Characterization of a

factor(s) from partially purified human gonadotrophin preparations which inhibit(s) the binding of radiolabelled human LH and human chorionic gonadotrophin to Candida albicans T. A. Bramley, G. S. D. J. Adams

Menzies, R. J. Williams, O. S. Kinsman and

Department of Obstetrics and Gynaecology, Centre for Reproductive Biology, University of Edinburgh,

37 Chalmers Street, Edinburgh eh3 9ew *Department of Microbiology, University of Leeds, Leeds ls2 9JT tChemotherapy Department, Glaxo Group Research, Greenford, Middlesex ubóohe received 26 April 1990 ABSTRACT

We have shown previously that partially purified human chorionic gonadotrophin (hCG) preparations inhibited the specific binding of I-labelled hLH or hCG to Candida albicans membranes at much lower concentrations than did highly purified hLH or hCG preparations. We now describe the characterization and partial purification of a heat-labile glycoprotein from commercially available gonadotrophin preparations. The factor strongly inhibited LH binding to Candida membranes, but not to sheep or pig luteal LH receptors. This material had a molecular weight of 16 000\p=n-\21 daltons, bound strongly to CM\x=req-\ Sepharose at physiological pH, and could be resolved completely from hCG and from epidermal growth factor-like factors present in commercial gonadotrophin preparations. Its activity was not attenuated by a range of inhibitors specific for the four major classes of proteolytic enzymes, nor did it inhibit hormone binding by causing degradation of 125 I\x=req-\ labelled hLH or hCG tracers. Factors which inhibited hLH binding to Candida membranes were also present

partially purified human urinary and equine serum gonadotrophin preparations and in placental extracts, but were not detected in highly purified CG of hLH preparations. The properties of this factor were similar to those described for \g=b\-coreprotein, a cleavage product of the \g=b\subunit of hCG which is a contaminant of commercial gonadotrophin preparations. Highly purified \g=b\-coreprotein inhibited 125I-labelled hLH binding to Candida membranes, but not to sheep luteal binding sites. Preparations of hCG depleted of inhibitor activity could stimulate adenylate cyclase activity in Candida membranes almost five fold. In contrast, partially purified inhibitor preparations strongly inhibited basal adenylate cyclase activity (to 18% of control levels). These observations suggest that endogenous LH-like factors, perhaps similar to \g=b\-core proteins of hCG, may play a role in the regulation of morphogenesis in Candida species. Journal ofEndocrinology (1991) 128, 139\p=n-\151

INTRODUCTION

Candida. Indeed, we have shown that hyphal growth of Candida albicans (believed to be important in the development of pathogenicity) was stimulated by mammalian steroid hormones and by luteinizing hor¬ mone (LH) (Kinsman, Pitblado & Coulson, 1988). Recently, we have demonstrated the presence of specific, high-affinity binding sites for 125I-labelled human LH (hLH) and human chorionic gonado¬ trophin (hCG) in two species of Candida (Bramley,

000

Candida albicans is a dimorphic, pathogenic fungus which commonly infects the female reproductive tract. The incidence of Candida infection is increased markedly during the third trimester of pregnancy (Odds, 1988), and it has jeen suggested that high levels of steroid and gonadotrophic hormones during late pregnancy may predispose to infection with

in

Menzies, Williams et al. 1990; authors' unpublished observations). Binding sites were specific for hLH and hCG; other radiolabelled hormones tested failed to bind to Candida membranes, and failed to displace binding of l25I-labelled hLH or hCG from Candida binding sites (Bramley et al. 1990; authors' unpub¬

lished observations). LH-binding sites appeared to be involved in germ tube development, and hLH added to Candida albicans yeast cells in culture stimu¬ lated morphogenesis and increased cyclic adenosine

3',5'-monophosphate production. Furthermore, hLH added to Candida membrane fractions rapidly stimu¬ lated adenylate cyclase activity in a dose-dependent fashion (Williams, Dickinson, Kinsman et al. 1990). Enhancement of basal adenylate cyclase activity by the GTP analogue GTPyS, and inhibition of activity by GDPyS, suggested G-protein involvement in the regulation of Candida adenylate cyclase activity and we provided the first demonstration of GTP-binding proteins in Candida (Williams et al. 1990). Scatchard plots of hormone binding to Candida cytosol and membrane fractions demonstrated the presence of two distinct hLH/hCG-binding sites, one with a binding equilibrium constant (KJ of high affinity (01—0-3 litres/pmol) and low capacity, and a second site of lower affinity (Ka, 5-7 litres/nmol) and higher capacity. Low concentrations of partially puri¬ fied gonadotrophin preparations competed for bind¬ ing to Candida membranes and cytosol binding sites at concentrations roughly 20 times greater than those required to inhibit binding to the sheep luteal LH receptor (Bramley et al. 1990, authors' unpublished observations). A range of other proteins, peptides and protease inhibitors failed to compete for these binding

sites. However, we observed that highly purified preparations of hCG or hLH competed poorly for Candida hLH-binding sites compared with partially

purified gonadotrophin preparations (authors' unpub¬ lished observations), suggesting that crude prep¬ arations may contain contaminants (removed in the final stages of purification) which are more potent than purified gonadotrophins in blocking binding

LH-binding sites. We now describe the partial purification and characterization of such a factor from commercial chorionic gonadotrophin to Candida

preparations.

MATERIALS AND METHODS

Materials

Sephadex G-100 (Fine), CM-Sepharose CL-6B and Concanavalin A-Sepharose CL-4B were purchased from Pharmacia Ltd, Milton Keynes, Bucks, U.K. and washed and stored as instructed by the manufacturers.

