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Revista mexicana de ciencias pecuarias
versión On-line ISSN 2448-6698versión impresa ISSN 2007-1124
Rev. mex. de cienc. pecuarias vol.7 no.1 Mérida ene./mar. 2016
Articles
Biological and immunological activity in bovine luteinizing hormone charge isoforms
a Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México. Circuito exterior de Ciudad Universitaria S/N, CP.04510. México.
b Unidad de Investigación Médica en Medicina Reproductiva, UMAE Hospital de Gineco Obstetricia no. 4 Luis Castelazo Ayala, IMSS, México.
c Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México. México.
Luteinizing hormone (LH) undergoes posttranslational modifications that originate different charge isoforms. The study evaluated the differences in biological (B) and immunological (I) activity between isoforms of bovine LH. Isoforms were isolated by chromatofocusing from anterior pituitary glycoprotein extract. The biological activity was evaluated in an in vitro bioassay. Immunological activity was measured with a radioimmunoassay (RIA) specific for LH. The USDA-bLH-B5 standard was utilized as the reference. LH isoforms were grouped by their pH range of elution, in basic (A, pH, 10.75-9.75; B, pH 9.58-8.41), neutral (C pH, 7.98-6.89) and acidic (D, pH, 6.88-5.41; E, pH 5.36-3.46). The molecular weight of the heterodimer of each isoform was similar to the LH standard, estimated to be 36.5 kDa. Immunological and biological activity behaved in a dose-dependent manner. With respect to the LH standard, all isoforms required higher protein concentration to reach the IC50 of the inhibition curve. In the bioassay EC50 value for cAMP production was significantly different among isoforms; the neutral isoform showed a lower EC50 which was interpreted as more bioactive, the acidic isoform E showed an EC50 being the least bioactive and the basic was intermediate (P<0.05). In conclusion, the results suggest a quantitative effect of LH charge isoforms on the cAMP production, per unit of immunoreactive LH in the bioassay.
Keywords: Luteinizing hormone; Biological activity; Immunological activity
La hormona luteinizante (LH) sufre modificaciones postraduccionales que dan origen a isoformas de carga. El estudio determinó la actividad biológica (B) e inmunológica (I) de distintas isoformas de LH bovina. Las isoformas se aislaron mediante el cromatoenfoque a partir del extracto glicoprotéico obtenido de lóbulos anteriores de hipófisis de bovino. La actividad biológica se evaluó en un bioensayo in vitro midiendo la producción de AMPc. La actividad inmunológica se midió con un radioinmunoensayo (RIA) específico para LH. El USDA-bLH-B5 se utilizó como referencia. Las isoformas se agruparon tomando como referencia el rango de pH de elución, en básicas (A, pH, 10.75-9.75; B, pH, 9.58-8.41), neutras (C, pH, 7.98-6.89) y ácidas (D, pH, 6.88-5.41; E, pH, 5.36-3.46). El peso molecular del heterodímero de cada isoforma y del estándar fue similar, estimado en 36.5 kDa. La actividad inmunológica y biológica se comportó de forma dosis-dependiente. Con respecto al estándar, se requirió una mayor concentración de proteína de cada isoforma para obtener el IC50 en la curva de inhibición. En el bioensayo, el valor EC50 para la producción de AMPc fue significativamente diferente entre isoformas; la isoforma neutra mostró un EC50 inferior lo que se interpretó como la proteína más bioactiva, en contraste, la isoforma ácida, mostró un valor de EC50 superior y resultó ser la menos bioactiva; la básica tuvo un comportamiento intermedio (P<0.05). En conclusión, los resultados sugieren un efecto diferenciado de las isoformas de carga, sobre la producción cuantitativa de AMPc por unidad de LH inmunoreactiva.
Palabras clave: Hormona luteinizante; Actividad biológica; Actividad inmunológica
Introduction
Luteinizing hormone (LH) is a glycoprotein present in the anterior lobe of the hypophysis. It participates in follicular maturation1, ovulation2, and luteal body formation and maintenance3. During LH synthesis, the hormone experiences post-translational modifications such as incorporation of N-type oligosaccharide residues linked to different proportions of sulfate and sialic acid4. This specific variation is considered the principal biochemical foundation for differentiation between hypophysis and circulating LH charge isoforms5.
