Capturing protein interactions in the secretory pathway of living cells

The secretory pathway is composed of membrane compartments specialized in protein folding, modification, transport, and sorting. Numerous transient protein-protein interactions guide the transport-competent proteins through the secretory pathway. Here we have adapted the yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) to detect protein-protein interactions in the secretory pathway of living cells. Fragments of YFP were fused to the homooligomeric cargo-receptor lectin endoplasmic reticulum Golgi intermediate compartment (ERGIC)-53, to the ERGIC-53-interacting multi-coagulation factor deficiency protein MCFD2, and to ERGIC-53's cargo glycoprotein cathepsin Z. YFP PCA analysis revealed the oligomerization of ERGIC-53 and its interaction with MCFD2, as well as its lectin-mediated interaction with cathepsin Z. Mutation of the lectin domain of ERGIC-53 selectively decreased YFP complementation with cathepsin Z. Using YFP PCA, we discovered a carbohydrate-mediated interaction between ERGIC-53 and cathepsin C. We conclude that YFP PCA can detect weak and transient protein interactions in the secretory pathway and hence is a powerful approach to study luminal processes involved in protein secretion. The study extends the application of PCA to carbohydrate-mediated protein-protein interactions of low affinity.     

A naturally occurring genetic variant in the human chorionic gonadotropin-beta gene 5 is assembly inefficient.

The hCGbeta gene family is composed of six homologous genes linked in tandem repeat on chromosome 19; the order of the genes is 7, 8, 5, 1, 2, and 3. Previous studies have shown that hCGbeta gene 5 is highly expressed during the first trimester of pregnancy. The purpose of our study was to identify naturally occurring polymorphisms in hCGbeta gene 5 and determine whether these alterations affected hCG function. The data presented here show that hCGbeta gene 5 was highly conserved in the 334 asymptomatic individuals and 41 infertile patients examined for polymorphisms using PCR followed by single stranded conformational polymorphism analysis. Most of the polymorphisms detected were either silent or located in intron regions. However, one genetic variant identified in beta gene 5 exon 3 was a G to A transition that changed the naturally occurring valine residue to methionine in codon 79 (V79M) in 4.2% of the random population studied. The V79M polymorphism was always linked to a silent C to T transition in codon 82 (tyrosine). To determine whether betaV79M hCG had biological properties that differed from those of wild-type hCG, a beta-subunit containing the V79M substitution was created by site-directed mutagenesis and was coexpressed with the glycoprotein hormone alpha-subunit in Chinese hamster ovary cells and 293T cells. When we examined betaV79M hCG biosynthesis, we detected atypical betaV79M hCG folding intermediates, including a betaV79M conformational variant that resulted in a beta-subunit with impaired ability to assemble with the alpha-subunit. The inefficient assembly of betaV79M hCG appeared to be independent of beta-subunit glycosylation or of the cell type studied, but, rather, was due to the inability of the betaV79M subunit to fold correctly. The majority of the V79M beta-subunit synthesized was secreted as unassembled free beta. Although the amount of alphabeta hCG heterodimer formed and secreted by betaV79M-producing cells was less than that by wild-type beta-producing cells, the hCG that was secreted as alphabeta V79M heterodimer exhibited biological activity indistinguishable from that of wild-type hCG.     

Diagnosis of hydatidiform mole and persistent trophoblastic disease: diagnostic accuracy of total human chorionic gonadotropin (hCG), free hCG {alpha}- and {beta}-subunits, and their ratios

