General and Comparative Endocrinology xxx (2014) xxx–xxx
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Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures Maya Haj Hassan 1, Claire Cahoreau, Gwenhael Jégot, Camille Jouanny, Julie Mariot 2, François Lecompte, Danièle Klett, Yves Combarnous ⇑ Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS), Unit « Physiologie de la Reproduction et des Comportements », 37380 Nouzilly, France
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Article history: Available online xxxx Keywords: Gonadotropin Reproduction Structure–function ELISA
a b s t r a c t Quaternary structure of human, bovine and ovine Follicle-Stimulating Hormones (hFSH, bFSH and oFSH) and Luteinizing Hormone was assessed in sandwich ELISAs using monoclonal anti-oFSHb or anti-oLHb antibodies, respectively, for capture and a biotinylated anti-hFSHa (a4 epitope) for detection. Neither free subunit gave any signal in this assay so that it was possible to measure the residual heterodimeric fraction after thermal treatment of the gonadotropins under study. The hormones were subjected to 5-min heating between 37 and 90 °C before rapid cooling in melting ice before ELISA. The data show half-dissociation of natural and recombinant human and ovine FSH preparations between 68 and 74 °C whereas bovine FSH preparations exhibited lower stability in these conditions with half-dissociation between 61 and 64 °C. Moreover, whereas all human and bovine as well as most ovine FSH preparations were fully dissociated at temperatures above 80 °C, one natural oFSH and one recombinant hLH preparations contained an important fraction that resisted dissociation even at 93 °C and retained in vitro bioactivity. This suggests the existence of gonadotropin ab heterodimer with covalently linked subunits. Similarly, about 20% of the recombinant hLH preparation was also found withstand heat denaturation and also probably to have cross-linked subunits. The origin and chemical nature of these inter-subunit bonds remain to be determined. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Glycoprotein hormones (Luteinizing Hormone LH, FollicleStimulating Hormone FSH, Chorionic Gonadotropin CG and Thyroid-Stimulating Hormone TSH) are comprised of two dissimilar, non-covalently bound glycoprotein subunits, named a and b (Pierce and Parsons, 1981). The a-subunit is common to all of them and it is encoded by a unique gene whereas b-subunits are different and confer hormonal specificity to each ab heterodimer (Combarnous, 1992). The 1:1 association of a and b subunits is mandatory for the expression of hormonal activity and the conformational stability of the heterodimers is thus of particular interest. ⇑ Corresponding author. Fax: +33 247427743. 1 2
E-mail address: [email protected] (Y. Combarnous). Present address: Faculty of Science, Lebanese University, Baalbek, Lebanon. Present address: Ceva Santé Animale SA, 33500 Libourne, France.
In a previous study, we have shown that short-term incubation of glycoprotein hormones at various temperatures and measurement of residual dimer either by micro-HPLC associated with radioreceptor assay (RRA), or sandwich ELISA provide similar values as microcalorimetry for the measurement of their conformational stability (Burova et al., 2001; Galet et al., 2004; Legardinier et al., 2008). Compared to physico-chemical techniques, sandwich ELISA allows us determination of this parameter using hormones that are not highly purified thanks to the high specificity of the antibodies towards the a and b subunits, respectively (Legardinier et al., 2008). In the present work, the short-term thermal stability of FSH and LH from human, bovine and ovine species was studied and compared to our previous data concerning the equine gonadotropins eLH and eCG as well as eLH/CG (Legardinier et al., 2005, 2008).
http://dx.doi.org/10.1016/j.ygcen.2014.03.033 0016-6480/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: Haj Hassan, M., et al. Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.03.033
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M. Haj Hassan et al. / General and Comparative Endocrinology xxx (2014) xxx–xxx
2. Materials and methods 2.1. Hormones oFSH RP1 and oFSH RP2 (NIH, NICDD), oFSH CY1771-II (28 NIH FSH S1) (our lab), bFSH NIH I2 (NIH), bFSH phe-III, bFSH CY1131, bFSH CY1132-Ia, bFSH CY2672-III, bFSH CY2683-I and bFSH CY2683-II (our lab) hFSH HP1398141201 (Ferring), hFSH-I3 (AFP-4822B; NIDDK Bethesda), hFSH SIAFP-1 (AFP-5720D NIDDK Bethesda) and recombinant hFSH-AFP8468A (6650 IU/mg) (Prof. A.F. Parlow, NHPP, NIDDKD, Harbor-UCLA Medical Center, Torrance CA, USA) pFSH 25UA batch 07J30 (Prof J.F. Beckers, Univ Liège, Belgium), pFSH CY1890-II (our lab) eFSH CY1368 (our lab) oLH CY1055 (2.68 NIH LH S1) (our lab) bLH CY1042 (1.19 bLH LER1072.2) (our lab) hLH rec Ulg (3.8 LH S1) (Dr. J. Closset, Univ Liège, Belgium) carp LH EBG (Dr. E. Burzawa-Gérard & S. Dufour, MNHN-CNRS, Paris, France)
2.2. Incubation Heat treatments were performed in 50 ll PBS buffer (pH 7.4) at a concentration of 10 lg/100 ll in 200 ll-PCR tubes that were incubated into a water bath at fixed temperatures between 37 and 93 °C. These temperatures were measured in parallel by a graduated alcohol thermometer and an electronic thermometer (CheckTemp 1, Hanna). After 5 min of incubation at each temperature, the tubes were immediately plunged into melting ice. For the sake of comparison, different hormones were incubated in parallel. In some instances, longer incubations were performed at the same hormone concentration to follow the kinetics of dissociation at a fixed temperature. At each selected time, a tube was removed from the heat bath and transferred into melting ice (or cooled off at room temperature in few cases).
Finally, Neutravidin–Horseradish peroxidase conjugate (Pierce, Thermo Scientific, Brebières, France) was used before peroxidase activity measurement with 100 ll TMB substrate (Interchim, Montluçon, Franc). After 20 min, the absorbance of each well at 450 nm (specific) and 660 nm (non-specific) was detected after acidification of the medium with 50 ll 0.2 N H2SO4 using a Tecan Sunrise 96-well spectrometer and the Magellan software package. The DA450-A660 values were used for calculations.
2.4. In vitro bioassays 2.4.1. FSH HEK cells stably expressing both the human FSH receptor gene (Tranchant et al., 2011) and a cAMP-responsive reporter gene (GloSensor™ -22F cAMP plasmid, Promega) kindly provided by G.Jégot and E. Reiter (INRA Nouzilly) were plated at about 8 104 cells per well on a white clear-bottom plate (Bio-One, Greiner) in 100 ll supplemented MEM growth medium (10% fetal bovine serum, hygromycin). At about 80% confluency, the cells were incubated in 100 ll serum-free medium containing 4 ll GloSensor cAMP reagent (E1291, Promega) for 2 h at room temperature). Luminescence was recorded using a POLARstar OPTIMA luminometer (BMG Labtech) just before that the cells were challenged with increasing concentration of FSH preparations previously submitted or not to heat treatment (4–90 °C; 5–40 min). Luminescence kinetics were then immediately recorded over a 90 min period of time. The initial slopes of the curves were calculated and used for measurement of bioactivities of hormones.
2.4.2. LH Mouse Leydig Tumor cells (mLTC) were cultured and challenged with increasing concentrations of LH or hCG for 3 h. At the end of incubation, the concentration of progesterone in each well was measured by EIA as previously described (Legardinier et al., 2008).