Polymorphism in gonadotropins occurs in ruminants6,7,8. Using the chromatofocusing protein separation technique, different proportions of LH isoforms have been identified in the serum9,10 and hypophysis11,12. Indeed, the proportion of these isoforms in circulation differs in the different stages of the estrus cycle9,10.
Variability in the in vivo biological activity of LH isoforms in ruminants is the cause of inconsistent results, although acidic isoforms are regularly reported to have the highest bioactivity13,14,15. This variability in biological response has been attributed to each isoform's depuration rate, low assay sensitivity (micrograms of protein are required), and biological variability in experimental animals. In in vitro bioassays, the highest biological activity is present in the basic isoforms16,17. Results are not always consistent due to the origin (rat or mouse) of the Leydig cells, since the question remains of how species specific they can be when ligands from other species are used. A recently developed alternative is bioassays based on gonadotropin receptor cloning18,19. Using a stable cell line derived from HEK-293 cells transfected with rat LH receptor cDNA, evaluations have been done of cAMP production associated with distinct rat hypophysis extracts at a basic pH20,21. This same biological model has been used to prove the constant affinity of bovine LH to the rat LH receptor22.
Over the last decade, our research group has focused on isolating and characterizing different ruminant LH isoforms to study their structure-function relationship, their secretion patterns in various physiological conditions and their biological activity. Biological characterization has been especially challenging due to low yields of acidic and neutral isoforms. Advent of the LH receptor cloning bioassay has opened the possibility of quantifying bioactivity in these isoform groups, which is difficult to monitor due to their low yield.
The present study objective was to evaluate cAMP production by HEK-293 cells transfected with rat LH receptor cDNA in response to different bovine hypophysis LH isoforms.
Material and methods
This study involved four main stages: 1) Isolation and purification of sufficient amounts of the different bovine LH isoforms; 2) Identification of each isoform's isoelectric point using chromatofocusing; 3) Quantification of each isoform's molecular weight by SDS-PAGE; and 4) Immunological characterization using a LH-specific immunoassay and immunotransference analysis.
Hypophysis collection
Hypophyses were extracted from adult bovines regardless of age, sex and physical condition. Prior to slaughter, each animal was stunned with a captive bolt gun. Once dead, the head was removed from each animal, the hypophysis extracted and deposited in phosphate buffer (0.05 M, pH 7.2). Excess tissue was removed from the gland, the posterior lobe discarded, and the anterior lobe lyophilized. All gland extraction and LH purification and isoform isolation steps were done at 4 °C.
Obtaining of glycoprotein extract from the hypophysis anterior lobe
An established protocol13 was used to extract glycoprotein from 375 anterior lobes. Briefly, lyophilized anterior lobes were hydrated for 48 h with 10% ammonium acetate (pH 7.0) containing 0.001 M phenylsulfonyl methyl fluoride. They were blended and homogenized with the same solution. The resulting extract was kept under constant agitation for 24 h and centrifuged at 10,000 xg for 45 min. The precipitate (R0) was stored. Supernatant was adjusted to pH 7.0 and a volume of ethanol equivalent to 40 % total volume added by drops under constant agitation. After 16 h agitation, the mixture was centrifuged, and the precipitate (protein fraction; R1) was discarded and stored. A volume of ethanol equal to that in the previous step was added to the supernatant, although in this case it was equal to 85 % of total volume. This mixture remained undisturbed for 48 h and the precipitated proteins recovered by centrifugation. This group of proteins Identified was named glycoprotein extract (GPE), was re-suspended in deionized water, dialyzed (Spectra/Por # 4, 12-14 kDa cut-off) for 24 h with the water changed every 8 h, and lyophilized. A second extraction was done to the R0 precipitate from the first extraction step, following the above process. This second GPE was mixed with the first before purification.
