A glycosynapse in myelin?

Myelin, the multilayered membrane which surrounds nerve axons, is the only example of a membranous structure where contact between extracellular surfaces of membrane from the same cell occurs. The two major glycosphingolipids (GSLs) of myelin, galactosylceramide (GalC) and its sulfated form, galactosylceramide I(3)-sulfate (SGC), can interact with each other by trans carbohydrate-carbohydrate interactions across apposed membranes. They occur in detergent-insoluble lipid rafts containing kinases and thus may be located in membrane signaling domains. These signaling domains may contact each other across apposed extracellular membranes, thus forming glycosynapses in myelin. Multivalent forms of these carbohydrates, GalC/SGC-containing liposomes, or galactose conjugated to albumin, have been added to cultured oligodendrocytes (OLs) to mimic interactions which might occur between these signaling domains when OL membranes or the extracellular surfaces of myelin come into contact. These interactions between multivalent carbohydrate and the OL membrane cause co-clustering or redistribution of myelin GSLs, GPI-linked proteins, several transmembrane proteins, and signaling proteins to the same membrane domains. They also cause depolymerization of the cytoskeleton, indicating that they cause transmission of a signal across the membrane. Their effects have similarities to those of anti-GSL antibodies on OLs, shown by others, suggesting that the multivalent carbohydrate interacts with GalC/SGC in the OL membrane. Communication between the myelin sheath and the axon regulates both axonal and myelin function and is necessary to prevent neurodegeneration. Participation of transient GalC and SGC interactions in glycosynapses between the apposed extracellular surfaces of mature compact internodal myelin might allow transmission of signals throughout the myelin sheath and thus facilitate myelin-axonal communication.     

Myelin glycosphingolipids,galactosylceramide and sulfatide,participate in carbohydrate–carbohydrate interactions between apposed membranes and may form glycosynapses between oligodendrocyte and/or myelin membranes

Glycosphingolipids (GSLs) can interact with each other by homotypic or heterotypic trans carbohydrate–carbohydrate interactions across apposed membranes, resulting in cell–cell adhesion. This interaction can also provide an extracellular signal which is transmitted to the cytosolic side, thus forming a glycosynapse between two cells. The two major GSLs of myelin, galactosylceramide (GalC) and its sulfated form, galactosylceramide I³-sulfate (SGC), are an example of a pair of GSLs which can participate in these trans carbohydrate–carbohydrate interactions and trigger transmembrane signaling. These GSLs could interact across apposed oligodendrocyte membranes at high cell density or when a membranous process of a cell contacts itself as it wraps around the axon. GalC and SGC also face each other in the apposed extracellular surfaces of the multilayered myelin sheath. Communication between the myelin sheath and the axon regulates both axonal and myelin function and is necessary to prevent neurodegeneration. Participation of transient GalC and SGC interactions in glycosynapses between the apposed extracellular surfaces of mature myelin might allow transmission of signals throughout the myelin sheath and thus facilitate myelin-axonal communication.     

Participation of galactosylceramide and sulfatide in glycosynapses between oligodendrocyte or myelin membranes

The two major glycosphingolipids of myelin, galactosylceramide (GalC) and sulfatide (SGC), interact with each other by trans carbohydrate-carbohydrate interactions. They face each other in the apposed extracellular surfaces of the multilayered myelin sheath produced by oligodendrocytes (OLs). Multivalent galactose and sulfated galactose, in the form of GalC/SGC-containing liposomes or silica nanoparticles conjugated to galactose and galactose-3-sulfate, interact with GalC and SGC in the membrane sheets of OLs in culture. This stimulus results in transmembrane signaling, loss of the cytoskeleton and clustering of membrane domains, suggesting that GalC and SGC could participate in glycosynapses between apposed OL membranes or extracellular surfaces of mature myelin. Such glycosynapses may be important for myelination and/or myelin function.     

