Structural Insights into Molecular Recognition by Human Chemokine CCL19.

The human chemokines CCL19 and CCL21 bind to the G protein-coupled receptor (GPCR) CCR7 and play an important role in the trafficking of immune cells as well as cancer metastasis. Conserved binding sites for sulfotyrosine residues on the receptor contribute significantly to the chemokine/GPCR interaction and have been shown to provide promising targets for new drug-discovery efforts to disrupt the chemokine/GPCR interaction and, consequently, tumor metastasis. Here, we report the first X-ray crystal structure of a truncated CCL19 (residues 7-70) at 2.50 Å resolution, revealing molecular details crucial for protein-protein interactions. Although the overall structure is similar to the previously determined NMR model, there are important variations, particularly near the N terminus and the so-called 30's and 40's loops. Computational analysis using the FTMap server indicates the potential importance of these areas in ligand binding and the differences in binding hotspots compared to CCL21. NMR titration experiments using a CCR7-derived peptide (residues 5-11, TDDYIGD) further demonstrate potential receptor recognition sites, such as those near the C terminus and 40's loop, which consist of both positively charged and hydrophobic residues that may be important for receptor binding. Taken together, the X-ray, NMR, and computational analysis herein provide insights into the overall structure and molecular features of CCL19 and enables investigation into this chemokine's function and inhibitor development.

CCL20 is a novel ligand for the scavenging atypical chemokine receptor 4.

The chemokine CCL20 is broadly produced by endothelial cells in the liver, the lung, in lymph nodes and mucosal lymphoid tissues, and recruits CCR6 expressing leukocytes, particularly dendritic cells, mature B cells, and subpopulations of T cells. How CCL20 is systemically scavenged is currently unknown. Here, we identify that fluorescently labeled human and mouse CCL20 are efficiently taken-up by the atypical chemokine receptor ACKR4. CCL20 shares ACKR4 with the homeostatic chemokines CCL19, CCL21, and CCL25, although with a lower affinity. We demonstrate that all 4 human chemokines recruit ß-arrestin1 and ß-arrestin2 to human ACKR4. Similarly, mouse CCL19, CCL21, and CCL25 equally activate the human receptor. Interestingly, at the same chemokine concentration, mouse CCL20 did not recruit ß-arrestins to human ACKR4. Further cross-species analysis suggests that human ACKR4 preferentially takes-up human CCL20, whereas mouse ACKR4 similarly internalizes mouse and human CCL20. Furthermore, we engineered a fluorescently labeled chimeric chemokine consisting of the N-terminus of mouse CCL25 and the body of mouse CCL19, termed CCL25_19, which interacts with and is taken-up by human and mouse ACKR4.

Structural Features of an Extended C-Terminal Tail Modulate the Function of the Chemokine CCL21.

The chemokines CCL21 and CCL19, through binding of their cognate receptor CCR7, orchestrate lymph node homing of dendritic cells and naïve T cells. CCL21 differs from CCL19 via an unstructured 32 residue C-terminal domain. Previously described roles for the CCL21 C-terminus include GAG-binding, spatial localization to lymphatic vessels, and autoinhibitory modulation of CCR7-mediated chemotaxis. While truncation of the C-terminal tail induced chemical shift changes in the folded chemokine domain, the structural basis for its influence on CCL21 function remains largely unexplored. CCL21 concentration-dependent NMR chemical shifts revealed weak, nonphysiological self-association that mimics the truncation of the C-terminal tail. We generated a series of C-terminal truncation variants to dissect the C-terminus influence on CCL21 structure and receptor activation. Using NMR spectroscopy, we found that CCL21 residues 80-90 mediate contacts with the chemokine domain. In cell-based assays for CCR7 and ACKR4 activation, we also found that residues 92-100 reduced CCL21 potency in calcium flux, cAMP inhibition, and ß-arrestin recruitment. Taken together, these structure-function studies support a model wherein intramolecular interactions with specific residues of the flexible C-terminus stabilize a less active monomer conformation of the CCL21. We speculate that the autoinhibitory intramolecular contacts between the C-terminal tail and chemokine body are disrupted by GAG binding and/or interactions with the CCR7 receptor to ensure optimal functionality.

