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BOR - Papers in Press, published online ahead of print September 22, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.033027
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BIOLOGY OF REPRODUCTION 72, 365–372 (2005)
DOI: 10.1095/biolreprod.104.033027
© 2005 by the Society for the Study of Reproduction, Inc.

Isolation and Sequence of an Interferon-{tau}-Inducible, Pregnancy- and Bovine Interferon-Stimulated Gene Product 15 (ISG15)-Specific, Bovine Ubiquitin-Activating E1-Like (UBE1L) Enzyme1

Lea A. Rempel, Brian R. Francis, Kathleen J. Austin, and Thomas R. Hansen2

Department of Animal Science, University of Wyoming, Laramie, Wyoming 82071


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bovine (bov) interferon-stimulated gene product 15 (ISG15) is produced in the endometrium in response to conceptus-secreted interferon (IFN)-{tau}. ISG15 conjugates to endometrial proteins through an enzymatic pathway that is similar to ubiquitinylation. Ubiquitin-activating enzyme 1-like protein (UBE1L) initiates enzymatic conjugation by forming a thioester bond with ISG15, thus preparing it for transfer to the next series of enzymes. The bovUBE1L has not been described. We hypothesized that bovUBE1L was induced by pregnancy and IFN-{tau} in the endometrium. A 110-kDa protein was purified from bovine endometrial (BEND) cells based on affinity with recombinant (r) glutathione S-transferase (GST)-ISG15. This protein was digested in gel with trypsin. Seven peptides were purified using HPLC, sequenced using liquid chromatography-mass spectroscopy-mass spectroscopy and found to share 43–100% identity with human UBE1L. The full-length bovUBE1L cDNA was isolated from a BEND cell cDNA library, sequenced, and found to share 83% identity with human UBE1L cDNA. Northern blot revealed two mRNAs that were detected in greater (P < 0.05) concentrations in endometrium from Day 17–21 pregnant versus nonpregnant cows. Western blots using antihuman UBE1L antibody revealed a similar pattern of pregnancy-associated expression of UBE1L protein in the uterus. The bovUBE1L mRNA was localized, using in situ hybridization, primarily to glandular and luminal epithelium, with more diffuse localization to stroma of the endometrium from pregnant cows. Because bovUBE1L was purified through its interaction with rGST-ISG15 and shares significant amino acid and cDNA sequence identity with human UBE1L, it is concluded that it mediates conjugation of ISG15 to uterine proteins in response to the developing and attaching conceptus.

bovine, cytokines, endometrium, female reproductive tract, implantation, ISG15, pregnancy, UBE1L, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One of the most abundantly expressed proteins in response to interferons (IFN) is IFN-stimulated gene product 15 (ISG15). This response is not only seen during times of antiviral challenge but also during early pregnancy within the uterus of bovine [1–3], ovine [4], porcine [5], murine [6], human and baboon [7] species. ISG15 has two ubiquitin (UB)-like domains and a C-terminal Leu-Arg-Gly-Gly sequence that is identical to that of UB. ISG15 is capable of conjugating to intracellular proteins (ISG15ylation) and this process is thought to occur through an enzymatic pathway that is similar to, yet distinct from, that of ubiquitinylation [8]. Conjugation of ISG15 to a target protein is theorized to alter function. Extended conjugation of proteins to ISG15 has lethal consequences, as evidenced by deletion of UBP43, an ISG15-specific protease [9]. Recent putative proteins that become conjugated to ISG15 include serpins [10] and several signal-transduction proteins [11, 12]. However, targeted uterine proteins still remain elusive; therefore, the true function of ISG15 in the uterus remains to be determined.

The ISG15 conjugation pathway is believed to be similar to that described for UB, relying on a series of sequential events and involving three enzymes: E1, the UB activating enzyme; E2s, the UB conjugating enzymes; and E3s, the UB ligases [13].

