Frédéric Borlat

Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines

During organogenesis, immunosurveillance, and inflammation, chemokines selectively recruit leukocytes by activating seven-transmembrane-spanning receptors. It has been suggested that an important component of this process is the formation of a haptotactic gradient by immobilization of chemokines on cell surface glycosaminoglycans (GAGs). However, this hypothesis has not been experimentally demonstrated in vivo. In the present study we investigated the effect of mutations in the GAG binding sites of three chemokines, monocyte chemoattractant protein-1/CC chemokine ligand (CCL)2, macrophage-inflammatory protein-1β/CCL4, and RANTES/CCL5, on their ability to recruit cells in vivo. These mutant chemokines retain chemotactic activity in vitro, but they are unable to recruit cells when administered intraperitoneally. Additionally, monomeric variants, although fully active in vitro, are devoid of activity in vivo. These data demonstrate that both GAG binding and the ability to form higher-order oligomers are essential for the activity of particular chemokines in vivo, although they are not required for receptor activation in vitro. Thus, quaternary structure of chemokines and their interaction with GAGs may significantly contribute to the localization of leukocytes beyond migration patterns defined by chemokine receptor interactions.

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-1α (MIP-1α) 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 [I]RANTES and [I]MIP-1α 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.

Identification of the Glycosaminoglycan Binding Site of the CC Chemokine, MCP-1 IMPLICATIONS FOR STRUCTURE AND FUNCTION IN VIVO

In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/CCL2, RANTES/CCL5, and MIP-1β/CCL4 (1). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAG-induced oligomerization beyond retention of chemokines at the site of production.

An engineered monomer of CCL2 has anti-inflammatory properties emphasizing the importance of oligomerization for chemokine activity in vivo

We demonstrated recently that P8A‐CCL2, a monomeric variant of the chemokine CCL2/MCP‐1, is unable to induce cellular recruitment in vivo, despite full activity in vitro. Here, we show that this variant is able to inhibit CCL2 and thioglycollate‐mediated recruitment of leukocytes into the peritoneal cavity and recruitment of cells into lungs of OVA‐sensitized mice. This anti‐inflammatory activity translated into a reduction of clinical score in the more complex inflammatory model of murine experimental autoimmune encephalomyelitis. Several hypotheses for the mechanism of action of P8A‐CCL2 were tested. Plasma exposure following s.c. injection is similar for P8A‐CCL2 and wild‐type (WT) CCL2, ruling out the hypothesis that P8A‐CCL2 disrupts the chemokine gradient through systemic exposure. P8A‐CCL2 and WT induce CCR2 internalization in vitro and in vivo; CCR2 then recycles to the cell surface, but the cells remain refractory to chemotaxis in vitro for several hours. Although the response to P8A‐CCL2 is similar to WT, this finding is novel and suggests that despite the presence of the receptor on the cell surface, coupling to the signaling machinery is retarded. In contrast to CCL2, P8A‐CCL2 does not oligomerize on glycosaminoglycans (GAGs). However, it retains the ability to bind GAGs and displaces endogenous JE (murine MCP‐1) from endothelial surfaces. Intravital microscopy studies indicate that P8A‐CCL2 prevents leukocyte adhesion, while CCL2 has no effect, and this phenomenon may be related to the mechanism. These results suggest that oligomerization‐deficient chemokines can exhibit anti‐inflammatory properties in vivo and may represent new therapeutic modalities.

A high-throughput protein refolding screen in 96-well format combined with design of experiments to optimize the refolding conditions

Production of correctly folded and biologically active proteins in Escherichiacoli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions. After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen. The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS-PAGE. The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives. The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests, which confirmed the reliability of the predictions. Finally, the refolded protein was purified and its biological activity was tested in vitro. The screen was applied for the refolding of Interleukin 17F (IL-17F), stromal-cell-derived factor-1 (SDF-1α/CXCL12), B cell-attracting chemokine 1 (BCA-1/CXCL13), granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a. This procedure identified refolding conditions for all the tested proteins. For the proteins where refolding conditions were already available, the optimized conditions identified in the screening process increased the yields between 50% and 100%. Thus, the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein.

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

The CC chemokines RANTES and MIP‐1α 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‐1α stably in the HEK 293 cell line. We have examined the effects of RANTES and MIP‐1α 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‐1α 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.

The role of γ-Secretase Activating Protein, GSAP and imatinib in the regulation of γ-secretase activity and amyloid-β generation

Background: γ-Secretase activating protein, GSAP, was identified as a novel regulator of γ-secretase and amyloid-β production. Results: Reducing GSAP expression in cells decreased amyloid-β generation. However, overexpression of GSAP in cells and recombinant GSAP in vitro did not modulate amyloid-β generation. Conclusion: The relationship between GSAP and γ-secretase is unclear. Significance: GSAP is not a validated therapeutic target for Alzheimer disease. γ-Secretase is a large enzyme complex comprising presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1 that mediates the intramembrane proteolysis of a large number of proteins including amyloid precursor protein and Notch. Recently, a novel γ-secretase activating protein (GSAP) was identified that interacts with γ-secretase and the C-terminal fragment of amyloid precursor protein to selectively increase amyloid-β production. In this study we have further characterized the role of endogenous and exogenous GSAP in the regulation of γ-secretase activity and amyloid-β production in vitro. Knockdown of GSAP expression in N2a cells decreased amyloid-β levels. In contrast, overexpression of GSAP in HEK cells expressing amyloid precursor protein or in N2a cells had no overt effect on amyloid-β generation. Likewise, purified recombinant GSAP had no effect on amyloid-β generation in two distinct in vitro γ-secretase assays. In subsequent cellular studies with imatinib, a kinase inhibitor that reportedly prevents the interaction of GSAP with the C-terminal fragment of amyloid precursor protein, a concentration-dependent decrease in amyloid-β levels was observed. However, no interaction between GSAP and the C-terminal fragment of amyloid precursor protein was evident in co-immunoprecipitation studies. In addition, subchronic administration of imatinib to rats had no effect on brain amyloid-β levels. In summary, these findings suggest the roles of GSAP and imatinib in the regulation of γ-secretase activity and amyloid-β generation are uncertain.

Characterization of endogenous and exogenous gamma-secretase activating protein (GSAP) expression in cells

that BIN1 is a genuine genetic determinant of AD. The Amphiphysins (Amph 1 and 2) are expressed in the brain and are involved in endocytosis processes. However, little is known about their potential implications in the pathological process. We aimed at characterizing how BIN1 may act in the AD process. Methods: mRNA levels of Amph 1 and BIN1 were measured in the frontal area of 64 AD brains and 64 controls using the Quantigene technology. The SKNSH-SY5Yand HEK cell lines, stably over-expressing APP695wt were transitorily transfected with a vector expressing BIN1. After 48 hours, supernatants were recovered and cells were lysed. Holo-APP and s-actin were measured by protein blotting. As1-40, As1-42, sAPPa and sAPPs were measured by sandwich ELISA. Finally, we assessed BIN1/Amph 1modulation of Tau and As -mediated neurotoxicity in D. melanogaster. using rough-eye and bristle phenotypes.Results:We observed an increase in expression of both Amph 1 and BIN1 genes in the brains of AD cases compared to controls. BIN1 over-expression in the two cell lines did not modify the APP metabolism whereas BIN1/Amph 1 knockdown suppressed Tau neurotoxicity in D. melanogaster. Conclusions: Our preliminary results indicated that BIN1 is over-expressed in AD brain and might play a role in AD process not by modulating APP metabolism but by controlling Tau proteinopathy-mediated neurotoxicity.