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A conserved sugar bridge connected to the WSXWS motif has an important role for transport of IL-21R to the plasma membrane
Abstract
Interleukin-21 (IL-21) is a class I cytokine that belongs to the γc-subfamily of cytokines and regulates immune responses. It signals through a heterodimeric receptor complex composed of the IL-21R1 and γc-receptor chains. A characteristic feature of class I cytokine receptors is the presence of a consensus motif WSXWS (WS motif) in the membrane proximal fibronectin type III domain (FNIII) of these receptors. We recently described the structure of the IL-21R:IL-21 complex and showed that the first tryptophan of the WS motif of IL-21R is mannosylated and involved in formation of a sugar bridge that connects the two FNIII domains of the receptor. Furthermore, a mutation within the WS motif of IL-21R was recently shown to cause a novel kind of primary immunodeficiency syndrome (PID). Here, we report the structure of IL-21R alone, which shows that the sugar bridge forms independently of whether IL-21R binds IL-21 or not, and we furthermore investigate the role of this bridge in the export of IL-21R and γC to the plasma membrane. Thus, we provide a molecular explanation for how mutations in the WS motif may cause PIDs.Genes and Immunity advance online publication, 4 June 2015; doi:10.1038/gene.2015.22.
... R201 is spatially adjacent to the first tryptophan residue (W214) in the canonical WSXWS motif present in the extracellular domain of all class I cytokine receptors. W214 undergoes post-translational mannosylation, while its side chain is in close proximity to a complex carbohydrate that bridges across from N73 in the N-terminal fibronectin domain [37,38]. This carbohydrate bridge may stabilize the IL-21R fold. ...
... The R201L and R201Q variants will result in a loss of a positively charged IL-21 (blue cartoon and transparent surface) binds the hinge between the two receptor fibronectin domains. A complex N-linked carbohydrate (gray sticks, extending from N73) bridges the two fibronectin domains and has been postulated to stabilize IL-21R [37,38]. Residues W138, L158, D179, and R201 (orange sticks) are positioned within the C-terminal fibronectin domain proximal to the extracellular membrane. ...
... This variant causes a frameshift at D179, thereby removing the WSXWS motif (aa 214-218) and introducing a premature stop codon 51 amino acids downstream [23]. The carbohydrate bridge formed between N73 and the WSXWS motif is critical for transport of IL-21R from the Golgi to the plasma membrane [38]. Thus, this truncating variant would dramatically reduce, if not abolish, cell surface expression of IL-21R, consistent with a lack of phosphorylation of STAT3 in CD4 + T cells from P1 in response to stimulation with IL-21, but not IL-10 [23]. ...
Biallelic inactivating mutations in IL21R causes a combined immunodeficiency that is often complicated by cryptosporidium infections. While eight IL-21R-deficient patients have been reported previously, the natural course, immune characteristics of disease, and response to hematopoietic stem cell transplantation (HSCT) remain to be comprehensively examined. In our study, we have collected clinical histories of 13 patients with IL-21R deficiency from eight families across seven centers worldwide, including five novel patients identified by exome or NGS panel sequencing. Eight unique mutations in IL21R were identified in these patients, including two novel mutations. Median age at disease onset was 2.5 years (0.5–7 years). The main clinical manifestations were recurrent bacterial (84.6%), fungal (46.2%), and viral (38.5%) infections; cryptosporidiosis-associated cholangitis (46.2%); and asthma (23.1%). Inflammatory skin diseases (15.3%) and recurrent anaphylaxis (7.9%) constitute novel phenotypes of this combined immunodeficiency. Most patients exhibited hypogammaglobulinemia and reduced proportions of memory B cells, circulating T follicular helper cells, MAIT cells and terminally differentiated NK cells. However, IgE levels were elevated in 50% of IL-21R-deficient patients. Overall survival following HSCT (6 patients, mean follow-up 1.8 year) was 33.3%, with pre-existing organ damage constituting a negative prognostic factor. Mortality of non-transplanted patients (n = 7) was 57.1%. Our detailed analysis of the largest cohort of IL-21R-deficient patients to date provides in-depth clinical, immunological and immunophenotypic features of these patients, thereby establishing critical non-redundant functions of IL-21/IL-21R signaling in lymphocyte differentiation, humoral immunity and host defense against infection, and mechanisms of disease pathogenesis due to IL-21R deficiency. Outcome following HSCT depends on prior chronic infections and organ damage, which should thus be considered as early as possible following molecular diagnosis.
... Similarly to EPOR, the W-x-x-W motif of IL-21R was important for intracellular transport [71]. When wild-type IL-21R and mutants (W195A and R182A) were expressed in HEK293 cells having C-mannosyltransferase activity, the wild-type IL-21R was localized in the Golgi apparatus and on the borders of cells. ...
... In addition to UNC5A and ADAMTS16, C-mannosyltransferase-deficient cell lines and experimental substitution of Trp for non-C-mannosylated amino acids caused disturbance in the secretion of proteins, such as mucins (MUC5AC and MUC5B) [78,79], TSP-1 [50], mindin (Spondin2) [52], ADAMTSL1 [53], MIG-21 [54], ADAMTS13 [56], MIC2 [60], R-spondin1 [62], R-spondin3 [63], ADAMTS4 [67], Isthmin-1 [68], lipoprotein lipase [86], and microfibril-associated glycoprotein 4 (MFAP4) [87]. C-Mannosylation also regulated cell surface localization of IL-21R [71] and thrombopoietin receptor (TPOR) [72]. ...
C-Mannosylation is a post-translational modification of proteins in the endoplasmic reticulum. Monomeric α-mannose is attached to specific Trp residues at the first Trp in the Trp-x-x-Trp/Cys (W-x-x-W/C) motif of substrate proteins, by the action of C-mannosyltransferases, DPY19-related gene products. The acceptor substrate proteins are included in the thrombospondin type I repeat (TSR) superfamily, cytokine receptor type I family, and others. Previous studies demonstrated that C-mannosylation plays critical roles in the folding, sorting, and/or secretion of substrate proteins. A C-mannosylation-defective gene mutation was identified in humans as the disease-associated variant affecting a C-mannosylation motif of W-x-x-W of ADAMTSL1, which suggests the involvement of defects in protein C-mannosylation in human diseases such as developmental glaucoma, myopia, and/or retinal defects. On the other hand, monomeric C-mannosyl Trp (C-Man-Trp), a deduced degradation product of C-mannosylated proteins, occurs in cells and extracellular fluids. Several studies showed that the level of C-Man-Trp is upregulated in blood of patients with renal dysfunction, suggesting that the metabolism of C-Man-Trp may be involved in human kidney diseases. Together, protein C-mannosylation is considered to play important roles in the biosynthesis and functions of substrate proteins, and the altered regulation of protein C-manosylation may be involved in the pathophysiology of human diseases. In this review, we consider the biochemical and biomedical knowledge of protein C-mannosylation and C-Man-Trp, and introduce recent studies concerning their significance in biology and medicine.
... The domains of FN3 are important extracellular components of human type I cytokine receptors that play an important role in binding cytokines [70,71]. One of the distinctive features of type I cytokine receptors is the type III fibronectin domain containing a WS-WS consensus motif (WS-motif, cytokine receptor motif, any amino acid in the middle of the motif) [72]. This motif plays a role in receptor folding, cytokine binding and signal transduction [73]. ...
... The results of computer modeling suggest that the region of interaction between interleukins and type I cytokine receptors is adjacent to the WS-WS motif [74]. Mutations in this motif alter the binding affinity between the receptor and the cytokine [72]. ...
Bifidobacteria are some of the major agents that shaped the immune system of many members of the animal kingdom during their evolution. Over recent years, the question of concrete mechanisms underlying the immunomodulatory properties of bifidobacteria has been addressed in both animal and human studies. A possible candidate for this role has been discovered recently. The PFNA cluster, consisting of five core genes, pkb2, fn3, aaa-atp, duf58, tgm, has been found in all gut-dwelling autochthonous bifidobacterial species of humans. The sensory region of the species specific serine-threonine protein kinase (PKB2), the transmembrane region of the microbial transglutaminase (TGM), and the type-III fibronectin domain-containing protein (FN3) encoded by the I gene imply that the PFNA cluster might be implicated in the interaction between bacteria and the host immune system. Moreover, the FN3 protein encoded by one of the genes making up the PFNA cluster, contains domains and motifs of cytokine receptors capable of selectively binding TNF-α. The PFNA cluster could play an important role for sensing signals of the immune system. Among the practical implications of this finding is the creation of anti-inflammatory drugs aimed at alleviating cytokine storms, one of the dire consequences resulting from SARS-CoV-2 infection.
... Given the presumed role of Cmannosylation in protein folding and/or stabilization (44,45,49,50), we examined whether the cellular level of MIC2 was influenced by the loss of C-mannosylation. Lysates of wild type, Δdpy19 and Δdpy19comp tachyzoites were analyzed by Western blot using an anti-MIC2 antibody and an antitubulin antibody to normalize loading in all lanes ( Fig. 2A). ...
... Cmannosylation might be co-translational, since folded proteins have been shown to be poor acceptor substrates in vitro (51). Indeed, expression of TSR-containing proteins in Cmannosylation deficient cells is often associated with poor yield, suggesting that this glycosylation process assists protein folding and/or contributes to protein stability (44,45,49,50,56,57). The involvement of Cmannosylation in TSR folding and stability was lately confirmed for the netrin receptor UNC5 (Shcherbakova et al., unpublished). ...
