Elucidation of the Binding Regions of PAI-1 Neutralizing Antibodies Using Chimeric Variants of Human and Rat PAI-1

Bijnens, AP; Ngo, Thu Hoa; Gils, Ann; Dewaele, J; Knockaert, I; Stassen, Jean Marie; Declerck, Paul

doi:10.1055/s-0037-1615761

Cited by 22 publications

(26 citation statements)

References 37 publications

(63 reference statements)

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“…However, two monoclonal antibodies, 33B8 (41) and mAb-7 (42), blocked binding of PAI-1 to r⌬sBVN, with 33B8 and mAb-7 reducing the binding of latent PAI-1 to 37 or 22% of its original value, respectively, and reducing the binding of active PAI-1 to less than 50% of its original value. The epitopes for these antibodies are composed of residues from the loop connecting ␣-helix D and ␤-strand 2A, from ␤-strand 3A, and from ␤-strand 2B; the epitope for mAb-7 was mapped to Trp-88, Lys-90, Asp-91, Lys-178, and His-231 (42,43).…”

Section: Mapping the Binding Site In Pai-1 That Recognizes Residues Omentioning

confidence: 99%

“…Although the core of the SMB domain, including residues 18 -41, is well structured, NMR measurements (26) and x-ray crystallography (15) indicate that the flanking regions extending beyond residue 42 are more disordered. It is therefore also possible that the slight SMB domain inhibition is due to the overlap of the SMB domain (residues 1-51 for the cyanogen bromide fragment (26) or residues 1-47 for the recombinant SMB (29)) and r⌬sBVN (residues and that this flexible region (amino acids [42][43][44][45][46][47] in the isolated SMB domain partially interferes with the binding of the PAI-1-SMB complex to fulllength vitronectin and r⌬sBVN.…”

Section: R Smentioning

confidence: 99%

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Characterization of a Site on PAI-1 That Binds to Vitronectin Outside of the Somatomedin B Domain

Schar

Jensen

Christensen

et al. 2008

Journal of Biological Chemistry

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Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are proteins that interact in the circulatory system and pericellular region to regulate fibrinolysis, cell adhesion, and migration. The interactions between the two proteins have been attributed primarily to binding of the somatomedin B (SMB) domain, which comprises the N-terminal 44 residues of vitronectin, to the flexible joint region of PAI-1, including residues Arg-103, Met-112, and Gln-125 of PAI-1. A strategy for deletion mutagenesis that removes the SMB domain demonstrates that this mutant form of vitronectin retains PAI-1 binding (Schar, C. R., Blouse, G. E., Minor, K. M., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297-10309). In the current study, the complementary binding site on PAI-1 was mapped by testing for the ability of a battery of PAI-1 mutants to bind to the engineered vitronectin lacking the SMB domain. This approach identified a second, separate site for interaction between vitronectin and PAI-1. The binding of PAI-1 to this site was defined by a set of mutations in PAI-1 distinct from the mutations that disrupt binding to the SMB domain. Using the mutations in PAI-1 to map the second site suggested interactions between ␣-helices D and E in PAI-1 and a site in vitronectin outside of the SMB domain. The affinity of this second interaction exhibited a K D value ϳ100-fold higher than that of the PAI-1-somatomedin B interaction. In contrast to the PAI-1-somatomedin B binding, the second interaction had almost the same affinity for active and latent PAI-1. We hypothesize that, together, the two sites form an extended binding area that may promote assembly of higher order vitronectin-PAI-1 complexes.Vitronectin is a circulatory protein that interacts with several other plasma components that regulate coagulation and fibrinolysis, cell lysis via the complement cascade, and cell binding and migration in an integrin-dependent and urokinase-type plasminogen activator receptor-dependent fashion (1, 2). An interaction with vitronectin that has been widely studied occurs with the serine protease inhibitor (or serpin), PAI-1, 2 the primary inhibitor of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). Most circulating PAI-1 is found in a complex with vitronectin (3). The formation of the complex between vitronectin and PAI-1 is presumed to be physiologically significant in several respects. Binding of vitronectin to PAI-1 stabilizes the active conformation of the serpin, increasing the half-life for its conversion to the inactive, so-called latent state, and maintains its protease inhibitory properties for a prolonged period (4, 5). PAI-1 binds to the N-terminal ϳ50-amino acid somatomedin B (SMB) domain of vitronectin. The binding of PAI-1 competes with the binding of the urokinase-type plasminogen activator receptor to this domain and with the binding of integrins to the adjacent RGD sequence (for a review, see Ref. 6). Moreover, PAI-1 has been reported to induce oligomerization of vitronectin (7-10).Th...

