Human Molecular Genetics, 2002, Vol. 11, No. 23 2941-2950
© 2002 Oxford University Press
New insights into the molecular basis of progressive myoclonus epilepsy: a multiprotein complex with cystatin B
1Department of Biology, Bologna University, 40126 Bologna, Italy, 2Istituto di Citomorfologia N.P., CNR, 40136 Bologna, Italy, and 3Chiron Vaccines, 53100 Siena, Italy
Received July 17, 2002; Accepted September 9, 2002
DDBJ/EMBL/GenBank accession numbers AAN03958, AAN03959
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONCONCLUSIONSMATERIALS AND METHODSREFERENCES |
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Cystatin B is an anti-proteolytic polypeptide implicated in progressive myoclonus epilepsy (EPM1), a degenerative disease of the central nervous system. The knock-out mouse model of the disease shows apoptosis of the cerebellar granule cells. We have identified five recombinant proteins interacting with cystatin B and none of them is a protease. We show that three of these proteins (RACK-1, ß-spectrin and NF-L) co-immunoprecipitate with cystatin B in rat cerebellum. Confocal immunofluorescence analysis shows that the same proteins are present in the granule cells of developing cerebellum, as well as in Purkinje cells of adult rat cerebellum. We propose that a cystatin B multiprotein complex has a specific cerebellar function and that the loss of this function might contribute to the disease in EPM1 patients.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONCONCLUSIONSMATERIALS AND METHODSREFERENCES |
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The progressive myoclonus epilepsy of the Unverricht–Lundborg type (EPM1) is an autosomal recessive disease characterized by progressive myoclonic jerks and decline in cognition. Genetic linkage studies, suggest the involvement of the cystatin B gene, located on human chromosome 21 (1–3). A decreased amount of cystatin B mRNA is a common finding in EPM1 patients and it may be due to: 1) a mutation in the promoter region causing a decrease in the rate of transcription of the gene (1,2); or 2) mutations of the coding region/splice sites that may inhibit translation or diminish the half-life of the transcript and/or of the protein. However, the main molecular damage seems to be the decreased amount/absence of functional cystatin B protein. Pennacchio et al. (4) have shown that the knock-out of the cystatin B gene generates a neurological disorder in mice, characterized by symptoms very similar to those observed in humans, thus providing a good model for the disease. The main cytological alteration described by the authors is the loss of the cerebellar granule cells that show apoptotic bodies, chromatin condensation and other changes typical of programmed cell death. This result shows that cystatin B has an essential role in the cerebellum, protecting granule cells against apoptosis.
Cystatin B belongs to the cystatin superfamily that includes a large number of proteins, originating from an ancestral peptide (5). The main structural features conserved in evolution are the type A and B disulfide loop structures, the proline-tryptophane (PW) sequence and the glutamine–valine–valine–alanine–glycine (QVVAG) sequence, corresponding to the consensus QxVxG. This latter sequence and the glycine at position 4 (6) represent the site where cystatins bind to cathepsin and inhibit its proteolytic function (7,8). In vitro, cystatin B binds to cathepsins B, L and H, the lysosomal cysteine proteases of the papain family (6). Cathepsin B and the related proteins are found in lysosomes (9,10).
