Chemical synthesis, purification, and folding of C-X-C and C-C chemokines.
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Chemokine CCL2↗
Biomedical subjects
I Clark-Lewis
Publications and source records attributed to I Clark-Lewis.
The purified myxoma virus gamma interferon receptor homolog M-T7 interacts with the heparin-binding domains of chemokines.
The myxoma virus T7 protein M-T7 is a functional soluble gamma interferon receptor homolog that has previously been shown to bind gamma interferon and inhibit its antiviral activities in a species-specific manner, but gene knockout analysis has suggested a further role for M-T7 in blocking leukocyte influx into infected lesions. We purified M-T7 to apparent homogeneity and showed that M-T7 is an N-linked glycoprotein that appears to be a stable homotrimer with a molecular mass of approximately 113 kDa in solution. M-T7, in addition to forming inhibitory complexes with rabbit gamma interferon, was also shown to bind to human interleukin-8, a prototypic member of the chemokine superfamily. Moreover, M-T7 was able to interact promiscuously with all members of the CXC, CC, and C chemokine subfamilies tested. Binding of human RANTES to M-T7 can be competed by rabbit gamma interferon and also by cold RANTES competitor with a 50% inhibitory concentration of 900 nM. Although M-T7 retains binding to a number of interleukin-8 N-terminal (ELR) deletion mutants, binding to mutants containing deletions in the C-terminal heparin-binding domain of interleukin-8 is abrogated. Furthermore, heparin effectively competes the interaction of M-T7 with the chemokine RANTES but not with rabbit gamma interferon. We propose that this novel M-T7 interaction with members of the chemokine superfamily may be facilitated through the conserved heparin-binding domains found in a wide spectrum of chemokines and that M-T7 may function by modulating chemokine-glycosaminoglycan interactions in virus-infected tissues.
Animals↗
The GM-CSF analogue E21R induces apoptosis of normal and activated eosinophils.
There is evidence that eosinophils have an important role in the pathogenesis of allergy and asthma. These cells are regulated by two classes of polypeptides, the colony-stimulating factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), and the chemokines, such as RANTES and eotaxin. GM-CSF is involved in the production, survival, and functional activation of eosinophils. RANTES and eotaxin regulate the migration of eosinophils to inflammatory sites, but any effect of these chemokines on eosinophil survival is not known. In this study we demonstrate that although GM-CSF promoted eosinophil survival, the specific GM-CSF analogue E21R induced apoptosis of eosinophils. Apoptosis was observed with unstimulated as well as with chemokine (RANTES and eotaxin)-activated eosinophils. Neither RANTES nor eotaxin supported eosinophil survival, and a RANTES antagonist did not affect either cell survival or apoptosis. E21R also induced apoptosis of eosinophils from asthmatic patients. These findings suggest that the GM-CSF receptor may actively control the death as well as the survival of eosinophils, and thus precisely regulate their numbers and activities. Our data also indicate that chemokines are not involved in regulating the lifespan of eosinophils. The introduction of the GM-CSF analogue E21R may offer a novel therapy in inflammatory diseases associated with eosinophil infiltration of different etiologies.
Apoptosis↗
Phosphorylation site substrate specificity determinants for the Pim-1 protooncogene-encoded protein kinase.
Pim-1 is an oncogene-encoded serine-threonine kinase that is expressed primarily in cells of the hematopoietic system and germ line. The full-length coding regions of both human and Xenopus laevis Pim-1 were expressed as recombinant bacterial fusion proteins that autophosphorylated in vitro and exhibited phosphotransferase activity towards various exogenous substrates. The consensus sequence for phosphorylation by Pim-1 was defined by stepwise replacement of the amino acids in peptide substrate analogues based on the carboxyl-terminal segment of human ribosomal protein S6 (residues 229-249). The optimal substrate peptide for Pim-1 was determined to be Lys/Arg-Lys/Arg-Arg-Lys/Arg-Leu-Ser/Thr-X, where X is an amino acid residue with a small side chain. These results were confirmed using X. laevis Pim-1 expressed in COS cells. These findings could permit the identification of physiological substrates of Pim-1 and predict the location of phosphorylation sites within these proteins.
