ALBERT

Description: VWF assembles into multimers in the Golgi by forming disulfide bonds between adjacent D’D3 domains using an oxidoreductase mechanism that requires the VWF propeptide (D1D2). During multimerization, free thiols are predicted to be important in the oxidoreductase mechanism either as the cysteine residues that form interchain disulfide bonds in the VWF multimer or as the cysteine residues that rearrange the disulfide-bonded intermediate between the propeptide and D’D3. To characterize free thiols involved in VWF multimerization, a model construct containing domains D1D2D’D3 was expressed in baby hamster kidney (BHK) cells and the secreted products were alkylated with thiol-reactive biotin-maleimide reagents and purified on avidin agarose. Both the D’D3 dimer and the D1D2 remained in the avidin agarose unbound fraction, indicating the absence of reduced Cys residues. The D’D3 monomer was bound to the avidin agarose beads, indicating the presence of reduced and alkylated cysteines. The reduced cysteines in the D’D3 monomer were alkylated with N-ethylmaleimide (NEM) for identification by mass spectrometry. NEM-labeled D’D3 monomer was reduced and alkylated with 4-vinylpyridine (4-VP). NEM increases the mass of Cys to 228.05686 Da, while 4-VP shifts the mass of Cys to 208.06703 Da. Peptides were generated with various proteases and analyzed by a linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FTMS). A theoretical peptide list was generated including either NEM or 4-VP modification of all Cys residues. The mass spectra were analyzed for m/z signals including up to the triply charged species of the theoretical digest. The MS/MS spectra were searched against a database with MASCOT. Using this method 98% of the 52 Cys residues within the D’D3 region were identified as either 4-VP- or NEM-modified. Cys1142 was identified in a tryptic peptide (ENGYECEWR+NEM, VWF residues 1137–1145) as a doubly charged ion at m/z 655.76031, confirming the previous identification of the Cys1142-Cys1142 interchain disulfide bond linking VWF multimers. A second NEM-modified peptide was identified from an Asp-N protease digest (DCACFC+2 4-VP+1 NEM, residues 1096–1101 of VWF). This species was observed as a doubly charged ion at m/z 498.67584. Analysis of the MS/MS revealed that Cys1099 was NEM-modified, suggesting that it may form an interchain disulfide bond between VWF multimers or may be important in the oxidoreductase mechanism of VWF multimerization. To determine if C1099 and/or C1142 are required to form D’D3 dimers, constructs D1D2D’D3 (C1099A), (C1142A), and (C1099A/C1142A) were expressed in BHK cells and the assembly of D’D3 dimers was assayed by Western blotting. All three mutant VWF proteins were secreted efficiently from BHK cells. However, the levels of D’D3 dimer were reduced markedly in the single mutants when compared with the dimer produced by the wild type D1D2D’D3 expression system. Further, mutation of both C1142 and C1099 (C1099A/C1142A) completely inhibited D’D3 dimerization, evidence that these specific cysteine residues in the D’D3 monomer are involved in the interchain disulfide bonds between VWF multimers and are essential for the oxidoreductase mechanism of VWF multimerization.