Binding of progesterone to the proteins of the uterine luminal fluid. Identification of uteroglobin as the binding protein.

1. The uterine luminal fluid of rabbits treated with estradiol and progesterone contains a protein factor with high affinity for [3-H] progesterone which is not present in the uterine secretion of control rabbits treated with estradiol. 2. This progesterone dependent factor is shown by gel filtration and polyacrylamide gel electrophoresis to be identical with the uterus specific protein uteroglobin, which seems to be required during the preimplantation phase. Uteroglobin specific antiserum, prepared in guinea pigs, completely inhibits the progesterone binding activity of the proteins of the uterine fluid. 3. Progesterone binding to uteroglobin is dependent upon millimolar concentrations of dithioerythritol. At saturation, one molecule of progesterone binds per uteroglobin molecule and the apparent association constant is 2 x 10-6 M-1 at 0 degrees C. 4. The progesterone binding species of uteroglobin exhibits a molecular weight of around 12 000 on polyacrylamide gels containing dodecylsulfate, and of 15 000 upon gel filtration, indicating a non-globular shape. This molecule is compased of two subunits of similar molecular size which are held together by a disulfide bridge among other forces.     

Isolation and characterization of uteroglobin from the lung of the hare (Lepus capensis)

Uteroglobin has been purified from hare lung by gel filtration and chromatography on carboxymethyl-cellulose. Hare uteroglobin appears homogeneous by electrophoresis under both denaturing and nondenaturing conditions. Its chemical and immunological properties as well as its ability to bind progesterone are compared to those of rabbit uteroglobin. The two proteins have the same N-terminal residue (glycine) and both lack tryptophan but differ in amino acid composition. Sodium dodecyl sulfate-gel electrophoresis shows that hare uteroglobin is composed of two subunits of identical Mr (about 7000) held together by disulfide bridges. The amino acid composition indicates a subunit composed of 65-67 residues, which is compatible with the apparent Mr observed. Thus, hare uteroglobin appears to be slightly smaller than the rabbit protein. Hare uteroglobin partially reacts with anti-rabbit uteroglobin in a radioimmunoassay and also binds progesterone, although this binding is relatively unaffected by dithiothreitol. The synthesis of hare uteroglobin in the uterus appears to be rather insensitive to ovarian steroid hormones.     

Uterine and lung uteroglobins in the rabbit: Two similar proteins with differential hormonal regulation

Previous studies have shown that several rabbit tissues contain proteins which cross-react in the radioimmunoassay for uteroglobin, a progestin-regulated protein in rabbit uterus (Torkkeli et al. (1977) Mol. Cell. Endocrinol. 9, 101–118). In the present study, a uteroglobin-like protein was purified to an apparent homogeneity from an extra-uterine tissue, rabbit lung, by successive chromatographies on hydroxyapatite, Sephadex G-75, SP-Sephadex, DEAE-cellulose and CM-cellulose. The final preparation behaved homogeneously in various polyacrylamide gel electrophoretic systems and in isoelectric focusing. The uteroglobin-like protein isolated from the lung had very similar physico-chemical and immunological properties to those of uteroglobin present in the rabbit uterine fluid. The two proteins had: (i) the same molecular weight, of approx. 13 000, with a two subunit structure (each approx. M_r 7000); (ii) identical behavior in polyacrylamide gel electrophoresis under non-denaturing and denaturing conditions; (iii) the same isoelectric point at pH 5.4; (iv) absence of carbohydrate in the molecule; (v) very similar amino acid compositions; (vi) lack of tryptophan among the amino acids; (vii) the same N-terminal amino acid (glycine), and (viii) indistinguishable immunological characteristics. Collectively, these data strongly suggest that uterine and lung uteroglobins are identical proteins.In contrast to the induction of the uterine uteroglobin by steroids with progestational activity, the synthesis of extra-uterine uteroglobins was not affected by these steroid hormones to any major extent. In keeping with the concept that lung is a target tissue for glucocorticoid action, cortisol and dexamethasone were capable of increasing the concentration of lung uteroglobin 3-fold (from 3 to 9 μg/mg soluble protein). These compounds did not, however, alter the secretion of the uterine protein. Administration of high doses of testosterone and 5α-dihydrotestosterone elevated significantly the content of both uterine and lung uteroglobin. Only approx. one-fifth of the adult pulmonary uteroglobin levels were present in lungs of newborn rabbits indicating that developmental changes occur in the lung uteroglobin content.     

