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D A Lomas

Publications and source records attributed to D A Lomas.

At least 19 recordsLinked to original sources
PubMed1999

A kinetic mechanism for the polymerization of alpha1-antitrypsin.

The mutation in the Z deficiency variant of alpha1-antitrypsin perturbs the structure of the protein to allow a unique intermolecular linkage. These loop-sheet polymers are retained within the endoplasmic reticulum of hepatocytes to form inclusions that are associated with neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. The process of polymer formation has been investigated here by intrinsic tryptophan fluorescence, fluorescence polarization, circular dichroic spectra and extrinsic fluorescence with 8-anilino-1-naphthalenesulfonic acid and tetramethylrhodamine-5-iodoacetamide. These biophysical techniques have demonstrated that alpha1-antitrypsin polymerization is a two-stage process and have allowed the calculation of rates for both of these steps. The initial fast phase is unimolecular and likely to represent temperature-induced protein unfolding, while the slow phase is bimolecular and associated with loop-sheet interaction and polymer formation. The naturally occurring Z, S, and I variants and recombinant site-directed reactive loop and shutter domain mutants of alpha1-antitrypsin were used to demonstrate the close association between protein stability and rate of alpha1-antitrypsin polymerization. Taken together, these data allow us to propose a kinetic mechanism for alpha1-antitrypsin polymer formation that involves the generation of an unstable intermediate, which can form polymers or generate latent protein.

Circular Dichroism↗
PubMed1999

Identification of DNA polymorphisms associated with the V type alpha1-antitrypsin gene.

alpha1-Antitrypsin (alpha1-AT) is a highly polymorphic protein. The V allele of alpha1-AT has been shown to be associated with focal glomerulosclerosis (FGS) in Negroid and mixed race South African patients. To identify mutations and polymorphisms in the gene for the V allele of alpha1-AT in five South African patients with FGS nephrotic syndrome DNA sequence analysis and restriction fragment length polymorphisms of the coding exons were carried out. Four of the patients were heterozygous for the BstEII RFLP in exon III [M1(Val213)(Ala213)] and one patient was a M1(Ala213) homozygote. The mutation for the V allele was identified in exon II as Gly-148 (GGG)-->Arg (AGG) and in all patients was associated with a silent mutation at position 158 (AAC-->AAT). The patient who was homozygous for (Ala213) also had a silent mutation at position 256 in exon III (GAT-->GAC) which was not present in any of the other four patients. Although the V allele of alpha1-AT is not associated with severe plasma deficiency, it may be in linkage disequilibrium with other genes on chromosome 14 that predispose to FGS. Furthermore, the associated silent mutation at position 158 and the Ala213 polymorphism are of interest, as these could represent an evolutionary intermediate between the M1(Ala213) and M1(Val213) subtypes.

Alleles↗
PubMed1998

Clinical outcome in relation to care in centres specialising in cystic fibrosis: cross sectional study.

OBJECTIVES: To assess the effect on clinical outcome of managing paediatric and adult patients with cystic fibrosis at specialised cystic fibrosis centres. DESIGN: Cross sectional study. SETTING: Two adult cystic fibrosis centres in the United Kingdom. SUBJECTS: Patients from an adult cystic fibrosis centre in Manchester were subdivided into those who had received continuous care from paediatric and adult cystic fibrosis centres (group A), and those who had received paediatric care in a centre not specialising in cystic fibrosis followed by adult care in a cystic fibrosis centre (group B). Group C were referrals to the new adult cystic fibrosis centre in Cambridge who had received neither paediatric nor adult centre care for their cystic fibrosis. MAIN OUTCOME MEASURES: Body mass index (weight (kg)/height (m2)), lung function (forced expiratory volume in one second (FEV1 percentage of predicted)), the Northern chest x ray film score, and age at colonisation with Pseudomonas aeruginosa. RESULTS: A prominent stepwise increase in body mass index was associated with increasing amounts of care at a cystic fibrosis centre; 18.3, 20.2, and 21.3 for groups C, B, and A respectively (P<0.001). Improved nutritional status was correlated with a higher FEV1 and better (lower) chest x ray film scores; r=0. 52 and -0.45 respectively (P<0.001 for both). CONCLUSION: These findings provide the first direct evidence that management of cystic fibrosis in paediatric and adult cystic fibrosis centres results in a better clinical outcome, and strongly supports the provision of these specialist services.

Adult↗
PubMed1998

Latent alpha1-antichymotrypsin. A molecular explanation for the inactivation of alpha1-antichymotrypsin in chronic bronchitis and emphysema.

alpha1-Antichymotrypsin is an acute phase protein that protects the tissues from damage by proteolytic enzymes, but previous studies have shown that alpha1-antichymotrypsin within the lungs of patients with chronic bronchitis and emphysema is intact but inactive as an inhibitor. Ammonium sulfate fractionation followed by blue Sepharose and DNA-Sepharose chromatography was used to isolate small amounts of intact, monomeric but inactive alpha1-antichymotrypsin from the plasma of 30 healthy blood donors. This species had a higher DNA binding affinity with more anodal electrophoretic mobility than native alpha1-antichymotrypsin and was conformationally stable against thermal denaturation, 8 M urea, and 7 M guanidinium chloride. The protein was unable to accept synthetic reactive loop peptides, and the reactive loop was resistant to proteolytic cleavage at the P5-P4 bond but could be cleaved between P1' and P3'. These data suggest that this new alpha1-antichymotrypsin species was in a conformation similar to those of the crystallographically determined latent serpins, plasminogen activator inhibitor-1 and antithrombin. alpha1-Antichymotrypsin from lung lavage migrated with the same electrophoretic mobility as the putative latent alpha1-antichymotrypsin, suggesting that this is the inactive conformation described previously in the lungs of patients with chronic bronchitis and emphysema. This conformational transition of alpha1-antichymotrypsin, from an active to an inactive state, within the lung may play an important role in the pathogenesis of chronic lung disease.

