Biomedical subjects

L D Ward

Publications and source records attributed to L D Ward.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1993

Epidermal growth factor induces serine phosphorylation of stathmin in a human colon carcinoma cell line (LIM 1215).

Changes in protein phosphorylation in the human colon carcinoma cell line LIM 1215 after stimulation with epidermal growth factor (EGF) have been analyzed by two-dimensional gel electrophoresis and phosphoamino acid analysis. In addition to a number of tyrosine-phosphorylated proteins, a family of small proteins (M(r) 19,000-20,000) is maximally phosphorylated on serine within 5 min of EGF stimulation. One member of the family has been purified by a combination of two-dimensional electrophoresis and reversed-phase high performance liquid chromatography and identified by amino acid sequence analysis as stathmin. Although phosphorylation of stathmin has been reported previously in leukemia cells and following stimulation of hemopoietic or lymphoid cells with several mitogenic agents, this is the first report of stathmin phosphorylation in response to EGF.

Amino Acid Sequence↗

PubMed1993

Effect of pH and denaturants on the folding and stability of murine interleukin-6.

The conformation and stability of a recombinant mouse interleukin-6 (mIL-6) has been investigated by analytical ultracentrifugation, fluorescence spectroscopy, urea-gradient gel electrophoresis, and near- and far-ultraviolet circular dichroism. On decreasing the pH from 8.0 to 4.0, the tryptophan fluorescence of mIL-6 was quenched 40%, the midpoint of the transition occurring at pH 6.9. The change in fluorescence quantum yield was not due to unfolding of the molecule because the conformation of mIL-6, as judged by both urea-gradient gel electrophoresis and CD spectroscopy, was stable over the pH range 2.0-10.0. Sedimentation equilibrium experiments indicated that mIL-6 was monomeric, with a molecular mass of 22,500 Da over the pH range used in these physicochemical studies. Quenching of tryptophan fluorescence (20%) also occurred in the presence of 6 M guanidine hydrochloride upon going from pH 7.4 to 4.0 suggesting that an amino acid residue vicinal in the primary structure to one or both of the two tryptophan residues, Trp-36 and Trp-160, may be partially involved in the quenching of endogenous fluorescence. In this regard, similar results were obtained for a 17-residue synthetic peptide, peptide H1, which corresponds to an N-terminal region of mIL-6 (residues Val-27-Lys-43). The pH-dependent acid quenching of endogenous tryptophan fluorescence of peptide H1 was 30% in the random coil conformation and 60% in the presence of alpha-helix-promoting solvents. Replacement of His-33 with Ala-33 in peptide H1 alleviated a significant portion of the pH-dependent quenching of fluorescence suggesting that the interaction of the imidazole ring of His-33 with the indole ring of Trp-36 is a major determinant responsible for the quenching of the endogenous protein fluorescence of mIL-6.

Amino Acid Sequence↗

PubMed1993

Role of the C-terminus in the activity, conformation, and stability of interleukin-6.

Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183-187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was < 0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6. Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis. These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding. A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.

Amino Acid Sequence↗

PubMed1993

Identification of canary grass (Phalaris aquatica) pollen allergens by immunoblotting: IgE and IgG antibody-binding studies.

The pollen of canary grass, which was introduced as a pasture grass from Europe, is a major allergen in the external environment of southern Australia. Seventeen allergenic fractions of canary grass pollen, ranging in mol. mass from 14 to 100 kDa, have been identified by immunoblotting, using IgE antibodies from sera of 24/30 grass-pollen-allergic subjects. The highest frequency of IgE binding (77%) was to a major 34-kDa fraction (tentatively designated Pha a I). This protein has been partially purified and identified as a group I allergen by immunodepletion experiments, with partially purified Lol p I (from rye-grass pollen), atopic serum, and Lol p I-specific MAb. In addition, microsequencing of the N-terminus of Pha a I showed an amino acid sequence identical to Lol p I. In a separate study, IgE binding to Western blots of Pha a I, Lol p I, and Cyn d I was investigated in 24 sera and found to occur in 19/24, 18/24, and 9/24, respectively. IgE binding to all three major allergens, and to both Pha a I and Lol p I, occurred in 8/24 sera. Our findings suggest that while the N-terminal sequence of Pha a I is identical to Lol p I, there may be specific allergenic epitopes exclusive to this allergen that are important for allergenicity in southern Australia.

