Characterization of gp 50, a major glycoprotein present in rat brain synaptic membranes, with a monoclonal antibody

Brain Research, 408 (1987) 65-78 Elsevier

65

BRE 12476

Characterization of gp 50, a major glycoprotein present in rat brain synaptic membranes, with a monoclonal antibody Philip W. Beesley!, Toni Paladino 1, Claude Gravel 2, Richard A. Hawkes 2 and James W. Gurd 1 1Department of Biochemistry, Scarborough College, Universityof Toronto, WestHill, Ont. (Canada) and 2Departmentof Biochemistry and Laboratory of Neurobiology, Laval University, Que. (Canada) (Accepted 19 August 1986)

Key words: Synaptic membrane; Glycoprotein; Monoclonal antibody

Several cell lines secreting monoclonal antibodies (Mabs) against a major forebrain synaptic membrane (SM) glycoprotein, gp 50, have been raised. Western blots show that the Mabs react with a polypeptide doublet of Mrs 49 and 45 kDa. These polypeptides exist solely in a concanavalin A (Con A) binding form. Removal of the Con A receptors by digestion with endo-fl-N-acetylglucosaminidase H (endo H) lowers the Mrs of the glycoprotein doublet to 36.5 and 34 kDa. Western blots of 2D polyacrylamide gels indicate that gp 50 exists in several isoforms. Solid phase radioimmunoassay (RIA) and Western blots of brain subcellular fractions show the antigenic material to be concentrated in the SM fraction, but to be present in much lower amounts in synaptic junctions and postsynaptic densities. Gp 50 appears to be brain specific. Regional distribution studies show that it is present in all brain regions but is two-fold concentrated in cerebellum, brainstem and midbrain compared to forebrain. Immunocytochemical studies of several brain regions show that gp 50-like immunoreactivity is neuron specific and is concentrated in selected neuronal species, particularly granule cells. In both cerebellar and hippocampal granule cells gp 50-like immunoreactivity is localized in the perikarya and primary dendrites. Though immunocytochemistry did not show staining of synaptic regions this may be due to masking of the reactive epitope. The results are discussed in terms of the molecular properties of gp 50 and its subcellular localization in brain tissue. INTRODUCTION Much current interest has focused on m e m b r a n e glycoproteins and their proposed role in the formation and maintenance of specific neuronal and synaptic connections (see refs. 1, 3, 14, 15, 20). Histochemical techniques have shown that concanavalin A (Con A) binding glycoproteins are enriched in the synaptic cleft and are particularly associated with the junctional complex 2.41. The Con A binding moieties are present in both isolated synaptic m e m b r a n e (SM) and synaptic junction (S J) fractions 9. Binding of [125I]Con A to sodium dodecyl sulphate (SDS) polyacrylamide gels of SM show that they contain 13-14 major Con A binding glycoproteins ranging in M r from greater than 200 to 30 k D a 16,2t,34. Of these, 4 high molecular weight species (Mrs close to 180, 145,

130 and 110 kDa) are specifically localized in the synaptic apparatus being enriched in both SJ and postsynaptic density (PSD)fractions 16,22,23,34. Several studies have been carried out to further characterize and elucidate the function of these glycoproteins. Developmental changes in both the level and biosynthesis of SM and SJ Con A binding glycoproteins have been studied using both sugar and amino acid precursors 16,35,48. The sugar composition of the oligosaccharides and their processing have also been investigated 13,17,27,51. More recently phosphorylation of SJ and postsynaptic density glycoproteins have been described 24-26. It seems likely that several of these glycoprotein species play a key role in neuronal cell surface or synaptic function, but no direct knowledge of this is yet available. Furthermore, these glycoproteins remain

Correspondence: P. Beesley. Present address: Department of Biochemistry, Royal Holloway and Bedford New College, Egham, Surrey TW20 OEX, U.K. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

66 poorly characterized and many important questions about them remain unanswered including their tissue specificity, cellular and subcellular localization and molecular properties. To answer these and related questions we have attempted to raise monoclonal antibodies (Mabs) which bind specifically to SM and SJ glycoproteins. The present paper gives new information on a major SM glycoprotein of Mr close to 50 kDa, gp 50, obtained using a highly specific Mab, SM gp 50. Although gp 50 has been previously identified by [125I]Con A overlay of SDS gels 16very little specific information has been available concerning this glycoprotein prior to the present study, MATERIALS AND METHODS

Preparation of subcellular fractions Synaptic membranes were prepared from 28-30day-old Wistar rats by a modification of the procedure of Cotman and Taylor s as previously described 13. Following sucrose density centrifugation of the P2 pellet SMs were harvested from the 1.0/1.2 M sucrose interface. The mitochondrial and microsoreal fractions were prepared as previously described 29. The cell soluble fraction corresponded to the postmicrosomal supernatant. Synaptosomes were prepared as described by Gurd et al.29. Synaptic junctions 8 and postsynaptic densities 2s were prepared as described. All fractions were resuspended in 0.32 M sucrose and stored a t - 2 0 °C.

