Desmin heterogeneity in the main electric organ of Electrophorus electricus

Biochimie 70 (1988) 783- 789 (~ Soci6t(~de Chimie biologique/ Elsevie(, Paris

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Research article

Desmin heterogeneity in the main electric organ of Electrophorus electricus Manuel Luis COSTA, Vivaldo M O U R A N E T O and Carlos C H A G A S * Instituto de Biofisica Carlos Chagas Filho, Bloco G, Centro de Cidncias da Smide, Universitlade Federal do Rio de Janeiro, llha do Funddo, 21941 Rio de Janeiro, Brasil (Received 1-7-1987, acceptedafter revision 18-1-1988)

Summary - - Desmin, the musclc-:pecific intermediate filament protein was purified from the main electric organ of Electrophorus electricus. It is shown that pure desmin can be separated into 5 isoforms presenting different isoelectric points. These isoforms have similar molecular weight, react with an antibody directed against desmin and generate identical peptides after digestion with protease V8 from Staphylococcus aureus. desmin isoforms / intermediate filaments / electric organs / E ~ o r u s

Introduction The main electric organ of Electrophorus electricus is composed of an association of electrocytes. This cellular unit is highly specialized and polarized° !ndeed~ the rostra! non-innervated face of the electrocyte is enriched in (Na + / K +) ATPase, while the acetylcholine receptors (ACh-R) are associated with the caudal membrane [1, 2]. Two lines of evidence indicate that this cellular polarity might be reflected at the level of the cytoskeleton. First, ACh-R of cultured muscle cells can be co-purified with cytoskeletal proteins [3]. Second, in the electric organ of Torpedo marmorata, the aggregated subsynaptic form of the ACh-R is linked to a 43 kDa polypeptide which strongly interacts with submembranous cytoskeleton [4-6]. Although tubulin and actin are present in the electrocyte [7-9], it seems that the study of the main intermediate filament protein present in this cell type (desmin) may be of particular interest. Indeed, the use of antibodies raised against the 43 kDa protein has demonstrated that this molecule is associated with a I0 nm intermediate

*Author to whom correspondence should be addressed.

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filament [6]. Moreover, ACh-R-enriched membrane fractions from Torpedo marmorata contain both actin and desmin [10]. Isoforms of cytoskelcte,! proteins, such as tubulin or the 200 kDa subunit of the neurofilament triplet, have been ~hown to be ass~iated with defined subcellular compartments [11]. The electrocyte might therefore ,"~nstitute a good model system for the study of t . : possible role of intermediate filament isoforms in the establishment a n d / o r the maintenance of the polarized state. In a first attempt to do so, we have undertaken the biochemical analysis of desrain in the electrocyte of Electrophorus electricus. We demonstrate that this cell contains 5 desmin isoforms which can be separated on the basis of their isoelectric points.

Materials and m e t h o d s Desmin preparation The main electric organ of E. electricus was minced and homogenized in 40 mM imidazol-HCI, pH 6.9, 0.6 M KCI, 1 mM ethyleneglycol-bis(/3-aminoethyl

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ether)N,N,N',N'-tetraacetic acid (EGTA), 1 mM 2mercaptoethanoi and 0.5% Triton X-100. All purification steps were performed according to Geisier and Webcr [12]. The ethanol-precipitated protein fractions were redissolved in urea buffer (6 M urea, 10 mM sod.:um phosphate, pH 7.5, 5 mM EGTn,, 0.1% 2-mercaptoethanol) and analyzed by gel electrophoresis. Protein content was measured according to Lowry et al. [13]. Cell labeling Fhe cerebellar glioma C8S was a kind gift of Drs. F. Alliot and B. Pessac (CNRS, France). Proteins were labeled with [35S]methionine and extracted as previously described [ 14}. Gel electrophoresis Two-dimensional (2D) polyacrylamide gel electrophorests (PAGE) was performed according to O'Farrell [15] a~ modified by Moura Neto et al. [16]. The first dimension was run in 9.6 M urea with 2% ( w / v ) LKB ampholines, pH ranges 3.5-10 and 5 - 8 (1:4). The second dimension was run in a 7% polyacrylamide gel containing sodium dodecyl sulfate (SDS). Slab-gel isoelectric focusing (IEF) electrophoresis was done in the same mixture of urea and ampholines according to the method of Ferreira and Eichinger [17]. Proteins were stained with Coomassie brilliant blue or with silver [18]. For autoradiography, the gels were vacuum dried and exposed to X-ray Sakura films.

Costa et al.