OBJECTIVE: Human chorionic gonadotropin (hCG) is widely used in the management of hydatidiform mole and persistent trophoblastic disease (PTD). Predicting PTD after molar pregnancy might be beneficial since prophylactic chemotherapy reduces the incidence of PTD. DESIGN: A retrospective study based on blood specimens collected in the Dutch Registry for Hydatidiform Moles. A group of 165 patients with complete moles (of which 43 had PTD) and 39 patients with partial moles (of which 7 had PTD) were compared with 27 pregnant women with uneventful pregnancy. METHODS: Serum samples from patients with hydatidiform mole with or without PTD were assayed using specific (radio) immunoassays for free alpha-subunit (hCGalpha), free beta-subunit (hCGbeta) and 'total' hCG (hCG + hCGbeta). In addition, we calculated the ratios hCGalpha/hCG + hCGbeta, hCGbeta/hCG + hCGbeta, and hCGalpha/hCGbeta. Specificity and sensitivity were calculated and paired in receiver-operating characteristic (ROC) curve analysis, resulting in areas under the curves (AUCs). RESULTS: hCGbeta, hCGbeta/hCG + hCGbeta and hCGalpha/hCGbeta show AUCs ranging between 0.922 and 0.999 and, therefore, are excellent diagnostic tests to distinguish complete and partial moles from normal pregnancy. To distinguish partial from complete moles the analytes hCGbeta, hCG + hCGbeta and the ratio hCGalpha/hCGbeta have AUCs between 0.7 and 0.8. Although hCGalpha, hCGbeta and hCG + hCGbeta concentrations are significantly elevated in patients who will develop PTD compared with patients with spontaneous regression after evacuation of their moles, in predicting PTD, these analytes and parameters have AUCs <0.7. CONCLUSIONS: Distinction between hydatidiform mole and normal pregnancy is best shown by a single blood specimen with hCGbeta, but hCGbeta/hCG + hCGbeta and hCGalpha/hCGbeta are also excellent diagnostic parameters. To predict PTD, hCGalpha, hCGbeta, hCG + hCGbeta and hCGalpha/hCGbeta are moderately accurate tests, although they are not accurate enough to justify prophylactic chemotherapy treatment for prevention of PTD.     

Human chorionic gonadotropin Molecular forms, detection, and clinical implications.

Different molecular forms of human chorionic gonadotropin (hCG) have been identified in biologic fluids of patients with various physiopathologic processes. These materials include (a) the intact heterodimer hCG comprising two mature a and beta subunits, and (b) the uncombined or free forms of the a (hCGalpha) and beta subunit (hCGbeta), and several fragments o f hCG such as the nicked forms o f both hCG and free hCGbeta and its ending degradation product, the beta-core fragment or hCGbetacf The determination of hCG and related molecules in biologic fluids is usually achieved by immunologic procedures, but discrepancies among kits remain a problem in clinical practice. Specific measurements of hCG and of, independently, its free beta subunit are important in the diagnosis and follow-up of either trophoblastic diseases or testicular cancers, whereas only the free hCGbeta has to be assayed for detection in nongonadal and nonplacental tumors.     

The beta-subunit of human chorionic gonadotrophin exists as a homodimer

The free beta-subunit of human chorionic gonadotrophin (hCGbeta) is well recognised as a product of many epithelial tumours. Recently, it has been shown that this ectopic production may have a functional relationship to tumour growth. The growth-promoting activity of hCGbeta may be explained by its structural similarity to a family of growth factors which all contain the same distinct topological fold known as the cystine-knot motif. Since the other members of this family all exhibit their activities as homo- and heterodimers, it is possible that the same may be true for hCGbeta. Using size-exclusion chromatography, low stringency SDS-PAGE and matrix assisted laser desorption/ionisation (MALDI) time-of-flight (TOF) mass spectrometry (MS) we have shown that pure preparations of hCGbeta contain hCGbetabeta homodimers. Size-exclusion chromatography revealed asymmetric elution profiles with a forward peak corresponding to the size-exclusion characteristic of a globular protein with an approximate mass of 44-54 kDa and a late shoulder centered around an elution position expected for a globular protein of approximately 29 kDa. Two immunoreactive hCGbeta species, of approximately 32 and 64 kDa, were clearly resolved by SDS-PAGE and Western blotting. When analysed by MALDI-TOF MS a |mf23 kDa monomer and a |mf46 kDa dimer were identified. Formation of hCGbetabeta homodimers is consistent with the behaviour of other cystine-knot growth factors and strengthens the inclusion of the glycoprotein hormones within this superfamily. It has yet to be determined whether it is this dimeric molecular species that is responsible for growth-promoting activity of hCGbeta preparations in tumours.     

Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalently associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hCGalpha and hCGbeta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Co-expression of both subunits in the same cell resulted in secretion of heterodimeric hCG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stimulating a biological response in vitro. Surprisingly, the maximal response obtained was twice that obtained with the native hCG. The level of expression of hCG achieved was 12-16 mg/l secretory medium and is expected to increase several-fold in a fermentor. Thus the Pichia expression system is capable of hyperexpressing properly folded, biologically active hCG and is suitable for structure-function studies of the hormone.     

