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Glycoprotein expression in foetal and adult mouse cerebellum
Comp. Biochem. Physiol. Vol. 95B, No. 4, pp. 855-860, 1990 Printed in Great Britain
0305-0491/90$3.00+ 0.00 © 1990PergamonPress pie
GLYCOPROTEIN EXPRESSION IN FOETAL A N D ADULT MOUSE CEREBELLUM MARK CARROLL* and MARGARET M. BIRD'~ Departments of *Biochemistry and tAnatomy, London Hospital Medical College, London E1 2AD, UK (Tel: 01 377 7000; Fax: 01 377 7677)
(Received 11 September 1989) Abstract--1. Explants of cerebellum from foetal mouse were cultured in vitro for up to 12 days. Some glycoprotein components displayed time-dependent changes in concentration in the cultured explants. 2. The specific activity of several enzymes involved in the biosynthesis or degradation of N-linked glycoproteins, increased markedly in the cerebellar explants as a function of time in culture. 3. Glycoprotein expression in foetal mouse cerebellum is compared with that in the adult tissue.
INTRODUCTION
Cell-surface glycoproteins are thought to play an essential role in the m a m m a l i a n nervous system, by mediating interactions of neuronal cells with other cells or with the extracellular matrix (Dodd and Jessell, 1988). Numerous cell adhesion molecules have been described (Edelman, 1983); they may play key roles in neuronal targeting and fasciculation, but in most cases the molecular mechanism of such developmental strategies has not been determined. These adhesion molecules are invariably N-linked glycoproteins associated with the extracellular face of the plasma membrane of growth cones and axons. The glycan portion of such glycoproteins is synthesised in vivo by the glycosyl transferases of the well-characterized dolichol-linked pathway (Kornfeld and Kornfeld, 1985), and it is degraded by the glycosidase enzymes of the lysosomes (von Figura and Hasilik, 1986). The cerebellum in rodents undergoes maturation in the early neonatal period (Altman, 1972). There is a major phase of cell proliferation that generates mainly granule cells with their associated processes. These fibres make important functional connections with Purkinje dendrites and stellate cells in the developing cerebellum. Previous studies have identified a burst of glycoprotein expression on the plasma membrane of the parallel fibres (Zanetta et al., 1978). This developmental phase was associated with a corresponding increase in activity of some glycosidases (Zanetta et aL, 1980). However, such studies failed to address two important points: firstly, the identity of individual glycoprotein moieties; and secondly, the existence of isoenzymes of glycosidases, some of which are not located in lysosomes.
*Abbreviations used:--Tes, N-Tris [hydroxymethyl]methyl2-aminoethanesulphonic acid; EDTA, ethylenediaminetetraacetic acid, disodium salt; SDS-PAGE, electrophoresis in polyacrylamide gel containing sodium dodecyl sulphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid. CBP(B) 95/4~N
855
It is possible to maintain explants of foetal brain under tissue-culture conditions such that they survive in a viable state (Bird, 1986). We have exploited this approach to study the expression of glycoproteins in cerebellum from foetal mouse. The results are compared with those from a corresponding analysis of adult mouse cerebellum. MATERIALS AND METHODS
All reagents were of the highest possible grade obtainable from Sigma Chemicals, Poole BH17 7NH, UK, unless otherwise stated. Tissue culture The cerebellum of foetal mice (18 days gestation) was dissected under sterile conditions so as to generate explants of about 1 mm 3 in size. Such explants were cultured in Dulbecco's modification of Eagle's minimal essential medium supplemented with 10% (v/v) horse serum (Bird, 1986). Morphological analysis of cultured explants The gross morphology of the living explants was studied by phase-contrast microscopy. Their ultrastructure was examined by transmission electron microscopy as described by Bird (1986). Preparation of membrane and soluble fractions Cerebellar explants (typically, material corresponding to about 200/zg of tissue protein) were transferred in culture medium to microfuge tubes. After centrifugation (3000g, 1 min) at room temperature, the supernatant was discarded, and the pellet washed three times with 1 ml 0.25 M sucrose in Tes buffer (I mM Tes*, pH 7.0, containing l mM EDTA, 1 mM mercaptoethanol, 1 mM NaN 3, and 0.1 mM phenylmethylsulphonyl fluoride. Subsequent steps were performed at 4°C. The tissue was disrupted in 0.2ml Tes buffer containing 25/ag/ml each of leupeptin and pepstatin (protease inhibitors), by means of a micro-homogenizer (Biomedix, Pinner, UK). The homogenate was centrifuged (105g, 15min), and the supematant (soluble fraction) removed with a Hamilton syringe. The pellet (membrane fraction) was washed twice, once with 1 ml Tes buffer and once with 1 ml Tes buffer containing 0.1 M (NH4)2SO4, before being suspended in 0.2 ml Tes buffer containing leupeptin and pepstatin (25/ag/ml each). Membrane and soluble fractions were stored at -20°C.
