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BOR - Papers in Press, published online ahead of print June 7, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.051458
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BIOLOGY OF REPRODUCTION 75, 395–406 (2006)
DOI: 10.1095/biolreprod.106.051458
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

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

Michael H. Melner 2 34 , Arthur L. Haas 7, Jennifer M. Klein 7, Alan R. Brash 5, William E. Boeglin 5, Subir K. NagDas 6, Virginia P. Winfrey 4, and Gary E. Olson 4

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 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 63/4 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 63/4 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.

female reproductive tract, implantation, pregnancy, uterus

INTRODUCTION

Endometrial epithelial cells play critical roles in embryo implantation, and their apical surface mediates the initial contact with and attachment to the embryo [1–6]. Following embryo attachment, the functions and fates of endometrial epithelial cell populations vary, depending on the species as well as their location in the implantation site. In the rabbit, mesometrial luminal epithelial cells of implantation sites undergo dramatic differentiation, and following attachment of the blastocyst, they ultimately fuse with the syncytial trophoblast knobs, forming the invasive cells that penetrate the endometrial stroma [2]. In contrast, luminal epithelial cells on the antimesometrial surface of implantation chambers fuse to form a symplasm that subsequently demonstrates apoptotic characteristics and is sloughed [7]. In studies of the mechanisms controlling these distinct developmental fates, we identified a cohort of endometrial genes that were differentially expressed specifically at implantation sites [8]. Among these implantation site-specific endometrial genes was the putative novel E2 ubiquitin-conjugating enzyme UBE2Q2 (also known as UBCi) [8].

The ubiquitination pathway is known to regulate critical control points in multiple cellular processes by targeting proteins for activation, degradation, or localization at specific intracellular sites. The pathway (reviewed in [9–11]) involves an enzymatic cascade in which the first step activates ubiquitin by forming a high-energy thiolester intermediate to the ubiquitin activating enzyme (E1) that is subsequently transferred to members of an E2 ubiquitin-conjugating enzyme superfamily [12, 13]. The E2 ubiquitin-conjugating enzymes function together with a larger superfamily of E3 ubiquitin ligases to transfer activated ubiquitin to specific cellular proteins. The existence of E2 and E3 superfamilies reflects the specificity of each E2-E3 cognate pair for a specific group of cellular substrates. UBE2Q2 is a potentially unique member of the ubiquitin-conjugating enzyme superfamily that includes at least 17 phenotypically different subfamilies, many of which contain isozymes [14, 15]. Members of the E2/Ubc superfamily share a common 150-amino acid core catalytic domain that contains the conserved cysteine required for E2-ubiquitin thiolester formation and binding site(s) for E1 and its cognate E3 [14]. The E2/Ubc families are distinguished by minor sequence differences within the core domain as well as amino and/or carboxyl terminal extension domains and, less commonly, sequence insertions within surface loops [9, 14]. In contrast, UBE2Q2 with 369 amino acids is over twice the size of the typical E2 core domain [8]. Interestingly, the size difference between UBE2Q2 and the E2 core domain principally resides in a large amino terminal extension that may represent a substrate recognition domain or direct other functional roles for the protein. The sequence homology of UBE2Q2 to bona fide ubiquitin-conjugating enzymes and the presence and spacing of critical, invariant amino acids within the putative catalytic domain suggest that it is either an active conjugating enzyme for ubiquitin or another type I ubiquitin-like protein, such as GIP2 (ISG15), NEDD8, or SUMO, since sequence and structural features distinguishing these parallel ligation cascades are poorly understood at present [16]. Indeed, a preliminary report of the recent crystal structure for human UBE2Q2 (Protein Data Bank entry 1ZUO) conforms approximately to the conserved E2 fold, except for marked divergence in the carboxyl terminal helix-loop-helix motif of the domain [17–20].

Recent evidence indicates significant changes in the expression of members of the ubiquitin and ubiquitin-related protein pathways during implantation and early pregnancy in the endometrium of several species [21–27]. In studies of implantation-induced changes in endometrial gene expression in the rabbit, UBE2Q2, as well as the oncogenic Tre2 ubiquitin-specific protease, was identified [8]. In addition, the ubiquitin-like protein GIP2 (ISG15) is upregulated in the mouse and sheep uterus in response to the implanting conceptus [28–30]. Given the fundamental roles of these enzyme pathways in regulating specific cellular processes, we have further characterized UBE2Q2. In the present study, we examined the expression and localization of rabbit endometrial UBE2Q2 protein in the peri-implantation period. In addition, in vitro kinetic studies with specific components of the ubiquitination pathway were undertaken to define the catalytic specificity of the UBE2Q2 protein.