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Section: Mapping the Binding Site In Pai-1 That Recognizes Residues Omentioning

confidence: 99%

Section: R Smentioning

confidence: 99%

Characterization of a Site on PAI-1 That Binds to Vitronectin Outside of the Somatomedin B Domain

Schar

Jensen

Christensen

et al. 2008

Journal of Biological Chemistry

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“…K A at least 400-fold less) for porcine, murine, and rat PAI-1 (Table II). Alignment of the sequence of human PAI-1 between residue 81 and 187, the region previously suggested to harbor the major interaction sites for MA-44E4 (24), with the corresponding segment of the latter three species reveals the presence of only two charged residues, i.e. Asp 181 and Arg 186 , that are not conserved in any of the other species.…”

Section: Affinity Of Monoclonal Antibodies With Pai-1 From Different mentioning

confidence: 89%

“…The affinity constants of both antibodies for porcine PAI-1 are reduced 6-and 9-fold, respectively, and no binding to murine or rat PAI-1 was observed. In the stretch of residues in human PAI-1 C-terminal from position 327 (24), only one charged amino acid in human PAI-1, Glu 350 , differs from the corresponding residues in murine and rat PAI-1, whereas it is conserved in porcine PAI-1. Therefore, we hypothesized that Glu 350 is involved in the interaction of PAI-1 with MA-42A2F6 and MA-56A7C10.…”

Section: Affinity Of Monoclonal Antibodies With Pai-1 From Different mentioning

confidence: 97%

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The Distal Hinge of the Reactive Site Loop and Its Proximity

Bijnens¹,

Gils²,

Stassen³

et al. 2001

Journal of Biological Chemistry

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The serpin plasminogen activator inhibitor type 1 (PAI-1) plays a regulatory role in various physiological processes (e.g. fibrinolysis and pericellular proteolysis) and forms a potential target for therapeutic interventions. In this study we identified the epitopes of three PAI-1 inhibitory monoclonal antibodies (MA-44E4, MA-42A2F6, and MA-56A7C10). Differential cross-reactivities of these monoclonals with PAI-1 from different species and sequence alignments between these PAI-1s, combined with the three-dimensional structure, revealed several charged residues as possible candidates to contribute to the respective epitopes. The production, characterization, and subsequent evaluation of a variety of alanine mutants using surface plasmon resonance revealed that the residues provides a molecular explanation for the differential exposure of this epitope in the different conformations of PAI-1 and for the effect of these antibodies on the kinetics of the formation of the initial PAI-1-proteinase complexes. The localization of the epitopes of MA-44E4, MA42A2F6, and MA-56A7C10 elucidates two previously unidentified molecular mechanisms to modulate PAI-1 activity and opens new perspectives for the rational development of PAI-1 neutralizing compounds.Plasminogen activator inhibitor type 1 (PAI-1), 1 a member of the serpin (serine proteinase inhibitor) superfamily (1-4), controls the plasminogen system at the level of tissue-type and urokinase-type plasminogen activator (t-PA and u-PA, respectively). Because PAI-1 is the main physiological inhibitor of t-PA in plasma (5), increased levels of PAI-1 result in a hypofibrinolytic state and are correlated with various vascular disorders such as venous thrombo-embolism, coronary artery disease, myocardial infarction, and atherosclerosis (6 -9). The u-PA-inhibiting effect of PAI-1 has its main physiological implications in processes outside of the circulation (10, 11).PAI-1 is a unique serpin because of its functional and conformational flexibility (12). PAI-1, synthesized as an active molecule, converts spontaneously into a nonreactive, latent form, which can be partially reactivated by denaturing reagents (13). Additionally, a third distinct, noninhibitory form (substrate) is identified that is reactive toward its target proteinases without the formation of a stable complex (14 -16).Elucidation of the three-dimensional structure of active PAI-1 (17, 18) reveals that the N-terminal side of the reactive site loop (extended from P16 to P3Ј and including the bait peptide bond Arg 345 -Met 346 (P1-P1Ј)) is exposed and accessible for the target proteinase. The C-terminal side of the reactive site loop (P4Ј-P13Ј) forms strand s1C in ␤-sheet C.Conversion to the latent state implies the insertion of the N-terminal side of the reactive site loop into ␤-sheet A, the loss of strand s1C from ␤-sheet C, and the formation of an unusual extended loop by the C-terminal side of the reactive site loop, resulting in the distortion of the P1-P1Ј "bait" peptide bond (19). It is hypothesized that th...

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Survey of the year 2001 commercial optical biosensor literature

Rich

Myszka

2002

J of Molecular Recognition

101

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We have assembled references of 700 articles published in 2001 that describe work performed using commercially available optical biosensors. To illustrate the technology's diversity, the citation list is divided into reviews, methods and specific applications, as well as instrument type. We noted marked improvements in the utilization of biosensors and the presentation of kinetic data over previous years. These advances reflect a maturing of the technology, which has become a standard method for characterizing biomolecular interactions.

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Elucidation of the Binding Regions of PAI-1 Neutralizing Antibodies Using Chimeric Variants of Human and Rat PAI-1

Cited by 22 publications

References 37 publications

Characterization of a Site on PAI-1 That Binds to Vitronectin Outside of the Somatomedin B Domain

Characterization of a Site on PAI-1 That Binds to Vitronectin Outside of the Somatomedin B Domain

The Distal Hinge of the Reactive Site Loop and Its Proximity

Survey of the year 2001 commercial optical biosensor literature

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