Type 1 von Willebrand disease mutation Cys1149Arg causes intracellular retention and degradation of heterodimers: a possible general mechanism for dominant mutations of oligomeric proteins.

Some families affected by von Willebrand disease type 1 show high penetrance with exceptionally low von Willebrand factor (VWF) levels. Previously, a mutation associated with this dominant phenotype, Cys1149Arg, was found to decrease the secretion of coexpressed normal VWF, and the mutation was proposed to cause intracellular retention of pro-VWF heterodimers. To demonstrate heterodimer formation, a model was developed in which subunits could be distinguished immunologically and by size. Recombinant VWF lacking domain A1 (dA1), A3 (dA3), or both (dA13) was secreted efficiently as a full range of multimers. Cotransfection of Cys1149Arg and dA13 resulted in the secretion of multimeric VWF containing about 250 kd (Cys1149Arg) and about 210 kd (dA13). Cell lysates contained pro-VWF forms of Cys1149Arg and dA13. Immunoprecipitation with an antidomain A1 antibody recovered both subunits in heterodimers, and subunit ratios were consistent with random dimerization. Similar results were obtained for cotransfection of Cys1149Arg and dA1. Normal VWF has a Cys1149-Cys1169 intrachain bond. When cotransfected with normal VWF, Cys1149Arg or the double mutant Cys1149Arg+Cys1169Ser caused a similar decrease in VWF secretion, suggesting that an unpaired Cys1169 does not explain the intracellular retention of Cys1149Arg. VWF Cys1149Arg was not secreted from BHK cells but was degraded intracellularly within about 4 hours, and the proteasome inhibitor lactacystin delayed its clearance more than 16 hours. Thus, dominant von Willebrand disease type 1 may be caused by heterodimerization of mutant and normal subunits in the endoplasmic reticulum followed by proteasomal degradation in the cytoplasm. A similar dominant negative mechanism could cause quantitative deficiencies of other multisubunit proteins.