analysis of the secretory glycoproteins of the ... · in the present work, the brain of 4500...

| ELSEVIER Molecular Brain Research 26 (1994) 299-308

MOLECULAR BRAIN

RESEARCH

Research Report

Analysis of the secretory glycoproteins of the subcommissural organ of the dogfish ( Scyliorhinus canicula)

J.M. Grondona ~, J. P6rez a, M. Cifuentes a M.D. L6pez-Avalos a, F.J. Nualart b, B. Peruzzo h, P. Fernfindez-LLebrez a, E.M. Rodrlguez b,,

a Departamento de BiologEa Animal, Facultad de Ciencias, Universidad de Mdlaga, Mdlaga, Spain b Instituto de Histologla y PatologEa, UniversidadAustral de Chile, Valdivia, Chile

Accepted 17 May 1994

Abstract

The subcomissural organ (SCO) is an ancient and conserved brain gland secreting glycoproteins into the cerebrospinal fluid which condense to form Reissner's fiber (RF). The SCO of an elasmobranch species, the dogfish Scyliorhinus canicula, was investigated applying morphological and biochemical methods. The SCO of 34 dogfishes were processed for the following techniques: (1) conventional transmission electron microscopy; (2) light and electron microscopy lectin histochemistry (Con- canavalin A, Con A; wheat germ agglutinin, WGA; Limax flavus agglutinin, LFA); (3) light and electron microscopy immunocytochemistry using antisera raised against the glycoproteins of the bovine RF (anti-bovine RF), and the secretory material of the dogfish SCO (anti-dogfish SCO). The former reacts with the SCO of virtually all vertebrate species [19] (conserved epitopes); the latter reacts only with the SCO of elasmobranchs [Cell Tissue Res., 276 (1994) 515-522] (class-specific epitopes). At the light microscopic level both antisera immunoreacted selectively with the SCO and RF; no other structure of the central nervous system was reactive. Within the SCO the binding sites for WGA (affinity = glucosamine, sialic acid) and LFA (affinity = sialic acid) displayed the same density and intracellular distribution. At the ultrastructural level two types of granules were distinguished. Type I granules (200-400 nm) were numerous, reacted with both antisera, bound WGA but not Con A. Type II granules (0.8-1.8 ~zm) reacted with the anti-bovine RF serum but not with the anti-dogfish SCO serum, bound Con A and WGA. The content of dilated cisternae of the rough endoplasmic reticulum reacted with both antisera and bound Con A; it did not bind WGA. The SCOs of 4500 dogfishes were extracted in ammonium bicarbonate. This extract was used for SDS-PAGE and blotting. Blots were processed for immunolabeling using anti-bovine RF and anti-dogfish SCO sera, and for lectin binding (Con A, WGA and LFA). The anti-bovine RF revealed four compounds with apparent molecular weights of 750, 380, 145 and 35 kDa. The two former also reacted with the anti-dogfish SCO serum and bound Con A. Only the 380 kDa compound bound WGA and LFA. The findings indicate that both the conserved and the class-specific epitopes are part of the same compounds (780, 380 kDa), which would be stored in type I granules. The lectin binding properties of these compounds point to the 780 kDa compound as a precursor form and the 380 kDa polypeptide as a processed form.

Keywords: Subcommissural organ; Secretory glycoprotein; Biochemistry; Immunocytochemistry; Dogfish; Scyliorhinus canicula

1. Introduction

The subcommissural organ (SCO) belongs to the group of periventricular brain structures known as circumventricular organs [8]. It can be regarded as a truly glandular structure. It is located in the dorso-caudal region of the third ventricle, at the entrance of the Sylvian aqueduct. It consists of a secretory ependyma

* Corresponding author. Fax: (56) 63-22 16 04.

0169-328X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0169-328X(94)001 14-T

with one or two layers of columnar cells lining the posterior commissure. Histochemical, autoradiographic and ultrastructural studies have revealed that the SCO is a structure highly specialized in the secretion of glycoproteins. The secretory material is released into the cerebrospinal fluid (CSF) of the third ventricle and condenses to form a thread-like structure, the Reiss- ner's fiber (RF). RF extends along the aqueduct, four ventricle and central canal of the spinal cord. The SCO is an ancient and persistent organ of the brain of vertebrates (for review see Rodriguez et al. [23,26]).

300 J.M. Grondona et aL / Molecular Brain Research 26 (1994) 299-308

During the last decade specific antisera against the secretory material of the SCO have been raised. These are antisera against: (i) bovine RF [21,31,32]; (ii) bovine SCO extracts [27]; (iii) chick embryo SCO extracts [10]. Also monoclonal antibodies against extracts of bovine SCO [14] and chick embryo SCO [3] have been obtained.

Since antisera against the bovine RF recognize the secretory material of the SCO of all vertebrate classes [21,32], it was thought that the secretory material of the SCO was highly conserved. This assumption was chal- lenged after the demostration that antisera developed against different SCO glycoproteins of the bovine SCO displayed different immunocytochemical properties when used to immunostain the SCO of several mammalian and reptilian species [16,24]. Recently, we have raised antisera against various types of extracts of the dogfish SCO. These antisera immunostained the SCO of elasmobranch species but did not react with the SCO of other group of vertebrates [5]. On the other hand the elasmobranch SCO does react with the anti- bovine-RF serum. These finding and those of Nualart et al. [16] have led to the suggestion that in the material secreted by the SCO there are epitopes (or compounds) which are class-specific, and others which are conserved and synthetized by the SCO of most vertebrate species [5].