856
MARK CARROLL a n d MARGARET M. BIRD
Enzyme assays
Glycosidase activity in the appropriate fraction at the optimal pH, was assayed with the corresponding 4-methylumbelliferyl substrate (Carroll and McCrorie, 1980): "neutral" ct-glucosidase of the endoplasmic reticulum (membrane fraction, pH 6.5); "acid" ct-glucosidaseof lysosomes (soluble fraction, pH4.0); "acid" fl-glucosidase of lysosomes (membrane fraction, pH 5.5); ~t-mannosidaseII of the Golgi complex (membrane fraction, pH 5.5); "acid" ct-mannosidase of lysosomes (soluble fraction, pH 4.0); Nacetyl-fl-glucosaminidaseof lysosomes (membrane and soluble fraction, pH 4.5). Ouabain-sensitive Na ÷, K+-ATPase of the plasma membrane was assayed as described by Morand and Kent (1986). Lactate dehydrogenase of the cytosol was measured at 340 nm (Pesce et al., 1964). Protein concentration was determined by a sensitive dye-binding method (Read and Northcote, 1981). Glycoprotein composition o f membrane and soluble fractions
Membrane-bound proteins were solubilized in 1% (w/v) CHAPS in Tes buffer at 4°C, as described by Hjelmeland (1980). The glycoprotein composition of the fractions was determined essentially according to the method of Thompson et al. (1987). Briefly, protein (5 #g) was mixed with agarose-bound concanavalin A (20 #1 bed volume) equilibrated at 4°C with Tes buffer containing 0.6% (w/v) Tween 20, 25 mM NaCI, 5 mM CaCI2and 5 mM MgC1z. Unbound non-glycoproteins were recovered in the supernatant after centrifugation (3000g, 1 min). The gel was washed three times with the buffer, before bound glycoproteins were eluted with 20pl SDS-PAGE sample buffer (Laemmli, 1970). The polypeptide composition of the unbound and bound fractions was assessed by SDS-PAGE (Laemmli, 1970); the resolved bands were visualized with a sensitive silver stain (Tsang et al., 1984). Analysis o f adult mouse cerebellum
Preparation of membrane and soluble fractions, analysis of their glycoprotein composition, and assays of enzyme activity, were carried out on small samples (typically, 2 5 mg wet wt) of adult mouse cerebellumprocessed exactly as described above for the foetal tissue. RESULTS
The specific activity of several glycoprotein- or glycan-metabolizing enzymes was measured in both foetal and adult mouse cerebellum (Table 1). In general, the foetal activity was closely comparable with, or somewhat higher than, the corresponding adult activity. The lysosomal enzyme N-acetyl-fl-glucosaminidase (hexosaminidase) was predominantly membrane-bound in the foetal tissue, but predominantly soluble in the adult tissue. For comparison, Table 1. Glycoprotein- and glycan-metabolizing enzymes of foetal and adult mouse cerebellum Enzyme ~-Glucosidase, pH 6.5 ~t-Glucosidase, pH 4.0 B-Glucosidase ~-Mannosidase II 0t-Mannosidase, pH 4.0 Hexosaminidase: membrane-bound soluble Na +, K+-ATPase~ Lactate dehydrogenaset
Specific activity* Foetal Adult 5.48 0.453 1.52 1.11 0.171 10.4 6.67 55.0 1760
3.60 0.619 0.429 0.553 0.250 5.42 6.72 296) 3230)
*Expressed as nmol min-~ mg protein-t (mean of duplicate results which agreed within 10%). ]'Enzyme not involved in glycoprotein metabolism.
Table 2. Time-course of enzyme activity in cultured explants of foetal cerebellum Enzyme ~t-Glucosidase, pH 6.5 ct-Glucosidase, pH 4.0 fl-Glucosidase ct-Mannosidase II ct-Mannosidase, pH4.0 Hexosaminidase: membrane-bound soluble Na +, K+-ATPase Lactate dehydrogenase
Day 0t
Specific activity* Day 5 Day 10
5.48 0.453 1,52 1.11 0.171 10.4 6.67 55.0 1760
5.89 1.31 3.09 1.96 0.193 44.5 7.82 76.3 2500
6.11 0.90 3.53 3.33 0.760 12 I 68.4 119 2540
*Expressed as in Table 1. tDays in culture.
two important enzymes not associated with glycoprotein metabolism were also assayed: Na +, K +ATPase (a glycoprotein of the plasma membrane) and lactate dehydrogenase (LDH, a non-glycoprotein of the cytosol). The ATPase was considerably more active in the adult tissue (Table 1), the LDH somewhat more so. It was of interest to follow the time-course of enzyme activity in foetal cerebellar explants maintained in culture, since genetically programmed changes in glycoprotein expression might be reflected in the activity of glycoprotein-metabolising enzymes. The results are presented in Table 2. In most cases, there was a gradual increase in specific activity as a function of time in culture. This rise was especially marked for some of the lysosomal enzymes, particularly hexosaminidase, which had increased 10-fold by day 10 in culture. However, the time-course of the increase in specific activity varied from one lysosomal enzyme to another (compare ~-glucosidase, pH 4.0 with ~t-mannosidase, pH 4.0). For the two enzymes not connected with glycoprotein metabolism (Na ÷, K+-ATPase and LDH), the specific activity increased with time in culture towards the corresponding adult value. Lectin affinity chromatography and SDS-PAGE were exploited in order to determine the glycoprotein composition of membrane and soluble fractions from both foetal and adult mouse cerebellum. Results of a typical experiment are depicted in Fig. 1. The lectin used (concanavalin A) has an affinity for ~t-linked residues of D-glucose and D-mannose. It would thus be expected to bind the high-mannose type of Nlinked glycoproteins, as well as most of the complex type and hybrid type (Osawa and Tsuji, 1987); Olinked glycoproteins and non-glycoproteins would not bind. The results (Fig. 1) suggest: (a) foetal and adult mouse cerebellum share many N-linked glycoproteins in common; (b) some such glycoproteins are found exclusively in the foetal tissue, and some only in the adult tissue. For example, a membrane-bound glycoprotein with an apparent molecular mass of about 64,000 is associated only with adult cerebellum, whereas a soluble polypeptide of about 60,000 is present only in foetal cerebellum. Glycoprotein composition was followed in the foetal cerebellum explants as a function of time in culture (Fig. 2). In some cases, there was little change in the concentration of a given glycoprotein component. For example, a membrane-bound polypeptide of mol. wt approximately 85,000 was present at about
