Use of a Biosensor with Surface Plasmon Resonance Detection for the Determination of Binding Constants: Measurement of Interleukin-6 Binding to the Soluble Interleukin-6 Receptor

Ward, Larry D.; Howlett, Geoffrey J.; Hammacher, Annet; Weinstock, Janet; Yasukawa, Kiyoshi; Simpson, Richard J.; Winzor, Donald J.

doi:10.1021/bi00009a021

Cited by 63 publications

(50 citation statements)

References 37 publications

(79 reference statements)

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“…to obtain as two curve-fitting parameters the magnitudes of K d and R max , the maximal response associated with saturation of all immobilized hnRNP A2 sites (37). An equivalent analysis was performed for experiments in which oligodeoxyribonucleotide was immobilized on the biosensor cuvette.…”

Section: Methodsmentioning

confidence: 99%

Binding of an RNA Trafficking Response Element to Heterogeneous Nuclear Ribonucleoproteins A1 and A2

Shan

Moran-Jones

Munro

et al. 2000

Journal of Biological Chemistry

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Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 binds a 21-nucleotide myelin basic protein mRNA response element, the A2RE, and A2RE-like sequences in other localized mRNAs, and is a trans-acting factor in oligodendrocyte cytoplasmic RNA trafficking. Recombinant human hnRNPs A1 and A2 were used in a biosensor to explore interactions with A2RE and the cognate oligodeoxyribonucleotide. Both proteins have a single site that bound oligonucleotides with markedly different sequences but did not bind in the presence of heparin. Both also possess a second, specific site that bound only A2RE and was unaffected by heparin. hnRNP A2 bound A2RE in the latter site with a K d near 50 nM, whereas the K d for hnRNP A1 was above 10 M. UV cross-linking assays led to a similar conclusion. Mutant A2RE sequences, that in earlier qualitative studies appeared not to bind hnRNP A2 or support RNA trafficking in oligodendrocytes, had dissociation constants above 5 M for this protein. The two concatenated RNA recognition motifs (RRMs), but not the individual RRMs, mimicked the binding behavior of hnRNP A2. These data highlight the specificity of the interaction of A2RE with these hnRNPs and suggest that the sequence-specific A2RE-binding site on hnRNP A2 is formed by both RRMs acting in cis.The family of more than 20 heterogeneous nuclear ribonucleoproteins (hnRNPs) 1 appears to play diverse roles in the post-transcriptional processing of hnRNA and subsequent packaging, transport, and translation of mRNA (1-4). hnRNPs A1, A2, B1, B2, C1, and C2 are the major components of 40S "core particles," studied primarily in HeLa cells, which are thought to package nascent ssRNA in the nucleus in a histonelike fashion (5-10). The hnRNP A/B proteins have a modular structure with two N-terminal RNA recognition motifs (RRMs) followed by a glycine-rich region ( Fig. 1) (11). The three-dimensional structures of the tandem RRMs of hnRNP A1, which are assumed to be the principal mediators of the RNA-protein interactions, have been determined (12, 13). Although the three-dimensional structures of other hnRNPs of the A/B family have not been determined, their RRMs share sufficient sequence identity with those of hnRNP A1 to suggest that they share the same tertiary fold: a four-stranded antiparallel ␤-sheet flanked by two helices. This module adopts a comparable fold in the RRMs of more distantly related proteins, such as U1A (14 -17).Although a number of the hnRNPs have a general role in intranuclear packaging of RNA, some also manifest sequencespecific binding (1, 4, 18 -22). In addition to being a major component of core particles (23), hnRNP A2 binds a telomeric sequence (24, 25, 43) and a small RNA segment known to be necessary and sufficient for transport of the message encoding myelin basic protein in the cytoplasm of oligodendrocytes, the A2RE (26 -28). The binding of hnRNP A2 to A2RE has been shown, by mutational and antisense approaches, to be essential for cytoplasmic RNA trafficking in oligodendrocytes (29).Although hnRNP A2 has been shown to ...

