Neurochemical Research, VoL 16, No. 2, 1991, pp. 123-128
The Phylogenic Expression of Plasmolipin in the Vertebrate Nervous System Victor S. Sapirstein 1, Charles E. Nolan 1, Itzhak Fischer 2, Elizabeth Cochary 2, Susana Blau 1, and Cheryl J. Flynn 1 (Accepted December 17, 1990)
Plasmolipin is a plasma membrane proteolipid is a major myelin membrane component (Cochary et al., 1990). In this study we report the phytogenic expression of plasmolipin in the vertebrate nervous system. Using Western blot analysis with polyclonat antibodies, we have analyzed membrane fractions, including myelin, from elasmobranchs, teleosts, amphibians, reptiles, birds and mammals. On the basis of immune detection, plasmolipin appears to be restricted to the mammalian nervous system. Comparison of the central and peripheral nervous systems of mammals showed only minor differences in the level of plasmolipin in these two regions. Within mammals, little quantitative differences were observed when rat, human and bovine membrane fractions were compared. The late evolutionary expression of plasmolipin which results in its restriction to mammals makes it unique among the (major) myelin proteins. The potential physiologic significance of these ,data are discussed. KEY WORDS: Plasmamembrane;proteotipid;myelin;evolution;mammalianbrain.
INTRODUCTION
between their CNS myelin proteins and mammalian PNS myelin proteins. The major CNS myelin proteins in these lower species (5,6) show cross-reactivity with antibodies to PO and an abundance of other glycosylated proteins (7). Amphibians and higher vertebrates show a different CNS pattern. For example, myelin proteolipid protein (PLP) and basic protein (MBP) are highly conserved and antibodies to these proteins isolated from mammals show cross-reactivity towards homologous proteins in amphibians and reptiles. In the case of PLP, this cross reactivity can be attributed to a high degree of C terminal sequence homology seen from tetrapods through mammals (8). Although the 18.5kD form of MBP is present in amphibians, the 21.5kD form is restricted to mammals (3). DM-20 protein, a protein closely related to PLP, is not observed in frogs and toads but is present as one advances to reptiles and higher vertebrates. The present study has focussed on the expression of plasmolipin. This protein is an 18kD hydrophobic
The evolution of the major myelin proteins has been examined from agnathans and elasmobranchs through primates (1-4). The pattern that has developed is that the central nervous systems (CNS) of elasmobranchs and teleosts express a pattern of myelin proteins distinct from that of amphibians and higher vertebrates. Although there are differences among these lower vertebrates, immunochemical and biochemical studies indicate a similarity 1 The Divisionof Neurobiology,The NathanKlineInstitute,Orangeburg, NY; and the Departmentof Psychiatry,New York University School of Medicine,New York, NY. 2 The Departmentof Biochemistry,The EuniceKennedyShriverCenter, Waltham,MA and Departmentof Neurology,HarvardMedical School, Boston, MA. Abbreviations:EDTA,EthylenediamineN,,N'N'tetraceticacid;EGTA, E~hyleneglycolbis-(B-AminoethylEther) N,,N'N'tetraceticacid; MES, (-[N-Morpholino] ethanesulfonicacid)DCCD, N,' Dicyclohexylcarbodiimidc 123
0364-3190/91/0200-0123506.50/0 9 1991 Plenum Publishing Corporation
124 plasma membrane protein which in vitro forms cation channels (9,10) and is abundant in the nervous system (11,12) and renal tubular epithelial cells (10) of mammals. The protein is comprised of two subunits of similar molecular weight; their parallel in vitro synthesis (11) and the requirement of both subunits for channel formation (9) suggests that they form a single biological unit. In brain, we have reported the abundance of this protein in oligodendrocytes (13) and several purified membrane fractions (14) including the membrane bilayer of clathrin coated vesicles (12) and isolated myelin (15). In myelin, plasmolipin represents 2.2-4.8% of the membrane protein. The present study has exclusively utilized Western blot analysis since this protein co-migrates with one of the MBP forms (18.5kD) and stains poorIy with Coomassie blue. The use of Western Blot with polyclonal antibodies provide a valid comparison with the expression of other major CNS myelin proteins which have been studied similarly (5-7). In this study we show that the expression of immunoreactive plasmolipin is restricted to mammals where it is found in both the CNS and PNS.
