Vol. 59, No. 7

INFECTION AND IMMUNITY, JUlY 1991, p. 2245-2251 0019-9567/91/072245-07$02.00/0 Copyright © 1991, American Society for Microbiology

An Immunoreactive Apoglycoprotein Purified from Coccidioides immitis KRIS 0. DUGGER,1'2 JOHN N. GALGIANI,12* NEIL M. AMPEL,"2 SUNG H. SUN,3 D. MITCHELL MAGEE,3 JEFF HARRISON,3 AND JOHN H. LAW4 Medical and Research Services, Veterans Affairs Medical Center, Tucson, Arizona, 857231; Department of Medicine, College of Medicine,2 and Department of Biochemistry,4 University of Arizona, Tucson, Arizona 85724; and Research Service, Veterans Affairs Medical Center, San Antonio, Texas 782843 Received 3 January 1991/Accepted 5 April 1991 Deglycosylation of glycoproteins in a lysate of spherules of Coccidioides immitis has permitted purification and partial characterization of a proline-rich pronase-sensitive antigen. Moreover, soluble antigen specifically stimulated lymphocytes from persons with dermal delayed-type hypersensitivity to coccidioidal antigens. When related to reference coccidioidin by tandem two-dimensional immunoelectrophoresis, the antigen fused in the anodal region with a specific reference antigen (antigen 2). It did not show identity with coccidioidal antigens used in conventional serologic assays. Although immunoblots of the purified protein with monospecific rabbit antiserum showed a single antigen at 33 kDa, the parent spherule lysate bound the same antibody in a broad band between 70 and >200 kDa, which could be explained by microheterogeneity of glycosylation. Immunoelectron microscopy using affinity-purified human antibodies localized the antigen to the cell wall and internal septa of spherules. These findings suggest that the apoglycoprotein may be important in human immune responses to coccidioidal infection.

from strain Silveira as described previously (24). The lyophilized extract (2 to 15 mg of protein) was chemically deglycosylated with anhydrous HF at the Biotechnology Core Facility at the University of Arizona, Tucson, as described by Shively (37), utilizing an HF Apparatus (Immunodynamics, La Jolla, Calif.). It should be noted that anhydrous HF is extremely toxic and must be handled in a closed system under an approved fume hood. The resulting material was dialyzed against phosphate-buffered saline (PBS, pH 7.2), concentrated, and further dialyzed against PBS. Insoluble material was removed by centrifugation at 10,000 x g for 5 min. The supematant from this step is subsequently referred to as the soluble protein fraction. The soluble protein fraction was applied to a Sephacryl S-200 HR column (1.8 by 90 cm; Pharmacia, Piscataway, N.J.) and eluted with 50 mM phosphate buffer, pH 7.2, containing 300 mM NaCl, at a flow rate of 0.2 ml/min. The Sephacryl column was calibrated by using high- and low-range gel filtration molecular weight calibration markers (Pharmacia). The peak of interest eluted from this column is subsequently referred to as the chromatographically purified protein. Analytic procedures. Protein concentration was determined with the BCA Protein Determination Kit (Pierce, Rockford, Ill.) (39), as modified (24). Total carbohydrate as glucose was determined by the phenol-sulfuric acid method of Dubois et al. (19), except that all volumes were reduced by a factor of 5. Sodium dodecyl sulfate (SDS)-polyacrylamide (6 to 18% gradient) and native polyacrylamide (6 to 20% gradient) gels were prepared and run as previously described (4) except that a minigel (6 by 8 by 0.075 cm) format (Hoeffer, San Francisco, Calif.) was used for some experiments, as noted. SDS gels in the minigel format were run at 20 mA for 40 min, and native gels were run at 20 mA for 40 min and then at 5 mA for 2 h. Rainbow molecular weight markers, "'C-labeled molecular weight markers (Amersham, Arlington Heights, Ill.), and gel electrophoresis markers (Pharmacia) were used to estimate Mr, as noted in Results. Gradient (8 to 25%)