Allergens↗

PubMed1993

Preparation of highly purified human myelin oligodendrocyte glycoprotein in quantities sufficient for encephalitogenicity and immunogenicity studies.

Myelin oligodendrocyte glycoprotein, (MOG), a quantitatively minor central nervous system (CNS) myelin component, is a candidate target antigen for autoimmune-mediated demyelination. It is a highly hydrophobic protein present in very small amounts in CNS tissue and thereby difficult to purify. Our aim was to devise a purification procedure that would yield sufficient quantities of highly purified MOG to subsequently test its potential encephalitogenic activity, as well as investigate the humoral and cell-mediated responses to this antigen in naturally occurring and experimentally induced autoimmune demyelinating diseases. MOG was purified from human CNS white matter using immunoaffinity chromatography, a procedure that gave a final yield of MOG corresponding to 0.02% total white matter protein. The final product, which migrated as two bands of molecular weight 28 kDa and 58 kDa, was highly pure as shown also by specific reactivity with monoclonal anti-MOG antibodies on immunoblots in the absence of any detectable reactivity with antibodies specific for myelin basic protein, proteolipid protein and myelin-associated glycoprotein. Partial amino acid sequence was obtained from both MOG bands separated by SDS-PAGE and electroblotted onto PVDF. The sequence of the first 17 N-terminal amino acids had approximately 55% homology with the reported rat MOG sequence deduced from the cloned cDNA sequence; small internal sequences obtained showed also very high homology. Our purified MOG preparations have been used to investigate T cell response to MOG by peripheral blood lymphocytes of multiple sclerosis patients and to induce a relapsing remitting demyelinating disease in Lewis rats.

Amino Acid Sequence↗

PubMed1992

A major binding protein for leukemia inhibitory factor in normal mouse serum: identification as a soluble form of the cellular receptor.

A protein that specifically binds leukemia inhibitory factor (LIF) has been isolated from normal mouse serum by using four successive fractionation steps: chromatography on a LIF affinity matrix, anion-exchange chromatography, size-exclusion chromatography, and preparative native gel electrophoresis. The purified LIF-binding protein (LBP) is a glycoprotein with an apparent molecular mass of 90 kDa that specifically binds 125I-labeled murine LIF with an affinity comparable to that of the low-affinity cellular LIF receptor (Kd = 600 pM). N-terminal sequencing has identified this protein as a soluble truncated form of the alpha chain of the cellular LIF receptor. LBP is present in normal mouse serum at high levels (1 microgram/ml) and these levels are elevated in pregnant mice and reduced in neonatal mice. Since normal serum concentrations of LBP can block the biological actions of LIF in culture, LBP may serve as an inhibitor of the systemic effects of locally produced LIF.

Amino Acid Sequence↗

PubMed1992

Purification and characterization of a recombinant murine interleukin-6. Isolation of N- and C-terminally truncated forms.

Murine interleukin-6 (IL-6), when expressed in Escherichia coli using the pUC9 vector, accumulated as insoluble aggregates or 'inclusion bodies'. After selective urea washing of the inclusion bodies, to remove extraneous proteins, murine IL-6 was solubilized with 8 M guanidine hydrochloride and then rapidly purified to homogeneity by gel-permeation chromatography followed by reversed-phase HPLC. It was demonstrated that complete disulfide bond formation in murine IL-6 occurred during the early urea washing/guanidine hydrochloride extraction steps, so no refolding step was required. When fully reduced murine IL-6 was dissolved in 8 M guanidine hydrochloride and allowed to air-oxidize, complete disulfide bond formation, monitored by analytical reversed-phase HPLC, was shown to occur within 13 h at 6 degrees C. About 25 mg pure protein was obtained from 37 g wet cells. This recombinant murine IL-6 had a specific activity in the hybridoma growth factor assay of 2 x 10(8) U/mg, which is equivalent to that of native murine IL-6. During the purification procedure, a number of variant forms of murine IL-6 were isolated and partially characterized. Two of these forms, T1 and T3, were C-terminal deletants of murine IL-6 lacking about 60 and 20 amino acids from the C-terminus, respectively, while the other form, T2, was an N-terminal deletant lacking 37 amino acids from the N-terminus. None of these variant forms of murine IL-6 bound to the murine IL-6 receptor and, consequently, all were inactive in the hybridoma growth factor assay.

