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A model for lamellar myelin
J. theor. Biol. (1979) 80, 101-113
A Model for Lamellar Myelin RODMAN G. MILLER
The Salk Institute,
P.O. Box 1809, San Diego, Ca. 92112, U.S.A.
(Received 23 September 1977, and in revised,for 7 March 1979) A model for the arrangement of the proteolipid and basic proteins in lamellar myelin is presented. The model suggests that the basic protein extends from the major dense line to interact with the proteolipid beyond the second major dense line. Observations from a variety of techniques are compatible with the model.
1. Introduction
Lamellar myelin is formed by an extension of the plasma membrane of the Schwann cell in the peripheral nervous system and the oligodendrocyte in the central nervous system. Cytoplasm is extruded from this extension allowing a close apposition of the cytoplasmic surfaces of the membranes which is seen in thin section electron microscopy as the main dense line (MDL). The extension forms a spiral wrapping around a segment (an internode) of an axon with the extracellular surfaces of the membrane in a relatively close apposition. This structure is seen in thin section electron microscopy and defined as the interperiod line (IPL). The mechanism whereby myelin is stabilized in this characteristic structure is an unknown. Studies from this laboratory and interpretation of data in the literature have led to a model for the interactions of the two major proteins in central myelin that explains the stabilization of myelin structure. Research on the structure of membranes has indicated that proteins play a major role in the determination of the shape and organization of membranes. In most cells, shape is determined by cytoplasmic proteins which are attached to the membrane (e.g., microfilaments and spectrin). In the case of myelin membranes, the close apposition of the membranes seems to exclude the more traditional sorts of proteins and call for protein(s) which Present address: Institute of Histology & Embryology, University of Geneva Medical School, 1211-Geneva 4, Switzerland. 101
R. G. MILLER 102 are more specialized to accomplish the task of holding two or more membranes together with a specific spacing. Lamellar myelin membrane is biochemically unique. It contains an unusual quantity of lipids (about 65% vs. 40-50X for most other membranes) which are unusually varied relative to other membranes. On the other hand, two proteins in CNS myelin are present in roughly equimolar quantities and constitute about 90% of the total protein on a molar basis. One of these proteins, the proteolipid (PL), is extremely hydrophobic, has a molecular weight of 25,000 and seems to contain covalently bound lipid. The other major protein in CNS myelin is the basic protein (BP) which has a molecular weight of 18,000. Since autoimmunity to BP produces a disease (experimental allergic encephalites) which bears some resemblance to multiple sclerosis, considerable effort has been devoted to the study of BP. The primary sequence of BP has been determined for a number of species (for reviews, see Braun & Brostoff, 1977; Carnegie & Dunkley, 1975). Central (A,) and peripheral (Pi) basic proteins have identical amino acid sequences.
2. The Model The proposed model claims that the BP and the PL are associated to form a unit will bridge both the MDL and IPL. The PL is globular and resides within the hydrophobic matrix of the membrane. The BP on the other hand, is considered to be in an extended form and traversing more than one membrane; it will be shown below that traversing two membranes and interacting with a third is sufficient to account for many properties of myelin without invocation of dimers. The BP can be divided into several functional IPL
MD1
IPL
IPL FIG. 1. The proposed model for lamellar myelin. Bar represents the BP; stippled circle represents the proteolipid; dark circles with tails represent lipid; functional regions and BP designated A through F. The elements in the figure are not drawn to scale (see text).
LAMELLAR
MYELIN
103 regions labeled A through F in Fig. 1. Region A resides in the main dense line (MDL) and can be considered as an anchor for the rest of the protein which goes through the adjacent two membranes. Regions B and D pass through the hydrophobic matrix of the membranes. Region C takes the protein across the interperiod line (IPL). Region E takes the protein into the MDL adjacent to the MDL in which A resides and region F enters a third membrane where the BP interacts with other major protein, the proteolipid (PL). This model, albeit unusual in suggesting that a protein may traverse two membranes and enter a third, seems to reconcile data on myelin and its constituents obtained through a variety of techniques. 3. Intact Myelin Thin section electron microscopy and diffraction studies of myelin clearly demonstrate asymmetry of the myelin membranes. Osmium deposition is most intense in the intracellular MDL. Although the mechanisms of osmium fixation are not well understood, its deposition is usually correlated with high protein concentration and thus by this criteria there is more protein associated with the MDL than the IPL. When it was believed that there were no proteins in the center (hydrophobic region) of the membrane, asymmetries within the membrane demonstrated by diffraction techniques were assigned to lipid asymmetries (especially cholesterol) (e.g. Casper & Kirschner, 1971). Indications of particles on the fracture faces of myelin (Pinto da Silva & Miller, 1975) and reconstituted bilayer membrane systems which contain either the PL (Vail, Papahadjopoulos & Moscarello, 1974; Boggs, Vail & Moscarello, 1976; Paphadjopoulos, Vail & Moscarello, 1975) or even BP (Guilik Krzywicki, 1975, Fig. 10A lower portion of the micrograph) make it clear that either or both of these proteins can be invoked to account for asymmetries (see below). Since there exists an asymmetry to myelin membranes, there have been many attempts to localize one of the proteins, BP, on an ultrastructural level in either the MDL or IPL. The results of these studies have led to seemingly contradictory conclusions (e.g. see Adams, Bayliss, Halpike & Turner, 1971; Bubis & Wolman, 1968; Cullen, Peterson & Webster, 1977; Dickinson, Jones, Aparicio & Lumsden, 1970; Golds & Braun, 1976; Hemdon, Rauch & Einstein, 1973; Stemberger, Tabira, Webster & Kies, 1977; Wood, Epand & Moscarello, 1977). In the intact myelin membrane, the model shown in Fig. 1 suggests that the reason for ambiguity in the location of the BP is that it is situated throughout the myelin membrane. Thus if one looks for antigenic sites, one
