Uromodulin. An immunosuppressive 85-kilodalton glycoprotein isolated from human pregnancy urine is a high affinity ligand for recombinant interleukin 1 alpha

Uromodulin is an 85-kDa immunosuppressive glycoprotein originally isolated from human pregnancy urine. It exhibits immunosuppressive activity in vitro at concentrations between 10(-9) and 10(-11) M. Recent data demonstrate that uromodulin is able to specifically inhibit in vitro assays dependent upon interleukin 1 (IL-1). We now present evidence that uromodulin is a high affinity ligand for recombinant murine IL-1 alpha. Since uromodulin has been purified to homogeneity, this should allow extensive further characterization of the mechanism of action of both uromodulin and IL-1.     

The lectin-like interaction between recombinant tumor necrosis factor and uromodulin

The polypeptide of uromodulin, an immunosuppressive glycoprotein isolated from human urine, has been shown to be identical to that of Tamm-Horsfall glycoprotein and is synthesized exclusively in the kidney (Hession, C., Decker, J. M., Sherblom, A. P., Kumar, S. (1987) Science 237, 1479-1484). Uromodulin binds recombinant murine interleukin 1 alpha with high affinity, and this binding can be inhibited by addition of specific saccharides (Muchmore, A. V., and Decker, J. M. (1987) J. Immunol. 138, 2541-2546). We now report that uromodulin binds recombinant human tumor necrosis factor (rTNF) with high affinity. Both diacetylchitobiose and Man(alpha 1-6)(Man(alpha 1-3]-Man-O-ethyl are effective inhibitors of the binding, whereas a wide variety of other saccharides are not inhibitory. Although Tamm-Horsfall glycoprotein contains predominantly tetraantennary N-linked chains, the binding to rTNF is unaffected by removal of terminal sialic acid, galactose, and N-acetylhexosamine residues. Fractionation of a Pronase digest of uromodulin by gel filtration yields material that inhibits the binding of uromodulin to rTNF but is of lower molecular weight than the major oligosaccharide. Uromodulin does not inhibit the cytotoxic activity of rTNF as monitored by lysis of tumor cell targets but effectively protects mice from lethal challenge with lipopolysaccharide, an event that may involve lymphokine toxicity. We have previously shown that rTNF binds to sections of human kidney and is localized in the same region as uromodulin. Thus, rTNF interacts with uromodulin via carbohydrate chains that are less processed than the major tetraantennary chain, and this interaction may be critical in promoting clearance and/or reducing toxicity of TNF and other lymphokines.     

In vitro evidence that carbohydrate moieties derived from uromodulin, an 85,000 dalton immunosuppressive glycoprotein isolated from human pregnancy urine, are immunosuppressive in the absence of intact protein

Our laboratory recently reported the purification of a unique immunosuppressive glycoprotein isolated from human pregnancy urine (7). This glycoprotein, which we term uromodulin, has a m.w. of 85,000 as assessed on SDS-PAGE and is 30% carbohydrate. Uromodulin blocks in vitro antigen-specific T cell proliferation to recall antigens such as tetanus toxoid at concentrations as low as 100 pM. This glycoprotein also blocks the in vitro generation of spontaneous monocyte-mediated cytotoxicity (7, 36). Recent evidence strongly suggests that the primary action of uromodulin is to act as a specific ligand and modulator of IL 1 (10, 33). We now report additional biochemical characterization of uromodulin, and based on three independent lines of evidence, find that its immunologic activity appears to result from its glycosylation. First, measures to alter the tertiary folding of the protein backbone of uromodulin, including succinylation or reduction and carboxymethylation, fail to significantly affect its in vitro bioactivity. Second, after extensive digestion of intact uromodulin with pronase, the majority of the in vitro bioactivity can be recovered in a single carbohydrate-rich fraction. Finally, digestion with N-glycanase (N-glycosidase F-, an enzyme specific for N-asparagine-linked oligosaccharides) and subsequent purification on thin layer chromatography yields a single complex oligosaccharide that appears to be responsible for the majority of the in vitro immunosuppression mediated by uromodulin. These data suggest that uromodulin displays N-linked carbohydrate sequences capable of down-regulating antigen-specific T cell responses in vitro. It has been suggested that endogenous lectins may play an important role as recognition molecules in mammalian, as well as more primitive immune systems (23, 24). Our in vitro biologic data strongly suggest that the carbohydrate portion of uromodulin is an excellent candidate to function as a potential lectin receptor.     

