Defining the native disulfide topology in the somatomedin B domain of human vitronectin.
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).
Li
Xiangqun
X
Institute of Human Virology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201, USA.
Zou
Guozhang
G
Yuan
Weirong
W
Lu
Wuyuan
W
D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013002:Somatomedins / Q000737:chemistry* / Q000378:metabolism; D019096:Vitronectin / Q000737:chemistry* / Q000378:metabolism
D004220:Disulfides; D017395:Plasminogen Activator Inhibitor 1; C051321:SERPINE1 protein, human; D013002:Somatomedins; D019096:Vitronectin; C016756:somatomedin B; D003545:Cysteine