GPE purification for LH isolation
The GPE was purified in an ionic exchanger (CM-Sepharose) to isolate LH13. One gram GPE containing 256 mg protein was resuspended in 0.005 M ammonium acetate (pH 5.1; elution buffer), agitated for 24 h, and centrifuged (10,000 xg for 30 min). The protein solution was applied to a pre-packed column (27.0 x 1.5 cm i.d.) with the cation exchanger previously adjusted with elution buffer, and stored at 4 °C. The protein fraction was eluted at a 23 ml/h flow through an ammonium acetate gradient (0.005 M, pH 5.1; 0.1 M, pH 6.8; and 1.0 M-glycine 0.1 M, pH 9.5). Two-milliliter fractions were collected during the chromatography run, and the protein elution pattern monitored at 280 nm. When effluent optical density was close to zero, the buffer was changed. After elution, a 1 M NaCl solution was run through the column. Elution of the GPE produced protein peaks identified based on the buffer. Fractions eluted with 0.005 M ammonium acetate (pH 5.1) were called CM-1ab, CM-1cd and CM-1ef. The protein eluted with 0.1 M ammonium acetate (pH 6.8) was called CM-2ab, and that eluted with 1.0 M ammonium acetate and 0.1 M glycine (pH 9.5), corresponding to raw LH, was called CM-3ab. The protein peak recovered after the NaCl run was called the salt peak (S). All the protein peaks were dialyzed and lyophilized until analysis.
CM-3ab purification for LH isoform isolation
Isolation of LH isoforms was done by purifying the CM-3ab fraction. During GPE purification in CM-Sepharose, CM-3ab stood out for its high LH content and for having an electrophoretic pattern similar to that of the bovine LH standard (USDA-bLH-B5). For this reason, it was processed for separation by electrical charge8,11. Briefly, 56 mg protein were resuspended in Pharmalyte (pH 8.0-10.5, Pharmalyte 0.36 meq/ml pH, Pharmacia Biotech Piscataway, NY, USA), diluted to 1:45 with deionized water and HCl added to adjust suspension pH to 7.0. This was then added to a column (27 x 0.7 cm i.d.) prepacked with PBE-118 ionic exchanger (Polybuffer exchanger for chromatofocusing, capacity: 50.4 μmol, pH unit-1 ml-1, Pharmacia, Biotech, Piscataway, NY, USA), balanced with 0.025 M triethylamine-HCl (pH 11.0) and stored at 4 °C. Three milliliters Pharmalyte buffer (pH 7.0) were added to the column before protein elution to prevent exposure of the sample to an extreme pH.
Protein fraction elution was done at a 7 ml/h flow rate, and 2 ml fractions collected. Each fraction's pH was measured, and when a pH above 7.0 was detected in more than ten consecutive samples the elution buffer was changed to polybuffer 74 (Pharmacia, Biotech). This was diluted to 1:8 with deionized water, and adjusted to pH 3.5 to elute proteins between pH 7.0 and 3.5. Any protein clinging to the column after elution at pH 3.5 was recovered by adding 1.0 M NaCl. Each fraction was then neutralized based on the pH record and the known protein elution pattern at 280 nm. Fractions collected between pH 11.0 and 7.0 were neutralized with 1.1 M Tris-HCl, and those eluded between pH 6.99 and 3.5, as well as those collected with 1 M NaCl, were neutralized with 1.1 M imidazole. After all fractions were neutralized, they were grouped according to the pH range of the elution protein peak: basic (A, pH 10.75-9.75; B, pH 9.58-8.41); neutral (C, pH 7.98-6.89); and acidic (D, pH 6.88-5.41; E, pH 5.36-3.46). The different protein peaks were dialyzed separately and then lyophilized.
Electrophoretic (SDS-PAGE) analysis
The protein fractions isolated during LH purification and their isoforms were analyzed with SDS-PAGE23, and their electrophoretic patterns compared to the LH reference (USDAbLH-B5). The resulting gels were stained following manufacturer instructions (Silver Stain Kit, Bio-Rad Laboratories, Inc.). Prestained, low molecular-weight markers were used as references (Bio-Rad Laboratories, Inc.).
Total protein quantification
Protein content was quantified in each fraction isolated during the LH purification and their isoforms, according to Bradford method24. Bovine serum albumin (BSA) was used as a reference standard.
Immunological analysis of LH and its isoforms
Identification of LH-immunoreactive proteins
The molecular weight of proteins immunoreactive to the LH was determined through immunoblotting25 The reference standard (USDA-bLH-B5), a protein fraction, or an isoform were analyzed by SDS-PAGE at a concentration of 100 ng of protein. At the end of electrophoresis, the proteins present in the gel were transferred (200 mA/75 min: Trans-Blot Semi-Dry, Bio-Rad, USA) to a nitrocellulose membrane (0.45 μm Trans-Blot, Bio-Rad). The membranes were incubated at 4 °C for 16 h with a 1:1000 dilution of rabbit-generated primary antibody (anti-oLH-26). They were then incubated for 60 min at room temperature with a 1:20,000 dilution of the secondary antibody (rabbit anti-IgG generated in goat and conjugated to peroxidase; Jackson Immuno Research). The membranes were viewed with chemiluminescence (ImmobilonTM Western Chemiluminescent HRP Substrate, Millipore Corporation, Billerica, MA, USA).
