Genetic fusion of an alpha-subunit gene to the follicle-stimulating hormone and chorionic gonadotropin-beta subunit genes: production of a bifunctional protein.

The human glycoprotein hormones, hCG, TSH, LH, and FSH, are composed of a common alpha-subunit assembled to a hormone-specific beta-subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers but not as multimers. LH/FSH are synthesized in the pituitary gonadotrophs, and several of the alpha-subunit sequences required for association with either the LHbeta or FSHbeta subunits are different. Thus, it is intriguing that no ternary complexes are observed for LH and FSH in vivo (e.g. two different beta-assembled to a single alpha-subunit). To examine whether the alpha-subunit can interact with more than one beta-subunit, and to study the conformational relationships between the ligand and the receptor, we constructed a vector encoding two tandemly arranged beta-subunits fused to a single alpha-subunit gene (FSHbeta-CGbeta-alpha). This approach permitted structure-function analyses of alpha/beta domain complexes without the possibility of subunit dissociation. We reported previously that the CGbeta or FSHbeta subunit gene can be genetically fused to the alpha-gene and the resulting single chains (CGbetaalpha and FSHbetaalpha, respectively) were biologically active. Here we demonstrate that a triple-domain single chain bearing the configuration FSHbeta-CGbeta-alpha is efficiently secreted from transfected Chinese hamster ovary (CHO) cells and exhibits high-affinity receptor binding to both FSH and LH/hCG receptors, comparable to the native heterodimers. These results indicate that the alpha-subunit can interact with each beta-subunit in the same complex and that an alpha-domain fused to a beta-domain can still interact with an additional beta-subunit. The data also demonstrate the remarkable flexibility of the receptor to accommodate the increased bulkiness of the triple-domain ligand. In addition, the formation of intrachain FSH- and CG-like complexes observed in a triple-domain single chain suggests that the alpha-subunit can resonate, i.e. shuttle between alpha-beta heterodimeric intermediates during the early stages of synthesis and accumulation in the endoplasmic reticulum. Such model compounds could be useful as substrates to generate a new class of analogs in which the ratio of the LH/FSH activity is varied. This could aid in the design of analogs that could be used to mimic the in vivo hormonal profiles.     

Three brain sodium channel alpha-subunit genes are clustered on the proximal segment of mouse chromosome 2.

We have used long-range physical mapping and restriction fragment length polymorphisms between two mouse species to determine the chromosomal organization and location of the genes encoding three distinct isoforms of the alpha-subunit of the brain sodium channel. Physical mapping by pulsed-field gel electrophoresis has established that Scn2a and Scn3a (genes encoding type II and type III sodium channel alpha-subunit isoforms) are physically linked and are separated by a maximum distance of 600 kb. The segregation of restriction fragment length variations in backcross progeny of a Mus musculus and Mus spretus mating indicates that Scn 1 a (gene encoding the type I sodium channel alpha subunit) and Scn2a are tightly linked and are separated by a distance of 0.7 cM. Linkage analysis in backcross and recombinant inbred (BXD and AKXD) strains of mice localized the three sodium channel genes to the proximal segment of mouse chromosome 2 and suggested the probable gene order centromere-Hc-Neb-Pmv7-Scn2a/Scn3a-Scn1a-Mpmv 14. These results indicate that the three isoforms of the brain sodium channel alpha-subunit are encoded by three distinct genes that share a common ancestral origin.     

Structural homologies between the amino acid sequence of Clostridium pasteurianum MoFe protein and the DNA sequences of nifD and K genes of phylogenetically diverse bacteria

The complete amino acid sequence of the larger (alpha-) subunit and about 70% of the total sequence of the smaller (beta-) subunit of the MoFe protein from Clostridium pasteurianum was determined by analyses of peptides derived from BrCN cleavage and by digestions with trypsin, staphylococcal protease and lysylendo-peptidase of the separated subunits. The alpha-subunit has 529 amino acid residues, giving an Mr value of 58 774. This is the first complete sequence for the alpha-subunit of an isolated MoFe protein. In comparing the sequences of both subunits to those from other sources, 5 out of 9 cysteines in the alpha-subunit and 3 out of 6 in the beta-subunit are invariant, thus suggesting a function as ligands to FeS and MoFeS clusters in the MoFe protein. All of these cysteines are located in the amino terminal halves of both subunits.     

