Gonadotropin alpha subunit. Differential processing of free and combined forms in human trophoblast and transfected mouse cells

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.     

Site-specific processing of the N-linked oligosaccharides of the human chorionic gonadotropin alpha subunit

Two forms of the gonadotropin alpha subunit are synthesized in placenta and in human chorionic gonadotropin (hCG)-producing tumors: an uncombined (monomer) form and a combined (dimer) form. These forms show differences in their migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The slower migration of the monomeric form on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been attributed to a different glycosylation pattern. Previous studies demonstrated different roles of each of the two alpha N-linked glycosylation sites (Asn-52 and Asn-78) in secretion of the uncombined subunit and the biologic activity of hCG dimer. To assess the influence of formation of dimer on the processing pattern at the individual sites, we characterized the N-linked oligosaccharides of monomer and dimer forms of recombinant human choriogonadotropin alpha subunit. Two approaches were employed. First, site-directed mutagenesis was used to alter the two N-linked oligosaccharide attachment sites, thus allowing the expression of alpha subunits containing only one glycosylation site. Second, tryptic glycopeptides of the wild-type subunits were examined. Concanavalin A (ConA) binding and sialic acid content indicated that the oligosaccharides at each glycosylation site of the uncombined alpha subunit are processed differently. Oligosaccharides present at Asn-52 are almost exclusively ConA-unbound and contain three sialic acid residues. The majority of Asn-78-linked oligosaccharides are ConA-bound and disialylated. Both sites are processed independently because no significant differences were observed between the oligosaccharides at the same sites in wild-type and mutant monomeric alpha subunits. By contrast, the majority of the oligosaccharides at both glycosylation sites of the dimer alpha are bound to ConA. Thus, combination primarily affects the processing pattern of the Asn-52-linked species. Because glycosylation at this site is essential for hCG assembly and signal transduction, these data imply a critical link between the site-specific processing and hormone function.     

Cell-surface expression of a membrane-anchored form of the human chorionic gonadotropin alpha subunit

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.     

Characterization of mouse integrin alpha3 subunit gene.

Integrin alpha3beta1 (VLA-3) is an adhesion receptor for extracellular matrix proteins including various isoforms of laminin. We have isolated mouse genomic clones encoding the integrin alpha3 subunit and deduced the exon/intron organization. The mouse integrin alpha3 subunit gene is encoded by 26 exons spanning 40 kb. The exon/intron structure of the integrin alpha3 subunit gene resembles that of the integrin alpha6 subunit gene, but differs somewhat from those of other members of the integrin family. We have demonstrated that the cytoplasmic domain splicing variants of the alpha3 subunits (alpha3A and alpha3B) are generated by alternative exon usage. We also cloned the 5'-flanking region and performed a preliminary analysis of its promoter activity in various tumor cell lines with different degrees of integrin alpha3 expression. Following transfection, activity in the luciferase assay was found to be roughly correlated with the expression level of integrin alpha3 as measured by flow cytometry. Furthermore, the luciferase assay was performed with normal and SV-40- or polyoma virus-transformed fibroblasts. In mouse, human, and hamster fibroblasts, higher levels of luciferase expression were observed in transformed cells than in normal cells. This result is consistent with our previous finding that integrin alpha3 expression at both the protein and mRNA levels is enhanced upon oncogenic transformation of fibroblasts by tumor viruses.     

Expression of PiM-and PiZ-mutated forms of the human alpha 1-antitrypsin gene in transfected monkey COS1 cells

The PiZ mutation of the gene coding for alpha 1-antitrypsin results in a serum deficiency of this protein leading to early onset emphysema and liver disease. The PiZ gene has a Z-specific point mutation in exon V together with a point mutation in exon III which is also present in some normal (PiM) individuals. There has thus far been no system to study the effects of PiZ point mutations in tissue culture. We constructed plasmids containing alpha 1-antitrypsin cDNA synthetically altered at either exon III or exon V mutation sites and linked to simian virus 40 promoter sequences. Such constructs with the exon V mutation were transfected into monkey COS1 cells followed by analysis of expression of alpha 1-antitrypsin gene products. COS1 cells normally synthesize virtually no alpha 1-antitrypsin mRNA or protein. alpha 1-Antitrypsin mRNA is transcribed at high levels in cells transfected with either M or Z plasmids. Immunologic staining of COS1 cells within 48 h of transfection localizes alpha 1-antitrypsin protein to specific regions of the cytoplasm. This extranuclear localization is also observed with human HepG2 hepatoma cells, which synthesize alpha 1-antitrypsin at high levels, and with human SK-Hep1 hepatoma cells transfected with an M plasmid. The cloned synthetically altered alpha 1-antitrypsin genes provide a system for dissecting contributions of distinct point mutations to the pathological effects of the PiZ protein.     

