Previous studies have demonstrated an imbalance in placental levels of the human choriogonadotropin (hCG) alpha and beta subunits. Free alpha subunit was present in first trimester placentae, and the imbalance was accentuated as gestation approached parturition. Two sets of experiments were performed to assess the control on production levels of each subunit. Synthesis of the alpha and beta subunits was assessed by labeling the nascent chains of polysomes derived from first trimester placenta. The products of these reactions were immunoprecipitated with subunit-specific antisera and the labeled subunits were quantitated; the ratio of alpha to beta subunit synthesized was 1.7. To examine whether this imbalanced synthesis reflected differences in the amount of subunit mRNAs, or differing mRNA translational efficiencies, the ratio of the steady state levels of these mRNAs was also determined. Total first trimester placental RNA was hydrolyzed with alkali, 5'-end-labeled with 32P, and hybridized in DNA excess to cloned alpha and beta cDNAs. These experiments demonstrated the presence of twice as much hCG-alpha mRNA as hCG-beta mRNA. In term placenta, the amounts of excess alpha subunit are greater than at first trimester; the ratio of alpha to beta mRNAs in term RNA was about 12:1. Thus, the subunit mRNA levels are independently regulated and their imbalance accounts for differences in the quantities of alpha and beta subunits seen in placental tissue. 相似文献
RNA derived from bovine steer pituitary was translated in wheat germ cell-free extracts containing [35S]methionine. Antisera generated against purified denatured alpha and beta subunits of lutropin were used to demonstrate the synthesis of both proteins in vitro. The immunoprecipitated products of the cell-free system were resolved on sodium dodecyl sulfate/polyacrylamide gels and it was observed that the molecular weight of the immunoprecipitated alpha subunit protein was approximately 14,000, while that of the beta protein was estimated to be 16,000. Since the molecular weights of authentic alpha and beta subunits are 10,600 and 14,000 respectively, the cell-free products presumably represented their pre-protein forms. The ratio of the immunoprecipitated subunit pre-proteins was dependent on the magnesium concentration in the translation mixtures; at 2.1 mM, translation of lutropin alpha and beta mRNAs was comparable. RNA isolated from cow pituitary tissue directed the synthesis of fivefold less of the alpha and beta immunoprecipitated proteins than did steer RNA. Since the blood levels of gonadal steroids are higher in the cow, the results supported the hypothesis that lutropin alpha and beta mRNA biosynthesis is repressed by these steroids. The data also suggest that synthesis of lutropin alpha and beta subunits is coordinately expressed in certain physiological situations. 相似文献
Pro-opiomelanocortin (POMC) is glycosylated and proteolytically cleaved to produce a number of smaller peptide hormones including adrenocorticotropic hormone (ACTH) and endorphin in mammalian pituitary and the mouse pituitary cell line AtT-20/D16v. When glycosylation of POMC is inhibited in AtT-20 cells with the drug tunicamycin, a 26,000-dalton protein appears in place of the glycosylated 29,000- and 32,000-dalton forms of POMC. The 26,000-dalton form found in tunicamycin-treated cells has the same [35S]methionine tryptic peptides as 29,000- and 32,000-dalton POMC, indicating that the decrease in apparent mass is most likely due to loss of carbohydrate and not to changes in the peptide backbone. The 4,500-dalton form of alpha(1-39)ACTH and the 3,000- and 11,000-dalton forms of endorphin are all present in tunicamycin-treated cells. The glycosylated form of alpha(1-39)ACTH, however, is missing and the glycosylated ACTH intermediates are replaced by unglycosylated ACTH intermediates. Pulse-chase studies demonstrate that the 26,000-dalton unglycosylated POMC is the precursor of the smaller ACTH and endorphin molecules in tunicamycin-treated cells. Furthermore, all of the forms of ACTH and endorphin found in tunicamycin-treated cells are secreted. Thus, it appears that glycosylation is not an essential step for correct cleavage or secretion of POMC or its products. 相似文献
