Efficient production of fluorophore-labeled CC chemokines for biophysical studies using recombinant enterokinase and recombinant sortase.

Chemokines are important immune system proteins, many of which mediate inflammation due to their function to activate and cause chemotaxis of leukocytes. An important anti-inflammatory strategy is therefore to bind and inhibit chemokines, which leads to the need for biophysical studies of chemokines as they bind various possible partners. Because a successful anti-chemokine drug should bind at low concentrations, techniques such as fluorescence anisotropy that can provide nanomolar signal detection are required. To allow fluorescence experiments to be carried out on chemokines, a method is described for the production of fluorescently labeled chemokines. First, a fusion-tagged chemokine is produced in Escherichia coli, then efficient cleavage of the N-terminal fusion partner is carried out with lab-produced enterokinase, followed by covalent modification with a fluorophore, mediated by the lab-produced sortase enzyme. This overall process reduces the need for expensive commercial enzymatic reagents. Finally, we utilize the product, vMIP-fluor, in binding studies with the chemokine binding protein vCCI, which has great potential as an anti-inflammatory therapeutic, showing a binding constant for vCCI:vMIP-fluor of 0.37 ± 0.006 nM. We also show how a single modified chemokine homolog (vMIP-fluor) can be used in competition assays with other chemokines and we report a Kd for vCCI:CCL17 of 14 µM. This work demonstrates an efficient method of production and fluorescent labeling of chemokines for study across a broad range of concentrations.

Convergent inactivation of the skin-specific C-C motif chemokine ligand 27 in mammalian evolution.

The appearance of mammalian-specific skin features was a key evolutionary event contributing for the elaboration of physiological processes such as thermoregulation, adequate hydration, locomotion, and inflammation. Skin inflammatory and autoimmune processes engage a population of skin-infiltrating T cells expressing a specific C-C chemokine receptor (CCR10) which interacts with an epidermal CC chemokine, the skin-specific C-C motif chemokine ligand 27 (CCL27). CCL27 is selectively produced in the skin by keratinocytes, particularly upon inflammation, mediating the adhesion and homing of skin-infiltrating T cells. Here, we examined the evolution and coding condition of Ccl27 in 112 placental mammalian species. Our findings reveal that a number of open reading frame inactivation events such as insertions, deletions, and start and stop codon mutations independently occurred in Cetacea, Pholidota, Sirenia, Chiroptera, and Rodentia, totalizing 18 species. The diverse habitat settings and lifestyles of Ccl27-eroded lineages probably implied distinct evolutionary triggers rendering this gene unessential. For example, in Cetacea, the rapid renewal of skin layers minimizes the need for an elaborate inflammatory mechanism, mirrored by the absence of epidermal scabs. Our findings suggest that the convergent and independent loss of Ccl27 in mammalian evolution concurred with unique adaptive roads for skin physiology.

Two glycosaminoglycan-binding domains of the mouse cytomegalovirus-encoded chemokine MCK-2 are critical for oligomerization of the full-length protein.

Chemokines are essential for antimicrobial host defenses and tissue repair. Herpesviruses and poxviruses also encode chemokines, copied from their hosts and repurposed for multiple functions, including immune evasion. The CC chemokine MCK-2 encoded by mouse CMV (MCMV) has an atypical structure consisting of a classic chemokine domain N-terminal to a second unique domain, resulting from the splicing of MCMV ORFs m131 and m129 MCK-2 is essential for full MCMV infectivity in macrophages and for persistent infection in the salivary gland. However, information about its mechanism of action and specific biochemical roles for the two domains has been lacking. Here, using genetic, chemical, and enzymatic analyses of multiple mouse cell lines as well as primary mouse fibroblasts from salivary gland and lung, we demonstrate that MCK-2 binds glycosaminoglycans (GAGs) with affinities in the following order: heparin > heparan sulfate > chondroitin sulfate = dermatan sulfate. Both MCK-2 domains bound these GAGs independently, and computational analysis together with site-directed mutagenesis identified five basic residues distributed across the N terminus and the 30s and 50s loops of the chemokine domain that are important GAG binding determinants. Both domains were required for GAG-dependent oligomerization of full-length MCK-2. Thus, MCK-2 is an atypical viral chemokine consisting of a CC chemokine domain and a unique non-chemokine domain, both of which bind GAGs and are critical for GAG-dependent oligomerization of the full-length protein.

