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Purification and Partial Characterization of Aspermatogenic Antigen*
FERTIUTY AND STERILITY
Copyright
@
Vol. 23, No.9, September 1972
1972 by The Williams & Wilkins Co.
Printed in U.S.A.
PURIFICATION AND PARTIAL CHARACTERIZATION OF ASPERMATOGENIC ANTIGEN* SEYMOUR KATSH, PH.D., ANTHONY R. AGUIRRF.. M.S., FREDRICK W. LEAVER, PH.D., AND GRACE F. KATSH, PH.D. Department of Pharmacology, University of Colorado Medical School, Denver, Colorado 80220
Activity in the area of immunoreproduction has intensified in recent years. 1 - S Perusal of the literature will reveal that it is possible to induce infertility in male and female experimental animals by immunization. Moreover, certain cases of idiopathic infertility in humans may be attributable to immune responses. Despite these advances, several key problems remain to be resolved if major progress is to be made in controlling fertility by immunologic methods and in correcting infertility attributable to immune responses. Prominent among these problems is the imperative one of extracting, purifying, and characterizing antigens causing infertility. Until even one such antigen has been identified chemically and immunologically, the entire field of immunoreproduction will continue to languish in the speculative limbo in which it has been cast for the past 70 years since Landsteiner, Metchnikoff, and Metchnikoff (see reviews) first described the antigenicity of sperm. Without such information, critical data relative to cross-reactivity and uniqueness of reproductive antigens, for example, will be lacking. Further, it will not be possible to determine the nature of the antibodies involved. Moreover, without such information, one can only speculate as to mechanisms of action. Therefore, studies were undertaken to isolate, purify, and, at least, partially characterize one reproductive antigen. Initiation of Received January 17, 1972; revised April 21, 1972. * Supported by funds from the National Science Foundation (GB 31555X) and the Ford Foundation.
studies with this goal in mind requires selection of the appropriate experimental model. The aspermatogenic syndrome induced in guinea pigs was selected because it has been well characterized and because it appears to provide a laboratory model for certain clinical cases of testicular dyscrasia. MATERIALS AND METHODS
The following abbreviations are used in this report: C.ASA (crude aspermatogenic antigen) refers to the material extracted from tissues prior to digestion with pepsin. P.D.ASA (pepsin digested aspermatogenic antigen) is C.ASA which has been subjected to digestion with pepsin. F.2aASA refers to a biologically active material obtained after two passages through Sephadex G-50 of P.D.ASA. A.E.ASA refers to F.2aASA which has been precipitated with antibody and eluted from the antigen-antibody complex.
Extraction of C.ASA
From Testes. Frozen guinea pig testes (obtained from commercial vendors or from previously sacrificed animals) were decapsulated and the fat pads as well as the epididymides removed. Decapsulated testes (150 gm./batch) were homogenized for 1 min. at high speed in a Waring blendor with an equal volume of 0.1 M acetic acid. The homogenate was stirred overnight at 4 0 C. and centrifuged for 20 min. at 8000 g; the supernatant solution was reserved and the precipitate rehomogenized in 0.1 M acetic acid (50%
644
September 1972
PURIFICATION OF ASPERMATOGENIC ANTIGEN
645
of the original volume), stirred for 2 hr. of Sephadex gels ranging from G-50-G-2oo. and recentifuged as before. This process From these preliminary experiments, it was repeated once more before discarding was determined that optimal fractionathe precipitate. To the combined super- tion was obtained by an 18-hr. pepsin natants was added enough 30% trichloro- digest followed by filtration on G-50. Sephadex Fractionation. P.D.ASA, acetic acid (TCA) to bring the final TCA mg., was dissolved in 2 ml. of 100-150 concentration to 5%, the solution was 0.09 M acetic acid pH 3.0 and loaded on allowed to stand in the cold for 1 hr. and 85 cm. column of Sephadex G-50 a 1.5 x was then centrifuged for 10 min. at 8000 g. (Fine). The sample was washed on with The precipitates were washed twice with and eluted with 0.09 M 1 ml. of acetic acid small portions of 5% TCA, were freed acetic acid; 2-ml. fractions were collected from dialyzable impurities by overnight at a flow rate of 10-12 ml./hr. The column dialysis against running tap water and effluents were monitored by Ultraviolet residual TCA and possible lipids were reat 280 m~. with a Model UV absorption moved by extraction with 1/4 volume of 280 IF recording ultraviolet absorption N-butanol (three times) and chloroform (three times). The resulting aqueous meter and collected by a Gilson Medical solution on lyophilization gave a white Electronics Model LB-1 fractionator. fluffy material which could be stored at Four peaks were observed in the column room temperature for several years with- effluent; the tubes were pooled into five fractions numbered from I-V, Fraction ill out loss of biologic activity. From Epididymides. Frozen epididy- being the overlap region between the two mides (200-400 gm.) were allowed to major peaks. The fractions were lyophipartially thaw and then homogenized lized and tested for antigenic activity using with 1.5 volumes of acetone (Waring the passive hemagglutination (PHA) inhiblendor, 5 min. at high speed), the homog- bition test (below). The fractions (II and III) showing high enate was filtered under vacuum and the residue homogenized with 1.5 volumes PHA activity (Table 1) were pooled, reof chloroform. After filtration, the residue dissolved in 0.09 M acetic acid, and rerun was again treated with acetone, filtered and allowed to air dry. The dry material TABLE 1. Purification of Aspermatogenic Antigen TesteS Epididymis (33-35 gm.) was homogenized with 10 volumes of 0.1 M acetic acid and stirred Treatment PHA PHA inhibition· BioasSayt inhibition· Bioassayt overnight at 4 0 C. Subsequent steps in the procedure were the same as for testes. 11-14 mg. 30-40 mg. Extract before pep+ + sin digest Pepsin Digest. Lyophilized C.ASA 2 6 Pepein + + (from testes or epididymides) was dis- Sepbsdex G-50 121 60 ± Fraction I 0* solved in 5.0 M acetic acid at a concen4 0.9 Fraction II + + tration of 5 mg./ml. The solution was 0.3 10.0 Fraction ill + + 0.9 0 70 Fraction IV + brought to 30 0 C. on a temperature-con0 0 Fraction V trolled magnetic stirrer (Sargent Model Recycled fractions 5.0 3.0 + la + T.E.) and crystalline pepsin added (2% 0.2 0.2 2a + + of C.ASA weight). Digestions were carried 3.0 11.0 + 3a + 0.10 0.09 + + out for intervals of 4, 8, 18, and 24 hr. Eluted from complex • Defmed as milligrams of material required to neutralize 1 ml. of Then each solution was immediately antiserum titering 10-. frozen and lyophilized. Each preparation t Causes aspermatogenesis, +. Partial damage to testes, ±. *Does not cause aspermatogenesis, O. was fractionated on 0.9 x 30 cm. columns
646
KATSH ET AL.
on the same column. The increased concentration of antigenic material at this point permitted the direct assay of tubes by PHA inhibition. This test showed outstanding correlation with the biologic results. The use of optical density at 280 m~ in the original monitoring is really fortuitous since measurements of highly active fractions show only modest density at 280 m~ (low aromatics) and the tubes with maximum density are practically void of aspermatogenic activity. When column eluates were assayed by PHA inhibition, the antigen was eluted as a single sharp peak which was pooled and lyophilized. This material was designated F .2aASA. Passive Hemagglutination (PHA) Tests. Passive hemagglutination (PHA) tests were performed to determine the titers of antibody preparations as described elsewhere. 6 PHA Inhibition Tests. Previously titered antiserum obtained from aspermatogenic guinea pigs 8 weeks after immunization was diluted with phosphate-saline buffer pH 5.9-6.1 to give a titer of 10,000. If the initial titration was greater than 1 million, the antiserum was diluted 1: 100 with phosphate-saline and then further diluted with normal guinea pig serum (diluted 1: 100 with phosphate-saline) to give a titer of 10,000. ThE:n 0.025 ml. of diluted antiserum was added to each well. A weighed amount (0.2-20 mg., depending on purity) of antigen was dissolved in 1.0 ml. of phosphate-saline and serially diluted in the antiserum. The dilution at which a dot failed to form was considered the end-point. From this dilution the number of milligrams of antigen required to neutralize 1 ml. of serum which titered 1 x 106 was calculated. Thus, lower numbers indicate increasing antigen activity. Column eluates were directly titrated as follows: 0.1 M phosphate buffer was prepared and adjusted such that addition
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of an equal volume of 0.09 M acetic acid would give a pH of 6.0-6.1. The first well of each series of dilutions contained 0.025 ml. of buffer in addition to the 0.025 ml. of antiserum; a 0.025 ml. aliquot from each tube to be assayed was then serially diluted in the antiserum. Biologic Assay of ASA. Depending on the stage of purification, 0.5-10.0 mg. of ASA were dissolved in 0.5 ml. of physiologic saline and emulsified with 0.5 ml. of complete Freund's adjuvant. The emulsion was injected intracutaneously in six sites into the shaved nuchal region of mature (500-600 gm.) male guinea pigs (Hartley strain). After 8 weeks, the animals were sacrificed by exsanguination and the testes excised and weighed. A weight of 1.2 gm. or less was indicative of complete deletion of the spermatogenic cell line as confirmed by histology. Preparation of Antibody to ASA. Guinea pigs injected 8 weeks previously with C.ASA were exsanguinated and the blood allowed to clot overnight at 4 0 C. in centrifuge tubes. After rimming the tubes, the blood was centrifuged for 20 min. at 3000 g, the serum obtained was titered and utilized for further purification only if the titers exceeded 100,000. The serum was mixed with an equal volume of a saturated ammonium sulfate solution, allowed to stand at room temperature for 10 min., then centrifuged for 5 min. at 1000 g. The precipitate was dissolved in distilled water (50% of the original serum volume) and again precipitated with ammonium sulfate and centrifuged. This process was repeated once more, the final precipitate was dissolved in distilled water and was dialyzed for 36 hr. against distilled water. The dialysate was clarified by centrifugation and then adjusted to pH 5.9 with 0.1 M acetic acid. After standing at room temperature briefly, the mixture was centrifuged and the supernatant was lyophilized. The material thus obtained corresponds to
September 1972
PURIFICATION OF ASPERMATOGENIC ANTIGEN
the pseudoglobulin fraction of serum proteins. Precipitation of Antigen-Antibody Complex. The lyophilized antibody preparation was dissolved in distilled water, the pH adjusted to 6.1 (final volume equal to that of the serum from which the antibody was obtained), and the solution titered by PHA. A known weight of F.2aASA was dissolved in distilled water (generally 0.2-0.3 mg./ml.) and also titered by PHA inhibition. From both titrations, the amount of antigen required to neutralize the antibody at hand was calculated, and this amount of antigen was dissolved in distilled water and adjusted to pH 6.1, the final volume of the antigen solution being held to 10% of the volume of the antibody solution. An aliquot of the antigen solution (about 25%) was mixed with an appropriate amount of pseudoglobulin which neutralized at 100% (see above) and allowed to remain at room temperature until a precipitate formed (at least 1 hr.), after which the solution was centrifuged at 3000 g for 10 min. and the supernatant retitered (PHA). The procedure was repeated with further aliquots of antigen solution until the supernatant indicated slight antigen excess (PHA inhibition). The pooled precipitates were then washed twice with 5- to 10-ml. portions of distilled water, dissolved in 2 ml. of 0.1 M acetic acid to dissociate the antigen-antibody complex 7 and immediately loaded on a 2.5 x 60 cm. column of Sephadex G-75. Fractions (4 ml.) were collected by eluting with 0.1 M acetic acid, the eluates being monitored by 280 m,u absorption and by PHA inhibition. The antibody was recovered as a sharp peak in the void volume, while the antigen (A.E.ASA) was eluted as a broad peak in the bed volume. The recovered antibody was dialyzed against distilled water until neutral and could then be utilized for further precipitations with antigen. This recovered antibody
647
was stored under refrigeration (4 0 C., thymol). Further purification of the A.E.ASA was obtained by reprecipitating the antigen with the appropriate amount of pseudoglobulin (see above) and then redissolving in 0.1 M acetic acid and filtering through a 0.025 ,u Millipore filter (Catalog listing VSWPO). Estimation of the Molecular Weight of ASA. The elution volume of F.2aASA was determined during the various fractionation runs on Sephadex G-50 (see above). The same column used for the fractionation of P.D.ASA was calibrated by applying various proteins and polypeptides of known molecular weight to the column and eluting them under the same conditions used for ASA. The markers used were pig thyroglobulin (Kit. No. 8109, Mann Research Laboratories), ribonuclease b, cytochrome c, adrenocorticotropic hormone (ACTH), and glucagon (all from Sigma Chemical Company). Acrylamide Gel Electrophoresis. All procedures were carried out using the Canalco Research Disc Electrophoresis apparatus with a Buchler D.C. Power Supply Catalog No. 3-1014. The tubes used were 5 mm. i.d. and 75 mm. long in which separating, stacking, and sample gels were formulated. Separating gel (15%, pH 4.3) 1 part A; 2 parts B; 1 part water. Solution A: 48 ml. of 1 M KOH, 17.2 ml. of glacial acetic acid, 4.0 ml. of N, N, Nl, Nl-tetramethyl ethylenediamine (TEMED) all diluted to 100 ml. with water. Solution B: 60 gm. of acrylamide; 0.4 gm. of N, Nl_ methylenebisacrylamide (Bis) diluted to 100 ml. with water. The four parts of solution described were mixed with four parts of catalyst (0.28% ammonium persulfate), 1.2-1.5 ml. added to each tube and allowed to polymerize for 30 min. Stacking and sample gels 1 part C; 1 part D; 1 part catalyst; 5 parts water. Solution C: 48 ml. of 1 M KOH, 2.87 ml. of glacial acetic acid, 0.46 ml. of TEMED all di-
648
KATSH ET AL.