Alkaline phosphatase activity in human placental cells transformed by a tsA mutant of simian virus 40 (SV40) can be greatly induced by growing these cells at 40 degrees C, the temperature at which the tsA transformants regain their nontransformed phenotype. The induction of alkaline phosphatase in these cells requires the synthesis of both RNA and protein. The induced alkaline phosphatase from a SV40 tsA30 mutant-transformed term placental cell line (TPA30-1) was purified, characterized, and compared with alkaline phosphatase from term placenta and first trimester placenta. The form of alkaline phosphatase found in TPA30-1 cells differs from the phosphatase of term placenta in physiochemical and immunological properties. The TPA30-1 phosphatase is, however, indistinguishable from the alkaline phosphatase of human first trimester placenta by several criteria, including electrophoretic mobility, apparent molecular weight (Mr = 165,000), size of monomeric subunit (Mr = 77,000), heat lability, and sensitivity to inhibition by amino acids and EDTA. In addition, alkaline phosphatase from both TPA30-1 cells and first trimester placenta can be inactivated by antiserum to liver alkaline phosphatase but not by antiserum to term placental alkaline phosphatase. The induction of first trimester phosphatase in cells derived from term placenta provides a system for the study of alkaline phosphatase gene regulation in human placenta. 相似文献
Using as a starting material either a detergent extract or a protein fraction eluted from membranes with ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, we have isolated from human placental membranes a major substrate for the epidermal growth factor (urogastrone) receptor kinase (EGF kinase). The substrate was isolated both in an intact form, having a molecular mass of approximately 38-kDa (p38), and in a 35-kDa form (p35) representing a proteolytic cleavage product of p38. Both p38 and p35 cross-reacted with antibodies directed against bovine retinal transducin, but did not cross-react with antibodies directed against the 35-kDa beta subunit of human placental G-protein. Antisera directed against the placental EGF kinase substrate failed to react with either bovine or human placental src kinase substrate, p36. Conversely, antisera directed against p36 reacted only poorly with placental p38 or p35. Although p38 had a blocked amino terminus that precluded sequence analysis, p35 yielded an N-terminal sequence that was identical with residues 13-36 of human lipocortin. Our data clearly distinguish p38 from the previously described intestinal calcium binding protein calpactin I or p36 that is also a tyrosine kinase substrate, and our work points to a close relationship (if not identity) between p35 and a 35-kDa EGF receptor kinase substrate previously characterized in A431 cells. We conclude that p38 and p35, which very likely represent human placental lipocortin, may share only limited epitope homology with transducin alpha subunit; however, the possibility that p38, along with intestinal p36 and with a family of related calcium binding proteins, may, like transducin, play a role in receptor-mediated transmembrane signaling is discussed. 相似文献
The gonadotropins luteinizing hormone, follicle-stimulating hormone, and human chorionic gonadotropin are composed of two noncovalently linked subunits, alpha and beta. The alpha subunit, identical in all three hormones, is produced in excess over the unique beta subunits by pituitary and placenta, and is secreted as uncombined, or free subunit. Free alpha subunit from both tissues has a larger molecular weight than the dimer form. In bovine pituitary an extra O-linked oligosaccharide is added to free alpha subunit, and this modification has recently been detected at an analogous position (threonine 39) on human alpha subunit secreted by choriocarcinoma cells. To assess the contribution of N-linked and O-linked oligosaccharides to the heterogeneity of human free alpha subunit, we have compared free alpha with human chorionic gonadotropin alpha secreted by explants and cultured cytotrophoblasts of human first trimester placenta. We have also examined the free and combined forms of human alpha subunit expressed in transfected C-127 mouse mammary tumor cells. Processing of the alpha subunit in placental and C-127 cells was similar. Tryptic mapping of placental-derived and transfected alpha subunits indicated that O-glycosylation at threonine 39 was not a major modification. In the presence of the oligosaccharide processing inhibitor swainsonine the difference in size between the free and combined forms of alpha was eliminated in both placental and C-127 cells, indicating that the two forms of alpha differed in their N-linked oligosaccharides. Furthermore, the oligosaccharides of free alpha subunits from placental and transfected cells were resistant to endoglycosidase H, but the combined forms of alpha were partially sensitive to the enzyme. Thus, in human first trimester placenta and mouse C-127 cells, combination of alpha with human chorionic gonadotropin beta alters the processing of N-linked oligosaccharides on alpha subunit. 相似文献