EXPERIMENTAL PROCEDURE Membrane Isolation. Membranes were prepared from various species obtained either from The Mount Desert Island Biological Research Laboratories, (Bar Harbour ME.), Western Scientific (Sacramento CA), Bronx Reptile Distributors (Mt. Vernon, NY), local slaughterhouses and the Nathan Kline Institute Animal Facility. Normal human cortical and subcortical material was obtained fresh from surgery at New York University Medical Center, University Hospital, during the excision of a brain tumor. Membranes were isolated in the presence of 10 U/ml of aprotinin (Sigma Chemical Co., St. Louis MO) and 25 ~xg/ml of leupeptin (Sigma Chemical Co.). Total membrane fractions were prepared from forebrain or upper brain stem, spinal cord or sciatic nerve (defined as peripheral nerve). Tissues were homogenized in 20 volumes of 0.32 M sucrose (BRL, Gaithersberg, MD) containing 10mM Tris (BRL) pH 7.5 and lmM EDTA (Sigma Chemical Co.). The homogenates were centrifuged at 500 g for 5 rain. in a Beckman J2-21 Centrifuge to remove nuclei and a post nuclear membrane fraction was obtained by centrifugation at 40,000 g for 30 min. Myelin was prepared by the Method of Waenheldt and Mandel (16) as modified by Reiss et al (17). Where beth myelin and axolemma were required, they were prepared by a modification of the procedure of Detskey et al (18). The myelin and axolemma obtained by this procedure were repurified. The myelin was osmotically shocked, sedimented at 12,000 g to remove residual microsomal contaminants and layered over 0.8M sucrose. The band above the 0.8 M sucrose was taken as purified myelin. The axolemma was repurified after lysis in 5 mM Tris-HC1, pH 8.5, containing 0.1mM EDTA. The lysate was made isotonic by the addition of 1/4 volume of 40% sucrose, layered over 0.8 and 1.0M sucrose and centrifuged at 75,000 g for 1 hour. The material obtained at the 0.8/1.0M sucrose interface was defined as purified axolemma. Characterization of this axolemmal material has been described elsewhere
Sapirstein, Nolan, Fischer, Coehary, Blau, and Flynn (19). Synaptic plasma membranes (SPM) were isolated as previously described (20). Gel Electrophoresis and Western Blot Analysis. All electrophoretic analysis was carried out using the procedure of Laemmli (22) with a 4-18% acrylamide gradient or 13% isocratic gels. Acrylamide and Bis acrylamide were obtained from National Diagnostics {Manville, NJ), while all other etectrophoretic reagents were purchased from BioRad (Riverside, CA). Preparation and scanning of Western blots were previously described (12). Isolation of plasmolipin and production of anti-plasmolipin antisera were described by Sapirstein and Rounds, (10) and by Fischer and Sapirstein (11). Antibodies to Na, K ATPase were a generous gift of Dr. W. Stahl, University of Washington. Protein determinations were performed on SDS solubilized membrane preparations using the dye binding method of Bradford with reagents from BioRad. All additional reagents were reagent grade and were purchased from either Fischer Scientific (Pittsburgh, PA) or Sigma Chemical Co.
RESULTS Distribution of Plasmolipin in the CNS and PNS. The distribution and phylogenic profile of plasmolipin was examined in brain, spinal cord and peripheral nerves. In the rat, total membrane fractions from these three areas were found to have roughly equivalent levels of plasmolipin, (Figure 1). In this analysis, the upper brain stem area was used instead of forebrain in order to obtain grey to white matter ratios similar to those found in other areas of the nervous system. The Western blot analysis indicated that the levels of plasmolipin in the CNS and PNS were very similar. Scanning of the bands showed the levels to be within a 20% range of each other with brain and spinal cord levels being higher than the peripheral nerve.
Fig.1. Distribution of plasmolipin in mammalian (rat) brain, spinal cord and peripheral nerve. Total membrane fraction (10 p.g) from brain (upper brain stem), lane 1; spinal cord (total spinal cord), lane 2; and peripheral nerve (sciatic nerve), lane 3 were analyzed by Western blot as described in Methods. Gel electrophoresis was performed on a 418% polyacylamide gradient.
Phylogenie Expression of Plasmolipin
125
In the CNS, plasmolipin is found predominantly in white matter tracts and is enriched in isolated myelin (15). Myelin was therefore isolated from the forebrain and peripheral nerve and each was analyzed for plasmolipin content, (Figure 2). Visual inspection of these Western blots indicate that the level of the protein in CNS and PNS were similar, with CNS myelin levels being slightly higher than PNS.
Phytogenic Expression of Plasmolipin in Vertebrates. The phylogenic diversity of plasmolipin was analyzed in the CNS and PNS of different vertebrate species by examining total membrane fractions isolated from amphibians (frog), reptile (lizard), birds (chicken) and mammals (rat). The abundance of plasmolipin usually allows for its detection in relatively small samples of total brain membrane fractions. However, because preliminary studies yielded negative results in lower animals, the experiment illustrated in Figure 3 compared 20 ug of mammalian protein to 100 ug from the other species. The results indicated that based on immune reactivity, plasmolipin was absent in forebrain of nonmammalian species (Figure 3A). From these results and our estimated limits of detection of pIasmolipin in rat membranes, (2-5 ~g), we calculated that these lower
Fig. 2. The level of plasmolipin in purified rat myelin isolated from CNS and PNS. Myelin was isolated as described in Methods; 1,2 and 5 ~g were analyzed from CNS (whole forebrain), lanes 1,2, and 3, respectively; and PNS (sciatic nerve), lanes 4,5, and 6, respectively, were analyzed by Western blot.
Fig. 3. Phylogenic expression of plasmolipin. Total membrane fraction from whole brain (A), spinal cord (B) and sciatic nerve (C) were analyzed by Western blot. Each of these regions were studied in rat, lane 1; chicken, lane 2; lizard, lane 3; and frog, lane 4. Rat was analyzed using 20 ~g protein while other fractions were tested at 100 Ixg.
species had less than 2-5% of the level of plasmolipin in the rat brain. The forebrain is both ontologically and phylogenetically younger than the spinal cord and peripheral nervous system; however attempts to detect plasmolipin in these two regions in amphibians, reptiles and birds yielded similar negative results, (Figures 3B and C). The results described here were extended to other non-mammalian species, including pigeon, turtle, toads, mud puppy and salamander with identical negative results (data not shown). As noted earlier, plasmolipin is highly enriched in isolated myelin and in an attempt to amplify the plasmolipin signal in lower vertebrates, we examined this protein in purified myelin isolated from these animals, (Figure 4). Five ~g of rat myelin was used and compared with 10 ~g of myelin from the other species. Purification of myelin failed to yield any detectable levels of plasmolipin in any non-mammalian species. In a previous report from this laboratory we described a proteolipid protein extracted from preparations of chicken synaptic plasma membrane (11). At the time
126
Sapirstein, Nolan, Fischer, Cochary, Blau, and Flynn 69
K--
46
K--
30
K--
21.5 K-14.3 K--
A
B C D E
F G H
I J
K
Fig. 4. Phylogenic analysis of plasmolipin in vertebrate myelin. Myelin was prepared as described in Methods and analyzed for the presence of plasmolipin by Western blot. Human, lane A; bovine, lane B; rat, lane C; chicken, lane D; lizard, lane E; turtle, lane F; frog, lane G; goldfish, lane H; shark, lane I, skate, lane J; human PNS, lane K. All lanes contained 10 p-g of myelin protein, except lane 3 that contained 5 p-g of rat myelin. All myelin was derived from CNS except for human PNS in lane 11.
