Hydrophobic Regions in Myelin Proteins Characterized Through Analysis of "Hydropathic"Profiles

Computer-generated "hydropathic" profiles were constructed for graphic comparison of the amino acid sequences for P2 protein, 18.5 kilodalton (kDa) myelin basic protein (BP), and myelin proteolipid protein (PLP). Profiles were also obtained for cytochrome b5, a membrane protein known to be capable of reversible association with lipid bilayers and of a size comparable to that of the myelin BPs. Analysis of the PLP sequence produced profiles generally compatible with the suggestions that PLP has three transbilayer and two bilayer intercalating segments. Profiles for P2 and 18.5 kDa BP were found to contain hydrophilic segments separated by relatively short hydrophobic regions. Whereas hydropathic indices in hydrophobic regions of P2, 18.5 kDa BP, and PLP fall in the value ranges recently reported for cores of globular proteins and intrabilayer domains of membrane proteins, hydrophobic sections of P2 and 18.5 kDa BP have hydropathic indices similar to those in the hydrophobic core (transprotein) regions of globular proteins. None of them are comparable to the region of cytochrome b5 known to anchor that protein in its membrane or to the segments of PLP sequence proposed as intrabilayer domains. This comparison suggests that neither BP has structural characteristics compatible with insertion into the hydrocarbon core of the myelin lipid bilayer, a conclusion that is consistent with a recently published study that identified the bilayer penetrating proteins of myelin with a hydrophobic probe. The above findings suggest an enhancement for some details of myelin architecture and a cautious approach to interpreting data for BP intercalation into bilayers.     

Disorder in nerve myelin: Phasing the higher order reflections by means of the diffuse scatter

Disorder in the stacking of membrane layers in nerve myelin has an important effect on the X-ray diffraction pattern. In a previous report we found broadening of the Bragg reflections and diffuse intensity between them. A model for the disorder was presented to account for these effects, and some of the model parameters were evaluated by analysis of the X-ray diffraction pattern in the region of Bragg reflections 1 to 6, where the phasing is well established. In this paper, we present further data and derive a more complete and precise set of values for the parameters. Then, assuming these values, all possible combinations of phases for reflections 7 to 15 are used to calculate the corresponding diffraction patterns for comparison with observation. The 20 best fitting phase combinations are considered in detail. Sixteen of these are eliminated, leaving four possible combinations which differ only in the phases for orders 8 and 15.Electron density profiles computed using the four possible phase combinations are not very different from one another. All four profiles show a markedly higher electron density in the extracellular half of the membrane bilayer than in the cytoplasmic half. Our calculations indicate that the cytoplasmic half can be accounted for entirely by myelin lipids, including cholesterol. However, an unreasonably high proportion of cholesterol would be needed to account for the extracellular half of the bilayer, and we tentatively conclude that an appreciable part of the myelin protein is inserted into this half of the bilayer.Analytical methods used by other investigators are considered in light of our experimental observations and model predictions in order to reconcile conflicting results. Failure to account properly for diffuse intensity arising from the disorder makes previous determinations of myelin membrane structure at moderate resolution questionable.     

Marker enzymes,phospholipids and acyl group composition of a somal plasma membrane fraction isolated from rat cerebral cortex: a comparison with microsomes and synaptic plasma membranes

Subjecting brain homogenates to differential speed and sucrose density gradient centrifugation resulted in the isolation of a membrane fraction from the post-mitochondrial supernatant with properties and marker enzyme profiles typical of plasma membranes. This membrane fraction is compared with the microsomes and the synaptic plasma membranes isolated from synaptosomes. Like the synaptic plasma membranes, membranes obtained from the post-mitochondrial supernatant were enriched five-fold in 5′-nucleotidase activity. However, the latter membranes were lower in (Na⁺, K⁺)-ATPase activity and higher in NADPH-cytochrome C reductase activity as compared to the synaptic plasma membranes. The post-mitochondrial plasma membranes were also different from the microsomes in their respective marker enzyme activities. Electron microscopic examination indicated largely membranous vesicles for both plasma membrane fractions with little contamination by myelin, mitochondra and intact synaptosomes. The phospholipid and acyl group profiles of the two plasma membrane fractions were surprisingly similar, but they were different from the characteristic profiles of myelin and mitochondria. It is concluded that plasma membranes isolated from the post-mitochondrial supernatant fraction are derived largely from neuronal and glial soma and are thus designated the somal plasma membrane fraction.     