Isolation ofglycoproteins Freshly prepared SMs (15-20 mg protein) were solubilized in a final concentration of 1% SDS (w/v) containing 4 M urea and 10 mM fl-mercaptoethanol by heating at 100 °C for 5 min and then dialyzed against 3 changes of 0.1 M sodium phosphate buffer, pH 7.0 containing 0.1% (w/v) SDS (column buffer), The sample was applied to a column of Con A-agarose (5 ml packed volume). Material which did not bind to the column (Con A-negative fraction) was eluted in column buffer (2 x 12 ml) and the column washed with a further 80 ml of buffer. Glycoproteins which bound to the column (Con A-positive fraction) were eluted in column buffer (2 x 12 ml) containing 5% (w/v) a-methyl-D-mannopyranoside as described by Gurd 23. The Con A-positive and Con A-negative material was allowed to precipitate overnight at

-20 °C following the addition of 75 ul glacial acetic acid and 24 ml methanol to each fraction. The precipitate was pelleted by centrifugation at 6il.~!0{Ig for 30 rain.

Digestion of SM with neuraminidase and endoglycosidase H Prior to neuraminidase digestion, SMs were centrifuged at 48,000 g for 10 rain and the pellet resuspended in 5 ml 10 mM Tris-HCl pH 7.6. The pellet was washed two times in Tris-HCl buffer and resuspended in 2 ml of the Tris buffer containing neuraminidase (Sigma Chemicals, type V). Four x 1(I-2 units of enzyme activity were added/5 mg SM and digestion carried out at 37 °C for 3{) min. The SMs were pelleted prior to further use. Preliminary experiments established that neuraminidase removed sialic acid residues from SM glycoproteins under these conditions. For treatment with endoglycosidase H SMs were solubilized in 0.1 M citrate-phosphate buffer pH 5.5 containing 1% SDS (w/v). Ten milliunits of endo-H (Miles Laboratories Inc.) were added for 2 mg SM and the digestion allowed to proceed for 16 h at 37 °C.

Raising and screening of Mabs BALB/c mice (Jackson Laboratories) were immunized with 200-300 ktg each of SM Con A-positive material administered intraperitoneally in Freund's complete adjuvant. The mice were immunized a further 3 times at 14-day intervals but with Freund's incomplete adjuvant as vehicle. Three days after the fihal immunization the spleen was removed and the cells fused with Sp2/0 myeloma cells at a ratio of 3:1 essentially as described by Galfre and Milstein Is. The resulting hybrids were grown in 96 well culture plates over a feeder layer of 5 × 105 splenocytes. Of the 240 wells plated out most (>90%) showed positive growth of clones. The cells were maintained at 37 °C in 93% air/7% CO2 and fed with fresh H A T medium at 7-day intervals. Hybridoma supernatants were tested for antibody activity when the culture medium yellowed (usually 14-21 days after fusion). Positive clones were subsequently grown up in 24 well Linbro plates (Gibco Inc.) and culture flasks. Cell lines of interest were cloned by limiting dilution. Hybridoma supernatants were screened for Mab

67 production by a dot binding assay 31 using a Bio-dot filtration apparatus (Bio-Rad Laboratories). Mab binding to 1 pg of SM or SM Con A-positive fractions spotted on nitrocellulose was detected with Vectastain reagents (Vector Laboratories) comprising biotinylated horse anti-mouse IgG and a complex of avidin-biotinylated peroxidase (Vectastain ABC reagent). Color was developed with 4-chloro-l-naphthol and positive clones identified by visual inspection.

Gel electrophoresis and Western blotting Both 1-D SDS polyacrylamide gel electrophoresis and Con A overlay procedures were carried out as previously described e3. Two-dimensional gels were prepared and run by the method of O'Farrel144. Western blotting was essentially as described by Towbin et al. 52. The blotting buffer comprised gel reservoir buffer containing 20% methanol (v/v). Electrophoretic transfer was carried out at 20-V constant voltage for 17 h. Following reaction with antibody the nitrocellulose strips were developed using the Vectastain ABC reagent kit. Total blotted protein was stained with India ink as described by Hancock and Tsang 3°. In some experiments gels and Western blots were treated with [125I]Con A for the identification of glycoproteins21.