Bronchitis↗
PubMed1998

Wild-type alpha 1-antitrypsin is in the canonical inhibitory conformation.

alpha 1-Antitrypsin is the archetypal member of the serine proteinase inhibitor or serpin superfamily. Members of the family show structural homology based on a dominant A beta-sheet and a mobile reactive centre loop. Our recent crystal structure of alpha 1-antitrypsin stabilized with a point mutation showed the loop to be in a canonical inhibitory conformation in the absence of significant insertion into the A beta-sheet. It could be argued that the stabilizing mutation may induce the reactive centre loop to adopt an artificial, and unrepresentative, conformation and the finding seems to be at variance with studies assessing rates of peptide insertion into the A beta-sheet and limited proteolysis of the reactive loop. Here we present a 2.9 A structure of recombinant wild-type alpha 1-antitrypsin with no stabilizing mutations. Again, the reactive loop is in a canonical conformation in the absence of significant insertion into the A beta-sheet. A stabilizing salt bridge between P5 glutamate and arginine residues 196, 223 and 281, already identified in the mutant, provides strong evidence that this conformation is not an artefact of crystallization but represents the conformation of the circulating inhibitor in vivo. Comparison with the structure of alpha 1-antitrypsin stabilized with the Phe51Leu mutation indicates that the increased thermal stability of the mutant results from enhanced packing of aromatic residues in the hydrophobic core of the molecule. The structure of wild-type alpha 1-antitrypsin reveals a hydrophobic pocket between s2A and helices D and E that is filled on reactive loop insertion and the formation of biologically relevant loop-sheet polymers. This pocket may provide a target for rational drug design to prevent the formation of polymers and the associated plasma deficiency, liver cirrhosis and emphysema.

Computer Simulation↗
PubMed1998

Implications for function and therapy of a 2.9 A structure of binary-complexed antithrombin.

The crystal structure of a binary complex of human antithrombin with a peptide of the same sequence as its reactive loop (P14-P3) has been determined at 2.9 A. The peptide binds as the middle strand s4A in the A beta-sheet, homologously to that of the reactive loop in the latent and cleaved forms of antithrombin. Peptide binding results in the complete expulsion of the hinge region of the loop from the A beta-sheet although the conformation differs from that of heparin-activated antithrombin. The 36-fold increase in the rate of reaction of the binary complex with factor Xa indicates that full loop expulsion alone is not sufficient for complete heparin activation of antithrombin but that this is also dependent on the overall conformation of the molecule. Previous studies have demonstrated that reactive loop peptides can block or reverse the polymerisation of serpins associated with cirrhosis and thrombosis. The antithrombin binary complex structure defines the precise localisation of the blocking peptide in a serpin and provides the basis for rational drug design for mimetics that will prevent polymerisation in vivo and so ameliorate the associated disease.

Amino Acid Sequence↗
PubMed1998

Lung polymers in Z alpha1-antitrypsin deficiency-related emphysema.

Patients with alpha1-antitrypsin (alpha1-AT) deficiency are at risk of developing early-onset panlobular basal emphysema, which has been attributed to uncontrolled proteolytic activity within the lung. Severe genetic deficiency of alpha1-AT is most commonly due to the Z mutation (342Glu--> Lys), which results in a block in alpha1-AT processing within the endoplasmic reticulum of hepatocytes. The retained alpha1-AT forms inclusions, which are associated with neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. Our recent studies have shown that the accumulation of alpha1-AT is due to the Z mutation perturbing the structure of alpha1-AT to allow polymer formation, with a unique linkage between the reactive center loop of one alpha1-AT molecule and the A beta-pleated sheet of a second. The detection of loop-sheet polymers and other conformations of alpha1-AT in the lungs of patients with emphysema has been technically difficult. We show here that transverse urea-gradient-gel (TUG) electrophoresis and Western blot analysis may be used to characterize conformations of alpha1-AT in dilute samples of bronchoalveolar lavage fluid (BALF). This technique was used to demonstrate loop-sheet polymers in the lungs of patients with Z alpha1-AT-deficiency-related emphysema. Polymers were the predominant conformational form of alpha1-AT in BALF from the lungs of two of five Z homozygotes with emphysema, but were not detectable in any of 13 MM, MS, or MZ alpha1-AT controls. Because alpha1-AT loop-sheet polymers are inactive as proteinase inhibitors, this novel conformational transition will further reduce the levels of functional proteinase inhibitor in the lungs of the Z alpha1-AT homozygote, and so exacerbate tissue damage.

Bronchoalveolar Lavage Fluid↗
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↗