Amino Acid Sequence↗
1H NMR evidence that Glu-38 interacts with the N-terminal functional domain in interleukin-8.
In order to assess the importance of the buried Glu-38 observed in the structure of interleukin-8, an analog in which Glu-38 was replaced with Ala (E38A analog) was investigated by 1H NMR spectroscopy and neutrophil activation. Detailed analysis of the NMR NOESY data showed that the solution structure of the E38A analog is essentially the same as that for the native protein. Also, the neutrophil elastase activity of the E38A analog was similar to that of the native protein. However, the Gln-8 and Cys-9 amide proton chemical shifts, which are significantly downfield-shifted in the native protein, exhibit more 'normal' values. This observation indicates that in the native protein, Glu-38 side-chain carboxylate interacts with Gln-8 and Cys-9 amide protons. Although the N-terminal residues are critical for function, this interaction is not essential for neutrophil activation.
Amino Acid Sequence↗
Efficient interaction of HIV-1 with purified dendritic cells via multiple chemokine coreceptors.
HIV-1 actively replicates in dendritic cell (DC)-T cell cocultures, but it has been difficult to demonstrate substantial infection of purified mature DCs. We now find that HIV-1 begins reverse transcription much more efficiently in DCs than T cells, even though T cells have higher levels of CD4 and gp120 binding. DCs isolated from skin or from blood precursors behave similarly. Several M-tropic strains and the T-tropic strain IIIB enter DCs efficiently, as assessed by the progressive formation of the early products of reverse transcription after a 90-min virus pulse at 37 degrees C. However, few late gag-containing sequences are detected, so that active viral replication does not occur. The formation of these early transcripts seems to follow entry of HIV-1, rather than binding of virions that contain viral DNA. Early transcripts are scarce if DCs are exposed to virus on ice for 4 h, or for 90 min at 37 degrees C, conditions which allow virus binding. Also the early transcripts once formed are insensitive to trypsin. The entry of a M-tropic isolates is blocked by the chemokine RANTES, and the entry of IIIB by SDF-1. RANTES interacts with CCR5 and SDF-1 with CXCR4 receptors. Entry of M-tropic but not T-tropic virus is ablated in DCs from individuals who lack a functional CCR5 receptor. DCs express more CCR5 and CXCR4 mRNA than T cells. Therefore, while HIV-1 does not replicate efficiently in mature DCs, viral entry can be active and can be blocked by chemokines that act on known receptors for M- and T-tropic virus.
Cells, Cultured↗
Structural characterization of a monomeric chemokine: monocyte chemoattractant protein-3.
1H-NMR spectroscopy and analytical ultracentrifugation studies reveal that monocyte chemoattractant protein-3 (MCP-3) is a monomer. NMR solution structure shows that MCP-3 adopts an alphabeta fold similar to what is observed in structures of other known chemokines. However, MCP-3 is unique in that it does not show a propensity to form dimers. The closely related chemokines MCP-1 and MCP-2 show a monomer-dimer equilibrium in sedimentation equilibrium studies (approximately 0.2-2 mg/ml). As these proteins are present at nanomolar concentrations in vivo, the results suggest that they are monomeric at functional concentrations and that the monomer is the functionally significant form of MCP-1, MCP-2 and MCP-3.
Amino Acid Sequence↗
HIV blocked by chemokine antagonist.
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Anti-HIV Agents↗
Chemokine receptor specific for IP10 and mig: structure, function, and expression in activated T-lymphocytes.
