Purification of receptor complexes of interleukin-10 stoichiometry and the importance of deglycosylation in their crystallization.

Interleukin-10 (IL-10) is a pleiotropic immunosuppressive cytokine that has a wide range of effects in controlling inflammatory responses. Viral IL-10 (vIL-10) is a homologue of human IL-10 (hIL-10) produced by Epstein-Barr virus (EBV). Both hIL-10 and vIL-10 bind to the soluble extracellular fragment of the cytokine receptor IL-10R1 (shIL-10R1). The stoichiometry of the vIL-10 : shIL-10R1 complex has been found to be the same as hIL-10 : shIL-10R1, with two vIL-10 dimers binding to four shIL-10R1 monomers. Complexes of both hIL-10 and vIL-10 with glycosylated shIL-10R1 could not be crystallized. Controlled deglycosylation using peptide : N-glycosidase F and endo-beta-N-acetylglucosaminidase F3 resulted in the formation of crystals of both hIL-10 : shIL-10R1 and vIL-10 : shIL-10R1 complexes, indicating that the difficulty in the crystal formation was largely due to the presence of complex carbohydrate side chains. The availability of the structure of the ligand-receptor complexes should facilitate our understanding of the basis of the interaction between IL-10 and the IL-10 receptor.     

Conformational changes mediate interleukin-10 receptor 2 (IL-10R2) binding to IL-10 and assembly of the signaling complex

Interleukin-10 receptor 2 (IL-10R2) is a critical component of the IL-10.IL-10R1.IL-10R2 complex which regulates IL-10-mediated immunomodulatory responses. The ternary IL-10 signaling complex is assembled in a sequential order with the IL-10.IL-10R1 interaction occurring first followed by engagement of the IL-10R2 chain. In this study we map the IL-10R2 binding site on IL-10 using surface plasmon resonance and cell-based assays. Critical IL-10R2 binding residues are located in helix A adjacent to the previously identified IL-10R1 recognition surface. Interestingly, IL-10R2 binding residues located in the N-terminal end of helix A exhibit large structural differences between unbound cIL-10 and cIL-10.IL-10R1 crystal structures. This suggests IL-10R1-induced conformational changes regulate IL-10R2 binding and assembly of the ternary IL-10.IL-10R1.IL-10R2 complex. The basic mechanistic features of the assembly process are likely shared by six additional class-2 cytokines (viral IL-10s, IL-22, IL-26, IL-28A, IL28B, and IL-29) to promote IL-10R2 binding to six additional receptor complexes. These studies highlight the importance of structure in regulating low affinity protein-protein interactions and IL-10 signal transduction.     

Differential IL-10R1 expression plays a critical role in IL-10-mediated immune regulation.

In this study, we characterized the differential receptor-binding specificity, affinity, and Janus kinase-STAT activation of cellular IL-10 (cIL-10) compared with viral IL-10 (vIL-10). Only cells expressing IL-10R1 bind human IL-10 or vIL-10. IL-10R2 does not bind to cIL-10 or vIL-10 alone and its presence does not enhance the receptor-binding affinity of cIL-10 or vIL-10, but it is essential for both cIL-10- and vIL-10-mediated signal transduction and immune regulation. Responses initiated by cIL-10 and vIL-10 were compared in B cell and mast cell lines, and demonstrated that the inability of vIL-10 to stimulate immune responses, as compared with human IL-10, is due to failure to initiate signaling. Absent signal transduction is due to low level expression of cell surface IL-10R1, since overexpressing IL-10R1 allows vIL-10 to initiate cIL-10-like signals and subsequent biological responses. These results are similar in primary cells, since splenocytes respond to both cIL-10 and vIL-10, while thymocytes respond only to cIL-10 and have very low mouse IL-10R1 but not mouse IL-10R2 expression. These data demonstrate that IL-10R1 expression plays a critical role in determining whether cells respond to IL-10. Modulation of cell surface IL-10R1 density might be an important mechanism for determining whether IL-10 leads to immunostimulation or immunosuppression in vivo.     

