A potent dimeric peptide antagonist of interleukin-5 that binds two interleukin-5 receptor alpha chains.

Two series of peptides that specifically bind to the extracellular domain of the alpha chain of the human interleukin-5 receptor (IL-5Ralpha), but share no primary sequence homology to IL-5, were identified from libraries of random recombinant peptides. Affinity maturation procedures generated a 19-aa peptide that binds to the IL-5 receptor alpha/beta heterodimer complex with an affinity equal to that of IL-5 and is a potent and specific antagonist of IL-5 activity in a human eosinophil adhesion assay. The active form of the peptide is a disulfide-crosslinked dimer that forms spontaneously in solution. Gel filtration analysis, receptor-binding studies, and analytical ultracentrifugation reveal that the dimeric peptide binds simultaneously to two receptor alpha chains in solution. Furthermore, the dimer peptide, but not IL-5, can activate a chimeric receptor consisting of the IL-5Ralpha extracellular domain fused to the intracellular domain of the epidermal growth factor receptor, thus demonstrating that the peptide also promotes receptor dimerization in a cellular context. The functional antagonism produced by the bivalent interaction of the dimeric peptide with two IL-5R alpha chains represents a distinctive mechanism for the antagonism of cytokines that use heteromeric receptors.

Gene gun delivered DNA-based immunizations mediate rapid production of murine monoclonal antibodies to the Flt-3 receptor.

Class-switched, affinity-matured murine monoclonal antibody (MAb)-producing cell lines were generated against the Flt-3 receptor in less than 4 weeks following polynucleotide immunizations, used in conjunction with repetitive immunizations, multiple sites (RIMMS). Plasmid DNA encoding Flt-3/Fc was coated onto gold particles, which were subsequently propelled into the epidermis of mice using biolistic particle bombardment using the Accell gene gun. Pools of immune peripheral lymph node cells were somatically fused 13 days after the onset of delivery of DNA encoding the target antigen. To determine if early responses could be augmented, DNA-encoding murine GM-CSF was delivered 3 days prior to the Flt-3/Fc DNA immunizations. The data presented demonstrates the successful identification and characterization of class-switched, affinity-matured MAbs that bind to the Flt-3 receptor. When compared to conventional methodologies or intramuscular targeted DNA-based immunization for the generation of MAbs, use of the gene gun in conjunction with RIMMS allows for a more rapid production of affinity-matured MAb-producing cell lines.

A generic method for the production of cell lines expressing high levels of 7-transmembrane receptors.

G-protein-coupled or 7-transmembrane receptors (7TMRs) are often studied after heterologous expression in mammalian cells such as COS-7, CHO-K1, or HEK-293s. In this paper, we describe the development of a rapid and generic method for producing stable Chinese hamster ovary cell lines expressing high levels of recombinant 7TMRs by N-terminal tagging these proteins with the hemagglutinin (HA) sequence. To illustrate the broad applicability of this technique, we have presented data from cell lines expressing a glycoprotein hormone receptor for follicle-stimulating hormone (FSHR), CXC- (CXCR-2), and CC-chemokine (CCR-1) receptors and peptide receptors from the somatostatin (SSTR1, 2, 5) and neuropeptide Y (NPY-Y2, -Y4 Rs) families. Typically, cell lines with a receptor density of 1 to 15 pmol/mg protein are produced with this method. The presence of the HA tag does not adversely affect the binding or functional activity of the receptors.

Receptor-mediated endocytosis of CC-chemokines.

Chemokines are chemotactic proteins which play a central role in immune and inflammatory responses. Chemokine receptors are members of the seven transmembrane G-protein coupled family and have recently been shown to be involved in the entry of human immunodeficiency virus (HIV) into target cells. To study chemokine endocytosis in detail we have used novel site-specific chemistry to make a fluorescently labeled CC-chemokine agonist (rhodamine-MIP-1alpha) and antagonist (NBD-RANTES). We have also generated a CHO cell line stably expressing a hemagglutinin-tagged version of the CC-chemokine receptor 1 (CCR1), and using these reagents we have examined the receptor-mediated endocytosis of CC-chemokines by confocal microscopy. Our studies reveal that the agonist was internalized and accumulated in transferrin receptor-positive endosomes whereas the antagonist failed to internalize. However, receptor-bound antagonist could be induced to internalize by co-administration of agonist. Analysis of receptor redistribution following chemokine addition confirmed that sequestration was induced by agonists but not by antagonists.

In vitro selection of peptides acting at a new site of NMDA glutamate receptors.