In the present investigation the antiserum recognizing conserved epitopes (anti-bovine RF) and the antiserum recognizing elasmobranch specific epitopes were used for immunocytochemical and immunoblotting studies of the dogfish SCO in order to establish: (i) whether the class-specific and conserved epitopes are constituents of the same compound; or they represent different compounds; (ii) the number and molecular mass of the secretory compounds present in the SCO proper; (iii) the subcellular distribution of the secretory material. This analysis was complemented by a lectin binding study in tissue sections and in blots with the aim to further characterize the materials secreted by the dogfish SCO.

dissected out and kept in pure acetone containing the following protease inhibitors: 0.5 mM phenylmethylsulfonyl fluoride (PMSF, Merck, Darmstadt, FRG), 1 mM ethylenediamine tetraacetate (EDTA, Sigma, St. Louis, MO), 1 t tM pepstatin (Sigma), 1 ttM leupeptin (Sigma). Under a dissecting microscope a block of tissue containing the SCO and the posterior commissure was dissected out. Each single extract was prepared on the same day the SCOs were collected, and usually contained 150 SCOs. The extraction medium contained 50 mM ammonium bicarbonate, 0.1% sodium dodecyl sulphate (SDS, Sigma) and all the protease inhibitors mentioned above. The pool of SCOs was homogeneized in 1 ml of extraction medium using an Ultra-turrax T-25 (Janke-Kunkel, Staufen Ger- many), at 24000 rpm, for 1 min. Then it was sonicated (Sonics & Materials Inc., Danbury CT) (50 Watts) for 15 s and cooled on ice for 15 s. This sonication-cooling cycle was repeated 4 times. The extract was centrifuged at 8,500 × g for 20 min at 4°C (Biofuge RS17, Heraeus, Osterode Germany) and the supernatant at 19,500× g for 30 min at 4°C. This second supernatant was regarded as a crude extract of dogfish SCO. The protein content was determined by the Bradford's method [1]. Aliquots were lyophilized and stored at

- 20°C. Tissue samples from the telencephalon, cerebellum and optic

tectum were extracted following the same procedure used for the preparation of SCO extracts. These extracts were used for SDS- PAGE and blotting. Additionally, the optic tectum extract was used for immunoabsorption of AFRU as described previously [5]. Collec- tion and extraction of bovine SCO and RF were performed as previously described [16].

2.3. Polyclonal antibodies

The following antisera were used: (1) Anti-dogfish SCO extracted in ammonium bicarbonate (ADSO: A = antiserum, D = dogfish, SO = subcommissural organ; Grondona et al. [5]), developed in rats (ADSO-3) and in a rabbit (ADSO-4). These antisera were sequentially immunoabsorbed with extracts of dogfish telencephalon, cerebellum and optic tectum to eliminate unwanted antibodies [5]. The immunoabsorbed antisera immunostained the SCO-RF complex of elasmobranch exclusively [5]. (2) An antiserum raised in rabbits against bovine Reissner's fiber extracted in a medium containing urea, EDTA and DTT (AFRU, A = antiserum, FR = fiber of Reiss- ner, U = urea, Rodrlguez et al. [21]); (3) anti-Limax flauus agglutinin (anti-LFA) obtained in our laboratory (see Rodrlguez et al. [25]); (4) anti-Concanavalin A (anti-Con A)(Sigma); (5) anti-wheat germ agglutinin (anti-WGA)(Sigma); (6) anti-rat IgG developed in rabbits obtained in our laboratory; (7) anti-rabbit IgG developed in goat obtained in our laboratory.

2.4. Polyacrylamide gel electrophoresis (PAGE) and blotting

2. Materials and methods

2.1. Animals

In the present work, the brain of 4500 dogfishes, Scyliorhinus canicula, have been used for the biochemical study. Additionally, 34 specimens were used for light and electron microscopy, immunocytochemistry, and lectin histochemistry.

2.2. Subcommissural organ: dissection and extraction

The dogfishes were obtained in the fishing harbor of Mfilaga. Post-mortem times ranged between 0 and 8 h. The brains were

SDS-PAGE was performed following the procedure of Laemmli [11] using 100× 100 mm slab gels containing a 5-15% polyacrylamide linear gradient; bisacrylamide was used at a concentration of 2.6%. Samples (100 ~g proteins) of the SCO and other brain areas were dissolved in 125 mM Tris-HC1 (Sigma) (pH 6.8) containing 2% SDS (Serva, Heidelberg, Germany), 5% /3-mercaptoethanol (Sigma) and 10% glycerol (Sigma), heated at 95°C for 2 min and loaded on the stacking gel. Some gels were stained with Coomassie blue or silver nitrate [15]. Most gels were electrotrasferred onto nitrocellulose sheets (Millipore, Bedford MA) according to the method of Towin et al. [34]. This transfer was carried out in 25 mM Tris-HCl (pH 8.3) containing 192 mM glycine, 0.2% SDS and 20% methanol at 0.1 A during 8 h. Some strips of nitrocellulose were stained with Amido black (Sigma) to control the protein transfer. Molecular weight standards were glycoproteins of the bovine subcommissural organ (540, 450, and 320 kDa [16]), and myosin (205 kDa), /3-galactosidase

J.M. Grondona et al. / Molecular Brain Research 26 (1994) 299-308 301

(116 kDa), phosphorylase b (97 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa) (Sigma).