R. G. MILLER 104 will localize it in the MDL or possibly the IPL. Most immunolabeling has localized the BP in the MDL in myelin which has been disrupted to allow penetration of the antibody. Thus while explicit localization of the BP and/or the PL is difficult through thin section electron microscopy, it is likely that a major portion of the protein in myelin is associated with the MDL. In this laboratory, much of the initial work of freeze fracture of myelin utilized central and peripheral myelin from the rat. This myelin, when fixed with glutaraldehyde, demonstrates no difference between the P (A) and E (B) faces. The particles seen under these conditions are pleomorphic, ranging in size from 3 nm to 7 nm. On the other hand Mugniani & Schnapp (1976) have found major differences between the P and E faces of aldehyde fixed frog central myelin; on the P face they find many particles which are bar shaped and relatively fewer particles on the E face. The sedimentation coefficient for the PB in aqueous media is consistent with a prolate spheroid with dimensions of 15 x 1.5 nm (Epand, Moscarello, Zierenberg & Vail, 1974). Schnapp & Mugniani point out that the bar shaped particles they find are of about these dimensions. In the present report, I have used another preparative technique for freeze fracture: fresh freezing. In freshly frozen rat myelin, there is a great difference between the P and E faces (Miller, in preparation). One of these faces appears covered with bar shaped particles while the other contains fewer particles and few bar shaped particles (see Plate 1). By counting fracture planes from the outside or from the axonal membrane, it is possible to determine that the particulate face is the E face. While it has been shown that the perferred cleavage plane in freeze fracture is within the hydrophobic matrix of the membrane (Pinto da Silva & Branton, 1970) it would seem that a structure which passes through a membrane could either cleave in the center of the membrane, breaking that structure in two, or be pulled through the membrane. If the structure pulled through is flexible, it is likely to “fall” to the surface and become partially embedded in the hydrophobic tails of the lipid rather than remain standing in the vacuum. I (1977) have suggested that bar shaped particles on freshly frozen axolemmal (E) face of the rat are created by this mechanism. Assuming that structures can be pulled through membranes, these changes in face morphology associated with the different preparative procedures accord well with the proposed model: in the freshly frozen tissue the membrane transversing portion of the BP is pulled through the bilayer adjacent to the observed fracture face. It is postulated that the BP would then “fall” and be shadowed as a bar-shaped (as opposed to a perpendicular post-shaped) particle. Fixation with a cross-linking agent (glutaraldehyde) is expected to inhibit the ability of a protein to be pulled through a membrane and thus the
LAMELLAR
MYELIN
105
fracture must cleave the peptide, creating the observed variations in the particle morphology. There is a major constraint that must be placed on the arrangement of proteins in myelin: under most preparative procedures (freeze fracture, and thin section electron microscopic and light microscopic), myelin undergoes “neurokeratinization” (see Nageotte, 19 10 ; Tewari & Bourne, 1960 ; Wolman, 1969). In light microscopy, especially with cross polarized iight, this effect produces a beaded appearance of the sheath and in thin section electron microscopy it produces regions where the lamellar structure is absent or distorted, probably depending upon the degree of delipidation during dehydration for embedment. Neurokeratinization has received little attention by electron microscopists and is rarely presented in the recent literature probably because it is recognized as an artifact. On the light microscopic level, Nageotte considered neurokeratinization an artifact, but one which could potentially tell us a great deal about myelin. In terms of freeze fracture, neurokeratinization manifests itself as particle free regions which are propagated radially through many myelin lamallae (Pinto da Silva & Miller, 1975). Since both the BP and PL may be visible through freeze fracture (see below), the propagation of the regions devoid of particles suggested an intimate connection between the two proteins. Therefore there must be interactions which extend across both the MDL and the IPL. The paucity of particles on the freshly frozen A face implies that, under these conditions, the PL is more closely associated with the IPL. Given the hydrophobicity of the PL, this association could be accomplished by interaction of the PL with the BP at regions B, C or F in Fig. 1. Some of the original studies of diffraction of myelin membranes demonstrated that the normal repeat distance was 170 A. Upon osmotic swelling the repeat distance increases to 250-270 A (Finean & Millington, 1957 ; see also McIntosh & Robertson, 1976). In terms of the present model, this might be represented as shown in Fig. 2. Figure 2 suggests that the region of the BP labeled F is reinserted into the adjacent membrane increasing the width of the IPL by the thickness of one membrane. The repeat distance predicted by this model (assuming no configurational changes occur in BP upon swelling) is 255 A.