Same structure, different function crystal structure of the Epstein-Barr virus IL-10 bound to the soluble IL-10R1 chain

Human IL-10 (hIL-10) is a cytokine that modulates diverse immune responses. The Epstein-Barr virus (EBV) genome contains an IL-10 homolog (vIL-10) that shares high sequence and structural similarity with hIL-10. Although vIL-10 suppresses inflammatory responses like hIL-10, it cannot activate many other immunostimulatory functions performed by the cellular cytokine. These functional differences have been correlated with the approximately 1000-fold lower affinity of vIL-10, compared to hIL-10, for the IL-10R1 receptor chain. To define the structural basis for these observations, crystal structures of vIL-10 and a vIL-10 point mutant were determined bound to the soluble IL-10R1 receptor fragment (sIL-10R1) at 2.8 and 2.7 A resolution, respectively. The structures reveal that subtle changes in the conformation and dynamics of the vIL-10 AB and CD loops and an orientation change of vIL-10 on sIL-10R1 are the main factors responsible for vIL-10's reduced affinity for sIL-10R1 and its distinct biological profile.     

A new DNA marker (D10S94) very tightly linked to the multiple endocrine neoplasia type 2A (MEN2A) locus.

Combined somatic cell hybrid and linkage studies between D10S94 and five pericentromeric loci (FNRB, D10Z1, MEN2A, RBP3, and D10S15) have localized the new DNA sequence pcl1/A1S-6-c23 at D10S94 to 10q11.2. No recombinants were observed between D10S94 and D10Z1 or MEN2A. D10S94 maps in proximal 10q11.2 very near to MEN2A. There are three possible orders for the six loci that we investigated from the centromeric region of chromosome 10. At present the genetic data do not allow us to order MEN2A with respect to D10Z1 and D10S94. The three possible orders are FNRB-D10Z1-D10S94-MEN2A-RBP3-D10S15, FNRB-D10Z1-MEN2A-D10S94-RBP3-D10S15, and FNRB-MEN2A-D10Z1-D10S94-RBP3-D10S15. In view of the fact that no recombinants between D10S94 and MEN2A or between D10S94 and D10Z1 were observed, the combined haplotypes formed from RFLPs and D10Z1 and D10S94 will increase the informativeness and accuracy of genotype prediction for at-risk members of the families having the MEN 2A syndrome, particularly when the affected parent is female. The localization of D10S94 with respect to MEN2A will prove valuable in experiments directed toward cloning the MEN2A locus.     

H-41, a new minor histocompatibility locus. I. Histogenetic analysis

The B10.STA12 mouse congenic line inherited from the wild mouse parent not only the H-2w13 haplotype but also an allele at a minor H locus, which we designate H-41. This allele (H-41a) differentiates the B10.STA12 line from B10.STA10 and B10.LIB55, which carry identical H-2w13 haplotypes but a different H-41 allele (the H-41b, also present in the background strain C57BL/10Sn). The B10.STA12 and B10.STA10 lines reject each other's skin grafts and generate cytolytic T lymphocytes (CTL) after in vivo immunization and in vitro restimulation with cells of the partner strain. The B10.STA12 anti-B10.STA10 CTL react with B10.STA10, B10.LIB55, and B10.STA39 target cells and with cells of F1 hybrids between the responder strain B10.STA12 and strains C57BL/6, C57BL/10, C57L, BALB/c, A, AKR, WB, DBA/1, and DBA/2 but fail to react with (C3H x B10.STA12) F1 and (CBA x B10.STA12) F1 cells. The B10.STA10 anti-B10.STA12 CTL react with B10.STA12, B10.P, and C3H.NB cells but fail to react to (B6 x B10.STA10) F1 target cells. The CTL reactivity in both combinations is Dp restricted. The B10.STA10 anti-B10.STA12 CTL exhibit, in addition, a cross-reactivity with B10.SAA48 cells that may be directed at one of the alloantigens controlled by the H-2 haplotype of this strain.     