Interactions between glucosylceramide and galactosylceramide I(3) sulfate and microstructures formed

The monohexoside glycosphingolipids (GSLs), galactosylceramide (GalC), glucosylceramide (GluC), and their sulfated forms are abundant in cell membranes from a number of tissues. Carbohydrate-carbohydrate interactions between the head groups of some GSLs can occur across apposed membranes and may be involved in cell-cell interactions. In the present study, the ability of GluC to participate in trans interactions with galactosylceramide I(3) sulfate (CBS) was investigated by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy. Gaucher's spleen GluC had polymorphic phase behavior; in its metastable state, it formed large wrinkled vesicles. It transformed to a stable state via an intermediate state in which the surface of the vesicles consisted of narrow ribbons. In the stable state, the narrow ribbons split off from the surface to form membrane fragments and flat and helical ribbons. The strength of the intermolecular hydrogen bonding interactions between the carbonyls increased in the order metastable相似文献   

Trans interactions between galactosylceramide and cerebroside sulfate across apposed bilayers

The two glycosphingolipids galactosylceramide (GalC) and its sulfated form, cerebroside sulfate (CBS), are present at high concentrations in the multilayered myelin sheath and are involved in carbohydrate-carbohydrate interactions between the lipid headgroups. In order to study the structure of the complex of these two glycolipids by Fourier transform infrared (FTIR) spectroscopy, GalC dispersions were combined with CBS dispersions in the presence and absence of Ca(2+). The FTIR spectra indicated that a strong interaction occurred between these glycolipids even in the absence of Ca(2+). The interaction resulted in dehydration of the sulfate, changes in the intermolecular hydrogen bonding interactions of the sugar and other oxygens, decreased intermolecular hydrogen bonding of the amide C==O of GalC and dehydration of the amide region of one or both of the lipids in the mixture, and disordering of the hydrocarbon chains of both lipids. The spectra also show that Ca(2+) interacts with the sulfate of CBS. Although they do not reveal which other groups of CBS and GalC interact with Ca(2+) or which groups participate in the interaction between the two lipids, they do show that the sulfate is not directly involved in interaction with GalC, since it can still bind to Ca(2+) in the mixture. The interaction between these two lipids could be either a lateral cis interaction in the same bilayer or a trans interaction between apposed bilayers. The type of interaction between the lipids, cis or trans, was investigated using fluorescent and spin-label probes and anti-glycolipid antibodies. The results confirmed a strong interaction between the GalC and the CBS microstructures. They suggested further that this interaction caused the CBS microstructures to be disrupted so that CBS formed a single bilayer around the GalC multilayered microstructures, thus sequestering GalC from the external aqueous phase. Thus the CBS and GalC interacted via a trans interaction across apposed bilayers, which resulted in dehydration of the headgroup and interface region of both lipid bilayers. The strong interaction between these lipids may be involved in stabilization of the myelin sheath.     

Divalent cation-mediated interaction between cerebroside sulfate and cerebrosides: an investigation of the effect of structural variations of lipids by electrospray ionization mass spectrometry.

Divalent cations mediate a carbohydrate-carbohydrate association between the two major glycolipids, galactosylceramide (GalCer) and its sulfated form, cerebroside sulfate (CBS), of the myelin sheath. We have suggested that interaction between these glycolipids on apposed extracellular surfaces of myelin may be involved in the stability or function of this multilayered structure. A mutant mouse lacking galactolipids because of a disruption in the gene that encodes a galactosyltransferase forms myelin that initially appears relatively normal but is unstable. This myelin contains glucosylceramide (GlcCer) instead of GalCer. To better understand the role of GlcCer in myelin in this mutant, we have compared the ability of divalent cations to complex CBS (galactosyl form) with GlcCer or GalCer in methanol solution by using positive ion electrospray ionization mass spectrometry. Because both the alpha-hydroxylated fatty acid species (HFA) and the nonhydroxylated fatty acid species (NFA) of these lipids occur in myelin, we have also compared the HFA and NFA species. In addition to monomeric Ca2+ complexes of all three lipids and oligomeric Ca2+ complexes of both GalCer and GlcCer, Ca2+ also caused heterotypic complexation of CBS to both GalCer and GlcCer. The heterotypic complexes had the greatest stability of all oligomers formed and survived better at high declustering potentials. Complexes of CBS with GlcCer were less stable than those with GalCer. This was confirmed by using the free sugars and glycosides making up the carbohydrate headgroups of these lipids. HFA species of CBS and GalCer formed more stable complexes than NFA species, but hydroxylation of the fatty acid of GlcCer had no effect. The ability of GlcCer to also complex with CBS, albeit with lower stability, may allow GlcCer to partially compensate for the absence of GalCer in the mouse mutant.     

