Vol. 167, No. 3, 1990 March 30, 1990
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AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1050-1056
SPECIFIC, HIGH-AFFINITY BINDING SITES FOR HUMAN LUTEINIZING HORMONE (hLH) AND HUMAN CHORIONIC GONADOTROPHIN (hCG) IN CANDIDA SPECIES ThomasA. Bramley*, Garry S. Menzies*, Robert J. Williams+, David J. Adams+ and OonaghS. Kinsman * University of Edinburgh, Departmentof Obstetricsand Gynaecology, Centrefor Reproductive Biology, 37 ChalmersStreet, Edinburgh EH3 9EW, Scotland, U.K. + Departmentof Microbiology, University of Leeds,LeedsLS2 9JT, U.K. Glaxo Group Research,ChemotherapyDepartment, Greenford, Middlesex,U.K. Received
February
7, 1990
The presenceof specific binding sitesfor [ I251]-labelledhLH and hCG is describedin Condidu species.Binding was presentin three strainsof Candidaalbicans, and in Candida tropicalis, and was greatest in microsomes,though binding was alsopresentin cytosol fractions. hLH and hCG mutually competedfor thesebinding sites. Other hormonesdid not bind and did not competefor hLH binding sites. Scatchard plots showed two classesof binding sites, one with high affinity, low capacity and the other with lower affinity, high capacity binding in both microsomesand cytosol. This is the first report of specific binding sitesfor mammalianpeptide hormonesin a yeast. 0 1990
Academic
Press,
Inc.
The pathogenic,dimorphic fungusCandidualbicuns is commonly found in the humanreproductive tract. Specific, high-affinity binding proteins for both corticosteroids (l-3) and oestrogens(4-6) have been demonstrated in cytosol fractions from Candida species. Furthermore, there is increasingevidence that other microorganismsmay expressmessengermoleculesandreceptors(7). Saccharomycescerevisiae producesan oestrogenicfactor which can bind to oestrogenreceptorsin the rat uterus cytosol (8), and S. cerevisiae (8,9) and Paracoccidioides brusiliensis (10,ll) possessoestrogen binding proteins. Moreover, expressionof the human oestrogen receptor in S. cerevisiae can stimulate the initiation of transcription in a strictly hormone-dependentfashion (12). S. cerevisiae can also phosphorylate certain proteins in a cyclic adenosine 3’S’-monophosphate(CAMP) dependent fashion (13,14) and several speciesof yeast possess adenylate cyclase activity (15) and other elementsof transmembranesignalling mechanisms (16,17) suchasthe RAS proteinswhich appearto be analogousto the family of guaninenucleotide proteins (G-proteins) important in the coupling of a wide rangeof mammalianhormonereceptors to their intracellular effector systems(18). Moreover, yeast mating factor ~1,the tridecapeptide mating pheromoneof S. cerevisiue (19,20) can inhibit adenylatecyclasein yeast membranes(2 1). It has been known for sometime that pregnancy predisposeswomen to Candida infections, particularly in the third trimester (22), suggesting that elevated steroid and/or chorionic gonadotrophin levels at this time may influence growth and morphogenesis.Indeed, we have shownthat germinationof Candida albicans is affected by mammaliansteroidhormonesand LH (23). If gonadotrophinsinfluence Candida albicans growth and morphogenesis,it should be possible to demonstrate specific binding sites for gonadotrophins in appropriate subcellular 0006-291x/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
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fractions. We now report the presence of specific, high-affinity binding sites for LH and hCG in Candida which may be involved in the stimulation of germ tube formation by these hormones.
METHODS Candicia strains were maintained on slopes of Sabouraud’s dextrose agar and cultured at monthly intervals. C. albicuns cells (3x107) were inoculated into 11 of Sabouraud’s dextrose broth in a 21 Erlenmeyer flask, and shaken (120 r-pm in an orbital incubator) for 16h at 37’C. Cells were harvested by centrifugation (15OOg, 10 min. in a Sorvall RCSB centrifuge) and the cell pellet washed twice with distilled water. Harvested cells were washed twice with O.lM potassium phosphate, pH 7.2 and resuspended in O.lM potassium phosphate-O.lM 2-mercaptoethanol-O.lM EDTA, pH 7.12at 2x109 cells per ml for 30 min at room temperature. Organisms were washed twice with O.lM potassium phosphate, pH 7.2 and resuspended in O.lM potassium phosphate0.9M sorbital,-1OmM EDTA, pH 7.2 (PSE buffer) containing zymolyase 20T (10 units per ml; Kit-in Brewery, Japan) for 90 min at 37°C with gentle agitation. Sphaeroplasts were harvested by centifugation at 6COg for 5 min, washed with PSE buffer and resuspended in disruption buffer (1OmM Tris-I SmM EDTA-0.25M sucrose-12 mM monothioglycerol-1OmM sodium molybdate, pH 7.4: TESHMo) to a concentration of 1010 cells per ml. Breakage of sphaeroplasts was effected using 15 complete strokes of a hand-held Teflon-glass homogeniser at 4°C. The procedure resulted in approximately 90% cell breakage. The resulting homogenate was centrifuged at 15,000g for 20 min (4°C) to remove unbroken cells and debris, then the supernatant was recentrifuged at 109,OOOg for lh (4°C). Cytosol (supematant) was immediately aliquoted and stored at -20X The microsomal pellet was washed once with TESHMo buffer, respun at 109,OOOgfor lh, resuspended in 3ml of ice-cold TESHMo buffer and stored at -20°C. Aliquants of cytosol