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Regular Article |
a Department of Anatomy and Reproductive Biology,
b Department of Biochemistry, Medical School, RWTH University of Aachen, D-52057 Aachen, Germany
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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female reproductive tract, implantation, male reproductive tract, progesterone, prostate, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After its original description in the rabbit uterus, UGB was detected in other organs, particularly in epithelia of the respiratory, gastrointestinal, and genitourinary tissues of both genders. In rabbits [21] and later in humans [22], UGB equivalent molecules were described in the pulmonary epithelium and secretion of the lung, where the origin of UGB could be attributed to Clara cells. In order to determine and understand the function of UGB, we considered its structure, how its quantitative appearance varies in organs, that it is conserved in mammalian species, and how the tissue-specific expression of the molecule could be traced. With this aim in mind, we cloned and sequenced the full-length cDNA to encode equine UGB. The nucleotide sequence enabled us to deduce and characterize the primary structure of the protein and, on the basis of crystallographic data from rabbit and human UGB molecules [23, 24], we created a computer-based three-dimensional model of horse UGB.
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MATERIALS AND METHODS |
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Tissues and organs (liver, kidney, heart, thyroid gland, adrenal gland, spleen, pituitary gland, lung, uterus, and prostate) from adult horses were obtained from a local slaughterhouse immediately after the animals were killed. Tissues were cut into small pieces, frozen in liquid N2, and stored until RNA preparation at -70°C. Lung lavage was obtained by flushing the entire organ with 0.9% sodium chloride solution. Samples were frozen in 1-ml aliquots at -70°C until they were analyzed.
Western Blot Analysis
For Western blot analysis we used endometrial biopsies and lung lavage. The protein content of all samples was determined as described by Lowry et al. ([25]; modified with a DC Protein Assay from Bio-Rad, Munich, Germany). The samples were subjected to SDS-PAGE using a 5% stacking gel and a 15% resolving gel under nonreducing conditions. The endometrial biopsies were homogenized and diluted in nonreducing (without mercaptoethanol) or reducing loading buffer (5% [v/v] mercaptoethanol, 30 mM Tris pH 6.8, 1.5 M urea, 7.5% [v/v] glycerol, 0.5% [w/v] SDS, and 0.05% [w/v] bromophenol blue). Lung lavage samples of 70 µg protein were freeze-dried and resolved in loading buffer. The samples were heated at 100°C for 5 min, microcentrifuged, and 30 µg of total protein in 20 µl of loading buffer was loaded onto the gel. After electrophoresis, the gel was blotted onto a polyvinylidene difluoride membrane (Sartorius, Epsom, U.K.) by semidry blotting according to Kyhse-Andersen [26] for 45 min at 900 mA. Following blotting, the membrane was blocked with 5% (w/v) milk powder in PBS-Tween-20 (0.1% w/v) overnight at 4°C. The primary antibody (rabbit anti-human uteroglobin, a donation from Dr. Klug, Marburg, Germany) was diluted 1:1000 in 1% milk powder in PBS and incubated with the blot at room temperature for 1 h. After three washes in PBS/Tween, the second antibody (donkey anti-rabbit labeled with horseradish peroxidase diluted 1:5000 in PBS/Tween; DAKO, Glostrup, Denmark) was added and left at room temperature for 1 h. After further rigorous washing, detection of the second antibody was performed using enhanced chemiluminescence reagents (Amersham, Little Chalfont, U.K.; horseradish peroxidase induced the chemiluminescent reaction) and exposure to x-ray film (X-Omat AR; IBI-Kodak Ltd, Cambridge, U.K.) for 1–3 min.
Preparation and Analysis of RNA
Total RNA was extracted using RNAzol (WAK Chemie, Bad Soden, Germany) according to the method of Chomczynski and Sacchi [27], and according to the manufacturer's directions. RNA concentrations were determined spectrophotometrically by absorption at 260 nm. For Northern blot analysis, RNA (4–8 µg) was separated by electrophoresis using a 1.2% agarose gel containing formaldehyde and ethidium bromide. After electrophoresis, gels were photographed under UV light to estimate the amount of ribosomal 28S and 18S, and the integrity of the RNA. The RNA was transferred by capillary blot to nylon membranes and immobilized by UV radiation. Blots were hybridized using a horse cDNA probe (400 base pairs [bp], a result of the rapid amplification of cDNA ends and polymerase chain reaction; RACE-PCR). The probe was random primed with digoxy dUTP with the digoxigenin DNA labeling kit (Roche, Germany) according to the manufacturer's directions. Hybridization with the random primed labeled probe (10 ng probe/ml hybridization solution) was carried out at 42°C overnight. After stringently washing the blots (0.5x SSC and 0.1% SDS), the specific hybridized probe was detected by an antidigoxigenin antibody conjugated with alkaline phosphatase (Roche, Germany) and the chemiluminescence substrate CSPD (Roche, Germany).