Shiverer and Normal Peripheral Myelin Compared: Basic Protein Localization, Membrane Interactions, and Lipid Composition

We have correlated membrane structure and interactions in shiverer sciatic nerve myelin with its biochemical composition. Analysis of x-ray diffraction data from shiverer myelin swollen in water substantiates our previous localization of an electron density deficit in the cytoplasmic half of the membrane. The density loss correlates with the absence of the major myelin basic proteins and indicates that in normal myelin, the basic protein is localized to the cytoplasmic apposition. As in normal peripheral myelin, hypotonic swelling in the shiverer membrane arrays occurs in the extracellular space between membranes; the cytoplasmic surfaces remain closely apposed notwithstanding the absence of basic protein from this region. Surprisingly, we found that the interaction at the extracellular apposition of shiverer membranes is altered. The extracellular space swells to a greater extent than normal when nerves are incubated in distilled water, treated at a reduced ionic strength of 0.06 in the range of pH 4-9, or treated at constant pH (4 or 7) in the range of ionic strengths 0.02-0.20. To examine the biochemical basis of this difference in swelling, we compared the lipid composition of shiverer and normal myelin. We find that sulfatides, hydroxycerebroside, and phosphatidylcholine are 20-30% higher than normal; nonhydroxycerebroside and sphingomyelin are 15-20% lower than normal; and ethanolamine phosphatides, phosphatidylserine, and cholesterol show little or no change. A higher concentration of negatively charged sulfatides at the extracellular surface likely contributes to an increased electrostatic repulsion and greater swelling in shiverer. The cytoplasmic surfaces of the apposed membranes of normal and shiverer myelins did not swell apart appreciably in the pH and ionic strength ranges expected to produce electrostatic repulsion. This stability, then, clearly does not depend on basic protein. We propose that P0 glycoprotein molecules form the stable link between apposed cytoplasmic membrane surfaces in peripheral myelin.     

Galactolipids are molecular determinants of myelin development and axo-glial organization

Myelination is a developmentally regulated process whereby myelinating glial cells elaborate large quantities of a specialized plasma membrane that ensheaths axons. The myelin sheath contains an unusual lipid composition in that the glycolipid galactosylceramide (GalC) and its sulfated form sulfatide constitute a large proportion of the total lipid mass. These glycolipids have been implicated in a range of developmental processes such as cell differentiation and myelination initiation, but analyses of mice lacking UDP-galactose:ceramide galactosyltransferase (CGT), the enzyme required for myelin galactolipid synthesis, have more recently demonstrated that the galactolipids more subtly regulate myelin formation. The CGT mutants display a delay in myelin maturation and axo-glial interactions develop abnormally. By interbreeding the CGT mutants with mice that lack myelin-associated glycoprotein, it has been shown that these specialized myelin lipids and proteins act in concert to promote axo-glial adhesion during myelinogenesis. The analysis of the CGT mutants is helping to clarify the roles myelin galactolipids play in regulating the development, and ultimately the function of the myelin sheath.     

