The BBXB motif of RANTES is the principal site for heparin binding and controls receptor selectivity.

The chemokine RANTES (regulated on activation normal T cell expressed and secreted; CCL5) binds selectively to glycosaminoglycans (GAGs) such as heparin, chondroitin sulfate, and dermatan sulfate. The primary sequence of RANTES contains two clusters of basic residues, (44)RKNR(47) and (55)KKWVR(59). The first is a BBXB motif common in heparin-binding proteins, and the second is located in the loop directly preceding the C-terminal helix. We have mutated these residues to alanine, both as point mutations as well as triple mutations of the 40s and 50s clusters. Using a binding assay to heparin beads with radiolabeled proteins, the (44)AANA(47) mutant demonstrated an 80% reduction in its capacity to bind heparin, whereas the (55)AAWVA(59) mutant retained full binding capacity. Mutation of the (44)RKNR(47) site reduced the selectivity of RANTES binding to different GAGs. The mutants were tested for their integrity by receptor binding assays on CCR1 and CCR5 as well as their ability to induce chemotaxis in vitro. In all assays the single point mutations and the triple 50s cluster mutation caused no significant difference in activity compared with the wild type sequence. However, the triple 40s mutant showed a 80-fold reduction in affinity for CCR1, despite normal binding to CCR5. It was only able to induce monocyte chemotaxis at micromolar concentrations. The triple 40s mutant was also able to inhibit HIV-1 infectivity, but consistent with its abrogated GAG binding capacity, it no longer induced enhanced infectivity at high concentrations.

Amino-terminally modified RANTES analogues demonstrate differential effects on RANTES receptors.

Modification of the amino terminus of regulated on activated normal T-cell expressed (RANTES) has been shown to have a significant effect on biological activity and produces proteins with antagonist properties. Two amino-terminally modified RANTES proteins, Met-RANTES and aminooxypentane-RANTES (AOP-RANTES), exhibit differential inhibitory properties on both monocyte and eosinophil chemotaxis. We have investigated their binding properties as well as their ability to activate the RANTES receptors CCR1, CCR3, and CCR5 in cell lines overexpressing these receptors. We show that Met-RANTES has weak activity in eliciting a calcium response in Chinese hamster ovary cells expressing CCR1, CCR3, and CCR5, whereas AOP-RANTES has full agonist activity on CCR5 but is less effective on CCR3 and CCR1. Their ability to induce chemotaxis of the murine pre-B lymphoma cell line, L1.2, transfected with the same receptors, consolidates these results. Monocytes have detectable mRNA for CCR1, CCR2, CCR3, CCR4, and CCR5, and they respond to the ligands for these receptors in chemotaxis but not always in calcium mobilization. AOP-RANTES does not induce calcium mobilization in circulating monocytes but is able to do so as these cells acquire the macrophage phenotype, which coincides with a concomitant up-regulation of CCR5. We have also tested the ability of both modified proteins to induce chemotaxis of freshly isolated monocytes and eosinophils. Cells from most donors do not respond, but occasionally cells from a particular donor do respond, particularly to AOP-RANTES. We therefore hypothesize that the occasional activity of AOP-RANTES to induce leukocyte chemotaxis is due to donor to donor variation of receptor expression.

Multiple forms of angiostatin induce apoptosis in endothelial cells.

Angiostatin is a circulating inhibitor of angiogenesis generated by proteolytic cleavage of plasminogen. In this study we have used recombinant human and murine angiostatins (kringles 1-4) as well as native human angiostatin (prepared by elastase digestion of plasminogen [kringles 1-3] or by plasmin autocatalysis in the presence of a free sulfhydryl donor [kringles 1-4]). We report that angiostatin reduces endothelial cell number in a 4-day proliferation assay without affecting cell cycle progression into S-phase (as determined by bromodeoxyuridine labeling). This suggested that the reduction in cell number in the proliferation assay might in part be due to cytotoxicity. This was confirmed by the observation that ethidium homodimer incorporation (a measure of plasma membrane integrity) into endothelial cells was increased by angiostatin in a manner similar to that seen with tumor necrosis factor- (TNF-) and transforming growth factor-beta1 (TGF-beta1), both of which induce apoptosis in endothelial cells. In contrast to TNF- and TGF-beta1, angiostatin did not induce cytotoxicity in human MRC-5 fibroblast, rat smooth muscle, canine MDCK epithelial, or murine B16-F10 melanoma cell lines. Angiostatin-induced apoptosis was confirmed by endothelial cell nuclear acridine orange incorporation as well as by annexin V and TUNEL staining. These in vitro findings point to endothelial cell apoptosis as a mechanism for the antiangiogenic effect of angiostatin in vivo.