Cloning and structural characterization of the genes coding for adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii.

The structural genes coding for both subunits of adenosylcobalamin-dependent methylmalonyl-CoA mutase from the Gram-positive bacterium Propionibacterium shermanii have been cloned, with the use of synthetic oligonucleotides as primary hybridization probes. The genes are closely linked and are transcribed in the same direction. Nucleotide sequence analysis of 4.5 kb of DNA encompassing both genes allowed us to infer the complete amino acid sequence of the two subunits: the beta-subunit is the product of the upstream gene, and consists of 638 amino acid residues (Mr 69465) and the alpha-subunit consists of 728 amino acid residues (Mr 80,147). There is a very close structural homology between the two subunits, reflecting the probable duplication of a common ancestral gene. A sequence present only in the alpha-subunit is significantly homologous to a portion of the sequence of the methylmalonyl-CoA-binding subunit of transcarboxylase from P. shermanii [Samols, Thornton, Murtif, Kumar, Haase & Wood (1988) J. Biol. Chem. 263, 6461-6464], and this homologous region may form part of the CoA ester-binding site in both enzymes.     

Evolution of placenta-specific gene expression: comparison of the equine and human gonadotropin alpha-subunit genes.

Primate and equine species are thought to be unique among mammals in synthesizing placental gonadotropin glycoprotein hormones. Human chorionic gonadotropin (CG) and equine pregnant mare's serum gonadotropin (PMSG) are produced in placenta by the specific activation of a glycoprotein hormone alpha-subunit gene and a corresponding beta-subunit gene. The evolutionary mechanisms for the apparently independent acquisition of tissue specificity were investigated by cloning the 5' flanking region of the equine alpha-subunit gene and comparing the DNA elements and trans-acting factors involved in placental expression. We find that though the equine gene is expressed and induced by cAMP, it does not contain the elements known to confer tissue-specific expression to the human gene, the cAMP response element (CRE) and the trophoblast-specific element (TSE), nor does it bind to the trans-acting factors CREB and TSEB. Instead, an additional factor (alpha-ACT) is found which binds to the equine and human, but not the murine, alpha-subunit genes in a region between the positions of the CRE and TSE and confers cAMP responsiveness.     

Expression and regulation of testicular inhibin alpha-subunit gene in vivo and in vitro

Inhibin, a gonadal peptide, selectively suppresses FSH release from the pituitary. The cDNAs coding for ovarian inhibin have been isolated and characterized. However, little is known about testicular inhibin. In this study we have isolated inhibin alpha-subunit cDNA from human testicular cDNA libraries and determined inhibin alpha-subunit mRNA levels in testes. The longest cDNA isolated from human testis was 1380 nucleotides long and contained a nucleotide sequence identical to that of human placental inhibin alpha-subunit and isolated human inhibin alpha-subunit gene, but different from human ovarian inhibin alpha-subunit in two amino acids in the signal peptide. A single 1.5-kilobase species of inhibin alpha-subunit mRNA was identified in the testes of several species. This mRNA was the same size as those in human ovary and placenta. The regulation of inhibin alpha-subunit mRNA in rat testis was next examined. The concentration of testicular inhibin alpha-subunit mRNA peaked between 20-25 days of age and gradually declined thereafter. Hypophysectomy decreased testicular inhibin alpha-subunit mRNA levels. Supplementation of hypophysectomized animals with FSH restored inhibin alpha-subunit mRNA levels to those in intact controls. By contrast, treatment with testosterone had no effect. Similarly, in Sertoli cell-enriched cultures, FSH, but not testosterone, increased inhibin alpha-subunit mRNA levels. We conclude that 1) human testicular inhibin alpha-subunit mRNA is similar to that of human ovary and placenta; and 2) inhibin alpha-subunit mRNA in Sertoli cells is regulated by FSH, but not testosterone, both in vivo and in vitro.     

