Vol. 29, No. 8JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1991, p. 1610-16150095-1137/91/081610-06$02.00/0Copyright C) 1991, American Society for Microbiology

43-Kilodalton Glycoprotein from Paracoccidioides brasiliensis:Immunochemical Reactions with Sera from Patients with

Paracoccidioidomycosis, Histoplasmosis,or Jorge Lobo's Disease

ROSANA PUCCIA AND L. R. TRAVASSOS*

Disciplina de Biologia Celular, Escola Paulista de Medicina,Rua Botucatu 862, Sao Paulo, SP 04023, Brazil

Received 10 September 1990/Accepted 7 May 1991

Sera from patients with paracoccidioidomycosis (PCM), histoplasmosis (HP), or Jorge Lobo's disease (JL)were titrated against purified gp43 from Paracoccidioides brasiliensis by using both enzyme-linked immuno-sorbent assay (ELISA) and immunoprecipitation (IPP) reactions with 1251-labeled antigens. In IPP, PCM seraand other sera could be distinguished on the basis of serum titers, whereas in ELISA, 53% of the HP sera and29% of the JL sera reacted similarly to the PCM sera. To investigate the possible role of the carbohydrateepitopes in these reactions, we compared the reactivities of sera from several patients with native anddeglycosylated gp43. Competition experiments were carried out with monosaccharides as inhibitors. Theresults suggest that >85% of the reactions of the PCM sera with gp43 involved peptide epitopes. Cross-reactions with HP and JL sera in ELISA were predominantly attributed to periodate-sensitive carbohydrateepitopes containing galactosyl residues. HP and JL sera which reacted strongly with gp43 in ELISA were onlyweakly reactive or did not react in IPP with labeled antigens in solution. Moreover, ELISA reactions could besignificantly inhibited either by monosaccharides or by periodate treatment. Apparently, carbohydrateepitopes in gp43 are more accessible to the antibodies when the molecule is bound to a plastic substrate thanwhen it is in solution. Structural changes in the gp43 antigen arising by N deglycosylation abolish reactivitywith PCM sera and support the existence of conformational peptide epitopes.

Human paracoccidioidomycosis (PCM) is a deep mycosiscaused by Paracoccidioides brasiliensis, a dimorphic funguswhich grows in the mycelial phase at room temperature andin the yeast phase at 35 to 37°C or in infected tissues. Theimmunological test currently used for the diagnosis of thismycosis is based on a double immunodiffusion (ID) testrecently standardized by Camargo et al. (2). By using ID andexoantigens from 7-day-old cultures in the yeast form, 97%sensitivity with 100% specificity was obtained for the diag-nosis of PCM. This is in contrast with other immunologicalmethods, such as complement fixation (10, 16, 20) andenzyme-linked immunosorbent assay (ELISA) (1, 4, 14),with unfractionated reagents. With these methods, cross-reactions with sera from patients with other deep mycoses,such as histoplasmosis (HP), Jorge Lobo's disease (JL),candidiasis, and blastomycosis, frequently occur.

Previously (17), we identified the antigenic molecule re-sponsible for both the band 1 specific immunoprecipitation(IPP) in ID (19), and the arc E precipitation band formed inimmunoelectrophoresis (27). This antigen, called gp43, is aconcanavalin A-binding glycoprotein of 43,000 Da whichwas isolated from the supernatant fluid of yeast cultures byaffinity chromatography.

Later reports (2, 3) confirmed the specificity of gp43 forthe diagnosis of PCM by ID and Western blotting (immuno-blotting) with a greater number of sera. Previous results fromour laboratory (18, 25) showed that sera from patients withsystemic mycoses other than PCM also reacted with theaffinity-purified gp43 in an ELISA format, sometimes withhigh titers. We therefore started analysis of the gp43 mole-

* Corresponding author.

cule to search for P. brasiliensis-specific and cross-reactingepitopes and to determine the reactivity of this molecule indifferent immunological assays.

In the present work, we studied gp43 reactivity with serafrom patients with systemic mycoses, focusing on the pos-sible role of carbohydrate epitopes. The gp43 isolated byaffinity chromatography usually copurified with smallamounts of both a high-molecular-weight (high-MW) com-plex, which included a galactomannan component (17), anda 72-kDa glycoconjugate (3), as detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Further purification with gel filtration provided a highlypurified gp43 antigen. The gp43 antigen was used either inthe native state or deglycosylated to form a 38-kDa polypep-tide. Several sera from patients with PCM, HP, or JL werestudied by ELISA and liquid-phase IPP reactions, using thepurified gp43 and deglycosylated preparations of this anti-gen.