Additional evidence from swelling of myelin comes from thin section electron microscopy of cultured myelin which has been exposed to heat inactivated serum from experimental allergic encephalitis animals. Bornstein & Raine (1976) have shown that, under these conditions the IPL, which is usually seen as a pair of thin electron dense lines, separates to form four thin
106
R. G. MILLER
LAMELLAR
MYELIN
107
lines as the myelin period doubles. Of these lines, the pair that are adjacent to the MDL are the most consistent and dense; in many regions, the four lines are quite distinct and suggest that another bilayer membrane has been formed. As seen in the light of the present model, a factor in the serum induces the BP to withdraw region F from one membrane and reinsert it in the adjacent membrane. If specific lipids bind to the BP at D, there may exist regions where an additional membrane is formed. This model predicts that there may be boundary effects associated with the initial (internal and external) layers of myelin membranes. Such effects have been noted by Mugniani, Osen, Schnapp and Friedric (1978) on the external layer of frog peripheral myelin. Effects of this sort have been observed in this laboratory in peripheral myelin of the rat which has been freshly frozen (in preparation).
4. Reconstitution
of Myelin Components
London and his coworkers (Demel, London, Geurts van Kessel, Vossenberg & van Deenen, 1973 ; London & Vossenberg, 1973) have shown that, at a lipid monolayer at the air-water interface, the BP is partially inserted into the hydrophobic tails of the lipid (see also Gould & London, 1972). This insertion can be measured as a change in surface tension, a change in surface radioactivity due to labeled BP, and a protection of certain portions of the BP from proteolytic attack from beneath the monolayer. In the monolayer system, the C-terminal portion of the BP (residues 106170) are available to lytic attack. Taken with the open configuration of the BP (e.g. see Block, Brady & Joffe, 1973 ; Epand, Moscarello, Zierenberg & Vail, 1974; Krigbaum & Hsu, 1975, but c.f. Anthony & Moscarello, 1971; Chapman & Moore, 1976, for reviews see Carnegie & Dunkley, 1975 ; Braun & Brostoff, 1977), these authors suggest that in the monolayer system, the BP must go in and out of the monolayer at least two times and up to 5 times (Fig. 3):
FIG. 3. Lipid
monolayer
with
BP partially
inserted
(after
London
er al.. 1973).
R. G. MILLER 108 However, when one extrapolates these results to a bilayer or multilayer system, other possible configurations emerge (Fig. 4):
FIG. 4. Alternative locations for BP in bilayers and multiiayers suggested by monolayer studies.
In all of these cases, the peptide chain passes through the membrane; in the monolayer this option is not available. Other possible configurations which are consistent with the monolayer studies are hybrids of Figs. 3 and 4 where some portions of the BP traverse the whole membrane and other portions of the BP are partially inserted into the membrane. The present model predicts that lytic attack of BP in a multilayered system will produce different peptides than lytic attack in the London monolayer system. X-ray diffraction of reconstituted membrane lipids (Mateu et al., 1973) clearly shows that BP induces a repeate distance indicative of two bilayer membranes. These data can be explained in terms of the BP residing predominantly between the two bilayers and inducing a conformational change in all four leaflets such that association of other BP to the outside surface of the double bilayer is not possible (see Mateu et al., 1973). Another interpretation which makes less stringent assumptions upon exclusive association of the BP with the four lipid monolayers is that the BP extends through two (or more) of the membranes (see Fig. 5). This would have the effect of a two dimensional zipper which would inhibit insertion of the BP from between the double bilayer and thus inhibit the formation of multilayered repeat patterns. The PL is present in roughly equimolar quantities to the BP (Mehl and Halaris, 1970) and is extremely hydrophobic. A priori, we might expect that the PL will reside within the hydrophobic matrix of the membrane and be visible as particles in freeze fracture (but c.f. Golds & Braun, 1976). Freeze fracture of reconstituted membranes containing PL clearly demonstrate particles (see above). These particles, however, are globular and do not look
LAMELLAR
MYELlN
109
FIG. 5. Suggested insertion of BP into multilayer lipids to produce double bilayer repeat distance.