Injectable Polymer-Nanoparticle Hydrogels for Local Immune Cell Recruitment.

The ability to engineer immune function has transformed modern medicine, highlighted by the success of vaccinations and recent efforts in cancer immunotherapy. Further directions in programming the immune system focus on the design of immunomodulatory biomaterials that can recruit, engage with, and program immune cells locally in vivo. Here, we synthesized shear-thinning and self-healing polymer-nanoparticle (PNP) hydrogels as a tunable and injectable biomaterial platform for local dendritic cell (DC) recruitment. PNP gels were formed from two populations of poly(ethylene glycol)-block-polylactide (PEG-b-PLA) NPs with the same diameter but different PEG brush length (2 or 5 kDa). PEG-b-PLA NPs with the longer PEG brush exhibited improved gel formation following self-assembly and faster recovery after shear-thinning. In all cases, model protein therapeutics were released via Fickian diffusion in vitro, and minor differences in the release rate between the gel formulations were observed. PNP hydrogels were loaded with the DC cytokine CCL21 and injected subcutaneously in a murine model. CCL21-loaded PNP hydrogels recruited DCs preferentially to the site of injection in vivo relative to non-CCL21-loaded hydrogels. Thus, PNP hydrogels comprise a simple and tunable platform biomaterial for in vivo immunomodulation following minimally invasive subcutaneous injection.

CCL19 with CCL21-tail displays enhanced glycosaminoglycan binding with retained chemotactic potency in dendritic cells.

CCL19 is more potent than CCL21 in inducing chemotaxis of human dendritic cells (DC). This difference is attributed to 1) a stronger interaction of the basic C-terminal tail of CCL21 with acidic glycosaminoglycans (GAGs) in the environment and 2) an autoinhibitory function of this C-terminal tail. Moreover, different receptor docking modes and tissue expression patterns of CCL19 and CCL21 contribute to fine-tuned control of CCR7 signaling. Here, we investigate the effect of the tail of CCL21 on chemokine binding to GAGs and on CCR7 activation. We show that transfer of CCL21-tail to CCL19 (CCL19CCL21-tail ) markedly increases binding of CCL19 to human dendritic cell surfaces, without impairing CCL19-induced intracellular calcium release or DC chemotaxis, although it causes reduced CCR7 internalization. The more potent chemotaxis induced by CCL19 and CCL19CCL21-tail compared to CCL21 is not transferred to CCL21 by replacing its N-terminus with that of CCL19 (CCL21CCL19-N-term ). Measurements of cAMP production in CHO cells uncover that CCL21-tail transfer (CCL19CCL21-tail ) negatively affects CCL19 potency, whereas removal of CCL21-tail (CCL21tailless ) increases signaling compared to full-length CCL21, indicating that the tail negatively affects signaling via cAMP. Similar to chemokine-driven calcium mobilization and chemotaxis, the potency of CCL21 in cAMP is not improved by transfer of the CCL19 N-terminus to CCL21 (CCL21CCL19-N-term ). Together these results indicate that ligands containing CCL21 core and C-terminal tail (CCL21 and CCL21CCL19-N-term ) are most restricted in their cAMP signaling; a phenotype attributed to a stronger GAG binding of CCL21 and defined structural differences between CCL19 and CCL21.

CCR7 Sulfotyrosine Enhances CCL21 Binding.