The absence of a co-localization of cystatin B with proteases of the cathepsin family in different cell types (11) prompted us to search for different partners of cystatin B using the two hybrid system technique. Five interacting proteins were isolated and, among those, two were present exclusively in the central nervous system (CNS): the neurofilament light chain (NF-L) and brain ß-spectrin. Furthermore, cystatin B interacts with both of them and with RACK-1 (the protein kinase C receptor), specifically in the cerebellum. The other two interacting proteins do not have a known function. The results of the confocal microscopy immunofluorescence analysis show that cystatin B and at least three of the identified proteins are expressed in the granule cells during cerebellar development. In contrast, in the adult cerebellum, cystatin B and its partners are not detectable in granule cells but are expressed in the Purkinje cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONCONCLUSIONSMATERIALS AND METHODSREFERENCES |
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Identification and characterization of cDNAs coding for peptides interacting with cystatin B
By confocal microscopy analysis, Riccio et al. have shown that cystatin B and cathepsin B are concentrated in different cell compartments (11). This observation suggests that, in vivo, cystatin B may have other functions beside inhibiting cysteine proteases. We have therefore used the yeast two-hybrid system (12) to identify protein(s) that interact with cystatin B. The screening in yeast of a cDNA library from mRNA of developing cerebella, generated 220 positive colonies. All recombinant clones were sequenced and the five cDNAs listed in Table 1 were further characterized. Only three of them code for known proteins: RACK-1, NF-L and ß-spectrin. One of the unknown proteins belongs to the myotubularin family and, at AA position 203–379, contains the consensus sequence of the serine/threonine and tyrosine dual-specificity phosphatase. This motif is also found in laforin, the protein mutated in the Lafora disease (EPM2A) (13). However the Lafora gene does not belong to the myotubularin family of proteins.
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Thus, the results indicate the presence of two proteins specific to the nervous system (NS), ß-spectrin and NF-L. Although cathepsin B, H and L mRNAs are expressed in developing cerebellum and present in the cDNA library (not shown), none of the isolated cystatin B partners belongs to a protease family or contains a protease motif.
As granule cells have been causally implicated in EPM1, we have studied in these cells the expression of the proteins interacting with cystatin B. The northern blot analysis of granule cells RNA (Fig. 1A) shows that the mRNAs coding for the cystatin B binding proteins are all expressed in primary granule cells differentiated for 10 days in vitro.
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Additional evidence comes from the immunofluorescence experiment in Figure 1B–J. So far, we have analyzed only the four proteins for which antibodies are commercially available. Primary cerebellar granule cells differentiated in vitro for 10 days were immunostained with antibodies against cystatin B, RACK-1, ß-spectrin and NF-L. The merging of the double staining with the cystatin B antibodies is shown in the H–J panels. The fluorescence intensity obtained with anti-ß-spectrin and anti-NF-L antibodies is weak, compared to that of the RACK-1 and cystatin B antibodies, suggesting that in vitro differentiating granule cells do not contain large amounts of either protein. In general, the cellular distribution of the proteins overlaps only partially, suggesting that a small proportion of the total protein may co-localize and interact. RACK-1 is cytoplasmic and ß-spectrin is also cytoplasmic but with a spread over the nuclear region. Cystatin B, as previously shown, is diffused over the entire cell (11) and NF-L, as expected, is distributed mainly along the neurofilaments. We have compared the results obtained by in vitro differentiation of primary granule cells with those observed in the cerebellum during development. The western blot analysis (Fig. 1K) shows that the cystatin B and RACK-1 proteins are already present in protein extracts of developing cerebellum from 7 day old rats and their levels remain constant up to 90 days (Lanes 1–4). The brain spectrin and neurofilament bands are very faint up to 13 days of differentiation and increase in the adult rat. This is in good agreement with the data obtained in the primary granule cells after 10 days differentiation in vitro (Fig. 1B–J). In fact, these cells are isolated from 7 day old cerebella and differentiated in culture for 10 days.
