Distribution of the Native Strain in Human α1-Antitrypsin and Its Association with Protease Inhibitor Function

Seo, Eun Joo; Im, Ha Na; Maeng, Jin Soo; Kim, Kyoon Eon; Yu, Myeong Hee

doi:10.1074/jbc.m001006200

Cited by 51 publications

(66 citation statements)

References 36 publications

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“…A number of studies have shown that this region is proposed to play a crucial role in establishing and maintaining serpin stability (Seo et al 2000;Tew and Bottomley 2001a). The structures described here suggested a number of residues that may be responsible for binding the citrate ion as well as identifying residues that make up the binding pocket.…”

Section: Citrate Stabilizes Aat Against Unfolding and Polymerizationmentioning

confidence: 92%

Preventing serpin aggregation: The molecular mechanism of citrate action upon antitrypsin unfolding

et al. 2008

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The aggregation of antitrypsin into polymers is one of the causes of neonatal hepatitis, cirrhosis, and emphysema. A similar reaction resulting in disease can occur in other human serpins, and collectively they are known as the serpinopathies. One possible therapeutic strategy involves inhibiting the conformational changes involved in antitrypsin aggregation. The citrate ion has previously been shown to prevent antitrypsin aggregation and maintain the protein in an active conformation; its mechanism of action, however, is unknown. Here we demonstrate that the citrate ion prevents the initial misfolding of the native state to a polymerogenic intermediate in a concentration-dependent manner. Furthermore, we have solved the crystal structure of citrate bound to antitrypsin and show that a single citrate molecule binds in a pocket between the A and B b-sheets, a region known to be important in maintaining antitrypsin stability.

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Section: Citrate Stabilizes Aat Against Unfolding and Polymerizationmentioning

confidence: 92%

Preventing serpin aggregation: The molecular mechanism of citrate action upon antitrypsin unfolding

et al. 2008

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“…An interaction between AAT and its target enzyme results in a conformational transition of AAT that allows the opening of ␤-sheet A and insertion of the cleaved reactive site loop (45). The insertion of the reactive site strand into ␤-sheet A can also be induced under mild denaturation conditions and during intermolecular polymerization of AAT (46).…”

Section: Detection Of Various Molecular Forms Of Aat By the Mono-mentioning

confidence: 99%

Detection of Circulating and Endothelial Cell Polymers of Z and Wild Type α1-Antitrypsin by a Monoclonal Antibody

Janciauskiene¹,

Dominaitiene²,

Sternby³

et al. 2002

Journal of Biological Chemistry

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Globular inclusions of abnormal ␣1-antitrypsin (AAT)in the endoplasmic reticulum of hepatocytes are a characteristic feature of AAT deficiency of the PiZZ phenotype. Monoclonal antibodies, which contain constant specificity and affinity, are often used for the identification of Z-mutation carriers. A mouse monoclonal antibody (ATZ11) raised against PiZZ hepatocytic AAT was successfully used in enzyme-linked immunosorbent assays (ELISA) and in identification of Z-related AAT globular inclusions by immunohistochemical techniques. Using electrophoresis, Western blotting, and ELISA procedures, we have shown in the present study that this monoclonal antibody specifically detects a conformationdependent neoepitope on both polymerized and elastase-complexed molecular forms of AAT. The antibody has no apparent affinity for native, latent, or cleaved forms of AAT. The antibody ATZ11 illustrates the structural resemblance between the polymerized form of AAT and its complex with elastase and provides evidence that Z-homozygotes beyond the native form may have at least one more circulating molecular form of AAT, i.e. its polymerized form. In addition, staining of endothelial cells with ATZ11 antibody in both M-and Z-AAT individuals shows that AAT attached to endothelial cells is in a polymerized form. The antibody can be a powerful tool for the study of the molecular profile of AAT, not only in Z-deficiency cases but also in other (patho)physiological conditions.The capacity to undergo conformational changes is crucial for the physiological function of many proteins; the serine proteinase inhibitors (serpins) 1 are a clear case for such changes having been exploited during evolution as a means of modulating inhibitory activity (1). Serpins possess two structural elements that are conformationally labile and are essential for efficient proteinase inhibition: a reactive center loop and a ␤-sheet (␤-sheet A) that is able to open and accommodate the reactive center loop after its cleavage by the attacking proteases (2-5). The x-ray crystal structures of native, cleaved, cleaved in complex with protease, and polymerized forms of serpins have confirmed the abilities of these proteins to undergo profound conformational changes under certain environmental conditions (6 -8). The crystal structure of the ␣1-antitrypsin-trypsin complex showed complete insertion of the reactive site loop, which substantially increases thermal and conformational stability of the serpin and results in the irreversible inhibition and then the structural destruction of the proteinase (9).The conformational changes that occur in the serpin molecule during the formation of the enzyme-serpin complexes, interaction with other molecules, polymerization, cleavage, and oxidation lead to the generation of conformation-dependent neoepitopes (10). Immunochemical analysis by means of monoclonal antibodies is a useful approach for study of the formation of new epitopes that occur as a result of the conformational polymorphism of serpins (11,12). For example, the sugge...