Chemokines are secreted proteins that direct the migration of immune cells and are involved in numerous disease states. For example, CCL21 (CC chemokine ligand 21) and CCL19 (CC chemokine ligand 19) recruit antigen-presenting dendritic cells and naïve T-cells to the lymph nodes and are thought to play a role in lymph node metastasis of CCR7 (CC chemokine receptor 7)-expressing cancer cells. For many chemokine receptors, N-terminal posttranslational modifications, particularly the sulfation of tyrosine residues, increases the affinity for chemokine ligands and may contribute to receptor ligand bias. Chemokine sulfotyrosine (sY) binding sites are also potential targets for drug development. In light of the structural similarity between sulfotyrosine and phosphotyrosine (pY), the interactions of CCL21 with peptide fragments of CCR7 containing tyrosine, pY, or sY were compared using protein NMR (nuclear magnetic resonance) spectroscopy in this study. Various N-terminal CCR7 peptides maintain binding site specificity with Y8-, pY8-, or sY8-containing peptides binding near the α-helix, while Y17-, pY17-, and sY17-containing peptides bind near the N-loop and ß3-stand of CCL21. All modified CCR7 peptides showed enhanced binding affinity to CCL21, with sY having the largest effect.

A microfluidic device for measuring cell migration towards substrate-bound and soluble chemokine gradients.

Cellular locomotion is a central hallmark of eukaryotic life. It is governed by cell-extrinsic molecular factors, which can either emerge in the soluble phase or as immobilized, often adhesive ligands. To encode for direction, every cue must be present as a spatial or temporal gradient. Here, we developed a microfluidic chamber that allows measurement of cell migration in combined response to surface immobilized and soluble molecular gradients. As a proof of principle we study the response of dendritic cells to their major guidance cues, chemokines. The majority of data on chemokine gradient sensing is based on in vitro studies employing soluble gradients. Despite evidence suggesting that in vivo chemokines are often immobilized to sugar residues, limited information is available how cells respond to immobilized chemokines. We tracked migration of dendritic cells towards immobilized gradients of the chemokine CCL21 and varying superimposed soluble gradients of CCL19. Differential migratory patterns illustrate the potential of our setup to quantitatively study the competitive response to both types of gradients. Beyond chemokines our approach is broadly applicable to alternative systems of chemo- and haptotaxis such as cells migrating along gradients of adhesion receptor ligands vs. any soluble cue.

Crystallographic Structure of Truncated CCL21 and the Putative Sulfotyrosine-Binding Site.

CCL21 chemokine binds the G protein-coupled receptor CCR7, aiding not only in immune response but also in cancer metastasis. Compared with other chemokines, CCL21 has a unique extended unstructured C-terminus that is truncated in some naturally occurring variants. We have determined the X-ray crystallographic structure of a truncated CCL21 (residues 1-79) lacking the extended C-terminus and identified, via two-dimensional nuclear magnetic resonance (NMR), a putative sulfotyrosine-binding site that may recognize such post-translationally modified tyrosine residues on the receptor. Compared to the previously determined NMR structure of full-length CCL21, the crystal structure presents new druggable binding hot spots resulting from an alternative N-loop conformation. In addition, whereas the previous NMR structure did not provide any structural information after residue 70, the C-terminus of the truncated CCL21, ordered up to Ala77 in our crystal structure, is placed near the N-loop and sulfotyrosine-binding site, indicating that the extended C-terminus of full-length CCL21 can interact with this important region for receptor binding. These observations suggest a potential origin for the autoinhibition of CCL21 activity that was recently described. The new crystal structure and binding hot spot analysis have important implications for the function of the CCL21 C-terminus and drug discovery.

Plasmin and regulators of plasmin activity control the migratory capacity and adhesion of human T cells and dendritic cells by regulating cleavage of the chemokine CCL21.

The homeostatic chemokine CCL21 has a pivotal role in lymphocyte homing and compartment localisation within the lymph node, and also affects adhesion between immune cells. The effects of CCL21 are modulated by its mode of presentation, with different cellular responses seen for surface-bound and soluble forms. Here we show that plasmin cleaves surface-bound CCL21 to release the C-terminal peptide responsible for CCL21 binding to glycosaminoglycans on the extracellular matrix and cell surfaces, thereby generating the soluble form. Loss of this anchoring peptide enabled the chemotactic activity of CCL21 and reduced cell tethering. Tissue plasminogen activator did not cleave CCL21 directly but enhanced CCL21 processing through generation of plasmin from plasminogen. The tissue plasminogen activator inhibitor neuroserpin prevented processing of CCL21 and blocked the effects of soluble CCL21 on cell migration. Similarly, the plasmin-specific inhibitor α2-antiplasmin inhibited CCL21-mediated migration of human T cells and dendritic cells and tethering of T cells to APCs. We conclude that the plasmin system proteins plasmin, tissue plasminogen activator and neuroserpin regulate CCL21 function in the immune system by controlling the balance of matrix- and cell-bound CCL21.

