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Demonstration of Ubiquitin Thiolester Formation of UBE2Q2 (UBCi), a Novel Ubiquitin-Conjugating Enzyme with Implantation Site-Specific Expression¹

Departments of Obstetrics and Gynecology,³ Cell Biology,⁴ and Pharmacology,⁵ Vanderbilt University School of Medicine, Nashville, Tennessee 37232 Department of Natural Sciences,⁶ Fayetteville State University, Fayetteville, North Carolina 28301 Department of Biochemistry and Molecular Biology,⁷ Louisiana State University School of Medicine, New Orleans, Louisiana 70112

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 mass, 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 dendrogram 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 K_m 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 with purified recombinant UBE2Q2 showed that the protein was undetectable by immunoblot analysis in endometrial lysates from estrous and Day 6 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 implantation sites of Day 6 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.

female reproductive tract, implantation, pregnancy, uterus

¹ Supported through National Institutes of Health (NIH) Cooperative Agreement U54 HD 37321 as part of the Specialized Cooperative Centers Program in Reproductive Research and NIH grants AR 51968 to A.R.B. and GM 34009 to A.L.H.

² Correspondence. FAX: 615 343 8881; mike.melner{at}vanderbilt.edu

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