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Abstract
The N-terminal 44 amino acid residues of the human plasma glycoprotein vitronectin, known as the somatomedin B (SMB) domain, mediates the interaction between vitronectin and plasminogen activator inhibitor 1 (PAI-1) in a variety of important biological processes. Despite the functional importance of the Cys-rich SMB domain, how its four disulfide bridges are arranged in the molecule remains highly controversial, as evidenced by three different disulfide connectivities reported by several laboratories. Using native chemical ligation and orthogonal protection of selected Cys residues, we chemically synthesized all three topological analogs of SMB with predefined disulfide connectivities corresponding to those previously published. In addition, we oxidatively folded a fully reduced SMB in aqueous solution, and prepared, by CNBr cleavage, the N-terminal segment of 51 amino acid residues of intact vitronectin purified from human blood. Proteolysis coupled with mass spectrometric analysis and functional characterization using a surface plasmon resonance based vitronectin-PAI-1-SMB competition assay allowed us to conclude that 1) only the Cys(5)-Cys(21), Cys(9)-Cys(39), Cys(19)-Cys(32), and Cys(25)-Cys(31) connectivity is present in native vitronectin; 2) only the native disulfide connectivity is functional; and 3) the native disulfide pairings can be readily formed during spontaneous (oxidative) folding of the SMB domain in vitro. Our results unequivocally define the native disulfide topology in the SMB domain of human vitronectin, providing biochemical as well as functional support to the structural findings on a recombinant SMB domain by Read and colleagues (Zhou, A., Huntington, J. A., Pannu, N. S., Carrell, R. W., and Read, R. J. (2003) Nat. Struct. Biol. 10, 541-544).
Highlights
Urinary-type plasminogen activators [3,4,5,6]
Added to the brewing controversy was a recent finding by Peterson and colleagues [33] that the first 51 amino acid residues cleaved by CNBr from natively purified vitronectin3 had a third type of disulfide topology (Cys5– Cys9, Cys19–Cys31, Cys21–Cys32, and Cys25–Cys39), which matched neither the pattern reported for rVN1–51-1 nor for rVN1–51-2
After the disulfide linkages were discerned for all three species by mass mapping of peptide fragments generated by proteolysis, the folding intermediate with desired disulfide bridges was selected for Acm deprotection and simultaneous formation of two remaining disulfide bonds, in three unique combinations
Summary
Urinary-type plasminogen activators [3,4,5,6]. PAI-1 plays important roles in thrombosis and fibrinolysis and has been implicated in hemostasis, angiogenesis, and tumor metastasis [7,8,9,10,11]. Loskutoff and colleagues (24 –27) first pinpointed a high affinity PAI-1-binding site in vitronectin to the N-terminal somatomedin B (SMB) domain of the adhesive glycoprotein. Kamikubo et al [32] reported that an N-terminal fragment of VN of 97 amino acid residues, expressed in the cytoplasm of Escherichia coli and purified by immuno-affinity chromatography, showed activity in PAI-1 binding and antibody recognition similar to urea-activated vitronectin (uVN) purified from human blood. Added to the brewing controversy was a recent finding by Peterson and colleagues [33] that the first 51 amino acid residues cleaved by CNBr from natively purified vitronectin (we term nVN1–51-3) had a third type of disulfide topology (Cys5– Cys, Cys19–Cys, Cys21–Cys, and Cys25–Cys39), which matched neither the pattern reported for rVN1–51-1 nor for rVN1–51-2. None of the folding intermediates accumulated showed any biological activity as judged by PAI-1 binding and antibody recognition [34]
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