Results Desmin electrophoretic profile Fig. 1 illustrates the analysis by 2D electrophoresis of the desmin purified from electrocytes. Protein spots were revealed by Coomassie brilliant blue staining. Several spots are clearly visible which migrate in the region expected for this intermediate filament protein [ 2 2 - 2 4 , 10]. These spots mainly differ by their electric charges and appear to havc similar molecular weights. Such a pattern suggests the presence of several desmin isoforms. In order to compare its isoelectric point and molecular weight with others cytoskeletal pro-

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lmmunoblotting Purified desmin was run on an IEF gel. The 5 Coomassie blue-stained bands were cut out of the gel, loaded onto a one dimensional slab-gel in 5 different slots, and run in the presence of SDS [16]. Proteins were electrophoretically transferred onto a nitrocellulose sheet according to Towbin et al. [19]. Reversible staining with Ponceau red was used to ensure that an efficient transfer had taken place [20]. Incubation with the antibodies was performcd as described earlier [25]. The anti-desmin antibody was provided by Dr. L. Rappaport (INSERM, U127, France) and diluted 1:200 before use. Peptide mapping analysis Desmin was isolated by IEF slab-gel electrophoresis [17] and the 5 desmin polypeptides were cut out of the gel. Each strip was minced and the fragments were loaded onto a one dimensional slab-gel. The 5 desmin polypeptides were characterized by digestion with Staphylococcus aureus V8 protease (Miles, 2 n g / spot). The peptide analysis was carried out on a 15% acrylamide slab-gel containing 0.1% SDS. The general procedure for peptide analysis was performed according to Cleveland et al. [21]. Peptides were visualized with the silver staining method described by Oakley et al. I18].

Fig. 1. Separation af electric organ desmin by 2D electrophoresis. 1EF was achieved in the first dimension (horizontal arrow), the acidic part of the gel being on the right side. Separation according to the size of the polypeptides was made in the second dimension (vertical arrow) in an SDScontaining 8% polyacrylamide g~l. Note the presence of several spots stained with Coomassie brilliant blue.

Desmin heterogeneity in E. electricus

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teins, cold desmin was mixed with an extract of [35S]methionine-labeled glial cells (glioma C8S). These glial cells are known to synthesize tubulins (a and /3), vimentin, the glial fibrillary acidic protein (GFAP) and actin [14]. The mixture of putative pure desmin and radioactive glial proteins was analyzed by 2D electrophoresis followed by silver staining (Fig. 3) and autoradiography (Fig. 2). Comparison of the two figures demonstrated that the silver stained spots which appear in the desmin region (indicated D) are not labeled on the autoradiogram and therefore are not synthesized by the glial cell line. On the contrary, proteins which are expected to be synthesized by the glioma do show up on the autoradiogram.

Fig. 3. Silver staining ol the same gel from which Fig. 2 autorad_iogram was obtaJne0. This figure mu~t be compared with Fig. 2. Note the presence of several unseparated spots in the 51 kDa region. These spots revealed by silver staining were not plesent in the autoradiogram (Fig. 2). They are therefore likely to correspond to muscle specific intermediate filament" desmin (D). Several spots present in both of the insets in Figs. 2 and 3 were not identified.

Analysis of desmin heterogeneity .

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Fig. 2. Autoradiogram of [3-SSlmethionine-labeled proteins extracted from g!ioma C8S and mixed ~vith non-labeled purified desmin before 2D electrophoresis. The protocol was the same as that used for Fig. 1, except that the polyacrylamide concentration in the second dimension was raised to 10%. The inset is an enlargement of the region of the gel which contains most cytoskeletal proteins, such as: aT: a-tubulin; bT:/3-tubulin; V: vimentin and Ac: actin.

Purified desmin preparations were run on an IEF gel and the proteins were stained with Coomassie brilliant blue. As shown in Fig. 4, 5 apparently homogeneous bands could clearly be separated. All 5 peptides niigrated in a close pl region which ranges between 5.6 and 5.75 and the most acidic one was less abundant than the 4 others. In order to compare their molecular weights, each band was cut out of the gel as precisely as possible and loaded onto an SDS-con-

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Discussion

Fig. 4. Separation of desmin isoforms by IEF. Separated proteins were stained with Coomassie brilliant blue. The acidic region of the gel is on the right side of the picture. Five polypeptides stained with different intensities are present.

taining polyacrylamide gel, as schematized in Fig. 5. Coomassie blue staining of the gel (Fig. 5) demonstrates that the 5 proteins separated by IEF have very similar molecular weights. In a parallel experiment, the proteins cut out of the IEF gel and run on a polyacrylamide slabgel were electrophoretically transferred onto nitrocellulose and incubated with a specific antibody directed against desmin [26]. Fig. 6 demonstrates that the 5 separated proteins react with ihe antibody.

Desmin is the main intermediate filament present in muscle cells. Genetic analyses have demonstrated that it is coded by a single gene [28]. These studies together with structural analyses indicate that avian and mammalian desmin molecules are very similar [12]. Desmin preparations from chick, duck and quail contain 2 variants (o~- and /3-desmin) of identical molecular weights but presenting dis-

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Peptide mapping The best demonstration that the 5 protein bands represent different desmin isoforms is to compare the electrophoretic mobilities of the peptides generated by specific proteolysis. In order to do so, one needs to purify a rather large amount of each isoform. This was achieved by running desmin purified from the main electric organ on a preparative IEF slab-gel. The pattern obtained with one of these gels is shown in Fig. 7. The 5 desmin bands are well separated. The nature of the very acidic contaminating peptides which migrate far ahead of the 5 putative desmin isoforms was not elucidated. These 5 bands were cut out of the gel, minced and loaded into 5 separate slots of an SDS-polyacrylamide gel (15%) in the presence of protease V8 from S. aureus (2 ng/slot). A control sample of purified desmin [27] was aso loaded with protease V8 and run in parallel (Fig. 8, slot 6). Fig. 8 shows that the 5 proteins separated by preparative IEF and the control desmin preparation generate identical peptides when incubated with protease V8.

