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S P Bottomley

Publications and source records attributed to S P Bottomley.

35 records · Page 2Linked to original sources
PubMed1999

The intracellular serpin proteinase inhibitor 6 is expressed in monocytes and granulocytes and is a potent inhibitor of the azurophilic granule protease, cathepsin G.

The monocyte and granulocyte azurophilic granule proteinases elastase, proteinase 3, and cathepsin G are implicated in acute and chronic diseases thought to result from an imbalance between the secreted proteinase(s) and circulating serpins such as alpha1-proteinase inhibitor and alpha1-antichymotrypsin. We show here that the intracellular serpin, proteinase inhibitor 6 (PI-6), is present in monocytes, granulocytes, and myelomonocytic cell lines. In extracts from these cells, PI-6 bound an endogenous membrane-associated serine proteinase to form an sodium dodecyl sulfate (SDS)-stable complex. Using antibodies to urokinase, elastase, proteinase 3, or cathepsin G, we demonstrated that the complex contains cathepsin G. Native cathepsin G and recombinant PI-6 formed an SDS-stable complex in vitro similar in size to that observed in the extracts. Further kinetic analysis demonstrated that cathepsin G and PI-6 rapidly form a tight 1:1 complex (ka = 6.8 +/- 0.2 x 10(6) mol/L-1s-1 at 17 degrees C; Ki = 9.2 +/- 0.04 x 10(-10) mol/L). We propose that PI-6 complements alpha1-proteinase inhibitor and alpha1-antichymotrypsin (which control extracellular proteolysis) by neutralizing cathepsin G that leaks into the cytoplasm of monocytes or granulocytes during biosynthesis or phagocytosis. Control of intracellular cathepsin G may be particularly important, because it has recently been shown to activate the proapoptotic proteinase, caspase-7.

Cathepsin G↗
PubMed1999

The production and characterisation of an immobilised chaperonin system.

Here we report a method of immobilising the chaperonins GroEL and GroES to a glass matrix. The immobilised chaperone system has been used to successfully refold target proteins denatured by guanidine hydrochloride and produce substantially higher levels of active protein than occur on dilution into aqueous solution alone. The chaperone system has been shown to refold proteins from each of the three categories of GroEL substrate. The refolding of the enzyme glycerol dehydrogenase from Bacillus stearothermophilus shows a two-fold increase in activity in the presence of immobilised GroEL compared to that in free solution. The lactate dehydrogenase from B. stearothermophilus also shows a two-fold higher yield of activity in the presence of the immobilised GroEL and ATP. The presence of immobilised GroEL in the absence of ATP arrests the refolding of LDH. The enzyme citrate synthetase from porcine heart demonstrates a three-fold increase in activity when refolded in the presence of immobilised GroEL, ATP and free GroES. Similar results are obtained in the presence of free GroEL, immobilised GroES and ATP. The matrix-bound chaperone can be removed from the refolding mixture by centrifugation, producing a reusable system that can be easily isolated and purified from the refolded substrate.

Adenosine Triphosphatases↗
PubMed1998

Alpha 1-antitrypsin polymerisation can occur by both loop A and C sheet mechanisms.

A number of disease states are attributable to alpha1-antitrypsin polymerisation within the endoplasmic reticulum of hepatocytes and subsequent plasma deficiency. Two distinct mechanisms describing the process of alpha1-antitrypsin polymerisation have been proposed, the loop A-sheet and C-sheet mechanisms. We report fluorescence studies using alpha1-antitrypsin covalently modified with pyrene maleimide. These results in conjunction with detailed molecular modelling studies, show that alpha1-antitrypsin is capable of undergoing both loop A-sheet and loop C-sheet mechanisms of polymerisation, depending upon the in vitro buffer conditions.

Models, Molecular↗
PubMed1998

The mechanism of alpha 1-antitrypsin polymerization probed by fluorescence spectroscopy.