Evidence for the identity of anti-proteinase pulmonary protein CCSP and uteroglobin

Purified Clara cell secretory protein (CCSP) from rabbit lung was analyzed by SDS gel electrophoresis, and immunoblotting with a specific anti-uteroglobin antibody as well as for its ability to bind [3H]progesterone. The results obtained indicate that proteins CCSP and uteroglobin are identical.     

Partial purification of a binding protein for polychlorinated biphenyls from rat lung cytosol: physicochemical and immunochemical characterization

A binding protein for certain methyl sulfone metabolites of polychlorinated biphenyls (PCB) was partially purified from lung cytosol of untreated female rats. The protein has an Mr of 13,000 and a pI of 5.3 in the absence of reducing agents. In the presence of dithioerythreitol or beta-mercaptoethanol, the protein is split into subunits with a more basic pI. The 13-kDa protein was electroeluted from SDS-polyacrylamide gels, and an antiserum against the protein was raised in rabbit. The immunoglobulin fraction was shown to contain monospecific antibodies against the 13-kDa protein as determined by Western immunoblots. The antibodies retained partially purified binding protein labeled with radioactive ligand when subjected to protein A-Sepharose chromatography and caused a shift in the elution of the labeled protein from Sephadex G-75 and a shift in its sedimentation behavior on sucrose gradients. Due to striking similarities in physicochemical characteristics of the 13-kDa protein and a protein purified from rabbit lung and uterus, uteroglobin, the anti 13-kDa protein antibodies were tested for cross-reactivity. As judged by Western immunoblots, the anti 13-kDa protein antibodies did not cross-react with uteroglobin and the two proteins, although similar, do not seem to be identical. The 13-kDa protein is proposed to be responsible for the accumulation of certain methylsulfonyl-PCBs in lung tissue of rats. Monospecific antibodies against the 13-kDa protein should constitute immunochemical probes of great value in attempts to elucidate the physiological role of the protein as well as its possible role in PCB-induced respiratory toxicity.     

Glucocorticoid and developmental regulation of uteroglobin synthesis in rabbit lung.

The synthesis of uteroglobin in rabbit lung was studied after the administration of glucocorticoids to intact adult animals as well as during the late stages of rabbit development. The synthesis of uteroglobin was compared with levels of translatable uteroglobin mRNA in the lung. Uteroglobin synthesis was determined both by incorporation of [25S]methionine into the protein by lung explants incubated in vitro and by radioimmunoassay measurements of uteroglobin concentration in lung. Lung poly(A)-containing mRNA, isolated by oligo(dT)--cellulose chromatography, was translated in cell-free systems and the activity of uteroglobin mRNA was determined after immunoprecipitation. Dexamethasone administration increased about 2-fold the synthesis of lung uteroglobin compared with the controls. The effect of cortisol was more moderate. Both glucocorticoids did not affect the degradation rate of lung uteroglobin, but produced increases in the translatable levels of uteroglobin mRNA parallel to those observed for uteroglobin synthesis. During the late stages of rabbit development, both the synthesis of lung uteroglobin and the translatable levels of its mRNA increase in parallel about 12-fold in a biphasic fashion. A first increase occurred between 2 days before and 2 days after birth. Starting at 5 days of age, there was a second increase in both parameters, which at 12 days of age reached values close to those observed in adult rabbits. Our results suggest that the rate of lung uteroglobin synthesis could be mainly determined by the translatable levels of its mRNA.     

Uteroglobin mRNA from the lung of the hare (Lepus capensis): cell-free translation and properties

Poly(A)-containing RNA was obtained from the lung of hares (Lepus capensis) and uteroglobin mRNA was characterized by cell-free translation and molecular hybridization to a rabbit uteroglobin cDNA probe. In the cell-free system, hare uteroglobin mRNA was preferentially translated as compared to the whole lung mRNA and it directed the synthesis of a precursor, preuteroglobin, containing about twenty additional amino acids. Hare uteroglobin mRNA was about 40 nucleotides larger than the homologous rabbit one, as analyzed by electrophoresis on agarose gel. Thermal stability of the hybrids formed between rabbit or hare uteroglobin mRNAs and the rabbit cDNA probe indicated differences in the nucleotide sequence of both mRNAs. The levels of uteroglobin mRNA and uteroglobin synthesis in lung are about two-fold higher in hare than in rabbit lung.     