Vol. 23
luted to 100 ml. with water (pH 6.7). Enzyme Preparations. Amidase, (l-asSolution D: 20 gm. of acrylamide, 5.0 gm. partamido-B-N -acetylglucosamine amido of Bis, water to 100 ml. Catalyst: 4.0 mg. % hydrolase) was prepared from rat liver by of riboflavin. The stacking gel was added the procedure of Conchie and Strachan. 9 in 0.2-ml. aliquots to each tube and photo- The synthetic substrate used for the aspolymerized. Sample was then added say of this enzyme was 2-acetamido-1-B (200 p,g. in 5 p,l. of water), mixed with (L-B aspartamido)-1,2-dideoxy-n-glucose, 0.2 ml. of sample gel, and polymerized. obtained from the Cyc10 Chemical ComThe electrophoresis buffer was prepared pany, Los Angeles, California. Pepsin was as follows: 31.2 gm. of B-alanine, 8.0 ml. obtained from the Sigma Chemical Comof glacial acetic acid and water to 1000 pany, and almond emulsin (B-glucosidase) ml., pH 5.0. The solution was diluted 1 : 10 from Mann Research Laboratories. Sugar Analyses. Total neutral sugars before use. Electrophoresis was performed with were determined by the anthrone method the tubes in a vertical position, sample gel of Roe. 10 Fucose was assayed by the at the top (anode), separating gel at the Dische-Shettles 11 cysteine-sulfuric acid bottom (cathode). Methyl green, 1-2 ml. reaction, reducing sugars were determined of 0.005%, was added to the top buffer by the Park-Johnson 12 ferricyanide techreservoir as a tracking dye. Electrophore- nic, and acetylated amine sugars were sis was continued with a current of 5 ma.! assayed according to Levvy and Mctube until the green dye band had just Allan.13 The complete sugar analysis was run off the end of the gels. The gels performed by vapor-phase chromatogwere then removed and stained with 0.5% raphy according to a modification of the aniline blue-black in 7% acetic acid for procedure of Sweeley, Wells, and Bent1 hr. after which they were electrophoreti- ley. 14 cally destained in 7% acetic acid using a Enzyme Degradation of Carbohydrate Canalco quick gel destainer. Gels were Moiety. F.2aASA (9.2 mg.) was dissolved scanned at 560 mp, using a Gilford Model in 1.0 ml. of 0.1 M Na-acetate buffer, pH 2000 spectrophotometer-recorder with a 4.2; 3.0 mg. of almond emulsin 15 Model 2410 linear transport. Slit width were added (together with 50 p,l. of for scanning was 0.2 mm. toluene to prevent bacterial growth), and Immunoelectrophoresis was performed the solution incubated at 37° C. Aliquots on 25 x 75 mm. microscope slides coated (0.1 ml.) were removed at 25, 43, 68, and with 1% agarose in 0.015 M barbital buffer 96 hr. and spotted on Whatman No.1 pH 7.0. The electrophoresis buffer was paper which had been washed for 2 days the same as that used to dissolve the in n-butanol-ethanol-water (10: 1: 2) and agarose. Antigen (F.2aASA or A.E.ASA, then for 2 days in distilled water, and sub75-100 p,g.) was dissolved in 3 p,l. of jected to descending chromatography in buffer and placed in the wells, the slides the same solvent for 96 hr. The spots were then electrophoresed for 2 hr. in a were visualized with alkaline silver ni12.5 volt/cm. gradient. Following electro- trate. 16 Another sheet was run in parallel phoresis, the troughs were charged with with the first. On this sheet only the inantibody solution and diffusion carried dividual standards were visualized; the out in a moist chamber for 6-8 hr. at room spotted aliquots as well as a standard temperature. The slides were washed mixture consisting of 20 p,g. of galactose, overnight in distilled water, dried, and 15.5 p,g. of mannose, and 13.6 p,g. of stained with Azocarmine b according to N-acetylglucosamine were left unstained. the procedure of Uriel. 8 Areas of the sheet corresponding to the
September 1972
PURIFICATION OF ASPERMATOGENIC ANTIGEN
visualized standards were cut out, divided into small pieces, and eluted overnight in 5 ml. of distilled water. The solutions obtained were analyzed for reducing sugar.12 Recoveries were calculated with reference to the standard mixture. Amino acid analysis was performed by standard procedures. 17 Glycopeptide Linkage Investigations. (a) Treatment with amidase: 5.0 mg. of F.2aASA and 2.0 mg. of A.E.ASA were separately dissolved in 2.5 ml. of distilled water; to each of these were added 1.25 ml. of 0.2 M Na2HP04-KH2P04 buffer pH 7.0, and 1.25 ml. of an amidase preparation assaying 4.26 ~M N-acetylhexosamine released per hour per milliliter. The solution was incubated at 37° C.; 1-ml. aliquots were removed at 4, 7, 18, and 24 hr. The reaction was stopped by boiling for 3 min.; the mixture was centrifuged at 1500 g for 10 min. The supernatant was then assayed for N-acetylhexosamine content. (b) Alkaline hydrolysis: 11.2 mg. of F.2aASA and 1.229 mg. of A.E.ASA were separately dissolved in 2 ml. of 0.5 N NaOH and left for 15 hr. at 4° C. The solutions were neutralized with 1 N HCl and an aliquot assayed for N-acetylhexosamine content. Aliquots of the remainder of the solutions were assayed by PHA inhibition to determine if loss of activity had occurred. The balance of the F .2aASA hydrolyzate was placed in overnight dialysis against distilled water. The bag contents and outer fluid were assayed for neutral sugar content. As controls for this experiment, 0.55 mg. (1.58 ~M) of the synthetic aspartyl-glucosylamine (a model for the N-glucosidic bond) was also subjected to alkaline hydrolysis. Another control consisted of dialyzing a sample of F.2aASA which had not been hydrolyzed. (c) Treatment with lithium borohydride: 1.2 mg. of A.E.ASA were dissolved in 2 ml. of 0.3 M LiBH4 in tetrahydrofuran and the solution was refluxed for 8 hr. After cooling, the reaction was ter-
649
minated by addition of 0.8 ml. of 1 N methanolic HCl and the solution lyophilized. The dried material was redissolved in water and analyzed for N-acetylglucosamine content. RESULTS
Studies in this laboratory on the induction of aspermatogenesis and the biochemical consequences thereof have heretofore involved the use of relatively crude aspermatogenic antigen (C.ASA). This material is biologically active when injected in milligram quantities. Upon assay by the PHA inhibition test, activities of 30-40 mg. for testicular preparations and 11-14 mg. for epididymal preparations are observed. Further purification of C.ASA has now been achieved. Table 1 shows 440and 140-fold increases in activity, respectively, for testicular and epididymal preparations, based on PHA inhibition titers. Two criteria were used to assess biologic activity and/or degree of purification throughout the procedures described. All products were injected into guinea pigs to test their ability to produce aspermatogenesis. Complete aspermatogenesis (as determined by histology) was characterized by a testicular weight of 0.8-1.2 gm. (normal weight = 3.5-4.5 gm.). In addition, all products were also tested by the PHA inhibition test. It was found that preparations which gave values below 60 mg. in this test invariably caused aspermatogenesis. Filtration on Sephadex gels as well as ion-exchange resins of various types (DEAE cellulose, Dowex 1, Dowex 50, and QAE-Sephadex) failed to fractionate C.ASA since it was removed as a single large peak (by absorption at 280 m~). Similar results were obtained following hydrolysis with 1 N HCI for 4 hr. at 37° C. and with 1 N acetic acid for 18 hr. at 102° C. A 4-hr. hydrolysis with pepsin at 37° C. gave a pattern of three poorly re-
650
Vol. 23
KATSH ET AL.