A human receptor that is selective for the CXC chemokines IP10 and Mig was cloned and characterized. The receptor cDNA has an open reading frame of 1104-bp encoding a protein of 368 amino acids with a molecular mass of 40,659 dalton. The sequence includes seven putative transmembrane segments characteristic of G-protein coupled receptors. It shares 40.9 and 40.3% identical amino acids with the two IL-8 receptors, and 34.2-36.9% identity with the five known CC chemokine receptors. The IP10/Mig receptor is highly expressed in IL-2-activated T lymphocytes, but is not detectable in resting T lymphocytes. B lymphocytes, monocytes and granulocytes. It mediates Ca2+ mobilization and chemotaxis in response to IP10 and Mig, but does not recognize the CXC-chemokines IL-8, GRO alpha, NAP-2, GCP-2. ENA78, PF4, the CC-chemokines MCP-1, MCP-2, MCP-3, MCP-4, MIP-1 alpha, MIP-1 beta. RANTES, 1309, eotaxin, nor lymphotactin. The exclusive expression in activated T-lymphocytes is of high interest since the receptors for chemokines which have been shown so far to attract lymphocytes, e.g., MCP-1, MCP-2, MCP-3, MIP-1 alpha, MIP-1 beta, and RANTES, are also found in monocytes and granulocytes. The present observations suggest that the IP10/Mig receptor is involved in the selective recruitment of effector T cells.
Amino Acid Sequence↗
The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry.
Chemokines are chemotactic cytokines that activate and direct the migration of leukocytes. There are two subfamilies, the CXC and the CC chemokines. We recently found that the CXC-chemokine stromal cell-derived factor-1 (SDF-1) is a highly efficacious lymphocyte chemoattractant. Chemokines act on responsive leukocyte subsets through G-protein-coupled seven-transmembrane receptors, which are also used by distinct strains of HIV-1 as cofactors for viral entry. Laboratory-adapted and some T-cell-line-tropic (T-tropic) primary viruses use the orphan chemokine receptor LESTR/fusin (also known as fusin), whereas macrophage-tropic primary HIV-1 isolates use CCR-5 and CCR-3 (refs 7-11), which are receptors for known CC chemokines. Testing of potential receptors demonstrated that SDF-1 signalled through, and hence 'adopted', the orphan receptor LESTR, which we therefore designate CXC-chemokine receptor-4 (CXCR-4). SDF-1 induced an increase in intracellular free Ca2+ and chemotaxis in CXCR-4-transfected cells. Because SDF-1 is a biological ligand for the HIV-1 entry cofactor LESTR, we tested whether it inhibited HIV-1. SDF-1 inhibited infection by T-tropic HIV-1 of HeLa-CD4 cells, CXCR-4 transfectants, and peripheral blood mononuclear cells (PBMCs), but did not affect CCR-5-mediated infection by macrophage-tropic (M-tropic) and dual-tropic primary HIV-1.
Animals↗
The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cell-line-adapted HIV-1.
A putative chemokine receptor that we previously cloned and termed LESTR has recently been shown to function as a co-receptor (termed fusin) for lymphocyte-tropic HIV-1 strains. Cells expressing CD4 became permissive to infection with T-cell-line-adapted HIV-1 strains of the syncytium-inducing phenotype after transfection with LESTR/fusin complementary DNA. We report here the indentification of a human chemokine of the CXC type, stromal cell-derived factor 1 (SDF-1), as the natural ligand for LESTR/fusin, and we propose the term CXCR-4 for this receptor, in keeping with the new chemokine-receptor nomenclature. SDF-1 activates Chinese hamster ovary (CHO) cells transfected with CXCR-4 cDNA as well as blood leukocytes and lymphocytes. In cell lines expressing CXCR-4 and CD4, and in blood lymphocytes, SDF-1 is a powerful inhibitor of infection by lymphocyte-tropic HIV-1 strains, whereas the CC chemokines RANTES, MIP-1 alpha and MIP-1 beta, which were shown previously to prevent infection with primary, monocyte-tropic viruses, are inactive. In combination with CC chemokines, which block the infection with monocyte/macrophage-tropic viruses, SDF-1 could help to decrease virus load and prevent the emergence of the syncytium-inducing viruses which are characteristic of the late stages of AIDS.
