Single molecule characterization of the binding kinetics of a transcription factor and its modulation by DNA sequence and methylation.

The interaction of transcription factors with their response elements in DNA is emerging as a highly complex process, whose characterization requires measuring the full distribution of binding and dissociation times in a well-controlled assay. Here, we present a single-molecule assay that exploits the thermal fluctuations of a DNA hairpin to detect the association and dissociation of individual, unlabeled transcription factors. We demonstrate this new approach by following the binding of Egr1 to its consensus motif and the three binding sites found in the promoter of the Lhb gene, and find that both association and dissociation are modulated by the 9 bp core motif and the sequences around it. In addition, CpG methylation modulates the dissociation kinetics in a sequence and position-dependent manner, which can both stabilize or destabilize the complex. Together, our findings show how variations in sequence and methylation patterns synergistically extend the spectrum of a protein's binding properties, and demonstrate how the proposed approach can provide new insights on the function of transcription factors.

A vital region for human glycoprotein hormone trafficking revealed by an LHB mutation.

Glycoprotein hormones are complex hormonally active macromolecules. Luteinizing hormone (LH) is essential for the postnatal development and maturation of the male gonad. Inactivating Luteinizing hormone beta (LHB) gene mutations are exceptionally rare and lead to hypogonadism that is particularly severe in males. We describe a family with selective LH deficiency and hypogonadism in two brothers. DNA sequencing of LHB was performed and the effects of genetic variants on hormone function and secretion were characterized by mutagenesis studies, confocal microscopy and functional assays. A 20-year-old male from a consanguineous family had pubertal delay, hypogonadism and undetectable LH. A homozygous c.118_120del (p.Lys40del) mutation was identified in the patient and his brother, who subsequently had the same phenotype. Treatment with hCG led to pubertal development, increased circulating testosterone and spermatogenesis. Experiments in HeLa cells revealed that the mutant LH is retained intracellularly and showed diffuse cytoplasmic distribution. The mutated LHB heterodimerizes with the common alpha-subunit and can activate its receptor. Deletion of flanking glutamic acid residues at positions 39 and 41 impair LH to a similar extent as deletion of Lys40. This region is functionally important across all heterodimeric glycoprotein hormones, because deletion of the corresponding residues in hCG, follicle-stimulating hormone and thyroid-stimulating hormone beta-subunits also led to intracellular hormone retention. This novel LHB mutation results in hypogonadism due to intracellular sequestration of the hormone and reveals a discrete region in the protein that is crucial for normal secretion of all human glycoprotein hormones.

Identification, cDNA Cloning, and Characterization of Luteinizing Hormone Beta Subunit (lhb) Gene in Catla catla.

Reproductive hormones play a significant role in the gonadal development and gametogenesis process of animals. In the present study luteinizing hormone beta, (lhb) subunit gene was cloned and characterized from the brain of Catla catla. The lhb full-length of cDNA sequence is 629 bp which consists of 43bp 5'-UTR (untranslated region) 447bp, ORF(open reading frame) and 139 bp of 3'-UTR respectively. The coding region of lhb gene encoded a peptide of 148 amino acids. The coding sequence of lhb gene consist of a single N-linked glycosylation site (NET) and 12 cysteine knot residues. Phylogenetic analysis of C. catla Lhß deduced amino acid sequence showed high similarity with Carassius auratus followed by Gobiocypris rarus. 3D structure Lhß protein comprises of five ß-sheets and six coils/loops. The qPCR results revealed lhb mRNA is mainly expressed in the pituitary, ovary while moderate expression was observed in brain and testis. To best our knowledge, this is the first report on the identification, molecular characterization and structural information regarding luteinizing hormone in Indian major carp.

Chemical synthesis of the ß-subunit of human luteinizing (hLH) and chorionic gonadotropin (hCG) glycoprotein hormones.