solved peaks upon filtration through Sephadex G-50 with the biologic activity being found in the second peak. The point of maximal degradation with pepsin was determined by digestion for different periods of time, and the 18-hr. interval was chosen since digestion for longer periods failed to produce any further changes in the elution pattern obtained from Sephadex G-50. Filtration of P.D.ASA on gels ranging from G-75 through G-200 failed to markedly improve the peak resolution over that obtained with G-50. Consequently, the final conditions chosen were those described in "Materials and Methods." The type of elution diagram routinely obtained from a fractionation of either testicular or epididymal P.D.ASA is shown in Fig. 1. PHA inhibition titers obtained on the lyophilized fractions are shown in Table 1. Dealing first with testicular preparations: Fraction I showed slight activity, Fractions II and III caused full aspermatogenesis, and Fractions IV and V were devoid of aspermatogenic activity. Epididymal preparations exhibit a slightly different pattern in that Fractions I and V are inactive; activity was concentrated in Fractions III and IV. The activities in the epididymal Fractions III and IV were considerably higher than those of the testicular preparations. Those peaks which showed a higher activity (according to PHA inhibition) than the P.D.ASA were routinely pooled and rerun on the same column. The elution pattern thus obtained showed only two large central peaks (Fractions I and V were eliminated) when monitored by 280 m~ absorption. However, when the tubes were assayed by PHA inhibition, only a single peak was obtained (shown by dashes in Fig. 1). Fractions la and 3a in Table 1 and Fig. 1 represent the initial and final portions of the antigen peak, respectively. Fraction 2a represents that portion of the peak showing a reciprocal
003
~
I I
~
>- 002
l-
v;
2:
~
--' <:(
U
00
I I~ 1II
0(
0
"Sl
i" \
f::
Q
I
101:20\130
.I 80
\. 100
120
ELUTION VOLUME (m/)
FIG. 1. Fractionation of P.D.ASA on a 1.5 x 90 cm. column of Sephadex G·50 in 0.09 M acetic acid. 0-0-0, fractions monitored by absorption at 280 mIL. e--e--e, fractions monitored by PHA inhibition test. The ordinate indicates the fold dilution at end· point.
titer greater than 16 by PHA inhibition. After preliminary determinations of purity by gel electrophoresis and immunoelectrophoresis, this fraction (F.2aASA) was further purified by precipitation with antibody. Acrylamide Gel Electrophoresis. Electrophoresis separation of F.2aASA gave a pattern of six to seven bands moving towards the cathode (Fig. 2, left). These bands were eluted from unstained gels and tested by PHA. It was found that the band nearest the anode was the only one which gave a positive PHA inhibition test. Furthermore, gel electrophoresis of the antigen recovered from the antigenantibody complex (A.E.ASA) gave only one band of identical mobility to the slowmoving band in the electropherogram of F.2aASA. (Fig. 2, right.) This material caused aspermatogenesis. Immunoelectrophoresis. The patterns obtained with F.2aASA showed all of the material moving towards the cathode. After reaction with and elution from antibody, only one precipitin band was obtained (Fig. 3). Estimation of Molecular Weight of Antigen. F.2aASA was eluted from Sephadex G-50 as a sharp peak which permitted accurate determination of the elution volume. Calibration of the column
I
September 1972
PURIFICATION OF ASPERMATOGENIC ANTIGEN
0050
.50
IMO
IMO
.30
o~o
.
::
i
:
!
~
i
651
! 0020
0020
0010
0·10
Dlstlnce towards Clthode In em
Distance towards cathode in em
FIG. 2. Acrylamide gel electrophoresis scans of F.2aASA and A.E.ASA. Left, F.2aASA. Right, A.E.ASA.
FIG. 3. Immunoelectrophoretic analysis of F.2aASA. Trough filled with antibody solution. Upper and lower wells filled with F.2aASA. Cathode to the right.
with a series of peptides and proteins of known molecular weight (Table 2) and the calibration curve subsequently obtained (Fig. 4) permitted the estimation of a molecular weight range approximating 11,000-14,000 for the antigen. Amino Acid and Carbohydrate Analyses. The results obtained from the carbo-
hydrate and amino acid analyses of F.2aASA and A.E.ASA are shown in Tables 3 and 4. Emulsin Digest. In order to gain some insight as to the sequence of attachment of the carbohydrate residues, F .2aASA was digested with almond emulsin. This is a crude enzyme preparation composed
652
TABLE 4. Amino Acid Analysis of Aspermatogenic Antigen
TABLE 2. Gel Filtration of Molecular Weight Markers and F.2aASA on Sephadex G-50 PrOtein
Molecular weight
Elution volume
Elution volume/ Void
mi.
Thyroglobulin * Ribonuclease b Cytochrome c ACTH Glucagon ASA
42.5 1.00 670,OOOt 14,700" 81.3 1.91 13,400" 83.5 1.96 5,250'· 2.18 93.0 3,670'· 96.3 2.27 12,600:j: 84.0 1.97 * Thyroglobulin was assumed to be completely excluded from the gel. Hence, void volume = elution volume. t Obtained from Mann Research Laboratories Molecular Weight Markers Kit. :j: Determined from Fig. 4.
THYROGlOOULN
~~O----------74.on---~----~50~--------~60
LOG" MOlECULAR WEIGHT
FIG. 4. Estimation of molecular weight of aspermatogenic antigen on Sephadex G-50 (see Table 2). TABLE 3. Carbohydrate Analysis of Aspermatogenic Antigen F.2aASA Total neutral sugars (Anthrone) Total sugars Fucose
A.E.ASA
4.2%
10.46%
5.07%" 0.43%
12.67% (V.P.C.) 0
Molar ratiot
Distribution of monosaccharide residues (as percentage of total carbohydrate)
Fucose Galactose Glucosamine N·Acetylglucosamine N·Acetylgaiactosa·
9.26 35.31 30.95 7.35 12.94 3.39
mine N-Acetylneuraminic
0.79
Mannose
0 32.75 42.15 0 24.94 0
2.90 (3)* 3.73 (4) 1.80 (2)
0
acid ... Based on anthrone results and carbohydrate distribution by V.P.C. t Based on carbohydrate content of 12.67% and molecular weight of 12,600.
t Numbers
in parentheses are the nearest integral number of resi-
A.E.ASA
F.2aASA
volume
dues.
Vol. 23
KATSH ET AL.
Ala Arg Asp Cys Glu Gly His He Leu Lys Met Phe Pro Ser Thr Tyr Val
pM/mg.
Residues
I'M/mg.
Residues
1.298 0.414 0.810 0.247 1.482 0.931 0.221 0.210 0.489 1.116 0.144 0.160 0.841 0.848 0.624 0.094 0.653
12.27 3.91 7.65 2.33 14.00 8.78 2.09 1.98 4.62 10.55 1.36 1.51 7.95 8.01 5.90 8.88 6.17
0.587 0.328 0.650 0.397 0.940 0.690 0.250 0.190 0.561 0.518 0.095 0.173 0.733 0.569 0.431 0.285 0.569
7.38 4.10 8.11 4.96 11.85 8.70 3.09 2.35 7.01 6.51 1.19 2.12 9.23 7.22 5.39 3.59 7.18
Total
107.96
Total
99.98
of a mixture of mannosidase, galactosidase' and N-acetylglucosaminidase which sequentially releases sugar residues from the nonreducing end of a polysaccharide. The results obtained suggest that the galactose residues are located at the nonreducing end of the polysaccharide chain. Further work is necessary to elucidate this point. The results of emulsin digestion also revealed that all sugars were released. Nature of the Glycopeptide Linkage. Three main types of linkage between the carbohydrate and peptide moieties of glycoproteins in general have been described: 21 - 23 (a) an N-glycosidic linkage between N-acetylglucosamine and the amide nitrogen of asparagine; (b) the hydroxyl group of serine or threonine in an O-glycosidic bond with C-l of a sugar; and (c) glycosidic ester linkages. NGlycosidic linkages are generally degraded by amidase. 24 - 26 This enzyme failed to cause the release of detectable N-acetylhexosamine from any of the antigen preparations. This cannot, however, be re-
September 1972
PURIFICATION OF ASPERMATOGENIC ANTIGEN
garded as conclusive evidence for the absence of this type of bond. Makino, Kojima, and Yamashina 26 have shown, for example, that ovalbumin was not attacked by amidase, whereas a glycopeptide prepared from it was cleaved. It is possible, then, that amidase alone cannot release N-acetylhexosamine unless the attached carbohydrate chain is removed. The results with emulsin digestion provide such evidence. Thus, in order to obtain further information of linkage of carbohydrate to the peptide, hydrolysis by NaOH was performed because Nuenke and Cunningham 27 have shown the N -glycosidic linkage to be stable to alkaline hydrolysis. We have confirmed these results employing synthetic aspartylglucosylamine as a model for the linkage. On the other hand, 0glycosidic linkages and ester linkages are subject to alkaline hydrolysis. When A.E.ASA was treated with NaOH, 46% of the N-acetylglucosamine was released with a loss of 98% of the PHA inhibition activity. This indicates that one of the two N-acetylglucosamines is subject to alkaline hydrolysis and the presence of this bond is necessary for PHA inhibition activity. However, one N-acetylglucosamine is not readily hydrolyzed by NaOH, suggesting that it is involved in an N-glycosidic linkage. The observation that treatment with LiBH4 failed to release any N-acetylglucosamine further indicates the absence of ester linkages. DISCUSSION
Throughout these studies the biologic activity of the antigen at any given stage of purification has been determined by injection into guinea pigs. It was necessary, however, to have some means of screening for biologic activity as well as degree of purification. Within limits, the PHA inhibition test provides a way of
653
doing this. It must be recognized that the test is a subjective one, in that the endpoint is determined by the first well in which a dot fails to form; thus one could expect a variation of at least one well to either side of the chosen end-point. In addition, the effectiveness of the coated red cells can vary considerably amongst preparations. These limitations have been controlled as far as possible by running many samples with a given cell preparation, by having only one operator perform the PHA tests, and by coding so that the operator was unaware of which preparation was under test. Regarding the precipitation of the antigen-antibody complex and elution of antigen therefrom, we have found it necessary to proceed with the elution immediately following precipitation and processing of the complex. Delays result in incomplete separation of antigen from antibody. The emulsin digestion was performed on F .2aASA which was still a relatively crude preparation. The results obtained must therefore be regarded as tentative, subject to confirmation by digestion of the purified antigen with pure preparations of the individual glycosidases. The results do strongly suggest, however, that the galactose residues are found at the nonreducing end of the polysaccharide chain. Reference to Table 3 indicates that the ratios in which galactose, mannose, and N-acetylglucosamine were released by emulsin are very close to the ratios found for these three sugars by gas chromatographic analysis of F.2aASA. The comparative carbohydrate and amino acid contents of F .2aASA and the A.E.ASA (Tables 3 and 4) indicate a considerable difference in chemical composition; the results obtained with F.2aASA no doubt reflect contamination of the antigen by other glycopeptides. The composition found for the antigen eluted
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KATSH ET AL.