2.3. Antibodies and ELISA Monoclonal anti-oFSH (Henderson et al., 1995) was provided by Drs. Keith Henderson and Jenny Juengel (AgResearch, Invermay Agricultural Centre, Mosgiel NZ); Monoclonal anti-oLHb 518B7 (Matteri et al., 1987) was obtained from Dr. Roser (University of California, Davis). Monoclonal anti-hFSH (INN-hFSH-132) directed against the a4 epitope (Dirnhofer et al., 1994a) was purchased from Serotec (MCA1026; Oxford, UK). Its biotinylation was performed by biotinamidohexanoic acid N-hydroxysuccinimide ester (Sigma, France) coupling on lysine residues as recommended by the manufacturer. The specific anti-oFSH (50 ng/well) and anti-oLH (20 ng/well) immunoglobulins were used for coating of Maxisorb 96-well microtiter plates (Nunc, Denmark) overnight at 4 °C in 100 ll carbonate buffer, pH 9.6. All wells were subsequently overcoated with 150 ll SeaBlock saturating agent (Uptima, Interchim, Montluçon, France). After elimination of the buffer, 100 ll of the media to be assayed, diluted in PBS-tween-SeaBlock saturating agent were introduced into wells in triplicate and incubated for 3 h at room temperature. After 4 washes with 150 ll PBS-tween, the detection step was performed. For detection, the biotinylated anti-hFSH specific for the a4 epitope present in the common a-subunit of glycoprotein hormones from numerous species was used in all assays. It was used at a concentration of 20 ng/100 ll and incubated for 1 h at 37 °C.
Fig. 1. Specificity of the sandwich ELISA making use of AgResearch anti-ovine FSH (anti-b) monoclonal antibody for capture and of biotinylated Serotec MCA-1026 anti-human FSH (anti-a) monoclonal antibody for detection. The values represent the means of triplicate assays at each hormone dose. This figure is representative of two independent experiments.
Please cite this article in press as: Haj Hassan, M., et al. Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.03.033
M. Haj Hassan et al. / General and Comparative Endocrinology xxx (2014) xxx–xxx
3. Results 3.1. Characterization of the assays The LH and FSH assays were evaluated for their species specificity as well as for their specificity for ab heterodimers. It was found that the FSH assay worked for human, bovine and ovine FSH but not for porcine and equine FSH preparations (Fig. 1). The assay was also evaluated to establish that it recognized only FSH heterodimer and not a and b subunits (Fig. 2). Similar experiments were carried out to validate the LH and hCG ELISA (data not shown). 3.2. Validation of the assay for dissociation measurement In order to validate the assay for the measurement of heterodimer dissociation, we tested it with known ratios of hFSH heterodimers and subunits (Fig. 2 – top). It can be observed that there is a close correlation of the slopes as a function of heterodimer proportion (Fig. 2 – bottom) thus allowing residual heterodimer proportion to be determined from the slope. 3.3. Measurement of heat-promoted dissociation of heterodimers 3.3.1. FSH Fig. 3 shows the thermal stability of the heterodimeric structure of natural and recombinant human FSH preparations after 5 min incubation at temperatures between 37 and 80 °C as determined by sandwich ELISA using anti-a and anti-b antibodies. Fig. 4 shows the same experiment for two natural bovine FSH preparations and Fig. 5 for two natural ovine FSH preparations. It can be seen that
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human and ovine FSH preparations exhibit half dissociation in the conditions used around 72 °C whereas the bovine FSH preparations were half-dissociated around 62 °C. In Fig. 5, the top panel shows the thermal stability of the heterodimeric structure of two pituitary ovine FSH preparations between 37 and 80 °C using the same ELISA. Since oFSH CY2178 was found only half-dissociated after 5 min at 80 °C, its quaternary structure stability was further studied by following its kinetics of dissociation at 93 °C (bottom panel) over 7 h. It can be seen that around 30% of the initial material still gave a positive signal in the sandwich ELISA at the end of this incubation. In order to ascertain whether this positive signal is really due to covalently-linked ab heterodimers and not to heat-aggregated material, we measured the residual in vitro bioactivity of the hormone after heating. These data (Fig. 5) showed that 28–34% bioactivity of oFSH CY2178 was retained after prolonged heating at 87.5 °C in contrast to oFSH CY1758 that was totally inactivated in 5-min time. The residual in vitro bioactivities of both oFSH preparations were thus well correlated to their respective heterodimer contents as determined by sandwich EIA. An overestimation of heterodimer content was only observed in oFSHCY2178 when the hormone was cooled off quickly by putting the tube directly from the bath at 87.5 °C into melting ice. The apparent heterodimer content (77%) was even higher than that measured after heating at 80 °C only (55%). When the solution was cooled more slowly, the residual immunoactivity and in vitro activity were much better correlated (28–34%) indicating that around 30% of oFSHCY2178 exists as a covalently-linked ab heterodimer, but a too rapid drop in temperature promotes aggregation of material leading to an overestimation of the hormone undissociable fraction.
Fig. 2. Validation of sandwich ELISA for the measurement of heterodimer ratio. Left top: Native hFSH and fully heat-dissociated hFSH were mixed in the proportions shown in the figure and the mixtures tested in triplicates. The slopes (±SD) were calculated using the linear regression program in GraphPad Prism. Left bottom: Slope values (±SD) from the top figure were plotted as a function of heterodimer proportion. A linear regression (R2 = 0.999; slope = 0.018 ± 0.00057) was performed using the GraphPad Prism package. Right: Example of dose–response in sandwich EIA after heating of bLH at the indicated temperatures for 5 min. Residual activities are determined from the ratio of slopes compared to the control maintained at 4 °C.
Please cite this article in press as: Haj Hassan, M., et al. Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.03.033
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M. Haj Hassan et al. / General and Comparative Endocrinology xxx (2014) xxx–xxx
Fig. 3. Thermal stability of natural and recombinant human FSH preparations. Both hormone preparations were incubated for 5 min at the indicated temperatures at 10 lg/ml concentration in PBS. The proportion of residual heterodimer was determined in triplicate from the slope of each assay after incubation at each temperature. These proportions (±SD) are plotted as a function of the 5-min incubation temperature.
3.3.2. LH Fig. 6 shows the thermal stability of the heterodimeric structure of urinary human CG and recombinant human LH. It can be seen that both hormones dissociate over the same range of temperatures and also over the same range as human FSH preparations (Fig. 3). Interestingly, about 20% of recombinant hLH molecules retained their quaternary structure after 5 min at 90 °C whereas it was not the case for hCG. This high thermal resistance of quaternary structure is reminiscent of the one observed for oFSH CY2178 (Fig. 5). Moreover, in keeping with this result, recombinant hLH was found to retain 20% of its activity after 5 min at 90 °C in the in vitro bioassay in mLTC. It can thus be concluded that the residual activity measured in the sandwich EIA is indeed due to undissociated ab heterodimer and not to aggregated material. For the sake of comparison, the transition temperatures (50% dissociation) are tabulated in Table 1. 4. Discussion The sandwich ELISAs described here allowed us to measure the concentration of heterodimeric FSH and LH from several but not all species (Fig. 1) and were insensitive to free uncombined a and b subunits from these hormones (Fig. 2). Indeed, increasing concentrations of totally dissociated hormone did not promote any signal (Fig. 2 left top) and the response was strictly proportionnal to the percentage of heterodimeric hormone (Fig. 2 left bottom). These assays were thus well suited to follow the heat stability of the quaternary structure of human, ovine and bovine gonadotropins by measuring the percentage of intact hormone in the
Fig. 4. Thermal stability of two natural bovine FSH preparations. Both hormones were incubated for 5 min at the indicated temperatures at 10 lg/ml concentration in PBS. The proportion of residual heterodimer was determined in triplicate from the slope of each assay after incubation at each temperature. These proportions are plotted as a function of the 5-min incubation temperature.