Human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) are human glycoprotein hormones each consisting of two subunits, an identical α-subunit and a unique ß-subunit, that form noncovalent heterodimers. Structurally, ß-hCG shares a high degree of sequence similarity with ß-hLH, including a common N-glycosylation site at the N-terminus but differs mainly in the presence of an extended C-terminal portion incorporating four closely spaced O-linked glycans. These glycoproteins play important roles in reproduction and are used clinically in the treatment of infertility. In addition, the role of hCG as a tumor marker in a variety of cancers has also attracted significant interest for the development of cancer vaccines. In clinical applications, these hormones are administered as mixtures of glycoforms due to limitations of biological methods in producing homogeneous samples of these glycoproteins. Using the powerful tools of chemical synthesis, the work presented herein focuses on the highly convergent syntheses of homogeneous ß-hLH and ß-hCG bearing model glycans at all native glycosylation sites. Key steps in these syntheses include a successful double Lansbury glycosylation en route to the N-terminal fragment of ß-hCG and the sequential installation of four O-linked glycosyl-amino acid cassettes into closely spaced O-glycosylation sites in a single, high-yielding solid-supported synthesis to access the C-terminal portion of the molecule. The final assembly of the individual glycopeptide fragments involved a stepwise native chemical ligation strategy to provide the longest and most complex human glycoprotein hormone (ß-hCG) as well as its closely related homologue (ß-hLH) as discrete glycoforms.

A novel carboxyl-terminal heptapeptide initiates the regulated secretion of LH from unique sub-domains of the ER.

The coordinated secretion of LH and FSH are critical for reproductive functions. After translocation into the endoplasmic reticulum (ER), their biosynthetic routes diverge at a determinative step prior to sorting in the regulated (LH) and constitutive (FSH) secretion pathways. Recently, we identified a C-terminal heptapeptide sequence, present only in the LHß subunit, as a critical signal for entry of the LH dimer into the regulated pathway. We showed that an LHß mutant lacking the heptapeptide (LHßΔT) assembled more efficiently with the α subunit than wild-type LHß subunit, and this LHΔT dimer was secreted constitutively. Thus, an association exists between the presence of the C-terminal heptapeptide and sorting of the LH heterodimer to the regulated pathway. To study how this delayed LHß subunit assembly is related to the trafficking of LH, we exploited the single subunit transfection model in rat somatotrope-derived GH3 cells with the use of immunofluorescence confocal microscopy. The LHß subunit showed a distinct immunofluorescent localization as compared to the FSHß subunit and LHß mutants. The wild-type LHß subunit exhibited a perinuclear staining corresponding to the ER/nuclear envelope region. In contrast, the wild-type FSHß subunit and the mutants LHßΔT and LHßL119A displayed no detectable perinuclear staining; only peripheral ER puncta were observed. Also, no perinuclear fluorescence was detected in cells expressing the LH heterodimer. We propose that the C-terminal heptapeptide is responsible for delayed heterodimer assembly within an ER sub-domain of the nuclear envelope, as an early partitioning event necessary for the entrance of LH into the regulated secretory pathway, whereas FSHß does not traverse the nuclear envelope region. These data suggest that, at least for LH, the molecular decision to enter the regulated secretory pathway is a pre-Golgi event controlled by the novel C-terminal heptapeptide.

Up-regulation of FSHR expression during gonadal sex determination in the frog Rana rugosa.

In vertebrates, gonadal production of steroid hormones is regulated by follicle-stimulating hormone (FSH) and luteinizing hormone (LH) via their receptors designated FSHR and LHR, respectively. We have shown recently that steroid hormones are synthesized in the differentiating gonad of tadpoles during sex determination in the frog Rana rugosa. To elucidate the role of gonadotropins (GTHs) and their receptors in the production of gonadal steroid hormones during sex determination, we isolated the full-length FSHß, LHß, FSHR and LHR cDNAs from R. rugosa and determined gonadal expression of FSHR (FSH receptor) and LHR (LH receptor) as well as brain expression of FSHß and LHß during sex determination in this species. The molecular structures of these four glycoproteins are conserved among different classes of vertebrates. FSHß expression was observed at similar levels in the whole brain (including the pituitary) of tadpoles, but it showed no sexual dimorphism during gonadal sex determination. By contrast, LHß mRNA was undetectable in the whole brain of tadpoles. FSHß-immunopositive cells were observed in the pituitary of female tadpoles with a differentiating gonad. Furthermore, FSHR expression was significantly higher in the gonad of female tadpoles during sex determination than in that of males, whereas LHR was expressed at similar levels in males and females. The results collectively suggest that FSHR, probably in conjunction with FSH, is involved in the steroid-hormone production during female-sex determination in R. rugosa.

