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.Chemokines selectively recruit and activate leukocyte populations, both during routine immunosurveillance and also during inflammation. The migration of cells is believed to require immobilization of the chemokines on proteoglycans in the extracellular matrix and on the endothelial cell (2, 3). The glycosaminoglycan (GAG) 1 moiety of the proteoglycan shows a wide range of structures, with heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate being important members of the family. Changes in the type of intensity of proteoglycan expression are known to happen in a wide variety of inflammatory diseases. It has been suggested that these changes in glycosaminoglycan expression play a role in the localization of the inflammatory response, by localizing inflammatory cytokines and chemokines (4 -7).Chemokines are a large family of small proteins with a remarkably highly conserved three-dimensional monomeric structure (Ref. 8 and Fig. 1). This conserved structure is mediated by the formation of two disulfide bridges imposed by the conserved 4-cysteine motif rather than identity at the level of primary sequence, which can be as low as 20%. The majority of chemokines (MIP-1␣ and -1 being the exceptions) 2 are highly basic proteins with an isoelectric point around pH 9.0. All chemokines are able to bind heparin, although with varying affinities. We have previously shown that selectivity exists for the chemokine/GAG interaction for four chemokines investigated: IL-8, RANTES, MIP-1␣, ...
Chemokines are a group of small proteins that have a variety of functions, including the activation and recruitment of immune cells during episodes of inflammation. In common with many cytokines, it has been observed that chemokines have the potential to bind heparin-like glycosaminoglycan molecules, which are normally expressed on proteoglycan components of the cell surface and extracellular matrix. The significance of this interaction for chemokine activity remains a subject of debate. In this study, Chinese hamster ovary cells were transfected separately with the human chemokine receptors CCR1 and CCR5, and these receptors were shown to induce an intracytoplasmic Ca 2؉ flux and cellular chemotaxis following stimulation with the natural CC chemokine ligands (MIP-1␣, RANTES (regulated on activation normal T cell expressed), and MIP-1). In further experiments, mutant CHO cells, with a defect in normal glycosaminoglycan (GAG) expression, were also transfected with, and shown to express similar levels of, CCR1 and CCR5. Although these receptors were functional, it was found that the mutant cells required exposure to higher concentrations of ligands than the wildtype cells in order to produce the same intracytoplasmic Ca 2؉ flux. Radioligand binding experiments demonstrated that specific chemokine receptors expressed by wild-type cells had a significantly greater affinity for MIP-1␣ than similar receptors expressed by GAG-deficient mutants. However, there was no significant difference between these cells in their affinity for RANTES or MIP-1. In conclusion, it has been demonstrated clearly that GAG expression is not necessary for the biological activity of the chemokines MIP-1␣, RANTES, or MIP-1. However, the presence of cell surface GAGs does enhance the activity of low concentrations of these chemokines by a mechanism that appears to involve sequestration onto the cell surface.
Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membranetargeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membranelocalizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli-derived protein were obtained and diffracted to 2.2 Å, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.
The chemokines are a family of small chemoattractant proteins that have a range of functions, including activation and promotion of vectorial migration of leukocytes. Regulation on activation, normal T cell expressed and secreted (RANTES; CCL5), a member of the CC-chemokine subfamily, has been implicated in a variety of immune responses. In addition to the interaction of CC-chemokines with their cognate cell-surface receptors, it is known that they also bind to glycosaminoglycans (GAGs), including heparan sulfate. This potential for binding to GAG components of proteoglycans on the cell surface or within the extracellular matrix might allow formation of the stable chemokine concentration gradients necessary for leukocyte chemotaxis. In this study, we created a panel of mutant RANTES molecules containing neutral amino acid substitutions within putative, basic GAG-binding domains. Despite showing reduced binding to GAGs, it was found that each mutant containing a single amino acid substitution induced a similar leukocyte chemotactic response within a concentration gradient generated by free solute diffusion. However, we found that the mutant K45A had a significantly reduced potential to stimulate chemotaxis across a monolayer of microvascular endothelial cells. Significantly, this mutant bound to the CCR5 receptor and showed a potential to mobilize Ca(2+) with an affinity similar to the wild-type protein. These results show that the interaction between RANTES and GAGs is not necessary for specific receptor engagement, signal transduction, or leukocyte migration. However, this interaction is required for the induction of efficient chemotaxis through the extracellular matrix between confluent endothelial cells.
SUMMARYIFN-g is a pleiotropic cytokine that is primarily involved in the regulation of immune cell activation and the development of tissue inflammation. It is capable of activating a range of non-immune cells, including those of the vascular endothelium. These cells respond by increasing the expression of intracellular and cell-surface molecules such as class II MHC antigens and adhesion molecules that, together, increase the tendency for interaction with immune cells. It is known that IFN-g can bind cell surface and extracellular heparan sulphate. Furthermore, soluble heparin can inhibit the function of this cytokine, presumably by competitive displacement from the cell surface, resulting in the failure of normal receptor signal transduction. In this study it is shown that heparin can prevent normal induction of the class II transactivator and heat shock cognate protein-70 in an IFN-g-treated endothelial cell line. Both of these molecules are dependent on the activation of intracytoplasmic STAT-1, which is the most receptor proximal component of their respective induction pathways. This provides further evidence for the blockade by heparin of ligand activation of the specific IFN-g receptor.
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