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L D Ward

Publications and source records attributed to L D Ward.

At least 19 recordsLinked to original sources
PubMed1998

Identification and characterization of two distinct truncated forms of gp130 and a soluble form of leukemia inhibitory factor receptor alpha-chain in normal human urine and plasma.

Leukemia inhibitory factor (LIF) is a polyfunctional cytokine known to require at least two distinct receptor components (LIF receptor alpha-chain and gp130) in order to form a high affinity, functional receptor complex. In this report, we present evidence that there are two distinct truncated forms of gp130 in normal human urine and plasma: a large form with a molecular weight of approximately 100, 000, which is similar to a previously described form of soluble gp130 in human serum, and a previously undescribed small form with a molecular weight of approximately 50,000. Using a panel of monoclonal antibodies raised against the extracellular domain of human gp130, we were able to show that the small form of the urinary gp130 probably contained only the hemopoietin domain. Both forms of gp130 bound LIF specifically and were capable of forming heterotrimeric complexes with soluble human LIF receptor alpha-chain in the presence of human LIF. In addition to the soluble forms of gp130, a soluble form of LIF receptor alpha-chain was also detected in human urine and plasma.

Amino Acid Sequence↗
PubMed1997

Evidence for the formation of a heterotrimeric complex of leukaemia inhibitory factor with its receptor subunits in solution.

Leukaemia inhibitory factor (LIF) is a polyfunctional cytokine that is known to require at least two distinct receptor components (LIF receptor alpha-chain and gp130) in order to form a high-affinity, functional, receptor complex. Human LIF binds with unusually high affinity to a naturally occurring mouse soluble LIF receptor alpha-chain, and this property was used to purify a stable complex of human LIF and mouse LIF receptor alpha-chain from pregnant-mouse serum. Recombinant soluble human gp130 was expressed, with a FLAG(R) epitope (DYKDDDDK) at the N-terminus, in the methylotropic yeast Pichia pastoris and purified using affinity chromatography. The formation of a trimeric complex in solution was established by native gel electrophoresis, gel-filtration chromatography, sedimentation equilibrium analysis, surface plasmon resonance spectroscopy and chemical cross-linking. The stoichiometry of this solution complex was 1:1:1, in contrast with that of the complex of interleukin-6, the interleukin-6-specific low-affinity receptor subunit and gp130, which is 2:2:2.

Antigens, CD↗
PubMed1997

Roles of histidine 31 and tryptophan 34 in the structure, self-association, and folding of murine interleukin-6.

Interleukin-6 (IL-6) is a multifunctional cytokine which is involved in a broad spectrum of activities such as immune defense, hematopoiesis, and the acute phase response, as well as in the pathogenesis of multiple myeloma. A series of murine IL-6 (mIL-6) mutants, H31A, W34A, and H31A/W34A, were constructed to investigate the roles of His31 and Trp34 in the structure, conformational stability, time-dependent aggregation, folding, and spectral properties of mIL-6. The characteristic pH-dependent quenching of fluorescence of mIL-6 at low pH was shown to be caused by an interaction between Trp34 and protonated His31 at low pH and not associated with Trp157. Denaturant-induced equilibrium unfolding experiments monitored by fluorescence and far-UV CD showed that the increased quantum yield and blue shift of the wavelength of the emission maximum observed for mIL-6 at moderate denaturant concentrations were also associated with Trp34, rather than Trp157. The tendency to form aggregation-prone unfolding intermediates, as judged by poor fits to a two-state unfolding mechanism, low m values (slopes of the unfolding curve in the transition region), and the range of denaturant concentrations over which these intermediates formed, was shown to be higher for H31A than mIL-6 but significantly lower for W34A and H31A/W34A. These differences were most pronounced at pH 7.4 and correlated with the tendencies of the proteins to aggregate at high protein concentrations in the absence of denaturant. As judged by the 1H NMR chemical shifts of the aromatic residues, the global conformations of H31A and W34A were not significantly different from that of mIL-6. Nuclear Overhauser effects (NOE) between the side chains of His31 and Trp34 were consistent with the indole side chain of Trp34 being oriented toward the face of the imidazolium side chain of His31, an arrangement consistent with our estimates of a low interaction energy (0.4-0.6 kcal/mol) between these side chains. A shift in the pKa of the His31 side chain in W34A (+0.3 unit) suggested that, in the absence of Trp34, His31 could interact with other residues. Further mutations in this region should yield forms of mIL-6, even less prone to aggregation, which would be more suitable for NMR studies. Mutation of His31 and Trp34 to alanine did not significantly alter the mitogenic activity of the mutants on mouse hybridoma 7TD1 cells, even though the corresponding region of human IL-6 has been shown to be important for biological activity.