When human ubiquitin-activating E1-like (UBE1L) protein was first identified, its function was unknown [14, 15]. Recently, it has been shown that UBE1L is the activating enzyme for huISG15 rather than the UB protein [16]. Human UBE1L has 45% amino acid sequence identity to the human ubiquitin-activating enzyme E1 [14]. A UBE1L for ISG15 has also been identified in the mouse [17]. Comparison of huUBE1L sequence with those for other UBlp E1 enzymes shows that the ATP-binding site and the active site cysteine are conserved [18].

We hypothesized that a UBE1L enzyme would be induced by IFN-{tau} in bovine endometrial (BEND) cells as well as in endometrium of pregnant cows and be involved in conjugation of bovine ISG15 to target proteins. In this study, bovine (bov) UBE1L was purified from BEND cells that were cultured with IFN-{tau} based on interaction affinity with recombinant glutathione S-transferase (GST)-ISG15. Protein sequencing liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) was used to confirm identity. To verify presence of UBE1L in the endometrium during early pregnancy and following culture with IFN-{tau} in BEND cells, Northern and Western blotting techniques were used. In situ hybridization also was employed to localize UBE1L mRNA to cell types within the endometrium.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant GST-Ovine ISG15

Recombinant ovine ISG15 cDNA was generated by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers based on the previously published cDNA sequence [19]. The coding region for ovISG15 cDNA was initially subcloned into pGEX-4T-1 (Amersham Pharmacia Biotech, Piscataway, NJ) using primers containing BamHI and EcoRI restriction sites situated behind a LacI promoter and a thrombin cleavage site for the Escherichia coli-expression system. Recombinant engineered pGEX-4T-1 vector was then transformed into BL21-RIL cells (Stratagene, La Jolla, CA) for protein production.

The coding region for ovISG15 cDNA was then cleaved from the pGEX-4T-1 vector using BamHI and EcoRI restriction enzymes and subcloned into pAcG2T (BD PharMingen, San Diego, CA). This plasmid was used to generate a recombinant baculovirus (AcGST-ovISG15) by the allelic transplacement method [20, 21]. AcGST-ovISG15 was plaque purified twice, amplified in Sf9 cells, and titered by plaque assay. AcGST-ovISG15 became toxic to Sf9 cells over a period of 100 h (Fig. 1); therefore, expression was terminated after 48 h. GST-ovISG15 fusion protein was released from Sf9 cells by lysis as a soluble protein.



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  		FIG. 1. Generation of recombinant ovine ISG15 using a baculovirus expression system. A) Expression of GST-ovISG15 (identified by arrow) in Sf9 cells infected for 48 h at 28°C. GST-ovISG15 was detected by Western blot using polyclonal antibodies against bovISG15. Lanes 1 and 2 show production of GST-ovISG15 from AcGST-ovISG15, clones 1 and 2. No production of GST-ovISG15 was found in Sf9 cells infected with wild-type virus (strain E2; lane 3) or mock infected cells (lane 4). B) Coomassie stain and purification of rovISG15. Lane 1: infected Sf9 cell extract before loading the GSH column; lane 2: proteins that had no affinity for GSH; lane 3: purified rovISG15 (identified by arrow) following cleavage of ISG15 from GST-ISG15 by treatment with thrombin. C) Toxicity of AcGST-ovISG15 infection in Sf9 cells. Numbers of live cells (solid circles) and dead cells (open circles) as a function of time postinfection with AcGST-ovISG15 were plotted. GST-ovISG15 was produced in Sf9 cells infected with AcGST-ovISG15, but slowly became toxic to Sf9 cells. For subsequent experiments, GST-ovISG15 was extracted from Sf9 cells 48 h postinfection or GST-rovISG15 produced in E. coli

Acquisition of Amino Acid Sequence of Bovine Ubiquitin E1-Like Enzyme (UBE1L)