Quantification of immunoreactive LH
Immunoreactive LH was quantified with liquid-phase radioimmunoassay (RIA) after validation with bovine LH11. Each protein fraction from LH purification and their isoforms were analyzed at six doses (0.2, 0.4, 0.8, 1.6, 3.2 and 6.4 ng protein/tube), with four replicates per dose. The LH reference (USDA-bLH-B5) was used as a standard at eight doses (0.01, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 and 10 ng/tube). To create a tracer, NaI125 was incorporated into the USDAbLH-B5 following the IODO-GEN method11. Primary antibody (anti-oLH-26) was used at a 1:40,000 dilution in the presence of normal rabbit serum (1:1600). Precipitation of the antigen-antibody complex occurred after 24 h at 4 °C with the secondary antibody (anti-IgG from rabbit generated in burro, 1:80). After addition of 1 ml 0.05 M PBS buffer (pH 7.2) containing 0.1% BSA, the immunoprecipitated fraction was separated from the unbonded fraction by centrifuging at 1,500 xg for 15 min at 4 °C. A gamma radiation counter was used to analyze the immunoprecipitated fraction. Assay sensitivity was 0.1 ng/tube. Using the reference curve EC50 as the dose value parameter, intra-assay variation was 2 % and interassay variation was 5 %.
Quantitative LH concentration was measured by calculating IC50, defined as the protein concentration (ng protein or LH/tube) that caused 50 % inhibition of the %B/Bo response.
This calculation was done using the Prism 6.0 (Graph Pad Software, Inc., USA) program, which includes the Hill equation. The IC50 and Hill slope (h) values were compared with the sumsquared F-test using a null hypothesis of the parameters being identical between each evaluated hormone pair; if a P>0.05 value is generated the parameter is deemed the same for both fits26. A one-way ANOVA and a Tukey multiple comparison test were applied to identify significant differences (P<0.05) between the tested isoforms27,28.
Measuring LH isoform biological activity
Biological activity was quantified by measuring cAMP production in three replicates per dose in three independent assays. HEK-293 cells20 transfected with rat LH recombinant receptor cDNA (provided by Dr. Mario Ascoli, Iowa University, Iowa City, IA,USA) were stimulated with the reference pattern (USDA-bLH-B5) or one of the isoforms. These were differentiated when their isoelectric points (IEP) were sufficiently separated between proteins, and each represented a LH polymorphism. Based on these criteria, three isoforms were analyzed: basic (B, pH 9.58-8.41); neutral (C, pH 7.98-6.89); and acidic (E, pH 5.36-3.46).
The HEK-293 cells were cultured in a 162 cm2 culture box (Costar, Cambridge, MA, USA) containing high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5 % lamb fetal serum, 0.002 M L-glutamine, 100 μg/ml geneticine, 50 UI/ml penicillin and 100 μg/ml streptomycin. Cells with 90% confluence were reseeded in 24-well boxes (Gibco, BRL) at a 5 X 104 concentration per well and incubated in a 5% CO2 atmosphere for 24 h at 37 °C. The medium was then removed and the cells exposed for 24 h to increasing doses (6.25, 12.5 25.0, 50.0, 100.0 and 200.0 ng/ml) of immunoreactive LH from USDA-bLH-B5 or an isoform. Each hormone was diluted in culture medium containing 0.0125 M 3-isobutyl-1-methylxantine (phosphodiesterase inhibitor).