The polymerization of alpha1-antitrypsin within the hepatic cell leads to alpha1-antitrypsin deficiency. Both the conformational changes and the kinetics of the polymerization process are poorly understood. Here we describe fluorescence experiments investigating the polymerization reaction using the fluorescent probe4, 4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (bis-ANS) which bound to both native and polymerized alpha1-antitrypsin. Biphasic changes in bis-ANS fluorescence were observed during formation of alpha1-antitrypsin polymers. Initially a rapid increase in fluorescence signal was observed; it was followed by a gradual reduction in fluorescence signal. The first phase is a conformational change in which the A beta-sheet of alpha1-antitrypsin opens, whereas the second phase represents the insertion of the reactive center loop into the A beta-sheet of another molecule and therefore determines the rate of the polymerization process.

Biopolymers↗
PubMed1998

Expression and purification of the human thrombin receptor.

The human thrombin receptor has been overexpressed in Sf9 (Spodoptera frugiperda) insect cells using a baculovirus vector. Cell surface expression of the receptor was confirmed by immunocytochemistry with polyclonal antibodies raised against the extracellular domain of the receptor. The expressed receptor was functional; both thrombin and the thrombin receptor agonist peptide produced increases in intracellular calcium in transfected cells. The concentration of thrombin causing the half-maximal increase (EC50) in intracellular calcium was 3.9 nM, whereas the EC50 for the agonist peptide was 2.7 microM. However, the observed maximum increase in intracellular calcium concentration with the agonist peptide (547 nM) was twofold greater than that observed with thrombin (258 nM). The recombinant receptor was purified by immunoaffinity chromatography using a monoclonal antibody raised against the receptor extracellular domain. The purified preparation contained two species with apparent molecular masses of 48 and 90 kDa, both of which were recognized by mono- and polyclonal antibodies against the thrombin receptor. The yield of the purified receptor was 0.78 mg/liter of insect cells suspension culture (10(6) cells/ml). The purified thrombin receptor will be useful in future structural and functional studies.

Amino Acid Sequence↗
PubMed1998

A study of the interactions between an IgG-binding domain based on the B domain of staphylococcal protein A and rabbit IgG.

The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein A from Staphylococcus aureus (termed SpA1) and the Fc fragment of rabbit IgG has been investigated. The contribution to binding of four putative hydrogen bond contacts between SpA1 and IgG-Fc were examined by the individual substitution of the residues in SpA1 involved in these interactions by others unable to form hydrogen bonds. It was found that the most important of the hydrogen bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity. The residues of SpA1 proposed to make close, mainly hydrophobic, contacts with Fc were replaced by residues with potential electrostatic charge to establish the importance of the hydrophobic interaction in the complex. The IgG-binding affinities of the mutant proteins were compared to the wild-type protein by a competitive enzyme-linked immunosorbent assay. The replacement of individual hydrophobic residues by His generated a number of novel IgG-binding proteins with reduced binding affinity at pH 5.0 but which maintained strong binding affinities at pH 8.0. The elution profile of human IgG1-Fc (Fc fragment of human IgG1) from a column made from an immobilized two-domain mutant protein shows that the complex dissociates at a higher pH relative to that of the non-mutated protein thus offering favorable elution characteristics.

Animals↗
PubMed1998

Protein engineering of chimeric Serpins: an investigation into effects of the serpin scaffold and reactive centre loop length.

The exposed Serpin reactive centre loop controls the specificity of the serpin proteinase interaction. Mutations within this region have been used to generate novel potentially therapeutic inhibitors. In this study we examine the effect of the serpin scaffold and reactive centre loop length upon the generation of such inhibitors. The reactive centre loop regions, P7-P3', of alpha1-antitrypsin and alpha1-antichymotrypsin were replaced by the corresponding residues of the viral serpin, Serp1, to form AT/Serp1 and ACT/Serp1, respectively. AT/Serp1 formed SDS stable complexes with a range of proteinases with association rate constants for plasmin, tissue plasminogen activator, urokinase, thrombin and factor Xa of approximately 10(4) M(-1)s(-1) and a stoichiometry of inhibition of approximately 1 for all of them. ACT/Serp1, however, formed SDS-stable complexes with only plasmin and thrombin with association rate constant 100-fold slower than AT/Serp1 and an increased stoichiometry of inhibition. The reactive centre loop of ACT/Serp1 is four amino acid residues longer than AT/Serp1. These four additional residues (VETR) were inserted into AT/Serp1 to form AT/Serp1(VETR). AT/Serp1(VETR) formed SDS stable complexes with plasmin, thrombin and tissue plasminogen activator similar to AT/Serp1, however, the association rate constants were 10-fold slower than those observed with AT/Serp1, while the stoichiometry of inhibition remained around 1. These results suggest that the additional reactive centre loop residues effect the rate of initial complex formation by placing the reactive centre loop in a non-ideal conformation. This study demonstrates that both reactive centre loop length and serpin scaffold are important in defining the inhibitory characteristics of a serpin.