THE FINE STRUCTURAL ORGANIZATION OF NERVE FIBERS, SHEATHS, AND GLIAL CELLS IN THE PRAWN, PALAEMONETES VULGARIS

In view of reports that the nerve fibers of the sea prawn conduct impulses more rapidly than other invertebrate nerves and look like myelinated vertebrate nerves in the light microscope, prawn nerve fibers were studied with the electron microscope. Their sheaths are found to have a consistent and unique structure that is unlike vertebrate myelin in four respects: (1) The sheath is composed of 10 to 50 thin (200- to 1000-A) layers or laminae; each lamina is a cellular process that contains cytoplasm and wraps concentrically around the axon. The laminae do not connect to form a spiral; in fact, no cytoplasmic continuity has been demonstrated among them. (2) Nuclei of sheath cells occur only in the innermost lamina of the sheath; thus, they lie between the sheath and the axon rather than outside the sheath as in vertebrate myelinated fibers. (3) In regions in which the structural integrity of the sheath is most prominent, radially oriented stacks of desmosomes are formed between adjacent laminae. (4) An ~200-A extracellular gap occurs around the axon and between the innermost sheath laminae, but it is separated from surrounding extracellular spaces by gap closure between the outer sheath laminae, as the membranes of adjacent laminae adhere to form external compound membranes (ECM's). Sheaths are interrupted periodically to form nodes, analogous to vertebrate nodes of Ranvier, where a new type of glial cell called the "nodal cell" loosely enmeshes the axon and intermittently forms tight junctions (ECM's) with it. This nodal cell, in turn, forms tight junctions with other glial cells which ramify widely within the cord, suggesting the possibility of functional axon-glia interaction.     

Differential Ultrastructural Localization of Myelin Basic Protein, Myelin/Oligodendroglial Glycoprotein, and 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase in the CNS of Adult Rats

In a light and electron microscopic immunocytochemical study we have examined the distribution of myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), and myelin/oligodendroglial glycoprotein (MOG) within CNS myelin sheaths and oligodendrocytes of adult Sprague-Dawley rats. Ultrastructural immunocytochemistry allowed quantitative analysis of antigen density in different myelin and oligodendrocyte zones: MBP was detectable in high density over the whole myelin sheath, but not in regions of loops, somata, or the oligodendrocyte plasma membrane. CNP reactivity was highest at the myelin/axon interface, and found in lower concentration over the outer lamellae of myelin sheaths, at the cytoplasmic face of oligodendrocyte membranes, and throughout the compact myelin. MOG was preferentially detected at the extracellular surface of myelin sheaths and oligodendrocytes and in only low amounts in the lamellae of compacted myelin and the myelin/axon border zone. Our studies, thus, indicate further the presence of different molecular domains in compact myelin, which may be functionally relevant for the integrity and maintenance of the myelin sheath.     

The Ultrastructure of Schmidt-Lanterman Clefts and Related Shearing Defects of the Myelin Sheath

Schmidt-Lanterman clefts in frog sciatic nerves have been studied in thin sections by electron microscopy utilizing permanganate fixation and araldite embedding. It is shown that they are shearing defects in myelin in which the lamellae are separated widely at the major dense lines. Each lamella consisting of two apposed Schwann cell unit membranes ~ 75 A across traverses the cleft intact. The unit membranes composing each lamella sometimes are slightly (~ 50 to 100 A) separated in the clefts. The layers between the lamellae contain membranous structures which may be components of the endoplasmic reticulum. These layers are continuous with the outer layer of Schwann cytoplasm and the thin and inconstant cytoplasmic layer next to the axon (Mauthner's sheath). Each of these layers in perfect clefts constitutes a long helical pathway through the myelin from the axon. One of these is connected with Schwann cytoplasm and the other directly with the outside. A type of cross-sectional shearing defect, not hitherto recognized, is described and shown to be a kind of Schmidt-Lanterman cleft. Incomplete clefts are seen and interpreted as representing stages in a dynamic process whereby the myelin lamellae may be constantly separating and coming together again in life.     