Quantitative Analysis of Dendritic Cell Haptotaxis.

Chemokines are the main guidance cues directing leukocyte migration. Opposed to early assumptions, chemokines do not necessarily act as soluble cues but are often immobilized within tissues, e.g., dendritic cell migration toward lymphatic vessels is guided by a haptotactic gradient of the chemokine CCL21. Controlled assay systems to quantitatively study haptotaxis in vitro are still missing. In this chapter, we describe an in vitro haptotaxis assay optimized for the unique properties of dendritic cells. The chemokine CCL21 is immobilized in a bioactive state, using laser-assisted protein adsorption by photobleaching. The cells follow this immobilized CCL21 gradient in a haptotaxis chamber, which provides three dimensionally confined migration conditions.

A CCL21 chemokine of tongue sole (Cynoglossus semilaevis) promotes host resistance against bacterial infection.

Chemokines are a large family of chemotactic cytokines. Based on the arrangement of the first two cysteine residues, chemokines are divided into four groups, one of which is the CC chemokine group. In this study, we characterized a CC chemokine, CsCCL21, from half-smooth tongue sole (Cynoglossus semilaevis), and analyzed its activity. CsCCL21 contains two conserved N-terminal cysteine residues in a NCCL motif and is phylogenetically related to the CCL19/21/25 subgroup of CC chemokines. CsCCL21 was constitutively expressed in nine tissues and significantly upregulated by bacterial and viral infection. The recombinant CsCCL21 (rCsCCL21) induced migration of peripheral blood leukocytes. When the two conserved cysteine residues in the NCCL motif were mutated, the chemotactic activity of rCsCCL21 was abolished. rCsCCL21 enhanced the resistance of tongue sole against bacterial infection, but the mutant protein with NCCL mutation lacked this antibacterial effect. Taken together, these results suggest that CsCCL21 is a functional CC chemokine with the ability to recruit leukocytes and is involved in antibacterial immunity in a manner that requires the conserved NCCL motif.

An efficient one-pot four-segment condensation method for protein chemical synthesis.

Successive peptide ligation using a one-pot method can improve the efficiency of protein chemical synthesis. Although one-pot three-segment ligation has enjoyed widespread application, a robust method for one-pot four-segment ligation had to date remained undeveloped. Herein we report a new one-pot multisegment peptide ligation method that can be used to condense up to four segments with operational simplicity and high efficiency. Its practicality is demonstrated by the one-pot four-segment synthesis of a plant protein, crambin, and a human chemokine, hCCL21.

A general method for site specific fluorescent labeling of recombinant chemokines.

Chemokines control cell migration in many contexts including development, homeostasis, immune surveillance and inflammation. They are also involved in a wide range of pathological conditions ranging from inflammatory diseases and cancer, to HIV. Chemokines function by interacting with two types of receptors: G protein-coupled receptors on the responding cells, which transduce signaling pathways associated with cell migration and activation, and glycosaminoglycans on cell surfaces and the extracellular matrix which organize and present some chemokines on immobilized surface gradients. To probe these interactions, imaging methods and fluorescence-based assays are becoming increasingly desired. Herein, a method for site-specific fluorescence labeling of recombinant chemokines is described. It capitalizes on previously reported 11-12 amino acid tags and phosphopantetheinyl transferase enzymes to install a fluorophore of choice onto a specific serine within the tag through a coenzyme A-fluorophore conjugate. The generality of the method is suggested by our success in labeling several chemokines (CXCL12, CCL2, CCL21 and mutants thereof) and visualizing them bound to chemokine receptors and glycosaminoglycans. CXCL12 and CCL2 showed the expected co-localization on the surface of cells with their respective receptors CXCR4 and CCR2 at 4 °C, and co-internalization with their receptors at 37 °C. By contrast, CCL21 showed the presence of large discrete puncta that were dependent on the presence of both CCR7 and glycosaminoglycans as co-receptors. These data demonstrate the utility of this labeling approach for the detection of chemokine interactions with GAGs and receptors, which can vary in a chemokine-specific manner as shown here. For some applications, the small size of the fluorescent adduct may prove advantageous compared to other methods (e.g. antibody labeling, GFP fusion) by minimally perturbing native interactions. Other advantages of the method are the ease of bacterial expression, the versatility of labeling with any maleimide-fluorophore conjugate of interest, and the covalent nature of the fluorescent adduct.