GST pull down assays
To investigate further the interaction between cystatin B and the partner proteins, we have used the GST pull-down assay. Figure 2A shows the autoradiography of SDS–PAGE of the B42 fusion peptides, labelled with [35S]-methionine, eluted as GST-cystatin B/protein complex by binding to Sepharose–glutathione beads. All peptides examined interact to various degrees with cystatin B. Under the conditions used, only 24% of cathepsin B binds to cystatin B indicating low affinity binding between the two proteins. The same experiment was carried out with the pro-cathepsin protein, giving similar results (not shown). We have also tested the interaction of cystatin B with cathepsin B (both precursor and processed peptides) in yeast. Only pale blue colonies were observed, suggesting a weak interaction. If similar results were to be obtained with the other members of the cathepsin family, we could consider them all excluded by our selection procedure. As already pointed out by Wakamatsu et al. (14) and Zerovnik et al. (15) cystatin B dimerizes, although with low efficiency. The B42 activation domain alone does not show interaction above the background level. To confirm that the fusion with the B42 peptide does not affect the interaction of cystatin B with its partners, we have repeated the GST pull-down experiments using proteins without the B42 transactivating domain. Interaction between cystatin B and the recombinant peptides occurred under these conditions too (Fig. 2B).
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We conclude that cystatin B interacts in vitro with the five peptides isolated with the two-hybrid screening technique.
Interaction between the cystatin B exon fusion products and the recombinant peptides
The structure of the cystatin B gene involves three exons (1), coding for the amino terminal, central and carboxyl terminal regions. In an attempt to characterize further the binding between cystatin B and its partners, we have fused a GST peptide with each exon of the cystatin B gene (Fig. 2C). The GST–exon I fusion peptide does not interact with the recombinant proteins. The GST–exon II and III fusion peptides interact with all five proteins, but all proteins bind preferentially to the GST–exon II fusion peptide. We conclude that cystatin B, in vitro, interacts with the recombinant proteins through the domains encoded by the second and third exon of the gene.
Interactions of cystatin B in the cerebellum
The results of the in vitro binding experiments prompted the analysis of the possible in vivo interactions of cystatin B with the identified proteins. The organ of choice was the cerebellum, which is the main target of the EPM1 disease.
The experiment in Figure 3A shows the western blot of a protein extract from the cerebellum of 10 day old rats, immunoprecipitated from cerebellar protein extract using antibodies against RACK-1 (Lane 2), NF-L (Lane 3), or brain spectrin (Lane 4). Lane 1 contains total cerebellar protein extract. Half of the blot was stained with antibodies against RACK-1, while the other half was stained with antibodies against cystatin B. In Lane 1, a 35 kDa band corresponds to the position of RACK-1 and a fast running 12 kDa band migrates to the position of cystatin B. Following immunoprecipitation, a weak band corresponding to the position of cystatin B and a stronger one corresponding to RACK-1, are also visible (Lanes 2–4). This suggests that each complex obtained by immunoprecipitation with antibodies against RACK-1, NF-L and brain spectrin contains cystatin B and RACK-1. Furthermore, the presence of RACK-1 and cystatin B in anti NF-L and anti ß-spectrin immunoprecipitates strongly suggests that all four proteins are present in the same complex. Unfortunately we could not carry out the immunoreaction directly with the anti-cystatin B antibodies, as they are not suitable for immunoprecipitation.
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Figure 3B shows a western blot of the immunoprecipitate obtained with antibodies against RACK-1 from protein extracts from 10 day old rat cerebellum (Lanes 1 and 2) and brain hemispheres (Lanes 3 and 4). The immunoprecipitated proteins were run on SDS gels, blotted and stained with antibodies against the proteins indicated in the figure. Lane 2 shows immunostained bands for the four different proteins, with the same mobility as in the total cerebellar protein extract (Lane 1). When brain hemisphere cell extract was used, only two of these bands were detectable (Lane 4 versus Lane 3), corresponding to the immunoprecipitated RACK-1 and NF-L. Cystatin B and ß-spectrin, although present in the cell extract of brain hemispheres, are not detectable in the immunoprecipitate.
Figure 3C shows a western blot of the immunoprecipitate obtained with antibodies against ß-actin and against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the same cerebellar extract used in A and B. The western blot analysis shows the presence of the immunoprecipitated specific proteins (Lanes 2 and 4) co-migrating with their control (Lanes 1 and 3) and the absence of cystatin B (Lanes 2 and 4), which is present in the cell extract (Lanes 1 and 3) but is not spuriously trapped by the immunoprecipitation procedure.
