Lara Izotova

Identification, Cloning, and Characterization of a Novel Soluble Receptor That Binds IL-22 and Neutralizes Its Activity

With the use of a partial sequence of the human genome, we identified a gene encoding a novel soluble receptor belonging to the class II cytokine receptor family. This gene is positioned on chromosome 6 in the vicinity of the IFNGR1 gene in a head-to-tail orientation. The gene consists of six exons and encodes a 231-aa protein with a 21-aa leader sequence. The secreted mature protein demonstrates 34% amino acid identity to the extracellular domain of the IL-22R1 chain. Cross-linking experiments demonstrate that the protein binds IL-22 and prevents binding of IL-22 to the functional cell surface IL-22R complex, which consists of two subunits, the IL-22R1 and the IL-10R2c chains. Moreover, this soluble receptor, designated IL-22-binding protein (BP), is capable of neutralizing IL-22 activity. In the presence of the IL-22BP, IL-22 is unable to induce Stat activation in IL-22-responsive human lung carcinoma A549 cells. IL-22BP also blocked induction of the suppressors of cytokine signaling-3 (SOCS-3) gene expression by IL-22 in HepG2 cells. To further evaluate IL-22BP action, we used hamster cells expressing a modified IL-22R complex consisting of the intact IL-10R2c and the chimeric IL-22R1/γR1 receptor in which the IL-22R1 intracellular domain was replaced with the IFN-γR1 intracellular domain. In these cells, IL-22 activates biological activities specific for IFN-γ, such as up-regulation of MHC class I Ag expression. The addition of IL-22BP neutralizes the ability of IL-22 to induce Stat activation and MHC class I Ag expression in these cells. Thus, the soluble receptor designated IL-22BP inhibits IL-22 activity by binding IL-22 and blocking its interaction with the cell surface IL-22R complex.

The interferon gamma (IFN-γ) receptor: a paradigm for the multichain cytokine receptor

Abstract With the purification and cloning of the interferon gamma (IFN-γ) receptor chains the mechanism of IFN-γ action and the resultant signal transduction events were delineated in remarkable detail. The interferon gamma (IFN-γ) receptor complex consists of two chains: IFN-γR1, the ligand-binding chain, and IFN-γR2, the accessory chain. Binding of IFN-γ causes oligomerization of the two IFN-γ receptor subunits, IFN-γR1 and IFN-γR2, which initiates the signal transduction events: activation of Jakl and Jak2 receptor associated protein tyrosine kinases, phosphorylation of the IFN-γR1 intracellular domain on Tyr 440 followed by phosphorylation and activation of Stat1α, the latent transcriptional factor. With all these steps established, the IFN-γ receptor complex has provided the basic model for understanding the receptors for other members of the family of class II cytokine receptors.

Interferon protects mice against inhalation anthrax.

Interferons (IFNs) play a role in innate immunity during many viral, bacterial, and protozoal infections. With the increasing threat of bioterrorist attacks with Bacillus anthracis, its high lethality, and the limited effectiveness of antibiotics, alternative treatments are being studied. Antibodies to protective antigen (PA) are promising, as is IFN. During many bacterial infections, production of and protection by IFNs has been reported, including B. anthracis in vitro. In vivo, we find that (1) the type I IFN inducer, Poly-ICLC, strongly and rapidly protects mice; (2) the protection is IFN-mediated since recombinant murine IFN-beta can protect, and protection by Poly-ICLC is abrogated in IFN type I receptor knockout mice. The greatest protection by Poly-ICLC was conferred by intranasal treatment. A delay in death was observed with the intramuscular route alone, but was not significant. Together, the results suggest the IFN defense could protect mice, up to 60%, against lethal inhalational anthrax, and thus have important medical implications for therapy of human anthrax.

Design and construction of a phosphorylatable chimeric monoclonal antibody with a highly stable phosphate.

A recognition site for the cAMP-dependent protein kinase was introduced into the MAb-chCC49 by site-directed mutation of the coding sequence to make a variant of MAb-chCC49 containing a highly stable phosphate. To design this monoclonal antibody (MAb) without changing its immunoreactivity or biological properties, molecular modeling was used to locate appropriate regions for introduction of the cAMP-dependent phosphorylation site with desirable properties. We selected one position to mutate on the heavy chain based on molecular dynamics study of the solvated antibody. A vector expressing the mutant was constructed and transfected into mouse myeloma NS0 cells that expressed a high level of the resultant MAb-WW5. MAb-WW5 contained the cAMP-dependent phosphorylation site at the hinge region of the heavy chain, could be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase with [gamma-32P]ATP to high specific activity, and retained the phosphate stably. Compared with MAb-chCC49K1, another phosphorylatable variant of MAb-chCC49, the phosphate attached to MAb-WW5 showed much improved stability: about a 10-fold increase in resistance to hydrolysis. MAb-WW5 exhibited the same binding specificity to the TAG-72 antigen on MCF-7 4C10 breast cancer cells as we observed with MAb-chCC49K1. The improved stability of the attached phosphate provides a MAb with potential to be used in diagnosis and therapy of adenocarcinomas.

Use of phosphorylation site tags in proteins

Publisher Summary This chapter discusses the use of phosphorylation site tags in proteins. Labeled proteins are used in a variety of applications including ligand-receptor interactions, pharmacokinetics, diagnostic imaging, and therapy. In particular, monoclonal antibodies labeled with many different isotopes have been used in diagnostic imaging as well as therapy. Two general methods are used for introduction of phosphorylation sites into proteins: site-specific mutations and fusions. Phosphorylation sites can be introduced into proteins as fusion segments to the N terminus or the C terminus, or even as internal segments. Also, it is possible to introduce phosphokinase recognition sites within the protein itself. This can often be accomplished by changing one amino acid with little or no modification of the function or biological activity of the protein. It is desirable to have some useful information about the protein before choosing a site to modify. Before phosphorylation, the proteins are purified by standard procedures. Because the introduction of the phosphorylation sites does not materially change the structure of the proteins, the usual procedures used for the unmodified proteins have been found to be effective.