Glycoprotein in the cerebellum
857
sion electron microscopy (Fig. 3). After 12 days in culture, explants appeared pale, with bundles of nerve fibres and a dense mat of heterogeneous cells extending from them. These fibres developed initially from undifferentiated cell types, which transformed into neuron-like cells with processes resembling dendrites and axons. Numerous glial cells and some macrophages were also apparent (Fig. 3a). The electron microscope provided images of well-differentiated neurons having the usual complement of subcellular organelles (Fig. 3b). The number of synaptic contacts increased with time in culture (results not shown). DISCUSSION
We have exploited the ability to culture explants of mammalian nervous tissue in order to address the significance of glycoprotein expression in neuronal development. The system we have chosen is foetal mouse cerebellum, which displays certain advantages from the point of view of biochemical analysis. This part of the brain comprises a relatively small number of morphologically distinguishable cell types; and it undergoes a phase of rapid maturation (cell proliferation and specific synapse formation) in the early neonatal period. Thus, one might expect to see genetically programmed changes in glycoprotein composition as cerebellar explants from 18-day mouse embryos are maintained in culture for a further 10-12 days. In exploiting this system, we have had to bear in mind some inherent constraints. Firstly, the artificial nature of the culture conditions may induce changes in cellular composition and/or gene expression. (However, in similar systems, organotypic development is evident; Bird, 1986). Secondly, Fig. 1. Glycoprotein composition of membrane and soluble the very small amounts of tissue available have fractions from foetal and adult mouse cerebellum. Glycoprotein composition was assessed by SDS-PAGE of those necessitated the use of analytical methods of extreme proteins which bound to concanavalin A. The samples were sensitivity. For example, we have optimized the tech(from left to right): membrane fraction from adult cerebel- niques necessary for: homogenizing small tissue lum; membrane fraction from foetal cerebellum; soluble samples (2-5rag wet wt); measuring microgram fraction from adult cerebellum; soluble fraction from foetal quantities of protein; the sensitive fluorimetric assay cerebellum; protein standards (with molecular weight): of glycosidase activity; and determining the glycomyosin (200,000), fl-galactosidase (116,000), phosphorylase protein composition of microgram samples of b (97,000), bovine serum albumin (66,000), ovalbumin protein. These biochemical approaches have been (43,000), and carbonic anhydrase (30,000). (The band at about 30,000 in some lanes is an artifact generated by release complemented by those of light microscopy and of concanavalin A subunits from the agarose-bound lectin). electron microscopy, in order to visualize the appearance of the cultured explants at both the gross and the subcellular level. the same level in the non-cultured tissue (day 0) as at At day 18 of gestation, mouse cerebellum has day 5 and day 11 in culture. Conversely, some active systems for synthesizing and degrading Nglycoprotein components were present preferentially linked glycoproteins (Fig. 1 and Table 1). The lysosoat day 0 or day 5 or day 11. These differences were mal acid hydrolases (glycosidases) are invariably just more marked for the membrane-bound polypeptides as active in the foetal tissue as they are in the adult. than for the soluble ones. From amongst the numer- These activities reflect the cells' ability to degrade the ous examples evident on the original gel (Fig. 2), glycan portion of senescent glycoproteins, both memone can highlight the following: a polypeptide of brane-bound and soluble (von Figura and Hasilik, mol. wt approximately 66,000 present in the mem- 1986). Indeed, lysosomes were evident in electron brane fraction at day 11 but apparently absent at micrographs of neurones from cultured explants day 5 and day 0; a polypeptide of mol. wt approxi- (Fig. 3b). Also apparent in such micrographs were mately 114,000 present at day 0 but apparently well-developed profiles of rough endoplasmic reticumissing at day 5 and day 11; and a polypeptide of lum and Golgi complex, the two major organelles mol. wt approximately 100,000 present at a higher which participate in the biosynthesis of N-linked concentration at day 5 than at either day 0 or day 11. glycoproteins (Kornfeld and Kornfeld, 1985). One of The morphology, cellular composition and ultra- the "trimming" glycosidases involved in this pathway structure of the cultured cerebellar explants, were in the Golgi complex, ~t-mannosidase II, was present revealed by phase-contrast microscopy and transmis- in foetal cerebellum at a level (measured in vitro)
Fig. 2. Time-course of glycoprotein composition of foetal cerebellar explants in culture. Legend as for Fig. 1, except that the samples were (from left to right): protein standards; membrane fraction, day 0; membrane fraction, day 5, membrane fraction, day 11; soluble fraction, day 0; soluble fraction, day 5; soluble fraction, day 11; protein standards.