Comparison of different myelin isolation methods by use of nonequilibriumpH gradient gel electrophoresis

Myelin was isolated from bovine white matter by five published procedures and several modifications of two of them. Comparison of the protein profiles of the preparations by nonequilibrium pH gradient gel electrophoresis, revealed clear differences in myelin protein content and composition between preparations obtained by different methods. In isolation methods where the medium contained salts, some of the myelin proteins were solubilized, the phenomenon being most pronounced in long-period isolations in buffered CsCl solution.     

Myelin Organization in the Nodal,Paranodal, and Juxtaparanodal Regions Revealed by Scanning X-Ray Microdiffraction

X-ray diffraction has provided extensive information about the arrangement of lipids and proteins in multilamellar myelin. This information has been limited to the abundant inter-nodal regions of the sheath because these regions dominate the scattering when x-ray beams of 100 µm diameter or more are used. Here, we used a 1 µm beam, raster-scanned across a single nerve fiber, to obtain detailed information about the molecular architecture in the nodal, paranodal, and juxtaparanodal regions. Orientation of the lamellar membrane stacks and membrane periodicity varied spatially. In the juxtaparanode-internode, 198–202 Å-period membrane arrays oriented normal to the nerve fiber axis predominated, whereas in the paranode-node, 205–208 Å-period arrays oriented along the fiber direction predominated. In parts of the sheath distal to the node, multiple sets of lamellar reflections were observed at angles to one another, suggesting that the myelin multilayers are deformed at the Schmidt-Lanterman incisures. The calculated electron density of myelin in the different regions exhibited membrane bilayer profiles with varied electron densities at the polar head groups, likely due to different amounts of major myelin proteins (P0 glycoprotein and myelin basic protein). Scattering from the center of the nerve fibers, where the x-rays are incident en face (perpendicular) to the membrane planes, provided information about the lateral distribution of protein. By underscoring the heterogeneity of membrane packing, microdiffraction analysis suggests a powerful new strategy for understanding the underlying molecular foundation of a broad spectrum of myelinopathies dependent on local specializations of myelin structure in both the PNS and CNS.     

LOCATING THE MAJOR GLYCOPROTEIN (Po PROTEIN) IN THE X-RAY PROFILE OF FROG SCIATIC-NERVE MYELIN

Some detailed X-ray profiles have been put forward for frog sciatic-nerve myelin in the past 8 y. In this paper we interpret one of these. Much of the myelin protein is located in the extracellular half of the membrane. On the basis of a chemical analysis and other recently published data indicating the sidedness of some myelin proteins, we suggest that the major glycoprotein (P_o protein) is inserted into the extracellular half of the myelin bilayer and that it extends into the extracellular space. Our interpretation is consistent with known physical properties of the P_o protein. Other myelin proteins have not yet been located. Electron densities and average hydrophobicities of some myelin proteins have been calculated, as well as electron densities and scattering powers of some myelin lipids.     

Membrane structure in isolated and intact myelins.

The biochemical composition of myelin and the topology of its constituent lipids and proteins are typically studied using membranes that have been isolated from whole, intact tissue using procedures involving hypotonic shock and sucrose density gradient centrifugation. To what extent, however, are the structure and intermembrane interactions of isolated myelin similar to those of intact myelin? We have previously reported that intact and isolated myelins do not always show identical myelin periods, indicating a difference in membrane-membrane interactions. The present study addresses the possibility that this is due to altered membrane structure. Because x-ray scattering from isolated myelin sometimes consists of overlapping Bragg reflections or is continuous, we developed nonlinear least squares procedures for analyzing the total intensity distribution after film scaling, background subtraction, and Lorentz correction. We calculated electron density profiles of isolated myelin for comparison with membrane profiles from intact myelin. The change in the width of the extracellular space and the relative invariance of the cytoplasmic space as a function of pH and ionic strength that we previously found for intact nerve was largely paralleled by isolated myelin. There were two exceptions: isolated CNS myelin was resistant to swelling under all conditions, and isolated PNS myelin in hypotonic saline showed indefinite swelling at the extracellular apposition. However, electron density profiles of isolated myelins, calculated to 30 A resolution, did not show any major change in structure compared with intact myelin that could account for the differences in interactions.     

Biochemical profiles of membranes from x-ray and neutron diffraction.