Various biosynthetic forms of rat alpha 1-antitrypsin (alpha 1AT) have been isolated by immunoprecipitation of in vitro and in vivo synthesized products. Rat alpha 1AT is synthesized in a rabbit reticulocyte system as a 45,000-Da preprotein with a 23-amino acid signal sequence. The majority of the amino acids in the signal sequence have been identified and resemble the signal peptides of other secretory proteins with respect to the abundance and positions of hydrophobic amino acids. Evidence from the translation of rat liver RNA in the presence of dog pancreas microsomes, from the translation of rat liver polysomes, and from tunicamycin-treated rat hepatocytes established that cleavage of the signal peptide of pre-alpha 1AT results in the formation of a 42,000-Da protein, the polypeptide backbone of mature alpha 1AT. A 50,000-Da glycoprotein is immunoprecipitated from translations programmed with rat liver microsomes or with rat liver mRNA and dog pancreas microsomes. Cotranslational glycosylation of alpha 1AT appears to occur in a stepwise fashion since three glycosylated forms of alpha 1AT (approximately 45,000, 47,000, and 50,000 Da) can be detected in polysome translations. These proteins are susceptible to cleavage by endo-beta-N-acetylglucosaminidase H and are digested to the same product, indicating that they have identical polypeptide chains. Two intracellular forms of alpha 1AT were detected in cultured rat hepatocytes, a 50,000- and a 52,000-Da protein; only the larger protein was immunoprecipitated from the medium of these cells. Digestion with endo-beta-N-acetylglucosaminidase H indicated that the 50,000-Da protein is a core glycosylated processing intermediate, whereas the 52,000-Da protein, which comigrated with purified serum alpha 1AT, appears to contain complex carbohydrate sidechains. When glycosylation was inhibited by incubation of hepatocytes with tunicamycin, a nonglycosylated 42,000-Da protein was immunoprecipitated from the cells and the culture medium, indicating that glycosylation of alpha 1AT is not essential for its secretion. 相似文献
The effects of N-linked glycosylation on the activation and secretion of lipoprotein lipase were studied in Ob17 cells. The cells were first depleted of any activity and enzyme content by cycloheximide treatment and of precursors of oligosaccharide chains by tunicamycin. The repletion of lipoprotein lipase content was studied in these cells maintained in the presence of tunicamycin after cycloheximide removal. During the repletion phase, the EC50 values of inhibition by tunicamycin (approx. 0.2 microgram/ml) of the incorporation of labeled glucose, mannose or galactose into trichloroacetic acid-insoluble material were found to be identical. Under these conditions, the rate of protein synthesis was maximally decreased by 30%. The results showed clearly that the recovery in lipoprotein lipase activity was parallel to the recovery in hexose incorporation, no activity being recovered in the absence of glycosylation. An inactive form of lipoprotein lipase from tunicamycin-treated cells was detected by competition experiments with mature active lipoprotein lipase for the binding to immobilized antilipoprotein lipase antibodies, as well as by immunofluorescence staining. SDS-polyacrylamide gel electrophoresis and Western blots of cellular extracts and of extracellular media, obtained after tunicamycin-treated cells were exposed to heparin, revealed a single immunodetectable Mr 52 000 protein, whereas a single Mr 57 000 protein was detected in control cells. Therefore, the results indicate that the acquisition by lipoprotein lipase of a catalytically active conformation is linked directly or indirectly to glycosylation. Despite this lack of activation, the lipoprotein lipase molecule was able to migrate intracellularily and to undergo secretion after heparin stimulation of the tunicamycin-treated cells. 相似文献
