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Chapter 44: The bovine subcommissural organ: cytochemical and immunochemical characterization of the secretory process
R. Landgraf and H.-J. Ruhle (Eds.) Progress in Brain Research, Vol. 91 0 1992 Elsevier Saeiice Publishers B.V. All rights reserved A. Ermisch,
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CHAPTER 44
The bovine subcommissural organ: cytochemical and immunochemical characterization of the secretory process Annie Meiniel, Robert Meiniel, Abdelaziz Karoumi, Nadia Duchier-Liris and JeanLouis Molat Laboratoire de Biochimie Mddicale, CJF INSERM 88.06, Universitd d’Auvergne, Clermont-Ferrand, France
Specific glycoproteins of the bovine subcommissural organ (SCO) were studied by means of various techniques: light and electron microscopy, immunoaffinity chromatography, electrophoresis and Western blotting. Use of lectins (Con A, WGA, PHA-E and -L, LCA) allowed to specify the synthesis and release of complex-type glycoproteins that bear high-mannosecarbohydrate chains in their precursor forms and probably triantennary carbohydrate chains in their mature forms. Antibodies raised against SCO extracts were characterized by means of various tests and used to purify specific compounds. Immunopurified fractions using A99 polyclonal antibody contained numerous polypeptides reactive with Con A, their apparent
molecular weight (MW) ranging from 240 to SO kDa. Only two glycopeptides were strongly labeled with WGA (98 and 52/54 kDa MW). lmmunopurified fractions using C,B,A, monoclonal antibody, specific of the complex-type glycoproteins at different steps of glycosylation, showed three specific Con A-reactive polypeptides at 88, 54 and 34 kDa MW. Only the 34 kDa glycopeptide was strongly labeled with WGA. The latter could correspond to the monomeric form of the secreted compound. Electrophoretical analyses of Reissner’s fiber material allowed the detection of a WGA-positive smear in the upper part of the blots, suggesting that the complex-type glycoproteins, when released into the CSF, constitute a stable polymer.
Introduction
Use of polyclonal antibodies raised against RF has provided evidence that RF is constituted by secretory products elaborated in the SCO (Sterba et al., 1982; Rodriguez et al., 1984). In addition, immunocytochemical studies (Losecke et al., 1984; Rodriguezet al., 1986; Meiniel et al., 1991) strongly support the concept of a basal secretory pathway (Oksche, 1969). The aim of our studies has been to contribute to the knowledge of the molecular aspects of the subcommissural organ. Our interest has focused on the secretory activity and the characterization of the glycoproteins released into the ventricular cavity. In this respect we used lectins to analyze the carbohydrate part of the secretory compound and antibodies to purify specific products.
The subcommissural organ (SCO) belongs to a series of specialized areas in the brain also called ‘‘circumventricular organs” (see Leonhardt, 1980). Located caudally to the pineal organ, at the border of the diencephalic and mesencephalic roof, this organ corresponds to a differentiation of the ependymal lining. Present in all vertebrates, it shows the peculiarity to develop early in the course of ontogeny. A secretory activity in the SCO has been suspected for a long time (Studnicka, 1900).The secretory products are released permanently into the ventricular cavity (Ermisch, 1973) and contribute to the formation of Reissner’s fiber (RF), a thread-like structure running along the central canal of the spinal cord.
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Fig. 1. Histochemical staining of the bovine subcommissural organ. A . Aldehyde fuchsin staining. The apical borderline (arrow) of the ependymal formation (E) is stained as well as the “rosettes” (R) in the hypendymal formation (H); V, third ventricle. B. After exposure to Con A, the cytoplasm of ependymal and hypendyrnal cells is strongly fluorescent. C. WGA-positive material is observed in the apical lining of the ependymal cells (arrow), in the ventricular cavity (pre-Reissner’s fiber) (double arrow) and in the “rosettes” (R). D.After exposure to PHA-E, the pattern of labeling is similar to that observed after WGA exposure in the apical lining of the ependymal cells (arrow) and in secreted material located in the ventricular cavity (double arrow). Note, in addition, the presence of fluorescent material in thearea of the Golgi complexes (G).E . Using C,B,A, Mab, diffuse immunoreactive material is observed in the cytoplasm of the ependymal (E) and hypendymal (H) cells while granular structures at the borderline of the ependymal formation (arrow) and in “rosettes” (R) are strongly reactive. F. C,B,A, Mab. Ependymal formation and immunoreactive material secreted into the ventricular cavity (arrow). Bar, 100 prn (A,B,C), 20 pn (D), 50 pm (E,F).
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Lectin cytochemistry and biochemistry The glycoprotein nature of the SCO secretions that give rise to Reissner’s fiber was first demonstrated by means of classical staining procedures. Both the SCO epithelium and RF were stained using Alcian blue, PAS or AF reactions (cf. Fig. 1A). Recent studies of glycoproteins have focused attention on the use of lectins for the characterization of carbohydrate moieties. The application of these tools in the study of the SCO secretory glycoproteins was introduced by our research group (Meiniel and Meiniel, 1985) and has helped to determine: (i) the synthesis of complex-type glycoproteins showing Nlinked (or Asn-linked) oligosaccharides and (ii) the probable structure of the oligosaccharidic chains at different steps of the process of glycosylation (Rodriguez et al., 1986; Meiniel et al., 1988a). Con A After exposure to concanavalin A (Con A) a strong fluorescence was detected in the entire epithelium of the SCO (Fig. 1B) (in the ependymal and hypendymal cells) while Reissner’s fiber remained negative (Meiniel and Meiniel, 1985). This lectin, specific to mannopyranosyl and glucosyl residues is known to link, in living organisms, with high-mannose-type glycoproteins (Debray et al., 1981; Kornfeld et al., 1981; Sharon and Lis, 1981). In addition, it was established that glycosylation of high-mannose-type glycoproteins takes place in the reticular compartment through the dolichol phosphate cycle (Sharon and Lis, 1981). The observation at the subcellular level that Con A-reactive material was located in the perinuclear spaces and cisternae of the rough endoplasmic reticulum (Rodriguez et al., 1986;Meinielet al., 1988a)strengthened theidea that high-mannose-type glycoproteins were synthesized in the SCO secretory ependymocytes. LCA, PHA-E and PHA-L Using Lens culinaris agglutinin (LCA), Phaseolus vulgaris erythro: and leuko-agglutinin (PHA-E and -L), reactive granular structures were detected in the Golgi areas, and in the borderline of the SCO
epithelium close to the ventricular cavity (Fig. ID). These threelectins are known to have a strong affinity to complex-type glycoproteins bearing either bi, tri- or tetra-antennary oligosaccharides (Debray et al., 1981; Kornfeld et al., 1981; Cummings and Kornfeld, 1982). Presence of LCA-, PHA-E- and L-reactive material in RF speaks in favor of a release of complex-type glycoproteins by the secretory ependymocytes contributing to the formation of Reissner’s fiber (Meiniel et al., 1988a). WGA A similar labeling pattern was observed after exposure to wheat germ agglutinin (WGA) (Fig. 1C). Regarding the high affinity of WGA to neuraminic acid (Bhavanandan and Katlic, 1979) the carbohydrate chains of the complex-type glycoproteins probably include terminal neuraminic acid residues. Presence at the subcellular level of WGA-labeled material in the Golgi apparatus, known to be the site of transformation of the carbohydrate chains in complex-type glycoproteins (Sharon and Lis, 1981), as well as in secretory vacuoles and in pre-Reissner’s fiber supports the concept of the synthesis and release of complex-type glycoproteins by the SCO secretory ependymocytes (Meiniel et al., 1988a).