Confluent BEND cells (ATCC, Manassas, VA [22]) were cultured with media (control; n = 8, T75 flasks) or 50 ng/ml rbovIFN-{tau} (n = 8, T75 flasks) for 24 h at 37°C, 5% CO2 to induce components of the ISG15-conjugation enzyme complex. Soluble components of BEND cells were collected by lysis in 1x PBS for interaction with either E. coli or baculovirus-generated GST-recombinant ovine (rov) ISG15 that was attached to glutathione Sepharose columns. UBE1L present in the BEND cell extract was then covalently linked to the GST-rovISG15 in the presence of ATP and Mg2+. A similar method of isolation was previously used to purify the human UBE1L protein and was the basis for our methodology [16]. Briefly, BEND cells were harvested in 1x PBS and lysed via Dounce homogenization and sonication. Insoluble and soluble fractions were separated by high-speed centrifugation. Ten mM Mg2+ and 2 mM ATP were added to soluble fractions (control and IFN-{tau}) before applying to individual glutathione sepharose/GST-rovISG15 columns. Proteins covalently linked to ISG15 via thioester bonds were eluted from the columns using 40 mM dithiothreitol (DTT) in 20 mM Tris-HCl, pH 7.5. Eluted protein products were concentrated (30 000 molecular weight cut-off) and separated by one-dimensional SDS 10% PAGE. Protein bands were visualized by staining with Coomassie blue reagent (Sigma, St. Louis, MO). Partial amino acid sequences of a 110-kDa protein were determined by the University of California–Davis (Dr. Yong Moo Lee) using in-gel trypsin digestion followed by liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS). Seven amino acid profiles were compared with human and mouse UBE1L sequences using BLAST.

Nucleotide Sequence of Bovine UBE1L

Comparison of the bovUBE1L amino acid sequence with known sequences from human and mouse species provided enough data to generate PCR primers. RT-PCR techniques were used in conjunction with designated primers (5' primer 5'-TGCACCCACTATCTGAAGCCACTGCTGGAGGC-3'; 3' primer 5'-GCATACAGGTTGGCAGCTGCCAGTACGTAGAGG-3') and IFN-{tau} treated BEND cell RNA (isolated using Tri-Reagent; Molecular Research Center, Inc., Cincinnati, OH) to amplify segments of bovUBE1L cDNA.

The RT-PCR product was used to screen a Lambda Zap cDNA library [2] that was generated from RNA of IFN-{tau}-treated BEND cells. Briefly, agar plates containing lambda phage were incubated overnight at 37°C. Plaques were then transferred to 0.45µ nitrocellulose membranes (Schleicher and Schuell, Keene, NH). Membranes were probed using [{alpha}-32P(dCTP)]-labeled bovUBE1L RT-PCR product. Autoradiography was used to visualize those clones that bound to the labeled amplicon. Corresponding clones were isolated and replated twice to increase purity of the clone.

Complementary DNA was isolated from purified clones and submitted for sequence analysis (UC Davis DNA Sequence Facility, Davis, CA). Because UBE1L is approximately 3200 base pairs (bp), several submissions were required to obtain the entire sequence. The complete cDNA sequence was compared with sequences available at the National Center for Biotechnology Information (NCBI: Bethesda, MD) to determine percent identity to the E1 enzyme of ubiquitin or other ubiquitin-like proteins.

In Situ Hybridization for Bovine UBE1L

After fixation in 4% paraformaldehyde, cross-sections of uterine horns ipsilateral to the corpus luteum were dehydrated and then embedded in paraffin for sectioning. The in situ hybridization and detection methods were performed using digoxygenin (DIG) reagents from Roche (Indianapolis, IN). DIG-labeled bovUBE1L antisense and sense riboprobes were prepared using 1 µg DNA and T3 or T7 polymerase, respectively. Sections were deparaffinized and rehydrated through a series of washes (2x 5 min each in xylene; 2 min in absolute ETOH, 95% ETOH, 80% ETOH, and 70% ETOH; and 2x 2 min each in 0.3 M NaCl/0.03 M sodium citrate [2x SSC]). To increase RNA exposure, slides were treated with 25 µg/ ml proteinase K for 30 min followed by two washes in 2x SSC for 5 min. To neutralize any positive charges that may interfere with probe penetration, slides were incubated for 10 min in 0.1 M triethanolamine and 0.5% acetic anhydride and washed 2 times in 2x SSC for 5 min.

