Vitronectin and substitution of a beta-strand 5A lysine residue potentiate activity-neutralization of PA inhibitor-1 by monoclonal antibodies against alpha-helix F.
Some monoclonal antibodies against plasminogen activator inhibitor-1 (PAI-1) are able to inhibit its reaction with its target proteinases. We have characterized the effect on PAI-1 of two monoclonal antibodies, Mab-2 and Mab-6, with overlapping epitopes in a sequence encompassing beta-strand 1A, alpha-helix F, and the loop connecting alpha-helix F and beta-strand 3A (the hF/s3A loop). Mab-2 reduced the inhibitory activity of wild type PAI-1 and almost totally abolished the inhibitory activity of a PAI-1 variant harboring an Ala substitution of Lys 325 (335 in the alpha1-proteinase inhibitor template residue numbering) in beta-strand 5A. In both cases, the neutralizing effect of the antibody was strongly potentiated by vitronectin. Mab-6 had no effect on wild type PAI-1, but reduced the inhibitory activity of the K325A variant. The effect of Mab-6 was not potentiated by vitronectin. With both Mab-2 and Mab-6, the neutralization of PAI-1 activity was associated with PAI-1 substrate behaviour. Mab-2, but not Mab-6, prevented vitronectin from rescuing PAI-1 from cold-induced substrate behaviour. We propose that the antibodies act by weakening the anchoring of alpha-helix F to the adjacent structures, resulting in an increased flexibility of beta-strand 5A and the hF/s3A loop and a changed conformational response to the binding of vitronectin in the alpha-helix E region. The potentiating effect of vitronectin on neutralization of PAI-1 by antibodies is a novel concept in the development of compounds for neutralizing PAI-1 in vivo.
Schousboe
S L
SL
Department of Molecular and Structural Biology, Aarhus University, Denmark.
Egelund
R
R
Kirkegaard
T
T
Preissner
K T
KT
Rodenburg
K W
KW
Andreasen
P A
PA
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D000918:Antibody Specificity; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D006860:Hydrogen Bonding; D046911:Macromolecular Substances; D008958:Models, Molecular; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D015843:Serpins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D019096:Vitronectin / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D046911:Macromolecular Substances; D017395:Plasminogen Activator Inhibitor 1; D015843:Serpins; D019096:Vitronectin