Interstitial dendritic cell guidance by haptotactic chemokine gradients.

Directional guidance of cells via gradients of chemokines is considered crucial for embryonic development, cancer dissemination, and immune responses. Nevertheless, the concept still lacks direct experimental confirmation in vivo. Here, we identify endogenous gradients of the chemokine CCL21 within mouse skin and show that they guide dendritic cells toward lymphatic vessels. Quantitative imaging reveals depots of CCL21 within lymphatic endothelial cells and steeply decaying gradients within the perilymphatic interstitium. These gradients match the migratory patterns of the dendritic cells, which directionally approach vessels from a distance of up to 90-micrometers. Interstitial CCL21 is immobilized to heparan sulfates, and its experimental delocalization or swamping the endogenous gradients abolishes directed migration. These findings functionally establish the concept of haptotaxis, directed migration along immobilized gradients, in tissues.

Cloning, expression and characterization of CCL21 and CCL25 chemokines in zebrafish.

Chemokines are a large group of proteins implicated in migration, activation, and differentiation of leukocytes. They are well-surveyed in mammals, but less is known in lower vertebrates about their spatiotemporal expressions and functions. From an evolutionary point of view, comparative analyses may provide some fundamental insights into these molecules. In mammals, CCL21 and CCL25 are crucial for thymocyte homing. Herein, we identified and cloned the zebrafish orthologues of CCL21 and CCL25, and analyzed their expression in embryos and adult fish by in situ hybridization. We found that CCL21 was expressed in the craniofacial region, pharynx, and blood vessels in embryos. In adult fish, CCL21 transcripts were located in the kidney, spinal cord, and blood cells. In contrast, expression of CCL25 was only detected in the thymus primordia in embryos. In adult fish, transcripts of CCL25 were maintained in the thymus, and they were also found in the brain and oocytes. Furthermore, we performed an antisense oligonucleotide experiment to evaluate the biological function of CCL25. Results showed that the recruitment of thymocytes was impeded by morpholino-mediated knockdown of CCL25, suggesting that CCL25 is essential for colonization of T-cells in the thymus in early development. Together, our results demonstrate the basic profiles of two CCL chemokines in zebrafish. The tissue-specific expression patterns may pave the way for further genetic dissection in this model organism.

Solution structure of CCL21 and identification of a putative CCR7 binding site.

CCL21 is a human chemokine that recruits normal immune cells and metastasizing tumor cells to lymph nodes through activation of the G protein-coupled receptor CCR7. The CCL21 structure solved by NMR contains a conserved chemokine domain followed by an extended, unstructured C-terminus that is not typical of most other chemokines. A sedimentation equilibrium study showed CCL21 to be monomeric. Chemical shift mapping indicates that the CCR7 N-terminus binds to the N-loop and third ß-strand of CCL21's chemokine domain. Details of CCL21-receptor recognition may enable structure-based drug discovery of novel antimetastatic agents.

Engineering chemoattractant gradients using chemokine-releasing polysaccharide microspheres.

