The D173G mutation in ADAMTS-13 causes a severe form of congenital thrombotic thrombocytopenic purpura. A clinical, biochemical and in silico study.
Congenital thrombotic thrombocytopenic purpura (TTP) is a rare form of thrombotic microangiopathy, inherited with autosomal recessive mode as a dysfunction or severe deficiency of ADAMTS-13 (A Disintegrin And Metalloprotease with ThromboSpondin 1 repeats Nr. 13), caused by mutations in the ADAMTS-13 gene. About 100 mutations of the ADAMTS-13 gene were identified so far, although only a few characterised by in vitro expression studies. A new Asp to Gly homozygous mutation at position 173 of ADAMTS-13 sequence was identified in a family of Romanian origin, with some members affected by clinical signs of TTP. In two male sons, this mutation caused a severe (< 3%) deficiency of ADAMTS-13 activity and antigen level, associated with periodic thrombocytopenia, haemolytic anaemia and mild mental confusion. Both parents, who are cousins, showed the same mutation in heterozygous form. Expression studies of the mutant ADAMTS-13, performed in HEK293 cells, showed a severe decrease of the enzyme's activity and secretion, although the protease was detected inside the cells. Molecular dynamics found that in the D173G mutant the interface area between the metalloprotease domain and the disintegrin-like domain significantly decreases during the simulations, while the proline-rich 20 residues linker region (LR, 285-304) between them undergoes extensive conformational changes. Inter-domain contacts are also significantly less conserved in the mutant compared to the wild-type. Both a decrease of the inter-domain contacts along with a substantial conformational rearrangement of LR interfere with the proper maturation and folding of the mutant ADAMTS-13, thus impairing its secretion.
Stefano
S
Lancellotti
Flora
F
Peyvandi
Maria Teresa
MT
Pagliari
Andrea
A
Cairo
Safwat
S
Abdel-Azeim
Edrisse
E
Chermak
Ilaria
I
Lazzareschi
Stefano
S
Mastrangelo
Luigi
L
Cavallo
Romina
R
Oliva
Raimondo De Cristofaro, Hemostasis Research Center - Internal Medicine, Largo F. Vito 1, Roma 00168, Italy, Tel.: +39 06 30154438, Fax: +39 06 30155915, E-mail: rdecristofaro@rm.unicatt.it.
Raimondo
R
De Cristofaro
D051722:ADAM Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000071120:ADAMTS13 Protein; D000293:Adolescent; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003241:Consanguinity; D004252:DNA Mutational Analysis; D005260:Female; D005819:Genetic Markers; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D040941:Heredity; D006801:Humans; D008297:Male; D008875:Middle Aged; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D010375:Pedigree; D010641:Phenotype; D011487:Protein Conformation; D011697:Purpura, Thrombotic Thrombocytopenic / Q000097:blood / Q000175:diagnosis / Q000201:enzymology / Q000235:genetics*; D013329:Structure-Activity Relationship; D014162:Transfection; D055815:Young Adult; D014841:von Willebrand Factor / Q000378:metabolism
D005819:Genetic Markers; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human
ADAMTS-13; Congenital thrombotic thrombocytopenic purpura; von Willebrand factor