medium after heat treatment (Fig. 2 right). However, they were not suited for porcine or equine hormones. The insensitivity of the present assay to porcine and equine FSH was due to the inability of the anti-a used, which was directed against the human a4 epitope (Dirnhofer et al., 1994b), to detect the same region in pa and ea subunits. Indeed the presence of a proline residue in this epitope (FSQP in human and FSKP in ovine and bovine a) appears mandatory for detection by this antibody. Since this amino-acid residue is absent and replaced by a leucine in the corresponding porcine (FSKL) and equine (FFKL) sequences, the gonadotropins from these two species cannot be detected in sandwich EIA making use of this anti-ha antibody. Proline residues are well known to induce breaks and changes of orientation in polypeptide secondary structures. It is thus not surprising that the absence of proline in the equine and porcine sequences at this location modify the three-dimensional structure of the epitope. The comparison between natural pituitary hFSH and recombinant hFSH preparations indicated that the latter exhibits a less stable quaternary structure than the former with transition temperatures of 74 and 68 °C, respectively. This might suggest that recombinant FSH preparations exhibit a lower thermal stability relative to pituitary hormones, and this difference could be attributed to the carbohydrate moieties (Erbel et al., 2002; Galet et al., 2004; Manjunath and Sairam, 1983) synthesized in pituitary gonadotrope cells (Bousfield et al., 2000; Green and Baenziger, 1988a,b) and CHO cells, respectively (Gawlitzek et al., 2009). Nevertheless, this observation must be taken with care and should not be generalized too fast as we compared only one batch of each type of hormone. The heterodimeric structure of human gonadotropins was found to be resistant to short-term heating above 70 °C whereas bovine FSH quaternary structure was found to be largely less stable
Please cite this article in press as: Haj Hassan, M., et al. Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.03.033
M. Haj Hassan et al. / General and Comparative Endocrinology xxx (2014) xxx–xxx
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Fig. 5. (A) Thermal stability of two preparations of pituitary ovine FSH (CY1758 and CY2178) after 5-min heating at the indicated temperature and arrest in melting ice. (B) Effect of stopping temperature (ice or room temperature) on residual activity of both oFSH preparations in sandwich EIA (top) and in vitro bioassay (bottom). (C) Kinetics of inactivation of oFSH CY2178 at 87.5 °C as measured by in vitro bioassay.
with transition temperatures of 61–64 °C (Fig. 4). This observation was puzzling as bovine and ovine FSH share a large proportion of identical polypeptide sequences. The identity is 100% for their a subunits whereas there are only 8 differences between their b subunit sequences (117 aa). Among these differences, three are located at the C-terminus after the last cystine residue and this region is known to be exposed in the heterodimer and thus not implied in the ab association (Combarnous, 1992; Fan and Hendrickson, 2005; Fox et al., 2001). More interestingly, two differences between the ovine and bovine bFSH amino acid sequences are found in the seatbelt region that wraps around the a partner. The sequence in the ovine FSHb is 90CGKCDRDSTDC100 and that in the bovine FSHb is 90CSKCDSDSTDC100. It is thus tempting to postulate that these two differences in the seatbelt region can affect the stability of the quaternary structure of the heterodimer as the formation and fastening of the seatbelt is mandatory for it. Another possibility is that ovine and bovine FSH differ in their carbohydrate structures and these differences in saccharide side chains could be responsible of the lower stability observed for the bovine hormone (Galet et al., 2004). A fraction of one of the ovine FSH preparations was found to retain partial activity in the sandwich EIA after long-term treatment at temperatures above 90 °C (Fig. 5). At the hormone concentration used, which is much below the equilibrium Kd, recombination of subunits upon cooling after heating is impossible. As it is unlikely that the gonadotropins preserve their folding above 80 °C, it can be hypothesized that the fraction withstanding very high temperature does refold upon cooling but reformation of the heterodimeric molecule is possible only if the subunits are covalently linked so that low concentration has no impact. Another possibility is that the hormone partially aggregates at these temperatures so that it can still be detected in the sandwich EIA. This was indeed observed when FSH CY2178 is cooled off fastly from 87.5 to 0 °C in melting ice but not when the sample is cooled off more slowly. Nevertheless, in both cases the residual FSH bioactivity in vitro was found to be 15–20% of non-heated control. Even more surprising, we found that the same residual FSH activity was retained after heating at 87.5 °C over 40 min (Fig. 5C).
A similar behaviour was observed for recombinant hLH (Fig. 6) and can also be interpreted either as the presence of a covalent bond between the a and b subunits of this hormone in part of the isoforms or as aggregation. In this case, the hypothesis of a covalent bond between the a and b subunits is the most likely as the hormone retained about 15% of its initial activity in the in vitro LH bioassay on mLTC (Fig. 6 insert) after a long term incubation at 90 °C in agreement with the proportion of undissociable heterodimer measured in the sandwich EIA. For the moment, we do not know the chemical nature of these putative inter-subunit links:disulfide bridge(s)? pseudo-peptide bond between the side chains of a Lys and Glu or Asp residue? dityrosine crosslink (Malencik and Anderson, 2003)? bonds between carbohydrate chains (Liu et al., 1984)? Also we don’t know whether this/these bond(s) is/are natural or formed during the extraction and purification steps of the hormone. If it is natural, it would be interesting to study the quantitative importance of this fraction and its possible specific biological role. It must be kept in mind that the 5-min heating and sandwich EIA protocol used in this paper is used to mimick microcalorimetric studies (Burova et al., 2001) but cannot be considered as a full substitute for it as it gives no access to thermodynamic parameters concerning the association–dissociation of a and b subunits. Nevertheless, it permits to indirectly study the stability of the cooperative folding of the hormone and, compared to microcalorimetry, it has two advantages: one is that it can be performed using non-purified hormones and another is that it requires much lower quantities of material. Consequently, the comparison of data concerning a large number of different preparations from various species and of different purities will allow to get a larger survey than would be possible by microcalorimetry. The data summarized in Table 1 show that the transition temperatures for mammalian gonadotropins are in the 61–77 °C range. They do not permit to point out obvious differences between LH and FSH preparations or between natural and recombinant hormones. Such comparisons would require the study of more numerous different preparations of each hormone. Nevertheless, it
Please cite this article in press as: Haj Hassan, M., et al. Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.03.033
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M. Haj Hassan et al. / General and Comparative Endocrinology xxx (2014) xxx–xxx
100
hCG 8149 hLH rec bLH CY1240 oLHCY1055
90
residual activity in sdw EIA
80 70 60
Acknowledgments
50 40 30 20 10 0 50
60
70
80
90
residual in vitro bioactivity (%) after 5min at 90°C
temperature (°C)
We are indebted to Dr. A.F. Parlow (NHPP, NIDDKD, HarborUCLA Medical Center, Torrance CA, USA), to Dr. J. Closset and J.F. Beckers (University of Liège, Belgium), to Dr. S. Dufour (MNHNCNRS, Paris, France) for their gifts of hormones and to Drs. Keith Henderson and Jenny Juengel (AgResearch, Invermay Agricultural Centre, Mosgiel NZ) for their gift of anti-oFSHb monoclonal antibody and to Dr. S. Dufour (MNHN-CNRS, Paris, France) for her gift of anti-carpLHb antiserum. We are grateful to Dr. E. Reiter (INRA Nouzilly) for the gift of HEK cells stably transfected with both hFSH receptor and cAMP-GLO reporter gene. We also thank INRA and the Region Centre (France) for financial support of MHH PhD thesis (2008-2011) presented at the University of Tours (France).
