Molecular Human Reproduction, Vol. 8, No. 3, 201-212,
March 2002
© 2002 European Society of Human Reproduction and Embryology
Reproductive endocrinology |
A novel Ala–3Thr mutation in the signal peptide of human luteinizing hormone ß-subunit: potentiation of the inositol phosphate signalling pathway and attenuation of the adenylate cyclase pathway by recombinant variant hormone
1 Department of Physiology, Institute of Biomedicine and 2 Department of Biotechnology, University of Turku, 20520 Turku, Finland and 3 Department of Biological Sciences, Florida International University, Miami, FL 33199, USA
Abstract
Upon screening for polymorphisms in the human luteinizing hormone ß-subunit (LHß) gene, we discovered a novel mutation in the LHß signal peptide with functional consequences for signal transduction in mouse Leydig tumour cells (mLTC-1). This G52A point mutation in exon 2 of the LHß gene, detected in heterozygous form in several normal DNA samples, caused an Ala–3Thr amino acid substitution. Recombinant forms of wild-type (WT) and Ala–3Thr variant (V) LH were produced in human embryonic kidney (HEK) 293 cells and purified. The immunoreactivities of the recombinant LH were determined by immunofluorometric assays and in-vitro bioactivities in mLTC-1 cells were assessed by using cAMP, progesterone and inositol trisphosphate (IP3), and activation of mitogen-activated protein kinase (MAPK) as end-points. Whereas both LH forms stimulated progesterone production and MAPK in similar fashion, WT-LH was more potent in stimulating cAMP, and V-LH was more potent in stimulating IP3 generation. Both LH forms bound to LH receptors with similar affinities. No evidence was found for influence of the signal peptide mutation on efficacy of 
- and ß-subunit dimerization. Sequencing of the recombinant V-LHß protein also revealed that the mutation did not interfere with signal peptide cleavage. In summary, the present findings indicate that the Ala–3Thr mutation in the LHß-subunit signal peptide has functional consequences, in the form of dissociation of stimulatory potency for different signal transduction pathways in vitro.
luteinizing hormone/LHß variant/mutation/PCR/purification
Introduction
LH is a heterodimeric glycoprotein that belongs to the family of glycoprotein hormones, including FSH, thyroid-stimulating hormone (TSH) and chorionic gonadotrophin (CG). These glycoprotein hormones are composed of a common ß-subunit and a unique -subunit that defines the functional specificity (Pierce and Parsons, 1981
; Fiddes and Talmadge, 1984).
Clinical studies have shown that point mutations, with consequent single amino acid alterations, in the ß-subunit genes of LH, TSH, and FSH can cause clinical disorders (Dacon-Voutetakis et al., 1990; Weiss et al., 1992; Matthews et al., 1993; Mederios-Neto et al., 1996). A point mutation resulting in a Gln54Arg change in the LHß-subunit has been reported (Weiss et al., 1992) in a hypogonadal male. This homozygous missense mutation (A
G) prevents the molecule from binding to its receptor, arrests the development of puberty, and is associated with low testosterone and arrested spermatogenesis.
A common genetic variant (V) of LH has been detected in apparently healthy individuals, and shown to cause point mutation-based substitutions of two amino acids (Trp8Arg and Ile15Thr) in the LHß-subunit (Pettersson et al., 1992; Furui et al., 1994; Okuda et al., 1994; Suganuma et al., 1996; Nilsson et al., 1997, 1998). The two mutations alter the biological function of the V-LH molecule which has higher in-vitro but lower in-vivo bioactivity than WT-LH, the latter due to its shorter circulatory half-life (Haavisto et al., 1995). Homo- or heterozygous expression of the V-LHß allele has distinct physiological and pathophysiological consequences, as evidenced by slight but significant alterations in ovarian steroidogenesis (Rajkhowa et al., 1995), and delayed progression of puberty and gain of height in boys (Raivio et al., 1996). In addition, disturbances of menstrual cycle have been reported in women homo- and heterozygous for the V-LHß gene (Furui et al., 1994; Okuda et al., 1994; Suganuma et al., 1995). It has been reported that there is increased prevalence of V-LHß in Japanese infertility patients and in women with premature ovarian failure (Takahashi et al., 1998, 1999). In addition, a heterozygous mutation (Gly102Ser) of the HLHß-subunit has been reported from Singapore in cases of female and male infertility (Liao et al., 1998; Ramanujam et al., 1999, 2000).
In our attempts to characterize the extent of LHß gene polymorphisms in various populations, we investigated the frequency of the G1502A (Gly102Ser) mutation in exon 3 of the HLHß-subunit in Finnish, Bengali, Danish and Rwandan populations by restriction fragment length polymorphism (RFLP) using the restriction enzyme EcoO 109I. This mutation was not found in the populations studied, but serendipitously, in three out of the 100 samples from Rwanda, the digestion products subjected to electrophoresis displayed another type of altered RFLP fragmentation pattern. Direct sequencing revealed a heterozygous mutation (G52A) in exon 2 of the HLHß-subunit gene [the nucleotides are numbered according to the start site of translation, excluding intronic sequences (Fiddes and Talmadge, 1984)], causing an Ala–3Thr amino acid substitution in the signal peptide of the HLHß-subunit.