The blots were processed for immunostaining and lectin binding.

Saturation of protein-binding sites with 5% non-fat milk in 0.1 M phosphate-buffered saline (PBS) containing 0.15 mM NaCl, was followed by immunoperoxidase (PAP) staining by use of ADSO-4

~ i ~ ¸

I

~ ~ ~ i~ ~i:!: ~ ~i~ ! t i ~

Figs. 1-4. Transverse sections through similar levels of the dogfish subcommissural organ (SCO) immunostained with antisera against dogfish SCO (ADSO-3) (Fig. 1) and bovine Reissner's fiber (AFRU) (Fig. 2); and stained with concanavalin A (Con A) and wheat germ agglutinin (WGA) (Figs. 3 and 4). AFRU and ADSO showed similar staining patterns. Con A bound throughout the cytoplasm of the SCO ependymal cells, with the exception of the most apical region; WGA labeled material located in the supranuclear and most apical regions of these cells. RF, Reissner's fiber, x 110.

302 J.M. Grondona et al. / Molecular Brain Research 26 (1994) 299-308

(dilution 1:500) and AFRU (dilution 1:1000); and 4-chloro-l-naph- tol (Sigma) as electron donor. Control transfers were incubated with preimmune serum or processed for the PAP method but omitting incubation in the primary antiserum.

For lectin binding, the blots were sequentially incubated with oxidized BSA (Sigma), Con A (affinity = mannose, glucose) 2 /zg /ml (Sigma), anti-Con A (dilution 1:800, Sigma) and then processed as for immunostaining. Control transfers were incubated with Con A in the presence of 1 M a-methyl-mannoside (Sigma); other blots were sequentially incubated with oxidized BSA, Limax flavus agglutinin (LFA, affinity = sialic acid; 2.0/~g/ml; Caibiochem, San Diego CA), anti-LFA (dilution 1:500) and then processed as for immunostaining.

[31]. The sections were sequentially incubated in (i) the primary antiserum (ADSO-3, developed in rats; ADSO-4, developed in a rabbit, both at a dilution 1:500; AFRU, developed in rabbits, dilution 1:1000) for 18 h; (ii) the secondary antibody (anti-rat IgG or anti-rabbit IgG) diluted 1:15, for 30 min; (iii) rat PAP (Serva) or rabbit PAP (Sigma), 1:75 dilution, for 30 min. DAB was used as electron donor. All antisera and the PAP complex were diluted in Tris buffer, pH 7.8, containing 0.7% non-gellin seaweed h-carra- geenan (Sigma) as saturating agent, and 0.5% Triton X-100 [29] (Sigma). Omission of incubation in the primary antiserum in the immunostaining procedure was used as a control test. Under this condition no immunoreaction was seen in the SCO.

2.5. Conventional transmission electron microscopy

The brain was quickly dissected out, and after exposing the cerebral ventricles it was immerssed in 2% paraformaldehyde and 1% glutaraldehyde in elasmobranch buffer [12] (0.15 M PO4H2Na H 20, 0.36 M urea, 0.07 M CINa) for 2 h. After washing in the same buffer for 30 min, tissue blocks containing the SCO were postfixed in 1% osmium tetroxide (Polyscience, Northampton UK), prepared in elasmobranch buffer, for 2 h at 4°C. Dehydration was in a graded series of ethanol and embedding was in Araldite (Polyscience). Ultrathin sections were stained with uranyl acetate (Polyscience) and lead citrate (Polaron, Herts, UK).

2.6. Lectin histochemistry

2. 7.2. Electron microscopy The brain was fixed by immersion in a mixture containing 2%

paraformaldehyde, 0.5% glutaraldehyde and 15% of a saturated picric acid solution, buffered in elasmobranch buffer during 2 h at 4°C. Embedding was in Lowicryl K4M (Polyscience) polymerized under UV light at - 30°C and using benzoyl peroxide (Fluka, Buchs, Switzerland) as acelerator [19]. Ultrathin sections were immunostained according to the protein A gold method [28]. Briefly, the sections, once incubated in primary antisera (AFRU, ADSO-4; both raised in rabbits), were immersed in a suspenssion of protein-A gold complexes in PBS buffer, pH 7.4, for 45 rain at room temperature. Gold particles were in the range of 15 nm. The sections were counterstained with aqueous uranyl acetate and lead citrate. Incuba- tion with non-immune serum instead of the primary antiserum was used as a control test. No labeling was detected in this case.

2.6.1. Light microscopy The brain was fixed by immersion in Bouin's fluid, for 2 days.

Embedding was in paraffin. For Con A and wheat germ agglutinin (WGA) binding, the sections were incubated with peroxidase-labeled lectin (Con A, 5 tzg/ml; WGA, 3 /zg/ml; Sigma) dissolved in Tris buffer, pH 7.8, for 45 rain. 3, 3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) was used as electron donor. For LFA binding the sections were sequentially incubated with (i) non-labeled LFA (1 ~g/ml; Calbiochem); (ii) anti-LFA (1:5000 dilution); (iii) anti-rabbit IgG (1:15 dilution); (iv) PAP (Sigma) (1:75 dilution); (v) DAB. For details see Peruzzo and Rodrlguez [19].