MATERIALS AND METHODSSera. The 50 PCM sera and 32 HP sera used were from

Brazil, Venezuela, Argentina, and the United States. Eachserum was checked for specificity via ID with P. brasiliensisand Histoplasma capsulatum exocellular antigens (2). PCMand HP sera with high titers in ID tests were used as positivecontrols. All patients exhibited various clinical forms of thedisease and were either untreated or in different stages ofantifungal treatment. The 27 JL sera were from clinicallydiagnosed Brazilian Indians from the Amazon region(Xingu).

Antigen preparation. Cultures of P. brasiliensis B339,from the Mycology Section, CBI-Hospital Pablo Tobon

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43-kDa ANTIGEN OF P. BRASILIENSIS 1611

Uribe, Medellin, Colombia, were grown in a liquid oragar-solidified complex medium enriched with tomato juicemedium. This medium was modified from the commercialUniversal Beer Agar medium (GIBCO Laboratories), whichwas originally used for the selection of Saccharomycesstrains. It consisted of 240 ml of gauze-filtered homogenizedpulp from fresh, peeled red tomatoes supplemented with 6.1g of yeast extract, 16.1 g of glucose, 15 g of casein peptone,0.31 g of K2HPO4, 0.31 g of KH2PO4, 0.12 g of MgSO47H20, and 0.006 g (each) of MnSO4. H20, NaCl, andFe2SO4 and made up to 1 liter with distilled water. The pHwas adjusted to 6.3. For the solid medium, 15 g of agar perliter was added. For preparation of the liquid medium, theyeast extract was first dialyzed in 500 ml of distilled waterovernight at 4°C. The dialysate, free of soluble yeast man-nan, was used to dissolve the other medium constituents andto dilute the tomato juice. The medium was sterilized byautoclaving at 120°C for 15 min. For preparation of theexocellular antigen, the cell mass from 3 to 4 slanted culturesin solid tomato juice medium was inoculated in 100 ml ofliquid tomato juice medium and incubated for 4 to 5 days at35°C. The resulting culture in the yeast phase was trans-ferred to fresh medium (1 liter) and further incubated for 5 to10 days. Cultures were then killed with 0.02% (wt/vol)merthiolate and filtered through filter paper (Klabin 80 filterpaper; Klabin Industries, Sao Paulo, Brazil). The superna-tant fluid was concentrated 10 times in vacuo at 45°C anddialyzed against 0.9% NaCl-0.01 M phosphate buffer (PBS)for 2 days (two changes of 2 liters of buffer) at 4°C. It wasthen affinity chromatographed in columns of protein A-puri-fied rabbit anti-gp43 immunoglobulin G coupled to Affi-Gel10 (Bio-Rad), as described previously (17). The gp43 waseluted from this column with 50 mM citrate buffer, pH 2.8,and was immediately neutralized with 1 M Tris-HCl, pH 9.0,and then concentrated at 4°C under N2 in an Amicon cellwith a PM10 Diaflo membrane. Aliquots of the final prepa-ration were kept frozen, or at 4°C, for short-term use.Further purification of affinity-purified gp43 was achieved bygel filtration in a Sephacryl S-200 column (1.65 by 83 cm;PBS-0.02% Na azide buffer at a flow rate of 6 ml/h) toeliminate high-MW contaminants.

Radiolabeling. Affinity-purified gp43 (30 Rg) was iodinatedwith 125I-Na (13 mCi/mmol) to a final specific activity of 3 x106 cpm/lpg by the method using insoluble 1,3,4,6-tetra-chloro-3a,6a-diphenylglycoluril (6). Free iodine was elimi-nated by exhaustive dialysis against PBS, and aliquots werekept frozen. In contrast to the affinity-purified gp43, theSephacryl-purified gp43 preparations degraded after iodina-tion and therefore could not be used.