Fig. 3(a)
Glycoprotein in the cerebellum
859
Fig. 3(b) Fig. 3. Morphology and ultrastructure of cultured explant of foetal mouse cerebellum. (a) Phase-contrast micrograph of explant (E) maintained in culture for 12 days. Bundles of nerve fibres are apparent (large arrows), as well as individual neurites (small arrows). × 800. (b) Electron micrograph of a portion of a neuron in the cultured explant seen in (a). Some of the major organelles are indicated: N, nucleus; RER, rough endoplasmic reticulum; G, Golgi complex; L, lysosome; M, mitochondria, x 50,000.
which was considerably higher than that of the lysosomal isoenzyme (ct-mannosidase, pH 4.0). The ratio of these two activities was closer to unity in the adult tissue; this change presumably reflects the reduced turnover of glycoproteins in the mature organ. The "neutral" ~-glucosidase measured here (Table 1) may be related to the isoenzyme which catalyses the initial step in the processing of N-linked glycoproteins in the rough endoplasmic reticulum. The expression of an individual glycoprotein species will depend on the relative rates of its biosynthesis (presumably genetically determined) and its degradation (possibly non-specific in nature). Our experimental approach only reveals the major glycoprotein moieties (Fig. 1). However, even at this rather gross level, it is clear that there are differences between the glycoprotein composition of foetal and adult mouse cerebellum. Our particular interest lies in the glycoprotein components of the neuronal plasma membrane and of the extracellular matrix. Further analysis will demand techniques of greater resolution
(such as subcellular fractionation and two-dimensional electrophoresis) or of greater discrimination (such as immunochemical methods). Since SDS-PAGE separates polypeptides only on the basis of their apparent molecular mass, we have insufficient information to assign an identity to individual bands on the gels. As the foetal cerebellar explants are maintained in culture, the enzymes of glycoprotein metabolism increase substantially in activity (Table 2), and the glycoprotein pattern changes somewhat (Fig. 2). Such changes may reflect genetically programmed development of the cerebellum in the early neonatal period, which corresponds approximately to days 4-10 in culture for the explants. Similar changes have been observed before (Zanetta et al., 1978, 1980), but these earlier studies did not take into account the existence of isoenzymes of the gtycosidases involved in glycoprotein metabolism. For example, we have taken care to differentiate the ~t-mannosidase active in the processing of nascent glycoproteins (by measuring the
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MARKCARROLLand MARGARETM. BIRD
membrane-bound enzyme at pH 5.5) from the isoenzyme active in their lysosomal degradation (by assaying the soluble enzyme at pH 4.0). The generalized increase in acid hydrolase activity cannot be explained simply by proliferation of macrophages in the cultured explants. Such cells represented a tiny fraction of the total; furthermore, the time-course for the increase in activity was not uniform for all the enzymes studied (Table 2). The results of the lectin affinity chromatography/SDS-PAGE analysis should be interpreted with some caution. Appearance of a glycoprotein component may reflect: (a) genetically determined switching on of its biosynthesis; or (b) glycosylation of a previously non-glycosylated polypeptide; or (c) a non-specific effect (experimental artefact). Nevertheless, there appears to be strong evidence for the developmental regulation of glycoprotein expression in mouse cerebellum. In culture, the cerebellum explants extend growth cones of neurites, which become consolidated into bundles of nerve fibres (Fig. 3a). Glial cells also proliferate. We have not yet determined the precise nature of any changes in the cellular composition of the cultured explants. Furthermore, the extracellular matrix in vitro would certainly differ from that in vivo. Such modifications would probably be reflected in the activity of enzymes involved in glycoprotein metabolism, and in the glycoprotein pattern (particularly in the soluble fraction). The results of our biochemical analysis should therefore be interpreted with care. Nevertheless, the cells of the cultured explants remain viable (up to day 12 at least), and they make synaptic contact with their neighbours. We do not know if such synapses are physiologically active, but it is clear that the components required to establish and maintain such contacts are expressed in the appropriate subcellular location. Cell-surface glycoproteins are likely to be involved (Dodd and Jessell, 1988). In conclusion, we have developed methods for the analysis of glycoprotein expression in small samples of foetal cerebellum maintained in culture for up to 12 days. This system should be useful for studying the effect of inhibitors of the N-linked glycosylation pathway in neuronal tissue. SUMMARY Numerous glycoprotein components were present in the cerebellum of both adult and foetal mouse, whereas a few glycoproteins were present predominantly in either the adult tissue or the foetal tissue. Both tissues had active systems for the biosynthesis and degradation of N-linked glycoproteins. Glycoprotein composition varied in cultured explants of foetal mouse cerebellum, dependent on the time in culture. In general, the glycoprotein-metabolizing enzymes increased in their specific activity as a function of time in culture.
Glycoprotein expression in foetal mouse cerebellum is compared with that in the adult tissue. Acknowledgement--We thank Miss Barbara Stevenson for
expert technical assistance with the electron microscopy.
