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A C Willis

Publications and source records attributed to A C Willis.

At least 19 recordsLinked to original sources
PubMed1992

Molecular analysis of the association of HLA-B53 and resistance to severe malaria.

The protective association between the human leukocyte antigen HLA-B53 and severe malaria was investigated by sequencing of peptides eluted from this molecule followed by screening of candidate epitopes from pre-erythrocytic-stage antigens of Plasmodium falciparum in biochemical and cellular assays. Among malaria-immune Africans, HLA-B53-restricted cytotoxic T lymphocytes recognized a conserved nonamer peptide from liver-stage-specific antigen-1 (LSA-1), but no HLA-B53-restricted epitopes were identified in other antigens. These findings indicate a possible molecular basis for this HLA-disease association and support the candidacy of liver-stage-specific antigen-1 as a malaria vaccine component.

Amino Acid Sequence↗
PubMed1992

Activity, disulphide mapping and structural modelling of the fifth domain of human beta 2-glycoprotein I.

Complexes formed by the interaction of negatively charged phospholipids and beta 2-glycoprotein I (beta 2-I) are the target of autoantibodies in systemic lupus erythematosus. The highly positively charged fifth (C-terminal) domain of human beta 2-I was produced as a fusion protein in an Escherichia coli expression system and was shown to bind to the negatively charged phospholipid, cardiolipin, almost as well as the intact protein. In an attempt to define the 3D structure of this domain, the disulphide linkage pattern was determined and shown to be Cys 1-4, Cys 2-5 and Cys 3-6 in contradiction to an earlier report. In the light of this information, the sequence of the fifth domain of beta 2 I (beta 2-I-5) is readily aligned with that of the 16th repeat of factor H, of which the 3D structure is known, and a model of beta 2I-5 has been built by homology. On the basis of the model we suggest residues that might be the target of profitable site-directed mutagenesis in structure-function studies.

Amino Acid Sequence↗
PubMed1992

Human interleukin-1 receptor antagonist. High yield expression in E. coli and examination of cysteine residues.

The human IL-1 receptor antagonist (IL-1ra) was produced in a high yield E. coli expression system, and was purified in a rapid two-step purification. This recombinant IL-1ra molecule possessed full binding activity to the IL-1 receptor (type I) and totally inhibited IL-1-induced PGE2 production by human dermal fibroblasts. Radioalkylation and analysis of V8-derived IL-1ra peptides indicate that the four cysteines present in the IL-1ra are not disulphide-linked.

Amino Acid Sequence↗
PubMed1992

Purification, characterization and cDNA cloning of human lung surfactant protein D.

Human pulmonary surfactant protein D (SP-D) was identified in lung lavage by its similarity to rat SP-D in both its molecular mass and its Ca(2+)-dependent-binding affinity for maltose [Persson, Chang & Crouch (1990) J. Biol. Chem. 265, 5755-5760]. For structural studies, human SP-D was isolated from amniotic fluid by affinity chromatography on maltose-Sepharose followed by f.p.l.c. on Superose 6, which showed it to have a molecular mass of approx. 620 kDa in non-dissociating conditions. On SDS/PAGE the human SP-D behaved as a single band of 150 kDa or 43 kDa in non-reducing or reducing conditions respectively. The presence of a high concentration of glycine (22%), hydroxyproline and hydroxylysine in the amino acid composition of human SP-D indicated that it contained collagen-like structure. Collagenase digestion yielded a 20 kDa collagenase-resistant globular fragment which retained affinity for maltose. Use of maltosyl-BSA as a neoglycoprotein ligand in a solid-phase binding assay showed that human SP-D has a similar carbohydrate-binding specificity to rat SP-D, but a clearly distinct specificity from that of other lectins, such as conglutinin, for a range of simple saccharides. Amino acid sequence analysis established the presence of collagen-like Gly-Xaa-Yaa triplets in human SP-D and also provided sequence data from the globular region of the molecule which was used in the synthesis of oligonucleotide probes. Screening of a human lung cDNA library with the oligonucleotide probes, and also with rabbit anti-(human SP-D), allowed the isolation of two cDNA clones which overlap to give the full coding sequence of human SP-D. The derived amino acid sequence indicates that the mature human SP-D polypeptide chain is 355 residues long, having a short non-collagen-like N-terminal section of 25 residues, followed by a collagen-like region of 177 residues and a C-terminal C-type lectin domain of 153 residues. Comparison of the human SP-D and bovine serum conglutinin amino acid sequences indicated that they showed 66% identity despite their marked differences in carbohydrate specificity.