X-ray and neutron diffraction methods provide some information about the distribution of mass in biological membranes and lipid-water systems. Scattering density profiles obtained from these systems, however, usually are not directly interpretable in terms of the relative amounts of chemical constituents (e.g., lipid, protein, and water) as a function of position in the membrane. We demonstrate here that the combined use of x-ray and neutron-scattering profiles, together with information on the total amounts of each of the major membrane components, are sufficient to calculate unambiguously the volume fractions of these components at well-defined regions of the lamellar unit. Three cases are considered: a calculated model membrane pair, dipalmitoylphosphatidylcholine-water multilayers, and rabbit sciatic nerve myelin. For the model system, we discuss the limitations imposed by finite resolution in the diffraction patterns. For the lipid-water multilayers, we calculate water volume fractions in the hydrocarbon tail, lipid headgroup, and interlamellar regions; estimates of these values by various methods are in good agreement with our results. For the nerve myelin, we predict new results for the distribution of protein through the membrane.     

An X-ray study of the condensed and separated states of sciatic nerve myelin

Low-angle X-ray diffraction patterns of peripheral nerve myelin after modification by either rehydration in various solutions or by chemical treatment have been recorded. These X-ray patterns and the previously reported modified nerve myelin patterns demonstrate that nerve myelin has at least five different states: the normal state, condensed state I and II and separated state I and II. There are two membranes per unit cell in the normal state and in states II whereas there is one membrane per unit cell in states I. Under certain conditions normal nerve can go reversibly into either of states II. With continued treatment the nerve myelin structure moves irreversibly from state II to state I and, once in state I, the nerve myelin layers cannot return to the normal state. Our results demonstrate that there is a reversible transformation between condensed state I and separated state I. Fourier profiles of nerve myelin in the normal state, condensed state I and separated state I are presented.     

The ultrastructure of the “difference factor” in the myelin

Summary Electron-microscopic studies of peripheral nerves as prepared by the freezeetching method show the myelin lamella to be 185 Å thick. This is the same dimension found by x-ray diffraction analysis of natural myelin. In contrast to the appearance of osmiumfixed material, the cytoplasmic surfaces of the paired membranes in the myelin lamella are apposed to two fine, separate lines, while the outer membrane sides are fused into a broader single line. The finding of a decidedly different structure for the outer and for the inner membrane surfaces appears to be the cause of the difference factor.This work was supported by the Swiss National Foundation (Nr. 4065). — Acknowledgement: We thank the Balzers AG. (9496 Balzers, Fürstentum Liechtenstein) for providing us with the High Vakuum Device.     

Isolation of non-myelin plasma membranes unique to white matter

A procedure is described for isolating two membrane fractions from rabbit spina-cord white matter enriched with 5′-nucleotidase, a nonspecific plasma membrane marker, 2′, 3′-cyclic nucleotide phosphohydrolase, an oligodendroglial plasma membrane marker, and acetylcholinesterase, an axonal plasma membrane marker. While the two membrane fractions exhibited similar enrichments with respect to cyclic nucleotide phosphohydrolase, enrichments of 5′-nucleotidase and acetylcholinesterase were significantly greater in the heavier membranes were not detected. Moreover, gray matter did not yield homologous membrane fractions in the gradient when subjected to the identical procedure, indicating that the two membrane fractions were unique to white matter. While electronmicroscopic examination revealed that both membrane fractions were contaminated with myelin, the heavier fraction was least contaminated and exhibited a fair degree of homogeneity with respect to single membrane vesicular profiles. It was concluded that both membrane fractions were enriched with oligodendroglial and axonal plasma membranes, with the heavier fraction containing significantly more axolemma.     

On phasing the small-angle x-ray diffraction pattern from nerve myelin.

Using a method they developed, Stamatoff and Krimm (1976) have phased swelling data from nerve myelin. Although most phases agree with those I determined previously, there are a few differences. In this letter the two different phasings, theirs and my own, are used to compute the corresponding electron-density profiles, which are then closely compared. For both phasings, small differences are seen in the membrane profile at different degrees of swelling. The explanation that these differences are due simply to errors in measuring intensity is shown to be quite improbable; thus the differences indicate a real change in the profile. It follows that the assumption of a constant membrane profile appears to be invalid in the case of myelin swelling. The differences therefore are assumed to indicate a real change in the profile. It is shown that this change can be attributed consistently to interdigitation of protein molecules at the surfaces of neighboring membranes, while the membrane structure itself remains unchanged. In this case, valid phases still can be determined by swelling, but the phases determined by Stamatoff and Krimm are not valid.     