Animals↗
RANTES and MCP-3 antagonists bind multiple chemokine receptors.
Antagonists of multiple chemokines could be more effective than inhibitors of specific chemokines for controlling cell migration and inflammation. To attempt to identify such antagonists we characterized a number of truncated analogs of regulated on activation normal T cell expressed protein (RANTES), monocyte chemoattractant protein (MCP)-3, and MCP-1. On the basis of their ability to compete for binding of their parent chemokines, three analogs were selected for cross-reactivity studies: RANTES (9-68), MCP-3 (10-76), and MCP-1 (9-76). These analogs bound to THP-1 monocytic cells with dissociation constants that were within 4-6-fold of their native counterparts, but they did not promote detectable chemotaxis of THP-1 cells or enzyme release from purified human monocytes. The RANTES (9-68) analog competed for the binding and inhibited the activities of all three chemokines. In contrast, native RANTES was specific for RANTES binding sites. However, truncation of either MCP-1 or MCP-3 did not change their respective binding specificity. MCP-3 and MCP-3 (10-76) competed for binding of all three labeled chemokines. MCP-1 (9-76) competed strongly for binding of labeled MCP-1, but only weakly for the other two labeled ligands and inhibited the activities induced by MCP-1 and MCP-3 but not RANTES. Although RANTES (9-68) and MCP-3 (10-76) inhibited all three chemokines, the RANTES analog was significantly more potent for RANTES-induced activity. The results indicate that NH2-terminal residues partly determine the receptor specificity of RANTES, and deletions within this region permit binding to multiple chemokine receptors. The findings suggest the feasibility of design of high affinity multi-specific CC chemokine antagonists.
Amino Acid Sequence↗
Deletion of the NH2-terminal residue converts monocyte chemotactic protein 1 from an activator of basophil mediator release to an eosinophil chemoattractant.
Chemotactic cytokines of the CC subfamily (CC chemokines) are considered as major mediators of allergic inflammation owing their actions on basophil and eosinophil leukocytes. The monocyte chemotactic protein (MCP) 1 is a potent inducer of mediator release from basophils but is inactive on eosinophils. To obtain information on the structural determinants of the activities of MCP-1, we have synthesized several NH2-terminally truncated analogues and tested their effects on basophils and eosinophils. Through deletion of the NH2-terminal residue, MCP-1(2-76) was obtained, which was a potent activator of eosinophils, as assessed by chemotaxis, cytosolic free Ca2+ changes, actin polymerization, and that induction of the respiratory burst. In contrast, the activity of MCP-1(2-76) on basophil leukocytes was dramatically decreased (50-fold) compared with that of full-length MCP-1. Deletion of the next residue led to total loss of activity on eosinophil and basophil leukocytes. Analogues with three or four residue deletions, MCP-1(4-76) and MCP-1(5-76), were again active on both cells, whereas all further truncation analogues, MCP-1(6-76) through MCP-1(10-76), were inactive. Thus, a minimal structural modification can change receptor and target cell selectivity of MCP-1. Our observations indicate that the recognition sites of CC chemokine receptors on eosinophils and basophils are similar, although they discriminate between MCP-1 and MCP-1(2-76) and suggest NH2-terminal processing as a potential mechanism for the regulation of CC chemokine activities.
Actins↗
Cloning of the human eosinophil chemoattractant, eotaxin. Expression, receptor binding, and functional properties suggest a mechanism for the selective recruitment of eosinophils.
