May 17, 1982 - Inhibition of cyclic. AMP accumulation was complete whereas inhibition of steroidogenesis was about 75%. DG-hCG had no effect.
THEJOURNALOF B~OLOCICAL CHEMISTRY Vol. 258. No. 1, Issue of January 10, pp. 445-449, 1983 Printed in U.S.A.
Hormonal Antagonistic Propertiesof Chemically Deglycosylated Human Choriogonadotropin* (Received for publication, May17, 1982)
M. R. SairamS andP. Manjunath From the Reproduction Research Laboratory, Clinical Research Institute of Montreal, Montreal, Quebec H2W lR7, Canada
The biological properties of chemically deglycosylated human choriogonadotropin (DG-hCG)preparations were examined in collagenase-dispersed rat interstitial cells in uitro. Despite effective receptor binding activity in membrane preparations, DG-hCG failed to induce cyclic AMP accumulation in the cells when incubated in the presence or absence of a phosphodiesterase inhibitor. The steroidogenic ability as assessed by testosterone accumulation in the medium was less than 0.5%of the native hormone with a failure to attain maximal steroid production. Time course experiments have revealed that altered kinetics could not be responsible for the loss of hormone response. Consistent with its property of good receptor bindingandpoor cell activation, DG-hCG antagonized the action of native hCG.When added to the cells at the same time, DGhCG inhibited the action of a maximal stimulatory dose of hCG in a dose-dependentmanner. Inhibitionof cyclic AMP accumulation was complete whereas inhibition of steroidogenesis was about 75%. DG-hCG had no effect on the stimulatory action of cholera toxin in interstitial cells or that of follitropin in rat seminiferous tubular preparations. Thedata suggest that DG-hCG has a conformation conducive for effective interaction with the receptor, but its ability to activate the adenylate cyclase is either lost or weakly expressed.
less complete loss of the ability t o i n d u c e t a r g e tcell response in vitro (3). In view of some differences noted in the properties of the enzymatically deglycosylated hCG' (2) and chemically deglycosylated hCG (3),it w a s of interest to e v a l u a t e its a c t i o n in vitro. The results of the present s t u d y s h o wthat DG-hCG is an effective and specific inhibitor of the a c t i o n of the native h o r m o n e in vitro and exerts its a c t i o n b y occupation of the r e c e p t o r sites on the cell surface. MATERIALS AND METHODS
Hormone Preparations-hCG was purified and subjected to chemical deglycosylation with anhydrousH F a t0 "C for 60 min. Details of the preparation and chemical characterization of the deglycosylated hormone(DG-hCG)havebeenreportedrecently (3). The use of concanavalin A-Sepharose chromatography step and gel filtration on Sephadex G-100 eliminated the possibility of native hCG being present in theDG-hCGpreparationsastheirbehavioriscompletely different under these conditions. The preparationsused in the present study had been stored in the lyophilized form at 4 "C for6-18 months. In some instances hCG (CR-119) supplied by the hormone distribution officer at the National Institutesof Health, Bethesda, MD, was also employed as the standard. Highly purified follitropin and lutropin were isolated in our laboratory from sheep (4) and human pituitary glands(5). OtherMaterials-Bovineserumalbumin, cyclic AMP binding protein, and cholera toxin were purchased from Sigma; collagenase, lima bean trypsin inhibitor and IBMX were obtained from Worthington and Aldrich, respectively. Dulbecco's modified Eagle's medium was purchased in powder form from Gibco, Long Island, NY, and reconstituted in the laboratory. ["HIAdenosine 3':5'-phosphate (NETThe p l a c e n t a l h o r m o n e human choriogonadotropin has a 275), [:'H]testosterone (NET-l87),Omnifluor, and TritonX-100 were much higher content of carbohydrate than pituitary lutropin obtained from New England Nuclear. Preparation of Interstitial Cells and Zncuhation-Testicular in(1).Recent studies h a v e shown that the s u g a r residues in the terstitialcellsfromadultrats (200-250 g, Charles River, Canada) bulky carbohydrate c h a i n s p r e s e n tin both the subunits of the were prepared by collagenase digestion and mechanical dispersion(6, i n t a c t hormone can be r e m o v e d b y p r o l o n g esequential d treat- 7). The cells were suspended in Dulbecco's modified Eagle's medium m e n t w i t h e x o g l y c o s i d a s e (2) s or in a single s t e p by treatment 0.01V limabeantrypsin containing 0.1%' bovineserumalbumin, inhibitor, and0.05 M IBMX. In some experiments IBMX was omitted w i t h anhydrous h y d r o g e n fluoride (3). We h a v e reported that short chemical