C-Mannosylation is a common modification of thrombospondin type 1 repeats present in metazoans and recently identified also in apicomplexan parasites. This glycosylation is mediated by enzymes of the DPY19 family that transfer α-mannoses to tryptophan residues in the sequence WX2WX2C, which is part of the structurally essential tryptophan ladder. Here, deletion of the dpy19 gene in the parasite Toxoplasma gondii abolished C-mannosyltransferase activity and reduced levels of the micronemal protein MIC2. The loss of C-mannosyltransferase activity was associated with weakened parasite adhesion to host cells and with reduced parasite motility, host cell invasion, and parasite egress. Interestingly, the C-mannosyltransferase–deficient Δdpy19 parasites were strongly attenuated in virulence and induced protective immunity in mice. This parasite attenuation could not simply be explained by the decreased MIC2 level and strongly suggests that absence of C-mannosyltransferase activity leads to insufficient level of additional proteins. In summary, our results indicate that T. gondii C-mannosyltransferase DPY19 is not essential for parasite survival, but is important for adhesion, motility and virulence.
... Hence, we investigated Thr66Ile effects on sIL-7Rα secretion. Since Nglycosylation of the D 1 domain of type I cytokine receptors has proven important for receptor trafficking [19,20], we included mutants defective for N-glycosylation in the D 1 domain of IL-7Rα (i.e. N49Q and N65Q) as controls. ...
... In addition, we found decreased secretion of the sIL-7Rα and expression of the mIL-7Rα. Because of the well-known importance of glycosylation for intracellular trafficking of the IL-21 receptor and the common-γ chain receptor [19], we assumed that the IL-7Rα Ile66 variant impaired IL-7Rα intracellular trafficking especially under glycosylation-deficient conditions. The Ile66 amino acid exchange is located in an IL-7Rα region distant from the IL-7 binding site, rendering differences in IL-7 binding affinities unlikely. ...
Interleukin-7 receptor α-chain (IL7RA) haplotypes are associated with susceptibility to autoimmune diseases including type 1 diabetes (T1D). Previous studies found lower soluble IL-7Rα (sIL-7Rα) serum levels of the protection-associated IL7RA haplotype assumed to reduce IL-7 availability for self-reactive T cells. Also, a risk-associated IL7RA haplotype is accompanied by lower sIL-7Rα serum concentrations but no underlying mechanisms have been described and the causative polymorphism remains unknown.
Here, we characterized functional implications of the nonsynonymous rs1494558 (Thr66Ile), which tags the protection-associated IL7RA haplotype, in HEK293T cells and serum samples of T1D patients with different haplotype carriers. Influence of risk- and protection-associated haplotypes on IL-7Rα was analyzed.
The risk-associated Ile66 variant affected gel mobility and impaired secretion of the sIL-7Rα as well as expression of the membrane-associated (m)IL-7Rα in HEK293T cells. Serum sIL-7Rα analyses confirmed differential gel mobility of the Ile66 variant and found decreased sIL-7Rα serum levels of T1D patients carrying the Ile66-tagged haplotype. Differences in glycosylation were not causative for differential mobility but enhanced the effects on impaired secretion. Comparison of protection- and risk-associated haplotypes in a cell line-based in vitro model identified dominant effects of the protective haplotype tagged by rs6897932 (Ile244) on mIL-7Rα expression, whereas the risk haplotype mainly affected the sIL-7Rα.
This study identified novel functional effects of the Ile66 IL7RA variant and characterized features of autoimmunity risk- and protection-associated haplotypes. The findings add to our understanding of how these haplotypes regulate sIL-7Rα and mIL-7Rα expression in T cells causing differential susceptibility to autoimmune diseases.
... Absence or decrease of C-mannosylation has been shown to affect protein secretion, which suggests a role for this glycosylation in proper folding, stability and/or export of proteins (Munte et al. 2008;Wang et al. 2009;Buettner et al. 2013;Siupka et al. 2015;Niwa et al. 2016;Shcherbakova et al. 2017). However, the dependence of proteins on C-mannosylation seems variable (Buettner et al. 2013;Shcherbakova et al. 2017). ...
... Olsen and Kragelund proposed that this structural motif represents a binding site for glycans and principally for sulfated glycosaminoglycans of the extracellular matrix, as shown for example in TRAP (Tossavainen et al. 2006;Olsen and Kragelund 2014). Since the C-mannose has been shown to influence the orientation of the tryptophan ring (Munte et al. 2008), C-mannosylation may play an indirect role in ligand binding (Li et al. 2009;Sasazawa et al. 2015;Siupka et al. 2015;Fujiwara et al. 2016;Niwa et al. 2016;Pronker et al. 2016). ...
In many metazoan species, an unusual type of protein glycosylation, called C-mannosylation, occurs on adhesive thrombospondin type 1 repeats (TSRs) and type I cytokine receptors. This modification has been shown to be catalyzed by the Caenorhabditis elegans DPY-19 protein and orthologues of the encoding gene were found in the genome of apicomplexan parasites. Lately, the micronemal adhesin thrombospondin-related anonymous protein (TRAP) was shown to be C-hexosylated in Plasmodium falciparum sporozoites. Here, we demonstrate that also the micronemal protein MIC2 secreted by Toxoplasma gondii tachyzoites is C-hexosylated. When expressed in a mammalian cell line deficient in C-mannosylation, P. falciparum and T. gondii Dpy19 homologues were able to modify TSR domains of the micronemal adhesins TRAP/MIC2 family involved in parasite motility and invasion. In vitro, the apicomplexan enzymes can transfer mannose to a WXXWXXC peptide but, in contrast to C. elegans or mammalian C-mannosyltransferases, are inactive on a short WXXW peptide. Since TSR domains are commonly found in apicomplexan surface proteins, C-mannosylation may be a common modification in this phylum.
... FN3 domains have been found in various eukaryotic extracellular and intracellular protein families, including extracellular matrix molecules, enzymes, muscle tissue proteins, and cell surface receptors [including motifs of cytokine receptors (MCRs)] [5,6] . FN3 domains often contain the consensus motif WSXWS (WS motif; MCR; random amino acid in the middle of the motif) [7,8] . This motif plays a role in receptor folding, ligand (cytokine) binding, and signal transduction [9,10] . ...
Aim: This study is mainly devoted to determining the ability of ∆FN3.1 protein fragments of Bifidobacterium (B.) longum subsp. longum GT15, namely two FN3 domains (2D FN3) and a C-terminal domain (CD FN3), to bind to tumor necrosis factor-alpha (TNF-α).
Methods: Fragments of the fn3 gene encoding the 2D FN3 and CD FN3 were cloned in Escherichia (E.) coli. In order to assess the binding specificity between 2D FN3 and CD FN3 to TNFα, we employed the previously developed sandwich ELISA system to detect any specific interactions between the purified protein and any of the studied cytokines. The trRosetta software was used to build 3D models of the ∆FN3.1, 2D FN3, and CD FN3 proteins. The detection of polymorphism in the amino acid sequences of the studied proteins and the analysis of human gut-derived bacterial proteins carrying FN3 domains were performed in silico.
Results: We experimentally showed that neither 2D FN3 nor CD FN3 alone can bind to TNFα. Prediction of the 3D structures of ΔFN3.1, 2D FN3, and CD FN3 suggested that only ΔFN3.1 can form a pocket allowing binding with TNFα to occur. Polymorphism analysis of amino acid sequences of ΔFN3.1 proteins in B. longum strains uncovered substitutions that can alter the conformation of the spatial structure of the ΔFN3.1 protein. We also analyzed human gut-derived bacterial proteins harboring FN3 domains which allowed us to differentiate between those containing motifs of cytokine receptors (MCRs) in their FN3 domains and those lacking them.
Conclusion: Only the complete ∆FN3.1 protein can selectively bind to TNFα. Analysis of 3D models of the 2D FN3, CD FN3, and ΔFN3.1 proteins showed that only the ΔFN3.1 protein is potentially capable of forming a pocket allowing TNFα binding to occur. Only FN3 domains containing MCRs exhibited sequence homology with FN3 domains of human proteins.
... C-Mannosylation might be co-translational, because folded proteins have been shown to be poor acceptor substrates in vitro (51). Indeed, expression of TSR-containing proteins in C-mannosylation-deficient cells is often associated with poor yield, suggesting that this glycosylation process assists protein folding and/or contributes to protein stability (44, 45,49,50,56,57). The involvement of C-mannosylation in TSR folding and stability was lately confirmed for the netrin receptor UNC5 (64). ...
C-Mannosylation is a common modification of thrombospondin type 1 repeats present in metazoans and recently identified also in apicomplexan parasites. This glycosylation is mediated by enzymes of the DPY19 family that transfer α-mannoses to tryptophan residues in the sequence WX2WX2C, which is part of the structurally essential tryptophan ladder. Here, deletion of the dpy19 gene in the parasite Toxoplasma gondii abolished C-mannosyltransferase activity and reduced levels of the micronemal protein MIC2. The loss of C-mannosyltransferase activity was associated with weakened parasite adhesion to host cells and with reduced parasite motility, host cell invasion, and parasite egress. Interestingly, the C-mannosyltransferase–deficient Δdpy19 parasites were strongly attenuated in virulence and induced protective immunity in mice. This parasite attenuation could not simply be explained by the decreased MIC2 level and strongly suggests that absence of C-mannosyltransferase activity leads to an insufficient level of additional proteins. In summary, our results indicate that T. gondii C-mannosyltransferase DPY19 is not essential for parasite survival, but is important for adhesion, motility, and virulence.
... This stalk region acts as a spacer to position the CHR at a defined distance from the plasma membrane to enable signal transduction [79]. Domains of the CHR are characterized by conserved cysteine residues for interstrand disulfide bonds and the WSXWS motif, which might be involved in receptor complex formation, ligand binding, signaling, receptor folding and export [80]. The cysteine residues and the WSXWS motif are also present in the β subunits of heterodimeric IL-12 type cytokines ( Figure 1). ...