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References
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Bousfield, G.R., Baker, V.L., Gotschall, R.R., Butnev, V.Y., 2000. Carbohydrate analysis of glycoprotein hormones. Methods 21, 15–39. Burova, T., Lecompte, F., Galet, C., Monsallier, F., Delpech, S., Haertle, T., Combarnous, Y., 2001. Conformational stability and in vitro bioactivity of porcine luteinizing hormone. Mol. Cell. Endocrinol. 176, 129–134. Combarnous, Y., 1992. Molecular basis of the specificity of binding of glycoprotein hormones to their receptors. Endocr. Rev. 13, 670–691. Dirnhofer, S., Lechner, O., Madersbacher, S., Klieber, R., de Leeuw, R., Wick, G., Berger, P., 1994a. Free alpha subunit of human chorionic gonadotrophin: molecular basis of immunologically and biologically active domains. J. Endocrinol. 140, 145–154. Dirnhofer, S., Madersbacher, S., Bidart, J.M., Ten Kortenaar, P.B., Spottl, G., Mann, K., Wick, G., Berger, P., 1994b. The molecular basis for epitopes on the free betasubunit of human chorionic gonadotrophin (hCG), its carboxyl-terminal peptide and the hCG beta-core fragment. J. Endocrinol. 141, 153–162. Erbel, P.J., Haseley, S.R., Kamerling, J.P., Vliegenthart, J.F., 2002. Studies on the relevance of the glycan at Asn-52 of the alpha-subunit of human chorionic gonadotropin in the alphabeta dimer. Biochem. J. 364, 485–495. Fan, Q.R., Hendrickson, W.A., 2005. Structure of human follicle-stimulating hormone in complex with its receptor. Nature 433, 269–277. Fox, K.M., Dias, J.A., Van Roey, P., 2001. Three-dimensional structure of human follicle-stimulating hormone. Mol. Endocrinol. 15, 378–389. Galet, C., Lecompte, F., Combarnous, Y., 2004. Association/dissociation of gonadotropin subunits involves disulfide bridge disruption which is influenced by carbohydrate moiety. Biochem. Biophys. Res. Commun. 324, 868–873. Gawlitzek, M., Estacio, M., Furch, T., Kiss, R., 2009. Identification of cell culture conditions to control N-glycosylation site-occupancy of recombinant glycoproteins expressed in CHO cells. Biotechnol. Bioeng. 103, 1164–1175. Green, E.D., Baenziger, J.U., 1988a. Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. I. Structural elucidation of the sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J. Biol. Chem. 263, 25–35. Green, E.D., Baenziger, J.U., 1988b. Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. II. Distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J. Biol. Chem. 263, 36–44. Henderson, K.M., Camberis, M., Hardie, A.H., 1995. Generation of monoclonal antibody to ovine FSH and its application in immunoneutralization and enzymeimmunoassay. J. Reprod. Fertil. Suppl. 49, 511–515. Legardinier, S., Duonor-Cerutti, M., Devauchelle, G., Combarnous, Y., Cahoreau, C., 2005. Biological activities of recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) expressed in Sf9 and Mimic insect cell lines. J. Mol. Endocrinol. 34, 47–60. Legardinier, S., Poirier, J.C., Klett, D., Combarnous, Y., Cahoreau, C., 2008. Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants. J. Mol. Endocrinol. 40, 185–198.
15 10 5 0 0
hCG
rec hLH
Fig. 6. Thermal stability of recombinant human LH, urinary human CG and pituitary bovine and ovine LH preparations. The hormones were incubated for 5 min at the indicated temperatures at 10 lg/ml concentration in PBS. The proportion of residual heterodimer was determined from the slope of each EIA assay and plotted as a function of the 5-min incubation temperature (top). Residual in vitro LH bioactivity of hCG and rec hLH after 5 min incubation at 90 °C (bottom).
Table 1 Comparison of transition temperatures (Tm).
*
association in pituitary at a much lower, and more variable temperature in poïkilotherms than in homeotherms such as mammals. The high Tm of carp LH is in good agreement with the observation that carp LH subunits have been found to recombine much faster than oLH subunits (Marchelidon et al., 1979). In conclusion, the systematic study of Tm of different preparations or isoforms of glycoprotein hormones from a large number of vertebrate species should help to understand the molecular thermodynamic bases of their subunits cooperative folding and quaternary association.
Hormone hFSH SIAFP1 rec hFSH bFSH NIHB2 bFSH CY2672-II oFSH CY1758
Tm (°C) 68 ± 2 74 ± 2 61 ± 2 64 ± 2 70 ± 2
rec hLH bLH CY1042 oLH CY1055 eLH* eCG* carp LH EBG
65 ± 2 67 ± 2 65 ± 2 75 ± 2 77 ± 1 83 ± 4
Legardinier et al. (2008).
appears that bovine FSH exhibits a weaker stability than the FSH preparations from other mammalian species. In addition, the first experiments with carp LH give a dissociation Tm of 83 °C which is much higher than those of all mammalian gonadotropins. The high stability of the cooperative folding of this hormone could be related to its folding and subunit
Please cite this article in press as: Haj Hassan, M., et al. Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.03.033
M. Haj Hassan et al. / General and Comparative Endocrinology xxx (2014) xxx–xxx Liu, W.K., Young, J.D., Ward, D.N., 1984. Deglycosylated ovine lutropin: preparation and characterization by in vitro binding and steroidogenesis. Mol. Cell. Endocrinol. 37, 29–39. Malencik, D.A., Anderson, S.R., 2003. Dityrosine as a product of oxidative stress and fluorescent probe. Amino Acids 25, 233–247. Manjunath, P., Sairam, M.R., 1983. Enhanced thermal stability of chemically deglycosylated human choriogonadotropin. J. Biol. Chem. 258, 3554–3558. Marchelidon, J., Salesse, R., Garnier, J., Burzawa-Gerard, E., Fontaine, Y.A., 1979. Zoological origin of gonadotropin subunits and association kinetics. Nature 281, 314–315.
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Matteri, R.L., Roser, J.F., Baldwin, D.M., Lipovetsky, V., Papkoff, H., 1987. Characterization of a monoclonal antibody which detects luteinizing hormone from diverse mammalian species. Domest. Anim. Endocrinol. 4, 157–165. Pierce, J.G., Parsons, T.F., 1981. Glycoprotein hormones: structure and function. Annu. Rev. Biochem. 50, 465–495. Tranchant, T., Durand, G., Gauthier, C., Crepieux, P., Ulloa-Aguirre, A., Royere, D., Reiter, E., 2011. Preferential beta-arrestin signalling at low receptor density revealed by functional characterization of the human FSH receptor A189 V mutation. Mol. Cell. Endocrinol. 331, 109–118.