MATERIALS AND METHODS

Animals

Timed pregnant and estrous New Zealand White rabbits [31] were obtained from Myrtle's Rabbitry (Franklin, TN). The animals were housed and treated at the Vanderbilt University Medical Center Animal Care Center (Nashville, TN) in accordance with National Institutes of Health and U.S. Department of Agriculture standards. Animals were killed with sodium pentobarbital, and the uterus was immediately removed. Pregnant uteri from triplicate Day 8 pregnant animals were subdivided into implant and interimplant segments. Uteri from triplicate estrous stage animals were also collected. Uterine segments either were prepared for immunohistochemistry (see below) or were slit along the mesometrial axis, to expose the mucosa, and the mesometrial and antimesometrial endometrium was dissected from the underlying myometrium and frozen on dry ice for SDS-PAGE (see below).

Expression and Purification of UBE2Q2 Protein

Full-length rabbit and mouse UBE2Q2 cDNAs were subcloned into the pET3a vector with N-terminal 6-His tags. The inserts were sequenced to exclude cloning artifacts prior to expression. The UBE2Q2 proteins were expressed at 37°C in Escherichia coli BL21 cells by isopropylthiogalactoside induction [32, 33]. His-tagged UBE2Q2 was purified by affinity chromatography with Ni-NTA agarose columns (Qiagen, Foster City, CA). An additional step of anion exchange chromatography on a Pharmacia Mono-Q column (Pharmacia, Uppsala, Sweden) was used to purify the protein to apparent homogeneity [34]. By these methods, 10 mg of UBE2Q2 was prepared for antibody production and enzymology studies.

Preparation of UBE2Q2 Antibodies

Antibodies were prepared by standard procedures [35]. Male guinea pigs were immunized with recombinant UBE2Q2 emulsified with Freund adjuvant. Animals received a primary injection and then two booster injections at 3-wk intervals; each injection contained 200 µg of UBE2Q2. Three weeks following the final injection, animals were anesthetized with sodium pentobarbital, and blood was collected by cardiac puncture. A serum fraction was collected, and anti-UBE2Q2 antibodies were purified by antigen affinity with a column of recombinant UBE2Q2 covalently linked to Amino-Link Plus coupling gel (Pierce, Rockford, IL). Specifically bound antibodies were eluted with 0.1 M glycine-HCl, pH 2.5, and the eluate was immediately neutralized with 1 M Tris-HCl, pH 8, and then dialyzed against Tris-buffered saline (TBS; 150 mM NaCl and 20 mM Tris-HCl [pH 8.0]).

Immunohistochemistry

Uterine segments either were immersed in optimal cutting temperature compound (Fisher Scientific, Atlanta GA) and frozen in liquid nitrogen for cryosectioning or were fixed in 4% formaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, for paraffin embedding. Cryosections were fixed for 15–30 min at 4°C with 4% formaldehyde and 0.1 M sodium phosphate buffer, pH 7.4, and then rinsed in PBS. Paraffin sections were dewaxed with xylene, rehydrated in an ethanol series, and subjected to antigen retrieval by incubation at 95°C for 30 min in 25 mM Tris-HCl, pH 9.0, 5 mM EDTA, 0.1% dithiothreitol (DTT), and 0.1% Triton X-100. Sections were next incubated in TBS-T (TBS containing 0.05% Tween 20) and blocked in TBS-T containing 5% normal goat serum and 2.5% BSA. Sections were then incubated in primary antibody diluted in blocking solution: these included affinity-purified anti-UBE2Q2, monoclonal mouse anti-ubiquitin (cat. no. SC 8017; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or, for controls, identical levels of nonimmune guinea pig or mouse immunoglobulin G (IgG). After several buffer washes, sections were incubated in a secondary antibody of affinity-purified, CY3-conjugated, goat anti-guinea pig IgG or peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA) diluted in blocking solution containing Hoechst 33258 (1 µg/ml; Molecular Probes Inc., Eugene, OR). Enzymatic detection of peroxidase-conjugated secondary antibodies utilized color development with diaminobenzidine and H2O2.