Surfactant protein D is a divalent cation-dependent carbohydrate-binding protein

Surfactant protein D (SP-D, CP4) is a collagenous surfactant-associated glycoprotein synthesized by lung type II epithelial cells. SP-D can be selectively and efficiently eluted from isolated rat surfactant with glucose, maltose, and certain other saccharides. We therefore examined the ability of the purified protein to interact with carbohydrates in vitro. Saccharide-substituted bovine serum albumins (BSA neoglycoproteins) were adsorbed to plastic wells, and binding of purified SP-D was quantified with monospecific antibodies to SP-D using an indirect immunoassay. SP-D showed specific calcium-dependent binding to alpha-D-glucosidophenyl isothiocyanate-BSA and maltosyl-BSA, but negligible binding to beta-D-glucosidophenyl isothiocyanate-BSA or unconjugated BSA. The most efficient inhibitors of SP-D binding were alpha-glucosyl-containing saccharides (e.g. isomaltose, maltose, malotriose). SP-D showed quantitative binding to maltosyl-agarose and was specifically eluted with maltose or EDTA. High affinity binding to maltosyl-BSA was also demonstrated using a solution-phase polyethylene glycol precipitation assay. These studies demonstrate that SP-D is a calcium dependent lectin-like protein and that the association of SP-D with surfactant is mediated by carbohydrate-dependent interactions with specificity for alpha-glucosyl residues.     

IL-2, a lectin with specificity for high mannose glycopeptides

Utilizing a solid phase binding assay, we have demonstrated that rIL-2 binds with high affinity to the human urinary glycoprotein uromodulin. This binding is specifically inhibited by the saccharides diacetylchitobiose and Man(alpha 1-3)(Man(alpha 1-6]Man-O-methyl and by the high mannose glycopeptides Man5GlcNAc2-R and Man6GlcNAc2-R, but not by Man9GlcNAc2-R. rIL-2 also binds OVA, a glycoprotein which contains approximately 50% high mannose chains at a single glycosylation site, and to yeast mannan. This binding is inhibited by the same battery of saccharides which inhibit the binding to uromodulin. The conclusion that rIL-2 is a lectin is further supported by the observation that the sequence of IL-2 shares 27% homology with a 33-residue sequence of the carbohydrate-binding domain of human mannose-binding protein. The potential physiologic relevance of the carbohydrate binding activity is further elucidated by studies which show that 1) binding of soluble rIL-2 to immobilized uromodulin is enhanced at a pH of 4 to5 in the presence of divalent cations, and 2) neither uromodulin nor the high mannose glycopeptide Man5GlcNAc2Asn blocks the binding of rIL-2 to the IL-2R. Thus the carbohydrate-binding site of rIL-2 is distinct from the cell surface receptor-binding site, and might function preferentially in acidic microenvironments.     

Crystal structure of the Nogo-receptor-2

The inhibition of axon regeneration upon mechanical injury is dependent on interactions between Nogo receptors (NgRs) and their myelin-derived ligands. NgRs are composed of a leucine-rich repeat (LRR) region, thought to be structurally similar among the different isoforms of the receptor, and a divergent "stalk" region. It has been shown by others that the LRR and stalk regions of NgR1 and NgR2 have distinct roles in conferring binding affinity to the myelin associated glycoprotein (MAG) in vivo. Here, we show that purified recombinant full length NgR1 and NgR2 maintain significantly higher binding affinity for purified MAG as compared to the isolated LRR region of either NgR1 or NgR2. We also present the crystal structure of the LRR and part of the stalk regions of NgR2 and compare it to the previously reported NgR1 structure with respect to the distinct signaling properties of the two receptor isoforms.     