Sections were prehybridized in buffer (50% formaldehyde, 0.18 M NaCl/10 mM phosphate, pH 7.4/1 mM EDTA [5x SSPE], 20 mM DTT, 1x Denhardt, 20 µg/ml yeast tRNA) for 2 h at 50°C. Slides were then hybridized with 1 µl DIG-labeled probe per 1 ml prehybridization buffer for 15 h at 50°C. Slides were washed 30 min each in 2x SSC at 37°C, 20 µg/ml RNase A in 2x SSC at 37°C, 2x SSC at 37°C, 2x SSC at 50°C, and then 0.5x SSC at 50°C.

To detect DIG labeling, slides were blocked (100 mM Tris-Cl, pH 7.5; 150 mM NaCl; 0.3% tritonX-100; 2% normal sheep serum) for 1 h. Goat anti-DIG antibody (1:100) conjugated to alkaline phosphatase was incubated on slides for 4 h at room temperature. DIG-labeled cells were visualized using NBT/BCIP substrate solution. Positive hybridization signal was purple, and sections were counterstained with nuclear fast red.

Northern Analysis for Bovine UBE1L

Tissues from bovine uterine horns of nonpregnant or pregnant cows (Days 17–21 of estrous cycle and pregnancy and Days 23–50 of pregnancy) were homogenized in Tri Reagent (Molecular Research Center, Inc.). Ten micrograms of total RNA from each sample were loaded per well and separated on a 1.5% denaturing agarose gel. Separated RNA was transferred to nitrocellulose by capillary transfer and baked at 80°C for 2 h. Membranes were prehybridized for 3 h at 42°C in buffer (50% formamide, 5x SSC, 50 mM Na2PO4, 5x Denhardt, 0.1% SDS, 0.1 mg/ml salmon sperm DNA). Blots were then hybridized in the same buffer with radiolabeled probe.

To prepare a radiolabeled probe, a partial bovine UBE1L cDNA (551 bp) was synthesized using RT-PCR and total RNA from IFN-{tau}-treated BEND cells, and then radiolabeled using 50 µCi [{alpha}-32P] dCTP and Klenow in a standard random primer labeling reaction. Northern blots were washed 3x, 5 min each at 42°C (2x SSC/0.1% SDS or 1x SSC/0.1% SDS). Membranes were exposed to film for 48 h at –80°C. Autoradiograms were scanned and quantitated using UNSCANIT (Silk Scientific, Orem, UT).

Western Blot Analysis for Bovine UBE1L

An anti-human UBE1L antibody (provided by A.L. Haas, Medical College of Wisconsin) was evaluated for cross-reactivity and optimal dilution using nontreated (control) or IFN-{tau}-treated BEND cell extracts. Cell extracts from confluent T25 flasks collected in 1 ml of 1x Laemmli buffer [23] were loaded at 10 µl per lane onto one-dimensional-SDS 12.5% PAGE gels, electrophoretically transferred to 0.2 µ nitrocellulose (Schleicher and Schuell, Keene, NH), and Western blot detection was performed using 1:5000 dilution of rabbit polyclonal anti-human UBE1L antibody incubated with gentle rocking at room temperature for 1 h. A secondary antibody against rabbit IgG (1:10 000) conjugated to alkaline phosphatase (Sigma) was incubated with blots for 30 min with gentle rocking at room temperature. To visualize immunoreacting bands, membranes were incubated in NBT/BCIP (Promega Corp, Madison, WI) substrate solution. Differences between control and IFN-{tau}-treated cellular extracts were determined using UNSCANIT.

Uterine tissue lysates from nonpregnant and pregnant cows were prepared by homogenizing 100 mg of endometrium in 1 ml of 1x Laemmli buffer. Lysates (100 µg per lane) were loaded onto one-dimensional-SDS 10% PAGE gels, electrophoretically transferred to 0.2 µ nitrocellulose, and Western blot detection was performed as previously described.