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Fig. 5. Comparison of the molecularweights of the five peptides separated by IEF. The bands visualized with Coomassie blue (as in Fig. 4) were cut out and loaded into separate slots of a one-dimensional polyacrylamide gel. This technique allows the comparison of the electrophoretic mobility of each peptide. The 5 peptides have similar weights. Letters S and R indicate the stacking part and the running part of the gel, respectively.

Desmin heterogeneity in E . electricus

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Fig. 7. Preparative slab-gel IEF of purified desmin. Separated bands were stained with Coomassie brilliant blue. The acidic region of the slab-gei is on the right side. Note the presence of 4 strongly stained bands and 1 weakly labeled band (the most acidic one). A non-characterized contaminant with a low isoelectric point was clearly present in this preparation.

Fig. 6. lmmunoblotting of peptides separated as indicated for Fig. 5. Proteins cut out of the IEF gel and run in separate slots on a polyacD,lamide slab-ge! were electrically transferred onto nitrocellulose. The cellulose sheet was then incubated with an antibody directed againgt chick desmin and raised in the rabbit. The antibody-antigen complex was vi.~,a!ized with peroxidase-conjugated goat anti-rabbit immunoglobulins, using DAB.

tinct isoelectric p o i n t s , 0~-desmin b e i n g slightly m o r e acidic t h a n ~ - d e s m i n [22, 24]. It is p o s s i b l e b u t n o t p r o v e n t h a t cz-desmin is a p h o s p h o r y l a t e d f o r m o f ~ - d e s m i n . In fact, in vivo p h o s p h o r y lation o f c h i c k d e s m i n f o l l o w e d b y tryptic a n a l y sis d e m o n s t r a t e s t h a t t w o p e p t i d e s g e n e r a t e d from pure desmin are phosphorylated on serine r e s i d u e s [29]. T h e p r e s e n c e o f t h e s e p h o s p h o r y lation sites d o e s n o t p r e c l u d e t h e possibility t h a t o t h e r r e s i d u e s c o u l d also b e p h o s p h o r y l a t e d . I n d e e d , w h e n purified d e s m i n is p h o s p h o r y l a t e d in vitro w i t h c A M P - d e p e n d e n t d e s m i n k i n a s e , it is c l e a r t h a t a d d i t i o n a l p h o s p h o r y l a t i o n sites c a n b e r e v e a l e d [29].

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Fig. 8. Slab-gel analysis of peptide products generated by protease V8. The 5 putative desmin isoforms were separated as described for Fig. 7 and digested by protease V8 during their loading into the 5 first slots of the SDS-containing polyacrylamide 15% gel. Slot 6 (indicated D) contains the produets generated by incubating characterized control desmin with the protease. Note the similarity of the 6 proteolitic patterns.

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The question can therefore be raised of whether several (more than 2) isoforms of desmin can be normally generated in vivo through post-transcriptional (differential splicing) events or post-translational (including phosphorylation) modifications. In the present report, we demonstrate that 5 desmin variants co-exist in the main electric organ of E. electricus. Indeed, purified desmin can be resolved into 5 isoforms by IEF gel electrophoresis. The 5 proteins have molecular weights of ~51 kDa, are recognized by the same anti-desmin antibody and give rise to the same proteolytic pattern after hydrolysis by protease V8 from S. aureus. This report is the first one in which 5 isoforms of desmin are shown to co-exist in vivo in the electric organs. This could be due to the tissue studied which is different from those normally analyzed (avian and mammalian muscles). A more interesting possibility is that the high number of desmin isovariants relates to the function of the electrocyte and to its highly specialized cellular organization, as has been shown in bovine Purkinje fibers in which "the development of nerve-like properties has taken precedence over the development of contractility" [30]. It could therefore be envisaged that specific post-translational modifications play a role in the establishment and the maintenance of the electric properties and the polarized state. In fact, post-translational modifications associated with the cellular compartmentalization of cytoskeletal proteins have been reported in other biological systems 131, 32, 11]. Although it is likely that phosphorylation events are implied in the generation of some desmin variants, other types of post-transcriptional and post-translational modifications could also be involved. This point is presently being studied in our laboratory.

Acknowledgments We want to thank D. Katz, M. H. L6vi and A. Prochiantz for helpful discussions and for help with the manuscript; and Miss A. M. Alves for the excellent technical assistance rendered during the experimental work. This work was supported by grants from Ci~Pq, FINEP, and CEPG-UFRJ.

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