2.6.2. Electron microscopy The brains were fixed in a mixture containing: 2% paraformalde-

hyde (Merck), 0.5% glutaraldehyde (Merck) and 15% of a saturated picric acid solution, buffered in elasmobranch buffer, during 2 h at 4°C. After washing in the same buffer for 30 min, the tissue blocks containing the SCO were post-fixed in 0.5% osmium tetroxide in elasmobranch buffer, for 2 h. Subsequently, the tissue were washed in distilled water for 15 min, dehydrated and embedded in LR White resin (TAAB, Berkshire, England). Ultrathin sections were incubated with the non-labeled lectins (Con A, WGA, Sigma; 1 ~g /ml in Tris buffer, pH 7.8) for 45 min at room temperature. Then they were washed and incubated with antisera against the respective lectin (anti-Con A, anti-WGA; 1 : 1000 dilution in PBS, pH 7.3) for 1 h at room temperature. After washing in PBS the sections were treated with protein A-gold complex, particle size 15 nm (dilution 1 : 10), for 45 min at room temperature [19]. The sections were counterstained with aqueous uranyl acetate and lead citrate. In control sections, incubation with the lectins was omitted; no labeling was detected in these cases.

2. 7. lmmunocytochemistry

2. 7.1. Light microscopy Eight /xm-thick paraffin sections of Bouin fixed brains were

processed for the immunoperoxidase method of Sternberger et al.

3. Results

3.1. L igh t microscopy

3.1.1. I m m u n o c y t o c h e m i s t r y T h e a n t i s e r u m aga ins t t h e b o v i n e R e i s s n e r ' s f ibe r

( A F R U ) s t rong ly s t a i ned t h e dogf i sh S C O a n d Re i s s -

n e r ' s f ibe r at a d i l u t i on o f 1 : 1 0 0 0 (Fig. 2). N o o t h e r

s t r u c t u r e o f t h e dogf i sh b r a i n a p p e a r e d s ta ined . T h e

S C O a p p e a r e d as a p s e u d o s t r a t i f i e d e p e n d y m a . T h e

i m m u n o r e a c t i v e m a t e r i a l o c c u p i e d t h e ap ica l a n d basa l

r eg ions o f t h e c y t o p l a s m o f t h e cel l bod ies , as we l l as

t he e p e n d y m a l p r o c e s s e s e n d i n g o n t h e l e p t o m e n i n g e

a n d b l o o d vessels . T h e p a t t e r n o f s t a in ing o f t h e S C O

and t h e R F us ing t h e a n t i s e r a aga ins t c r u d e ex t rac t s o f

t h e dogf i sh S C O ( A D S O - 3 , A D S O - 4 ) a n d p u r i f i e d by

i m m u n o a b s o r p t i o n w i t h ex t rac t s o f t h r e e d i f f e r e n t b r a i n

r eg ions , was s imi la r to t ha t o b t a i n e d w i t h A F R U (com-

p a r e Figs. 1 a n d 2).

3.1.2. Lec t in his tochemistry

C o n A b i n d i n g s i tes w e r e n u m e r o u s t h r o u g h o u t t h e cel l b o d y o f t he e p e n d y m a l cells, w i th t h e e x c e p t i o n o f

t he a p i c a l - m o s t p o r t i o n o f t h e s e cells, w h i c h was de -

vo id o f label . T h i s l a t t e r r e g i o n a p p e a r e d as a consp ic -

u o u s c l ea r b a n d b o r d e r i n g t h e v e n t r i c l e (Fig. 3). Re i s s -

n e r ' s f ibe r d id n o t b i n d C o n A. W G A a n d L F A s t rong ly

s t a ined t h e c y t o p l a s m e x t e n d i n g f r o m the n u c l e a r re-

g ion to t he v e n t r i c u l a r cel l po le . T h e subap ica l r e g i o n

wh ich d id n o t b i n d C o n A was s t rong ly l a b e l e d by

W G A (Fig. 4) a n d L F A . R F b o u n d L F A a n d W G A .

J.M. Grondona et al. ~Molecular Brain Research 26 (1994) 299-308 303

Fig. 5. Electron microscopic view of dogfish SCO ependymal cells. The perinuclear cytoplasms (PC) contain many distended cisternae of rough cndoplasmic reticulum. The supranuclear cytoplasms (SC) display Golgi apparatus (GA) and large dense granules (type II granulesXarrowheads). The apical cytoplasms (AC) contain most of type I secretory granules (arrows). x 2900.

Figs. 6-8. Detail of the apical cytoplasm immunostained with ADSO-4, AFRU and stained with WGA. Type I granules are labeled by all three procedures (arrows). x 19000.

Figs. 9-11. Detail of the supranuclear cytoplasm immunostained with ADSO-4, AFRU and stained with Con A. Type II granules are labeled by AFRU and Con A but not by ADSO (arrows). x 10000.

Figs. 12-14. Detail of the perinuclear cytoplasm immunostained with ADSO-4, AFRU and stained by Con A. Distended cisternae of RER are labeled with all three procedures (asterisk). Note mitochondria close to the RER membranes (arrows). × 9500.