Coccidioidomycosis is a systemic infection which is caused by the dimorphic fungus Coccidioides immitis, the control of which is critically dependent on cellular immunity (1, 2, 16, 18, 23, 27, 30, 31, 40). In addition, delayed-type hypersensitivity and serum antibody production have been useful epidemiologic and diagnostic markers (34, 38). We and others have sought to identify, purify, and characterize antigens from C. immitis that might stimulate cellular or humoral immune responses during infection (8, 10, 11, 13, 17, 36, 45-47). In previous studies, treatment of coccidioidal spherules with toluene produced soluble antigens which stimulated lymphocytes and bound serum antibodies from humans who had acquired coccidioidal infections (24). Our antigen preparation, like others (6, 7, 11, 15, 29, 34-36), contained very high amounts of carbohydrate, suggesting that many, and possibly all, relevant antigens of C. immitis are glycoproteins or families of related glycoproteins differing in sugar content. Such heterogeneous glycosylation poses technical difficulties in separating and characterizing individual protein antigens. In an effort to simplify this problem, we have resorted to a chemical treatment to remove sugars from glycoproteins. Exposure to anhydrous hydrogen fluoride at 0°C for 1 h cleaves all the 0 linkages of neutral and acidic sugars, leaving peptide bonds intact (33, 43). Using this approach we have been able to identify, isolate, and partially characterize an apoglycoprotein that retains antibody affinity and lymphocyte-stimulating activity. (This work was presented in part at the 91st General Meeting of the American Society for Microbiology, Dallas, Tex., 5 to 9 May 1991.) MATERIALS AND METHODS Protein purification. A toluene-induced spherule lysate (TSL) was prepared from first-generation, 96-h spherules *

Corresponding author. 2245

2246

DUGGER ET AL.

Phast gels (Pharmacia; SDS and native) were run, and the silver stain was developed as suggested by the manufacturer in the PhastSystem Separation Technique Files 110 and 120 and the PhastGel Silver Stain Kit Instruction Manual. The isoelectric point of chromatographically purified protein was estimated on a Phast isoelectric focusing gel with a pl range of 3 to 9 and broad-range IEF markers (Pharmacia) according to the protocol suggested in the PhastSystem Separation Technique File 100. The amino acid composition was determined at the Macromolecular Structure Facility of the Biotechnology Program, University of Arizona, Tucson. Chromatographically purified protein was dissolved in high-performance liquid chromatography (HPLC)-grade water and filtered through a prerinsed 0.2-,im-pore-size filter (Gelman Scientific, Ann Arbor, Mich.). Duplicate samples (66 pmol) were applied to an automated amino acid analyzer (model 420A; Applied Biosystems, Inc., Foster City, Calif.). Pronase (0.08 U; Boehringer Mannheim, Indianapolis, Ind.) was added to the soluble protein fraction (25 jig) in 100 mM Tris-15 mM CaCl2 (pH 8.0) and incubated for 24 h at 37°C. A 10-,ul aliquot was mixed with an equal volume of SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer, boiled for 5 min, applied to an SDS-polyacrylamide gel, and immunoblotted with rabbit antispherulin serum (described below). Analytical reverse-phase HPLC was performed with a Beckman (Fullerton, Calif.) C8 column (0.1 by 25 cm; 5-,um particles), using a Beckman detector (model 166), integrator (model 427), and pump (model 126). The chromatographically purified protein was suspended in HPLC-quality water containing 0.1% trifluoroacetic acid and eluted with a 0 to 70% gradient of acetonitrile in water containing 0.05% trifluoroacetic acid. Sources of sera and preparation of antiserum in rabbits. Human sera were obtained from patients with active coccidioidomycosis and from whom C. immitis had been recovered. Sera were also obtained from healthy persons without prior history of coccidioidal infection and who lacked a dermal delayed-type hypersensitivity response to spherulin (Berkeley Biologics, Berkeley, Calif.). Each of two female New Zealand White rabbits was injected subcutaneously with 29 jig of chromatographically purified apoglycoprotein suspended in saline in the RIBI adjuvant system (catalog no. 730; Immunochem Research, Inc., Hamilton, Mont.) according to the manufacturer's directions (revised August 1989). The rabbits were boosted on days 17 (14.5 jig), 38 (14.5 jig), and 56 (3.5 ,ug) and bled on day 70. Rabbit antiserum against a complex soluble spherule extract (spherulin) was described previously (25). All animal protocols were approved by the University of

Arizona Institutional Animal Care and Use Committee. Burro anticoccidioidin serum was as previously described

(9).