Interleukin 2 mediates an alteration in the T200 antigen expressed on activated B lymphocytes

We have examined the effect of exogenous IL 2 on cell surface antigen expression in LPS/dextran sulfate-activated murine B cells with the use of a panel of fluorescein-conjugated lectins. Elevated binding of the lectins PNA and SBA to activated B cells was found to be mediated by IL 2-containing supernatants from stimulated EL4 cells as well as by recombinant IL 2. These lectins have specificity for terminal beta-(1-3)-N-acetylgalactosaminyl residues; thus, the quantity or accessibility of these moieties is mediated by IL 2 in activated B lymphocytes. PNA binding in all strains tested, regardless of MHC or background genes, was found to be elevated fivefold to 15-fold by exogenous IL 2. To observe this effect, IL 2 must be added during the first 24 hr of culture. Based on anti-Thy-1 + complement depletion studies, T cells were not required, suggesting a direct effect of IL 2 on B cells. The glycoprotein responsible for this elevated binding of PNA has an Mr of approximately 220K and by immunodepletion was shown to belong to the T200 (Ly-5) family of cell surface antigens. These data demonstrate that exogenous IL 2 can mediate alterations in T200 expression on activated B cells that may be related to IL 2-driven modulation of B cell proliferation and/or differentiation.     

Two distinct monokines, interleukin 1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells

A murine monoclonal antibody (H4/18) raised against cultured human endothelial cells (HEC) prestimulated by the monokine interleukin 1 (IL 1) recognizes a cell surface molecule inducible by IL 1 or by the distinct monokine tumor necrosis factor (TNF) in primary or serially passaged HEC. H4/18 binding is not basally expressed or inducible by IL 1 in an SV-40 transformed HEC line, in human dermal fibroblasts, or in blood leukocytes. Expression of this molecule by HEC in response to IL 1 can be blocked by protein and RNA synthesis inhibitors but not by cyclooxygenase inhibitors. In addition, H4/18 can immunoprecipitate two biosynthetically labeled polypeptides (Mr 100,000 and 120,000) from HEC stimulated with IL 1 but not from control HEC. Thus, the H4/18 binding site appears to be an inducible surface protein specific for HEC. The majority of HEC in a culture can be induced to express the H4/18 binding protein, but expression is transient (peak 4 to 6 hr) and over the next 24 hr declines to near basal levels either in the continued presence of or upon removal of IL 1. The magnitude of the peak response depends upon IL 1 concentration (peak 5 to 10 U/ml), and the response is optimized by the continued presence of IL 1 during the initial 4- to 6-hr induction period. The time of peak H4/18 binding does not appear to be a function of IL 1 concentration. The decline of H4/18 binding from peak levels is prevented by cycloheximide, a protein synthesis inhibitor. HEC maintained in the presence of IL 1 for 24 hr become refractory to restimulation by IL 1; however, IL 1-stimulated cells rested in the absence of IL 1 for 20 hr can be stimulated by fresh IL 1. HEC expression of the H4/18 binding protein is not induced by interleukin 2 or by interferon-alpha, -beta, or -gamma. Induction of H4/18 binding by TNF is also concentration dependent, transient, and dependent upon protein and RNA synthesis. Several observations suggest that IL1 and TNF act independently on HEC. Our TNF is a recombinant protein, expressed from a cloned cDNA and thus free of IL 1 contamination; it also has no activity in a highly sensitive IL 1 assay. Our standard IL 1 preparation is affinity purified and lacks TNF activity on L929 cells. Thus, our monokine preparations are not cross-contaminated. Most interestingly, HEC incubated with IL 1 and refractory to IL1 restimulation can be restimulated by TNF to express H4/18 binding and vice versa.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interleukin 1 alpha and interleukin 1 beta bind to the same receptor on T cells