REFERENCES
Altman J. (1972) Post-natal development of the cerebellar cortex in the rat. J. comp. Neurol. 145, 353 514. Bird M. M. (1986) An ultrastructural study of embryonic chick retinal neurones in culture. Cell Tiss. Res. 245, 563-577. Carroll M. and McCrorie P. (1980) Glycosidases in bloodstream forms of Trypanosoma brucei brucei. Comp. Biochem. Physiol. 67B, 685-688. Dodd J. and Jessell T. M. (1988) Axon guidance and the patterning of neuronal projections in vertebrates. Science 242, 692-699. Edelman G. M. (1983) Cell adhesion molecules. Science 219, 450~,57. Figura K. von and Hasilik A. (1986) Lysosomal enzymes and their receptors. A. Rev. Biochem. 55, 167-193. Hjelmeland L. M. (1980) A non-denaturing zwitterionic detergent for membrane biochemistry: design and synthesis. Proc. natn. Aead. Sci. USA 77, 6368-6370. Kornfeld R. and Kornfeld S. (1985) Assembly of asparagine-linked oligosaccharides. A. Rev. Biochem. 54, 631-664. Laemmli W. K. (1970) Cleavage of the structural proteins during the assembly of the head of the bacteriophage T4. Nature, Lond. 227, 680-685. Morand J. N. and Kent C. (1986) A one-step technique for the subcellular fractionation of total cell homogenates. Analyt. Biochem. 159, 157 162. Osawa T. and Tsuji T. (1987) Fractionation and structural assessment of oligosaccharides and glycopeptides by use of immobilised lectins. A. Rev. Biochem. 56, 21~42. Pesce A., McKay R. H., Stolzenbach F., Cahn R. D. and Kaplan N. O. (1964) The comparative enzymology of lactic dehydrogenases. J. biol. Chem. 239, 1753 1761. Read S. M. and Northcote D. H. (1981) Minimisation of variation in the response to different proteins of the Coomassie blue G dye-binding assay for protein. Analyt. Biochem. 116, 53-64. Thompson W., Latham J. A. E. and Turner G. A. (1987) A simple, reproducible and cheap batch method for the analysis of serum glycoproteins by using Sepharosecoupled lectins and silver staining. Clinica chim. Aeta 167, 217-223. Tsang V. C. W., Hancock K., Maddison S. E., Beatty A. L. and Moss D. M. (1984) Demonstration of species-specific and cross-reactive components of the adult microsomal antigens from Schistosoma mansoni and S. japonicum. J. Immun. 132, 2607-2613. Zanetta J.-P., Roussel G., Ghandour M. S., Vincendon G. and Gombos G. (1978) Post-natal development of rat cerebellum: massive and transient accumulation of concanavalin A-binding glycoproteins in parallel fibre axolemma. Brain Res. 142, 301-309. Zanetta J.-P., Frederico A. and Vincendon G. (1980) Glycosidases and cerebellar ontogenesis in the rat. J. Neurochem. 34, 831 834.
0305-0491/90$3.00+ 0.00 © 1990PergamonPress pie
GLYCOPROTEIN EXPRESSION IN FOETAL A N D ADULT MOUSE CEREBELLUM MARK CARROLL* and MARGARET M. BIRD'~ Departments of *Biochemistry and tAnatomy, London Hospital Medical College, London E1 2AD, UK (Tel: 01 377 7000; Fax: 01 377 7677)
(Received 11 September 1989) Abstract--1. Explants of cerebellum from foetal mouse were cultured in vitro for up to 12 days. Some glycoprotein components displayed time-dependent changes in concentration in the cultured explants. 2. The specific activity of several enzymes involved in the biosynthesis or degradation of N-linked glycoproteins, increased markedly in the cerebellar explants as a function of time in culture. 3. Glycoprotein expression in foetal mouse cerebellum is compared with that in the adult tissue.
INTRODUCTION
Cell-surface glycoproteins are thought to play an essential role in the m a m m a l i a n nervous system, by mediating interactions of neuronal cells with other cells or with the extracellular matrix (Dodd and Jessell, 1988). Numerous cell adhesion molecules have been described (Edelman, 1983); they may play key roles in neuronal targeting and fasciculation, but in most cases the molecular mechanism of such developmental strategies has not been determined. These adhesion molecules are invariably N-linked glycoproteins associated with the extracellular face of the plasma membrane of growth cones and axons. The glycan portion of such glycoproteins is synthesised in vivo by the glycosyl transferases of the well-characterized dolichol-linked pathway (Kornfeld and Kornfeld, 1985), and it is degraded by the glycosidase enzymes of the lysosomes (von Figura and Hasilik, 1986). The cerebellum in rodents undergoes maturation in the early neonatal period (Altman, 1972). There is a major phase of cell proliferation that generates mainly granule cells with their associated processes. These fibres make important functional connections with Purkinje dendrites and stellate cells in the developing cerebellum. Previous studies have identified a burst of glycoprotein expression on the plasma membrane of the parallel fibres (Zanetta et al., 1978). This developmental phase was associated with a corresponding increase in activity of some glycosidases (Zanetta et aL, 1980). However, such studies failed to address two important points: firstly, the identity of individual glycoprotein moieties; and secondly, the existence of isoenzymes of glycosidases, some of which are not located in lysosomes.
*Abbreviations used:--Tes, N-Tris [hydroxymethyl]methyl2-aminoethanesulphonic acid; EDTA, ethylenediaminetetraacetic acid, disodium salt; SDS-PAGE, electrophoresis in polyacrylamide gel containing sodium dodecyl sulphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid. CBP(B) 95/4~N
855
It is possible to maintain explants of foetal brain under tissue-culture conditions such that they survive in a viable state (Bird, 1986). We have exploited this approach to study the expression of glycoproteins in cerebellum from foetal mouse. The results are compared with those from a corresponding analysis of adult mouse cerebellum. MATERIALS AND METHODS
All reagents were of the highest possible grade obtainable from Sigma Chemicals, Poole BH17 7NH, UK, unless otherwise stated. Tissue culture The cerebellum of foetal mice (18 days gestation) was dissected under sterile conditions so as to generate explants of about 1 mm 3 in size. Such explants were cultured in Dulbecco's modification of Eagle's minimal essential medium supplemented with 10% (v/v) horse serum (Bird, 1986). Morphological analysis of cultured explants The gross morphology of the living explants was studied by phase-contrast microscopy. Their ultrastructure was examined by transmission electron microscopy as described by Bird (1986). Preparation of membrane and soluble fractions Cerebellar explants (typically, material corresponding to about 200/zg of tissue protein) were transferred in culture medium to microfuge tubes. After centrifugation (3000g, 1 min) at room temperature, the supernatant was discarded, and the pellet washed three times with 1 ml 0.25 M sucrose in Tes buffer (I mM Tes*, pH 7.0, containing l mM EDTA, 1 mM mercaptoethanol, 1 mM NaN 3, and 0.1 mM phenylmethylsulphonyl fluoride. Subsequent steps were performed at 4°C. The tissue was disrupted in 0.2ml Tes buffer containing 25/ag/ml each of leupeptin and pepstatin (protease inhibitors), by means of a micro-homogenizer (Biomedix, Pinner, UK). The homogenate was centrifuged (105g, 15min), and the supematant (soluble fraction) removed with a Hamilton syringe. The pellet (membrane fraction) was washed twice, once with 1 ml Tes buffer and once with 1 ml Tes buffer containing 0.1 M (NH4)2SO4, before being suspended in 0.2 ml Tes buffer containing leupeptin and pepstatin (25/ag/ml each). Membrane and soluble fractions were stored at -20°C.