Mab NC-I In the nervous system several complex-type glycoproteins are known to bear carbohydrate chains including a terminal 3-sulfoglucuronyl residue that reacts with Mab HNK-1 or Mab NC-1 (Tucker et al., 1984), e.g., MAG (myelin-associated glycoprotein, Mc Garry et al., 1983), L1 and NCAM (neural-cell adhesion molecules, Nieke and Schachner, 1985) ependymins 6 and y (Shashoua et al., 1986). Absence of labeling after exposure to Mab NC-1 in the SCO epithelium shows that the oligosaccharides of the complex-type glycoproteins do not include this glycan sequence (Meiniel et al., 1990). In conclusion, regarding the lectin reactivity to the SCO glycoproteins and on the basis of the known biosynthetic pathway of complex-type glycoproteins (Sharon and Lis, 1981), the precursor
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forms synthesized in the reticular compartment probably correspond to high-mannose-type glycoproteins while the mature forms released into the ventricular cavity appear to be complex-type glycoproteins. A structure of the oligosaccharidic chains at different steps of the process of glycosylation has been proposed (Fig. 2). Nevertheless, biochemical techniques must now be undertaken to precise the exact carbohydrate composition of the SCO complex-type glycoproteins and whether 0-linked oligosaccharides also occur. Lectins were also used to identify glycopeptides specific to SCO extracts after SDS-polyacrylamide gel electrophoresis and Western blotting. From the comparison of soluble extracts of the SCO' with other brain tissues (cerebral hemispheres, cerebellum, pineal organ) it appeared that in the sheep, a Con A-positive 54 kDa glycopeptide was specific to the SCO profile (Meiniel et al., 1986) (Fig. 3A). Comparative two-dimensional electrophoresis of various brain tissue extracts also led to the characterization of polypeptides specific to the bovine SCO (Fig. 3B,C). Most of these polypeptides can be regarded as acidic compounds as their
isoelectric points range from 6 to 5.5. Few were found to be Con A-positive (Duchier-Liris, 1991). Such a complex electrophoretic pattern is to date difficult to analyze in details. Nevertheless, it points to a particular biochemical evolution of the secretory ependymocytes.
Immunocytochemistry It is clear that lectins, in addition to the SCO complex-type glycoproteins, can recognize many ubiquitous glycosylated products, e.g., endoplasmin (Koch et al., 1986), a Con A-binding glycoprotein probably involved in the calciumbinding function of the RER, or glycoproteins of the cell coat that often strongly react with WGA (Huet and Garrido, 1972). Thus, more specific probes, such as antibodies, were necessary to analyze the particular phenotype of the SCO secretory ependymocytes and to characterize their secretory product@). Recently, we obtained specific immunological probes that served for investigation of the cellular and molecular aspects of the SCO. These poly- and monoclonal antibodies were produced after im-
A Man +Man Man gluc +gluc
+glut +Man
\
+N a n
+Wan
+Man
+Man
\
f
Wan a GlcNAc +GlcNAc
+ Asn
B NeuAc +Gal
+GlcNAc
NeuAc +Gal
+ GlcNAc
Y +Man PI-2
y
GlcNAc +Man NeuAc + G a l
+ GlcNAc
-Man
L
Fuc
4
+ GlcNAc + GlcNAc
+ ASn
Fig. 2. Oligosaccharides of the SCO complex-type glycoproteins at different steps of glycosylation. A . Proposed structure for the precursor form (high-mannose-type glycoprotein). B. Proposed structure for the mature form (complex-type glycoprotein). (From Meiniel et al., 1988a.)
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1
2
3
4
7 I
6
5.5
Fig. 3. Electrophoretical analysis of crude soluble extracts of SCO compared to other brain tissues. A. Ovine SCO: SDS-PAGE and Western blot stained with Con A. (1) Standards of molecular weight (ovalbumin 45 kDa); (2) profile of the SCO. Note the presence of a specific 54 kDa polypeptide (arrow); (3) profile of the pineal organ; (4) profile of the cerebellum. (From Meiniel et al., 1986.) B. Bovine SCO: two-dimensional gel electrophoresis of soluble extracts stained with silver nitrate. Arrows indicate specific polypeptides. C Bovine ependyma: two-dimensional gel electrophoresis of soluble extracts (silver staining).
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munization with SCO extracts. Accordingly, the present results can be considered as complementary to those obtained with antibodies raised against RF by the research groups of Professors Sterba and Rodriguez (Sterba et al., 1982; Loseckeet al., 1984, 1986; Rodriguez et al., 1984, 1986, 1987). A99 Polyclonal antibodies raised against crude extracts from the bovine SCO were adsorbed with brain crude extracts to precipitate antibodies raised against common compounds present in both tissues. By means of various tests, these antibodies were shown to be specific of SCO glycoproteins (Karoumi, 1990). After application of the immunofluorescence technique, the reactive material was located in the cytoplasm of the ependymal and hypendymal cells of the SCO as well as in Reissner’s fiber (Karoumi et al., 1990a). c,B$I8 Mab This monoclonal antibody was selected on the basis of its histochemical reactivity with SCO and Reissner’s fiber material (Fig. lE,F) (Meiniel et al., 1988b). Using a competitive test between lectins (Con A and WGA) and the monoclonal antibody it was demonstrated that both the high-mannose-type glycoproteins (Con A-reactive) and the complextype glycoproteins (WGA-reactive) were recognized by the antibody (Meiniel et al., 1988b). In addition, electron microscopical studies revealed the presence of C,B,A, immunoreactive material in the reticular compartment of the secretory ependymocytes (site of synthesis of the precursor form), in the Golgi area (site of transformation of the carbohydrate chains) and in secretory vacuole (site of transport and/or storage of the secreted form). These antibodies were thus excellent tools to try to characterize by immunochemical techniques specific compounds related to the particular phenotype of the secretory ependymocytes (polyclonal antibody) and to the various glycosylated forms of the complex-type glycoproteins (monoclo nal antibody).
