The nucleotide and deduced amino acid sequences of a cDNA encoding a new species of Pregnancy-Specific β1-Glycoprotein (PSβG)

We screened a cDNA library of a human placenta with cDNA for nonspecific cross-reacting antigen, a member of the carcinoembryonic antigen gene family. One of the positive clones, PS34, was found to encode a 426 amino acid protein belonging to pregnancy-specific β₁-glycoprotein (PSβG). The mature PS34 protein consisted of domains, N, A1, A2, B2 and C. The domain-N of PS34 showed sequence similarities of 79.8–83.5% to those of the PSβG members so far reported, indicating PS34 is a new member of PSβG and also of the carcinoembryonic antigen gene family.     

Carcinoembryonic antigen gene family: molecular cloning of cDNA for a PS beta G/FL-NCA glycoprotein with a novel domain arrangement

Human fetal liver contains a family of carcinoembryonic antigen related glycoproteins called the pregnancy specific beta 1 glycoprotein/fetal liver non specific cross-reactive antigen (PS beta G/FL-NCA) glycoprotein family. The sequence for the major FL-NCA species has been reported [Khan et al., Proc. Natl. Acad. Sci. USA 1989, 86 in press]. Two additional CEA-related fetal liver glycoproteins FL-NCA-2 and 3 were cloned, sequenced and expressed. FL-NCA-3 is a new gene family member. It has a unique domain arrangement (N-A2-B2-T) and contains a hydrophobic tail. FL-NCA-3 has a molecular weight of approximately 54 kD and is released from the transfected cells. Like three other members of the family, FL-NCA-3 contains the Arg-Gly-Asp sequence in a position in the N-domain corresponding to complementarity determining region 3 of immunoglobulin. FL-NCA-2 is identical in structure to PS beta G-D previously found in placenta. The PS beta G/FL-NCA glycoprotein family may be involved in processes related to cell adhesion and cell differentiation.     

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.     

Effects of sodium butyrate on the synthesis of human pregnancy-specific beta 1-glycoprotein

Human pregnancy-specific beta 1-glycoprotein (PS beta G) is a polymorphic placental protein which shows strong sequence similarity with the oncofetal protein, carcinoembryonic antigen. To better understand the role of PS beta G in pregnancy, we examined its synthesis and regulation in placental fibroblasts, which had been shown to express the PS beta G gene. The major placental PS beta G is a 72-kDa glycoprotein, while the major fibroblast PS beta G is a 62-kDa species. Administration of sodium butyrate to these fibroblasts slightly stimulated the synthesis of the 62-kDa species but markedly increased the production of two additional PS beta Gs of 72 and 48 kDa. The similarity between the PS beta Gs synthesized by butyrate-treated fibroblasts and human placenta was confirmed by cell-free protein synthesis. Poly(A)+ RNA from butyrate-treated fibroblasts and placenta directed the synthesis of two polypeptides of 48 and 36 kDa, which form the polypeptide backbone of the 72- and 48-kDa glycoproteins. Moreover, the predicted molecular weights of PS beta Gs encoded by the two types of PS beta G cDNA clones were 48,000 and 36,000. Most PS beta G cDNAs identified to date, including the three cDNAs (PSG16, PSG93, and PSG95) isolated in this laboratory, share strong sequence similarity at the 5' region (designated PSG-5') but differ in sequences at their 3' regions. The PSG-5', PSG93-specific, PSG16/PSG93-3', and PSG95-3' probes, which identify the majority of PS beta G mRNAs, hybridized with three PS beta G mRNAs of 2.3, 2.2, and 1.7 kilobases from placental fibroblasts. Butyrate increased the steady-state levels of all three mRNAs. Ribonuclease protection analysis showed that butyrate increased the PS beta G mRNAs containing the PSG-5' or PSG93-specific sequence to approximately 20% of human placental levels. However, unlike human term placenta, which predominantly expressed PS beta G mRNAs with 3'-sequences similar to PSG16/PSG93, the butyrate-treated fibroblasts expressed roughly equal levels of PS beta G mRNAs with the PSG16/PSG93-3' and PSG95-3' ends. All PS beta G cDNAs identified encode proteins with distinct carboxyl termini, suggesting that the composition of the 72-kDa species in placenta and butyrate-treated fibroblasts is likely to be different. Placental fibroblasts provide a unique model for the study of the mechanisms responsible for the differential expression of the PS beta G gene.     

