Journal: Journal of Biological Chemistry

Article Title: Kinetic Interaction Analysis of Human Interleukin 5 Receptor α Mutants Reveals a Unique Binding Topology and Charge Distribution for Cytokine Recognition

doi: 10.1074/jbc.m309327200

Figure Lengend Snippet: FIG. 1. Extracellular domain construct of IL5R used in biosensor analysis of mutational variants. A, comparison of soluble form of IL5R (lower) used in antibody-capture binding assay with full-length IL5R (upper) composed of an extracellular region (D1, D2, and D3), a transmembrane (TM) region and a cytoplasmic (CP) region. The soluble form of IL5R (sIL5R) corresponds to the extracellular region. V5-His tag was fused at the C-terminal region of D3 that normally adjoins the TM region. B, sequence alignment of extracellular regions of human and mouse IL5R, and rat prolactin receptor. Identical amino acid residues are marked by asterisks, and similar amino acid residues are marked by the “:” symbol. Multiple sequence alignments were performed by using PSI-BLAST (27) and ClustalW 1.60 (43), both of which provided similar results. Alignments were further adjusted manually (see “Experimental Procedures”). Groups of similar amino acid residues were categorized according to ClustalW 1.60. Underlines in the prolactin receptor sequence show the -strand regions determined by its crystal structure (26). An alignment of the human growth hormone and human IL5R was initially used to design the position of mutagenesis, whereas crystal structure of rat prolactin receptor was used for homology modeling. The -strand regions estimated from human growth hormone and rat prolactin receptor were essentially the same, except for the -strand G in D2 domain. Shaded are the residues that were substituted by alanine in this study.

Article Snippet: Levels of expression were measured for both cell cultures and precipitants by Western blot using anti-human IL5R polyclonal antibody (R&D Systems) and anti-histidine tag polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Construct, Comparison, Binding Assay, Sequencing, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Kinetic Interaction Analysis of Human Interleukin 5 Receptor α Mutants Reveals a Unique Binding Topology and Charge Distribution for Cytokine Recognition

doi: 10.1074/jbc.m309327200

Figure Lengend Snippet: FIG. 2. Basic features and Ramachandran plot of modeled structure of sIL5R. A, homology model of the tertiary structure of the extracellular region of IL5R. Loops are represented as coils and -strands as ribbons. In this structure, each fibronectin type III domain of IL5R is termed D1, D2, and D3 from the N to the C termini, which are labeled N-ter and C-ter, respectively. D2 and D3 domains have sequence homology with the classic cytokine recognition motif (CRM). Seven cysteine residues are shown as CPK (Corey Pauling Kultin) models and labeled by residue type and number. One disulfide bond (Cys114-Cys135) links the neighboring strands A and B and another disulfide bond (Cys162-Cys176) links strands D and E. In this modeled structure, a disulfide bond between Cys249 and Cys296 in D3 domain was introduced (see “Experimental Procedures”). The guanidinium group of Arg290 was intercalated between the two indole rings from the WSXWS motif in D3 domain. The tryptophan residues are shown as CPK mod- els, and the arginine residue is shown as a ball-and-stick model. All the molecular graphic figures in this article were prepared with the pro- gram MOLMOL (42). B, the Ramachandran plot analysis by PRO- CHECK (30) showed that 85.5% of non-glycine and non-proline residues fell into the most favored regions, 12.4% into the additional allowed regions, 2.1% into the generously allowed regions, and no non-glycine residues in disallowed regions.

Article Snippet: Levels of expression were measured for both cell cultures and precipitants by Western blot using anti-human IL5R polyclonal antibody (R&D Systems) and anti-histidine tag polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Labeling, Sequencing, Residue

Journal: Journal of Biological Chemistry

Article Title: Kinetic Interaction Analysis of Human Interleukin 5 Receptor α Mutants Reveals a Unique Binding Topology and Charge Distribution for Cytokine Recognition

doi: 10.1074/jbc.m309327200

Figure Lengend Snippet: FIG. 3. Western blotting of sIL5R variants. A, Western blotting of secreted wild type sIL5R. Lane 1 shows the band stained by anti- human IL5R polyclonal antibody; lane 2, the band stained by anti- histidine tag polyclonal antibody. B, Western blotting of the superna- tants (upper) and precipitates (lower) of non-secretion variants by anti- histidine tag polyclonal antibody: lane 1, positive control (wild type IL5R); lane 2, negative control (empty vector transfection); lane 3, H71A; lane 4, K167A/D168A; lane 5, R172A; lane 6, D189A; lane 7, R260A; and lane 8, D279A/D280A.

