Molecular characterization of antithrombin III (ATIII) variants using polymerase chain reaction. Identification of the ATIII Charleville as an Ala 384 Pro mutation.

Molho-Sabatier, P; Aiach, Martine; Gaillard, Isabelle; Fiessinger, Jean‐Noël; Fischer, Anne‐Marie; Chadeuf, Gilliane; Clauser, Éric

doi:10.1172/jci114290

Cited by 57 publications

(21 citation statements)

References 41 publications

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“…As mentioned above, amino acid substitutions in the RCL hinge regions of some serpins interfere with their inhibitory activity, and naturally occurring mutations within serpin RCLs have been linked to disease, such as at P10 of anti-thrombin III (35)(36)(37) as well as at P10 (38), P12 (39), and P14 (40) of C1 inhibitor. The rat CBG crystal structure (13) has revealed how several naturally occurring CBG variants could account for defects in steroid binding (13), including human CBG variants L93H (41,42) located in helix D and D367N (43) located in strand s4B as well as the rat CBG variant M276I (44) in strand s6A and the mouse CBG variant K201E (45) in strand s2C.…”

Section: Discussionmentioning

confidence: 99%

Residues in the Human Corticosteroid-binding Globulin Reactive Center Loop That Influence Steroid Binding before and after Elastase Cleavage

Lin

Underhill

Gardill

et al. 2009

Journal of Biological Chemistry

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Corticosteroid-binding globulin (CBG) is a non-inhibitory serine proteinase inhibitor (serpin) that transports cortisol and progesterone in blood. Crystal structures of rat CBG and a thrombin-cleaved human CBG:anti-trypsin (Pittsburgh) chimera show how structural transitions after proteolytic cleavage of the CBG reactive center loop (RCL) could disrupt steroid binding. This ligand release mechanism is assumed to involve insertion of the cleaved RCL into the ␤-sheet A of the serpin structure. We have, therefore, examined how amino acid substitutions in the human CBG RCL influence steroid binding before and after its cleavage by neutrophil elastase. Elastase-cleaved wild-type CBG or variants with substitutions at P15 and/or P16 (E334G/G335N or E334A) lost steroid binding completely, whereas deletion of Glu-334 resulted in no loss of steroid binding after RCL cleavage, presumably because this prevents its insertion into ␤-sheet A. Similarly, the steroid binding properties of CBG variants with substitutions at P15 (G335P), P14 (V336R), or P12 (T338P) in the RCL hinge were largely unaffected after elastase cleavage, most likely because the re-orientation and/or insertion of the cleaved RCL was blocked. Substitutions at P10 (G340P, G340S) or P8 (T342P, T342N) resulted in a partial loss of steroid binding after proteolysis which we attribute to incomplete insertion of the cleaved RCL. Remarkably, several substitutions (E334A, V336R, G340S, and T342P) increased the steroid binding affinities of human CBG even before elastase cleavage, consistent with the concept that CBG normally toggles between a high affinity ligand binding state where the RCL is fully exposed and a lower affinity state in which the RCL is partly inserted into ␤-sheet A. Corticosteroid-binding globulin (CBG)2 is the major carrier protein for natural glucocorticoids (cortisol and corticosterone) and progesterone in blood (1), and it regulates the bioavailability of steroids that control numerous physiological processes including reproduction, inflammation, stress responses, and tissue development (2). Because CBG binds up to 90% of the glucocorticoids in blood plasma, it serves as a reservoir of anti-inflammatory steroids that can be released at their sites of action (3). The latter concept was proposed when it was discovered that human CBG exhibits remarkable sequence identity with the archetypal serine proteinase inhibitor, ␣1-anti-trypsin (AAT), and was based on the realization that CBG might also be a target of specific classes of proteinases (4).The close structural relationship between CBG and AAT defines it as a clade A serine proteinase inhibitor (serpin) family member (5). Many of these serpin A family members, including CBG, are encoded by genes in the human 14q21.1 chromosome cluster and are thought to have arisen by gene duplication to produce serpins with similar properties and physiological functions (6). Unlike most clade A serpins, CBG is not known to act as a proteinase inhibitor but appears to be a suicide substrate of specific protei...

