Characterization of pregnancy-specific beta 1-glycoprotein synthesized by human placental fibroblasts

We have previously demonstrated that human placental fibroblasts produce a pregnancy-specific beta 1-glycoprotein (PS beta G) immunologically indistinguishable from placental PS beta G. This was confirmed by the immunocytochemical localization of PS beta G in these fibroblasts. In addition, placental fibroblasts contain all three PS beta G mRNAs of 2.3, 2.2, and 1.7 kilobases which hybridize with the three PS beta G cDNAs (PSG16, PSG93, and PSG95) identified, although at 1.4-2.5% of the levels in human term placenta. The major PS beta G species synthesized by placental fibroblasts is a 62K glycopolypeptide formed from a 58K intracellular precursor polypeptide. However, the PS beta G species found in human placenta are one major glycoprotein of 72K and two minor ones of 64K and 54K. Poly(A)+ RNA from placental fibroblasts directed the synthesis of two polypeptides of 48K and 46K (major), whereas, poly(A)+ RNA from human placenta directed the synthesis of higher levels of four polypeptides of 50 K, 48 K (major), 46 K, and 36 K. Thus, the major PS beta G species found in fibroblasts and human placenta differ. The carbohydrate side-chains are essential for the stability of fibroblast PS beta G, because PS beta G synthesis in these fibroblasts could not be detected in the presence of tunicamycin, a protein glycosylation inhibitor which did not affect PS beta G mRNA expression. Our finding that a variant PS beta G species is produced in placental fibroblasts raises the possibility that the authentic placental PS beta G species may have different functions.     

Isolation and characterization of complementary DNAs encoding human pregnancy-specific beta 1-glycoprotein

Pregnancy-specific beta 1-glycoprotein (PS beta G) isolated from human placenta consists of a set of at least three glycoproteins with apparent molecular masses of 72, 64, and 54 kDa, respectively. This heterogeneity is confirmed by the detection of three nonglycosylated polypeptides of 50, 48, and 36 kDa, which can be immunoprecipitated by antiserum to placental PS beta G obtained by in vitro translation of placental poly(A)+ RNA. To examine the structural relationships between these proteins, two cDNA clones of 1912 base pairs (PSG16) and 2131 base pairs (PSG93) encoding human PS beta Gs were isolated from a human placental lambda gt11 cDNA library. The sequenced portions of these two cDNAs are identical with the exception that clone PSG93 contains an additional 86 base pairs at the end of the common 3'-coding region. This insertion could result in the generation of a PS beta G species of 419 amino acid residues instead of the 417 amino acid residues predicted by the sequence of clone PSG16. The calculated molecular masses of the two polypeptides encoded by PSG16 and PSG93 are 46.9 and 47.2 kDa, close to the size of the major nonglycosylated PS beta G of 48 kDa. The identity of proteins coded for by these cDNA clones was confirmed by comparing the predicted amino acid sequences to sequences determined from endoproteinase Lys-C peptides obtained from human placental PS beta G. Two placental PS beta G mRNAs of 2200 bases (major) and 1700 bases (minor) have been detected by Northern hybridization analysis. Primer extension and S1 nuclease mapping experiments demonstrated that PS beta G mRNAs have heterogeneous 5' termini.     

Exon-intron organization of a gene for pregnancy-specific beta 1-glycoprotein, a subfamily member of CEA family: implications for its characteristic repetitive domains and C-terminal sequences

A fragment of human gene for pregnancy-specific beta 1-glycoprotein(s), recently identified CEA family member(s), has been cloned. Analyses of nucleotide and deduced amino acid sequences revealed that it carried, from 5' to 3' direction, exons IA, IB, IIA, IIB, C3, C1 and C2, the first four encoding peptides distinct from but highly similar to domains of PS beta Gs. The lack of consensus 3' splice site sequence ahead of IB indicated that it was an abortive exon, which would explain the peculiar domain construction of PS beta Gs, i.e. N-IA-IIA-IIB-C1, 2 or 3. Apparently, the multiple C-terminal sequences for a PS beta G were generated by alternative splicing among C1, C2 and C3 exons. Furthermore, sequences which overlapped partly with Cexons, were found to be similar to parts of 3'-UTR of CEA and NCA, indicating further the close relationship of CEA/NCA and PS beta G subfamily genes.     

Human pregnancy-specific beta 1-glycoproteins are coded within chromosome 19.