Article Snippet: Levels of expression were measured for both cell cultures and precipitants by Western blot using anti-human IL5R polyclonal antibody (R&D Systems) and anti-histidine tag polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Western Blot, Staining, Positive Control, Negative Control, Plasmid Preparation, Transfection

Journal: Journal of Biological Chemistry

Article Title: Kinetic Interaction Analysis of Human Interleukin 5 Receptor α Mutants Reveals a Unique Binding Topology and Charge Distribution for Cytokine Recognition

doi: 10.1074/jbc.m309327200

Figure Lengend Snippet: FIG. 4. Epitope analysis and mapping for a non-neutralizing anti human IL5R antibody 16. A, Biacore-derived real-time sen- sorgrams of the interactions of wild type IL5R and receptor variants, which impaired the high affinity interaction with 16. Receptor sam- ples were injected onto the immobilized 16 surface at zero time, and arrows shows the time when the injections reverted to buffer alone. B, mapping of the 16 epitope on the modeled structures of IL5R. Resi- dues that impaired, through alanine substitution, the high affinity interaction with 16 are shown as CPK models and labeled by residue type and number. Residues that caused non-secretion through alanine substitutions (Table II) are shown as ball-and-stick models.

Article Snippet: Levels of expression were measured for both cell cultures and precipitants by Western blot using anti-human IL5R polyclonal antibody (R&D Systems) and anti-histidine tag polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Derivative Assay, Injection, Labeling, Residue

Journal: Journal of Biological Chemistry

Article Title: Kinetic Interaction Analysis of Human Interleukin 5 Receptor α Mutants Reveals a Unique Binding Topology and Charge Distribution for Cytokine Recognition

doi: 10.1074/jbc.m309327200

Figure Lengend Snippet: FIG. 5. Biosensor binding analysis of sIL5R. A, anti-V5 tag monoclonal an- tibody was covalently immobilized to the dextran matrix on a sensor chip gold sur- face. sIL5R was captured on the surface via tight interaction of V5 tag and the antibody, providing the similar configura- tion to cell surface receptors. IL5 was then injected to the sIL5R-captured sur- face, and its kinetic interaction was exam- ined. B, the effect of flow rate. The asso- ciation rates were measured at flow rates of 10, 20, 40, or 80 l/min (cyan, blue, green, or red, respectively). C, overlay of real-time sensorgrams shows sequential injections of cell-free media containing sIL5R (a), injection of running buffer (b), injections of 0, 12.5, 25, and 50 nM of IL5 (c), injection of running buffer alone (d), and injections of the regeneration buffer (e). D, global fitting of wild type IL5R. Various concentrations of IL5 (0, 12.5, 25, and 50 nM) were injected. The rate con- stants were calculated by fitting the asso- ciation phase and dissociation phase to a model for 1:1 Langmuir binding with mass transfer. Residuals are shown in the lower panel of D. Magenta dots show ex- perimental sensorgrams, and black lines show calculated sensorgrams.

Article Snippet: Levels of expression were measured for both cell cultures and precipitants by Western blot using anti-human IL5R polyclonal antibody (R&D Systems) and anti-histidine tag polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Binding Assay, Injection

Journal: Journal of Biological Chemistry

Article Title: Kinetic Interaction Analysis of Human Interleukin 5 Receptor α Mutants Reveals a Unique Binding Topology and Charge Distribution for Cytokine Recognition

doi: 10.1074/jbc.m309327200

Figure Lengend Snippet: FIG. 6. Mutations that diminished the high affinity interaction of IL5R with IL5. Binding curves of (A) the D1 mutants and (B) the D2D3 mutants, which markedly decreased the binding af- finity. Each sensorgram shows the associ- ation (0–60 s) and dissociation (60–180 s) of IL5 (50 nM) to sIL5R or the mutational variants captured by anti-V5 tag anti- body. For comparison, sensorgrams were normalized depending on the amount of captured protein. Various concentrations of IL5 (0, 50, 100, and 250 nM) were in- jected to captured D55A variant (C) or R188A variant (D). The interaction curves were globally fit using Langmuir 1:1 bind- ing with mass transfer. Residuals are shown in the lower panels in C and D. Magenta dots show experimental sensor- grams, and black lines show calculated sensorgrams.

Article Snippet: Levels of expression were measured for both cell cultures and precipitants by Western blot using anti-human IL5R polyclonal antibody (R&D Systems) and anti-histidine tag polyclonal antibody (Santa Cruz Biotechnology).

Techniques: Binding Assay, Comparison, Variant Assay

Journal: Journal of Biological Chemistry

Article Title: Kinetic Interaction Analysis of Human Interleukin 5 Receptor α Mutants Reveals a Unique Binding Topology and Charge Distribution for Cytokine Recognition

doi: 10.1074/jbc.m309327200

Figure Lengend Snippet: FIG. 7. Hypothetical structure of the IL5 complex with IL5R. Both IL5 (R91 and E110) and IL5R (D55, D56, E58, K186, R188, and R297) epitope residues are expressed in the CPK model.

Article Snippet: Levels of expression were measured for both cell cultures and precipitants by Western blot using anti-human IL5R polyclonal antibody (R&D Systems) and anti-histidine tag polyclonal antibody (Santa Cruz Biotechnology).

Techniques: