Identification of molecules involved in the 'early pregnancy factor' phenomenon.
An isolated preparation from ovine placental extracts which was active in the rosette inhibition assay mimicking the activity of the so-called 'early pregnancy factor' (EPF) has been shown to contain a 12 kDa polypeptide which could be partially resolved from low-molecular-weight active moieties. N-terminal amino acid sequence analysis of the polypeptide indicated that it was ovine thioredoxin, an identification confirmed by isolation and complete sequence analysis of the corresponding cDNA. The cDNA for human thioredoxin was expressed in Escherichia coli and the recombinant protein isolated and purified. Pure recombinant thioredoxin alone did not induce the expression of increased rosette inhibition titres (RITs) when tested in the rosette inhibition assay; but, when tested in combination with cell stimuli such as platelet-activating factor (PAF) or serum, it allowed the expression of increased RITs where none was achieved in its absence. Thioredoxin acted in the assay to reverse a refractory state normally induced by these stimuli, allowing lipoxygenase-dependent moieties also induced by the stimuli to exert their effects, resulting in the expression of increased RITs. Antibodies to recombinant thioredoxin removed from pregnancy sera the capacity to induce increased RITs, i.e. to express EPF activity, thus establishing a role for thioredoxin or thioredoxin-like proteins and associated molecules in the mechanisms which allow pregnancy sera to induce increased RITs. Based on a consideration of these and other results, a new model for the study of the EPF phenomenon is presented and discussed.
Clarke
F M
FM
Division of Science and Technology, Griffith University, Brisbane, Queensland, Australia.
Orozco
C
C
Perkins
A V
AV
Cock
I
I
Tonissen
K F
KF
Robins
A J
AJ
Wells
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D018835:Chaperonin 10; D015342:DNA Probes; D004270:DNA, Circular / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006801:Humans; D007166:Immunosuppressive Agents / Q000737:chemistry*; D051379:Mice; D010455:Peptides / Q000737:chemistry*; D010972:Platelet Activating Factor / Q000378:metabolism; D011257:Pregnancy Proteins; D012397:Rosette Formation; D012756:Sheep / Q000378:metabolism; D013491:Suppressor Factors, Immunologic; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D018835:Chaperonin 10; D015342:DNA Probes; D004270:DNA, Circular; D007166:Immunosuppressive Agents; D010455:Peptides; D010972:Platelet Activating Factor; D011257:Pregnancy Proteins; D013491:Suppressor Factors, Immunologic; C025124:early pregnancy factor; D013879:Thioredoxins