Role of Conserved Disulfide Bridges and Aromatic Residues in Extracellular Loop 2 of Chemokine Receptor CCR8 for Chemokine and Small Molecule Binding.

Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr(184) (Cys + 1) and Tyr(187) (Cys + 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr(187) in TMIV (Phe(171)) and TMV (Trp(194)). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr(187) This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors.

Molecular cloning and expression analysis of CCL25 and its receptor CCR9s from Epinephelus coioides post Cryptocaryon irritans infection.

Among other functions, CCL25/CCR9 has an important role in regulating the trafficking of developing T cells in the thymus, and in homing memory T cells to the small intestine. The function of this chemokine-receptor complex is not well studied in fish. We identified a CCL25-like (EcCCL25, 108 aa) and two CCR9-like sequences (EcCCR9aa 373 aa; and EcCCR9b, 375 aa) from a transcriptome database of orange-spotted grouper (Epinephelus coioides). EcCCL25, EcCCR9a, and EcCCR9b shared conserved structural features with homologs from mammals and from other fish, and a consistent relationship with phylogenetic trees and sequence identities. In healthy grouper, EcCCL25, EcCCR9a, and EcCCR9b were highly expressed in the thymus, and the gills, were expressed at lower levels in the stomach, and had different expression levels in other tissues. After infection with Cryptocaryon irritans, EcCCL25 expression was up-regulated at early time points in the spleen and head kidney, and in the skin, and gills at later time points; EcCCR9a expression was increased in the gill, spleen, and head kidney. After infection with C. irritans, EcCCR9b expression was reduced in all tissues tested. These results suggested that grouper CCL25/CCR9a complex may be involved in host defense against C. irritans infection.

The Effect of N-Terminal Cyclization on the Function of the HIV Entry Inhibitor 5P12-RANTES.

Despite effective treatment for those living with Human Immunodeficiency Virus (HIV), there are still two million new infections each year. Protein-based HIV entry inhibitors, being highly effective and specific, could be used to protect people from initial infection. One of the most promising of these for clinical use is 5P12-RANTES, a variant of the chemokine RANTES/CCL5. The N-terminal amino acid of 5P12-RANTES is glutamine (Gln; called Q0), a residue that is prone to spontaneous cyclization when at the N-terminus of a protein. It is not known how this cyclization affects the potency of the inhibitor or whether cyclization is necessary for the function of the protein, although the N-terminal region of RANTES has been shown to be critical for receptor interactions, with even small changes having a large effect. We have studied the kinetics of cyclization of 5P12-RANTES as well as N-terminal variations of the protein that either produce an identical cyclized terminus (Glu0) or that cannot similarly cyclize (Asn0, Phe0, Ile0, and Leu0). We find that the half life for N-terminal cyclization of Gln is roughly 20 h at pH 7.3 at 37 °C. However, our results show that cyclization is not necessary for the potency of this protein and that several replacement terminal amino acids produce nearly-equally potent HIV inhibitors while remaining CC chemokine receptor 5 (CCR5) antagonists. This work has ramifications for the production of active 5P12-RANTES for use in the clinic, while also opening the possibility of developing other inhibitors by varying the N-terminus of the protein.

Structure-function analysis of CCL28 in the development of post-viral asthma.