Deglycosylation procedures. gp43 was deglycosylated bythe following methods: (i) with endo-p-N-acetylglucosamini-dase H (endo H; Sigma Chemical Co.) in 50 mM sodiumacetate buffer, pH 5.5, 50 mU of enzyme per ml (670,ug) ofcold antigen or 1.5 mU of enzyme per 100,ul (20 x 106 cpm)of labeled antigen was used; (ii) with N-glycanase (peptide:N-glycanase F; Genzyme) at 15 U/ml, the labeled antigen (20x 106 cpm/100 RI) was first denatured by boiling in 0.5%SDS, and the detergent in the reaction mixture was thendiluted to 0.17% SDS with 0.1 M sodium phosphate buffer,pH 8.6, containing 1.25% Nonidet P-40 before addition of theenzyme. Enzymatic digestions were carried out for 18 h at37°C, and the products were kept frozen. Endo H- andN-glycanase-treated gp43 were called EH38 and NG38,respectively, because they migrated in SDS-PAGE with anapparent molecular mass of 38 kDa.

Periodate oxidation of iodinated antigens. 125I-labeled gp43

or EH38 (3 x 106 cpm/,ug) was oxidized with 10 mM sodiummetaperiodate (Merck) in 50 mM acetate buffer, pH 4.5, for30 min at 28°C in the dark. The reaction was interrupted byadding 0.5% glycerol (10 min, room temperature). Aldehydegroups were blocked with 1% (final concentration) glycine in0.1 M Tris-HCl buffer, pH 7.4, with 0.2% bovine serumalbumin for 1 h at room temperature. The reaction mixturewas dialyzed against PBS for 2 h at 4°C. It was thenaliquoted and kept at -20°C for further use. Control reac-tions followed the same protocol except that periodate wasnot added.

IPP. Sera were serially diluted (10-fold dilutions), startingfrom 300-1, in PBS with 0.05% bovine serum albumin and1% Triton X-100. Diluted sera (final volume, 200 pA) wereincubated for 1 h at 37°C with 4,000 to 5,000 cpm of125I-labeled gp43 or EH38. A 10% suspension of Staphylo-coccus aureus (Cowan 1 strain) was added (50 ,ul) andallowed to react for 30 min at room temperature. Boundimmunocomplexes were separated by centrifugation (Ep-pendorf microcentrifuge, 12,000 rpm, 1 min) and washedtwice in the PBS-BSA-Triton X-100 buffer described above.Pellets were counted for total radioactivity (counts perminute) in a gamma counter. The negative control corre-sponded to a pool of 10 sera from healthy individuals, and itsreactivity was taken as the background value. The resultswere calculated as percentages of counts per minute precip-itated, as follows: (Pcpm - Bcpm)ITcpm x 100, where Pcpm,Bcpm, and Tcpm are the counts per minute corresponding,respectively, to the immunoprecipitate, background reactiv-ity (negative sera), and total radioactivity added. Serumtiters were recorded as the greatest serum dilutions immu-noprecipitating more than 10% of the total radioactivity.Two determinations were made simultaneously for eachserum, using the same antigen preparation. The positivecontrol serum (from a patient) at a 300-1 dilution immuno-precipitated ca. 95% of the total radioactivity added,whereas 2 to 5% precipitation was obtained with the negativecontrol serum. The PCM sera immunoprecipitated at least50% (2,000 to 2,500 cpm) of the total radioactivity at dilu-tions higher than 300-1.The antigenicities of EH38, periodate-treated EH38, and

gp43 in the presence of carbohydrate were tested with alimited number of sera (seven PCM sera). The workingdilution of these sera was that sufficient to immunoprecipi-tate 50% of the 1251-gp43. Results are means of threedeterminations, with standard deviations not higher than 5%of the mean. When autoradiograms were to be taken, over30,000 cpm of the total antigen was added in each reaction,and the immunocomplexes were processed as describedpreviously (17). Competitive assays were carried out withthe following monosaccharides at 0.1 or 0.2 M: D(+)-galac-tose, a-methyl-D-galactopyranoside, P-methyl-D-galactopy-ranoside, a-methyl-D-mannopyranoside, D( - )-arabinose,and D(+)-xylose (all from Sigma). Inhibition values for bothIPP and ELISA were the percent differences between theresults obtained with the control and the experimental sys-tems.ELISA. ELISA was carried out as described previously

(1), but with the following minor changes. ELISA plateswere coated with 4 ,ug of gp43 or EH38 per ml (50 ,ul perwell) overnight at 4°C in 0.1 M sodium carbonate-bicarbon-ate buffer, pH 9.6. Alternatively, a 4-h incubation at 37°Cwas used when gp43 was tested alone. Blockade of the freesites on the plate was achieved with PBS-0.1% Tween20-0.7% gelatin for 2 h at 37°C. Diluted sera and a 500-1dilution of anti-human immunoglobulin G conjugated with

