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Biomedical subjects

D A Lomas

Publications and source records attributed to D A Lomas.

At least 55 records · Page 3Linked to original sources
PubMed1998

Alpha1-antitrypsin deficiency alleles and the Taq-I G-->A allele in cystic fibrosis lung disease.

Cystic fibrosis (CF) is characterized by progressive and ultimately fatal pulmonary disease although there are notable variations in clinical features. This heterogeneity is thought to lie outside the cystic fibrosis transmembrane regulator (CFTR) gene locus and may stem from deficiencies in the antiproteinase screen that protects the lung from proteolytic attack. One hundred and fifty seven patients were recruited from two UK CF centres. The serum concentrations of alpha1-antitrypsin, alpha1-antichymotrypsin and C-reactive protein (CRP) were determined and patients were screened for the common S and Z deficiency alleles of alpha1-antitrypsin and the G-->A mutation in the 3' noncoding region of the alpha1-antitrypsin gene (Taq-I G-->A allele). Alpha1-antitrypsin deficiency phenotypes were detected in 20 (16 MS, 1 S and 3 MZ) out of 147 unrelated tested CF patients and were, surprisingly, associated with significantly better lung function (adjusted mean forced expiratory volume in one second (FEV1) 62.5% of predicted for deficient group and 51.1% pred for normal alleles; p=0.043). The Taq-I G-->A allele was found in 21 out of 150 unrelated patients and had no significant effect on CF lung disease or on levels of alpha1-antitrypsin during the inflammatory response. We show here that, contrary to current thinking, common mutations of alpha1-antitrypsin that are associated with mild to moderate deficiency of the protein predict a subgroup of cystic fibrosis patients with less severe pulmonary disease. Moreover, the Taq-I G-->A allele has no effect on serum levels of alpha1-antitrypsin in the inflammatory response, which suggests that the previously reported association of the Taq-I G-->A allele with chronic obstructive pulmonary disease is not mediated by its effect on the serum level of alpha1-antitrypsin.

Adult↗
PubMed1997

Conformational disease.

Several diverse disorders, including the prevalent dementias and encephalopathies, are now believed to arise from the same general disease mechanism. In each, there is abnormal unfolding and then aggregation of an underlying protein. The gradual accumulation of these aggregates and the acceleration of their formation by stress explain the characteristic late or episodic onset of the clinical disease. The understanding of these processes at the molecular level is opening prospects of more rational approaches to investigation and therapy.

Amyloid↗
PubMed1997

Commercial plasma alpha1-antitrypsin (Prolastin) contains a conformationally inactive, latent component.

Fractionated plasma alpha1-antitrypsin is widely-used as replacement therapy in patients with Z alpha1-antitrypsin deficiency-related emphysema. We have recently shown that purified antitrypsin may be induced to adopt an inactive latent conformation by heating at high temperatures in stabilizing concentrations of sodium citrate. Such a conformation was predicted to be present in commercial preparations of antitrypsin, as these require heating under similar conditions for viral inactivation. Native antitrypsin was purified from plasma, and commercial antitrypsin (Prolastin) was obtained from Bayer Corporation. Western blot analysis of transverse urea gradient (TUG) gels showed that commercial antitrypsin migrated as two bands: one with an unfolding profile of native antitrypsin and the second with a profile of latent antitrypsin. A latent fraction, comprising approximately 8% of the total antitrypsin, was separated from the native antitrypsin in Prolastin by anion exchange chromatography. The specific activity of this latent form against bovine alpha-chymotrypsin increased from 1 to 2% to 50% over 3 h after refolding from 6 M guanidine hydrochloride. These data show that commercial antitrypsin contains a latent component. The significance of this conformation in vivo is unknown, although Prolastin has shown few adverse side-effects in prolonged clinical usage.

Animals↗
PubMed1996

The house dust mite allergen Der p1 catalytically inactivates alpha 1-antitrypsin by specific reactive centre loop cleavage: a mechanism that promotes airway inflammation and asthma.

Der p1, a cysteine proteinase derived from the house dust mite (HDM) Dermatophagoides pteronyssinus, is a major component of the allergic immune response in HDM atopic individuals. Recent evidence suggests that cysteine proteinase activity is important in the disease process as it increases the permeability of the allergen in the respiratory tract and disrupts the regulation of IgE synthesis. Der p1 is found in high concentrations in the faecal pellets of mites which are aerosolised and inhaled via the respiratory tract. The serine proteinase inhibitor, alpha 1-antitrypsin, protects the lower respiratory tract against damage by proteinases released in the lung during inflammation. Der p1 catalytically inactivates alpha 1-antitrypsin by a thiol-dependent mechanism involving specific cleavage of the reactive centre loop and we propose that this mechanism may be important in the pathogenesis of asthma.