like those seen in intact myelin membranes (e.g. see Vail er al., 1974). This suggests that either the reconstitution is not biologically accurate, or the barshaped particles and pleomorphism of the particles seen in intact myelin results from a combination of the PL with BP. Lipid specificities have been described for both the proteolipid and the BP (e.g. see Schafer & Franklin, 1975; Hemminga & Post, 1976; Palmer & Dawson, 1969; Demel et al., 1973; London et al., 1974; Paphadjopoulos, Moscarello, Eylar & Isac, 1975). Sequestration of non-cholsterol lipids by protein binding may play an important role in altering the phase transition properties of myelin membranes (see Miller & Torreyson, 1977). I have proposed that the two proteins interact with one another; since both have lipid specificities, this interaction could be through mutual binding to a lipid. 5 Biochemistry Since the amino acid sequence of the BP is well known, it might be possible to predict which regions correspond to regions A through F in Fig. 1. There are few protein sequences known that take a protein across a membrane (Tomita & Marchesi, 1975 ; 0~01s & Gerrard, 1977). In all documented cases to date, a protein traverses a membrane in a helix with the peptide bonds in the center of the helix and hydrophobic moieties inserted into the hydrophobic matrix of the membrane. To do this requires a sequence of about 20 hydrophobic amino acids. The amino acid sequences of BP is high in helix breaking residues. One region in which a secondary structure can be surmised on the basis of the sequence is the proline rich region (residues 96-101 : numeration is according to Carnegie & Dunkley, 1975) which seems to be bent back upon itself. Physical studies have shown that the BP in aqueous solutions contains little or no helical structure. There are two regions of about 10 residues which are predominantly hydrophobic (residues 34-46 and 85-101 have 9 and 11 hydrophobic residues respectively). If both of these extend the chain across a
R. G. MILLER 110 membrane, the BP would form a structural bridge across the IPL. It should be noted, however, that these regions also contain charged amino acids which have to be neutralized either by interaction with lipids or interaction with the PL (c.f. Hemminga 8~ Post, 1976). Thus, although the amino acid sequence of the BP is well known, unequivocal assignment of specific sequences to regions A through F in Fig. 1 is not possible. This may be because the generalization that proteins which traverse membranes do so in a hydrophobic helix is not universally applicable. One possible arrangement for the peptide would be for the BP to be in a largely open configuration with residues 100-170 acting as an anchor in the MDL and residues I-100 extending perpendicularly to the MDL. There are enough residues to traverse 200, 280 and 350 A if these residues are in a 3,, helix, 2, ribbon and /? antiparallel sheet respectively (Dickerson & Geis, 1969). Any combination of these configurations would be capable of producing a peptide long enough to extend beyond the myelin period (180 A). There are indications of polymerization of the BP (e.g. Martenson & Deibler, 1975; which might allow for the possible b antiparallel sheet in some regions.?
6 Other Models Another model which has been considered (see London et al., 1973) places the BP in either the MDL or IPL with hydrophobic regions of the BP extending into the adjacent membranes. The BP in this model is in the same configuration as it assumes in the monolayer system. A variant of this model would allow the hydrophobic regions to insert into both of the adjacent membranes (see also Smith, 1977). In either of these variants, the BP can form a structural link across either the MDL or IPL. To obtain neurokeratinization, the PL would have to form the structural link across the other. The PL, being extremely hydrophobic, is not expected to bridge the aqueous IPL (but c.f. Golds & Braun, 1976). The ambiguity of localization studies of BP do not argue strongly for any assignment. Thus the likely assignment of positions would be that the BP is in the IPL and the PL forms the link across the MDL. The pleomorphism of the freeze fracture particles and similarity of P and E fracture faces seen in fixed mammalian tissue, and t Since submission of this manuscript, E. E. Golds & P. E. Braun (J. Biol. Chem. 253,8162) have reported the use of bifunctional reagents to cross-link lysine residues in intact central myelin. In this manner they have shown that major quantities of BP dimers (and not multimers) are formed by linkage of lysine residues at the terminal regions of the BP while the center region of the BP is arranged in such a way as to separate lysine moieties by more than 9 A. On the basis of this and other evidence, the authors propose that, in viva, the BP is an antiparallel dimer which forms a structural link across and within the MDL.
LAMELLAR
111 associated with fresh freezing are
MYELIN
the alteration of particle morphology difficult to explain under this model. In the present model, I have assumed that one PL is associated with one BP. Using the same arguments, more efficient, and at this time more speculative, models could be made assuming valencies of more than one for either or both of the proteins. Dimerization of either the PL or the BP would all permit the BP to perform the same tasks proposed by traversing a single membrane. There is evidence of dimer or trimer forms of the PL. Unfortunately experiments which would determine the associations and/or valencies of the PL and BP are difficult to perform due to their differences is solubilities (c.f. Block et al., 1973). This model does not make any predictions concerning the configuration of the C terminal region of the BP (region A in Fig. 1); it is possible that there is an interaction in this region with the same or another BP, with the PL (see Smythies, Benington & Morin, 1972) or that this region is in an extended form interacting with charged groups of the lipids. Since central myelin is biochemically better characterized and contains fewer proteins than peripheral myelin, the present model has been formulated on the basis of data obtained from central myelin. Although the model applies most directly to central myelin, the similarity in ultrastructure and function and the identity of one of the proteins (BP) suggests that similar mechanisms are operating to stabilize the lamellar organization in both types of myelin. In conclusion, I am proposing a model for lamellar myelin in which the basic protein has an organizing role by passing through two membranes and interacting with the proteolipid in a third. The C-terminal region of the basic protein acts as an anchor in the MDL for the rest of the protein and interaction of the N-terminal end of the protein with the proteolipid forms a structural link which binds the myelin membranes together in their characteristic pattern.
I thank Fred Westall for encouragement and helpful discussions,Dave Schubert for his generoussupport and useof his laboratory, Bonne Harkins and Nadine Maalaoui for preparation of the manuscript,Gary Jonesand JamieSimon for art work, and FrancisCrick, Jim Patrick and Joe-Henry Steinbachfor comments on the manuscript.This work wassupportedby the National Institutesof Health (to D. S.) and wasdone during the tenure of a ResearchFellowshipof the Muscular Dystrophy Association. REFERENCES C. W. hf., 389.