Amino Acid Sequence↗
PubMed1997

The effects of reactive centre loop length upon serpin polymerisation.

The clinical effects of serpin polymerisation include thromboembolism, emphysema, and liver disease. A through understanding of serpin polymerisation mechanisms and the structures involved will permit the rational design of therapeutic polymerisation inhibitors. Here we show that serpin polymerisation can be delayed by extending the length of the serpin reactive centre loop. The heat stability of three chimeric serpins was examined. One of them, an active alpha 1-antitrypsin variant with a reactive centre loop C-terminal extension of four amino acid residues, was shown to have increased resistance to inactivation by polymerisation. This variant could also form serpin/peptide binary complexes with a reactive centre loop peptide, which indicates that the increase in thermostability was not due to the A-beta-sheet being unable to accept reactive centre loop residues, an essential requirement for polymerisation. Rather, we conclude that the additional residues within the reactive centre loop delay the release of strand 1C from the C-sheet, a process essential for polymer formation.

Amino Acid Sequence↗
PubMed1997

Recombinant caspase-3 expressed in Pichia pastoris is fully activated and kinetically indistinguishable from the native enzyme.

Intracellular cysteine proteinases (caspases) play key roles in inflammation and apoptosis. Recombinant caspases are typically produced in Escherichia coli expression systems with the attendant problems of solubilization, re-folding and activation of the protease. Here we describe the expression of hexahistidine-tagged caspase-3 (CPP32/Yama/Apopain) in the methylotropic yeast Pichia pastoris, and the purification of soluble enzyme from yeast lysates using cobalt affinity chromatography. The recombinant protease is fully activated, stable, and cleaves the synthetic substrate DEVD-AFC (Km 16.8 microM) but not YVAD-AFC. It mediates the cleavage of the apoptotic death substrate poly(ADP-ribose) polymerase in cell extracts, but does not cleave pro-interleukin-1beta. It is inhibited by the peptide DEVD-CHO (Ki 2.2 nM), far less efficiently by YVAD-CMK (Ki 0.3 microM), and not detectably by CrmA. By these criteria, recombinant caspase-3 is indistinguishable from native caspase-3 purified from apoptotic cell extracts. Activation of recombinant caspase-3 occurs in yeast in the absence of any intrinsic caspase activity, suggesting that caspase-3 can auto-activate. However, the purified enzyme was incapable of cleaving pro-caspase-3 indicating that autoactivation of caspase-3 in vivo is not likely to occur unless very high concentrations are achieved.

Caspase 3↗
PubMed1995

Equilibrium and pre-equilibrium fluorescence spectroscopic studies of the binding of a single-immunoglobulin-binding domain derived from protein G to the Fc fragment from human IgG1.

A single-immunoglobulin-binding protein based upon the C2 domain of Protein G from Streptococcus has been shown to bind tightly to the Fc fragment of IgG1. The binding interaction results in a decrease in the fluorescence intensity from the sole Trp residue (Trp-48) in this domain. This spectral change has been used to monitor the binding interactions between the two proteins using equilibrium and pre-equilibrium fluorescence spectroscopy. Comparison of the data from the two techniques suggests that a conformational change occurs after the initial formation of the complex. Mutagenesis studies have shown that the Trp residue is important for binding and that replacement by a Phe residue is important for binding and that replacement by a Phe residue leads to a 300-fold decrease in the affinity for Fc gamma 1. Determination of the rate constants kon and koff at different values of pH between 4.0 and 9.0 suggest that variations in Kd are mediated predominantly by changes in kon. Competition experiments between SpG1 and a single-IgG-binding domain from Protein A from Staphylococcus aureus have been used to determine the affinity of the latter for Fc gamma 1.

