Identification of viral macrophage inflammatory protein (vMIP)-II as a ligand for GPR5/XCR1

Lymphotactin is unique among chemokines in that it contains only two of four conserved cysteines and may possess a structure less constrained than other chemokines. The viral chemokine vMIP-II, which presumably has a structure similar to that of CC chemokines has been shown to inhibit many chemokine receptors, but its activity at GPR5/XCR1 has not been described. Interestingly, vMIP-II (but not vMIP-I) was found to be a potent antagonist of lymphotactin activity at GPR5/XCR1, extending the range of chemokine classes that this viral protein is known to inhibit to include the C class chemokine. In addition, we have extended previous analyses of GPR5/XCR1 expression and show that this receptor is expressed in leukocyte cells previously shown to be responsive to lymphotactin.     

Cloning of BRAK, a novel divergent CXC chemokine preferentially expressed in normal versus malignant cells

Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in many disease processes. Chemokines are divided into two major groups, CC or CXC, based on their sequence around the amino terminal cysteines. We report the PCR cloning of a novel human chemokine termed BRAK for its initial isolation from breast and kidney cells. This novel chemokine is distantly related to other CXC chemokines (30% identity with MIP-2alpha and beta) and shares several biological activities. BRAK is expressed ubiquitously and highly in normal tissue. However, it was expressed in only 2 of 18 cancer cell lines. BRAK is located on human chromosome 5q31.     

Isolation of ALP, a novel divergent murine CC chemokine with a unique carboxy terminal extension.

Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in many disease processes. Chemokines are divided into two major groups, CC or CXC, based on their sequence around the amino terminal cysteines. We report here, the isolation of a novel murine CC chemokine termed ALP for its amino terminal peptide sequence. This novel chemokine is distantly related to other CC chemokines (37% identity with murine Exodus-1/LARC/Mip-3alpha), but has a unique carboxy terminal extension. It is expressed preferentially in testis, heart, and liver, which is atypical for CC chemokines.     

Truncation of NH2-terminal amino acid residues increases agonistic potency of leukotactin-1 on CC chemokine receptors 1 and 3

Leukotactin-1 (Lkn-1) is a human CC chemokine that binds to both CC chemokine receptor 1 (CCR1) and CCR3. Structurally, Lkn-1 is distinct from other human CC chemokines in that it has long amino acid residues preceding the first cysteine at the NH(2) terminus, and contains two extra cysteines. NH(2)-terminal amino acids of Lkn-1 were deleted serially, and the effects of each deletion were investigated. In CCR1-expressing cells, serial deletion up to 20 amino acids (Delta20) did not change the calcium flux-inducing activity significantly. Deletion of 24 amino acids (Delta24), however, increased the agonistic potency approximately 100-fold. Deletion of 27 or 28 amino acids also increased the agonistic potency to the same level shown by Delta24. Deletion of 29 amino acids, however, abolished the agonistic activity almost completely showing that at least 3 amino acid residues preceding the first cysteine at the NH(2) terminus are essential for the biological activity of Lkn-1. Loss of agonistic activity was due to impaired binding to CCR1. In CCR3-expressing cells, Delta24 was the only form of Lkn-1 mutants that revealed increased agonistic potency. Our results indicate that posttranslational modification is a potential mechanism for the regulation of biological activity of Lkn-1.     

Monocyte chemotactic protein-3.

Monocyte chemotactic protein-3 (MCP-3) belongs to the MCP subgroup of CC chemokines that are structurally closely related but, which differ in receptor usage and hence in biological activities. MCP-3 is one of the most pluripotent chemokines since it activates all types of leukocytes, by binding to at least four different chemokine receptors. The natural protein is heterogeneous due to glycosylation and NH2-terminal processing. Only small amounts of MCP-3 are induced in various cell types by endogeneous (cytokines) or exogeneous (bacteria, viruses) agents. Nevertheless, this omnipotent chemokine, inducible in most body compartments, might play an important role in normal homeostasis as well as in various pathologies including cancer, auto-immune diseases and chronic inflammation.     