Amino Acid Sequence↗
PubMed1997

Disruption of the disulfide bonds of recombinant murine interleukin-6 induces formation of a partially unfolded state.

A chemical modification approach was used to investigate the role of the two disulfide bonds of recombinant murine interleukin-6 (mIL-6) in terms of biological activity and conformational stability. Disruption of the disulfide bonds of mIL-6 by treatment with iodoacetic acid (IAA-IL-6) or iodoacetamide (IAM-IL-6) reduced the biological activity, in the murine hybridoma growth factor assay, by 500- and 200-fold, respectively. Both alkylated derivatives as well as the fully reduced (but not modified) molecule (DTT-IL-6) retained a high degree of alpha-helical structure as measured by far-UV CD (37-51%) when compared to the mIL-6 (59%). However, the intensity of the near-UV CD signal of the S-alkylated derivatives was very low relative to that of mIL-6, suggesting a reduction in fixed tertiary interactions. Both IAA-IL-6 and IAM-IL-6 exhibit native-like unfolding properties at pH 4.0, characteristic of a two-state unfolding mechanism, and are destabilized relative to mIL-6, by 0.3 +/- 1.6 and 2.4 +/- 1.2 kcal/mol, respectively. At pH 7.4, however, both modified proteins display stable unfolding intermediates. These intermediates are stable over a wide range of GdnHCl concentrations (0.5-2 M) and are characterized by increased fluorescence quantum yield and a blue shift of lambda(max) from 345 nm, for wild-type recombinant mIL-6, to 335 nm. These properties were identical to those observed for DTT-IL-6 in the absence of denaturant. DTT-IL-6 appears to form a partially unfolded and highly aggregated conformation under all conditions studied, as showed by a high propensity to self-associate (demonstrated using a biosensor employing surface plasmon resonance), and an increased ability to bind the hydrophobic probe 8-anilino-1-naphthalenesulfonic acid. The observed protein concentration dependence of the fluorescence characteristics of these mIL-6 derivatives is consistent with the aggregation of partially folded forms of DTT-IL-6, IAM-IL-6, and IAA-IL-6 during denaturant-induced unfolding. For all forms of the protein studied here, the aggregated intermediates unfold at similar denaturant concentrations (2.1-2.9 M GdnHCl), suggesting that the alpha-helical structure and nonspecific hydrophobic interprotein interactions are of similar strength in all cases.

Alkylation↗
PubMed1997

Interleukin-6: structure-function relationships.

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-11, 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-affinity 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.

Amino Acid Sequence↗
PubMed1997

The immediate treatment of pelvic ring disruption with the pelvic stabilizer.

The management of the hemodynamically unstable patient with a severe pelvic ring disruption remains one of the most serious trauma emergencies. Standard resuscitation protocols may include attempted closure of the pelvic ring by the use of pneumatic anti-shock trousers, external fixation applied in the operating room, or a sheet wrapped around the patient in the emergency room. We report a case of pelvic ring disruption in which a successful clinical outcome was achieved with the emergent use of the Pelvic Stabilizer in the emergency room. The Pelvic Stabilizer is a device that can be effectively applied in the emergency room for the acute reduction and early stabilization of the displaced pelvis in a hemodynamically unstable patient. The use of a pelvic clamp can also be effective in the acute setting for a stable trauma patient with pelvic ring disruption. It rapidly reduces and stabilizes a potential cause for patient decompensation without obstructing access to further concomitant diagnostic or therapeutic interventions in the abdomen and perineum.

Accidents, Traffic↗
PubMed1996

Influence of interleukin-6 (IL-6) dimerization on formation of the high affinity hexameric IL-6.receptor complex.