eNOS基因在JAR细胞中的表达

本研究应用脂质体介导转染技术将人内皮型一氧化氮合酶 (eNOS)基因转入绒癌细胞系JAR细胞 ,获得转染阳性细胞。用RT PCR和Westernblot技术从基因及蛋白水平对表达产物进行鉴定 ,结果显示 :有较高水平的mRNA转录和特异目的蛋白表达 ;通过免疫细胞化学方法证实 ,转染eNOS基因的阳性JAR细胞与对照组相比 ,有外源eNOS蛋白高表达 ;但是一氧化氮合酶 (NOS)活性及其催化产物NO并没有直接升高 ;使用A2 3187处理则能增加NOS的活性 ,说明在转染的JAR细胞中 ,NOS没有被直接激活     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Construction, purification and characterization of a recombinant baculovirus containing the gene for alpha subunit of human chorionic gonadotropin.

A cDNA encoding the alpha subunit of human chorionic gonadotropin, a placental glycoprotein hormone, was cloned downstream to the viral polyhedrin gene promoter of Autographa california nuclear polyhedrosis virus and the recombinant transfer vector was used to co-transfect Spodoptera frugiperda cells growing in culture. Recombinant baculovirus carrying the alpha hCG gene was detected and isolated after dot hybridization using supernatant from co-transfected cells. Recombinant vAc alpha hCG having a replacement of the viral polyhedrin gene, which is hyper-transcribed very late in the infection cycle, with the alpha hCG cDNA was purified after a single round of plaque purification. Insect cell culture infected with vAc alpha hCG, secreted high levels of hCG which was biologically active.     

Kinetics of folding and assembly of the human chorionic gonadotropin beta subunit in transfected Chinese hamster ovary cells.

We have employed Chinese hamster ovary (CHO) cell lines transfected with either the wild type human chorionic gonadotropin beta (hCG-beta) gene alone (CHO beta cells) or in conjunction with the gene expressing the alpha subunit (CHO alpha,beta cells) to study the folding pathway of the hCG-beta subunit. In both CHO beta and CHO alpha,beta cells, the earliest detectable hCG-beta precursor, p beta 1, which had two of six potential disulfide bonds (34-88 and 38-57) formed, was converted to p beta 2, a form that, following the formation of disulfide bonds between cysteines 9-90 and 23-72, migrated more slowly than p beta 1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. The t1/2 for the conversion of p beta 1 to p beta 2 in CHO alpha,beta and CHO beta cells was 5 min, demonstrating that the alpha subunit had no effect on the rate of this conversion. Furthermore, the tryptic-releasable peptides generated from nonreduced p beta 1 or p beta 2 were the same in both CHO alpha,beta and CHO beta cells. Thus, both the rate and order of disulfide bond formation during the conversion of the folding intermediate p beta 1 into p beta 2 were the same whether or not the alpha subunit was present. A comparison between cell types expressing different alpha/beta subunit ratios revealed that the higher the glycoprotein hormone alpha subunit to beta subunit ratio, the greater the rate and extent of hCG heterodimer assembly.     

Expression of the human angiotensinogen gene in transgenic mice and transfected cells.

We have generated two lines of transgenic mice with integrated copies of a 14-kilobase pair (kb) human DNA fragment containing the angiotensinogen gene, which includes 1.3 kb of 5'- and 3'-flanking regions. In both transgenic lines, a considerable quantity of the correctly initiated and processed angiotensinogen mRNA was detected in the liver and it was detectable in heart. Unexpectedly, mRNA for the transgene was accumulated in the kidney, where is normally the minor source of angiotensinogen, to levels comparable to that in the liver. In addition, an in vitro transfection analysis suggested that the 1.3-kb 5'-flanking sequences are essential for expression of the angiotensinogen gene in hepatic and renal cells and that neither DNA segment within the 14-kb construct contributes significantly to repression of the gene expression in renal cells.     