Hormone

preparations

Preparations of partially purified hCG were pur¬ chased from Intervet Laboratories, Cambridge, Cambs, U.K. (Chorulon; 1500IU hCG/vial), Sigma Chemical Co. Ltd, Poole, Dorset, U.K. (CG-5; 5000 IU hCG/vial), Organon Laboratories Ltd, Maiden, Surrey, U.K. (Pregnyl; 5000IU/vial) and Serono Laboratories (UK) Ltd, Welwyn Garden City, Herts, U.K. (Profasi; lOOOOIU/vial). Human urinary

gonadotrophin preparations, Pergonal (a partially purified mixture of human urinary post-menopausal gonadotrophins containing 75 IU hLH; 75 IU human follicle-stimulating hormone (hFSH)/vial) and Metrodin (a highly purified hFSH preparation containing 75 IU hFSH/vial with very low hLH contamination) were purchased from Serono. Highly purified ovine hormones (NIH-FSH-S14, 75 IU/mg; NIH-PRL-S13, 30-0 IU/mg; NIH-LH-S21, 2-5 IU/ mg) and human growth hormone (NIH-HS-2160E

iodination grade, 32-0 IU/mg) were generous gifts from the Hormone Distribution Officer, NIAMDDK, Bethesda, MD, U.S.A. Highly purified hCG (Cr 119; 11 600 IU/mg) and hCG subunits (Cr 119-la, hCG-a subunit; Cri 19-Iß, hCG-ß subunit) were kind gifts from Dr R. Canfield through the Center for Population Research, National Institute of Child Health and Human Development (NICHHD), NIH, Bethesda, MD, U.S.A. Highly purified human pitu¬ itary FSH (CPDS-33; 2925 IU/mg), human pituitary LH

(hLH-4;

5110 IU/mg) and

polyclonal

rabbit

antisera to the and ß subunits of hCG were gener¬ ous gifts from Dr S. S. Lynch, Women's Hospital,

Birmingham, U.K. The antibody to hCG-a subunit (MHO) was highly specific, with a titre for radio¬ immunoassay (RIA) of 1:5 xlO6, whereas the anti¬ body to hCG-ß subunit (F98: titre for RIA, 1:5 IO5)

cross-reacted somewhat with intact hCG and with subunit at the concentrations (1:100 dilution) used for

immunoblotting. Highly purified hCG-ß-core protein the kind gift of Drs B. C. Nisula and D. Blithe,

was

NICHHD, MD, U.S.A.

Radioiodinated hLH tracer (100Ci/g) was pur¬ chased from Dr . Ferguson, Hammersmith Hospital, London, U.K. Unlabelled and 125I-labelled mouse epidermal growth factor (mEGF) were generous gifts from Dr . Brown, Institute for Animal Physiology and Genetics Research, Babraham, Cambridge, U.K. l25I-Labelled hCG (specific binding activity, 3055 mCi/mg by self-displacement assay) was prepared

by a lactoperoxidase procedure (Miyachi, Vaitukaitis, Nieschlag & Lipsett, 1972).

An extract of hCG was prepared from a pool of early pregnancy urine by benzoic acid extraction. Briefly, urine was filtered, adjusted to pH 4-2 with glacial acetic acid, and saturated benzoic acid (in

acetone; 50 ml/litre urine) was added with stirring and allowed to settle overnight at 4 °C. Benzoic acid was collected by centrifugation, and the cake was extracted with 50 ml acetone. Supernatant fractions were used to re-extract urine, and the extracts were combined. The activity of the extracted hCG prep¬ aration was 100 IU/mg by both bioassay and receptor binding assay (authors' unpublished results). Tissue

preparation

Microsomal fractions of Candida albicans (serotype A; NCPF 3153) were prepared from cells harvested in

the late-exponential growth phase, as described in detail previously (Bramley et al. 1990). Ovine corpus luteum tissue was obtained from ovaries of animals slaughtered at a local abattoir within 2 h of death. Corpora lutea from mid-luteal phase ovaries were dissected, minced with scissors, and homogenized in ice-cold sucrose (0-3mol/l)EDTA

(1 mmol/l)-Tris-HCl (10 mmol/1; pH 7-4) (SET medium; 10 ml/g) using a Poly tron homogenizer (Kinematica, Lucerne, Switzerland) with two 10-s bursts at full speed separated by a 1-min cooling period in ice. After filtration through four layers of cheesecloth, homogenates were stored in 2 ml aliquots

-20°C. Porcine ovaries were collected from a local abattoir and transported to the laboratory within 2 h of slaughter. Ovaries were kept on ice whilst all bloodfree, clear follicles of 2-5 mm diameter were aspirated using a syringe fitted with a fine-gauge needle. Granu¬ losa cells were recovered by centrifugation at 800 g for 10 min, and washed twice in isotonic phosphatebuffered saline, before being resuspended in ice-cold SET medium (10 ml/ml packed cell pellet). Cells were homogenized using 12 complete strokes of an all-glass Dounce homogenizer, and 2 ml aliquots were stored 20 °C until required. Porcine corpora lutea (midat luteal phase of the cycle) were processed as described for ovine corpus luteum tissue. Human placental tissue was obtained from women undergoing elective Caesarian section at full term. Informed consent was obtained from each patient. Placental villi were excised, washed extensively with phosphate-buffered saline to remove blood, then homogenized (Polytron homogenizer) in ice-cold SET medium (10 ml/g) using two bursts at full power (10 s) separated by a 1-min cooling period in ice. Homogenates were filtered through four layers of cheesecloth, then centrifuged at 1000# for 10 min. The pellet was discarded, and the supernatant recen¬ trifuged at 30 000 g for 30 min. The supernatant fraction was then respun at 109 000 g for 60 min (4 °C), and the pellet (microsomes) resuspended in SET by gentle homogenization, and the supernatant at