and microsornal fractions were assayed for protein content by the method of Lowry (24). Hormones (hCG, hFSH, hGH, oFSH, oPRL) were iodinated by a lactoperoxidase method (25) and purified by chromatography on Sephadex GlOO (1x45 cm; Pharmacia PL Ltd. U.K.). Specific binding was calculated for each tracer by self-displacement assay in the standard receptor systems shown below. Sheep and pig ovaries were collected from a local abattoir within 2h of slaughter, and kept on ice. Sheep corpora lutea were dissected free of stroma and connective tissue, minced, and homogenized (Polytron homogenizer, Kinematika, Sweden) in ice-cold 0.3M sucrose-l mM EDTA-10 mM Tris-HCl, pH 7.4 (SET medium; 10 ml/g) using two 10s bursts at full speed, separated by a 1 min cooling period in ice. Homogenates were filtered through cheese-cloth, and 2ml aliquants stored at -20°C until required. Mammary glands were dissected from a late-pregnant New Zealand White rabbit, minced and homogenized in ice-cold SET medium as described above. Homogenates were filtered to remove fat and connective tissue, centrifuged at 1,OOOgfor 10 min, and the supematant then recentrifuged at 109,OOOg for lh (4°C) in a Sorvall 65Ti rotor. The supematant was discarded, and the pellet (microsomes) was resuspended by gentie homogenization in SET medium, and 2ml aliquants stored at -20°C. Porcine granulosa cells were collected by aspiration of small (3-5mm diameter) ovarian follicles using a syringe with a narrow gauge needle. Cells were collected by centrifugation at 1GOOgfor 10 min, washed twice in 10 vol of isotonic phosphate-buffered saline, then resuspended in SET medium (lOml/ ml packed pellet) and homogenized using 15 complete strokes of an all-glass Dounce homogenizer. Aliquants (2ml) were stored at -20’C. Term placentae were obtained from women having elective Caesarean section 1051
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at full term. Placental villi were dissected, washed extensively with isotonic saline to remove blood, minced and homogenized (Polytron) in SET medium (lOml/g). After centrifugation at 1,OOOgfor 10 min, the supematant was centrifuged at 109,OOOg for lh. The pellet (microsomes) was resuspended by gentle homogenization in SET medium, and stored at -2O’C. Binding of [1251]-labelled hLH was measured by incubation of aliquants of cytosol or membrane fractions in lml ice-cold buffer (0.5 g bovine serum albumin per lOOml4OmM Trisacetate buffer, pH 6.5) containing 100,OOOcpm of [ 12511~hLH (100 Ci/g)for 30 min at 4°C. Icecold bovine gamma-globulin (0.5 ml; 0.5 g/lOOml in 40mM Tris-acetate buffer) was added, followed immediately by Iml of polyethyleneglycol ( MW 8,000, Sigma Chemical Co. Ltd: 25 g/lOOml in Tris-acetate buffer). Tubes were vortexed vigorously, then centrifuged at 2,500g for 10 min (4’C). Supematants were aspirated by vacuum suction, and pellets counted for [ 1251]in a Packard ‘Crystal’ gamma-counter at an efficiency of 75%. Non-specific binding was measured in duplicate by the inclusion of 50 i.u. hCG (Chorulon) in the incubation buffer. The difference between total and non-specific binding represented specific hormone binding. All separation steps were carried out as rapidly as possible using ice-cold reagents, in order to minimise dissociation of bound hormone. RESULTS Specific binding of radiolabelled hLH to microsomal and cytosol fractions from Cundidu albicans (serotype A; NCPF 3153) increasedwith increasing protein concentration (Fig la). [125Ij hLH binding increasedlinearly at low microsomeconcentrations,whereasbinding to cytosol fractions reacheda plateauat high protein levels. This may be a reflection of decreasedefficiency of the polyethyleneglycol precipitation technique for the separationof bound from free hormone at high protein levels. Alternatively, it may reflect inactivation or degradation of the LH tracer or binding site, or the presenceof inhibitors of LH binding present in cytosol fractions. Similar binding curves to thoseobservedfor Candida ufbicuns (serotypeA; NCPF 3153) were obtained
100' ,
?
a
mgprotem
m g protein
Fig. 1: Specific binding of [l*s I]-labelled human LH (hLH) to Candida species. Aliquots of fractions of Candida were incubated with [**%I-labelled hLH, and specific binding measured as described in Materials and Methods. Points represent means f range for triplicate estimates of specific binding of [**5r]-hLH binding to microsomes (clod symbols) and cytosol (open symbols) prepared from late-exponential cultures of C. albicum (serotype A: NCPF 3 153, Fig la; Glaxo 2402E, Fig 1b; square symbols : 87; 9714, Fig lb; triangular symbols; and C. fropicalis (Fig lb, circles ).
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Standard receptor
Suecific binding (pmproteinl) C. albicans C. albicans Microsomes Cytosol
tTiiCCer
hLH hCG hGH hFSH oFSH mEGF
sheep corpus luteum sheep corpus luteum pregnant rabbit marmnary gland pig granulosa cells pig granulosa cells human placenta
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(serotype A: NCPF 3153) fractions
Table 1: Hormone binding to Candida albicanr Hormone
RESEARCH
3380 1050 62
148 140 n.d.
;4 n.d.
42
n.d. n.d. n.d.
n.d. n.d. n.d.
12,;:
n.d., specific hormone binding not detectable under all conditions tested. Figures are means of l-3 separate experiments in triplicate.