from the antibody is consistent with the molecular weight obtained by Sephadex filtration. The values obtained for the carbohydrate content, based on a molecular weight of 12,600 indicate the presence of three mannose residues, four galactose residues, and two N -acetylglucosamine residues. The release of 46% of the N-acetylglucosamine by alkaline hydrolysis permits the inference that one of the two postulated residues of this sugar is located within the polysaccharide chain, and therefore not susceptible to alkaline hydrolysis. A carbohydrate content of approximately 13% is consistent with the great solubility of the antigen in aqueous solvents. The amino acid content of the antigen merits some discussion. The small number of aromatic amino acids explains the low absorption at 280 mJL which we have observed. In order to account for a slight mobility of A.E.ASA towards the cathode, the dicarboxylic amino acids must be present predominantly as their amide derivatives. Further detailed determinations are necessary regarding the carbohydrate sequence, the residues involved in the glycopeptide linkage, and the amino acid sequence. These studies are now under way. Of critical importance in further studies is the purification of antibody to ASA as well as an evaluation of the differences (if any) between the antigen which is injected into the animals, and the antigen released from the testes during the autoimmune phase of the aspermatogenic response. In this connection, it is pertinent to recall the hypothesis presented which accounts for the genesis of experimentally induced autoimmune aspermatogenesis. 28 Briefly, the injection of specific antigen in adjuvant elicits antibodies which interact with antigen-
Vol. 23
containing cells (in testes and epididymides), causing release of antigen. The released antigen incites further antibody formation until all endogenous antigenic units have been released. Confirmatory evidence including antibody titers has been presented. 6 It now becomes possible to determine if the "native" antigen released during the immune response is or is not identical with the extracted antigen by eluting the "native" antigen from circulating antigen-antibody complex. With respect to purification and characterization of reproductive antigens in general, very little appears in the literature. So far only the aspermatogenic antigen has been accorded consistent effort. For example, Brown, Glynn, and Holborrow 29 and Brown, Holborrow, and Glynn 30 employed papain digestion and phenol extraction in attempts to purify the antigen. Katsh and Katsh 31 employed enzymatic and electrophoresis experiments to note the presence of polypeptide and polysaccharide. Kirkpatrick and Katsh 32 noted the amino acid content of the antigen. Katsh, Aguirre, and Katsh 33 were able to demonstrate inactivation of antigen by enzymes and by tissues and sera. Katsh 5 noted that pepsin could be employed as a first important step in purification of the antigen. Voisin and Toullet 34 have separated four antigens from sperm of which two appeared to be involved in aspermatogenesis. We believe the material described in this report is of far greater purity than any yet obtained and that this material is of sufficient purity to warrant extensive investigation of its chemical structure. The possibility of extracting a lower molecular weight aspermatogenic antigen is already under study. These studies are considered significant from the point of view of application to reproductive antigens of human origin. Ultimately, it is the objective to isolate and characterize not only
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PURIFICATION OF ASPERMATOGENIC ANTIGEN
aspermatogenic antigen but also immobilizing and agglutinating antigens. SUMMARY
Aspermatogenic antigen has been obtained from guinea pig testes and epididymides by extraction of the tissues with acetic acid, precipitation with trichloroacetic acid and extraction with butanolchloroform. The material thus obtained has been purified by digestion with pepsin and fractionation on Sephadex G-50 followed by precipitation with antibody and elution from the antigen-antibody complex. This material induces autoimmune aspermatogenesis in guinea pigs when injected in microgram amounts with Freund complete adjuvant. The purified antigen shows a single band on acrylamide gel electrophoresis, yields a single band upon immunoelectrophoresis, and has an estimated molecular weight of approximately 12,600. The antigen is a glycopeptide containing approximately 13% carbohydrate (galactose, mannose, and N-acetylglucosamine). Information is provided that the galactose residues appear to be at the nonreducing end of the polysaccharide. Suggestive evidence is provided that the polysaccharide moiety is attached to a polypeptide of 100 residues through an N-glycosidic linkage. The importance of these studies relates to the groundwork being laid for extraction, purification, and characterization of reproductive antigens of human origin. Acknowledgments. The authors would like to thank Drs. Sanford Markey and Lewis Johnson for performing the carbohydrate analyses by vapor-phase chromatography, and Drs. Ernest Borek and Barry Starcher for the amino acid analyses. REFERENCES 1. KATSH, S. Immunology, fertility and infertility: A historical survey. Amer J Obstet Gynec 77:946, 1959.
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2. TYLER, A. Approaches to the control of fertility based on immunological phenomena. J Reprod Fertil 2:473, 1961. 3. KATS", S., AND KATS", G. F. Perspectives in immunological control of reproduction: past, present and future. Paci! Med Surg 73 (IA):28, 1965.
4. KATSH, S. "Immunological Aspects of Infertility and Conception Control." In Advances in Obstetrics and Gynecology (Vol. I), Marcus, S. L., and Marcus, C. C., Eds. Williams & Wilkins, Baltimore, 1967. 5. KATSH, S. "Immunological Aspects of Reproduction." In Ovum Implantation. Shelesnyak, M. C., and Marcus, G. J., Eds. Gordon and Breach, Science Publishers, New York, 1969. 6. KATSH, S., LEAVER, F. W., KATSH, G. F., AND WILLSON, J. T. Circulating antibody titers in relation to fertility. I. Male guinea pigs. Fertil Steril 22:456, 1971. 7. CAMPBELL, D. H, AND WELIK, N. "Immunadsorbents: Preparation and Use of Cellulose Derivatives." In Methods in Immunology and Immunochemistry (Vol. I), Williams, C. A., and Chase, M. W., Eds. Academic Press, New York, 1967, p. 371. 8. URIEL, J. "Color Reactions for the Identification of Antigen-Antibody Precipitates in Gels." In Methods in Immunology and Immunochemistry (Vol. 111), Williams, C. A., and Chase, M. W., Eds. Academic Press, New York, 1971, p. 298. 9. CONCHIE, J., AND STRACHAN, I. Distribution, purification and properties of 1-aspartamido-B-Nacetylglucosamine amidohydrolase. Biochem J 115:709, 1969. 10. RoE, J. H. The determination of sugar in blood and spinal fluid with anthrone reagent. J Bioi Chem 212:335, 1955. 11. DlScHE, Z., AND SHETTLES, L. B. A specific color reaction of methylpentoses and a spectrophotometric micromethod for their determination. J Bioi Chem 175:595, 1948. 12. PARK, J. T., AND JOHNSON, M. J. A submicrodetermination of glucose. J Bioi Chem 181:149, 1949. 13. LEvVY, G. A., AND McALLAN, A. The N-acetylation and estimation of hexosamines. Biochem J 73:127, 1959. 14. SWEELEY, C. C., WELLS, W. W., AND BENTLEY, R. "Gas Chromatography of Carbohydrates." In Methods in Enzymology (Vol. VIII), Neufeld, E. F., and Ginsburg, V., Eds. Academic Press, New York, 1966, p. 95. 15. SPIRO, R. G. "Characterization of Carbohydrate Units of Glycoproteins." In Methods in Enzymology (Vol. VIII), Neufeld, E. F., and Ginsburg, V., Eds. Academic Press, New York, 1966, p. 40.
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16. 'TREVELYAN, W. E., PROCTER, D. P. AND HARRISON, J. S. Detection of sugars on paper chromato· grams. Nature (London) 166:444, 1950. 17. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S. Automatic recording apparatus for use in the chromatography of amino acids. Anal Chem 30: 1190,1958. 18. PLUMMER, T. H., AND HIRS, C. H. W. The isolation of ribonuclease B, a glycoprotein, from bovine pancreatic juice. J Bioi Chem 238:1396, 1963. 19. EHRENBERG, A. Determination of molecular weights and diffusion coefficients in the ultracentrifuge. Acta Chem Scand 11:1257, 1957. 20. DAYHOFF, M. Atlas of Protein Sequence and Structure (Vol. 4). National Biomedical Research Foundation, Silver Spring, Md. 1969. 21. NEUBERGER, A., G01TSCHALK, A. AND MARSHALL, R. D. Glycoproteins. American Elsevier, New York,l966. 22. SHARON, N. Polysaccharides. Ann Rev Biochem 35:485, 1966. 23. YAMASHINA, I. The Amino Sugars (Vol. lIB), Balazs, E. A., and Jeanloz, R. W., Eds. Academic Press, New York, 1966, p. 83. 24. MAHADEVAN, S., AND TAPPEL, A. L. B-aspartylglucosylamine amido hydrolase of rat liver and kidney. J Bioi Chem 242:4568, 1967. 25. WATANABE, K., AND YASUNOBU, K.. T. Carbohydrate content of bovine plasma amine oxidase and isolation of a carbohydrate-containing frag-
26.
27.
28.
29.
30.
31. 32.
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ment attached to asparagine. J Bioi Chem 245: 961,1970. MAKINO, M., KOJIMA, T., AND YAMASHINA, I. Enzymatic cleavage of glycopeptides. Biochem Biophys Res Commun 24:961, 1966. NUENKE, R. H., AND CUNNINGHAM, L. W. Preparation and structural studies of ovalbumin glycopeptides. J Bioi Chem 236:2452, 1961. KATSH, S. Localization and identification of antispermatogenic factor in guinea pig testicles. Int Arch Allerg 16:241, 1960. BROWN, P. C., GLYNN, L. E., AND HOLBORROW, E. J. The pathogenesis of experimental allergic orchitis in guinea pigs. J Path Bact 86:505, 1963. BROWN, P. C., HOLBORROW, E. J., AND GLYNN, L. E. The aspermatogenic antigen in experimental allergic orchitis in guinea pigs. Immunology 9:255, 1965. KATSH, S., AND KATSH, G. F. Antigenicity of spermatozoa. Fertil Steril 12:522, 1961. KIRKPNI'RlCK, C. H., AND KATSH, S. Amino acid content of aspermatogenic antigen. Nature
(London) 301:197, 1964. 33. KATSH, S., AGUIRRE, A., AND KATSH, G. F. inactivation of sperm antigens by tissues and sera of the female reproductive tract. Fertil Steril 19:740, 1968. 34. VOISIN, G. A., AND TOULLET, F. Etude sur l'orchite aspermatogenetique autoimmune et les antigenes de spermatozoides chez Ie cobaye. Ann Inst Pasteur (Paris) 114:727, 1968.