Allergens↗
PubMed1996

Probing serpin reactive-loop conformations by proteolytic cleavage.

Several crystal structures of intact members of the serine proteinase inhibitor (or serpin) superfamily have recently been solved but the relationship of their reactive-loop conformations to those of circulating forms remains unclear. Here we examine reactive-loop conformational changes of anti-trypsin and anti-thrombin by using limited proteolysis and binary complex formation with synthetic homologous reactive-loop peptides. Proteolysis at the P10-P9, P8-P7 and P7-P6 of anti-trypsin was distorted by binary complex formation. The P1'-P2' bond in anti-thrombin was more accessible to proteolysis after binary complex formation, whereas cleavage at the P4-P3 bond was variably altered by synthetic peptide insertion. The proteolytic accessibility of the reactive-site P1-P1' bond of anti-trypsin and anti-thrombin binary complexes was identical with that of the native form and no cleavage was observed in the hinge region (P15-P10) of either protein, whether native or as binary complexes. these results fit with the proposal that the hydrophobic reactive loop of serpins adopts a modified helical conformation in the circulation, with the hinge region being partly incorporated into the A beta-pleated sheet. This loop can be displaced by peptides and induced to adopt a new conformation similar to the three-turn helix of ovalbumin. Both the native and binary complexed forms of anti-thrombin showed a greatly increased proteolytic sensitivity in the presence of heparin, indicating that heparin either induces a conformational change in the local structure of the helical reactive loop or facilitates the approximation of enzyme and inhibitor.

Amino Acid Sequence↗
PubMed1996

Inhibitory conformation of the reactive loop of alpha 1-antitrypsin.

The reactive site loop of the serpin family of serine proteinase inhibitors is flexible and can adopt a number of diverse conformations. A 2.9 A resolution structure of alpha 1-antitrypsin-the principal proteinase inhibitor in human plasma-shows the loop in a stable canonical conformation matching that found in all other families of serine proteinase inhibitors. This unexpected finding in the absence of loop insertion into the body of the molecule favours a two-stage mechanism of inhibition and provides a model for the heparin activation of antithrombin. The beta-pleated strand conformation of the loop also accounts for the polymerization of the serpins in disease and for their association with other beta-sheet structures, most notably the beta-amyloid of Alzheimer's disease.

Crystallography, X-Ray↗
PubMed1996

New insights into the structural basis of alpha 1-antitrypsin deficiency.

The serpin superfamily of serine proteinase inhibitors contains many members but the best-characterized is the plasma protein alpha 1-antitrypsin. its genetic deficiency is associated, in the homozygote, with hepatic damage that may progress to cirrhosis and hepatocellular carcinoma. Low levels of circulating alpha 1-antitrypsin fail to protect the lungs against proteolytic attack and predispose the homozygote to early onset pan-lobular emphysema, bronchiectasis and asthma. The major cause of alpha 1-antitrypsin deficiency, the Z mutation (Glu342Lys), results in the accumulation of protein in the endoplasmic reticulum of the liver. Using a structural approach, we have shown that the hepatic inclusions result from a protein-protein interaction between the reactive centre loop of one molecule and the beta-pleated sheet of a second. This loop-sheet polymerization is now also recognized to be the basis of deficiencies associated with mutations of C1-inhibitor, antithrombin and alpha 1-antichymotrypsin. Our recent solution of a crystal structure of a thermostable mutant of alpha 1-antitrypsin shows the detailed interactions that result in loop-sheet linkage and helps to explain the mechanism of action of this family of proteinase inhibitors.

Genetic Linkage↗
PubMed1996

Alpha 1-antitrypsin deficiency. A conformational disease.

The serpin family of protease inhibitors, to which alpha 1-antitrypsin belongs, has the unique feature of a mobile reactive center. Mutations within the critical regions of the molecule that control this mobility can allow premature changes in conformation with consequent abnormalities in folding and accompanying polymer formation. These abnormalities explain the plasma deficiency and liver inclusions associated with the common Z variant, as well as other variants of alpha 1-antitrypsin. The understanding of the molecular mechanisms provides a satisfying explanation for the clinical findings associated with these deficiency variants.