Cytokines of the IL-12 family show structural similarities but have distinct functions in the immune system. Prominent members of this cytokine family are the pro-inflammatory cytokines IL-12 and IL-23. These two cytokines share cytokine subunits and receptor chains but have different functions in autoimmune diseases, cancer and infections. Accordingly, structural knowledge about receptor complex formation is essential for the development of new therapeutic strategies preventing and/or inhibiting cytokine:receptor interaction. In addition, intracellular signaling cascades can be targeted to inhibit cytokine-mediated effects. Single nucleotide polymorphisms can lead to alteration in the amino acid sequence and thereby influencing protein functions or protein–protein interactions. To understand the biology of IL-12 and IL-23 and to establish efficient targeting strategies structural knowledge about cytokines and respective receptors is crucial. A highly efficient therapy might be a combination of different drugs targeting extracellular cytokine:receptor assembly and intracellular signaling pathways.
... The WSxWS motif, also called the WS motif, is well-known and has been actively investigated, but it is still enigmatic. In the case of the IL-21 receptor, it has been linked to sugar binding [55,56]. It is conserved in the family in protodomain 2. The symmetry-related pattern SxWS in protodomain 1 however is not as conserved. ...
We will consider in this chapter supersecondary structures (SSS) as a set of secondary structure elements (SSEs) found in protein domains. Some SSS arrangements/topologies have been consistently observed within known tertiary structural domains. We use them in the context of repeating supersecondary structures that self-assemble in a symmetric arrangement to form a domain. We call them protodomains (or protofolds). Protodomains are some of the most interesting and insightful SSSs. Within a given 3D protein domain/fold, recognizing such sets may give insights into a possible evolutionary process of duplication, fusion, and coevolution of these protodomains, pointing to possible original protogenes. On protein folding itself, pseudosymmetric domains may point to a “directed” assembly of pseudosymmetric protodomains, directed by the only fact that they are tethered together in a protein chain. On function, tertiary functional sites often occur at protodomain interfaces, as they often occur at domain-domain interfaces in quaternary arrangements.
... In mammals, C-mannosylation is suggested to play an important role in stabilization and/or folding of some but not all proteins (Munte et al., 2008;Wang et al., 2009;Buettner et al., 2013;Sasazawa et al., 2015;Siupka et al., 2015;Fujiwara et al., 2016;Niwa et al., 2016;Okamoto et al., 2017;Shcherbakova et al., 2017). Using a CRISPR/Cas9 genetic screen or a piggyBac transposon insertional mutagenesis, DPY19 was predicted to confer fitness to T. gondii tachyzoites or P. falciparum asexual stages, respectively (Sidik et al., 2014;Zhang et al., 2018). ...
Apicomplexan parasites are amongst the most prevalent and morbidity-causing pathogens worldwide. They are responsible for severe diseases in humans and livestock and are thus of great public health and economic importance. Until the sequencing of apicomplexan genomes at the beginning of this century, the occurrence of N - and O -glycoproteins in these parasites was much debated. The synthesis of rudimentary and divergent N -glycans due to lineage-specific gene loss is now well established and has been recently reviewed. Here, we will focus on recent studies that clarified classical O -glycosylation pathways and described new nucleocytosolic glycosylations in Toxoplasma gondii, the causative agents of toxoplasmosis. We will also review the glycosylation of proteins containing thrombospondin type 1 repeats by O -fucosylation and C -mannosylation, newly discovered in Toxoplasma and the malaria parasite Plasmodium falciparum. The functional significance of these post-translational modifications has only started to emerge, but the evidence points towards roles for these protein glycosylation pathways in tissue cyst wall rigidity and persistence in the host, oxygen sensing, and stability of proteins involved in host invasion.
... Mass spectrometry analyses of P. falciparum TRAP and circumsporozoite protein (CSP) indicate that a hexose (Hex) residue elongates the fucose on Apicomplexa TSRs; this sugar may be glucose, but has not yet been unequivocally identified (21,25). TSRs glycosylation is carried out in the endoplasmic reticulum (22,26) and, in studies performed in mammalian cell and C. elegans, defects in both C-mannosylation and Ofucosylation have been shown to affect protein stabilization and secretion, albeit with protein-toprotein variability (19,23,(27)(28)(29)(30)(31). A knockout of pofut2 is embryonically lethal in mice (31). ...
Toxoplasma gondii is an intracellular parasite that causes disseminated infections that can produce neurological damage in fetuses and immunocompromised individuals. Microneme protein 2 (MIC2), a member of the thrombospondin-related anonymous protein (TRAP) family, is a secreted protein important for T. gondii motility, host cell attachment, invasion, and egress. MIC2 contains six thrombospondin type I repeats (TSRs) that are modified by C-mannose and O-fucose in Plasmodium spp. and mammals. Here, using MS analysis, we found that the four TSRs in T. gondii MIC2 with protein O-fucosyltransferase 2 (POFUT2) acceptor sites are modified by a dHexHex disaccharide, while Trp residues within three TSRs are also modified with C-mannose. Disruption of genes encoding either POFUT2 or the putative GDP-fucose transporter (NST2) resulted in loss of MIC2 O-fucosylation, as detected by an antibody against the GlcFuc disaccharide, and in markedly reduced cellular levels of MIC2. Furthermore, in 10%-15% of the Δpofut2 or Δnst2 vacuoles, MIC2 accumulated earlier in the secretory pathway rather than localizing to micronemes. Dissemination of tachyzoites in human foreskin fibroblasts was reduced for these knockouts, which both exhibited defects in attachment to and invasion of host cells comparable to the Δmic2 phenotype. These results, indicating that O-fucosylation of TSRs is required for efficient processing of MIC2 and for normal parasite invasion, are consistent with the recent demonstration that P. falciparum Δpofut2 strain has decreased virulence and also support a conserved role for this glycosylation pathway in quality control of TSR-containing proteins in eukaryotes.
... TSRs glycosylation is carried out in the endoplasmic reticulum (22,26) and, in studies performed in mammalian cell and C. elegans, defects in both C-mannosylation and Ofucosylation have been shown to affect protein stabilization and secretion, albeit with protein-toprotein variability (19,23,(27)(28)(29)(30)(31). A knockout of pofut2 is embryonically lethal in mice (31). ...
Toxoplasma gondii is an intracellular parasite that causes disseminated infections which can lead to neurological damage in fetuses and immunocompromised individuals. Microneme protein 2 (MIC2), a member of the thrombospondin-related anonymous protein (TRAP) family, is a secreted protein important for motility, host cell attachment, invasion, and egress. MIC2 contains six thrombospondin type I repeats (TSRs) that are modified by C-mannose and O-fucose in Plasmodium spp. and mammals.
Here we used mass spectrometry to show that the four TSRs in T. gondii MIC2 with protein O-fucosyltransferase 2 (POFUT2) acceptor sites are modified by a dHexHex disaccharide, while Trp residues within three TSRs are also modified with C-mannose. Disruption of genes encoding either pofut2 or nucleotide sugar transporter 2 (nst2), the putative GDP-fucose transporter, results in loss of MIC2 O-fucosylation, as detected by an antibody against the GlcFuc disaccharide, and markedly reduced cellular levels of MIC2. Furthermore, in 10-15% of the Δpofut2 or Δnst2 vacuoles, MIC2 accumulates earlier in the secretory pathway rather than localizing to micronemes. Dissemination of tachyzoites in human foreskin fibroblasts is reduced in these knockouts, which both show defects in attachment to and invasion of host cells comparable to the phenotype observed in the Δmic2.
These results, which show O-fucosylation of TSRs is required for efficient processing of MIC2 and for normal parasite invasion, are consistent with the recent demonstration that P. falciparum Δpofut2 has decreased virulence and support a conserved role for this glycosylation pathway in quality control of TSR-containing proteins in eukaryotes.
... This Val200Met polymorphism is located in the second fibronectin domain of IL-21R adjacent to the class I cytokine receptor signature motif (WSXWS, amino acid position 214-218) (24,25). This motif is involved in proper protein folding, receptor internalization and transport to the plasma membrane, ligand binding, and signal transduction (25)(26)(27). Based on the crystal structure of the extracellular domain of IL-21R that complexes with IL-21 (Research Collaboratory for Structural Bioinformatics Protein Data Bank [RCSB PDB]: 3TGX) (28), we predicted slight structural differences between IL-21R Val200 and IL-21R Met200 that might affect the function of the protein (Supplemental Figure 10). ...
Risk for ischemic stroke has a strong genetic basis, but heritable factors also contribute to the extent of damage after a stroke has occurred. We previously identified a locus on distal mouse chromosome 7 that contributes over 50% of the variation in postischemic cerebral infarct volume observed between inbred strains. Here, we used ancestral haplotype analysis to fine-map this locus to 12 candidate genes. The gene encoding the IL-21 receptor (Il21r) showed a marked difference in strain-specific transcription levels and coding variants in neonatal and adult cortical tissue. Collateral vessel connections were moderately reduced in Il21r-deficient mice, and cerebral infarct volume increased 2.3-fold, suggesting that Il21r modulates both collateral vessel anatomy and innate neuroprotection. In brain slice explants, oxygen deprivation (OD) activated apoptotic pathways and increased neuronal cell death in IL-21 receptor-deficient (IL-21R-deficient) mice compared with control animals. We determined that the neuroprotective effects of IL-21R arose from signaling through JAK/STAT pathways and upregulation of caspase 3. Thus, natural genetic variation in murine Il21r influences neuronal cell viability after ischemia by modulating receptor function and downstream signal transduction. The identification of neuroprotective genes based on naturally occurring allelic variations has the potential to inform the development of drug targets for ischemic stroke treatment.