Amino Acids↗

PubMed1992

Isolation and characterization of a novel receptor-type protein tyrosine kinase (hek) from a human pre-B cell line.

In this report we describe the identification and characterization of a novel tumor-associated receptor-type tyrosine kinase (hek). We produced a monoclonal antibody (III.A4) that detected a novel glycoprotein on the immunizing pre-B cell acute lymphoblastic leukemia cell line (LK63). This antigen was shown to be expressed sporadically on hemopoietic tumor cell lines and on ex vivo tumors. However, using antibody staining, the molecule was undetectable on normal tissues. Further biochemical characterization showed this molecule (hek) to be a phosphoroprotein. This observation taken together with the tumor-associated nature of hek expression suggested that hek might be a receptor-type protein tyrosine kinase. This was demonstrated by affinity purification of hek. In in vitro kinase experiments the purified hek protein was autophosphorylated on tyrosine and also mediated tyrosine phosphorylation of casein. Purified hek was subjected to N-terminal amino acid sequence analysis which showed that hek had a unique N terminus. Amino acid sequence determination of peptides from a V8 protease digest of hek yielded one 21-amino acid stretch of sequence which showed close homology with the eph subfamily of protein tyrosine kinases. These studies show hek to be a novel human tumor-associated protein tyrosine kinase, which by analogy with previously characterized protein tyrosine kinase proto-oncogenes, may have a role in tumorigenesis.

Amino Acid Sequence↗

PubMed1992

Characterization of bovine heparin-binding neurotrophic factor (HBNF): assignment of disulfide bonds.

The topology of the disulfides in native heparin-binding neurotrophic factor (HBNF) isolated from bovine brain was studied by proteolytic digestion using trypsin, Asp-N endoproteinase and chymotrypsin and peptide mapping. Disulfide-linked peptides were identified by automated Edman degradation. It has been shown that there are disulfide bonds between Cys15-Cys44, Cys23-Cys53, Cys30-Cys57, Cys67-Cys99 and Cys77-Cys109.

Amino Acid Sequence↗

PubMed1992

The group III allergen from the house dust mite Dermatophagoides pteronyssinus is a trypsin-like enzyme.

Faecally enriched extracts of Dermatophagoides pteronyssinus were shown to contain a trypsin-like enzyme which was allergenic. Chromatofocusing studies revealed the presence of nine major isoforms in D. pteronyssinus, with pI in the range 4 to greater than 8, but only two (range 4-5) in D. farinae. Trypsin isolated from D. pteronyssinus by benzamidine-Sepharose 6B affinity chromatography and gelfiltration was found to be a 31-kDa protein which was enzymatically similar to both invertebrate and vertebrate trypsins. The N-terminal sequence obtained (IVGGEXALAGEXPYQISL) was identical to that reported for the mite allergen Der p III and showed homology with crayfish trypsin and Der f III from D. farinae. Mite trypsin underwent autolysis and the N-terminal sequences of two fragments were found to be ALAGEXPYQI and NNQVXGI respectively. Both showed homology with crayfish trypsin, and the former sequence was identical to residues 7-18 of the native enzyme and Der p III. All isoforms of mite trypsin were showed to be allergenic by radioallergosorbent assay and further studies indicated that the trypsin degradation products were also allergenic. The enzyme was compared with other mite allergens and the rank order of allergenic potency was shown to be: whole mite extract greater than Der p I greater than trypsin. However, all sera from a panel of mite allergic individuals showed IgE reactivity to trypsin, comparable to that seen using whole mite extract and Der p I. These data indicate that mite trypsin is a major allergen corresponding to the previously described allergen, Der p III.