Copyright
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Vol. 23, No.9, September 1972
1972 by The Williams & Wilkins Co.
Printed in U.S.A.
PURIFICATION AND PARTIAL CHARACTERIZATION OF ASPERMATOGENIC ANTIGEN* SEYMOUR KATSH, PH.D., ANTHONY R. AGUIRRF.. M.S., FREDRICK W. LEAVER, PH.D., AND GRACE F. KATSH, PH.D. Department of Pharmacology, University of Colorado Medical School, Denver, Colorado 80220
Activity in the area of immunoreproduction has intensified in recent years. 1 - S Perusal of the literature will reveal that it is possible to induce infertility in male and female experimental animals by immunization. Moreover, certain cases of idiopathic infertility in humans may be attributable to immune responses. Despite these advances, several key problems remain to be resolved if major progress is to be made in controlling fertility by immunologic methods and in correcting infertility attributable to immune responses. Prominent among these problems is the imperative one of extracting, purifying, and characterizing antigens causing infertility. Until even one such antigen has been identified chemically and immunologically, the entire field of immunoreproduction will continue to languish in the speculative limbo in which it has been cast for the past 70 years since Landsteiner, Metchnikoff, and Metchnikoff (see reviews) first described the antigenicity of sperm. Without such information, critical data relative to cross-reactivity and uniqueness of reproductive antigens, for example, will be lacking. Further, it will not be possible to determine the nature of the antibodies involved. Moreover, without such information, one can only speculate as to mechanisms of action. Therefore, studies were undertaken to isolate, purify, and, at least, partially characterize one reproductive antigen. Initiation of Received January 17, 1972; revised April 21, 1972. * Supported by funds from the National Science Foundation (GB 31555X) and the Ford Foundation.
studies with this goal in mind requires selection of the appropriate experimental model. The aspermatogenic syndrome induced in guinea pigs was selected because it has been well characterized and because it appears to provide a laboratory model for certain clinical cases of testicular dyscrasia. MATERIALS AND METHODS
The following abbreviations are used in this report: C.ASA (crude aspermatogenic antigen) refers to the material extracted from tissues prior to digestion with pepsin. P.D.ASA (pepsin digested aspermatogenic antigen) is C.ASA which has been subjected to digestion with pepsin. F.2aASA refers to a biologically active material obtained after two passages through Sephadex G-50 of P.D.ASA. A.E.ASA refers to F.2aASA which has been precipitated with antibody and eluted from the antigen-antibody complex.
Extraction of C.ASA
From Testes. Frozen guinea pig testes (obtained from commercial vendors or from previously sacrificed animals) were decapsulated and the fat pads as well as the epididymides removed. Decapsulated testes (150 gm./batch) were homogenized for 1 min. at high speed in a Waring blendor with an equal volume of 0.1 M acetic acid. The homogenate was stirred overnight at 4 0 C. and centrifuged for 20 min. at 8000 g; the supernatant solution was reserved and the precipitate rehomogenized in 0.1 M acetic acid (50%
644
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PURIFICATION OF ASPERMATOGENIC ANTIGEN
645
of the original volume), stirred for 2 hr. of Sephadex gels ranging from G-50-G-2oo. and recentifuged as before. This process From these preliminary experiments, it was repeated once more before discarding was determined that optimal fractionathe precipitate. To the combined super- tion was obtained by an 18-hr. pepsin natants was added enough 30% trichloro- digest followed by filtration on G-50. Sephadex Fractionation. P.D.ASA, acetic acid (TCA) to bring the final TCA mg., was dissolved in 2 ml. of 100-150 concentration to 5%, the solution was 0.09 M acetic acid pH 3.0 and loaded on allowed to stand in the cold for 1 hr. and 85 cm. column of Sephadex G-50 a 1.5 x was then centrifuged for 10 min. at 8000 g. (Fine). The sample was washed on with The precipitates were washed twice with and eluted with 0.09 M 1 ml. of acetic acid small portions of 5% TCA, were freed acetic acid; 2-ml. fractions were collected from dialyzable impurities by overnight at a flow rate of 10-12 ml./hr. The column dialysis against running tap water and effluents were monitored by Ultraviolet residual TCA and possible lipids were reat 280 m~. with a Model UV absorption moved by extraction with 1/4 volume of 280 IF recording ultraviolet absorption N-butanol (three times) and chloroform (three times). The resulting aqueous meter and collected by a Gilson Medical solution on lyophilization gave a white Electronics Model LB-1 fractionator. fluffy material which could be stored at Four peaks were observed in the column room temperature for several years with- effluent; the tubes were pooled into five fractions numbered from I-V, Fraction ill out loss of biologic activity. From Epididymides. Frozen epididy- being the overlap region between the two mides (200-400 gm.) were allowed to major peaks. The fractions were lyophipartially thaw and then homogenized lized and tested for antigenic activity using with 1.5 volumes of acetone (Waring the passive hemagglutination (PHA) inhiblendor, 5 min. at high speed), the homog- bition test (below). The fractions (II and III) showing high enate was filtered under vacuum and the residue homogenized with 1.5 volumes PHA activity (Table 1) were pooled, reof chloroform. After filtration, the residue dissolved in 0.09 M acetic acid, and rerun was again treated with acetone, filtered and allowed to air dry. The dry material TABLE 1. Purification of Aspermatogenic Antigen TesteS Epididymis (33-35 gm.) was homogenized with 10 volumes of 0.1 M acetic acid and stirred Treatment PHA PHA inhibition· BioasSayt inhibition· Bioassayt overnight at 4 0 C. Subsequent steps in the procedure were the same as for testes. 11-14 mg. 30-40 mg. Extract before pep+ + sin digest Pepsin Digest. Lyophilized C.ASA 2 6 Pepein + + (from testes or epididymides) was dis- Sepbsdex G-50 121 60 ± Fraction I 0* solved in 5.0 M acetic acid at a concen4 0.9 Fraction II + + tration of 5 mg./ml. The solution was 0.3 10.0 Fraction ill + + 0.9 0 70 Fraction IV + brought to 30 0 C. on a temperature-con0 0 Fraction V trolled magnetic stirrer (Sargent Model Recycled fractions 5.0 3.0 + la + T.E.) and crystalline pepsin added (2% 0.2 0.2 2a + + of C.ASA weight). Digestions were carried 3.0 11.0 + 3a + 0.10 0.09 + + out for intervals of 4, 8, 18, and 24 hr. Eluted from complex • Defmed as milligrams of material required to neutralize 1 ml. of Then each solution was immediately antiserum titering 10-. frozen and lyophilized. Each preparation t Causes aspermatogenesis, +. Partial damage to testes, ±. *Does not cause aspermatogenesis, O. was fractionated on 0.9 x 30 cm. columns
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KATSH ET AL.
on the same column. The increased concentration of antigenic material at this point permitted the direct assay of tubes by PHA inhibition. This test showed outstanding correlation with the biologic results. The use of optical density at 280 m~ in the original monitoring is really fortuitous since measurements of highly active fractions show only modest density at 280 m~ (low aromatics) and the tubes with maximum density are practically void of aspermatogenic activity. When column eluates were assayed by PHA inhibition, the antigen was eluted as a single sharp peak which was pooled and lyophilized. This material was designated F .2aASA. Passive Hemagglutination (PHA) Tests. Passive hemagglutination (PHA) tests were performed to determine the titers of antibody preparations as described elsewhere. 6 PHA Inhibition Tests. Previously titered antiserum obtained from aspermatogenic guinea pigs 8 weeks after immunization was diluted with phosphate-saline buffer pH 5.9-6.1 to give a titer of 10,000. If the initial titration was greater than 1 million, the antiserum was diluted 1: 100 with phosphate-saline and then further diluted with normal guinea pig serum (diluted 1: 100 with phosphate-saline) to give a titer of 10,000. ThE:n 0.025 ml. of diluted antiserum was added to each well. A weighed amount (0.2-20 mg., depending on purity) of antigen was dissolved in 1.0 ml. of phosphate-saline and serially diluted in the antiserum. The dilution at which a dot failed to form was considered the end-point. From this dilution the number of milligrams of antigen required to neutralize 1 ml. of serum which titered 1 x 106 was calculated. Thus, lower numbers indicate increasing antigen activity. Column eluates were directly titrated as follows: 0.1 M phosphate buffer was prepared and adjusted such that addition
Vol. 23
of an equal volume of 0.09 M acetic acid would give a pH of 6.0-6.1. The first well of each series of dilutions contained 0.025 ml. of buffer in addition to the 0.025 ml. of antiserum; a 0.025 ml. aliquot from each tube to be assayed was then serially diluted in the antiserum. Biologic Assay of ASA. Depending on the stage of purification, 0.5-10.0 mg. of ASA were dissolved in 0.5 ml. of physiologic saline and emulsified with 0.5 ml. of complete Freund's adjuvant. The emulsion was injected intracutaneously in six sites into the shaved nuchal region of mature (500-600 gm.) male guinea pigs (Hartley strain). After 8 weeks, the animals were sacrificed by exsanguination and the testes excised and weighed. A weight of 1.2 gm. or less was indicative of complete deletion of the spermatogenic cell line as confirmed by histology. Preparation of Antibody to ASA. Guinea pigs injected 8 weeks previously with C.ASA were exsanguinated and the blood allowed to clot overnight at 4 0 C. in centrifuge tubes. After rimming the tubes, the blood was centrifuged for 20 min. at 3000 g, the serum obtained was titered and utilized for further purification only if the titers exceeded 100,000. The serum was mixed with an equal volume of a saturated ammonium sulfate solution, allowed to stand at room temperature for 10 min., then centrifuged for 5 min. at 1000 g. The precipitate was dissolved in distilled water (50% of the original serum volume) and again precipitated with ammonium sulfate and centrifuged. This process was repeated once more, the final precipitate was dissolved in distilled water and was dialyzed for 36 hr. against distilled water. The dialysate was clarified by centrifugation and then adjusted to pH 5.9 with 0.1 M acetic acid. After standing at room temperature briefly, the mixture was centrifuged and the supernatant was lyophilized. The material thus obtained corresponds to
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PURIFICATION OF ASPERMATOGENIC ANTIGEN
the pseudoglobulin fraction of serum proteins. Precipitation of Antigen-Antibody Complex. The lyophilized antibody preparation was dissolved in distilled water, the pH adjusted to 6.1 (final volume equal to that of the serum from which the antibody was obtained), and the solution titered by PHA. A known weight of F.2aASA was dissolved in distilled water (generally 0.2-0.3 mg./ml.) and also titered by PHA inhibition. From both titrations, the amount of antigen required to neutralize the antibody at hand was calculated, and this amount of antigen was dissolved in distilled water and adjusted to pH 6.1, the final volume of the antigen solution being held to 10% of the volume of the antibody solution. An aliquot of the antigen solution (about 25%) was mixed with an appropriate amount of pseudoglobulin which neutralized at 100% (see above) and allowed to remain at room temperature until a precipitate formed (at least 1 hr.), after which the solution was centrifuged at 3000 g for 10 min. and the supernatant retitered (PHA). The procedure was repeated with further aliquots of antigen solution until the supernatant indicated slight antigen excess (PHA inhibition). The pooled precipitates were then washed twice with 5- to 10-ml. portions of distilled water, dissolved in 2 ml. of 0.1 M acetic acid to dissociate the antigen-antibody complex 7 and immediately loaded on a 2.5 x 60 cm. column of Sephadex G-75. Fractions (4 ml.) were collected by eluting with 0.1 M acetic acid, the eluates being monitored by 280 m,u absorption and by PHA inhibition. The antibody was recovered as a sharp peak in the void volume, while the antigen (A.E.ASA) was eluted as a broad peak in the bed volume. The recovered antibody was dialyzed against distilled water until neutral and could then be utilized for further precipitations with antigen. This recovered antibody
647
was stored under refrigeration (4 0 C., thymol). Further purification of the A.E.ASA was obtained by reprecipitating the antigen with the appropriate amount of pseudoglobulin (see above) and then redissolving in 0.1 M acetic acid and filtering through a 0.025 ,u Millipore filter (Catalog listing VSWPO). Estimation of the Molecular Weight of ASA. The elution volume of F.2aASA was determined during the various fractionation runs on Sephadex G-50 (see above). The same column used for the fractionation of P.D.ASA was calibrated by applying various proteins and polypeptides of known molecular weight to the column and eluting them under the same conditions used for ASA. The markers used were pig thyroglobulin (Kit. No. 8109, Mann Research Laboratories), ribonuclease b, cytochrome c, adrenocorticotropic hormone (ACTH), and glucagon (all from Sigma Chemical Company). Acrylamide Gel Electrophoresis. All procedures were carried out using the Canalco Research Disc Electrophoresis apparatus with a Buchler D.C. Power Supply Catalog No. 3-1014. The tubes used were 5 mm. i.d. and 75 mm. long in which separating, stacking, and sample gels were formulated. Separating gel (15%, pH 4.3) 1 part A; 2 parts B; 1 part water. Solution A: 48 ml. of 1 M KOH, 17.2 ml. of glacial acetic acid, 4.0 ml. of N, N, Nl, Nl-tetramethyl ethylenediamine (TEMED) all diluted to 100 ml. with water. Solution B: 60 gm. of acrylamide; 0.4 gm. of N, Nl_ methylenebisacrylamide (Bis) diluted to 100 ml. with water. The four parts of solution described were mixed with four parts of catalyst (0.28% ammonium persulfate), 1.2-1.5 ml. added to each tube and allowed to polymerize for 30 min. Stacking and sample gels 1 part C; 1 part D; 1 part catalyst; 5 parts water. Solution C: 48 ml. of 1 M KOH, 2.87 ml. of glacial acetic acid, 0.46 ml. of TEMED all di-
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KATSH ET AL.