A dileucine determinant in the carboxyl terminal sequence of the LHß subunit is implicated in the regulated secretion of lutropin from transfected GH3 cells.

LH and FSH are essential for control of gonadal function. They are synthesized in the same gonadotrope but differ in their mode of secretion. LH release is regulated, while FSH is secreted constitutively. One unique feature of LHß is a carboxyl terminal hydrophobic heptapeptide. We demonstrated that deleting the heptapeptide diverted the truncated LH dimer to the constitutive pathway in vitro. To examine if the residues of this heptapeptide play a role in LH sorting, leucines 118-119 were substituted with alanine (L118A and L119A, respectively). The intracellular pool of the L118A mutant protein decreased with a corresponding increase in constitutive secretion. Moreover, immunofluorescence microscopy revealed that the L118A mutant exhibited fewer puncta as compared to wild-type LH. L119A behaved similar to wild-type LH, indicating that a single leucine residue at position 118, rather than a dileucine motif, contributes to the process that sorts LH into the regulated pathway.

Expression of three gonadotropin subunits in Southern catfish gonad and their possible roles during early gonadal development.

The three gonadotropin (GtH) subunit cDNAs, GtHalpha, FSHbeta and LHbeta, which contain complete open reading frames were isolated from Southern catfish (Silurus meridionalis Chen) ovary. RT-PCR revealed that GtHalpha, FSHbeta and LHbeta mRNA were expressed in ovary, female and male pituitaries, but not in testis. Ontogeny study showed that GtHalpha and FSHbeta expressed in ovary from 25 dah (days after hatching) and LHbeta expressed from 40 dah onwards. The expression levels of these genes in all-female Southern catfish gonad were down-regulated after treatment with tamoxifen from 5 to 25 dah when measured at 65 dah. These results indicated the involvement of the three subunits in gonadal development and sexual differentiation.

Molecular characterization of gonadotropin subunits and gonadotropin receptors in black porgy, Acanthopagrus schlegeli: effects of estradiol-17beta on mRNA expression profiles.

The cDNAs of three gonadotropin (GTH) subunits (GTHalpha, FSHbeta, and LHbeta) and two GTH receptors (FSHR and LHR) from pituitary and gonads of black porgy were cloned. The nucleotide sequences of the GTHalpha, FSHbeta, and LHbeta cDNA were 354, 363, and 414 base pairs (bps) in length with open reading frames (ORF) encoding peptides of 117, 120, and 137 amino acids, respectively. The FSHR and LHR cDNA was 2118 and 2076 bps in length with ORFs encoding peptides of 705 and 691 amino acids, respectively. To study the mechanism of the estradiol-17beta (E(2)) action, we examined the expression pattern of GTH subunit mRNAs in pituitary and GTH-receptor mRNAs in gonads, and the changes of plasma E(2) level when E(2) treatment was applied to immature black porgy. E(2) treatment increased mRNA expression levels of the genes and plasma E(2) levels, indicating that E(2) stimulated the increases in GTH subunit and GTH-receptor mRNAs. These data indicate that E(2) plays an important regulatory role in the brain-pituitary-gonad axis of immature black porgy. We provide the molecular characterization and expression of the GTH subunits and GTH receptors during sex change in the protandrous black porgy.

A carboxyl-terminal sequence in the lutropin beta subunit contributes to the sorting of lutropin to the regulated pathway.

Although synthesized in the same pituitary gonadotropes, the secretion profiles of lutropin (LH) and follitropin (FSH) differ. LH is secreted through a regulated pathway and associated with a bolus release at mid-estrous cycle. In contrast, the majority of FSH is secreted constitutively with an incremental increase until ovulation. Both share an identicalalpha subunit, and thus thebeta subunit contains determinants for sorting into the regulated pathway. Previously, we demonstrated that a hydrophobic carboxyl-terminal heptapeptide of the LHbeta subunit (Leu-Ser-Gly-Leu-Leu-Phe-Leu), not found in the FSHbeta subunit, influences the intracellular behavior of the LH dimer. To test the hypothesis that the peptide contributes to differential sorting, we monitored the fates of LH and LHDeltaT (LHbeta subunit lacking the carboxyl-terminal seven amino acids) dimers in the rat somatotrope-derived GH(3) cell line in which both the regulated and constitutive secretory pathways operate. Pulse-chase labeling demonstrated that the LHDeltaT dimer was diverted to the constitutive pathway, resulting in a significant decrease in the corresponding intracellular pool. Forskolin stimulated LH dimer release 3-fold, which was accompanied by a parallel decrease of intracellular LH; only marginal forskolin stimulation of LHDeltaT was seen. Immunofluorescence after cycloheximide treatment demonstrated decreased retention of LHDeltaT compared with LH, consistent with increased constitutive secretion of LHDeltaT. We also demonstrated that fusing the heptapeptide to the carboxyl terminus of the FSHbeta subunit resulted in an increased regulated secretion of this FSH analog compared with wild-type FSH. These data are the first to identify a novel structural determinant responsible for the sorting of a member of the glycoprotein hormone family into the regulated secretory pathway.

