Edward R. Appelbaum

Osteoprotegerin Is a Receptor for the Cytotoxic Ligand TRAIL

TRAIL is a tumor necrosis factor-related ligand that induces apoptosis upon binding to its death domain-containing receptors, DR4 and DR5. Two additional TRAIL receptors, TRID/DcR1 and DcR2, lack functional death domains and function as decoy receptors for TRAIL. We have identified a fifth TRAIL receptor, namely osteoprotegerin (OPG), a secreted tumor necrosis factor receptor homologue that inhibits osteoclastogenesis and increases bone density in vivo. OPG-Fc binds TRAIL with an affinity of 3.0 nm, which is slightly weaker than the interaction of TRID-Fc or DR5-Fc with TRAIL. OPG inhibits TRAIL-induced apoptosis of Jurkat cells. Conversely, TRAIL blocks the anti-osteoclastogenic activity of OPG. These data suggest potential cross-regulatory mechanisms by OPG and TRAIL.

Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.

Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin‐2, MPIF‐2, or CKβ‐6, is a human CC chemokine with low amino acid sequence identity to other chemo‐ kines. Eotaxin‐2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross‐desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP‐4, but not RANTES, MIP‐lα, or MCP‐3, can completely cross‐desensitize the calcium response to eotaxin‐2 on these cells, indicating that eotaxin‐2 shares the same receptor used by eotaxin and MCP‐4. Eotaxin‐2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin‐2 also displaced 125I‐eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO‐CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP‐4. l25I‐Eotaxin‐2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin‐2 displace the ligand with equal affinity. Eotaxin and eotaxin‐2 promote a Ca2+ transient in RBL‐2H3 cells stably transfected with CCR3 (RBL‐2H3‐CCR3) and both ligands cross‐desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin‐2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor. J. Leukoc. Biol. 62: 667–675; 1997.

Cloning, in vitro expression, and functional characterization of a novel human CC chemokine of the monocyte chemotactic protein (MCP) family (MCP-4) that binds and signals through the CC chemokine receptor 2B.

Here we describe the characterization of a novel human CC chemokine, tentatively named monocyte chemotactic protein (MCP-4). This chemokine was detected by random sequencing of expressed sequence tags in cDNA libraries. The full-length cDNA revealed an open reading frame for a 98-amino acid residue protein, and a sequence alignment with known CC chemokines showed high levels of similarity (59–62%) with MCP-1, MCP-3, and eotaxin. MCP-4 cDNA was cloned into Drosophila S2 cells, and the mature protein (residues 24–98) was purified from the conditioned medium. Recombinant MCP-4 induced a potent chemotactic response (EC50 = 2.88 ± 0.15 nm) and a transient rise in cytosolic calcium concentration in fresh human peripheral blood monocytes but not in neutrophils. Binding studies in monocytes showed that MCP-4 and MCP-3 were very potent in displacing high affinity binding of125I-MCP-1 (IC50 for MCP-4, MCP-3, and unlabeled MCP-1 of 2.1 ± 1.4, 0.85–1.6, and 0.7 ± 0.2 nm respectively), suggesting that all three chemokines interact with the CC chemokine receptor-2 (MCP-1 receptor). This was confirmed in binding studies with Chinese hamster ovary cells, stably transfected with the CC chemokine 2B receptor. Northern blot analysis in extracts of normal human tissues showed expression of mRNA for MCP-4 in small intestine, thymus, and colon, but the level of protein expression was too low to be detected in Western blot analysis. However, expression of MCP-4 protein was demonstrated by immunohistochemistry in human atherosclerotic lesion and found to be associated with endothelial cells and macrophages.

Binding Interactions of Human Interleukin 5 with Its Receptor α Subunit LARGE SCALE PRODUCTION, STRUCTURAL, AND FUNCTIONAL STUDIES OF DROSOPHILA-EXPRESSED RECOMBINANT PROTEINS