CCL28 is a human chemokine constitutively expressed by epithelial cells in diverse mucosal tissues and is known to attract a variety of immune cell types including T-cell subsets and eosinophils. Elevated levels of CCL28 have been found in the airways of individuals with asthma, and previous studies have indicated that CCL28 plays a vital role in the acute development of post-viral asthma. Our study builds on this, demonstrating that CCL28 is also important in the chronic post-viral asthma phenotype. In the absence of a viral infection, we also demonstrate that CCL28 is both necessary and sufficient for induction of asthma pathology. Additionally, we present the first effort aimed at elucidating the structural features of CCL28. Chemokines are defined by a conserved tertiary structure composed of a three-stranded ß-sheet and a C-terminal α-helix constrained by two disulfide bonds. In addition to the four disulfide bond-forming cysteine residues that define the traditional chemokine fold, CCL28 possesses two additional cysteine residues that form a third disulfide bond. If all disulfide bonds are disrupted, recombinant human CCL28 is no longer able to drive mouse CD4+ T-cell chemotaxis or in vivo airway hyper-reactivity, indicating that the conserved chemokine fold is necessary for its biologic activity. Due to the intimate relationship between CCL28 and asthma pathology, it is clear that CCL28 presents a novel target for the development of alternative asthma therapeutics.

Osteopontin binds and modulates functions of eosinophil-recruiting chemokines.

BACKGROUND: Allergic asthma is characterized by eosinophilic inflammation and airway obstruction. There is also an increased risk of pulmonary infection caused by Streptococcus pneumoniae, in particular during severe asthma where high levels of the glycoprotein, osteopontin (OPN), are present in the airways. Eosinophils can be recruited by chemokines activating the receptor CCR3 including eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26, RANTES/CCL5, and MEC/CCL28. In addition to inducing chemotaxis, several of these molecules have defensin-like antibacterial properties. This study set out to elucidate the functional consequences of OPN binding to eosinophil-recruiting chemokines. METHODS: Antibacterial activities of the chemokines were investigated using viable count assays and electron microscopy. Binding studies were performed by means of surface plasmon resonance. The potential interference of OPN with antibacterial, receptor-activating, and lipopolysaccharide-neutralizing abilities of these chemokines was investigated. RESULTS: We found that OPN bound all eosinophil-recruiting chemokines with high affinity except for CCL5. The eosinophil-recruiting chemokines all displayed bactericidal activity against S. pneumoniae, but only CCL26 and CCL28 retained high antibacterial activity in the presence of sodium chloride at physiologic concentrations. Preincubation of the chemokines with OPN strongly inhibited their antibacterial activity against S. pneumoniae but did not affect their ability to activate CCR3. All chemokines investigated showed LPS-neutralizing activity that was impaired by OPN only in the case of CCL24. CONCLUSIONS: The data suggest that OPN may impair host defense activities of the chemokines without affecting their eosinophil-recruiting properties. This could be one mechanism explaining the increased vulnerability to acquire pneumococcal infection in parallel with sustained allergic inflammation in asthma.

Identification of the pharmacophore of the CC chemokine-binding proteins Evasin-1 and -4 using phage display.

To elucidate the ligand-binding surface of the CC chemokine-binding proteins Evasin-1 and Evasin-4, produced by the tick Rhipicephalus sanguineus, we sought to identify the key determinants responsible for their different chemokine selectivities by expressing Evasin mutants using phage display. We first designed alanine mutants based on the Evasin-1·CCL3 complex structure and an in silico model of Evasin-4 bound to CCL3. The mutants were displayed on M13 phage particles, and binding to chemokine was assessed by ELISA. Selected variants were then produced as purified proteins and characterized by surface plasmon resonance analysis and inhibition of chemotaxis. The method was validated by confirming the importance of Phe-14 and Trp-89 to the inhibitory properties of Evasin-1 and led to the identification of a third crucial residue, Asn-88. Two amino acids, Glu-16 and Tyr-19, were identified as key residues for binding and inhibition of Evasin-4. In a parallel approach, we identified one clone (Y28Q/N60D) that showed a clear reduction in binding to CCL3, CCL5, and CCL8. It therefore appears that Evasin-1 and -4 use different pharmacophores to bind CC chemokines, with the principal binding occurring through the C terminus of Evasin-1, but through the N-terminal region of Evasin-4. However, both proteins appear to target chemokine N termini, presumably because these domains are key to receptor signaling. The results also suggest that phage display may offer a useful approach for rapid investigation of the pharmacophores of small inhibitory binding proteins.

The viral chemokine MCK-2 of murine cytomegalovirus promotes infection as part of a gH/gL/MCK-2 complex.