Confirmation of the presence of uteroglobin in tubal secretions]

The acrylamide gel electrophoresis of rabbit serum, of rabbit tubal secretions at the oestrus stage (STO) and at the luteal stage (STL), followed by immunodiffusion with anti-serum, was done to detect any tubal proteins which were not found in the rabbit serum. The anti-rabbit STO goat anti-serum was absorbed with rabbit plasma in order to purify antibodies to proteins found specifically in tubal secretions. This technique revealed the presence of a protein (STx) in STO and STL but not in the rabbit serum. The electrophoretic migration (Rf = 0.72) of the tubal protein (STx) is similar to uteroglobin (Rf = 0.74). The tubal protein was detected immunologically only in tissues where uteroglobin had been found by other workers: lungs, semen, Fallopian tubes and uterine secretions (5 days post-ovulation); further more a complete identity for the precipitating line existed between these extracts. These results corroborate the presence of uteroglobin or of a very similar protein in oestrus of luteal tubal secretions and in some other rabbit tissues.     

Cloning, structure, and expression of a rat binding protein for polychlorinated biphenyls. Homology to the hormonally regulated progesterone-binding protein uteroglobin

Certain metabolites of polychlorinated biphenyls (PCBs) are retained in the Clara cells and in the airway lumen of rodent lung due to their interaction with a secretory 13-kDa protein. Here, we report the isolation of a cDNA encoding the rat lung PCB-binding protein. The identity of the PCB-binding protein is supported by expression of the cDNA in Cos-1 cells where the homogenates from transfected cells show specific binding of 4,4'-bis([ 3H]methylsulfonyl)-2,2',5,5'-tetrachlorobiphenyl, a high affinity ligand for the PCB-binding protein. Also a monospecific antiserum to the PCB-binding protein recognizes a 13-kDa protein in the homogenates of transfected cells but not in the corresponding fraction of mock-transfected cells. Northern blot analysis of total RNA from different rat tissues demonstrates that the cDNA detects a approximately 600-base pair mRNA which appears to be solely expressed in lung. Interestingly, DNA sequence analysis and prediction of the amino acid sequence reveals that the PCB-binding protein shares 53% positional amino acid identity with uteroglobin, a progesterone-binding protein found in rabbit uterus and lung. Furthermore, amino acids shown by x-ray crystallography to delineate the central cavity of uteroglobin, which fits progesterone, are highly conserved in the two proteins.     

Clara cell 10 kDa protein (CC10): comparison of structure and function to uteroglobin

The cellular localization, functional activities and structures of rat and human Clara cell 10 kDa proteins (CC10) are compared to rabbit uteroglobin. CC10 is present exclusively in the non-ciliated cells of the surface epithelium of the pulmonary airways, whereas uteroglobin is reported to be present in the lung and reproductive organs. There is about 55% identity between the amino acid sequences of rat CC10 and either rabbit uteroglobin or human CC10. The latter two have 61% identity. Using the known structure of uteroglobin as the model, correlations between the structure and function for this group of proteins are made. Substitution of the residues for the rat and human CC10 into the structure of uteroglobin suggests that these proteins may be members of a structurally homologous family. Some of the functional differences may be due to distortion of the hydrophobic pocket in the dimeric protein and a surface hypervariability located on one contiguous helix and beta turn. Rat CC10 and rabbit uteroglobin both, nearly equally, inhibit papain and bind progesterone. Human CC10 does not inhibit papain and has markedly lower progesterone binding (4.6% of rabbit uteroglobin). Antiinflammatory activity of synthetic peptides corresponding to a homologous sequence region of uteroglobin and the two Clara cell proteins was tested. The region chosen has sequence similarity to lipocortin I. The peptides not only failed to inhibit carrageenan-induced foot pad swelling but exacerbated it. All three proteins inhibit pancreatic phospholipase A2. The phospholipase A2 inhibitory effect of CC10 may be important in regulating the inflammatory responses in the lung.     

Ultrastructural localization of uteroglobin immunoreactivity in rabbit lung and endometrium,and rat ventral prostate

Summary Recent biochemical studies have demonstrated amino acid sequence homologies between uteroglobin from rabbit endometrium and prostatic binding protein from rat ventral prostate. We have studied the ultrastructural distribution of uteroglobin-immunoreactive material in rabbit lung and endometrium and rat ventral prostate using an uteroglobin antibody raised in guinea pigs. Secretory granules of bronchiolar Clara cells, endometrial non-ciliated cells and rat prostate secretory cells gave a positive immunoreaction when this antibody was used. The results indicate a close relationship of immunoreactive epitopes of proteins present in those secretory cells. The functional properties of these proteins (glycoproteins, steroid binding, androgen-dependent secretion) suggest a close functional relationship, for instance a surface action such as coating, capping, masking or lubrication.Supported by grants Au 48/7-8 and Ki 154/9-3 from the Deutsche Forschungsgemeinschaft     

Primary structure of rabbit lung uteroglobin as deduced from the nucleotide sequence of a cDNA

Double-stranded cDNA was synthesized from partially purified uteroglobin mRNA from rabbit lung. A cDNA coding for lung uteroglobin was then cloned in the plasmid pUC18 and both the nucleotide sequence and the derived amino acid sequence were determined. This allowed us to demonstrate unequivocally that uteroglobins from lung and uterus are identical proteins.     

