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PREGNANCY INDUCES THE UBIQUITIN PARALOG, ISG17, BUT HAS NO EFFECT ON CONJUGATED UBIQUITIN OR UBIQUITIN CONJUGATING ENZYME IN BOVINE UTERINE CROSS-SECTIONS.
Austin, Kathy1, Carr, Amy1, Pru, James1, Belden, E2, Hansen, Thomas1, 1 2
ABSTRACT- A 17-kDa product of an interferon (IFN) stimulated gene, called ISG17, is a ubiquitin paralog that is induced in the bovine endometrium in response to pregnancy (days 15 to 30) and conceptus-derived IFN-tau. ISG17 becomes covalently conjugated to targeted proteins that are distinct from those conjugated to ubiquitin. The goal of the present work was to generate a monoclonal antibody against recombinant (r)ISG17 that could be used to test the hypothesis that ISG17 and its conjugates were induced in uterine cross-sections in response to pregnancy. Also, it was hypothesized that conjugated ubiquitin and a ubiquitin conjugating enzyme required in the N-end rule pathway (E2-14K), the major cellular degradative pathway, would not change in uterine cross-sections in response to pregnancy. Monoclonal antibody (clone 5F10; IgG1) was generated against rISG17 using standard procedures. Polyclonal rabbit antibodies against the E2 and conjugated ubiquitin were provided by A.L. Haas (Medical College of Wisconsin, Milwaukee). Using immunohistochemical procedures, very little staining of ISG17 was found in sections derived from day 17 non-pregnant cows (n = 3 cows). However, light localization of ISG17 was found in the luminal epithelium (LE) and deep stromal (ST) cells and very dense localization was found in the glandular epithelium (GE) as well as the ST immediately beneath the LE from day 17 pregnant cows (n = 3). In contrast, staining of the E2 and conjugated ubiquitin was not affected by pregnancy status and was localized primarily to GE and LE with a more diffuse pattern of staining to ST cells. One-dimensional PAGE and western blot of endometrial extracts confirmed that pregnancy induced (P < 0.05) ISG17 and its conjugates, but had no effect on levels of the E2 enzyme or conjugated ubiquitin. A monoclonal antibody against rISG17 was made and used to show pregnancy-specific induction of ISG17 and its conjugates in uterine cross-sections and western blots. The induction of this ubiquitin paralog by pregnancy is a specific response that was not observed with components of the ubiquitin/proteasome-dependent degradation pathway. NIH HD 32475-07.
KEY WORDS: Bovine, Pregnancy, Uterus, Ubiquitin