The Lateral Membrane Organization and Dynamics of Myelin Proteins PLP and MBP Are Dictated by Distinct Galactolipids and the Extracellular Matrix

In the central nervous system, lipid-protein interactions are pivotal for myelin maintenance, as these interactions regulate protein transport to the myelin membrane as well as the molecular organization within the sheath. To improve our understanding of the fundamental properties of myelin, we focused here on the lateral membrane organization and dynamics of peripheral membrane protein 18.5-kDa myelin basic protein (MBP) and transmembrane protein proteolipid protein (PLP) as a function of the typical myelin lipids galactosylceramide (GalC), and sulfatide, and exogenous factors such as the extracellular matrix proteins laminin-2 and fibronectin, employing an oligodendrocyte cell line, selectively expressing the desired galactolipids. The dynamics of MBP were monitored by z-scan point fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), while PLP dynamics in living cells were investigated by circular scanning FCS. The data revealed that on an inert substrate the diffusion rate of 18.5-kDa MBP increased in GalC-expressing cells, while the diffusion coefficient of PLP was decreased in sulfatide-containing cells. Similarly, when cells were grown on myelination-promoting laminin-2, the lateral diffusion coefficient of PLP was decreased in sulfatide-containing cells. In contrast, PLP''s diffusion rate increased substantially when these cells were grown on myelination-inhibiting fibronectin. Additional biochemical analyses revealed that the observed differences in lateral diffusion coefficients of both proteins can be explained by differences in their biophysical, i.e., galactolipid environment, specifically with regard to their association with lipid rafts. Given the persistence of pathological fibronectin aggregates in multiple sclerosis lesions, this fundamental insight into the nature and dynamics of lipid-protein interactions will be instrumental in developing myelin regenerative strategies.     

Myelin P0-Glycoprotein: Predicted Structure and Interactions of Extracellular Domain

Protein zero (P₀), a transmembrane glycoprotein, accounts for over 50% of the total protein in PNS myelin. The extracellular domain of P₀ (P₀-ED) is similar to the immunoglobulin variable domain, carrying one acceptor sequence for N-linked glycosylation. The x-ray diffraction analysis of PNS myelin has demonstrated reversible transitions that depend on pH and ionic strength, resulting in three distinct structures characterized by widths of about 36 Å, 50 Å (native), and 90 Å between the extracellular surfaces of the membranes. In the current work, we considered the constraints imposed by these x-ray diffraction data on the orientation of P₀-ED, and we propose how this immunoglobulin-like domain could be accommodated in the variable widths of the extracellular space between myelin membranes. The modeling made use of the finding that β-strand predictions for P₀-ED are virtually superimposable with those of the V_H domain of the phosphocholine-binding immunoglobulin M603 of mouse, which has a similar number of residues as P₀-ED and a structure that has been solved crystallographically. The dimensions of P₀-ED from the space-filling model, developed using PC- based molecular modeling software, were found to be 44 Å× 25 Å× 23 Å. On the assumption that neither the shape nor the orientation of P₀-ED changes appreciably, then the different widths at the extracellular apposition would easily accommodate P₀-ED from apposed membranes if the molecules were oriented so that the β- strands were approximately perpendicular to the membrane surface. The apposed P₀-EDs would fully overlap at the closest apposition of the membranes, partially overlap in the native state, and align end to end in the incompletely swollen state. The P₀-ED regions analogous to the complementarity-determining regions of immunoglobulins can account for the recognition of P₀-ED from apposed membranes in the incompletely swollen state. Two of the faces of P₀-ED that show charge complementarity could account for the homophilic interactions of P₀-ED from apposed membranes in the native state. This association can be stabilized further by hydrophobic interactions. The N- linked nonasaccharide after energy minimization fit into a cavity, which was at the base of P₀-ED and which was lined with three positively charged residues. Thus, the carbohydrate may help to maintain the orientation of P₀ at the membrane surface. Our model shows how the single immunoglobulin-like domain of P₀ can account for distinct structural states of myelin membrane packing by homophilic interactions.     

Myelin abnormalities in mice deficient in galactocerebroside and sulfatide

Myelin sheath formation depends on appropriate axo-glial interactions that are mediated by myelin-specific surface molecules. In this study, we have used quantitative morphological analysis to determine the roles of the prominent myelin lipids galactocerebroside (GalC) and sulfatide in both central and peripheral myelin formation, exploiting mutant mice incapable of synthesizing these lipids. Our results demonstrate a significant increase in uncompacted myelin sheaths, the frequency of multiple cytoplasmic loops, redundant myelin profiles, and Schmidt-Lanterman incisures in the CNS of these mutant mice. In contrast, PNS myelin appeared structurally normal in these animals; however, at post-natal day 10, greater than 10% of the axons withered and pulled away from their myelin sheaths. These results indicate that GalC and sulfatide are critical to the formation of CNS myelin. In contrast, PNS myelin formation is not dependent on these lipids; however, GalC and sulfatide appear to be instrumental in maintaining Schwann cell-axon contact during a specific developmental window.     