Total cAMP content was measured in the culture medium with liquid phase RIA21. The cAMP tracer 2-0-monosuccinyl tyrosyl-methyl ester (Sigma) was labelled with Nal125 (Amersham International Limited, UK) following the chloramine-T method29. The primary antibody (anti-cAMP, CV-27, NIADDK, Bethesda, MD, USA) was used at a 1:70,000 dilution in the presence of 0.005 M sodium and 0.1 % BSA (pH 6.1). Each reaction tube was incubated for 24 h at 4 °C, and then the bonded antibody separated from the free cAMP by adding cold ethanol, and then centrifuging at 1,500 xg for 30 min at 4 °C. The immunoprecipitated fraction was analyzed in a gamma radiation counter. Assay sensitivity was 2.0 pmol/ml with a 2 % intraassay variation coefficient, and a 5 % interassay variation coefficient. Production of cAMP was calculated by interpolating from the cAMP 2-0-monosuccinyl tyrosyl-methyl ester reference curve.
Each isoform's biological activity was measured by calculating the EC50 parameter, defined as the amount of LH (ng/ml) required to generate a response equal to 50 % of the maximum response under assay conditions26,27. For this purpose, the experimental data were fitted in four-parameter dose-stimulation response curves in the tested LH immunoreactive dose range for the standard and for each isoform. These calculations were done with the Prism 6.0 program (GraphPad Software, Inc., USA).
Using the fit curves, statistical comparisons were made of the EC50 parameters and Hill slope (h). These comparisons were done with the sum-of-squares F-test, testing the null hypothesis of the parameters being identical between each pair of evaluated hormones; a value of P>0.05 indicated the parameter was the same in both fits27. A one-way ANOVA and the Tukey multiple comparison test were applied to identify significant differences (P<0.05) between the relative bioactivity strength of each assayed isoform.
Results
Three fractions were generated during the anterior lobe extraction process. The first fraction, R0, corresponded to 63.2 % (77.16 g) of the original weight. The second, R1, was obtained with 40% ethanol with a 9.03 % (11.02 g) yield. The third was the GPE, recovered using 85% ethanol with a 8.24 % (10.06 g) yield and a 226 μg LH/mg protein concentration.
Five protein fractions resulted from GPE purification in the ionic exchanger (Figure 1(I)). Fraction CM-3ab exhibited the highest protein content and high immunoreactive LH content (Figure 1(II)). In contrast, fractions CM-1cd and CM-1ef had low LH and protein contents, and were excluded from the analysis.
Figure 1 I) Elution pattern of the glycoproteic extract (GPE) during its cation-exchange chromatography with CMSepharose. II) Table summarizing the protein concentration (mg) and concentration of LH (μg LH/mg protein) of each fraction. III) Electrophoretic pattern of the reference LH (USDA-bLH-B5), the GPE and the fractions with LH immunological activity under non-reducing (NR) and reducing (R) conditions. Arrows indicated at proteins with molecular weights of 72, 60, 36.5, 23.4, 20.8, 17.0 kDa, respectively.
In the electrophoresis patterns generated under non-reducing (NR) conditions (i.e. absence of β-mercaptoethanol) (Figure 1(II)), CM-3ab exhibited a pattern similar to that of the reference, consisting of two proteins with molecular weights of 36.5 and 23.4 kDa, respectively (Figure 1(III)). Although both these proteins were present in the GPE, as well as CM-1ab and CM-2ab, the pattern in the latter two fractions was more heterogeneous with an additional predominant, higher weight (56.0 kDa) protein. Under reducing (R) conditions (i.e. presence of β-mercaptoethanol), CM-3ab exhibited three proteins with weights of 23.4, 20.8 and 17.0 kDa, respectively. Fractions CM-1ab and CM-2ab had a similar pattern, with addition of an even more dominant protein at 72.0 kDa. Based on their patterns, the latter two fractions were excluded, and LH charge isoforms were isolated only from fraction CM-3ab (pH 9.5).
In the PBE-118 ionic exchanger, the CM-3ab elution pattern exhibited five protein peaks along the pH gradient, each peak corresponding to an isoform (Figure 2(I)). Of the total recovered immunoreactive LH, 72.8 % was obtained at a basic pH (≥7.5, fractions A and B), 6.3 % at a neutral pH (7.4 - 6.6, fraction C), and 20.1 % at an acidic pH (≤6.5, fractions D and E). Each isoform had different protein and immunoreactive LH contents (Figure 2(II)).
*Estimated by Bradford. **Estimated by RIA.
Figure 2 I) Elution pattern of the CM-3ab fraction in chromatofocusing. Protein was monitored at 280 nm and each protein peak was identified with a letter, starting with the elution protein at basic pH (A) and ending with the eluted protein at acid pH (E). The protein that did not eluted with the gradient was obtained after, using 1.0M NaCl and was designated as S. II) Table summarizing the protein concentration and LH-specific for each isoform.