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A Model for Lamellar Myelin RODMAN G. MILLER
The Salk Institute,
P.O. Box 1809, San Diego, Ca. 92112, U.S.A.
(Received 23 September 1977, and in revised,for 7 March 1979) A model for the arrangement of the proteolipid and basic proteins in lamellar myelin is presented. The model suggests that the basic protein extends from the major dense line to interact with the proteolipid beyond the second major dense line. Observations from a variety of techniques are compatible with the model.
1. Introduction
Lamellar myelin is formed by an extension of the plasma membrane of the Schwann cell in the peripheral nervous system and the oligodendrocyte in the central nervous system. Cytoplasm is extruded from this extension allowing a close apposition of the cytoplasmic surfaces of the membranes which is seen in thin section electron microscopy as the main dense line (MDL). The extension forms a spiral wrapping around a segment (an internode) of an axon with the extracellular surfaces of the membrane in a relatively close apposition. This structure is seen in thin section electron microscopy and defined as the interperiod line (IPL). The mechanism whereby myelin is stabilized in this characteristic structure is an unknown. Studies from this laboratory and interpretation of data in the literature have led to a model for the interactions of the two major proteins in central myelin that explains the stabilization of myelin structure. Research on the structure of membranes has indicated that proteins play a major role in the determination of the shape and organization of membranes. In most cells, shape is determined by cytoplasmic proteins which are attached to the membrane (e.g., microfilaments and spectrin). In the case of myelin membranes, the close apposition of the membranes seems to exclude the more traditional sorts of proteins and call for protein(s) which Present address: Institute of Histology & Embryology, University of Geneva Medical School, 1211-Geneva 4, Switzerland. 101
R. G. MILLER 102 are more specialized to accomplish the task of holding two or more membranes together with a specific spacing. Lamellar myelin membrane is biochemically unique. It contains an unusual quantity of lipids (about 65% vs. 40-50X for most other membranes) which are unusually varied relative to other membranes. On the other hand, two proteins in CNS myelin are present in roughly equimolar quantities and constitute about 90% of the total protein on a molar basis. One of these proteins, the proteolipid (PL), is extremely hydrophobic, has a molecular weight of 25,000 and seems to contain covalently bound lipid. The other major protein in CNS myelin is the basic protein (BP) which has a molecular weight of 18,000. Since autoimmunity to BP produces a disease (experimental allergic encephalites) which bears some resemblance to multiple sclerosis, considerable effort has been devoted to the study of BP. The primary sequence of BP has been determined for a number of species (for reviews, see Braun & Brostoff, 1977; Carnegie & Dunkley, 1975). Central (A,) and peripheral (Pi) basic proteins have identical amino acid sequences.
2. The Model The proposed model claims that the BP and the PL are associated to form a unit will bridge both the MDL and IPL. The PL is globular and resides within the hydrophobic matrix of the membrane. The BP on the other hand, is considered to be in an extended form and traversing more than one membrane; it will be shown below that traversing two membranes and interacting with a third is sufficient to account for many properties of myelin without invocation of dimers. The BP can be divided into several functional IPL
MD1
IPL
IPL FIG. 1. The proposed model for lamellar myelin. Bar represents the BP; stippled circle represents the proteolipid; dark circles with tails represent lipid; functional regions and BP designated A through F. The elements in the figure are not drawn to scale (see text).
LAMELLAR
MYELIN
103 regions labeled A through F in Fig. 1. Region A resides in the main dense line (MDL) and can be considered as an anchor for the rest of the protein which goes through the adjacent two membranes. Regions B and D pass through the hydrophobic matrix of the membranes. Region C takes the protein across the interperiod line (IPL). Region E takes the protein into the MDL adjacent to the MDL in which A resides and region F enters a third membrane where the BP interacts with other major protein, the proteolipid (PL). This model, albeit unusual in suggesting that a protein may traverse two membranes and enter a third, seems to reconcile data on myelin and its constituents obtained through a variety of techniques. 3. Intact Myelin Thin section electron microscopy and diffraction studies of myelin clearly demonstrate asymmetry of the myelin membranes. Osmium deposition is most intense in the intracellular MDL. Although the mechanisms of osmium fixation are not well understood, its deposition is usually correlated with high protein concentration and thus by this criteria there is more protein associated with the MDL than the IPL. When it was believed that there were no proteins in the center (hydrophobic region) of the membrane, asymmetries within the membrane demonstrated by diffraction techniques were assigned to lipid asymmetries (especially cholesterol) (e.g. Casper & Kirschner, 1971). Indications of particles on the fracture faces of myelin (Pinto da Silva & Miller, 1975) and reconstituted bilayer membrane systems which contain either the PL (Vail, Papahadjopoulos & Moscarello, 1974; Boggs, Vail & Moscarello, 1976; Paphadjopoulos, Vail & Moscarello, 1975) or even BP (Guilik Krzywicki, 1975, Fig. 10A lower portion of the micrograph) make it clear that either or both of these proteins can be invoked to account for asymmetries (see below). Since there exists an asymmetry to myelin membranes, there have been many attempts to localize one of the proteins, BP, on an ultrastructural level in either the MDL or IPL. The results of these studies have led to seemingly contradictory conclusions (e.g. see Adams, Bayliss, Halpike & Turner, 1971; Bubis & Wolman, 1968; Cullen, Peterson & Webster, 1977; Dickinson, Jones, Aparicio & Lumsden, 1970; Golds & Braun, 1976; Hemdon, Rauch & Einstein, 1973; Stemberger, Tabira, Webster & Kies, 1977; Wood, Epand & Moscarello, 1977). In the intact myelin membrane, the model shown in Fig. 1 suggests that the reason for ambiguity in the location of the BP is that it is situated throughout the myelin membrane. Thus if one looks for antigenic sites, one