Pure, E. coli-derived recombinant murine interleukin 1 alpha (IL 1 alpha) was labeled with 125I and used for receptor binding studies. The 125I-IL 1 binds to murine EL-4 thymoma cells in a specific and saturable manner. Scatchard plot analysis for binding studies carried out at 4 degrees C reveals a single type of high affinity binding site with an apparent dissociation constant of approximately 2.6 X 10(-10) M and the presence of approximately 1200 binding sites per cell. The rate of association of the 125I-IL 1 with EL-4 cells is slow, requiring more than 3 h to reach apparent steady state at 4 degrees C. Cell-bound 125I-IL 1 cannot be dissociated from EL-4 cells upon removal of unbound 125I-IL 1 and incubation of the cells at 4 degrees C in the presence or absence of unlabeled IL 1. Unlabeled recombinant murine IL 1 competes for 125I-IL 1 binding in a dose-dependent manner, whereas interferon-alpha A, interleukin 2 (IL 2), epidermal growth factor, and nerve growth factor have no effect. The 125I-IL 1 binding site is sensitive to trypsin, suggesting that it is localized on the cell surface. We have also examined the ability of purified recombinant human IL 1 alpha and IL 1 beta to compete for binding of the radiolabeled murine IL 1 to its receptor and to stimulate IL 2 production by EL-4 cells. Previous reports have shown that human IL 1 alpha is approximately 60% homologous in amino acid sequence with murine IL 1, but that human IL 1 beta is only about 25% homologous with either murine IL 1 or human IL 1 alpha. Despite these marked differences, however, we report here that both human IL 1 proteins are able to recognize the same binding site as mouse IL 1. In addition, murine as well as both human IL 1 proteins stimulate IL 2 production by EL-4 cells.     

Pregnancy-associated changes in the glycosylation of tamm-horsfall glycoprotein. Expression of sialyl Lewis(x) sequences on core 2 type O-glycans derived from uromodulin

Tamm-Horsfall glycoprotein (THP) is a major glycoprotein associated with human urine that binds pro-inflammatory cytokines and also inhibits in vitro T cell proliferation induced by specific antigens. THP derived from human pregnancy urine (designated uromodulin) has previously been shown to be 13-fold more effective as an inhibitor of antigen-induced T cell proliferation than THP obtained from other sources. Structural analysis of human THP and uromodulin has for the first time revealed that these glycoproteins are O-glycosylated. THP from nonpregnant females and males expresses primarily core 1 type O-glycans terminated with either sialic acid or fucose but not the sialyl Lewis(x) epitope. By contrast, the O-glycans linked to uromodulin include unusual core 2 type glycans terminated with one, two, or three sialyl Lewis(x) sequences. The specific association of these unusual carbohydrate sequences with uromodulin could explain its enhanced immunomodulatory effects compared with THP obtained from males and nonpregnant females. Analysis of THP from one of the pregnant females 2 months postpartum showed a reversion of the O-glycan profile to that found for a non-pregnant female. These data suggest that the glycosylation state of uromodulin could be under the regulation of steroidal hormones produced during pregnancy. The significant physiological implications of these observations are discussed.     

The hyalinosis-associated 90 kD glycoprotein of human skin has lectin-like characteristics

90 kD glycoprotein is a sialylated, mannose-rich protein which accumulates predominantly in the skin in massive hyalinosis. This study demonstrates that purified 90 kD glycoprotein induces agglutination of A, B and O red cells which is inhibitable by alpha-D-glucose and alpha-D-fucose. The binding of various 125I-labelled proteins to 90 kD glycoprotein is both carbohydrate and calcium dependent. The results show that 90 kD glycoprotein is a lectin-like carbohydrate-binding haemagglutinin.     