The high affinity interleukin-6 (IL-6) signaling complex consists of IL-6 and two membrane-associated receptor components: a low affinity but specific IL-6 receptor and the affinity converter/signal transducing protein gp130. Monomeric (IL-6M) and dimeric (IL-6D) forms of Escherichia coli-derived human IL-6 and the extracellular ("soluble") portions of the IL-6 receptor (sIL-6R) and gp130 have been purified in order to investigate the effect of IL-6 dimerization on binding to the receptor complex. Although IL-6D has a higher binding affinity for immobilized sIL-6R, as determined by biosensor analysis employing surface plasmon resonance detection, IL-6M is more potent than IL-6D in a STAT3 phosphorylation assay. The difference in potency is significantly less pronounced when measured in the murine 7TD1 hybridoma growth factor assay and the human hepatoma HepG2 bioassay due to time-dependent dissociation at 37 degrees C of IL-6 dimers into active monomers. The increased binding affinity of IL-6D appears to be due to its ability to cross-link two sIL-6R molecules on the biosensor surface. Studies of the IL-6 ternary complex formation demonstrated that the reduced biological potency of IL-6D resulted from a decreased ability of the IL-6D (sIL-6R)2 complex to couple with the soluble portion of gp130. These data imply that IL-6-induced dimerization of sIL-6R is not the driving force in promoting formation of the hexameric (IL-6 IL-6R gp130)2 complex. A model is presented whereby the trimeric complex of IL-6R, gp130, and IL-6M forms before the functional hexamer. Due to its increased affinity for the IL-6R but its decreased ability to couple with gp130, we suggest that a stable IL-6 dimer may be an efficient IL-6 antagonist.

Antigens, CD↗
PubMed1995

Equilibrium denaturation of recombinant murine interleukin-6: effect of pH, denaturants, and salt on formation of folding intermediates.

The equilibrium denaturation of an Escherichia coli-derived recombinant murine interleukin-6 (mIL-6) was studied using fluorescence and circular dichroism spectroscopy. The urea-induced unfolding of mIL-6 at pH 4.0 can be described by a two-state unfolding mechanism based on the superimposibility of the CD and fluorescence unfolding transitions. Assuming a two-state mechanism and a linear dependence of the free energy of unfolding on denaturant concentration, a value of 6.9-9.0 kcal/mol was calculated for the free energy of unfolding in the absence of denaturant [delta GU(H2O)]. However, when GuHCl was used as a denaturant at pH 4.0, a biphasic unfolding transition was observed. This unfolding transition has a distinct midpoint occurring at 2.5 M GuHCl, which is indicative of the formation of stable folding intermediates. Similar intermediate folded species were also observed at pH 7.4 when either urea or GuHCl were used as denaturants. The intermediate folded states of mIL-6 exhibited a tendency to aggregate, as judged by the concentration dependence of their fluorescence characteristics. The fluorescence emission maximum of mIL-6 at pH 7.4 in the presence of 1.5 M GuHCl, for example, was blue-shifted from 343 nm at a protein concentration of 50 micrograms/mL to 336 nm at 500 micrograms/mL. Intermediate formation at pH 4.0, using 10 mM sodium acetate buffer and urea as the denaturant, was facilitated by the addition of 0.4 and 0.8 M salt, where the salt was either NaCl or GuHCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Effects of solute multivalence on the evaluation of binding constants by biosensor technology: studies with concanavalin A and interleukin-6 as partitioning proteins.

The interaction of concanavalin A with immobilized carboxylmethyldextran has been characterized by means of a biosensor based on surface plasmon resonance detection. Adsorption and desorption of this bivalent lectin to/from the biosensor surface are shown to deviate markedly from pseudo-first-order kinetics, an assumption inherent in the usual kinetic approach to the characterization of interactions by biosensor technology. Similar results for the interaction of a dimeric and hence bivalent form of human interleukin-6 with its receptor immobilized on the biosensor plate support the conclusion that this deviation from pseudo-first-order kinetics originates from multivalence of the partitioning protein. Use of the kinetic approach to characterize the binding of multivalent proteins to immobilized affinity sites on the biosensor chip is therefore precluded because of nonconformity with the model on which the quantitative analysis is based. Instead, an intrinsic binding constant of 2.5 x 10(5) M-1 for the interaction of concanavalin A with the carboxymethylated dextran layer coating the biosensor chip has been obtained by interpreting the equilibrium biosensor responses in terms of expressions developed in the context of quantitative affinity chromatography of multivalent partitioning solutes.