Statistics

To determine differences among pregnancy status (pregnant or nonpregnant) or treatment effect (control or rbovIFN-{tau}), statistical analyses were performed using the General Linear Model of the Statistical Analysis System [24]. The effects of pregnancy status on endometrial concentrations of UBE1L and IFN-{tau} treatment on BEND cell concentrations of UBE1L were analyzed using one-way ANOVA, followed by protected preplanned t-test [24].

Use of Animals

Investigations using vertebrate animals were approved by the University of Wyoming Animal Care and Use Committee (Assurance A-3216-01) and were conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Recombinant GST-ISG15 was successfully produced for the first time herein using a baculoviral expression system (Fig. 1). However, expression of ISG15 in Sf9 cells became lethal after 48 h (Fig. 1). This also occurred in previous experiments, when ISG15 was expressed using a yeast system [25]. Therefore, production of GST-ISG15 in Sf9 insect cells was concluded at 48 h. Binding of a 110-kDa protein to GST-ISG15 required prior treatment of BEND cells with IFN-{tau} (Fig. 2, A and B, lane 2 compared with media-treated cells, lane 1) and was specific to ISG15 because glutathione Sepharose alone failed to retain this protein (Fig. 2A, lane 3).



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  		FIG. 2. Purification of bovine UBE1L. A, B) Representative Coomassie-stained gels of proteins eluted from GSH-agarose bound GST-ISG15 when using baculoviral (A) or E. coli (B) expression systems. Extracts of BEND cells were applied to individually prepared columns. Lane 1: extracts of control/media treated BEND cells; lane 2: extracts of BEND cells treated with rbovIFN-{tau} (50 ng/ml for 24 h); and lane 3 (A only): eluate buffer passed over a GSH-agarose column. Gels were loaded based on equal volumes of DTT-eluted protein. Binding of GST-ISG15 to the 110-kDa UBE1L required prior treatment of cells with IFN-{tau} (lane 2 [A and B], identified by arrows) and specific to ISG15 because GSH alone failed to retain a 110-kDa protein (A, lane 3)

Because the 110-kDa protein was similar in size to that reported for huUBE1L, it was subjected to in-gel trypsin digestion followed by LC-MS-MS. Seven peptide sequences were identified that had homology to human UBE1L peptides (Table 1). Peptides varied in size from 6 to 11 amino acids and exhibited 63–100% identity to human UBE1L (BC006378). These sequences enabled the identification of the 110-kDa protein as bovUBE1L. All sequences were later verified to have 100% identity with amino acid sequences derived from computer translation of the full-length bovine cDNA (data not shown).


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  		TABLE 1. Bovine peptide sequence analysis of the 110-kDa protein using LC-MS-MS

Screening of a BEND cell lambda phage cDNA library with an RT-PCR product yielded 20 potential clones for bovUBE1L. Two clones were completely sequenced to provide the full bovUBE1L cDNA sequence. The bovine cDNA sequence of 3220 bp had 83% identity to human and 73% identity to mouse UBE1L enzymes, and reduced identity to human UB-activating E1 (UBE1) enzyme (60%). The isolated bovine cDNA (AY597816) had stronger sequence identity to UBE1L when compared with UBE1 sequences and was, therefore, the bovine ortholog to UBE1L.

Translation of the nucleotide sequence to the protein sequence revealed 83% and 70% identity of bovUBE1L with the human and murine counterparts, respectively. Comparison of the bovine UBE1L protein sequence to that of human and murine UBE1L, human UBE1, and two UBlp E1s (NEDD8 UBA3 and SUMO subunit 2) revealed highly conserved regions that corresponded to interactive sites of these proteins with the initiating enzyme of their respective conjugation pathways (Fig. 3).