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Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures Maya Haj Hassan 1, Claire Cahoreau, Gwenhael Jégot, Camille Jouanny, Julie Mariot 2, François Lecompte, Danièle Klett, Yves Combarnous ⇑ Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS), Unit « Physiologie de la Reproduction et des Comportements », 37380 Nouzilly, France
a r t i c l e
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Article history: Available online xxxx Keywords: Gonadotropin Reproduction Structure–function ELISA
a b s t r a c t Quaternary structure of human, bovine and ovine Follicle-Stimulating Hormones (hFSH, bFSH and oFSH) and Luteinizing Hormone was assessed in sandwich ELISAs using monoclonal anti-oFSHb or anti-oLHb antibodies, respectively, for capture and a biotinylated anti-hFSHa (a4 epitope) for detection. Neither free subunit gave any signal in this assay so that it was possible to measure the residual heterodimeric fraction after thermal treatment of the gonadotropins under study. The hormones were subjected to 5-min heating between 37 and 90 °C before rapid cooling in melting ice before ELISA. The data show half-dissociation of natural and recombinant human and ovine FSH preparations between 68 and 74 °C whereas bovine FSH preparations exhibited lower stability in these conditions with half-dissociation between 61 and 64 °C. Moreover, whereas all human and bovine as well as most ovine FSH preparations were fully dissociated at temperatures above 80 °C, one natural oFSH and one recombinant hLH preparations contained an important fraction that resisted dissociation even at 93 °C and retained in vitro bioactivity. This suggests the existence of gonadotropin ab heterodimer with covalently linked subunits. Similarly, about 20% of the recombinant hLH preparation was also found withstand heat denaturation and also probably to have cross-linked subunits. The origin and chemical nature of these inter-subunit bonds remain to be determined. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Glycoprotein hormones (Luteinizing Hormone LH, FollicleStimulating Hormone FSH, Chorionic Gonadotropin CG and Thyroid-Stimulating Hormone TSH) are comprised of two dissimilar, non-covalently bound glycoprotein subunits, named a and b (Pierce and Parsons, 1981). The a-subunit is common to all of them and it is encoded by a unique gene whereas b-subunits are different and confer hormonal specificity to each ab heterodimer (Combarnous, 1992). The 1:1 association of a and b subunits is mandatory for the expression of hormonal activity and the conformational stability of the heterodimers is thus of particular interest. ⇑ Corresponding author. Fax: +33 247427743. 1 2
E-mail address: [email protected] (Y. Combarnous). Present address: Faculty of Science, Lebanese University, Baalbek, Lebanon. Present address: Ceva Santé Animale SA, 33500 Libourne, France.
In a previous study, we have shown that short-term incubation of glycoprotein hormones at various temperatures and measurement of residual dimer either by micro-HPLC associated with radioreceptor assay (RRA), or sandwich ELISA provide similar values as microcalorimetry for the measurement of their conformational stability (Burova et al., 2001; Galet et al., 2004; Legardinier et al., 2008). Compared to physico-chemical techniques, sandwich ELISA allows us determination of this parameter using hormones that are not highly purified thanks to the high specificity of the antibodies towards the a and b subunits, respectively (Legardinier et al., 2008). In the present work, the short-term thermal stability of FSH and LH from human, bovine and ovine species was studied and compared to our previous data concerning the equine gonadotropins eLH and eCG as well as eLH/CG (Legardinier et al., 2005, 2008).
http://dx.doi.org/10.1016/j.ygcen.2014.03.033 0016-6480/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: Haj Hassan, M., et al. Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.03.033
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M. Haj Hassan et al. / General and Comparative Endocrinology xxx (2014) xxx–xxx
2. Materials and methods 2.1. Hormones oFSH RP1 and oFSH RP2 (NIH, NICDD), oFSH CY1771-II (28 NIH FSH S1) (our lab), bFSH NIH I2 (NIH), bFSH phe-III, bFSH CY1131, bFSH CY1132-Ia, bFSH CY2672-III, bFSH CY2683-I and bFSH CY2683-II (our lab) hFSH HP1398141201 (Ferring), hFSH-I3 (AFP-4822B; NIDDK Bethesda), hFSH SIAFP-1 (AFP-5720D NIDDK Bethesda) and recombinant hFSH-AFP8468A (6650 IU/mg) (Prof. A.F. Parlow, NHPP, NIDDKD, Harbor-UCLA Medical Center, Torrance CA, USA) pFSH 25UA batch 07J30 (Prof J.F. Beckers, Univ Liège, Belgium), pFSH CY1890-II (our lab) eFSH CY1368 (our lab) oLH CY1055 (2.68 NIH LH S1) (our lab) bLH CY1042 (1.19 bLH LER1072.2) (our lab) hLH rec Ulg (3.8 LH S1) (Dr. J. Closset, Univ Liège, Belgium) carp LH EBG (Dr. E. Burzawa-Gérard & S. Dufour, MNHN-CNRS, Paris, France)
2.2. Incubation Heat treatments were performed in 50 ll PBS buffer (pH 7.4) at a concentration of 10 lg/100 ll in 200 ll-PCR tubes that were incubated into a water bath at fixed temperatures between 37 and 93 °C. These temperatures were measured in parallel by a graduated alcohol thermometer and an electronic thermometer (CheckTemp 1, Hanna). After 5 min of incubation at each temperature, the tubes were immediately plunged into melting ice. For the sake of comparison, different hormones were incubated in parallel. In some instances, longer incubations were performed at the same hormone concentration to follow the kinetics of dissociation at a fixed temperature. At each selected time, a tube was removed from the heat bath and transferred into melting ice (or cooled off at room temperature in few cases).
Finally, Neutravidin–Horseradish peroxidase conjugate (Pierce, Thermo Scientific, Brebières, France) was used before peroxidase activity measurement with 100 ll TMB substrate (Interchim, Montluçon, Franc). After 20 min, the absorbance of each well at 450 nm (specific) and 660 nm (non-specific) was detected after acidification of the medium with 50 ll 0.2 N H2SO4 using a Tecan Sunrise 96-well spectrometer and the Magellan software package. The DA450-A660 values were used for calculations.
2.4. In vitro bioassays 2.4.1. FSH HEK cells stably expressing both the human FSH receptor gene (Tranchant et al., 2011) and a cAMP-responsive reporter gene (GloSensor™ -22F cAMP plasmid, Promega) kindly provided by G.Jégot and E. Reiter (INRA Nouzilly) were plated at about 8 104 cells per well on a white clear-bottom plate (Bio-One, Greiner) in 100 ll supplemented MEM growth medium (10% fetal bovine serum, hygromycin). At about 80% confluency, the cells were incubated in 100 ll serum-free medium containing 4 ll GloSensor cAMP reagent (E1291, Promega) for 2 h at room temperature). Luminescence was recorded using a POLARstar OPTIMA luminometer (BMG Labtech) just before that the cells were challenged with increasing concentration of FSH preparations previously submitted or not to heat treatment (4–90 °C; 5–40 min). Luminescence kinetics were then immediately recorded over a 90 min period of time. The initial slopes of the curves were calculated and used for measurement of bioactivities of hormones.
2.4.2. LH Mouse Leydig Tumor cells (mLTC) were cultured and challenged with increasing concentrations of LH or hCG for 3 h. At the end of incubation, the concentration of progesterone in each well was measured by EIA as previously described (Legardinier et al., 2008).