deglycosylation resultsin the r e m o v a l of more and these are identified in the appropriate figures. All incubations than 75% of the oligosaccharide units w i t h o u t any effects on were performed in duplicate or triplicate in disposable glass tubes (12 X 75 mm). The cell suspension (0.5 ml)wasaddedtothetubes the q u a t e r n a r y s t r u c t u r e o f the h o r m o n e . This was demon- containing 0.1 ml of thehormonesolutionspreparedin 0.025 M strated by an evaluation of the receptor binding, immunolog- phosphate buffer, pH 7.4, containing 0.l? bovine serum albumin and ical, biophysical, and biochemical properties of the deglycoheld in an ice-water bath. The reaction was initiatedby transferring sylated h o r m o n e ( 3 ) . The specific loss of s u g a r residues in- the tubes to a metabolic incubator a t 37 O C with constant gassing under 95% 0,,5%1 COZ.The tubeswere incubated for30 min-2 h. The d u c e d i n t e r e s t i n g differential effects on the biological properties of the hormone. The interaction of the hormone w i t h reaction was terminated by dilution or by transferring the tubes to a "C for about 15 min. The tubeswere then centrifuged water bath at 80 m e m b r a n e receptor is e n h a n c e d significantly w i t h a more or a t 2900 X g for 10-15 min a t room temperatureandtheclear supernatant was savedfor the estimation of cyclic AMP or testosterone or both. When the products were not estimated immediately, the * This work received financial support from the Medical Research supernatants were stored at -20 "C until assayed. Council of Canada and the Special Program of Research in Human Estimatton of Cyclic AMP-Cyclic AMP in themediumwas ReproductionandTraining,WorldHealthOrganization,Geneva, estimated by the protein binding assay ( 8 ) as employed earlier (7) Switzerland. The costs of publication of this article were defrayed in using the Sigma binding protein. These incubations were performed part by the payment of page charges. This article must therefore be at pH 4.0, overnight at 4 "C, and details of processing have already 18 U.S.C. Section hereby marked "adoertisernent" in accordance with "_ ". 1734 solely to indicate this fact. $ T o whom correspondence should be addressed a t Clinical ~ .Re~ " . . ' T h e abbreviationsusedare:hCG,humanchoriogonadotropin; search Institute of Montreal, 100 Pine Ave. W., Montreal, Quebec DG-hCG, chemically deglycosylated human choriogonadotropin; H2W 1R7, Canada. IBMX, isobutylmethylxanthine.
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hCG Antagonist
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been reported (7). In some experiments, a binding protein fraction prepared frombovine adrenalsandsupplied by Dr. A. Goff (St. Hyacinthe. Quebec)wasalsousedfor cyclic AMP assay. In this instance, the incubationswere done at pH7.0 in a total volume of 300 pl at 4 "C for 16 h. Bound and free cyclic AMP were separated by the addition of a n equal volume of charcoal (Norit A, 5 mg/ml) in the incubating buffer. The tubeswere mixed and centrifuged immediately a t 4 "C for 15 min at 2900 X g . The radioactivity in 0.3 ml of the supernatant was determined by mixing it with5 ml of scintillant prepared in the laboratory (4% Omnifluor in 2:1 mixture of toluene and Triton X-100) and counting in a Beckman liquidscintillation counter. The sensitivity of the cyclic AMP assaywith the Sigma binding protein was 0.1 pmol/tube and with the adrenal protein it was 0.05 pmol/tube.Eachstudycontained a set of appropriate controls to ensure thatcomparisons could be made within the experiment. Testosterone was estimated by a specific radioimmunoassay (7) which could detect 25 pg/tube under the conditions employed. Preparations of Seminiferous Tubular Suspensions and Incubation-In order to check the specificity of action of DG-hCG, experiments were carried out with seminiferous tubular suspensions which are responsive to follitropin. These suspensions were prepared from the testes of 19-day-old immature rats by collagenase digestion and dispersion as described by Rao and Ramachandran (9) with some modifications. The suspensionswere incubated with thehormone samples (see"Results") for 30 min at 37 "C in the presence of 3.3 mM IBMX. After termination of the reaction by immersion in water bath a t 80 "C for 15 min, the amount of cyclic AMP in the supernatant was estimated by theprotein binding assay(7) using theSigma binding protein. Analysis of Data"G11 cyclic AMP and testosterone assay results a logit-logbasis using a Hewlett-Packard prowerecalculatedon grammable desk top calculator. Wherever necesary, the results were analyzed for statistical significance by Student's t test.