黄淮麦区地方小麦品种籽粒颜色相关基因Tamyb10-1等位变异检测?

Tamyb10-1基因属于MYB家族的一种转录因子,决定着小麦种皮的颜色,同时对穗发芽抗性也具有一定影响。本研究以来自我国黄淮麦区的地方小麦品种为材料,利用功能标记对参试小麦品种Tamyb10-1基因位点在3A、3B和3D染色体上的等位变异类型进行了检测。结果表明,参试材料中上述每一位点均有2种等位变异类型,由此形成了7种基因型组合,分别为Tamyb10-A1a/Tamyb10-B1a/Tamyb10-D1a、Tamyb10-A1a/Tamyb10-B1a/Tamyb10-D1b、Tamyb10-A1a/Tamyb10-B1b/Tamyb10-D1a、Tamyb10-A1b/Tamyb10-B1a/Tamyb10-D1a、Tamyb10-A1b/Tamyb10-B1b/Tamyb10-D1a、Tamyb10-A1b/Tamyb10-B1a/Tamyb10-D1b和Tamyb10-A1b/Tamyb10-B1b/Tamyb10-D1b,其分布频率分别为38.0%、15.0%、1.0%、8.0%、1.0%、33.0%和4.0%。进一步研究结果表明,种皮颜色为白色时,Tamyb10-1基因在3个位点均为野生型,而当任何一个位点发生突变时均表现为红色。由于该基因也影响穗发芽的抗性,且子粒颜色与其抗氧化能力密切相关,因此本研究对以子粒颜色性状为育种目标的优异种质资源筛选具有一定参考价值。     

Quantitative analysis of the elemental composition and the mass of bacterial polyphosphate bodies using STEM EDX

The quantitative analysis of laboratory grown organisms (Plectonema boryanum and Staphylococcus aureus) revealed that a typical in vivo polyphosphate body (PPB) contains O (4.3 x 10(-8) microg), C (1.2 x 10(-8) microg), P (6.7 x 10(-9) microg), Mg (1.3 x 10(-9) microg), Ca (6.7 x 10(-10) microg), K (6.7 x 10(-10) microg), Fe (6.0 x 10(-10) microg), S (5.4 x 10(-10) microg) and Al (5.9 x 10(-10) microg). Quantitative X-ray analysis of samples from nature showed PPB contain O (1.63 x 10(-8) microg), C (4.75 x 10(-9) microg), P (2.50 x 10(-9) microg), Mg (5.0 x 10(-10) microg), Ca (2.50 x 10(-10) microg), K (2.50 x 10(-10) microg), Fe (2.25 x 10(-10) microg) and S (2.0 x 10(-10) microg). The mass of an average polyphosphate body was 6.7 x 10(-8) microg for P. boryanum, 2.5 x 10(-8) microg for S. aureus and for microbes from the natural environment 6.3 x 10(-8) microg. The results indicate that the PPB may have other unknown functions in addition to essential element storage, acting as a detoxification method by sequestering heavy metals and providing a homeostasis system in the cell.     

Design and analysis of an engineered human interleukin-10 monomer

A monomeric form of human interleukin 10 (IL-10M1) has been engineered for detailed structure-function studies on IL-10 and its receptor complexes. Wild type IL-10 (wtIL-10) is a domain swapped dimer whose structural integrity depends on the intertwining of two peptide chains. wtIL-10 was converted to a monomeric isomer by inserting 6 amino acids into the loop connecting the swapped secondary structural elements. Characterization of IL-10M1 by mass spectroscopy, size exclusion chromatography, cross-linking, and circular dichroism shows that IL-10M1 is a stable alpha-helical monomer at physiological pH whose three-dimensional structure closely resembles one domain of wtIL-10. As previously reported, incubation of wtIL-10 with a soluble form of the IL-10Ralpha (sIL-10Ralpha) generates a complex that consists of 2 wtIL-10 molecules and 4 sIL-10Ralphas. In contrast, IL-10M1 forms a 1:1 complex with the sIL-10Ralpha. Characterization of the interaction using isothermal titration calorimetry confirmed the 1:1 stoichiometry and yielded a dissociation constant of 30 nm with an apparent binding enthalpy of -12.2 kcal/mol. Despite forming a 1:1 complex, IL-10M1 is biologically active in cellular proliferation assays. These results indicate that the 1:1 interaction between IL-10M1 and IL-10Ralpha is sufficient for recruiting the signal transducing receptor chain (IL-10Rbeta) into the signaling complex and eliciting IL-10 cellular responses.     