Aminooxypentane-RANTES induces CCR5 internalization but inhibits recycling: a novel inhibitory mechanism of HIV infectivity.

CCR5, a chemokine receptor expressed on T cells and macrophages, is the principal coreceptor for M-tropic HIV-1 strains. Recently, we described an NH2-terminal modification of the CCR5 ligand regulated on activation, normal T cell expressed and secreted (RANTES), aminooxypentane-RANTES (AOP-RANTES), that showed potent inhibition of macrophage infection by HIV-1 under conditions where RANTES was barely effective. To investigate the mechanism of AOP-RANTES inhibition of HIV infectivity we examined the surface expression of CCR5 using a monoclonal anti-CCR5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused >90% decrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. RANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycling of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged periods of time in the presence of RANTES, CCR5 expression is comparable to that seen on cells treated with control medium, whereas there is no CCR5 surface expression on cells cultured in the presence of AOP-RANTES. Immunofluorescence indicated that both AOP-RANTES and RANTES induced downmodulation of cell surface CCR5, and that the receptor was redistributed into endocytic organelles containing the transferrin receptor. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence of AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOST) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infection by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differences between AOP-RANTES and RANTES in their effect on receptor downregulation and recycling suggest a mechanism for the potent inhibition of HIV infection by AOP-RANTES. Moreover, these results support the notion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.

Glycosaminoglycans mediate cell surface oligomerization of chemokines.

Chemokines are 8-10 kDa proteins involved in the control of leukocyte trafficking and activation. In free solution, chemokines are monomers at physiologic concentrations, although many multimerize at higher concentrations. Cell surface heparan sulfate may sequester chemokines, increasing their local concentrations and facilitating their binding to receptors expressed on leukocytes. In competitive binding assays using immobilized heparin, a 2-3-fold increase in the bound radiolabeled chemokine was seen with increasing concentrations of unlabeled chemokine in the nanomolar range. Unlabeled chemokine concentrations between 0.25 and 50 microM were needed to compete the bound radioactivity. This biphasic competition curve was not seen for N-methyl-L25 IL-8, a variant of IL-8 which is unable to dimerize. In addition, complexes of chemokine and heparin eluted from gel filtration columns with apparent molecular masses of 33-60 kDa, suggesting that chemokine multimerization had occurred. The physiological relevance of this multimerization process was seen from studies using human endothelial cells. The endothelial cell binding sites for IL-8, RANTES, and MCP-1 were deduced to be glycosaminoglycans since competition assays showed the biphasic curves and micromolar IC50 values seen in studies with immobilized heparin, and mRNA for known chemokine receptors was not detected. Furthermore, digestion of endothelial cell monolayers with glycosaminidases decreased chemokine binding by up to 80%. Glycosaminoglycans can act as modulators of the ligand binding affinity of chemokine receptor-bearing cells. Removal of glycosaminoglycans from CHO cells expressing chemokine receptors CXCR1, CCR1, or CCR2 resulted in 40-70% decreases in the binding of RANTES, MCP-1, IL-8, and MIP-1alpha. Our data show that cell surface glycosaminoglycans induce polymerization of chemokines, increasing their local concentration and therefore enhancing their effects on high-affinity receptors within the local microenvironment.

Spatial orientation of the alpha and betac receptor chain binding sites on monomeric human interleukin-5 constructs.

Interleukin-5 (IL-5), a disulfide-linked homodimer, can be induced to fold as a biological active monomer by extending the loop between its third and fourth helices (Dickason, R. R., and Huston, D. P. (1996) Nature 379, 652-655). We have designed eight monomeric IL-5 proteins to optimize biological activity and stability of the monomer. This was achieved by (i) inserting the joining loop at three different positions, (ii) by introducing an additional intramolecular disulfide bridge onto these backbones, and (iii) by creating circular permutations to fix the position of the carboxyl-terminal helix relative to the three other helices. The proteins dimerize with Kd values ranging from 20 to 200 microM and are therefore monomeric at the picomolar concentrations where they are biologically active. Introduction of a second disulfide confers increased stability, but this increased rigidity results in lower activity of the protein. Contrary to wild type IL-5, mutation of the betac contact residue on the first helix, Glu12, to Lys, into the circularly permutated constructs, did not abolish TF-1 proliferative and eosinophil activation activities. These results indicate that activation of the IL-5 receptor complex is not mediated solely by Glu12 on the first helix, and alternative mechanisms are discussed.