Human cytomegalovirus (HCMV) forms two gH/gL glycoprotein complexes, gH/gL/gO and gH/gL/pUL(128,130,131A), which determine the tropism, the entry pathways and the mode of spread of the virus. For murine cytomegalovirus (MCMV), which serves as a model for HCMV, a gH/gL/gO complex functionally homologous to the HCMV gH/gL/gO complex has been described. Knock-out of MCMV gO does impair, but not abolish, virus spread indicating that also MCMV might form an alternative gH/gL complex. Here, we show that the MCMV CC chemokine MCK-2 forms a complex with the glycoprotein gH, a complex which is incorporated into the virion. We could additionally show that mutants lacking both, gO and MCK-2 are not able to produce infectious virus. Trans-complementation of these double mutants with either gO or MCK-2 showed that both proteins can promote infection of host cells, although through different entry pathways. MCK-2 has been extensively studied in vivo by others. It has been shown to be involved in attracting cells for virus dissemination and in regulating antiviral host responses. We now show that MCK-2, by forming a complex with gH, strongly promotes infection of macrophages in vitro and in vivo. Thus, MCK-2 may play a dual role in MCMV infection, as a chemokine regulating the host response and attracting specific target cells and as part of a glycoprotein complex promoting entry into cells crucial for virus dissemination.

Eotaxin-3 (CCL26) exerts innate host defense activities that are modulated by mast cell proteases.

BACKGROUND: During bacterial infections of the airways, a Th1-profiled inflammation promotes the production of several host defense proteins and peptides with antibacterial activities including ß-defensins, ELR-negative CXC chemokines, and the cathelicidin LL-37. These are downregulated by Th2 cytokines of the allergic response. Instead, the eosinophil-recruiting chemokines eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26 are expressed. This study set out to investigate whether these chemokines could serve as innate host defense molecules during allergic inflammation. METHODS: Antibacterial activities of the eotaxins were investigated using viable count assays, electron microscopy, and methods assessing bacterial permeabilization. Fragments generated by mast cell proteases were characterized, and their potential antibacterial, receptor-activating, and lipopolysaccharide-neutralizing activities were investigated. RESULTS: CCL11, CCL24, and CCL26 all showed potent bactericidal activity, mediated through membrane disruption, against the airway pathogens Streptococcus pneumoniae, Staphylococcus aureus, Nontypeable Haemophilus influenzae, and Pseudomonas aeruginosa. CCL26 retained bactericidal activity in the presence of salt at physiologic concentrations, and the region holding the highest bactericidal activity was the cationic and amphipathic COOH-terminus. Proteolysis of CCL26 by chymase and tryptase, respectively, released distinct fragments of the COOH- and NH2 -terminal regions. The COOH-terminal fragment retained antibacterial activity while the NH2 -terminal had potent LPS-neutralizing properties in the order of CCL26 full-length protein. An identical fragment to NH2 -terminal fragment generated by tryptase was obtained after incubation with supernatants from activated mast cells. None of the fragments activated the CCR3-receptor. CONCLUSIONS: Taken together, the findings show that the eotaxins can contribute to host defense against common airway pathogens and that their activities are modulated by mast cell proteases.

Efficient production of FAM19A4, a novel potential cytokine, in a stable optimized CHO-S cell system.

FAM19A4 is a novel potential cytokine identified by our group, which can chemoattract macrophages, promote phagocytosis against zymosan and increase reactive oxygen species (ROS) release. To further explore the role of FAM19A4 in immune system, abundant recombinant protein with high quality is indispensable. For efficient production of FAM19A4, we used an improved CHO-S cell expression system on the basis of pMH3 vector containing GC-rich regions which were novel ubiquitous chromatin opening elements (UCOEs). We selected CHO-S cells stably expressing FAM19A4 with G418 and screened cell clones with high level of FAM19A4 expression by immune blot and his-ELISA, adapted cell clones to serum-free suspension culture. Afterwards, we obtained the highest FAM19A4 expressing cell clone (2#) through 40 ml batch culture. We optimized the fed-batch culture condition and discovered the final cell viability was critical for FAM19A4 production successfully. Then we scaled 2# clone up to 3 L in fed-batch culture and obtained 22 mg (7.33 mg/L, averagely) endotoxin free FAM19A4 protein with purity over 95% using Ni affinity chromatography and size exclusion chromatography. The final yield was increased 3.6-folds compared to that of our previously reported transient system. Besides, the purified FAM19A4 protein showed chemotactic activity on macrophages. In summary, we developed a stable optimized fed-batch CHO-S cell system to produce FAM19A4, which not only provided sufficient bioactive FAM19A4 protein for further research but also offered an efficient strategy for other recombinant protein production.