Under NR electrophoretic conditions (Figure 3(I), upper panel), each LH isoform exhibited a homogeneous pattern with two dominant protein bands (36.5 and 23.4 kDa) in a pattern similar to those of USDA-bLH-B5 and CM-3ab. Although the GPE and isoform E (pH; 5.36-3.46) had a pattern similar to those above, they also included a 55.0 kDa protein. Under R electrophoretic conditions (Figure 3(I), lower panel), all the isoforms exhibited the same pattern as the GPE, CM-3ab and USDA-bLH-B5. Of interest is the presence of a 17.0 kDa protein in CM-3ab and the B, C and D isoforms.
Figure 3 Electrophoretic pattern (I) and immunoblotting electrophoretic pattern (II) for the standard bLH (USDAbLH-B5), glycoprotein extract (GPE), CM-3ab fraction, isoform A, pH 10.75-9.75; B, pH 9.58-8.41; C, pH 7.98-6.89; D, pH 6.88-5.41; E, pH 5.36-3.46 in non-reducing and reducing conditions.
Immunotransference analysis under NR conditions (Figure 3(II), upper panel) showed that the different isoforms maintained a LH immunoreactive protein pattern similar to that of USDA-bLH-B5 with the 36.5 kDa protein most notable. Under R conditions, this protein almost disappears while lower weight proteins (23.4 and 20.8 kDa) increase in intensity.
When the LH isoform and CM-3ab fraction are compared to the reference standard in the RIA displacement curves (Figure 4), immunological activity (IC50) is similar between USDA-bLH-B5 and CM-3ab (Figure 4(II)). However, the IC50 for the reference was lower than those of the isoforms, indicating that the standard is immunologically more active versus the antibody used in the assay. The dose-response curve slopes of the standard and the isoforms did not differ, indicating the proteins' identity (Figure 4(III)).
Figure 4 Inhibitory dose-response curves of a LH-specific RIA for the standard bLH (USDA-bLH-B5) and isoforms. I) The reference standard and CM-3ab fraction. II) Standard and basic isoforms (A, pH, 10.75-9.75, B, pH, 9.58-8.41); Standard and neutral isoform (C, pH 7.98-6.89); Standard and acidic isoforms (D, pH 6.88-5.41 and E, pH 5.36-3.46). III) Table summarizing the analysis parameters of the B/Bo vs dose.
Production of cAMP by HEK-239 cells in response to the immunoreactive LH standard and the LH isoforms was dose-dependent (Figure 5(I)). Calculation of EC50 as a proxy for biological activity on the normalized dose-response curve showed the standard and the isoforms to differ (Figure 5(II)). The neutral isoform had the lowest EC50 value, meaning it was the most biologically active, whereas the acidic isoform had the highest EC50 value and therefore the lowest bioactivity (Figure 5(III)). For the standard, the Hill slope (h) value differed from those of the basic and acidic isoforms, but not from that of the neutral isoform (Figure 5(III)).
* Indicates p< 0.05 vs USDA-bLH-B5.
a,b,c Different letter supercripts in the same columns indicate significant differentces (p <0.05).
Figure 5 AMPc production in HEK-293 cells after stimulation with different isoforms of LH. I) Dose-response pattern isoforms to basic (B), neutral (C) and acidic (E). II) Normalized response pattern in cAMP production by LH immunoreactive dose. III) Analysis parameters of the dose-response curve. Each point represents the mean of three independent assays for each isoform.
Discussion
Polymorphism in mammal LH is reflected in its physicochemical, immunological and biological properties. In vivo13,14,15 and in vitro16,17 bioassays have been done of biological activity in different LH isolates from ruminant hypophyses. The results indicate that basic isoforms are the most bioactive proteins in vitro, whereas acidic isoforms are more active in vivo. Recent developments include bioassays using cell lines that express gonadotropin receptors, which allow study of biologically functional proteins20,30.
The present study is the first time an in vitro bioassay using HEK-293 cells transfected with rat LH receptor cDNA has been applied to evaluate biological activity in different charge isoforms of bovine hypophysis20. Quantitative cAMP production per immunoreactive LH unit differed between isoforms, with the neutral isoform having more activity than the basic and acidic isoforms.