Interleukin‐11 (IL‐11) is a member of the IL‐6 family of cytokines and is an important factor for bone homeostasis. IL‐11 binds to and signals via the membrane‐bound IL‐11 receptor (IL‐11R, classic signaling) or soluble forms of the IL‐11R (sIL‐11R, trans‐signaling). Mutations in the IL11RA gene, which encodes the IL‐11R, are associated with craniosynostosis, a human condition in which one or several of the sutures close prematurely, resulting in malformation of the skull. The biological mechanisms of how mutations within the IL‐11R are linked to craniosynostosis are mostly unexplored. In this study, we analyze two variants of the IL‐11R described in craniosynostosis patients: p.T306_S308dup, which results in a duplication of three amino‐acid residues within the membrane‐proximal fibronectin type III domain, and p.E364_V368del, which results in a deletion of five amino‐acid residues in the so‐called stalk region adjacent to the plasma membrane. The stalk region connects the three extracellular domains to the transmembrane and intracellular region of the IL‐11R and contains cleavage sites for different proteases that generate sIL‐11R variants. Using a combination of bioinformatics and different biochemical, molecular, and cell biology methods, we show that the IL‐11R‐T306_S308dup variant does not mature correctly, is intracellularly retained, and does not reach the cell surface. In contrast, the IL‐11R‐E364_V368del variant is fully biologically active and processed normally by proteases, thus allowing classic and trans‐signaling of IL‐11. Our results provide evidence that mutations within the IL11RA gene may not be causative for craniosynostosis and suggest that other regulatory mechanism(s) are involved but remain to be identified.
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C‐mannosylation is a unique type of protein glycosylation via C‐C linkage between an α‐mannose and a tryptophan residue. This modification has been identified in about 30 proteins and regulates several functions, such as protein secretion and intracellular localization, as well as protein stability. About half of C‐mannosylated proteins are categorized as proteins containing thrombospondin type 1 repeat domain or type I cytokine receptors. To evaluate whether C‐mannosylation broadly affects protein functions regardless of protein domain or family, we have sought to identify other types of C‐mannosylated protein and analyze their functions. In this study, we focused on receptor activity modifying protein 1, which neither contains thrombospondin type 1 repeat domain nor belongs to the type I cytokine receptors. Our mass spectrometry analysis demonstrated that RAMP1 is C‐mannosylated at Trp56. It has been shown that RAMP1 transports to the plasma membrane after dimerization with calcitonin receptor‐like receptor and is important for ligand‐dependent downstream signaling activation. Our results showed that C‐mannosylation has no effect on this transport activity. On the other hand, C‐mannosylation did enhance protein stability and cell‐migration activity. Our data may provide new insight into both C‐mannosylation research and novel RAMP1 analysis.
The Trp-x-x-Trp (W-x-x-W) peptide motif, a consensus site for C-mannosylation, is the functional motif in cytokine type I receptors or thrombospondin type I repeat (TSR) superfamily proteins. W-x-x-W motifs are important for physiological and pathological functions of their parental proteins, but effects of C-mannosylation on protein functions remain to be elucidated. By using chemically synthesized WSPW peptides and C-mannosylated WSPW peptides (C-Man-WSPW), we herein investigated whether C-mannosylation of WSPW peptides confer additional biological functions to WSPW peptides. C-Man-WSPW peptide, but not non-mannosylated WSPW, reduced E-cadherin levels in A549 cells. Via peptide mass fingerprinting analysis, we identified actinin-4 as a C-Man-WSPW-binding protein in A549 cells. Actinin-4 partly co-localized with E-cadherin or β-catenin, despite no direct interaction between actinin-4 and E-cadherin. C-Man-WSPW reduced co-localization of E-cadherin and actinin-4; non-mannosylated WSPW had no effect on localization. In actinin-4-knockdown cells, E-cadherin was upregulated and demonstrated a punctate staining pattern in the cytoplasm, which suggests that actinin-4 regulated cell-surface E-cadherin localization. Thus, C-mannosylation of WSPW peptides is required for interaction with actinin-4 that subsequently alters expression and subcellular localization of E-cadherin and morphology of epithelial-like cells. Our results therefore suggest a regulatory role of C-mannosylation of the W-x-x-W motif in interactions between the motif and its binding partner and will thereby enhance understanding of protein C-mannosylation.
A highly unique type of protein glycosylation was identified, in which a mannose residue is linked to tryptophan as C-glycoside. C-Mannosyl tryptophan (CMW) has been discovered in various proteins, such as RNase 2, complements, properdin, thrombospondin, F-spondin, erythropoietin receptor, mucins, ADAMTSs, and myelin-associated glycoprotein. However, the physiological function of CMW has remained unclear. In this article, chemical (structural determination, detection, and synthesis) and biological (biosynthesis, protein targets, and relationship with diseases) aspects of CMW are summarized.
Background:
Mindin (spondin2), a secretory protein related to neural development and immunity, is a member of thrombospondin type I repeat (TSR) superfamily proteins, and has a unique glycosylation of C-mannosylation in its structure. However, it remains unclear whether C-mannosylation plays a functional role in the biosynthesis of mindin in cells.
Methods:
Protein C-mannosylation was analyzed by mass spectrometry. Mindin expression was examined by immunoblot and immunofluorescence analyses in COS-7 cells transfected with the expression vectors for wild type (mindin-WT) or C-mannosylation-defective mutant of mindin (mindin-mutF). The redox status was examined in mindin by using 4-acetoamide-4'-maleimidylstilbene-2,2'-disulfonate.
Results:
When mindin cDNA was expressed in COS-7 cells, C-mannosylation of mindin was confirmed at Trp257 by mass spectrometry. In cells expressing a mindin-mutF, secretion of the mutant was significantly inhibited compared with mindin-WT. In immunofluorescence analysis, mindin-mutF was accumulated in the endoplasmic reticulum (ER), whereas mindin-WT was detected in the Golgi. In addition, mindin-mutF showed an enhanced interaction with calreticulin, an ER-resident chaperone, in cells. In cells, reduced forms were increased in mindin-mutF, compared with a mostly oxidized form of mindin-WT. In the presence of chemical chaperones such as dimethylsulfoxide or 4-phenylbutyrate, inhibited secretion of mindin-mutF was ameliorated in cells, although redox-dependent folding was not affected.
Conclusions:
C-Mannosylation of mindin facilitates its secretion especially through modulating disulfide bond formation in mindin in cells.
General significance:
These results suggest that C-mannosylation plays a functional role in the redox-dependent folding and transport of TSR superfamily proteins in cells.
Integral membrane proteins in eukaryotes are central to various cellular processes and key targets in structural biology, biotechnology and drug development. However, the number of available structures for eukaryotic membrane protein belies their physiological importance. Recently, the number of available eukaryotic membrane protein structures has been steadily increasing due to the development of novel strategies in construct design, expression and structure determination. Here, we examine the major expression systems exploited for eukaryotic membrane proteins. Additionally we strive to tabulate and describe the recent expression strategies in eukaryotic membrane protein structural biology. We find that a majority of targets have been expressed in advanced host systems and modified from their wild-type form with distinct focus on conformation and thermostabilisation. However, strategies for native protein purification should also be considered where possible, particularly in light of the recent advances in single particle cryo electron microscopy.
Members of the cytokine receptor family have a consensus WSXWS sequence (WS motif) in the extracellular domain. With the interleukin-2, erythropoietin, and prolactin receptors, alteration of the WS sequence disrupts ligand binding and receptor signaling. The structural basis for these effects is unclear. To examine the role of the WS equivalent sequence (Y222GEFS226) in the function of the growth hormone receptor, each residue was mutated to alanine or to the WS consensus sequence. Although we used stable cell lines expressing all of these mutants, we show only three mutants, Y222A, G223A, and S226A, which display lower ligand affinity. Using conformation-specific monoclonal antibodies, we show that Y222A and S226A receptors have structural perturbations, which result in decreased signal transduction. This was shown by a decreased ability of growth hormone to stimulate protein synthesis and to transactivate the c-fos promoter with these mutants. The crystal structure of the ligand-occupied extracellular domain of growth hormone receptor indicates that Tyr222 and Ser226 have important interactions within the second beta-barrel domain, providing a structural basis for our results. The WS segment is not involved in sequence-specific accessory protein interaction, as mutation of residues Gly223, Glu224, and Phe225 does not alter receptor function.
In this study the gene for the murine interleukin-11 receptor α chain (IL-11Rα) has been characterized. The gene spans 9 kilobase pairs of DNA, and the organization of its 14 exons conforms to the pattern observed for other members of the hematopoietin receptor family. Analysis of the 5′ end of the cDNA using 5′ RACE showed that the first two exons, designated exons 1a and 1b, are spliced to form alternate transcripts. Transcripts initiating from exon 1b were not found in adult tissues but were present in embryonic stem cells. S1 nuclease and 5′ rapid amplification of cDNA ends assays demonstrated multiple major and minor sites of transcription initiation for each exon. The putative promoter regions of both exons lacked TATA boxes, although potential recognition sites for several transcription factors including Sp1, AP1, and AP2 were present. A comparison of the murine and human IL-11Rα revealed that the 5′ sequence upstream of the major site of transcription initiation site for exon 1b is highly conserved. Northern analysis showed that IL-11Rα is expressed in many adult murine tissues. A second IL-11Rα-like locus containing a sequence homologous to exons 2-13 was also identified.