Signal sequences are present within precursors of most secreted proteins and their task is to mediate the targeting of nascent secreted proteins to membranes, thereby playing multiple roles in the protein secretory pathway. The functional importance of the signal sequence has been established through functional characterization of mutant proteins. Some mutations in human signal sequences have been reported to cause defective protein synthesis, with a consequent phenotype. Such a mutation in the preproparathyroid hormone (preproPTH) gene has been proposed as the cause of familial isolated hypoparathyroidism (FIH). One study (Arnold et al., 1990) reported a T to C point mutation changing the codon at position –18 of the 31 amino acid prepro sequence from cysteine to arginine within the hydrophobic core of prepro-PTH in one FIH kindred, causing a demonstrated functional defect. The mutation causes a disruption of the core that leads into impaired interaction of the nascent protein with the signal recognition particle (SRP), the translocation machinery, and signal peptidase cleavage (Karaplis et al., 1995). A G279A mutation, causing a Thr (ACG) to Ala (GCG) substitution in the C-terminus of the signal peptide in preprovasopressin (preproVP) has been identified in patients with familial central diabetes insipidus (Ito et al., 1993). The results indicated that inefficient processing of preproVP produced by the mutant allele is possibly involved in the pathogenesis of diabetes insipidus in the affected individuals. A novel missense mutation (L9P) in the signal peptide region of cathepsin K (CK) has been studied in patients with pycnodysostosis (Fujita et al., 2000). Expression of the mutant protein was found to be markedly reduced due to dysfunction of the signal peptide, providing evidence that a structural change in the signal peptide of the CK protein is involved in the pathogenesis of pycnodysostosis.
Since there is ample evidence that mutations in the signal peptide can affect the synthesis and/or function of proteins, we considered it important to determine the functional consequences of the novel signal peptide mutation (Ala–3Thr) of the human LHß-subunit.
Materials and methods
Amplification of the HLHß gene by PCR and subcloning
DNA amplification was carried out using PCR with specific primers designed on the basis of the known HLHß gene sequence, and selected with specific mismatches in order to discriminate between LHß and the highly homologous HCGß gene (Fiddes and Talmadge, 1984). First, an 826 bp PCR fragment (Figure 1) was amplified using primers FI (forward) and RIII (reverse), each 0.5 µmol/l, in a total reaction volume of 25 µl, containing thermostable DNA polymerase (1 unit), deoxynucleotide triphosphates (dNTP, 0.4 mmol/l of each), in buffer containing KCl (50 mmol/l), Tris–HCl (10 mmol/l, pH 8.8), Triton X-100 (0.1%) and MgCl2 (1.5 mmol/l). Thirty-five PCR cycles were performed as follows: denaturation (96°C, 1 min), annealing (65°C, 1 min), extension (72°C, 2 min). The DNA polymerase (DyNAZymeTM II; Finnzymes OY, Espoo, Finland) was added after an initial denaturation step (5 min). Later, the three samples displaying an altered RFLP fragmentation pattern (see below) were further amplified by primer pairs FI–RI and FI–RII (Table I; Figure 1) to obtain different lengths of fragments in several individual PCR runs under optimal conditions. These PCR products were subsequently used in sequencing (see below). For further confirmation of sequences of the PCR products, the FI–RIII fragment was subcloned into the pGEM®-T easy vector according to the manufacturer's recommendations (Promega, Madison, WI, USA). The other pairs of primers (FIII–RI; FII–RIII; FIV–RI) were used to amplify other parts of the LHß gene (Table I; Figure 1).
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Analysis of the Gly102Ser mutations in the HLHß-subunit by RFLP
In all, 383 genomic DNA samples were collected anonymously and randomly from apparently healthy donors screened for metabolic and endocrinological diseases, or collected for anthropological studies, in populations from Finland (n = 60), Bengali/North-East India (n = 78), Denmark (n = 145) and Rwanda (n = 100). Appropriate local guidelines for ethics of sample collection were followed. The genomic DNA amplified by PCR with primers FI–RIII (see above) was digested by the restriction enzyme EcoO 109I to screen for the Gly102Ser (G1502A) mutation in the HLHß-subunit (Roy et al., 1996
; Liao et al., 1998). According to the previously published LHß gene sequence (Fiddes and Talmadge, 1984), EcoO 109I digestion in WT-LH individuals results in two DNA fragments of 707 and 119 bp. In the presence of the mutation at codon 102, the enzyme recognition site is lost so that in heterozygotes there are three bands identified, i.e. 826, 707 and 119 bp, whereas in homozygotes there is only one band of 826 bp. A DNA sample containing the G1502A mutation was used as a positive control for the method.
DNA sequencing
DNA from the PCR reaction was purified using agarose gel electrophoresis prior to the Qiaex II Agarose Gel Extraction Kit (Qiagen GmbH, Hilden, Germany). Purification of the plasmids was carried out by using the Nucleobond AX plasmid purification protocol (Macherey-Nagel GmbH, Düren, Germany). The genotypes were confirmed by sequencing from several individual PCR runs, in order to cover the whole LHß gene sequence, or from DNA extracted after cloning the PCR products into the pGEM®-T easy vector. The PCR primers (Figure 1
and Table I) were used as sequencing primers with the dideoxy chain termination method. Sequencing was performed on an ABI PrismTM377 DNA Sequencer (Perkin Elmer, Norwalk, CT, USA).
