Kitty Moores

Purification, Properties, Sequencing, and Cloning of a Lipoprotein-Associated, Serine-Dependent Phospholipase Involved in the Oxidative Modification of Low-Density Lipoproteins

A novel LDL-associated phospholipase A2 (LDL-PLA2) has been purified to homogeneity from human LDL obtained from plasma apheresis. This enzyme has activity toward both oxidized phosphatidylcholine and platelet activating factor (PAF). A simple purification procedure involving detergent solubilization and affinity and ion exchange chromatography has been devised. Vmax and Km for the purified enzyme are 170 micromol.min-1.mg-1 and 12 micromol/L, respectively. Extensive peptide sequence from LDL-PLA2 facilitated identification of an expressed sequence tag partial cDNA. This has led to cloning and expression of active protein in baculovirus. A lipase motif is also evident from sequence information, indicating that the enzyme is serine dependent. Inhibition by diethyl p-nitrophenyl phosphate and 3,4-dichloroisocoumarin and insensitivity to EDTA, Ca2+, and sulfhydryl reagents confirm that the enzyme is indeed a serine-dependent hydrolase. The protein is extensively glycosylated, and the glycosylation site has been identified. Antibodies to this LDL-PLA2 have been raised and used to show that this enzyme is responsible for >95% of the phospholipase activity associated with LDL. Inhibition of LDL-PLA2 before oxidation of LDL reduces both lysophosphatidylcholine content and monocyte chemoattractant ability of the resulting oxidized LDL. Lysophosphatidylcholine production and monocyte chemoattractant ability can be restored by addition of physiological quantities of pure LDL-PLA2.

Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.

Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin‐2, MPIF‐2, or CKβ‐6, is a human CC chemokine with low amino acid sequence identity to other chemo‐ kines. Eotaxin‐2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross‐desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP‐4, but not RANTES, MIP‐lα, or MCP‐3, can completely cross‐desensitize the calcium response to eotaxin‐2 on these cells, indicating that eotaxin‐2 shares the same receptor used by eotaxin and MCP‐4. Eotaxin‐2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin‐2 also displaced 125I‐eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO‐CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP‐4. l25I‐Eotaxin‐2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin‐2 displace the ligand with equal affinity. Eotaxin and eotaxin‐2 promote a Ca2+ transient in RBL‐2H3 cells stably transfected with CCR3 (RBL‐2H3‐CCR3) and both ligands cross‐desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin‐2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor. J. Leukoc. Biol. 62: 667–675; 1997.

Fractalkine Is Expressed by Smooth Muscle Cells in Response to IFN-γ and TNF-α and Is Modulated by Metalloproteinase Activity

Fractalkine/CX3C-chemokine ligand 1 is expressed as a membrane-spanning adhesion molecule that can be cleaved from the cell surface to produce a soluble chemoattractant. Within the vasculature, fractalkine is known to be generated by endothelial cells, but to date there are no reports describing its expression by smooth muscle cells (SMC). In this study we demonstrate that IFN-γ and TNF-α, but not IL-1β, cooperate synergistically to induce fractalkine mRNA and protein expression in cultured aortic SMC. We also report the release of functional, soluble fractalkine from the membranes of stimulated SMC. This release is inhibited by the zinc metalloproteinase inhibitor batimastat, resulting in the accumulation of membrane-associated fractalkine on the SMC surface. Therefore, an SMC-derived metalloproteinase activity is involved in fractalkine shedding. While soluble fractalkine present in SMC-conditioned medium is capable of inducing calcium transients in cells expressing the fractalkine receptor (CX3CR1), blocking experiments using neutralizing Abs reveal that it can be inactivated without affecting the chemotactic activity of SMC-conditioned media on monocytes. However, membrane-bound fractalkine plays a major role in promoting adhesion of monocytic cells to activated SMC. This fractalkine-mediated adhesion is further enhanced in the presence of batimastat, indicating that shedding of fractalkine from the cell surface down-regulates the adhesive properties of SMC. Hence, during vascular inflammation, the synergistic induction of fractalkine by IFN-γ and TNF-α together with its metalloproteinase-mediated cleavage may finely control the recruitment of monocytes to SMC within the blood vessel wall.

Cloning, in vitro expression, and functional characterization of a novel human CC chemokine of the monocyte chemotactic protein (MCP) family (MCP-4) that binds and signals through the CC chemokine receptor 2B.