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.     

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.     

Primary structure of nonspecific crossreacting antigen (NCA), a member of carcinoembryonic antigen (CEA) gene family, deduced from cDNA sequence

A cDNA containing the entire coding region for a member of carcinoembryonic antigen (CEA) gene family has been cloned from cDNA library of HLC-1 cells by immunochemical screening with the antibody specific to nonspecific crossreacting antigen (NCA). The cDNA encodes a precursor form of a polypeptide consisting of a 34-residue signal sequence, a 108-residue N-terminal (N-) domain, a 178-residue domain (NCA-I domain) and a 24-residue domain rich in hydrophobic amino acids (M-domain). Each domain has a distinct but homologous amino acid sequence to that of the corresponding domain of CEA. Unlike the coding sequences, the 3'-untranslated sequences differ markedly in the NCA and CEA cDNAs facilitating the preparation of probes that will discriminate between nucleotide sequences for CEA and NCA.     

The A beta 6w302 gene and molecular mechanisms of resistance to the spread of radiation-induced lymphoma in a mouse mutant, survivor-27

Metastatic tumors escape from immune response and spread in the body; survivors are very rare. Novel single exon genes A beta 4-7 and a pseudogene A beta 8 psi have been cloned from survivors. Their protein coding sequences are similar to MHC class II beta H2-Ab cDNA while their promoter is different from MHC promoters. The A beta 4 protein was demonstrated on macrophages (antigen presenting cells). The A beta gene family is genetically unstable in germ line and somatic cells of survivors. Mutants S-27 and S-87/1 lost the A beta 5s5 and acquired the A beta 6w302 gene; the Ab gene mutated in S-27. The proposed mechanism of resistance is molecular instability of the A beta gene family resulting in somatic mutations and wandering immune responses that destroy the tumor in the survivor.     

Molecular cloning of a cDNA for human pregnancy-specific beta 1-glycoprotein:homology with human carcinoembryonic antigen and related proteins

Human pregnancy-specific beta 1-glycoprotein (SP1) plays an essential role in normal pregnancy. It is also a well-characterized oncodevelopmental antigen, expressed aberrantly by all trophoblastic tumors and some other malignant cell types. Here we report the identification of a human placental cDNA encoding the SP1 polypeptide sequence. The coding sequence shows 95% identity at the nucleotide level with a distinct, recently published SP1 cDNA sequence (PSG16). Unexpectedly, the sequence is also highly homologous to the published sequence of human carcinoembryonic antigen (CEA). SP1, CEA and CEA-related nonspecific cross-reacting species thus belong to a group of closely related though antigenically diverse tumor-associated glycoproteins. Comparison of the deduced amino acid sequence of the SP1 cDNA with that of CEA provides insight into the modular nature of these related proteins. This may have implications for the genomic organization and evolution of the CEA gene family.     

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.     

A pregnancy-specific beta 1-glycoprotein, a CEA gene family member, expressed in a human promyelocytic leukemia cell line, HL-60: structures of protein, mRNA and gene

Both genomic and cDNA clones encoding a precursor for a pregnancy-specific beta 1-glycoprotein (PS beta G) belonging to the CEA family, expressed in a human promyelocytic leukemia cell line, HL-60, have been isolated and the entire primary structure of the precursor is deduced. The 335-AA precursor has a 34-AA signal peptide followed by domains of N, IIA, IIB and C, which are encoded by separate exons. The genomic sequence contains extra exons IA and IB between exons N and IIA. Apparently, exon IA is excluded from the mRNA by alternative splicing while IB is a pseudo-exon having a stop codon formed by a deletion of dinucleotide in the middle of the sequence. This provides another mechanism to render exon IB abortive and is different from that we reported for another PS beta G (Biochem. Biophys. Res. Comm. (1988) 156, 68-77).     