Solidphase radioimmunoassay Solid phase radioimmunoassay was carried out by binding antigen to 96 well Titertek plates (Flow Laboratories). Samples were solubilized as for gel electrophoresis and diluted in phosphate-buffered saline (PBS) such that the SDS concentration was reduced to 0.005% (w/v). Fifty-microliter aliquots of appropriately diluted samples (containing up to a maximum of 1.2/ag of protein) were applied to each well and the liquid evaporated under a cool stream of air. Dried plates were washed 4 times with PBS containing 0.05% (v/v) Tween-20. A primary blocking step of 200/A 1% (w/v) bovine serum albumin in PBS was applied to each well for 30 min followed by 100/~1 hybridoma supernatant (diluted 5 times in blocking solution) for 2 h. The plates were washed 4 times with PBS/Tween, a secondary blocking step applied and then 100 pl of [125I]sheep F(ab')2 anti-mouse Ig (New England Nuclear) containing 4 × 105 cpm added to each well for 2 h. Plates were washed 4

times with 0.05% Tween (v/v) in PBS followed by a further four 10-min washes with 0.1% Triton X-100 (v/v) in PBS. Each well was cut from the plate and radioactivity determined using a Beckman gamma counter. Four separate concentrations of each test fraction (up to 1.2 pg well) were assayed, each concentration being assayed in triplicate. For control wells blocking agent was added to each triplicate concentration series. Binding curves were constructed by subtracting the appropriate control values from each sample value and plotting cpm bound against antigen concentration. The binding curves were linear under the conditions described and cpm bound/ pg sample was determined from the slope of the curve.

Immunocytochemicalmethods For immunocytochemistry Lewis rats were anesthetized with sodium pentobarbital and then fixed by transcardiac perfusion with 4% paraformaldehyde, 0.2% glutaraldehyde in 0.l M phosphate buffer, pH 7.4 followed by overnight fixation by immersion without the glutaraldehyde. The brain was stored in phosphate buffer plus 10 mM sodium azide until sectioning (up to 4 days later). Sections were cut at 50 /~m with a freezing stage microtome and were incubated in Mab SMgp 50 overnight at room temperature on a rocker table. SMgp 50 was used directly from spent culture medium and diluted as necessary into 10% normal horse serum in phosphate buffer. Antibody binding was detected with a second incubation for 2 h in rabbit anti-mouse immunoglobulin conjugated to horseradish peroxidase (Cedarlane Inc.). For light microscopy peroxidase activity was revealed with a 15-min incubation in 0.5 mg/ml 4chloro-l-naphthol, 0.01% v/v hydrogen peroxide in 0.1 M NaCI, 20 mM Tris-HCl, pH 7.5. For electron microscopy, diaminobenzidine was used as chromogen. Sections were washed for 15 min in 3 changes of buffer between incubations. When the Mab was replaced by either myeloma-conditioned medium or irrelevant mouse immunoglobulins, there was no specific staining. RESULTS

Identification and preliminary characterization of the gp 50 antigen

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Fig. 1. A: Coomassie blue-stained 8% SDS polyacrylamide gel of SM Con A-positive material. The gp 50 band is prominently stained. B, C: localization of gp 50 in SM Con A-positive material. Samples of SM on A-negative material (lane 1), Con Apositive material (lane 2) and total SM (lane 3) were immunodeveloped against SMgp 50 (B) or India ink stained (C) as described in the text. Seventy-microgram samples of SM and SM Con A-negative fractions were separated on an 8% SDS polyacrylamide gel prior to blotting. SM Con A-positive material was estimated to be equivalent in loading to 70/~g of Con Anegative material. The major Con A-positive glycoprotein present in SM fractions as determined by the Con A overlay procedure is a diffuse band with an apparent M~ of close to 50 kDa and designated gp 50 (ref. 16). Coomassie blue staining of polyacrylamide gels of SM Con A-positive material shows that this band is the most densely staining component and resolves into a doublet of apparent M~s 49 and 45 kDa, respectively (Fig. 1A). Several hybridoma clones secreting Mabs that react with this glycoprotein doublet have been identified by immunodevelopment of Western blots of total SM, and SM Con A-positive and -negative material. A typical blot for the clone with which we have mainly worked, SMgp 50 is shown in Fig. lB. The two immunoreactive bands present in both SM and SM Con A-positive fractions (lanes 2 and 3) have apparent M~s of 49 and 45 kDa and correspond to a prominent polypeptide doublet observed on an India ink-stained blot of SM Con A-positive material (Fig. 1C, lane 2). Though the M~ of this doublet varies slightly between gels it will be referred to as gp 50 in the present paper. In order to determine whether gp 50 exists solely in a Con A binding form Western blot analysis of SM Con A-positive and Con A-negative fractions was

