Identification of an Epitope in Antithrombin Appearing on Insertion of the Reactive-Bond Loop into the A β-Sheet

Nordling, Kerstin; Björk, Ingemar

doi:10.1021/bi9603579

Cited by 9 publications

(9 citation statements)

References 38 publications

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“…However, in contrast to the results presented here on m A b # 2H5, most of these studies were able to precisely determine the linear peptide sequence recognised by the antibodies. For example, Nordling and Bjork (1996) identified a hexapeptide sequence near Helix I of antithrombin that is exposed in all forms of the serpin in which the RCL had been inserted into P-sheet A. Similar to the results presented in this thesis, Picard et al…”

Section: General Discussion and Conclusionsupporting

confidence: 74%

“…These include the proximal and distal hinge regions, F helix, and psheets A and C. Furthermore, several studies have used monoclonal antibodies to investigate the nature of conformational changes associated with the S->R transition in a variety of serpins (eg. PAI-1, antithrombin) (Debrock & Declerck 1995;Nordling & Bjork 1996;Bjorquist et al 1997;Bjorquist et al 1999;Picard et al 1999). These studies used monoclonal antibodies to show the unavailability of epitopes on serpimprotease complexes that were present in the native serpin or appearance of new epitopes on serpin:protease complexes or proteolytically cleaved serpins.…”

Section: General Discussion and Conclusionmentioning

confidence: 99%

“…Using this approach, they estimated the conformational stabilit (AG[H 2 OJ) of inhibitory serpins in the S state to be 5 -15 kJ.mol" 1 , significantly lo than that of most globular proteins. Further evidence of large scale conformational changes accompanying the S->R transition is provided by immunological studies showing the expression of neoepitopes on a range of serpins following formation of stable serpin-protease complexes, Pl-Pl' cleavage, or insertion of synthetic RCL peptides (Schulze et al 1990;Bjork et al 1993;Dawes et al 1994;Eldering et al 1995;Debrock & Declerck 1995;Nordling & Bjork 1996).…”

Section: Overall Conformational Changesmentioning

confidence: 99%

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39 Structure function relationships of plasminogen activator inhibitor type-2 (PAI-2)

1998

Fibrinolysis and Proteolysis

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Section: General Discussion and Conclusionsupporting

confidence: 74%

Section: General Discussion and Conclusionmentioning

confidence: 99%

Section: Overall Conformational Changesmentioning

confidence: 99%

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39 Structure function relationships of plasminogen activator inhibitor type-2 (PAI-2)

1998

Fibrinolysis and Proteolysis

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“…Unexpectedly, mouse AT also contains the motif DAFHK and was recognized in immunoblotting, implying that 12A5 was an autoantibody. However, mammalian synthesis of an autoantibody directed against a cryptic region of a self-protein is not implausible; in fact, Nordling and Björk (33) have characterized a rabbit serum that recognizes the sequence VDLFSP shared by rabbit and human AT.…”

Section: A5 a Monoclonal Antibody That Distinguishes At-proteasementioning

confidence: 99%

“…Based on functional studies of serpin variants and immunochemical investigations, a number of reports nevertheless suggest that, in the stable complex, the RCL is inserted into ␤-sheet A. Antibodies have been characterized that fail to react with native serpin, but recognize binary complexes with a synthetic tetradecapeptide corresponding to residues P 14 to P 1 of the RCL, as well as consumed inhibitors (27)(28)(29)(30)(31)(32)(33). Thus, antibodies revealed that insertion of the RCL in ␤-sheet A exposes neoepitopes that are not present in the intact inhibitor.…”

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confidence: 99%

Topology of the Stable Serpin-Protease Complexes Revealed by an Autoantibody That Fails to React with the Monomeric Conformers of Antithrombin