= z 100 ; 60
8
80
5 p
60
0 .g D
40
.o ki ::
20 0 0
Hormone
concentration
1o-3
(IUlng)
Hormone
concentration
10-2 Hormone
16’ 10 10’ concentration
102 lo3 (IU/ng)
(IUlng)
Fig. 2: Comparison of hormonal specificities of LH/hCG binding sites of Candida albicans (serotype A: NCPF 3153) microsomes (b) and cytosol (c) with sheep corpus luteum (a). Specific binding of [l25I]-hLH (solid symbols) or [12%]-hCG (open symbols) was measured in the absence or the presence of increasing concentrations of unlabelled gonadotrophin preparations: ( l ,O), Chorulon (Intervet Laboratories, Cambridge, U.K.: 1,500 i.u.hCG /vial): (A, A ), CG-5 (Sigma Chemical Co. Ltd., Poole, Dorset, U.K.; 5,000 i.u.hCG /vial); ( W ,O) Pergonal (Serono Laboratories Ltd., Welwyn Garden City. U.K.; 75 iu. hLH and 75i.u. hFSH/ vial). Binding was expressed as percent control binding remaining. Points represent means, and vertical bars SEM for l-6 separate competition experiments in triplicate. Other highly-purified hormones including hFSH (CPDS-33; 2925 iu. FSH per mg), oFSH (NIH-FSH-S14; 75 i.u. per mg), oPRL (NIHP-S13; 30 i.u. per mg). hGH (NIH-HS-2160E; iodination grade; 32 i.u. per mg) and mEGF failed to compete for LH or hCG binding sites [ v 1. 1053
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for specific binding of [1251]-labelled hLH to microsomesand cytosol fractions from Candida tropicalis (Fig. lb), and for two further strains of Candida albicans, isolated from vaginal infections (87:9714, Regional Mycology Laboratory, University of Leeds,U.K. and Glaxo 2402 E, Glaxo Group Research,Greenford, Middlesex, U.K.: Fig. lb). The hormonal specificity of Candida binding siteswasestablishedin two ways. Firstly, a number of radiolabelledhormonetracerswere preparedand testedfor their ability to bind to wellcharacterisedreceptor systems.All hormone tracers bound well to their respective standardtest systems(Table l), demonstratingthat each tracer was biologically active, and that the conditions usedfor incubation and recovery of boundhormonewere effective. Thesetracerswere then usedto measurespecific binding to C. albicans (serotypeA: NCPF 3153) microsomes.Only radiolabelled hLH and hCG bound significantly to C. albicans microsomesand cytosol (Table 1). In contrast, there was no significant specific binding of hFSH, oFSH, hGH, and EGF tracers(nor oPRL; not shown), whether measured in the presence or absenceof divalent metal ions, and whether incubated at 4°C for 30 min (conditions optimal for hLH binding to C. albicans microsomes),for 16hat 20°C (conditions optimal for binding of FSH and lactogensto mammalianreceptor systems) or 37°C for 3h (optimal for binding of EGF tracer to placentalmembranes). Secondly, hormonal specificity was assessed by measuringthe abilities of a number of hormonepreparationsto competefor binding of [ 125r]-labelledhLH and hCG to either C. albicans microsomes (Fig. 2b) or cytosol fractions (Fig. 2c), or to sheepcorpus luteum homogenate receptors (Fig. 2a). Similar levels of hLH and hCG tracers were bound by sheepluteal LH-
lz51
0
10 hLH
20 added
30 (PM)
40
! 0
5
10 hLH
bound
15
20
(PM)
Fig. 3: Scatchard plots of [**%I-hLH binding to Candidu albicam (serotype A: NCPF 3153) microsomes. Aliquantsof a late-exponential microsomalfractionof C. ulbicuns (serotypeA: NCPF 3153) were incubated with twelve different concentrations of [I*sI]-hLH for 30 min at 4°C as described in the legend to Fig. 1. Bound and free hormone were separated by polyethyleneglycol precipitation, and bound hormone measured by gamma-counting. Non-specific binding was measured by inclusion of excess hCG (50 i.u. Chorulon/ tube at 100,OOOcpm hLH tracer), and Scatchard plots were constructed. Microsomes demonstrated two distinct binding components. Similar results were obtained for cytosol fractions (not shown). The inset figure shows the curve for specific binding of [IzIj-hLH from which the Scatchard plot was consmnzed. Numbers of binding sites and Ka for the two binding sites were. estimated from the dotted curves calculated from the Scatchard plot as described by Zierler (26).