carried out (Fig. 1B). The gp 50 doublet was detected only in the Con A-positive fraction (lane 2). 1his resuIt was confirmed by immtmoblotting of SM proteins which had been digested with endo t4. This enzyme cleaves only N-linked high mannose and hybrid, but not complex oligosaccharides and thus oilgosaccharides resistant to endo H digestion would not react with Con A. The immunoblot of control and endo H digested SM (Fig. 2A) showed that all of the gp 50 doublet is shifted from apparent Mrs of 49 and 45 to 36.5 and 34 kDa, respectively. Thus very close to 25% of the apparent molecular mass of each glycoprotein is attributable to Con A binding oligosaccharides. Further, these results indicate that the difference in M~ of the two polypeptides present in the gp 50 doublet is not attributable solely to differences in the Con A receptors linked to the polypeptide chains. A Con A overlay of a Western blot of control and endo H-treated SM (Fig. 2B) confirmed that the enzyme treatment abolished all Con A binding activity.

Tissue specificity and subcellular distribution of gp 50 Western blots (results not shown) of homogenates from brain, peripheral nerve trunk, skeletal muscle, heart, liver, kidney, spleen and lung showed gp 50 to be detectable only in brain tissue. Thus this glycopro-

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Fig. 3. Quantitation of gp 50 in brain subcellular fractions by solid phase RIA. Typical binding curves for solid phase RIA of forebrain fractions are shown. A A, SM; [] •, light m e m b r a n e fraction; • • , washed (2 x) synaptosomes; O O, homogenate; ! - , SJ.

tein appears to be highly specific to the CNS. The relative amounts of gp 50 present in a series of subcellular fractions isolated from forebrain and cerebellum were estimated by solid phase radioimmunoassay. Typical binding curves for washed synaptosomes, SMs, SJs, homogenate and light membranes are shown in Fig. 3. The enrichment of gp 50 in each fraction relative to forebrain homogenate was determined by dividing the cpm bound//~g protein for the individual fractions by the corresponding value for forebrain homogenate.

The results for forebrain fractions (Table I) clearly showed the glycoprotein to be membrane bound and not present in the soluble fraction. Gp 50 is most concentrated in SMs followed by lesser amounts in light membrane and microsomal fractions. Despite the high enrichment of gp 50 in SMs, only relatively low amounts are present in fractions enriched in postsynaptic structures i.e., SJs and PSDs. Gp 50 is absent from the myelin fraction and is neither substantially enriched nor depleted in the P2 and mitochondrial fractions. Whilst gp 50 is concentrated (1.5-2-fold) in unwashed synaptosomes the degree of enrichment is reduced by twice washing the P2 pellet prior to applying the sample to the Ficoll gradient. This is consistent with gp 50 being a constituent of synaptosomes themselves and also being present on microsomal membranes contaminating the unwashed synaptosome fraction. The cerebellar fractions show essentially the same trends as for the forebrain excepting that this brain region contains close to 2.5 times the forebrain level of gp 50. Immunoblotting of the subcellular fractions was performed to ensure that Mab SMgp 50 only reacts with gp 50 in all brain fractions and to validate the radioimmunoassay results. The results (not shown) confirmed both of these points.

Immunocytochemicallocalization ofgp 50 The cellular and subcellular localization of gp 50

TABLE I

Distribution of gp 50 in forebrain and cerebellar subcellular fractions Subcellular fractions were prepared as described and aliquots assayed by solid phase R I A at 4 separate concentrations, cpm//~g fraction protein was calculated as described in the text. Relative enrichment is the ratio of cpm/~ug fraction protein to cpm/Itg h o m o g e n a t e protein. E n r i c h m e n t of cerebellar and forebrain fractions are each expressed relative to their own h o m o g e n a t e s .

Fraction

Homogenate Cell soluble Pe Microsomes Myelin Mitochondria Unwashed synaptosomes W a s h e d (2 x ) s y n a p t o s o m e s Light m e m b r a n e s SM SJ PSD

Forebrain

Cerebellum

cpm bound/l~g protein

Relative enrichment

cpm bound/l~g protein

Relative enrichment

541 21 503 1370 0 514 931 666 1136 2664 335 471

1.00 0.04 0.93 2.53 0.00 0.95 1.72 1.23 2. l 4.92 0.62 0.87

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e-• Fig. 4. A shows a low power view of the adult rat cerebellar cortex in sagittal section immunoperoxidase-stained with Mab SMgp 50. Reaction product is found primarily in the granule cell layer (GCL) with light staining in the molecular layer (ML) extending from the Purkinje cell layer out to the pia (P). The white matter (WM) is unstained. The scale bar is 250 urn. B shows a higher power view of the cerebellar cortex. Peroxidase reaction product in the granule cell layer resolves to sharply defined deposits at the perimeter of the granule cells. The synaptic glomeruli are unstained. In the Purkinje cell layer (PCL) the somata of the Purkinje cells are encircled by dense reaction product. In the molecular layer, reaction product is distributed amorphously throughout with some highlighting of fibrous elements which extend radially from the Purkinje cell layer to the pia. Staining of the Bergmann glia is non-specific. The scale bar indicates 100/~m.