M W I

J.M. Grondona et al. / Molecular Brain Research 26 (1994) 299-308

540 - 750~ ~ , . - ~ 450- 380• ~ ~. 320 -

205 .......

116 - 9 7 - -

6 6 - -

4 5 -

29-

D O G F I S H SCO

145 i

DOGFISH

i

I

OT

I

305

SILVER Con A WGA AFRU ADSO AFRU ADSO SILVER STAIN STAIN

Fig. 18. SDS-PAGE of dogfish subcommissural organ (SCO) and optic tectum (OT) extracts stained with silver nitrate. Blots were processed for Con A and WGA binding and for immunostaining with AFRU and ADSO-4. MW, molecular weight markers; small arrowheads, secretory compounds with an apparent molecular mass of 750 and 380; asterisk, absence of the latter bands in the optic tectum lanes; arrows, 145 kDa band; large arrowheads, 35 kDa band.

3.2. Electron microscopy

A clear zonation of the cytoplasm of the ependymal cells of the dogfish SCO can be distinguished at the ultrastructural level: (i) apical region; (ii) supranuclear region; (iii) perinuclear region and; (iv) basal processes and endings (Fig. 5). The apical region corresponds to the ventricular cell pole. It has an approximate thickness of 12 /~m and contained many granules with a content of a modera te electron density, and ranging in size between 200 and 400 nm. These will be regarded as type I granules. The supranuclear cytoplasm has a thickness of 35/~m and displayed: (i) many flat parallel cisternae of rough endoplasmic reticulum (RER); (ii) a well developed Golgi complex; (iii) granules of high electron density, ranging between 0.8 and 1.8 /~m in

diameter; these will be regarded as type II granules (Figs. 5 and 17); (iv) type I granules were also present in the supranuclear cytoplasm but in a lower number as compared with the apical cytoplasm (Fig. 17). The perinuclear cytoplasm was occupied by large and dilated cisternae of R E R filled with a flocculent material. Usually one single cisternum occupied most of the infranuclear area. The basal processes displayed mitochondria, microfilaments, glycogen particles, and R E R cisternae.

3.3. Ultrastructural lectin histochemistry and immunocytochemistry

Type I granules were immunolabeled with the anti- bovine RF (AFRU) and the anti-dogfish SCO (ADSO-

s '

Fig. 15. Apical region of the ependymal cells of the dogfish SCO processed for WGA binding. Type I granules (arrows) and the plasma membrane, especially at microvilli (MV), appeared labeled, x 10000.

Fig. 16. Supranuclear cytoplasm of the ependymal cells of the dogfish SCO. WGA binding. Type I (solid arrows) and Type II (arrow profiles) granules appeared labeled. × 20000.

Fig. 17. Supranuclear region of the ependymal cells of the dogfish SCO processed for conventional transmission electron microscopy. Type I (solid arrows) and type II (arrow profiles) granules are distinct. × 30000.


Table 1 Reactivity of the ependymal cells of the dogfish subcommissural organ to antisera and lectins

AFRU ADSO Con A WGA

Type 1 granules + + + Type I1 granules + - + + Dilated RER cisternae + + + -

AFRU, anti-bovine RF serum developed in rabbits; ADSO-4, anti- dogfish SCO serum developed in a rabbit; Con A, concanavalin A (affinity = mannose, glucose); WGA, wheat germ agglutinin (affinity = N-acetyl glucosamine, sialic acid); + , positive; - , negative.

4) sera (Fig. 6 and 7). They bound WGA (Figs. 8, 15) but not Con A. Type II granules were labeled by AFRU (Fig. 10), Con A (Fig. 11) and WGA (Fig. 16), but did not react with ADSO-4 (Fig. 9). The content of the dilated cisternae of RER located in the perinuclear region reacted with ADSO-4 (Fig. 12), AFRU (Fig. 13) and Con A (Fig. 14), but not with WGA (Table 1).

3.4. SDS-PAGE and blotting

Coomassie blue staining of SDS-PAGE gels of dogfish subcommissural organ and optic tectum extracts revealed one band (380 kDa) present in the SCO and missing in the optic tectum. Silver staining of the gels revealed a few additional bands present in the SCO and not detectable in the optic tectum. One of them was distinct and had an apparent molecular mass of 750 kDa. A band of 145 kDa was distinct in the SCO and poorly detectable in the optic tectum (Fig. 18).

SDS-PAGE of extracts of the dogfish SCO, followed by immunoblotting using AFRU revealed four immunoreactive bands with apparent molecular weights of 750, 380, 145 and 35 kDa (Fig. 18). AFRU also revealed a band of 35 kDa in the optic tectum extract. Immunoabsorption of AFRU with an extract of optic tectum abolished the immunostaining of the 145 kDa and 35 kDa bands of the SCO extracts; the 750 and 380 kDa continued to be reactive. Parallel runs of the SCO extracts immunostained by use of ADSO-3 and ADSO- 4 revealed the 750 and 380 kDa bands, but not the 145

Table 2 Immunoblotting and lectin binding of the secretory material of the dogfish subcommissural organ

AFRU ADSO Con A LFA WGA

750 750 750 - - 380 380 380 380 380

145 . . . . 35 . . . .