SDS-PAGE and Western Blotting

Frozen endometrial samples were weighed and lysed at 95°C for 5 min in SDS sample buffer (50 mg/ml) under reducing conditions. Identical volumes of the lysates were then fractionated on 7%–15% polyacrylamide gradient gels [36]. Protein loads were determined by the method of Bradford [37]. Polypeptides were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes [38] for immunoblot analysis. Immunoblots were blocked with TBS-Tween (150 mM NaCl, 20 mM Tris-HCl [pH 7.5], and 0.1% Tween 20) containing 1% BSA and then incubated with immune or nonimmune IgG diluted in TBS-Tween containing 1% BSA. After three washes in TBS-Tween, the blots were incubated in an affinity-purified peroxidase-conjugated secondary antibody diluted in TBS-Tween containing 5% nonfat dry milk; following several TBS-Tween washes, immunoreactive bands were identified by chemiluminescence detection with SuperSignal (Pierce) and Kodak BioMax film (Kodak, Rochester, NY).

In Vitro Kinetic Assay of UBE2Q2-125I-Ubiquitin Thiolester Formation

Activity of recombinant UBE2Q2 was determined at 37°C by the E1-dependent stoichiometric formation of UBE2Q2-125I-ubiquitin thiolester in end-point assays of 25-µl final volume containing 50 mM Tris-HCl (pH 7.5), 2 mM ATP, 10 mM MgCl2, 1 mM DTT, 5 µM 125I-ubiquitin (specific radioactivity = 9200 cpm/pmol), 10 mM creatine phosphate, creatine phosphokinase (1 IU/ml), 20 nM human erythrocyte E1, and the indicated amount of UBE2Q2 protein [39]. The kinetics of E1-UBE2Q2 transthiolation were determined from initial rate studies under similar conditions in 1-min assays containing 0.3 nM human erythrocyte E1 and the indicated concentrations of active recombinant UBE2Q2 protein. Kinetic constants were determined from nonlinear regression analysis of the resulting UBE2Q2 concentration dependence data [39]. Human erythrocyte E1, radioiodinated ubiquitin, and recombinant human Ubc2b were prepared as described previously [39].

RESULTS

Relationship of UBE2Q2 to the E2 Ubiquitin Conjugating Enzyme Superfamily

UBE2Q2 was previously designated a ubiquitin-conjugating enzyme on the basis of its sequence homology and the presence of critical amino acids in the putative catalytic domain. However, some proteins that are assigned to the E2 superfamily by sequence homology transfer other small ubiquitin-like proteins, such as SUMO or GIP2 (ISG15), to protein substrates. Therefore, it is necessary both to evaluate the homology of any new putative ubiquitin-conjugating enzyme to the E2 superfamily and to experimentally determine its ability to accept ubiquitin thiolester from the E1 ternary complex to form the corresponding E2-ubiquitin thiolester. The known E2 enzymes and sequences from databases with homology to E2 enzymes have detected over 120 E2 sequences that segregate into distinct families (Fig. 1). The primary structural determinants of the family have been discussed previously [8, 15]. These include the presence of the catalytic site cysteine with spatially conserved upstream prolines and downstream tryptophan. While UBE2Q2 contains these spatially conserved residues of the catalytic domain, its unique aspect is its size in relation to most members of the superfamily, 369 amino acids compared to approximately 200. This size difference is in an extended amino terminus.


Figure 1
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  		FIG. 1. Dendrogram of the reported E2 sequences compared by the clustal method with a PAM250 residue weight table. Families were named for the corresponding S. cerevisiae member, except where no yeast ortholog has been identified. Sequences are noted with GenBank acquisition numbers and are preceded by a two-letter genus/species abbreviation. *Asterisk denotes a database ambiguity—UBC10 and UBC11 are both listed as UBE2D4

A dendrogram constructed from the homologous E2 sequences is shown in Figure 1. The nomenclature follows that of the original Saccharomyces cerevisiae member, except where no yeast ortholog has been identified [15]. As shown in Figure 1, UBE2Q2 is within the UBC2-like clade and is most closely related to the UBC2 (UBE2B) family of E2 enzymes. Also evident from the dendrogram is the presence of UBE2Q2 isoforms in a broad range of species from the closely related rabbit, human, and mouse isoforms to the more distantly related rat, chicken, and Xenopus isoforms. UBE2Q2 isoforms are also identified in Caenorhabditis elegans and Drosophila.