2.3. Antibodies and ELISA Monoclonal anti-oFSH (Henderson et al., 1995) was provided by Drs. Keith Henderson and Jenny Juengel (AgResearch, Invermay Agricultural Centre, Mosgiel NZ); Monoclonal anti-oLHb 518B7 (Matteri et al., 1987) was obtained from Dr. Roser (University of California, Davis). Monoclonal anti-hFSH (INN-hFSH-132) directed against the a4 epitope (Dirnhofer et al., 1994a) was purchased from Serotec (MCA1026; Oxford, UK). Its biotinylation was performed by biotinamidohexanoic acid N-hydroxysuccinimide ester (Sigma, France) coupling on lysine residues as recommended by the manufacturer. The specific anti-oFSH (50 ng/well) and anti-oLH (20 ng/well) immunoglobulins were used for coating of Maxisorb 96-well microtiter plates (Nunc, Denmark) overnight at 4 °C in 100 ll carbonate buffer, pH 9.6. All wells were subsequently overcoated with 150 ll SeaBlock saturating agent (Uptima, Interchim, Montluçon, France). After elimination of the buffer, 100 ll of the media to be assayed, diluted in PBS-tween-SeaBlock saturating agent were introduced into wells in triplicate and incubated for 3 h at room temperature. After 4 washes with 150 ll PBS-tween, the detection step was performed. For detection, the biotinylated anti-hFSH specific for the a4 epitope present in the common a-subunit of glycoprotein hormones from numerous species was used in all assays. It was used at a concentration of 20 ng/100 ll and incubated for 1 h at 37 °C.
Fig. 1. Specificity of the sandwich ELISA making use of AgResearch anti-ovine FSH (anti-b) monoclonal antibody for capture and of biotinylated Serotec MCA-1026 anti-human FSH (anti-a) monoclonal antibody for detection. The values represent the means of triplicate assays at each hormone dose. This figure is representative of two independent experiments.
Please cite this article in press as: Haj Hassan, M., et al. Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.03.033
M. Haj Hassan et al. / General and Comparative Endocrinology xxx (2014) xxx–xxx
3. Results 3.1. Characterization of the assays The LH and FSH assays were evaluated for their species specificity as well as for their specificity for ab heterodimers. It was found that the FSH assay worked for human, bovine and ovine FSH but not for porcine and equine FSH preparations (Fig. 1). The assay was also evaluated to establish that it recognized only FSH heterodimer and not a and b subunits (Fig. 2). Similar experiments were carried out to validate the LH and hCG ELISA (data not shown). 3.2. Validation of the assay for dissociation measurement In order to validate the assay for the measurement of heterodimer dissociation, we tested it with known ratios of hFSH heterodimers and subunits (Fig. 2 – top). It can be observed that there is a close correlation of the slopes as a function of heterodimer proportion (Fig. 2 – bottom) thus allowing residual heterodimer proportion to be determined from the slope. 3.3. Measurement of heat-promoted dissociation of heterodimers 3.3.1. FSH Fig. 3 shows the thermal stability of the heterodimeric structure of natural and recombinant human FSH preparations after 5 min incubation at temperatures between 37 and 80 °C as determined by sandwich ELISA using anti-a and anti-b antibodies. Fig. 4 shows the same experiment for two natural bovine FSH preparations and Fig. 5 for two natural ovine FSH preparations. It can be seen that
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human and ovine FSH preparations exhibit half dissociation in the conditions used around 72 °C whereas the bovine FSH preparations were half-dissociated around 62 °C. In Fig. 5, the top panel shows the thermal stability of the heterodimeric structure of two pituitary ovine FSH preparations between 37 and 80 °C using the same ELISA. Since oFSH CY2178 was found only half-dissociated after 5 min at 80 °C, its quaternary structure stability was further studied by following its kinetics of dissociation at 93 °C (bottom panel) over 7 h. It can be seen that around 30% of the initial material still gave a positive signal in the sandwich ELISA at the end of this incubation. In order to ascertain whether this positive signal is really due to covalently-linked ab heterodimers and not to heat-aggregated material, we measured the residual in vitro bioactivity of the hormone after heating. These data (Fig. 5) showed that 28–34% bioactivity of oFSH CY2178 was retained after prolonged heating at 87.5 °C in contrast to oFSH CY1758 that was totally inactivated in 5-min time. The residual in vitro bioactivities of both oFSH preparations were thus well correlated to their respective heterodimer contents as determined by sandwich EIA. An overestimation of heterodimer content was only observed in oFSHCY2178 when the hormone was cooled off quickly by putting the tube directly from the bath at 87.5 °C into melting ice. The apparent heterodimer content (77%) was even higher than that measured after heating at 80 °C only (55%). When the solution was cooled more slowly, the residual immunoactivity and in vitro activity were much better correlated (28–34%) indicating that around 30% of oFSHCY2178 exists as a covalently-linked ab heterodimer, but a too rapid drop in temperature promotes aggregation of material leading to an overestimation of the hormone undissociable fraction.
Fig. 2. Validation of sandwich ELISA for the measurement of heterodimer ratio. Left top: Native hFSH and fully heat-dissociated hFSH were mixed in the proportions shown in the figure and the mixtures tested in triplicates. The slopes (±SD) were calculated using the linear regression program in GraphPad Prism. Left bottom: Slope values (±SD) from the top figure were plotted as a function of heterodimer proportion. A linear regression (R2 = 0.999; slope = 0.018 ± 0.00057) was performed using the GraphPad Prism package. Right: Example of dose–response in sandwich EIA after heating of bLH at the indicated temperatures for 5 min. Residual activities are determined from the ratio of slopes compared to the control maintained at 4 °C.
Please cite this article in press as: Haj Hassan, M., et al. Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.03.033
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Fig. 3. Thermal stability of natural and recombinant human FSH preparations. Both hormone preparations were incubated for 5 min at the indicated temperatures at 10 lg/ml concentration in PBS. The proportion of residual heterodimer was determined in triplicate from the slope of each assay after incubation at each temperature. These proportions (±SD) are plotted as a function of the 5-min incubation temperature.
3.3.2. LH Fig. 6 shows the thermal stability of the heterodimeric structure of urinary human CG and recombinant human LH. It can be seen that both hormones dissociate over the same range of temperatures and also over the same range as human FSH preparations (Fig. 3). Interestingly, about 20% of recombinant hLH molecules retained their quaternary structure after 5 min at 90 °C whereas it was not the case for hCG. This high thermal resistance of quaternary structure is reminiscent of the one observed for oFSH CY2178 (Fig. 5). Moreover, in keeping with this result, recombinant hLH was found to retain 20% of its activity after 5 min at 90 °C in the in vitro bioassay in mLTC. It can thus be concluded that the residual activity measured in the sandwich EIA is indeed due to undissociated ab heterodimer and not to aggregated material. For the sake of comparison, the transition temperatures (50% dissociation) are tabulated in Table 1. 4. Discussion The sandwich ELISAs described here allowed us to measure the concentration of heterodimeric FSH and LH from several but not all species (Fig. 1) and were insensitive to free uncombined a and b subunits from these hormones (Fig. 2). Indeed, increasing concentrations of totally dissociated hormone did not promote any signal (Fig. 2 left top) and the response was strictly proportionnal to the percentage of heterodimeric hormone (Fig. 2 left bottom). These assays were thus well suited to follow the heat stability of the quaternary structure of human, ovine and bovine gonadotropins by measuring the percentage of intact hormone in the
Fig. 4. Thermal stability of two natural bovine FSH preparations. Both hormones were incubated for 5 min at the indicated temperatures at 10 lg/ml concentration in PBS. The proportion of residual heterodimer was determined in triplicate from the slope of each assay after incubation at each temperature. These proportions are plotted as a function of the 5-min incubation temperature.