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Affinities and numbers of binding sites for [ *2sIJ-hLH in membranes and cytosol fractions from Candi& albicuns (serotype A: NCPF 3153) Micmsomes B max Ka (x1010M-t) (pmol.mgprot-l)
Site1 Site2
RESEARCH
2.5 iz 0.4 (6) 0.05 * 0.02 (4)
24 f 8 (6) 1570 f 950 (3)
CYtosol Ka
Bmax
(x1OtuM-t) 1.3 * 0.4 0.07 * 0.02
(pmol.mgprot-l) (3) (5)
2+1 202 + 52
(2)
(3)
receptors, and binding of both tracers was displaceable in a dose-dependentfashion by low concentrationsof gonadotrophin preparationscontaining hLH or hCG, but not by hFSH, oFSH, oPRL, hGH or EGF (Fig 2~). Both tracers also bound similarly to C albicans microsomes (Fig 2b) and cytosol (Fig 2c), and both were displaced by hLH or hCG preparations (though somewhat higher concentrations of these hormones were required to give half-maximal displacement[ IC50, 0.3-2 i.u./tube] compared to sheepluteal tissue [0.06-0.1 i.u./tube]). Once again, no displacement was observed with much higher concentrations of the other hormones tested,demonstratingthat, like the sheepluteal receptor, C. albicans binding sitesappearedto be LH/hCG-specific. Scatchardplots derived from binding isothermsof [ I]-hLH binding to microsomal fractions from C. albicans (serotypeA:NCPF 3153) showedthe presenceof two distinct hormone binding sites(Fig. 3), one of low capacity and high affinity, and one of high capacity and lower affinity (Table 2). LH-binding to cytosol fractions also showedthe presenceof two binding sites with similar affinities to thoseof microsomalfractions ( Table 2). DISCUSSION We have described the presenceof specific, high-affinity LH/hCG binding proteins in microsomal and cytosol fractions from three sub-strainsof Candida albicans and in Candida tropicalis . These binding sites may function as receptors which mediate the stimulation of morphogenesisin Candida by thesehormones(23). Theseobservationsmay be of interestin three important areas. Firstly, high affinity binding sites for LH/hCG in this yeast may representan early evolutionary form of the mammalian LH/hCG receptor. Studiesof the structureand function of the Candida receptor would allow comparisonsto be made with LH receptors from other mammalianspecies,allowing the evolution of this receptor to be mapped.Furthermore, Candida receptorscould be grown in bulk, enablingeasierpurification of the receptor than from mammalian gonadal tissu,es. Secondly, since the transition from yeast to mycelial form is thought to be associatedwith increased pathogenicity in Candida (22), studies of the involvement of these receptors will further our understandingof morphogenesisin dimorphic yeasts like Candida. Thirdly, in the light of the important link between morphogenesisand pathogenesis,substances targeted to interfere with the function of thesereceptorsmay be of greatimportancetherapeutically asantifungal agents. ACKNOWLEDGMENTS This work was supportedby a grant from the Medical ResearchCouncil (TAB; G8711653 SB) and the SERC and Glaxo Group Research(DJA, RJW and OSK). We shouldlike to thank the 1055
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Hormone Distribution Officer, NIADDK, NM, Bethesda, MD, USA for the generous gifts of highly-purified human and ovine gonadotrophins and human growth hormone, and Dr. K. Brown, Institute for Animal Physiology and Genetics Research, Babraham, Cambridge, U.K. for the generous gift of [12~I]-labelled and unlabelled mEGF. Human FSH and LH tracers (lOOmCi/mg) were purchased from Dr. K. Ferguson, Chelsea Hospital for Women, London, U.K. REFERENCES ;* 3: 4. 5. 76: 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
19. 2 ii. 24: 25. 26.
Loose, D.S., Schurman, D.J. & Feldman, D. Nature, (London) 293,477-479 (1981). Loose, D.S. 8z Feldman, D. J. Biol. Chem. 257,4925-4930 (1982). Loose, D.S., Stevens, D.A., Schurman, D.J. & Feldman, D. J. Gen. Microbial. 129,2379-2385 (1983). Powell, B.L., Frey, C.L. 8z Drutz, D.J. Experimental Mycol. 8, 304-313, (1984). Othman, Y.H.S., Adams, D.J., Hitchcock, C.A. & Oakey, R.E. Biochem. Sot. Trans. 16,789-790, (1988). Skowronski, R. & Feldman, D. Endocrinology 124, 1965-1972 (1989). LeRoith, D., Roberts, C. Jr., Lesniak, M.A. & Roth, J. Experientia 42,782-788 (1986). Feldman, D., Do. Y., Burshell, A., Stathis, P. & Loose, D.S. Science 218,297-298, (1982). Burshell, A., Stathis, P.A., Do. Y., Miller, S.C. & Feldman, D. J. Biol. Chem. 259, 3450-3456. Loose, D.S., Stover, E.P., Restrepo, A., Stevens, D.A. & Feldman, D. Proc. Natl. Acad. Sci. (USA) 80,7659-7663, (1983). Stover, E.P., Schar, G., Clemons, K.V., Stevens, D.A. & Feldman, D. Infection and Immunity, 51, 199-203, (1986) Metzger, D., White, J.H. & Chambon, P. Nature (London) 334, 31-36 (1988). Brownlee, A.G., Phillips, D.R. & Polya, G.M. Eur. J. Biochem. 109, 39-49 (1980). Hixson, C.S. & Krebs, E.G. J. Biol. Chem. 255, 2137-2145 (1980). Levitzki, A. Trends in B&hem. Sci. 13,298-301 (1988). Janssens, P.M.W. Trends in Biochem. Sci. 12,456-459 (1987). Engelberg, D., Perlman, R. & Levitzki, A. Cellular Signalling 1, l-7 (1989). Bimbaumer, L., Codina, J., Mattera, R., Cerione, R.A., Hildebrandt, J.D., Sunyer, T., Rojas, F.J., Caron, M.C., Lefkowitz, R.J. and Iyengar, R. In Molecular mechanisms of transmembrane signalling, ( Cohen J. & Houslay M, eds), 131-182. (Elsevier Science Publishing, Amsterdam), 1985. Loumaye, E., Thomer, J. & Catt, K.J. Science 218, 1323-1325 (1982). Betz, R., Manney, T.R. & Duntze, W. Gamete Res. 4, 57 l-584 (1981). Liao, H. 8z Thomer, J. Proc. Natl. Acad. Sci. USA. 77, 1898-1902 (1980). Odds, F.C. In; ‘Candida and candidosis” 2nd edition, Bailliere Tindall, London, (1988). Kinsman, O.S., Pitlado, K. & Coulson, C.J. Mycoses 12, 617-626, (1988) . Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. J. Biol. Chem. 193,265-275, (1951) . Miyachi,Y., Vaitukaitis, J.L., Nieschlag, E. & Lipsett, M.B. J. Clin. Endocr. Metab. 34,23-28, (1972). Zierler, K. Trends in Biochem. Sci. 14, 314-317, (1989).