856
MARK CARROLL a n d MARGARET M. BIRD
Enzyme assays
Glycosidase activity in the appropriate fraction at the optimal pH, was assayed with the corresponding 4-methylumbelliferyl substrate (Carroll and McCrorie, 1980): "neutral" ct-glucosidase of the endoplasmic reticulum (membrane fraction, pH 6.5); "acid" ct-glucosidaseof lysosomes (soluble fraction, pH4.0); "acid" fl-glucosidase of lysosomes (membrane fraction, pH 5.5); ~t-mannosidaseII of the Golgi complex (membrane fraction, pH 5.5); "acid" ct-mannosidase of lysosomes (soluble fraction, pH 4.0); Nacetyl-fl-glucosaminidaseof lysosomes (membrane and soluble fraction, pH 4.5). Ouabain-sensitive Na ÷, K+-ATPase of the plasma membrane was assayed as described by Morand and Kent (1986). Lactate dehydrogenase of the cytosol was measured at 340 nm (Pesce et al., 1964). Protein concentration was determined by a sensitive dye-binding method (Read and Northcote, 1981). Glycoprotein composition o f membrane and soluble fractions
Membrane-bound proteins were solubilized in 1% (w/v) CHAPS in Tes buffer at 4°C, as described by Hjelmeland (1980). The glycoprotein composition of the fractions was determined essentially according to the method of Thompson et al. (1987). Briefly, protein (5 #g) was mixed with agarose-bound concanavalin A (20 #1 bed volume) equilibrated at 4°C with Tes buffer containing 0.6% (w/v) Tween 20, 25 mM NaCI, 5 mM CaCI2and 5 mM MgC1z. Unbound non-glycoproteins were recovered in the supernatant after centrifugation (3000g, 1 min). The gel was washed three times with the buffer, before bound glycoproteins were eluted with 20pl SDS-PAGE sample buffer (Laemmli, 1970). The polypeptide composition of the unbound and bound fractions was assessed by SDS-PAGE (Laemmli, 1970); the resolved bands were visualized with a sensitive silver stain (Tsang et al., 1984). Analysis o f adult mouse cerebellum
Preparation of membrane and soluble fractions, analysis of their glycoprotein composition, and assays of enzyme activity, were carried out on small samples (typically, 2 5 mg wet wt) of adult mouse cerebellumprocessed exactly as described above for the foetal tissue. RESULTS
The specific activity of several glycoprotein- or glycan-metabolizing enzymes was measured in both foetal and adult mouse cerebellum (Table 1). In general, the foetal activity was closely comparable with, or somewhat higher than, the corresponding adult activity. The lysosomal enzyme N-acetyl-fl-glucosaminidase (hexosaminidase) was predominantly membrane-bound in the foetal tissue, but predominantly soluble in the adult tissue. For comparison, Table 1. Glycoprotein- and glycan-metabolizing enzymes of foetal and adult mouse cerebellum Enzyme ~-Glucosidase, pH 6.5 ~t-Glucosidase, pH 4.0 B-Glucosidase ~-Mannosidase II 0t-Mannosidase, pH 4.0 Hexosaminidase: membrane-bound soluble Na +, K+-ATPase~ Lactate dehydrogenaset
Specific activity* Foetal Adult 5.48 0.453 1.52 1.11 0.171 10.4 6.67 55.0 1760
3.60 0.619 0.429 0.553 0.250 5.42 6.72 296) 3230)
*Expressed as nmol min-~ mg protein-t (mean of duplicate results which agreed within 10%). ]'Enzyme not involved in glycoprotein metabolism.
Table 2. Time-course of enzyme activity in cultured explants of foetal cerebellum Enzyme ~t-Glucosidase, pH 6.5 ct-Glucosidase, pH 4.0 fl-Glucosidase ct-Mannosidase II ct-Mannosidase, pH4.0 Hexosaminidase: membrane-bound soluble Na +, K+-ATPase Lactate dehydrogenase
Day 0t
Specific activity* Day 5 Day 10
5.48 0.453 1,52 1.11 0.171 10.4 6.67 55.0 1760
5.89 1.31 3.09 1.96 0.193 44.5 7.82 76.3 2500
6.11 0.90 3.53 3.33 0.760 12 I 68.4 119 2540
*Expressed as in Table 1. tDays in culture.