Humans↗
PubMed1995

The control of neutrophil chemotaxis by inhibitors of cathepsin G and chymotrypsin.

Neutrophil chemotaxis plays an important role in the inflammatory response and when excessive or persistent may augment tissue damage. The effects of inhibitors indicated the involvement of one or more serine proteinases in human neutrophil migration and shape change in response to a chemoattractant. Monospecific antibodies, chloromethylketone inhibitors, and reactive-site mutants of alpha 1-antitrypsin and alpha 1-antichymotrypsin were used to probe the specificity of the proteinases involved in chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis. Moreover, rapid inhibitors of cathepsin G and alpha-chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose. The concentrations of antichymotrypsin mutants that reduced chemotaxis by 50% would inactivate free cathepsin G with a half-life of 1.5-3 s, whereas the concentrations of chloromethylketones required to produce a similar inhibition of chemotaxis would inactivate cathepsin G with a half-life of 345 s. These data suggest different modes of action for these two classes of inhibitors. Indeed the chloromethylketone inhibitors of cathepsin G (Z-Gly-Leu-Phe-CMK) and to a lesser extent of chymotrypsin (Cbz-Gly-Gly-Phe-CMK) mediated their effect by preventing a shape change in the purified neutrophils exposed to fMLP. Antichymotrypsin did not affect shape change in response to fMLP even at concentrations that were able to reduce neutrophil chemotaxis by 50%. These results support the involvement of cell surface proteinases in the control of cell migration and show that antichymotrypsin and chloromethylketones have differing modes of action. This opens the possibility for the rational design of anti-inflammatory agents targeted at neutrophil membrane enzymes.

Amino Acid Chloromethyl Ketones↗
PubMed1995

alpha 1-Antitrypsin Mmalton (Phe52-deleted) forms loop-sheet polymers in vivo. Evidence for the C sheet mechanism of polymerization.

The Z (Glu342-->Lys) and Siiyama (Ser53-->Phe) deficiency variants of alpha 1-antitrypsin result in the retention of protein in the endoplasmic reticulum of the hepatocyte by loop-sheet polymerization in which the reactive center loop of one molecule is inserted into a beta-pleated sheet of a second. We show here that antitrypsin Mmalton (Phe52-deleted), which is associated with the same liver inclusions, is also retained at an endoglycosidase H-sensitive stage of processing in the Xenopus oocyte and spontaneously forms polymers in vivo. These polymers, obtained from the plasma of an Mmalton/QO (null) bolton heterozygote, were much shorter than other antitrypsin polymers and contained a reactive center loop-cleaved species. Monomeric mutant antitrypsin was also isolated from the plasma. The monomeric component had a normal unfolding transition on transverse urea gradient gel electrophoresis and formed polymers in vitro more readily than M, but less readily than Z, antitrypsin. The A beta-sheet accommodated a reactive center loop peptide much less readily than Z antitrypsin, which in turn was less receptive than native M antitrypsin. The nonreceptive conformation of the A sheet in antitrypsin Mmalton had little effect on kinetic parameters, the formation of SDS-stable complexes, the S to R transition, and the formation of the latent conformation. Comparison of the results with similar findings of short chain polymers associated with the antithrombin variant Rouen VI (Bruce, D., Perry, D., Borg, J.-Y., Carrell, R. W., and Wardell, M. R. (1994) J. Clin. Invest. 94, 2265-2274) suggests that polymerization is more complicated than the mechanism proposed earlier. The Z, Siiyama, and Mmalton mutations favor a conformational change in the antitrypsin molecule to an intermediate between the native and latent forms. This would involve a partial overinsertion of the reactive loop into the A sheet with displacement of strand 1C and consequent loop-C sheet polymerization.