Human interleukin 5 (hIL5) and soluble forms of its receptor α subunit were expressed in Drosophila cells and purified to homogeneity, allowing a detailed structural and functional analysis. B cell proliferation confirmed that the hIL5 was biologically active. Deglycosylated hIL5 remained active, while similarly deglycosylated receptor α subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-Å resolution and found to be similar to that of the protein produced in Escherichia coli. Human IL5 was shown by analytical ultracentrifugation to form a 1:1 complex with the soluble domain of the hIL5 receptor α subunit (shIL5Rα). Additionally, the relative abundance of ligand and receptor in the hIL5·shIL5Rα complex was determined to be 1:1 by both titration calorimetry and SDS-polyacrylamide gel electrophoresis analysis of dissolved cocrystals of the complex. Titration microcalorimetry yielded equilibrium dissociation constants of 3.1 and 2.0 n M, respectively, for the binding of hIL5 to shIL5Rα and to a chimeric form of the receptor containing shIL5Rα fused to the immunoglobulin Fc domain (shIL5Rα-Fc). Analysis of the binding thermodynamics of IL5 and its soluble receptor indicates that conformational changes are coupled to the binding reaction. Kinetic analysis using surface plasmon resonance yielded data consistent with the Kdvalues from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor α subunit. Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5Rα in Drosophila membranes was found to be 6 n M, in accord with the affinities measured for the soluble receptor forms. Hence, most of the binding energy of the α receptor is supplied by the soluble domain. Taken with other aspects of hIL5 structure and biological activity, the data obtained allow a prediction for how 1:1 stoichiometry and conformational change can lead to the formation of hIL5·receptor αβ complex and signal transduction.

Neuromedin U elicits cytokine release in murine Th2-type T cell clone D10.G4.1

Neuromedin U (NmU), originally isolated from porcine spinal cord and later from other species, is a novel peptide that potently contracts smooth muscle. NmU interacts with two G protein-coupled receptors designated as NmU-1R and NmU-2R. This study demonstrates a potential proinflammatory role for NmU. In a mouse Th2 cell line (D10.G4.1), a single class of high affinity saturable binding sites for 125I-labeled NmU (KD 364 pM and Bmax 1114 fmol/mg protein) was identified, and mRNA encoding NmU-1R, but not NmU-2R, was present. Competition binding analysis revealed equipotent, high affinity binding of NmU isopeptides to membranes prepared from D10.G4.1 cells. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular Ca2+ concentration (EC50 4.8 nM for human NmU). In addition, NmU also significantly increased the synthesis and release of cytokines including IL-4, IL-5, IL-6, IL-10, and IL-13. Studies using pharmacological inhibitors indicated that maximal NmU-evoked cytokine release required functional phospholipase C, calcineurin, MEK, and PI3K pathways. These data suggest a role for NmU in inflammation by stimulating cytokine production by T cells.

Radioligand Binding and Functional Characterization of Recombinant Human NmU1 and NmU2 Receptors Stably Expressed in Clonal Human Embryonic Kidney-293 Cells

Neuromedin U (NmU) is a smooth muscle contracting peptide. Recently, two G-protein-coupled receptors for NmU (NmU1R and NmU2R) have been cloned having approximately 50% homology. They have distinct patterns of expression suggesting they may have different biological functions. This study provides a comprehensive characterization of both NmU receptors expressed in human embryonic kidney 293 cells. [125I]hNmU binding to the recombinant NmU receptors was rapid, saturable, of high affinity and to a single population of binding sites. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular [Ca2+]i release (EC50 value of 0.50 ± 0.10 nmol/l) and inositol phosphate formation (EC50 1.6 ± 0.2 and 1.50 ± 0.4 nmol/l for NmU1R and NmU2R respectively). Furthermore, hNmU inhibited forskolin (3 µmol/l)-stimulated accumulation of cAMP in intact HEK-293 cells expressing either NmU1R or NmU2R. The inhibitory effect was significant for the cells expressing NmU2R with IC50 value of 0.80 ± 0.21 nmol/l. In summary, both NmU1R and NmU2R in HEK-293 cells have similar signaling capability.

Optimization of a Saccharomyces cerevisiae fermentation process for production of a therapeutic recombinant protein using a multivariate Bayesian approach.

Various approaches have been applied to optimize biological product fermentation processes and define design space. In this article, we present a stepwise approach to optimize a Saccharomyces cerevisiae fermentation process through risk assessment analysis, statistical design of experiments (DoE), and multivariate Bayesian predictive approach. The critical process parameters (CPPs) were first identified through a risk assessment. The response surface for each attribute was modeled using the results from the DoE study with consideration given to interactions between CPPs. A multivariate Bayesian predictive approach was then used to identify the region of process operating conditions where all attributes met their specifications simultaneously. The model prediction was verified by twelve consistency runs where all batches achieved broth titer more than 1.53 g/L of broth and quality attributes within the expected ranges. The calculated probability was used to define the reliable operating region. To our knowledge, this is the first case study to implement the multivariate Bayesian predictive approach to the process optimization for the industrial application and its corresponding verification at two different production scales. This approach can be extended to other fermentation process optimizations and reliable operating region quantitation.