Fig. 5. Frontal section through the adult rat cerebral cortex lrnmunoperoxidase stained with SMgp 50. Stained neuronal profiles are found throughout all cortical layers (I-V1) with rcaction product in both the cell bodies and the dendrites (e.g. the pyramidal neurons of layer V). The underlying white matter axon tract (WM) is not immunoreactivc and there is no cvidence of specifically synaptic reaction product. The apparent high density of staining in layer I is artefactual. The scale bar is 2l)O~m.

71 has been studied immunocytochemically at both the light and electron microscope levels in the cerebellum and at the light microscope level in cerebral cortex, hippocampus, striate cortex and A m m o n s horn. Localization of gp 50-like immunoreactivity in the adult cerebellar cortex by light .microscopy gives a characteristic and highly reproducible pattern of staining (Fig. 4). Reaction product is deposited primarily in the granular layer where it sharply outlines the nuclei of the granule cells (Fig. 4B). There is no prominent staining of axons, either in the granular layer or in the white matter tracts. The synaptic glomeruli, the Golgi cells and the Lugaro cells are not immunoreactive. There is no staining of fibrous astrocytes or of oligodendrocytes. In the Purkinje cell layer, reaction product is especially prominent around the base of the Purkinje cell somata but does not extend beyond the axon hillock and there is no immunoreactivity in the basket cell axon pinceaux. The Purkinje cells themselves appear to be unstained. A similar highlighting of the Purkinje cell periphery is also often seen around the primary dendrites. Other than this, there is little or no immunoreactivity in the molecular layer: the parallel fibers in the deeper regions may contain a little reaction product but there is no staining of basket or stellate cells and the Bergmann glia are unreactive, The distribution of r e a c t i o n product as s e e n by light microscopy does not suggest a concentration of gp 50-like immunoreactivity in synaptic structures. Specifically, the two most conspicuous classes of synapses, between granule cell axons and Purkinje cell dendritic spines on the one hand and granule cell dendrites and mossy fiber terminals on the other, are non-reactive. The pattern of gp 50-like immunoreactivity in other regions of the rat brain is consistent with this interpretation, In the cerebral cortex, stained neuronal profiles are found in all laminae (Fig. 5) with reaction product deposited both in the cell somata and in the dendrites. There is no immunoreactivity associated with the synaptic terminals, the axons or the non-neuronal cells. Likewise, in the hippocampus the staining is also exclusively neuronal (Fig. 6). Reaction product is confined primarily to the intensely stained neuron somata of the granular layer but immunoreactive neuronal profiles are also readily found in the molecular layer and the hilus. The neuronal specificity is

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Fig. 6. The distribution of Mab SMgp 50 immunoreactivity in

the adult rat hippocampus. Frontal sections show that immunoreactivity is confined primarily to the granular layer (GL) but that stained neuronal profiles are found in both the molecular layer (ML) and the hilus (H). Reaction product is associated with neuronal profiles in all cases. Scale bar is 150pm in A and 50/~m in B.

underlined in Fig. 7 which shows immunoreactive somata and dendrites in the striate cortex (Fig. 7A) and A m m o n s horn (Fig. 7B): the synaptic terminal regions are not marked. Some immunoperoxidase-stained sections of cerebellum, were e m b e d d e d and resectioned for electron microscopy (Fig. 8). The ultrastructural studies confirmed and extended the observations made by light microscopy. The reaction product seen in the granular layer by light microscopy is confined exclusively to the granule cells (Fig. 8A). The cytoplasm surrounding the nucleus, the dendrites and portions of the ascending axons are all immunoreactive. There is no staining of adjacent vellate glial processes or gliai somata. Reaction product in the granule cell dendrites does not extend far into the synaptic glomeruli, which are generally not immunoreactive, and no-

