Murine pregnancy-specific glycoproteins (PSGs) are encoded by 17 different genes. Different family members have different expression levels at different stages of embryonic development. It is currently unknown whether all members of this family of placentally secreted proteins have the same function and bind to the same receptor. Furthermore, the requirement of post-translational modifications for the activity of these highly glycosylated proteins remains undetermined.
Method of study
Recombinant PSG17 and PSG19 were generated and purified by affinity chromatography. An expression library was screened to identify the receptor for mouse PSG19. Binding to the receptor by proteins generated in different expression systems and mapping of the binding domain were analyzed by pull-down assays. Analysis of the carbohydrate composition of the receptor-binding domain was performed with the DIG glycan differentiation kit.
PSG19 binds to the tetraspanin CD9, specifically to extra cellular loop 2 and can induce secretion of TGFβ1 by a macrophage cell line. The receptor-binding domain of PSG17 and PSG19 is post-translationally modified by the addition of N-linked carbohydrates and, when expressed in CHO cells, terminal sialic acids are detected. PSGs produced in bacteria do not bind CD9.
PSG19, as previously determined for PSG17, binds to the second extracellular loop 2 of the tetraspanin CD9. The first immunoglobulin variable-like domain of PSG19 is sufficient for receptor binding and function. Analysis of receptor usage by the remaining 15 murine PSGs will most likely require that the proteins be generated in eukaryotic expression systems, as we have demonstrated that the addition of carbohydrates is essential for PSG-receptor interaction.