Glucocorticoids induce the expression of the uteroglobin gene in rabbit foetal lung explants cultured in vitro.

In 27-day-old rabbit foetal lung explants cultured in vitro, the synthesis of the protein uteroglobin decreased progressively during several days of culture. Addition of glucocorticoids to the medium progressively induced the synthesis of uteroglobin in a dose-dependent manner without affecting the synthesis of total proteins. The glucocorticoid-mediated induction of uteroglobin appears mainly due to increased amounts of uteroglobin mRNA and seems to be independent of simultaneous cell proliferation, suggesting a glucocorticoid-triggered differentiation of pre-existing cells. The results suggest a major role of glucocorticoids in the developmental regulation of the uteroglobin gene in the lung.     

Evidence that progesterone binding uteroglobin is similar to myosin alkali light chain

Using a computer program designed to detect evolutionary relationships between proteins, I find that exon 2 of rabbit uteroglobin, a progesterone binder, and part of myosin alkali light chain have a comparison score that is 7.2 standard deviations higher than that obtained with a comparison of randomized sequences of these proteins. The probability (p) of getting this score by chance is less than 10(-12). This theoretical finding that these sequences are similar has led to the experimental finding that copper, calcium and the tranquilizer trifluoperazine, a calmodulin binding ligand, affect progesterone binding to uteroglobin.     

The structural homology between uteroglobin and the pore-forming domain of colicin A suggests a possible mechanism of action for uteroglobin.

Although the exact physiological function of uteroglobin is not known, it has been suggested that it may function by inhibiting phospholipase A2. We have found that the uteroglobin fold is embedded in that of the poreforming domain of colicin A. Colicin A is an antibiotic protein that kills sensitive Escherichia coli cells by forming a pore in their phospholipid membrane. The RMS deviation between the C alpha atoms after the structural alignment is 2.39 A for the 52 superimposed residues. In the alignment, uteroglobin helices 1, 2, 3, and 4 align with colicin A helices 6, 7, 3, and 4, respectively. The motif is strongly amphipathic in both proteins. On the basis of this common structural motif and of known experimental data on both proteins, we propose that UG binds to the membrane surface by lying on it monotopically. The phospholipase A2 inhibition would follow this initial binding step.     

The mouse salivary androgen-binding protein (ABP) alpha subunit closely resembles chain 1 of the cat allergen Fel dI

Androgen-binding protein (ABP) is found in the salivas of a wide variety of rodents and it has been proposed that ABP functions in sex and/or subspecies recognition (Karn and Dlouhy,J. Hered. 82, 453, 1991). This is a report of significant identity between the alpha subunit of mouse salivary ABP and Chain 1 of cat allergen Fel dI (50% identity), as well as with two other proteins that share identity with Chain 1 of Fel dI, rabbit uteroglobin (27% identity with ABP alpha) and human lung Clara 10 (27% identity with ABP alpha). The secondary structure predicted for the mouse ABP alpha subunit is a very good fit with the secondary structure determined by X-ray crystallography for rabbit uteroglobin, a protein that shares with mouse ABP the capability of binding steroid. Fel dI is found in cat saliva, sebaceous glands, and pelt. Its function is not known but it has been proposed to be involved in protecting dry epithelia, a parallel to uteroglobin protecting wet epithelia. Since mice, like cats, lick themselves and each other extensively, coating their pelts with ABP may be part of this or another biological function.     

Immunohistochemical localization of secretory proteins in the endometrial epithelium of the rabbit

Summary Proteins of uterine fluid and lung homogenates of the rabbit were separated by gel and ion exchange chromatography. Purified protein fractions were used for immunisation and antiserum production. By means of several absorptions, six monospecific antisera against uteroglobin and five other proteins were obtained. Using immunohistochemistry, four of them could be localised in the uterine epithelium from oestrus and the first and the seventh day post coitum, and also in the blastocyst. The present study indicates the involvement of different endometrial cells in the synthesis and release of the various proteins of uterine secretion.Supported by grant Ki 154/6-7 from the Deutsche Forschungsgemeinschaft