We have isolated and characterized cDNAs that code for apoproteins having amino acid sequences highly similar to pregnancy-specific beta 1-glycoproteins (PS beta G). cDNAs coding for PS beta Gs, as well as the cDNA clone reported here, are members of the carcinoembryonic antigen (CEA) gene family. The previous localization of CEA-related genes to human chromosome 19, and the high level of DNA sequence conservation in the CEA family, suggested that the PS beta G genes are also located on this chromosome. We demonstrate here that chromosome 19 is indeed the site of PS beta G sequences. Our finding is in contrast to the recently reported indication that pregnancy-specific glycoproteins are encoded in chromosomes X and 6.     

Imbalanced synthesis of human choriogonadotropin alpha and beta subunits reflects the steady state levels of the corresponding mRNAs

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.     

Molecular basis for two forms of the G protein that stimulates adenylate cyclase

Most cells contain two forms of the alpha subunit of the G protein (Gs) that stimulates adenylate cyclase; their apparent molecular weights are 45,000 and 52,000. Two cDNAs that correspond to distinct mRNAs for the alpha subunit of Gs have been cloned from a bovine adrenal library and sequenced. The sequences of the two cDNAs, designated pGs-l and pGs-S, are identical except for a single stretch of 46 nucleotides in the coding region, where four are altered and 42 are deleted in pGs-S. Expression of pGs-S and pGs-l in COS-m6 cells yields protein products with apparent molecular weights of 45,000 and 52,000, respectively, based on their mobility in sodium dodecyl sulfate-polyacrylamide gels. We conclude that pGs-S and pGs-l encode the 45- and 52-kDa forms of Gs alpha, respectively, and propose that the mRNAs encoding these proteins arise from a single gene by internal alternative RNA splicing.     

Expression of CEA-related genes in the first trimester human placenta

Eight cDNA clones, closely related to the carcino-embryonic antigen gene family, have been isolated from a cDNA library representing genes expressed in the first trimester human placenta. Sequence analysis of one clone shows it to be a pregnancy-specific beta 1-glycoprotein (PS beta G) closely related to three other PS beta G cDNA recently characterised from a term placenta library. The protein encoded by the cDNA is predicted to be less high glycosylated than those reported previously and differs markedly in the C-terminal sequence. The 3' untranslated region of the cDNA is very similar to the equivalent region of beta 1-glycoprotein PS beta G E except that it contains the 12bp repeat sequence found flanking the Alu sequence in CEa and an additional 67bp of sequence that appears to be derived from CEA.     

Characterization of new members of the pregnancy-specific β1-glycoprotein family

Three cDNAs encoding members of the pregnancy-specific 1-glycoprotein (PSG) family were isolated from human term placental cDNA library. All three cDNAs encode proteins with similar domain structure. There is a leader sequence of 34 amino acids followed by an N-domain of 109 amino acids. Immediately after the N-domain are one or two copies of a repeating A-domain of 93 amino acids, a B-domain of 85 amino acids and a C-domain of variable size. The proteins are highly hydrophilic. However, one of them has an 81-amino acid C-domain which is very hydrophobic and could potentially serve as a membrane attachment site. The putative cell-cell recognition tripeptide, Arg-Gly-Asp, is present in the N-domain of two of the proteins. Partial sequence of one of the cDNAs has been found in HeLa cells while cDNAs highly homologous to two of the cDNAs have been found in the fetal liver. Functional roles of the PSG proteins basing on their structure are proposed.Abbreviations PSG Pregnancy-Specific 1-Glycoprotein, according to nomenclature recommended at the ISOBM XVII Meeting, 1989 [31] - CEA Carcinoembryonic Antigen - bp base-pair - kb kilo-base-pair - nt nucleotide - aa amino acid - UTR Untranslated Region - RGD Arg-Gly-Asp The nucleotide sequence of two of the cDNAs presented in this article have been submitted to GenBank under the accession numbers M37102 (hPS91) and M37103 (hPS133). The nucleotide sequence of hPS176 has also been submitted. No accession number is available yet.     