—

fraction (cytosol) and microsomes aliquots) at 20 °C until required.

were

stored

(2 ml

—

Measurement of specific hormone

binding

Specific binding of l25I-labelled hLH to aliquots of sheep and pig luteal homogenates and to Candida microsomes was measured in triplicate as described in detail previously (Bramley et al. 1990; authors' unpublished observations). Candida membranes were incubated at 4 °C for 30-45 min, and sheep and pig luteal homogenates and membranes for 16-20 h at 20 °C (pH 6-5) with 100 000 c.p.m. of l25I-labelled hLH or hCG. Bound and free hormone were then separated by polyethyleneglycol precipitation, and l25I in the pellet was counted in a Packard Crystal counter at an efficiency of 75%. Non-specific binding was measured in duplicate in the presence of Chorulon (50IU/tube). The difference between binding in the presence and absence of excess Chorulon was taken as specific binding. Non-specific binding accounted for 6-12% of total counts added, and specific binding of >60% of total counts added could be achieved at high ovine luteal or Candida membrane levels (data not

shown).

The specific binding of mEGF tracers was measured as described previously (Bramley & Menzies, 1988). Specific binding activity of EGF tracers was measured by self-displacement assay, with human term placen¬ tal membranes as a receptor source and ranged from 8 to 18 Ci/g. Protein content of membrane fractions was measured by the method of Lowry, Rosebrough, Farr & Randall (1951). Methods for the measurement of the specific binding of other tracers have been described in detail previously: l25I-labelled human growth hormone (hGH) tracer to ovine luteal homogenates (Bramley, Menzies, McNeilly & Friesen, 1987), radiolabelled gonadotrophin-releasing hormone (GnRH) agonist

(buserelin)

Menzies &

to rat

pituitary homogenates (Bramley,

Baird, 1986), and l25I-labelled hFSH

porcine granulosa etal. 1987).

cells

(Bramley, Stirling,

to

Swanston

Concentrations of unlabelled hormones required reduce specific hormone binding by 50% (IC50) were calculated from regression analysis of compe¬ tition curves performed over 5-6 log orders of concentration. to

Measurement of Candida

adenylate cyclase activity Adenylate cyclase activity was measured by a modifi¬

cation of the method of Salomon, Londos & Rodbell (1974), method C, using Pipes buffer (50 mmol/1; pH 6-5) instead of Tris and l-methyl,3-isobutyl xanthine (1 mmol/1). Incubation was for 30 min at 37 °C.

Chromatography

permeation chromatography was performed using a 1 45 cm column of Sephadex G-100, eluted with 0-2 mol ammonium bicarbonate/1 buffer, pH 7-8. Partition coefficients (Kav) were calculated by the ratio of ( Ve VQ) (elution volume of the unknown ( Ke) minus void volume of the gel (V0) to (Vt— V0) (total volume of the gel ( Vt) minus void volume of the gel Gel

—

volume of the Blue (mol. wt 2 106) and 125I-labelled sodium iodide respectively. A number of standard proteins (bovine serum albumin, bovine -globulin, cytochrome C, l25I-labelled hCG and 125I-labelled ovine prolactin) eluted with Kav values consistent with their molecular weights (data

(V0)). The void volume and total gel were estimated using Dextran

not

shown).

Ion

exchange chromatography Fractions from Sephadex G-100 chromatography which were enriched in Candida LH-binding inhibitor activity were pooled, lyophilized, reconstituted in

distilled water, then stirred at 20 °C for 2 h with 15 ml well-washed, packed CM-Sepharose. The gel was packed into a 20 ml plastic syringe, then eluted sequentially with distilled water, Tris-HCl buffer (40 mmol/1; pH 7-4), then a gradient of NaCl (from 0 to 0-3 mol/1 in Tris buffer) and finally a salt gradient from 0-3 to 1-0 mol/1 in Tris buffer. Fractions (2 ml) were collected, and assayed for inhibition of binding, as described in the figure legends. The osmolality of each fraction was measured using a vapour pressure osmometer (model 5100S; Wescor Ine, Hornchurch,

Essex, U.K.).

A-Sepharose chromatography Fractions from Sephadex G-100 which were enriched in Candida inhibitor (or hCG) were mixed with 1 ml well-washed, packed Concanavalin A-Sepharose gel (20 °C for 90 min). After packing into the barrel of a 2 ml syringe, the gel was eluted sequentially with 40 mmol Tris-HCl/1-0-05% (w/v) bovine serum albumin (BSA), 0-5 mol NaCl/1 in Tris-BSA and 0-2 mol -methylmannoside/l in NaCl-Tris-BSA buffer. Fractions (1 ml) were collected and assayed for inhibition of l25I-labelled hLH binding to aliquots of ovine luteal homogenate and Candida membranes. Concanavalin

