A Novel Protease Involved in the Pathogenesis of Recurrent Pregnancy Loss

Background: In humans, serine proteases are responsible for various cellular processes including reproduction, immune response, and blood coagulation. In a previous study, a novel protease gene was identified to be more expressed in chorionic villi from the normal group than in those from the recurrent pregnancy loss (RPL) group.

Objecive: The aim of this study is to identify the binding partners of a novel protease we identified and to investigate whether the regulation of a novel protease and its substrates is associated with RPL.

Methods: The full length a novel protease and human cDNA library were used for two-hybrid Matchmaker GAL4 Two-Hybrid System 3. A novel protease gene was transfected into HeLa cells, cellular lysates from these cells were separated on SDS-PAGE, and proteins in these lysates were immunoblotted with antibodies including Flag, Myc, and β-actin. In addition, bioinformatics tools were employed to identify putative substrates of a novel protease.

Results: In a previous study, we identified eight differentially expressed genes in chorionic villi between the normal control group and the RPL group. Of these, a novel protease gene is less expressed in chorionic villi from RPL patients than in those from controls. By performing yeast two hybrid screening, 22 different genes encoding proteins, which interacted with a novel protease, were identified. In addition, we tried to identify the interaction between a novel protease and known substrates through the bioinformatics tools. Immunoprecipitation assay revealed that putative substrates including immunoglobulin transporter (IGT) and inhibitor of apoptosis (IOA) interact with a novel protease. Exogenous and endogenous levels of substrates were decreased by a novel protease in a dose dependent manner.

Conclusions: This study suggests that the interaction between a novel protease and its binding partners may be involved in the pathogenesis of RPL.