CXC and CC chemokines form mixed heterodimers: association free energies from molecular dynamics simulations and experimental correlations

CXC and CC chemokines are involved in numerous biological processes, and their function in situ may be significantly influenced by heterodimer formation, as was recently reported, for example, for CXC chemokines CXCL4/PF4 and CXCL8/IL8 that interact to form heterodimers that modulate chemotactic and cell proliferation activities. Here we used molecular dynamics simulations to determine relative association free energies (overall average and per residue) for homo- and heterodimer pairs of CXC (CXCL4/PF4, CXCL8/IL8, CXCL1/Gro-alpha, and CXCL7/NAP-2) and CC (CCL5/RANTES, CCL2/MCP-1, and CCL8/MCP-2) chemokines. Even though structural homology among monomer folds of all CXC and CC chemokines permits heterodimer assembly, our calculated association free energies depend upon the particular pair of chemokines in terms of the net electrostatic and nonelectrostatic forces involved, as well as (for CC/CXC mixed chemokines) the selection of dimer type (CC or CXC). These relative free energies indicate that association of some pairs of chemokines is more favorable than others. Our approach is validated by correlation of calculated and experimentally determined free energies. Results are discussed in terms of CXC and CC chemokine function and have significant biological implications.     

An inflammatory CC chemokine of Cynoglossus semilaevis is involved in immune defense against bacterial infection

Chemokines are a family of small cytokines that regulate leukocyte migration. Based on the arrangement of the first two cysteine residues, chemokines are classified into four groups called CXC(α), CC(β), C, and CX(3)C. In this study, we identified a CC chemokine, CsCCK1, from half-smooth tongue sole (Cynoglossus semilaevis) and analyzed its biological activity. The deduced amino acid sequence of CsCCK1 contains 111 amino acid residues and is phylogenetically belonging to the CCL19/21/25 group of CC chemokines. CsCCK1 possesses a DCCL motif that is highly conserved among CC chemokines. Quantitative real time RT-PCR analysis showed that the expression of CsCCK1 was relatively abundant in immune organs under normal physiological conditions and was upregulated by experimental infection of a bacterial pathogen. Purified recombinant CsCCK1 (rCsCCK1) induced chemotaxis in peripheral blood leukocytes (PBL) of both tongue sole and turbot (Scophthalmus maximus) in a dose-dependent manner. Mutation of the CC residues in the DCCL motif by serine substitution completely abolished the biological activity of rCsCCK1. When rCsCCK1, but not the mutant protein, was added to the cell culture of PBL, it enhanced cellular resistance against intracellular bacterial infection. Taken together, these results indicate that CsCCK1 is a functional CC chemokine whose biological activity depends on the DCCL motif and that CsCCK1 plays a role in host immune defense against bacterial infection.     

Probing the Role of CXC Motif in Chemokine CXCL8 for High Affinity Binding and Activation of CXCR1 and CXCR2 Receptors

All chemokines share a common structural scaffold that mediate a remarkable variety of functions from immune surveillance to organogenesis. Chemokines are classified as CXC or CC on the basis of conserved cysteines, and the two subclasses bind distinct sets of GPCR class of receptors and also have markedly different quaternary structures, suggesting that the CXC/CC motif plays a prominent role in both structure and function. For both classes, receptor activation involves interactions between chemokine N-loop and receptor N-domain residues (Site-I), and between chemokine N-terminal and receptor extracellular/transmembrane residues (Site-II). We engineered a CC variant (labeled as CC-CXCL8) of the chemokine CXCL8 by deleting residue X (CXC → CC), and found its structure is essentially similar to WT. In stark contrast, CC-CXCL8 bound poorly to its cognate receptors CXCR1 and CXCR2 (K_i > 1 μm). Further, CC-CXCL8 failed to mobilize Ca²⁺ in CXCR2-expressing HL-60 cells or recruit neutrophils in a mouse lung model. However, most interestingly, CC-CXCL8 mobilizes Ca²⁺ in neutrophils and in CXCR1-expressing HL-60 cells. Compared with the WT, CC-CXCL8 binds CXCR1 N-domain with only ∼5-fold lower affinity indicating that the weak binding to intact CXCR1 must be due to its weak binding at Site-II. Nevertheless, this level of binding is sufficient for receptor activation indicating that affinity and activity are separable functions. We propose that the CXC motif functions as a conformational switch that couples Site-I and Site-II interactions for both receptors, and that this coupling is critical for high affinity binding but differentially regulates activation.     