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Section: Discussionmentioning

confidence: 99%

Residues in the Human Corticosteroid-binding Globulin Reactive Center Loop That Influence Steroid Binding before and after Elastase Cleavage

Lin

Underhill

Gardill

et al. 2009

Journal of Biological Chemistry

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“…Letters 348 (1994) [14][15][16] accommodated in the site occupied by alanine in the wild type (Wright, H.T. and Scarsdale, J.N., submitted).…”

Section: Ht Wright Ma Blajchmanlfebsmentioning

confidence: 99%

Proteolytically cleaved mutant antithrombin‐Hamilton has high stability to denaturation characteristic of wild type inhibitor serpins

Wright

Blajcliman

1994

FEBS Letters

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The serpin family of proteins consists primarily of proteinase inhibitors which form tight complexes with target proteinases. Inhibitor serpins are cleaved by proteinase and undergo a large conformational change in which the polypeptide segment terminating at the target reactive site, at which cleavage takes place, inserts itself as an additional strand, s4A, in the center of a preexisting/?-sheet. This change in conformation increases the stability towards denaturation of the cleaved serpin relative to the native uncleaved form. Mutant serpins with single amino acid changes in the s4A strand have been identified, and in most cases these are proteinase substrates but not inhibitors. We have measured the stability to denaturation of one of these non-inhibitor substrate mutants, antithrombin-Hamilton, which has an Ala + Thr change at position P12 in strand s4A. We find that it undergoes the transformation to the more stable form which is observed for inhibitor serpins, from which we conclude that the Ala + Thr change in antithrombin-Hamilton does not prevent insertion of s4A into B-sheet A in the cleaved form.

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“…7). Substitution ofPI residue 393 Arg by Cys, His, and Pro, and P2 residue 392 Gly by Asp, all completely block the inhibitor-proteinase reaction (20,(34)(35)(36)(37)(38)(39). Substitution of P1' residue 394 Ser by Leu (40,41) drastically impairs the function of the reactive site when thrombin is the target proteinase, but has less effect upon inhibitory function against Factor Xa (42).…”

Section: -']-'----mentioning

confidence: 99%

“…Substitution of P1' residue 394 Ser by Leu (40,41) drastically impairs the function of the reactive site when thrombin is the target proteinase, but has less effect upon inhibitory function against Factor Xa (42). Substitution of PlO0 and P12 residues, 384 Ala and 382 Ala, respectively (38,43,44), causes the inhibitory reaction to be transformed into a substrate reaction in which the reactive bond is completely cleaved by the proteinase (23,45,46). Substrate reactions have also been demonstrated for other serpins that have amino acid substitutions or insertions in the P1I2-PlO region (47,48).…”

Section: -']-'----mentioning

confidence: 99%

Pleiotropic effects of antithrombin strand 1C substitution mutations.

Lane

Olds²,

Conard³

et al. 1992

J. Clin. Invest.

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Six different substitution mutations were identified in four different amino acid residues of antithrombin strand 1C and the polypeptide leading into strand 4B (F402S, F402C, F402L, A404T, N405K, and P407T), and are responsible for functional antithrombin deficiency in seven independently ascertained kindreds (Rosny, Torino, Maisons-Laffitte, Paris 3, La Rochelle, Budapest 5, and Oslo) affected by venous thromboembolic disease. In all seven families, variant antithrombins with heparin-binding abnormalities were detected by crossed immunoelectrophoresis, and in six of the kindreds there was a reduced antigen concentration of plasma antithrombin. Two of the variant antithrombins, Rosny and Torino, were purified by heparin-Sepharose and immunoaffinity chromatography, and shown to have greatly reduced heparin cofactor and progressive inhibitor activities in vitro. The defective interactions of these mutants with thrombin may result from proximity of s1C to the reactive site, while reduced circulating levels may be related to siC proximity to highly conserved internal ft strands, which contain elements proposed to influence serpin turnover and intracellular degradation. In contrast, siC is spatially distant to the positively charged surface which forms the heparin binding site of antithrombin; altered heparin binding properties of siC variants may therefore reflect conformational linkage between the reactive site and heparin binding regions of the molecule. This work demonstrates that point mutations in and immediately adjacent to strand 1C have multiple, or pleiotropic, effects on this serpin, leading ultimately to failure of its regulatory function. (J.

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Molecular characterization of antithrombin III (ATIII) variants using polymerase chain reaction. Identification of the ATIII Charleville as an Ala 384 Pro mutation.

Cited by 57 publications

References 41 publications

Residues in the Human Corticosteroid-binding Globulin Reactive Center Loop That Influence Steroid Binding before and after Elastase Cleavage

Residues in the Human Corticosteroid-binding Globulin Reactive Center Loop That Influence Steroid Binding before and after Elastase Cleavage

Proteolytically cleaved mutant antithrombin‐Hamilton has high stability to denaturation characteristic of wild type inhibitor serpins

Pleiotropic effects of antithrombin strand 1C substitution mutations.

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