In Vitro E1-Dependent UBE2Q2-Ubiquitin Thiolester Formation

The initial step in ubiquitin conjugation requires the ATP-coupled E1-dependent activation of the ubiquitin carboxyl terminus to form an E1 ternary complex and the subsequent transfer of the E1-ubiquitin thiolester high-energy intermediate to form an E2/Ubc thiolester in a process termed transthiolation [39]. Analogous steps are presumably required in the proposed role of UBE2Q2 as a ubiquitin-conjugating enzyme, given its sequence conservation [8]. To test the predicted biological function of UBE2Q2 as a ubiquitin-conjugating enzyme, an in vitro assay with recombinant UBE2Q2, purified human E1, 125I-ubiquitin, and an ATP-regenerating system demonstrated a time-dependent formation of UBE2Q2-125I-ubiquitin thiolester formation, as detected by nonreducing SDS-PAGE (Fig. 2A). At approximately equimolar human E1 and recombinant UBE2Q2, the corresponding UBE2Q2-125I-ubiquitin thiolester formation was complete within 1 min at 37°C, as was also found for the formation of the analogous intermediate with recombinant UBE2B, the human ortholog of S. cerevisiae Rad6 [39] (Fig. 2A, left panel). Quantitation of the 125I-associated radioactivity within the thiolester band provided stoichiometric quantitation of active recombinant UBE2Q2 protein [12], which represented 18% of the total UBE2Q2 protein determined spectrophotometrically with a theoretical 280-nm extinction coefficient of 0.77 (mg/ml)–1. That the radiolabeled adduct bands in Figure 2A (left panel) represent thiolesters is demonstrated by the lability of the Ubc2b- and UBE2Q2-125I-ubiquitin thiolesters to a brief incubation at 100°C in the presence of 0.1% (v/v) ß-mercaptoethanol [12, 13] (Fig. 2A, right panel). At a lower E1 concentration, the kinetics of E1-UBCi transthiolation were quantitated by examining initial rates for UBE2Q2-125I-ubiquitin thiolester formation at the indicated concentrations of UBE2Q2 [39] (Fig. 2B). Under the E1-limiting conditions and saturating concentrations of ATP and 125I-ubiquitin cosubstrates, the initial velocity for UBE2Q2 thiolester formation was hyperbolic with respect to UBE2Q2 concentration, as demonstrated by the linearity of the corresponding double-reciprocal plot (Fig. 2B). Nonlinear regression analysis was used to fit the data to a hyperbolic dependence from which the values of Km = 814 ± 77 nM for the UBCi binding to the E1 ternary complex and of kcat = 1.0 ± 0.1 s–1 (defined as Vmax/[E1]0) for the rate-limiting transfer of E1-ubiquitin thiolester to UBE2Q2 within the corresponding Michaelis complex were determined. These data demonstrate an E1-dependent UBE2Q2-ubiquitin thiolester formation, indicating that UBE2Q2 is a bona fide functional member of the E2 ubiquitin-conjugating enzyme superfamily.


Figure 2
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  		FIG. 2. E1-catalyzed ubiquitin thiolester formation with recombinant UBE2Q2. End-point thiolester assays were conducted as described in Materials and Methods in the presence of 20 nM active human erythrocyte E1 and either recombinant human Ubc2b (0.4 µg/ml) or recombinant rabbit UBE2Q2 (2.8 µg/ml) (A). After a 1-min incubation at 37°C, the incubations were quenched by the addition of SDS sample buffer, from which ß-mercaptoethanol had been omitted, and were resolved by a nonreducing 10% SDS-PAGE [65] (left frame of A). Another aliquot of each reaction was adjusted to 1% (v/v) ß-mercaptoethanol and boiled for 1 min before being resolved in parallel by reducing 10% SDS-PAGE (right frame of A). Mobilities for free 125I-ubiquitin and its thiolesters to E1, UBE2Q2, and Ubc2b are shown to the right of panel A. Mobilities of molecular mass markers are shown to the left of A. The kinetics for UBE2Q2-125I-ubiquitin formation were determined under identical conditions, except that the E1 concentration was decreased to 0.3 nM to allow measurement of initial rates [39]. The dependence of initial velocity for transthiolation on functional UBE2Q2 concentration (determined by end-point thiolester formation) is shown in the double-reciprocal plot of panel B

Endometrial UBE2Q2 Expression

The examination of UBE2Q2 protein expression required the preparation of specific, high-titer antisera. The rabbit UBE2Q2 protein was expressed with a 6-His tag in prokaryotic cells and was purified with sequential nickel affinity and Mono-Q columns. Antisera were prepared against the expressed protein in guinea pigs by standard techniques, and the resulting antisera were purified with a UBE2Q2 affinity column. The affinity-purified antibodies recognized prokaryotic-expressed rabbit and mouse UBE2Q2 (Fig. 3). The antibodies also recognized the UBE2Q2 46-kDa protein in endometrial lysates from Day 8 pregnant rabbit implantation chambers and displayed no detectable immunoreactivity with other polypeptides (Fig. 3).