Electrophoresis Sodium dodecylsulphate-polyacrylamide gel electro¬ phoresis (SDS-PAGE) was performed on 7-15% SDSPAGE gels (pH 8-8) with a 3-6% stacking gel (pH 6-8). Aliquots of hCG and gel chromatography fractions (50 pi) were applied in sample buffer (0-374 mol Tris/1, 2% (w/w) SDS, 10% (w/w) sucrose with tracking dye). Aliquots for immunoblotting were first

by boiling for 3 min in sample buffer con¬ 2-mercaptoethanol (2%, v/v). Gels were run taining at 40 mA/gel during stacking, then 25 mA/gel for the reduced

remainder ofthe run. Gels were stained with Coomassie Brilliant Blue 250, or proteins were transferred electrophoretically (Novablot apparatus; Pharmacia LKB Ltd, Milton Keynes, Bucks, U.K.; 0-8mA/cm2 gel) to nitrocellulose membranes overnight. Membranes were then incubated for 1 h at room temperature in 3% (w/v) gelatin in Tris (0-3 mol/l)-NaCl (0-5 mol/1), pH 7-5 (GTS buffer) to block non-specific binding, rinsed, then incubated for 4 h in 1 % gelatin in Trissaline buffer containing antibody to hCG-a (MHO) or hCG-ß (F98) diluted 1:100. After briefly rinsing in distilled water, the membranes were washed with two 10-min washes of GTS buffer containing 0-05% (w/v) Tween 20. Membranes were then incubated for 1 h with sheep anti-rabbit IgG linked to horseradish per¬ oxidase (1:3000 dilution in 1% gelatin-Tris-saline buffer; BioRad Ltd, Hemel Hempstead, Herts, U.K.), and washed twice more in Tween 20-GTS buffer, then developed at room temperature for 15 min in BioRad colour development solution (60 mg horseradish per¬ oxidase colour development reagent, 20 ml methanol, 60 pi H,02 in 100 ml GTS buffer). Development was stopped by transfer to distilled water. RESULTS

Fractionation of commercial hCG

preparations Gel permeation chromatography of a partially purified preparation of hCG (Chorulon; 9000 IU) on Sephadex G-100 resolved a single sharp peak which was identified as hCG since: (i) it eluted with the same value as both highly purified hCG standards and 12T-labelled hCG (data not shown); (ii) it inhibited binding of both hLH and hCG tracers to sheep luteal LH receptors (Fig. 1) and (iii) it immunoblotted against antisera to hCG-a and hCG-ß subunits on reduced SDS-PAGE gels (Fig. 2). When eluted frac¬ tions were tested for their ability to inhibit 125Ilabelled hLH/hCG binding to Candida membranes, a broad major peak of activity with a Km of 0-6 was observed which was clearly resolved from hCG (although a minor peak of inhibitory activity chromatographed in the same regions as hCG (Fig. 1)). The factor inhibited the binding of both hLH and hCG tracers to Candida microsomes. However, specific binding of l25I-labelled GnRH agonist to rat pituitary GnRH receptors, of hFSH to pig granulosa cell recep¬ tors, and specific binding of hGH to ovine luteal lactogenic receptors was unaffected by incubation with fivefold higher levels of inhibitor fractions than those which decreased 125I-labelled hLH binding to Candida by >90% (data not shown).

c

120

S

100

g> 80 60 c

4020

Q.

10

20 30 Fraction number

40

50

1. Gel permeation chromatography of Chorulon on Sephadex G-100. Chorulon (9000 IU) was dissolved in 1 ml ammonium bicarbonate buffer (40 mmol/1; pH 7-8) and applied to a 1 45 cm column of Sephadex G-100. Fractions (1 ml) were collected at a flow rate of 5 ml/h, and frozen at 20 °C until assayed. Aliquots of each fraction were incu¬ bated in triplicate in a 1 ml system containing Tris-acetate buffer (40 mmol/1, pH 6-5) bovine serum albumin (0-5 g/ 100 ml), hormone tracer (100 000 c.p.m./tube of i:5I-labelled hLH (circles) or 125I-labelled hCG (triangles)) and either Candida microsomal fraction (0-5 mg protein/tube; solid symbols; 5 µ fraction/tube) or sheep luteal homogenate (0-2 mg protein/tube; open symbols; 1 µ fraction/tube). After incubation for 30 min at 4 °C (Candida) or 16 h at 20 °C (sheep), bound and free hormone were separated by polyethyleneglycol precipitation, supernatants were aspirated and pellets counted for 125I by gamma counting at an efficiency of 75%. Points shown are means of triplicate determinations. Similar profiles were obtained in several separate experiments. figure

—

SDS-PAGE of highly purified hCG (Cr 119, Fig. 2b; lane 1) under non-reducing conditions demon¬ strated a band which stained faintly with Coomassie Blue (Mr 46 000) corresponding to hCG. In contrast, the partially purified preparation of hCG used as the starting material for fractionation on Sephadex G-100 (Fig. 2b, lane 2) demonstrated the presence of a number of additional bands which stained strongly

with Coomassie Blue with Mr values of 24 000, 21 000 and 16 000. Fractions from the peak of inhibition of LH binding to sheep luteal receptors from Sephadex G-100 chromatography of partially purified hCG (Fig. 2a; fractions A and B) were enriched in hCG (band not well visualized because of poor contrast with Coomassie Blue), with little contamination by the lower Mr components (Fig. 2b; lanes A, B). How¬ ever, fractions from the peak of inhibition of 125Ilabelled LH binding to Candida membranes (Fig. 2a; fractions C, D, E and F) were enriched in the lower Mr components, but showed minimal hCG content (Fig. 2b; lanes C, D, E and F).