Site-directed mutagenesis and DNA construct preparation
Oligonucleotides used for site-directed mutagenesis and as sequencing primers were prepared by Genosys Biotechnologies, Inc. (Cambridge, Cambs, UK). The full length WT-LHß gene (1511 bp) was inserted into the eukaryotic expression vector pM2, downstream of the Harvey murine sarcoma virus long terminal repeat, as described previously (Suganuma et al., 1996). The expression vectors were kindly provided by Dr N.Suganuma (Dept of Obstetrics and Gynecology, Nagoya University School of Medicine, Nagoya, Japan). The mutant (G52A) LHß gene was constructed by site-directed mutagenesis (QuickChangeTM Site-Directed Mutagenesis Kit, Stratagene Cloning System, La Jolla, CA, USA) and sequenced. All the plasmids were purified by using the Nucleobond AX plasmid purification protocol (Macherey-Nagel GmbH, Düren, Germany).
Transient transfection and in-vitro bioassays of recombinant WT- and V-LH using unpurified media
Before making the stable transfected cell lines, the gene constructs (2:1 ratio of the -subunit gene to either the WT- or V-LHß gene) were transiently co-transfected into HEK 293 cells using Lipofectamine (Gibco BRL, Life Technologies, Gaithersburg, MD, USA). Following 48 h of transfection, the level of recombinant LH in the medium was determined by immunofluorometric assay (IFMA, Delfia LH Spec; Wallac OY, Turku, Finland). The crude media (from WT- and V-LHß transfected cells) were collected and studied by in-vitro bioassays using mLTC-1 cells (see below).
Transfection, clone selection and production of WT- and V-recombinant LH
Human embryonic kidney (HEK) 293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM)/F-12 medium (1:1) (Life Technologies, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS; Bioclear, Berkshire, UK) containing penicillin (50x103 IU/l) and streptomycin (50 mg/l; Sigma Chemical Co., St Louis, MO, USA).
The gene constructs (2:1 ratio of -subunit gene to either the WT or V-LHß gene) were co-transfected into HEK 293 cells using Lipofectamine (Gibco). Following 48 h of transfection, the level of recombinant LH in the medium was determined by immunofluorometric assay (Delfia® LH Spec Kit). Cells containing the expression vector were selected by growing them in selection medium containing 800 mg/l of the active form of neomycin antibiotic analogue G-418 sulphate (Promega) in a humidified 5% CO2 incubator. After 2 weeks, resistant clones were selected, expanded and screened for expression of LH by measuring the LH level in medium using Delfia LH Spec. The selected clonal lines (WT-A3 and mut-A5) were subsequently cultured at 200 mg/l of G-418 in 15 cm diameter culture dishes. The culture was initiated in medium containing 200 mg/l of G-418 using NunclonTM Delta TripleFlask Culturing Technique (Nunc® Brand Products, Rochester, NY, USA). After 2 days, perfusion was started with serum-free DMEM/F-12 medium. The production rates of human recombinant WT- and V-LH were determined by Delfia (see above).
Measurement of intact LH-dimer, and free - and ß-subunits from culture medium
Five time-resolved IFMA (Delfia) for LH, employing different combinations of monoclonal antibodies (mAb) were used. (i) Delfia LH Spec assay, using two ß-subunit specific mAbs and codetecting intact LH and free ß-subunits (Pettersson and Söderholm, 1990). (ii) Delfia LH assay (no. 1244-017), using as the capture antibody, a mAb directed against the ß-subunit of HLH, and, as the detection antibody, one directed against the HLH -subunit. This assay was used to measure intact LH concentrations in the HEK 293 cell media. (iii) An IFMA with a mAb recognizing an epitope present in the LH/ß-dimer used as the capture mAb (I3), and the detecting mAb (8D10) recognizing an epitope in the -subunit. This assay recognizes only the LH/ß-dimers, but not the Trp8Arg/Ile15Thr variant form of LH (Pettersson et al., 1992). (iv) An IFMA for free -subunit measurement, using a specific mAb (2G11) directed against the -subunit as the capture mAb, and another -specific antibody (7E10) for detection (Korhonen et al., 1997). The mAbs were kindly provided by Dr H.Alfthan (Dept of Clinical Chemistry, Helsinki University Central Hospital). (v) Assay for LHß-subunit determination. First, an intact LH specific mAb (I3) was used to capture the intact LH molecules from the samples during an overnight incubation at 4°C; the samples were then measured by Delfia LH Spec, which detects the free LHß-subunit. In all assays, the standards were calibrated against the World Health Organization (WHO) 2nd international standard for pituitary LH for immunoassay (code 82/552).
Purification of human recombinant WT- and mut-LH
The media collected from HEK 293 cells stably expressing WT- and V-LH were first concentrated through a PelliconTM-2 ultrafitration system (Millipore Corporation, Bedford, MA, USA). The concentrated human recombinant LH were then desalted and re-concentrated by a Centricon® Plus-80 Centrifugal Filter Device (Amicon Inc., Beverly, MA, USA). The purification was carried out by immunoaffinity chromatography, using cyanogen bromide (CNBr)-activated Sepharose 4 Fast Flow matrix (Amersham Pharmacia Biotech AB, Uppsala, Sweden). The four different monoclonal anti-HLH (codes 5301, 5302, 5303 and 5304, Medix Biochemica, Kauniainen, Finland) with different specificities were tested in immunoaffinity chromatography in small-scale samples. These four mAb react with the ß-subunit (codes 5301, 5302), the intact dimer (code 5303), and both the intact dimer and ß-subunit (code 5304), respectively. Anti-HLH mAb 5303 showed the highest immunological binding activity to recombinant LH in the medium and it was therefore used for final purification.