Vol. 23
luted to 100 ml. with water (pH 6.7). Enzyme Preparations. Amidase, (l-asSolution D: 20 gm. of acrylamide, 5.0 gm. partamido-B-N -acetylglucosamine amido of Bis, water to 100 ml. Catalyst: 4.0 mg. % hydrolase) was prepared from rat liver by of riboflavin. The stacking gel was added the procedure of Conchie and Strachan. 9 in 0.2-ml. aliquots to each tube and photo- The synthetic substrate used for the aspolymerized. Sample was then added say of this enzyme was 2-acetamido-1-B (200 p,g. in 5 p,l. of water), mixed with (L-B aspartamido)-1,2-dideoxy-n-glucose, 0.2 ml. of sample gel, and polymerized. obtained from the Cyc10 Chemical ComThe electrophoresis buffer was prepared pany, Los Angeles, California. Pepsin was as follows: 31.2 gm. of B-alanine, 8.0 ml. obtained from the Sigma Chemical Comof glacial acetic acid and water to 1000 pany, and almond emulsin (B-glucosidase) ml., pH 5.0. The solution was diluted 1 : 10 from Mann Research Laboratories. Sugar Analyses. Total neutral sugars before use. Electrophoresis was performed with were determined by the anthrone method the tubes in a vertical position, sample gel of Roe. 10 Fucose was assayed by the at the top (anode), separating gel at the Dische-Shettles 11 cysteine-sulfuric acid bottom (cathode). Methyl green, 1-2 ml. reaction, reducing sugars were determined of 0.005%, was added to the top buffer by the Park-Johnson 12 ferricyanide techreservoir as a tracking dye. Electrophore- nic, and acetylated amine sugars were sis was continued with a current of 5 ma.! assayed according to Levvy and Mctube until the green dye band had just Allan.13 The complete sugar analysis was run off the end of the gels. The gels performed by vapor-phase chromatogwere then removed and stained with 0.5% raphy according to a modification of the aniline blue-black in 7% acetic acid for procedure of Sweeley, Wells, and Bent1 hr. after which they were electrophoreti- ley. 14 cally destained in 7% acetic acid using a Enzyme Degradation of Carbohydrate Canalco quick gel destainer. Gels were Moiety. F.2aASA (9.2 mg.) was dissolved scanned at 560 mp, using a Gilford Model in 1.0 ml. of 0.1 M Na-acetate buffer, pH 2000 spectrophotometer-recorder with a 4.2; 3.0 mg. of almond emulsin 15 Model 2410 linear transport. Slit width were added (together with 50 p,l. of for scanning was 0.2 mm. toluene to prevent bacterial growth), and Immunoelectrophoresis was performed the solution incubated at 37° C. Aliquots on 25 x 75 mm. microscope slides coated (0.1 ml.) were removed at 25, 43, 68, and with 1% agarose in 0.015 M barbital buffer 96 hr. and spotted on Whatman No.1 pH 7.0. The electrophoresis buffer was paper which had been washed for 2 days the same as that used to dissolve the in n-butanol-ethanol-water (10: 1: 2) and agarose. Antigen (F.2aASA or A.E.ASA, then for 2 days in distilled water, and sub75-100 p,g.) was dissolved in 3 p,l. of jected to descending chromatography in buffer and placed in the wells, the slides the same solvent for 96 hr. The spots were then electrophoresed for 2 hr. in a were visualized with alkaline silver ni12.5 volt/cm. gradient. Following electro- trate. 16 Another sheet was run in parallel phoresis, the troughs were charged with with the first. On this sheet only the inantibody solution and diffusion carried dividual standards were visualized; the out in a moist chamber for 6-8 hr. at room spotted aliquots as well as a standard temperature. The slides were washed mixture consisting of 20 p,g. of galactose, overnight in distilled water, dried, and 15.5 p,g. of mannose, and 13.6 p,g. of stained with Azocarmine b according to N-acetylglucosamine were left unstained. the procedure of Uriel. 8 Areas of the sheet corresponding to the
September 1972
PURIFICATION OF ASPERMATOGENIC ANTIGEN
visualized standards were cut out, divided into small pieces, and eluted overnight in 5 ml. of distilled water. The solutions obtained were analyzed for reducing sugar.12 Recoveries were calculated with reference to the standard mixture. Amino acid analysis was performed by standard procedures. 17 Glycopeptide Linkage Investigations. (a) Treatment with amidase: 5.0 mg. of F.2aASA and 2.0 mg. of A.E.ASA were separately dissolved in 2.5 ml. of distilled water; to each of these were added 1.25 ml. of 0.2 M Na2HP04-KH2P04 buffer pH 7.0, and 1.25 ml. of an amidase preparation assaying 4.26 ~M N-acetylhexosamine released per hour per milliliter. The solution was incubated at 37° C.; 1-ml. aliquots were removed at 4, 7, 18, and 24 hr. The reaction was stopped by boiling for 3 min.; the mixture was centrifuged at 1500 g for 10 min. The supernatant was then assayed for N-acetylhexosamine content. (b) Alkaline hydrolysis: 11.2 mg. of F.2aASA and 1.229 mg. of A.E.ASA were separately dissolved in 2 ml. of 0.5 N NaOH and left for 15 hr. at 4° C. The solutions were neutralized with 1 N HCl and an aliquot assayed for N-acetylhexosamine content. Aliquots of the remainder of the solutions were assayed by PHA inhibition to determine if loss of activity had occurred. The balance of the F .2aASA hydrolyzate was placed in overnight dialysis against distilled water. The bag contents and outer fluid were assayed for neutral sugar content. As controls for this experiment, 0.55 mg. (1.58 ~M) of the synthetic aspartyl-glucosylamine (a model for the N-glucosidic bond) was also subjected to alkaline hydrolysis. Another control consisted of dialyzing a sample of F.2aASA which had not been hydrolyzed. (c) Treatment with lithium borohydride: 1.2 mg. of A.E.ASA were dissolved in 2 ml. of 0.3 M LiBH4 in tetrahydrofuran and the solution was refluxed for 8 hr. After cooling, the reaction was ter-
649
minated by addition of 0.8 ml. of 1 N methanolic HCl and the solution lyophilized. The dried material was redissolved in water and analyzed for N-acetylglucosamine content. RESULTS
Studies in this laboratory on the induction of aspermatogenesis and the biochemical consequences thereof have heretofore involved the use of relatively crude aspermatogenic antigen (C.ASA). This material is biologically active when injected in milligram quantities. Upon assay by the PHA inhibition test, activities of 30-40 mg. for testicular preparations and 11-14 mg. for epididymal preparations are observed. Further purification of C.ASA has now been achieved. Table 1 shows 440and 140-fold increases in activity, respectively, for testicular and epididymal preparations, based on PHA inhibition titers. Two criteria were used to assess biologic activity and/or degree of purification throughout the procedures described. All products were injected into guinea pigs to test their ability to produce aspermatogenesis. Complete aspermatogenesis (as determined by histology) was characterized by a testicular weight of 0.8-1.2 gm. (normal weight = 3.5-4.5 gm.). In addition, all products were also tested by the PHA inhibition test. It was found that preparations which gave values below 60 mg. in this test invariably caused aspermatogenesis. Filtration on Sephadex gels as well as ion-exchange resins of various types (DEAE cellulose, Dowex 1, Dowex 50, and QAE-Sephadex) failed to fractionate C.ASA since it was removed as a single large peak (by absorption at 280 m~). Similar results were obtained following hydrolysis with 1 N HCI for 4 hr. at 37° C. and with 1 N acetic acid for 18 hr. at 102° C. A 4-hr. hydrolysis with pepsin at 37° C. gave a pattern of three poorly re-
650
Vol. 23
KATSH ET AL.