The results obtained with cerebellar extracts from 10 day old rats were similar to those obtained from adult rats (not shown).
The experiments in Figure 3 show that NF-L, ß-spectrin, cystatin B and RACK-1 co-immunoprecipitate from a cerebellar cell extract and that the reaction is specific. In fact, in the cell extract of brain hemispheres, the four proteins are present but they do not co-immunoprecipitate, suggesting that the binding is not an artifact due to protein solubilization during the extraction procedure. We conclude that the co-immunoprecipitation of cystatin B with NF-L, RACK-1 and brain spectrin observed in rat cerebellum is specific and should be due to the in vivo interaction of the four proteins.
Expression of cystatin B and its partners in the cerebellum
To achieve better characterization of the proteins under scrutiny, we have studied expression and distribution patterns in the developing and adult rat cerebellum. Figure 4 shows the double-immunofluorescence analysis of cryostatic sections from a 7 days old rat cerebellum. The haematoxylin-eosin staining in A shows that, at this stage of development, the cerebellum is very rich in granule cells, while the formation of the molecular layer is just beginning. The green immunofluorescence of the anti-cystatin B antibodies (B) is intense over the granule cell layer. A detailed analysis of the sections is shown in C–K. RACK-1 (C), NF-L (E), and cystatin B (F, G and H) are clearly visible in the granule cells. In contrast, ß-spectrin (D) is barely detectable, suggesting that in the 7 day old cerebellum the synthesis of this protein is just beginning. The red fluorescence of cystatin B is seen merging with the green fluorescence of RACK-1 (I), ß-spectrin (J) and NF-L (K), to yield images of some mainly green cells, some mainly red and some yellow, indicating the mixing of the two colours.
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The proteins are not equally distributed in all cells: all four are present mainly in the cells localized at the inner edge of the granule layer, in the vicinity of the molecular layer. The granule cells in the outer layer are mainly stained by the anti-cystatin B antibodies, while the fluorescence of RACK-1, NF-L and ß-spectrin antibodies is weak.
Figure 5 shows the double-immunofluorescence analysis of cryostatic sections from adult rat cerebellum. The haematoxylin-eosin staining in A shows the expected structure, with a well developed molecular layer rich in fibers and the inner layer rich in granule cells. In B, the green fluorescence of cystatin B shows up almost exclusively in the molecular layer, with higher intensity at the border between granule cells and the molecular layer. The granule cells show little or no green fluorescence and this is in marked contrast with the picture in Figure 4, where the granule cells are characterized by bright green fluorescence. Details of the sections at higher magnification are shown in C–K. RACK-1, ß-spectrin and NF-L show a strong staining in the body and filaments of cells that are at the border between the molecular and cellular layer and are clearly identifiable as Purkinje cells. The red fluorescence of cystatin B merges well with the green fluorescence of the proteins, generating a yellow colour. As in the case of cystatin B, the staining of the three interactive proteins is not detectable in the granule cells. We cannot exclude the presence of trace amounts of the four proteins but the technique is not sensitive enough to detect them. In conclusion, these results show that cystatin B and the three partners analyzed so far, are expressed mostly in granule cells in the developing cerebellum and almost exclusively in the Purkinje cells in the adult cerebellum.
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DISCUSSION |
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The proteins isolated by the two-hybrid screening
The recent developments in the molecular genetics of EPM1 have important implications for the diagnosis of the disease. Furthermore, the knowledge of the gene responsible for EPM1 has allowed accurate prenatal and presymptomatic diagnosis and detection of carriers. The availability of a cystatin B knock-out mouse as a model for the disease has allowed identification of the presence of severe apoptotic damage to the cerebellar granule cells (4). This observation combined with the anti-protease function of the ablated protein has suggested that cystatin B may have an anti-apoptotic function in the cerebellum. However, the molecular basis of such function is not clear, since the cellular damage, at least initially, seems to be restricted to the cerebellum, while cystatin B is a ubiquitous protein, detected in most cell types, possibly protecting the cells against inappropriate cellular degradation by proteases that leak from lysosomes (16). The experiments reported here show that a number of proteins that are not proteases can interact specifically with cystatin B. The absence of proteases among the identified proteins may be explained if the weak interaction between cystatin B and cathepsin B observed in yeast and in vitro and/or a fast exchange rate of the anti-protease/protease binding are taken into account.