Amino Acid Sequence↗
PubMed1995

Mutations which impede loop/sheet polymerization enhance the secretion of human alpha 1-antitrypsin deficiency variants.

alpha 1-Antitrypsin plasma deficiency variants which form hepatic inclusion bodies within the endoplasmic pathway include the common Z variant (Glu342-->Lys) and the rarer alpha 1-antitrypsin Siiyama (Ser53-->Phe). It has been proposed that retention of both abnormal proteins is accompanied by a common mechanism of loop-sheet polymerization with the insertion of the reactive center loop of one molecule into a beta-pleated sheet of another. We have compared the biosynthesis, glycosylation, and secretion of normal, Z and Siiyama variants of alpha 1-antitrypsin using Xenopus oocytes. Siiyama and Z alpha 1-antitrypsin both duplicated the secretory defect seen in hepatocytes that results in decreased plasma alpha 1-antitrypsin levels. Digestion with endoglycosidase H localized both variants to a pre-Golgi compartment. The mutation Phe51-->Leu abolished completely the intracellular blockage of Siiyama alpha 1-antitrypsin and reduced significantly the retention of Z alpha 1-antitrypsin. The secretory properties of M and Z alpha 1-antitrypsin variants containing amino acid substitutions designed to decrease loop mobility and sheet insertion were investigated. A reduction in intracellular levels of Z alpha 1-antitrypsin was achieved with the replacement of P11/12 alanines by valines. Thus a decrease in Z and Siiyama alpha 1-antitrypsin retention was observed with mutations which either closed the A sheet or decreased loop mobility at the loop hinge region.

Animals↗
PubMed1995

Preparation and characterization of latent alpha 1-antitrypsin.

Members of the serine proteinase inhibitor or serpin superfamily have a common molecular architecture based on a dominant five-membered A beta-pleated sheet and a mobile reactive center loop. The reactive center loop has been shown to adopt a range of conformations from the three turn alpha-helix of ovalbumin to the cleaved or latent inhibitor in which the reactive center loop is fully inserted into the A sheet of the molecule. While the cleaved state can be achieved in all inhibitory serpins only plasminogen activator inhibitor-1 and, more recently, antithrombin have been shown to adopt the latent conformation. We show here that the archetypal serpin, alpha 1-antitrypsin, can also be induced to adopt the latent conformation by heating at high temperatures in 0.7 M citrate for 12 h. The resulting species elutes at a lower sodium chloride concentration on an anion-exchange column and has a more cathodal electrophoretic mobility on non-denaturing polyacrylamide gel electrophoresis and isoelectric focusing than native M antitrypsin. Latent antitrypsin is inactive as an inhibitor of bovine alpha-chymotrypsin, is stable to unfolding with 8 M urea, and is more resistant to heat-induced loop-sheet polymerization than native but less resistant than cleaved antitrypsin. The reactive center loop of latent antitrypsin is inaccessible to proteolytic cleavage, and its occupancy of the A sheet prevents the molecule accepting an exogenous reactive center loop peptide. The activity of latent antitrypsin may be increased from < 1% to approximately 35% by refolding from 6 M guanidinium chloride.

Amino Acid Sequence↗
PubMed1994

The effect of the Z mutation on the ability of alpha 1-antitrypsin to prevent neutrophil mediated tissue damage.

Recent studies have shown that alpha 1-antitrypsin (alpha 1-AT) from Z antitrypsin deficiency subjects has a slightly lower association rate constant with neutrophil elastase (NE) than alpha 1-AT from normal subjects, although it is unknown whether this is of clinical importance. We have purified alpha 1-AT from a normal (M alpha 1-AT) and from a deficient (Z alpha 1-AT) subject and have confirmed that the association rate constants for NE are different (5.28; S.E. 0.06.10(7) M-1 s-1 and 1.2; S.E. 0.2.10(7) M-1 s-1, respectively). We have assessed the ability of both of these proteins to inhibit neutrophil mediated fibronectin (FN) degradation in vitro. Both proteins inhibited FN degradation in a dose dependant manner although Z alpha 1-AT was less effective than M alpha 1-AT at equivalent concentrations of active inhibitor (P < 0.05). Inhibition by M alpha 1-AT was 28.5% S.E. 3.9 at 0.01 microM; 35.5% S.E. 7.3 at 0.1 microM and 37% S.E. 8.4 at 0.5 microM, whereas inhibition by Z alpha 1-AT was 9.25% S.E. 3.9; 19.25% S.E. 7.7 and 21.2% S.E. 9.7, respectively. When the time course of inhibition of FN degradation was studied the difference (although less at 1.0 microM) became greater over the 3 h period of the assay. These results suggest that Z alpha 1-AT is less able than the M phenotype to inhibit connective tissue degradation by neutrophils at equivalent concentrations. This is probably due to the lower association rate constant although the reduced stability of the Z molecule may play a role. The differences, together with the reduced plasma concentration, may accentuate the susceptibility of deficient subjects to the development of emphysema.