A biologically active single chain human chorionic gonadotropin analog with altered receptor binding properties

hCG is a heterodimer consisting of an alpha-subunit common among all members of the glycoprotein hormone family, LH, FSH, and TSH, and a unique beta-subunit responsible for receptor specificity. Biologically active single chain analogs of these hormones have been engineered in which the C-terminus of the beta-subunit was fused to the N-terminus of the alpha-subunit (N-beta-alpha-C) either with or without a linker such as the hCGbeta C-terminal peptide (CTP). This tandem order of subunits was chosen based on studies suggesting that the N-terminal region of hCGbeta and particularly the C-terminal region of the alpha-subunit are important in receptor binding and activation. Single chain hCG (YhCG1) can, in turn, be fused to the LH receptor to yield a hormone-receptor complex that is biologically active in transfected cells. Herein, we report the construction of a new single chain hCG analog (YhCG3) in which the C-terminus of the alpha-subunit is fused to the N-terminus of hCGbeta via a CTP (N-alpha-CTP-beta-C). Compared with YhCG1, this analog binds receptor with a 25- to 30-fold lower affinity, but, surprisingly, is capable of stimulating intracellular cAMP levels to the same extent. Furthermore, YhCG3 can be covalently linked to its receptor to produce a biologically active complex that results in elevated levels of basal cAMP in transfected cells. These results suggest that free N- and C-termini of hCGbeta and the alpha-subunit, respectively, are not essential for receptor binding and activation and that YhCG3 is in a more efficacious conformation for receptor activation than YhCG1.     

Processing of O-linked glycosylation in the chimera consisting of alpha-subunit and carboxyl-terminal peptide of the human chorionic gonadotropin beta-subunit is affected by dimer formation with follicle-stimulating hormone beta-subunit

hCG, LH, FSH, and TSH are a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit, but differ in their hormone-specific beta-subunits. hCGbeta is unique among beta-subunits due to a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharides. We previously reported that there were differences in O-glycosylation between two chimeras consisting of alpha-subunit and CTP, i.e. a variant with CTP at the N-terminal region (Calpha) and another analog with CTP at the C-terminus (alphaC) of the alpha-subunit. To address whether O-glycosylation is influenced by the heterodimer formation, Calpha and alphaC were expressed alone or with FSHbeta-subunit in Chinese hamster ovary cells. The O-linked glycosylation was assessed by continuous labeling with [(35)S]methionine/cysteine, immunoprecipitation with anti-alpha or anti-FSH serum, serial digestion with endoglycosidase-F and neuraminidase, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The decrease in molecular weight of dimeric chimeras digested with endoglycosidase-F was greater in Calpha than that in alphaC after treatment with neuraminidase, revealing that both chimeras have different numbers of sialic acids on O-linked carbohydrates. By treating with endoglycosidase-F, the dimeric alphaC migrated faster than its free form, whereas the mobility difference between assembled and unassembled forms of Calpha was very little. These data indicate that processing of O-glycosylation is affected by the backbone polypeptide chain(s).     

Evidence for altered synthesis of human chorionic gonadotropin in gestational trophoblastic tumors.

Tissue extracts of molar tissue or choriocarcinoma, blood, or urine of 21 women with gestational trophoblastic disease were analyzed for hCG and its subunits. The extracts or biologic fluids were initially chromatographed through a standardized Sephadex G-100 column. Each fraction was radioimmunoassayed in homologous hCG, hCGalpha, and hCGbeta assays. Extracts of four hydatidiform moles contained primarily hCG but no free alpha subunit of that hormone. One of the molar extracts contained a small amount of free hCGbeta not observed in the small amount of free hCGbeta not observed in the extracts of the other three moles. Plasma and urine samples from 18 women with localized or metastatic gestational trophoblastic disease contained hCG but no free alpha or beta subunits; the neoplasms of those patients readily responded to chemotherapy, and all patients have had no evidence of disease for at least one year. The major portion of immunologic hCG present in the biologic samples was indistinguishable from native hCG in its physical behavior on Sephadex G-100 chromatography. On the other hand, three women with widely metastatic tumors died in spite of extensive chemotherapy. Extracts of tumors from two of those patients contained hCG and free subunits of hCG. The urine and plasma of the third patient contained hCG and hCGalpha with the concentration of hCGalpha far exceeding that for hCG in urine. The results of these studies clearly show a disparity between the forms of hCG found in the normal placenta and pregnancy sera, as previously reported, and those found in the neoplastic trophoblast with varying degrees of anaplasia. The most striking finding was the absence of free circulating hCGalpha in the sera in patients with gestational trophoblastic disease which responded to chemotherapy, since free hCGalpha is readily detectable in the sera of normally pregnant women.     

