SUMMARYThe type 2 helper T cell (T H 2) cytokine interleukin (IL)-4 is thought to play a central role in the early stages of asthma. In an effort to develop an antibody treatment for asthma that neutralizes the effects of IL-4, a murine monoclonal antibody, 3B9, was generated with specificity for human IL-4. In vitro studies demonstrated that 3B9 inhibited IL-4-dependent events including IL-5 synthesis, T H 2 cell activation and up-regulation of immunoglobulin E expression. 3B9 was then humanized (pascolizumab, SB 240683) to reduce immunogenicity in humans. SB 240683 demonstrated species specificity for both monkey and human IL-4 with no reactivity to mouse, rat, cow, goat or horse IL-4. Pascolizumab inhibited the response of human and monkey T cells to monkey IL-4 and effectively neutralized IL-4 bioactivity when tested against several IL-4-responsive human cell lines. Affinity studies demonstrated rapid IL-4 binding by pascolizumab with a slow dissociation rate. In vivo pharmacokinetic and chronic safety testing in cynomolgus monkeys demonstrated that pascolizumab was well tolerated, and no adverse clinical responses occurred after up to 9 months of treatment. Three monkeys developed an anti-idiotypic response that resulted in rapid pascolizumab clearance. However, in the chronic dosing study the antibody response was transient and not associated with clinical events. In conclusion, pascolizumab is a humanized anti-IL-4 monoclonal antibody that can inhibit upstream and downstream events associated with asthma, including T H 2 cell activation and immunoglobulin E production. Clinical trials are under way to test the clinical efficacy of pascolizumab for asthma.
Human interleukin 5 (hIL5) and soluble forms of its receptor alpha 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 alpha subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-A 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 alpha subunit (shIL5R alpha). Additionally, the relative abundance of ligand and receptor in the hIL5.shIL5R alpha 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 nM, respectively, for the binding of hIL5 to shIL5R alpha and to a chimeric form of the receptor containing shIL5R alpha fused to the immunoglobulin Fc domain (shIL5R alpha-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 Kd values from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor alpha subunit. Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5R alpha in Drosophila membranes was found to be 6 nM, in accord with the affinities measured for the soluble receptor forms. Hence, most of the binding energy of the alpha 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 alpha beta complex and signal transduction.
Cassette mutagenesis was used to identify side chains in human interleukin 5 (hIL-5) that mediate binding to hIL-5 receptor a chain . A series of single alanine substitutions was introduced into a stretch of residues in the C-terminal region, including helix D, which previously had been implicated in receptor ca chain recognition and which is aligned on the IL-5 surface so as to allow the topography of receptor binding residues to be examined. hIL-5 and single site mutants were expressed in COS cells, their interactions with hIL-5Ra were measured by a sandwich surface plasmon resonance biosensor method, and their biological activities were measured by an IL-5-dependent cell proliferation assay. A pattern of mutagenesis effects was observed, with greatest impact near the interface between the two four-helix bundles of IL-5, in particular at residues , and least at the distal ends of the D helices. This pattern suggests the possibility that residues near the interface of the two four-helix bundles in hIL-5 comprise a central patch or hot spot, which constitutes an energetically important a chain recognition site. This hypothesis suggests a structural explanation for the 1:1 stoichiometry observed for the complex of hIL-5 with hIL-5Ra.Interleukin 5 (IL-5) plays a central role in the control of eosinophilia and as such is a major contributor to the tissue damage seen in asthma and other eosinophil-related disorders (1-4). The high-resolution crystallographic structure of human IL-5 (hIL-5) has been determined (5, 6). It contains a core of two four-helix bundles in the IL-5 dimer, with each of the four-helix units similar to that in other cytokines (7-11). However, the arrangement of bundles in IL-5 is unusual in that helix D of one monomer combines with helices A, B, and C of the second monomer and vice versa.The hIL-5 receptor is composed of two types of subunits, denoted a and ,3 (hIL-SRa and -13) (12). observed (6, 14).The mechanism of IL-5 recognition of receptor a chain and the structural origin of 1:1 stoichiometry have formed a major focus for our mechanistic studies of hIL-5R recognition. In this study, we chose to investigate the effects of mutagenesis of residues in the C-terminal region of hIL-5 that includes helix D. This was based on a previous report (19) that used data from hybrid molecules of mouse-human IL-5 to suggest that the C-terminal region interacts directly with IL-5Rca and confers the species specificity of IL-5. We made sequence changes over a stretch of surface that extends from the distal ends of the two four-helix bundles of the IL-5 dimer inward to the interface between the bundles. This mutagenesis series allowed us to investigate both the importance of specific residues in the C-terminal region and the overall topography of the receptor recognition site on IL-5. The results show that the greatest impact of mutagenesis on receptor binding occurs for residues close together at the bundle interface, in particular Glu-110 and Trp-111. This observation suggests that a central reco...