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SPECIFIC, HIGH-AFFINITY BINDING SITES FOR HUMAN LUTEINIZING HORMONE (hLH) AND HUMAN CHORIONIC GONADOTROPHIN (hCG) IN CANDIDA SPECIES ThomasA. Bramley*, Garry S. Menzies*, Robert J. Williams+, David J. Adams+ and OonaghS. Kinsman * University of Edinburgh, Departmentof Obstetricsand Gynaecology, Centrefor Reproductive Biology, 37 ChalmersStreet, Edinburgh EH3 9EW, Scotland, U.K. + Departmentof Microbiology, University of Leeds,LeedsLS2 9JT, U.K. Glaxo Group Research,ChemotherapyDepartment, Greenford, Middlesex,U.K. Received
February
7, 1990
The presenceof specific binding sitesfor [ I251]-labelledhLH and hCG is describedin Condidu species.Binding was presentin three strainsof Candidaalbicans, and in Candida tropicalis, and was greatest in microsomes,though binding was alsopresentin cytosol fractions. hLH and hCG mutually competedfor thesebinding sites. Other hormonesdid not bind and did not competefor hLH binding sites. Scatchard plots showed two classesof binding sites, one with high affinity, low capacity and the other with lower affinity, high capacity binding in both microsomesand cytosol. This is the first report of specific binding sitesfor mammalianpeptide hormonesin a yeast. 0 1990
Academic
Press,
Inc.
The pathogenic,dimorphic fungusCandidualbicuns is commonly found in the humanreproductive tract. Specific, high-affinity binding proteins for both corticosteroids (l-3) and oestrogens(4-6) have been demonstrated in cytosol fractions from Candida species. Furthermore, there is increasingevidence that other microorganismsmay expressmessengermoleculesandreceptors(7). Saccharomycescerevisiae producesan oestrogenicfactor which can bind to oestrogenreceptorsin the rat uterus cytosol (8), and S. cerevisiae (8,9) and Paracoccidioides brusiliensis (10,ll) possessoestrogen binding proteins. Moreover, expressionof the human oestrogen receptor in S. cerevisiae can stimulate the initiation of transcription in a strictly hormone-dependentfashion (12). S. cerevisiae can also phosphorylate certain proteins in a cyclic adenosine 3’S’-monophosphate(CAMP) dependent fashion (13,14) and several speciesof yeast possess adenylate cyclase activity (15) and other elementsof transmembranesignalling mechanisms (16,17) suchasthe RAS proteinswhich appearto be analogousto the family of guaninenucleotide proteins (G-proteins) important in the coupling of a wide rangeof mammalianhormonereceptors to their intracellular effector systems(18). Moreover, yeast mating factor ~1,the tridecapeptide mating pheromoneof S. cerevisiue (19,20) can inhibit adenylatecyclasein yeast membranes(2 1). It has been known for sometime that pregnancy predisposeswomen to Candida infections, particularly in the third trimester (22), suggesting that elevated steroid and/or chorionic gonadotrophin levels at this time may influence growth and morphogenesis.Indeed, we have shownthat germinationof Candida albicans is affected by mammaliansteroidhormonesand LH (23). If gonadotrophinsinfluence Candida albicans growth and morphogenesis,it should be possible to demonstrate specific binding sites for gonadotrophins in appropriate subcellular 0006-291x/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
1050
Vol.
167,
No.
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fractions. We now report the presence of specific, high-affinity binding sites for LH and hCG in Candida which may be involved in the stimulation of germ tube formation by these hormones.
METHODS Candicia strains were maintained on slopes of Sabouraud’s dextrose agar and cultured at monthly intervals. C. albicuns cells (3x107) were inoculated into 11 of Sabouraud’s dextrose broth in a 21 Erlenmeyer flask, and shaken (120 r-pm in an orbital incubator) for 16h at 37’C. Cells were harvested by centrifugation (15OOg, 10 min. in a Sorvall RCSB centrifuge) and the cell pellet washed twice with distilled water. Harvested cells were washed twice with O.lM potassium phosphate, pH 7.2 and resuspended in O.lM potassium phosphate-O.lM 2-mercaptoethanol-O.lM EDTA, pH 7.12at 2x109 cells per ml for 30 min at room temperature. Organisms were washed twice with O.lM potassium phosphate, pH 7.2 and resuspended in O.lM potassium phosphate0.9M sorbital,-1OmM EDTA, pH 7.2 (PSE buffer) containing zymolyase 20T (10 units per ml; Kit-in Brewery, Japan) for 90 min at 37°C with gentle agitation. Sphaeroplasts were harvested by centifugation at 6COg for 5 min, washed with PSE buffer and resuspended in disruption buffer (1OmM Tris-I SmM EDTA-0.25M sucrose-12 mM monothioglycerol-1OmM sodium molybdate, pH 7.4: TESHMo) to a concentration of 1010 cells per ml. Breakage of sphaeroplasts was effected using 15 complete strokes of a hand-held Teflon-glass homogeniser at 4°C. The procedure resulted in approximately 90% cell breakage. The resulting homogenate was centrifuged at 15,000g for 20 min (4°C) to remove unbroken cells and debris, then the supernatant was recentrifuged at 109,OOOg for lh (4°C). Cytosol (supematant) was immediately aliquoted and stored at -20X The microsomal pellet was washed once with TESHMo buffer, respun at 109,OOOgfor lh, resuspended in 3ml of ice-cold TESHMo buffer and stored at -20°C. Aliquants of cytosol and microsornal fractions were assayed for protein content by the