Antigenic analysis. SDS-polyacrylamide gels were electroblotted onto nitrocellulose paper (0.1-jim pore size; Schleicher & Schuell, Inc., Keene, N.H.) with a Genie blotter (Idea Scientific, Minneapolis, Minn.) at 12 V for 90 min (3). Blots were allowed to air dry briefly, blocked in 1% nonfat dry milk in PBS (MPBS) for 30 min, and incubated in serum diluted 1:50 in MPBS for 1 h. After three 5-min washes in MPBS, the blots were incubated in '25I-protein A (Amersham) for 1 h, washed three times, dried briefly, and exposed to X-Omat film (Eastman Kodak Co., Rochester, N.Y.) at -800C. All incubations were at room temperature. Analogous transfer blots were developed with a panel of

INFECT. IMMUN.

lectins (40 jig/ml) conjugated to fluorescein isothiocyanate (Vector Laboratories, Burlingame, Calif.) in 20 mM TrisHCl (pH 7.4) containing 0.5 M NaCl, 1 mM CaCl2, and 1 mM MgCl2. Blocking and wash steps for these studies were performed in PBS containing 0.2% Tween 20 (Sigma). Tandem two-dimensional immunoelectrophoresis (IEP) followed the method of Reiss et al. (35) and was performed as previously described (24). The soluble protein fraction was run in tandem with reference coccidioidal antigen against burro anticoccidioidin reference serum. With this particular reference system, 26 antigens are detectable. Peaks are numbered according to the distance migrated toward the anode in the first dimension, i.e., the most anodally migrating antigen is number 1 (29). Conventional tube precipitin-type antigen activity (TP activity) was measured by an immunodiffusion technique (IDTP; Meridian Diagnostics, Cincinnati, Ohio). Complement fixing-type antigen activity was also measured by an immunodiffusion technique, as previously described (44). Purification of antiapoglycoprotein antibodies and immunoelectron microscopy (IEM). An affinity column was prepared with chromatographically purified apoglycoprotein by a slight modification of a previously described method (21). Briefly, 75 jig of protein was mixed with an equal weight of N-hydroxysuccinimido-biotin (Sigma) in 0.1 M NaHCO3 for 3 to 4 h at room temperature. Unreacted N-hydroxysuccinimido-biotin was removed by dialysis, and the biotinylated protein was mixed with excess agarose-avidin D (Vector) with rocking for 3 h. The agarose was placed in a 10-ml column and washed extensively with PBS to remove unbound protein. Serum from a patient with culture-proven coccidioidal infection or pooled from patients without evidence of coccidioidal infection was circulated over the column for 30 to 60 min to obtain human affinity-purified antibody. Bound antibody was eluted from the column with 3 M MgCl2, desalted, and concentrated by ultrafiltration. Spherules that had developed after 96 h on Converse agar from arthroconidia of strain C-735 were prepared for IEM as described previously (45). Staining and microscopy were performed as described by Coalson et al. (5) except that the primary (human affinity-purified) antibody was diluted 1:1,000 in Tris buffer (pH 8.2) containing 1% ovalbumin and colloidal gold particles (10-nm diameter; Amersham) conjugated to goat anti-human antibody (Amersham) were used. Lymphocyte transformation assay. As previously described (24), peripheral blood mononuclear cells were obtained from healthy volunteers with known skin test reactions to spherulin by a one-step method (22). Culture conditions, tritiated thymidine incorporation, and harvesting were as described previously (24) except that 5 x 105 cells per well and 20% autologous serum were used. To appropriate wells in triplicate was added concanavalin (ConA) A (5 jig/ml; Sigma), TSL (86 jig of protein per ml), or soluble protein fraction (6.2 jig/ml). No stimulant was added to a set of three wells which served as controls. Results were expressed as mean counts per minute of samples minus the mean counts per minute of control wells, and the significance was assessed by the Mann-Whitney U test. RESULTS Isolation of a 33-kDa apoglycoprotein. Treatment of TSL with anhydrous hydrogen fluoride resulted in an average 95% reduction in soluble carbohydrate. Approximately 9% of the initial protein was recovered in the soluble protein fraction. Much of the apparent protein loss was due to

VOL. 59, 1991 TABLE 1. Amino acid analysis of the 33-kDa protein as determined on an ABI 420A automated amino acid analyzer Mol% Residue Alanine ................ 9.0 3.1 Arginine ................ 10.3 Asparagine + aspartate ................ 10.2 Glutamine + glutamate ................ 10.1 Glycine ................ 1.5 Histidine ................ Isoleucine ................ 2.6 3.3 Leucine ................ 3.5 Lysine ................ 1.3 Methionine ................ 2.9 Phenylalanine ................ 17.9 Proline ................ 6.0 Serine ................ 13.8 Threonine ................ 0.7 Tyrosine ................ Valine ................ 3.7 ND' Cysteine ................ ND Tryptophan ................ a ND, not determined.