Primary immunodeficiencies (PIDs) represent exquisite models for studying mechanisms of human host defense. In this study, we report on two unrelated kindreds, with two patients each, who had cryptosporidial infections associated with chronic cholangitis and liver disease. Using exome and candidate gene sequencing, we identified two distinct homozygous loss-of-function mutations in the interleukin-21 receptor gene (IL21R; c.G602T, p.Arg201Leu and c.240_245delCTGCCA, p.C81_H82del). The IL-21R(Arg201Leu) mutation causes aberrant trafficking of the IL-21R to the plasma membrane, abrogates IL-21 ligand binding, and leads to defective phosphorylation of signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5. We observed impaired IL-21-induced proliferation and immunoglobulin class-switching in B cells, cytokine production in T cells, and NK cell cytotoxicity. Our study indicates that human IL-21R deficiency causes an immunodeficiency and highlights the need for early diagnosis and allogeneic hematopoietic stem cell transplantation in affected children.
Interleukin 15 (IL-15) and IL-2 have distinct immunological functions even though both signal through the receptor subunit IL-2Rβ and the common γ-chain (γ(c)). Here we found that in the structure of the IL-15-IL-15Rα-IL-2Rβ-γ(c) quaternary complex, IL-15 binds to IL-2Rβ and γ(c) in a heterodimer nearly indistinguishable from that of the IL-2-IL-2Rα-IL-2Rβ-γ(c) complex, despite their different receptor-binding chemistries. IL-15Rα substantially increased the affinity of IL-15 for IL-2Rβ, and this allostery was required for IL-15 trans signaling. Consistent with their identical IL-2Rβ-γ(c) dimer geometries, IL-2 and IL-15 showed similar signaling properties in lymphocytes, with any differences resulting from disparate receptor affinities. Thus, IL-15 and IL-2 induced similar signals, and the cytokine specificity of IL-2Rα versus IL-15Rα determined cellular responsiveness. Our results provide new insights for the development of specific immunotherapeutics based on IL-15 or IL-2.
The prolactin receptor (PRLR) is activated by binding of prolactin in a 2:1 complex, but the activation mechanism is poorly understood. PRLR has a conserved WSXWS motif generic to cytokine class I receptors. We have determined the nuclear magnetic resonance solution structure of the membrane proximal domain of the human PRLR and find that the tryptophans of the motif adopt a T-stack conformation in the unbound state. By contrast, in the hormone bound state, a Trp/Arg-ladder is formed. The conformational change is hormone-dependent and influences the receptor-receptor dimerization site 3. In the constitutively active, breast cancer-related receptor mutant PRLR(I146L), we observed a stabilization of the dimeric state and a change in the dynamics of the motif. Here we demonstrate a structural link between the WSXWS motif, hormone binding, and receptor dimerization and propose it as a general mechanism for class 1 receptor activation.
IL-21 is a class I cytokine that exerts pleiotropic effects on both innate and adaptive immune responses. It signals through
a heterodimeric receptor complex consisting of the IL-21 receptor (IL-21R) and the common γ-chain. A hallmark of the class
I cytokine receptors is the class I cytokine receptor signature motif (WSXWS). The exact role of this motif has not been determined yet; however, it has been implicated in diverse functions, including
ligand binding, receptor internalization, proper folding, and export, as well as signal transduction. Furthermore, the WXXW motif is known to be a consensus sequence for C-mannosylation. Here, we present the crystal structure of IL-21 bound to IL-21R and reveal that the WSXWS motif of IL-21R is C-mannosylated at the first tryptophan. We furthermore demonstrate that a sugar chain bridges the two fibronectin domains that
constitute the extracellular domain of IL-21R and anchors at the WSXWS motif through an extensive hydrogen bonding network, including mannosylation. The glycan thus transforms the V-shaped receptor
into an A-frame. This finding offers a novel structural explanation of the role of the class I cytokine signature motif.
The structure of the cytokine-binding homology region of the cell surface receptor gp130 has been determined by X-ray crystallography at 2.0 A resolution. The beta sandwich structure of the two domains conforms to the topology of the cytokine receptor superfamily. This first structure of an uncomplexed receptor exhibits a similar L-shaped quaternary structure to that of ligand-bound family members and suggests a limited flexibility in relative domain orientation of some 3 degrees. The putative ligand-binding loops are relatively rigid, with a phenylalanine side chain similarly positioned to exposed aromatic residues implicated in ligand binding for other such receptors. The positioning and structure of the N-terminal portion of the polypeptide chain have implications for the structure and function of cytokine receptors, such as gp130, which contain an additional N-terminal immunoglobulin-like domain.
A hallmark of human immunodeficiency virus type 1 (HIV-1) pathogenesis is the rapid loss of CD4 T cells leading to generalized immune dysfunction, including an exhausted CD8 T cell phenotype. Understanding the necessary factors that govern the functional quality and protective potential of antiviral T cell responses would facilitate rational vaccine design and improve therapeutic strategies to combat persistent infections. Mouse models of chronic viral infection demonstrate that interleukin-21 (IL-21), produced primarily by CD4 T cells, is required for the generation and maintenance of functionally competent CD8 T cells and viral containment. We reasoned that preserved IL-21 production during HIV-1 infection would be associated with enhanced CD8 T cell function, allowing improved viral control. Here we analyzed the ability of CD4 and CD8 T cells to produce several cytokines in addition to IL-21 ex vivo following stimulation with overlapping HIV-1 peptides. Both CD4 and CD8 T cells were able to produce IL-21 in response to HIV-1 infection, with the latter cell type more closely associated with viral control. Furthermore, IL-21-producing HIV-1-specific CD4 T cells (compared to those producing other cytokines) were the best indicator of functional CD8 T cells. Our results demonstrate that HIV-1-specific IL-21-producing CD8 T cells are induced following primary infection and enriched in elite controllers, suggesting a critical role for these cells in the maintenance of viremia control.
Functional defects in cytotoxic CD8+ T cell responses arise in chronic human viral infections, but the mechanisms involved are not well understood. In mice, CD4
cell-mediated interleukin-21 (IL-21) production is necessary for the maintenance of CD8+ T cell function and control of persistent viral infections. To investigate the potential role of IL-21 in a chronic human
viral infection, we studied the rare subset of HIV-1 controllers, who are able to spontaneously control HIV-1 replication
without treatment. HIV-specific triggering of IL-21 by CD4+ T cells was significantly enriched in these persons (P = 0.0007), while isolated loss of IL-21-secreting CD4+ T cells was characteristic for subjects with persistent viremia and progressive disease. IL-21 responses were mediated by
recognition of discrete epitopes largely in the Gag protein, and expansion of IL-21+ CD4+ T cells in acute infection resulted in lower viral set points (P = 0.002). Moreover, IL-21 production by CD4+ T cells of HIV controllers enhanced perforin production by HIV-1-specific CD8+ T cells from chronic progressors even in late stages of disease, and HIV-1-specific effector CD8+ T cells showed an enhanced ability to efficiently inhibit viral replication in vitro after IL-21 binding. These data suggest that HIV-1-specific IL-21+ CD4+ T cell responses might contribute to the control of viral replication in humans and are likely to be of great importance
for vaccine design.
Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
Chronic viral infection is often associated with the dysfunction of virus-specific T cells. Our studies using Il21r-deficient (Il21r–/–) mice now suggest that interleukin-21 (IL-21) is critical for the long-term maintenance and functionality of CD8+ T cells and the control of chronic lymphocytic choriomeningitis virus infection in mice. Cell-autonomous IL-21 receptor (IL-21R)–dependent
signaling by CD8+ T cells was required for sustained cell proliferation and cytokine production during chronic infection. Il21r–/– mice showed normal CD8+ T cell expansion, effector function, memory homeostasis, and recall responses during acute and after resolved infection with
several other nonpersistent viruses. These data suggest that IL-21R signaling is required for the maintenance of polyfunctional
T cells during chronic viral infections and have implications for understanding the immune response to other persisting antigens,
such as tumors.
The terminal development of erythroid progenitor cells is promoted in part through the interaction of erythropoietin (EPO) with its cell surface receptor. This receptor and a growing family of related cytokine receptors share homologous extracellular features, including a well-conserved WSXWS motif. To explore the functional significance of this motif in the murine EPO receptor, five WSAWSE mutants were prepared and their signal-transducing, ligand binding, and endocytotic properties were compared. EPO receptors mutated at tryptophan residues (W-232, W-235----G; W-235----G; W-235----F) failed to mediate EPO-induced growth or pp100 phosphorylation, while S-236----T and E-237----K mutants exhibited partial to full activity (50 to 100% of wild-type growth and induced phosphorylation). Ligand affinity was reduced for mutant receptors (two- to fivefold), yet expression at the cell surface for all receptors was nearly equivalent. Also, the ability of mutated receptors to internalize ligand was either markedly reduced or abolished (W-235----F), indicating a role for the WSAWSE region in hormone internalization. Interestingly, receptor forms lacking 97% of the cytosolic domain (no signal-transducing capacity; binding affinity reduced two- to threefold) internalized EPO efficiently. This and all WSAWSE receptor forms studied also mediated specific cross-linking of 125I-EPO to three accessory membrane proteins (M(r)s, 120,000, 105,000, and 93,000). These findings suggest that the WSAWSE domain of the EPO receptor is important for EPO-induced signal transduction and ligand internalization. In contrast, although the cytosolic domain is required for growth signaling, it appears nonessential for efficient endocytosis.
Recent studies have identified a new family of cytokine receptors, which is primarily characterized by the conservation of periodically interspersed four cysteine residues and the W-S-X-W-S sequence ('WS motif') within the extracellular domain. However, the role of such conserved structures still remains elusive, in particular that of the WS motif. Interleukin-2 (IL-2) is known to play a critical role in the clonal expansion of antigen-stimulated T lymphocytes, and the IL-2 signal is delivered by one of the receptor components, the IL-2 receptor beta (IL-2R beta) chain. The IL-2R beta chain, unlike the IL-2R alpha chain, belongs to this receptor family. In the present study, we analyzed the function of the WS motif of IL-2R beta (Trp194-Ser195-Pro196-Trp197-Ser198) with the use of site-directed mutagenesis. Our results indicate the critical role of the two Trp residues in the proper folding of the IL-2R beta extracellular domain and point to the general functional importance of the WS motif in the new cytokine receptor family.