Unmasking a new recognition signal for O-linked glycosylation in the chorionic gonadotropin beta subunit

hCGbeta subunit is distinguished among the other members of the family of the glycoprotein hormones by the presence of four serine O-linked oligosaccharide units in the last 25 amino acids. This carboxy terminal peptide (CTP) influences the intracellular behavior of the subunit and is important for maintaining the biological half-life of hCG. To examine how the O-linked oligosaccharides affect the metabolic behavior of hCG, we generated a CGbeta mutant devoid of the native O-linked acceptor sites. An alternative site not used in the native subunit was glycosylated and the structure of this oligosaccharide differed from the wild-type O-linked carbohydrates. This glycosylation occurred at serine 130 in the CTP and in contrast to the wild type O-linked oligosaccharides, sialic acid is a major component of the alternatively linked carbohydrate. The data show that deleting the native acceptor sites exposes a new site for O-glycosylation and promotes a differential intracellular processing of the beta subunit. These results support the hypothesis that the CTP participates in the folding of the newly synthesized subunit, which is manifested by the post-translational changes reported here.     

Synthesis of chorionic gonadotropin subunits in human choriocarcinoma clonal cell line JEG-3: carbohydrate differences in glycopeptides from free and combined alpha-subunits

The glycoprotein hormone hCG and its free alpha-subunit are secreted by the clonal choriocarcinoma cell line JEG-3. Free hCG alpha has a larger apparent mol wt (22,000-24,000) than the combined hCG alpha (18,000-19,000) obtained by dissociation of the hCG secreted by these cells. Techniques developed for the specific isolation and purification of the free and combined hCG alpha forms and for the preparation of glycopeptides from these subunits have permitted detection of the incorporation of D-[3H]glucosamine [( 3H]GlcN) and L-[3H]fucose into both alpha-subunit forms. Relative to their [35S]methionine content, 2.3-fold more [3H]GlcN and 6-fold more L-[3H]fucose were incorporated into free hCG alpha than into combined hCG alpha. Analyses of [3H]GlcN glycopeptides prepared from free and combined hCG alpha indicate that the 22,000- to 24,000-dalton subunit form contained more [3H]GlcN and 27% more of the GlcN metabolite N-acetylneuraminic acid than the 18,000- to 19,000-dalton hCG alpha-subunit, than both hCG alpha forms contained two major N-linked oligosaccharide chains differing primarily in their NeuAc content, and that most of the [3H]GlcN was incorporated as GlcN or metabolites of GlcN other than N-acetylneuraminic acid. These studies provide direct chemical evidence of a higher content of carbohydrate in the larger free alpha-subunit form.     

Effect of individual N-glycosyl chains in the beta-subunit on the conformation of human choriogonadotropin

Human choriogonadotropin is a heterodimeric glycoprotein hormone comprised of noncovalently associated alpha- and beta-subunits. Each of the subunits has two N-glycosyl chains. In our previous communication, we investigated the role of individual carbohydrate chains in the alpha-subunit on the signal transduction function and conformation of the hormone. This paper deals with the effect of individual or both N-glycosyl chains in the beta-subunit on the function and conformation of the monomer as well as of the heterodimer. Three mutants each of hCGbeta and hCG lacking N-glycosyl chains at beta13Asn, beta30Asn and beta13,30Asn were prepared by site-directed mutagenesis by replacing Thr residues in the recognition triplet sequence at beta15 and beta32 positions with Gln. All mutant heterodimers had receptor binding and cAMP and progesterone stimulating activities comparable to wild type hCG. While the loss of carbohydrate at beta13Asn or beta13,30Asn in the case of hCGbeta monomer resulted in a 4-6%, decrease in the ordered structure, the loss of the glycosyl chain at beta 30Asn did not alter the conformation as compared with the wild type hCGbeta. Similarly, all carbohydrate deficient hCG heterodimers had a decrease of 6-8%) in the ordered structure as compared with hCG. Thus, while the individual N-glycosyl chains did not affect the function of the hormone, they did have marked effect on its conformation but the conformational changes were localized and did not perturb the receptor binding and signal transduction sites.     