Figure 3
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  		FIG. 3. Purified recombinant rabbit UBE2Q2 and mouse UBE2Q2 (5 ng of protein per lane) and 5 µl of endometrial lysate of rabbit Day 8 pregnant implantation chamber (d8I). Fifty micrograms of tissue per microliter was fractionated by SDS-PAGE and transferred to PVDF. Immunoblots were probed with affinity-purified anti-UBE2Q2, and bands were detected by enhanced chemiluminescence

To analyze implantation-dependent UBE2Q2 expression, endometrial lysates from estrous and from Days 63/4 and 8 pregnant rabbits were fractionated by SDS-PAGE and transferred to PVDF membranes for immunoblot analyses. UBE2Q2 was not detected in estrous endometrium, in implant and nonimplant site Day 63/4 pregnant endometrium, or in Day 8 pregnant nonimplant site endometrium (Fig. 4). However, UBE2Q2 expression was apparent in Day 8 pregnant implant site endometrium; interestingly, at Day 8, higher UBE2Q2 expression was detected in antimesometrial than in mesometrial endometrium (Fig. 4). Parallel blot lanes immunostained with nonimmune guinea pig IgG, instead of anti-UBE2Q2, showed no immunoreactive bands (Fig. 4). These data demonstrate an upregulated implantation-dependent UBE2Q2 expression pattern in the rabbit endometrium.


Figure 4
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  		FIG. 4. Immunoblots probed with anti-UBE2Q2 (upper panel) or, as a control, nonimmune guinea pig IgG (lower panel) and detection by enhanced chemiluminescence. Shows 5 ng of recombinant rabbit UBE2Q2 and identical quantities (5 µl of 50 µg tissue per microliter) of endometrial lysates from estrus and Days 63/4 and 8 endometrial lysates. NI, Nonimplant; I-M, implant mesometrial; I-AM, implant antimesometrial

UBE2Q2 Localization During Implantation

Sections of uteri from estrous and Days 63/4 and 8 pregnant rabbits were immunostained with anti-UBE2Q2 and, for controls, with nonimmune guinea pig IgG. UBE2Q2 expression was not detectable in either estrous (Fig. 5, A and B) or Day 63/4 pregnant uteri (data not shown). Day 8 implantation chambers showed strong UBE2Q2 expression in the endometrial luminal epithelium. Mesometrial placental folds displayed specific UBE2Q2 expression in the luminal epithelium (Fig. 5C); however, the overall staining was less intense and more variable than that detected antimesometrially (Fig. 5D). Most cells displayed cytoplasmic UBE2Q2 localization, but in cells with low expression, UBE2Q2 appeared to be concentrated at the cell margins. Antimesometrially, the endometrial symplasm, which is destined to undergo cell death and sloughing, exhibited strong cytoplasmic UBE2Q2 expression (Fig. 5D). Staining diminished in the endometrial glands, and no staining was detectable in the stroma, the myometrium, or the implanting embryo. The luminal epithelium of the lateral walls of the implantation chamber also exhibited prominent UBE2Q2 expression, and its expression was greatly diminished in the glandular epithelium (Fig. 6A). Control sections of Day 8 implantation site endometrium stained with nonimmune guinea pig IgG were null (Fig. 6, B and C). While the Day 8 implantation site luminal epithelium displayed prominent UBE2Q expression, no staining was detectable in the adjacent interimplantation site endometrium (Fig. 6, D and E). These data demonstrate that UBE2Q2 exhibits a luminal epithelium-specific expression pattern and is present in both the mesometrial and antimesometrial aspects of implantation sites.