Connective Tissue↗
PubMed1994

Loop-sheet polymerization: the structural basis of Z alpha 1-antitrypsin accumulation in the liver.

1. The Z deficiency variant of alpha 1-antitrypsin predisposes the homozygote to early-onset panlobular emphysema and results in the accumulation of antitrypsin within the hepatocyte, which leads to hepatocellular damage and cirrhosis. The mechanism of this accumulation has been shown to be due to the Z mutation (Glu-342-->Lys) perturbing the structure of the protein, allowing a unique interaction between the reactive-centre loop of one molecule and the A sheet of a second. This loop-sheet polymerization occurs spontaneously at 37 degrees C in purified plasma Z but not M antitrypsin. The rate of polymerization is greatly accelerated at 41 degrees C and is blocked by the insertion of a specific peptide into the A sheet of the antitrypsin molecule. Electron microscopy and circular dichroic spectral analysis confirm that the Z antitrypsin polymers formed in vitro have structural identity with those isolated from the liver of a Z homozygote. 2. That loop-sheet polymerization is a more general phenomenon was shown by the examination of a second deficiency variant, antitrypsin Siiyama (Ser-53-->Phe), which is also associated with liver inclusions. Electron microscopy confirmed that isolated antitrypsin Siiyama from the plasma of a homozygote was present as long chains of polymers identical with those of Z antitrypsin.

Humans↗
PubMed1994

Potential role of recombinant secretory leucoprotease inhibitor in the prevention of neutrophil mediated matrix degradation.

BACKGROUND: Neutrophil elastase is able to degrade connective tissue matrices and is thought to be involved in the pathogenesis of destructive lung diseases. METHODS: The ability of recombinant secretory leucoprotease inhibitor (rSLPI) to inhibit neutrophil mediated degradation of fibronectin in vitro is demonstrated and its efficacy compared with native alpha-1-proteinase inhibitor (n alpha 1-PI), recombinant alpha-1-proteinase inhibitor (r alpha 1-PI), and the chemical elastase inhibitor ICI 200,355. RESULTS: When preincubated with neutrophils both rSLPI and r alpha 1-PI were effective inhibitors of fibronectin degradation although n alpha 1-PI and ICI 200,355 were less effective. Recombinant SLPI was the most effective inhibitor when the cells were allowed to adhere to fibronectin before the addition of the inhibitors. Preincubation of rSLPI (0.1 mumol/l) with the fibronectin plate resulted in almost total inhibition of fibronectin degradation (reduced to 3.3 (SE 0.9)% of control). Pretreating the fibronectin plate with 1 mumol/l rSLPI, r alpha 1-PI and ICI 200,355 followed by thorough washing before the addition of cells resulted in no inhibition of fibronectin degradation with r alpha 1-PI and the ICI inhibitor, but rSLPI retained its inhibitory effect. This effect could be reduced by adding rSLPI in high pH buffer or 2 mol/1 NaCl. CONCLUSIONS: It is postulated that rSLPI binds to fibronectin to form a protective layer which prevents its degradation by neutrophil elastase. It may prove to be the most useful therapeutic agent in the prevention of neutrophil mediated lung damage.

Fibronectins↗
PubMed1994

Conformational changes in serpins and the mechanism of alpha 1-antitrypsin deficiency.

alpha 1-Antitrypsin is a member of the serine proteinase inhibitor, serpin, family of protease inhibitors, which have their reactive centers situated on a mobile peptide loop. This reactive loop can adopt varied conformations and perturbations of molecular structure to allow the pathological linking of the loop of one molecule to a beta-pleated sheet of another. This linkage has been shown to be the cause of the polymerization and aggregation within the hepatocyte of the common Z mutant of antitrypsin. The occurrence of loop-sheet polymerization has been confirmed with other deficiency variants of antitrypsin that accumulate in the liver (Mmalton, Siiyama) and also shown to occur in pathological mutants of C1-inhibitor and antithrombin. Deductive evidence indicates that the loop is inserted into the A-sheet of the next molecule, but recent structural findings raise the possibility of insertion into the C-sheet. This detail of loop-sheet polymerization is important for the design of strategies to interfere with insertion and hence lesson the accumulation of Z antitrypsin that is responsible for associated liver damage.

Humans↗