Cloning of cDNA
Reverse transcription and cDNA synthesis of total RNA from horse lung and endometrial samples was performed using the Biometra Personal Cycler (Göttingen, Germany). All additives (without the oligo(dT) primer) used for the mixture for reverse transcription (20 µl/tube) were from Roche (Germany). The RNA (1 µg/tube) was heated for 10 min at 65°C and then kept at 4°C for 5 min. After adding the RNA to the reaction mixture (4 µl 5x DNA reaction buffer, 1 mM dNTP mixture, 1.6 µg oligo(dT) primer, 20 units RNase inhibitor, and 20 units of reverse transcriptase) the samples were reverse-transcribed at 37°C for 1 h.
The specific cDNA for horse UGB was cloned using the FirstChoice RLM-RACE kit (Ambion) for 5' RACE-PCR and NotI(dT)18 (Pharmacia) for 3' RACE-PCR. The 3' end of UGB was amplified first by using NotI(dT)18 oligonucleotides and the UGB wooble primer, UG1FW (5'-ACCATGAAG(AC)TCGCCATCAC-3'). UG1FW resulted from the mostly conserved nucleotide sequences that resulted from an alignment with the already described UGB cDNA from other species. The reaction was carried out in a final volume of 50 µl of the PCR reaction buffer (1.5 mM MgCl2, 0.2 mM dNTP mixture, 0.2 pmol UG1FW primer, 2 pmol NotI-d(T)18 primer, 0.5 units Taq polymerase, and 2 µl of cDNA). Thermocycling was performed in 35 cycles, with 30 sec at 94°C, 45 sec at 50°C, 60 sec at 72°C, and a final extension of 3 min at 72°C. Ten microliters of each PCR product was subjected to electrophoresis on a 1.2% agarose gel and stained with ethidium bromide. After electrophoresis the gel was placed on a UV transilluminator and photographed. The amplified DNA band of approximately 400 bp was cut out and the DNA recovered. The DNA was ligated to the plasmid pGEM-T easy (Promega) and propagated into E. coli DH5
. Plasmid DNA was prepared and the DNA insert was sequenced (SeqLab, Göttingen, Germany). From the obtained sequence we made use of the last 21 nucleotides for a specific reverse primer that we used in the 5' RACE-PCR protocol according to the manufacturer's instructions. The PCR product was subjected to electrophoresis and the same procedure was created as described before, with the 3' RACE-PCR product (cloning and sequencing). The first amino acids of mature equine UGB were verified by Edman degradation (Knauer protein sequencer).
Sequence Data Analysis
The cDNA and the deduced amino acid sequence of horse UGB was compared with those of other species using the ClustalW computer program. The phylogenetic tree was created with the JalView computer program. The computer design of the tertiary structure of UGB protein was composed using the coordinates published by Umland et al. [24] on the human UGB molecule.
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RESULTS |
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Earlier studies on UGB in the horse supported the present research goal of analyzing the tissue-specific expression of UGB. Northern blot analysis of RNAs isolated from horse liver, kidney, heart, thyroid gland, adrenal gland, spleen, pituitary gland, lung, uterus, and prostate indicated that its highest expression was in lung tissue, followed by uterus, with weak expression in the prostate. UGB mRNA was not detected in other organs. Figure 1 demonstrates the remarkable expression in lung and uterus, whereas kidney and liver did not express detectable levels of UGB mRNA.
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Western Blot Analysis
Western blot analysis (Fig. 2) indicates that horse UGB is a dimer (16 kDa) just as in other mammalian species. The monomer form (8 kDa) was detected in homogenized endometrial biopsies diluted in a reducing loading buffer (with mercaptoethanol) before SDS-PAGE electrophoresis. To conserve the natural dimer form of UGB, the same homogenate was diluted in a nonreducing loading buffer (without mercaptoethanol) before SDS-PAGE electrophoresis.