Bacterial Proteins↗
PubMed1995

Elution of human IgG from affinity columns containing immobilised variants of protein A.

Immobilised analogues of protein A have been used for affinity chromatographic separation of human IgG. Truncation of the C-terminal region of an engineered IgG-binding domain based upon the B domain from protein A, in combination with site-directed mutagenesis, has led to the generation of a number of proteins with a decreased affinity for IgG. The elution of human IgG from these proteins when immobilised onto a solid support occurs over the pH range 3.2-5.0 with 0.5 M acetate buffer. These proteins may be effective alternatives to standard protein A columns when milder elution conditions are required.

Binding Sites↗
PubMed1995

Crystallization and X-ray analysis of a single fab binding domain from protein L of Peptostreptococcus magnus.

Protein L is a multidomain cell wall constituent of certain strains of Peptostreptococcus magnus which binds to the variable domain of immunoglobulin kappa-light chains. A single immunoglobulin-binding domain of M(r) = 9000 from this protein has been isolated and crystallized. The crystals are of space group P4(2)2(1)2, with cell dimensions a = b = 66.9 A, c = 68.3 A, and diffract to at least 2.2 A resolution. The asymmetric unit of the crystal contains two molecules of the protein L domain, related by a noncrystallographic 2-fold axis, as revealed by a self-rotation function calculated with native diffraction data.

Bacterial Proteins↗
PubMed1995

Cloning, expression and purification of Ppl-1, a kappa-chain binding protein, based upon protein L from Peptostreptococcus magnus.

Protein L is a multi-domain, cell wall constituent of certain strains of Peptostreptococcus magnus, which binds to the variable domain of the L-chains of Ig. A gene fragment which codes for a single Ig-binding domain of protein L (Ppl-1) was cloned into a modified pKK223-3 vector and over-expressed in E. coli JM103. A rapid protein purification protocol is described. In these studies, purified Ppl-1 was immobilised on to an agarose gel and tested against an array of Igs and Ig fragments. It was found that Ppl-1-bound Igs from a number of different sources via interactions with the L kappa-chain. An enzyme linked immunosorbant assay has been developed to assay the binding of Ppl-1 to IgG. The incubation of Ppl-1 with human serum does not produce an immunoprecipitate, thus suggesting one unique interaction per binding domain which has been confirmed by ELISA. These experiments demonstrate the potential value of Ppl-1 as an immunological tool and as an affinity chromatography ligand for the purification of Igs.

Amino Acid Sequence↗
PubMed1994

The stability and unfolding of an IgG binding protein based upon the B domain of protein A from Staphylococcus aureus probed by tryptophan substitution and fluorescence spectroscopy.

The stability and unfolding of an immunoglobulin (Ig) G binding protein based upon the B domain of protein A (SpAB) from Staphylococcus aureus were studied by substituting tryptophan residues at strategic locations within each of the three alpha-helical regions (alpha 1-alpha 3) of the domain. The role of the C-terminal helix, alpha 3, was investigated by generating two protein constructs, one corresponding to the complete SpAB, the other lacking a part of alpha 3; the Trp substitutions were made in both one- and two-domain versions of each of these constructs. The fluorescence properties of each of the single-tryptophan mutants were studied in the native state and as a function of guanidine-HCl-mediated unfolding, and their IgG binding activities were determined by a competitive enzyme-linked immunosorbent assay. The free energies of folding and of binding to IgG for each mutant were compared with those for the native domains. The effect of each substitution upon the overall structure and upon the IgG binding interface was modelled by molecular graphics and energy minimization. These studies indicate that (i) alpha 3 contributes to the overall stability of the domain and to the formation of the IgG binding site in alpha 1 and alpha 2, and (ii) alpha 1 unfolds first, followed by alpha 2 and alpha 3 together.

Base Sequence↗