AFRU, anti-bovine RF serum; ADSO-4, anti-dogfish SCO serum; Con A, concanavalin A (affinity = mannose, glucose); LFA, Limax flavus agglutinin (affinity = sialic acid); WGA, wheat germ agglutinin (affinity = glucosamine, sialic acid); Numbers, molecular weight in kDa; - . negative reaction.

and 35 kDa bands (Fig. 18). The 750 kDa band displayed affinity for Con A, but not for WGA or LFA. The 380 kDa band had affinity for Con A and LFA (Table 2).

4. Discussion

The anti-bovine Reissner's fiber (AFRU) and the anti-dogfish subcommissural organ (ADSO) sera have been used previously for a comparative immunocytochemical study of the SCO in several vertebrate classes [5,18]. While AFRU recognized the SCO secretory material in all studied species, ADSO reacted only with the secretory material of the SCO of elasmobranchs. These results led to the suggestion that: (i) there should be class-specific epitopes (or compounds) in the secretory material of the SCO of elasmobranchs, and which are revealed by ADSO; (ii) there should be conserved epitopes present in the material secreted by the SCO of most of the vertebrate species, and which are revealed by AFRU [5].

The important question whether the class-specific and the conserved epitopes are present in the same compound or represent different compounds could not be solved by the immunocytochemical study referred above. In this respect, the present immunoblotting study has provided valuable evidence. The antiserum revealing, in immunocytochemistry, class-specific epitopes (ADSO) reacted with two bands of 750 and 380 kDa. The same bands were revealed by the antiserum (AFRU) containing the antibody against the conserved epitope(s). This result supports the possibility that both types of epitopes are part of the same molecules. The alternative that there may be a conserved compound different from the 750 and 380 kDa compounds should also be considered. This putative compound should be ADSO-negative and AFRU-positive. The 145 and 35 kDa compounds present in the dogfish SCO meet this requirement. The fact that preabsorption of AFRU with an optic tectum extract abolished the reactivity of the 145 and 35 kDa bands but not that of the 750 and 380 kDa bands suggests that the two former would correspond to tissue components different from the SCO secretory material. However, in none of the numerous immunoblotting studies carried out before on the mammalian SCO, AFRU reacted with a non-secretory compound [6,16,26]. Furthermore, the AFRU- positive 145 kDa band present in the SCO was missing in the optic tectum. It is therefore puzzling that ab- sorption of AFRU with an extract of optic tectum abolished the reactivity of this band in the SCO. Exper- iments are in progress to raise antibodies against the 145 and 35 kDa bands of the SCO. At present, and as a working hypothesis, the 145 kDa compound could be regarded as a secretory non-glycosylated polypeptide

J.M. Grondona et al. / Molecular Brain Research 26 (1994) 299-308 307

containing conserved epitopes. A third alternative should consider the possibility that type II granules are not secretory in nature but lysosomes; this could ex- plain their ultrastructural appearance, intracellular distribution, and their affinity for Con A and WGA. The affinity of these granules for AFRU could be explained on the following graounds. The conservative epitope revealed by AFRU appears to be a conformational epitope associated to the integrity of the disulfide bonds [17]. On the other hand, after tryptic digestion, small peptides associated to disulfide bonds remain intact (unpublished observation). Furthermore, it has been shown that misfolded glycoproteins are retained in the RER and then degraded [13]. It seems, therefore, possible that part of the glycoproteins secreted by the dogfish SCO are transported to lysosomes, where some of the degradation products would retain their affinity for AFRU.

Considering the information available on glycosylation of proteins [9,30,35], and taking into account pre- vious results in the rat SCO [7,22,23], and the results of the present investigation, it can be postulated that the secretory compounds of the dogfish SCO are N-linked complex type glycoproteins with sialic acid as a terminal residue.

Sialilated, core-glycosylated glycoproteins bind Con A when extracted from the tissue and blotted, but do not bind Con A in tissue sections, despite the existence of internal mannose residues [7,16]. This makes this lectin a useful tool to identify, in tissue sections, core- glycosylated glycoproteins with terminal mannose residues [7]. The exclusive affinity of LFA for sialic acid [16] makes this lectin an excellent marker for sialilated glycoproteins that have passed through the Golgi complex [16,25,26]. WGA does not have affinity for the glucosamine residues of the core of core-glycosylated proteins, but it does display affinity for the internal glucosamine residues, and the terminal sialic acid residues added to the terminal chain in the Golgi complex [7,16]. Therefore, WGA is also a good marker for glycoproteins located in, or extracted from post- Golgi compartments.

The 750 kDa band revealed by AFRU and ADSO, binds Con A but it does neither bind WGA nor LFA. It seems likely that this compound was extracted from a pre-Golgi compartment, that is, the distended RER cisternae of the perinuclear region. Indeed, the content of these cisternae is labeled by AFRU, ADSO, Con A but not by WGA or LFA. Considering all these charac- teristics, and being the 750 kDa glycoprotein the largest immunoreactive secretory compound, it may be postulated that it represents a precursor form of the dogfish SCO secretion.