Under the studied bioassay conditions, the response patterns for the different bovine LH isolates were dose-dependent between 6.5 and 100 ng immunoreactive LH. Production of cAMP was unaltered at higher doses and even exhibited a decline. This response pattern could indicate desensitizing of the limited number of receptors in the HEK-293 cells through a relatively slow down-regulation process of the number of receptors in each cell22. However, it could also be a product of the relatively rapid process of a decrease in the LH receptor's capacity to interact with and activate its related G proteins31. Time-dependent cAMP production was not analyzed in the present study, but 24 h of stimulus allowed an evaluation of differential changes in cAMP production between isoforms.
The greater response of the basic isoform compared to that of the acidic isoform coincided with the biological effect reported after placing different doses of diabetic and obese rat hypophysis eluted at basic and acidic pHs in HEK-293 cells20,21. It is a response pattern also seen in vitro in primary Leydig cell cultures after stimulus with human32, rat33 and ovine17 LH hypophysis isoforms, as well as with basic isoforms present in circulation16.
Why biological activity differed between the tested isoforms is not completely clear. Partial or total removal of some isoform oligosaccharide components by chemical or enzymatic means modifies their activity34,35, suggesting that one explanation may be the type of oligosaccharides in each isoform4. For example, removal of the sulphate group in bovine LH containing only oligosaccharides with sulphated N-acetylgalactosamine terminals (thus exposing the N-acetylgalactosamine) produces greater LH depuration from the blood36. This occurs due to easy recognition of the desulphated LH by the specific receptor in the liver (Gal/GalNAcspecific receptor), which proportionally reduces this protein's in vivo biological activity36,37.
Total removal of the oligosaccharide from LH has shown that deglycosilated hormones can no longer stimulate adenylate cyclase activity, although no apparent change occurs in receptor affinity38. Deglycosilated hormones exhibit antagonistic functions in second messenger formation and production39, and therefore on production of steroidal hormones40. Variation in receptor affinity for each isoform may also have had an effect. Studies of bonding in HEK-239 cells transfected with rat LH receptor cDNA have shown that there is a constant association at different orders of magnitude between different mammal LHs22. Bovine LH exhibits the lowest affinity for the LH receptor, even though it has repeated leucine-rich regions (LRR; specifically leucine 3, 7, 8 and 9) in the receptor's extracellular domain that are specifically for recognizing this hormone22.
It can therefore be inferred that cAMP production after treatment with the different isoforms could also depend on differentiated affinity in the receptor. This makes it highly probable that the LH's glycosylation pattern is what regulates the differential pattern in biological response measured as cAMP production by HEK-239 cells after stimulation with different LH isoforms. Proteins with distinct receptor affinities can be produced as a result, the interaction of which can be reflected in changes in biological response quantified as cAMP production by cells.
The present data on isoform biological activity will complement the limited data currently available. To date, the neutral isoform in LH from ruminants and other species has not been immunologically or biologically characterized, possibly because it is difficult to purify and characterize due to its low content in the adenohypophysis13,40,41 and the blood6. The bioactivity recorded in the neutral isoform in the present study is a valuable addition to biological evaluation of the bovine LH isoform spectrum. For the basic isoform, the observed biological activity complements previous reports on ovine17, bovine13 and rat LH33 in which the basic isoform exhibits greater in vitro bioactivity than the acidic isoform. Under the in vitro bioassay conditions used here, the isoforms exhibited differential bioactivity, but use of a homologous bioassay is desirable because it would provide superior analysis of LH isoforms in ruminants.
Quantitative immunoreactive LH concentration in the LH isoforms isolated from the bovine adenohypophysis was measured using USDAbLH-B5 as a reference. The RIA response pattern depended in the amount of assayed protein, exhibiting similar values between the slopes, indicating the proteins are analogous. Values for IC50 were also similar among elution proteins in the same basic pH range (USDAbLH-B5; CM-3ab; and basic isoform B, pH 9.58-8.41). However, they increased significantly in the neutral and acidic isoforms, suggesting a progressive decrease in LH immunological activity.