Fish chemokines 14, 20 and 25: A comparative statement on computational analysis and mRNA regulation upon pathogenic infection.

In this study, we reported a molecular characterization of three CC chemokines namely, CsCC-Chem14, CsCC-Chem20 and CsCC-Chem25 which are were identified from the established cDNA library of striped murrel Channa striatus. Multiple sequence alignment of all the three chemokines revealed the presence of gene specific domains and motifs including small cytokine domain, IL8 like domain, receptor binding site and glycosaminoglycan (GAG) binding sites. Three dimensional structures of the chemokines under study showed an important facet on their anti-microbial property. Tissue specific mRNA expression showed that the CsCC-Chem14 is highly expressed in spleen, CsCC-Chem20 in liver and CsCC-Chem25 in trunk kidney. On challenge C. striatus with oomycete fungus Aphanomyces invadans, both CsCC-Chem20 and CsCC-Chem25 showed significant (P < 0.05) up-regulation compared to CsCC-Chem14. The increase in the expression levels of CsCC-Chem20 and CsCC-Chem25 due to infection showed that they are antimicrobial proteins. But considering the CsCC-Chem14 expression, it is found to be a constitutive chemokine and is involved in homeostatic function in spleen of C. striatus. C. striatus challenged with bacteria Aeromonas hydrophila also exhibited different up-regulation pattern in all the three chemokines at various time points. However, extensive studies are required to determine the functional activities of CsCC-Chem14, CsCC-Chem20 and CsCC-Chem25 in vitro and in vivo to gain more knowledge at the molecular and proteomic levels.

Cloning and expression analysis of three novel CC chemokine genes from Japanese flounder (Paralichthys olivaceus).

Chemokines are small cytokines secreted by various cell types. They not only function in cell activation, differentiation and trafficking, but they also have influences on many biological processes. In this study, three novel CC chemokine genes Paol-SCYA105, 106 and 107 in Japanese flounder (Paralichthys olivaceus) were cloned and characterized. Paol-SCYA105 was mainly detected in gill, kidney and spleen, Paol-SCYA106 was detected in all tissues examined and Paol-SCYA107 was mainly detected in the spleen and kidney. Paol-SCYA105 and Paol-SCYA106 gene expressions peaked in kidney at day 3 after viral hemorrhagic septicemia virus infection and decreased at day 6, but Paol-SCYA106 still remained at a high level at day 6. Paol-SCYA107 gene expression was significantly up-regulated in kidney at day 6 after viral hemorrhagic septicemia virus infection. In response to infection by Gram-negative Edwardsiella tarda and Gram-positive Streptococcus iniae in kidney, only Paol-SCYA106 gene expression significantly increased. Together, these results indicate that these three novel CC chemokines are involved in the immune response against pathogen infections.

Characterization of structure, dynamics, and detergent interactions of the anti-HIV chemokine variant 5P12-RANTES.