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where is gp 50-like immunoreactivity concentrated in synaptic regions (Fig. 8C). Likewise, the presynaptic mossy fiber terminals are without reaction product. The nuclei of the granule cells are not stained. Within the granule cell cytoplasm, reaction product is associated primarily with microtubular profiles but this may well reflect an adventitious association due to fixation of the antigen or diffusion of the diaminobenzidine reaction product. In the Purkinje cell layer there are numerous, small, immunoreactive profiles (Fig. 8B) close by but not apposed to the Purkinje cells themselves, which resemble a subset of the parallel fiber axons: the cells of origin of these fibers have not been identified. The basket cell axons and synaptic terminals are not immunoreactive, neither is the glial sheath which surrounds the Purkinje cell body and dendrites. The Purkinje cells themselves are never immunoreactive. As in the granular layer, there is no reaction product associated with synaptic profiles. In an attempt to expose additional antigenic sites and perhaps reveal staining of synaptic structures tissues were treated with O. 1% (v/v) Triton X-I(IO prior to immunodevelopment. The detergent treatment reduced the general quality of the tissue sections but did not alter the staining pattern. Postembedding staining was also tried but resulted in a complete loss of gp 50-like immunoreactivity.

Two-dimensional polyacrylamide gel analysis of gp

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The apparent discrepancy between the localization of gp 50 in SMs and synaptic structures by biochemical methods and the non-synaptic immunocytochemical staining made it essential to confirm that Mab SMgp 50 was reacting with gp 50 and not a minor comigrating contaminant. The most conclusive way to show this was by comparing an immunoblot of gp 50 with the Con A binding pattern of SM proteins

Fig. 7. Mab SMgp 50 immunoreactive neurons in the rat brain. A: a frontal section through area 18 of the adult rat striate cortex immunoperoxidase stained with Mab SMgp 50 showing reaction product deposited in large neurons of layers III and IV. Scale bar is 50/tm. B: CA l region of Ammons horn shows an intensely immunoreactive layer of small neurons in the straturn pyramidale (P) with scattered stained cell bodies in the stratum oriens (O) and reaction product in dendritiC profiles in the stratum radiatum (R). Scale bar is 50,urn

73

Fig. 8. Electromicrographs from rat cerebellar cortex immunoperoxidase stained with Mab SMgp 50. A shows a cluster of granule cells. There is no reaction product in the prominent nuclei (N) but deposits are scattered throughout the thin layer of cytoplasm (e.g. arrowheads). B shows a region of molecular layer near the Purkinje cell layer. Arrowheads indicate deposits of reaction product in parallel fiber axons. Neighboring presynaptic profiles (indicated by the arrows) are never immunostained. C shows a detail from a synaptic glomerulus in the granule cell layer. Reaction product is found in the granule cell dendrites (arrowheads) but never in the presynaptic mossy fiber terminals (arrows). The scale bar is 1.0 ~m in each case.

74

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separated on 2D-polyacrylamide gels. Comparison of the Con A overlay with the Coomassie blue staining pattern of the 2D gels (Fig. 9A and C, respectively) show that whilst the SM polypeptides are well resolved the glycoprotein species are more diffuse consistent with heterogeneity of their carbohydrate moieties. The immunoblot (Fig. 9B) shows that the species recognized by Mab SMgp 50 ran as a diffuse band between p H 3.9 and 4.9. Much of the immunoreactive material did not enter the pH gradient but a major antigenic determinant also focused at a pH close to 4.7. The species recognized by SMgp 50 cor-

responded to the large majority of the Con A binding material observed in this gel region (Fig. 9A). Two additional minor Con A binding spots of M r 4 5 - 5 0 kDa were also observed which were not detected with the antibody.

Regional distribution ofgp 50 The distribution of gp 50 in a series of rat brain regions was studied by solid phase radioimmunoassay. The results (Table II) clearly showed a marked regional variation in the distribution of gp 50. The highest concentration was localized in the cerebellum, con-

75 TABLE II

since neuraminidase treatment of samples prior to

Regionaldistribution ofgp 50in rat brain

1D and 2D polyacrylamide gel electrophoresis did not abolish the presence of the doublet or substantial-

Homogenate samples of the above brain regions were assayed by solid phase RIA as described in the text. Enrichment of gp 50 in each brain region was measured as the ratio of cpm bound/pg region homogenate protein to cpm bound/pg parietal cortex homogenate protein, Brain region

cpm bound/pg protein

Enrichment relative to parietal cortex

Parietal cortex Entorhinal cortex Occipital cortex Frontal cortex Hippocampus Striatum Thalamus Hypothalamus Cerebellum Midbrain Brainstem