Study of expression of Na+,K+-ATPase subunit genes in the human brain using cDNA libraries

Human brain cDNA libraries were screened with cDNA inserts corresponding to the mRNA for the Na+,K(+)-ATPase alpha-subunit from pig kidney. The results obtained demonstrate the existence of two highly homologous mRNAs encoding the alpha- and alpha III-isoforms of the Na+,K(+)-ATPase catalytic subunit.     

The characterization of alpha-subunit of glycoprotein hormone produced by undifferentiated carcinoma

The glycoprotein hormone alpha-subunit was extracted and purified from the urine of a patient with undifferentiated carcinoma producing isolated alpha-subunit. Its final specific immunoactivity was 0.92 (mg alpha-subunit/mg protein). The alpha-subunit exhibited virtually identical immunoantigenicity to hCG-alpha antiserum with standard hCG-alpha. The molecular weight of the alpha-subunit determined by gel chromatography on Sephadex G-100 was greater than that of standard hCG-alpha dissociated by urea in vitro. By SDS disc electrophoresis, however, the alpha-subunit moved faster than hCG-alpha separated by mercaptoethanol reduction. The amino acid composition of the alpha-subunit was quite similar to that of standard hCG-alpha. In the isoelectric focusing, the major components of the alpha-subunit from undifferentiated carcinoma and the alpha-subunit from urine of normal pregnant women (third trimester) were distributed over the range from pH 3.5 to 6.0, while standard hCG-alpha was distributed in the fractions ranging from pH 6.0 to 8.0. The result of a combination study in vitro indicated that both alpha-subunits from undifferentiated carcinoma and from urine of normal pregnant women did not actively combine with hCG-beta. These results suggest that the alpha-subunit secreted by undifferentiated carcinoma is virtually identical with standard hCG-alpha as the protein moiety but differs in regard to carbohydrate moiety, and also suggest that the excess of alpha-subunit, which is not associated with beta-subunit, may have undergone some intracellular modification, and consequently, the electric charge of the freely secreted alpha-subunit changes and it no longer has the ability to combine with the beta-subunit.     

Genomic structure and homoeologous relationship of the two alpha-subunit genes of a heterotrimeric GTP-binding protein in tobacco.

A heterotrimeric GTP-binding protein (G protein) plays a number of important roles in the signal-transduction pathways of eukaryotic cells. The allotetraploid tobacco genome has two alpha-subunit genes, NtGA1 and NtGA2, of the heterotrimeric G protein. In this study, we determined the nucleotide sequences and the exon-intron structures of the NtGA loci in tobacco and its ancestral diploid species. The genomic sequences of the NtGA loci were interrupted by 13 introns. The sizes of most exons (12 of 14) were completely conserved among the NtGA genes and the Arabidopsis alpha-subunit gene (GPA1), but most introns (11 of 13) in the NtGA genes were longer than those in GPA1. In comparison with the genomic sequences of the NtGA orthologues of ancestral Nicotiana sylvestris and Nicotiana tomentosiformis, the tobacco NtGA1 and NtGA2 were concluded to be homoeologous and assigned to the S and T genomes, respectively. More than 300 mutations including insertions-deletions (indels) and nucleotide substitutions were found in the intron regions between the NtGA1 and NtGA2 loci, whereas the exon sequences were highly conserved among these and GPA1. The structural comparison revealed larger divergence at the NtGA2 locus than at NtGA1.     

Sequence and molecular analysis of the rpoA cluster genes from Xanthomonas campestris pv. campestris

The Xanthomonas campestris rpsM (S13)-rpsK (S11)-rpsD (S4)-rpoA (alpha)-rplQ (L17) cluster, encoding RNA polymerase alpha-subunit and four ribosomal proteins, reside in a 3164-bp DNA region. The N-terminal sequence of the authentic alpha-protein determined chemically matches that predicted from the nucleotide sequence. rplQ is monocistronic, instead of being co-transcribed with the other genes as in Escherichia coli. Antiserum against the His-tagged alpha-protein cross-reacted with the E. coli alpha-protein.     