1 is a T cell-derived cytokine that specifically stimulates differentiation, proliferation, and activation of eosinophils (1) and appears to play an important role in the pathogenesis of asthma. IL-5 is a homodimer that folds into a cylindrical molecule containing two four-helix bundles (2), each of which resembles the four-helix bundle seen in the monomeric cytokines IL-3, IL-4, granulocyte-macrophage colony-stimulating factor, and growth hormone.The cell receptor for IL-5 is composed of two subunits, ␣ and  c . The ␣ chain is cytokine-specific and by itself can bind IL-5 with high affinity (3). Extensive site-directed mutagenesis studies have shown that residues clustered near the helix bundle interface, in the CD loop, including Glu 89 and Arg 91 , and at the carboxyl-terminal end of helix D, notably Glu 110 , engage in receptor ␣ chain binding (4 -6). Glu 13 , which is at each of the distal ends of the IL-5 cylinder away from the interface between the two four-helix bundles and is not involved directly in ␣ chain interaction, is a key residue mediating productive interaction of the  c chain of IL-5 receptor (4, 5). It is the  c recruitment into ␣ c that leads to triggering of the intracellular signaling cascade.Although site-directed mutagenesis studies have identified important residues for receptor binding, the one-residue-at-atime substitution allowed by this technique limits a full mechanistic understanding of the specific structural and electrostatic features of the IL-5 surface required for receptor-ligand interaction. However, through the use of randomly generated side chain libraries, it should be possible to measure the effect of replacing individual side chains or sets of side chains in local regions on the IL-5 surface and hence determine which combinations allow productive binding. Such random epitope mutagenesis can be achieved with sequence libraries formed by phage display. This technique has been applied successfully to in vitro antibody maturation and protein engineering (7). Typically a foreign gene is fused in frame with phage coat protein pIII encoded by a phagemid vector, and the surface-displayed recombinant foreign protein selected by affinity selection procedures (biopanning). Human growth hormone has been displayed on phage and higher receptor affinity variants selected through random mutagenesis and phage panning (8). Phage display of multichain proteins also has been achieved, for example for antibody Fab fragments (9) and vascular epidermal growth factor (10). Nonetheless, in our hands, phage display of the dimeric wild type IL-5 has proven difficult, perhaps reflecting folding difficulties that previously have led to insoluble inclusion bodies in intracellular Escherichia coli expression (11).We have sought to overcome limitations in phage display of IL-5 by using a single chain form of human IL-5 (scIL-5), in which two IL-5 molecules are tandemly linked by a Gly-Gly dipeptide linker (12). Single chain IL-5 and wtIL-5 have virtually identical biological activities and bindin...