Picard

Marque

Paolucci

et al. 1999

Journal of Biological Chemistry

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Solving the structure of the stable complex between a serine protease inhibitor (serpin) and its target has been a long standing goal. We describe herein the characterization of a monoclonal antibody that selectively recognizes antithrombin in complex with either thrombin, factor Xa, or a synthetic peptide corresponding to residues P 14 to P 9 of the serpin's reactive center loop (RCL, ultimately cleaved between the P 1 and P 1 residues). Accordingly, this antibody reacts with none of the monomeric conformers of antithrombin (native, latent, and RCL-cleaved) and does not recognize heparin-activated antithrombin or antithrombin bound to a non-catalytic mutant of thrombin (S195A, in which the serine of the charge stabilizing system has been swapped for alanine). The neoepitope encompasses the motif DAFHK, located in native antithrombin on strand 4 of ␤-sheet A, which becomes strand 5 of ␤-sheet A in the RCL-cleaved and latent conformers. The inferences on the structure of the antithrombin-protease stable complex are that either a major remodeling of antithrombin accompanies the final elaboration of the complex or that, within the complex, at the most residues P 14 to P 6 of the RCL are inserted into ␤-sheet A. These conclusions limit drastically the possible locations of the defeated protease within the complex. Serine protease inhibitors (serpins)1 are mainly composed of three ␤-sheets (A, B, and C) united by nine ␣-helices (A-I); indeed, many are inhibitors that neutralize their target(s) by forming a, stoichiometric, stable complex (1-3). Formation of the stable complex involves the charge stabilizing system of the target protease (4 -8) and a surface loop of the inhibitor called the reactive center loop (RCL). The RCL connects strand 4 of ␤-sheet A to strand 1 of ␤-sheet C; it is exposed to solvent in the inhibitory serpins. By analogy with protease substrates, the 20 amino acids constituting the RCL are numbered P n -. . . -P 1 -PЈ 1 -. . . -PЈ n , where P 1 -PЈ 1 is ultimately cleaved. The mechanism of protease inhibition involves multiple steps that initiate by the formation of a reversible association, converting to a stable complex, ultimately split into regenerated enzyme and RCL-cleaved (consumed) serpin (9 -12). The RCL sustains a variety of conformations (13-16). In antithrombin (AT) that is heparin-activated (17, 18) and other inhibitory serpins such as ␣1-antitrypsin (also called ␣1-proteinase inhibitor; 19 -21) or ␣1-antichymotrypsin (22), the RCL is wholly exposed, whereas in the AT monomer, residue P 14 of the RCL disrupts ␤-sheet A (23-25). In latent AT, an intact but non-inhibitory conformer (25), and in latent type-1 plasminogen activator inhibitor (16), residues P 14 to P 3 of the RCL are completely inserted into ␤-sheet A, constituting an additional, sixth, strand. The same conversion from a five-to six-stranded ␤-sheet A occurs in the inhibitory serpins, following cleavage of the RCL (14, 26).To date, no x-ray analysis of a serpin-protease complex has been reported; thus, its structure rema...

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Activated Protein C−Protein C Inhibitor Complex Formation: Characterization of a Neoepitope Provides Evidence for Extensive Insertion of the Reactive Center Loop

et al. 2000

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Protein C inhibitor, a serine proteinase inhibitor (serpin), is the physiologically most important inhibitor of activated protein C. We have made a monoclonal antibody (M36) that binds with equally high affinity to an epitope present in activated protein C-protein C inhibitor complexes and cleaved loop-inserted protein C inhibitor. Insertion of a synthetic N-acetylated tetradecapeptide (corresponding to residues P1-P14 of the reactive center loop) into beta-sheet A of the uncleaved inhibitor also exposed the epitope. The antibody had no apparent affinity for native uncleaved inhibitor or for the free peptide. Synthetic P1-P14 analogues, with Arg P13 or Ala P9 substituted to the residues found in mouse protein C inhibitor (Thr and Ile, respectively), were also inserted in beta-sheet A. The Arg P13/Thr substitution led to a greatly impaired reactivity with the antibody, whereas the Ala P9/Ile mutation resulted in a modest loss of reactivity with the antibody. These results indicate that complex formation leads to insertion of the reactive center loop in beta-sheet A from Arg P14 and presumably beyond Ala P9. Moreover, to the best of our knowledge, this is the first instance where the neoepitope of a complexation-specific monoclonal antibody has been localized to the loop-inserted part of beta-sheet A, the part of the serpin where the complexation-induced conformational change is most conspicuous.

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Identification of an Epitope in Antithrombin Appearing on Insertion of the Reactive-Bond Loop into the A β-Sheet

Cited by 9 publications

References 38 publications

39 Structure function relationships of plasminogen activator inhibitor type-2 (PAI-2)

39 Structure function relationships of plasminogen activator inhibitor type-2 (PAI-2)

Topology of the Stable Serpin-Protease Complexes Revealed by an Autoantibody That Fails to React with the Monomeric Conformers of Antithrombin

Activated Protein C−Protein C Inhibitor Complex Formation: Characterization of a Neoepitope Provides Evidence for Extensive Insertion of the Reactive Center Loop

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