method of Lowry (24). Hormones (hCG, hFSH, hGH, oFSH, oPRL) were iodinated by a lactoperoxidase method (25) and purified by chromatography on Sephadex GlOO (1x45 cm; Pharmacia PL Ltd. U.K.). Specific binding was calculated for each tracer by self-displacement assay in the standard receptor systems shown below. Sheep and pig ovaries were collected from a local abattoir within 2h of slaughter, and kept on ice. Sheep corpora lutea were dissected free of stroma and connective tissue, minced, and homogenized (Polytron homogenizer, Kinematika, Sweden) in ice-cold 0.3M sucrose-l mM EDTA-10 mM Tris-HCl, pH 7.4 (SET medium; 10 ml/g) using two 10s bursts at full speed, separated by a 1 min cooling period in ice. Homogenates were filtered through cheese-cloth, and 2ml aliquants stored at -20°C until required. Mammary glands were dissected from a late-pregnant New Zealand White rabbit, minced and homogenized in ice-cold SET medium as described above. Homogenates were filtered to remove fat and connective tissue, centrifuged at 1,OOOgfor 10 min, and the supematant then recentrifuged at 109,OOOg for lh (4°C) in a Sorvall 65Ti rotor. The supematant was discarded, and the pellet (microsomes) was resuspended by gentie homogenization in SET medium, and 2ml aliquants stored at -20°C. Porcine granulosa cells were collected by aspiration of small (3-5mm diameter) ovarian follicles using a syringe with a narrow gauge needle. Cells were collected by centrifugation at 1GOOgfor 10 min, washed twice in 10 vol of isotonic phosphate-buffered saline, then resuspended in SET medium (lOml/ ml packed pellet) and homogenized using 15 complete strokes of an all-glass Dounce homogenizer. Aliquants (2ml) were stored at -20’C. Term placentae were obtained from women having elective Caesarean section 1051
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at full term. Placental villi were dissected, washed extensively with isotonic saline to remove blood, minced and homogenized (Polytron) in SET medium (lOml/g). After centrifugation at 1,OOOgfor 10 min, the supematant was centrifuged at 109,OOOg for lh. The pellet (microsomes) was resuspended by gentle homogenization in SET medium, and stored at -2O’C. Binding of [1251]-labelled hLH was measured by incubation of aliquants of cytosol or membrane fractions in lml ice-cold buffer (0.5 g bovine serum albumin per lOOml4OmM Trisacetate buffer, pH 6.5) containing 100,OOOcpm of [ 12511~hLH (100 Ci/g)for 30 min at 4°C. Icecold bovine gamma-globulin (0.5 ml; 0.5 g/lOOml in 40mM Tris-acetate buffer) was added, followed immediately by Iml of polyethyleneglycol ( MW 8,000, Sigma Chemical Co. Ltd: 25 g/lOOml in Tris-acetate buffer). Tubes were vortexed vigorously, then centrifuged at 2,500g for 10 min (4’C). Supematants were aspirated by vacuum suction, and pellets counted for [ 1251]in a Packard ‘Crystal’ gamma-counter at an efficiency of 75%. Non-specific binding was measured in duplicate by the inclusion of 50 i.u. hCG (Chorulon) in the incubation buffer. The difference between total and non-specific binding represented specific hormone binding. All separation steps were carried out as rapidly as possible using ice-cold reagents, in order to minimise dissociation of bound hormone. RESULTS Specific binding of radiolabelled hLH to microsomal and cytosol fractions from Cundidu albicans (serotype A; NCPF 3153) increasedwith increasing protein concentration (Fig la). [125Ij hLH binding increasedlinearly at low microsomeconcentrations,whereasbinding to cytosol fractions reacheda plateauat high protein levels. This may be a reflection of decreasedefficiency of the polyethyleneglycol precipitation technique for the separationof bound from free hormone at high protein levels. Alternatively, it may reflect inactivation or degradation of the LH tracer or binding site, or the presenceof inhibitors of LH binding present in cytosol fractions. Similar binding curves to thoseobservedfor Candida ufbicuns (serotypeA; NCPF 3153) were obtained
100' ,
?
a
mgprotem
m g protein
Fig. 1: Specific binding of [l*s I]-labelled human LH (hLH) to Candida species. Aliquots of fractions of Candida were incubated with [**%I-labelled hLH, and specific binding measured as described in Materials and Methods. Points represent means f range for triplicate estimates of specific binding of [**5r]-hLH binding to microsomes (clod symbols) and cytosol (open symbols) prepared from late-exponential cultures of C. albicum (serotype A: NCPF 3 153, Fig la; Glaxo 2402E, Fig 1b; square symbols : 87; 9714, Fig lb; triangular symbols; and C. fropicalis (Fig lb, circles ).
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Standard receptor
Suecific binding (pmproteinl) C. albicans C. albicans Microsomes Cytosol
tTiiCCer
hLH hCG hGH hFSH oFSH mEGF
sheep corpus luteum sheep corpus luteum pregnant rabbit marmnary gland pig granulosa cells pig granulosa cells human placenta
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(serotype A: NCPF 3153) fractions
Table 1: Hormone binding to Candida albicanr Hormone
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3380 1050 62
148 140 n.d.
;4 n.d.
42
n.d. n.d. n.d.
n.d. n.d. n.d.
12,;:
n.d., specific hormone binding not detectable under all conditions tested. Figures are means of l-3 separate experiments in triplicate.