An Apical-Type Trafficking Pathway Is Present in Cultured Oligodendrocytes but the Sphingolipid-enriched Myelin Membrane Is the Target of a Basolateral-Type Pathway

Myelin sheets originate from distinct areas at the oligodendrocyte (OLG) plasma membrane and, as opposed to the latter, myelin membranes are relatively enriched in glycosphingolipids and cholesterol. The OLG plasma membrane can therefore be considered to consist of different membrane domains, as in polarized cells; the myelin sheet is reminiscent of an apical membrane domain and the OLG plasma membrane resembles the basolateral membrane. To reveal the potentially polarized membrane nature of OLG, the trafficking and sorting of two typical markers for apical and basolateral membranes, the viral proteins influenza virus–hemagglutinin (HA) and vesicular stomatitis virus–G protein (VSVG), respectively, were examined. We demonstrate that in OLG, HA and VSVG are differently sorted, which presumably occurs upon their trafficking through the Golgi. HA can be recovered in a Triton X-100-insoluble fraction, indicating an apical raft type of trafficking, whereas VSVG was only present in a Triton X-100-soluble fraction, consistent with its basolateral sorting. Hence, both an apical and a basolateral sorting mechanism appear to operate in OLG. Surprisingly, however, VSVG was found within the myelin sheets surrounding the cells, whereas HA was excluded from this domain. Therefore, despite its raft-like transport, HA does not reach a membrane that shows features typical of an apical membrane. This finding indicates either the uniqueness of the myelin membrane or the requirement of additional regulatory factors, absent in OLG, for apical delivery. These remarkable results emphasize that polarity and regulation of membrane transport in cultured OLG display features that are quite different from those in polarized cells.     

Tight Junction Proteins in Human Schwann Cell Autotypic Junctions

Tight junctions (TJs) form physical barriers in various tissues and regulate paracellular transport of ions, water, and molecules. Myelinating Schwann cells form highly organized structures, including compact myelin, nodes of Ranvier, paranodal regions, Schmidt-Lanterman incisures, periaxonal cytoplasmic collars, and mesaxons. Autotypic TJs are formed in non-compacted myelin compartments between adjacent membrane lamellae of the same Schwann cell. Using indirect immunofluorescence and RT-PCR, we analyzed the expression of adherens junction (E-cadherin) and TJ [claudins, zonula occludens (ZO)-1, occludin] components in human peripheral nerve endoneurium, showing clear differences with published rodent profiles. Adult nerve paranodal regions contained E-cadherin, claudin-1, claudin-2, and ZO-1. Schmidt-Lanterman incisures contained E-cadherin, claudin-1, claudin-2, claudin-3, claudin-5, ZO-1, and occludin. Mesaxons contained E-cadherin, claudin-1, claudin-2, claudin-3, ZO-1, and occludin. None of the proteins studied were associated with nodal inter-Schwann cell junctions. Fetal nerve expression of claudin-1, claudin-3, ZO-1, and occludin was predominantly punctate, with a mesaxonal labeling pattern, but paranodal (ZO-1, claudin-3) and Schmidt-Lanterman incisure (claudins-1 and -3) expression profiles typical of compact myelin were visible by gestational week 37. The clear differences observed between human and published rodent nerve profiles emphasize the importance of human studies when translating the results of animal models to human diseases. (J Histochem Cytochem 57:523–529, 2009)     

Two types of detergent-insoluble, glycosphingolipid/cholesterol-rich membrane domains from isolated myelin

Two different types of low-density detergent-insoluble glycosphingolipid-enriched membrane domain (DIG) fractions were isolated from myelin by extraction with Triton X-100 (TX-100) in 50 mM sodium phosphate buffer at room temperature (20 degrees C) (procedure 1), in contrast to a single low-density fraction obtained by extraction with TX-100 in Tris buffer containing 150 mM NaCl and 5 mM EDTA at 4 degrees C (procedure 2). Procedure 1 has been used in the past by others for myelin extraction to preserve the cytoskeleton and/or radial component of oligodendrocytes and myelin, whereas procedure 2 is now more commonly used to isolate myelin DIG fractions. The two DIG fractions obtained by procedure 1 gave opaque bands, B1 and B2, at somewhat lower and higher sucrose density respectively than myelin itself. The single DIG fraction obtained by procedure 2 gave a single opaque band at a similar sucrose density to B1. Both B1 and B2 had characteristics of lipid rafts, i.e. high galactosylceramide and cholesterol content and enrichment in GPI-linked 120-kDa neural cell adhesion molecule (NCAM)120, as found by others for the single low-density DIG fraction obtained by procedure 2. However, B2 had most of the myelin GM1 and more of the sulfatide than B1, and they differed significantly in their protein composition. B2 contained 41% of the actin, 100% of the tubulin, and most of the flotillin-1 and caveolin in myelin, whereas B1 contained more NCAM120 and other proteins than B2. The single low-density DIG fraction obtained by procedure 2 contained only low amounts of actin and tubulin. B1 and B2 also had size-isoform selectivity for some proteins, suggesting specific interactions and different functions of the two membrane domains. We propose that B1 may come from non-caveolar raft domains whereas B2 may derive from caveolin-containing raft domains associated with cytoskeletal proteins. Some kinases present were active on myelin basic protein suggesting that the DIGs may come from signaling domains.     

