Cloning, Characterization, and Functional Analysis of Murine Pregnancy Specific Glycoproteins
UNIFORMED SERVICES UNIV OF THE HEALTH SCIENCES BETHESDA MD
Pagination or Media Count:
see report Pregnancy Specific Glycoproteins PSG are a group of highly conserved placental proteins that are present 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 PSGg 17, 18, and 19 expression determined. The baculovirus expression system was used to generate recombinant PSG 17, 18, 18N, and 19 as fusion proteins. The lull 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 crosseactivity with the mouse proteins, a polyclonal antibody was generated to PSG18N. To determine the functional role of these proteins, the recombinant murine PSG18 and PSG 18N 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 lL-6 and IL-l0 mRNA. At the protein level, PSG18N stimulates both cell tepes to secrete of lL-6 and IL-l0 in a dose dependent manner. In addition, the PSG synergized with LPS to induce secretion of 1L-6 and IL-lO protein. The affect of PSG18N treatment on expression of 1L-1beta, TNF-alpha, TGF-beta, iNOS, and IL-12p0 mRNA in RAW 264.7 cell and C3HHeJ macrophages was also examined. My data show that PSG18N did not alter the expression of these immune mediators at the mRNA level.
- Genetic Engineering and Molecular Biology
- Medicine and Medical Research