The WSXWS motif in the extracellular domain defines members of the cytokine receptor family, yet its role in receptor structure and function remains unresolved. To address this question we have generated a panel of 100 mutants within the WSXWS motif of the erythropoietin receptor, which represents all single amino acid substitutions of these five amino acids. All mutants were synthesized at the same level; however, their passage from the endoplasmic reticulum to the Golgi apparatus differed. Because of this, expression of mutant receptors at the cell surface varied more than 300-fold. The tolerance of the tryptophan and serine residues to substitution was quite narrow; as a result, most of these mutants were retained in the endoplasmic reticulum and showed no cell surface expression or reduced cell surface expression. Although many mutants with substitutions at the middle residue of the motif reached the cell surface, it was notable that one mutant, A234E, was processed more efficiently than the wild type receptor and was expressed in elevated numbers at the cell surface. Despite this variation, all mutant receptors that reached the cell surface appeared able to bind erythropoietin and transduce a proliferative signal normally. These results are discussed in terms of a general model for WSXWS function in which the motif contributes to efficient receptor folding.
Cytokines are important in the regulation of haematopoiesis and immune responses, and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.
The cytokine potential of developing T helper (Th) cells is directly shaped both positively and negatively by the cytokines expressed by the effector Th cell subsets. Here we find that the recently identified cytokine, interleukin (IL)-21, is preferentially expressed by Th2 cells when compared with Th1 cells generated in vitro and in vivo. Exposure of naive Th precursors to IL-21 inhibits interferon (IFN)-gamma production from developing Th1 cells. The repression of IFN-gamma production is specific in that the expression of other Th1 and Th2 cytokines is unaffected. IL-21 decreases the IL-12 responsiveness of developing Th cells by specifically reducing both signal transducer and activator of transcription 4 protein and mRNA expression. These results suggest that Th2 cell-derived IL-21 regulates the development of IFN-gamma-producing Th1 cells which could serve to amplify a Th2 response.
Effector T cell responses have long been viewed in the context of the Th1/Th2 paradigm. Recently, a third major subset of nonpolarized effector T cells that provides help to B cells has been identified. These T cells, termed T follicular helper (T(FH)) cells, home to the B cell areas of secondary lymphoid tissue, through interactions mediated via the chemokine receptor CXCR5 and its ligand CXCL13. Affymetrix microarrays were used to identify transcription factors, cytokines, and cell surface molecules that underlie the differentiation pathways and functional properties of the T(FH) subset. The transcriptional profile of human CXCR5(+) T(FH) cells was compared with that of Th1 and Th2 cells, which enabled the identification of numerous genes expressed preferentially by T(FH) cells, over the other effector subsets. Certain T(FH) genes were also expressed by B cells and thus appear to be particularly relevant for humoral immunity. Abs were used to confirm the expression of several factors. In particular, CD84 and CD200, the cytokine IL-21, and the transcription factor BCL6 were all strongly associated with T(FH) cells. Gene microarrays reveal a highly distinctive transcriptional profile for a third subset of effector T cells that differs markedly from Th1 and Th2 cells.
Several cell lines and primary cultures benefit from the use of positively charged extracellular matrix proteins or polymers that enhance their ability to attach to culture plates. Polyethyleneimine is a positively charged polymer that has gained recent attention as a transfection reagent. A less known use of this cationic polymer as an attachment factor was explored with several cell lines.
Polyethyleneimine compared favorably to traditional attachment factors such as collagen and polylysine. PC-12 and HEK-293 cells plated on dishes coated with polyethyleneimine showed a homogeneous distribution of cells in the plate, demonstrating strong cell adhesion that survived washing procedures. The polymer could also be used to enhance the adherence and allow axonal outgrowth from zebrafish retinal explants. The effects of this coating agent on the transfection of loosely attaching cell lines were studied. Pre-coating with polyethyleneimine had the effect of enhancing the transfection yield in procedures using lipofection reagents.
Polyethyleneimine is an effective attachment factor for weakly anchoring cell lines and primary cells. Its use in lipofection protocols makes the procedures more reliable and increases the yield of expressed products with commonly used cell lines such as PC-12 and HEK-293 cells.
CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for X-ray structure solution, structure comparison and analysis, and publication-quality graphics. The map-fitting tools are available as a stand-alone package, distributed as 'Coot'.
Interleukin (IL)-21 is the most recently recognized of the cytokines that share the common cytokine receptor gamma chain (gamma(c)), which is mutated in humans with X-linked severe combined immunodeficiency. We now report that IL-21 synergistically acts with IL-15 to potently promote the proliferation of both memory (CD44high) and naive (CD44low) phenotype CD8+ T cells and augment interferon-gamma production in vitro. IL-21 also cooperated, albeit more weakly, with IL-7, but not with IL-2. Correspondingly, the expansion and cytotoxicity of CD8+ T cells were impaired in IL-21R-/- mice. Moreover, in vivo administration of IL-21 in combination with IL-15 boosted antigen-specific CD8+ T cell numbers and resulted in a cooperative effect on tumor regression, with apparent cures of large, established B16 melanomas. Thus, our studies reveal that IL-21 potently regulates CD8+ T cell expansion and effector function, primarily in a synergistic context with IL-15.
The common gamma-chain cytokine, IL-21, is produced by CD4(+) T cells and mediates potent effects on a variety of immune cells including NK, T, and B cells. NKT cells express the receptor for IL-21; however, the effect of this cytokine on NKT cell function has not been studied. We show that IL-21 on its own enhances survival of NKT cells in vitro, and IL-21 increases the proliferation of NKT cells in combination with IL-2 or IL-15, and particularly with the CD1d-restricted glycosphingolipid Ag alpha-galactosylceramide. Similar to its effects on NK cells, IL-21 enhances NKT cell granular morphology, including granzyme B expression, and some inhibitory NK receptors, including Ly49C/I and CD94. IL-21 also enhanced NKT cell cytokine production in response to anti-CD3/CD28 in vitro. Furthermore, NKT cells may be subject to autocrine IL-21-mediated stimulation because they are potent producers of this cytokine following in vitro stimulation via CD3 and CD28, particularly in conjunction with IL-12 or following in vivo stimulation with alpha-galactosylceramide. Indeed, NKT cells produced much higher levels of IL-21 than conventional CD4 T cells in this assay. This study demonstrates that NKT cells are potentially a major source of IL-21, and that IL-21 may be an important factor in NKT cell-mediated immune regulation, both in its effects on NK, T, and B cells, as well as direct effects on NKT cells themselves. The influence of IL-21 in NKT cell-dependent models of tumor rejection, microbial clearance, autoimmunity, and allergy should be the subject of future investigations.
CD4+ helper T cells can differentiate into several possible fates including: Th1, Th2, T regulatory, and Th17 cells. Although,
cytokine production by non-T cells is an important factor in helper T cell differentiation, a characteristic feature of both
Th1 and Th2 lineages is their ability to secrete cytokines that promote their respective differentiation. However, cytokines
produced by T cells that help to sustain Th17 cells have not yet been identified. Here we show that IL-21 is a product of
Th17 cells, which is induced in a Stat3-dependent manner. Additionally, Stat3 can directly bind the Il21 promoter. IL-21 also induces IL-17 production and expression of the transcription factor, RORγt. Furthermore, generation
of Th17 cells in the conventional manner is attenuated by blocking IL-21. IL-21 is known to activate Stat3 and its ability
to induce Th17 differentiation is abrogated in the absence of Stat3. These data argue that IL-21 serves as an autocrine factor
secreted by Th17 cells that promotes or sustains Th17 lineage commitment.
Interleukin (IL)-21 was recently discovered using a functional cloning approach based on expression of its receptor. It is similar in domain organization and primary sequence to IL-2 and IL-15. Like these cytokines, IL-21 uses the common γ chain of the IL-2/15 receptor, which forms a heterodimeric receptor complex with IL-21R. IL-21 is produced by activated T cells, and it influences proliferation of T and B cells and cytolytic activity of natural killer cells. The elucidation of the unique biological effects of IL-21 represents an intense area of interest in current cytokine biology.
The terminal development of erythroid progenitor cells is promoted in part through the interaction of erythropoietin (EPO) with its cell surface receptor. This receptor and a growing family of related cytokine receptors share homologous extracellular features, including a well-conserved WSXWS motif. To explore the functional significance of this motif in the murine EPO receptor, five WSAWSE mutants were prepared and their signal-transducing, ligand binding, and endocytotic properties were compared. EPO receptors mutated at tryptophan residues (W-232, W-235----G; W-235----G; W-235----F) failed to mediate EPO-induced growth or pp100 phosphorylation, while S-236----T and E-237----K mutants exhibited partial to full activity (50 to 100% of wild-type growth and induced phosphorylation). Ligand affinity was reduced for mutant receptors (two- to fivefold), yet expression at the cell surface for all receptors was nearly equivalent. Also, the ability of mutated receptors to internalize ligand was either markedly reduced or abolished (W-235----F), indicating a role for the WSAWSE region in hormone internalization. Interestingly, receptor forms lacking 97% of the cytosolic domain (no signal-transducing capacity; binding affinity reduced two- to threefold) internalized EPO efficiently. This and all WSAWSE receptor forms studied also mediated specific cross-linking of 125I-EPO to three accessory membrane proteins (M(r)s, 120,000, 105,000, and 93,000). These findings suggest that the WSAWSE domain of the EPO receptor is important for EPO-induced signal transduction and ligand internalization. In contrast, although the cytosolic domain is required for growth signaling, it appears nonessential for efficient endocytosis.
