Abstract
The NH(2)-terminal somatomedin B (SMB) domain (residues 1-44) of human vitronectin contains eight Cys residues organized into four disulfide bonds and is required for the binding of type 1 plasminogen activator inhibitor (PAI-1). In the present study, we map the four disulfide bonds in recombinant SMB (rSMB) and evaluate their functional importance. Active rSMB was purified from transformed Escherichia coli by immunoaffinity chromatography using a monoclonal antibody that recognizes a conformational epitope in SMB (monoclonal antibody 153). Plasmon surface resonance (BIAcore) and competitive enzyme-linked immunosorbent assays demonstrate that the purified rSMB domain and intact urea-activated vitronectin have similar PAI-1 binding activities. The individual disulfide linkages present in active rSMB were investigated by CNBr cleavage, partial reduction and S-alkylation, mass spectrometry, and protein sequencing. Two pairs of disulfide bonds at the NH(2)-terminal portion of active rSMB were identified as Cys(5)-Cys(9) and Cys(19)-Cys(21). Selective reduction/S-alkylation of these two disulfide linkages caused the complete loss of PAI-1 binding activity. The other two pairs of disulfide bonds in the COOH-terminal portion of rSMB were identified as Cys(25)-Cys(31) and Cys(32)-Cys(39) by protease-generated peptide mapping of partially reduced and S-alkylated rSMB. These results suggest a linear uncrossed pattern for the disulfide bond topology of rSMB that is distinct from the crossed pattern present in most small disulfide bond-rich proteins.
Highlights
Vitronectin (VN)1 is a 75-kDa adhesive glycoprotein that is present in plasma and the extracellular matrix, and it plays a significant role in a number of biological processes [1]
Molecular Characteristics of the Two recombinant VN1–97 (rVN1–97) Forms—Recombinant VN1–97 was expressed in E. coli and purified by immunoaffinity chromatography using the conformation-specific monoclonal antibody (mAb) 153 conjugated to a Cellufine column
Most of the recombinant SMB (rSMB) expressed in the cytoplasm of E. coli was misfolded and inactive, even though it was expressed as a fusion protein linked to the COOH terminus of thioredoxin
Summary
That conversion of any single Cys residue in the SMB domain into alanine destroyed its PAI-1 binding activity. We expressed the SMB domain of human VN in transformed Escherichia coli as a fusion protein containing 97 amino acid residues linked to the COOH terminus of thioredoxin (VN1–97). The disulfide linkages present in active rSMB were identified by partial reduction and S-alkylation under acidic conditions, chemical and proteolytic digestions, mass spectrometry, and protein sequencing. These results indicate that the disulfide bonds in active rSMB are arranged consecutively in a linear uncrossed pattern and suggest that the disulfide linkages within the SMB domain of human VN are arranged
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