= z 100 ; 60
8
80
5 p
60
0 .g D
40
.o ki ::
20 0 0
Hormone
concentration
1o-3
(IUlng)
Hormone
concentration
10-2 Hormone
16’ 10 10’ concentration
102 lo3 (IU/ng)
(IUlng)
Fig. 2: Comparison of hormonal specificities of LH/hCG binding sites of Candida albicans (serotype A: NCPF 3153) microsomes (b) and cytosol (c) with sheep corpus luteum (a). Specific binding of [l25I]-hLH (solid symbols) or [12%]-hCG (open symbols) was measured in the absence or the presence of increasing concentrations of unlabelled gonadotrophin preparations: ( l ,O), Chorulon (Intervet Laboratories, Cambridge, U.K.: 1,500 i.u.hCG /vial): (A, A ), CG-5 (Sigma Chemical Co. Ltd., Poole, Dorset, U.K.; 5,000 i.u.hCG /vial); ( W ,O) Pergonal (Serono Laboratories Ltd., Welwyn Garden City. U.K.; 75 iu. hLH and 75i.u. hFSH/ vial). Binding was expressed as percent control binding remaining. Points represent means, and vertical bars SEM for l-6 separate competition experiments in triplicate. Other highly-purified hormones including hFSH (CPDS-33; 2925 iu. FSH per mg), oFSH (NIH-FSH-S14; 75 i.u. per mg), oPRL (NIHP-S13; 30 i.u. per mg). hGH (NIH-HS-2160E; iodination grade; 32 i.u. per mg) and mEGF failed to compete for LH or hCG binding sites [ v 1. 1053
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for specific binding of [1251]-labelled hLH to microsomesand cytosol fractions from Candida tropicalis (Fig. lb), and for two further strains of Candida albicans, isolated from vaginal infections (87:9714, Regional Mycology Laboratory, University of Leeds,U.K. and Glaxo 2402 E, Glaxo Group Research,Greenford, Middlesex, U.K.: Fig. lb). The hormonal specificity of Candida binding siteswasestablishedin two ways. Firstly, a number of radiolabelledhormonetracerswere preparedand testedfor their ability to bind to wellcharacterisedreceptor systems.All hormone tracers bound well to their respective standardtest systems(Table l), demonstratingthat each tracer was biologically active, and that the conditions usedfor incubation and recovery of boundhormonewere effective. Thesetracerswere then usedto measurespecific binding to C. albicans (serotypeA: NCPF 3153) microsomes.Only radiolabelled hLH and hCG bound significantly to C. albicans microsomesand cytosol (Table 1). In contrast, there was no significant specific binding of hFSH, oFSH, hGH, and EGF tracers(nor oPRL; not shown), whether measured in the presence or absenceof divalent metal ions, and whether incubated at 4°C for 30 min (conditions optimal for hLH binding to C. albicans microsomes),for 16hat 20°C (conditions optimal for binding of FSH and lactogensto mammalianreceptor systems) or 37°C for 3h (optimal for binding of EGF tracer to placentalmembranes). Secondly, hormonal specificity was assessed by measuringthe abilities of a number of hormonepreparationsto competefor binding of [ 125r]-labelledhLH and hCG to either C. albicans microsomes (Fig. 2b) or cytosol fractions (Fig. 2c), or to sheepcorpus luteum homogenate receptors (Fig. 2a). Similar levels of hLH and hCG tracers were bound by sheepluteal LH-
lz51
0
10 hLH
20 added
30 (PM)
40
! 0
5
10 hLH
bound
15
20
(PM)
Fig. 3: Scatchard plots of [**%I-hLH binding to Candidu albicam (serotype A: NCPF 3153) microsomes. Aliquantsof a late-exponential microsomalfractionof C. ulbicuns (serotypeA: NCPF 3153) were incubated with twelve different concentrations of [I*sI]-hLH for 30 min at 4°C as described in the legend to Fig. 1. Bound and free hormone were separated by polyethyleneglycol precipitation, and bound hormone measured by gamma-counting. Non-specific binding was measured by inclusion of excess hCG (50 i.u. Chorulon/ tube at 100,OOOcpm hLH tracer), and Scatchard plots were constructed. Microsomes demonstrated two distinct binding components. Similar results were obtained for cytosol fractions (not shown). The inset figure shows the curve for specific binding of [IzIj-hLH from which the Scatchard plot was consmnzed. Numbers of binding sites and Ka for the two binding sites were. estimated from the dotted curves calculated from the Scatchard plot as described by Zierler (26).