Immunoaffinity chromatography Below we list a number of technical considerations that should be considered when analyzing the results. (1) Only soluble proteins were immunopurified as no detergents were added to the various tissue extracts; thus specific unsoluble proteins may occur in the SCO that were not revealed using our protocol. (2) The immunoaffinity chromatography was performed in the presence of inhibitors of proteases in order to avoid degradation of proteins. (3) Electrophoresis of extracts prepared in SDS-0mercaptoethanol separates proteins into subunits. The exact molecular weight of the native proteins must be determined by other techniques. (4) As immunopurified products were mainly glycoproteins, lectins (Con A and WGA) were used to reveal glycopeptides on Western blots. ( 5 ) Comparison with other brain tissues was carried out in order to specify the reactivity of our antibodies, and to detect eventual cross-reactivity. A99 immunoaffinity (Figs. 4A,B, 5)
Con A . Several Con A-positive bands were identified in the eluted fraction of the SCO, their apparent molecular weight (MW) ranging from 240 to 50 kDa. In the eluted fraction of the cerebral hemispheres polypeptides located at 52/54 and 50 kDa MW were also present while no polypeptides were immunopurified from soluble extracts of the classical ependymal lining. WGA. Only polypeptides at 98 and 52/54 kDa MW could be revealed in the eluted fraction of the SCO. In the eluted fraction of the cerebral hemispheres the 52/54 and 50 kDa polypeptides were also WGA-positive. C,B$18 immunoaffinity (Figs. 4C,D, 5)
Con A. Three glycopeptides were identified in
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Fig. 4. lmmunoaffinity chromatography and Reissner’s fiber. A . Con A-reactive polypeptides revealed after A99 immunoaffinity chromatography. (1) Eluted fraction of theependymal lining (no band); (2) profile of solubleextracts of the ependymal lining; (3) profile of solubleextracts of the SCO; (4) eluted fraction of the SCO (several bands, cf. Fig. 5 for exact molecular weights); ( 5 ) profile of soluble extracts of the cerebral hemispheres; (6) eluted fraction of the cerebral hemispheres (two bands 52/54 and 50 kDa); (7) standards of molecular weight stained with Coomassie blue (MW expressed in kDa). B. WGA-reactive polypeptides revealed after A99 immunoaffinity chromatography. (1) Profile of soluble extracts of the SCO; (2) eluted fraction of the SCO (two bands at 98 and 52/54 kDa); (3) profile of soluble extracts of the cerebral hemispheres; (4) eluted fraction of the cerebral hemispheres (one band 52/54 kDa). A and B: same electrophoresis and electrotransfer. C. Con A-reactive polypeptides revealed after ClB8A, immunoaffinity chromatography. Three bands are located at 88, 54 and 34 kDa MW. D. WGA-reactive polypeptides revealed after ClB8A8 immunoaffinity chromatography. One band is located at 34 kDa MW. Cand D:same electrophoresis and electrotransfer. E . Western blot of Reissner’s fiber (RF) stained with WGA. (I) RF pre-treated with urea 9 M; (2) RF pre-treated with SDS 4%. Note the presence of a positive smear in the stacking gel and in the migrating gel (arrow, top of the migrating gel). F. Western blot of RF polymer cleaved by trypsine 0.01 Yo. Three groups of bands were identified after WGA staining (arrow, top of the migrating gel).
the eluted fraction of the SCO having an apparent molecular weight of 88, 54 and 34 kDa. WGA. Only the glycopeptide located at 34 kDa was strongly positive. In eluted fractions of the classical ependymal lining no glycopeptides were revealed using either Con A or WGA.
These results can be compared with those obtained in the chick embryo (Karoumi et al., 1990b)using A74 IgG and the same technology. A tentative classification of the immunopurified glycopeptides is presented in Fig. 5 . Three groups were distinguished according to their reactivity to the various antibodies.
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Fig. 5 . Immunoaffinity chromatography using A99 (polyclonal antibody against bovine K O ) , C,B,A, (monoclonal antibody against bovine complex-typeglycoproteins) and A74 (polyclonal antibody against chick SCO). Con A- and WGA-positive bands observed after SDS-PAGE and electrotransfer. Glycopeptides linked to the complex-type glycoproteins. Number of sign + is proportional to intensity of the labeling. (CH), Glycopeptides also present in the cerebral hemispheres; (MO), glycopeptides present in the medulla oblongata.
(1) Glycopeptides related to the complex-type glycoproteins at different steps of glycosylation. These glycopeptides with an apparent molecular weight of 88, 52/54 and 32/34 kDa were immunopurified on C,B,A, immunoadsorbant. They were also detected using A99 and A74 IgG in different experiments (Karoumi et al., 1990b, 1991). (2) Glycopeptides linked to the particular phenotype of the secretory ependymocytes. These glycopeptides (240, 98 and 66 kDa MW) were present in both the chick embryo and the bovine SCO after A99 and A74 immunoaffinity chromatography. They can include enzymes involved in the secretory pathway and/or carrier proteins. These two groups of glycopeptides have a high intensity in the various experiments using either Con A or WGA. Their presence in both species suggests a certain stability of these products in the course of phylogenetical evolution.
(3) The third group of polypeptides could be species-dependent as they are present either in the chick embryo (150, 84, 77, 46, 42 kDa) or in the bovine (220,50 kDa) using respectivelyA74 and A99 immunoaffinity chromatography. Nevertheless, taking into account their low intensity and their inconstant presence in the various experiments, these glycopeptides could be at the limit of detection using the present technology. Their significance in each species requires further analysis. Regarding the putative glycopeptides linked to the complex-type glycoproteins (88, 52/54 and 32/34 kDa), differences in their affinity to the lectins Con A and WGA allow some speculation. It is possible that the 88 kDa band that is mainly Con Apositive represents the high-mannose-type glycoprotein (or a subunit), while the 52/54 kDa band that shows Con A- and WGA-positive reaction could be linked to an intermediate form. The 32/34
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kDa glycopeptide that is strongly WGA-positive could correspond to the secreted monomeric form of the complex-type glycoprotein. This glycopeptide was also isolated from soluble extracts of the medulla oblongata in the chick embryo (Karoumi et al., 1990b) and was discussed as the putative secreted glycoprotein present in Reissner’s fiber. The fact that a 52/54 kDa glycopeptide also occurred in immunopurified fractions of the cerebral hemispheres in both the chick embryo and bovine, raises the question whether a brain polypeptide, having similar properties, could cross-react with our polyclonal antibodies. Nevertheless, considering that this glycopeptide was not immunopurified from extracts of the ependymal lining using either A99 or C,B,A,, and that the site(s) of action of the secreted compound(s) is (are) still unknown, the possibility that the 52/54 kDa glycopeptide is present in the brain must be considered. This would indicate that other brain regions could be the target for SCO complex-type glycoproteins. Further analysis is required after purification of soluble extracts of cerebral hemispheres on C,B,A, immunoadsorbent (in progress).
Reissner’s fiber Reissner (1860) first described in the lamprey Petromyzonfluviatilis an axon-like structure in the central canal of the spinal cord. In 1900, Studnicka suspected that Reissner’s fiber (RF) was the aggregation of the specific compound secreted by the subcommissural organ. Immunohistochemical staining of both the subcommissural organ and RF using specific antibodies demonstrated that at least the major constituent(s) of RF arises from the SCO (Sterba et al., 1982; Rodriguez et al., 1984; Meiniel et al., 1988b;Karoumiet al., 1990a). The functional significance of RF is still a matter of speculation. Hofer et al. (1984) and Peruzzo et al. (1987) have postulated a degradation of RF material in the ampulla caudalis of lampreys and an absorbtion of this material by the surrounding blood capillaries. RF was collected from portions of the bovine spinal cord and rinced twice in distilled water. The
following procedures were then applied: (1) solubilization in 4% SDS or urea 9 M or urea 9 M + chaps 2% + SDS 2% (12 h); (2) digestion in pronase, pepsine, papaine, trypsine 0.5% (1 h); and (3) partial cleavage in trypsine 0.01% (2 h). The samples were then prepared for SDS-PAGE (cf. Meiniel et al., 1986), separated and transferred to nitrocellulose sheets. RF material and analyzed on Western blots stained with WGA. After treatment with SDS, urea or SDS + urea, a WGA-positive smear was detected in the upper part of the blots corresponding to the stacking gel and the top of the migrating gel (Fig. 4E). Such a smear probably indicates the presence of polymers that were not completely solubilized under our experimental conditions. The WGA-positive smear completely disappeared when RF material was preincubated in the various proteases. Incomplete cleavage of RF polymer was obtained after treatment with trypsine 0.01 %.Under these experimental conditions, three WGA-positive bands were visualized in the upper part of the blots as well as two groups of small bands ranging from 80 to 150 kDa and from 45 to 55 kDa (Fig. 4F). These WGApositive bands represent fragments of the RF polymer at different steps of digestion by trypsine. Thus, in the adult bovine, RF can be regarded as a stable polymer (insoluble in SDS and urea). This suggests that the monomeric form of the SCO complex-type glycoproteins, when secreted into the CSF, changes its conformation. Such a situation cannot be extended to all species (or all stages of development) as in the embryonic chicken a 32/34 kDa glycopeptide, suspected to represent the compound secreted by the SCO and present in Reissner’s fiber, could be isolated from soluble extracts of the medulla oblongata (Karoumi et al., 1990b). In the brain, other secreted complex-type glycoproteins involved in synaptic plasticity (ependymins CY and 0) are known to constitute either in vitro or in vivo polymers insoluble in SDS-0mercaptoethanol (Shashoua, 1988; Shashoua et al., 1990). The monomeric forms of these proteins have an apparent molecular weight of 38 and 32 kDa. A few biochemical properties of ependymins and of
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the SCO complex-type glycoproteins point to a large similarity of both products. Nevertheless, further analysis of the amino acid sequence of the complextype glycoproteins is needed to compare these proteins and to give a better insight into the molecular aspect of the compounds secreted by the SCO.
