Pregnancy

Demonstration of Ubiquitin Thiolester Formation of UBE2Q2 (UBCi), a Novel Ubiquitin Conjugating Enzyme with Implantation Site-Specific Expression

Abstract
We recently identified a differentially expressed gene in implantation stage rabbit endometrium encoding a new member of the ubiquitin conjugating enzyme family designated UBE2Q2 (also known as UBCi). Its unusually high molecular weight, novel N-terminus extension, and highly selective pattern of mRNA expression suggest a specific function in implantation. This study analyzes its relationship to the E2 ubiquitin conjugating enzyme superfamily, investigates its enzymatic activity, and examines its localization in implantation site endometrium. Construction of a dendogram indicated that UBE2Q2 is homologous to the UBC2 family of enzymes and isoforms are present in a broad range of species. In vitro enzymatic assays of ubiquitin thiolester formation demonstrated that UBE2Q2 is a functional ubiquitin-conjugating enzyme. The Km for transfer of ubiquitin thiolester from E1 to UBE2Q2 is 817 nM compared to 100 nM for other E2 paralogs; this suggests that the unique amino terminal domain of UBE2Q2 confers specific functional differences. Affinity-purified antibodies prepared using purified recombinant UBE2Q2 showed that the protein was undetectable by immunoblot analysis in endometrial lysates from estrous and day 6¾ pregnant (blastocyst attachment stage) rabbits but was expressed in both mesometrial and antimesometrial implantation site endometrium of day 8 pregnant animals: no expression was detected in adjacent interimplantion sites. Immunohistochemistry demonstrated UBE2Q2 expression exclusively in mesometrial and antimesometrial endometrial luminal epithelial cells of the day 8 implantation chamber. Immunohistochemical localization of ubiquitin mirrored UBE2Q2 expression with low to undetectable levels in implantion sites of day 6-3/4 pregnant endometrium but high levels in luminal epithelial cells of day 8 pregnant endometrium. This implantation site-specific expression of UBE2Q2 in luminal epithelial cells could play major roles in orchestrating differentiation events through the modification of specific protein substrates.

Key words: Female Reproductive Tract • Pregnancy • Implantation • Uterus

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