Demonstration of ubiquitin thiolester formation of UBE2Q2 (UBCi), a novel ubiquitin-conjugating enzyme with implantation site-specific expression.
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.
Melner
Michael H
MH
Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. mike.melner@vanderbilt.edu
Haas
Arthur L
AL
Klein
Jennifer M
JM
Brash
Alan R
AR
Boeglin
William E
WE
Nagdas
Subir K
SK
Winfrey
Virginia P
VP
Olson
Gary E
GE
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*
D013438:Sulfhydryl Compounds; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D044764:Ubiquitin-Activating Enzymes