Fig. 5. Comparison of plasmolipin in rat and chicken synaptic plasma membranes. Synaptic plasma membrane was purified from rat and chicken brain; rat SPM was taken as defined previously (12). Chicken synaptosomes were lysed as described (12) and membrane sedimenting on 0.8 M and 1.0 M sucrose were defined as SPM fractions. Lane 1, rat SPM (10 p-g); lanes 2 and 3, chicken SPM (20 p.g) from 0.8 and 1.0M sucrose respectively; lanes 4 and 5, chicken SPM fractions (50 ~xg) and lanes 6 and 7, chicken SPM fractions (100 Ixg). Plasmolipin analysis was as described in Figure 1.
the protein was isolated, anti-pIasmolipin antibodies were not available, but a relationship of the protein to plasmolipin was implied. The lack of reactivity of anti-plasmolipin with chicken brain membranes, (Figures 3 and 4), prompted a re-examination. Synaptic plasma membrane was isolated from chicken and 10-100 Ixg of protein was analyzed by western blot, (Figure 5). For this analysis, we employed a second polyclonal antibody against plasmolipin in order to exclude antibody bias. Since SPM sediments at 0.8 M and 1.0 M sucrose, we analyzed both fractions from the chicken in order to preclude sampling error. The data indicated that in all of the samples no
detectable reactivity between plasmolipin and chicken SPM could be observed. In contrast, the analysis of 10 txg of rat SPM gave a clear signal, lane 1. The restriction of plasmolipin to mammalian brain prompted us to look for phylogenic differences within this vertebrate class, examining rat, bovine and human material. We confined our analysis to purified membrane fractions so as not to avoid variation due to differential contributions of myelinated and non-myelinated regions. We isolated myelin and an axolemmal enriched fraction which is released from myelin by osmotic lysis in the presence of EDTA, (see Methods). The data in Figure 6 indicated that the level in myelin and the myelin derived axolemmal fractions were similar across the mammalian species studied. Scanning of these Western blots (Table 1) showed that quantitatively all three myelin fractions from these species were vitually identical. In all cases the levels of plasmolipin were identical in myelin and axolemma enriched fractions were indistinguishable. The only significant difference was that the human material in both membrane fractions exhibited a slightly higher molecular weight for one of the plasmolipin sub-
6. Plasmolipin levels in myelin and axolemma enriched fractions in different mammalian species. Plasmolipin in myelin and axolemma were compared from human, bovine and rat brainstem isolated as described in Methods. Three p-g of protein was analyzed from each fraction. Lanes 1 and 2 are human axolemma and myelin, lanes 3 and 4 are bovine axolemma and myelin, and lanes 5 and 6 are rat axolemma and myelin. Analyses were carried out as described in Figure 1. Fig.
Table
I. Relative Level of Plasmolipin in Myelin and Axolemma Enriched Fractions
Membrane Fraction Myelin Axolemma
Rat
Specie Human
Bovine
100"
93 _ 6
89 -+-8
102 +_+_8* *
98 +_.12
91-4-13
* Rat myelin was taken as 100% ** Values represent the mean of 3 Western blots -- the standard deviation.
Phylogenic Expression of Plasmolipin units, differing from the rat and bovine material by approximately 500 daltons.
DISCUSSION The results of this study indicate that the expression of plasmolipin is restricted to mammals in both the peripheral and central nervous systems. In mammals, the level of this protein in CNS and PNS is similar, suggesting that plasmolipin is important to both areas of the nervous system. The absence of plasmolipin in avian brain is striking, since the avian nervous system is more similar to the mammalian than reptilian nervous system. However, significant differences exist between mammalian and avian brains, including the absence of cortical layers in birds and the lack of a corpus callosum. The phylogenic pattern of plasmolipin may be a reflection of either limited antibody cross-reactivity or the absence of protein in the non-mammalian nervous systems. It is common to see variability in immune reactivity when using monoclonal antibodies, since these epitopes often show widely different phylogenic patterns (23). In contrast, polyclonal antibodies can detect even small regions of homo]!ogy. For example, the extensive phylogenic cross reactMty of PLP antisera with lungfish is reactivity with only a C.