Ontogenetical study and cell filiation With reference to the observation that aldehyde fuchsin staining (AF) revealed compounds in the SCO and in the pineal organ, Kelly and Van de Kamer (1 960) suggested that positive AF .elements present in the pineal organ have been detached from the ependymal roof during embryogenesis and could correspond to specialized ependymal cells coming from the SCO anlage. AF-positive material has been reported to occur in pinealocytes of various mammalian species (Quay, 1970; Lukaszyk and Reiter, 1975; Japha et al., 1976). In the rat, the AF-positive material appears to be more conspicuous during embryogenesis (Owman, 1960). On the other hand, using polyclonal antibodies raised against RF, Rodriguez et al. (1988) were able to detect immunoreactive material in the pineal organ of various vertebrate species. Using the monoclonal antibody ClBgA8, a comparative spatio-temporal analysis of both organs has been undertaken in the embryonic bovine (Meiniel et al., 1990). Immunoreactive material was detected as early as 2 months of development in the SCO anlage. The labeling increases rapidly in the secretory ependymocytes and extends in a caudal direction. Reissner’s fiber could be detected in the central canal of the spinal cord at 4 months of development. In the course of ontogeny, no immunoreactive material could be revealed in the bovine pineal organ. Glycoproteins present in pinealocytes require additional research to precise their exact nature and their biological role. In addition, identification of the cell type engaged in glycoprotein synthesis in the pineal organ would help to establish a parallel with the SCO secretory ependymocytes.
Several authors have mentioned the early expression of specific compounds in the subcommissural organ (Schoebitz et al., 1986; Naumannet al., 1987; Karoumi et al., 1990a). Theexact significance of the secretory process in the course of ontogenetical development remains enigmatic. A possible role of the complex-type glycoproteins in the maturation of other brain regions cannot be determined without experimental research.
Acknowledgements Thanks are due to C. Michaud for typing the manuscript and G. Ragonnaud for the illustrations. This research was supported by a grant from the “Association Francaise contre les Myopathies” (A.F.M.).
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cell-coat components by means of wheat germ agglutinin. Exp. Cell Res., 75: 523 - 527. Japha, J.L., Eder, T.J. and Goldsmith, E.D. (1976) A histochemical study of aldehyde fuchsin-positive material and “high-esterase cells” in the pineal gland of the Mongolian gerbil. Am. J. Anat., 149: 23 -38. Karoumi, A. (1990) Mise en Evidence et Caractdrisationpar des Mdthodes Immunologiquesde SPcrdtions Glycoprotdiques au Niveau du Diencdphale Dorsal. (Etude Particulikre d’une Structure Circumventriculaire: I’Organe Sous-commissural du B e u f Adulte et de I’Embryon de Poulet), Thesis No. UD 199. Faculte des Sciences, Universite Blaise Pascal, ClermontFerrand. Karoumi, A., Meiniel, R., Croisille, Y .,Belin, M.F. and Meiniel, A. (1 990a) Glycoprotein synthesis in the subcommissural organ of the chick embryo. I. An ontogenetical study using specific antibodies. J. Neural Transm., 79: 141 - 153. Karoumi, A., Croisille, Y .,Croisille, F., Meiniel, R., Belin, M.F. and Meiniel, A. (1990b) Glycoprotein synthesis in the subcommissural organ of the chick embryo. 11. An immunochemical study. J. Neural Transm.,, 80: 203 - 212. Karoumi, A., Meiniel, R., Belin, M. and Meiniel, A. (1991) A comparative immunocytochemical and immunochemical analysis of glycoproteins synthesized in the bovine subcommissural organ of the adult bovine using specific polyclonal antibody. J. Neural Transm., 86: 205 - 216. Kelly, E.D.and Van de Kamer, J.C. (1960) Cytological and histochemical investigations in the pineal organ of the adult frog. Z . Zellforsch., 52: 618-639. Koch, G., Smith, M., Macer, D., Webster, P. and Mortara, R. (1 986) Endoplasmic reticulum contains a common, abundant calcium-binding glycoprotein, endoplasmin. J. Cell Sci., 86: 2 17 - 232. Kornfeld, K., Reitmann, M.L. and Kornfeld, R. (1981) The carbohydrate binding specificity of pea and lentil lectins. Fucose is an important determinant. J. Biochem., 256: 6633 -6640. Leonhardt, H. (1980) Ependym und circumventriculare Organe. In: A. Oksche and L. Vollrath (Eds.), Neuroglial. Handbuch der Microskopischen Anatomie des Menschen, Vol. IV-10, Springer, Berlin, Heidelberg, New York, pp. 177 - 665. Losecke, W., Naumann, W. and Sterba, G. (1984) Preparation and discharge of secretion in the subcommissural organ of the rat. An electron-microscopic immunocytochemical study. Cell Tissue Res., 235: 201 - 206. Losecke, W., Naumann, W. and Sterba, G. (1986) Immunoelectron-microscopic analysis of the basal route of secretion in the subcommissural organ of the rabbit. Cell TissueRes., 224: 449-456.
Lukaszyk, K. and Reiter, R.J. (1975) Histophysiological evidence for the secretion of polypeptides by the pineal gland. Am. J. Anat., 143: 451 -464. Mc Carry, R.C., Helfand, S.L., Quarles, R.H. and Roder, J.C. (1983) Recognition of myelin-associated glycoprotein by the monoclonal antibody HNK-1. Nature, 306: 376- 378.
