Dissecting The Transmembrane Receptor Siglec-6 And Determining Its Functional Role In Gestational Trophoblastic Disease

Gestational trophoblastic disease (GTD) is a spectrum of pregnancy-related disorders in which placental trophoblasts develop abnormally, leading to molar pregnancies as well as in situ and metastatic cancers.  While molar pregnancies are benign, they carry a risk of developing into malignant and invasive choriocarcinomas (CCAs).  Our lab’s previously published microarray data, determined that sialic acid binding immunoglobulin-like lectin 6 (Siglec-6) is upregulated in preeclampsia, another pregnancy-related disorder thought to be a result of dysregulated trophoblast invasion.  BeWOs (brain metastasis CCA cell line) overexpressing Siglec-6 display an increased invasion and protection from apoptosis.  Siglec-6 has three distinct Ig extracellular domains as well as an intracellular domain with signaling motifs.  We hypothesize that mutagenesis of Siglec-6 will determine the critical domains involved in downstream signaling and alterations in invasion, proliferation and apoptosis.  Using site-directed mutagenesis, we created specific Siglec-6 domain mutations.  Plasmid DNA containing each of the mutants will be transfected into BeWO cells and unique clones will be selected and grown.  Invasion, proliferation and apoptosis assays will be performed.  We have successfully mutated a binding domain mutation, which has been transfected into BeWO cells.  If ligand binding at this domain is necessary, then we predict that invasion and apoptotic protection will be abrogated.  By differentially mutating the other domains, we will be able to determine critical functional domains and their potential cellular functions.  Our findings on the function of Siglec-6 will provide knowledge that will assist in understanding Siglec-6 role in the pathogenesis of GTD and preeclampsia.