formation of a precipitate which was insoluble in pyridine, 1 or 10% acetic acid, and 6 M guanidine hydrochloride. A major 33-kDa band and a much weaker 70-kDa band were evident on silver-stained SDS-polyacrylamide gels of the soluble protein fraction. Gel filtration chromatography of the soluble protein fraction yielded an absorbance peak (A280) which produced a single silver-stained band at 33 kDa on SDS-PAGE. Analysis of the chromatographically purified 33-kDa protein by reverse-phase HPLC showed a single antigenically active A220 peak. However, the chromatographically purified protein displayed anomalous migration in nondenaturing PAGE, with an apparent Mr of 50,000, and eluted from the gel filtration column as if the molecular mass were >100 kDa. The isoelectric point of the chromatographically purified protein was 4.0, and the amino acid analysis is given in Table 1. Carbohydrate was unmeasurable in this fraction. The purified 33-kDa protein blotted onto nitrocellulose failed to bind a panel of fluorescein isothiocyanateconjugated lectins, whereas TSL bound fluorescein isothiocyanate-ConA (only) in the 70- to 200-kDa region and at 21.5 kDa. Silver-stained SDS-polyacrylamide gels of the HF-insoluble precipitate showed bands at 55, 33, and 16 kDa and a diffuse staining in the high-molecular-mass region (>200 kDa). These proteins have not been analyzed further. Antigenic activity. The chromatographically purified apoglycoprotein showed a single band at 33 kDa when immunoblotted either with homologous rabbit antiserum or with rabbit antiserum prepared against spherulin (Fig. 1). Moreover, sera from all of nine patients with active coccidioidomycosis demonstrated some degree of binding to the 33-kDa antigen, whereas no binding was evident with sera from uninfected subjects (Fig. 2). In contrast, neither monospecific rabbit anti-33-kDa apoglycoprotein serum (Fig. 1) nor any of the patient sera (data not shown) detected a 33-kDa band in untreated spherule lysate (TSL). With extended exposures of the immunoblots to radiographic film, however, the rabbit anti-33-kDa serum was found to react with untreated spherule lysate in a diffuse band in the highermolecular-mass region (70 to >200 kDa). Following pronase digestion of the soluble protein fraction, no bands were detected on an immunoblot with rabbit antispherulin serum.

ANTIGENIC APOGLYCOPROTEIN FROM C. IMMITIS

1

3

2

4

5

6

7

2247

8 9

200-

925-_

6946-

-

30-

14.3 -

-

FIG. 1. Immunoblot of TSL and deglycosylated protein fractions following SDS-PAGE. Lane 1, 14C-labeled molecular weight markers; lanes 2 and 6, 0.5 ,ug of soluble protein fraction; lanes 3 and 7, 0.7 ,ug of chromatographically purified apoglycoprotein; lanes 4 and 8, 3.7 ,ug of insoluble fraction; lanes 5 and 9, 6.4 jig of TSL. Lanes 2 through 5 were incubated with rabbit antispherulin serum and '25I-labeled protein A. Lanes 6 through 9 were incubated with monospecific rabbit anti-33-kDa protein serum and "25I-labeled protein A.

In tandem two-dimensional IEP, the major protein of the soluble protein fraction fused with antigen 2 in the anodal region (Fig. 3). None of the other 25 antigens identified in reference coccidioidin (see Materials and Methods) showed cross-reactivity with the soluble protein fraction. Moreover, the purified 33-kDa apoglycoprotein did not show identity in standard immunodiffusion tests for tube precipitin- or com-

1

2 3 4 5

6 7 8 9 10

200-

6946-

30-

.j:

FIG. 2. Human serum immunoblots of 33-kDa apoglycoprotein following SDS-PAGE. Seven micrograms of chromatographically purified protein was loaded into a single well in a minigel (6 by 8 by 0.075 cm) format, subjected to SDS-PAGE, and electroblotted onto nitrocellulose paper. The nitrocellulose was cut into 4-mm strips and incubated with human serum and 125I-protein A. Strips 1 to 9, sera from patients with active coccidioidomycosis; strip 10, normal human serum.

. XK, .~ ~ .

INFECT. IMMUN.

DUGGER ET AL.

2248

TABLE 2. Lymphocyte transformation using mononuclear cells obtained from skin test-positive and -negative donors stimulated with ConA, spherule lysate, or soluble protein fraction cpma with stimulation by: Donor skin test

Subject

ConA

Con(5AigmI (5 iLg/ml)

Spherule lysate

Soluble

(86 jig of

protein/ml)

protein (6.2 p.g/ml)

Positive

1 2 3 4 5 Mean SEM

23,030 14,421 18,048 14,343 49,439 23,856 6,590

NDb 10,395 16,579 12,052 23,952 15,744 3,032

3,099 4,830 2,147 7,295 10,912 5,656 1,579

Negative

6 7 8 9 10 Mean SEM

10,512 7,956 66,156 5,990 23,811 22,885 11,258

ND ND -221 249 68 32 349

1,623 441 315 856 -53 636 286

0.037

0.009

I Flo `'^. ,t,,,^I_ .