Integrins play a key role in cellular immune responses in a variety of organisms; however, knowledge of integrins and their effects on cell signalling and functional responses in molluscan defence reactions is poor. Using integrin-mediated cell adhesion kits, alphaVbeta3 and beta1 integrin-like subunits were identified on the surface of Lymnaea stagnalis haemocytes. Haemocyte binding via these integrins was found to be dependent on Ca2+/Mg2+. Western blotting with an anti-phospho (anti-active) focal adhesion kinase (FAK) antibody revealed a 120-125 kDa FAK-like protein in these cells; this protein was transiently phosphorylated upon haemocyte adhesion over 90 min, with maximal phosphorylation occurring after 30 min binding. Also, integrin engagement with the tetrapeptide Arg-Gly-Asp-Ser (RGDS) resulted in a rapid increase in phosphorylation of the FAK-like protein; however, RGDS did not affect the phosphorylation of extracellular signal-regulated kinase. Treatment of haemocytes with RGDS (2 mM) inhibited phagocytosis of E. coli bioparticles by 88%. Moreover, at this concentration, RGDS reduced cell spreading by 61%; stress fiber formation was also impaired. Taken together, these results demonstrate a role for integrins in L. stagnalis haemocyte adhesion and defence reactions and, for the first time, link integrin engagement to FAK activation in molluscs.
Wild type human (h) interleukin 5 (wt IL5) Human interleukin 5 (hIL5)1 is a T cell-derived cytokine which plays an important role in the differentiation, proliferation, and activation of eosinophils (Sanderson et al., 1992;Bentley et al., 1992). Natural hIL5 is a disulfide-linked, homodimeric glycoprotein with 115 residues per chain. The high resolution crystal structures of both Escherichia coli-expressed (Milburn et al., 1993) and Drosophila-expressed hIL5 (Johanson et al., 1995) have revealed a core of two four-helix bundles. Each four-helix bundle resembles the four-helix bundle seen in IL2 (Bazan and McKay, 1992), IL4 (Smith et al., 1992), and GM-CSF (Diederichs et al., 1991). However, the bundle organization in IL5 is unique in that helix D of one monomer combines with helices A, B, and C of the second monomer, and vice versa. In the two-bundle structure, the A and D helices form one face, the B and C helices a second. The organization of structural features on each face is palindromic, as observed for restriction sites in DNA.Human IL5 receptor is composed of two different chains, denoted ␣ and  (Tavernier et al., 1991). The ␣ chain is specific for IL5 (Murata et al., 1992) and has a K d in the 0.1-1 nM range depending on the assays and receptor forms examined (Li et al., 1996). In contrast, the  chain of hIL5R is not cytokine-specific but is shared with the receptors for IL3 and GM-CSF (Tavernier et al., 1991) and appears needed for signal transduction. A soluble form of hIL5 receptor ␣ chain (shIL5R␣), which has only the extracellular domain of IL5R␣, also has been described. This binds to hIL5 with nanomolar affinity (Tavernier et al., 1991;Johanson et al., 1995). Despite the dimeric nature of IL5, a 1:1 binding stoichiometry between shIL5R␣ and hIL5 has been reported (Devos et al., 1993;Johanson et al., 1995).New insights into the binding mechanism of IL5 to its receptor are emerging with the availability of high resolution structure and mutagenesis techniques. Data from hybrid constructs of mouse/human IL5 suggest that the carboxyl-terminal 36 residues of IL5 interact directly with the IL5R␣ and confer species specificity (McKenzie et al., 1991). By Ala-scanning mutagenesis of the carboxyl-terminal region of hIL5, we found previously that Glu-110 and Trp-111 contribute significantly to receptor binding . In addition, mutation of residues (Glu-89 and Arg-91) in the loop between helices C and D have been found to affect binding to IL5R␣ (Tavernier et al., 1995;Graber et al., 1995). All of these residues cluster around the interface between the two 4-helix bundles of hIL5 and appear to constitute a central patch for binding to a single molecule of hIL5R␣ . In contrast, residue Glu-13, which is at the distal ends of the IL5 dimer away from the helix bundle interface, was suggested to interact with the  chain of IL5R, since mutation at this position resulted in loss of biological activity but did not affect the binding affinity to the ␣ chain (Tavernier et al., 1995;Graber et al., 1995).While emerging...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.