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1612 PUCCIA AND TRAVASSOS

horseradish peroxidase (Sigma) were added in successiveincubation steps of 1 h at 37°C in the same buffer. Betweenthe incubation steps, the wells were washed four times withPBS-0.1% Tween 20. The substrate o-phenylenediamine(0.04%) and 0.05% H202 were added to the complexedconjugate, and the reaction was allowed to develop for 5 minat room temperature in 0.04 M phosphate-0.025 M citratebuffer, pH 5.0. The A492 was read in a Titertek MultiskanMCC/340.

Sera were serially diluted (threefold dilutions) startingfrom 300-1, and the titer was that which gave an A492 at least0.1 U higher than that for the negative control serum at thesame dilution. The negative and positive controls were thesame as described for IPP. All sera were also tested forbackground reactivity without the antigen. Two or threetitrations were carried out for each serum on different days.

In competition assays, reactions were run with monosac-charides at 0.07 or 0.2 M (final concentration) as describedfor IPP. Treatment of gp43 and EH38 with 10 mM periodatewas carried out in the ELISA plates, essentially as describedpreviously (26), for 30 min at 28°C in the dark.PAGE of labeled antigens. SDS-PAGE of the I251-antigens

was carried out by the method of Laemmli (13). Gels withradiolabeled samples were dried and autoradiographed onKodak XK-1 films at -70°C with an intensifying screen.

RESULTS

Titration of sera for IPP and ELISA. Homologous sera andheterologous sera (from patients other than those with PCM)(50 PCM, 32 HP, and 27 JL sera) were titrated with gp43 byELISA; for IPP, 40 PCM, 30 HP, and 26 JL sera were used.Quantitative differences were seen in the distribution of theend-point dilutions of these sera (Fig. 1). In the case of thePCM sera, this was expected from the heterogeneity of thespecimens and the fact that they had been collected ingeographically diverse locations. For the same PCM serum,however, the titers obtained by ELISA and IPP were similarin 80% of the cases.

In ELISA (Fig. 1B), 53% of HP sera and 29% of JL serahad titers higher than 2,700'. These reactions were similarto those of the PCM sera and contrasted with the resultsobtained in IPP reactions, in which a distinct separation ofthe serum reactivities was clear (Fig. 1A). A few HP and JLsera reactive with gp43 in ELISA gave IPP reactions of 10 to18% of the total radioactivity added when they were diluted300-1. They differed from the PCM sera, which immunopre-cipitated at least 50% of the total radioactivity at the samedilution. Among HP sera reactive in ELISA (titer higherthan 2,700-1), none was from Venezuela, 25% were from theUnited States, and 75% were from Argentina. The lowreactivity of 10 sera from Venezuela differed significantly (P< 0.01) from that of the other HP sera. Substituting 1251_EH38 for 1251-gp43 gave the same distribution as in Fig. 1A.

Antigenicity of carbohydrate structures in ELISA. To as-sess the possible involvement of the carbohydrate epitopesof gp43 in the reactions obtained by ELISA, we tested somesera with periodate-treated gp43, EH38, and periodate-treated EH38. When the protocol for binding antigen de-scribed in Materials and Methods was used, 60% of the totalprotein bound to the plates, as was determined with iodi-nated gp43 and EH38. Oxidation with periodate was carriedout in the plates, as described previously (26). A 10 mM finalconcentration of periodate was chosen, since at this concen-tration most concanavalin A-binding residues in the antigenwere destroyed (data not shown), leaving the protein core

_ 300.

0, 30-iwu

1-3.

'0.3.