two important enzymes not associated with glycoprotein metabolism were also assayed: Na +, K +ATPase (a glycoprotein of the plasma membrane) and lactate dehydrogenase (LDH, a non-glycoprotein of the cytosol). The ATPase was considerably more active in the adult tissue (Table 1), the LDH somewhat more so. It was of interest to follow the time-course of enzyme activity in foetal cerebellar explants maintained in culture, since genetically programmed changes in glycoprotein expression might be reflected in the activity of glycoprotein-metabolising enzymes. The results are presented in Table 2. In most cases, there was a gradual increase in specific activity as a function of time in culture. This rise was especially marked for some of the lysosomal enzymes, particularly hexosaminidase, which had increased 10-fold by day 10 in culture. However, the time-course of the increase in specific activity varied from one lysosomal enzyme to another (compare ~-glucosidase, pH 4.0 with ~t-mannosidase, pH 4.0). For the two enzymes not connected with glycoprotein metabolism (Na ÷, K+-ATPase and LDH), the specific activity increased with time in culture towards the corresponding adult value. Lectin affinity chromatography and SDS-PAGE were exploited in order to determine the glycoprotein composition of membrane and soluble fractions from both foetal and adult mouse cerebellum. Results of a typical experiment are depicted in Fig. 1. The lectin used (concanavalin A) has an affinity for ~t-linked residues of D-glucose and D-mannose. It would thus be expected to bind the high-mannose type of Nlinked glycoproteins, as well as most of the complex type and hybrid type (Osawa and Tsuji, 1987); Olinked glycoproteins and non-glycoproteins would not bind. The results (Fig. 1) suggest: (a) foetal and adult mouse cerebellum share many N-linked glycoproteins in common; (b) some such glycoproteins are found exclusively in the foetal tissue, and some only in the adult tissue. For example, a membrane-bound glycoprotein with an apparent molecular mass of about 64,000 is associated only with adult cerebellum, whereas a soluble polypeptide of about 60,000 is present only in foetal cerebellum. Glycoprotein composition was followed in the foetal cerebellum explants as a function of time in culture (Fig. 2). In some cases, there was little change in the concentration of a given glycoprotein component. For example, a membrane-bound polypeptide of mol. wt approximately 85,000 was present at about
Glycoprotein in the cerebellum
857
sion electron microscopy (Fig. 3). After 12 days in culture, explants appeared pale, with bundles of nerve fibres and a dense mat of heterogeneous cells extending from them. These fibres developed initially from undifferentiated cell types, which transformed into neuron-like cells with processes resembling dendrites and axons. Numerous glial cells and some macrophages were also apparent (Fig. 3a). The electron microscope provided images of well-differentiated neurons having the usual complement of subcellular organelles (Fig. 3b). The number of synaptic contacts increased with time in culture (results not shown). DISCUSSION
We have exploited the ability to culture explants of mammalian nervous tissue in order to address the significance of glycoprotein expression in neuronal development. The system we have chosen is foetal mouse cerebellum, which displays certain advantages from the point of view of biochemical analysis. This part of the brain comprises a relatively small number of morphologically distinguishable cell types; and it undergoes a phase of rapid maturation (cell proliferation and specific synapse formation) in the early neonatal period. Thus, one might expect to see genetically programmed changes in glycoprotein composition as cerebellar explants from 18-day mouse embryos are maintained in culture for a further 10-12 days. In exploiting this system, we have had to bear in mind some inherent constraints. Firstly, the artificial nature of the culture conditions may induce changes in cellular composition and/or gene expression. (However, in similar systems, organotypic development is evident; Bird, 1986). Secondly, Fig. 1. Glycoprotein composition of membrane and soluble the very small amounts of tissue available have fractions from foetal and adult mouse cerebellum. Glycoprotein composition was assessed by SDS-PAGE of those necessitated the use of analytical methods of extreme proteins which bound to concanavalin A. The samples were sensitivity. For example, we have optimized the tech(from left to right): membrane fraction from adult cerebel- niques necessary for: homogenizing small tissue lum; membrane fraction from foetal cerebellum; soluble samples (2-5rag wet wt); measuring microgram fraction from adult cerebellum; soluble fraction from foetal quantities of protein; the sensitive fluorimetric assay cerebellum; protein standards (with molecular weight): of glycosidase activity; and determining the glycomyosin (200,000), fl-galactosidase (116,000), phosphorylase protein composition of microgram samples of b (97,000), bovine serum albumin (66,000), ovalbumin protein. These biochemical approaches have been (43,000), and carbonic anhydrase (30,000). (The band at about 30,000 in some lanes is an artifact generated by release complemented by those of light microscopy and of concanavalin A subunits from the agarose-bound lectin). electron microscopy, in order to visualize the appearance of the cultured explants at both the gross and the subcellular level. the same level in the non-cultured tissue (day 0) as at At day 18 of gestation, mouse cerebellum has day 5 and day 11 in culture. Conversely, some active systems for synthesizing and degrading Nglycoprotein components were present preferentially linked glycoproteins (Fig. 1 and Table 1). The lysosoat day 0 or day 5 or day 11. These differences were mal acid hydrolases (glycosidases) are invariably just more marked for the membrane-bound polypeptides as active in the foetal tissue as they are in the adult. than for the soluble ones. From amongst the numer- These activities reflect the cells' ability to degrade the ous examples evident on the original gel (Fig. 2), glycan portion of senescent glycoproteins, both memone can highlight the following: a polypeptide of brane-bound and soluble (von Figura and Hasilik, mol. wt approximately 66,000 present in the mem- 1986). Indeed, lysosomes were evident in electron brane fraction at day 11 but apparently absent at micrographs of neurones from cultured explants day 5 and day 0; a polypeptide of mol. wt approxi- (Fig. 3b). Also apparent in such micrographs were mately 114,000 present at day 0 but apparently well-developed profiles of rough endoplasmic reticumissing at day 5 and day 11; and a polypeptide of lum and Golgi complex, the two major organelles mol. wt approximately 100,000 present at a higher which participate in the biosynthesis of N-linked concentration at day 5 than at either day 0 or day 11. glycoproteins (Kornfeld and Kornfeld, 1985). One of The morphology, cellular composition and ultra- the "trimming" glycosidases involved in this pathway structure of the cultured cerebellar explants, were in the Golgi complex, ~t-mannosidase II, was present revealed by phase-contrast microscopy and transmis- in foetal cerebellum at a level (measured in vitro)
Fig. 2. Time-course of glycoprotein composition of foetal cerebellar explants in culture. Legend as for Fig. 1, except that the samples were (from left to right): protein standards; membrane fraction, day 0; membrane fraction, day 5, membrane fraction, day 11; soluble fraction, day 0; soluble fraction, day 5; soluble fraction, day 11; protein standards.