Identification of a carbohydrate-based endothelial ligand for a lymphocyte homing receptor

Lymphocyte attachment to high endothelial venules within lymph nodes is mediated by the peripheral lymph node homing receptor (pnHR), originally defined on mouse lymphocytes by the MEL-14 mAb. The pnHR is a calcium-dependent lectin-like receptor, a member of the LEC-CAM family of adhesion proteins. Here, using a soluble recombinant form of the homing receptor, we have identified an endothelial ligand for the pnHR as an approximately 50-kD sulfated, fucosylated, and sialylated glycoprotein, which we designate Sgp50 (sulfated glycoprotein of 50 kD). Recombinant receptor binding to this lymph node-specific glycoprotein requires calcium and is inhibitable by specific carbohydrates and by MEL-14 mAb. Sialylation of the component is required for binding. Additionally, the glycoprotein is precipitated by MECA-79, an adhesion-blocking mAb reactive with lymph node HEV. A related glycoprotein of approximately 90 kD (designated as Sgp90) is also identified.     

Characterization of sugar binding by the mannose receptor family member,Endo180

Members of the mannose receptor family, the mannose receptor, the phospholipase A(2) receptor, DEC-205, and Endo180, contain multiple C-type lectin-like domains (CTLDs) within a single polypeptide. In addition, at their N termini, all four family members contain a cysteine-rich domain similar to the R-type carbohydrate recognition domains of ricin. However, despite the common presence of multiple lectin-like domains, these four endocytic receptors have divergent ligand binding activities, and it is clear that the majority of these domains do not bind sugars. Here the functions of the lectin-like domains of the most recently discovered family member, Endo180, have been investigated. Endo180 is shown to bind in a Ca(2+)-dependent manner to mannose, fucose, and N-acetylglucosamine but not to galactose. This activity is mediated by one of the eight CTLDs, CTLD2. Competition assays indicate that the monosaccharide binding specificity of Endo180 CTLD2 is similar to that of mannose receptor CTLD4. However, additional experiments indicate that, unlike the cysteine-rich domain of the mannose receptor, the cysteine-rich domain of Endo180 does not bind sulfated sugars. Thus, although Endo180 and the mannose receptor are now both known to be mannose binding lectins, each receptor is likely to have a distinct set of glycoprotein ligands in vivo.     

Isolation and characterization of human urine extracellular vesicles

Extracellular vesicles (ECV) reflect physiological or pathological conditions, emerging as potential biomarkers for disease. They can be obtained from a variety of body fluids, particularly urine that is an ideal source because it can be obtained in great quantities, recurrently and with minimal intervention. However, the characterization of urine ECV is challenging because the preparation is usually contaminated with soluble proteins, such as uromodulin (UMOD) or Tamm-Horsfall glycoprotein that forms large extracellular filaments co-sedimenting with ECV. We developed a method to obtain human urine ECV free of UMOD by the addition of ZnSO₄ prior to vesicle isolation by differential centrifugation. Treatment with ZnSO₄ did not affect the size and concentration of the vesicle preparation and preserved the storage of the samples at low temperatures. We did not observe a variation in the number of vesicles isolated during different times of the day or different days between different donors. The glycoprotein pattern of urine ECV was characterized by binding to concanavalin A (Con A) and mass spectroscopy. Several markers were found, including dipeptidyl peptidase IV (CD26), vacuolar protein sorting factor 4A (VPS4A) and dipeptidase 1 (DPEP1), and galectin 3 binding protein (G3-BP). The levels of VPS4A and DPEP1 were similar in ECV preparations obtained from several donors of both sexes. Con A binding pattern and monosaccharide composition were also comparable between subjects. In summary, our method for the isolation of highly pure ECV derived from human urine is likely to help in the use of these vesicles as potential biomarkers.     

Change in lactate production in Myc-transformed cells precedes apoptosis and can be inhibited by Bcl-2 overexpression

Interleukin-18 binding protein is a novel glycoprotein that we successfully cloned and expressed. First, murine interleukin-18 binding protein was purified from the sera of mice with endotoxin shock using ligand affinity chromatography. The murine interleukin-18 binding protein cDNA was cloned after RT-PCR using mixed primer pair sequences based on partial murine interleukin-18 binding protein amino acid sequence analysis. Subsequently, human interleukin-18 binding protein cDNA was cloned from cDNA libraries of normal human liver using murine interleukin-18 binding protein cDNA as a probe. Next, we transiently expressed recombinant human and murine interleukin-18 binding proteins in COS-1 cells and purified them from culture supernatants. Both recombinant interleukin-18 binding proteins did not exhibit species specificity and prevented interleukin-18 binding to its receptor. In addition, they inhibited interleukine-18 dependent IFN-gamma production from KG-1 cells effectively. These results suggest that the interleukin-18 binding protein may possess interleukine-18 antagonist activity.     