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  		FIG. 3. Translation of the bovUBE1L cDNA sequence and comparison with other UBE1Ls (hu—BC006378; mu—AAG03060), UBE1 (hu—AAA61246), and ubiquitin-like E1 proteins (NEDD8 UBA—NP 003959; SUMO subunit 2—NP 005490). Identity of protein sequences were approximately 83% and 73% for bovine when compared with human and murine UBE1L protein, respectively. Arginine69 (A) (box with solid black line) was conserved among UBE1Ls and UBE1 and has an active role in ATP hydrolysis. Amino acids involved in adenine binding are highlighted with a dashed line (B). Identification of the glycine rich P-loop that binds {alpha}-phosphate of ATP (GXGXXG; (B), solid-black lined box) within the UBE1L protein shows great similarity among various species and also with the human UBE1 protein and ubiquitin-like E1 proteins; NEDD 8 UBA and SUMO subunit 2. Other amino acids involved with ATP binding and/or hydrolysis are indicated by boxes with solid black lines (B). Amino acids that are known to bind to residue 72 of UB or corresponding residues of NEDD8 or SUMO are identified with a box shaded black (B). Comparative amino acids from 635 to 642 to these residues in the UBE1Ls are very different from UB, NEDD8, or SUMO. The active cysteine residue (B, solid grey box) that interacts with ISG15, UB, or UBlps also is conserved. However, the sequence just N-terminal (643–647) to the cysteine residue is dissimilar. Grey outlined boxes (C) indicate residues believed to contact NEDD8 from model building studies [29]. The full-length bovUBE1L sequence is available on Genbank (AY597816)

Northern analysis revealed the presence of two mRNA bands within endometrial tissue from pregnant versus nonpregnant cows (P < 0.05; Fig. 4). These mRNAs were expressed throughout pregnancy until Day 40, when both bands were no longer observed. Detection of 18S rRNA was similar across all samples as evaluated by Northern blot. In situ hybridization using DIG-labeled bovUBE1L amplicon riboprobes localized bovUBE1L expression to the luminal and glandular epithelium and to a lesser extent within stroma of endometrium from pregnant cows (Fig. 5). Endometrial tissue from nonpregnant cows (Days 16.5–20) and sense RNA probed sections showed no expression of UBE1L.



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  		FIG. 4. UBE1L mRNA in endometrial tissues. Northern blot was used to analyze UBE1L mRNA in total endometrial RNA isolated from nonpregnant and pregnant cows. A 551-bp (1,735–2,285 bp) UBE1L antisense probe hybridized to two mRNA bands at approximately 3 kb. These mRNAs were present in pregnant but not in nonpregnant endometrial tissue during early pregnancy (Days 17–21). No differences (P > 0.05) in expression were found between the two mRNAs; therefore, the values were pooled for size of mRNA so that day and pregnancy status could be analyzed. Values represent means ± SEM. * denotes P < 0.05 means are different across pregnancy status within day. NP identifies nonpregnant and PREG identifies pregnant



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  		FIG. 5. Localization of UBE1L mRNA in bovine uterine cross-sections. Bovine UBE1L mRNA was localized to luminal and glandular epithelium and stromal tissue from pregnant cows (Day 17) but was not present in uterine cross-sections from nonpregnant cows (Day 16.5). Sense probe served as a negative control in which no staining was observed in any tissue. L, Luminal epithelium; G, glandular epithelium; and S, stromal tissue. NP identifies nonpregnant and P identifies pregnant. Original magnification x100

Lysates from BEND cells treated with IFN-{tau} had greater (P < 0.05) concentrations of bovUBE1L when compared with control BEND cell lysates (Fig. 6). Bovine UBE1L was present in all endometrial lysates but was greater (P < 0.05) in lysates from pregnant when compared with nonpregnant cows (Fig. 7). Detection of a secondary antibody reactive protein was used to verify loading. No differences were seen among samples (data not shown). Thus, bovUBE1L was present in the endometrium at all times but was upregulated during pregnancy.