R. G. MILLER 104 will localize it in the MDL or possibly the IPL. Most immunolabeling has localized the BP in the MDL in myelin which has been disrupted to allow penetration of the antibody. Thus while explicit localization of the BP and/or the PL is difficult through thin section electron microscopy, it is likely that a major portion of the protein in myelin is associated with the MDL. In this laboratory, much of the initial work of freeze fracture of myelin utilized central and peripheral myelin from the rat. This myelin, when fixed with glutaraldehyde, demonstrates no difference between the P (A) and E (B) faces. The particles seen under these conditions are pleomorphic, ranging in size from 3 nm to 7 nm. On the other hand Mugniani & Schnapp (1976) have found major differences between the P and E faces of aldehyde fixed frog central myelin; on the P face they find many particles which are bar shaped and relatively fewer particles on the E face. The sedimentation coefficient for the PB in aqueous media is consistent with a prolate spheroid with dimensions of 15 x 1.5 nm (Epand, Moscarello, Zierenberg & Vail, 1974). Schnapp & Mugniani point out that the bar shaped particles they find are of about these dimensions. In the present report, I have used another preparative technique for freeze fracture: fresh freezing. In freshly frozen rat myelin, there is a great difference between the P and E faces (Miller, in preparation). One of these faces appears covered with bar shaped particles while the other contains fewer particles and few bar shaped particles (see Plate 1). By counting fracture planes from the outside or from the axonal membrane, it is possible to determine that the particulate face is the E face. While it has been shown that the perferred cleavage plane in freeze fracture is within the hydrophobic matrix of the membrane (Pinto da Silva & Branton, 1970) it would seem that a structure which passes through a membrane could either cleave in the center of the membrane, breaking that structure in two, or be pulled through the membrane. If the structure pulled through is flexible, it is likely to “fall” to the surface and become partially embedded in the hydrophobic tails of the lipid rather than remain standing in the vacuum. I (1977) have suggested that bar shaped particles on freshly frozen axolemmal (E) face of the rat are created by this mechanism. Assuming that structures can be pulled through membranes, these changes in face morphology associated with the different preparative procedures accord well with the proposed model: in the freshly frozen tissue the membrane transversing portion of the BP is pulled through the bilayer adjacent to the observed fracture face. It is postulated that the BP would then “fall” and be shadowed as a bar-shaped (as opposed to a perpendicular post-shaped) particle. Fixation with a cross-linking agent (glutaraldehyde) is expected to inhibit the ability of a protein to be pulled through a membrane and thus the
LAMELLAR
MYELIN
105
fracture must cleave the peptide, creating the observed variations in the particle morphology. There is a major constraint that must be placed on the arrangement of proteins in myelin: under most preparative procedures (freeze fracture, and thin section electron microscopic and light microscopic), myelin undergoes “neurokeratinization” (see Nageotte, 19 10 ; Tewari & Bourne, 1960 ; Wolman, 1969). In light microscopy, especially with cross polarized iight, this effect produces a beaded appearance of the sheath and in thin section electron microscopy it produces regions where the lamellar structure is absent or distorted, probably depending upon the degree of delipidation during dehydration for embedment. Neurokeratinization has received little attention by electron microscopists and is rarely presented in the recent literature probably because it is recognized as an artifact. On the light microscopic level, Nageotte considered neurokeratinization an artifact, but one which could potentially tell us a great deal about myelin. In terms of freeze fracture, neurokeratinization manifests itself as particle free regions which are propagated radially through many myelin lamallae (Pinto da Silva & Miller, 1975). Since both the BP and PL may be visible through freeze fracture (see below), the propagation of the regions devoid of particles suggested an intimate connection between the two proteins. Therefore there must be interactions which extend across both the MDL and the IPL. The paucity of particles on the freshly frozen A face implies that, under these conditions, the PL is more closely associated with the IPL. Given the hydrophobicity of the PL, this association could be accomplished by interaction of the PL with the BP at regions B, C or F in Fig. 1. Some of the original studies of diffraction of myelin membranes demonstrated that the normal repeat distance was 170 A. Upon osmotic swelling the repeat distance increases to 250-270 A (Finean & Millington, 1957 ; see also McIntosh & Robertson, 1976). In terms of the present model, this might be represented as shown in Fig. 2. Figure 2 suggests that the region of the BP labeled F is reinserted into the adjacent membrane increasing the width of the IPL by the thickness of one membrane. The repeat distance predicted by this model (assuming no configurational changes occur in BP upon swelling) is 255 A.