>2,1 87 -

2,187-

729-

243-0

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43-kDa ANTIGEN OF P. BRASILIENSIS 1613

** **

**AA

** ** 0* *

* 00000 ** 00000 * * @0000-0 0000. *@000

**

A *&*

@0*6^ **- AA00000 * * 00000 * *

gp43+ per I EH38 I EH38+ per I GaI I M a n I Ara / Xyl

FIG. 2. Distribution ofPCM (0), HP (*), and JL (A) sera according to the percentages of inhibition of their reactivities with gp43 in ELISAby the modified antigens periodate-treated gp43 (gp43 + per), EH38, and periodate-treated EH38 (EH38 + per) or by the carbohydrateD-galactose (Gal), a-methylmannoside (Man), D(-)arabinose (Ara), or D(+)xylose (Xyl). The percentage of inhibition of the reaction of eachserum was calculated as the difference between the mean A492 for the original reaction with gp43 (taken as 100%) and the A492 of the inhibitedreactions with modified antigens or carbohydrates. Each point gives the results for one serum sample.

reactions, which were common when monosaccharides wereused at high concentration (0.2 M), the monosaccharidesbeing tested as inhibitors were used at 0.07 M. The resultsshown in Fig. 2 are means of three independent determina-tions with standard deviations not higher than 5% of themean value. Six of eight heterologous sera had their reac-tions inhibited by 50 to 100% in the presence of galactose,but the same pattern of inhibition was obtained with either a-or ,-methylgalactopyranoside. Other sugars were generallypoor inhibitors. In two cases, inhibition was observed withmannose and arabinose as well as with galactose.Another exception was one HP serum whose reactivity

was strongly inhibited by EH38 and by periodate-treatedgp43 or EH38 but only weakly reduced by 0.2 M galactose.In IPP, the reaction of this serum was also poorly inhibitedby monosaccharides.

Generally, the ELISA reactions of the PCM sera werelittle affected by addition of monosaccharides. The reactionsof two sera with periodate-treated EH38, which were re-duced by 35 to 45% in comparison with that of the untreatedEH38 control, were not inhibited by any of the carbohy-drates tested.To explain the differences in the reactivities of heterolo-

gous sera with gp43 either in solution or immobilized inplastic, we carried out inhibitions of ELISA reactions withsoluble gp43 at two concentrations (0.1 and 0.5 ,uM). Thereactivities of two HP sera were not significantly affected byaddition of highly purified gp43 at 0.1 ,uM, whereas the sameantigen solution strongly decreased the reactions of PCMsera (50 to 75% inhibition). Inhibition of the reactions ofPCM and HP sera with gp43 at 0.5 1xM was 70 to 95% and 30to 40%, respectively.

Importance of carbohydrate epitopes in IPP reactions. Asalready mentioned, the serum IPP titration curves with125I-gp43 (Fig. 1A) were the same when 125I-EH38 wastested in parallel. In a second set of experiments, we studiedthe reactions of 7 PCM sera at a single dilution with EH38,periodate-treated EH38, and gp43 in the presence ofmonosaccharides. The pattern of the labeled antigens andtheir deglycosylated derivatives used in IPP can be seen inFig. 3. '25I-labeled preparations of gp43 contained smallamounts of a high-MW antigenic complex described previ-ously (17). Removal of the periodate-sensitive high-MWcomplex from gp43 by gel filtration did not significantlyinterfere with the reactivity of the PCM sera with gp43 butdid clearly contribute to the responses of HP sera in IPPreactions. Periodate-treated purified 1251-gp43 could not be

tested in these studies because after dialysis its recovery wasmuch reduced (Fig. 3, lane 3). In contrast, the concentrationof periodate-treated 1251-labeled EH38 (Fig. 3, lane 6) wasnot significantly affected by dialysis.The reactivities of the PCM sera in IPP reactions were

basically the same when gp43, EH38, or periodate-treatedEH38 was used as the antigen. Monosaccharides at 0.2 Malso failed to inhibit IPP of gp43 with PCM sera. In one case,a 25% inhibition was obtained with a-methylmannopyrano-side. The IPP reaction of this serum was weakly (14%)inhibited by EH38, whereas a 30% inhibition was observedin ELISA with periodate-treated gp43. The IPP reactivitiesof seven PCM sera decreased by 35% after gp43 wasdenatured with 0.5% SDS. They were further reduced tobasal levels (those obtained with normal human sera) whenthe antigen was deglycosylated with N-glycanase (notshown), suggesting that conformation alterations leading toinactivation of the antigen probably took place.Although the IPP reactions of HP and JL sera were

quantitatively not comparable to those with PCM sera, westudied a few HP sera which reacted weakly with gp43 todetermine their reactivities with periodate-treated EH38 andalso with gp43 in the presence of monosaccharides at 0.1 and0.2 M. When the results were quantitatively significant interms of counts per minute in the immunoprecipitates,antigens in the immunocomplexes were checked by SDS-

1 2 3 4 5 6 7 8

FIG. 3. Autoradiogram of iodinated antigens prepared for IPP.Lanes: 1, native gp43; 2, control for gp43 in the periodate reaction;3, periodate-treated gp43; 4, EH38; 5, control for EH38 in theperiodate reaction; 6, periodate-treated EH38; 7, NG38; 8, controlfor NG38 (see Materials and Methods) in the N-glycanase reaction.The arrow indicates a molecular mass of 43 kDa.

