyl derivative data sets were collected to 3 A resolution at SPring-8 and the Photon Factory.

Bacterial Proteins↗

PubMed2005

Extensive polymorphisms of LILRB1 (ILT2, LIR1) and their association with HLA-DRB1 shared epitope negative rheumatoid arthritis.

Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1/LIR1/ILT2) is an inhibitory receptor broadly expressed on leukocytes and recognizes HLA-class I and human cytomegalovirus UL18. LILRB1 is encoded within the leukocyte receptor complex on 19q13.4, previously implicated to be a susceptibility region to systemic lupus erythematosus (SLE). In this study, we screened for polymorphisms of LILRB1 and examined their association with SLE and rheumatoid arthritis (RA). In the 5' portion of LILRB1, three haplotypes containing four non-synonymous substitutions within the ligand-binding domains and two single nucleotide polymorphisms within the promoter region were identified and designated as PE01-03. In the 3' portion, two haplotypes (CY01, 02) containing a non-synonymous substitution of the cytoplasmic region were identified. CY01 and 02 did not co-segregate with PE01-03. Significant association with susceptibility to SLE or RA was not observed; however, among the subjects not carrying RA-associated HLA-DRB1 shared epitope (SE), LILRB1.PE01/01 diplotype was significantly associated with RA (odds ratio 2.05, P = 0.019 and Pc = 0.038). Gross difference was not observed in the crystal structures, thermostabilities and binding affinities to HLA-class I ligands among LILRB1.PE01-03 haplotype products; however, surface expression of LILRB1 was significantly decreased in lymphocytes and monocytes from the carriers of PE01 haplotype. These findings demonstrated that LILRB1 is highly polymorphic and is associated with susceptibility to RA in HLA-DRB1 SE negative subjects, possibly by insufficient inhibitory signaling in leukocytes. In addition, these observations suggested that the polymorphisms of LILR family members may be substantially involved in the diversity of human immune responses.

Adult↗

PubMed2005

Crystallization and preliminary X-ray analysis of mitochondrial presequence receptor Tom20 in complexes with a presequence from aldehyde dehydrogenase.

Most mitochondrial proteins are synthesized in the cytosol and must be imported into the mitochondria. Many mitochondrial precursor proteins have an extra leader sequence at their N-terminus called a presequence. Presequences are recognized by the Tom20 receptor protein. Based on the previously determined NMR structure of rat Tom20, a fragment corresponding to the core structure was generated. A cysteine residue was added at the C-terminus of the rat aldehyde dehydrogenase presequence to fix the presequence peptide onto the Tom20 fragment via an intermolecular disulfide bond. Two crystal forms of the complex were successfully obtained with different designs of the linker sequence which diffracted to 2.1 and 1.9 A. Crystal dehydration and subsequent annealing was essential to obtain good diffraction data for the 2.1 A crystal form.

Aldehyde Dehydrogenase↗

PubMed2005

Crystallization and preliminary X-ray analysis of the mRNA-binding domain of elongation factor SelB in complex with RNA.

In bacteria, the selenocysteine-specific elongation factor SelB is necessary for incorporation of selenocysteine, the 21st amino acid, into proteins by the ribosome. SelB binds to an mRNA hairpin formed by the selenocysteine-insertion sequence (SECIS) and delivers selenocysteyl-tRNA (Sec-tRNASec) at the ribosomal A site. The minimum fragment (residues 512-634) of Moorella thermoacetica SelB (SelB-M) required for mRNA binding has been overexpressed and purified. The complex of SelB-M with 23 nucleotides of the SECIS mRNA hairpin was crystallized at 293 K using the hanging-drop vapour-diffusion or oil-batch methods. The crystals diffract to 2.3 A resolution using SPring-8 BL41XU and belong to the space group P2(1)2(1)2, with unit-cell parameters a = 81.69, b = 169.58, c = 71.69 A.

Bacterial Proteins↗

PubMed2005

Crystal structure of a biologically functional form of PriB from Escherichia coli reveals a potential single-stranded DNA-binding site.

PriB is not only an essential protein necessary for the replication restart on the collapsed and disintegrated replication fork, but also an important protein for assembling of primosome onto PhiX174 genomic DNA during replication initiation. Here we report a 2.0-A-resolution X-ray structure of a biologically functional form of PriB from Escherichia coli. The crystal structure revealed that despite a low level of primary sequence identity, the PriB monomer, as well as the dimeric form, are structurally identical to the N-terminal DNA-binding domain of the single-stranded DNA-binding protein (SSB) from Escherichia coli, which possesses an oligonucleotides-binding-fold. The oligonucleotide-PriB complex model based on the oligonucleotides-SSB complex structure suggested that PriB had a DNA-binding pocket conserved in SSB from Escherichia coli and might bind to single-stranded DNA in the manner of SSB. Furthermore, surface plasmon resonance analysis and fluorescence measurements demonstrated that PriB binds single-stranded DNA with high affinity, by involving tryptophan residue. The significance of these results with respect to the functional role of PriB in the assembly of primosome is discussed.

Amino Acid Sequence↗

PubMed2005

Structural basis for mRNA recognition by elongation factor SelB.

In bacteria, incorporation of selenocysteine, the 21(st) amino acid, into proteins requires elongation factor SelB, which has the unusual property of binding to both transfer RNA (tRNA) and mRNA. SelB binds to an mRNA hairpin formed by the selenocysteine insertion sequence (SECIS) with extremely high specificity, the molecular basis of which has been unknown. We have determined the crystal structure of the mRNA-binding domain of SelB in complex with SECIS RNA at a resolution of 2.3 A. This is the first example of a complex between an RNA and a winged-helix (WH) domain, a motif found in many DNA-binding proteins and recently discovered in RNA-binding proteins. Notably, RNA binding does not induce a major conformational change in the WH motif. The structure reveals a new mode of RNA recognition with a geometry that allows the complex to wrap around the small ribosomal subunit.

Amino Acid Motifs↗

PubMed2005

Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system.

Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression of silkworm. By using this system, the green fluorescence protein was successfully expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses.

Animals↗

PubMed2005

Genetic variability of the major histocompatibility complex class I homologue encoded by human cytomegalovirus leads to differential binding to the inhibitory receptor ILT2.

Human cytomegalovirus carries a gene, UL18, that is homologous to cellular major histocompatibility complex (MHC) class I genes. Like MHC class I molecules, the protein product of the UL18 gene associates with beta2-microglobulin, and the stability of this complex depends on peptide loading. UL18 protein binds to ILT2 (CD85j), an inhibitory receptor present on B cells, monocytes, dendritic cells, T cells, and NK cells that also recognizes classical and nonclassical MHC molecules. These observations suggest that UL18 may play a role in viral immune evasion, but its real function is unclear. Since this molecule has similarity with polymorphic MHC proteins, we explored whether the UL18 gene varied between virus isolates. We report here that the UL18 gene varies significantly between virus isolates: amino acid substitutions were found in the predicted alpha1, alpha2, and alpha3 domains of the UL18 protein molecule. We also studied the ability of several variant UL18 proteins to bind to the ILT2 receptor. All of the variants tested bound to ILT2, but there were marked differences in the affinity of binding to this receptor. These differences were reflected in functional assays measuring inhibition of the cytotoxic capacity of NK cells via interaction with ILT2. In addition, the variants did not bind other members of the CD85 family. The implications of these data are discussed.

Antigens, CD↗