Under reducing conditions, highly purified hCG showed two bands, with Mr values of 23 000 and 32 000 respectively (Fig. 2c; lane 1). Immunoblotting of identical SDS-PAGE gels with antisera specific for the subunit (Fig. 2d) or the ß subunit of hCG (Fig. 2e) identified the 23 000 band as the subunit (Fig. 2d; lane 1) and the 32 000 band as the ß subunit (Fig. 2e; lane 1). Under reducing conditions, partially purified hCG also showed bands corresponding to the subunits of hCG (Fig. 2c,d,e; lane 2). Intense bands were also present at 21 000 and 16 000 (Fig. 2c; lane 2). Fractions A and (Fig. 2a) were enriched in hCG subunits (Fig. 2c,d,e; lanes A and B) with little contamination by lower Mr components. However, fractions D, E and F were enriched in the 21 000 com¬ ponent (Fig. 2c; lanes D, E and F), but had little or no hCG or hCG subunits (Fig. 2d,e; lanes D, E and F). Inhibitor fractions from Sephadex G-100 chromato¬ graphy of a commercial hCG preparation (Chorulon; Fig. 1; fractions 18-24) were pooled, lyophilized and applied to CM-Sepharose. Step-wise elution with buffers of increasing ionic strength completely resolved the inhibitor of LH binding to Candida from any remaining hCG (Fig. 3). A major peak of hCG was observed in the material which was recovered in the distilled-water wash, representing material not adsorbed by the gel. This material inhibited l25Ilabelled hLH/hCG binding to sheep and pig luteal LH receptors (Fig. 3a), and immunoblotted against antisera to hCG-a and hCG-ß subunits on SDS-PAGE (data not shown). A second minor peak of hCG was eluted by Tris-HCl buffer alone, as judged by the inhibition of LH binding to sheep and pig luteal LH receptors (Fig. 3a) and immunoblotting against hCG subunits (not shown). The proportion of material in this minor peak and its elution characteristics varied between preparations. Material in these two peaks had only weak activity when tested for inhibition of hLH/hCG binding to Candida membranes. In con¬ trast, a broad major peak of Candida LH-binding inhibitor was eluted as the ionic strength of the elution buffer increased (Fig. 3b). Fractions from this peak did not immunoblot against antisera to hCG subunits (data not shown) and did not inhibit binding to sheep and pig luteal LH receptors (Fig. 3a). Factors with EGF-like

preparations

activity present in crude hCG

The presence of factors with EGF-like activity in commercial preparations of hCG has been described (Knox, Reynolds, Cohen & Alford, 1978; Hofmann, Holzel, Hackenberg & Schulz, 1989). EGF-like activity was present in unfractionated crude hCG preparations (Chorulon), but a highly purified preparation of hCG (Profasi) failed to compete for placental EGF-binding

(ß)

[125]hLH bound (c.p.m.

x

10~3)

AB C DEF

120-

100-

60—

40-

3

O

.2

J

20-

10

20 Fraction

30

40

no.

figure 2. SDS-PAGE separation of fractions from Sephadex G-100 chromatography of Chorulon. Aliquots of fractions obtained by chromatography of Chorulon (9000 IU) on a 1 45 cm column of Sephadex G-100 were assayed for (a) inhibition of 125I-labelled hLH binding to Candida membranes (·) or to sheep corpus luteum (O) or were taken for SDS-PAGE under (b) non-reducing or (c.d.e) reducing conditions on 7-15% polyacrylamide gels. Gels were stained with Coomassie Blue (b,c) or were immunoblotted using antisera to the subunit (d) or the ß subunit (e) of human chorionic gonado¬ trophin (hCG) diluted 1:100. The mobilities of hCG (right-hand side) and a range of molecular weight markers used to calibrate the gels (left-hand sides) are also shown. Lanes marked A-F in (b-e) correspond to fractions A-F shown in (a).

sites

(Table 1). Because of the lack of activity in Profasi, and because the EGF-like activity eluted from

Sephadex G-100 with an elution volume (data shown) similar to that of the Candida LH inhibitor (Fig. 1), we considered the possibility that not

this EGF-like molecule may inhibit hLH binding to Candida non-competitively. However, although these activities eluted similarly on gel permeation chromato¬ graphy, EGF-like activity and Candida LH-binding inhibitor were resolved completely by chromatography

CM-Sepharose (Fig. 3c). EGF-like activity was recovered quantitatively in the fractions which failed to adsorb to CM-Sepharose; no EGF-like activity being detectable in fractions with maximal Candida on

120

LH-binding inhibitory activity. Thus, inhibition of LH binding to Candida cannot be attributed to EGF-like contaminants of crude gonadotrophin

100 80

preparations.

60 40

20

0

a

Contamination of the crude hCG protease (Mr 14 000-20000) could

degrade hormone

tracer

120

or

protease

preparation by

a

receptor, leading to an apparent inhibition of

125I-labelled hLH and hCG binding to Candida mem¬ branes. However, fractions with maximal inhibitory

100

activity against Candida LH-binding sites did not inhibit binding to ovine luteal receptors, despite the much longer duration and higher temperature of incubation (see Fig. 1). Moreover, similar levels of inhibition of l25I-labelled hLH binding were observed whether Candida membranes were preincubated with

80

60-1 40

20

0 120

Candida LH-binding inhibitor is not

(c) 1-2

100

-1-0 0-8

80 60

ç.

g

0-6 r 0-4 I

40200-

10

20 30 40 Fraction number

50

0-2 0

3. Fractionation of inhibitor from Sephadex G-100 chromatography on CM-Sepharose. Fractions from the peak of Candida hLH-binding inhibitory activity following Sephadex G-100 chromatography of Chorulon (9000 IU; fractions 18-24, Fig. 1) were pooled and freeze-dried, then figure

reconstituted in 3 ml distilled water and mixed with CMSepharose gel. The gel was then packed into a 20 ml plastic syringe (10 ml gel) and washed with distilled water, TrisHCl buffer (40 mmol/1; pH 7-4) then Tris-HCl buffer containing a sodium chloride gradient running from 0 to 0-3 mol/1, and finally Tris-HCl buffer containing a sodium chloride gradient running from 0-3 to 1 mol/1. Media changes are indicated by the arrows. The osmolality of each fraction (2 ml) was measured using an osmometer (c) (O). Aliquots of each fraction (5 µ ) were assayed for the ability to inhibit the specific binding of 125I-labelled hLH to (a) mammalian LH receptors (ovine corpora lutea, O; porcine corpora lutea, ·) or (b) Candida membrane fractions (O) and (c) inhibition of 125I-labelled mouse epidermal growth factor binding to human placental membranes (50 µ fraction/tube; ·). Values shown are means; s.e.m. values were always less than 10% of specific binding levels. Similar profiles were obtained in a number of other fractionation

experiments.