The 380 kDa band reacted with AFRU, and ADSO, and bound both Con A and LFA. This compound could therefore be characterized as a glycoprotein con-

taining sialic acid and that has been extracted from a post-Golgi cgmpartment, i.e.; the secretory granules. All these features point to the 380 kDa compound as a processed form. Recently we have developed an antiserum against the 380 kDa band; when used for light microscopy immunocytochemistry this antiserum stained, exclusively, the most apical region of the ependymal cells of the SCO, where type I granules are accumulated.

The SCO seems to be a unique type of gland with regard to the storage of its secretory products. A good body of evidence points to the existence in the SCO of two pools of secretory material. One of them is released into the ventricle within 1 h after synthesis; the other being retained in the dilated cisternae of RER and progressively processed and released over a period of 3-5 days [4,7,26]. In the mammalian SCO the bulk of the secretion of the SCO is stored in the RER; only a minor part is stored in the secretory granules [26]. In the snake [20] and the dogfish SCO (present report) both the RER and the secretory granules represent important storage sites. This may reflect an important functional difference between the mammalian SCO and the SCO of non-mammalian species. It could be postulated that the latter contains a larger pool of readily releasable material as compared to the SCO of mammals. Interestingly, an inhibitory serotoninergic input to the SCO is present in mammalas and missing in non-mammalian species [2,26].

Acknowledgements

Supported by Grants DGICYT PB 90-0804, Madrid, Spain to P.F.LL and Grants 91/0956 from CONICYT (Chile) and from the Direcci6n de Investigaciones, Universidad Austral de Chile, Chile, to E.M.R.

References

[1] Bradford, M.M., A rapid and sensitive method for the quantita- tion of microgram quantities of protein utilizing the principle of protein-dye binding, Anal, Biochem., 72 (1976) 248-254.

[2] Bouchaud, C. and Bosler, O., The circumventricular organs of the mammalian brain with special reference to monoaminergic inervation, Int. Rev. Cytol., 105 (1986) 283-327.

[3] Didier, R., Meiniel, R. and Meiniel, A., Monoclonal antibodies as probes for the analysis of the secretory ependymal differenti- ation in the subcommissural organ of the chick embryo, Dev. Neurosci., 14 (1992) 44-52.

[4] Diederen, J.H.B., Vulling, H.G.B. and Legerstee-Oostveen, G.G., Autoradiographic study of the production of secretory material by the subcommissural organ of frogs (Rana tempo- raria) after injection of several radioactive precursors, with special reference to the glycosylation and turnover rate of the secretory material, Cell Tissue Res., 248 (1987) 215-222.

[5] Grondona, J.M., Fernfindez-LLebrez, P., P~rez, J., Cifuentes, M., P6rez-Ffgares, J.M. and Rodrfguez, E.M., Class-specific


epitopes detected by polyclonal antibodies against the secretory products of the subcommissural organ of the dogfish (Scyliorhinus canicida), Cell Tissue Res., 276 (1994) 515-522.

[6] Hein, S., Nualart, F.J., Rodrlguez, E.M. and Oksche, A., Partial characterization of the secretory products of the subcommissural organ. In A. Oksche, E.M. Rodr~guez and P. Fern~ndez- Llebrez (Eds.), The Subcommissural Organ. An Ependymal Brain Gland, Springer, Berlin, 1993, pp. 79-88.

[7] Herrera, H. and Rodrlguez, E.M., Secretory glycoproteins of the rat subcommissural organ are N-linked complex-type glycopro- reins. Demonstration by combined use of lectins and specific gtycosidases, and by the administration of Tunicamycin, Histo- chemistry, 93 (1990) 607-615.

[8] Hofer, H., Zur Morphologie der circumventricul~iren Organe des Zwischenhirns der S~iugetiere, Verh. Deutsch. Zod. Ges., 22 (1959) 202-251.

[9] Hunt, L.A., Biosynthesis and maturation of cellular membrane glycoproteins, J. Supramol. Struct., 12 (1979) 209-226.

[10] Karoumi, A., Meiniel, R., Croisille, Y., Belin, M.F. and Meiniel, A., Glycoprotein synthesis in the subcommissural organ of the chick embryo, J. Neural Transm., 79 (1990) 141-153.

[1 l] Laemmli, U.K., Cleavage of structural proteins during the as- sembly of the head of bacteriophage T4, Nature, 227 (1970) 680-685.

[12] Long, D.M., Bodenheimer, T.S., Hartmann, J.F. and Klatzo, 1., Ultrastructural features of the shark brain, Am. Z Anat., 122 (1968) 209-236.

[13] Marquardt, T.M. and Helenius, A., Misfolding and aggregation of newly synthesized proteins in the endoplasmic reticulum, J. Cell Biol. 117 (1992) 505-513.

[14] Meiniel, R., Duchier, N. and Meiniel, A., Monoclonal antibody CIBsA s recognizes a ventricular secretory product elaborated in the bovine subcommissural organ, Cell Tissue Res., 254 (1988) 611-615.

[15] Merril, C.R., Switzer, R.C. and Van Keuren, M.L., Trace polypeptides in cellular extracts and human body fluids detected by two dimensional electrophoresis and a highly sensitive silver stain, Proc. Natl. Acad. Sci. USA, 76 (1979) 4335-4339.

[16] Nualart, F., Hein, S., Rodr~guez, E.M. and Oksche, A., Identifi- cation and partial characterization of the secretory glycoproteins of the bovine subcommissural organ-Reissner's fiber complex. Evidence for the existence of two precursor forms, Mol. Brain Res., 11 (199t) 227-238.