687 937 895 770 666 604 937 979 1666 1583 1562

1.00 1.36 1.30 1.12 0.96 0.88 1.36 1.42 2.42 2.30 2.27

sistent with the immunocytochemical localization of gp 50 in cerebellar granule cells. The midbrain and brainstem regions contained comparable amounts of gp 50 to cerebellum. All other brain regions examined possessed considerably lower amounts of gp 50. There appeared to be some variation in the level of the glycoprotein in the 4 cortical regions examined, The lowest level of the antigen was present in the striatum. The general trend of the results was to indicare that the highest levels of gp 50 were present in the evolutionary older brain regions. DISCUSSION In the present paper we have described a Mab with a high degree of specificity for a major SM Con A binding glycoprotein. Although others have reported Mabs that react strongly with brain Con A binding material these Mabs have low specificity, binding to many SM peptides 11. Mab SMgp 50 has been used to show that gp 50, quantitatively the major SM glycoprotein, is composed of at least two immunologically related polypeptides of M r 49 and 45 kDa. Two-dimensional gel blots suggest that there may be additional isoforms of gp 50. Different molecular forms of some glycoproteins, such as the embryonic and adult forms of NCAM, can be largely attributed to difference in their degree of sialylation 47. This is not the case for gp 50

ly alter the M r of each species. The difference between the two forms cannot be attributed solely to a difference in the number of Con A receptors bound to each polypeptide since the doublet is not abolished by endo H treatment, though this does alter the Mrs of both species by 11-13 kDa. SM Con A binding oligosaccharides comprise on average 7 - 8 mannose residues and two N-acetylglucosamine residues thus having a M r of approximately 1500 Da 51. This suggests that each form of gp 50 contains 6 - 7 Con A receptors. Although we have not ascertained whether gp 50 contains any oligosaccharides which do not react with Con A , SMgp 50 binding to gp 50 is not abolished by endo H or neuraminidase treatment suggesting that the epitope is located on the polypeptide portion of the molecule. Furthermore, the results clearly show that all forms of gp 50 detected by SMgp 50 contain Con A binding moieties. The biochemical data on tissue specificity suggests that gp 50 is a brain-specific glycoprotein and may therefore play an important role in CNS function. Indeed the immunocytochemical data suggests an even more specific localization of gp 50-like immunoreactivity, predominantly to the perikarya and primary dendrites of granule cells in the cerebellum and hippocampus and pyramidal cells in the cerebral cortex. It is of interest that the granule cells in cerebellum and hippocampus both contain high amounts of gp 50-like immunoreactivity. The other prominently staining area in cerebellum, the small fibers adjacent to the Purkinje cells, is difficult to identify. These structures may be granule cell processes but if so have an atypical spatial arrangement. This might be caused by distortion of the granule cell processes as they migrate past Purkinje cell bodies thus giving rise to an array of fibers which would outline the Purkinje cell perikarya. Staining of microtubule-like profiles in the granule cell cytoplasm is likely to be artefactual. The majority of microtubules are cold-labile and as the tissue samples for biochemical experiments were all prepared at 4 °C it seems likely that any gp 50 associated with these structures would also become soluble, as is the case with MAPs 5°. Our data, however, indicates that gp 50 does not occur in the soluble fraction. Fur-

76 thermore, gp 50 is not present in an isolated cytoskeletal preparation enriched in tubulin and actin (results not shown). Others have provided evidence for some non-specific localization of the immunoperoxidase reaction products. Staining of synapsin I within synaptosomes using immunoferritin specifically localizes the molecule to synaptic vesicles whereas immunoperoxidase staining showed a more widespread distribution of reaction product to include membranes close to the vesicles 12. Furthermore, immunoperoxidase staining for clathrin 4, a 65-kDa synaptic vesicle membrane protein 3s and tyrosine hydroxylase a~ all gave similar patterns despite the differing subcellular localization of these proteins. It has been suggested that this is due to accumulation of peroxidase reaction product in the cytoplasmic space followed by its non-specific adsorption onto any nearby particulate structure ~2. Several other Mabs recognizing antigens which are either specific to or highly concentrated in cerebellar granule cells have been reported. The Mab anti-BSP3 reacts with a cerebellar glycoprotein of M r 48 kDa 32. This Mab, in contrast to anti-gp 50, stains both astrocytes and neurons including granule cells and Purkinje cells. Furthermore anti-BSP-3 is only reported as reacting with a single polypeptide band rather than a doublet, Ghandour et al. 19 have reported 3 Mabs which heavily stain granule cells, including the molecular layer and the synaptic glomeruli. One of these also stains the periphery of Purkinje cell perikarya. No data on the nature of the antigenic species recognized by these Mabs is given, but the staining pattern does not correspond to that given by SMgp 50 which does not heavily stain either the molecular layer or the cerebellar glomeruli. This group has also reported a granule cell marker which heavily stains parallel fibers in adult cerebellum and also the perikaryal cytoplasm in young animals 36. This Mab binds to antigens of Mr 120 and 185 kDa. A Mab reacting with a triplet of cerebellar glycoproteins, Mr 41, 38 and 36 kDa, has been reported but this Mab predominantly stains large projection cells such as Purkinje cells, rather than granule cells 54. Several Mabs and polyclonal antisera to NgCAM and L1 have been used to show that these molecules are likely to play a role in guiding granule cell migration during development, but clearly the distri-