inhibitor for 30 min or 16 h at 4 °C; furthermore, incubation of l25I-labelled hLH with fractions which inhibited hLH binding to Candida membranes by >90% had no effect on the integrity of the tracer on Sephadex G-100 (data not shown). Finally, addition of a cocktail of inhibitors specific for the four major classes of proteases (phenylmethylsulphonyl fluoride,

N-ethyl maleimide,

EDTA and pepstatin) had no the inhibition of l25I-labelled hLH/hCG binding to either sheep luteal or Candida LH-binding sites by Chorulon fractions (data not shown). Thus, inhibition of hLH binding to Candida does not appear to be due to proteolysis of hormone tracers or binding sites.

effect

on

Properties of the Candida LH-binding inhibitor Incubation of inhibitor preparations at different tem¬ peratures for 30 min showed that the factor was stable at

temperatures up

to 60

°C, but labile

at 80 °C

or

shown). Incubation at 37 °C with trypsin, papain, collagenase-dispase or pronase failed to decrease inhibitory activity significantly (data not shown). Fractions enriched in hCG adsorbed strongly to Concanavalin A-Sepharose, and hCG was eluted only by buffers containing -methyl mannoside (Fig. Ab). In contrast, although fractions enriched 100 °C

(data

not

in Candida LH-binding inhibitor also adsorbed to Concanavalin A-Sepharose, inhibitor was eluted by 0-5 mol NaCl/1 alone (Fig. Ad). Presence of LH-binding inhibitor in other commercial

gonadotrophin preparations Inhibition of binding of 125I-labelled hLH to both Candida and ovine luteal LH-binding sites (and of

1. Competition for Candida l25I-labelled hLH binding sites and placental l25I-labelled epidermal growth factor (mEGF) binding sites by gonadotrophin preparations. The concentrations required to inhibit specific l25I-labelled hLH and 125I-labelled mEGF by 50% are shown (ng or IU*). Values are means ± s.e.m. for 2-12 separate determinations in triplicate (figures in parentheses) table

"I-labelled mEGF

-labelled hLH

Sheep luteal receptor Chorionic hormones Chorulon* Sigma CG-5*

Pregnyl*

Profasi* Equine CG (PMSG)*

Pituitary/urinary hormones Pergonal* Metrodin* hLH-4 hFSH-CPDS 33

Candida albicans microsomes

4±3

(3)

1-2 + 0-2 5-4 + 0-3 3-2 + 0-5 >1000 12 + 4

0-45 ±0-2 > 100 9-2 + 60 >1000

(4) (4) (3) (2)

1-2 + 0-3 0-5 + 0-2 > 10000 >1000

0-03±0-01 (12) 0-04 ±0-02 (3) 003 ±001(4) 006 ±0-02 (4)

Human placenta

(8) (4) (7) (5) (3)

8+3 n.d. n.d.

(6)

>100(2) >100(3)

(4)

(4) (3)

(5)

>100(2)

n.d.

n.d. n.d.

Aliquots of Candida albicans (serotype A;NCPF3153) microsomes (2—10 µ ) were incubated (4 X for 30 min) in 1 ml BSA-Tris buffer, pH 6-5, containing 100 000 c.p.m. of l35I-labelled hLH, in the absence or presence of increasing concentrations of unlabelled gonadotrophin preparations, n.d., Not done.

125I-labelled EGF to human placental receptors) was measured for a number of highly purified and par¬ tially purified hCG preparations over a wide range of dilutions, and concentrations required to inhibit specific binding by 50% were calculated (Table 1). All hCG preparations inhibited binding to sheep corpus luteum with high potency. Highly purified hCG (Profasi) did not inhibit binding to Candida mem¬ branes; however, partially purified hCG preparations inhibited binding to Candida membranes in a dosedependent fashion (Table 1). l25I-Labelled hLH binding to Candida was also inhibited by pregnant mare serum gonadotrophin (equine CG) and by Pergonal (a human post-menopausal urinary gonadotrophin preparation with equal activities of hLH and hFSH). Highly purified pituitary hLH and hFSH prep¬ arations failed to inhibit hLH binding to Candida (though the hLH preparation was a potent inhibitor of 125I-labelled LH binding to ovine luteal LH recep¬ tors; Table 1). However, Metrodin (a purified urinary

hFSH shown

preparation with little LH-like activity, as by its lack of inhibition of hLH binding to sheep luteal LH receptors; Table 1) had significant activity against Candida LH-binding sites. Chorulon (but not Profasi, Pergonal and Metrodin) also had significant EGF-like activity (Table 1). Gel permeation chromatography of various par¬ tially purified hCG preparations demonstrated the presence of inhibitory activity against Candida hLHbinding sites (Fig. 5a,b,c) which eluted with KdV values (0-6) distinct from that of hCG (Km, 0-2). In contrast, the highly purified hCG preparation Profasi had little or no activity against Candida membranes (though

fractions eluting at a ATav of about 0-2 strongly inhibited LH binding to sheep luteal receptors) and contained little EGF-like activity (Fig. 5a"). Gel permeation chromatography of the human