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Full-Length cDNA Cloning of Equine UGB
One goal of this investigation was to use equine UGB to construct a computer-based alignment with the nucleotide sequences of the UGB cDNAs from other species. The cDNA alignment showed that the ATG start codon and a few following nucleotides are very well conserved among species. A synthetic nucleotide with this conserved sequence (UG1FW) and an oligo(dT) primer was used in a 3' RACE-PCR with cDNA from equine lung and uterus. The product resulted in a band of approximately 400 bp. Cloning and sequencing of this product indicated that an almost full-length cDNA for UGB had been isolated. To obtain the 5' end of the cDNA we used a specific reverse primer from the 3' end in a 5' RACE-PCR protocol. The product resulted in a band of approximately 500 bp. Cloning and sequencing of this product indicated that the full-length cDNA (submitted to GenBank, accession number AF372660) for equine UGB had been isolated (Fig. 3).
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Amino Acid Sequence of Equine UGB
The cloned cDNA for equine UGB codes for a protein of 91 amino acids. The first 21 amino acids correspond to the signal peptide and the last 70 amino acids to the mature secretory peptide. The site of rupture of the signal peptide was deduced by comparing it with the typical features of UGB from other species and by amino acid sequencing the first four amino acids of the mature equine protein (Fig. 4). Because the first four sequenced amino acids (glycine, isoleucine, cysteine, and glutamic acid) are identical to the deduced first amino acids of the mature protein as coded from the cDNA, we confirmed the cutting site of the signal peptide and, at the same time, the correctness of our deduced amino acid sequence derived from the cloned full-length cDNA.
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Amino Acid Sequence Homology Study
The amino acid sequence of horse UGB shows homology to UGB from pigs (60%), monkeys (57%), humans (53%), rabbits (51%), hares (49%), rats (45%), mice (41%), and hamsters (41%). The amino acid alignment of the protein (Fig. 4) and the phylogenetic tree (Fig. 5) revealed that the structure of horse UGB is most similar to the families of Artiodactyla (pigs), Primates (humans and macaques) and Lagomorpha (hares and rabbits). But it significantly differs from that of Rodentia (mice, rats, hamsters), in which the signal peptide is shorter and the mature peptide consists of 77 amino acids compared to that of horses and other species, for which the mature protein consists of 70 amino acids.
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With the known peptide sequences of mature UGB we created a phylogenetic tree (Fig. 5) for UGB with the JalView computer program. The Rodentia, Primates, and Lagomorpha were grouped together. Artiodactyla (pigs) and Perissodactyla (horses) could not be grouped together because for each, only one UGB peptide sequence has been described. As shown in Figure 5, primates are more closely related to pigs and horses than to mice or rats.
Completely Conserved Amino Acids Among Species
The alignment of the amino acid sequences indicated that some amino acids are completely conserved in UGB in all species. These completely conserved amino acids were in the following positions in horse UGB: cysteine at 3 and 69; phenylalanine at 6; leucine at 13, 48, 59, and 68; proline at 30; glutamine at 40; lysine at 42 and 58; aspartic acid at 46; isoleucine at 63; and serine at 66. The computer design of the tertiary structure of UGB protein was determined using the crystallographic data of Morize et al. [23] and Umland et al. [24], to demonstrate the equally globular character of horse UGB (Fig. 6). Every completely conserved amino acid (not considering cysteines at positions 3 and 69, which are required for the disulfide bridges to form a dimer) are allocated at the centrally formed pouch. It is interesting that other investigators found that this "pouch" is able to bind progesterone or similar lipophilic molecules [24]. Figure 4 shows these 14 amino acids located mainly between positions 40 and 68. The characteristic cysteine residues at positions 3 and 69 are essential for connecting the two monomeric chains to form the antiparallel chains in dimeric UGB.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression and regulation of UGB in equine endometrium shows remarkable similarities to its well-known production and secretion by rabbit endometrium. No other mammalian species (other than rabbits) are known to express UGB in comparable abundant amounts during progesterone dominance during the ovarian cycle and during early pregnancy [20]. Further striking similarities have been demonstrated before on the experimental findings after progesterone substitution in ovarectomized females [20]. As in rabbits, the endometrial production and release of UGB into the uterine secretion is induced within a few days after initiation of treatment with progesterone or a synthetic progestagen. This striking similarity in UGB expression in the horse, compared to the rabbit, led to our detailed studies on the molecular structure of equine UGB and on its full-length cDNA.
One comparison of the equine UGB amino acid sequence was made with the UGB amino acid sequences from other species. As Figure 4 demonstrates, 14 amino acids of the mature UGB are completely conserved among all 9 species investigated. The cysteines at positions 3 and 69 are completely conserved, because they combine the 2 UGB monomers by their disulfide bridges. Lysine at position 42 has been shown to be extremely important for structural stability, because in silico mutations resulted in a total loss of molecular stability [28].
