Isolation and purification of an organ-specific autoantigen from rabbit seminal plasma

Pergamoa Press

Life Sciences Vol. 14, pp " 1695-1704 Printed is II .S .A .

ISOLATION AND PURIFICATION OF AN ORGAN-SPECIFIC AUTOANTIGEN FROM RABBIT SEMINAL PLASMA Miguel A . Yides#, Elsa Vottero-Cime and Carlos Yantorno Department of Clinical Biochemistry : Faculty of Chemical Sciences, C6rdoba National University . CGrdoba, Argentina . (Received is

fuel

form 26 March 1974)

SUMMARY A specific autoantigen of rabbit accessory glands was isolated and purified from seminal plasma using DEAESephadex batch adsorption and Sephadex G-100 chromato graphic fractionationa . The autoantigen appeared homogeneous by polyacrylamide electrophoresis and by immunodiffusion . It has en extinction coefficient at 284 mN (E ~xm) of 6.42 . It contains 87 .73 ~ of polypeptidea .The carbohydrate moiety is composed of 9 .21 ~ hexose, 1 .03 ~ hexoseminea, 0 .33 % fucose . From these chemical values the autoantigen has been identified as glycoprotein . It has been established that organ and species specific autoantibodiea against male accessory glands can be induced in rabbits by a variety of procedures (1,2,3) . On the other hand,

the presence of cellular immunity, together with autoimmune damage of the accessory glands has been demonstrated in auto and isoimmunized rabbits (4) . The availability of homogeneous preparations of chemically

defined autoantigens is an unavoidable requirement for further

exploration of the immunogenesis and pathogenesis of experimental autoimmune diseases ($) . The present work was undertaken in order

Present address : Laboratorio Central-Hospital National de Clfnicas . Santa Rosa I$64 .C6rdoba .Argentina .' 1695

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to purify a specific autoantigen of the rabbit male accessory glands . A simple procedure hes been devised which yields a

homogeneous preparation. Polyacrylamide gel electrophoresis and double immunodiffusion gel precipitation were used as homogeneity criteria . Preliminary studies on the chemical composition of the specific autoantigen are also reported . MATERIALS AND METHODS Antitaena : Semen was obtained from rabbits by using an artificial vagina (6) . Individual

samples were pooled and centrifuged at

12,300 g for I$ min, at 4°C in order to obtain the seminal plasma . All specimens were stored at -18°C until used . Extracts from several tissues were homogenized with an equal

weight of 0.1$ M NaCI following procedure described elsewhere (7) . The protein content of the supernatants was determined by the Biuret method of Gornall et al (8) . The cluster comprising the

seminal vesicle, the coagulating gland, the bulbo uretral and the prostate gland will be referred to as the ~ rabbit male accessory glands~ (7,9) . Antisera :

Heterologous anti-rabbit semen serum was prepared by

immunizing periodically a sheep with pooled fresh rabbit semen emulsified in complete Freund~s adjuvant following a procedure described elsewhere (10) . Autologous anti-rabbit seminal plasma serum was prepared by autoimmunizing several adult male rabbits with seminal plasma emulsified with an equal volume of complete Freund~s adjuvant

following the procedure already described (II) . Rabbit 134 serum was selected due to its high antibody titer an a high precipitating

activity when confronted with rabbit male accessory glands extract or seminal plasma .

Pre~aration of DEAE-Seohadex : I$ g of DEAE Sephadex A-$0 (Pharma-

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cie, Uppsala) were allowed to swell in excess of 0 .05 M Tria-HCI buffer pH 8 + 0 .0$ M NeCI + 0.001 M EDTA . The supernatant was decanted end replaced with fresh buffer several times during the swelling period until it acquired the correct pH and counter ion concentration . The get was filtered and washed on a Buchner funnel with the buffer solution and the Liquid excess was then removed . Finally the gel was taken with a spatula, placed in a beaker, and kept at 4°C . Column chromatos:raohv :

Sephadex G-100 (Pharmacia, Uppsala) was

prepared according to manufacturer s instructions . The buffer consisted of 0 .1 M Tris-HCI + I M NeCI + 0 .001 M EDTA . All columns were run at room temperature (22° ± 2° C) .The protein concentration in the effluents was determined by reading O .D . at 280 mN in a Beckman Mod . DU spectrophotometer . Immunolo~aical assays :

The antigenic activity was evaluated by

tanned cell hemagglutination (12), inhibition of tanned cell hemagglutination (II) and double immunodiffuaion gel precipitation

Electroehoretic analysis ; Polyacrylamide gel electrophoresis was performed by the Devis (14) procedure in 9 x 0 .8 cm size gels with Tris-glycine buffer, applying 2 .$ mA current per tube during the time required for an albumin standard 4 cm migration .