-terminal sequence of PLP (8). The present study with two different polyclonal antibodies indicated that, if present, pIasmolipin must have undergone significant evolutionary change. Chick brain synaptic plasma membrane has a proteolipid protein of molecular weight similar to plasmolipin which might exist in other non-mammalian species (11). This proteolipid was nor. analyzed for reactivity with antiplasmolipin. However, since our report it has become clear that other proteolipids exist in the plasmolipin molecular weight range. A 16-17kD (19) proteolipid protein is present in several tissues and was isolated from keratinocytes (23) and showed no cross-reactivity with anti-plasmolipin, (data now shown). In addi'Iion, the major DCCD binding protein of golgi and plas;ma membrane derived vesicles is a also proteolipid and has a molecular weight of 16-17kD (20). However, we have been unable to detect DCCD binding to plasmolipin (data not shown). This finding confirms the report of Lin and Lees (25) which indicates that in myelin, only the myelin PLP reacts with DCCD. These data, along with the lack of immunereactivity of plasmoIipin antibodies with chicken SPM, suggest that the proteolipid present in these preparations is unrelated to plasmolipin. Comparison of rat, bovine and human material indicates that the high level of plasmolipin is sustained across
127 mammalian species. Rat preparations showed tighter plasmolipin bands in Western blots than did human or bovine. This may have been due to the rapid post mortem processing of the rodent material that minimized degradation. The minor difference in one of the human plasmolipin subunits, although 500 daltons or less, suggests some small compositional difference in human plasmolipin in relation to other mammals exists. Proteolipids are acylated (26) and the electrophoretic variations observed in humans may result from fatty acid differences. The restricted pattern of phylogenic expression of plasmolipin is unique among major myelin proteins. The major CNS myelin proteins are not exclusive to mammals, but are present in all air breathing vertebrates (15). The exception to this is the 21.5kD form of MBP which is restricted to mammals (3). The absence or low level of these CNS myelin proteins in aquatic species can be explained, in part, by the nature of myelin among teleosts and elasmobranchs. Their myelin is comprised mostly of PNS-like proteins, since antibodies to PO protein react with proteins in teleost and elasmobranch CNS myelin (1,5-7). However, unlike the PLP and MBP, plasmolipin is present in equal abundance in the PNS and CNS and could, therefore, be expected to have a broader phylogenic range. This is not the case. In fact, plasmolipin appears to have the most restricted phylogenic range among the myelin proteins studied so far, with mammals representing a sharp transition in the expression of plasmotipin. The relative conservation of PLP and MBP and Wolgran protein as well as the major myelin lipids (27) have been considered as evidence for their importance in myelin structural integrity. We propose that the restriction of plasmolipin to mammals reflects a specific physiologic function that is characteristic of the mammalian nervous system. The ability of plasmolipin to form K § channel (9) suggests that its function may be related to the regulation of membrane excitability. This hypothesis is consistent with recent physiologic studies which have demonstrated K + channels of oligodendrocytes (27). This hypothesis is also consistent with the increased complexity of the mammalian nervous system. The demands of increased neural activity requires complex factors to modulate the flow of information between brain regions. Plasmolipin and other potential factors may therefore be important for the higher integrative neural function associated with the mammalian brain. ACKNOWLEDGMENTS The authors would like to acknowledgethe technical assistance of Gladys Gray-Boardand Roz Cohen. The authors also thank Dr.
128 Oscar Bizzozero for his helpful comments on the manuscript. This work was supported by NS24894, NS25950 and HD05515.
Sapirstein, Nolan, Fischer, Cochary, Blau, and Flynn
13. 14.
REFERENCES 15. 1. Franz, T., Waehneldt, T.V., Neuhoff, V., and Wachtler, K. 1981. Central nervous system myelin proteins and glycoproteins in vertebrates: a phylogenetic study. Brain Res. 226:245-258. 2. Saavedra, R.A., Fors, L., Aebersold, R.H., Arden, B., Horvath, S., Sanders, J., and Hood, L. 1989. The myelin proteins of the shark brain are similar to the myelin proteins of the mammalian peripheral nervous system. J. MoI. Evol. 29(2):149-156. 3. Tai, F.L., Smith, Ross, Bernard, C.C.A., and Hearn, M.W.T. 1986. Evolutionary divergence in the structure of myelin basic protein: comparison of chondrichthye basic proteins with those from higher vertebrates. J. Neurochem. 46(4):1050-1057. 4. Waehneldt, T.V., Matthieu, J.M., Malotka, J., and Joss, J. 1987. A glycosylated proteolipid protein is common to CNS myelin of recent lungfish (ceratodidae, lepidosirenidae). Comp. Biochem. Physiol. 88(4):1209-1212. 5. Waehneldt, T.V., Malotka, J., Karin, N.J., and Matthieu, J.M. 1985. Phylogenetic examination of vertebrate central nervous system myelin proteins by electro-immunoblotting. Neurosci. Lett. 57:97-102. 6. Waehneldt, T.V., Stoklas, S., Jeserich, G., and Matthieu, J. 1986. Central nervous system myelin of teleosts: comparative electrophoretic analysis of its proteins by staining and immunoblotting. Comp. Biochem. Physiol. 84:273-278. 