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  		FIG. 6. Upregulation of UBE1L in response to culture with interferon-{tau} in BEND cells. Recombinant bovine IFN-tau (50 ng/ml; ~5000 IU/ml) upregulated the production of UBE1L protein (identified by arrow) when compared with BEND cells treated with media only (control). Values represent means ± SEM; * denotes P < 0.05



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  		FIG. 7. Expression of UBE1L in endometrial extracts during the estrous cycle and pregnancy. Endometrial protein lysates from nonpregnant and pregnant cows were analyzed by Western blot using an anti-huUBE1L polyclonal antibody. Bovine UBE1L (identified by arrow) was present in all samples, but was greater in endometrium from pregnant when compared with nonpregnant cows on Days 18, 19, and 21. Values represent means ± SEM; * denotes P < 0.05. NP identifies nonpregnant and PREG identifies pregnant


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
ISG15 becomes conjugated to numerous target proteins in response to IFNs [8, 26] through an enzymatic pathway that is believed to resemble that of UB. The initiating enzyme for this pathway, UBE1L, has been identified, and putative E2s, 1–8U and Leu13, have been proposed [27]. At the present time, ligating enzymes (E3s) have not been identified. Bovine UBE1L is a 110-kDa protein that was isolated from BEND cells incubated with IFN-{tau} or IFN-{alpha} but not in untreated or IFN-{gamma}-treated cells (data not shown). The cDNA for bovUBE1L codes for amino acid sequences that correspond to peptide sequences obtained from the 110-kDa protein by LC-MS-MS. The bovUBE1L amino acid sequence has high overall identity with human and murine UBE1L (83% and 73%, respectively) and less identity with the E1s for UB or other UBlps.

E1s appear to have evolved from homodimeric bacterial enzymes such as MoeB and ThiF that adenylate MoaD and ThiS proteins, respectively (homologs of UB and UBlps [28]). NEDD8 and SUMO E1s are heterodimers, whereas the E1s for UB and ISG15 have evolved into monomeric proteins that retain domains that are similar to those found in NEDD8 and SUMO E1 heterodimers. The subunits of the NEDD8 and SUMO heterodimers are structurally related, as are the two halves of UBE1 and ISG15 UBE1L. Our results indicate that bovUBE1L has sequence data related to a variant of the Rossman fold structure, with an ATP binding site and an adenylation site that is similar to UBE1. In the structure of the NEDD8 E1 heterodimer ATP binding, hydrolysis and adenylation are catalyzed by one subunit (UBA3), but Arg15 from the other subunit (APPBP1) is required for the {gamma}-ATP salt bridge to form [29, 30]. Similarly, a conserved Arg residue is located in both UBE1 and bovUBE1L near the N-terminal portion with adenylation that is catalyzed by the C-terminal half of the protein.

Presence of multiple mRNA bands on Northern blots was interpreted to mean that bovine UBE1L may be encoded by multiple genes or is differentially processed from a single gene. The human UBE1L gene, first reported by Kok et al. [15], was absent in certain lung cancer cell lines. The human UBE1L gene is distributed over 8.75 kilobases (kb) (NM_003335) and is capable of producing 10 unique transcripts by alternative splicing (NCBI/IEB/Research/ Acembly). The various transcripts encode nine distinct protein isoforms, seven of which contain the UBA/THIF-type binding fold motif. The E1s for UB (UBE1C), NEDD8 (APPBP1 subunit), and SUMO-1 (UBA2 subunit) also contain this binding fold motif. Based on human UBE1L gene data, we suspect that bovine UBE1L may produce multiple transcripts from a single gene and each isoform may determine specificity to ISG15. Further research is warranted.

Recombinant ISG15 was synthesized for the first time using a baculovirus expression system. Over-expression of GST-ISG15 in SF9 cells became lethal over time in culture. Insects do not have an ISG15 ortholog. Thus, the lethality of over-expression of ISG15 may have been due to interference of GST-ISG15 with ubiquitin pathways that are critical for survival in insect cells. Regardless, GST-ISG15 generated in baculovirus and in E. coli expression systems was able to interact with and pull down the first enzyme, UBE1L, in the ISG15ylation pathway. The 110-kDa protein that was purified in this manner was confirmed to be bovine UBE1L through sequencing seven internal peptides that shared 63–100% identity with human UBE1L. The full-length cDNA and inferred amino acid sequence of bovine UBE1L are also reported for the first time (AY597816).