Hormonal regulation of expression of the endogenous and transfected human growth hormone gene

Expression of the endogenous human GH (hGH) gene in response to glucocorticoids, thyroid hormone, and insulin was studied in cultures of dispersed GH-secreting human pituitary adenomas. Results were compared to those obtained when the hGH gene was transfected into rat pituitary tumor cells (GC). In the human pituitary cells the glucocorticoid dexamethasone [(Dex) 10(-6) M] increased the release of GH and the levels of GH mRNA by 2 to 4-fold (P less than 0.05). T3 (10(-8) M) had no effect on GH mRNA but increased hGH release by 2- to 6-fold (P less than 0.01). Insulin (5 x 10(-9) M) alone had no significant effect on either hGH mRNA or protein, but blunted the effect of Dex. Among 11 of 18 GC cell clones transfected with the hGH gene with detectable hGH mRNA expression, Dex increased hGH mRNA levels in seven and T3 treatment reduced hGH mRNA levels in eight. Conversely, rat GH mRNA levels from the endogenous rat gene were increased by either Dex or T3 in all 18 clones. Insulin alone or in combination with T3 or Dex was found to increase hGH mRNA levels in some cell lines and to decrease hGH mRNA levels in others; these effects were correlated strongly (r = 0.88; P less than 0.001) with the influence of insulin on the endogenous rat GH gene, implying that individual cellular differences can simultaneously affect the insulin responsiveness of both genes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression of the human gonadotropin alpha gene in ectopic and eutopic cells.

We have analyzed the regulation of the alpha gonadotropin gene in eutopic placental cells and ectopic tumor cells by constructing a series of plasmid vectors containing alpha genomic 5' flanking DNA placed upstream of the gene encoding the bacterial enzyme chloramphenicol acetyltransferase (CAT). These plasmid DNAs were transfected into a eutopic (JAr) and an ectopic (HeLa) cell line. Both cell types expressed the CAT gene from plasmid constructs containing as much as 1,500 base pairs (bp) and as little as 140 bp of alpha 5' flanking DNA; JAr cells were considerably more efficient than HeLa cells. Ectopic and eutopic cells differed qualitatively in their expression from these alpha-CAT constructs when cells were treated with cAMP or butyrate. Butyrate induced alpha expression in HeLa cells but not in JAr cells, while cAMP induced expression in JAr cells. These results are consistent with and extend previous observations suggesting that there are cell-specific differences in the regulation of alpha gene expression in ectopic and eutopic cells. However, by using deletion constructs of the alpha-CAT gene, we found that the basal expression and cell-specific induction of the alpha gene in ectopic and eutopic cells were dependent on the same 140 bp of alpha 5' flanking DNA. These 140 bp were sequenced and found to contain a 9-bp stretch of DNA homologous with the consensus viral enhancer sequence. Such features of alpha expression common to both ectopic and eutopic cells may be involved in the coordinate expression of the alpha gene and the tumorigenic phenotype observed in each cell type.     

Mapping of the phosphorylase kinase alpha subunit gene on the mouse X chromosome

Phosphorylase kinase is a glycogenolytic enzyme in several animal tissues. Within the last few years all four subunits of the enzyme have been cloned. The beta, gamma, and delta subunits are known to be autosomal. We have mapped the alpha subunit of phosphorylase kinase, recently cloned by Zander et al. (1988), in an interspecific mouse pedigree and localized it on the X chromosome, where it maps between the X-linked zinc finger protein and phosphoglycerate kinase genes, close to the latter. In man and mouse several X-linked disorders of this enzyme have been described. Although the X-linked phosphorylase kinase deficiency in mice may be caused by a mutation in the structural gene for the alpha subunit, mapped here, the existence of a separate regulatory locus, important in the normal expression or function of the enzyme in muscle, still remains a possibility.