[17] Nualart, F. and Rodrlguez, E.M., Conservative epitopes of glycoproteins of the bovine Reissner's fiber involve the integrity of disulfide bonds, submitted.

[18] P6rez, J., Grondona, J.M., Cifuentes, M., Nualart, F.J., Fern~ndez-Llebrez, P. and Rodrlguez, E.M., Immunochemical analysis of the dogfish subcommissural organ. In A. Oksche, E.M. Rodriguez and P. Fern~indez-Llebrez (Eds.), The Subcom- missural Organ. An Ependymal Brain Gland, Springer, Berlin, 1993, pp. 99-110.

[19] Peruzzo, B. and Rodrlguez, E.M., Light and electron microscop- ical demostration of concanavalin A and wheat-germ-agglutinin binding sites by use of antibodies against the lectin or its label (peroxidase), Histochemistry, 92 (1989) 505-513.

[20] Peruzzo, B., P~rez, J., Fern~ndez-Llebrez, P., P6rez-Figares, J.M., Rodriguez, E.M. and Oksche, A., Ultrastructural immunocytochemistry and lectin histochemistry of the subcommissural organ in the snake Natrix maura with particular emphasis on its

vascular and leptomeningeal projections, Histochemistry, 93 (1990) 269-277.

[21] Rodriguez, E.M., Oksche, A., Hein, S., Rodrlguez, S. and Yulis, R., Comparative immunocytochemical study of the subcommissural organ, Cell Tissue Res., 237 (1984) 427-441.

[22] Rodrlguez, E.M., Herrera, H., Peruzzo, B., Rodriguez, S., Hein, S. and Oksche, A., Light-and electron-microscopic lectin histochemistry of the subcommissural organ: evidence for processing of the secretory material, Cell Tissue Res., 243 (1986) 545-559.

[23] Rodrfguez, E.M., Oksche, A., Rodrlguez, S., Hein, S., Peruzzo, B., Schoebitz, K. and Herrera, H., The subcommissural organ and Reissner's fiber: fine structure and cytochemistry. In P.M. Gross (Ed.), Circumventricular Organs and Body Fluids, 1t"ol. 2, CRC Press, Boca Raton, FL, 1987, pp. 1-41.

[24] Rodrlguez, E.M., Oksche, A., Rodrlguez, S., Nualart, F., Irigoin, C., Fernfindez-Llebrez, P. and P6rez-Figares, J.M., Subcommis- sural organ and cerebrospinal fluid. A biochemical and experi- mental study. In A. Epple (Ed.), Progress in Comparatil~e En- docrinology, Wiley-Liss, New York, 1990, pp. 282-292.

[25] Rodriguez, E.M., Garrido, O. and Oksche, A., Lectin histochemistry of the fetal subcommissural organ, Cell Tissue Res., 262 (1990) 105-113.

[26] Rodrlguez, E.M., Oksche, A., Hein, S. and Yulis, C.R., Cell biology of the subcommissural organ, Int. Rev. Cytol., 135 (1992) 39-121.

[27] Rodrlguez, S., Hein, S., Yulis, R., Delannoy, L., Siegmund, 1. and Roddguez, E.M., Reissner's fiber and the wall of the central canal in the lumbo-sacral region of the bovine spinal cord. Comparative immunocytochemical and ultrastructural study, Cell Tissue Res., 240 (1985) 649-662.

[28] Roth, J., Application of lectin-gold complexes for electron microscopic localization of glycoconjugates on thin sections, J. Histochem. Cytochem., 31 (1983) 987-999.

[29] Sofroniew, M.V., Weindl, A., Schinko, I. and Wetzstein, R., The distribution of vasopressin-oxytocin, and neurophysin-producing neurons in the guinea pig brain: I. The classical hypothalamo- neurohypophyseal system, Cell Tissue Res., 197 (1979) 367-384.

[30] Staneloni, R.J. and Leloir, L.F., The biosynthetic pathway of the asparagine-linked oligosaccharides of glycoproteins, CRC Crit. Rev. Biochem., 12 (1982) 289-326.

[31] Sterba, G., Kleim, I., Naumann, W. and Petter, H., Immunocy- tochemical investigation of the subcommissural organ in the rat, Cell Tissue Res., 218 (1981) 659-662.

[32] Sterba, G., Kiessing, C., Naumann, W., Petter, H. and Kleim, 1., The secretion of the subcommissural organ. A comparative immunocytochemical investigation, Cell Tissue Res., 226 (1982) 427-439.

[33] Sternberger, L.A., Hardy, P.H., Jr., Cuculis, J.J. and Meyer, H.G., The unlabeled antibody enzyme method of immunohisto- chemistry. Preparation and properties of soluble antigen-antibody complex (horseradish-peroxidase-anti peroxidase) and its use in identification of spirochetes, J. Histochem. Cytochem, 18 (1970) 315-333.

[34] Towbin, H., Staehelin, T. and Gordon, J., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA, 76 (1979) 4350-4354.

[35] Zilberstein, A., Snider, M.D., Poter, M. and Lodish, N.F., Mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein, Cell, 21 (1980) 417-427.

analysis of the secretory glycoproteins of the ... · in the present work, the brain of 4500...

Documents