Ph sical and chemical anal ais : Ultraviolet extinction coefficient was determined in a Beckman Mod. DU spectrophotometer in a (E I cm) 0 .) N NaOH at 0 .$ mg/ml concentration . Dry weight was determined in vacuo over phosphorus pentoxide up to constant weight . Polypeptides were determined by the biuret method of Gornall et al (8) using bovine serum albumin (Sigma Chemical Co) as standard . Neutral sugars were determined by the method of Winzler

(I$), modified by Frencois et al (16), using galactose and mannose

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as standard in a ratio of I :I . Fucose was determined by the method of Dische and Shettles (I~) with fucose as standard and the O .D .

was determined at 396 and 430 mN . Aminosugars were determined after hydrolysis of the glycoprotein in 4 N HCI at 100° C for 3 hs by

the method of Rondle and Morgen (18) with glucosamine and

galactosamine hydrochloride as standard in a ratio of I :I as a free base, Sialic acid was determined after hydrolysis of the glycoprotein in O.IN H2 S0 4 for 30,60 and 90 minutes at 80° C by

the method of Warren (19) .

EXPERIMENTAL PROCEDURE AND RESULTS Rabbit seminal plasma was employed as a source of the male

accessory glands autoantigens (3,10) .

The autoantigenie activity of the original material and the

specimens obtained at several steps of the purification was checked

by double immunodiffuaion gel precipitation using rabbit 134 antiserum . This selected serum is in fact an specific autoantiserum as demonstrated in the experiment presented in Table I . TABLE I Reaction of Tanned Cell Hemagglutination of Antiserum from Rabbit 134 (Autoimmunized with Seminal Plasma) Against Human Red Blood Cells Coated with Various Tissue Extracts and Fluids at 0 .1 g % Protein Concentration . ANTISERUM RECIPROCAL TITRE AGAINST RED BLOOD CELLS COATED WITH : Rabbit seminal plasma 4096

Autologous seminal plasma

Rabbit accessory glands extract

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Guinea pigs# accessory glands extract <8

Rabbit## Kidney extract < 8

Male accessory Blends of cat, pig, mouse, rat, dog end human seminal plasma were negative when tested in the same way .

Other rabbit preparations tested, including extracts of testis, vagina, bladder, uterous, liver, spleen and brain. gave negative results .

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Purification procedure: Approximately $0 ml of seminal plasma, containing I g of proteins, were used for each preparation . The first step consisted in dialyzing the seminal plasma sample overnight at 4° C against 0 .0$ M Tris-HCI buffer pH 8 + 0.05 M NaCI + 0 .001 M EDTA . The precipitate formed during dialysis was removed by centrifugation at

12,000 g for 30 min . The supernatant

was added to 2$ g aliquot of DEAE Sephadex gel prepared as

described ebova and mixed with the aid ôf a magnetic stirrer for I h at 4° C . The slurry was then filtered through a Buchner funnel (Whatman N° I filter paper) and washed four times with 25 ml of the buffer used for the gel equilibration . Finally, the excess liquid was removed . The filtrates were transfered to a beaker containing 25 9 of cold DEAE Sephadex gel . Close contact with the exchanger was continued for I h mixing with the aid of a magnetic stirrer . The suspension containing crude autoantigen was filtered

through a Buchner funnel as described above, but only two 2$ ml

portions were used to wash the gel . After the final rinse the gel was drained again to ensure maximal removal of products . The

filtrates were then immediately dialyzed against 0 .1 M potassium chloride, followed by exhaustive dialysis against distilled water . A small precipitate was usually formed during dialysis, which was removed by centrifugation . The supernatant was liophylized. The yield of this materiel was approximately 340 m9~100 ml of seminal plasma . 80 mg of liophylized material from the preeed,ing steps was run through Sephadex G-100 columns (2 .2 x II$ em) and eluted at a flow rate of 8-10 ml per h . Fig . IA shows a characteristic elution profile . Three major fractions were present . The first corresponds to the excluded volume and the remaining two to different volumes of penetration into the gel . The second peak denominated FII A, contained the autoantigen as deduced by double immunodiffusion gel precipitation assays, and this was therefore chosen for further purification . Recycling of the FII A was done in Sephadex G-100 columns under the above described experimental conditions . Fig. IB