Allergens↗

PubMed1992

Binding of anti-human-interleukin-6 monoclonal antibodies to synthetic peptides of human interleukin-6 studied using surface plasmon resonance.

Biosensor technology employing surface plasmon resonance (SPR) detection provides a highly-sensitive (sub ng), non-extrinsic labelling approach for monitoring protein interactions in real-time. We have used this approach to map the binding sites on human interleukin-6 (hIL-6) for a series of anti-hIL-6 monoclonal antibodies (mAbs). Epitopes were localised by monitoring the ability of ten synthetic peptides, spanning the sequence of hIL-6, to inhibit the binding of anti-hIL-6 mAbs to immobilised hIL-6. Peptide P8 (Pro139-Gln153) inhibited binding of anti-IL-6-mAbs 1, 2 and 7. To increase the sensitivity of detection of antibody-synthetic peptide interactions, a procedure was developed for immobilising the synthetic peptides directly to the sensor surface of the SPR instrument. From this study, association equilibrium constants of 2.1 x 10(6)M-1 and 3.6 x 10(4)M-1 were calculated for the mAb7-immobilised P8 and mAb7-free P8 interactions, respectively.

Antibodies, Monoclonal↗

PubMed1991

Cloning and sequencing of Lol pI, the major allergenic protein of rye-grass pollen.

We have isolated a full length cDNA clone encoding the major glycoprotein allergen Lol pI. The clone was selected using a combination of immunological screening of a cDNA expression library and PCR amplification of Lol pI-specific transcripts. Lol pI expressed in bacteria as a fusion protein shows recognition by specific IgE antibodies present in sera of grass pollen-allergic subjects. Northern analysis has shown that the Lol pI transcripts are expressed only in pollen of rye-grass. Molecular cloning of Lol pI provides a molecular genetic approach to study the structure-function relationship of allergens.

Allergens↗

PubMed1991

Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen: intracellular targeting to the amyloplast.

We have identified a major allergenic protein from rye-grass pollen, tentatively designated Lol pIb of 31kDa and with pI 9.0. A cDNA clone encoding Lol pIb has been isolated, sequenced, and characterized. Lol pIb is located mainly in the starch granules. This is a distinct allergen from Lol pI, which is located in the cytosol. Lol pIb is synthesized in pollen as a pre-allergen with a transit peptide targeting the allergen to amyloplasts. Epitope mapping of the fusion protein localized the IgE binding determinant in the C-terminal domain.

Allergens↗

PubMed1991

House dust mite-derived amylase: allergenicity and physicochemical characterization.

Amylase activity was found in extracts of both Dermatophagoides pteronyssinus whole mite (0.16 U/mg) and spent growth medium (0.01 U/mg) but not in unused growth medium. It was also detected in all extracts of house dust obtained from mattresses (n = 20; geometric mean, 1.95 U/gm) and in 18 extracts of dust obtained from lounge room carpets (n = 20; geometric mean, 0.54 U/gm). Although the origins of amylase in dust are unclear, enzyme activity correlated with mite counts (n = 40; r = 0.35; p less than 0.05) and Der p I concentrations (r = 0.41; p less than 0.01). Mite amylase was purified from spent growth medium by affinity chromatography, gel filtration, and chromatofocusing. It was physicochemically similar to mammalian amylase with regard to molecular weight (60,000), charge heterogeneity (isoelectric point, 5 to 7) and the capacity to bind to an organomercurial affinity matrix. The optimum pH for enzymatic activity was revealed to be 6.4. IgE immunoblot studies demonstrated that the enzyme was allergenic and that its expression was dependent on the integrity of intrachain disulfide bonds. Sera from 25% of mite-allergic children and 46% of mite-allergic adults contained specific IgE to mite amylase. IgE to amylase was associated (p less than 0.01) with increased concentrations of total mite-specific IgE determined with a direct RAST assay.

Adult↗