The configuration of the alpha and beta subunit domains in single-chain bovine LH analogs influences the secretion and steroidogenic response.

The gonadotrophins LH, FSH and human (h) CG are non-covalent heterodimers composed of a common alpha and the hormone-unique beta subunit. LH regulates the production of androgens and progestins in the follicle, and the levels of these steroids are critical for the ovarian function. Structural features of the gonadotrophins involved in the steroidogenic response of the ovary are not completely understood. As an approach to address how the topology of the ligand affects steroidogenesis we exploited the single-chain (SC) gonadotrophin methodology because manipulating the relative position of the tethered subunit domains in SC hCG analogs enabled to change in the conformation, secretion, receptor binding and adenylyl cyclase activity. We genetically engineered a SC bovine LH analog with a linker derived from the CTP domain of the hCGbeta subunit, NH2-alpha-CTP-LHbeta-COOH (denoted as alphaCTPLHbeta; AB configuration) and evaluated the secretion form transfected CHO cells and steroidogenesis in follicular derived cells in comparison to the variant NH2-LHbeta-CTP-alpha-COOH (LHbetaCTPalpha; BA configuration). The secretion of the analogs from CHO cells was quantitative, and that of alphaCTPLHbeta was more efficient than that of LHbetaCTPalpha The experiments suggested that both variants were N- and O- glycosylated, though the posttranslational modifications are likely to be non-identical in the AB and BA analogs. The analogs stimulated progesterone secretion by immortalized rat granulosa cells that express the rat LH receptor but the EC50 of alphaCTPLHbeta (AB orientation) was higher by 20 fold, as compared to LHbetaCTPalpha (BA). In primary cultures of bovine theca cells, alphaCTPLHbeta stimulated progesterone release with a reduced sensitivity (by at least 50 folds) and smaller magnitude over the basal levels (about 3 folds) relative to LHbetaCTPalpha. In contrast, the accumulation of androstenedione in the media of the same primary cultures appeared to be nearly identical. As a result, the androstenedione/progesterone ratio for the alphaCTPLHbeta analog was significantly increased relative to LHbetaCTPalpha (2-3 folds). This unequal response suggests a distinct regulation of progesterone and androstenedione biosynthesis. Our data demonstrate major differences in steroid balance following stimulation of the receptor with structural LH analogs and provide further insight into gonadotrophin regulation of ovarian steroid production.

Molecular cloning and histological localization of LH-like substances in a bottlenose dolphin (Tursiops truncatus) placenta.

All mammals exhibit pituitary-specific expression of LH and FSH, whereas placental expression of gonadotropins has been reported only in primates and equids. Some cetaceans, such as dolphins, have a long gestational period and a sexual cycle of about 27 days almost comparable with that of humans. Histologically, dolphins have an epitheliochorial placentae that resembles placentas of Perissodactyla including horses. In the present study, we cloned cDNAs encoding gonadotropins and observed their immunohistochemical localization in the placenta of bottlenose dolphin. The cDNAs obtained encoded 120 amino acids for the alpha-subunit (including 96 amino acids of mature proteins), and 141 amino acids for the beta-subunit (including 121 amino acids of mature proteins). The sequence of the alpha-subunit was similar to that in the pig (Artiodactyla) pituitary glycoprotein hormone [96.7% homology at amino acids (aa) level], and the sequence of the beta-subunit was similar to that of luteinizing hormone (LH) in the pig [94.3% homology at aa level] and white rhinoceros (Perissodactyla) [93.3% homology at aa level]. Of interest, dolphin LHbeta lacks carboxyl-terminal-peptides (CTP). This fact suggests that CTP are not essential for placental expression of gonadotropin in dolphins. Immunohistochemical observations employing anti-ovine LHbeta antibody revealed positive staining in the villositycal tissue. Our observations suggest placental expression of gonadotropin homologues in cetaceans and possible evolutionary conservation of placentae-derived hormonal control of ovarian functions during pregnancy.