G-protein subunit mRNA levels in rat heart, liver, and adipose tissues: analysis by DNA-excess solution hybridization

The steady-state levels of mRNAs for the G-proteins Gi alpha 2, Go alpha, and the G beta-subunits common to each were established in rat adipose, heart and liver. Uniformly-radiolabeled, single-stranded antisense probes were constructed from cDNAs or assembled from oligonucleotides. Direct comparison of the steady-state levels of the G-protein mRNAs was performed under identical assay conditions, and on a molar basis. In adipose, liver and heart, Gs alpha mRNA was more abundant than mRNA for Go alpha, Gi alpha, and G beta. In adipose tissue, mRNA levels were as follows: 19.4, 7.6, 7.0, and 2.3 amol mRNA per micrograms total cellular RNA for Gs alpha, G beta, Gi alpha 2, and Go alpha, respectively. In heart Gs alpha mRNA was less abundant than in adipose, but the relative trend among the G-protein subunits was the same. In liver, G beta mRNA was more abundant than either Go alpha or Gi alpha 2. Go alpha mRNA levels ranged from 1.2 to 2.3 amol/micrograms total RNA in liver and adipose, respectively. The present work demonstrates the many advantages of this strategy when applied to the study of a family of homologous, low-abundance proteins and establishes for the first time the molar levels of Gi alpha 2, Gs alpha, Go alpha, and G beta-subunit mRNAs in several mammalian tissues.     

Influence of thyroid hormone status on expression of genes encoding G protein subunits in the rat heart.

We have studied the influence of thyroid hormone status in vivo on expression of the genes encoding guanine nucleotide-binding regulatory protein (G protein) alpha-subunits Gs alpha, Gi alpha(2), Gi alpha(3), and both the 36-kDa form (beta 1) and the 35-kDa form (beta 2) of the beta-subunit in rat ventricle. The relative amounts of immunoactive Gi alpha(2) and Gi alpha(3) were greater in ventricular membranes from hypothyroid animals than from euthyroid animals (1.9- and 2.6-fold, respectively). A corresponding 2.3-fold increase in Gi alpha(2) mRNA was observed as well as a 1.5-fold increase in Gi alpha(3) mRNA. The relative amounts of immunoactive beta 1 and beta 2 polypeptides were also increased (2.8- and 1.8-fold, respectively) in the hypothyroid state and corresponded with comparable increases in the relative levels of beta 1 and beta 2 mRNAs. No difference was seen between the amounts of Gi alpha(2), Gi alpha(3), beta 1, and beta 2 in the euthyroid state and the hyperthyroid state. In contrast to these effects of thyroid hormone status on Gi alpha and beta, the steady-state amounts of Gs alpha protein and mRNA were not altered by thyroid hormone status. Thyroid hormone status did not alter sensitivity of adenylyl cyclase to stimulation by sodium fluoride or guanyl-5'-yl imidodiphosphate (GppNHp), nor did it influence GppNHp-induced inhibition of forskolin-stimulated enzyme activity. These results demonstrate that thyroid hormone status in vivo can regulate expression of specific G protein subunits in rat myocardium. However, the physiological consequences of these changes remain unclear.     

Studies on the interaction of alpha subunits of GTP-binding proteins with beta gamma dimers.

The interaction of several preparations of purified beta gamma dimers with two types of guanosine-nucleotide-binding-regulatory-(G)-protein alpha subunits, a recombinant bv alpha i3, made in Sf9 Spodoptera frugiperda cells by the baculovirus (bv) expression system, and alpha s, either purified from human erythrocyte Gs-type GTP-binding protein, and activated by NaF/AlCl3, or unpurified as found in a natural membrane, were studied. The beta gamma dimers used were from bovine rod outer segments (ROS), bovine brain, human erythrocytes (hRBC) and human placenta and contained distinct ratios of beta subunits that, upon electrophoresis, migrated as two bands with approximate M(r) of 35,000 and 36,000, as well as distinct complements of at least two gamma subunits each. When tested for their ability to recombine at submaximal concentrations with bv alpha i3, ROS, brain, hRBC and placental beta gamma dimers exhibited apparent affinities that were the same within a factor of two. When bovine brain, placental and ROS beta gamma dimers were tested for their ability to promote deactivation of Gs, brain and placental beta gamma dimers were equipotent and at least 10-fold more potent than that of ROS beta gamma dimers; likewise, brain beta gamma and placental dimers were equipotent in inhibiting GTP-activated and GTP-plus-isoproterenol-activated adenylyl cyclase, while ROS beta gamma dimers were less potent when assayed at the same concentration. The possibility that different alpha subunits may distinguish subsets of beta gamma dimers from a single cell was investigated by analyzing the beta gamma composition of three G proteins, Gs, Gi2 and Gi3, purified to near homogeneity from a single cell type, the human erythrocyte. No evidence for an alpha-subunit-specific difference in beta gamma composition was found. These findings suggests that, in most cells, alpha subunits interact indistinctly with a common pool of beta gamma dimers. However, since at least one beta gamma preparation (ROS) showed unique behavior, it is clear that there may be mechanisms by which some combinations of beta gamma dimers may exhibit selectivity for the alpha subunits they interact with.