Oxygen profiles in membranes

Transmembrane profiles of molecular oxygen in lipid bilayers are not only significant for membrane physiology and pathology, but also are essential to the determination of membrane protein structure by site-directed spin labeling. Oxygen profiles obtained with spin-labeled lipid chains have a Boltzmann sigmoidal dependence on the depth into each lipid leaflet, which represents a two-compartment distribution between outer and inner regions of the membrane, with a transfer free energy that depends linearly on distance from the dividing planes. Transmembrane profiles for intramembrane polarity, and for water penetration into the membrane, have an identical form, but are of the reverse sign. Comparison with recently published oxygen profiles from a site-specifically spin-labeled alpha-helical transmembrane peptide validates the use of spin-labeled lipids for all these profiles and provides the necessary bridge to generate the full bilayer from a single lipid leaflet.     

The intrinsic fluorescence of isolated central-nervous-system myelin-sheath preparations.

The intrinsic fluorescence characteristics of tyrosine and tryptophan residues in the proteins of isolated central-nervous-system myelin were investigated to gain information concerning the location of these residues within the intact membrane system. Tryptophan fluorescence from isolated myelin has an emission maximum at 325 nm that appears to arise from at least two different populations of tryptophan residues. Further evidence for heterogeneity of tryptophan location in the membrane is obtained from quenching studies with chloroform and acrylamide. It is speculated that one tryptophan population is hydrophobically situated and may be derived from the proteolipid protein of myelin, whereas the other tryptophan population is located at the membrane surface and may arise from the extrinsic basic protein. A significant tyrosine fluorescence is detected from isolated myelin, indicating that some of these residues are not quenched by structural interactions within the lipid--protein membrane system. Studies with freeze-dried resuspended myelin suggest that the structural arrangement of protein components in the dried rehydrated membrane system differs significantly from that of the freshly isolated myelin membrane.     

X-ray diffraction study of myelin structure in immature and mutant mice

X-ray diffraction patterns were obtained from freshly dissected central and peripheral nerves of quaking, myelin synthesis deficiency ($\text{msd}$), and trembler mutants, as well as immature and adult normal mice. The patterns were compared with respect to strength of myelin diffraction, background scatter level, repeat period, and intensity and linewidth of Bragg reflections. The deficiency of myelin in optic nerves was found to be (in decreasing severity): quaking > immature > trembler ? normal adult; and in sciatic nerves: $\text{trembler > immature > quaking}\text{msd ?}\text{normal}$ adult. Repeat periods about 3 Å less than that for normal adult sciatic myelin were detected in corresponding nerves from immature, quaking, and trembler mice. In some trembler sciatic nerves a second phase having a 190–200 Å period and accounting for about 60% of the total ordered myelin was also evident. Comparison of electron density profiles of membrane units calculated from the repeat periods and diffracted intensities for sciatic myelins indicate structural differences at the molecular level. The main findings are: (1) quaking myelin shows a significant elevation of density in the external protein-water layer between membrane bilayers; (2) the membrane bilayer of immature myelin is ≈ 2 Å thinner than that for normal adult; (3) the membrane bilayer of the more compact phase in trembler myelin is ≈ 5 Å thinner than for normal; and (4) the difference in repeat periods for the two phases present in some of the trembler nerves can be accounted for predominantly by distinct membrane bilayer separations at the external boundary.     

Renaturation of the brain myelin proteins by octyl glucoside detergent.

The secondary structure of myelin proteins undergoes a deep change when the membrane is delipidated and suspended in an aqueous buffer containing phosphate and sulfate anions. However, when increasing concentrations of octyl glucoside are dissolved in this saline medium, proteins recover gradually its native secondary structure, reaching a maximum for a detergent/protein ratio which, in addition, is optimal for maximal membrane solubilization. Larger amounts of detergent, however, reverted the effect. Results are explained in terms of anion-lipid and detergent-lipid interactions. Quantitative estimates on the spectral profiles let us find the optimal detergent-protein stoichiometry for preserving almost completely the native secondary structure of myelin proteins while keeping maximal solubilization. These findings are of great importance for reconstitution experiments designed with the goal of determining the biological functions of myelin proteins.