Nitro-thiocyanobenzoic acid (NTCB) reactivity of cysteines beta100 and beta110 in porcine luteinizing hormone: metastability and hypothetical isomerization of the two disulfide bridges of its beta-subunit seatbelt.

Luteinizing hormone (LH) like all other glycoprotein hormones is composed of two dissimilar subunits, alpha and beta, that are non-covalently associated. The heterodimer is stabilized by a region of the beta-subunit called the "seatbelt" because it wraps around the alpha-subunit and it is fastened by a disulfide bridge between cysteines beta26 and beta110. Although all 22 cysteines of porcine LH (pLH) are engaged in disulfide bridges, we previously showed that the free cysteine-specific reagent NTCB could react with pLH: it slowly cyanylated two cysteines in pLH and there was a close relationship between NTCB reaction with pLH and association/dissociation kinetics of its subunits. Therefore, cysteines beta26 and beta110 were considered as the best candidates for NTCB reaction. In order to identify the NTCB-reactive cysteines in pLH we have performed a mass spectroscopic analysis of the peptides released after mild basic hydrolysis of S-cyanylated pLH and its subunits. Only cysteines beta100 and beta110 were found to react with NTCB. Since these residues are not linked by a disulfide bridge in the crystallographic 3D structure of gonadotropins, it is proposed that their respective counterparts (Cysbeta93 and beta26) do not react with NTCB either because they are shielded from solvent or because they form a transient bridge. In the first hypothesis, both seatbelt bridges would be independently metastable; in the second one, a fast reversible isomerization between bridges beta26-beta110 and beta93-beta100 would occur. Such a reaction could be catalyzed by the previously recognized intrinsic protein disulfide isomerase (PDI) activity of gonadotropins.

Molecular cloning of cDNAs and structural model analysis of two gonadotropin beta-subunits of snakehead fish (Channa maculata).

The cDNAs encoding beta-subunits of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) have been cloned from the pituitary of snakehead fish, Channa maculata, and the three-dimensional structural models of the encoded FSH and LH were investigated. The cloned cDNAs, including 5'-untranslated region (UTR), open-reading frame, and 3'-UTR followed by a poly(A) tail, were obtained by reverse transcription-polymerase chain reaction and rapid amplification of cDNA end methods. The open-reading frames of FSH-beta cDNA encodes a 120-amino acid protein with a signal peptide of 18 amino acids and a mature protein of 102 amino acids; while LH-beta cDNA encodes a 140-amino acid protein with a signal peptide of 33 amino acids and a mature protein of 115 amino acids. The amino acid sequence identities of snakehead fish FSH-beta and LH-beta in comparison with other fish are 27.8-81.9% and 45.2-88.8%, respectively; while in comparison with tetrapods are 26.2-28.9% and 37.5-51.2%, respectively. Both FSH-beta and LH-beta of snakehead fish resemble most to those of Perciformes, implying their closer phylogenetic relationship. All 12 cysteine residues are conserved in snakehead fish LH-beta; while 11 cysteine residues are conserved in its FSH-beta. The third cysteine is absent in snakehead fish FSH-beta; instead, a positionally shifted cysteine residue is present at the N-terminus, as found in some phylogenetic related fish. The structure models of snakehead fish FSH and LH, constructed by using the crystal structures of human FSH and human chorionic gonadotropin as respective template, showed that the positionally shifted N-terminal cysteine residue of snakehead fish FSH-beta likely can substitute the third cysteine to form a disulfide bond with the 12th cysteine.

The LHbeta gene of several mammals embeds a carboxyl-terminal peptide-like sequence revealing a critical role for mucin oligosaccharides in the evolution of lutropin to chorionic gonadotropin in the animal phyla.