First, the coupling of the ligand, anti-HLH mAb 5303, to the CNBr-activated Sepharose 4 Fast Flow matrix was conducted according to the manufacturer's instructions and carried out at 4°C overnight. After coupling, non-reacted groups in the medium were blocked by keeping the coupled medium in 0.1 mmol/l Tris buffer (pH 8.0). The anti-LH tagged Sepharose was then loaded onto the HR 10/10 column (Amersham Pharmacia Biotech), with 8 cm bed height. The coupled medium was washed using alternate low (100 mmol/l sodium acetate, pH 4.6) and high pH (10 mmol/l Tris–HCl, pH 8.5) buffers. This cycle was repeated three to six times. The equilibration of the coupled medium was done with buffer A (10 mmol/l sodium phosphate with 0.6% NaCl, pH 7.2). The concentrated, desalted recombinant LH were loaded onto the column and incubated for 6 h at 4°C. Bound LH were eluted with buffer B (buffer A containing 3 mol/l sodium iodide) as a chaotrophic agent. The immunoreactive peak fractions (500 µl each) were pooled, then concentrated, desalted using Centricon® Plus-80 Centrifugal Filter Device, and stored at –70°C.
Reversed phase chromatography
After immunoaffinity chromatography, the recombinant V-LH sample was further purified by reversed phase chromatography on a Vydac C4, 2.1 mmx150 mm column (The Separations Group, Hesperia, CA, USA). The solvent gradient used was 2–30% B (0–63 min) [A = 0.1% trifluoroacetic acid (TFA)/H2O, B = 0.08% TFA/acetonitrile]; 30–60% B (63–75 min); 60–80% B (95–105 min); manual steps to >95% B. The buffer flow rate was 150 µl/min, and the detection wavelength was 280 nm. Peak fractions were manually collected and their immunological activities were determined by Delfia LH Spec.
Protein sequencing
Amino-terminal amino acid sequence analysis was performed with an Applied Biosystems (Foster City, CA, USA) model 477A protein sequencer equipped with an on-line Applied Biosystems model 120A phenylthiohydantoin amino acid analyser.
Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE)
The recombinant LH preparations and the purification steps were analysed by PhastGel® gradient system (8–25%) using SDS buffer strips (Amersham Pharmacia Biotech). The molecular mass was determined by using RainbowTM coloured protein mol. wt markers (Amersham Pharmacia Biotech). The gels were silver-stained in an automatic developing device (Amersham Pharmacia Biotech).
Cyclic AMP and progesterone bioassays
The murine Leydig tumour (mLTC-1) cells were cultured in Waymouth's growth medium (Gibco, Paisley, UK), supplemented with 9% heat-inactivated horse serum (Gibco) and 4.5% heat-inactivated fetal calf serum (FCS; Bioclear, Berkshire, UK), containing 50 mg/l gentamycin (Biological Industries, Haemek, Israel), in humidified air containing 5% CO2 at 37°C. The mLTC-1 cells were plated on 24-well culture plates (Greiner, Frickenhansen, Germany) at a density of 80 000 cells/well 18–24 h before stimulation.
The cells were stimulated with increasing doses (0–1000 IU/l) of recombinant WT- and V-LH for 2 h in serum-free Waymouth's medium, containing 0.1% BSA, 50 mg/l gentamycin and 0.2 mmol/l 1-methyl-3-isobutylxanthine (MIX, a phosphodiesterase inhibitor; Aldrich, Steinheim, Germany). The units of measurement (IU/l or ng/tube) of recombinant LH were set in accordance with the WHO 2nd international standard luteinizing hormone (Code 80/552). The media were collected and boiled for 3 min to inactivate the phosphodiesterase activity. Extracellular cAMP was measured using a standard radioimmunoassay method (Harper and Brooker, 1975). Both purified and unpurified (crude media) recombinant LH were used in the in-vitro bioassays.
The cells were stimulated for 6 h with different concentrations of recombinant LH (0–1000 IU/l) in serum-free Waymouth's medium, containing 0.1% BSA and gentamycin (50 mg/l). The media were collected, stored at –20°C and subjected to progesterone measurement by radioimmunoassay (Vuorento et al., 1989). All stimulation experiments were performed in quadruplicate and repeated at least four times.
Ligand binding assays
Highly purified HCG (CR-127; NIDDK, Rockville, MD, USA) was iodinated with Na [125I]iodide (IMS 300; Amersham Pharmacia Biotech), using a solid-phase lactoperoxidase method (Karonen et al., 1975) to a specific activity of 37 000 c.p.m./ng and 28% specific binding to an excess of LH receptors, as determined according to a published method (Catt et al., 1976). For binding measurements, intact mLTC-1 cells were washed twice with ice-cold PBS and scraped off into Dulbecco's PBS containing 0.1% BSA (Sigma). The cells were pelleted by centrifugation at 450 x g, washed twice, and resuspended in Dulbecco's PBS-0.1% BSA. Equal aliquots of cells were then incubated (16–18 h, room temperature) with increasing amounts (0.01–1000 ng/tube) of unlabelled recombinant HCG, recombinant HLH (both from Organon, Oss, The Netherlands), recombinant WT- or V-LH in the presence of 100 000 c.p.m. [125I]HCG, in a total volume of 300 µl. After overnight incubation at room temperature, the cells were washed with 3 ml ice-cold Dulbecco's PBS–0.1% BSA and centrifuged at 1850 x g for 30 min at 4°C, the supernatant was discarded and the radioactivity bound to cell pellets was counted in a 
-spectrometer (1260 Multigamma II, Wallac). Non-specific binding was determined in the presence of 50 IU HCG (Pregnyl; Organon), and specific binding in the pellet was determined by subtracting non-specific from total binding.