However, it is clear that cystatin B binds a number of cytoplasmic proteins that are involved in the regulation of cytoskeletal functions. At least two of them are expressed exclusively in cells of the nervous system, mainly neurons. ß-spectrin (17), a cytoskeletal protein that belongs to the spectrin superfamily, is bound to the neuronal cell membrane through the ankyrin protein forming different types of complexes and membrane domains. NF-L is a structural element that belongs to the family of intermediate filaments, with a role in the specification of cytoarchitecture and cytodynamics. Interaction between ß-spectrin and the amino terminus domain of NF-L was described by Frappier et al. (18). Macioce et al. (19) have shown such interaction is not direct but requires a linker protein. It is worth remembering that the NF-L cDNA that we have isolated spans from amino acid 388 and lacks the amino terminus region. Our results show that this fragment is sufficient to achieve interaction with cystatin B.
Another protein identified by the two hybrid procedure is the RACK-1 receptor protein that binds the active conformation of some of the isoforms of PKC and mediates the interaction between the activated kinase and the cell membrane (20,21). The highest affinity is for the ß isoform of PKC (22). In addition, RACK-1 interacts with the cytoplasmic domain of the integrin ß subunit, indicating an involvement in the interaction between plasma membrane and cytoskeleton (23) and is abundant during cell proliferation and differentiation. The decrease of the PKCß activity with ageing correlates with the decrease of membrane bound RACK-1 (24,25). Of interest is the interaction between the PKC anchoring protein RACK-1 and the pleckstrin homology domains of ß-spectrin, described by Rodriguez et al. (26).
Finally, TCrp has no known function while Mtrp is almost identical to the human myotubularin related protein 8 also of unknown function but phylogenetically related to the myotubularin family of proteins (27), some of which are abundant in motor and sensory neurons (28). The hybridization of the Mtrp and TCrp probes to total RNA from several tissues including brain and cerebellum shows that the corresponding genes are ubiquitously expressed in the rat (not shown). The myotubularin (MTM) family of proteins is characterized by the presence of a conserved dual-specificity phosphatase and the SET interacting domain (29,30), which is missing in the protein that we have isolated. Mutations of the myotubularin related gene 2 (MTMR2) are associated with the Charcot–Marie–Tooth disease type 4B1, a severe hereditary motor and sensory neuropathy (28). The known substrate of the MTM and MTMR2 proteins is phosphatidylinositol-3-phosphate which becomes dephosphorylated. Phosphoinositides are involved in number of cellular processes such as proliferation, differentiation and cytoskeleton reorganization (31). In conclusion, at least four of the five proteins interacting with cystatin B in yeast have known functions related to cell growth and differentiation.
Interaction of cystatin B and its partners in vitro and in vivo
Significantly, the five identified proteins bind cystatin B in the GST pull-down assay and the interaction occurs with the GST–exon II and GST–exon III fusion peptides. The second and third exons of the cystatin B gene also contain the binding site of cystatin B to the cathepsin proteases. The involvement of these two exons in the interaction correlates with a stop codon mutation at amino acid position 68 (exon 3) of the cystatin B gene found by Pennacchio et al. (1) in EPM1 patients. If cystatin B requires binding to any of the proteins under scrutiny to function, interruption of cystatin B at amino acid 68 might be consistent with alterations to the binding site. The search for homologies between the cystatin B interacting proteins and cathepsin proteases was negative. Furthermore, within the sequences of the five proteins, no common motif was found that could justify their binding to the peptides encoded by the second and third exons of the gene.