7. Waehneldt, T.V., Matthieu, J.M., and Jeserich, G. 1986. Major central nervous system myelin glycoprotein of the african lungfish (protopterus dolloi) cross-reacts with myelin proteolipid protein antibodies, indicating a close phylogenetic relationship with amphibians. J. Neurochem. 46:1387-1391. 8. Linington, C., and Waehneldt, T.V. 1990. Conservation of the carboxyl terminal epitope of myelin proteolipid protein in the tetrapods and lobe-finned fish. J. Neurochem. 54:1354-1359. 9. Tosteson, M.T., and Sapirstein, V.S. 1981. Protein Interactions with Lipid Bilayers. The channels of kidney plasma membrane proteolipids. J. Membr. Biol. 63:77-84. 10. Sapirstein, V.S., and Rounds, T.C. 1983. Circular dichroism and fluorescence studies on a cation channel forming plasma membrane proteolipid. Biochem. 22:3330-3335. 11. Fischer, I., and Sapirstein, V.S. 1986. Characterization and biosynthesis of the plasma membrane proteolipid protein in neural tissue. J. Neurochem. 47:232-238. 12. Sapirstein, V.S., Nolan, C., Stern, Ciocci, M., and Masur, S.K. 1988. Identification of the plasma membrane proteolipid protein
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as a constituent of brain coated vesicles and synaptic plasma membrane. J. Neurochem. 51(3):925-933. Fischer, I., Cochary, E., Bizzozero, O., and Sapirstein, V.S. 1989. The plasma membrane proteolipid is enriched in myelin and oligodendrocytes. Trans. Amer. Soc. for Neurochem. 20(1):343. Sapirstein, V.S., Nolan, C., Puszkin, S., Marks, N., and Braun P. 1990. Regulation of white matter coated vesicles. Trans. Amer. Soc. for Neurochem. 21(1):233. Cochary, E.F., Bizzozero, O.A., Sapirstein, V.S., Nolan, C.E., and Fischer, I. 1990. Presence of the plasma membrane proteolipid (plasmolipin) in myelin. J. Neurochem. 55:602-610. Waehneldt, T.V., and Mandel, P. 1972. Isolation of rat-brain myelin monitored by polyacrylamide gel electrophoresis of dodecyl sulfate-extracted proteins. Brain Res. 40:419-436. Reiss, D.S., Lees, M.B., and Sapirstein, V.S. 1980. Is Na+K ATPase a myelin-associated enzyme? J. Neurochem. 36(4):14181426. Detskey, P.Z., Bigbee, J.W., and DeVries, G.H. 1988. Isolation and characterization of axolemma-enriched fractions from discrete areas of bovine CNS. Neurochem. Res. 13:449-454. Flynn, C.J., Nolan, C.E. and Sapirstein, V.S. 1989. Analysis of membrane constituents in purified rat brain axolemma. Trans. Amer. Soc. Neurochem. 20:139. Cotman, C.W. and Matthews, D.A. 1971. Synaptic plasma membranes from rat brain symaptosomes: isolation and partial characterization. Biochim. Biophys. Acta 249:380-394. Laemmli, U.K. 1970. Cleavage of structural proteins of the head of bacteriophage T4. Nature 227:680-685. Fischer, I., Kosik, K.S., and Sapirstein, V.S., 1987. Heterogeneity of microtubule-associatedprotein (MAP2) in vertebrate brains. Brain Res. 436:39-48. Grayson, S. 1988. Human epidermal proteolipids: isolation, partial characterization, and subcellular localization. J. Invest. Derm. 90:185-192. Arai, H. Berne M. and Forgac, M. 1987. Inhibition of the coated vesicle proton pump and labelling of a 17,000 dalton polypeptide by N,N'-dicyclohexyl-cabodiimide. J. Biol. Chem. 262:11000611011. Hou Lin, L.F., and Lees, M.B. 1982. Interactions of dicyclohexylcarbodiimide with myelin proteolipid. Proc. natl. Acad. Sci. 79:941-945. Bizzozero, O.A., McGarry, J.F., and Lees, M.B. 1987. Autoacylation of myelin proteolipid protein with acyl coenzyme A. J. Biochem. 262(8):13550-7. Burgisser, P., Matthieu, J.M., Jeserich, G., and Waehneldt, T.V. 1986. Myelin lipids: a phylogenetic study. Neurochem. Res. 11(9):1261-1272. Barres, B.A., Chun, L.L.Y., and Corey, D.P. 1990. Ion channels in vertebrate glia. Pp. 441.474 in Cowan, Shooter, Stevens and Thompson (eds.), Annual Review of Neuroscience, Annual Reviews, Inc. Palo Alto, CA.
The Phylogenic Expression of Plasmolipin in the Vertebrate Nervous System Victor S. Sapirstein 1, Charles E. Nolan 1, Itzhak Fischer 2, Elizabeth Cochary 2, Susana Blau 1, and Cheryl J. Flynn 1 (Accepted December 17, 1990)
Plasmolipin is a plasma membrane proteolipid is a major myelin membrane component (Cochary et al., 1990). In this study we report the phytogenic expression of plasmolipin in the vertebrate nervous system. Using Western blot analysis with polyclonat antibodies, we have analyzed membrane fractions, including myelin, from elasmobranchs, teleosts, amphibians, reptiles, birds and mammals. On the basis of immune detection, plasmolipin appears to be restricted to the mammalian nervous system. Comparison of the central and peripheral nervous systems of mammals showed only minor differences in the level of plasmolipin in these two regions. Within mammals, little quantitative differences were observed when rat, human and bovine membrane fractions were compared. The late evolutionary expression of plasmolipin which results in its restriction to mammals makes it unique among the (major) myelin proteins. The potential physiologic significance of these ,data are discussed. KEY WORDS: Plasmamembrane;proteotipid;myelin;evolution;mammalianbrain.
