THE CLUSTER OF BASIC-AMINO-ACIDS IN VITRONECTIN CONTRIBUTES TO ITS BINDING OF PLASMINOGEN-ACTIVATOR INHIBITOR-1 - EVIDENCE FROM THROMBIN-CLEAVED, ELASTASE-CLEAVED AND PLASMIN-CLEAVED VITRONECTINS AND ANTIPEPTIDE ANTIBODIES
Z. Gechtman et al., THE CLUSTER OF BASIC-AMINO-ACIDS IN VITRONECTIN CONTRIBUTES TO ITS BINDING OF PLASMINOGEN-ACTIVATOR INHIBITOR-1 - EVIDENCE FROM THROMBIN-CLEAVED, ELASTASE-CLEAVED AND PLASMIN-CLEAVED VITRONECTINS AND ANTIPEPTIDE ANTIBODIES, Biochemical journal, 325, 1997, pp. 339-349
Derivatives of vitronectin obtained by specific cleavage at its cluste
r of basic amino acids with thrombin, elastase and plasmin are shown t
o have a decreased ability to bind plasminogen activator inhibitor-1 (
PAI-I). The identification and localization of the segment involved in
the binding of PAI-1 (Lys(348)-Arg(379)) were carried out by purifica
tion of these cleaved vitronectins and their subsequent structural cha
racterization (sequence analysis, phosphorylation of Ser(378) with cAM
P-dependent protein kinase and immunostaining with peptide-specific an
tibodies), then measurement of the vitronectin-PAI-l interaction by (a
) a two-phase system (ELISA); (b) co-precipitation of the vitronectin-
PAI-1 complex out of solution, and (c) analysis of the stereospecific
interaction between the active conformation of PAI-1 and a peptide der
ived from the above-mentioned cluster; this interaction occurs when th
e peptide is composed of all-L-amino acids but not when it is composed
of all-D-amino acids. Our results explain why workers who have used i
mmobilized vitronectin to study this interaction could not have observ
ed the involvement of the cluster of basic amino acids in PAI-1 bindin
g, since the immobilization of vitronectin is shown to render this clu
ster inaccessible for interaction. We propose that vitronectin binds a
ctive PAI-I by interaction via amino acid residues that originate from
distal locations in the N- and C-termini of vitronectin.