Hypermethylation of the inhibin alpha-subunit gene in prostate carcinoma

Inhibin is composed of an alpha- and a beta-subunit. Transgenic studies assigned a tumor-suppressive role to the inhibin alpha-subunit, and in human prostate cancer inhibin alpha-subunit gene expression was down-regulated. This study examined the inhibin alpha-subunit gene promoter and gene locus to determine whether promoter hypermethylation or LOH occurred in DNA from prostate cancer. The 5'-untranslated region of the human inhibin alpha-subunit gene was sequenced and shown to be highly homologous to the bovine, rat, and mouse inhibin alpha-subunit promoter sequences. A 135-bp region of the human promoter sequence that continued a cluster of CpG sites was analyzed for hypermethylation. Significant (P < 0.001) hypermethylation of the inhibin alpha-subunit gene promoter occurred in DNA from Gleason pattern 3, 4, and 5 carcinomas compared with nonmalignant tissue samples. A subset of the carcinomas with a cribriform pattern were unmethylated. LOH at 2q32-36, the chromosomal region harboring the inhibin alpha-subunit gene, was observed in 42% of prostate carcinomas. These data provide the first demonstration that promoter hypermethylation and LOH are associated with the inhibin alpha-subunit gene and gene locus in prostate cancer.     

Characterization of glycoprotein hormone free alpha-subunit from human pituitary and placenta extracts

Standard alpha-subunit dissociated from glycoprotein hormones differs from individual (free) alpha-subunit found in sera or in cell culture media; secreted free alpha-subunit is larger, more acidic and lacks the ability of recombining in vitro with standard hCG-beta. It is unclear whether the large free alpha-subunit is only a secretory product or whether it is also present in tissue. Herein were studied the molecular size, the isoelectric pH, and the recombining activity of free alpha-subunit obtained from pituitary and placenta extracts. Sephadex exclusion chromatography showed the presence of both a large and a small form, and a changing large/small free alpha-subunit ratio in the various extracts. Most of the large form obtained from placenta extracts electrofocused into two peaks of pI 4.8 and 5.1. The large form showed no recombining activity with standard hCG-beta while the small free alpha-subunit recombined as well as did standard hCG-alpha. The observation of three common characteristics (a larger size, a pI 4.8, and a lack of recombining activity) suggests a similarity between the large secreted form and a fraction of the free alpha-subunit in tissue.     

Na+,K+-ATPase: tissue-specific expression of genes coding for alpha-subunit in diverse human tissues

The expression of genes coding for alpha and alpha III isoforms of Na+,K+-ATPase alpha-subunit has been studied in human kidney, brain, thyroid and liver cells. The expression was shown to be subjected to a tissue-specific control and also depended on the developmental stage. The tissue-specific expression of genes coding for different isoforms of the catalytic subunit of Na+,K+-ATPase perhaps may be attributed to various functions of proteins belonging to this family.     

Heterogeneity of free alpha-subunit in term placenta

Free alpha-subunit in normal term placenta was examined for molecular weight, electric charge and ability to combine with standard hCG-beta in comparison with extracellular free alpha-subunit and standard hCG-alpha dissociated from urinary hCG in vitro. The gel chromatography on Sephadex G-100 of the placental extract revealed three major immunoreactive hCG-alpha peaks, designated as P alpha-A (Kav = 0.32-0.46), P alpha-B (0.47-0.58) and P alpha-C (0.59-0.70), near the position of standard hCG-alpha. In the isoelectric focusing, while P alpha-A was mainly distributed over the acidic region, the major components of P alpha-B and P alpha-C were distributed over the basic region. Furthermore, in the combination study with standard hCG-beta, such a alpha-subunit with acidic pI scarcely showed any combining activity whereas alpha-subunit with basic pI revealed significant combining activity. These results suggest the following possibilities: that 1) the various size species of placental alpha-subunit may be responsible for the progressive glycosylation; 2) the small alpha-subunit with basic pI may combine with beta-subunit to form immunoreactive hCG; 3) the alpha-subunit, which has not associated with beta-subunit, may be converted to a large and incombinative form with acidic pI by further glycosylation, followed by secretion as a free alpha-subunit.