solved peaks upon filtration through Sephadex G-50 with the biologic activity being found in the second peak. The point of maximal degradation with pepsin was determined by digestion for different periods of time, and the 18-hr. interval was chosen since digestion for longer periods failed to produce any further changes in the elution pattern obtained from Sephadex G-50. Filtration of P.D.ASA on gels ranging from G-75 through G-200 failed to markedly improve the peak resolution over that obtained with G-50. Consequently, the final conditions chosen were those described in "Materials and Methods." The type of elution diagram routinely obtained from a fractionation of either testicular or epididymal P.D.ASA is shown in Fig. 1. PHA inhibition titers obtained on the lyophilized fractions are shown in Table 1. Dealing first with testicular preparations: Fraction I showed slight activity, Fractions II and III caused full aspermatogenesis, and Fractions IV and V were devoid of aspermatogenic activity. Epididymal preparations exhibit a slightly different pattern in that Fractions I and V are inactive; activity was concentrated in Fractions III and IV. The activities in the epididymal Fractions III and IV were considerably higher than those of the testicular preparations. Those peaks which showed a higher activity (according to PHA inhibition) than the P.D.ASA were routinely pooled and rerun on the same column. The elution pattern thus obtained showed only two large central peaks (Fractions I and V were eliminated) when monitored by 280 m~ absorption. However, when the tubes were assayed by PHA inhibition, only a single peak was obtained (shown by dashes in Fig. 1). Fractions la and 3a in Table 1 and Fig. 1 represent the initial and final portions of the antigen peak, respectively. Fraction 2a represents that portion of the peak showing a reciprocal
003
~
I I
~
>- 002
l-
v;
2:
~
--' <:(
U
00
I I~ 1II
0(
0
"Sl
i" \
f::
Q
I
101:20\130
.I 80
\. 100
120
ELUTION VOLUME (m/)
FIG. 1. Fractionation of P.D.ASA on a 1.5 x 90 cm. column of Sephadex G·50 in 0.09 M acetic acid. 0-0-0, fractions monitored by absorption at 280 mIL. e--e--e, fractions monitored by PHA inhibition test. The ordinate indicates the fold dilution at end· point.
titer greater than 16 by PHA inhibition. After preliminary determinations of purity by gel electrophoresis and immunoelectrophoresis, this fraction (F.2aASA) was further purified by precipitation with antibody. Acrylamide Gel Electrophoresis. Electrophoresis separation of F.2aASA gave a pattern of six to seven bands moving towards the cathode (Fig. 2, left). These bands were eluted from unstained gels and tested by PHA. It was found that the band nearest the anode was the only one which gave a positive PHA inhibition test. Furthermore, gel electrophoresis of the antigen recovered from the antigenantibody complex (A.E.ASA) gave only one band of identical mobility to the slowmoving band in the electropherogram of F.2aASA. (Fig. 2, right.) This material caused aspermatogenesis. Immunoelectrophoresis. The patterns obtained with F.2aASA showed all of the material moving towards the cathode. After reaction with and elution from antibody, only one precipitin band was obtained (Fig. 3). Estimation of Molecular Weight of Antigen. F.2aASA was eluted from Sephadex G-50 as a sharp peak which permitted accurate determination of the elution volume. Calibration of the column
I
September 1972
PURIFICATION OF ASPERMATOGENIC ANTIGEN
0050
.50
IMO
IMO
.30
o~o
.
::
i
:
!
~
i
651
! 0020
0020
0010
0·10
Dlstlnce towards Clthode In em
Distance towards cathode in em
FIG. 2. Acrylamide gel electrophoresis scans of F.2aASA and A.E.ASA. Left, F.2aASA. Right, A.E.ASA.
FIG. 3. Immunoelectrophoretic analysis of F.2aASA. Trough filled with antibody solution. Upper and lower wells filled with F.2aASA. Cathode to the right.
with a series of peptides and proteins of known molecular weight (Table 2) and the calibration curve subsequently obtained (Fig. 4) permitted the estimation of a molecular weight range approximating 11,000-14,000 for the antigen. Amino Acid and Carbohydrate Analyses. The results obtained from the carbo-
hydrate and amino acid analyses of F.2aASA and A.E.ASA are shown in Tables 3 and 4. Emulsin Digest. In order to gain some insight as to the sequence of attachment of the carbohydrate residues, F .2aASA was digested with almond emulsin. This is a crude enzyme preparation composed
652
TABLE 4. Amino Acid Analysis of Aspermatogenic Antigen
TABLE 2. Gel Filtration of Molecular Weight Markers and F.2aASA on Sephadex G-50 PrOtein
Molecular weight
Elution volume
Elution volume/ Void
mi.
Thyroglobulin * Ribonuclease b Cytochrome c ACTH Glucagon ASA
42.5 1.00 670,OOOt 14,700" 81.3 1.91 13,400" 83.5 1.96 5,250'· 2.18 93.0 3,670'· 96.3 2.27 12,600:j: 84.0 1.97 * Thyroglobulin was assumed to be completely excluded from the gel. Hence, void volume = elution volume. t Obtained from Mann Research Laboratories Molecular Weight Markers Kit. :j: Determined from Fig. 4.
THYROGlOOULN
~~O----------74.on---~----~50~--------~60
LOG" MOlECULAR WEIGHT
FIG. 4. Estimation of molecular weight of aspermatogenic antigen on Sephadex G-50 (see Table 2). TABLE 3. Carbohydrate Analysis of Aspermatogenic Antigen F.2aASA Total neutral sugars (Anthrone) Total sugars Fucose
A.E.ASA
4.2%
10.46%
5.07%" 0.43%
12.67% (V.P.C.) 0
Molar ratiot
Distribution of monosaccharide residues (as percentage of total carbohydrate)
Fucose Galactose Glucosamine N·Acetylglucosamine N·Acetylgaiactosa·
9.26 35.31 30.95 7.35 12.94 3.39
mine N-Acetylneuraminic
0.79
Mannose
0 32.75 42.15 0 24.94 0
2.90 (3)* 3.73 (4) 1.80 (2)
0
acid ... Based on anthrone results and carbohydrate distribution by V.P.C. t Based on carbohydrate content of 12.67% and molecular weight of 12,600.
t Numbers
in parentheses are the nearest integral number of resi-
A.E.ASA
F.2aASA
volume
dues.
Vol. 23
KATSH ET AL.
Ala Arg Asp Cys Glu Gly His He Leu Lys Met Phe Pro Ser Thr Tyr Val
pM/mg.
Residues
I'M/mg.
Residues
1.298 0.414 0.810 0.247 1.482 0.931 0.221 0.210 0.489 1.116 0.144 0.160 0.841 0.848 0.624 0.094 0.653
12.27 3.91 7.65 2.33 14.00 8.78 2.09 1.98 4.62 10.55 1.36 1.51 7.95 8.01 5.90 8.88 6.17
0.587 0.328 0.650 0.397 0.940 0.690 0.250 0.190 0.561 0.518 0.095 0.173 0.733 0.569 0.431 0.285 0.569
7.38 4.10 8.11 4.96 11.85 8.70 3.09 2.35 7.01 6.51 1.19 2.12 9.23 7.22 5.39 3.59 7.18
Total
107.96
Total
99.98
of a mixture of mannosidase, galactosidase' and N-acetylglucosaminidase which sequentially releases sugar residues from the nonreducing end of a polysaccharide. The results obtained suggest that the galactose residues are located at the nonreducing end of the polysaccharide chain. Further work is necessary to elucidate this point. The results of emulsin digestion also revealed that all sugars were released. Nature of the Glycopeptide Linkage. Three main types of linkage between the carbohydrate and peptide moieties of glycoproteins in general have been described: 21 - 23 (a) an N-glycosidic linkage between N-acetylglucosamine and the amide nitrogen of asparagine; (b) the hydroxyl group of serine or threonine in an O-glycosidic bond with C-l of a sugar; and (c) glycosidic ester linkages. NGlycosidic linkages are generally degraded by amidase. 24 - 26 This enzyme failed to cause the release of detectable N-acetylhexosamine from any of the antigen preparations. This cannot, however, be re-
September 1972
PURIFICATION OF ASPERMATOGENIC ANTIGEN
garded as conclusive evidence for the absence of this type of bond. Makino, Kojima, and Yamashina 26 have shown, for example, that ovalbumin was not attacked by amidase, whereas a glycopeptide prepared from it was cleaved. It is possible, then, that amidase alone cannot release N-acetylhexosamine unless the attached carbohydrate chain is removed. The results with emulsin digestion provide such evidence. Thus, in order to obtain further information of linkage of carbohydrate to the peptide, hydrolysis by NaOH was performed because Nuenke and Cunningham 27 have shown the N -glycosidic linkage to be stable to alkaline hydrolysis. We have confirmed these results employing synthetic aspartylglucosylamine as a model for the linkage. On the other hand, 0glycosidic linkages and ester linkages are subject to alkaline hydrolysis. When A.E.ASA was treated with NaOH, 46% of the N-acetylglucosamine was released with a loss of 98% of the PHA inhibition activity. This indicates that one of the two N-acetylglucosamines is subject to alkaline hydrolysis and the presence of this bond is necessary for PHA inhibition activity. However, one N-acetylglucosamine is not readily hydrolyzed by NaOH, suggesting that it is involved in an N-glycosidic linkage. The observation that treatment with LiBH4 failed to release any N-acetylglucosamine further indicates the absence of ester linkages. DISCUSSION
Throughout these studies the biologic activity of the antigen at any given stage of purification has been determined by injection into guinea pigs. It was necessary, however, to have some means of screening for biologic activity as well as degree of purification. Within limits, the PHA inhibition test provides a way of
653
doing this. It must be recognized that the test is a subjective one, in that the endpoint is determined by the first well in which a dot fails to form; thus one could expect a variation of at least one well to either side of the chosen end-point. In addition, the effectiveness of the coated red cells can vary considerably amongst preparations. These limitations have been controlled as far as possible by running many samples with a given cell preparation, by having only one operator perform the PHA tests, and by coding so that the operator was unaware of which preparation was under test. Regarding the precipitation of the antigen-antibody complex and elution of antigen therefrom, we have found it necessary to proceed with the elution immediately following precipitation and processing of the complex. Delays result in incomplete separation of antigen from antibody. The emulsin digestion was performed on F .2aASA which was still a relatively crude preparation. The results obtained must therefore be regarded as tentative, subject to confirmation by digestion of the purified antigen with pure preparations of the individual glycosidases. The results do strongly suggest, however, that the galactose residues are found at the nonreducing end of the polysaccharide chain. Reference to Table 3 indicates that the ratios in which galactose, mannose, and N-acetylglucosamine were released by emulsin are very close to the ratios found for these three sugars by gas chromatographic analysis of F.2aASA. The comparative carbohydrate and amino acid contents of F .2aASA and the A.E.ASA (Tables 3 and 4) indicate a considerable difference in chemical composition; the results obtained with F.2aASA no doubt reflect contamination of the antigen by other glycopeptides. The composition found for the antigen eluted
654
KATSH ET AL.