Meiniel, A., Molat, J.L. and Meiniel, R. (1988a) Complex-type glycoproteins synthesized in the subcornmissural organ of mammals. Light- and electron-microscopic investigations by use of lectins. Cell Tissue Res., 253: 383 - 395. Meiniel, R. and Meiniel, A. (1985) Analysis of the secretions of the subcommissural organs of several vertebrate species by use of fluorescent lectins. Cell Tissue Res., 239: 359 - 364. Meiniel, R., Molat, J.L. and Meiniel, A. (1986) Concanavalin Abinding glycoproteins in the subcommissural and the pineal organ of the sheep (Ovis aries). A fluorescence-microscopic and electrophoretic study. Cell Tissue Res., 245: 605 - 613. Meiniel, R., Duchier, N. and Meiniel, A. (1988b) Monoclonal antibody ClB8A8 recognizes a ventricular secretory product elaborated in the bovine subcommissural organ. Cell Tissue Res., 254: 611 -615. Meiniel, R., Molat, J.L., Duchier-Liris, N. and Meiniel, A. (1990) Ontogenesis of the secretory epithelium of the bovine subcommissural organ. A histofluorescence study using lectins and monoclonal antibodies. Dev. Brain Res., 55: 171 - 180. Meiniel, R., Duchier-Liris, N., Molat, J.L. and Meiniel, A. (1991) The complex-type glycoprotein secreted by the bovine subcommissural organ: an immunological study using C,B,A, monoclonal antibody. Cell Tissue Res., 266: 483 - 490. Naumann, W., Miiller, G. and Kloss, P. (1987) Immunoreactive glycoproteins of the subcommissural organ in the embryonic stages of the vertebrate brain. Wiss. Z . KarlMarx Univ. Leipzig, Math. Naturwiss. R , 36: 17 - 20. Nieke, J. and Schachner, M. (1985) Expression of the neural cell adhesion molecules L1 and N-CAM and their common carbohydrate epitope L2/HNK-1 during development and after transection of the mouse sciatic nerve. Differentiation, 30 (2): 141 - 151. Oksche, A. (1969) The subcommissural organ. J. Neuro-Vise. Rel., Suppl. 9: 11 - 139. Owman, C.H.(1%0/1961) Secretory activity of the fetal pineal gland of the rat. Acta Morphol. Neer. Scand., 3: 367 - 394. Peruzzo, M., Rodriguez, S . , Delannoy, L., Hein, S., Rodriguez, E.M. and Oksche, A. (1987) Ultrastructural immunocytochemical study of the massa caudalis of the subcommissural organ-Reissner’s fiber complex in lamprey larvae (Geotria australis). Evidence for a terminal vascular route of secretory material. Cell Tissue Res., 247: 367 - 376. Quay, W.B. (1970) Pineal structure and composition in the orang-utan (Pongo pygmaeus). Anat. Rec., 168: 93 - 104. Reissner, E. (1860) Beitrage zur Kenntnis vom Bau des Riickenmarks von Petromyzon fluviatilis L. Arch. Anat. Physiol., pp. 545 - 588. Rodriguez, E.M., Oksche, A., Hein, S., Rodriguez, S.and Yulis, R. (1984) Comparative immunocytochemical study of the subcommissural organ. Cell Tissue Res., 237: 427 - 441. Rodriguez, E.M., Herrera, H., Peruzzo, B., Rodriguez, S., Hein, S. and Oksche, A. (1986) Light- and electron-
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microscopic immunocytochemistry and lectin histochemistry of the subcommissural organ: evidence for processing of the secretory material. Cell Tissue Res., 243: 545 - 559. Rodriguez, E.M., Hein, S., Rodriguez, S., Herrera, H., Peruzzo, B., Nualart, F. and Oksche, A. (1987) Analysis of the secretory products of the subcommissural organ. In: B. Scharrer, H.W. Korf and H.G. Hartwig (Eds.), Functional Morphology of Neuroendocrine Systems, Springer, Berlin, Heidelberg, New York, pp. 189-202. Rodriguez, E.M., Korf, H.W., Oksche, A., Yulis, R. and Hein, S. (1988) Pinealocytes immunoreactive with antisera against secretory glycoproteins of the subcommissural organ: a comparative study. Cell Tissue Res., 245: 469 - 480. Schoebitz, K., Garrido, O., Heinrichs, M., Peer, L. and Rodriguez, E.M. (1986) Ontogenetical development of the chick and duck subcommissural organ. An immunocytochemical study. Hisrochemistry, 81 : 31 - 40. Sharon, N. and Lis, H. (1981) Glycoproteins: research booming on long-ignored ubiquitous compounds. C.E.N. (special report), Am. Chem. Soc., Washington, DC, pp. 21 -44. Shashoua, V.E. (1988) Monomeric and polymeric forms of epen-
dymin: a brain extracellular glycoprotein implicated in memory consolidation processes. Neurochem. Res., 13: 649 - 655. Shashoua, V.E., Daniel, P.F., Moore, M.E. and Jungwala, F.B. (1986) Demonstration of glucuronic acid on brain glycoproteins which react with HNK-1antibody. Eiochem. Biophys. Res. Commun., 138: 902 - 909. Shashoua, V.E., Hesse, G.W. and Milinazzo, B. (1990) Evidence for the in vivo polymerization of ependymin: a brain extracellular glycoprotein. Bruin Res., 522: 181 - 190. Sterba. G., Kiessig, C., Naumann, W. and Petter, H. (1982)The secretion of the subcommissural organ. A comparative immunocytochemical investigation. Cell Tissue Res., 226: 427 - 439. Studnicka, F.K. (1900) Untersuchungen uber den Bau des Ependyms der nervosen Centralorgane. Anat. Hefte, 15: 303 - 430. Tucker, G.C., Aoyama, H., Lipinsky, M., Tursz, T. and Thiery, J.P. (1984) Identical reactivity of monoclonal antibodies HNK-I and NC-I: conservation in vertebrates on cells derived from the neural primordium and on some leucocytes. Cell Diff., 14: 223 - 230.
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CHAPTER 44
The bovine subcommissural organ: cytochemical and immunochemical characterization of the secretory process Annie Meiniel, Robert Meiniel, Abdelaziz Karoumi, Nadia Duchier-Liris and JeanLouis Molat Laboratoire de Biochimie Mddicale, CJF INSERM 88.06, Universitd d’Auvergne, Clermont-Ferrand, France
Specific glycoproteins of the bovine subcommissural organ (SCO) were studied by means of various techniques: light and electron microscopy, immunoaffinity chromatography, electrophoresis and Western blotting. Use of lectins (Con A, WGA, PHA-E and -L, LCA) allowed to specify the synthesis and release of complex-type glycoproteins that bear high-mannosecarbohydrate chains in their precursor forms and probably triantennary carbohydrate chains in their mature forms. Antibodies raised against SCO extracts were characterized by means of various tests and used to purify specific compounds. Immunopurified fractions using A99 polyclonal antibody contained numerous polypeptides reactive with Con A, their apparent
molecular weight (MW) ranging from 240 to SO kDa. Only two glycopeptides were strongly labeled with WGA (98 and 52/54 kDa MW). lmmunopurified fractions using C,B,A, monoclonal antibody, specific of the complex-type glycoproteins at different steps of glycosylation, showed three specific Con A-reactive polypeptides at 88, 54 and 34 kDa MW. Only the 34 kDa glycopeptide was strongly labeled with WGA. The latter could correspond to the monomeric form of the secreted compound. Electrophoretical analyses of Reissner’s fiber material allowed the detection of a WGA-positive smear in the upper part of the blots, suggesting that the complex-type glycoproteins, when released into the CSF, constitute a stable polymer.
Introduction
Use of polyclonal antibodies raised against RF has provided evidence that RF is constituted by secretory products elaborated in the SCO (Sterba et al., 1982; Rodriguez et al., 1984). In addition, immunocytochemical studies (Losecke et al., 1984; Rodriguezet al., 1986; Meiniel et al., 1991) strongly support the concept of a basal secretory pathway (Oksche, 1969). The aim of our studies has been to contribute to the knowledge of the molecular aspects of the subcommissural organ. Our interest has focused on the secretory activity and the characterization of the glycoproteins released into the ventricular cavity. In this respect we used lectins to analyze the carbohydrate part of the secretory compound and antibodies to purify specific products.
The subcommissural organ (SCO) belongs to a series of specialized areas in the brain also called ‘‘circumventricular organs” (see Leonhardt, 1980). Located caudally to the pineal organ, at the border of the diencephalic and mesencephalic roof, this organ corresponds to a differentiation of the ependymal lining. Present in all vertebrates, it shows the peculiarity to develop early in the course of ontogeny. A secretory activity in the SCO has been suspected for a long time (Studnicka, 1900).The secretory products are released permanently into the ventricular cavity (Ermisch, 1973) and contribute to the formation of Reissner’s fiber (RF), a thread-like structure running along the central canal of the spinal cord.
