We investigated an Iranian patient with history of recurrent thrombotic thrombocytopenic purpura (TTP) and low plasma levels of ADAMTS13 activity (2.3%). Genetic analysis revealed a 6 bp homozygous deletion at nucleotides 2930-2935(GTGCCC), in exon 23 of ADAMTS13 gene, leading to replacement of Cys977 by a Trp and the deletion of Ala978-Arg979 residues, in the TSP1-6 repeat domain. To explore the mechanism of ADAMTS13 deficiency, HEK293 and COS-7 cells were transiently transfected using wild type (ADAMT13WT) and mutant (ADAMTS13del6bp) expression vectors. The enzymatic activity of the rADAMTS13WT and mutant proteins, evaluated by quantitative immunoblotting assay (Furlan, 1998) showed 100% and ~10%, respectively. Western blot analysis of the conditioned media and lysate showed a band of ~190 KDa in the medium, corresponding to rADAMT13WT protein; however a fainter band roughly estimated to be 5% of the WT was obtained for mutant protein. Therefore, pulse-chase labelling experiments were performed to evaluate the secretion pathway alteration. After 60’ pulse with [35S] methionine, the maximum level of rADAMT13WT in conditioned media was found at 24 hours, but a very weak band of rADAMTS13del6bp was present after 3 hours only and no band being detected after 7 hours of chase. Differential immunofluorescence studies in WT and mutant transfected cells showed that rADAMTS13WT was mostly localized in the perinuclear area, whereas rADAMTS13del6bp showed less intense staining diffusely throughout the cytoplasm, only a minimal amount of the mutant protein being localized in the Cis-Golgi and ER. This study suggests that the residue Cys977 could be involved in the formation of disulphide bonds responsible for a correct folding of one of the TSP1-like domains of ADAMTS13 and the deletion mutation could lead to uncorrected folding process and an intracellular degradation. This condition causes a secretion defect of the mutant protease reflecting the severe ADAMTS13 deficiency in the patient’s plasma

The first deletion mutation in the TSP1-6 repeat domain of ADAMTS13 leads to a secretion defect / S. Lavoretano, R. Palla, I. Garagiola, C. Valsecchi, R. Lombardi, M.T. Canciani, F. Peyvandi. - In: HAEMATOLOGICA. - ISSN 0390-6078. - 91:Suppl. 2(2006 Sep), pp. 38-39. ((Intervento presentato al 19. convegno Congress of the Società Italiana per lo studio dell’Emostasi e della Trombosi tenutosi a Milano nel 2006.

The first deletion mutation in the TSP1-6 repeat domain of ADAMTS13 leads to a secretion defect

S. Lavoretano
Primo
;
R. Palla
Secondo
;
I. Garagiola;C. Valsecchi;F. Peyvandi
Ultimo
2006-09

Abstract

We investigated an Iranian patient with history of recurrent thrombotic thrombocytopenic purpura (TTP) and low plasma levels of ADAMTS13 activity (2.3%). Genetic analysis revealed a 6 bp homozygous deletion at nucleotides 2930-2935(GTGCCC), in exon 23 of ADAMTS13 gene, leading to replacement of Cys977 by a Trp and the deletion of Ala978-Arg979 residues, in the TSP1-6 repeat domain. To explore the mechanism of ADAMTS13 deficiency, HEK293 and COS-7 cells were transiently transfected using wild type (ADAMT13WT) and mutant (ADAMTS13del6bp) expression vectors. The enzymatic activity of the rADAMTS13WT and mutant proteins, evaluated by quantitative immunoblotting assay (Furlan, 1998) showed 100% and ~10%, respectively. Western blot analysis of the conditioned media and lysate showed a band of ~190 KDa in the medium, corresponding to rADAMT13WT protein; however a fainter band roughly estimated to be 5% of the WT was obtained for mutant protein. Therefore, pulse-chase labelling experiments were performed to evaluate the secretion pathway alteration. After 60’ pulse with [35S] methionine, the maximum level of rADAMT13WT in conditioned media was found at 24 hours, but a very weak band of rADAMTS13del6bp was present after 3 hours only and no band being detected after 7 hours of chase. Differential immunofluorescence studies in WT and mutant transfected cells showed that rADAMTS13WT was mostly localized in the perinuclear area, whereas rADAMTS13del6bp showed less intense staining diffusely throughout the cytoplasm, only a minimal amount of the mutant protein being localized in the Cis-Golgi and ER. This study suggests that the residue Cys977 could be involved in the formation of disulphide bonds responsible for a correct folding of one of the TSP1-like domains of ADAMTS13 and the deletion mutation could lead to uncorrected folding process and an intracellular degradation. This condition causes a secretion defect of the mutant protease reflecting the severe ADAMTS13 deficiency in the patient’s plasma
Settore MED/09 - Medicina Interna
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/143403
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