The expression of a previously untranslated carboxylterminal sequence is associated with the ancestral lutropin (LH) beta to the beta-subunit gene evolution of choriogonadotropins (CG). The peptide extension (denoted as CTP) is rich in mucin-type O-glycans and confers new hormonal properties on CG relative to the LH. Although the LHbeta gene is conserved among mammals and only a few frameshift mutations account for the extension, it is merely seen in primates and equids. Bioinformatics identified a CTP-like sequence that is encrypted in the LHbeta gene of several mammalian species but not in birds, amphibians, or fish. We then examined whether or not decoding of the cryptic CTP in the bovine LHbeta gene (boCTP) would be sufficient to generate the LHbeta species of a ruminant with properties typical to the CGbeta subunit. The mutated bovine LHbeta-boCTP subunit was expressed and N-glycosylated in transfected Chinese hamster ovary cells. However, unlike human (h) CGbeta CTP, the cryptic boCTP was devoid of mucin O-glycans. This deficiency was further confirmed when the boCTP domain was substituted for the natural CTP in the human CGbeta subunit. Moreover, when expressed in polarized Madin-Darby canine kidney cells, this hCGbeta-boCTP chimera was secreted basolaterally rather than from the apical compartment, which is the route of the wild type hCGbeta subunit, a sorting function attributed to the O-glycans attached to the CTP. This result shows that the cryptic peptide does not orientate CG to the apical face of the placenta, to the maternal circulation as seen in primates. The absence of this function, which distinguishes CG from LH, provides an explanation as to why the LHbeta to CGbeta evolution did not occur in ruminants. We propose that in primates and equids, further natural mutations in the progenitor LHbeta gene resulted in the efficient O-glycosylation of the CTP, thus favoring the retention of an elongated reading frame.

Receptor-selective determinants in catfish gonadotropin seat-belt loops.

Mammalian gonadotropins are highly selective. Charge differences between the Cys(10-11) sequence of FSHbeta and LHbeta/CGbeta seat-belt loops determine the ability of these hormones to interact with the LH-R. Selective FSH-R binding is mainly dependent on the presence of an FSHbeta-specific sequence between Cys(11-12) of the seat-belt loop. Intriguingly, African catfish LHbeta (cfLHbeta) lacks a positively charged Cys(10-11) region and stimulates both catfish LH-R and FSH-R with comparable potencies. Our studies on the promiscuous behaviour of cfLH using chimeric gonadotropins revealed that the Cys(10-11) region of cfLHbeta contains cfLH-R-selective determinants, whereas the Cys(11-12) region of cfLHbeta confers FSH-R-stimulating activity to cfLH. Hence, the location of receptor-selective determinants appeared to be fairly well conserved throughout evolution, despite the low sequence identity between mammalian and catfish seat-belt loops. Moreover, various structure-function differences between gonadotropins are discussed in the context of the different (female) reproductive strategies between mammalian and non-mammalian species that required the divergence to a more specific LH-R-stimulating activity of one of the gonadotropins in mammals.

Identification of amino-acids in the alpha-subunit first and third loops that are crucial for the heterospecific follicle-stimulating hormone activity of equid luteinizing hormone/choriogonadotropin.

OBJECTIVE: To identify amino-acids in the alpha-subunit important for expression of heterospecific FSH activity of horse (e) LH/choriogonadotropin (CG) (eLH) and donkey (dk) LH/CG (dkLH) (FSH/LH ratio ten times higher for eLH than for dkLH); this FSH activity absolutely requires an equid (donkey or horse) alpha-subunit combined with an equid beta-LH subunit. DESIGN: Chimeric alpha-subunits possessing the first 63 amino-acids of the porcine (p) and the last 33 amino-acids of the donkey alpha-subunit (alphap-dk) and the inverse (alphadk-p) were constructed. Porcine-specific amino-acids were introduced by mutagenesis in donkey alpha-subunit at positions 70, 85, 89, 93 and 96 (alphadk5xmut), 18 (alphadkK18E) or 78 (alphadkI78A). METHODS: These different alpha-subunits were co-transfected in COS-7 cells with beta-eLH, beta-dkLH and beta-eFSH. The LH and FSH bioactivities of the dimers were then assessed in two heterologous in vitro bioassays. RESULTS: alphap-dk or alphadk-p exhibited FSH activity when co-expressed with beta-eLH but not with beta-dkLH. alphadkK18E or alphadkI78A gave hybrids with no FSH activity and important LH activity when expressed with beta-dkLH. alphadkI78A/betaeLH displayed an FSH/LH ratio as low as that of dkLH. However, mutation at 78 in alpha-dk had no effect on FSH bioactivity when co-expressed with beta-eFSH. CONCLUSIONS: Amino-acids present in both the first two-thirds and the last third of the alpha-subunit of equid LHs are involved in their heterologous biospecificity. Ile alpha78 exerts as strong an influence on it as the beta102-103 residues. By contrast, this residue plays no role in the FSH specificity of eFSH.