61 - 100 -

z0I-

31 - 60-

zz

* 30- *0*00*000000000

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1614 PUCCIA AND TRAVASSOS

W.' AN

l-

A a B C c Dd E e Ff

FIG. 4. Autoradiogram showing the gp43 IPP by four differentHP sera at a dilution of 3001 (lanes C through F and c through f) inthe presence (lowercase letters) or absence (uppercase letters) of 0.2M D-galactose. The serum in lane A (dilution, 6,000-1) is from aPCM patient; that in lane B (dilution, 300-') is from a healthyindividual. A total of 70,000 cpm of iodinated antigen was used ineach reaction. The arrowhead indicates the position of gp43.

PAGE to determine the relative contribution of the high-MWcomponent. In these experiments, the partially purifiedrather than the highly purified (by gel filtration) gp43 wasused.By measuring the radioactivities of the immunoprecipi-

tates (means ± standard deviations, with standard devia-tions < 5% of the mean) from three independent determina-tions for each serum, we verified that galactose was the bestinhibitor of most reactions with heterologous sera, causing a50% decrease in the total counts per minute of the precipi-tate. Inhibition studies with either a- or P-methylgalactopy-ranoside had similar results. With some sera (Fig. 4, serumin lane F), the radioactivity precipitated was entirely due tothe high-MW component, and this reaction probably in-volved epitopes containing galactose. Other sera (Fig. 4,lanes C, D, and E) immunoprecipitated both gp43 and thehigh-MW component. With the serum in Fig. 4, lane D,galactose inhibited only the reaction with gp43.The reactivities of various sera with periodate-treated

EH38 are shown in Fig. 5. The heterologous sera reactedonly with the high-MW component present in the EH38preparation, but not with EH38 itself. This reaction wastotally abolished by treatment with periodate. The PCMserum tested reacted strongly with EH38. Only a weakreaction observed with the high-MW component was inhib-ited by periodate treatment.

Sip:.., 04

EH38-

A a 8 C c D d E e

FIG. 5. Autoradiogram of IPP of periodate-treated EH38 (low-ercase letters) and its control (uppercase letters) by HP sera. Threerepresentative HP sera (lanes C through E and c through e) areshown. The serum in lane A (dilution, 2,000-') is from a patient withPCM; that in lane B (dilution, 300-') is from a healthy individual. Atotal of 35,000 cpm of iodinated antigen was used in each reaction.

DISCUSSION

To determine the role of carbohydrate epitopes in theantigenicity of gp43, we tested a number of sera frompatients with PCM, HP, or JL against native purified ordeglycosylated antigens from P. brasiliensis yeast phase byELISA and IPP. Since the gp43 preparations obtained fromaffinity columns could still contain small amounts ofhigh-MW and 72-kDa antigenic components, our first con-cern was to further purify the gp43 by Sephacryl S-200filtration to determine whether the strong reactivity of somesera in ELISA was due to those other exoantigens. Ourresults show that PCM sera recognize primarily gp43 peptideepitopes that are independent of carbohydrate epitopes.With a few sera, however, the carbohydrate epitopes ac-counted for up to 45% of the total reactivity in ELISA and15% of that in IPP. By competition studies with monosac-charides, we found that a-methylmannopyranoside was themain inhibitor of these reactions.Our results are in good agreement with those of Taba et al.