Extension of recombinant human RANTES by the retention of the initiating methionine produces a potent antagonist.

Extension of recombinant human RANTES by a single residue at the amino terminus is sufficient to produce a potent and selective antagonist. RANTES is a proinflammatory cytokine that promotes cell accumulation and activation in chronic inflammatory diseases. When mature RANTES was expressed heterologously in Escherichia coli, the amino-terminal initiating methionine was not removed by the endogenous amino peptidases. This methionylated protein was fully folded but completely inactive in RANTES bioassays of calcium mobilization and chemotaxis of the promonocytic cell line THP-1. However, when assayed as an antagonist of both RANTES and macrophage inflammatory polypeptide-1 alpha (MIP-1 alpha) in these assays, the methionylated RANTES (Met-RANTES) inhibited the actions of both chemokines. T cell chemotaxis was similarly inhibited. The antagonistic effect was selective since Met-RANTES had no effect on interleukin-8- or monocyte chemotractant protein-1-induced responses in these cells. Met-RANTES can compete with both [125I]RANTES and [125I]IMP-1 alpha binding to THP-1 cells or to stably transfected HEK cells recombinantly expressing their common receptor, CC-CKR-1. These data show that the integrity of the amino terminus of RANTES is crucial to receptor binding and cellular activation.

Characterisation of the RANTES/MIP-1 alpha receptor (CC CKR-1) stably transfected in HEK 293 cells and the recombinant ligands.

The CC chemokines RANTES and MIP-1 alpha are known to activate certain leucocytes and leucocytic cell lines. We have produced and fully characterised the recombinant proteins expressed in E. coli. They induce chemotaxis of the pro-monocytic cell line, THP-1 and T cells. THP-1 cells express three of the known CC chemokine receptors. In order to study the activation of a single receptor, we have expressed the shared receptor (CC CKR-1) for RANTES and MIP-1 alpha stably in the HEK 293 cell line. We have examined the effects of RANTES and MIP-1 alpha on the CC CKR-1 transfectants by equilibrium binding studies and in a chemotaxis assay. RANTES competes for [125I]RANTES with an IC50 of 0.6 +/- 0.23 nM, whereas MIP-1 alpha competes for its radiolabelled counterpart with an IC50 of 10 +/- 1.6 nM in the transfectants. These affinities are the same as those measured on the THP-1 cell line. The stably transfected HEK 293 cells respond to both these chemokines in the chemotaxis assay with the same EC50 values as those measured for THP-1 cells. This indicates that this cellular response can be mediated through the CC CKR-1 receptor.

Selenomethionine labelling of phosphomannose isomerase changes its kinetic properties.

Phosphomannose isomerase (PMI) is an essential enzyme in the early steps of the protein glycosylation pathway in both prokaryotes and eukaryotes. Lack of the enzyme is lethal for fungal organisms and it is thus a potential fungicidal target. To facilitate the solution of the three-dimensional structure of the enzyme from the pathogen Candida albicans, we have produced the recombinant selenomethionine-labelled enzyme (SeMet-PMI). DL41, a methionine auxotroph Escherichia coli strain, was transformed with a PMI expression plasmid and grown on an enriched selenomethionine-containing medium to high-cell densities. The SeMet-PMI protein has been purified and found by amino acid analysis to have its methionine residues replaced by selenomethionine residues. Electrospray mass spectroscopy showed a major species of 49,063 +/- 10 Da for SeMet-PMI compared to 48,735 +/- 6 Da for the normal recombinant enzyme, accounting for the incorporation of seven selenomethionine residues. SeMet-PMI crystallised isomorphously with the normal PMI protein and the crystals diffract to 0.23 nm. Kinetic characterisation of SeMet-PMI showed that its Km for the substrate mannose-6-phosphate was fourfold higher than that of its methionine-containing counterpart. The inhibition constant for zinc ions was also increased by a similar factor. However, the Vmax was unaltered. These results suggested that one or more methionine residues must be in close proximity to the substrate-binding pocket in the active site, rendering substrate access more difficult compared to the normal enzyme. This hypothesis was confirmed by the finding of four methionine residues lying along one wall of the active site.