Protein S secretion differences of missense mutants account for phenotypic heterogeneity.
To elucidate the molecular background for the heterogeneity in protein S plasma concentrations observed in protein S deficient individuals, the in vitro synthesis of recombinant protein S missense mutants was investigated. Six different naturally occurring mutations identified in the protein S gene (PROS1) of thrombosis patients were reproduced in protein S cDNA by site directed mutagenesis. Two mutants, G441C and Y444C (group A), were associated with low total plasma concentration of protein S. Modestly low protein S was found in families with R520G and P626L (group B) mutants. T57S and I518M (group C), which was associated with marginally low protein S, did not segregate with protein S deficiency in the respective families, raising doubts as to whether they were causative mutations or rare neutral variants. The 6 protein S mutants were transiently expressed in COS 1 cells. The Y444C mutant showed the lowest level of secretion (2.5%) followed by the G441C mutant (40%). Group B demonstrated around 50% reduction in secretion, whereas group C mutants showed normal secretion. Pulse-chase experiments demonstrated impaired protein S processing with intracellular degradation and decreased secretion into the culture media of group A and B mutants. Interestingly, there was a good correlation between in vitro secretion and the concentration of free protein S in the plasma of heterozygous carriers. These results demonstrate impaired protein S secretion to be an important mechanism underlying hereditary protein S deficiency and that variations in protein secretion is a major determinant of the phenotypic heterogeneity observed in protein S deficiency. (Blood. 2000;95:173-179)
Espinosa-Parrilla
Y
Y
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, Sweden.
Yamazaki
T
T
Sala
N
N
Dahlbäck
B
B
de Frutos
P G
PG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D005260:Female; D014644:Genetic Variation; D006801:Humans; D008297:Male; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010375:Pedigree; D010641:Phenotype; D017293:Protein S / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018455:Protein S Deficiency / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013030:Spain; D014162:Transfection
D017293:Protein S; D011994:Recombinant Proteins