Spatial and temporal concentration gradients of chemoattractants direct many biological processes, especially the guidance of immune cells to tissue sites during homeostasis and responses to infection. Such gradients are ultimately generated by secretion of attractant proteins from single cells or collections of cells. Here we describe cell-sized chemoattractant-releasing polysaccharide microspheres, capable of mimicking chemokine secretion by host cells and generating sustained bioactive chemokine gradients in their local microenvironment. Exploiting the common characteristic of net cationic charge and reversible glycosaminoglycan binding exhibited by many chemokines, we synthesized alginate hydrogel microspheres that could be loaded with several different chemokines (including CCL21, CCL19, CXCL12, and CXCL10) by electrostatic adsorption. These polysaccharide microspheres subsequently released the attractants over periods ranging from a few hours to at least 1 day when placed in serum-containing medium or collagen gels. The generated gradients were able to attract cells more than hundreds of microns away to make contact with individual microspheres. This versatile system for chemoattractant delivery could find applications in immunotherapy, vaccines and fundamental chemotaxis studies in vivo and in vitro.

Polysialylated neuropilin-2 enhances human dendritic cell migration through the basic C-terminal region of CCL21.

Dendritic cell (DC) migration to secondary lymphoid organs is a critical step to properly exert its role in immunity and predominantly depends on the interaction of the chemokine receptor CCR7 with its ligands CCL21 and CCL19. Polysialic acid (PSA) has been recently reported to control CCL21-directed migration of mature DCs. Here, we first demonstrate that PSA present on human mature monocyte-derived dendritic cells did not enhance chemotactic responses to CCL19. We have also explored the molecular mechanisms underlying the selective enhancing effect of PSA on CCL21-driven chemotaxis of DCs. In this regard, we found out that prevention of DC polysialylation decreased CCL21 activation of JNK and Akt signaling pathways, both associated with CCR7-mediated chemotaxis. We also report that the enhanced PSA-mediated effect on DC migration towards CCL21 relied on the highly basic C-terminal region of this chemokine and depended on the PSA acceptor molecule neuropilin-2 (NRP2) and on the polysialyltransferase ST8SiaIV. Altogether, our data indicate that the CCR7/CCL21/NRP2/ST8SiaIV functional axis constitutes an important guidance clue for DC targeting to lymphoid organs.

Synergy-inducing chemokines enhance CCR2 ligand activities on monocytes.

The migration of monocytes to sites of inflammation is largely determined by their response to chemokines. Although the chemokine specificities and expression patterns of chemokine receptors are well defined, it is still a matter of debate how cells integrate the messages provided by different chemokines that are concomitantly produced in physiological or pathological situations in vivo. We present evidence for one regulatory mechanism of human monocyte trafficking. Monocytes can integrate stimuli provided by inflammatory chemokines in the presence of homeostatic chemokines. In particular, migration and cell responses could occur at much lower concentrations of the CCR2 agonists, in the presence of chemokines (CCL19 and CCL21) that per se do not act on monocytes. Binding studies on CCR2(+) cells showed that CCL19 and CCL21 do not compete with the CCR2 agonist CCL2. Furthermore, the presence of CCL19 or CCL21 could influence the degradation of CCL2 and CCL7 on cells expressing the decoy receptor D6. These findings disclose a new scenario to further comprehend the complexity of chemokine-based monocyte trafficking in a vast variety of human inflammatory disorders.

[Cloning and expression in Escherichia coli of secondary lymphoid-tissue chemokine (SLC) gene].

Secondary lymphoid-tissue chemokine (SLC) is a type of CC chemokine identified by searching the Expressed Sequence Tag (EST) database. The full-length SLC gene was synthesized based on human SLC sequence using SOE-PCR. The sequenced SLC gene was cloned into expression vector pTMF and pALM, which used to transform Escherichia coli. Then the E. coli was cultured and induced according to protocol. The expressed target protein was identified by Western blotting. The target protein was expressed as soluble protein as well as inclusion bodies, the ratio of these two forms target protein varied with the difference conditions of culture and induction. The target protein was purified with the methods of nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography. The results of electrophoresis of the purified target protein showed that the molecular weight was larger than the predicted molecular weight.