medium after heat treatment (Fig. 2 right). However, they were not suited for porcine or equine hormones. The insensitivity of the present assay to porcine and equine FSH was due to the inability of the anti-a used, which was directed against the human a4 epitope (Dirnhofer et al., 1994b), to detect the same region in pa and ea subunits. Indeed the presence of a proline residue in this epitope (FSQP in human and FSKP in ovine and bovine a) appears mandatory for detection by this antibody. Since this amino-acid residue is absent and replaced by a leucine in the corresponding porcine (FSKL) and equine (FFKL) sequences, the gonadotropins from these two species cannot be detected in sandwich EIA making use of this anti-ha antibody. Proline residues are well known to induce breaks and changes of orientation in polypeptide secondary structures. It is thus not surprising that the absence of proline in the equine and porcine sequences at this location modify the three-dimensional structure of the epitope. The comparison between natural pituitary hFSH and recombinant hFSH preparations indicated that the latter exhibits a less stable quaternary structure than the former with transition temperatures of 74 and 68 °C, respectively. This might suggest that recombinant FSH preparations exhibit a lower thermal stability relative to pituitary hormones, and this difference could be attributed to the carbohydrate moieties (Erbel et al., 2002; Galet et al., 2004; Manjunath and Sairam, 1983) synthesized in pituitary gonadotrope cells (Bousfield et al., 2000; Green and Baenziger, 1988a,b) and CHO cells, respectively (Gawlitzek et al., 2009). Nevertheless, this observation must be taken with care and should not be generalized too fast as we compared only one batch of each type of hormone. The heterodimeric structure of human gonadotropins was found to be resistant to short-term heating above 70 °C whereas bovine FSH quaternary structure was found to be largely less stable
Please cite this article in press as: Haj Hassan, M., et al. Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.03.033
M. Haj Hassan et al. / General and Comparative Endocrinology xxx (2014) xxx–xxx
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Fig. 5. (A) Thermal stability of two preparations of pituitary ovine FSH (CY1758 and CY2178) after 5-min heating at the indicated temperature and arrest in melting ice. (B) Effect of stopping temperature (ice or room temperature) on residual activity of both oFSH preparations in sandwich EIA (top) and in vitro bioassay (bottom). (C) Kinetics of inactivation of oFSH CY2178 at 87.5 °C as measured by in vitro bioassay.
with transition temperatures of 61–64 °C (Fig. 4). This observation was puzzling as bovine and ovine FSH share a large proportion of identical polypeptide sequences. The identity is 100% for their a subunits whereas there are only 8 differences between their b subunit sequences (117 aa). Among these differences, three are located at the C-terminus after the last cystine residue and this region is known to be exposed in the heterodimer and thus not implied in the ab association (Combarnous, 1992; Fan and Hendrickson, 2005; Fox et al., 2001). More interestingly, two differences between the ovine and bovine bFSH amino acid sequences are found in the seatbelt region that wraps around the a partner. The sequence in the ovine FSHb is 90CGKCDRDSTDC100 and that in the bovine FSHb is 90CSKCDSDSTDC100. It is thus tempting to postulate that these two differences in the seatbelt region can affect the stability of the quaternary structure of the heterodimer as the formation and fastening of the seatbelt is mandatory for it. Another possibility is that ovine and bovine FSH differ in their carbohydrate structures and these differences in saccharide side chains could be responsible of the lower stability observed for the bovine hormone (Galet et al., 2004). A fraction of one of the ovine FSH preparations was found to retain partial activity in the sandwich EIA after long-term treatment at temperatures above 90 °C (Fig. 5). At the hormone concentration used, which is much below the equilibrium Kd, recombination of subunits upon cooling after heating is impossible. As it is unlikely that the gonadotropins preserve their folding above 80 °C, it can be hypothesized that the fraction withstanding very high temperature does refold upon cooling but reformation of the heterodimeric molecule is possible only if the subunits are covalently linked so that low concentration has no impact. Another possibility is that the hormone partially aggregates at these temperatures so that it can still be detected in the sandwich EIA. This was indeed observed when FSH CY2178 is cooled off fastly from 87.5 to 0 °C in melting ice but not when the sample is cooled off more slowly. Nevertheless, in both cases the residual FSH bioactivity in vitro was found to be 15–20% of non-heated control. Even more surprising, we found that the same residual FSH activity was retained after heating at 87.5 °C over 40 min (Fig. 5C).
A similar behaviour was observed for recombinant hLH (Fig. 6) and can also be interpreted either as the presence of a covalent bond between the a and b subunits of this hormone in part of the isoforms or as aggregation. In this case, the hypothesis of a covalent bond between the a and b subunits is the most likely as the hormone retained about 15% of its initial activity in the in vitro LH bioassay on mLTC (Fig. 6 insert) after a long term incubation at 90 °C in agreement with the proportion of undissociable heterodimer measured in the sandwich EIA. For the moment, we do not know the chemical nature of these putative inter-subunit links:disulfide bridge(s)? pseudo-peptide bond between the side chains of a Lys and Glu or Asp residue? dityrosine crosslink (Malencik and Anderson, 2003)? bonds between carbohydrate chains (Liu et al., 1984)? Also we don’t know whether this/these bond(s) is/are natural or formed during the extraction and purification steps of the hormone. If it is natural, it would be interesting to study the quantitative importance of this fraction and its possible specific biological role. It must be kept in mind that the 5-min heating and sandwich EIA protocol used in this paper is used to mimick microcalorimetric studies (Burova et al., 2001) but cannot be considered as a full substitute for it as it gives no access to thermodynamic parameters concerning the association–dissociation of a and b subunits. Nevertheless, it permits to indirectly study the stability of the cooperative folding of the hormone and, compared to microcalorimetry, it has two advantages: one is that it can be performed using non-purified hormones and another is that it requires much lower quantities of material. Consequently, the comparison of data concerning a large number of different preparations from various species and of different purities will allow to get a larger survey than would be possible by microcalorimetry. The data summarized in Table 1 show that the transition temperatures for mammalian gonadotropins are in the 61–77 °C range. They do not permit to point out obvious differences between LH and FSH preparations or between natural and recombinant hormones. Such comparisons would require the study of more numerous different preparations of each hormone. Nevertheless, it
Please cite this article in press as: Haj Hassan, M., et al. Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.03.033
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100
hCG 8149 hLH rec bLH CY1240 oLHCY1055
90
residual activity in sdw EIA
80 70 60
Acknowledgments
50 40 30 20 10 0 50
60
70
80
90
residual in vitro bioactivity (%) after 5min at 90°C
temperature (°C)
We are indebted to Dr. A.F. Parlow (NHPP, NIDDKD, HarborUCLA Medical Center, Torrance CA, USA), to Dr. J. Closset and J.F. Beckers (University of Liège, Belgium), to Dr. S. Dufour (MNHNCNRS, Paris, France) for their gifts of hormones and to Drs. Keith Henderson and Jenny Juengel (AgResearch, Invermay Agricultural Centre, Mosgiel NZ) for their gift of anti-oFSHb monoclonal antibody and to Dr. S. Dufour (MNHN-CNRS, Paris, France) for her gift of anti-carpLHb antiserum. We are grateful to Dr. E. Reiter (INRA Nouzilly) for the gift of HEK cells stably transfected with both hFSH receptor and cAMP-GLO reporter gene. We also thank INRA and the Region Centre (France) for financial support of MHH PhD thesis (2008-2011) presented at the University of Tours (France).