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Fig. 1. Histochemical staining of the bovine subcommissural organ. A . Aldehyde fuchsin staining. The apical borderline (arrow) of the ependymal formation (E) is stained as well as the “rosettes” (R) in the hypendymal formation (H); V, third ventricle. B. After exposure to Con A, the cytoplasm of ependymal and hypendyrnal cells is strongly fluorescent. C. WGA-positive material is observed in the apical lining of the ependymal cells (arrow), in the ventricular cavity (pre-Reissner’s fiber) (double arrow) and in the “rosettes” (R). D.After exposure to PHA-E, the pattern of labeling is similar to that observed after WGA exposure in the apical lining of the ependymal cells (arrow) and in secreted material located in the ventricular cavity (double arrow). Note, in addition, the presence of fluorescent material in thearea of the Golgi complexes (G).E . Using C,B,A, Mab, diffuse immunoreactive material is observed in the cytoplasm of the ependymal (E) and hypendymal (H) cells while granular structures at the borderline of the ependymal formation (arrow) and in “rosettes” (R) are strongly reactive. F. C,B,A, Mab. Ependymal formation and immunoreactive material secreted into the ventricular cavity (arrow). Bar, 100 prn (A,B,C), 20 pn (D), 50 pm (E,F).
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Lectin cytochemistry and biochemistry The glycoprotein nature of the SCO secretions that give rise to Reissner’s fiber was first demonstrated by means of classical staining procedures. Both the SCO epithelium and RF were stained using Alcian blue, PAS or AF reactions (cf. Fig. 1A). Recent studies of glycoproteins have focused attention on the use of lectins for the characterization of carbohydrate moieties. The application of these tools in the study of the SCO secretory glycoproteins was introduced by our research group (Meiniel and Meiniel, 1985) and has helped to determine: (i) the synthesis of complex-type glycoproteins showing Nlinked (or Asn-linked) oligosaccharides and (ii) the probable structure of the oligosaccharidic chains at different steps of the process of glycosylation (Rodriguez et al., 1986; Meiniel et al., 1988a). Con A After exposure to concanavalin A (Con A) a strong fluorescence was detected in the entire epithelium of the SCO (Fig. 1B) (in the ependymal and hypendymal cells) while Reissner’s fiber remained negative (Meiniel and Meiniel, 1985). This lectin, specific to mannopyranosyl and glucosyl residues is known to link, in living organisms, with high-mannose-type glycoproteins (Debray et al., 1981; Kornfeld et al., 1981; Sharon and Lis, 1981). In addition, it was established that glycosylation of high-mannose-type glycoproteins takes place in the reticular compartment through the dolichol phosphate cycle (Sharon and Lis, 1981). The observation at the subcellular level that Con A-reactive material was located in the perinuclear spaces and cisternae of the rough endoplasmic reticulum (Rodriguez et al., 1986;Meinielet al., 1988a)strengthened theidea that high-mannose-type glycoproteins were synthesized in the SCO secretory ependymocytes. LCA, PHA-E and PHA-L Using Lens culinaris agglutinin (LCA), Phaseolus vulgaris erythro: and leuko-agglutinin (PHA-E and -L), reactive granular structures were detected in the Golgi areas, and in the borderline of the SCO
epithelium close to the ventricular cavity (Fig. ID). These threelectins are known to have a strong affinity to complex-type glycoproteins bearing either bi, tri- or tetra-antennary oligosaccharides (Debray et al., 1981; Kornfeld et al., 1981; Cummings and Kornfeld, 1982). Presence of LCA-, PHA-E- and L-reactive material in RF speaks in favor of a release of complex-type glycoproteins by the secretory ependymocytes contributing to the formation of Reissner’s fiber (Meiniel et al., 1988a). WGA A similar labeling pattern was observed after exposure to wheat germ agglutinin (WGA) (Fig. 1C). Regarding the high affinity of WGA to neuraminic acid (Bhavanandan and Katlic, 1979) the carbohydrate chains of the complex-type glycoproteins probably include terminal neuraminic acid residues. Presence at the subcellular level of WGA-labeled material in the Golgi apparatus, known to be the site of transformation of the carbohydrate chains in complex-type glycoproteins (Sharon and Lis, 1981), as well as in secretory vacuoles and in pre-Reissner’s fiber supports the concept of the synthesis and release of complex-type glycoproteins by the SCO secretory ependymocytes (Meiniel et al., 1988a).
Mab NC-I In the nervous system several complex-type glycoproteins are known to bear carbohydrate chains including a terminal 3-sulfoglucuronyl residue that reacts with Mab HNK-1 or Mab NC-1 (Tucker et al., 1984), e.g., MAG (myelin-associated glycoprotein, Mc Garry et al., 1983), L1 and NCAM (neural-cell adhesion molecules, Nieke and Schachner, 1985) ependymins 6 and y (Shashoua et al., 1986). Absence of labeling after exposure to Mab NC-1 in the SCO epithelium shows that the oligosaccharides of the complex-type glycoproteins do not include this glycan sequence (Meiniel et al., 1990). In conclusion, regarding the lectin reactivity to the SCO glycoproteins and on the basis of the known biosynthetic pathway of complex-type glycoproteins (Sharon and Lis, 1981), the precursor
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forms synthesized in the reticular compartment probably correspond to high-mannose-type glycoproteins while the mature forms released into the ventricular cavity appear to be complex-type glycoproteins. A structure of the oligosaccharidic chains at different steps of the process of glycosylation has been proposed (Fig. 2). Nevertheless, biochemical techniques must now be undertaken to precise the exact carbohydrate composition of the SCO complex-type glycoproteins and whether 0-linked oligosaccharides also occur. Lectins were also used to identify glycopeptides specific to SCO extracts after SDS-polyacrylamide gel electrophoresis and Western blotting. From the comparison of soluble extracts of the SCO' with other brain tissues (cerebral hemispheres, cerebellum, pineal organ) it appeared that in the sheep, a Con A-positive 54 kDa glycopeptide was specific to the SCO profile (Meiniel et al., 1986) (Fig. 3A). Comparative two-dimensional electrophoresis of various brain tissue extracts also led to the characterization of polypeptides specific to the bovine SCO (Fig. 3B,C). Most of these polypeptides can be regarded as acidic compounds as their
isoelectric points range from 6 to 5.5. Few were found to be Con A-positive (Duchier-Liris, 1991). Such a complex electrophoretic pattern is to date difficult to analyze in details. Nevertheless, it points to a particular biochemical evolution of the secretory ependymocytes.
Immunocytochemistry It is clear that lectins, in addition to the SCO complex-type glycoproteins, can recognize many ubiquitous glycosylated products, e.g., endoplasmin (Koch et al., 1986), a Con A-binding glycoprotein probably involved in the calciumbinding function of the RER, or glycoproteins of the cell coat that often strongly react with WGA (Huet and Garrido, 1972). Thus, more specific probes, such as antibodies, were necessary to analyze the particular phenotype of the SCO secretory ependymocytes and to characterize their secretory product@). Recently, we obtained specific immunological probes that served for investigation of the cellular and molecular aspects of the SCO. These poly- and monoclonal antibodies were produced after im-
A Man +Man Man gluc +gluc
+glut +Man
\
+N a n
+Wan
+Man
+Man
\
f
Wan a GlcNAc +GlcNAc
+ Asn
B NeuAc +Gal
+GlcNAc
NeuAc +Gal
+ GlcNAc
Y +Man PI-2
y
GlcNAc +Man NeuAc + G a l
+ GlcNAc
-Man
L
Fuc
4
+ GlcNAc + GlcNAc
+ ASn
Fig. 2. Oligosaccharides of the SCO complex-type glycoproteins at different steps of glycosylation. A . Proposed structure for the precursor form (high-mannose-type glycoprotein). B. Proposed structure for the mature form (complex-type glycoprotein). (From Meiniel et al., 1988a.)