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t

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wa

~~oao

s

FIG . I Chromatography on Sephadex G-100. A :80 mg of material obtained by DEAE-Sephadex batch procedure. Bt and C : recycling of fraction II A separated as indicated in A. and Fig . IC show the elution profiles of the first and second recycling steps respectively . It is evident that the recycling was efficient in the elimination of the main contaminants present in the exclusion volume (FI) . The final yield of purified material was 68 mg weight for 100 ml of seminal plasma and is subsequently referred to as FII autoantigen . Criteria of ourity and aoecificitv :

In the polyacrylamide gel

electrophoresis assay presented in Fig . 2, seminal plasma shows I$ distinct bands, whereas in the final product

only one well-defined

band close to the origen is present . Thus, FII autoantigen behave es a homogeneous substance. Double immunodiffuaion gel precipitation

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experiments were performed in order to investigate the antigenic purity .Fig . 3 shows a representative experiment . It may be seen

that seminal plasma (well 3) shows a marked antigenic heterogeneity

Fig.2 . Polyacrylamide gel electrophoresis in Tria-glycine buffer(Amido Black 10 B stained) . I ; Rabbit seminal plaaAa (840 Ng) " 2 : Batch DEAE Sephadex fraction (200 Ng) " 3 : FII autoantigen (100 Ng) "

when confronted with sheep anti-rabbit seven serum (well $) . In contrast, a single precipitation line is produced by FII autoantigen (well 2) reacting with the same antiserum.Furthermore, the reaction of this fraction with the autoantiserum charged in well

I proved the a~toantigenic nature of the macromolecule under st udy. Fig .3 . Gel diffusion precipitation pattern . Wells charged as indicated . I : Autoantiserum from rabbit 134 . 2 : FII autoantigen I g ~. 3 : Rabbit seminal plasma 0 .~ 9 ~" 4: Saline . $ : Sheep anti-rabbit semen serum absorbed with liophylized rabbit serum .

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The organ-specificity of the FII autoantigen was explored using inhibition of hemagglutination reaction . Results presented in Table 2 show that among several tissue extracts only rabbit seminal plasma and extract from male accessory glands were able to inhibit the reaction, Thus, the purified macromolecule behaves as an organ-specific autoantigen, TABLE 2 Inhibition of Reaction Between Rabbit 134 Autoantiserum at Dilution Corresponding to 8 Hemagglutinating Units and Human Red Blood Cells Coated with FII Autoantigen at 0 .1 g ~ Using as Inhibitors Various Tissue Extracts and Fluids from Rabbits at an Initial Protein Concentration of I g % . RECIPROCAL TITRE OF THE INHIBITING SPECIMENS LISTED BELOW : Seminal plasma

Accessory glands extract

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2048

Testis extract < 4

Epididymis extract <4

Kidney extract(#) <4

(#) Other rabbit preparations tested were extracts from heart thyroid, liver, spleen, pancreas, submaxillary gland, salivary gland and brain ; all were negative as inhibitors, Preliminary studies on some physical properties and chemical composition of the isolated macromolecule are summarized in Table 2 . TABLE 2 E ~

~m

284 mN

6.42

Chemical compo~itian

Hexose Hexosamines Fucose Sialic acid Polypeptides

9 .61 1 .03 0 .33 0 .00 87 .73

From these chemical values, the autoentigen hes been identified as a glycoprotein .