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References
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Bousfield, G.R., Baker, V.L., Gotschall, R.R., Butnev, V.Y., 2000. Carbohydrate analysis of glycoprotein hormones. Methods 21, 15–39. Burova, T., Lecompte, F., Galet, C., Monsallier, F., Delpech, S., Haertle, T., Combarnous, Y., 2001. Conformational stability and in vitro bioactivity of porcine luteinizing hormone. Mol. Cell. Endocrinol. 176, 129–134. Combarnous, Y., 1992. Molecular basis of the specificity of binding of glycoprotein hormones to their receptors. Endocr. Rev. 13, 670–691. Dirnhofer, S., Lechner, O., Madersbacher, S., Klieber, R., de Leeuw, R., Wick, G., Berger, P., 1994a. Free alpha subunit of human chorionic gonadotrophin: molecular basis of immunologically and biologically active domains. J. Endocrinol. 140, 145–154. Dirnhofer, S., Madersbacher, S., Bidart, J.M., Ten Kortenaar, P.B., Spottl, G., Mann, K., Wick, G., Berger, P., 1994b. The molecular basis for epitopes on the free betasubunit of human chorionic gonadotrophin (hCG), its carboxyl-terminal peptide and the hCG beta-core fragment. J. Endocrinol. 141, 153–162. Erbel, P.J., Haseley, S.R., Kamerling, J.P., Vliegenthart, J.F., 2002. Studies on the relevance of the glycan at Asn-52 of the alpha-subunit of human chorionic gonadotropin in the alphabeta dimer. Biochem. J. 364, 485–495. Fan, Q.R., Hendrickson, W.A., 2005. Structure of human follicle-stimulating hormone in complex with its receptor. Nature 433, 269–277. Fox, K.M., Dias, J.A., Van Roey, P., 2001. Three-dimensional structure of human follicle-stimulating hormone. Mol. Endocrinol. 15, 378–389. Galet, C., Lecompte, F., Combarnous, Y., 2004. Association/dissociation of gonadotropin subunits involves disulfide bridge disruption which is influenced by carbohydrate moiety. Biochem. Biophys. Res. Commun. 324, 868–873. Gawlitzek, M., Estacio, M., Furch, T., Kiss, R., 2009. Identification of cell culture conditions to control N-glycosylation site-occupancy of recombinant glycoproteins expressed in CHO cells. Biotechnol. Bioeng. 103, 1164–1175. Green, E.D., Baenziger, J.U., 1988a. Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. I. Structural elucidation of the sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J. Biol. Chem. 263, 25–35. Green, E.D., Baenziger, J.U., 1988b. Asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. II. Distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J. Biol. Chem. 263, 36–44. Henderson, K.M., Camberis, M., Hardie, A.H., 1995. Generation of monoclonal antibody to ovine FSH and its application in immunoneutralization and enzymeimmunoassay. J. Reprod. Fertil. Suppl. 49, 511–515. Legardinier, S., Duonor-Cerutti, M., Devauchelle, G., Combarnous, Y., Cahoreau, C., 2005. Biological activities of recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) expressed in Sf9 and Mimic insect cell lines. J. Mol. Endocrinol. 34, 47–60. Legardinier, S., Poirier, J.C., Klett, D., Combarnous, Y., Cahoreau, C., 2008. Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants. J. Mol. Endocrinol. 40, 185–198.
15 10 5 0 0
hCG
rec hLH
Fig. 6. Thermal stability of recombinant human LH, urinary human CG and pituitary bovine and ovine LH preparations. The hormones were incubated for 5 min at the indicated temperatures at 10 lg/ml concentration in PBS. The proportion of residual heterodimer was determined from the slope of each EIA assay and plotted as a function of the 5-min incubation temperature (top). Residual in vitro LH bioactivity of hCG and rec hLH after 5 min incubation at 90 °C (bottom).
Table 1 Comparison of transition temperatures (Tm).
*
association in pituitary at a much lower, and more variable temperature in poïkilotherms than in homeotherms such as mammals. The high Tm of carp LH is in good agreement with the observation that carp LH subunits have been found to recombine much faster than oLH subunits (Marchelidon et al., 1979). In conclusion, the systematic study of Tm of different preparations or isoforms of glycoprotein hormones from a large number of vertebrate species should help to understand the molecular thermodynamic bases of their subunits cooperative folding and quaternary association.
Hormone hFSH SIAFP1 rec hFSH bFSH NIHB2 bFSH CY2672-II oFSH CY1758
Tm (°C) 68 ± 2 74 ± 2 61 ± 2 64 ± 2 70 ± 2
rec hLH bLH CY1042 oLH CY1055 eLH* eCG* carp LH EBG
65 ± 2 67 ± 2 65 ± 2 75 ± 2 77 ± 1 83 ± 4
Legardinier et al. (2008).
appears that bovine FSH exhibits a weaker stability than the FSH preparations from other mammalian species. In addition, the first experiments with carp LH give a dissociation Tm of 83 °C which is much higher than those of all mammalian gonadotropins. The high stability of the cooperative folding of this hormone could be related to its folding and subunit
Please cite this article in press as: Haj Hassan, M., et al. Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.03.033
M. Haj Hassan et al. / General and Comparative Endocrinology xxx (2014) xxx–xxx Liu, W.K., Young, J.D., Ward, D.N., 1984. Deglycosylated ovine lutropin: preparation and characterization by in vitro binding and steroidogenesis. Mol. Cell. Endocrinol. 37, 29–39. Malencik, D.A., Anderson, S.R., 2003. Dityrosine as a product of oxidative stress and fluorescent probe. Amino Acids 25, 233–247. Manjunath, P., Sairam, M.R., 1983. Enhanced thermal stability of chemically deglycosylated human choriogonadotropin. J. Biol. Chem. 258, 3554–3558. Marchelidon, J., Salesse, R., Garnier, J., Burzawa-Gerard, E., Fontaine, Y.A., 1979. Zoological origin of gonadotropin subunits and association kinetics. Nature 281, 314–315.
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Matteri, R.L., Roser, J.F., Baldwin, D.M., Lipovetsky, V., Papkoff, H., 1987. Characterization of a monoclonal antibody which detects luteinizing hormone from diverse mammalian species. Domest. Anim. Endocrinol. 4, 157–165. Pierce, J.G., Parsons, T.F., 1981. Glycoprotein hormones: structure and function. Annu. Rev. Biochem. 50, 465–495. Tranchant, T., Durand, G., Gauthier, C., Crepieux, P., Ulloa-Aguirre, A., Royere, D., Reiter, E., 2011. Preferential beta-arrestin signalling at low receptor density revealed by functional characterization of the human FSH receptor A189 V mutation. Mol. Cell. Endocrinol. 331, 109–118.
Please cite this article in press as: Haj Hassan, M., et al. Differential thermal stability of human, bovine and ovine Follicle-Stimulating Hormone (FSH) and Luteinizing Hormone (LH) quaternary structures. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.03.033
