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Mechanisms of the interaction between two ADAMTS13 gene mutations leading to severe deficiency of enzymatic activity
Author(s) -
Peyvandi Flora,
Lavoretano Silvia,
Palla Roberta,
Valsecchi Carla,
Merati Giuliana,
De Cristofaro Raimondo,
Rossi Edoardo,
Mannuccio Mannucci Pier
Publication year - 2006
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.20267
Subject(s) - adamts13 , missense mutation , endoplasmic reticulum , biology , mutant , mutation , protease , microbiology and biotechnology , secretion , unfolded protein response , compound heterozygosity , mutant protein , von willebrand factor , enzyme , gene , genetics , biochemistry , immunology , platelet
Abstract The inherited deficiency of the von Willebrand factor‐cleaving protease ADAMTS13 is associated with rare forms of thrombotic thrombocytopenic purpura (TTP). We investigated a woman with a family history of chronic recurrent TTP and undetectable plasma levels of ADAMTS13 activity. Genetic analysis revealed two missense mutations in the heterozygous state: p.Val88Met substitution in the metalloprotease domain and p.Gly1239Val substitution in the first CUB domain of ADAMTS13. To explore the mechanism of ADAMTS13 deficiency in this patient, the wild type (WT; ADAMT13 WT ) and each mutant construct (ADAMTS13 Val88Met , ADAMTS13 Gly1239Val ) were transiently expressed in HEK 293 and COS‐7 cells. To recapitulate the compound heterozygous state of the patient, both mutant ADAMTS13 proteins were also expressed together. The p.Val88Met mutation led to a defect of secretion of the protease associated with a reduction of enzymatic activity, the p.Gly1239Val mutation led to a secretion defect causing intracellular accumulation of the protease. The mechanistic effects of the mutations were further explored by means of differential immunofluorescence, that demonstrated an homogeneous distribution of ADAMTS13 WT in the Cis‐Golgi and endoplasmic reticulum (ER) compartments, a reduction of ADAMTS13 Val88Met in both compartments, while ADAMTS13 Gly1239Val failed to reach the Cis‐Golgi compartment and remained in the ER. Hum Mutat 27(4), 330–336, 2006. © 2006 Wiley‐Liss, Inc.