Epstein-Barr Virus IL-10 Engages IL-10R1 by a Two-step Mechanism Leading to Altered Signaling Properties

Human interleukin-10 (hIL-10) is a pleiotropic cytokine that is able to suppress or activate cellular immune responses to protect the host from invading pathogens. Epstein-Barr virus (EBV) encodes a viral IL-10 (ebvIL-10) in its genome that has retained the immunosuppressive activities of hIL-10 but lost the ability to induce immunostimulatory activities on some cells. These functional differences are at least partially due to the ～1000-fold difference in hIL-10 and ebvIL-10 binding affinity for the IL-10R1·IL-10R2 cell surface receptors. Despite weaker binding to IL-10R1, ebvIL-10 is more active than hIL-10 in inducing B-cell proliferation. To explore this counterintuitive observation further, a series of monomeric and dimeric ebvIL-10·hIL-10 chimeric proteins were produced and characterized for receptor binding and cellular proliferation on TF-1/hIL-10R1 cells that express high levels of the IL-10R1 chain. On this cell line, monomeric chimeras elicited cell proliferation in accordance with how tightly they bound to the IL-10R1 chain. In contrast, dimeric chimeras exhibiting the highest affinity for IL-10R1 exhibited reduced proliferative activity. These distinct activity profiles are correlated with kinetic analyses that reveal that the ebvIL-10 dimer is impaired in its ability to form a 1:2 ebvIL-10·IL-10R1 complex. As a result, the ebvIL-10 dimer functions like a monomer at low IL-10R1 levels, which prevents efficient signaling. At high IL-10R1 levels, the ebvIL-10 dimer is able to induce signaling responses greater than hIL-10. Thus, the ebvIL-10 dimer scaffold is essential to prevent activation of cells with low IL-10R1 levels but to maintain or enhance activity on cells with high IL-10R1 levels.     

Noncompetitive antibody neutralization of IL-10 revealed by protein engineering and x-ray crystallography

IL-10 is a dimeric cytokine that must engage its high-affinity cell surface receptor, IL-10R1, to induce multiple cellular activities. Here we report the 1.9 A crystal structure of an engineered IL-10 monomer (IL-10M1) in complex with a neutralizing Fab fragment (9D7Fab). 9D7Fab and IL-10R1 bind distinct nonoverlapping surfaces on IL-10M1. Antagonism of the IL-10M1/IL-10R1 interaction is the result of 9D7Fab-induced conformational changes in the CD loop of IL-10M1 that indirectly alter the structure of the IL-10R1 binding site. A single mutation (Ile87Ala) in the same CD loop region of the Epstein-Barr virus IL-10 (ebvIL-10) also reduces IL-10R1 binding affinity, suggesting that ebvIL-10 and 9D7Fab use similar allosteric mechanisms to modulate IL-10R1 affinity and biological activity.     

Linkage mapping of human chromosome 10 microsatellite polymorphisms.

Ten microsatellite DNA polymorphisms located on human chromosome 10 were regionally mapped using subchromosomal somatic cell hybrids and linkage analysis. The resulting order of the markers from pter-qter was [D10S89, D10S111], D10S107, D10S109, [D10S91, D10S110, D10S108, D10S88, D10S168], and D10S169. Order of the markers within brackets was uncertain, although the order given was most likely. The microsatellites were distributed along the chromosome from the proximal p arm to near qter, with an unlinked gap between D10S168 and D10S169.     

