The Cloning, Characterization, and Functional Analysis of Murine Pregnancy Specific Glycoproteins
[Technical Report, Doctoral Thesis]
Uniformed Services University of The Health Sciences
Pagination or Media Count:
Pregnancy Specific Glycoproteins PSG are a group of highly conserved placental proteins that are prescot at high concentrations in the serum of pregnant women. As a result, these proteins are postulated to have a critical role in maintaining a successful pregnancy. A murine model was established to examine whether recombinant PSGs regulate the expression of cytokines by macrophages, a cell type known to be prevalent in the uterus during pregnancy. To this end, full length Psg18 and 19 cDNAs were cloned, and the anatomical sites of Psg17, 18,and 19 expression determined. The baculovirus expression system was used to generate recombinant PSG17, 18, 18N, and 19 as fusion proteins. The full length proteins were generated as GST fusion proteins and the PSG18N, a truncated form of PSG18 containing only the N1-domain, was generated as GST and 6x His fusion proteins. Since no currently available antibody shows reactivity with murine PSGs and human anti-PSG antibodies do not demonstrate crossreactivity with the mouse proteins, a polyclonal antibody was generated to PSG18N. To determine the functional role of these proteins, the recombinant murine PSG18 and PSG18N were used to investigate whether PSGs regulate cytokine expression by thioglycollate-stimulated peritoneal macrophages and the murine macrophage cell line RAW 264.7. Both PSG18 and PSG18N were found to induce murine macrophages and RAW 264.7 cells to produce IL-6 and IL-10 mRNA. At the protein level, PSG18N stimulates both cell types to secrete of IL-6 and IL-10 in a dose dependent manner. In addition, the PSG synergized with LPS to induce secretion of IL-6 and IL-10 protein.