Amino Acid Sequence↗
PubMed1992

Biotinyl analogues of amylin as biologically active probes for amylin/CGRP receptor recognition.

Biotinyl analogues of rat amylin were synthesised with sulfosuccinimidyl 2-(biotinamido) ethyl-1,3-dithiopropionate (NHS-SS-Biotin). Biotinylated amylin peptides were purified by HPLC, quantitated, and the presence of the biotin group at Lys-1 confirmed by peroxidase-labelled avidin and FAB mass spectroscopy. Amylin-biotin retained a similar affinity for binding to rat liver plasma membranes compared with rat amylin and also completely inhibited insulin-stimulated glycogen synthesis in rat soleus muscle incubated in vitro. These biologically active amylin probes will enable a complete analysis of amylin/CGRP receptor expression in various cell types and facilitate the isolation and characterisation of the hormone-receptor complex.

Amyloid↗
PubMed1992

The hinge region of the CD8 alpha chain: structure, antigenicity, and utility in expression of immunoglobulin superfamily domains.

The lymphocyte surface CD8 antigen is a heterodimer with each chain containing a single Ig-related domain, a hinge-like sequence, a transmembrane segment, and a short cytoplasmic sequence. A soluble form of the rat CD8 alpha chain was produced by introducing a stop codon into the cDNA at the end of the region encoding the extracellular sequence and expressed in Chinese hamster ovary cells. sCD8 alpha was produced at 20 mg/l, and consisted of monomers, dimers, and higher aggregates. The latter could be minimized, but not eliminated, by removal of one of the two cysteine residues in the hinge region by mutation and by growth in serum-free medium. The positions of the N- and O-linked glycosylation sites and the disulphide bond in the Ig-like domain were determined. The MRC OX-8 antibody was shown to react with a region from the CD8 alpha hinge containing 24 amino acids and the antigenic determinant was sensitive to neuraminidase digestion. A construct encoding the Ig-like domain of rat CD8 alpha without the hinge was not expressed in CHO cells, indicating the importance of the hinge region for expression. It seemed possible that the CD8 alpha hinge might facilitate expression of other Ig-related domains and such expression could be detected using the MRC OX-8 antibody. To test the system cDNA constructs were made with the rat CD8 alpha hinge spliced to the V-like domain of mouse CD8 alpha, to the V alpha and V beta domains of a T lymphocyte antigen receptor, and to one or both of the Ig-like domains of the MRC OX-47 membrane antigen. All these forms were expressed as soluble proteins that were detected with the MRC OX-8 antibody. This method may prove useful for the expression of Ig superfamily domains for raising antibodies and other studies.

Aged↗
PubMed1991

Rapid histone H3 phosphorylation in response to growth factors, phorbol esters, okadaic acid, and protein synthesis inhibitors.

When quiescent cells are stimulated with growth factors, phorbol esters, okadaic acid, or protein synthesis inhibitors, the early-response genes, which include c-fos and c-jun, are rapidly induced. The earliest growth factor- and phorbol ester-stimulated nuclear signaling events concomitant with proto-oncogene induction are the rapid phosphorylation of two chromatin-associated proteins, pp33 and pp15. We show here that the tumor promoter okadaic acid, which inhibits protein phosphatases 1 and 2A, and the protein synthesis inhibitors anisomycin and cycloheximide also stimulate pp33 and pp15 phosphorylation. Using transcriptional inhibitors, we show that this response is not a consequence of early gene induction. By peptide mapping and microsequencing, chromatin-associated pp15 is identified as histone H3. Upon stimulation, histone H3 is rapidly phosphorylated on serine residues within its highly charged, basic N-terminal domain. Thus, these diverse agents elicit a common early nuclear signal modulating nucleosomal structure or function, potentially contributing to conformational regulation of proto-oncogene induction.

Amanitins↗
PubMed1991

Secondary structure of a complement control protein module by two-dimensional 1H NMR.

