Site-directed mutagenesis of human thioredoxin. Identification of cysteine 74 as critical to its function in the "early pregnancy factor" system.
Thioredoxin has been identified as a key component of the "early pregnancy factor" system, a system of components present in pregnancy sera which expresses a lymphocyte modifying activity in an assay known as the rosette inhibition assay. Although thioredoxin alone is inactive, addition of thioredoxin to lymphocytes in combination with nonpregnancy sera or platelet-activating factor results in a positive response. We have changed several amino acids of human thioredoxin by site-directed mutagenesis to investigate the residues required for this cooperative function. Conversion of the two active site residues (cysteines 32 and 35) to serines results in a protein devoid of classical redox activity; however, this protein retained its ability to cooperate with non-pregnancy sera or platelet-activating factor in the rosette inhibition assay. Vertebrate thioredoxins contain an additional conserved pair of cysteine residues in the C-terminal portion of the protein. Changing both to serines resulted in no change in redox activity but completely abolished function in the rosette inhibition assay. Further study revealed this function was solely dependent on cysteine 74 as conversion of only cysteine 74 to serine abolished function, whereas replacement of only cysteine 70 with serine had no effect. The nonfunctional mutants counteracted the action of pregnancy serum in the assay strongly supporting the hypothesis that thioredoxin is an integral part of the early pregnancy factor system with residue cysteine 74 having an important role.
Tonissen
K
K
Department of Biochemistry, University of Adelaide, Australia.
Wells
J
J
Cock
I
I
Perkins
A
A
Orozco
C
C
Clarke
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003545:Cysteine; D018076:DNA, Complementary / Q000378:metabolism; D005260:Female; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000187:drug effects / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011247:Pregnancy / Q000097:blood / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D012397:Rosette Formation; D013154:Spleen / Q000187:drug effects / Q000276:immunology; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013879:Thioredoxins; D003545:Cysteine