Figure 5
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  		FIG. 5. Phase-contrast (A) and fluorescence (B) images of estrous uterus immunostained with anti-UBE2Q2. No staining was detected in the luminal epithelium (LE). St, Stroma. C, D) Combined phase-contrast and fluorescence images showing the immunohistochemical localization of UBE2Q2 in the mesometrial (C) and antimesometrial (D) aspects of a Day 8 implantation chamber. C) In the mesometrial mucosal folds, UBE2Q2 staining (red color) is restricted to the luminal epithelial cells (LE) and that no specific staining is detected in the stroma (St). Note also that the luminal epithelial cells display variable staining intensity for UBE2Q2. N, Nuclei stained with Hoechst. D) In the antimesometrial endometrium, UBE2Q2 is highly expressed in the symplasm (LE) formed by the fusion of luminal epithelial cells, but it is not detectable in other endometrial cells, including the glandular epithelium (GE), stroma (St), and myometrium (MM), or in the embryo (Emb). N, Hoechst stained nuclei. Data are representative of triplicate animals and triplicate experiments


Figure 6
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  		FIG. 6. Immunohistochemical localization of UBE2Q in Day 8 pregnant implantation chambers and interimplantation sites. A) Combined fluorescence and differential interference contrast image showing UBE2Q2 localization in the lateral wall of the implantation chamber. Note the intense expression of UBE2Q2 in the endometrial luminal epithelium (LE) of the mucosal folds and the diminished UBE2Q2 expression in the glandular epithelium (GE). No staining is detected in the endometrial stroma (St). The nuclei (N) of luminal epithelial cells appear as negative images surrounded by the intense cytoplasmic UBE2Q2 staining. B, C) Matched differential interference contrast (DIC) (B) and fluorescence (C) images of Day 8 pregnant implantation site immunostained with nonimmune guinea pig IgG as the primary antibody. In control specimens, no fluorescence is detectable in the luminal (LE) or glandular (GE) epithelium. D, E) Matched DIC (D) and fluorescence (E) images of interimplantation region of a Day 8 pregnant uterus immunostained with anti-UBE2Q2. No staining of the luminal epithelium (LE) is detectable. Data are representative of triplicate animals and triplicate experiments

Upregulated Ubiquitin Expression in Implantation Site Endometrium

Since UBE2Q2 is highly upregulated during implantation, we predicted that there would be parallel changes in ubiquitin and/or ubiquitinated protein levels in the endometrium. To test this idea, we used immunohistochemistry to examine ubiquitin expression in peri-implantation stage uteri. The ubiquitin immunoreactivity in the Day 63/4 implantation site endometrium was low to undetectable (Fig. 7A), but at Day 8, increased staining was detected in the luminal epithelium (Fig. 7, B and C). This demonstrates that the pattern of ubiquitin immunoreactivity mirrored that of UBE2Q2 in Day 8 pregnant endometrium. These data indicate an increase in the content of ubiquitin and/or stably ubiquitinated proteins in the endometrial luminal epithelium during implantation.


Figure 7
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  		FIG. 7. Immunoperoxidase localization of ubiquitin in implantation site endometrium at Day 63/4 (A) and Day 8 (B, C) of pregnancy. A) At Day 63/4 of pregnancy, no ubiquitin immunoreactivity was detected in the luminal epithelium (LE) or stroma (St) of the mesometrial mucosal folds. L, Uterine lumen. B) At Day 8 of pregnancy, the luminal epithelium (LE) of the mesometrial mucosal folds shows detectable ubiquitin immunoreactivity, but the stroma (St) is devoid of detectable staining. C) At Day 8 of pregnancy, the luminal epithelium (LE) of the lateral wall of the implantation chamber displays intense ubiquitin immunoreactivity with diminished staining noted in the glandular epithelium (GE). The stroma (St) displays no detectable ubiquitin staining. L, Uterine lumen. Data are representative of triplicate animals and triplicate experiments

DISCUSSION

The present functional data demonstrate that UBE2Q2 (also known as UBCi) represents a novel, previously unidentified family of ubiquitin-conjugating enzymes. The E2 proteins functionally link the half-reactions of ATP-coupled, E1-catalyzed activation of ubiquitin and other type 1 ubiquitin-like polypeptides to the subsequent E3-catalyzed ligation to specific target proteins [9, 15]. Members of this superfamily are readily recognized by their characteristic 150-amino acid core catalytic domain, which harbors a highly conserved bipartite E2 sequence motif [15]. Because the parallel, but distinct, ligation pathways for ubiquitin and other type 1 ubiquitin-like proteins arose by divergent evolution, their respective E2/Ubc paralogs share marked sequence homology. Presently, however, it is not possible to distinguish between cognate versus noncognate E2 species within a given conjugation pathway given our rudimentary knowledge of the determinants within their primary sequences that define such specificity. Nonetheless, with the exception of UbcH8, which has been suggested to function in both the ubiquitin and GIP2 (ISG15) pathways [40, 41], other pathways exhibit absolute specificity for their respective cognate E2 paralogs.