ent increase in the accumulationof cyclic AMP. As compared to thehigh activity of DG-hCG in receptor binding assays ( 3 , its ability to cause cyclic AMP accumulationin rat interstitial 1) either in the presence or cells was extremely poor (Fig. absence of the phosphodiesterase inhibitor, IBMX, confirming of the DG-hCG preparations previousobservations.None affected thestandardcurve of cyclic AMP in the assay, excluding the possibility thatspuriousinterference in the estimation could be responsible for the observed lack of response. Kinetics of Cyclic AMP Accumulation-In the previous experiments (Fig. 1) cyclic AMP accumulation in the interstitial cells was measuredafter onlya 30-min incubation in which case DG-hCG preparationswere noted tobe completely inert. It was possible that this could have been the result of a lag time or altered kinetics in the presence of DG-hCG. This was tested by measuring the nucleotide at different intervals up to 2 h. In the presence of native hCG (Fig. 2A), cyclic 40
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Receptor Binding Actzuity-As reported recently (3) the DG-hCG preparations showed good ability to bind to testicular and ovarianreceptors. Theaveragereceptor binding activityincreased 150-200% (molar basis) ascomparedto native hCG. Cyclic AMP Accumulation by hCG a n d DG-hCG-The addition of hCG to the interstitial cells caused a dose-depend-
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FIG. 2. Kinetics of cyclic AMP accumulation and steroidogenesis in rat interstitial cells. A , Collagenase-dispersed cells were incubated in a total volume of 0.6 ml a t 37 "C in the presence of 0.05 FIG. 1. Cyclic AMP accumulation in rat interstitial cells. About 150,000 collagenase-dispersed cells were incubated with the samples in a total volume of 0.6 ml in the absence ( A )or thepresence ( B )of 0.05 mM IBMX. T h e incubation was a t 37 "C for 30 min. Cyclic AMP in the medium was assayed by using theadrenal binding protein.
M IBMX. Tubes in quadruplicateswere removeda t specified intervals and inactivated by immersion in water bath at 80 "C. Cyclic AMP in the supernatant was estimated by the binding protein method. B, the cells were incubated in the absenceof IBMX and a t selected intervals triplicateswereremoved for inactivation in waterbath at 80"C. Testosterone in the supernatant was estimated by radioimmunoassay. Cyclic AMP levels in all the incubations includinghCGwere not significantly different from the control values (1.5-3.0 pmol/tube).
hCG Antagonist
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AMP accumulation continued to rise up to 60 min and leveled off by 2 h resulting in approximately a 40-fold increase over basal levels. These characteristics were completely absent in I the presence of 30 ng of DG-hCG or100 ng of DG-hCG despite I I the inclusion of the phosphodiesterase inhibitor, IBMX. The I differences between the control cells and cells incubated with I I DG-hCG were not statistically significant. I Cyclic AMP Accumulation a n d Steroidogenesis Induced by hCG a n d DG-hCG-The addition of hCG to the rat interstitial cells caused a dose-dependent increase in the accumulation of cyclic AMP and testosterone production (Fig. 3 ) .The concentration of hCG required toelicit near maximal steroidogenesis in interstitial cells was about 100-200 times less than I I 10 100 1000 that required to elevatecyclic AMP levels in the same experng O G - h C G iment. These data again confirm previous studies obtained with hCG (2, 10) and ovine lutropin (6, 7, 11) in the same mode1 system. NO D G - h C G In marked contrast to the high activity observed in binding assays ( 3 ) , theability of DG-hCG toinducetestosterone synthesis or cyclic AMP accumulation was extremely poor. With respect to steroidogenesis, DG-hCG was not only less +llnq OG-hCG effective but also failed toproducethe maximalresponse typical of hCG, even at supramaximal doses. An estimate of potency in the narrow linear portion of the dose-response curve revealed an average activity of