Additional evidence from swelling of myelin comes from thin section electron microscopy of cultured myelin which has been exposed to heat inactivated serum from experimental allergic encephalitis animals. Bornstein & Raine (1976) have shown that, under these conditions the IPL, which is usually seen as a pair of thin electron dense lines, separates to form four thin
106
R. G. MILLER
LAMELLAR
MYELIN
107
lines as the myelin period doubles. Of these lines, the pair that are adjacent to the MDL are the most consistent and dense; in many regions, the four lines are quite distinct and suggest that another bilayer membrane has been formed. As seen in the light of the present model, a factor in the serum induces the BP to withdraw region F from one membrane and reinsert it in the adjacent membrane. If specific lipids bind to the BP at D, there may exist regions where an additional membrane is formed. This model predicts that there may be boundary effects associated with the initial (internal and external) layers of myelin membranes. Such effects have been noted by Mugniani, Osen, Schnapp and Friedric (1978) on the external layer of frog peripheral myelin. Effects of this sort have been observed in this laboratory in peripheral myelin of the rat which has been freshly frozen (in preparation).
4. Reconstitution
of Myelin Components
London and his coworkers (Demel, London, Geurts van Kessel, Vossenberg & van Deenen, 1973 ; London & Vossenberg, 1973) have shown that, at a lipid monolayer at the air-water interface, the BP is partially inserted into the hydrophobic tails of the lipid (see also Gould & London, 1972). This insertion can be measured as a change in surface tension, a change in surface radioactivity due to labeled BP, and a protection of certain portions of the BP from proteolytic attack from beneath the monolayer. In the monolayer system, the C-terminal portion of the BP (residues 106170) are available to lytic attack. Taken with the open configuration of the BP (e.g. see Block, Brady & Joffe, 1973 ; Epand, Moscarello, Zierenberg & Vail, 1974; Krigbaum & Hsu, 1975, but c.f. Anthony & Moscarello, 1971; Chapman & Moore, 1976, for reviews see Carnegie & Dunkley, 1975 ; Braun & Brostoff, 1977), these authors suggest that in the monolayer system, the BP must go in and out of the monolayer at least two times and up to 5 times (Fig. 3):
FIG. 3. Lipid
monolayer
with
BP partially
inserted
(after
London
er al.. 1973).
R. G. MILLER 108 However, when one extrapolates these results to a bilayer or multilayer system, other possible configurations emerge (Fig. 4):
FIG. 4. Alternative locations for BP in bilayers and multiiayers suggested by monolayer studies.
In all of these cases, the peptide chain passes through the membrane; in the monolayer this option is not available. Other possible configurations which are consistent with the monolayer studies are hybrids of Figs. 3 and 4 where some portions of the BP traverse the whole membrane and other portions of the BP are partially inserted into the membrane. The present model predicts that lytic attack of BP in a multilayered system will produce different peptides than lytic attack in the London monolayer system. X-ray diffraction of reconstituted membrane lipids (Mateu et al., 1973) clearly shows that BP induces a repeate distance indicative of two bilayer membranes. These data can be explained in terms of the BP residing predominantly between the two bilayers and inducing a conformational change in all four leaflets such that association of other BP to the outside surface of the double bilayer is not possible (see Mateu et al., 1973). Another interpretation which makes less stringent assumptions upon exclusive association of the BP with the four lipid monolayers is that the BP extends through two (or more) of the membranes (see Fig. 5). This would have the effect of a two dimensional zipper which would inhibit insertion of the BP from between the double bilayer and thus inhibit the formation of multilayered repeat patterns. The PL is present in roughly equimolar quantities to the BP (Mehl and Halaris, 1970) and is extremely hydrophobic. A priori, we might expect that the PL will reside within the hydrophobic matrix of the membrane and be visible as particles in freeze fracture (but c.f. Golds & Braun, 1976). Freeze fracture of reconstituted membranes containing PL clearly demonstrate particles (see above). These particles, however, are globular and do not look
LAMELLAR
MYELlN
109
FIG. 5. Suggested insertion of BP into multilayer lipids to produce double bilayer repeat distance.