Given the marked specificity of the parallel ligation pathways for their cognate ubiquitin-like proteins, the ability of the human Uba1 ternary complex to catalyze the formation of a 125I-ubiquitin-UBE2Q2 thiolester provides the first empirical evidence that UBE2Q2 is a bona fide ubiquitin-conjugating enzyme (Fig. 2A), previously inferred only from its sequence similarity to the E2 superfamily [8]. Kinetic studies summarized in Figure 2B demonstrate that the affinity of the E1 ternary complex for UBE2Q2 (Km = 817 ± 77 nM) is in the range of that determined previously for the binding of human UBE2B (Km = 123 ± 19 nM) and that for the binding of the heterodimeric AppBp1-Uba3 Nedd8 activating enzyme to its cognate Ubc12 (UBE2M) (Km = 43 ± 13 nM) ubiquitin carrier protein [39, 42]. Therefore, the binding interactions between UBE2Q2 and Uba1 appear to be largely conserved within the larger superfamily of E2 paralogs. The kcat derived from the maximal velocity of E1-catalyzed transthiolation reflects the structure and reactivity of the transition-state complex formed between the E1 ternary complex and the E2 during ubiquitin transfer [39]. The good correspondence between the kcat for Uba1-catalyzed transthiolation of 1.0 ± 0.1 s–1 for UBE2Q2 (Fig. 2B) versus 4.5 ± 0.3 s–1 for HsUbc2b (UBE2B) and the AppBp1-Uba3-catalyzed transthiolation of HsUbc12 (UBE2M) with the Nedd8 of 3.5 ± 0.2 s–1 [42] suggests a similarly conserved geometry for the E1-E2 transition state. The good correspondence between the kinetics of transthiolation of UBCi by the Uba1 ternary complex and those of either HsUbc2b or HsUbc12 largely precludes the formation of the 125I-ubiquitin-UBE2Q2 thiolester as arising by noncatalytic transfer from the highly reactive E1-ubiquitin intermediates.

Most E2 families are conserved across all eukaryotes, indicating an early divergence into the present forms [15]. However, some families, such as UBE2S and UbcH8, are found only in higher eukaryotes, indicating that these E2 families diverged to satisfy new functions not required in lower eukaryotes [15]. The human UBE2Q2 sequence (NM_173469) was used to search the genome for additional paralogs in order to determine the ubiquity of the UBE2Q2 orthologs (Fig. 8). This search identified 10 highly conserved paralogs among vertebrates and invertebrates; however, no orthologs were found in plants or yeast. This suggests that the UBE2Q2 family is more recently diverged than most of the E2 families but not as recent as the UBE2S and UbcH8 families. Moreover, the orthologs identified segregated into a rational rooted phylogeny (Fig. 8). Interestingly, the UBE2Q2 orthologs were most conserved within the carboxyl terminal E2 domain and least conserved within the amino terminal domain presumably required for substrate protein targeting or additional functions (Fig. 8).


Figure 8
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  		FIG. 8. Amino acid sequence comparisons of UBE2Q2 orthologs between species. The official database names of the genes are indicated in parentheses

The 10 UBE2Q2 orthologs were then subjected to a comprehensive sequence analysis against 125 known E2 sequences by the Clustal W method [15]. This analysis demonstrated that the UBE2Q2 family belongs to the Rad6-like clade of the E2 superfamily and is most similar in sequence to members of the Ubc2/Rad6 family (data not shown). Alignment of the human UBE2Q2 sequence with the other human E2 paralogs reveals considerable sequence conservation within the characteristic core catalytic domain (Fig. 9). The sequence around the catalytic cysteine is relatively well conserved, as is found with all E2 families [15], except that the HPN tripeptide found within one of the bipartite E2 motifs (red bar) is absent in human UBE2Q2 (Fig. 9) and its paralogs (Fig. 8). The absence of the HPN tripeptide probably accounts for the UBE2Q2 not being recognized as an E2 paralog in earlier sequence searches. The side chain of the asparagine occupying the third position of the E2 HPN motif functions as a catalytic group to stabilize the incipient oxyanion transition state during isopeptide bond formation but serves no role in E1-dependent thiolester formation [43]. Absence of the paralogous asparagine (Fig. 9) suggests that another group assumes this function within UBE2Q2. In addition, UBE2Q2 does not contain a well-conserved Y/F-X6-Y/F motif that constitutes the second half of the E2 bipartite motif [15]. The latter motif forms a buried aromatic-aromatic diad that is suggested to stabilize the carboxyl terminal helix-loop-helix of the core catalytic domain [15]. The first of these aromatic residues forms a buried histidine-aromatic diad with the histidine residue present within the HPN tripeptide. Notably, both the histidine of the HPN tripeptide and the first aromatic of the second half of the bipartite motif are absent in UBE2Q2. The presence of these differences in a functional UBE2Q2 indicates a new understanding of the basis of catalytic domains within the family of active ubiquitin-conjugating enzymes.