INTRODUCTION
between their CNS myelin proteins and mammalian PNS myelin proteins. The major CNS myelin proteins in these lower species (5,6) show cross-reactivity with antibodies to PO and an abundance of other glycosylated proteins (7). Amphibians and higher vertebrates show a different CNS pattern. For example, myelin proteolipid protein (PLP) and basic protein (MBP) are highly conserved and antibodies to these proteins isolated from mammals show cross-reactivity towards homologous proteins in amphibians and reptiles. In the case of PLP, this cross reactivity can be attributed to a high degree of C terminal sequence homology seen from tetrapods through mammals (8). Although the 18.5kD form of MBP is present in amphibians, the 21.5kD form is restricted to mammals (3). DM-20 protein, a protein closely related to PLP, is not observed in frogs and toads but is present as one advances to reptiles and higher vertebrates. The present study has focussed on the expression of plasmolipin. This protein is an 18kD hydrophobic
The evolution of the major myelin proteins has been examined from agnathans and elasmobranchs through primates (1-4). The pattern that has developed is that the central nervous systems (CNS) of elasmobranchs and teleosts express a pattern of myelin proteins distinct from that of amphibians and higher vertebrates. Although there are differences among these lower vertebrates, immunochemical and biochemical studies indicate a similarity 1 The Divisionof Neurobiology,The NathanKlineInstitute,Orangeburg, NY; and the Departmentof Psychiatry,New York University School of Medicine,New York, NY. 2 The Departmentof Biochemistry,The EuniceKennedyShriverCenter, Waltham,MA and Departmentof Neurology,HarvardMedical School, Boston, MA. Abbreviations:EDTA,EthylenediamineN,,N'N'tetraceticacid;EGTA, E~hyleneglycolbis-(B-AminoethylEther) N,,N'N'tetraceticacid; MES, (-[N-Morpholino] ethanesulfonicacid)DCCD, N,' Dicyclohexylcarbodiimidc 123
0364-3190/91/0200-0123506.50/0 9 1991 Plenum Publishing Corporation
124 plasma membrane protein which in vitro forms cation channels (9,10) and is abundant in the nervous system (11,12) and renal tubular epithelial cells (10) of mammals. The protein is comprised of two subunits of similar molecular weight; their parallel in vitro synthesis (11) and the requirement of both subunits for channel formation (9) suggests that they form a single biological unit. In brain, we have reported the abundance of this protein in oligodendrocytes (13) and several purified membrane fractions (14) including the membrane bilayer of clathrin coated vesicles (12) and isolated myelin (15). In myelin, plasmolipin represents 2.2-4.8% of the membrane protein. The present study has exclusively utilized Western blot analysis since this protein co-migrates with one of the MBP forms (18.5kD) and stains poorIy with Coomassie blue. The use of Western Blot with polyclonal antibodies provide a valid comparison with the expression of other major CNS myelin proteins which have been studied similarly (5-7). In this study we show that the expression of immunoreactive plasmolipin is restricted to mammals where it is found in both the CNS and PNS.
EXPERIMENTAL PROCEDURE Membrane Isolation. Membranes were prepared from various species obtained either from The Mount Desert Island Biological Research Laboratories, (Bar Harbour ME.), Western Scientific (Sacramento CA), Bronx Reptile Distributors (Mt. Vernon, NY), local slaughterhouses and the Nathan Kline Institute Animal Facility. Normal human cortical and subcortical material was obtained fresh from surgery at New York University Medical Center, University Hospital, during the excision of a brain tumor. Membranes were isolated in the presence of 10 U/ml of aprotinin (Sigma Chemical Co., St. Louis MO) and 25 ~xg/ml of leupeptin (Sigma Chemical Co.). Total membrane fractions were prepared from forebrain or upper brain stem, spinal cord or sciatic nerve (defined as peripheral nerve). Tissues were homogenized in 20 volumes of 0.32 M sucrose (BRL, Gaithersberg, MD) containing 10mM Tris (BRL) pH 7.5 and lmM EDTA (Sigma Chemical Co.). The homogenates were centrifuged at 500 g for 5 rain. in a Beckman J2-21 Centrifuge to remove nuclei and a post nuclear membrane fraction was obtained by centrifugation at 40,000 g for 30 min. Myelin was prepared by the Method of Waenheldt and Mandel (16) as modified by Reiss et al (17). Where beth myelin and axolemma were required, they were prepared by a modification of the procedure of Detskey et al (18). The myelin and axolemma obtained by this procedure were repurified. The myelin was osmotically shocked, sedimented at 12,000 g to remove residual microsomal contaminants and layered over 0.8M sucrose. The band above the 0.8 M sucrose was taken as purified myelin. The axolemma was repurified after lysis in 5 mM Tris-HC1, pH 8.5, containing 0.1mM EDTA. The lysate was made isotonic by the addition of 1/4 volume of 40% sucrose, layered over 0.8 and 1.0M sucrose and centrifuged at 75,000 g for 1 hour. The material obtained at the 0.8/1.0M sucrose interface was defined as purified axolemma. Characterization of this axolemmal material has been described elsewhere
Sapirstein, Nolan, Fischer, Coehary, Blau, and Flynn (19). Synaptic plasma membranes (SPM) were isolated as previously described (20). Gel Electrophoresis and Western Blot Analysis. All electrophoretic analysis was carried out using the procedure of Laemmli (22) with a 4-18% acrylamide gradient or 13% isocratic gels. Acrylamide and Bis acrylamide were obtained from National Diagnostics {Manville, NJ), while all other etectrophoretic reagents were purchased from BioRad (Riverside, CA). Preparation and scanning of Western blots were previously described (12). Isolation of plasmolipin and production of anti-plasmolipin antisera were described by Sapirstein and Rounds, (10) and by Fischer and Sapirstein (11). Antibodies to Na, K ATPase were a generous gift of Dr. W. Stahl, University of Washington. Protein determinations were performed on SDS solubilized membrane preparations using the dye binding method of Bradford with reagents from BioRad. All additional reagents were reagent grade and were purchased from either Fischer Scientific (Pittsburgh, PA) or Sigma Chemical Co.
RESULTS Distribution of Plasmolipin in the CNS and PNS. The distribution and phylogenic profile of plasmolipin was examined in brain, spinal cord and peripheral nerves. In the rat, total membrane fractions from these three areas were found to have roughly equivalent levels of plasmolipin, (Figure 1). In this analysis, the upper brain stem area was used instead of forebrain in order to obtain grey to white matter ratios similar to those found in other areas of the nervous system. The Western blot analysis indicated that the levels of plasmolipin in the CNS and PNS were very similar. Scanning of the bands showed the levels to be within a 20% range of each other with brain and spinal cord levels being higher than the peripheral nerve.
Fig.1. Distribution of plasmolipin in mammalian (rat) brain, spinal cord and peripheral nerve. Total membrane fraction (10 p.g) from brain (upper brain stem), lane 1; spinal cord (total spinal cord), lane 2; and peripheral nerve (sciatic nerve), lane 3 were analyzed by Western blot as described in Methods. Gel electrophoresis was performed on a 418% polyacylamide gradient.