menopausal gonadotrophin preparation Pergonal (Fig. 6a) demonstrated the presence of hLH (putatively identified by the peak of inhibition of hLH binding to sheep luteal LH receptors; Km, 0-25), and a factor eluted later than hLH which had little inhibitory activity in the sheep luteal system, but which strongly inhibited

binding

to

Candida membranes. Further¬

more, an extract of human pregnancy urine contained (in addition to hCG) a peak of activity which strongly inhibited binding to Candida LH-binding sites, eluting

with a ATav of 0-6 (Fig. 6b), along with high levels of EGF-like activity (as judged by inhibition of 125Ilabelled mEGF binding to placental binding sites). The presence of Candida LH-binding inhibitor in crude hCG preparations (Figs 1, 5) and in gonado¬

trophins isolated from urine extracts (Fig. 6a,b) suggested that the inhibitor may be an excretory product, only found in urinary materials. However, equine serum chorionic gonadotrophin fractions (Fig. 6c) and fresh human placental cytosol frac¬ tions also demonstrated the presence of the Candida LH-binding inhibitor (Fig. 6d). Effects of partially purified LH-binding inhibitor Candida adenylate cyclase activity

on

Fractions enriched in hCG or Candida LH-binding inhibitor were incubated with Candida membranes and adenylate cyclase activity was measured. The

120-

cleaved and nicked ß-subunits (Taliadouros, Amir, Louvet et al. 1982; Wehmann, Blithe, Akar & Nisula, 1988; Puisieux, Bellet, Troalen et al. 1990). The major processed form of hCG found in pregnancy urine is ß-core protein (Wehmann & Nisula, 1980). Binding of l25I-labelled hLH to sheep luteal receptors (Fig. 7a). was inhibited by low doses of purified hCG, but was unaffected by ß-core protein. In contrast, hLH bind¬ ing to Candida membranes was inhibited in a dosedependent fashion by ß-core protein (half-maximal inhibition, 60 ng/tube), but was unaffected by highly purified hCG (Fig. lb).

()

100 80

60

40-1 20 0 120

(b)

—

-2
). Gel permeation chromatography of Candida cytosol fractions resolved two peaks of binding with approximate molecular weights of > 106 and 30 00050 000 (authors' unpublished observations). The higher molecular weight fraction showed a single, high affinity hLH-binding site by Scatchard analysis, whereas the lower molecular weight peak consisted of a mixture of both high and low affinity sites. In the course of these studies, we observed that partially purified preparations of hLH and hCG competed at lower

doses than highly purified gonadotrophins, suggesting that crude urinary hCG preparations contained a factor which could compete for Candida LH-binding sites more effectively than purified hLH or hCG (authors' unpublished observations). We have now described the partial purification of a factor from commercial hCG preparations which inhibits LH binding to for Candida

LH-binding sites

Candida. Gel permeation chromatography of partially purified hCG preparations resolved hCG from the Candida binding inhibitor. hCG standard and 125Ilabelled hCG chromatographed with the same elution volume as the major peak which inhibited binding of hLH and hCG tracers to sheep luteal LH receptors (Figs 1,2d). In contrast, the factor which inhibited LH binding to Candida membranes eluted much later than 125I-labelled hCG or hCG standard (Fig. 1), and was

120-1

10080

60-1

o o s-

o

40

£-

20

o

120

O

e

(c)

(d)

3

2

íoo

I

80

ac

60-1

o

40

20

0

io

30

20

0 40 Fraction number

10

20

30

40

5. Gel permeation chromatography of human chorionic gonadotrophin (hCG) preparations. Aliquots of hCG preparations were dissolved in ammonium bicarbonate buffer (0-1 mol/1; pH 7-8) and applied to a precalibrated 1 45 cm column of Sephadex G-100. Fractions (1 ml) were eluted at a flow rate of 5-8 ml/h, and aliquots were tested for inhibition of l25I-labelled hLH binding to ovine luteal LH receptors (O; 1-5 µ /tube) and to Candida microsomes ( · ; 5-40 µ /tube). Points are means ± s.d. for separate fractionation experiments in triplicate and are expressed as percentage of specific binding of controls, (a) Chorulon (9000 IU applied; 6); (b) Pregnyl (9000 IU applied; 3); (c) Sigma CG-5 (1000 IU applied; 2); (d) Profasi (1000 IU applied; « 3). In (d) the triangles indicate lack of inhibition of 125I-labelled mouse epidermal growth factor binding to human placental membranes (50 µ /tube) by Profasi preparations. figure

=

=

=

=

associated with a contaminant(s) of crude hCG prep¬ arations with Mr values of 21 000 and 16 000 (Fig. 2b,c) which did not immunoblot against antisera to hCG subunits (Fig. 2a». Furthermore, whereas hCG was readily eluted from CM-Sepharose by low ionic strength buffers, the inhibitor of Candida binding was strongly adsorbed, and could only be eluted by high ionic strength buffer (Fig. 3b). Moreover, the Candida LH-binding inhibitor activity bound to Concanavalin A-Sepharose (indicating that it was glycosylated; Fig. 4a), but in contrast to hCG (which bound strongly to Concanavalin A-Sepharose gels and was eluted only by -methylmannoside; Fig. Ab), inhibitor was eluted by high salt only.