The complement control protein (CCP) module (also known as the short consensus repeat) is a consensus sequence of about 60 amino acid residues which is thought to fold independently. It occurs over 140 times in more than 20 extracellular mosaic proteins including 12 proteins of the complement cascade. An isolated CCP module, the 16th repeat from human complement factor H, has been expressed in a yeast vector and shown to fold with the same pattern of disulfide bond formation as is seen in the native protein. Two-dimensional 600-MHz 1H NMR spectra of this module have been recorded at pH 3.3 and 6.0 and analyzed to permit determination of secondary structure in solution. The CCP module comprises two predominantly extended segments (Glu1-His13 and Ala17-Glu27), two segments of double-stranded antiparallel beta-sheet (Gly14-Val16 paired with Tyr31-Cys33 and Gly38-Asp40 paired with Ser57-Ile59), and a short piece of triple-stranded beta-sheet (Glu27-Thr30, Ile44-Leu48, and Lys51-Ser53). Turns occur at Asp22, Gly36, and Glu50, while Gly41-Ala43 appear to form a looped-out segment or bulge. This structure is compared with a secondary structure prediction made on the basis of an alignment scheme of 101 sequences for CCP modules [Perkins, S. J., Haris, P. I., Sim, R. B., & Chapman, D. (1988) Biochemistry 27, 4004-4012]--the experimentally determined secondary structure bears an overall resemblance to the predicted one but differs in the number and position of turns. Some of those amino acid residues which are highly conserved throughout the range of CCP modules appear to play a role in stabilizing the global fold.

Amino Acid Sequence↗
PubMed1991

Adsorption of fetal surfactant protein SP-B on the human amnion at term and on amniocytes incubated with fetal surfactant in vitro.

Fetal surfactant stimulates the synthesis of prostaglandins by slices of human amnion at term and by a human amnion cell line, and these effects are partly dependent upon surfactant apoproteins. In this paper, methods are described for the purification of surfactant from human amniotic fluid and from post-mortem human lung. A procedure is described for the purification of surfactant protein SP-B from human amniotic fluid, and the sequence of 20 amino acids at the N-terminal has been determined. A monoclonal antibody generated against human lung surfactant has been shown to react with SP-B from amniotic-fluid surfactant, and the presence of SP-B on the surface of the amnion at term has been demonstrated by immunohistochemical methods. It has also been shown that SP-B from surfactant is present on the surface of amniocytes incubated with surfactant in vitro.

Adsorption↗
PubMed1991

Isolation and partial characterisation of ACV synthetase from Cephalosporium acremonium and Streptomyces clavuligerus. Evidence for the presence of phosphopantothenate in ACV synthetase.

delta-(L-alpha-Aminoadipoyl)-L-cysteinyl-D-valine (ACV) synthetase was isolated and partially characterised from Cephalosporium acremonium CO728 and Streptomyces clavuligerus. The purification procedure resulted in a 745- and 277-fold increase in specific enzyme activity, respectively. Both enzymes had similar apparent molecular masses of ca. 300 kdaltons by SDS-polyacrylamide electrophoresis, under reducing and denaturing conditions, and in excess of 600 kdaltons in the native state by gel filtration. Attempts to obtain an N-terminal amino acid sequence of ACV synthetase from C. acremonium were unsuccessful, hence internal amino acid sequence data were obtained after tryptic digestion of the protein. Phosphopantothenic acid was shown to be associated with the enzyme from both sources, which suggests the possible involvement of pantothenate as a 'swinging arm' in the formation of the tripeptide ACV.

Acremonium↗
PubMed1990

High level expression in Chinese hamster ovary cells of soluble forms of CD4 T lymphocyte glycoprotein including glycosylation variants.

The CD4 cell surface antigen is of interest as a marker of T lymphocytes that recognize foreign antigens in the context of MHC Class II antigen, as a receptor for the human immunodeficiency virus (HIV) and as a member of the immunoglobulin superfamily (IgSF) with four Ig-like domains present in the extracellular domain. In order to produce large amounts of soluble CD4 for x-ray crystallography and other molecular studies, a recently developed expression system based on selection via glutamine synthetase was used. Expression was attempted for rat CD4 corresponding to the full extracellular sequence (sCD4; domains 1-4), the NH2-terminal half (domains 1 and 2) and the first domain alone. Stable transfected Chinese hamster ovary cell lines were obtained that expressed sCD4 and sCD4 (half) at typical maximal levels in spent tissue culture supernatant of greater than 80 and 25 mg/liter, respectively. Domain 1 alone was not expressed and introduction of a N-linked glycosylation site did not facilitate expression. The role of glycosylation in the expression of sCD4 was investigated by mutagenesis of the constructs to remove each of the two N-linked glycosylation sites in turn and both together. All three forms were expressed at 60-120 mg/liter. The sCD4 (half) was not expressed after deletion of its N-linked site. The disulfide bonds of sCD4 were determined to be within domains 1, 2, and 4 and isolation of glycopeptides showed that both N-linked sites were glycosylated. Analysis of the hydrodynamic properties of sCD4 suggested that the molecule adopted an extended conformation in solution rather than folding to form a compact structure like an Fab. The possibility of dimerisation of CD4 was investigated but sCD4 dimers were not seen at an affinity cut-off of about 4 x 10(5) M-1.