The size of the five proteins interacting with cystatin B varies from approximately 30 to more than 200 kDa and cystatin B is a small protein of approximately 12 kDa. It seems therefore unlikely that all these proteins interact directly with cystatin B at once, unless cystatin B is in a dimeric or polymeric form (15). However, the possibility of interactions between NF-L and ß-spectrin and between RACK-1 and ß-spectrin suggests that these proteins have multiple sites of interaction with one another as well as with cystatin B.
The interaction with cystatin B is detectable in the cerebellum and not in the brain hemispheres, indicating the tissue specificity of the protein complex. In the cerebellum these proteins are localized in different cells, depending on the age of the animal: in the granule cells in the developing cerebellum and in the Purkinje cells in the adult cerebellum. This observation is consistent with the results of Pennacchio et al. (4) who show the loss of granule cells in the cerebellum of cystatin B knock-out mice. The latter finding correlates with the marked loss of Purkinje cells revealed by autopsy of patients affected by EPM1 (32,33). The presence in the cerebellum of a complex between cystatin B and one or more proteins specific to the NS would explain the specificity of the EPM1 symptoms. The multiprotein complex we have identified contains at least the four proteins we have tested, but may also contain the two cystatin B interacting peptides, identified by the two-hybrid assay, for which the antibodies are not available, as well as possibly further peptides which may not interact directly with cystatin B. Recently, Gavin et al. (34) have discussed cellular processes in terms of interaction between multiprotein complexes and have shown that the great majority of proteins do not act alone, but are assembled into complexes that define specific functions. By analogy, we think that cystatin B in the cerebellum is part of a specific complex which is not present in other organs. The disruption of this complex in EPM1 patients may be the cause of the disease.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONCONCLUSIONSMATERIALS AND METHODSREFERENCES |
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These results strongly suggest a specific role for a cystatin B multiprotein complex in the cerebellum during development and in the adult animal, although this role is not clear. We envisage several possibilities. The first hypothesis is that cystatin B may be active as antiprotease, protecting the complex against the attack of proteases. To test this hypothesis, we have analyzed the proteins immunoprecipitated with anti-RACK-1 antibodies for the presence of cathepsin B, H, and L, known to bind cystatin B and we have not detected them (not shown). An alternative hypothesis is that cystatin B may bind to the interacting proteins modifying the structure, thus allowing the correct formation of the complex. Both hypotheses are possible, as they are not mutually exclusive. A further hypothesis, is the sequestration of cystatin B by the multiprotein complex, thus impeding its interaction with cathepsins. Considering the nature of the cystatin B partners, it is possible that the complex is involved in the process of differentiation of granule cells and possibly Purkinje cells in the cerebellum. Mutation/absence of the cystatin B protein may cause disruption of the complex and hinder neuronal differentiation. Finally, our results suggest a correlation between the disruption of this complex and the ethiopathogenesis of EPM1.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONCONCLUSIONSMATERIALS AND METHODSREFERENCES |
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All routine techniques and DNA manipulations were carried out according to Sambrook et al. (35). Restriction modification enzymes and antibodies (anti-brain ß-spectrin: sc-7468 and anti-NF-L: sc12966 from Santa Cruz Biotechnology, Santa Cruz, CA, USA; anti-RACK-1: from BD Bioscience Lexington, UK. A-2066 from Sigma-Aldrich, Italy; anti-cystatin B: from Biogenesis, Poole, UK; anti-glyceraldehyde-3-phosphate dehydrogenase: MAB374 from Chemicon) were used according to the manufacturer's instructions.
cDNA library construction
Total RNA was isolated from the cerebellum of Sprague-Dawley rats according to Chomczynski and Sacchi (36) and poly-A+RNA was purified (35). The cDNA was synthesized from a pool of equal amounts of the poly-A+RNA obtained from rat cerebella at 1, 2, 4, 8, 13 days of age. Double stranded cDNAs were prepared according to standard protocols and cloned into pJG4–5 vector (Clontech, ??, CA, USA) as a B42 transactivating domain fusion. The recombinant plasmids were used to transform SURE electrocompetent cells (Stratagene, La Soll, CA, USA). A cDNA library of 5x106 independent clones was obtained.