An engineered second disulfide bond restricts lymphotactin/XCL1 to a chemokine-like conformation with XCR1 agonist activity

Chemokines adopt a conserved tertiary structure stabilized by two disulfide bridges and direct the migration of leukocytes. Lymphotactin (Ltn) is a unique chemokine in that it contains only one disulfide and exhibits large-scale structural heterogeneity. Under physiological solution conditions (37 degrees C and 150 mM NaCl), Ltn is in equilibrium between the canonical chemokine fold (Ltn10) and a distinct four-stranded beta-sheet (Ltn40). Consequently, it has not been possible to address the biological significance of each structural species independently. To stabilize the Ltn10 structure in a manner independent of specific solution conditions, Ltn variants containing a second disulfide bridge were designed. Placement of the new cysteines was based on a sequence alignment of Ltn with either the first (Ltn-CC1) or third disulfide (Ltn-CC3) in the CC chemokine, HCC-2. NMR data demonstrate that both CC1 and CC3 retain the Ltn10 chemokine structure and no longer exhibit structural rearrangement. The ability of each mutant to activate the Ltn receptor, XCR1, has been tested using an intracellular Ca2+ flux assay. These data support the conclusion that the chemokine fold of Ltn10 is responsible for receptor activation. We also examined the role of amino- and carboxyl-terminal residues in Ltn-mediated receptor activation. In contrast to previous reports, we find that the 25 residues comprising the novel C-terminal extension do not participate in receptor activation, while the native N-terminus is absolutely required for Ltn function.     

Molecular cloning and genomic structure of an interleukin-8 receptor-like gene from homozygous clones of rainbow trout (Oncorhynchus mykiss)

Chemokines are small proteins (70-100 amino acids) which play an important role in recruitment and activation of leucocytes to migrate to the site of inflammation. Based on the position of the first two conserved cysteines, chemokines are classified into four subfamilies: C, CC, CXC and CX3C. To date, many members of CC and CXC have been found and studied extensively [1]. Chemokines exert effects on their target cell via chemokine receptors, which are G-protein coupled receptors containing seven transmembrane domains with an extracellular N-terminus and an intracellular C-terminus [2]. Interleukin 8 (IL-8) belongs to the CXC chemokine subfamily. It can activate and attract migratory neutrophils to an inflammation site. Two IL-8 receptors, CXCR1 and CXCR2, have been identified in mammals [3-6]; both of these receptors have high affinity for IL-8 and are expressed on the neutrophil. CXCR1 just binds IL-8; however, CXCR2 binds IL-8 and other structurally related chemokines such as growth-related oncogene (GRO) a, GRObeta, GROgamma, neutrophil-activating peptide-2 (NAP-2) and epithelial cell-derived neutrophil activating peptide-78 (ENA-78) [7, 8]. Several studies on fish chemokine receptors have been reported [9-11]. Thus far, however, IL-8 and CXCR1 and CXCR2 proteins from rainbow trout have not been reported: however, the sequence of a rainbow trout IL-8 has been noted (GenBank Accession No. AJ279069 [12]). Cloning of the IL-8 receptor is important to study the function of IL-8/CXCR1 and (CXCR2) in inflammation and signal transduction in fish. This paper reports the molecular cloning and genomic structure of an IL-8 receptor-like gene from four homozygous clones of rainbow trout: Oregon State University (OSU), Hot Creek (HC), Arlee (AR) and Swanson (SW).     

CC类趋化因子亚家族与心肌梗死的研究进展

CC类趋化因子亚家族是趋化因子家族中成员最多、研究最广泛的一大类细胞因子,其主要功能参与炎症细胞激活、迁移、粘附等病理生理过程。大量研究表明,CC类趋化因子亚家族成员参与了心肌梗死后病理过程的各个阶段。其中研究最为深入的为单核细胞趋化蛋白-1（monocyte chemoattractant protein-1,MCP-1）及其受体CC趋化因子受体2（CC chemokine receptor 2,CCR2）,在心肌梗死后炎症期、增殖期及疤痕愈合期都发挥了重要作用从而影响梗死后心室重构。近年来,CC类趋化因子亚家族其他成员亦被逐渐揭示参与了心肌梗死的发展。本文结合以往大量文献将对CC类趋化因子亚家族在心肌梗死各个阶段中尤其是梗死后各期对于心室重构的影响进行综述,以期为今后的实验研究提供方向及疾病的预防和治疗提供药物靶点。     

Identification of chemokines and a chemokine receptor in cichlid fish,shark, and lamprey

