title: 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 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 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 with purified recombinant UBE2Q2 showed that the protein was undetectable by immunoblot analysis in endometrial lysates from estrous and Day 6(3/4) 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(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.
authors.affiliation: Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. mike.melner@vanderbilt.edu
authors.forename: Michael H
authors.initials: MH
authors.lastname: Melner
authors.forename: Arthur L
authors.initials: AL
authors.lastname: Haas
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Klein
authors.forename: Alan R
authors.initials: AR
authors.lastname: Brash
authors.forename: William E
authors.initials: WE
authors.lastname: Boeglin
authors.forename: Subir K
authors.initials: SK
authors.lastname: Nagdas
authors.forename: Virginia P
authors.initials: VP
authors.lastname: Winfrey
authors.forename: Gary E
authors.initials: GE
authors.lastname: Olson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D010064:Embryo Implantation / Q000502:physiology*; D004717:Endometrium / Q000378:metabolism; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D011817:Rabbits; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: D013438:Sulfhydryl Compounds; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D044764:Ubiquitin-Activating Enzymes