(22), who cloned in bacteria a fungal gene(s) coding forepitopes present in the gp43 molecule. Gene expression wasdetected by Western blotting and IPP with rabbit hyperim-mune anti-P. brasiliensis antiserum as well as with a pool of10 human PCM sera with high titers by ID. Since cloning andexpression of the P. brasiliensis gene(s) were carried out inEscherichia coli, the positive reactivity with human serainvolved only peptide epitopes.HP and JL sera, on the other hand, cross-reacted in

ELISA mainly with galactose-containing carbohydrateepitopes present in N-linked carbohydrate chains of gp43.When tested by IPP, the HP and JL sera reacted very wellwith the high-MW component, binding in all cases to perio-date-sensitive epitopes. The nature of the specific residuesinvolved, however, is unclear, since not all sera reactivewith this glycoconjugate were inhibited by galactose.The expression of carbohydrate epitopes recognizable by

heterologous antibodies depends on whether the gp43 isbound to the ELISA plates or is in solution. The strong HPand JL serum reactions observed with the plastic-immobi-lized antigen are not obtained when the soluble antigen isused in reactions such as IPP, inhibition-of-gp43 radioim-mune assay (21), and ID (2). Moreover, soluble gp43 signif-icantly inhibits ELISA reactions with PCM sera, whereasthe reactions with HP sera remain strong even in thepresence of excess soluble gp43. Obviously, gp43 assumesdifferent conformations when bound to the plastic or insolution. Immunological tests with the antigen in differentconformations, such as ELISA and immunoblotting, shouldbe analyzed with caution. As has been shown previously (5,9, 11, 15), globular protein and peptide antigens change theirconformations when immobilized in ELISA plates.The reactions with PCM sera decreased to the level of the

negative control when gp43 deglycosylated with N-glyca-nase was used as the antigen. Although this result may seemcontradictory when compared with those involving period-ate and endo H treatments, it is probable that in the case ofthe N-glycanase treatment a conformational change of theantigen occurs as a result of both the denaturation by SDSand the removal of carbohydrate chains by cleavage of theasparagine-N-acetylglucosamine bond (23). Such cleavage,which, as suggested elsewhere (7), causes a conformationalchange, is not observed in the deglycosylation reaction withendo H, which cleaves carbohydrate chains only betweentwo internal residues of N-acetylglucosamine (24).

In conclusion, the present work shows that PCM sera

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43-kDa ANTIGEN OF P. BRASILIENSIS 1615

recognize predominantly conformational peptide epitopes ofthe purified gp43 antigen, in contrast to sera from patientswith HP or JL, which react preferentially with the galactose-containing carbohydrate epitopes of this antigen.The identification of the specific peptide epitopes recog-

nized by the PCM sera is a logical complement to the presentwork. Recently, we have obtained mouse monoclonal anti-bodies specific for peptide epitopes of gp43 (unpublishedresults). These monoclonal antibodies can be used in com-petition experiments with the human antibodies and asreagents to help determine the structure of the gp43 antigenand, eventually, its sequence.

Practically, the intact gp43 molecule present in concen-trated culture filtrates of P. brasiliensis will continue to belargely used for the specific ID serodiagnosis of PCM. If theELISA method is introduced, the loss of specificity of thegp43 antigen can be minimized only by adding galactose tothe reaction mixture or by using a deglycosylated form of theantigen. However, since the deglycosylation procedure isexpensive, it is not a practical method for routine testing.Perhaps future efforts should be directed to obtaining bacte-rial recombinant clones with high expression of the ungly-cosylated antigen or to sequencing the protein core with theaim of producing a synthetic peptide. An attempt to clonegenes encoding epitopes of the gp43 antigen has already beensuccessful in our laboratory (22).

ACKNOWLEDGMENTS

We are extremely grateful for the generous gift of sera from Z. P.Camargo, R. Tewari, and R. Baruzzi, as well as for the gift of P.brasiliensis B339 culture from A. Restrepo-Moreno. We also thankM. M. Rodrigues for helpful discussions throughout this investiga-tion.

This work was supported by CNPq, FAPESP, and FINEP.

REFERENCES1. Camargo, Z. P., J. L. Guesdon, E. Drouhet, and L. Improvisi.

1984. Enzyme-linked immunosorbent assay (ELISA) in theparacoccidioidomycosis. Comparison with counterimmunoelec-trophoresis and erythro-immunoassay. Mycopathologia 88:31-37.

2. Camargo, Z. P., C. Unterkircher, S. P. Campoy, and L. R.Travassos. 1988. Production of Paracoccidioides brasiliensisexoantigens for immunodiffusion tests. J. Clin. Microbiol. 26:2147-2151.

3. Camargo, Z. P., C. Unterkircher, and L. R. Travassos. 1989.Identification of antigenic polypeptides of Paracoccidioidesbrasiliensis by immunoblotting. J. Med. Vet. Mycol. 27:407-412.

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