We have reported the presence of insulin-related poly A+RNA sequences in human placenta by RNA to DNA hybridization. In this study we have used a monoclonal antibody to somatomedin C/insulin-like growth factor I (Sm-C/IGF-I) to identify somatomedin-like proteins whose synthesis is directed by placental mRNA. Poly A+RNA from first trimester and term placenta was translated in a cell-free system using micrococcal nuclease-treated reticulocyte-lysate and [35S]methionine as a label. From 2.0×106 cpm of specifically incorporated [35S]methionine labeled protein, an immunoprecipitate with an apparent molecular weight of 14000 represented about 0.1% of total radioactivity in the translational products of poly A+RNA of first trimester placenta. A less prominent band (0.006%) of the same apparent molecular weight was also evident from translational products of term placental mRNAs. This protein could be competed with either acromegalic serum or synthetic Sm-C/IGF-I when added prior to immunoprecipitation. Translational products synthesized from mRNA of term placenta showed a second labeled band of 24000 daltons. This band was less effectively competed by acromegalic serum and not competed with either Sm-C/IGF-I or IGF-II and therefore its identity is uncertain. A protein similar to Sm-C/IGF-I is, therefore synthesized in first trimester placenta and to a lesser extent at term, suggesting developmental changes in Sm-C/IGF-I synthesis. Because Sm-C/IGF-I may act in a paracrine fashion, our findings suggest a role for Sm-C/IGF-I in growth of the placenta during early gestation. 相似文献
Two inhibitors of glycosylation, glucosamine and tunicamycin, were utilized to examine the effect of glycosylation inhibition in mouse neuroblastoma N18 cells on the degradation of membrane glycoproteins synthesized before addition of the inhibitor. Treatment with 10 mM-glucosamine resulted in inhibition of glycosylation after 2h, as measured by [3H]fucose incorporation into acid-insoluble macromolecules, and in a decreased rate of glycoprotein degradation. However, these results were difficult to interpret since glucosamine also significantly inhibited protein synthesis, which in itself could cause the alteration in glycoprotein degradation [Hudson & Johnson (1977) Biochim. Biophys. Acta 497, 567-577]. N18 cells treated with 5 microgram of tunicamycin/ml, a more specific inhibitor of glycosylation, showed a small decrease in protein synthesis relative to its effect on glycosylation, which was inhibited by 85%. Tunicamycin-treated cells also showed a marked decrease in glycoprotein degradation in experiments with intact cells. The inhibition of glycoprotein degradation by tunicamycin was shown to be independent of alterations in cyclic AMP concentration. Polyacrylamide-gel electrophoresis of isolated membranes from N18 cells, double-labelled with [14C]fucose and [3H]fucose, revealed heterogeneous turnover rates for specific plasma-membrane glycoproteins. Comparisons of polyacrylamide gels of isolated plasma membranes from [3H]fucose-labelled control cells and [14C]fucose-labelled tunicamycin-treated cells revealed that both rapidly and slowly metabolized, although not all, membrane glycoproteins became resistant to degradation after glycosylation inhibition. 相似文献
Since both prostaglandin (PG) F2 alpha and corticosteroids are elevated in mammals before the onset of parturition, we studied the effect of the synthetic corticosteroid dexamethasone on PGF2 alpha accumulation and cyclooxygenase (prostaglandin synthase, PGS) expression in the bovine fetal placenta. Cultures were prepared from cotyledons at different stages of gestation. The effect of dexamethasone on PGF2 alpha accumulation and PGS expression was determined by radioimmunoassay and [35S]methionine metabolic labeling followed by immunoprecipitation with specific anti-cyclooxygenase antibodies, respectively. Data demonstrate that in fetal placental cells at term, both PGF2 alpha accumulation and cyclooxygenase expression are significantly inhibited after 18 hours of dexamethasone treatment (150 nM). In contrast, neither first nor second trimester cells were sensitive to dexamethasone treatment. Dexamethasone inhibition of PGF2 alpha synthesis in fetal cells at term was abolished in the presence of RNA or protein synthesis inhibitors (actinomycin D or puromycin, 10 micrograms/ml each). Neither progesterone nor 17 beta-estradiol accumulation were affected by dexamethasone treatment at any stage of gestation. Data suggest that corticosteroids play a role in parturition through PGF2 alpha synthesis regulation by fetal placental cells. Since abnormalities during parturition e.g. retained placenta, are common following dexamethasone induction of labor in cows, we postulate that the local inhibition of PGF2 alpha accumulation by cotyledon cells after corticosteroid administration, may be involved in placental retention. 相似文献