Most of the other evolutionarily conserved amino acids have been shown to line the inside of a hydrophobic pocket that forms between the two subunits of the dimeric UGB. This hydrophobic pocket can bind several lipophilic molecules [24, 29–31]. The results described here support the hypothesis that UGB serves as a carrier protein, because 85.7% of the conserved amino acids over nine species are located within the hydrophobic pocket. Actually, the first described ligand was progesterone [32], but the biological significance of this binding is not known. After the first description and isolation in rabbit uterus [1–3] it was speculated that UGB might be a carrier or a scavenger of steroids [11]. In vitro experiments demonstrated that threonine 60 and tyrosine 21 are important for binding progesterone to UGB in rabbits, mice, and rats [29, 30]. In humans, pigs, and horses, UGB has no threonine at position 60. In human UGB, a limited binding capacity of progesterone could be shown in vitro, because the tyrosine at position 21 remains [30]. In amino acid sequence of equine UGB, the two amino acids are mutated from tyrosine at position 21 to phenylalanine, and from threonine at position 60 to methionine. This leads us to speculate that equine UGB may not bind progesterone, but may bind other lipophilic molecules.
As shown in Figure 6, the lower segment of the "binding pouch" in UGB is characterized by the phenolic side chains of phenylalanine (position 6 of each subunit). The other conserved amino acids are either strong acidic side chains (aspartic acid at position 46, glutamine at position 40) or nonpolarized side chains, such as leucine (positions 13, 48, 59, and 68) and proline (position 30) or isoleucine (position 63). Serine (position 66) contains an alcoholic hydroxyl side chain, which may be important for certain binding affinities. Lysine (positions 42 and 48) is characterized by its basic amino side chain, perhaps interacting with the acidic side chains at positions 40 and 46. In Table 1 these strong hydrophobic amino acids are shown lining the binding pouch of the dimeric UGB, clearly indicating that the biological ligands of UGB are strongly lipophilic, similar to phospholipids, steroids, and inositols. The six asterisks in Table 1 indicate six completely conserved amino acids among all UGB sequences reported.
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The most stimulating observations were made by Umland et al. [24], who indicated that the two phospholipid molecules, phosphatidylinositol and phosphatidylcholine, appear as ligands in isolated UGB molecules from human lung lavage. It may be scientifically rewarding to follow this lead, because the comparison of "topohydrophobic" positions of strong hydrophobic amino acids in the UGB molecule all line the so-called "binding pouch" of the dimeric UGB [33] (Table 1). It is interesting that UGB binds lipophilic polychlorinated biphenyls (PCBs). After intraperitoneal injection of PCBs, UGB knockout mice did not accumulate PCBs in the lung, compared with high accumulations in wild type controls [34], which emphasizes the function of UGB as a binding and carrier protein. These results are consistent with other reports that UGB is a secretory protein in Clara cells of all species investigated so far [15]. It has been speculated that UGB in the lung may serve as a binding protein for uptake of toxic agents to prevent further pathogenic actions on the organism.
Tissue-specific expression of UGB varies among species. In rabbits [1, 2], humans [35–37], and horses [20] UGB expression has been well-documented in the reproductive organs, particularly the endometrium, whereas in pigs it seems to show no or only extremely weak expression in the uterus [18]. Results of the present study revealed that horse and pig UGB have very similar amino acid sequences, therefore, tissue specificity is not necessarily correlated with the assumed amino acid sequence. Furthermore, the phylogenetic tree of UGB emphasizes that rodents are not the experimental model of the first choice because their amino acid sequence homology is dissimilar to that of humans. In contrast, horses, pigs, and rabbits seem to be more similar to humans and therefore much better experimental models.
One of the challenging research goals is the investigation of binding molecules (for example IGFBP 3 or haptoglobin; [38–40]) that may play a significant role in the events leading to endometrial receptivity and the establishment of pregnancy, and in their specific action within the so-called embryo-maternal dialogue. UGB, recognized now as a certain binding or carrier molecule for highly specific lipophilic ligands, could influence endometrial receptivity and blastocyst implantation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by START: Forschungsschwerpunkt "Molekulare Endokrinologie" (TP 6/2000) from the University of Aachen Medical School, with additional financial support from Schering Aktiengesellschaft, Berlin (Gender Health Care Research). 
2 Correspondence: Henning M. Beier, Department of Anatomy and Reproductive Biology, Medical School RWTH Aachen, Wendlingweg 2, D-52074 Aachen, Germany. FAX: 49 241-8082508; hmbeier{at}ukaachen.de
Accepted: January 8, 2002.
Received: September 24, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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