RANTES (CCL5) is a chemokine that recruits immune cells to inflammatory sites by interacting with the G-protein coupled receptor CCR5, which is also the primary coreceptor used together with CD4 by HIV to enter and infect target cells. Ligands of CCR5, including chemokines and chemokine analogs, are capable of blocking HIV entry, and studies of their structures and interactions with CCR5 will be key to understanding and optimizing HIV inhibition. The RANTES derivative 5P12-RANTES is a highly potent HIV entry inhibitor that is being developed as a topical HIV prevention agent (microbicide). We have characterized the structure and dynamics of 5P12-RANTES by solution NMR. With the exception of the nine flexible N-terminal residues, 5P12-RANTES has the same structure as wild-type RANTES but unlike the wild-type, does not dimerize via its N-terminus. To prepare the ground for interaction studies with detergent-solubilized CCR5, we have also investigated the interaction of RANTES and 5P12-RANTES with various commonly used detergents. Both RANTES variants are stable in Cymal-5, DHPC, Anzergent-3-12, dodecyltrimethylammonium chloride, and a DDM/CHAPS/CHS mixture. Fos-Cholines, dodecyldimethylglycine, and sodium dodecyl-sulfate denature both RANTES variants at low pH, whereas at neutral pH the stability is considerably higher. The onset of Fos-Choline-12-induced denaturation and the denatured state were characterized by circular dichroism and NMR. The detergent interaction starts below the critical micelle concentration at a well-defined mixed hydrophobic/positive surface region of the chemokine, which overlaps with the dimer interface. An increase of Fos-Choline-12 concentration above the critical micelle concentration causes a transition to a denatured state with a high α-helical content.

Biochemical analysis of matrix metalloproteinase activation of chemokines CCL15 and CCL23 and increased glycosaminoglycan binding of CCL16.

Leukocyte migration and activation is orchestrated by chemokines, the cleavage of which modulates their activity and glycosaminoglycan binding and thus their roles in inflammation and immunity. Early research identified proteolysis as a means of both activating or inactivating CXC chemokines and inactivating CC chemokines. Recent evidence has shown activating cleavages of the monocyte chemoattractants CCL15 and CCL23 by incubation with synovial fluid, although the responsible proteases could not be identified. Herein we show that CCL15 is processed in human synovial fluid by matrix metalloproteinases (MMPs) and serine proteases. Furthermore, a family-wide investigation of MMP processing of all 14 monocyte-directed CC chemokines revealed that each is precisely cleaved by one or more MMPs. By MALDI-TOF-MS, 149 cleavage sites were sequenced including the first reported instance of CCL1, CCL16, and CCL17 proteolysis. Full-length CCL15-(1-92) and CCL23-(1-99) were cleaved within their unique 31 and 32-amino acid residue extended amino termini, respectively. Unlike other CCL chemokines that lose activity and become receptor antagonists upon MMP cleavage, the prominent MMP-processed products CCL15-(25-92, 28-92) and CCL23-(26-99) are stronger agonists in calcium flux and Transwell CC receptor transfectant and monocytic THP-1 migration assays. MMP processing of CCL16-(1-97) in its extended carboxyl terminus yields two products, CCL16-(8-77) and CCL16-(8-85), with both showing unexpected enhanced glycosaminoglycan binding. Hence, our study reveals for the first time that MMPs activate the long amino-terminal chemokines CCL15 and CCL23 to potent forms that have potential to increase monocyte recruitment during inflammation.

The disulfide bond between cysteine 10 and cysteine 34 is required for CCL18 activity.

Asthma is a Th2-mediated disease that involves Th2 cell and eosinophil migration into the bronchial mucosa which is dependent upon the expression of a specific set of chemokines within the lung. Among them, CCL18 seems to play a key role because of its preferential expression in the lung, and its up-regulation by Th2 cytokines. Here, we show that the optimal naïve T cell and basophil chemotaxis, and basophil histamine release induced by rhCCL18 occurred at a 100 time lower concentration with CHO-derived rhCCL18 than with E. coli-derived rhCCL18. FT-ICR mass spectrometry of the intact chemokines showed that the rhCCL18 produced by CHO cells contained the 2 disulfide bonds Cys10-Cys34 and Cys11-Cys50, in clear contrast to the rhCCL18 derived from E. coli where the Cys10-Cys34 bond was absent. We found that reduction of the Cys10-Cys34 of the CHO-derived rhCCL18 resulted in a shift of its activity, reaching the same level as the E. coli-derived rhCCL18. These results demonstrate that the Cys10-Cys34 disulfide bond is involved in the function of CCL18.

Using glycosaminoglycan/chemokine interactions for the long-term delivery of 5P12-RANTES in HIV prevention.

