Epitope mapping for four monoclonal antibodies against human plasminogen activator inhibitor type‐1

Abstract

The inhibitory mechanism of serine proteinase inhibitors of the serpin family is based on their unique conformational flexibility. The formation of a stable proteinase-serpin complex implies insertion of the reactive centre loop of the serpin into the large central b-sheet A and a shift in the relative positions of two groups of secondary structure elements, the smaller one including a-helix F. In order to elucidate this mechanism, we have used phage-display and alanine scanning mutagenesis to map the epitopes for four monoclonal antibodies against a-helix F and its flanking region in the serpin plasminogen activator inhibitor-1 (PAI-1). One of these is known to inhibit the reaction between PAI-1 and its target proteinases, an effect that is potentiated by vitronectin, a physiological carrier protein for PAI-1. When combined with the effects these antibodies have on PAI-1 activity, our epitope mapping points to the mobility of amino-acid residues in a-helix F and the loop connecting a-helix F and b-strand 3A as being important for the inhibitory function of PAI-1. Although all antibodies reduced the affinity of PAI-1 for vitronectin, the potentiating effect of vitronectin on antibody-induced PAI-1 neutralization is based on formation of a ternary complex between antibody, PAI-1 and vitronectin, in which PAI-1 is maintained in a state behaving as a substrate for plasminogen activators. These results thus provide new details about serpin conformational changes and the regulation of PAI-1 by vitronectin and contribute to the necessary basis for rational design of drugs neutralizing PAI-1 in cancer and cardiovascular diseases.Keywords: PAI-1; vitronectin; monoclonal antibody; epitope mapping; phage-display.Plasminogen activator inhibitor-1 (PAI-1) is a fast and specific inhibitor of the serine proteinases urokinase-type (uPA) and tissue-type plasminogen activator (tPA), and, as such, an important regulator of extracellular proteolysis in turnover of extracellular matrix and in fibrinolysis (reviewed in [1]). PAI-1 binds with high affinity to vitronectin (reviewed in [2,3]) and inhibits vitronectin binding of integrins [4,5] and of the uPA receptor [6,7], and is thereby able to regulate cell migration and adhesion independently of its function as a proteinase inhibitor [4,5,7±9]. The PAI-1 level in malignant tumours is one of the most informative biochemical markers of a poor prognosis (reviewed in [10,11]), and PAI-1 seems to be causally involved in tumour invasion and angiogenesis [12]. PAI-1 is therefore a potential target for anticancer therapy.PAI-1 belongs to the serpin superfamily. Serpins are composed of three b-sheets and nine a-helices, which form a scaffold supporting a solvent-exposed, about 20 amino acids long peptide loop, the reactive centre loop (RCL). Serpins and proteinases form stable complexes by interaction of the active site of the proteinases with the reactive centre peptide bond (P 1 ±P 1 H ) in the RCL (reviewed in [1,13]). There is both biochemical and structural evidence that formation of a...

Modulation of Serpin Reaction through Stabilization of Transient Intermediate by Ligands Bound to α-Helix F

“…Active PAI-1 circulates in the bloodstream as a high affinity complex with vitronectin (Vn) (38), a cell adhesive glycoprotein, which is present in plasma in micromolar concentrations (39,40), and stabilizes the active conformation of PAI-1 through a decrease in the rate of its spontaneous inactivation (41,42). Although Vn does not significantly affect the SI for the reactions of PAI-1 with uPA and tPA (43)(44)(45), under physiological pH it decreases the k lim for both enzymes and dramatically increases the rate of the reaction with activated protein C (46). On the other hand, the binding of Vn results in an increase in both the rate and SI for the reaction of PAI-1 with thrombin (43,47,48).…”

“…On the other hand, the binding of Vn results in an increase in both the rate and SI for the reaction of PAI-1 with thrombin (43,47,48). Moreover, Vn dramatically enhances the fraction of the substrate reaction between tPA or uPA and complexes of PAI-1 with mAbs (MA-55F4C12, mAb-2) directed against ␣HF (44,45,49). Therefore, the binding of Vn and anti-␣HF mAbs to PAI-1 affects steps of the serpin mechanism, which contribute to distribution between substrate and inhibitory reactions.…”

Mechanisms of Conversion of Plasminogen Activator Inhibitor 1 from a Suicide Inhibitor to a Substrate by Monoclonal Antibodies

“…Despite attempts to develop small inhibitors of PAI-1 (27)(28)(29)(30), anti-PAI-1 monoclonal antibodies (mAbs), which combine high affinity and specificity of interaction, are major established models for studying intermolecular PAI-1 inactivation. A number of mAbs inactivating PAI-1 have been selected and characterized, and their epitopes have been identified as well as several possible mechanisms affecting PAI-1 activity (31)(32)(33)(34)(35)(36)(37). Two mechanisms of PAI-1 inactivation have been confirmed directly thus far: (i) acceleration of the active-to-latent transformation of PAI-1 by MA-33B8 (38) and (ii) prevention of MichaelisMenten complex formation by mAbs MA-44E4, MA-42A2F6, and MA-56A7C10 (32,39).…”

Mapping the Epitopes of Antibodies

“…Example [22]: Wind and colleagues (Wind, et al, 2001) used phage-displayed peptides and selected alanine mutants (SAMs) of human plasminogen activator inhibitor-1 (PAI-1) to identify the epitopes of four previously identified murine mAbs: Mab2, Mab6, AD3780, and AD3783. PAI-1 is a serpin comprising 3 ß sheets and 9 α helices which inhibits tissue-type plasminogen activator (tPA) and urinary-type plasminogen activator (uPA).…”

“…Rather, SAMs showed that R133, R135, and F136 are part of the Mab2 epitope. Based on the teaching of Jin and colleagues (Jin, et al, 1992), the structure of PAI-1, and the previous mapping, Wind et al (Wind, et al, 2001) picked 14 residues between 102 and 156 for SAM. The functional epitopes identified for AD3780 and AD3783 are essentially identical, involving residues Q102, R103, Q125, D127, E130, R133, F136, I137, and D140 (bold for EC 50 increases 100-fold, and underscore for EC 50 increases 10-fold, plain for EC 50 increases 3-fold).…”