Yeast two-hybrid screening
The cystatin B cDNA was used as bait in the interaction trap assay. Rat cerebellar mRNA was reverse transcribed and cloned into the pEG202 vector (Clontech) as a LexA DNA binding domain fusion. The plasmid cDNA library was used to transform the EGY-48 yeast strain containing pEG-cystatin B plasmid and the pSH18–34 reporter plasmid (Clontech). The screening of the library was carried out according to Brent and Finley (12). Two hundred and twenty lacZ+, Leu+ positive colonies from 108 yeast transformants were isolated. The pJG-cDNA clones were rescued from yeast colonies (37); the cDNAs were amplified by PCR using pJG4.5 specific primers. The PCR products were sequenced and characterized. Only the cDNA recombinants cloned in-frame with the fusion peptide were further analyzed. In particular, those found in more than one independent colony were studied.
Western blot analysis
Cerebella and hemispheres of Sprague-Dawley rats were homogenized in 50 mM Tris pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM ß-mercaptoethanol, 0.5 mM DTT, anti-protease and anti-phosphatase mixture (Sigma). 50 µg of protein extract were electrophoresed on SDS–PAGE, transferred to nitrocellulose membrane (MSI), and stained as indicated. The protocols of the western blot were as described by Sambrook et al. (35). Immunoreactive proteins were detected with ECL (Amersham Pharmogene Biotech, Milano, Italy).
GST pull-down assays
The GST pull down assays were carried out between either the entire cystatin B protein, or the peptides coded for by each of the three cystatin B exons fused to the glutathione S-transferase (GST) and the [35S]-methionine labelled peptides isolated with the yeast two-hybrid system. The full length rat cystatin B cDNA was obtained by PCR amplification from the cerebellum cDNA library. The three separate exons of the gene were synthesized from the cystatin-B cDNA using specific oligonucleotides for each exon (exon I: aa 1–22; exon II: aa 23–56; exon III: aa 57–98). The PCR products were inserted into pGEX-3X plasmid (Pharmacia) and the corresponding GST–fusion peptides were expressed and purified from bacterial lysates using glutathione-sepharose 4B beads (sigma).
[35S]-peptides were obtained by T3 RNA polymerase (Promega) transcription of the PCR amplification products of pJG-cDNA clones and translation with rabbit reticulocyte lysate (Promega, Italie ?? Milano, Italy) in the presence of [35S]-methionine (Amersham). Each GST–cystatin B/protein complex was purified by binding to sepharose-glutathione beads. A volume of beads suspension corresponding to 
20 µg of GST or GST–fusion proteins was incubated for 3 h at room temperature with 5 µl of reticulocyte extract containing the labelled peptides in a final volume of 100 µl. The incubation buffer was: 20 mM sodium phosphate pH 6, 100 mM NaCl, 10% glycerol, 1 mM PMSF, 1 mM DTT, 0.02% Nonidet P-40. After washing, the pelleted beads were resuspended in Laemmli sample buffer, boiled and electrophoresed on SDS–polyacrylamide gels with acrylamide concentration appropriate to the molecular weight of the peptides. The 35S labelled peptides fused to the B42 transactivation domain (38) were synthesized in vitro in a cell free system, and incubated with the GST–cystatin B fusion protein. Each GST–cystatin B/protein complex was purified by binding to sepharose-glutathione beads.