Oligomeric N-methyl D-aspartate receptor (NMDAR) in brain is a ligand-gated ion channel that becomes selectively permeable to ions upon binding to ligands. For NMDAR channel, the binding of glutamate and glycine results in opening of the calcium permeable channel. Because the calcium influx mediated by NMDAR is important for synaptic plasticity and excitotoxicity, the function of NMDA receptors has been implicated in both health and disease. Native NMDA receptors are thought to be heteromeric pentamers with a central ion conduction pathway. There are five genes (NR1, 2A, 2B, 2C, and 2D) encoding various subunits that have been cloned, and NR1 is thought to be the essential subunit since it forms a functional channel by itself. To study NMDAR structure and function, we have searched for peptide modulators of NR1 using random peptide bacteriophage libraries. The peptides were identified based on their specific association with a purified receptor fusion protein that contains the putative ligand binding domain. We report the identification of one group of cyclic peptides (Mag-1) with a consensus sequence of CDGLRHMWFC. Using biochemical binding analysis and patch clamp electrophysiological recording, we show that the synthetic Mag-1 peptides cause noncompetitive inhibition of the receptor channel activity.

High affinity type I interleukin 1 receptor antagonists discovered by screening recombinant peptide libraries.

Two families of peptides that specifically bind the extracellular domain of the human type I interleukin I (IL-1) receptor were identified from recombinant peptide display libraries. Peptides from one of these families blocked binding of IL-lalpha to the type I IL-1 receptor with IC50 values of 45-140 microM. Affinity-selective screening of variants of these peptides produced ligands of much higher affinity (IC50 approximately 2 nM). These peptides block IL-1-driven responses in human and monkey cells; they do not bind the human type II IL-1 receptor or the murine type I IL-1 receptor. This is the first example (that we know of) of a high affinity peptide that binds to a cytokine receptor and acts as a cytokine antagonist.

Improved green fluorescent protein by molecular evolution using DNA shuffling.

Green fluorescent protein (GFP) has rapidly become a widely used reporter of gene regulation. However, for many organisms, particularly eukaryotes, a stronger whole cell fluorescence signal is desirable. We constructed a synthetic GFP gene with improved codon usage and performed recursive cycles of DNA shuffling followed by screening for the brightest E. coli colonies. A visual screen using UV light, rather than FACS selection, was used to avoid red-shifting the excitation maximum. After 3 cycles of DNA shuffling, a mutant was obtained with a whole cell fluorescence signal that was 45-fold greater than a standard, the commercially available Clontech plasmid pGFP. The expression level in E. coli was unaltered at about 75% of total protein. The emission and excitation maxima were also unchanged. Whereas in E. coli most of the wildtype GFP ends up in inclusion bodies, unable to activate its chromophore, most of the mutant protein is soluble and active. Three amino acid mutations appear to guide the mutant protein into the native folding pathway rather than toward aggregation. Expressed in Chinese Hamster Ovary (CHO) cells, this shuffled GFP mutant showed a 42-fold improvement over wildtype GFP sequence, and is easily detected with UV light in a wide range of assays. The results demonstrate how molecular evolution can solve a complex practical problem without needing to first identify which process is limiting. DNA shuffling can be combined with screening of a moderate number of mutants. We envision that the combination of DNA shuffling and high throughput screening will be a powerful tool for the optimization of many commercially important enzymes for which selections do not exist.

Ligand binding kinetics of IL-2 and IL-15 to heteromers formed by extracellular domains of the three IL-2 receptor subunits.

Studies on the binding of IL-2 to its receptor (IL-2R) have generally been limited to receptors expressed on cell surfaces. This has hampered detailed kinetic and mechanistic studies at the molecular level. We have prepared the soluble extracellular domains of all three receptor subunits (called alpha, beta and gamma) by recombinant techniques and have used these to perform detailed kinetic studies of their binding properties using the technique of surface plasmon resonance. We describe a novel approach whereby the receptors are assembled on an antibody surface, being held by an epitope engineered into the C-terminus of each of these domains. Thus the receptors are oriented naturally leading to homogeneous ligand binding kinetics. We have characterized the interactions of the heteromeric complexes of these subunits with mouse and human IL-2 and their analogs, as well as the recently discovered cytokine, IL-15. We have also studied the extracellular domains of the mouse receptor subunits for the first time and have used these as well as mouse-human hybrid receptors to probe the mechanism of assembly of these complexes. We show that no additional proteins are required to reproduce the properties of these complexes in vitro. In addition, kinetic studies with site-specific analogs of IL-2 and the mouse-human receptor hybrids clearly indicate that the extracellular domains of alpha and beta can together readily bind ligand with kinetic properties distinct from those of the constituent subunits. In contrast, a complex containing ligand and the extracellular domains of beta and gamma was comparatively difficult to assemble and required prolonged exposure to IL-2. Our method enabled us to calculate the stoichiometry of these complexes and to determine that anchoring these subunits is necessary to efficiently drive complex formation. The kinetic and equilibrium differences between the mouse and human receptor complexes, and between IL-2 and IL-15 binding to these receptors clarify the roles of the alpha and gamma subunits in the differential response of cells to different cytokines that may be present simultaneously in the environment.