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1
2
3
4
7 I
6
5.5
Fig. 3. Electrophoretical analysis of crude soluble extracts of SCO compared to other brain tissues. A. Ovine SCO: SDS-PAGE and Western blot stained with Con A. (1) Standards of molecular weight (ovalbumin 45 kDa); (2) profile of the SCO. Note the presence of a specific 54 kDa polypeptide (arrow); (3) profile of the pineal organ; (4) profile of the cerebellum. (From Meiniel et al., 1986.) B. Bovine SCO: two-dimensional gel electrophoresis of soluble extracts stained with silver nitrate. Arrows indicate specific polypeptides. C Bovine ependyma: two-dimensional gel electrophoresis of soluble extracts (silver staining).
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munization with SCO extracts. Accordingly, the present results can be considered as complementary to those obtained with antibodies raised against RF by the research groups of Professors Sterba and Rodriguez (Sterba et al., 1982; Loseckeet al., 1984, 1986; Rodriguez et al., 1984, 1986, 1987). A99 Polyclonal antibodies raised against crude extracts from the bovine SCO were adsorbed with brain crude extracts to precipitate antibodies raised against common compounds present in both tissues. By means of various tests, these antibodies were shown to be specific of SCO glycoproteins (Karoumi, 1990). After application of the immunofluorescence technique, the reactive material was located in the cytoplasm of the ependymal and hypendymal cells of the SCO as well as in Reissner’s fiber (Karoumi et al., 1990a). c,B$I8 Mab This monoclonal antibody was selected on the basis of its histochemical reactivity with SCO and Reissner’s fiber material (Fig. lE,F) (Meiniel et al., 1988b). Using a competitive test between lectins (Con A and WGA) and the monoclonal antibody it was demonstrated that both the high-mannose-type glycoproteins (Con A-reactive) and the complextype glycoproteins (WGA-reactive) were recognized by the antibody (Meiniel et al., 1988b). In addition, electron microscopical studies revealed the presence of C,B,A, immunoreactive material in the reticular compartment of the secretory ependymocytes (site of synthesis of the precursor form), in the Golgi area (site of transformation of the carbohydrate chains) and in secretory vacuole (site of transport and/or storage of the secreted form). These antibodies were thus excellent tools to try to characterize by immunochemical techniques specific compounds related to the particular phenotype of the secretory ependymocytes (polyclonal antibody) and to the various glycosylated forms of the complex-type glycoproteins (monoclo nal antibody).
Immunoaffinity chromatography Below we list a number of technical considerations that should be considered when analyzing the results. (1) Only soluble proteins were immunopurified as no detergents were added to the various tissue extracts; thus specific unsoluble proteins may occur in the SCO that were not revealed using our protocol. (2) The immunoaffinity chromatography was performed in the presence of inhibitors of proteases in order to avoid degradation of proteins. (3) Electrophoresis of extracts prepared in SDS-0mercaptoethanol separates proteins into subunits. The exact molecular weight of the native proteins must be determined by other techniques. (4) As immunopurified products were mainly glycoproteins, lectins (Con A and WGA) were used to reveal glycopeptides on Western blots. ( 5 ) Comparison with other brain tissues was carried out in order to specify the reactivity of our antibodies, and to detect eventual cross-reactivity. A99 immunoaffinity (Figs. 4A,B, 5)
Con A . Several Con A-positive bands were identified in the eluted fraction of the SCO, their apparent molecular weight (MW) ranging from 240 to 50 kDa. In the eluted fraction of the cerebral hemispheres polypeptides located at 52/54 and 50 kDa MW were also present while no polypeptides were immunopurified from soluble extracts of the classical ependymal lining. WGA. Only polypeptides at 98 and 52/54 kDa MW could be revealed in the eluted fraction of the SCO. In the eluted fraction of the cerebral hemispheres the 52/54 and 50 kDa polypeptides were also WGA-positive. C,B$18 immunoaffinity (Figs. 4C,D, 5)
Con A. Three glycopeptides were identified in
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Fig. 4. lmmunoaffinity chromatography and Reissner’s fiber. A . Con A-reactive polypeptides revealed after A99 immunoaffinity chromatography. (1) Eluted fraction of theependymal lining (no band); (2) profile of solubleextracts of the ependymal lining; (3) profile of solubleextracts of the SCO; (4) eluted fraction of the SCO (several bands, cf. Fig. 5 for exact molecular weights); ( 5 ) profile of soluble extracts of the cerebral hemispheres; (6) eluted fraction of the cerebral hemispheres (two bands 52/54 and 50 kDa); (7) standards of molecular weight stained with Coomassie blue (MW expressed in kDa). B. WGA-reactive polypeptides revealed after A99 immunoaffinity chromatography. (1) Profile of soluble extracts of the SCO; (2) eluted fraction of the SCO (two bands at 98 and 52/54 kDa); (3) profile of soluble extracts of the cerebral hemispheres; (4) eluted fraction of the cerebral hemispheres (one band 52/54 kDa). A and B: same electrophoresis and electrotransfer. C. Con A-reactive polypeptides revealed after ClB8A, immunoaffinity chromatography. Three bands are located at 88, 54 and 34 kDa MW. D. WGA-reactive polypeptides revealed after ClB8A8 immunoaffinity chromatography. One band is located at 34 kDa MW. Cand D:same electrophoresis and electrotransfer. E . Western blot of Reissner’s fiber (RF) stained with WGA. (I) RF pre-treated with urea 9 M; (2) RF pre-treated with SDS 4%. Note the presence of a positive smear in the stacking gel and in the migrating gel (arrow, top of the migrating gel). F. Western blot of RF polymer cleaved by trypsine 0.01 Yo. Three groups of bands were identified after WGA staining (arrow, top of the migrating gel).
the eluted fraction of the SCO having an apparent molecular weight of 88, 54 and 34 kDa. WGA. Only the glycopeptide located at 34 kDa was strongly positive. In eluted fractions of the classical ependymal lining no glycopeptides were revealed using either Con A or WGA.
These results can be compared with those obtained in the chick embryo (Karoumi et al., 1990b)using A74 IgG and the same technology. A tentative classification of the immunopurified glycopeptides is presented in Fig. 5 . Three groups were distinguished according to their reactivity to the various antibodies.
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Fig. 5 . Immunoaffinity chromatography using A99 (polyclonal antibody against bovine K O ) , C,B,A, (monoclonal antibody against bovine complex-typeglycoproteins) and A74 (polyclonal antibody against chick SCO). Con A- and WGA-positive bands observed after SDS-PAGE and electrotransfer. Glycopeptides linked to the complex-type glycoproteins. Number of sign + is proportional to intensity of the labeling. (CH), Glycopeptides also present in the cerebral hemispheres; (MO), glycopeptides present in the medulla oblongata.