P valuec

a Mean counts per minute of samples minus the mean counts per minute of control wells. b ND, not done. c P values obtained by Mann-Whitney U test.

R

HF

FIG.

3.

Tandem 2-dimensional IEP studies of the soluble

fraction. Reference coccidioidin (R) and either PBS (A)

or

protein soluble

apoglycoprotein produced by hydrogen fluoride treatment (B) migrated to the right in the first dimension and then vertically into antibody-containing agar in the second dimension. 2 refers to antigen 2 in the reference system. Note the appearance of the HF arc, which fuses with antigen 2 in its anodal region.

plement fixing-type antigens. It did, however, show a line of nonidentity in the standard IDTP test. IEM. When thin sections of mature spherules were incubated with affinity-purified antibodies from a patient with coccidioidomycosis, bound antibodies were detected by colloidal gold particles in the cell wall and cleavage planes (Fig. 4A). In contrast, very little binding was apparent when pooled sera from uninfected patients were substituted (Fig. 4B). By particle enumeration, there were 873% more colloidal gold particles on the cell wall in sections treated with affinity-purified were

antibodies

from

the

infected

found with antibodies from control

patient

than

sera.

Lymphocyte transformation. The soluble protein fraction induced

lymphocyte

transformation

in

peripheral blood delayed-

mononuclear cells from humans who demonstrated

type dermal hypersensitivity to spherulin but not in cells no dermal sensitivity (Table 2). by the soluble protein fraction was less than that produced by the crude spherule lysate, which was used at an optimal concentration that was approximately 14-fold greater than that of the soluble apoglycoprotein.

from donors who showed Stimulation

DISCUSSION We have identified, purified, and partially characterized a protein antigen from spherules of C. immitis. We have

termed the isolated 33-kDa protein an apoglycoprotein because (i) carbohydrate is undetectable on the protein we isolated, (ii) the native protein behaves as a glycoprotein on chromatographic media, and (iii) treatment with anhydrous HF as we have done would be expected to strip carbohydrate side chains from glycoproteins. However, anhydrous HF treatment at 0°C for 1 h would not be expected to remove N-linked N-acetylated hexosamine (33), and thus it is possible that single sugar residues remain. The apoglycoprotein was readily apparent as a 33-kDa band by SDS-PAGE and immunoblotting and fused anodally with antigen 2 in the tandem two-dimensional IEP system of Huppert et al. (29). In contrast, the parent extract (TSL) displayed neither characteristic but rather exhibited diffuse binding of monospecific antiapoglycoprotein antibody in immunoblots (Fig. 1, lane 9) and poorly localized distortion of antigen 2 by tandem two-dimensional IEP (24). The improved electrophoretic and chromatographic resolution of the apoglycoprotein may be the result of eliminating microheterogeneity of glycosylation (28) of the antigens in the TSL. Deglycosylation also may have contributed to the identification of antigens by Cox and Britt, who have extensively characterized alkali-soluble, water-soluble (ASWS) extracts of C. immitis (10). Alkali extraction can result in removal of 0-linked carbohydrate from proteins (43), and one antigen in ASWS extracts exhibits anodal fusion with antigen 2, as does our apoglycoprotein (10, 12). Recently, anodal fusion with antigen 2 of alkali-extracted antigens from inner conidial walls of C. immitis has also been demonstrated by Kruse and Cole (32). ASWS extracts of C. immitis elicit dermal hypersensitivity in guinea pigs previously infected with C. immitis (15), stimulate human peripheral blood mononuclear cells from subjects with reactive coccidioidal skin tests (14), and, used as a vaccine, protect mice from intraperitoneal challenge (16). These findings are consistent with ours that

A

Li.

.'

:-i' : ,.,.,,,,. ''' ''',,'.''\ ~ ~ ~~~~~~~~-

f..~~~~~~~~~~~~*'

;:_r.rs ;*; ps ;_S_v>;'

_

_

i

P

rs_~~~~~

.~~~~~A -s ...' 4-si=

*Si!

B

.1I I

FIG. 4. IEM examination of a mature spherule of C. immitis. Sections from spherules were incubated with antibodies to the 33-kDa apoglycoprotein, which were affinity purified from a patient with culture-proven infection (A) or from a pool of sera from uninfected subjects (B). Goat anti-human immunoglobulin G conjugated to colloidal gold (10 nm) was used to detect bound antibodies. Magnification as shown,

x28,158.

