Degradation of two novel congenital TTP ADAMTS13 mutants by the cell proteasome prevents ADAMTS13 secretion.
BACKGROUND
Over 150 mutations have been identified in the ADAMTS13 gene in patients with congenital thrombotic thrombocytopenic purpura (TTP). The majority of these (86%), lead to reduced (<50%) secretion of mutant recombinant ADAMTS13. The mechanism by which this occurs has not been investigated in vitro. Two novel ADAMTS13 mutations (p.I143T and p.Y570C) identified in two congenital adolescence onset TTP patients were studied, to investigate their effects on ADAMTS13 secretion and subcellular localisation.


METHODS
HEK293T cells were transiently transfected with wild type or mutant ADAMTS13 cDNA. Immunofluorescence and confocal microscopy were used to study localisation within the endoplasmic reticulum (ER) and Golgi. The cell proteasome and lysosomes were inhibited in cells stably expressing ADAMTS13 to investigate degradation of ADAMTS13 by either organelle.


RESULTS
Both mutations severely impaired secretion and both mutants localised within the ER and Golgi. Proteasome inhibition led to the intracellular accumulation of both mutants, suggesting proteasome degradation. Lysosome inhibition on the other hand did not lead to increased intracellular accumulation of the mutants.


CONCLUSIONS
Proteasome degradation of these ADAMTS13 mutants contributed to their reduced secretion.
Haemostasis Research Unit, University College London, London, England; Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy. Electronic address: m.underwood@ucl.ac.uk.
Mary
M
Underwood
Haemostasis Research Unit, University College London, London, England; Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy; Angelo Bianchi Bonomi Hemophilia and Thrombosis Centre, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
Flora
F
Peyvandi
Angelo Bianchi Bonomi Hemophilia and Thrombosis Centre, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
Isabella
I
Garagiola
Haemostasis Research Unit, University College London, London, England.
Samuel
S
Machin
Haemostasis Research Unit, University College London, London, England.
Ian
I
Mackie
D000071120:ADAMTS13 Protein / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D017354:Point Mutation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D059748:Proteolysis; D011697:Purpura, Thrombotic Thrombocytopenic / Q000235:genetics* / Q000378:metabolism
D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human; D046988:Proteasome Endopeptidase Complex
ADAMTS13; Congenital; Mutation; Proteasome; Thrombotic thrombocytopenic purpura; Von Willebrand factor-cleaving protease