Two different types of oligosaccharides, designated type 1 and 2 carbohydrate residues, are present on the contact site A molecule, an 80-kDa glycoprotein involved in the formation of EDTA-stable cell adhesion during cell aggregation in Dictyostelium discoideum. The first precursor detected by pulse-chase labeling with [35S]methionine was a 68-kDa glycoprotein carrying type 1 carbohydrate. Conversion of the precursor into the 80-kDa form occurred simultaneously with the addition of type 2 carbohydrate. Tunicamycin inhibited type 1 glycosylation more efficiently than type 2 glycosylation. The first precursor detected in tunicamycin-treated cells by pulse-chase labeling was a 53-kDa protein lacking both carbohydrates, which was converted through addition of type 2 carbohydrate into a 66-kDa final product. Labeling of intact cells indicated that this 66-kDa glycoprotein is transported to the cell surface. Prolonged treatment with tunicamycin resulted in the accumulation within the cells of the 53-kDa precursor with no detectable exposure of this protein on the cell surface. It is concluded that type 1 carbohydrate, which is cotranslationally added in N-glycosidic linkages, is neither required for transport of the protein to the Golgi apparatus nor for type 2 glycosylation or protection of the protein against proteolytic degradation. Incapability of tunicamycin-treated cells of forming EDTA-stable cell contacts suggests a role for type 1 carbohydrate in cell adhesion. Type 2 carbohydrate is added posttranslationally. It is required in the absence of type 1 glycosylation for transport of the protein to the cell surface. 相似文献
We carried out experiments designed to generate a novel cell-surface protein from a small glycosylated secretory protein. DNA encoding the entire precursor of human chorionic gonadotropin (hCG, alpha subunit) was fused precisely to DNA encoding the transmembrane and cytoplasmic domains of the vesicular stomatitis virus glycoprotein. When expressed in animal cells this DNA encoded the 92-amino acid hCG-alpha subunit anchored in cellular membranes by an extension composed of the 49 carboxyl-terminal amino acids of vesicular stomatitis virus glycoprotein. This hybrid protein was transported efficiently to the plasma membrane of animal cells. The two asparagine-linked glycans on the anchored form of hCG-alpha were large and heterogeneous when compared to those on the secretory form. Experiments employing in vitro mutagenesis and the glycosylation inhibitor tunicamycin established that the presence of at least one of the two asparagine-linked glycans was required for expression of the anchored molecule on the cell surface. However, as reported previously, secretion of hCG-alpha occurred in the absence of glycosylation. Also, mutations eliminating the second glycosylation site (at amino acid 78) in both the anchored or secreted forms apparently led to partial denaturation or a conformational change interfering with transport of the protein. 相似文献
The in vitro synthesis of RNA in the human placental tissue, incubated in organ culture, was investigated. We followed the synthesis of the poly A(-) and poly A(+) RNA fractions, and investigated the distribution of the newly synthesized RNA among the subcellular fractions isolated from first and third trimester placentas.The poly A(-) RNA was the major fraction of the RNA synthesized in vitro. The incorporation of [3H]uridine into the poly A(+) RNA fraction was very low.As protein synthesis occurred during the entire incubation period, we suggest the presence of a pool of mRNA molecules in the form of mRNP particles. 相似文献
Induction of apoptosis in a variety of cell types leads to inhibition of protein synthesis. Recently, the cleavage of eukaryotic translation initiation factor 4G (eIF4G) by caspase 3 was described as a possible event contributing to translation inhibition. Here, we report the cleavage of another initiation factor in apoptotic cells, eIF2alpha, that could contribute to regulation of translation during apoptosis. This cleavage event could be completely inhibited by pretreatment of HeLa cells with Z-VAD-fmk. In vitro analysis using purified eIF2 and purified caspases showed cleavage of eIF2alpha by caspase 3, 6, 8, and 10 but not 9. Caspase 3 most efficiently cleaved eIF2alpha and this could be inhibited by addition of Ac-DEVD-CHO in vitro. Comparison of cleavage of phosphorylated versus nonphosphorylated eIF2alpha revealed a modest preference of the caspases for the nonphosphorylated form. When eIF2. 