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“…If the increase in the stability of the native state of ␣ 1 AT is manifested in the formation of the inhibitory complex, there should be a correlation between that increase in stability and the loss of inhibitory activity. Interestingly, however, only the stabilizing substitutions in the loop insertion region, such as Lys 335 (23) and Gly 117 (18) of sheet A, decreased the inhibitory activity, and the stabilizing mutations at most other sites of ␣ 1 AT did not cause the activity loss (17). It was shown recently that cavity filling substitutions designed at several sites of ␣ 1 AT increased the stability of the molecule, but activity affecting mutations among them were localized in the region that appears to be mobilized during the loop insertion (22 1 The abbreviations used are: serpin, serine protease inhibitor; ␣ 1 AT, ␣ 1 -antitrypsin; PPE, porcine pancreatic elastase; HLE, human leukocyte elastase; SI, stoichiometry of inhibition.…”

mentioning

confidence: 99%

“…In the crystal structure of the native ␣ 1 AT (19,20), unfavorable interactions such as side chain overpacking, buried polar groups, internal cavities, and surface hydrophobic pockets occur (16,17). These unfavorable interactions can be replaced by stabilizing substitutions (21,22).…”

mentioning

confidence: 99%

“…These unfavorable interactions can be replaced by stabilizing substitutions (21,22). We screened thermostable mutations over the entire ␣ 1 AT molecule and found stabilizing mutations that influence the flexibility of the native state distributed throughout the molecule (17). If the increase in the stability of the native state of ␣ 1 AT is manifested in the formation of the inhibitory complex, there should be a correlation between that increase in stability and the loss of inhibitory activity.…”

mentioning

confidence: 99%

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Concerted Regulation of Inhibitory Activity of α1-Antitrypsin by the Native Strain Distributed throughout the Molecule

Seo

Lee²,

2002

Journal of Biological Chemistry

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The native forms of common globular proteins are in their most stable state but the native forms of plasma serpins (serine protease inhibitors) show high energy state interactions. The high energy state strain of ␣ 1 -antitrypsin, a prototype serpin, is distributed throughout the whole molecule, but the strain that regulates the function directly appears to be localized in the region where the reactive site loop is inserted during complex formation with a target protease. To examine the functional role of the strain at other regions of ␣ 1 -antitrypsin, we increased the stability of the molecule greatly via combining various stabilizing single amino acid substitutions that did not affect the activity individually. The results showed that a substantial increase of stability, over 13 kcal mol ؊1 , affected the inhibitory activity with a correlation of 11% activity loss per kcal mol ؊1 . Addition of an activity affecting single residue substitution in the loop insertion region to these very stable substitutions caused a further activity decrease. The results suggest that the native strain of ␣ 1 -antitrypsin distributed throughout the molecule regulates the inhibitory function in a concerted manner.The native forms of common globular proteins are in their most stable state and protein folding is a spontaneous process (1). However, the native forms of some proteins are not in their most stable state: typical examples are the strained native structure of plasma serpins 1 (serine protease inhibitors) (2), the spring-loaded structure of the fusion protein of some viruses (3, 4), and heat shock transcription factors (5). The high energy state of the native structure of serpins is considered to be crucial to their physiological functions, such as plasma protease inhibition (1, 6), hormone delivery (7), Alzheimer filament assembly (8, 9), and extracellular matrix remodeling (10). The inhibition process of serpins can be described as a suicide substrate mechanism (11, 12), in which serpins, upon binding with proteases, partition between cleaved serpins (substrate pathway) and stable serpin-enzyme complexes (inhibitory pathway) as described in Scheme 1.In this scheme, I denotes the serpin; E, protease; EI, noncovalent Michaelis complex; E-I, a proposed intermediate prior to partitioning; E-I*, stable enzyme-inhibitor complex; and I*, cleaved serpin. The stoichiometry of inhibition (SI, the number of moles of inhibitors required to completely inhibit 1 mol of a target protease) is given by 1 ϩ k substrate /k inhibition , in which k substrate and k inhibition are the rate constants for the substrate and inhibitory pathways, respectively. The crystal structure of a serpin-protease complex revealed that the reactive site loop of the serpin is cleaved and inserted into the major ␤-sheet, sheet A, in the complex, whereas the target protease is attached to the cleaved loop of the serpin as an acyl intermediate (13). The conformational conversion during complex formation accompanies the distortion of the protease active site (13), ...

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Distribution of the Native Strain in Human α1-Antitrypsin and Its Association with Protease Inhibitor Function

Cited by 51 publications

References 36 publications

Preventing serpin aggregation: The molecular mechanism of citrate action upon antitrypsin unfolding

Preventing serpin aggregation: The molecular mechanism of citrate action upon antitrypsin unfolding

Detection of Circulating and Endothelial Cell Polymers of Z and Wild Type α1-Antitrypsin by a Monoclonal Antibody

Concerted Regulation of Inhibitory Activity of α1-Antitrypsin by the Native Strain Distributed throughout the Molecule

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