Phylogenie Expression of Plasmolipin
125
In the CNS, plasmolipin is found predominantly in white matter tracts and is enriched in isolated myelin (15). Myelin was therefore isolated from the forebrain and peripheral nerve and each was analyzed for plasmolipin content, (Figure 2). Visual inspection of these Western blots indicate that the level of the protein in CNS and PNS were similar, with CNS myelin levels being slightly higher than PNS.
Phytogenic Expression of Plasmolipin in Vertebrates. The phylogenic diversity of plasmolipin was analyzed in the CNS and PNS of different vertebrate species by examining total membrane fractions isolated from amphibians (frog), reptile (lizard), birds (chicken) and mammals (rat). The abundance of plasmolipin usually allows for its detection in relatively small samples of total brain membrane fractions. However, because preliminary studies yielded negative results in lower animals, the experiment illustrated in Figure 3 compared 20 ug of mammalian protein to 100 ug from the other species. The results indicated that based on immune reactivity, plasmolipin was absent in forebrain of nonmammalian species (Figure 3A). From these results and our estimated limits of detection of pIasmolipin in rat membranes, (2-5 ~g), we calculated that these lower
Fig. 2. The level of plasmolipin in purified rat myelin isolated from CNS and PNS. Myelin was isolated as described in Methods; 1,2 and 5 ~g were analyzed from CNS (whole forebrain), lanes 1,2, and 3, respectively; and PNS (sciatic nerve), lanes 4,5, and 6, respectively, were analyzed by Western blot.
Fig. 3. Phylogenic expression of plasmolipin. Total membrane fraction from whole brain (A), spinal cord (B) and sciatic nerve (C) were analyzed by Western blot. Each of these regions were studied in rat, lane 1; chicken, lane 2; lizard, lane 3; and frog, lane 4. Rat was analyzed using 20 ~g protein while other fractions were tested at 100 Ixg.
species had less than 2-5% of the level of plasmolipin in the rat brain. The forebrain is both ontologically and phylogenetically younger than the spinal cord and peripheral nervous system; however attempts to detect plasmolipin in these two regions in amphibians, reptiles and birds yielded similar negative results, (Figures 3B and C). The results described here were extended to other non-mammalian species, including pigeon, turtle, toads, mud puppy and salamander with identical negative results (data not shown). As noted earlier, plasmolipin is highly enriched in isolated myelin and in an attempt to amplify the plasmolipin signal in lower vertebrates, we examined this protein in purified myelin isolated from these animals, (Figure 4). Five ~g of rat myelin was used and compared with 10 ~g of myelin from the other species. Purification of myelin failed to yield any detectable levels of plasmolipin in any non-mammalian species. In a previous report from this laboratory we described a proteolipid protein extracted from preparations of chicken synaptic plasma membrane (11). At the time
126
Sapirstein, Nolan, Fischer, Cochary, Blau, and Flynn 69
K--
46
K--
30
K--
21.5 K-14.3 K--
A
B C D E
F G H
I J
K
Fig. 4. Phylogenic analysis of plasmolipin in vertebrate myelin. Myelin was prepared as described in Methods and analyzed for the presence of plasmolipin by Western blot. Human, lane A; bovine, lane B; rat, lane C; chicken, lane D; lizard, lane E; turtle, lane F; frog, lane G; goldfish, lane H; shark, lane I, skate, lane J; human PNS, lane K. All lanes contained 10 p-g of myelin protein, except lane 3 that contained 5 p-g of rat myelin. All myelin was derived from CNS except for human PNS in lane 11.
Fig. 5. Comparison of plasmolipin in rat and chicken synaptic plasma membranes. Synaptic plasma membrane was purified from rat and chicken brain; rat SPM was taken as defined previously (12). Chicken synaptosomes were lysed as described (12) and membrane sedimenting on 0.8 M and 1.0 M sucrose were defined as SPM fractions. Lane 1, rat SPM (10 p-g); lanes 2 and 3, chicken SPM (20 p.g) from 0.8 and 1.0M sucrose respectively; lanes 4 and 5, chicken SPM fractions (50 ~xg) and lanes 6 and 7, chicken SPM fractions (100 Ixg). Plasmolipin analysis was as described in Figure 1.
the protein was isolated, anti-pIasmolipin antibodies were not available, but a relationship of the protein to plasmolipin was implied. The lack of reactivity of anti-plasmolipin with chicken brain membranes, (Figures 3 and 4), prompted a re-examination. Synaptic plasma membrane was isolated from chicken and 10-100 Ixg of protein was analyzed by western blot, (Figure 5). For this analysis, we employed a second polyclonal antibody against plasmolipin in order to exclude antibody bias. Since SPM sediments at 0.8 M and 1.0 M sucrose, we analyzed both fractions from the chicken in order to preclude sampling error. The data indicated that in all of the samples no
detectable reactivity between plasmolipin and chicken SPM could be observed. In contrast, the analysis of 10 txg of rat SPM gave a clear signal, lane 1. The restriction of plasmolipin to mammalian brain prompted us to look for phylogenic differences within this vertebrate class, examining rat, bovine and human material. We confined our analysis to purified membrane fractions so as not to avoid variation due to differential contributions of myelinated and non-myelinated regions. We isolated myelin and an axolemmal enriched fraction which is released from myelin by osmotic lysis in the presence of EDTA, (see Methods). The data in Figure 6 indicated that the level in myelin and the myelin derived axolemmal fractions were similar across the mammalian species studied. Scanning of these Western blots (Table 1) showed that quantitatively all three myelin fractions from these species were vitually identical. In all cases the levels of plasmolipin were identical in myelin and axolemma enriched fractions were indistinguishable. The only significant difference was that the human material in both membrane fractions exhibited a slightly higher molecular weight for one of the plasmolipin sub-
6. Plasmolipin levels in myelin and axolemma enriched fractions in different mammalian species. Plasmolipin in myelin and axolemma were compared from human, bovine and rat brainstem isolated as described in Methods. Three p-g of protein was analyzed from each fraction. Lanes 1 and 2 are human axolemma and myelin, lanes 3 and 4 are bovine axolemma and myelin, and lanes 5 and 6 are rat axolemma and myelin. Analyses were carried out as described in Figure 1. Fig.