2B complex was used as substrate, only caspase 3 was capable of eIF2alpha cleavage, which was not affected by phosphorylation of the alpha subunit. The eIF2.GDP binary complex was cleaved much less efficiently by caspase 3. Sequence analysis of the cleavage fragment suggested that the cleavage site is located in the C-terminal portion of the protein. Analysis showed that after caspase cleavage, exchange of GDP bound to eIF2 was very rapid and no longer dependent upon eIF2B. Furthermore, in vitro translation experiments indicated that cleavage of eIF2alpha results in functional alteration of the eIF2 complex, which no longer stimulated upstream AUG selection on a mRNA containing a viral internal ribosome entry site and was no longer capable of stimulating overall translation. In conclusion, we describe here the cleavage of a translation initiation factor, eIF2alpha that could contribute to inhibition or alteration of protein synthesis during the late stages of apoptosis. 相似文献
Messenger RNA isolated from first trimester placentae was translated using radiolabeled amino acids in both the wheat germ and the ascites cell-free systems. The choriogonadotropin α subunit product was purified by immunoprecipitation with a subunit specific antiserum. Its amino acid sequence was partially determined by automated Edman degradation analysis. An NH2-terminal extension of 24 amino acids was found and its partial sequence is: The preprotein form of the subunit was cleaved by the addition of microsomal membranes resulting in a homogeneous NH2-terminal product. Hence, it is unlikely that this processing step accounts for the heterogeneity that has been observed previously in the structure of this region of the subunit. 相似文献
Human choriogonadotropin (hCG) is a placental glycoprotein hormone composed of a 92-amino acid alpha subunit noncovalently linked to a 145-amino acid beta subunit. We report here the expression of biologically active hCG in mouse C127 cells transfected with expression vectors containing the DNA coding for both subunits. In addition, the same cell line was used to express the alpha subunit alone. The expression products were purified by affinity chromatography using specific monoclonal antibodies to hCG or its subunits. The system secreting biologically active hCG also produced a 10-fold or greater molar excess of free beta subunit. The dimeric hormone, as well as the excess beta subunit, resembles the standard urinary hCG and beta subunit by chemical and biological criteria. In contrast, when the vector encoding for the alpha subunit was expressed alone, the alpha subunit had a higher molecular weight than both standard alpha and the alpha found in the expressed dimeric hormone. The molecular weight difference between expressed alpha subunit and standard alpha was found to reside in the alpha peptide consisting of residues 52-91 which contained all of the carbohydrate of the alpha subunit. The N-asparagine-linked carbohydrate moieties in the recombinant alpha were found to be triantennary in contrast to biantennary in urinary alpha, and this hyperglycosylation was responsible for the higher molecular weight of the alpha subunit when it was expressed alone. We found no evidence of O-threonine glycosylation at position alpha 39 reported to be present in free forms of the alpha subunit; however, the companion paper (Corless, C.L., Bielinska, M., Ramabhadran, T. V., Daniels-McQueen, S. Otani, T., Reitz, B. A., Tiemeier, D. C., and Boime, I. (1987) J. Biol Chem. 262, 14197-14203) finds a small quantity of O-glycosylation. Since the excess beta subunit appears to be of normal size and contains the expected complement of sugars, only free alpha subunit seems to be a potential substrate for addition of extra sugar moieties. No large beta subunit forms have been found by others, while large alpha subunits have been described both clinically and in tissue culture systems. These observations imply that the conformation of the free alpha subunit, in the regions of the glycosylation recognition sites, allows easier access for glycosyltransferases than those same sites in the beta subunit. When alpha is combined with beta, the local structures around the alpha glycosylation sites are apparently altered so as to make the synthesis of triantennary chains less favorable. 相似文献