Fig. 3(a)
Glycoprotein in the cerebellum
859
Fig. 3(b) Fig. 3. Morphology and ultrastructure of cultured explant of foetal mouse cerebellum. (a) Phase-contrast micrograph of explant (E) maintained in culture for 12 days. Bundles of nerve fibres are apparent (large arrows), as well as individual neurites (small arrows). × 800. (b) Electron micrograph of a portion of a neuron in the cultured explant seen in (a). Some of the major organelles are indicated: N, nucleus; RER, rough endoplasmic reticulum; G, Golgi complex; L, lysosome; M, mitochondria, x 50,000.
which was considerably higher than that of the lysosomal isoenzyme (ct-mannosidase, pH 4.0). The ratio of these two activities was closer to unity in the adult tissue; this change presumably reflects the reduced turnover of glycoproteins in the mature organ. The "neutral" ~-glucosidase measured here (Table 1) may be related to the isoenzyme which catalyses the initial step in the processing of N-linked glycoproteins in the rough endoplasmic reticulum. The expression of an individual glycoprotein species will depend on the relative rates of its biosynthesis (presumably genetically determined) and its degradation (possibly non-specific in nature). Our experimental approach only reveals the major glycoprotein moieties (Fig. 1). However, even at this rather gross level, it is clear that there are differences between the glycoprotein composition of foetal and adult mouse cerebellum. Our particular interest lies in the glycoprotein components of the neuronal plasma membrane and of the extracellular matrix. Further analysis will demand techniques of greater resolution
(such as subcellular fractionation and two-dimensional electrophoresis) or of greater discrimination (such as immunochemical methods). Since SDS-PAGE separates polypeptides only on the basis of their apparent molecular mass, we have insufficient information to assign an identity to individual bands on the gels. As the foetal cerebellar explants are maintained in culture, the enzymes of glycoprotein metabolism increase substantially in activity (Table 2), and the glycoprotein pattern changes somewhat (Fig. 2). Such changes may reflect genetically programmed development of the cerebellum in the early neonatal period, which corresponds approximately to days 4-10 in culture for the explants. Similar changes have been observed before (Zanetta et al., 1978, 1980), but these earlier studies did not take into account the existence of isoenzymes of the gtycosidases involved in glycoprotein metabolism. For example, we have taken care to differentiate the ~t-mannosidase active in the processing of nascent glycoproteins (by measuring the
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MARKCARROLLand MARGARETM. BIRD
membrane-bound enzyme at pH 5.5) from the isoenzyme active in their lysosomal degradation (by assaying the soluble enzyme at pH 4.0). The generalized increase in acid hydrolase activity cannot be explained simply by proliferation of macrophages in the cultured explants. Such cells represented a tiny fraction of the total; furthermore, the time-course for the increase in activity was not uniform for all the enzymes studied (Table 2). The results of the lectin affinity chromatography/SDS-PAGE analysis should be interpreted with some caution. Appearance of a glycoprotein component may reflect: (a) genetically determined switching on of its biosynthesis; or (b) glycosylation of a previously non-glycosylated polypeptide; or (c) a non-specific effect (experimental artefact). Nevertheless, there appears to be strong evidence for the developmental regulation of glycoprotein expression in mouse cerebellum. In culture, the cerebellum explants extend growth cones of neurites, which become consolidated into bundles of nerve fibres (Fig. 3a). Glial cells also proliferate. We have not yet determined the precise nature of any changes in the cellular composition of the cultured explants. Furthermore, the extracellular matrix in vitro would certainly differ from that in vivo. Such modifications would probably be reflected in the activity of enzymes involved in glycoprotein metabolism, and in the glycoprotein pattern (particularly in the soluble fraction). The results of our biochemical analysis should therefore be interpreted with care. Nevertheless, the cells of the cultured explants remain viable (up to day 12 at least), and they make synaptic contact with their neighbours. We do not know if such synapses are physiologically active, but it is clear that the components required to establish and maintain such contacts are expressed in the appropriate subcellular location. Cell-surface glycoproteins are likely to be involved (Dodd and Jessell, 1988). In conclusion, we have developed methods for the analysis of glycoprotein expression in small samples of foetal cerebellum maintained in culture for up to 12 days. This system should be useful for studying the effect of inhibitors of the N-linked glycosylation pathway in neuronal tissue. SUMMARY Numerous glycoprotein components were present in the cerebellum of both adult and foetal mouse, whereas a few glycoproteins were present predominantly in either the adult tissue or the foetal tissue. Both tissues had active systems for the biosynthesis and degradation of N-linked glycoproteins. Glycoprotein composition varied in cultured explants of foetal mouse cerebellum, dependent on the time in culture. In general, the glycoprotein-metabolizing enzymes increased in their specific activity as a function of time in culture.
Glycoprotein expression in foetal mouse cerebellum is compared with that in the adult tissue. Acknowledgement--We thank Miss Barbara Stevenson for
expert technical assistance with the electron microscopy.
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