from the antibody is consistent with the molecular weight obtained by Sephadex filtration. The values obtained for the carbohydrate content, based on a molecular weight of 12,600 indicate the presence of three mannose residues, four galactose residues, and two N -acetylglucosamine residues. The release of 46% of the N-acetylglucosamine by alkaline hydrolysis permits the inference that one of the two postulated residues of this sugar is located within the polysaccharide chain, and therefore not susceptible to alkaline hydrolysis. A carbohydrate content of approximately 13% is consistent with the great solubility of the antigen in aqueous solvents. The amino acid content of the antigen merits some discussion. The small number of aromatic amino acids explains the low absorption at 280 mJL which we have observed. In order to account for a slight mobility of A.E.ASA towards the cathode, the dicarboxylic amino acids must be present predominantly as their amide derivatives. Further detailed determinations are necessary regarding the carbohydrate sequence, the residues involved in the glycopeptide linkage, and the amino acid sequence. These studies are now under way. Of critical importance in further studies is the purification of antibody to ASA as well as an evaluation of the differences (if any) between the antigen which is injected into the animals, and the antigen released from the testes during the autoimmune phase of the aspermatogenic response. In this connection, it is pertinent to recall the hypothesis presented which accounts for the genesis of experimentally induced autoimmune aspermatogenesis. 28 Briefly, the injection of specific antigen in adjuvant elicits antibodies which interact with antigen-
Vol. 23
containing cells (in testes and epididymides), causing release of antigen. The released antigen incites further antibody formation until all endogenous antigenic units have been released. Confirmatory evidence including antibody titers has been presented. 6 It now becomes possible to determine if the "native" antigen released during the immune response is or is not identical with the extracted antigen by eluting the "native" antigen from circulating antigen-antibody complex. With respect to purification and characterization of reproductive antigens in general, very little appears in the literature. So far only the aspermatogenic antigen has been accorded consistent effort. For example, Brown, Glynn, and Holborrow 29 and Brown, Holborrow, and Glynn 30 employed papain digestion and phenol extraction in attempts to purify the antigen. Katsh and Katsh 31 employed enzymatic and electrophoresis experiments to note the presence of polypeptide and polysaccharide. Kirkpatrick and Katsh 32 noted the amino acid content of the antigen. Katsh, Aguirre, and Katsh 33 were able to demonstrate inactivation of antigen by enzymes and by tissues and sera. Katsh 5 noted that pepsin could be employed as a first important step in purification of the antigen. Voisin and Toullet 34 have separated four antigens from sperm of which two appeared to be involved in aspermatogenesis. We believe the material described in this report is of far greater purity than any yet obtained and that this material is of sufficient purity to warrant extensive investigation of its chemical structure. The possibility of extracting a lower molecular weight aspermatogenic antigen is already under study. These studies are considered significant from the point of view of application to reproductive antigens of human origin. Ultimately, it is the objective to isolate and characterize not only
September 1972
PURIFICATION OF ASPERMATOGENIC ANTIGEN
aspermatogenic antigen but also immobilizing and agglutinating antigens. SUMMARY
Aspermatogenic antigen has been obtained from guinea pig testes and epididymides by extraction of the tissues with acetic acid, precipitation with trichloroacetic acid and extraction with butanolchloroform. The material thus obtained has been purified by digestion with pepsin and fractionation on Sephadex G-50 followed by precipitation with antibody and elution from the antigen-antibody complex. This material induces autoimmune aspermatogenesis in guinea pigs when injected in microgram amounts with Freund complete adjuvant. The purified antigen shows a single band on acrylamide gel electrophoresis, yields a single band upon immunoelectrophoresis, and has an estimated molecular weight of approximately 12,600. The antigen is a glycopeptide containing approximately 13% carbohydrate (galactose, mannose, and N-acetylglucosamine). Information is provided that the galactose residues appear to be at the nonreducing end of the polysaccharide. Suggestive evidence is provided that the polysaccharide moiety is attached to a polypeptide of 100 residues through an N-glycosidic linkage. The importance of these studies relates to the groundwork being laid for extraction, purification, and characterization of reproductive antigens of human origin. Acknowledgments. The authors would like to thank Drs. Sanford Markey and Lewis Johnson for performing the carbohydrate analyses by vapor-phase chromatography, and Drs. Ernest Borek and Barry Starcher for the amino acid analyses. REFERENCES 1. KATSH, S. Immunology, fertility and infertility: A historical survey. Amer J Obstet Gynec 77:946, 1959.
655
2. TYLER, A. Approaches to the control of fertility based on immunological phenomena. J Reprod Fertil 2:473, 1961. 3. KATS", S., AND KATS", G. F. Perspectives in immunological control of reproduction: past, present and future. Paci! Med Surg 73 (IA):28, 1965.
4. KATSH, S. "Immunological Aspects of Infertility and Conception Control." In Advances in Obstetrics and Gynecology (Vol. I), Marcus, S. L., and Marcus, C. C., Eds. Williams & Wilkins, Baltimore, 1967. 5. KATSH, S. "Immunological Aspects of Reproduction." In Ovum Implantation. Shelesnyak, M. C., and Marcus, G. J., Eds. Gordon and Breach, Science Publishers, New York, 1969. 6. KATSH, S., LEAVER, F. W., KATSH, G. F., AND WILLSON, J. T. Circulating antibody titers in relation to fertility. I. Male guinea pigs. Fertil Steril 22:456, 1971. 7. CAMPBELL, D. H, AND WELIK, N. "Immunadsorbents: Preparation and Use of Cellulose Derivatives." In Methods in Immunology and Immunochemistry (Vol. I), Williams, C. A., and Chase, M. W., Eds. Academic Press, New York, 1967, p. 371. 8. URIEL, J. "Color Reactions for the Identification of Antigen-Antibody Precipitates in Gels." In Methods in Immunology and Immunochemistry (Vol. 111), Williams, C. A., and Chase, M. W., Eds. Academic Press, New York, 1971, p. 298. 9. CONCHIE, J., AND STRACHAN, I. Distribution, purification and properties of 1-aspartamido-B-Nacetylglucosamine amidohydrolase. Biochem J 115:709, 1969. 10. RoE, J. H. The determination of sugar in blood and spinal fluid with anthrone reagent. J Bioi Chem 212:335, 1955. 11. DlScHE, Z., AND SHETTLES, L. B. A specific color reaction of methylpentoses and a spectrophotometric micromethod for their determination. J Bioi Chem 175:595, 1948. 12. PARK, J. T., AND JOHNSON, M. J. A submicrodetermination of glucose. J Bioi Chem 181:149, 1949. 13. LEvVY, G. A., AND McALLAN, A. The N-acetylation and estimation of hexosamines. Biochem J 73:127, 1959. 14. SWEELEY, C. C., WELLS, W. W., AND BENTLEY, R. "Gas Chromatography of Carbohydrates." In Methods in Enzymology (Vol. VIII), Neufeld, E. F., and Ginsburg, V., Eds. Academic Press, New York, 1966, p. 95. 15. SPIRO, R. G. "Characterization of Carbohydrate Units of Glycoproteins." In Methods in Enzymology (Vol. VIII), Neufeld, E. F., and Ginsburg, V., Eds. Academic Press, New York, 1966, p. 40.
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16. 'TREVELYAN, W. E., PROCTER, D. P. AND HARRISON, J. S. Detection of sugars on paper chromato· grams. Nature (London) 166:444, 1950. 17. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S. Automatic recording apparatus for use in the chromatography of amino acids. Anal Chem 30: 1190,1958. 18. PLUMMER, T. H., AND HIRS, C. H. W. The isolation of ribonuclease B, a glycoprotein, from bovine pancreatic juice. J Bioi Chem 238:1396, 1963. 19. EHRENBERG, A. Determination of molecular weights and diffusion coefficients in the ultracentrifuge. Acta Chem Scand 11:1257, 1957. 20. DAYHOFF, M. Atlas of Protein Sequence and Structure (Vol. 4). National Biomedical Research Foundation, Silver Spring, Md. 1969. 21. NEUBERGER, A., G01TSCHALK, A. AND MARSHALL, R. D. Glycoproteins. American Elsevier, New York,l966. 22. SHARON, N. Polysaccharides. Ann Rev Biochem 35:485, 1966. 23. YAMASHINA, I. The Amino Sugars (Vol. lIB), Balazs, E. A., and Jeanloz, R. W., Eds. Academic Press, New York, 1966, p. 83. 24. MAHADEVAN, S., AND TAPPEL, A. L. B-aspartylglucosylamine amido hydrolase of rat liver and kidney. J Bioi Chem 242:4568, 1967. 25. WATANABE, K., AND YASUNOBU, K.. T. Carbohydrate content of bovine plasma amine oxidase and isolation of a carbohydrate-containing frag-
26.
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Vol. 23
ment attached to asparagine. J Bioi Chem 245: 961,1970. MAKINO, M., KOJIMA, T., AND YAMASHINA, I. Enzymatic cleavage of glycopeptides. Biochem Biophys Res Commun 24:961, 1966. NUENKE, R. H., AND CUNNINGHAM, L. W. Preparation and structural studies of ovalbumin glycopeptides. J Bioi Chem 236:2452, 1961. KATSH, S. Localization and identification of antispermatogenic factor in guinea pig testicles. Int Arch Allerg 16:241, 1960. BROWN, P. C., GLYNN, L. E., AND HOLBORROW, E. J. The pathogenesis of experimental allergic orchitis in guinea pigs. J Path Bact 86:505, 1963. BROWN, P. C., HOLBORROW, E. J., AND GLYNN, L. E. The aspermatogenic antigen in experimental allergic orchitis in guinea pigs. Immunology 9:255, 1965. KATSH, S., AND KATSH, G. F. Antigenicity of spermatozoa. Fertil Steril 12:522, 1961. KIRKPNI'RlCK, C. H., AND KATSH, S. Amino acid content of aspermatogenic antigen. Nature
(London) 301:197, 1964. 33. KATSH, S., AGUIRRE, A., AND KATSH, G. F. inactivation of sperm antigens by tissues and sera of the female reproductive tract. Fertil Steril 19:740, 1968. 34. VOISIN, G. A., AND TOULLET, F. Etude sur l'orchite aspermatogenetique autoimmune et les antigenes de spermatozoides chez Ie cobaye. Ann Inst Pasteur (Paris) 114:727, 1968.