Interfaces between microbes and membranes of host epithelial cells in hemipteran midguts

Certain families of plant-feeding insects in the order Hemiptera (infraorder Pentatomomorpha) have established symbiotic relationships with microbes that inhabit specific pouches (caeca) of their midgut epithelium. The placement of these caeca in a well-delineated region at the most posterior end of the midgut bordering the hindgut is conserved in these families; in situ the convoluted midgut is predictably folded so that this caecal region lies adjacent to the anterior-most region of the midgut. Depending on the hemipteran family, caeca vary in their number and configuration at a given anterior–posterior location. At the host-microbe interface, epithelial plasma membranes of midgut epithelial cells interact with nonself antigens of microbial surfaces. In the different hemipteran species examined, a continuum of interactions is observed between microbes and host membranes. Bacteria can exist as free living cells within the midgut lumen without contacting host membranes while other host cells physically interact extensively with microbial surfaces by extending numerous processes that interdigitate with microbes; and, in many instances, processes completely envelope the microbes. The host cells can embrace the foreign microbes, completely enveloping each with a single host membrane or sometimes enveloping each with the two additional host membranes of a phagosome.     

Membrane interactions in nerve myelin: II. Determination of surface charge from biochemical data.

In our accompanying paper (Inouye and Kirschner, 1988) we calculated the surface charge density at the extracellular surfaces in peripheral and central nervous system (PNS; CNS) myelins from observations on the dependency of the width of the extracellular space on pH and ionic strength. Here, we have determined the surface charge density of the membrane surfaces in myelin from its chemical composition and the localization of some of its molecular components. We then analyzed the attractive and repulsive forces between the apposed surfaces and calculated equilibrium periods for comparison with the measured values. The biochemical model accounts for the observed isoelectric range of the myelin period and, with the surface charge reduced (possibly by divalent cation binding or a space charge approximation), the model also accounts for the dependency of period on pH above the isoelectric range. At the extracellular (and cytoplasmic) surfaces the contribution of lipid (with pI approximately 2) to the net surface charge is about the same in both PNS and CNS myelin, whereas the contribution of protein depends on which ones are exposed at the two surfaces. The protein conformation and localization modulate the surface charge of the lipid, resulting in positively-charged cytoplasmic surfaces (pI approximately 9) and negatively-charged extracellular surfaces (pI approximately 2-4). The net negative charge at the extracellular surface is due in CNS myelin to lipid, and in PNS myelin to both lipid and (PO) glycoprotein. The net positive charge at the cytoplasmic surface is due in CNS myelin mostly to basic protein, and in PNS myelin to PO glycoprotein and basic protein. The invariance of the cytoplasmic packing may be due to specific short-range interactions. Our models demonstrate how the particular myelin proteins and their localization and conformation can account for the differences in inter-membrane interactions in CNS and PNS myelins.     

A convenient oxidation of natural glycosphingolipids to their "ceramide acids" for neoglycoconjugation. Bovine serum albumin-glycosylceramide acid conjugates as investigative probes for HIV gp120 coat protein-glycosphingolipid interactions.