bution of these antigens is not granule cell specific 37'46, Thus although a number of antibodies reacting with cerebellar proteins and glycoproteins have been reported none appear to have the properties of the anti-SMgp 50 Mabs. The regional distribution of gp 50 is consistent with the immunocytochemical localization of the antigenic species, for example in showing the high concentration of gp 50 in cerebellum. There is, however, a marked discrepancy between the immunocytochemical and biochemical results with respect to the subcellular localization of gp 50. The results obtained by RIA and Western blotting show unequivocally that gp 50 is particulate, being recovered in the P2 and microsomal pellets. The 5-fold enrichment of gp 50 in the SM fraction is similar to reported enrichment values for Na+,K+-ATPase 7,29,33,43, as well as nicotinic and muscarinic acetylcholine receptors and fladrenergic receptors in SM fractions w'a9 and is consistent with the presence of gp 50 in the synaptic region. In addition, the presence of gp 50 in SJ and PSD fractions indicates that it may be an integral component of the junctional complex although the possibility that it may be artefactually absorbed to these structures cannot be ruled out 4°. While these results support a synaptic location gp 50 is also enriched (2.5fold) in the microsomal fraction suggesting that it occurs in more than one subcellular compartment, a conclusion which is consistent with the immunocytochemical results. In contrast to the biochemical data, the immunocytochemical studies show no evidence for gp 50-like immunoreactivity in synaptic structures in any of the brain regions examined. Several possible explanations for this apparent discrepancy may be offered: (1) different epitopes are recognized in fixed tissue sections and solubilized brain samples (see for exampie ref. 42); (2) the occurrence of gp 50 in the SM fraction is due to the presence of contaminating membranes; or (3) the binding of antibody to gp 50 may be blocked or inhibited in the synaptic region in intact tissue. Of these 3 possibilities the first seems to be the least likely. The regional distribution of gp 50 monitored by RIA is quite consistent with the immunocytochemicai data suggesting that the antibody is binding to the same epitope in both cases. The second explanation is plausible. It is well-recognized that SM

77 contains contaminating membranes from a variety of sources, notably axons, dendrites and glia 7'29'33'39'43. If this explanation is correct, then gp 50 present in SM must be derived from m e m b r a n e fragments of granule and pyramidal cell perikarya and dendrites, which the immunocytochemical studies show to be enriched in this glycoprotein. Moreover, as gp 50 is concentrated jn isolated SM this explanation would require that the gp 50-rich granule and pyramidal cell membrane fragments must copurify in the SM fraction. The retention of gp 50 in a washed synaptosome fraction as well as in SJ and PSD fractions which are devoid of membrane and vesicular structures strongly support the contention that a portion of gp 50 is located at the nerve ending, With respect to the third explanation that the epitope may be blocked or masked in the synaptic region it has been noted elsewhere that antigenic determinants may be hidden by interaction with other molecules 53. Although Triton X-100 does not alter the immunocytochemical profile of gp 50, blocking or masking of the epitope is still not precluded since this may be due to a detergent-insensitive process such as covalent linkage. However, it should be noted that preliminary results indicate gp 50 is only detectable in low quantities in isolated SM unless the material is

first treated with SDS. It is also significant in this context that poor immunocytochemical staining of synapsin I in nerve endings in intact tissue sections has been reported 12 and that satisfactory staining of synapsin I in synaptosomes was achieved only when the majority of cytoplasmic proteins were extracted. Thus, whilst it is not clear at present whether a portion of gp 50 is synaptically located or not, the results indicate that gp 50 is a glycoprotein of considerable interest principally because of its specific localization in only certain neuronal types, notably cerebellar granule cells. Mab SMgp 50 has been used to give an initial characterization of the molecule and describe its localization. It is anticipated that this antibody will continue to be a useful tool for probing the structure and function of this nervous system specific glycoprotein.

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ACKNOWLEDGEMENTS P.W.B. gratefully acknowledges the receipt of Anglo-Canadian Exchange Fellowship from the Royal Society and the Canadian National Science and Engineering Research Council. The work was also supported by grants from the Canadian Medical Research Council to J.W.G. and R.H.

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