Interaction of uromodulin and complement factor H enhances C3b inactivation

Recent studies suggest that uromodulin plays an important role in chronic kidney diseases. It can interact with several complement components, various cytokines and immune system cells. Complement factor H (CFH), as a regulator of the complement alternative pathway, is also associated with various renal diseases. Thus, we have been suggested that uromodulin regulates complement activation by interacting with CFH during tubulointerstitial injury. We detected co‐localization of uromodulin and CFH in the renal tubules by using immunofluorescence. Next, we confirmed the binding of uromodulin with CFH in vitro and found that the affinity constant (K_D) of uromodulin binding to CFH was 4.07 × 10^?6M based on surface plasmon resonance results. The binding sites on CFH were defined as the short consensus repeat (SCR) units SCR1–4, SCR7 and SCR19–20. The uromodulin‐CFH interaction enhanced the cofactor activity of CFH for factor I‐mediated cleavage of C3b to iC3b. These results indicate that uromodulin plays a role via binding and enhancing the function of CFH.     

GMP-140 binds to a glycoprotein receptor on human neutrophils: evidence for a lectin-like interaction

GMP-140 is a rapidly inducible receptor for neutrophils and monocytes expressed on activated platelets and endothelial cells. It is a member of the selectin family of lectin-like cell surface molecules that mediate leukocyte adhesion. We used a radioligand binding assay to characterize the interaction of purified GMP-140 with human neutrophils. Unstimulated neutrophils rapidly bound [125I]GMP-140 at 4 degrees C, reaching equilibrium in 10-15 min. Binding was Ca2+ dependent, reversible, and saturable at 3-6 nM free GMP-140 with half-maximal binding at approximately 1.5 nM. Receptor density and apparent affinity were not altered when neutrophils were stimulated with 4 beta-phorbol 12-myristate 13-acetate. Treatment of neutrophils with proteases abolished specific binding of [125I]GMP-140. Binding was also diminished when neutrophils were treated with neuraminidase from Vibrio cholerae, which cleaves alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids, or from Newcastle disease virus, which cleaves only alpha 2-3- and alpha 2-8-linked sialic acids. Binding was not inhibited by an mAb to the abundant myeloid oligosaccharide, Lex (CD15), or by the neoglycoproteins Lex-BSA and sialyl-Lex-BSA. We conclude that neutrophils constitutively express a glycoprotein receptor for GMP-140, which contains sialic acid residues that are essential for function. These findings support the concept that GMP-140 interacts with leukocytes by a lectin-like mechanism.     

Shear-dependent capping of L-selectin and P-selectin glycoprotein ligand 1 by E-selectin signals activation of high-avidity beta2-integrin on neutrophils

Two adhesive events critical to efficient recruitment of neutrophils at vascular sites of inflammation are up-regulation of endothelial selectins that bind sialyl Lewis(x) ligands and activation of beta(2)-integrins that support neutrophil arrest by binding ICAM-1. We have previously reported that neutrophils rolling on E-selectin are sufficient for signaling cell arrest through beta(2)-integrin binding of ICAM-1 in a process dependent upon ligation of L-selectin and P-selectin glycoprotein ligand 1 (PSGL-1). Unresolved are the spatial and temporal events that occur as E-selectin binds to human neutrophils and dynamically signals the transition from neutrophil rolling to arrest. Here we show that binding of E-selectin to sialyl Lewis(x) on L-selectin and PSGL-1 drives their colocalization into membrane caps at the trailing edge of neutrophils rolling on HUVECs and on an L-cell monolayer coexpressing E-selectin and ICAM-1. Likewise, binding of recombinant E-selectin to PMNs in suspension also elicited coclustering of L-selectin and PSGL-1 that was signaled via mitogen-activated protein kinase. Binding of recombinant E-selectin signaled activation of beta(2)-integrin to high-avidity clusters and elicited efficient neutrophil capture of beta(2)-integrin ligands in shear flow. Inhibition of p38 and p42/44 mitogen-activated protein kinase blocked the cocapping of L-selectin and PSGL-1 and the subsequent clustering of high-affinity beta(2)-integrin. Taken together, the data suggest that E-selectin is unique among selectins in its capacity for clustering sialylated ligands and transducing signals leading to neutrophil arrest in shear flow.     