2249

2250

DUGGER ET AL.

human peripheral blood mononuclear cells from previously infected subjects are stimulated by apoglycoprotein. The similarities between the 33-kDa antigen and a component in ASWS lead us to speculate that the apoglycoprotein may be important in human recognition of coccidioidal infection and encourages us to study further its immunologic activity. ASWS also contains strong TP antigenic activity. However, the TP activity in ASWS extracts has been shown to reside in a fraction which does not show anodal fusion with antigen 2 (12, 32). Three findings from our studies indicate that the apoglycoprotein does not possess TP antigenic activity, either. First, the apoglycoprotein does not produce a line of identity with reference TP antigen in immunodiffusion. Second, unlike TP antigen (48), the apoglycoprotein is pronase sensitive. Third, in other studies, antigens recovered by ConA affinity chromatography from spherule culture supernatant are rich in TP antigen (26) but after deglycosylation with hydrogen fluoride demonstrate neither a 33-kDa band by SDS-PAGE nor TP antigenic activity in standard double immunodiffusion (unpublished data). Isolation of the putative holoprotein from TSL is of obvious interest and will provide unequivocal information about the nature of the precursor to the 33-kDa antigen. Even though the 33-kDa protein appears distinct from TP antigen, immunoblots with patient sera demonstrated binding. This raises the possibility that the protein may be of future use in serologic testing. Amino acid analysis showed unusually high proline (17%) and threonine (14%) contents. Proline-rich proteins have in some cases shown highly localized areas of immunodominance (41). In its relatively high contents of proline, threonine, serine, glycine, and alanine, the apoglycoprotein resembles the apomucins of mammalian submaxillary glands (42), in which the hydroxy amino acids serve as junction points for the many carbohydrate chains of the holoprotein. Apomucins have an extended polypeptide chain (20), and these and other proline-rich proteins have shown anomalous behavior in polyacrylamide gels and gel filtration media (41, 42), much like those we have observed for the 33-kDa apoglycoprotein. Structural studies now under way will determine if the C. immitis protein has a similar extended polypeptide structure and if immunodominance resides in proline-rich domains. ACKNOWLEDGMENTS Rosemary Hayden provided valuable technical assistance, and Ralph R. Martel provided useful comments during the course of our studies. We thank Mark Estes for his assistance with affinity chromatography and Ron Jasensky, Virlane Torbit, and Wallace Clark of the University of Arizona Division of Biotechnology Core Facility. This work was supported in part by the Department of Veterans Affairs and by the Arizona Disease Control Research Commission (grant 82-0689). REFERENCES 1. Ampel, N. M., C. L. Dols, and J. N. Galgiani. 1990. Coccidioidomycosis among HIV-infected subjects. Program Abstr. 30th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1165. 2. Beaman, L., D. Pappagianis, and E. Benjamini. 1979. Mechanisms of resistance to infection with Coccidioides immitis in mice. Infect. Immun. 23:681-685. 3. Burnette, W. N. 1981. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112:195-203. 4. Calhoun, D. L., E. 0. Osir, K. 0. Dugger, J. N. Galgiani, and

INFECT. IMMUN.

5. 6.

7.

8.

9.

10. 11. 12.

13. 14.

15.

16. 17.

18. 19. 20.

21.

22.

23.

24.

J. H. Law. 1986. Humoral antibody responses to specific antigens of Coccidioides immitis. J. Infect. Dis. 154:265-272. Coalson, J. J., V. T. Winter, H. M. Martine, and R. J. King. 1986. Colloidal gold immunoultrastructural localization of rat surfactant. Am. Rev. Respir. Dis. 133:230-237. Cole, G. T., T. N. Kirkland, M. Franco, S. Zhu, L. Luan, S. H. Sun, and V. M. Hearn. 1988. Immunoreactivity of a surface wall fraction produced by spherules of Coccidioides immitis. Infect. Immun. 56:2695-2701. Cole, G. T., T. N. Kirkland, and S. H. Sun. 1987. An immunoreactive, water-soluble conidial wall fraction of Coccidioides immitis. Infect. Immun. 55:657-667. Cole, G. T., D. Kruse, S. Zhu, K. R. Seshan, and R. W. Wheat.

