The somatomedin B domain of vitronectin. Structural requirements for the binding and stabilization of active type 1 plasminogen activator inhibitor.
We recently localized the high affinity binding site for activated type 1 plasminogen activator inhibitor (PAI-1) to the somatomedin B domain (i.e. amino acid (aa) 1-51) of vitronectin (Vn). In this study to further define this site, N-terminal Vn fragments of various lengths were expressed in Escherichia coli and tested for PAI-1 binding activity. Vn polypeptides containing aa 1-52 and 1-40 retained PAI-1 binding activity and stabilized PAI-1 to a similar extent as intact Vn, but polypeptides containing aa 1-30 did not bind to PAI-1 nor stabilize its activity. The effects of monoclonal antibodies (mAbs) to Vn on PAI-1 binding was also determined. One mAb bound to Vn and blocked its ability to bind to PAI-1. It also dissociated pre-existing PAI-1.Vn complexes, and prevented the incorporation of PAI-1 into extracellular matrix of HT 1080 cells. This mAb bound to the recombinant peptide containing aa 1-40, but not to the peptide consisting of aa 1-30. A second randomly chosen mAb with similar affinity for Vn was inactive in these assays and bound to the region between aa 52 and 239. These results indicate that the high affinity binding site for active PAI-1 in Vn is between aa 1 and 40, and that this domain may also stabilize active PAI-1.
Seiffert
D
D
Department of Vascular Biology, Scripps Research Institute, La Jolla, California.
Ciambrone
G
G
Wagner
N V
NV
Binder
B R
BR
Loskutoff
D J
DJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002461:Cell Line, Transformed; D017931:DNA Primers; D004926:Escherichia coli; D005109:Extracellular Matrix / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008168:Lung; D008969:Molecular Sequence Data; D017395:Plasminogen Activator Inhibitor 1 / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013002:Somatomedins / Q000737:chemistry* / Q000378:metabolism; D019096:Vitronectin
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D017395:Plasminogen Activator Inhibitor 1; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013002:Somatomedins; D019096:Vitronectin; C016756:somatomedin B