A generic method for expression and use of "tagged" soluble versions of cell surface receptors.

A general method for expression, purification, immobilization, detection and radiolabeling of extracellular domains (ECD) of type I membrane proteins. The type I interleukin-1 receptor (IL-1RtI), the alpha-subunit of interleukin-2 receptor (IL-2R alpha) and E-selectin are used as illustrative examples of cell surface receptors. DNA encoding the ECD of the proteins are fused at their 3' end to a chimeric DNA which serves to generically "tag" the recombinant ECD. The resulting fusion protein contains a substrate sequence for protein kinase-A (PKA) adjacent to the signal sequence from human placental alkaline phosphatase (HPAP), The HPAP signal sequence directs the formation of the phosphatidylinositol-glycan (PI-G) anchorage of the protein at the cell surface. When these chimeric genes are expressed in CHO cells, the ECDs are detected on the cell surface and can be released by treatment with phosphatidylinositol-specific phospholipase-C (PI-PLC). Based on protein processing known to occur for native HPAP, twenty amino acids from the HPAP signal sequence remain at the C-terminus of the ECD. A high affinity monoclonal antibody was generated against this common epitope. This antibody can be used to detect, purify and immobilize the ECDs. In addition, the ECDs can be radiolabeled with 32P by treatment with PKA and maintain the ability to bind their natural ligands. This "tagging" method has been successfully applied to many other type I proteins which serve as cell surface receptors.

Peptides which bind to E-selectin and block neutrophil adhesion.

E-selectin is an inducible cell adhesion molecule which mediates rolling of neutrophils on the endothelium, an early event in the development of an inflammatory response. Inhibition of selectin-mediated rolling is a possible means for controlling inflammation-induced diseases, and several classes of compounds have been tested for this use. We describe here the use of recombinant peptide library screening for identification and optimization of novel ligands which bind to E-selectin. Several of these peptides bind with Kd values in the low nanomolar range and block E-selectin-mediated adhesion of neutrophils in static and flow-cell assays. Administration of the peptide to mice undergoing an acute inflammatory response reduced the extent of neutrophil transmigration to the site of inflammation, demonstrating the utility of this compound as a potential therapeutic. The identification of a peptide ligand for E-selectin suggests that the complete natural ligand for this adhesion molecule may include protein as well as carbohydrate moieties.

Half-site editing: an in vitro mutagenesis procedure for truncating a DNA fragment and introducing a new restriction site.

Half-site editing is an in vitro mutagenesis procedure designed for use in making precise plasmid constructions. Like many in vitro mutagenesis techniques, this procedure involves hybridization of a mutagenic oligonucleotide primer to single-stranded template DNA followed by polymerization with DNA polymerase I (Klenow). Half-site editing differs from other techniques in two main ways. First, T4 DNA polymerase treatment truncates the target DNA at a point determined by the primer and repairs any mismatches to the sequence specified by the primer. Second, a blunt-end ligation step is included. This ligation exploits the symmetry inherent in most restriction sites to create a desired restriction site at the truncated end of the target DNA fragment. Half-site editing has been used to place ClaI restriction sites at the 3' end of the yeast pyruvate kinase promoter and at two positions at the 5' end of the yeast acetolactate synthase coding sequence.

Complete nucleotide sequence of the AIDS virus, HTLV-III.

The complete nucleotide sequence of two human T-cell leukaemia type III (HTLV-III) proviral DNAs each have four long open reading frames, the first two corresponding to the gag and pol genes. The fourth open reading frame encodes two functional polypeptides, a large precursor of the major envelope glycoprotein and a smaller protein derived from the 3'-terminus long open reading frame analogous to the long open reading frame (lor) product of HTLV-I and -II.

The effects of hybrid ribosome-binding-site variants on the expression of human interferon-beta in Escherichia coli.

Human beta-interferon (IFN-beta) has been expressed in Escherichia coli by using vectors that join the trp promoter and a hybrid ribosome-binding site (HRBS) to the mature IFN-beta coding sequence. Introduction of an NcoI site at the ATG initiation codon of IFN-beta by site-directed mutagenesis has facilitated the construction of a series of portable HRBSs, by utilizing oligodeoxynucleotide adapters. The spacing between the Shine-Dalgarno (S-D) sequence and the initiator ATG ranged from 7-13 bp. One spacer of 9 bp length was varied at a single position. IFN-beta expression by these HRBS variants, as analyzed by antiviral activity and SDS-PAGE, shows both nucleotide sequence and spacer length effects. In vitro transcription-translation experiments indicate that some single base changes in the HRBS significantly decrease the rate of translation of the IFN-beta mRNA.