1990. Composition, serologic reactivity, and immunolocalization of a 120-kilodalton tube precipitin antigen of Coccidioides immitis. Infect. Immun. 58:179-188. Cole, G. T., S. H. Sun, J. Dominguez, L. Luan, M. Franco, and T. N. Kirkland. 1989. Wall-associated antigens of Coccidioides immitis, p. 395-415. In J. P. Latge, G. T. Cole, and E. Drouhet (ed.), Isolation, purification, and characterization of fungal antigens. Plenum Press, New York. Cox, R. A., and L. A. Britt. 1985. Antigenic heterogeneity of an alkali-soluble, water-soluble cell wall extract of Coccidioides immitis. Infect. Immun. 50:365-369. Cox, R. A., and L. A. Britt. 1986. Isolation and identification of an exoantigen that is specific for Coccidioides immitis. Infect. Immun. 52:138-143. Cox, R. A., and L. A. Britt. 1986. Isolation of a coccidioidin component that reacts with immunoglobulin M precipitin antibody. Infect. Immun. 53:449-453. Cox, R. A., L. A. Britt, and R. A. Michael. 1987. Isolation of Coccidioides immitis F antigen by immunoaffinity chromatography with monospecific antiserum. Infect. Immun. 55:227-232. Cox, R. A., E. Brummer, and G. Lecara. 1977. In vitro lymphocyte responses of coccidioidin skin test-positive and -negative persons to coccidioidin, spherulin, and a Coccidioides cell wall antigen. Infect. Immun. 15:751-755. Cox, R. A., C. G. Mead, and E. F. Pavey. 1981. Comparisons of mycelia- and spherule-derived antigens in cellular immune assays of Coccidioides immitis-infected guinea pigs. Infect. Immun. 31:687-692. Cox, R. A., and J. R. Vivas. 1977. Spectrum of in vivo and in vitro cell-mediated immune responses in coccidioidomycosis. Cell. Immunol. 31:130-141. Deresinski, S. C., R. J. Applegate, H. B. Levine, and D. A. Stevens. 1977. Cellular immunity to Coccidioides immitis: in vitro lymphocyte response to spherules, arthrospores, and endospores. Cell. Immunol. 32:110-119. Deresinski, S. C., and D. A. Stevens. 1974. Coccidioidomycosis in compromised hosts. Medicine (Baltimore) 54:377-395. Dubois, M., K. A. Gilles, P. A. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350-356. Eckhardt, A. E., C. S. Timpte, J. H. Abernethy, A. Toumadje, W. C. Johnson, Jr., and R. L. Hill. 1987. Structural properties of porcine submaxillary gland apomucin. J. Biol. Chem. 262: 11339-11344. Estes, D. M., J. W. Templeton, D. M. Hunter, and L. G. Adams. 1990. Production and use of murine monoclonal antibodies reactive with bovine IgM isotype and IgG subisotypes (IgGl, IgG2a, and IgG2b) in assessing immunoglobulin levels in serum of cattle. Vet. Immunol. Immunopathol. 25:61-72. Ferrante, A., and Y. H. Thong. 1978. A rapid one-step procedure for purification of mononuclear and polymorphonuclear leukocytes from human blood using a modification of the Hypaque-Ficoll technique. J. Immunol. Methods 24:389-393. Fish, D. G., N. M. Ampel, J. N. Galgiani, C. L. Dols, P. C. Kelly, C. H. Johnson, D. Pappagianis, J. E. Edwards, R. B. Wasserman, R. J. Clark, D. Antoniskis, R. A. Larsen, S. J. Englander, and E. A. Petersen. 1990. Coccidioidomycosis during human immunodeficiency virus (HIV) infection: a retrospective review of 77 patients. Medicine (Baltimore) 69:384-391. Galgiani, J. N., K. 0. Dugger, N. M. Ampel, S. H. Sun, and

VOL. 59, 1991

25. 26.

27. 28.

29.

30. 31. 32.

33. 34. 35. 36.

37.