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Purificatioa of an Autoantigen DISCUSSION

Seminal plasma is regarded as en appropriate source of the male accessory glands autoantigen (3,10) . The present

investigations were iniciated for the purpose of preparing from

rabbit seminal plasma a homogeneous autoantigenic protein of these glands, in a sufficiently large quantity for both chemical

study

and immunological research .The first step in the method of purification by employing DEAE-Sephadex batch adsorption, as presented here, is extremely simple and rapid. The molarity of the buffer and the quantity of DEAE-Sephadex gel used per gram of

protein were critical as far as the recovery of the autoantigen was concerned . Furthermore, when the crude material obtained from the preceding step was fractionated through Sephadex G-100 column, two recyclings were necessary in order to remove contaminating proteins . The isolated and purified protein, called FII autoantigen, behaved as a homogeneous substance in polyacrylamide electrophoresis, as well as in double immunodiffuaion gel precipitation experiments where both heterologous and autologous antisera were used . Additionally, is was characterized as a glycoprotein by reason of ite chemical properties . The organ specific nature of the FII

autoantigen was proved using inhibition of tanned cell hemagglutination test, confirming and enlarging previous observations, (3,10,11) . Experiments on physico-chemical characterization and immunogenic properties of the purified autoantigen are now in progress in our laboratory . The object of this research project is to clarify the mechanism of experimental autoimmune damage to rabbit male accessory Blends (4) .This is part of a more general study on immunologic factors involved in the pathogenesis of I"esion observed in the human prostatic gland, as well as in other related tissues . Homologues of the rabbit male accessory glands, the prostate seminal vesicle and Cowper~s gland are found in human (9) .

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ACKNOWLEDGEMENT This Work Was sustained by a grant from the Consejo National de Investigaciones Cientificas y Técnicas (CNICT 5$06/72) . The authors Would like to thank Dr . Luis C . Patrito for performing the chemical studies . REFERENCES I . S . SHULMAN, C . YANTORNO, W.A . SOHNES, M.J . GONDER and E. WITEBSKY, Immunolocy 10, 99-113 (1966) .

2 . C . YANTORNO, W.A . SOHNES, M .J . GONDER and S . SHULMAN, Immunolo~ay 12, 395-410 (1967) . 3. S. SHULMAN, C. RIERA and C . YANTORNO, J . Immunol . 100, . 682-688 (1968) . 4. C . YANTORNO, E . VOTTERO-CIMA and M . GALMARINI, l0, 397-403 (1973) .

Invest . Urol .

5 . E. WITEBSKY, Cancer Res. 21, 1216-1222 (1961) . 6 . P.J . BREDDERMAN, R.H . FOOTE and A .M . JASSEN, J . Re,~rod. Fertil . Z . 401-403 (1964) . 7 . S. SHULMAN, C. YANTORNO, G .W . BARNES, M .J . GONDER, W .A . SOHNES and W . WITEBSKY, Ann . N .Y . Acad . Sci . Imo, 279-291 (1965) . 8 . A .G . GORNALL, C .S . BARDAWILL and M.M . DAVID, J . Biol . Chem . 177, 751-766 (1949) .

9 . D . PRICE, Comparative Aspects of Development and Structure in the Prostate . In : Biology of the Prostate and Related Tissues, p . I-28 . National ancer nst~tute onograp overnment Printing Office (1963) . IO .C . YANTORNO, M .A . VIDES and E . VOTTERO-CIMA, J . Reprod . Fertil . 25,, 229-238 (1972) . II .E . YOTTERO-CIMA, M .A . VIDES and C . YANTORNO, Ann . Immunol . slnst . Pasteur ) ~ C, 273-284 (1973) . 12 .S .V . BOYDEN, J, exo. Med. ~,

107-120 (19$I) .

13 .0 . OUCHTERLONY, Pro~ar . AIlerAV , ,~,,

I-78 (19$8) .

14 .ß .J . DAVIS, Ann . N .Y . Acad . .Sci . 121, 404-427 (1964) . 15 .R .J . WINZLER, Methods of Biochem Analysis 2, 293-294 (1955) . 16 . D. FRANCOIS, R .D . MARSHALL and A. NEUBERGER, Biochem. J . 8,~, 335-341 (1962) . 17 . Z . DISCHE and L .B . SHETTLES, J . Biol . Chem . ~, 595-603 (1948) .

18 . C.J .M . RONDL E and T .J . MORGAN, Biochem. J . 61, 586-589 (1955) .

19 . L. WARREN, J . Biol . Chem . 2~, 1971-1975 (1959) .