Figure 9
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  		FIG. 9. Amino acid sequence comparisons of the human UBE2Q2 ortholog with human E2 paralogs

Our ex vivo data demonstrate the cell-specific expression of the UBE2Q2 protein during implantation. The highly specific expression of UBE2Q2 protein in luminal, but not in glandular, epithelial cells of implantation sites highlights the functional difference of these two cell populations. Developmentally, uterine glands form from invaginations of the luminal epithelium [44]. The two cell populations differ in the expression of genes, with the luminal epithelial cells interacting directly with the trophoblast and the glandular epithelial cells expressing secretory proteins for conceptus recognition, survival, and development. During implantation, luminal epithelial cells also undergo progressive differentiation with reorganization of the cytoskeletal networks as invasion of the trophoblast occurs [4, 45–47]. Some of the peri-implantation cytoskeletal changes have been shown to involve an upregulated expression of cytokeratin 13, specifically in the luminal epithelial cells of both rabbits and humans [47], as well as changes in the localization and expression of other intermediate filament proteins [46, 48, 49]. Interestingly, the cytokeratin 13 is localized to the apical region of these cells that is immediately adjacent to the cell surface interacting with the embryo. The apical surface of these cells changes dramatically in the peri-implantation period [3–6, 46, 50], and these changes could be essential for the progression of implantation. In addition, there is a reduced expression of desmoplakin and a reduction in desmosomes, facilitating the penetration of the epithelium by the trophoblast [51]. The expression of UBE2Q2 in the antimesometrial epithelial cells is complex, as these cells have a function and fate that are different from the mesometrial cells. However, it is conceivable that the antimesometrial epithelial cells express a unique set of cellular protein substrates for UBE2Q2 that direct different cellular functions. Future experiments will need to compare the actions of UBE2Q2 in these two locations for potential differences. The involvement of UBE2Q2 and the ubiquitination pathway in the reorganization of luminal epithelial cells by targeting proteins for specific regions of the cells, activating specific proteins, or targeting proteins for turnover is of importance in understanding the cellular changes at this critical time during implantation.

The potential involvement of UBE2Q2 in the reorganization of the epithelial cytoskeleton is highlighted by the recent finding of human UBE2Q2 as an overexpressed gene in hypopharyngeal tumors and its protein as an interacting target for multiple cytoskeletal proteins [52]. The potential link between UBE2Q2 and the cytoskeleton provides a conceptual framework for testing hypotheses of ubiquitinated proteins and changing epithelial cell functions.

Collectively, the data indicate that activities of ubiquitin and ubiquitin-related pathways in the endometrium are significantly increased during implantation and early pregnancy. In the present study, epithelial immunoreactivity for ubiquitin was undetectable prior to implantation but became strongly positive in Day 8 pregnant rabbit endometrium, primarily in the same luminal epithelial cells where UBE2Q2 was detected. Similarly, the ubiquitin homolog GIP2 (ISG15) was detected during implantation and early pregnancy in the bovine endometrium, although mostly in cell populations different from UBE2Q2, the glandular epithelial cells, and the stromal cells [29]. It is not surprising that, during times of tissue and cellular restructuring and differentiation that altered protein functions, targeting, and turnover would be increased. Increases in ubiquitin conjugated pools have been previously observed during differentiation in tissues undergoing pronounced remodeling [53, 54]. In addition, the ubiquitin and ubiquitin-related pathways have been shown to be involved in the activation of key signaling proteins, such as the IkB kinase [55, 56]; the turnover of other essential signaling proteins, such as cell cycle control proteins and transcription factors [57–63]; and the intracellular targeting of proteins [64]. The specific protein substrates of UBE2Q2 will therefore be important to identify, since the control of these proteins and their targeting could define the cellular changes critical for endometrial epithelial cell functions in implantation.

FOOTNOTES

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. Back

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

Received: 9 February 2006.

First decision: 6 March 2006.

Accepted: 4 June 2006.

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