Distinct immunological activity between isoforms can be attributed to use of a polyclonal antibody in the RIA which is specifically focused against native oLH (NIDDK-oLH-26), a protein isolated using a procedure similar to that described here. A number of studies have shown that pure forms of LH contain different components. In a study using Nal125-labelled USDA-bLH-B5 analyzed using chromatofocusing9, this reference LH exhibited a basic type distribution pattern in which 80 % of the LH eluted in a basic pH range, while the rest of the labelled protein eluted in neutral and acidic pH gradients. This may indicate that the antibody generated for oLH, used in the immunodiagnostic, is mostly focused on basic isoforms with little emphasis on neutral and acidic proteins in LH. A clear advantage therefore exists for isoforms recovered at extremely basic pH (e.g. isoform A). Neutral and acid isoforms require more protein for this antibody to identify them.
The increasing amount of protein in each isoform could interfere in their biological response due to possible contamination from proteins structurally related to LH which remain present throughout purification and in the final products. However, this is unlikely since the anti-oLH antibody had been shown to be highly specific in quantifying LH in serum and ruminant hypophysis extracts without generating cross reactions with structurally related proteins such as FSH and TSH9,11.
All the isoforms were recognized by the same antibody, although specificity was greatest in the basic isoform group. This was reflected in the amount of protein required to attain the IC50 dose in the system, which was significantly lower in the other analyzed isoforms. Calculation of each isoform's immunological activity using the LH-specific heterologous assay may be adequate, but more accurate immunological evaluation will require developing a specific homologue system for each isoform.
Glycoprotein extract (GPE) was generated in the extraction process with the hypophysis anterior lobes. Purification of the GPE in the CM-Sepharose exchanger produced CM-3ab, a protein fraction containing the greatest amount of immunoreactive LH. Its physicochemical, immunological and biological characteristics were similar to those of the reference, and its chromatofocusing distribution pattern was similar to that of adenohypophysis tissue in bovines42,43,44, caprines14 and humans45. Use of the chromatofocusing technique resulted in higher LH yield (μg LH/mg protein) than reported in other species and using other techniques15,17,44.
Of the isoforms isolated from CM-3ab using chromatofocusing, LH immunoreactive proteins in the basic pH range dominated. Similar distribution patterns have been reported in ovine species42, and in the elution patterns of ovine46 and bovine hypophysis extracts11,47. It can therefore be assumed that the procedure developed here to isolate isoform groups from bovine LH does not interfere in the isoform distribution patterns of bovine LH.
In NR conditions, the isoform group isolated from the hypophysis anterior lobe had the same molecular weight as the reference standard, which corresponds to the 36.5 kDa heterodimer. Under R conditions, the dominant presence of proteins with 20.8 and 23.0 kDa molecular weights corresponds to alpha- and beta-LH, respectively, and with values reported for native LH and its subunits13,14,15. Pattern analysis of the acidic isoform under NR conditions showed a series of high molecular weight proteins that disappeared after treatment with the reducing agent β-mercaptoethanol. This suggests the presence of molecular aggregates of LH.
Electrophoretic analysis under R conditions of CM-3ab, and the C and D isoforms showed them all to be characterized by the presence of a 17 kDa protein, which was absent in isoform A and barely present in isoform B. Because this protein was not identified in the immunotransference analysis, it is probably a contaminating protein concentrated between pH 7.98 and 5.41. It may correspond to one of the isoforms of bovine FSH11, which elute in this pH range. Another possibility is that it is an immature form of one of the subunits. A protein with a molecular weight similar to that described here was identified in electrophoretic analysis under R conditions of deglycosilated ovine LH48. Some of the subunits may also have experienced proteolytic breakage, as described for fractionated proteins of human LH with a similar molecular weight49. Presence of this protein type in some isoform isolates does not correspond to LH, although it could interfere in the interaction between LH and it receptor, thus modifying the strength of its biological activity.
Conclusions and implications
Isoforms of bovine luteinizing hormone have differing biological activities. Differences in the strength of in vitro biological activity between LH isoforms suggest that some may experience changes during the isoform synthesis process. This may represent a fine-tuned regulation mechanism of gonadal function in the adenohypophysis. Isoform biological activity was quantified using an in vitro heterologous bioassay, which does not truly reflect each isoform's physiological response in the organism as a whole. However, this technique did prove the presence of differential biological response among the tested isoforms. Confirming each isoform's affinity and selective specificity will require use of HEK-239 cells transfected with bovine LH (homologue) receptor cDNA.
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Received: July 09, 2015; Accepted: November 27, 2015
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