5P12-RANTES is a recently developed chemokine analogue that has shown high level protection from SHIV infection in macaques. However, the feasibility of using 5P12-RANTES as a long-term HIV prevention agent has not been explored partially due to the lack of available delivery devices that can easily be modified for long-term release profiles. Glycosaminoglycans (GAGs) have been known for their affinity for various cytokines and chemokines, including native RANTES, or CCL5. In this work, we investigated used of GAGs in generating a chemokine drug delivery device. Initial studies used surface plasmon resonance analysis to characterize and compare the affinities of different GAGs to 5P12-RANTES. These different GAGs were then incorporated into drug delivery polymeric hydrogels to engineer sustained release of the chemokines. In vitro release studies of 5P12-RANTES from the resulting polymers were performed, and we found that 5P12-RANTES release from these polymers can be controlled by the amount and type of GAG incorporated. Polymer disks containing GAGs with stronger affinity to 5P12-RANTES resulted in more sustained and longer term release than did polymer disks containing GAGs with weaker 5P12-RANTES affinity. Similar trends were observed by varying the amount of GAGs incorporated into the delivery system. 5P12-RANTES released from these polymers demonstrated good levels of CCR5 blocking, retaining activity even after 30 days of incubation.

Molecular identification and expression analysis of the CC chemokine gene in rock bream (Oplegnathus fasciatus) and the biological activity of the recombinant protein.

We identified the CC chemokine cDNA designated as RbCC1 (CC chemokine 1 in rock bream, Oplegnathus fasciatus), which was isolated using expressed sequence tag (EST) analysis of a lipopolysaccharide (LPS)-stimulated rock bream liver cDNA library. The full-length RbCC1 cDNA (850 bp) contained an open reading frame (ORF) of 366 bp encoding 122 amino acids. Results from our phylogenetic analysis demonstrated that the RbCC1 was closest relationship to the orange-spotted grouper and Mi-iyu croaker CC chemokines located within the fish CC chemokine group. RbCC1 was significantly expressed in the intestine, spleen, liver, and PBLs (peripheral blood leukocytes). Rock bream PBLs were stimulated with several mitogens, LPS and Con A/PMA which significantly induced the expression of RbCC1 mRNA in the PBLs. The RbCC1 mRNA expression in several tissues under conditions of bacterial and viral challenge was examined. The experimental challenge revealed that the kidney and spleen of fish infected with Streptococcus iniae showed the most significant increases in RbCC1 expression compared to the control. In the case of RSIV infection, the RbCC1 mRNA expression was markedly up-regulated in the liver. In this study, recombinant RbCC1 (approximately 53 kDa) was produced using an Escherichia coli expression system followed by purification. Subsequently, the addition of purified rRbCC1 was examined to investigate the impact on the proliferative and chemotactic activity on kidney leukocytes from rock bream. The results demonstrated that the rRbCC1 induces significant biological activity on kidney leukocyte proliferation and attraction at concentrations in the range of 10-300 µg/mL and suggests that rRbCC1 could be utilized as an immune-stimulant and/or molecular adjuvant to enhance the immune effects of vaccines.

Molecular cloning and functional characterization of a mouse ccl6 analog gene in the rat.

Suppression subtractive hybridization was used to analyze differential expression of genes in rat peritoneal macrophages after granulocyte macrophage colony-stimulating factor treatment. We identified and cloned the mouse C10 analog gene in the rat, and named it as ccl6. The full-length cDNA of rat ccl6 was 467 bp, which contains a single-open reading frame and encodes 116 amino acid residues. Compared with other C-C chemokines, the rat ccl6 gene had an unusual four-exon genome structure instead of the typical three exons, it had the highest homology with murine ccl6. The rat ccl6 gene was localized on chromosome 10, where most of the C-C chemokine superfamily members are located. The recombinant rat C-C chemokine ligand 6 (CCL6) protein was expressed by the pGEX4T-1 plasmid in Escherichia coli BL21. The purified recombinant protein had bioactivity similar to that of mouse CCL6, which is a chemoattractant for macrophages and lymphocytes, but not for neutrophils.