Severe combined immunodeficiency (SCID), a rare type of genetic associated immune disorder, is poorly characterized in mainland China. We retrospectively reviewed 44 patients with SCID who received treatment from 2004 to 2011 in Shanghai, China, and herein summarize their clinical manifestations and immunological and preliminary genetic features. The male-to-female ratio was 10:1. Twenty five patients presented with X-SCID symptoms. Only one patient was diagnosed before the onset of symptoms due to positive family history. The mean time of delay in the diagnosis of X-SCID was 2.69 months (range, 0.5-8.67). Thirty-seven of the 44 patients died by the end of 2011 with the mean age of death being 7.87 months (range, 1.33-31). Six patients received hematopoietic stem cell transplantation (HSCT); only one of them survived, who was transplanted twice. The time between onset and death was shorter in the HSCT-treated group compared with the untreated group (2.87 ± 1.28 and 3.34 ± 0.59 months, respectively), probably due to active infections during transplantation. Bacillus Calmette-Guérin (BCG) complications occurred in 14 of the 34 patients who received BCG vaccination. Transfusion-induced graft-versus-host disease occurred in 5 patients. Total 20 mutations in interleukin-2 receptor subunit gamma (IL2RG) were identified in 22 patients, including 11 novel mutations. Most patients were misdiagnosed before referred to our SCID Center. Therefore, establishing more diagnostic centers dedicated to the care of PID and accessible by primary immunodeficiency patients will facilitate early, correct diagnosis and better care of SCID in China.
X-linked severe combined immunodeficiency (X-SCID) is a rare, life-threatening immune disorder, caused by mutations of the gene for the γ-chain (γc) of the interleukin-2 receptor, IL2RG. We analyzed the clinical, immunologic, and molecular characteristics of children with X-SCID, attempting to improve the diagnosis and treatment of X-SCID in China.
Methods
X-SCID was suspected in male infants with recurrent or persistent infections. Eleven male infants from ten unrelated Chinese families were included. The IL2RG gene was amplified and sequenced, followed by mutation analysis in these children and their female relatives. X-linked short tandem repeat (X-STR) typing was done to define the maternal lymphocyte engraftment.
Results
The 11 children exhibited recurrent infections and 10 of them had lymphopenia. B cells were present in all patients, T cells were markedly reduced in 10, and NK cells were markedly reduced in 9. Nine IL2RG gene mutations were identified in the 11 children, with 5 novel mutations. One patient was found to have the maternal lymphocyte engraftment.
Conclusion
The clinical presentations and immunologic characteristics of the X-SCID patients were accordingly quite uniform despite the heterogeneity of mutations locating almost in the entire γc gene.
Locus-Specific DataBases (LSDBs) store information on gene sequence variation associated with human phenotypes and are frequently used as a reference by researchers and clinicians. We developed the Leiden Open-source Variation Database (LOVD) as a platform-independent Web-based LSDB-in-a-Box package. LOVD was designed to be easy to set up and maintain and follows the Human Genome Variation Society (HGVS) recommendations. Here we describe LOVD v.2.0, which adds enhanced flexibility and functionality and has the capacity to store sequence variants in multiple genes per patient. To reduce redundancy, patient and sequence variant data are stored in separate tables. Tables are linked to generate connections between sequence variant data for each gene and every patient. The dynamic structure allows database managers to add custom columns. The database structure supports fast queries and allows storage of sequence variants from high-throughput sequence analysis, as demonstrated by the X-chromosomal Mental Retardation LOVD installation. LOVD contains measures to ensure database security from unauthorized access. Currently, the LOVD Website (http://www.LOVD.nl/) lists 71 public LOVD installations hosting 3,294 gene variant databases with 199,000 variants in 84,000 patients. To promote LSDB standardization and thereby database interoperability, we offer free server space and help to establish an LSDB on our Leiden server.
Hum Mutat 32:1–7, 2011.
In advanced renal cell cancer and malignant melanoma, the current FDA approved immune modulators, such as IL-2, are the only agents which provide a durable complete remission. These responses, however, occur in < 10% of treated patients and their applicability is limited to selected patients because of their toxicity. The identification of new immunotherapeutic agents with an improved response rate and toxicity profile would represent a significant advancement in the treatment of these malignancies.
This is a comprehensive review of IL-21 including its pharmacology and current developmental status. A literature review was performed using all PubMed listed publications involving IL-21, including original research articles, reviews and abstracts. It also includes a review of current ongoing trials and information from the official product website.
Recombinant IL-21 (rIL-21) is a new immune modulator currently undergoing Phase I and II testing. It is a cytokine with a four helix structure that has structural and sequence homology to IL-2 and -15, but also possesses many unique biological properties. In this review, we evaluate the development, pharmacologic properties, safety profile and current clinical efficacy of rIL-21.
rIL-21 has an acceptable safety profile and encouraging single agent activity in early phase renal cell carcinoma and melanoma clinical trials.
IL-21 enhances NK cell functions and survival in healthy and HIV-infected patients with inhibition of viral replication.
IL-21 plays an important role in regulating immune response and controlling chronic viral infections. Recently, we reported its decreased serum concentrations and their immunological consequences in HIV-infected persons. In this study, we have investigated how exogenous IL-21 enhances NK cell responses in these persons. We show that the cytokine receptors are expressed equally on all NK cell subsets defined by expression of CD16 and CD56; the cytokine activates STAT-3, MAPK, and Akt to enhance NK cell functions; the STAT-3 activation plays a key role in constitutive and IL-21-mediated enhancement of NK cell functions; the cytokine increases expression of antiapoptotic proteins Bcl-2 and Bcl-XL and enhances viability of NK cells but has no effect on their proliferation; the cytokine enhances HIV-specific ADCC, secretory, and cytotoxic functions, as well as viability of NK cells from HIV-infected persons; it exerts its biological effects on NK cells with minimal stimulation of HIV-1 replication; and the cytokine-activated NK cells inhibit viral replication in cocultured, HIV-infected, autologous CD4+ T cells in a perforin- and LFA-1-dependent manner. These data suggest that IL-21 may serve as a valuable therapeutic tool for enhancing NK cell responses and inhibiting viral replication in HIV-infected patients.
Cytokines are secreted signalling molecules with decisive effects on haematopoiesis, innate and adaptive immunity, and immunopathology. Interleukin (IL)-21 is a novel cytokine produced by activated CD4(+) T cells and natural killer T (NKT) cells. IL-21 is part of a family of cytokines which include IL-2, -4, -7, -9 and -15 that all share the common IL-2 receptor gamma chain (gamma(c)) in their individual receptor complexes. IL-21 receptor (IL-21R) is widely expressed on both myeloid and lymphoid cell lineages and IL-21 actions include co-stimulation of B cell differentiation and immunoglobulin (Ig) production, co-mitogen of T cells, and stimulation of NK and CD8(+) T cell cytotoxic function. Initially, IL-21 was recognized for its anti-tumour effects in several preclinical tumour models, warranting its currently ongoing clinical development as a cancer immunotherapeutic. More recently, IL-21 has been associated with the development of a panel of autoimmune and inflammatory diseases, where neutralization of IL-21 has been suggested as a potential new therapy. In this review, we will cover the latest discoveries of IL-21 as a cancer therapy and its implications in immunopathologies.
IL-7 and IL-7Ralpha bind the gamma(c) receptor, forming a complex crucial to several signaling cascades leading to the development and homeostasis of T and B cells. We report that the IL-7Ralpha ectodomain uses glycosylation to modulate its binding constants to IL-7, unlike the other receptors in the gamma(c) family. IL-7 binds glycosylated IL-7Ralpha 300-fold more tightly than unglycosylated IL-7Ralpha, and the enhanced affinity is attributed primarily to an accelerated on rate. Structural comparison of IL-7 in complex to both forms of IL-7Ralpha reveals that glycosylation does not participate directly in the binding interface. The SCID mutations of IL-7Ralpha locate outside the binding interface with IL-7, suggesting that the expressed mutations cause protein folding defects in IL-7Ralpha. The IL-7/IL-7Ralpha structures provide a window into the molecular recognition events of the IL-7 signaling cascade and provide sites to target for designing new therapeutics to treat IL-7-related diseases.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific alpha subunit and a betac subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.
The Trp-Ser-X-Trp-Ser motif commonly exists just outside the transmembrane domains of all cytokine receptors so far isolated. The role of this conserved motif in erythropoietin receptor was examined by assessing a series of mutant receptors on erythropoietin-induced signal transduction. Replacement of one of the two conserved Trp residues in the motif to Gly was found to completely abolish the binding of erythropoietin to the receptor and also to lose the ability to transduce the factor-dependent growth signal. While the mutants with one Ser residue converted to Gly or Ala retained full biological activities, the replacement of both conserved Ser residues diminished the functions of the receptor. Furthermore, the receptors lacking a part or all of the Trp-Ser-X-Trp-Ser motif did not respond to erythropoietin. The Trp-Ser-X-Trp-Ser motif, especially Trp residue, located in extracellular domains of the erythropoietin receptor thus appears to play a critical role in receptor-mediated signal transduction.
Interleukin-15 (IL-15) is a novel cytokine of the four-helix bundle family which shares many biological activities with IL-2, probably due to its interaction with the IL-2 receptor beta and gamma (IL-2R beta and gamma c) chains. We report here the characterization and molecular cloning of a distinct murine IL-15R alpha chain. IL-15R alpha alone displays an affinity of binding for IL-15 equivalent to that of the heterotrimeric IL-2R for IL-2. A biologically functional heteromeric IL-15 receptor complex capable of mediating IL-15 responses was generated through reconstruction experiments in a murine myeloid cell line. IL-15R alpha is structurally similar to IL-2R alpha; together they define a new cytokine receptor family. The distribution of IL-15 and IL-15R alpha mRNA suggests that IL-15 may have biological activities distinct from IL-2.