Human interleukin‐23 receptor antagonists derived from an albumin‐binding domain scaffold inhibit IL‐23‐dependent ex vivo expansion of IL‐17‐producing T‐cells

Engineered combinatorial libraries derived from small protein scaffolds represent a powerful tool for generating novel binders with high affinity, required specificity and designed inhibitory function. This work was aimed to generate a collection of recombinant binders of human interleukin‐23 receptor (IL‐23R), which is a key element of proinflammatory IL‐23‐mediated signaling. A library of variants derived from the three‐helix bundle scaffold of the albumin‐binding domain (ABD) of streptococcal protein G and ribosome display were used to select for high‐affinity binders of recombinant extracellular IL‐23R. A collection of 34 IL‐23R‐binding proteins (called REX binders), corresponding to 18 different sequence variants, was used to identify a group of ligands that inhibited binding of the recombinant p19 subunit of IL‐23, or the biologically active human IL‐23 cytokine, to the recombinant IL‐23R or soluble IL‐23R‐IgG chimera. The strongest competitors for IL‐23R binding in ELISA were confirmed to recognize human IL‐23R‐IgG in surface plasmon resonance experiments, estimating the binding affinity in the sub‐ to nanomolar range. We further demonstrated that several REX variants bind to human leukemic cell lines K‐562, THP‐1 and Jurkat, and this binding correlated with IL‐23R cell‐surface expression. The REX125, REX009 and REX128 variants competed with the p19 protein for binding to THP‐1 cells. Moreover, the presence of REX125, REX009 and REX115 variants significantly inhibited the IL‐23‐driven expansion of IL‐17‐producing primary human CD4⁺ T‐cells. Thus, we conclude that unique IL‐23R antagonists derived from the ABD scaffold were generated that might be useful in designing novel anti‐inflammatory biologicals. Proteins 2014; 82:975–989. © 2013 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Characterization of a recombinant herpes simplex virus 1 designed to enter cells via the IL13Ralpha2 receptor of malignant glioma cells

Malignant glioma tumor cells in situ exhibit on their surfaces the interleukin 13 (IL-13) receptor designated IL13Ralpha2. To target herpes simplex virus 1 to this receptor, we constructed a recombinant virus (R5111) in which the known heparan sulfate binding sites in glycoproteins B and C were deleted and IL-13 was inserted into both glycoproteins C and D. We also transduced a baby hamster kidney cell line lacking the known viral receptors (J1-1) and Vero cells with a plasmid encoding IL13Ralpha2. The J1-1 derivative (J-13R) cell line is susceptible to and replicates the R5111 recombinant virus but not the wild-type parent virus. We report the following. (i) Expression of IL13Ralpha2 was rapidly lost from the surface of transduced cells grown in culture. The loss appeared to be related to ligands present in fetal bovine serum in the medium. None of the malignant glioma cell lines cultivated in vitro and tested to date exhibited the IL13Ralpha2 receptor. (ii) Soluble IL-13 but not IL-4 or IL-2 blocked the replication of R5111 recombinant virus in J-13R cells. (iii) The endocytosis inhibitor PD98059 blocked the replication in J1-1 cells of a mutant lacking glycoprotein D (gD-/-) but not the replication of R5111 in the J-13R cells. We conclude that R5111 enters cells via its interaction with the IL13Ralpha2 receptor in a manner that cannot be differentiated from the interaction of wild-type virus with its receptors.