A model of the ternary complex of interleukin-10 with its soluble receptors

Background  

Interleukin-10 (IL-10) is a cytokine whose main biological function is to suppress the immune response by induction of a signal(s) leading to inhibition of synthesis of a number of cytokines and their cellular receptors. Signal transduction is initiated upon formation of a ternary complex of IL-10 with two of its receptor chains, IL-10R1 and IL-10R2, expressed on the cell membrane. The affinity of IL-10R1 toward IL-10 is very high, which allowed determination of the crystal structure of IL-10 complexed with the extracellular/soluble domain of IL-10R1, while the affinity of IL-10R2 toward either IL-10 or IL-10/sIL-10R1 complex is quite low. This so far has prevented any attempts to obtain structural information about the ternary complex of IL-10 with its receptor chains.     

Physical Analysis of Tn10- and Is10-Promoted Transpositions and Rearrangements

We have investigated by Southern blot hybridization the rate of IS10 transposition and other Tn10/IS10-promoted rearrangements in Escherichia coli and Salmonella strains bearing single chromosomal insertions of Tn10 or a related Tn10 derivative. We present evidence for three primary conclusions. First, the rate of IS10 transposition is approximately 10(-4) per cell per bacterial generation when overnight cultures are grown and plated on minimal media and is at least ten times more frequent than any other Tn10/IS10-promoted DNA alteration. Second, all of the chromosomal rearrangements observed can be accounted for by two previously characterized Tn10-promoted rearrangements: deletion/inversions and deletions. Together these rearrangements occur at about 10% the rate of IS10 transposition. Third, the data suggest that intramolecular Tn10-promoted rearrangements preferentially use nearby target sites, while the target sites for IS10 transposition events are scattered randomly around the chromosome.     

Association of endosperm reduction with parental imprinting in maize

Among 38 reciprocal translocations between the maize B chromosome and the proximal region of the long arm of chromosome 10 were six interchanges associated with reduced endosperm development. These six have breakpoints that are the most proximal of the set and constitute a graded series with those broken nearer the centromere which have the most abnormal phenotypes. The group of six defines three major regions that produce the endosperm effects. The remaining 32 translocations reduce kernel size very slightly, suggesting the presence of a fourth region distal to all break-points.-The affected class of kernels lacks a paternally derived representative of that segment of 10L translocated to the B centromeric element (B(10) chromosome; 10 10 B(10)). An accompanying class of kernel in which the paternal B(10) chromosome is duplicated in the endosperm (10 10 10(B) B(10) B(10)) is normal. Kernels of the same endosperm constitution synthesized by introducing both 10 and B(10) maternally, however, are defective, resembling 10 10 10(B). Maternal B(10)'s are therefore unable to compensate for the absence of a paternal B(10). Clearly expression of the 10L genes involved supports normal endosperm growth only following pollen transmission.     

The Arabidopsis IRX10 and IRX10-LIKE glycosyltransferases are critical for glucuronoxylan biosynthesis during secondary cell wall formation

Arabidopsis IRX10 and IRX10-LIKE (IRX10-L) proteins are closely related members of the GT47 glycosyltransferase family. Single gene knock-outs of IRX10 or IRX10-L result in plants with either a weak or no mutant phenotype. However irx10 irx10-L double mutants are severely affected in their development, with a reduced rosette size and infrequent formation of a small infertile inflorescence. Plants homozygous for irx10 and heterozygous for irx10-L have an intermediate phenotype exhibiting a short inflorescence compared with the wild type, and an almost complete loss of fertility. Stem sections of the irx10 homozygous irx10-L heterozygous or irx10 irx10-L double mutants show decreased secondary cell-wall formation. NMR analysis shows that signals derived from the reducing end structure of glucuronoxylan were detected in the irx10 single mutant, and in the irx10 homozygous irx10-L heterozygous combination, but that the degree of polymerization of the xylan backbone was reduced compared with the wild type. Additionally, xylans from irx10 stem tissues have an almost complete loss of the GlcUA side chain, whereas the level of 4- O -Me-GlcUA was similar to that in wild type. Deletion of the predicted signal peptide from the N terminus of IRX10 or IRX10-L results in an inability to rescue the irx10 irx10-L double mutant phenotype. These findings demonstrate that IRX10 and IRX10-L perform a critical function in the synthesis of glucuronoxylan during secondary cell-wall formation, and that this activity is associated with the formation of the xylan backbone structure. This contrasts with the proposed function of the tobacco NpGUT1, which is closely related to the Arabidopsis IRX10 and IRX10-L proteins, in rhamnogalacturonan II biosynthesis.     