Immunoprecipitation assays
Extracts from rat cerebella and brain hemispheres, at a final protein concentration of 10 mg/ml, were incubated overnight at 4°C with the appropriate antibodies in a final volume of 0.6 ml of the protein extraction buffer, containing 100 µl, 50% beads bound to Anti-IgM agarose, (Sigma) or protein–A–sepharose (Pharmacia), depending on the antibodies. Anti-protease and anti-phosphatase mixtures were added. Following incubation, the beads were extensively washed. The immunoprecipitated pellet(s) were electrophoresed half on a 15% and half on a 7% SDS–polyacrylamide gel. 50 µg protein extracts were loaded as a reference in all experiments. After blotting, the nitrocellulose membrane was cut into sections chosen according to the molecular weights of the proteins to be analyzed and each section was stained with the specific antibodies.
Immunofluorescence and confocal microscopy
Perfusion and dissection of the cerebellum were carried out on Sprague-Dawley rats, in agreement with the European Union guidelines and with Italian law. The rats were anaesthetized by intraperitoneal injection with sodium thiopental (45 mg/kg b.w.) and were perfused transcardially with PBS, followed by 4% paraformaldehyde in PBS (pH 7.4). Cerebella were dissected, postfixed in 4% paraformaldehyde/PBS, crioprotected in 10% sucrose/PBS and frozen in liquid nitrogen. Sagittal criosections were cut at 7 µm and mounted on slides. Haematoxylin and eosin staining (H&E) were carried out on the sections. Fixation of cells and tissues and purification of the granule cells were carried out as described by Riccio et al. (11).
The cerebellar granule cells were fixed in 4% paraformaldehyde in PBS for 30 min at 4°C and permeabilized with 0.1% Triton x100 in PBS for 10 min. After 30 min incubation in 3% BSA/PBS, the cells and the cerebellum sections were incubated for 1 h at room temperature with the primary antibodies (rabbit anti-cystatin B polyclonal Ab, Biogenesis; mouse anti-RACK-1 monoclonal Ab, Transduction Laboratories; mouse anti-ß1-spectrin monoclonal Ab, Oncogene, San Diego, CA, USA; goat anti-NF-L monoclonal Ab, Santa Cruz Biotechnology) diluted 1 : 50 in 3% BSA/PBS. After washing in 3% BSA/PBS the samples where incubated 1 h at room temperature with the secondary antibodies (FITC-conjugated donkey anti-mouse Ab F(ab)2 fragment, Sigma; FITC-conjugated mouse anti-goat Ab, Sigma; Cy5-conjugated donkey anti-rabbit Ab F(ab)2 fragment, Jackson, West Grove, PA, USA) diluted 1 : 20 in 3% BSA/PBS. Samples not incubated with the primary antibody were used as negative controls. When possible, antibody specificity was checked by pre-adsorption with the corresponding protein. Confocal imaging was obtained on a Radiance 2000 confocal laser scanning microscope (BioRad, Milano, Italy), equipped with a Nikon 40x, 1.4 N.A. objective and with Krypton and Red Diode lasers as previously described (11). Image processing was carried out with ImageSpace (Molecular Dynamics, CA, USA), Adobe Photoshop and Adobe Distiller Software.
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ACKNOWLEDGEMENTS |
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We are very grateful to Dr Elisabetta Ciani and Dr Antonio Contestabile for help in the isolation and purification of cerebellar granule cells; to Dr Patrizia Ambrogini for help in animal care; and to Dr Marialuisa Sartirana and Dr Angela Algeri for helpful discussions and reading of the manuscript. This work was supported by a Telethon grant E. 711, by a CNR grant 2002 and by a grant from the Italian Ministry of Technology and Scientific Research.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +39 0512094146; Fax: +39 051251208; Email: melli{at}alma.unibo.it
Present address: Department of Genetics and Molecular Biology at the Federico II University of Naples.Via Mezzocannone 8, 80134 Naples.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONCONCLUSIONSMATERIALS AND METHODSREFERENCES |
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