O-glycosylation of the alpha-subunit does not limit the assembly of chorionic gonadotropin alpha beta dimer in human malignant and nonmalignant trophoblast cells

Biosynthetic experiments were carried out in cultures of human malignant trophoblast cells (the JAR cell line) and in explants of normal first trimester human placental tissue to test the hypothesis that the O-glycosylation of the glycoprotein hormone alpha-subunit at Thr-39 regulates the assembly of the CG alpha beta dimer. This modification of alpha has been shown to ablate its ability to combine in vitro with the beta-subunit of bovine LH and might explain why JAR cells and placental explants secrete uncombined alpha- and beta-subunits in addition to the hCG alpha beta dimer. We have previously detected an O-linked carbohydrate chain at Thr-39 in preparations of secreted free alpha-subunit, but not dimer CG alpha from JAR culture medium. We report here evidence that the O-glycosylation of alpha does not regulate the biosynthetic assembly of the hCG dimer in cultures of JAR choriocarcinoma cells or first trimester placental explants. The intracellular precursor forms of alpha and beta that accumulate in the endoplasmic reticulum and combine in that compartment are not yet modified with O-linked carbohydrate, as determined by measurements of their [3H]galactosamine content after biosynthetic labeling of amino sugars with [3H]glucosamine. Furthermore, only half of the free alpha-subunit secreted by JAR cells and less than 10% of free alpha secreted by 10-week-old placental explants received the O-linked chain. This was shown by determining the ratio of the unglycosylated and glycosylated forms of the tryptic peptide from free alpha that contains the O-glycosylation site (residues 36-42). Based on these findings, we make the following conclusions. 1) O-Glycosylation of alpha-subunit is a late event in the secretory pathway of trophoblasts compared to the rapid combination in the rough endoplasmic reticulum of hCG subunit precursors to form alpha beta dimer. 2) Association of alpha with beta precludes the subsequent addition of the glycan to alpha at Thr-39. 3) The alpha molecules that fail to combine with beta in the endoplasmic reticulum are substrates for the later addition of O-linked carbohydrate, presumably in the Golgi complex, but only a fraction of the free alpha molecules are modified with O-linked carbohydrate.     

The development of a sialic acid specific lectin-immunoassay for the measurement of human chorionic gonadotrophin glycoforms in serum and its application in normal and Down's syndrome pregnancies

OBJECTIVE: We have developed and validated a lectin-immunoassay for the recognition of sialic acid residues on hCG glycoforms in serum. DESIGN: This assay employs a hCG specific capture antibody and a sialic acid specific lectin (Wheat Germ Agglutinin) labelled with horse radish peroxidase. RESULTS: The standard curve covered hCG concentrations of 0-4000 IU/l (3rd IS for hCG, 75/537) with an analytical sensitivity of 1.0 IU/l. The within and between batch coefficient of variation was < 7% for all doses. Cross-reactivity of < 1% with TSH, LH, FSH, hCGalpha, hCGbeta and desialylated hCG confirmed assay specificity. Dilutions of serum of < 10% final concentration were parallel to the standard curve (within and between batch CV, < 6%). The assay working range was 100 - > 500 000 IU/l and the recovery of hCG from serum was in the range of 94.5% to 115.4%, with a mean value of 102.1%. The assay detected a time dependent change in hCG sialylation during normal pregnancy with the relative abundance of sialylated hCG declining after week 9 and increasing after week 15 of gestation. In addition preliminary studies showed that maternal serum hCG concentrations measured with the lectin-immunoassay were elevated in high risk Down's pregnancies (as defined by conventional screening tests between weeks 16-18 gestation, median multiple of median, 3.14; range 1.81-19.12, P < 0. 001) and low risk (1.57, 0.49-6.14, P = 0.034) compared to normal (1. 00, 0.32-3.20) pregnancies. Furthermore, the lectin immunoassay had greater discriminatory power compared to conventional immunoassay of hCG and hCGbeta between normal and both low and high risk Down's pregnancies. CONCLUSION: This assay will allow analysis of serum samples for the investigation of sialylated variants of hCG glycoforms in various pathological and physiological situations.     