IP3 bioassay
To study the effects of recombinant WT- and V-LH on inositol trisphosphate (IP3) production, the experiments were performed as described previously (Tena-Sempere et al., 1999), with some modifications. mLTC-1 cells were plated at a density of 5x105 cells/well (6-well plates; Greiner, Frickenhansen, Germany) for 24 h, then labelled for 24 h using 1 µCi/l of [3H]inositol (NENTM Life Science Products, Inc., Boston, MA, USA). After 24 h labelling, the cells were washed twice with PBS and incubated in 0.1% BSA serum-free medium containing 13 mmol/l LiCl and 58 mmol/l NaCl (1 ml/ well) for 15 min, at 37°C. Thereafter the cells were stimulated for 45 min in the same medium at different concentrations of the recombinant LH (0, 1, 10, 100, 1000, 5000 IU/l). Accumulation of IP3 was terminated by placing the cells in an ice-bath and adding 200 µl of 10% ice-cold perchloric acid in 0.5 mmol/l EDTA to each well. The samples were then centrifuged at 4°C for 15 min, at 2500 x g, and the supernatant was collected and neutralized by adding 200 µl 1.5 mol/l KOH in 60 mmol/l HEPES buffer. The supernatant was recentrifuged at 4°C for 5 min (2500 x g) and collected for IP3 determination through an AG-1 anion exchange column (Bio-Rad, Hercules, CA, USA). Before adding samples, the columns were washed with 6 ml of distilled water. After application of samples, the columns were washed twice with distilled water and twice with 6 ml 5 mmol/l Na2 tetraborate and 60 mmol/M NH4 formate to remove the unbound materials. IP3 was eluted with 6 ml 0.1 mol/l formic acid and 1.0 mol/l ammonium formate. The eluates were mixed with 12 ml scintillation liquid and counted for radioactivity in a ß-spectrometer (Wallac WinSpectral, 1414 Liquid Scintillation Counter). All experiments were carried out in triplicates in three independent experiments.
MAPK bioassay
To study the involvement of recombinant LH in stimulation of the mitogen-activated protein kinase (MAPK) pathway, an in-vitro MAPK assay was performed using the PathDetect® Elk 1 trans-Reporting System (#219005), according to the manufacturer's suggested procedure (Stratagene). In brief, the gene constructs were transfected into mLTC-1 cells by using Lipofectamine (Gibco). Before transfection (18–24 h), mLTC-1 cells were plated at a density of 3x105 cells/well (6-well tissue culture plates; Greiner) in 2 ml of the appropriate complete growth medium. For the negative control, the mLTC-1 cells were co-transfected with 1 µg of pFR-Luc plasmid (reporter plasmid) and 50 ng of pFC2-dbd plasmid (negative control). For positive control, the mLTC-1 cells were co-transfected with 1 µg of pFR-Luc plasmid (reporter plasmid) and 50 ng of pFC-MEK1 plasmid (positive control). One µg of the pFR-Luc plasmid (reporter plasmid) and 50 ng of pFA2-Elk1 plasmid (fusion trans-activator plasmid) were cotransfected to study the effects of recombinant LH on MAPK signal transduction pathway. Ten µl of lipofectamine was used in each transfection reaction, according to the manufacturer's instructions. The mLTC-1 cells were washed twice with phosphate-buffered saline (PBS, pH 7.5, Gibco), 24 h after starting the transfection, then the cells were stimulated at increasing concentrations (0–1000 IU/l) of recombinant WT- and V-LH for 24 h in serum-free Waymouth's medium, containing 0.1% BSA and 50 mg/l gentamycin. Thereafter, the mLTC-1 cells were washed twice with PBS, incubated in 100 µl of cell lysis buffer containing 40 mmol/l tricine (pH 7.8), 50 mmol/ NaCl, 2 mmol/l EDTA, 1 mmol/l MgSO4, 5 mmol/l dithiothreitol, and 1% Triton X-100 at room temperature for 5 min, and harvested by scraping the cells into Eppendorf tubes. After centrifugation at 9750 x g (1 min, 4°C), 20 µl cell lysate was pipetted into a microtitration plate (96-well, Wallac), and 100 µl of luciferase assay buffer was added [40 mmol/l tricine (pH 7.8), 0.5 mmol/l ATP, 10 mmol/l MgSO4, 0.5 mmol/l EDTA, 10 mmol/l DTT, 0.5 mmol/l co-enzyme A, 0.5 mmol/l luciferin]. Luciferase activity was measured using the Vector2TM 1420 Multilabel Counter (Wallac OY, Turku, Finland). All the transfection experiments were carried out in triplicates, and in three independent experiments.
Data analysis
The data are expressed as mean ± SEM. Statistically significant differences were determined by one-factor analysis of variance (a Macintosh version of the super-ANOVA Program, Abacus Concepts, Inc., Berkeley, CA, USA), followed by Duncan's new multiple range test. P < 0.05 was considered statistically significant.