Crystals of the beta-subunit of bovine luteinizing hormone and indicators for the involvement of proteolysis in protein crystallization.

The beta-subunit of luteinizing hormone (LH), the subunit responsible for the physiological response, has been crystallized beginning with the intact alphabeta-heterodimeric hormone purified from bovine pituitary glands. The crystals were grown at 310 K in the presence of neutral detergents along with trypsin. The tetragonal bipyramidal crystals diffract to 3 A resolution and belong to space group I4(1)22, with unit-cell parameters a = b = 57, c = 207 A. It is noted that proteins exposed to proteases sometimes yield products that crystallize better than the native molecule and that the beta-subunit of LH represents yet another example. Some indicators of when proteolysis may be a factor in crystallization, as well as some consequences, are described.

Luteinizing hormone and follicle-stimulating hormone exhibit different secretion patterns from cultured Madin-Darby canine kidney cells.

LH, FSH, and chorionic gonadotropin (CG) are comprised of a common alpha subunit and a hormone-specific beta subunit. Using Madin-Darby canine kidney (MDCK) epithelial cells to examine the polarized secretion of human CG/LH, we previously reported that CG and LH were detected in the apical and basolateral compartments, respectively, and the carboxyl terminal end of the CGbeta subunit contains a strong apical signal. Here we show that the carboxyl seven amino acids in the LHbeta subunit contribute to the basolateral secretion of LH, and an LH chimera bearing the CGbeta apical signal is redirected from the basolateral to the apical compartments. Because LH and FSH are synthesized in the same cell, we also compared the secretion polarity of LH with FSH. MDCK cells expressing the FSH dimer displayed an almost equal distribution of protein into the apical and basolateral compartments. Given that the LHbeta and CGbeta carboxy terminal sequences, which differ from that in the FSHbeta subunit, occupy a pivotal role in their polarized behavior, the results support the hypothesis that pituitary exit of LH and FSH occur via different secretion pathways, and are released spatially from the pituitary via different circulatory routes.

Molecular characterization of sea bass gonadotropin subunits (alpha, FSHbeta, and LHbeta) and their expression during the reproductive cycle.

Reproduction is controlled by two pituitary gonadotropin hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). This study reports the cloning, sequence analysis, and gene expression of gonadotropin (GTH) subunits from the European sea bass (Dicentrarchus labrax). The GTH subunits were cloned from a sea bass brain-pituitary cDNA library. The nucleotide sequences of the common alpha, the FSHbeta, and the LHbeta subunit cDNAs were 625, 521, and 591 base pair (bp) long, respectively, encoding for mature peptides of 94, 105, and 115 amino acids (aa), respectively. Sequence analysis showed that sea bass FSHbeta is more similar to higher vertebrate FSHbeta's (35-37%) than to LHbeta's (26-30%), whereas sea bass LHbeta is more similar to LHbeta's (40-53%) than to FSHbeta's (26-41%). Phylogenetic analysis of fish GTH sequences grouped the beta subunits into two groups, FSH and LH, distributed into four classes, corresponding to the accepted divisions of Elopomorphs, Ostariophysis, Salmonids, and Percomorphs. A dot-blot technique was developed to analyze GTH pituitary mRNA levels during the reproductive cycle of male sea bass. From October (initiation of gametogenesis) to February (spermiation), the expression of all three subunits in the pituitary increased in parallel, concomitantly with the gonadosomatic index (GSI) and the accumulation of LH protein in the pituitary, all values declining sharply at post-spermiation. This study demonstrates that the pituitary of sea bass contains two gonadotropin hormones and that both gonadotropins are probably involved in the control of gametogenesis, gamete maturation, and spermiation.