Single-chain human chorionic gonadotropin analogs containing the determinant loop of the beta-subunit linked to the alpha-subunit

Human chorionic gonadotropin (hCG) is a member of the family of glycoprotein hormones containing a common alpha-subunit and distinct beta-subunits that confer hormonal specificity. hCG binds to the relatively large ectodomain of the human luteinizing hormone receptor (hLHR), a member of the G protein-coupled receptor superfamily, leading to increased intracellular production of cAMP. Using protein engineering, two miniaturized versions of hCGbeta have been separately fused to the N-terminus of the alpha-subunit to give N-des[1-91]hCGbeta-alpha-C and N-des[1-91,110-114]hCGbeta-alpha-C, i.e. fusion proteins of the hCGbeta determinant loop (extended to include the complete seat belt and carboxy-terminal peptide) coupled to the alpha-subunit. Bioactivity of these single-chain gonadotropin analogs was assessed in two systems following transient transfections into HEK 293 cells and subsequent cAMP measurements. In one, each mini-beta-alpha cDNA was fused to that of hLHR and transfected into cells to create yoked miniaturized hCG-hLHR complexes; in the other, the cDNA of each single chain mini-beta-alpha was co-transfected with that of hLHR in an effort to produce non-covalent miniaturized hCG-hLHR complexes. Using yoked hCG-hLHR and hLHR as positive and negative controls respectively, expression of each mini-hCG-hLHR complex was confirmed using antibody and ligand binding assays. The two mini-hCGs led to minimal activation of hLHR, suggesting weak intrinsic activity of the mini-beta-alpha fusion proteins. These results suggest that potent agonists and antagonists will require the presence of other portions of hCGbeta in addition to the determinant loop/seat belt.     

The effect of carbonyl cyanide trifluoromethoxyphenylhydrazone and methylamine on the processing and secretion of the glycoprotein hormone chorionic gonadotropin by human choriocarcinoma cells

Carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), a protonophore, and methylamine, a weak base, agents that dissipate hydrogen gradients across cellular membranes, were used to probe the coupling of hydrogen gradients to the processing and secretion of the glycoprotein hormone hCG by human choriocarcinoma cells (JAR) in culture. Both drugs disrupted the processing of asparagine-linked oligosaccharides such that the secreted hCG forms contained mostly high mannose rather than complex oligosaccharide chains. As the concentrations of FCCP were increased above 1 microgram/ml and those of methylamine above 12.5 mg/ml, the secretion of the labeled hCG dimer and free alpha-subunit was progressively inhibited. Both FCCP and methylamine also inhibited the incorporation of [35S] methionine and [3H]mannose into hCG subunits. Nevertheless, the inhibition of secretion was clearly apparent as an intracellular accumulation of the hCG subunit precursors in spite of the diminished incorporation of radioactive substrates. The intracellular hCG precursors that accumulated in the drug-treated cells contained predominantly Man8-9GlcNAc2 units, structures characteristic of glycoproteins localized in the endoplasmic reticulum. Both FCCP and methylamine inhibited hCG secretion at concentrations that did not lower the cellular content of ATP. We postulate on the basis of these results that a hydrogen gradient across the membrane either of the rough endoplasmic reticulum or the transitional vesicle is coupled to the rough endoplasmic reticulum to Golgi translocation step such that dissipation of the proton gradient blocks the secretion of hCG.     

Human chorionic gonadotropin beta-subunit affects the folding and glycosylation of alpha-cys mutants

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that contain a common alpha-subunit but differ in their hormone-specific beta-subunits. Both subunits have five and six disulfide bonds, respectively, which consist of cystine knot structure. It is evident from numerous studies that the structure of beta-subunits is rigid, whereas that of alpha-subunit is flexible and can be molded by a beta-subunit. Previously, we reported that secreted forms of a mutants where either cysteine residue in the disulfide bond 7-31 or 59-87 was converted to alanine contained a disulfide-linked homodimer in addition to a monomer. To study whether the hCGbeta-subunit affects the conformations of alpha mutants, alpha-subunits lacking either the 7-31 or 59-87 disulfide bond were expressed with wild-type (WT) hCGbeta in Chinese hamster ovary cells, and homodimer formation and glycosylation of dimerized alpha-subunit were assessed by continuous labeling with [35S]methionine/cysteine, immunoprecipitation with anti-alpha or -hCGbeta serum, digestion with endoglycosidase-H or -F, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a non-reducing condition. Our data showed that a homodimer was not observed in the half-Cys mutants except one, where cysteine at position 7 was converted to alanine, in the presence of beta-subunit. This finding indicated that hCGbeta-subunit rescued the a half-Cys mutants from the formation of intermolecular disulfide-linked homodimer by preferentially combining with the alpha mutants. In both free WT and all mutants treated with endoglycosidase-H, no or faint bands were recognized as the same migration as seen in endoglycosidase-F treatment. Even in the endoglycosidase-H sensitive cases, the amount of sensitive alpha-subunits was less than 5% of total alpha-subunits. In contrast to free alpha-subunits, distinct endoglycosidase-H sensitive bands were seen in both WT and mutants, although the ratio was various. We concluded that hCGbeta-subunit affects the folding and glycosylation of the alpha-subunit mutants.     