Results
Detection of a new HLHß gene polymorphism
To obtain further information about occurrence of the Gly102Ser polymorphism of the LHß gene, detected recently in Singapore (Liao et al., 1998), we attempted to detect it by RFLP in DNA samples (n = 383) collected from Rwanda, Bengali, Denmark and Finland. As demonstrated in Figure 2, the Gly102Ser (GGTAGT) mutation would have eliminated an EcoO 109I restriction site, and instead of two restriction fragments of the wild-type DNA, 707 and 119 bp, we would have observed a single 826 bp band. None of the samples analysed yielded this result, indicating that none of them contained the Gly102Ser mutation. Instead, almost all of the samples yielded three fragments, 447, 260 and 119 bp, and the positive control (LHß Gly102Ser heterozygous mutation) resulted in four bands, 447, 379, 260 and 119 bp, which was at variance with the published LHß sequence (Fiddes and Talmadge, 1984). Sequencing revealed that there was an additional EcoO 109I restriction site at position –689 of intron 2, which had remained unnoticed upon original sequencing of the WT-LHß gene. In addition, a different RFLP pattern was observed in PCR products of three DNA samples from Rwanda. It displayed four fragments, 707, 447, 260 and 119 bp (Figure 2), and in the rest of samples there were three fragments (447, 260 and 119 bp). Following either direct sequencing, or subcloning of the PCR products, it was found that the altered EcoO 109I digestion pattern in these three samples was due to heterozygous CAGTGA mutations in intron 2 of the LHß gene at nucleotides 687–689. These mutations destroyed an EcoO 109I restriction site (5' AG
GACCT5' GAGACCT) giving rise to the additional 707 bp DNA fragment. When the three variant amplicons were completely sequenced, they were also found to contain a heterozygous G to A transition at nucleotide 52 in exon 2 of the LHß gene, predicting an amino acid transition Ala–3 (GCA)Thr (ACA) in the LHß signal peptide (Figure 3). This substituted amino acid residue of the variant LH is identical to that of the HCGß signal sequence (Fiddes and Talmadge, 1984). When the whole LHß gene was sequenced, no other deviations from the published WT-LHß sequence were found in this newly identified LHß allele.
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Production of recombinant LH and determination of production rates of intact LH, and free
- and free ß-subunits in culture media by specific immunoassaysHEK 293 cells were transfected with the
LHß (WT-/V-LHß)/pM2 expression vectors (see Materials and methods) containing the bacterial neomycin resistance gene. Clones were isolated after selection with G-418. The highest LH-producing clones were used for large-scale production of recombinant LH. The intact LH, and free - and ß-subunit concentrations in the culture media of two cell lines stably transfected with the common and WT- or V-LHß clones were measured by the five IFMA for LH (see Materials and methods) employing different mAb combinations. The ratios of free /intact LH and free ß/intact LH were calculated. The results indicated that the secretory proportions of intact LH, free and free ß were not significantly different between culture media from WT- and V-LHß-expressing HEK 293 cells. The ratios of free :free ß:intact LH were 1.96 ± 0.3:0.19 ± 0.3:1 in WT-LHß media, and 3.05 ± 0.5:0.15 ± 0.2:1 (mean ± SEM, n = 5) in Ala–3Thr V-LHß media, and the differences of ratios were not statistically significant. These findings indicated that the mutation did not affect the dimerization efficiency of the LH-subunits.
Purification of recombinant WT- and V-LH
The human recombinant LH accumulated in the culture media was first concentrated and desalted. For in-vitro bioactivity measurement, the samples were purified by one-step immunoaffinity chromatography. For the purpose of protein sequencing, the recombinant V-LH sample was further purified by reversed phase chromatography. The purity and molecular sizes of recombinant WT- and V-LH were determined by SDS–PAGE (Figure 4), which demonstrated several bands with one major band in concentrated medium and one clear band of ~30 kDa in the purified samples.
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LH receptor-binding affinity
The receptor-binding affinities of the recombinant WT- and V-LH were studied, and compared with that of HCG, based on their displacement of specific binding of [125I] iodo-HCG from mLTC-1 cells. Displacement of [125I] iodo-HCG from the LH receptor was dose dependent and the concentrations at 50% displacement were not significantly different between WT- and V-LH, whereas HCG displayed significantly higher binding affinity (Table II
). The binding-inhibition curves of recombinant HCG, recombinant HLH (both from Organon), as well as that of the WT-LH and V-LH preparations are shown in Figure 5.
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Effects of recombinant WT- and V-LH on cyclic AMP and progesterone production
The in-vitro bioassay of LH was based on stimulation of cAMP and progesterone production in mLTC-1 cells. Progesterone and cAMP production were stimulated by recombinant WT- and V-LH in a dose-dependent manner (Figure 6A,B). The progesterone output was not significantly different, when comparing at ED50 values or maximal production rates, between WT-LH and V-LH (Table II
, Figure 6A), but the cAMP responses at each LH concentration tested were 30–50% higher with WT-LH than with V-LH (P < 0.05, Table II, Figure 6B). The cAMP responses plateaued, or decreased in some experiments, at LH concentrations >100 IU/l (data not shown), but the difference between the WT- and V-LH responses persisted. When two types of recombinant LH preparations (purified recombinant LH and cell culture supernatant) were tested in-vitro bioassays, the results showed a similar tendency.
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Effects of recombinant WT- and V-LH on IP3 production
The IP3 responses of mLTC-1 cells to the stimulation with recombinant WT- and V-LH displayed clear-cut differences. Although treatment of mLTC-1 cells with both forms of LH resulted in a dose-dependent increase in IP3 production, the response was 1.6-fold over the basal level with recombinant WT-LH, and 2.5-fold over recombinant V-LH (Figure 7
). The difference was statistically significant (P < 0.01).