The CC chemokine eotaxin, identified in guinea pigs and also recently in mice, may be a key element for the selective recruitment of eosinophils to certain inflamed tissues. Using a partial mouse eotaxin CDNA probe, the human eotaxin gene was cloned and found to be 61.8 and 63.2% identical at the amino acid level to guinea pig and mouse eotaxin. Human eotaxin protein was a strong and specific eosinophil chemoattractant in vitro and was an effective eosinophil chemoattractant when injected into the skin of a rhesus monkey. Radiolabeled eotaxin was used to identify a high affinity receptor on eosinophils (0.52 nM Kd), expressed at 4.8 x 10(4) sites per cell. This receptor also bound RANTES and monocyte chemotactic protein-3 with lower affinity, but not macrophage inflammatory protein-1 alpha. Eotaxin could desensitize calcium responses of eosinophils to RANTES and monocyte chemotactic protein-3, although RANTES was able to only partially desensitize eosinophil calcium responses to eotaxin. Immunohistochemistry on human nasal polyp with antieotaxin mAbs showed that certain leukocytes as well as respiratory epithelium were intensely immunoreactive, and eosinophil infiltration occurred at sites of eotaxin upregulation. Thus eotaxin in humans is a potent and selective eosinophil chemoattractant that is expressed by a variety cell types in certain inflammatory conditions.
Amino Acid Sequence↗
Activation of NK cells by CC chemokines. Chemotaxis, Ca2+ mobilization, and enzyme release.
The responses of cloned human NK cells (ERNK57) to seven CC chemokines (monocyte chemotactic protein-1 (MCP-1), MCP-2, MCP-3, RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein-1 alpha (MIP-1 alpha), MIP-1 beta, and 1309) and two CXC chemokines (IL-8 and IP-10) were studied. Except for 1309, all CC chemokines induced chemotaxis of the NK cells in vitro, whereas the CXC chemokines were inactive. Maximal activity was obtained at 1 nM for MCP-1 and 10 to 100 nM for the other CC chemokines. The response showed a typically bimodal concentration dependence in all cases, except for RANTES, which induced a linear increase of migration over the concentration range of 0.1 to 1000 nM. A transient rise of the cytosolic-free Ca2+ concentration ([Ca2+]i), which is characteristic for chemokine-stimulated leukocytes, was observed in NK cells after stimulation with all six active chemokines. Since granule exocytosis is required for NK cell-dependent target killing, the effect of CC chemokines on exocytosis was tested. All CC chemokines that induced chemotaxis and [Ca2+]i changes also induced the release of granzyme A and N-acetyl-beta-D-glucosaminidase from cloned and blood NK cells, as well as CD8+ T cells after pretreatment with cytochalasin B. Maximum release was obtained from NK cells, and amounted to 35% and 13% of the total content of granzyme A and N-acetyl-beta-D-glucosaminidase, respectively. The capacity of cloned NK cells and CD8+ T cells to respond to chemokines depended on the time in culture after stimulation with PHA in the presence of irradiated feeder cells, and maximum responses were observed after 10 to 16 days. Our results demonstrate that CC chemokines activate NK cells, and are, therefore, not only attractants for monocytes, T lymphocytes, and eosinophil and basophil granulocytes.
Calcium↗
1H NMR solution structure of an active monomeric interleukin-8.