Members of the cytokine receptor family have a consensus WSXWS sequence (WS motif) in the extracellular domain. With the interleukin-2, erythropoietin, and prolactin receptors, alteration of the WS sequence disrupts ligand binding and receptor signaling. The structural basis for these effects is unclear. To examine the role of the WS equivalent sequence (Y222GEFS226) in the function of the growth hormone receptor, each residue was mutated to alanine or to the WS consensus sequence. Although we used stable cell lines expressing all of these mutants, we show only three mutants, Y222A, G223A, and S226A, which display lower ligand affinity. Using conformation-specific monoclonal antibodies, we show that Y222A and S226A receptors have structural perturbations, which result in decreased signal transduction. This was shown by a decreased ability of growth hormone to stimulate protein synthesis and to transactivate the c-fos promoter with these mutants. The crystal structure of the ligand-occupied extracellular domain of growth hormone receptor indicates that Tyr222 and Ser226 have important interactions within the second beta-barrel domain, providing a structural basis for our results. The WS segment is not involved in sequence-specific accessory protein interaction, as mutation of residues Gly223, Glu224, and Phe225 does not alter receptor function.
Severe combined immunodeficiency (SCID) is a syndrome of profoundly impaired cellular and humoral immunity. In humans, SCID is most commonly caused by mutations in the X-linked gene IL2RG, which encodes the common gamma chain, gamma c, of the leukocyte receptors for interleukin-2 and multiple other cytokines. To investigate the frequency and variety of IL2RG mutations that cause SCID, we analyzed DNA, RNA, and B-cell lines from a total of 103 unrelated SCID-affected males and their relatives using a combination of molecular and immunologic techniques. Sixty-two different mutations spanning all eight IL2RG exons were found in 87 cases, making possible correlations between mutation type and functional consequences. Although skewed maternal X chromosome inactivation, single-strand conformation polymorphism, mRNA expression, and cell surface staining with anti-gamma c antibodies were all helpful in establishing IL2RG defects as the cause of SCID, only dideoxy fingerprinting and DNA sequence determination each detected 100% of the IL2RG mutations in our series. Abnormal gamma c chains may be expressed in the lymphocytes of as many as two thirds of patients with X-linked SCID. Specific mutation diagnosis thus remains technically challenging, but it is important for genetic counseling and perhaps for helping to select appropriate subjects for retroviral gene therapy trials, This is a US government work. There are no restrictions on its use.
Erythropoietin receptor (EPOR) is thought to be activated by ligand-induced homodimerization. However, structures of agonist
and antagonist peptide complexes of EPOR, as well as an EPO-EPOR complex, have shown that the actual dimer configuration is
critical for the biological response and signal efficiency. The crystal structure of the extracellular domain of EPOR in its
unliganded form at 2.4 angstrom resolution has revealed a dimer in which the individual membrane-spanning and intracellular
domains would be too far apart to permit phosphorylation by JAK2. This unliganded EPOR dimer is formed from self-association
of the same key binding site residues that interact with EPO-mimetic peptide and EPO ligands. This model for a preformed dimer
on the cell surface provides insights into the organization, activation, and plasticity of recognition of hematopoietic cell
surface receptors.
Interleukin-4 (IL-4) is a principal regulatory cytokine during an immune response and a crucial determinant for allergy and asthma. IL-4 binds with high affinity and specificity to the ectodomain of the IL-4 receptor alpha chain (IL4-BP). Subsequently, this intermediate complex recruits the common gamma chain (gamma c), thereby initiating transmembrane signaling. The crystal structure of the intermediate complex between human IL-4 and IL4-BP was determined at 2.3 A resolution. It reveals a novel spatial orientation of the two proteins, a small but unexpected conformational change in the receptor-bound IL-4, and an interface with three separate clusters of trans-interacting residues. Novel insights on ligand binding in the cytokine receptor family and a paradigm for receptors of IL-2, IL-7, IL-9, and IL-15, which all utilize gamma c, are provided.
Interleukin (IL)-21 was recently discovered using a functional cloning approach based on expression of its receptor. It is similar in domain organization and primary sequence to IL-2 and IL-15. Like these cytokines, IL-21 uses the common gamma chain of the IL-2/15 receptor, which forms a heterodimeric receptor complex with IL-21R. IL-21 is produced by activated T cells, and it influences proliferation of T and B cells and cytolytic activity of natural killer cells. The elucidation of the unique biological effects of IL-21 represents an intense area of interest in current cytokine biology.
The cytokine interleukin-21 (IL-21) is closely related to IL-2 and IL-15, and their receptors all share the common cytokine
receptor γ chain, γc, which is mutated in humans with X-linked severe combined immunodeficiency disease (XSCID). We demonstrate that, although
mice deficient in the receptor for IL-21 (IL-21R) have normal lymphoid development, after immunization, these animals have
higher production of the immunoglobulin IgE, but lower IgG1, than wild-type animals. Mice lacking both IL-4 and IL-21R exhibited
a significantly more pronounced phenotype, with dysgammaglobulinemia, characterized primarily by a severely impaired IgG response.
Thus, IL-21 has a significant influence on the regulation of B cell function in vivo and cooperates with IL-4. This suggests
that these γc-dependent cytokines may be those whose inactivation is primarily responsible for the B cell defect in humans with XSCID.
Type I helical cytokines are ligands for receptors structurally related by a common sequence signature. Here we analyze the 27 ligands and 34 human type I cytokine receptor encoded by the human genome. We compare these to ligands and receptors found in mouse and insects. We describe their structural relatedness to one another and discuss the evolution of these gene families.
Interleukin-2 (IL-2) is an immunoregulatory cytokine that acts through a quaternary receptor signaling complex containing alpha (IL-2Ralpha), beta (IL-2Rbeta), and common gamma chain (gc) receptors. In the structure of the quaternary ectodomain complex as visualized at a resolution of 2.3 angstroms, the binding of IL-2Ralpha to IL-2 stabilizes a secondary binding site for presentation to IL-2Rbeta. gammac is then recruited to the composite surface formed by the IL-2/IL-2Rbeta complex. Consistent with its role as a shared receptor for IL-4, IL-7, IL-9, IL-15, and IL-21, gammac forms degenerate contacts with IL-2. The structure of gammac provides a rationale for loss-of-function mutations found in patients with X-linked severe combined immunodeficiency diseases (X-SCID). This complex structure provides a framework for other gammac-dependent cytokine-receptor interactions and for the engineering of improved IL-2 therapeutics.
Class I cytokine receptors efficiently transfer activation signals from the extracellular space to the cytoplasm and play a dominant role in growth control and differentiation of human tissues. Although a significant body of literature is devoted to this topic, a consistent mechanistic picture for receptor activation in the membrane environment is still missing. Using the interleukin-4 receptor (IL-4R) as an example, we propose that the membrane-proximal stem-loop of the extracellular domains contains pivotal elements of a rotational switch. Interfacial energies of amino acid side-chains contained in the highly conserved WSXWS at the surface of the lipid bilayer suggest a new functional role for this motif. A generic activation mechanism for this receptor class is presented, which may impact the design of a new generation of biophysical assay systems.
Interleukin 15 (IL-15) and IL-2, which promote the survival of memory CD8(+) T cells and regulatory T cells, respectively, bind receptor complexes that share beta- and gamma-signaling subunits. Receptor specificity is provided by unique, nonsignaling alpha-subunits. Whereas IL-2 receptor-alpha (IL-2Ralpha) is expressed together in cis with the beta- and gamma-subunits on T cells and B cells, IL-15Ralpha is expressed in trans on antigen-presenting cells. Here we present a 1.85-A crystal structure of the human IL-15-IL-15Ralpha complex. The structure provides insight into the molecular basis of the specificity of cytokine recognition and emphasizes the importance of water in generating this very high-affinity complex. Despite very low IL-15-IL-2 sequence homology and distinct receptor architecture, the topologies of the IL-15-IL-15Ralpha and IL-2-IL-2Ralpha complexes are very similar. Our data raise the possibility that IL-2, like IL-15, might be capable of being presented in trans in the context of its unique receptor alpha-chain.
Interleukin-4 and Interleukin-13 are cytokines critical to the development of T cell-mediated humoral immune responses, which are associated with allergy and asthma, and exert their actions through three different combinations of shared receptors. Here we present the crystal structures of the complete set of type I (IL-4R alpha/gamma(c)/IL-4) and type II (IL-4R alpha/IL-13R alpha1/IL-4, IL-4R alpha/IL-13R alpha1/IL-13) ternary signaling complexes. The type I complex reveals a structural basis for gamma(c)'s ability to recognize six different gamma(c)-cytokines. The two type II complexes utilize an unusual top-mounted Ig-like domain on IL-13R alpha1 for a novel mode of cytokine engagement that contributes to a reversal in the IL-4 versus IL-13 ternary complex assembly sequences, which are mediated through substantially different recognition chemistries. We also show that the type II receptor heterodimer signals with different potencies in response to IL-4 versus IL-13 and suggest that the extracellular cytokine-receptor interactions are modulating intracellular membrane-proximal signaling events.
Mutation analysis of IL2RG in human X-linked severe combined immunodeficiency
- J M Puck
- A E Pepper
- P S Henthorn
- F Candotti
- J Isakov
- T Whitwam
- JM Puck
the next generation in gene variant databases
- If A C Fokkema
- Pem Taschner
- Gcp Schaafsma
- J Celli
- Jfj Laros
- IFAC Fokkema
PHENIX: a comprehensive Python-based system for macromolecular structure solution
- P D Adams
- P V Afonine
- G Bunkóczi
- V B Chen
- I W Davis
- N Echols
- PD Adams