Background:The class I cytokine IL-21 exerts pleiotropic effects on innate and adaptive immunity. Results: We obtained the crystal structure of the partially glycosylated IL-21 receptor (IL-21R) bound to IL-21. Conclusion: A sugar chain is an integral part of IL-21R. Significance: This structure offers an insight into the putative role of the class I cytokine receptor signature motif. 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 ␥-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.IL-21 is a class I cytokine with a four-helix bundle structure arranged in an up-up-down-down topology typical for the class I cytokines (1). It exerts pleiotropic effects on both innate and adaptive immune responses. IL-21 is secreted by activated CD4 ϩ T cells, in particular T H 17 and T follicular helper cells, as well as natural killer cells (2). Not only do both T H 17 and T follicular helper cells produce IL-21, but this cytokine also plays an important role in promoting the development of T H 17 and T follicular helper cells by a feed-forward mechanism (3-8). Furthermore, IL-21 cooperates with other cytokines to increase the cytotoxicity of CD8 ϩ T cells and promotes proliferation of CD8 ϩ cells in the presence of antigens (9). IL-21 also influences antibody production by B cells (10). Recent studies demonstrated that IL-21 produced by CD4 ϩ cells is critical for the ability of CD8 ϩ T cells to control viral infection (11-13). The ability of IL-21 to augment immunity has spurred substantial interest in the therapeutic use of IL-21, and it is currently being evaluated in a number of clinical trials against, for example, metastatic melanoma and renal cancer (14).IL-21 signals through a heterodimeric receptor complex consisting of the private chain IL-21 receptor (IL-21R) 2 and the common ␥-chain (␥C), the latter being shared by IL-2, IL-4, . Upon binding of IL-21 to the receptor complex and subsequent receptor activation, signaling occurs through the Jak-STAT ...

The high resolution three-dimensional structure of human interleukin (hIL)-21 has been resolved by heteronuclear NMR spectroscopy. Overall, the hIL-21 structure is dominated by a well defined central four-helical bundle, arranged in an up-updown-down topology, as observed for other cytokines. A segment of the hIL-21 molecule that includes the third helical segment, helix C, is observed to exist in two distinct and interchangeable states. In one conformer, the helix C segment is presented in a regular, ␣-helical conformation, whereas in the other conformer, this segment is largely disordered. A structure-based sequence alignment of hIL-21 with receptor complexes of the related cytokines, interleukin-2 and -4, implied that this particular segment is involved in receptor binding. An hIL-21 analog was designed to stabilize the region around helix C through the introduction of a segment grafted from hIL-4. This novel hIL-21 analog was demonstrated to exhibit a 10-fold increase in potency in a cellular assay. Interleukin (IL)2 -21 is a recently identified type 1 cytokine, which is secreted as a 133-amino acid protein by activated CD4 ϩ T cells (1). The IL-21 cytokine has been demonstrated to possess potent stimulatory effects on the proliferation, differentiation, and activation of several classes of hematopoietic cells, including B-cells, T-cells, and NK-cells. The biological effects of IL-21 are mediated via activation of the IL-21 receptor complex, which is composed of an IL-21 private receptor chain (IL-21R␣) in complex with the common ␥ chain (␥ c ), which similarly constitutes an essential component of the signaling receptor complex of the cytokines IL-2, IL-4, IL-7, IL-9, and IL-15. These cytokines thus constitute a subfamily referred to as common ␥ chain cytokines, with IL-21 being the most recently added member (2).Within the common ␥ chain family of cytokines, high resolution structural information has been obtained through x-ray crystallography and NMR spectroscopy for IL-2 and IL-4 (3-8). It is apparent from these studies that IL-2 and IL-4 along with other type 1 cytokines, including IL-6 and granulocyte-macrophage colony-stimulating factor, share a common overall topology in their structures despite a distant homology in sequence (9). The common structural motif of these proteins consists of a central four-helical bundle, arranged in an up-updown-down topology, connected by loops that are characterized by a high degree of structural freedom, a considerable difference in loop length, and variation in the number and positioning of stabilizing disulfide bridges.Crystal structures have also been reported for IL-2 and IL-4 in complex with the corresponding private chains and, in the case of IL-2, the common ␥ chain (10 -12). IL-2 is distinct from both IL-4 and IL-21 by having two private receptor chains, IL-2R␣ and IL-2R␤, where IL-2R␤ is homologous to IL-4R␣ and IL-21R␣. Only minor structural differences are observed between the free and receptor-bound forms of IL-2 and IL-4, indicating that only slight stru...

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Pannexin-1 (Panx1) forms nonselective large channel in cell plasma membrane and has been shown to be associated with NLRP3 inflammasome activation, ATP release and phagocytes recruitment. In the current study, by manipulation of Panx1 expression in human myeloid cells and application of Panx1 deficient mice, we failed to find a correlation between Panx1 and NLRP3 inflammasome activation, although an interaction between these two proteins was evident. However, in thioglycollate induced peritonitis, Panx1 deficient mice showed much more phagocytes infiltration. Further analyses showed that mice deficient for Panx1 exhibited enlarged F4/80(low)Gr1(-)Ly6C(-)cell population in the peritonea. Our study thus reveals an important role for Panx1 in regulation of peritoneal cell population and peritonitis development.

D-Alanine is a central component of the cell wall in most prokaryotes. D-Alanine synthesis in Escherichia coliis carried out by two different alanine racemases encoded by the alr and dadX genes. Deletion of alr and dadX from the E. coli genome results in a D-alanine auxotrophic phenotype. However, we have observed growth of prototrophic phenotypic revertants during routine culturing of a D-alanine auxotrophic strain. We present a detailed comparison of the proteome and transcriptome profiles of the D-alanine auxotroph and a prototrophic revertant strain. Most noticeably, a general upregulation of genes involved in methionine synthesis in the revertant strain was detected. The appearance of the revertant phenotype was genetically linked to point mutations in the methionine repressor gene (metJ). Our results reveal an alternative metabolic pathway which can supply essential D-alanine for peptidoglycan synthesis of alr-and dadX-deficient E. coli mutants and provide evidence for significant alanine racemase coactivity of the E. coli cystathionine beta-lyase (MetC).Alanine racemases (EC 5.1.1.1) are unique prokaryotic enzymes that catalyze the reversible racemization of L-and Dalanine, the latter one being an essential component in the biosynthesis of the bacterial peptidoglycan of Gram-positive and Gram-negative bacteria (47). The bacteria investigated to date have been found to possess either one or two distinct alanine racemase genes. The alr gene encodes a constitutively expressed alanine racemase, which provides D-alanine for sufficient cross-linking of adjacent peptidoglycan strands in the cell wall. The second gene encodes the so-called catabolic alanine racemase, which is essential for L-alanine catabolism (24,28,41,42,48). In Escherichia coli, the alr-encoded alanine racemase is constitutively expressed, whereas the dadX-encoded enzyme is essential only for L-alanine catabolism, providing a substrate for a D-alanine-specific dehydrogenase encoded by the dadA gene (51). The dadX gene product provides a secondary source of D-alanine for cell wall biosynthesis.D-Alanine auxotrophic E. coli, Bacillus subtilis, Corynebacterium glutamicum, Listeria monocytogenes, and Lactobacillus plantarum strains have been generated by inactivating genes encoding alanine racemases (15,17,24,42,43,45). A strong selective pressure for maintenance of an alanine racemase (Dal)-encoding plasmid in a chromosomal dal mutant of Bacillus subtilis was observed upon growth on rich medium. In Lactobacillus plantarum, plasmids encoding alanine racemase (Alr) were efficiently selected in an alr-deficient Lactobacillus plantarum strain (5). In Listeria monocytogenes, two genes, an alanine racemase gene (dal) and a D-amino acid aminotransferase gene (dat), which control the synthesis of D-alanine, had to be inactivated in order to achieve complete D-alanine auxotrophy (46).Under certain circumstances, the D-alanine auxotrophic phenotype was lost, indicating a redundancy of alanine racemase activity in bacteria. The D-alanine auxotrophic phenoty...