The solution structure of a monomeric form of interleukin-8 (IL-8) has been solved using 1H NMR spectroscopy. The chemically synthesized nonnatural analog [IL-8 (4-72) L25 NH-->NCH3] has the same activity as that of native IL-8. Thirty structures were generated using the hybrid distance geometry and simulated annealing protocol using the program X-PLOR. The structure is well-defined except for N-terminal residues 4-6 and C-terminal residues 67-72. The rms distribution about the average structure for residues 7-66 is 0.38 A for the backbone atoms and 0.87 A for all heavy atoms. The structure consists of a series of turns and loops followed by a triple-stranded beta sheet and a C-terminal alpha helix. The structure of the monomer is largely similar to the native dimeric IL-8 structures previously determined by both NMR and X-ray methods. The major difference is that, in the monomeric analog, the C-terminal residues 67-72 are disordered whereas they are helical in the two dimeric structures. The best fit superposition of the backbone atoms of residues 7-66 of the monomer structure on the dimeric IL-8 structures showed rms differences of 1.5 and 1.2 A respectively. The turn (residues 31-35), which is disulfide linked to the N-terminal region, adopts a conformation in the monomer similar to that seen in the dimeric X-ray structure (rms difference 1.4 A) and different from that seen in the dimeric NMR structure (rms difference 2.7 A).(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Comparison of the specificity of bacterially expressed cytoplasmic protein-tyrosine phosphatases SHP and SH-PTP2 towards synthetic phosphopeptide substrates.
SHP and SH-PTP2 are related cytoplasmic protein-tyrosine phosphatases having two tandem amino-terminal src homology 2 domains linked to a single catalytic domain. There is growing evidence that these two molecules may exhibit opposing effects within specific signaling pathways. However, the relative contributions of the src homology 2 domains or the catalytic domains to these opposing effects are not well known. To evaluate the potential contribution of the catalytic domains, we compared the substrate specificity of the two phosphatases. As seen previously, the catalytic activities of bacterially expressed SHP and SH-PTP2 were regulated by the presence of the linked src homology 2 domains. In addition, we characterized a cryptic thrombin cleavage site within the carboxy-terminus of SHP that led to a striking increase in the activity of the catalytic domain. Employing a panel of phosphopeptide substrates whose sequences were modeled after intracellular phosphorylation sites, both SHP and SH-PTP2 demonstrated a similar specificity pattern. Similar to SH-PTP2, SHP failed to elicit detectable phosphate release from several phosphopeptide substrates, while displaying catalytic efficiencies that ranged over approximately 40-1.6 x 10(3) M-1 s-1 towards other substrates. In contrast, the PTP-1B phosphatase dephosphorylated all of the phosphopeptide substrates tested with approximately equal ease. The overall similarity demonstrated by the catalytic domains of SHP and SH-PTP2 suggested that differences in the in vivo behavior of these two molecules might not stem from differences in the substrate specificity of the catalytic domains, suggesting instead that the specificity of the src homology 2 domains is more important in this regard.
Amino Acid Sequence↗
Monocyte chemotactic protein MCP-2 activates human basophil and eosinophil leukocytes similar to MCP-3.
It has been shown that CC chemokines activate basophil and eosinophil leukocytes with different selectivities and patterns of activity. The most effective are monocyte chemotactic protein-1 (MCP-1), a potent stimulus of mediator release in basophils without effects on eosinophils, RANTES, a weak stimulus of release and strong chemoattractant for basophils and eosinophils, and MCP-3, which combines the activities of MCP-1 and RANTES. We have now compared MCP-2, which has 62 and 60% of sequence identity with MCP-1 and MCP-3, respectively, with the other CC chemokines. MCP-2 induced mediator release by human basophils with lower efficacy and potency than MCP-1 and MCP-3. It promoted transient changes of cytosolic-free calcium concentration ([Ca2+]i) and chemotactic responses in both basophils and eosinophils, however somewhat less efficiently than MCP-3 and RANTES. Desensitization studies indicate that MCP-2 interacts with receptors recognizing MCP-1 as well as RANTES. These results demonstrate that MCP-2 and MCP-3 exert qualitatively similar biologic activities on basophils and eosinophils. In basophils that had not been treated with IL-3, MCP-2 induced minimal exocytosis only, but desensitized the cells toward MCP-1 and MCP-3, suggesting that MCP-2 may act as a functional inhibitor of CC chemokine actions. The results of this study further indicate that MCP analogues display partially distinct, partially overlapping bioactivities toward eosinophils and basophils, and may thus regulate inflammatory processes involving these effector cell types.
Basophils↗