J. H. Law. 1988. Extraction of serologic and delayed hypersensitivity antigens from spherules of Coccidioides immitis. Diagn. Microbiol. Infect. Dis. 11:65-80. Galgiani, J. N., K. 0. Dugger, J. I. Ito, and M. A. Wieden. 1984. Antigenemia in primary coccidioidomycosis. Am. J. Trop. Med. Hyg. 33:645-649. Galgiani, J. N., S. H. Sun, and G. G. Grace. 1990. Concanavalin A-bound spherule antigen (CAB) as an ELISA diagnostic reagent for coccidioidomycosis. Program Abstr. 30th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1196. Galgiani, J. N., and E. E. Wack. 1988. Coccidioidomycosis, p. 1785-1792. In A. P. Fishman, (ed.), Pulmonary diseases and disorders, 2nd ed. McGraw-Hill Book Co., New York. Horowitz, M. I. 1977. Purification of glycoproteins and criteria of purity, p. 15-34. In M. I. Horowitz and W. Pigman (ed.), The glycoconjugates: mammalian glycoproteins and glycolipids, vol. 1. Academic Press, Inc., New York. Huppert,.M., N. S. Spratt, K. R. Vukovich, S. H. Sun, and E. H. Rice. 1978. Antigenic analysis of coccidioidin and spherulin determined by two-dimensional immunoelectrophoresis. Infect. Immun. 20:541-551. Huppert, M., S. H. Sun, and J. L. Harrison. 1982. Morphogenesis throughout saprobic and parasitic cycles of Coccidioides immitis. Mycopathologia 78:107-122. Kirkland, T. N., and J. Fierer. 1983. Inbred mouse strains differ in resistance to lethal Coccidioides immitis infection. Infect. Immun. 40:912-916. Kruse, D., and G. T. Cole. 1990. Isolation of tube precipitin antibody-reactive fractions of Coccidioides immitis. Infect. Immun. 58:169-178. Mort, A. J., and D. T. A. Lamport. 1977. Anhydrous hydrogen fluoride deglycosylates glycoproteins. Anal. Biochem. 82:289309. Pappagianis, D., and B. L. Zimmer. Serology of coccidioidomycosis.'Clin. Microbiol. Rev. 3:247-268. Reiss, E., M. Huppert, and R. Cherniak. 1985. Characterization of protein 'and mannan polysaccharide' antigens of yeasts, moulds and actinomycetes. Curr. Top. Med. Mycol. 1:172-207. Resnick, S., B. Zimmer,' D. Pappagianis, A. Eakin, and J. McKerrow. 1990. Purification and amino-terminal sequence analysis of the complement-fixing and precipitin antigens from Coccidioides immitis. J. Clin. Microbiol. 28:385-388. Shively, J. E. 1986. Reverse-phase HPLC isolation and microsequencing, p. 60-64. in J. E. Shively', (ed.), Methods of protein

ANTIGENIC APOGLYCOPROTEIN FROM C. IMMITIS

38. 39.

40. 41.

42.

43. 44.

45. 46.

47.

48.

2251

microcharacterization. A practical handbook. Humana Press, Clifton, N.J. Smith, C. E., E. G. Whiting, E. E. Baker, H. G. Rosenberger, R. R. Beard, and M. T. Saito. 1948. The use of coccidioidin. Am. Rev. Tuberc. 57:330-360. Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk. 1985. Measurement of protein using bicinchonic acid. Anal. Biochem. 150:76-85. Sun, S. H., S. S. Sekhon, and M. Huppert. 1979. Electron microscopic studies of saprobic and parasitic forms of Coccidioides immitis. Sabouraudia 17:265-273. Thole, J. E. R., L. F. E. M. Stabel, M. E. G. Suykerbuyk, M. Y. L. De Wit, P. R. Klatser, A. H. J. Kolk, and R. A. Hartskeerl. 1990. A major immunogenic 36,000-molecularweight antigen form Mycobacterium leprae contains an immunoreactive region of proline-rich repeats. Infect. Immun. 58:8087. Timpte, C. S., A. E. Eckhardt, J. L. Abernathy, and R. L. Hill. 1988. Porcine submaxillary gland apomucin contains tandemly repeated, identical sequences of 81 residues. J. Biol. Chem. 263:1081-1088. Wagh, P. V., and 0. P. Bahl. 1981. Sugar residues on proteins. Crit. Rev. Biochem. 10:307-377. Weiden, M. A., J. N. Galgiani, and D. Pappagianis. 1983. Comparison of coccidioidal antibody quantitation by the complement fixation and the immunodiffusion methods. J. Clin. Microbiol. 18:529-534. Yuan, L., G. T. Cole, and S. H. Sun. 1988. Possible role of a proteinase in endosporulation of Coccidioides immitis. Infect. Immun. 56:1551-1559. Zimmer, B. L., and D. Pappagianis. 1986. Comparison of immunoblot analyses of spherule-endospore-phase extracellular protein and mycelial-phase antigen of Coccidioides immitis. Infect. Immun. 53:64-70. Zimmer, B. L., and D. Pappagianis. 1988. Characterization of a soluble protein of Coccidioides immitis with activity as an immunodiffusion-complement fixation antigen. J. Clin. Microbiol. 26:2250-2256. Zimmer, B. L., and D. Pappagianis. 1989. Immunoaffinity isolation and partial characterization of the Coccidioides immitis antigen detected by the tube precipitin and immunodiffusiontube precipitin tests. J. Clin. Microbiol. 27:1759-1766.