Plasma coenzyme Q10 response to oral ingestion of coenzyme Q10 formulations

Plasma coenzyme Q10 (CoQ10) response to oral ingestion of various CoQ10 formulations was examined. Both total plasma CoQ10 and net increase over baseline CoQ10 concentrations show a gradual increase with increasing doses of CoQ10. Plasma CoQ10 concentrations plateau at a dose of 2400 mg using one specific chewable tablet formulation. The efficiency of absorption decreases as the dose increases. About 95% of circulating CoQ10 occurs as ubiquinol, with no appreciable change in the ratio following CoQ10 ingestion. Higher plasma CoQ10 concentrations are necessary to facilitate uptake by peripheral tissues and also the brain. Solubilized formulations of CoQ10 (both ubiquinone and ubiquinol) have superior bioavailability as evidenced by their enhanced plasma CoQ10 responses.     

Epstein-Barr virus RNA. VI. Viral RNA in restringently and abortively infected Raji cells.

Nuclear and polyadenylated RNA fractions of Raji cells are encoded by larger fractions of Epstein-Barr virus DNA (35 and 18%, respectively) than encode polyribosomal RNA (10%). Polyribosomal RNA is encoded by DNA mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons. An abundant, small (160-base), non-polyadenylated RNA encoded by EcoRI fragment J (0.05 X 10(8) to 0.07 X 10(8) daltons) is also present in the cytoplasm of Raji cells. After induction of early antigen in Raji cells, there was a substantial increase in the complexity of viral polyadenylated and polyribosomal RNAs. Thus, nuclear RNA was encoded by 40% of Epstein-Barr virus DNA, and polyadenylated and polyribosomal RNAs were encoded by at least 30% of Epstein-Barr virus DNA. Polyribosomal RNA from induced Raji cells was encoded by Epstein-Barr virus DNAs mapping at 0.05 X 10(8) to 0.29 X 10(8), 0.63 X 10(8) to 0.66 X 10(8), and 1.10 X 10(8) to 0.03 X 10(8) daltons and also by DNAs mapping within the long unique regions of Epstein-Barr virus DNA at 0.39 X 10(8) to 0.49 X 10(8), 0.51 X 10(8) to 0.59 X 10(8), 0.66 X 10(8) to 0.77 X 10(8), and 1.02 X 10(8) to 1.05 X 10(8) daltons.     

IL-15-induced IL-10 increases the cytolytic activity of human natural killer cells

Interleukin 10 (IL-10) is a multifunctional cytokine that regulates diverse functions of immune cells. Natural killer (NK) cells express the IL-10 and IL-10 receptor, but little is known about the function of IL-10 on NK cell activation. In this study, we show the expression and role of IL-10 in human NK cells. Among the cytokines tested, IL-15 was the most potent inducer of IL-10, with a maximal peak expression at 5 h after treatment. Furthermore, IL-10 receptor was shown to be expressed in NK cells. IL-10 alone had a significant effect on NK cytotoxicity which additively increased NK cell cytotoxicity in the presence of IL-15. Neutralizing IL-10 with anti-IL-10 antibody suppressed the inductive effect of IL-10 on NK cell cytotoxicity; however, IL-10 had no effect on IFN-γ or TNF-α production or NK cell activatory receptor expression. STAT signals are implicated as a key mediator of IL-10/IL-15 cytotoxicity response. Thus, the effect of IL-10 on NK cells is particularly interesting with regard to the STAT3 signal that was enhanced by IL-10 or IL-15.