(1) Glycopeptides related to the complex-type glycoproteins at different steps of glycosylation. These glycopeptides with an apparent molecular weight of 88, 52/54 and 32/34 kDa were immunopurified on C,B,A, immunoadsorbant. They were also detected using A99 and A74 IgG in different experiments (Karoumi et al., 1990b, 1991). (2) Glycopeptides linked to the particular phenotype of the secretory ependymocytes. These glycopeptides (240, 98 and 66 kDa MW) were present in both the chick embryo and the bovine SCO after A99 and A74 immunoaffinity chromatography. They can include enzymes involved in the secretory pathway and/or carrier proteins. These two groups of glycopeptides have a high intensity in the various experiments using either Con A or WGA. Their presence in both species suggests a certain stability of these products in the course of phylogenetical evolution.
(3) The third group of polypeptides could be species-dependent as they are present either in the chick embryo (150, 84, 77, 46, 42 kDa) or in the bovine (220,50 kDa) using respectivelyA74 and A99 immunoaffinity chromatography. Nevertheless, taking into account their low intensity and their inconstant presence in the various experiments, these glycopeptides could be at the limit of detection using the present technology. Their significance in each species requires further analysis. Regarding the putative glycopeptides linked to the complex-type glycoproteins (88, 52/54 and 32/34 kDa), differences in their affinity to the lectins Con A and WGA allow some speculation. It is possible that the 88 kDa band that is mainly Con Apositive represents the high-mannose-type glycoprotein (or a subunit), while the 52/54 kDa band that shows Con A- and WGA-positive reaction could be linked to an intermediate form. The 32/34
339
kDa glycopeptide that is strongly WGA-positive could correspond to the secreted monomeric form of the complex-type glycoprotein. This glycopeptide was also isolated from soluble extracts of the medulla oblongata in the chick embryo (Karoumi et al., 1990b) and was discussed as the putative secreted glycoprotein present in Reissner’s fiber. The fact that a 52/54 kDa glycopeptide also occurred in immunopurified fractions of the cerebral hemispheres in both the chick embryo and bovine, raises the question whether a brain polypeptide, having similar properties, could cross-react with our polyclonal antibodies. Nevertheless, considering that this glycopeptide was not immunopurified from extracts of the ependymal lining using either A99 or C,B,A,, and that the site(s) of action of the secreted compound(s) is (are) still unknown, the possibility that the 52/54 kDa glycopeptide is present in the brain must be considered. This would indicate that other brain regions could be the target for SCO complex-type glycoproteins. Further analysis is required after purification of soluble extracts of cerebral hemispheres on C,B,A, immunoadsorbent (in progress).
Reissner’s fiber Reissner (1860) first described in the lamprey Petromyzonfluviatilis an axon-like structure in the central canal of the spinal cord. In 1900, Studnicka suspected that Reissner’s fiber (RF) was the aggregation of the specific compound secreted by the subcommissural organ. Immunohistochemical staining of both the subcommissural organ and RF using specific antibodies demonstrated that at least the major constituent(s) of RF arises from the SCO (Sterba et al., 1982; Rodriguez et al., 1984; Meiniel et al., 1988b;Karoumiet al., 1990a). The functional significance of RF is still a matter of speculation. Hofer et al. (1984) and Peruzzo et al. (1987) have postulated a degradation of RF material in the ampulla caudalis of lampreys and an absorbtion of this material by the surrounding blood capillaries. RF was collected from portions of the bovine spinal cord and rinced twice in distilled water. The
following procedures were then applied: (1) solubilization in 4% SDS or urea 9 M or urea 9 M + chaps 2% + SDS 2% (12 h); (2) digestion in pronase, pepsine, papaine, trypsine 0.5% (1 h); and (3) partial cleavage in trypsine 0.01% (2 h). The samples were then prepared for SDS-PAGE (cf. Meiniel et al., 1986), separated and transferred to nitrocellulose sheets. RF material and analyzed on Western blots stained with WGA. After treatment with SDS, urea or SDS + urea, a WGA-positive smear was detected in the upper part of the blots corresponding to the stacking gel and the top of the migrating gel (Fig. 4E). Such a smear probably indicates the presence of polymers that were not completely solubilized under our experimental conditions. The WGA-positive smear completely disappeared when RF material was preincubated in the various proteases. Incomplete cleavage of RF polymer was obtained after treatment with trypsine 0.01 %.Under these experimental conditions, three WGA-positive bands were visualized in the upper part of the blots as well as two groups of small bands ranging from 80 to 150 kDa and from 45 to 55 kDa (Fig. 4F). These WGApositive bands represent fragments of the RF polymer at different steps of digestion by trypsine. Thus, in the adult bovine, RF can be regarded as a stable polymer (insoluble in SDS and urea). This suggests that the monomeric form of the SCO complex-type glycoproteins, when secreted into the CSF, changes its conformation. Such a situation cannot be extended to all species (or all stages of development) as in the embryonic chicken a 32/34 kDa glycopeptide, suspected to represent the compound secreted by the SCO and present in Reissner’s fiber, could be isolated from soluble extracts of the medulla oblongata (Karoumi et al., 1990b). In the brain, other secreted complex-type glycoproteins involved in synaptic plasticity (ependymins CY and 0) are known to constitute either in vitro or in vivo polymers insoluble in SDS-0mercaptoethanol (Shashoua, 1988; Shashoua et al., 1990). The monomeric forms of these proteins have an apparent molecular weight of 38 and 32 kDa. A few biochemical properties of ependymins and of
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the SCO complex-type glycoproteins point to a large similarity of both products. Nevertheless, further analysis of the amino acid sequence of the complextype glycoproteins is needed to compare these proteins and to give a better insight into the molecular aspect of the compounds secreted by the SCO.
Ontogenetical study and cell filiation With reference to the observation that aldehyde fuchsin staining (AF) revealed compounds in the SCO and in the pineal organ, Kelly and Van de Kamer (1 960) suggested that positive AF .elements present in the pineal organ have been detached from the ependymal roof during embryogenesis and could correspond to specialized ependymal cells coming from the SCO anlage. AF-positive material has been reported to occur in pinealocytes of various mammalian species (Quay, 1970; Lukaszyk and Reiter, 1975; Japha et al., 1976). In the rat, the AF-positive material appears to be more conspicuous during embryogenesis (Owman, 1960). On the other hand, using polyclonal antibodies raised against RF, Rodriguez et al. (1988) were able to detect immunoreactive material in the pineal organ of various vertebrate species. Using the monoclonal antibody ClBgA8, a comparative spatio-temporal analysis of both organs has been undertaken in the embryonic bovine (Meiniel et al., 1990). Immunoreactive material was detected as early as 2 months of development in the SCO anlage. The labeling increases rapidly in the secretory ependymocytes and extends in a caudal direction. Reissner’s fiber could be detected in the central canal of the spinal cord at 4 months of development. In the course of ontogeny, no immunoreactive material could be revealed in the bovine pineal organ. Glycoproteins present in pinealocytes require additional research to precise their exact nature and their biological role. In addition, identification of the cell type engaged in glycoprotein synthesis in the pineal organ would help to establish a parallel with the SCO secretory ependymocytes.
Several authors have mentioned the early expression of specific compounds in the subcommissural organ (Schoebitz et al., 1986; Naumannet al., 1987; Karoumi et al., 1990a). Theexact significance of the secretory process in the course of ontogenetical development remains enigmatic. A possible role of the complex-type glycoproteins in the maturation of other brain regions cannot be determined without experimental research.
Acknowledgements Thanks are due to C. Michaud for typing the manuscript and G. Ragonnaud for the illustrations. This research was supported by a grant from the “Association Francaise contre les Myopathies” (A.F.M.).
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