Adsorption↗
PubMed1995

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.

The interaction of recombinant human interleukin-6 (IL-6) with the soluble extracellular form of its receptor (sIL-6R) has been characterized by the application of expressions developed for quantitative affinity chromatography to results obtained with a biosensor based on surface plasmon resonance detection. First, the interaction of sIL-6R with IL-6 covalently attached to the biosensor-chip was characterized from the dependence of the surface plasmon resonance response upon the concentration of receptor injected into the biosensor. A binding constant for the interaction between sIL-6R and IL-6 was then determined from the biosensor response observed for mixtures of IL-6 and receptor--a procedure that is shown to provide unequivocal characterization of the competing reaction, irrespective of the model used to describe the biphasic interaction between partitioning receptor and immobilized IL-6. A binding constant of 5 x 10(7) M-1 has been obtained for the interaction of sIL-6R with two equivalent and independent sites on an essentially dimeric IL-6 preparation produced using the pUC vector system, and also for the interaction of sIL-6R with a monomeric IL-6 preparation that was univalent in its interaction with receptor.

Amino Acid Sequence↗
PubMed1994

Cooperative multiple binding of bisANS and daunomycin to tubulin.

The binding of daunomycin and bisANS to tubulin was studied by direct equilibrium techniques. Both ligands generated abnormal Scatchard plots. Their concave-downward nature indicated positive cooperativity. The data conform to tubulin possessing ca. 35 daunomycin binding sites with a binding constant of 570-1430 M-1. The binding of bisANS is characterized by 1 strong binding site (KA = 4.5 x 10(5) M-1) and 40-50 lower affinity sites. Hill plots of both showed low degrees of cooperativity (m = 1.8 for daunomycin and 2.3 for bisANS). A detailed analysis was carried out of the cooperativity of binding of daunomycin to tubulin. Concentration differences spectra and sedimentation velocity analysis of daunomycin showed that this molecule undergoes self-association in the drug concentration range used in the binding study. The low level of polymerization (approximately tetramer), however, indicated that this could not be the source of the observed cooperativity between 35 molecules. Both the shape and concentration dependence of the daunomycin concentration difference spectra were strikingly similar to those generated on the binding of daunomycin to tubulin, which indicates the stacking of daunomycin in both cases. The observed Scatchard plot of the binding was found to be consistent with a process that involves in part ligand-ligand interactions when complexed to tubulin. Examination of the binding of bisANS in the presence of daunomycin revealed a strong increase of bisANS binding to tubulin, which suggests a loosening of tubulin structure with the exposure of new sites as these ligands bind. The mutual interaction between the two ligands in dilute solution was demonstrated by difference spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1994

Energy transfer studies of the distances between the colchicine, ruthenium red, and bisANS binding sites on calf brain tubulin.

Fluorescence energy transfer experiments were performed in order to measure the spatial separation between the colchine and Ruthenium Red binding sites, the high-affinity bisANS and Ruthenium Red sites, and the allocolchicine and high-affinity bisANS sites on calf brain tubulin. Energy transfer was observed between both colchicine and allocolchicine and Ruthenium Red, resulting in a distance of 40-45 A between these sites on the tubulin molecule. No detectable energy transfer could be observed when allocolchicine was used as fluorescence donor and bisANS as acceptor or when bisANS was used as donor and Ruthenium Red as acceptor. This indicates that the distance of separation between the allocolchicine and bisANS sites is greater than 50 A, while that between the bisANS and Ruthenium Red sites is greater than 72 A. On the basis of these and previous distance measurements (Ward & Timasheff, 1988), two triangles of binding sites have been defined (colchicine-bisANS-E-site and colchicine-bisANS-Ruthenium Red). Since the dihedral angle between them is not known, a schematic model has been drawn with all the sites located in a single plane.(ABSTRACT TRUNCATED AT 250 WORDS)

Anilino Naphthalenesulfonates↗
PubMed1994

High affinity interleukin-6 receptor is a hexameric complex consisting of two molecules each of interleukin-6, interleukin-6 receptor, and gp-130.