Brefeldin A affects early events but does not affect late events along the exocytic pathway in pancreatic acinar cells.

Brefeldin A (BFA) blocks protein export from the endoplasmic reticulum (ER) to Golgi complex and causes dismantling of the Golgi complex with relocation of resident Golgi proteins to the ER in some cultured cells. It is not known whether later steps in the secretory process are affected. We previously have shown that in BFA-treated rat pancreatic lobules, there is no detectable relocation of Golgi proteins to the ER and, although Golgi cisternae are rapidly dismantled, clusters of small smooth vesicles consisting of both bona fide Golgi remnants and associated vesicular carriers persist even with prolonged BFA exposure. We now report the effects of BFA on transport of proteins through the secretory pathway in exocrine pancreatic cells; we pulse-labeled pancreatic lobules with [35S]methionine and then chased for various times before adding BFA. When BFA was added at pulse, treated lobules released less than 10% of radioactive protein in comparison with controls, regardless of whether or not the lobule cultures were stimulated with carbamoylcholine. However, when lobules were pulsed and then chased for 30, 45, or 60 min before BFA addition, the amount of labeled protein released was comparable in both BFA-treated and untreated cultures. Furthermore, the kinetics and amounts of basal and carbamoylcholine-stimulated release of unlabeled alpha-amylase from storage in zymogen granules were similar in both control and BFA-treated lobules. Therefore, in the rat pancreas, BFA blocks ER to Golgi transport but does not affect later stages along the secretory pathway, including intra-Golgi transport, exit from the Golgi complex, formation and concentration of secretory granules, and exocytosis.     

Role of amino acid residues at the interface of alpha52asparginyl-N-glycosyl chain of human choriogonadotropin.

Of all the four N-glycosyl chains present in hCG, only one of them at alpha52Asn is located at the alpha/beta subunit interface and is crucial for the biological function of the hormone. The other three are exposed on the surface of the molecule and play only a minor role in the function of the hormone. The alpha52Asn oligosaccharide interacts with five amino acid residues in the beta-subunit, Tyr59, Val62, Phe64, Ala83, and Thr97. The present studies were undertaken to determine the role of the residues at the alpha52Asn-oligosaccharide and the beta-subunit interface in the mechanism of subunit association and downstream signaling events. Ten mutants, two of the alpha-subunit by the replacement of Asn52 and Thr80 with Gln and eight of the beta-subunit by multiple or single amino acid mutations, were prepared. These mutants included, hCGbeta59,62,64,97Ala, hCGbeta59,62,64Ala, hCGbeta62,64Ala, hCGbeta59Phe, hCGbeta62Ala or Thr, hCGbeta83Ile and hCGbeta97Ala. The mutation of the Asn52 to Gln resulted in a drastic change in its conformation and as a consequence in its weak affinity with the wild type beta as compared with that of the wild type hCGalpha and hCGalpha80Gln. The mutants with mutations in the four or three amino acids as well as both mutants of hCGbeta62Val almost failed to combine with hCGalpha again as a result of conformational changes shown by circular dichroism (CD) analysis and not due to their direct involvement in the subunit association. The double mutant combined with hCGalpha and the heterodimer behaved more like the wild type hCG. The mutation of Tyr to Phe resulted in a drop of 20% in the receptor binding and cAMP stimulation although Tyr is considered to be involved directly in subunit association. HCGbeta with mutations in the other amino acids, Phe64, Ala83, and Thr97, combined with the alpha subunit forming heterodimers with biological activity comparable to that of the wild type hCG. Thus, it appears that among the five amino acids in the vicinity of alpha52Asn carbohydrate, only beta59Tyr and beta62Val may be involved directly or indirectly in the alpha/beta beta dimer formation.