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Involvement of recombinant WT- and V-LH on MAPK pathway
When the PathDetect® Elk1 trans-Reporting System was used to study the influences of recombinant WT-LH and V-LH on activation of the MAPK pathway, the patterns of dose–response were almost identical with the two hormones in mLTC-1 cells (Figure 8
).
|
Protein sequencing
The purified V-LH sample was sequenced and the result showed that the signal peptide was cleaved in an identical fashion to WT-LH. The protein sequence demonstrated that the mature V-LH protein has the same sequence as WT-LH.
Discussion
The glycoprotein hormones are composed of two dissimilar, noncovalently associated subunits designated and ß. The human LHß-subunit gene is located on chromosome 19q13.32 and is close to the HCGß genes. Each of the glycoprotein hormone ß-subunits is encoded by a single gene except for the seven genes and pseudogenes for HCGß. The LHß and HCGß genes are ~1.5 kb in size and each is composed of three exons and two conserved introns (Fiddes and Talmadge, 1984). Within the species, the -subunit has an identical amino acid sequence in all four members of this hormone family (Gharib et al., 1990). The - and ß-subunits of HLH are secreted both as a non-covalently linked dimer form as well as uncombined free forms by pituitary gonadotrophin cells. The biosynthesis of gonadotrophin subunits by the usual ribosomal assembly of the peptide chains is followed by post-translational modifications of the expressed subunits prior to the final step of their secretion.
Unlike the many mutations reported in the LH and FSH receptor genes, only a few sporadic cases of mutations and polymorphisms of genes for the gonadotrophin ß-subunits have been reported (Themmen and Huhtaniemi, 2000; Table III). Only one loss-of-function mutation of the LHß-subunit (Gln54Arg) has been reported (Weiss et al., 1992). In addition, there is a common polymorphism (Trp8Arg and Ile15Thr) in the LHß-subunit with possible clinical significance as a contributing factor to pathologies of LH-dependent gonadal functions (Lamminen and Huhtaniemi, 2001). Recently, another polymorphism of LHß (Gly102Ser) was reported and it was suggested to be associated with menstrual disorders, and female and male infertility (Liao et al., 1998; Ramanujam et al., 1999, 2000). Our limited analysis of two European, one African and one Asian population (a total of 383 samples) was unable to detect this mutation. This finding suggests that it does not exist, or is very rare, in healthy subjects outside Singapore. In this study, we report a novel mutation (G52A) in exon 2 of the LHß gene, causing Ala–3Thr amino acid substitution in the signal peptide of the LHß-subunit.
|
With the possible exception of the egg white protein ovalbumin (Palmiter et al., 1978
), all secretory proteins are synthesized as precursors extended at the NH2 terminus by a sequence of 15 to 30 amino acids, termed the signal or leader sequence. The signal sequence is required for transport of the protein across the membrane of the endoplasmic reticulum (ER) (Blobel and Dobberstein, 1975). The signal sequence is recognized by the signal recognition particle (SRP), an evolutionarily conserved ribonucleoprotein complex, which then binds to the SRP receptor (docking protein) in the microsomal membrane, thus transferring the precursor protein to the membrane (Meyer et al., 1982). The signal sequence is then cleaved by signal peptidase embedded within the membrane (Rapoport, 1992). Typically, the signal peptides are composed of three domains: (i) a positively charged NH2-terminal region (N-region), which is important for translocation; (ii) a central hydrophobic core (H-region), forming the target for the SRP (Walter and Blobel, 1981; Walter et al., 2000), and (iii) a polar COOH-terminal region (C-region) which affects the efficiency and fidelity of signal peptidase cleavage (Perlman and Halvorson, 1983; von Heijne, 1985).
The LHß gene encodes a prohormone with a 20 amino acid leader sequence and a mature peptide of 121 amino acids. The G52A mutation of the LHß gene is located in the part of the LHß gene encoding the signal peptide, causing an Ala–3Thr amino acid substitution. According to the –3, –1 rule of the signal peptidase recognition site, the region around the cleavage site shows strong preferences for specific amino acids in particular positions (Perlman and Halvorson, 1983; von Heijne, 1983). Crystallographic analysis of the leader peptidase shows that amino acids –1 and –3 may help to position the signal peptide relative to the active site through their interactions with distinct binding pockets on the enzyme's surface. Position –3 is important for signal peptide recognition in the endoplasmic reticulum (von Heijne, 1983; Paetzel et al., 1998). Alterations near the cleavage site can also disrupt signal peptidase cleavage (Koshland et al., 1982; Haguenauer-Tsapis and Hinnen, 1984; Schauer et al., 1985; Inouye et al., 1986). A point mutation at position –3 in the signal peptide of the preproparathyroid hormone was found to be associated with autosomal recessive familial isolated hypoparathyroidism (Sunthornthepvarakul et al., 1999), apparently because the preprohormone mutant was not cleaved by signal peptidase at the normal position, with consequent degradation of the immature protein in rough endoplasmic reticulum.