The high affinity human interleukin-6 (IL-6) receptor complex consists of IL-6 and two membrane-associated receptor components, the IL-6 receptor (alpha-subunit) and the high affinity converter and signal transducing molecule, gp-130 (beta-subunit). Recombinant IL-6 and the extracellular ("soluble") components of the IL-6 receptor (sIL-6R) and gp-130 (sgp-130) have been prepared in order to investigate the stoichiometry and binding of these components in the low affinity (IL-6.sIL-6R) and high affinity (IL-6.sIL-6R.sgp-130) IL-6 receptor complexes. Using a combination of size-exclusion chromatography and analytical ultracentrifugation analysis, in the low affinity receptor complex, IL-6 was shown to bind sIL-6R in a stoichiometric ratio of 1:1, whereas the high affinity ternary complex is hexameric consisting of two molecules each of IL-6, sIL-6R, and sgp-130. This is the first direct demonstration of a higher order arrangement for receptor cytokine interactions that exhibit both high and low affinity complexes.

Amino Acid Sequence↗
PubMed1994

Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding.

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 alpha-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 (Ward LD et al., 1993, Protein Sci 2:1472-1481). To further probe the structure-function 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 alpha-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.

Amino Acid Sequence↗
PubMed1993

Specific covalent modification of the tryptophan residues in murine interleukin-6. Effect on biological activity and conformational stability.

Modification of recombinant murine interleukin-6 (mIL-6) with the tryptophan-specific reagent 2-nitrophenylsulfenyl chloride under mild acidic conditions, 0.1 M sodium acetate, pH 3.5, yielded a derivative containing 2.02 mol 2-nitrophenylsulfenyl tryptophan/mol protein. The sites of modification were identified as Trp36 and Trp160. No detectable side reactions occurred on other amino acids in the molecule, as indicated by the combination of endoproteinase Asp-N peptide mapping, Edman degradation and electrospray mass spectrometry. Sulfenylation of the two tryptophan residues in mIL-6 caused a 50% reduction in both the biological activity in the murine-hybridoma-growth-factor assay using 7TD1 cells and receptor-binding affinity to mIL-6 receptors. Sulfenylation of mIL-6 did not significantly affect the overall conformation of the protein as measured by farultraviolet circular dichroism and binding to the neutralizing anti-mIL-6 mAb 6B4. The sulfenylated protein was, however, significantly less stable [delta delta G(H2O) = 3.98 kJ/mol] than unmodified mIL-6 as measured by urea-gradient gel electrophoresis.

Amino Acid Sequence↗
PubMed1993

Proteins of the Golgi apparatus. Purification to homogeneity, N-terminal sequence, and unusually large Stokes radius of the membrane-bound form of UDP-galactose:N-acetylglucosamine beta 1-4galactosyltransferase from rat liver.

The Golgi marker enzyme, UDP-galactose:N-acetylglucosamine beta 1-4galactosyltransferase (beta 1-4GalT) was purified 44300-fold in its intact, membrane-bound form from rat liver membranes. The protein was isolated from detergent extracts as a high-M(r) form, having a Stokes radius approximating a globular protein of M(r) 440,000. It is comprised of a single protein component as observed on SDS/polyacrylamide gels, having an M(r) near 51,000, and does not have intermolecular disulfide cross-links. N-terminal sequencing of the enzyme demonstrated that it contains an N-terminal hydrophobic stretch deduced previously from cDNA encoding for the enzyme. Previous studies have indicated that the protein may be translated at either of two AUG sites near the 5' end of the mRNA [Russo, R. N., Shaper, N. L. & Shaper, J. H. (1990) J. Biol. Chem. 265, 3324-3331], giving rise to two polypeptides, one appended with 13 amino acids. In the work described here, evidence was only found for the sequence of the short form, missing a single methionine at the N-terminus. Mild proteolytic treatment cleaved the enzyme, giving rise to low-M(r) forms which were fully catalytically active and which, upon sequencing, were missing a 66-amino-acid stretch from the N-terminus (as compared to the mouse cDNA). Proteolytic treatment was accompanied by conversion of the form having a large Stokes radius to one approximating a globular protein with M(r) near 50,000. The N-terminal stretch appears to contribute to maintenance of the form having a large Stokes radius. This may be the result of interaction with a detergent micelle, dimerization or oligomerization, or interaction with some other large, non-protein molecule, although a detergent exchange still resulted in a form having a large Stokes radius.

Amino Acid Sequence↗