Here we describe the characterization of a novel human CC chemokine, tentatively named monocyte chemotactic protein (MCP-4). This chemokine was detected by random sequencing of expressed sequence tags in cDNA libraries. The full-length cDNA revealed an open reading frame for a 98-amino acid residue protein, and a sequence alignment with known CC chemokines showed high levels of similarity (59–62%) with MCP-1, MCP-3, and eotaxin. MCP-4 cDNA was cloned into Drosophila S2 cells, and the mature protein (residues 24–98) was purified from the conditioned medium. Recombinant MCP-4 induced a potent chemotactic response (EC50 = 2.88 ± 0.15 nm) and a transient rise in cytosolic calcium concentration in fresh human peripheral blood monocytes but not in neutrophils. Binding studies in monocytes showed that MCP-4 and MCP-3 were very potent in displacing high affinity binding of125I-MCP-1 (IC50 for MCP-4, MCP-3, and unlabeled MCP-1 of 2.1 ± 1.4, 0.85–1.6, and 0.7 ± 0.2 nm respectively), suggesting that all three chemokines interact with the CC chemokine receptor-2 (MCP-1 receptor). This was confirmed in binding studies with Chinese hamster ovary cells, stably transfected with the CC chemokine 2B receptor. Northern blot analysis in extracts of normal human tissues showed expression of mRNA for MCP-4 in small intestine, thymus, and colon, but the level of protein expression was too low to be detected in Western blot analysis. However, expression of MCP-4 protein was demonstrated by immunohistochemistry in human atherosclerotic lesion and found to be associated with endothelial cells and macrophages.

The role of fractalkine in the recruitment of monocytes to the endothelium.

Recombinant fractalkine possesses both chemoattractive and adhesive properties in vitro. Previous studies have demonstrated an upregulation of this molecule on the membranes of activated human endothelial cells and hypothesised that fractalkine plays a role in the recruitment and adherence of monocytes to the activated endothelium. Here we present data analysing both the adhesive and chemoattractive properties of this chemokine expressed by activated human umbilical vein endothelial cells. We demonstrate that both recombinant fractalkine and endogenously produced fractalkine function as adhesion molecules, tethering monocytes to the endothelium. However, our data demonstrate that although recombinant fractalkine has the potential to function as a potent monocyte chemoattractant, the endogenous fractalkine cleaved from activated human umbilical vein endothelial cells is not responsible for the observed chemotaxis in this model. Instead, we show that monocyte chemoattractant protein-1 (MCP-1), secreted from the activated human umbilical vein endothelial cells, is responsible for the chemotaxis of these monocytes.

CCR2B receptor antagonists : Conversion of a weak HTS hit to a potent lead compound

A weak HTS hit at the CCR2B receptor has been converted into a potent antagonist by array SAR studies. Selectivity over the closely related CCR5 receptor is also achieved.

N-1 substituted pyrimidin-4-ones: Novel, orally active inhibitors of lipoprotein-associated phospholipase A2

From two related series of 2-(alkylthio)-pyrimidones, a novel series of 1-((amidolinked)-alkyl)-pyrimidones has been designed as nanomolar inhibitors of human lipoprotein-associated phospholipase A2. These compounds show greatly enhanced activity in isolated plasma. Selected derivatives such as compounds 51 and 52 are orally active with a good duration of action.

Selective binding of the truncated form of the chemokine CKβ8 (25–99) to CC chemokine receptor 1 (CCR1)

Human CC chemokine receptor 1 (CCR1) has been proposed as a receptor for CKbeta8. To obtain conclusive evidence, binding-displacement studies of 125I-CKbeta8 (25-99) were performed on membranes of Chinese hamster ovary cells expressing human CCR1. The Ic50 for displacement of 125I-CKbeta8 (25-99) with CKbeta8 (25-99) was 0.22 nM. The longer forms of CKbeta8 (24-99 and 1-99) also displaced 125I-CKbeta8, with Ic50 values of 6.5 and 16 nM, respectively. Displacement profiles of 125I-CKbeta8 (25-99) on freshly prepared human monocytes indicated that CCR1 was the major receptor for CKbeta8. We conclude that CCR1 is a receptor for different-length CKbeta8 and that CKbeta8 (25-99) has a similar affinity for CCR1 as macrophage inflammatory protein-1alpha (MIP-1alpha). The longer variants of CKbeta8 are significantly less potent than CKbeta8 (25-99) and MIP-1a on CCR1 and monocytes (P < 0.05).