The novel G52A (Ala–3Thr) LHß mutation could possibly affect the hydrophobic interactions required during the earliest nucleation events, and disrupt signal peptidase cleavage. Since the signal sequence together with SRP plays an important role in protein synthesis, it was interesting to study whether this mutation would affect, in addition to signal peptide cleavage, the conformation of LHß during its synthesis and then possibly the bioactivity of the final LH molecules. To this end, a ß-subunit gene containing the G52A substitution was generated by site-directed mutagenesis and co-expressed along with the -subunit gene in human embryonic kidney (HEK) 293 cells. Immunoreactivities of the WT- and V-LH heterodimers, or free subunits, secreted by the cells were determined by an array of IFMA. The majority of the G52A variant ß-subunit synthesized was secreted as assembled /ß-dimers, and their relative proportion, in comparison to free - and ß-subunits, was the same as in cells expressing WT-LHß. In conclusion, the Ala–3Thr mutation in the LHß signal sequence did not alter the biosynthesis of ß-subunit or its assembly with glycoprotein hormone -subunit, or the secretion of LH from the transfected HEK 293 cells.
The biological activity of the recombinant WT- and V-HLH forms was determined by in-vitro bioassays based on induction of cAMP and progesterone production of mLTC-1 cells. Both recombinant hormones were biologically active, which provides further evidence that the mutation in the HLHß signal peptide does not influence the ß-subunit dimerization with -subunit. The bioassay also confirms that the recombinant hormones were properly glycosylated and assembled since free subunits or deglycosylated hormones are not bioactive (Flack et al., 1994). Finally, protein sequencing demonstrated that the Ala–3Thr signal peptide mutation did not disrupt signal peptide cleavage. When examined in more detail, the signal transduction of the two recombinant hormones differed to some extent. The cAMP production of mLTC-1 cells was more actively stimulated by WT- than V-LH. Conversely, a higher IP3 response was found in cells stimulated with V-LH. However, the progesterone and MAPK dose–response curves for both forms of LH were almost superimposable. The exact mechanism of these differences remains unclear, but it can be speculated that the altered signal peptide structure somehow influences the tertiary structure of the LH molecule and its association with the LH receptor. It should still be mentioned that the amino acid change observed in the variant signal peptide is the same as that observed in this position in HCGß (Fiddes and Talmadge, 1984). Generally, the IP3 pathway is less sensitive than the adenylate cyclase pathway to stimulation by LH or HCG (Cooke, 1999; Kuhn and Gudermann, 1999) and, therefore, it is even more remarkable that a more sensitive and higher maximum IP3 stimulation was observed with the V-LH than with WT-LH in mLTC-1 cells.
Recent studies have provided important insights into the genetic disorders affecting the function of the human hypothalamic–pituitary–gonadal axis (Achermann and Jameson, 1999; Themmen and Huhtaniemi, 2000). However, inherited disorders of LH and FSH are very rare, and no germline mutations of the common -subunit gene have been reported, since it is possible that they would be lethal to the embryo blocking the synthesis of HCG. A total of five cases (three women and two men) with different inactivating mutations of the FSHß gene have been reported in women with delayed puberty, lack of breast development, primary amenorrhoea and infertility and in men with azoospermia or delayed puberty, low FSH and small testes (Themmen and Huhtaniemi, 2000). As summarized in Table III, only one LHß gene mutation (Gln54Arg) has been reported in a male with delayed puberty, low testosterone and arrested spermatogenesis. A common polymorphism occurs in the LHß-subunit, with two point mutations altering the amino acid sequence (Trp8Arg and Ile15Thr) and introducing an extra glycosylation signal to Asn13. The frequency of this variant LHß allele differs widely between ethnic groups, being most common in aboriginal Australians (carrier frequency >50%; allelic frequency 28.3%). There are also clear functional differences between this variant and wild-type LH; variant LH possesses increased in-vitro bioactivity, whereas its half-life in circulation is shorter. Also the regulation of the variant LHß gene differs due to additional changes in its promoter sequence (Jiang et al., 1999). The LHß Gly102Ser mutation has been reported only in infertile subjects (Liao et al., 1998; Ramanujam et al., 1999, 2000) in Singapore, and this may explain why it was not found in our study, analysing randomly collected samples from apparently normal subjects. The novel LH variant (Ala–3Thr) in signal peptide, reported in this study, provides new information on structure–function relationships of the LHß-subunit. It would seem that the signal peptide mutation affects the final conformation of the secreted LH molecules which, in turn, influences anomalously the stoichiometry of activation of the different signalling cascades involved in LH action.
Acknowledgements
We thank Drs M.Poutanen, F.-P.Zhang, P.Ryhänen, R.Shariatmadari (Dept of Physiology, University of Turku), and C.Nilsson (Dept of Biotechnology, University of Turku) for advice and helpful discussions during this study. The technical assistance of Ms R.Kytömaa and Ms T.Laiho is gratefully acknowledged. We are indebted to Dr N. Suganuma (Dept of Obstetrics and Gynecology, Nagoya University School of Medicine, Nagoya, Japan) for the expression vectors pM2 and pM2ß. Dr H.Simonsen (Dept of Clinical Biochemistry, State Serum Institute, Copenhagen, Denmark) is acknowledged for providing us with the Danish DNA samples and Dr H.Alfthan (Dept of Clinical Chemistry, Helsinki University Central Hospital) for the gift of mAb (2G11 and 7E10). Drs A.C.Roy and W.X.Liao (Dept of Obstetrics and Gynaecology, Faculty of Medicine, National University of Singapore) are thanked for providing us with a positive sample of the Gly102Ser LHß mutation. This study was supported by grants from the Academy of Finland and the Sigrid Jusélius Foundation and National Institutes of Health (grant RRO8205 to R.J.Herrera).
Notes
4 To whom correspondence should be addressed at: Department of Physiology, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland. E-mail: ilpo.huhtaniemi{at}utu.fi 
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