Partially purified preparations of chorionic gonado¬ trophin have been shown to possess EGF-like activity (Knox et al. 1978; Hofmann et al. 1989). We con¬ firmed these observations, demonstrating that differ¬ ent Chorulon preparations contained between 200

and 800 ng mEGF

per vial of hCG have demonstrated the presence of two EGF-like components in urinary hCG extracts (Mr, 30 000 and 14 000; Fig. 6b). These values are approximate, but compare fairly well with the molecular weights of EGF-like components in crude hCG preparations (71000, 52 000, 11-12000 and 7000) reported by Hofmann et al. (1989). The smaller EGF-like component appeared to be the major form, and had a similar elution volume on Sephadex G-100 to the Candida LH-binding inhibitor (Fig. 6 ). Furthermore, the highly purified hCG prep¬ aration Profasi failed to inhibit l25I-labelled hLH binding to Candida LH-binding sites and failed to inhibit binding of 125I-labelled mEGF to placental EGF receptors (Fig. 5d), raising the possibility that this EGF-like factor was responsible for the inhibition ofLH binding to Candida. However, EGF-like activity could be resolved completely from the Candida LH-

equivalents

(1500 IU). Moreover,

we

40 0 Fraction number figure an

6. Gel permeation chromatography of a human post-menopausal urinary gonadotrophin preparation,

extract of human pregnancy urine, equine serum gonadotrophin and human term placental extract.

Aliquots

of (a) a human post-menopausal urinary gonadotrophin preparation Pergonal (300 IU applied) or (c) pregnant mare serum gonadotrophin (PMSG; equine CG; 5000 IU applied), (b) a human term placental extract ( 15 000 IU hCG applied) and (d) a human term placental cytosol preparation ( 1533 IU hCG equiv. applied) were chromatographed on a 1 45 cm precalibrated column of Sephadex G-100. Fractions (1 ml) were collected, and aliquots assayed for inhibition of 125I-labelled hLH binding to ovine luteal LH receptors ( O ; 0-5-3 µ fraction/tube) and Candida LH-binding sites (·; 5-50 µ fraction/tube), (b) Aliquots were also taken for inhibition of 125I-labelled mouse epidermal growth factor binding to human placental membranes ( ; 10 µ fraction/tube). Points are means ± s.d. for 1-6 separate fractionation experiments in triplicate and are expressed as a percentage of specific binding to controls.

table

2. Effects of Candida inhibitor fractions on adenylate s.d. for three complete

cyclase activity. Values are means ± experiments performed in triplicate

Adenylate cyclase activity Additions Control Peak I Peak III

10 4-8 + 0-2* 0-18 + 0-02*

*f90%) failed to inhibit binding of GnRH agonist to pituitary receptors, hGH to ovine luteal receptors, or hFSH binding to porcine granulosa cell receptors (data not shown). Although fractions from gel permeation chromatography enriched in Candida LH-binding inhibitor also inhibited EGF binding to placental EGF receptors (Fig. 6b), the inhibitor of Candida LH binding and EGF-like factors were quite distinct, and readily resolved by ion exchange chromatography (Fig. 3b,c). Finally, incubation of inhibitor fractions with a cocktail of inhibitors specific for the major classes of protease failed to abolish its activity (data not shown).

proteases.

The Candida inhibitor was adsorbed by Concanava¬ lin A-Sepharose indicating that it was a glycoprotein, but was eluted by moderate salt concentrations (Fig. 4a). In contrast, hCG could only be eluted by high salt solutions containing -methyl mannoside (Fig. Ab). ß-Core protein is known to be a glycoprotein (Wehmann & Nisula, 1980; Schroeder & Halter, 1983) which contains the two asparagine-linked carbo¬ hydrate sequences at residues 13 and 30 of the hCG-ß subunit, but lacks O-linked side chains. ß-Core protein binds less strongly to lectin-sepharose affinity columns than hCG, because of its lower carbohydrate content.

Finally, highly purified ß-core protein

inhibited l25I-labelled hLH binding to Candida mem¬ branes, but had no effect on ovine luteal LH binding (Fig. 7). This mimicked the activity of the LH-binding inhibitor described herein (see Figs l^t). Experiments are in progress to establish unequivocally identity between the Candida LH-binding inhibitor protein and hCG-ß-core protein (or to nicked forms of hCG) by their cross-reactions with a panel of specific mono¬ clonal and polyclonal antibodies to hCG subunits and ß-core protein and by determination of the amino acid sequence of our inhibitor. Addition of fractions enriched in hCG, but depleted in LH-binding inhibitor to Candida membranes stimu¬ lated adenylate cyclase activity by almost fivefold, extending previous observations that human gonado¬ trophin preparations could activate Candida cyclase activity (Williams et al. 1990). Interestingly, addition of fractions enriched in Candida LH-binding inhibitor suppressed basal adenylate cyclase by >80%, raising the possibility that a component of

adenylate

'basal' adenylate cyclase activity may be due to acti¬ vation of cyclase by an endogenous LH-like molecule. It will be of interest to study the specificity and mech¬ anism of action of the inhibitor and, in particular, to establish whether it can antagonize LH-stimulated adenylate cyclase activity and germ tube formation. If so, it may then be possible to engineer specific inhibi¬ tors which would be of use clinically for the control of candidosis.

Kato, Y. & Braunstein, G. D. (1988). ß-Core fragment is a major

ACKNOWLEDGEMENTS

Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193,265-274.

form of immunoreactive urinary chorionic gonadotropin in human pregnancy. Journal of Clinical Endocrinology and Metabolism 66,1197-1201. Kinsman, O. S., Pitblado, K. & Coulson, C. J. (1988). Effect of mammalian steroid hormones and luteinizing hormone on the germination of Candida albicans and implications for vaginal Candidosis. Mycoses 31,617-626. Knox, G. E., Reynolds, D. W., Cohen, S. & Alford, C. A. (1978). Alteration of the growth of cytomegalovirus and Herpes simplex virus type I by epidermal growth factor, a contaminant of crude human chorionic gonadotropin preparations. Journal of Clinical

Investigation 11,1635-1644.

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