Chromosomal localization of the genes encoding two forms of the G protein beta polypeptide, beta 1 and beta 3, in man

The signal-transducing G proteins are heterotrimers composed of three subunits, alpha, beta, and gamma. Multiple distinctive forms of the alpha, beta, and gamma subunits, each encoded by a distinct gene, have been described. To investigate further the structural diversity of the beta subunits, we recently cloned and characterized a novel cDNA encoding a third form of the G protein beta subunit, which we have termed beta 3. The protein corresponding to beta 3 has not yet been identified. The three forms of the beta subunit show 81-90% amino acid sequence identity. Previous studies had localized the human genes for the beta 1 and beta 2 subunits to chromosomes 1 and 7, respectively. The present studies were designed to determine whether the gene encoding beta 3 is linked to either the beta 1 or the beta 2 gene. Genomic DNA was isolated from a panel of rodent-human hybrid cell lines and analyzed by hybridization to cDNAs for beta 1 and beta 3. Discordancy analysis allowed assignment of the beta 3 gene to chromosome 12 and confirmed the previous assignment of the beta 1 gene to chromosome 1. These results were confirmed and extended by using in situ chromosome hybridization, which permitted the regional localization of the beta 1 gene to 1pter----p31.2 and the beta 3 gene to 12pter----p12.3. Digestion of human genomic DNA with 10 restriction enzymes failed to disclose a restriction fragment length polymorphism for the beta 3 gene. These data indicate that there is considerable diversity in the genomic organization of the beta subunit family.     

Characterization of cDNAs of the human pregnancy-specific beta 1-glycoprotein family, a new subfamily of the immunoglobulin gene superfamily

Three highly homologous cDNAs encoding human pregnancy-specific beta 1-glycoprotein (SP1) were isolated from a human placental cDNA library. These cDNAs share greater than 90% nucleotide homology in their coding sequences, and greater than 79% of the encoded amino acids are homologous. Proteins encoded by these cDNAs are very similar to members of the carcinoembryonic antigen family and contain repeating domains, conserved disulfide bridges, and beta-sheet structure typical of the immunoglobulin gene superfamily. However, the high degree of sequence homology and relatively lesser degree of glycosylation among the SP1 proteins suggest that they exist as a unique family instead of being members of the CEA family. Both soluble and potentially membrane-bound forms of SP1 proteins were present in the placenta. Northern blot analysis using specific probes confirmed the expression of multiple mRNA species in human term placenta.     

The 35- and 36-kDa beta subunits of GTP-binding regulatory proteins are products of separate genes

The wide range of functions attributed to GTP-binding regulatory proteins (G proteins) is reflected in the structural diversity which exists among the alpha, beta, and gamma subunits of G proteins. Recently two cDNA clones encoding beta subunits, beta 1 and beta 2, were isolated from bovine and human cDNA libraries. We report here that the beta 2 gene encodes the 35-kilodalton (kDa) component of the beta 35/beta 36 subunit of G proteins and that the beta 1 gene encodes the 36-kilodalton component. The in vitro translation product of the beta 2 cDNA co-migrates with the 35-kDa beta subunit (beta 35), while the in vitro product of the beta 1 cDNA co-migrates with the 36-kDa beta subunit (beta 36) on denaturing polyacrylamide gels. In addition, antisera generated against synthetic beta 2 peptides bind specifically to the beta 35 component of isolated G proteins and to a 35-kDa protein in myeloid cell membranes. Our results suggest that the two beta subunits could serve distinct functions, as they are derived from separate genes which have been highly conserved in evolution.     

Diversity among the beta subunits of heterotrimeric GTP-binding proteins: characterization of a novel beta-subunit cDNA.

Heterotrimeric guanine nucleotide binding proteins transduce signals from cell surface receptors to intracellular effectors. The alpha subunit is believed to confer receptor and effector specificity on the G protein. This role is reflected in the diversity of genes that encode these subunits. The beta and gamma subunits are thought to have a more passive role in G protein function; biochemical data suggests that beta-gamma dimers are shared among the alpha subunits. However, there is growing evidence for active participation of beta-gamma dimers in some G protein mediated signaling systems. To further investigate this role, we examined the diversity of the beta subunit family in mouse. Using the polymerase chain reaction, we uncovered a new member of this family, G beta 4, which is expressed at widely varying levels in a variety of tissues. The predicted amino acid sequence of G beta 4 is 79% to 89% identical to the three previously known beta subunits. The diversity of beta gene products may be an important corollary to the functional diversity of G proteins.