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Section: Methodsmentioning

confidence: 99%

Binding of an RNA Trafficking Response Element to Heterogeneous Nuclear Ribonucleoproteins A1 and A2

Shan

Moran-Jones

Munro

et al. 2000

Journal of Biological Chemistry

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“…1993;Frieling et al, 1994) (see below). Recombinant sIL-6R purified from CHO cells (Ward et a!., 1995a) and baculovirus-infected insect cells (Weiergraber et a!., 1995) bind IL-6 with similar affinity (-20 nM). A nonglycosylated sIL-6R expressed in E. coli, which was refolded from inclusion bodies to its native conformation, exhibits biological activity comparable to the sIL-6R expressed in eukaryotic cells .…”

Section: Rj Simpson Et Almentioning

confidence: 99%

Interleukin‐6: Structure‐function relationships

et al. 1997

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Interleukin-6 (IL-6) is a multifunctional cytokine that plays a central role in host defense due to its wide range of immune and hematopoietic activities and its potent ability to induce the acute phase response. Overexpression of IL-6 has been implicated in the pathology of a number of diseases including multiple myeloma, rheumatoid arthritis, Castleman's disease, psoriasis, and post-menopausal osteoporosis. Hence, selective antagonists of IL-6 action may offer therapeutic benefits. IL-6 is a member of the family of cytokines that includes interleukin-1 1, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, and ciliary neurotrophic factor. Like the other members of this family, IL-6 induces growth or differentiation via a receptor-system that involves a specific receptor and the use of a shared signaling subunit, gp130. Identification of the regions of IL-6 that are involved in the interactions with the IL-6 receptor and gp130 is an important first step in the rational manipulation of the effects of this cytokine for therapeutic benefit. In this review, we focus on the sites on IL-6 which interact with its low-affhity specific receptor, the IL-6 receptor, and the high-affinity converter gp130. A tentative model for the IL-6 hexameric receptor ligand complex is presented and discussed with respect to the mechanism of action of the other members of the IL-6 family of cytokines.

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“…The association equilibrium constant describing the interaction of shIL-6R with immobilized ligand (kAx) was calculated from the dependence of the equilibrium response ( R p ) upon the concentration of applied shIL-6R (Ci), according to the Scatchard relationship (Scatchard, 1949). The association equilibrium constant for the interaction between ligand and receptor in solution (kAs) was obtained from competition experiments, as described (Ward et al, 1995). The value for kAs was obtained from the slope of a plot of Q versus C; -[ ( Q -l ) / Q ] C i (Equation l), where Q = kAx/k,&, k i x is the constitutive binding constant measured in the presence of competing ligand, and Ci is the total concentration of competing ligand (Ward et al, 1995).…”

Section: Ligand-receptor Interaction Analysismentioning

confidence: 99%

“…The association equilibrium constant for the interaction between ligand and receptor in solution (kAs) was obtained from competition experiments, as described (Ward et al, 1995). The value for kAs was obtained from the slope of a plot of Q versus C; -[ ( Q -l ) / Q ] C i (Equation l), where Q = kAx/k,&, k i x is the constitutive binding constant measured in the presence of competing ligand, and Ci is the total concentration of competing ligand (Ward et al, 1995).…”

Section: Ligand-receptor Interaction Analysismentioning

confidence: 99%

Structure‐Function analysis of human IL‐6: Identification of two distinct regions that are important for receptor binding

et al. 1994

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Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 a-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability , Protein Sci 2:1472-1481). To further probe the structurefunction relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of a-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.

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Use of a Biosensor with Surface Plasmon Resonance Detection for the Determination of Binding Constants: Measurement of Interleukin-6 Binding to the Soluble Interleukin-6 Receptor

Cited by 63 publications

References 37 publications

Binding of an RNA Trafficking Response Element to Heterogeneous Nuclear Ribonucleoproteins A1 and A2

Binding of an RNA Trafficking Response Element to Heterogeneous Nuclear Ribonucleoproteins A1 and A2

Interleukin‐6: Structure‐function relationships

Structure‐Function analysis of human IL‐6: Identification of two distinct regions that are important for receptor binding

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