Chemokines are small, inducible, structurally related proteins that guide cells expressing the right chemokine receptors to sites of immune response. They have been identified and studied extensively in mammals, but little is known about their presence in other vertebrate groups. Here we describe seven new chemokines in bony fish and one in a cartilaginous fish, as well as one chemokine receptor in a jawless vertebrate. All eight chemokines belong to the SCYA (CC) subfamily characterized by four conserved cysteine residues of which the first two are adjacent. The chemokine receptor is of the CXCR4 type. Phylogenetic analysis does not reveal any clear evidence of orthology of fish and human chemokines. Although the divergence of the subfamilies began before the fish-tetrapod split, much of the divergence within the subfamilies took place separately in the two vertebrate groups. The existence of a chemokine receptor in the lamprey indicates that chemokines are apparently also present in the Agnatha.     

The crystal structure of the chemokine domain of fractalkine shows a novel quaternary arrangement

Fractalkine, or neurotactin, is a chemokine that is present in endothelial cells from several tissues, including brain, liver, and kidney. It is the only member of the CX(3)C class of chemokines. Fractalkine contains a chemokine domain (CDF) attached to a membrane-spanning domain via a mucin-like stalk. However, fractalkine can also be proteolytically cleaved from its membrane-spanning domain to release a freely diffusible form. Fractalkine attracts and immobilizes leukocytes by binding to its receptor, CX(3)CR1. The x-ray crystal structure of CDF has been solved and refined to 2.0 A resolution. The CDF monomers form a dimer through an intermolecular beta-sheet. This interaction is somewhat similar to that seen in other dimeric CC chemokine crystal structures. However, the displacement of the first disulfide in CDF causes the dimer to assume a more compact quaternary structure relative to CC chemokines, which is unique to CX(3)C chemokines. Although fractalkine can bind to heparin in vitro, as shown by comparison of electrostatic surface plots with other chemokines and by heparin chromatography, the role of this property in vivo is not well understood.     

Differentiation of CD34+ cells from human cord blood and murine bone marrow is suppressed by C6 beta-chemokines

Several recently identified chemokines, Lkn-1, CKbeta8-1, MRP-2, and Mu C10 (MRP-1), are classified as C6 beta-chemokines. All of these chemokines have been found to suppress colony formation by bone marrow (BM) myeloid progenitors. Since cord blood (CB), like BM, contains CD34-positive cells, we examined the effects of these chemokines on CD34+ cells isolated from human CB. Lkn-1 and CKbeta8-1 suppressed colony formation by multi-potential granulocyte erythroid mega-karyocyte macrophages (CFU-GEMM), granulocyte-macrophages (CFU-GM), and erythroid (BFU-E) cells among the CD34+ cells from CB. CC chemokine receptor 1 (CCR1) that is known to be a receptor for Lkn-1 and CKbeta8-1 in neutrophils, monocytes, and lymphocytes, was also present on the surface of CD34+ cells from CB. Taken together these results suggest that Lkn-1 and CKbeta8-1 are active in inhibiting myeloid progenitor cells from both BM and CB. Macrophage inflammatory protein related protein-2 (mMRP-2) and Mu C10 (mMRP-1), which are murine C6 beta-chemokines, also inhibited colony formation by CB CD34+ cells. The inhibitory activity of these chemokines suggests that they may protect hematopoietic progenitors from the cytotoxic effects of the antiblastic drugs used in cancer therapy.     

Peptide mimics of monocyte chemoattractant protein-1 (MCP-1) with an antagonistic activity

In this study, we attempted to analyze the peptide motifs recognized by 24822.111 and F9, monoclonal antibodies (mAbs) that inhibit the chemotactic activity of monocyte chemoattractant protein-1 (MCP-1), a member of the CC subfamily of chemokines. We isolated phage clones from a phage display library and identified six peptide motifs. One of these clones, C27, was strongly and specifically recognized by 24822.111 mAb, while another, G25, was similarly recognized by F9 mAb. Both the C27 motif and the G25 motif contain two cysteines in their sequences and have little homology to the primary amino acid sequence of MCP-1. These clones, however, bound to THP-1 cells, and the binding was competitively inhibited by MCP-1. The clones strongly inhibited the MCP-1-induced chemotaxis of human monocytes. The synthetic and intramolecularly disulfide-linked peptides of C27 and G25 (sC27 and sG25) also inhibited the chemotaxis induced by MCP-1, while their derivatives with serine in place of cysteine did not, suggesting the importance of the loop structure for the inhibition. These results suggest that sC27 and sG25 may mimic the MCP-1-binding domain to the MCP-1 receptor.     

Crystal structure of viral macrophage inflammatory protein I encoded by Kaposi's sarcoma-associated herpesvirus at 1.7A

Viral macrophage inflammatory protein I (vMIP-I) is a chemokine encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) that selectively activates the CC chemokine receptor 8 (CCR8), for which the endogenous ligand is CCL1. The crystal structure of vMIP-I was determined at 1.7A for comparison with other chemokines, especially those that bind CCR8, such as vMIP-II from KSHV, a CCR8 antagonist and the closest homolog (40% identical). vMIP-I has a typical chemokine fold consisting of an extended N-terminal loop, followed by a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix. The four molecules in the asymmetric unit comprise two MIP-1beta-like dimers. Electrostatic surface representations of CCR8-binding chemokines reveal only minor areas of correlating surface potential, which must be reconciled with promiscuity in receptor and glycosaminoglycan (GAG) binding. In addition, the biological relevance of chemokine oligomerization is examined by comparing the oligomeric states of all chemokine structures deposited to date in the RCSB PDB.     

The redox switch of gamma-glutamylcysteine ligase via a reversible monomer-dimer transition is a mechanism unique to plants

In plants, the first committed enzyme for glutathione biosynthesis, γ-glutamylcysteine ligase (GCL), is under multiple controls. The recent elucidation of GCL structure from Brassica juncea (BjGCL) has revealed the presence of two intramolecular disulfide bridges (CC1, CC2), which both strongly impact on GCL activity in vitro . Here we demonstrate that cysteines of CC1 are confined to plant species from the Rosids clade, and are absent in other plant families. Conversely, cysteines of CC2 involved in the monomer–dimer transition in BjGCL are not only conserved in the plant kingdom, but are also conserved in the evolutionarily related α- (and some γ-) proteobacterial GCLs. Focusing on the role of CC2 for GCL redox regulation, we have extended our analysis to all available plant (31; including moss and algal) and related proteobacterial GCL (46) protein sequences. Amino acids contributing to the homodimer interface in BjGCL are highly conserved among plant GCLs, but are not conserved in related proteobacterial GCLs. To probe the significance of this distinction, recombinant GCLs from Nicotiana tabacum (NtGCL), Agrobacterium tumefaciens (AtuGCL, α-proteobacteria) and Xanthomonas campestris (XcaGCL, γ-proteobacteria) were analyzed for their redox response. As expected, NtGCL forms a homodimer under oxidizing conditions, and is activated more than threefold. Conversely, proteobacterial GCLs remain monomeric under oxidizing and reducing conditions, and their activities are not inhibited by DTT, despite the presence of CC2. We conclude that although plant GCLs are evolutionarily related to proteobacterial GCLs, redox regulation of their GCLs via CC2-dependent dimerization has been acquired later in evolution, possibly as a consequence of compartmentation in the redox-modulated plastid environment.     

Structural insight into the evolution of a new chemokine family from zebrafish

The mammalian chemokine family is segregated into four families – CC, CXC, CX3C, and XC—based on the arrangement of cysteines and the corresponding disulfides. Sequencing of the Danio rerio (zebrafish) genome has identified more than double the amount of human chemokines with the absence of the CX3C family and the presence of a new family, CX. The only other family with a single cysteine in the N‐terminal region is the XC family. Human lymphotactin (XCL1) has two interconverting structures due to dynamic changes that occur in the protein. Similar to an experiment with XCL1 that identified the two structural forms, we probed for multiple forms of zCXL1 using heparin affinity. The results suggest only a single form of CXL1 is present. We used sulfur‐SAD phasing to determine the three‐dimensional structure CXL1. Zebrafish CXL1 (zCXL1) has three disulfides that appear to be important for a stable structure. One disulfide is common to all chemokines except those that belong to the XC family, another is similar to a subset of CC chemokines containing three disulfides, but the third disulfide is unique to the CX family. We analyzed the electrostatic potential of the zCXL1 structure and identified the likely heparin‐binding site for glycosaminoglycans (GAGs). zCXL1 has a similar sequence identity with human CCL5 and CXCL12, but the structure is more related to CCL5. Our structural analysis supports the phylogenetic and genomic studies on the evolution of the CXL family. Proteins 2014; 82:708–716. © 2013 Wiley Periodicals, Inc.