Table
I. Relative Level of Plasmolipin in Myelin and Axolemma Enriched Fractions
Membrane Fraction Myelin Axolemma
Rat
Specie Human
Bovine
100"
93 _ 6
89 -+-8
102 +_+_8* *
98 +_.12
91-4-13
* Rat myelin was taken as 100% ** Values represent the mean of 3 Western blots -- the standard deviation.
Phylogenic Expression of Plasmolipin units, differing from the rat and bovine material by approximately 500 daltons.
DISCUSSION The results of this study indicate that the expression of plasmolipin is restricted to mammals in both the peripheral and central nervous systems. In mammals, the level of this protein in CNS and PNS is similar, suggesting that plasmolipin is important to both areas of the nervous system. The absence of plasmolipin in avian brain is striking, since the avian nervous system is more similar to the mammalian than reptilian nervous system. However, significant differences exist between mammalian and avian brains, including the absence of cortical layers in birds and the lack of a corpus callosum. The phylogenic pattern of plasmolipin may be a reflection of either limited antibody cross-reactivity or the absence of protein in the non-mammalian nervous systems. It is common to see variability in immune reactivity when using monoclonal antibodies, since these epitopes often show widely different phylogenic patterns (23). In contrast, polyclonal antibodies can detect even small regions of homo]!ogy. For example, the extensive phylogenic cross reactMty of PLP antisera with lungfish is reactivity with only a C.-terminal sequence of PLP (8). The present study with two different polyclonal antibodies indicated that, if present, pIasmolipin must have undergone significant evolutionary change. Chick brain synaptic plasma membrane has a proteolipid protein of molecular weight similar to plasmolipin which might exist in other non-mammalian species (11). This proteolipid was nor. analyzed for reactivity with antiplasmolipin. However, since our report it has become clear that other proteolipids exist in the plasmolipin molecular weight range. A 16-17kD (19) proteolipid protein is present in several tissues and was isolated from keratinocytes (23) and showed no cross-reactivity with anti-plasmolipin, (data now shown). In addi'Iion, the major DCCD binding protein of golgi and plas;ma membrane derived vesicles is a also proteolipid and has a molecular weight of 16-17kD (20). However, we have been unable to detect DCCD binding to plasmolipin (data not shown). This finding confirms the report of Lin and Lees (25) which indicates that in myelin, only the myelin PLP reacts with DCCD. These data, along with the lack of immunereactivity of plasmoIipin antibodies with chicken SPM, suggest that the proteolipid present in these preparations is unrelated to plasmolipin. Comparison of rat, bovine and human material indicates that the high level of plasmolipin is sustained across
127 mammalian species. Rat preparations showed tighter plasmolipin bands in Western blots than did human or bovine. This may have been due to the rapid post mortem processing of the rodent material that minimized degradation. The minor difference in one of the human plasmolipin subunits, although 500 daltons or less, suggests some small compositional difference in human plasmolipin in relation to other mammals exists. Proteolipids are acylated (26) and the electrophoretic variations observed in humans may result from fatty acid differences. The restricted pattern of phylogenic expression of plasmolipin is unique among major myelin proteins. The major CNS myelin proteins are not exclusive to mammals, but are present in all air breathing vertebrates (15). The exception to this is the 21.5kD form of MBP which is restricted to mammals (3). The absence or low level of these CNS myelin proteins in aquatic species can be explained, in part, by the nature of myelin among teleosts and elasmobranchs. Their myelin is comprised mostly of PNS-like proteins, since antibodies to PO protein react with proteins in teleost and elasmobranch CNS myelin (1,5-7). However, unlike the PLP and MBP, plasmolipin is present in equal abundance in the PNS and CNS and could, therefore, be expected to have a broader phylogenic range. This is not the case. In fact, plasmolipin appears to have the most restricted phylogenic range among the myelin proteins studied so far, with mammals representing a sharp transition in the expression of plasmotipin. The relative conservation of PLP and MBP and Wolgran protein as well as the major myelin lipids (27) have been considered as evidence for their importance in myelin structural integrity. We propose that the restriction of plasmolipin to mammals reflects a specific physiologic function that is characteristic of the mammalian nervous system. The ability of plasmolipin to form K § channel (9) suggests that its function may be related to the regulation of membrane excitability. This hypothesis is consistent with recent physiologic studies which have demonstrated K + channels of oligodendrocytes (27). This hypothesis is also consistent with the increased complexity of the mammalian nervous system. The demands of increased neural activity requires complex factors to modulate the flow of information between brain regions. Plasmolipin and other potential factors may therefore be important for the higher integrative neural function associated with the mammalian brain. ACKNOWLEDGMENTS The authors would like to acknowledgethe technical assistance of Gladys Gray-Boardand Roz Cohen. The authors also thank Dr.
128 Oscar Bizzozero for his helpful comments on the manuscript. This work was supported by NS24894, NS25950 and HD05515.
Sapirstein, Nolan, Fischer, Cochary, Blau, and Flynn
13. 14.
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