Amino Acid Sequence↗
PubMed1990

Acyl coenzyme A: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum and Aspergillus nidulans.

A study of the final stages of the biosynthesis of the penicillins in Penicillium chrysogenum has revealed two types of enzyme. One hydrolyses phenoxymethyl penicillin to 6-aminopenicillanic acid (6-APA). The other, also obtained from Aspergillus nidulans, transfers a phenylacetyl group from phenylacetyl CoA to 6-APA. The acyltransferase, purified to apparent homogeneity, had a molecular mass of 40 kDa. It also catalyses the conversion of isopenicillin N (IPN) to benzylpenicillin (Pen G) and hydrolyses IPN to 6-APA. In the presence of SDS it dissociates, with loss of activity, into fragments of ca 30 and 10.5 kDa, but activity is regained when these fragments recombine in the absence of SDS.

Acyl Coenzyme A↗
PubMed1990

Different isoforms of human FcRII distinguished by CDw32 antibodies.

The Third and Fourth International Workshops on Leucocyte Differentiation Antigens identified six mAb, designated CDw32, reacting with human Ig FcR type II (FcRII). We have examined the immunohistochemical and immunocytologic reactivities of these antibodies and find that the antibodies could be divided into three classes of reactivity: 1) antibodies IV.3, CIKM3, and CIKM5 reacted with monocytes, macrophages and neutrophils; 2) antibodies KB61 and 41H.16 gave strong reactions with B lymphocytes, placental and hepatic endothelium, and weaker reactions with monocytes, macrophages, and neutrophils; 3) antibody 2E1 gave an intermediate reaction pattern. Immunoprecipitation from U937 cell lysates showed that antibodies KB61 and 41H.16 recognized Mr 41,000 and Mr 37,000 molecules whereas the other antibodies detected a Mr 42,000 molecule. Preclearing with antibody KB61 removed the Ag recognized by the other five antibodies confirming the identity of the Ag and demonstrating reactivity of KB61 with the Mr 42,000 molecule. Antibodies KB61 and 41H.16 precipitated a Mr 41,000 molecule from B lymphocytes. Flow cytometry and immunoprecipitation studies of cells transfected with cDNA clones coding for two isoforms of FcRII showed that all six of the antibodies react with both transfectants but the only immunoprecipitations were obtained using KB61 and 41H.16 and one of the transfectants. The protein sequence of KB61 Ag isolated from leukemic B cells showed close homology with the proteins encoded by the cDNA clones but diverged in the intracytoplasmic carboxyl-terminal region. It was concluded that preferential recognition of one or more of the numerous isoforms of FcRII underlies the differing reaction patterns of CDw32 antibodies.

Amino Acid Sequence↗
PubMed1990

Photoaffinity labelling of isopenicillin N synthetase.

The alpha-aminoadipoyl group of the natural substrate of isopenicillin N synthetase (IPNS), L-alpha-aminoadipoyl-L-cysteinyl-D-valine (ACV), has been replaced by a diazirinyl-containing group, which can be photoactivated. This has allowed investigation of the substrate binding site of IPNS by photoaffinity labelling. Laser flash photolysis of this analogue, [3H]DCV, in the presence of IPNS leads to the incorporation of radioactivity into the enzyme. Tryptic digestion of this labelled enzyme, followed by separation and sequencing of the resultant fragments, identified two labelled regions of the protein. These are the fragments Asp-40 to Arg-78 and Thr-237 to Gly-256.

Affinity Labels↗
PubMed1990

Investigation of the structural basis of the interaction of calpain II with phospholipid and with carbohydrate.

Two forms of pig kidney calpain II were isolated, both of which appeared to contain an intact 80 kDa large subunit, but which showed specific proteolytic degradation at the N-terminal end of the 30 kDa small subunit. The structure of each of these molecules was investigated by amino acid sequence analysis. The forms corresponded to molecules with small subunits starting at residue 38 (degraded calpain A) and at residue 62 (degraded calpain B) of the complete sequence. These molecules were tested for their ability to interact with phosphatidylinositol and with carbohydrate (agarose gel-filtration media). Calpain and degraded calpain A, but not degraded calpain B, would interact with phosphatidylinositol. Thus the sequence (G)17TAMRILG (residues 38-61) is essential for the interaction. Neither calpain nor the degraded forms of the enzyme showed specific interaction with carbohydrate.

Amino Acid Sequence↗