like those seen in intact myelin membranes (e.g. see Vail er al., 1974). This suggests that either the reconstitution is not biologically accurate, or the barshaped particles and pleomorphism of the particles seen in intact myelin results from a combination of the PL with BP. Lipid specificities have been described for both the proteolipid and the BP (e.g. see Schafer & Franklin, 1975; Hemminga & Post, 1976; Palmer & Dawson, 1969; Demel et al., 1973; London et al., 1974; Paphadjopoulos, Moscarello, Eylar & Isac, 1975). Sequestration of non-cholsterol lipids by protein binding may play an important role in altering the phase transition properties of myelin membranes (see Miller & Torreyson, 1977). I have proposed that the two proteins interact with one another; since both have lipid specificities, this interaction could be through mutual binding to a lipid. 5 Biochemistry Since the amino acid sequence of the BP is well known, it might be possible to predict which regions correspond to regions A through F in Fig. 1. There are few protein sequences known that take a protein across a membrane (Tomita & Marchesi, 1975 ; 0~01s & Gerrard, 1977). In all documented cases to date, a protein traverses a membrane in a helix with the peptide bonds in the center of the helix and hydrophobic moieties inserted into the hydrophobic matrix of the membrane. To do this requires a sequence of about 20 hydrophobic amino acids. The amino acid sequences of BP is high in helix breaking residues. One region in which a secondary structure can be surmised on the basis of the sequence is the proline rich region (residues 96-101 : numeration is according to Carnegie & Dunkley, 1975) which seems to be bent back upon itself. Physical studies have shown that the BP in aqueous solutions contains little or no helical structure. There are two regions of about 10 residues which are predominantly hydrophobic (residues 34-46 and 85-101 have 9 and 11 hydrophobic residues respectively). If both of these extend the chain across a
R. G. MILLER 110 membrane, the BP would form a structural bridge across the IPL. It should be noted, however, that these regions also contain charged amino acids which have to be neutralized either by interaction with lipids or interaction with the PL (c.f. Hemminga 8~ Post, 1976). Thus, although the amino acid sequence of the BP is well known, unequivocal assignment of specific sequences to regions A through F in Fig. 1 is not possible. This may be because the generalization that proteins which traverse membranes do so in a hydrophobic helix is not universally applicable. One possible arrangement for the peptide would be for the BP to be in a largely open configuration with residues 100-170 acting as an anchor in the MDL and residues I-100 extending perpendicularly to the MDL. There are enough residues to traverse 200, 280 and 350 A if these residues are in a 3,, helix, 2, ribbon and /? antiparallel sheet respectively (Dickerson & Geis, 1969). Any combination of these configurations would be capable of producing a peptide long enough to extend beyond the myelin period (180 A). There are indications of polymerization of the BP (e.g. Martenson & Deibler, 1975; which might allow for the possible b antiparallel sheet in some regions.?
6 Other Models Another model which has been considered (see London et al., 1973) places the BP in either the MDL or IPL with hydrophobic regions of the BP extending into the adjacent membranes. The BP in this model is in the same configuration as it assumes in the monolayer system. A variant of this model would allow the hydrophobic regions to insert into both of the adjacent membranes (see also Smith, 1977). In either of these variants, the BP can form a structural link across either the MDL or IPL. To obtain neurokeratinization, the PL would have to form the structural link across the other. The PL, being extremely hydrophobic, is not expected to bridge the aqueous IPL (but c.f. Golds & Braun, 1976). The ambiguity of localization studies of BP do not argue strongly for any assignment. Thus the likely assignment of positions would be that the BP is in the IPL and the PL forms the link across the MDL. The pleomorphism of the freeze fracture particles and similarity of P and E fracture faces seen in fixed mammalian tissue, and t Since submission of this manuscript, E. E. Golds & P. E. Braun (J. Biol. Chem. 253,8162) have reported the use of bifunctional reagents to cross-link lysine residues in intact central myelin. In this manner they have shown that major quantities of BP dimers (and not multimers) are formed by linkage of lysine residues at the terminal regions of the BP while the center region of the BP is arranged in such a way as to separate lysine moieties by more than 9 A. On the basis of this and other evidence, the authors propose that, in viva, the BP is an antiparallel dimer which forms a structural link across and within the MDL.
LAMELLAR
111 associated with fresh freezing are
MYELIN
the alteration of particle morphology difficult to explain under this model. In the present model, I have assumed that one PL is associated with one BP. Using the same arguments, more efficient, and at this time more speculative, models could be made assuming valencies of more than one for either or both of the proteins. Dimerization of either the PL or the BP would all permit the BP to perform the same tasks proposed by traversing a single membrane. There is evidence of dimer or trimer forms of the PL. Unfortunately experiments which would determine the associations and/or valencies of the PL and BP are difficult to perform due to their differences is solubilities (c.f. Block et al., 1973). This model does not make any predictions concerning the configuration of the C terminal region of the BP (region A in Fig. 1); it is possible that there is an interaction in this region with the same or another BP, with the PL (see Smythies, Benington & Morin, 1972) or that this region is in an extended form interacting with charged groups of the lipids. Since central myelin is biochemically better characterized and contains fewer proteins than peripheral myelin, the present model has been formulated on the basis of data obtained from central myelin. Although the model applies most directly to central myelin, the similarity in ultrastructure and function and the identity of one of the proteins (BP) suggests that similar mechanisms are operating to stabilize the lamellar organization in both types of myelin. In conclusion, I am proposing a model for lamellar myelin in which the basic protein has an organizing role by passing through two membranes and interacting with the proteolipid in a third. The C-terminal region of the basic protein acts as an anchor in the MDL for the rest of the protein and interaction of the N-terminal end of the protein with the proteolipid forms a structural link which binds the myelin membranes together in their characteristic pattern.
I thank Fred Westall for encouragement and helpful discussions,Dave Schubert for his generoussupport and useof his laboratory, Bonne Harkins and Nadine Maalaoui for preparation of the manuscript,Gary Jonesand JamieSimon for art work, and FrancisCrick, Jim Patrick and Joe-Henry Steinbachfor comments on the manuscript.This work wassupportedby the National Institutesof Health (to D. S.) and wasdone during the tenure of a ResearchFellowshipof the Muscular Dystrophy Association. REFERENCES C. W. hf., 389.
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