TY - JOUR
T1 - The dominant-negative von Willebrand factor gene deletion p.P1127_C1948delinsR: molecular mechanism and modulation
AU - Casari, Caterina
AU - Pinotti, Mirko
AU - Lancellotti, Stefano
AU - Adinolfi, Elena
AU - Casonato, Alessandra
AU - De Cristofaro, Raimondo
AU - Bernardi, Francesco
PY - 2010
Y1 - 2010
N2 - Understanding molecular mechanisms in the dominant inheritance of von Willebrand disease would improve our knowledge of pathophysiologic processes underlying its prevalence. Cellular models of severe type 2 von Willebrand disease, caused by a heterozygous deletion in the von Willebrand factor (VWF) gene, were produced to investigate the altered biosynthesis. Coexpression of the wild-type and in-frame deleted (p.P1127_C1948delinsR) VWF forms impaired protein secretion, high molecular weight multimer formation and function (VWF collagen-binding 1.9% ± 0.5% of wild-type), which mimicked the patient's phenotype. mRNA, protein, and cellular studies delineated the highly efficient dominant-negative mechanism, based on the key role of heterodimers as multimer terminators. The altered VWF, synthesized in large amounts with the correctly encoded "cysteine knot" domain, formed heterodimers and heterotetramers with wild-type VWF, in addition to deleted homodimers. Impaired multimerization was associated with reduced amounts of VWF in late endosomes. Correction of the dominant-negative effect was explored by siRNAs targeting the mRNA breakpoint, which selectively inhibited the in-frame deleted VWF expression. Although the small amount of the deleted protein synthesized after inhibition still exerted dominant, even though weakened, negative effects, the siRNA treatment restored secretion of large multimers with improved function (VWF collagen-binding 28.0% ± 3.3% of wild-type).
AB - Understanding molecular mechanisms in the dominant inheritance of von Willebrand disease would improve our knowledge of pathophysiologic processes underlying its prevalence. Cellular models of severe type 2 von Willebrand disease, caused by a heterozygous deletion in the von Willebrand factor (VWF) gene, were produced to investigate the altered biosynthesis. Coexpression of the wild-type and in-frame deleted (p.P1127_C1948delinsR) VWF forms impaired protein secretion, high molecular weight multimer formation and function (VWF collagen-binding 1.9% ± 0.5% of wild-type), which mimicked the patient's phenotype. mRNA, protein, and cellular studies delineated the highly efficient dominant-negative mechanism, based on the key role of heterodimers as multimer terminators. The altered VWF, synthesized in large amounts with the correctly encoded "cysteine knot" domain, formed heterodimers and heterotetramers with wild-type VWF, in addition to deleted homodimers. Impaired multimerization was associated with reduced amounts of VWF in late endosomes. Correction of the dominant-negative effect was explored by siRNAs targeting the mRNA breakpoint, which selectively inhibited the in-frame deleted VWF expression. Although the small amount of the deleted protein synthesized after inhibition still exerted dominant, even though weakened, negative effects, the siRNA treatment restored secretion of large multimers with improved function (VWF collagen-binding 28.0% ± 3.3% of wild-type).
KW - Cells, Cultured
KW - Endosomes
KW - Genes, Dominant
KW - Heterozygote
KW - Humans
KW - Protein Multimerization
KW - RNA, Small Interfering
KW - Sequence Deletion
KW - von Willebrand Diseases
KW - von Willebrand Factor
KW - Cells, Cultured
KW - Endosomes
KW - Genes, Dominant
KW - Heterozygote
KW - Humans
KW - Protein Multimerization
KW - RNA, Small Interfering
KW - Sequence Deletion
KW - von Willebrand Diseases
KW - von Willebrand Factor
UR - http://hdl.handle.net/10807/32858
U2 - 10.1182/blood-2010-02-268920
DO - 10.1182/blood-2010-02-268920
M3 - Article
SN - 0006-4971
VL - 116
SP - 5371
EP - 5376
JO - Blood
JF - Blood
ER -