Because ISG15 is synthesized and becomes conjugated to target proteins in endometrial tissue in a temporal manner during early pregnancy [31] and in response to the presence of an embryo [3, 26], it was reasoned that the enzymes involved in ISG15 conjugation would be expressed in a similar manner. UBE1L mRNA and protein were found to be upregulated in endometrium from pregnant when compared with nonpregnant cows. It is evident that bovine UBE1L is expressed in the uterus in response to IFN-{tau} and early pregnancy. UBE1L mRNA was localized within the luminal and glandular epithelium and more diffusely within the stroma in endometrial sections from pregnant cows only, which is similar to the pattern of ISG15 localization (glandular and stromal tissues) during early pregnancy in the cow. Thus, the timing and localization of UBE1L expression in uterine tissues corresponds to expression of ISG15, which has also been previously demonstrated for 1-8U and Leu13, putative E2 enzymes [27]. The fact that ISG15 and conjugation of ISG15 to target proteins is increased during early pregnancy or stimulation by type I IFNs [3, 4, 6, 7, 26] suggests that the conjugation pathway plays a significant role during this period of time.

Induction of ISG15 in the uterus during pregnancy in humans, mice, and cows indicates that some functions of ISG15 are universal across species. ISG15 also is induced by type-1 IFN and mediates antiviral responses. The gene for UBE1L has ISREs, or IFN-stimulated response elements [15]. The influenza B virus protein NS1B inhibits UBE1L and, consequently, conjugation of ISG15 to proteins. Yuan and Krug [16] proposed a function for ISG15 in controlling defense against virus. The UBE1L, UB processing protease (UBP43), and ISG15 genes are inducible by IFN and viral stimulation. Because these genes are coordinately induced, we hypothesize that conjugation of ISG15 is dynamic and needs to be balanced to maintain homeostasis. For example, over-expression of UBP43 in M1 monoblast cells inhibits terminal differentiation into macrophages [32]. This could be caused by differentiation or modulation of cell-cycle proteins/genes. UBE1L was absent in 14 different lung cancer cell lines [33], suggesting that a suppression of ISG15ylation may contribute to carcinogenesis. Some viruses specifically inhibit conjugation or synthesis of ISG15 [16]. This may be a protective mechanism to inhibit host-cell suicide and inflammatory response. In addition to antiproliferative actions of IFN, these observations support the hypothesis that differentiation of cells and suppression of growth might be consequences of ISG15ylation. Recently, it was determined that the ISG15 conjugation pathway shares a major E2 ligase enzyme, UbcH8, with the ubiquitin pathway [34]. This means that the conjugating pathways might overlap in context of some targeted proteins. So in addition to functioning as a pregnancy-associated response to the embryo, the ISG15 system may also play a role in other important physiological processes. As the actual functions of ISG15 are deciphered, it will be possible to determine the importance of the initiating enzyme, UBE1L, for embryonic viability, placentation, and implantation.


   ACKNOWLEDGMENTS
 
This research contributed to the 2003 Society for the Study of Reproduction Trainee Research Award to L.A.R. (First Place in Platform Presentations), that was sponsored by Serono Reproductive Biology Institute, Inc. (Rockland, MA). We thank Don Pratt and Don Jarvis in the Department of Molecular Biology at the University of Wyoming for assistance with generating recombinant ISG15 using the baculovirus system (NIH COBRE award to the University of Wyoming RR15640). Recombinant bovIFN-{tau} was kindly donated by Dr. R.M. Roberts (University of Missouri). Anti-huUBE1L polyclonal antibody was generously provided by Dr. A.L. Haas and Dr. K. Suresh (Medical College of Wisconsin).


   FOOTNOTES
 
1 Supported by grants from the National Institutes of Health (NIHCHDR01-32475 and NIHBRIN/INBRE 1P20RR16474-01). Back

2 Correspondence: Thomas R. Hansen, Animal Science, Department 3684, 1000 E. University Ave., Laramie, WY 82071. FAX: 307 766 2533; thansen{at}uwyo.edu Back

Received: 10 June 2004.

First decision: 29 June 2004.

Accepted: 10 September 2004.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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