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Affinities and numbers of binding sites for [ *2sIJ-hLH in membranes and cytosol fractions from Candi& albicuns (serotype A: NCPF 3153) Micmsomes B max Ka (x1010M-t) (pmol.mgprot-l)
Site1 Site2
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2.5 iz 0.4 (6) 0.05 * 0.02 (4)
24 f 8 (6) 1570 f 950 (3)
CYtosol Ka
Bmax
(x1OtuM-t) 1.3 * 0.4 0.07 * 0.02
(pmol.mgprot-l) (3) (5)
2+1 202 + 52
(2)
(3)
receptors, and binding of both tracers was displaceable in a dose-dependentfashion by low concentrationsof gonadotrophin preparationscontaining hLH or hCG, but not by hFSH, oFSH, oPRL, hGH or EGF (Fig 2~). Both tracers also bound similarly to C albicans microsomes (Fig 2b) and cytosol (Fig 2c), and both were displaced by hLH or hCG preparations (though somewhat higher concentrations of these hormones were required to give half-maximal displacement[ IC50, 0.3-2 i.u./tube] compared to sheepluteal tissue [0.06-0.1 i.u./tube]). Once again, no displacement was observed with much higher concentrations of the other hormones tested,demonstratingthat, like the sheepluteal receptor, C. albicans binding sitesappearedto be LH/hCG-specific. Scatchardplots derived from binding isothermsof [ I]-hLH binding to microsomal fractions from C. albicans (serotypeA:NCPF 3153) showedthe presenceof two distinct hormone binding sites(Fig. 3), one of low capacity and high affinity, and one of high capacity and lower affinity (Table 2). LH-binding to cytosol fractions also showedthe presenceof two binding sites with similar affinities to thoseof microsomalfractions ( Table 2). DISCUSSION We have described the presenceof specific, high-affinity LH/hCG binding proteins in microsomal and cytosol fractions from three sub-strainsof Candida albicans and in Candida tropicalis . These binding sites may function as receptors which mediate the stimulation of morphogenesisin Candida by thesehormones(23). Theseobservationsmay be of interestin three important areas. Firstly, high affinity binding sites for LH/hCG in this yeast may representan early evolutionary form of the mammalian LH/hCG receptor. Studiesof the structureand function of the Candida receptor would allow comparisonsto be made with LH receptors from other mammalianspecies,allowing the evolution of this receptor to be mapped.Furthermore, Candida receptorscould be grown in bulk, enablingeasierpurification of the receptor than from mammalian gonadal tissu,es. Secondly, since the transition from yeast to mycelial form is thought to be associatedwith increased pathogenicity in Candida (22), studies of the involvement of these receptors will further our understandingof morphogenesisin dimorphic yeasts like Candida. Thirdly, in the light of the important link between morphogenesisand pathogenesis,substances targeted to interfere with the function of thesereceptorsmay be of greatimportancetherapeutically asantifungal agents. ACKNOWLEDGMENTS This work was supportedby a grant from the Medical ResearchCouncil (TAB; G8711653 SB) and the SERC and Glaxo Group Research(DJA, RJW and OSK). We shouldlike to thank the 1055
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Hormone Distribution Officer, NIADDK, NM, Bethesda, MD, USA for the generous gifts of highly-purified human and ovine gonadotrophins and human growth hormone, and Dr. K. Brown, Institute for Animal Physiology and Genetics Research, Babraham, Cambridge, U.K. for the generous gift of [12~I]-labelled and unlabelled mEGF. Human FSH and LH tracers (lOOmCi/mg) were purchased from Dr. K. Ferguson, Chelsea Hospital for Women, London, U.K. REFERENCES ;* 3: 4. 5. 76: 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
19. 2 ii. 24: 25. 26.
Loose, D.S., Schurman, D.J. & Feldman, D. Nature, (London) 293,477-479 (1981). Loose, D.S. 8z Feldman, D. J. Biol. Chem. 257,4925-4930 (1982). Loose, D.S., Stevens, D.A., Schurman, D.J. & Feldman, D. J. Gen. Microbial. 129,2379-2385 (1983). Powell, B.L., Frey, C.L. 8z Drutz, D.J. Experimental Mycol. 8, 304-313, (1984). Othman, Y.H.S., Adams, D.J., Hitchcock, C.A. & Oakey, R.E. Biochem. Sot. Trans. 16,789-790, (1988). Skowronski, R. & Feldman, D. Endocrinology 124, 1965-1972 (1989). LeRoith, D., Roberts, C. Jr., Lesniak, M.A. & Roth, J. Experientia 42,782-788 (1986). Feldman, D., Do. Y., Burshell, A., Stathis, P. & Loose, D.S. Science 218,297-298, (1982). Burshell, A., Stathis, P.A., Do. Y., Miller, S.C. & Feldman, D. J. Biol. Chem. 259, 3450-3456. Loose, D.S., Stover, E.P., Restrepo, A., Stevens, D.A. & Feldman, D. Proc. Natl. Acad. Sci. (USA) 80,7659-7663, (1983). Stover, E.P., Schar, G., Clemons, K.V., Stevens, D.A. & Feldman, D. Infection and Immunity, 51, 199-203, (1986) Metzger, D., White, J.H. & Chambon, P. Nature (London) 334, 31-36 (1988). Brownlee, A.G., Phillips, D.R. & Polya, G.M. Eur. J. Biochem. 109, 39-49 (1980). Hixson, C.S. & Krebs, E.G. J. Biol. Chem. 255, 2137-2145 (1980). Levitzki, A. Trends in B&hem. Sci. 13,298-301 (1988). Janssens, P.M.W. Trends in Biochem. Sci. 12,456-459 (1987). Engelberg, D., Perlman, R. & Levitzki, A. Cellular Signalling 1, l-7 (1989). Bimbaumer, L., Codina, J., Mattera, R., Cerione, R.A., Hildebrandt, J.D., Sunyer, T., Rojas, F.J., Caron, M.C., Lefkowitz, R.J. and Iyengar, R. In Molecular mechanisms of transmembrane signalling, ( Cohen J. & Houslay M, eds), 131-182. (Elsevier Science Publishing, Amsterdam), 1985. Loumaye, E., Thomer, J. & Catt, K.J. Science 218, 1323-1325 (1982). Betz, R., Manney, T.R. & Duntze, W. Gamete Res. 4, 57 l-584 (1981). Liao, H. 8z Thomer, J. Proc. Natl. Acad. Sci. USA. 77, 1898-1902 (1980). Odds, F.C. In; ‘Candida and candidosis” 2nd edition, Bailliere Tindall, London, (1988). Kinsman, O.S., Pitlado, K. & Coulson, C.J. Mycoses 12, 617-626, (1988) . Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. J. Biol. Chem. 193,265-275, (1951) . Miyachi,Y., Vaitukaitis, J.L., Nieschlag, E. & Lipsett, M.B. J. Clin. Endocr. Metab. 34,23-28, (1972). Zierler, K. Trends in Biochem. Sci. 14, 314-317, (1989).
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