A new method to cleave the double bond of sphingolipids has been developed. Using limited concentrations of KMnO4 and an excess of NaIO4, in a neutral aqueous tert-butanol solvent system gave nearly quantitative yields of the oxidized product. A variety of natural glycosphingolipids (GSLs): GlcC, GalC, SGC, LC, Gb3C, Gb4C, Gg4C, Gb5C, and GM1C, gave the corresponding acids: 2-hydroxy-3-(N-acyl)-4-(O-glycosyl)-oxybutyric acids, i.e. "glycosyl ceramide acids" (GSL.CCOOH) in excellent yields (80-90%). Deacyl GSLs (dGSLs) were oxidized to acids containing the oligosaccharides devoid of hydrocarbon chains, i.e. "ceramide oligosaccharides" (dGSL. NRR1CCOOH, where R = R1 = H; R = H, R1 = CH3CO; or R = R1 = Me). The efficacy of this method was demonstrated by transforming natural GSLs: GlcC, GalC, GalS, SGC, LC, Gb3C, and Gb4C into neoglycoproteins via coupling glycosyl ceramide acids (except GalS, which was coupled directly) to bovine serum albumin (BSA). Mass spectroscopic analysis of GalC-BSA conjugates, (GalC.CONH)nBSA and (GalS.NHCO)nBSA gave a value of 9 +/- 1 and 16 +/- 2 for n. Neoglycoconjugates derived from GlcC, GalC (type I and II and the behenic analog), SGC, LC, and Gb3C were recognized by the recombinant human immunodeficiency virus coat protein gp120 (rgp120). The GalS conjugate showed significantly reduced binding, and the Gb4C conjugate showed no binding. Thus, rgp120/GSL-BSA interaction requires a terminal galactose and/or glucose residue. Terminal N-acetylgalactosamine containing GSLs are not bound. The ceramide acid conjugates provide a more effective scaffold for presentation of glycone for rgp120 binding than those derived from dGSLs. The retention of receptor specificity of the glycoconjugates was validated by retention of the expected binding specificity of VT1 and VT2e for Gb3C and Gb4C conjugates, respectively. These studies open a new vista in the generation of glycoconjugates from GSLs and further emphasize the role of aglycone in glycolipid recognition.     

Membrane Interactions Are Altered in Myelin Isolated from Central and Peripheral Nervous System Tissues

Isolated myelin has been used for determinations of membrane surface charge density and topographical mapping of components in the membrane. To determine how similar such myelin is to myelin of intact tissue, we have used x-ray diffraction to compare their intermembrane interactions. The interactions were monitored by measuring the myelin period in samples treated with distilled water, buffered saline at pH 4-9 and ionic strength 0.06-0.18, and saline containing HgCl2 or triethyl tin sulfate. Myelin was isolated from whole brains and sciatic nerves of mice by conventional methods involving sucrose gradient centrifugation and osmotic shock. Consistent with previous findings, electron microscopy showed that the multilamellar morphology, staining, and repeat periods of isolated myelin were essentially like those of intact myelin; however, the membrane stacks were less extensive than those in whole tissue. X-ray diffraction revealed that isolated CNS myelin was like intact myelin in showing reversible compaction in acidic media and in distilled water. However, unlike the myelin in whole tissue, isolated CNS myelin did not swell in hypotonic or alkaline media, or in the presence of HgCl2-saline or triethyl tin. The altered membrane interactions could result from an increase in adhesiveness of the apposed membrane surfaces. Reorganization of proteolipid protein and/or a reduction of surface charge could account for the change in surface properties of isolated CNS myelin. Isolated PNS myelin, like the membranes in whole tissue, showed both compaction and swelling; however, the membrane pairs were disordered in the swollen structure. This irregular membrane swelling could result from charge variation in the extracellular surfaces.     

Effects of ZnCl2 on membrane interactions in myelin of normal and shiverer mice

X-ray diffraction was used to record the effects of metal cations on the structure of peripheral nerve myelin. Acidic saline (pH 5.0) either with or without added metal cations caused myelin to swell by 10-20 A from its native period of 178 A. The X-ray patterns usually showed broad reflections, and higher orders were either weak or unobserved. With added ZnCl2, however, the swollen myelin gave diffraction patterns that retained sharp reflections to approx. 15 A spacing. Alkaline saline (pH 9.7) containing ZnCl2 produced a reduction of the myelin period by approx. 5 A which was at least twice as much as that produced by other metals. To examine the underlying chemical basis for these unique interactions of Zn2+ with myelin, we carried out parallel X-ray experiments on sciatic nerve from the shiverer mutant mouse, which lacks the major myelin basic proteins. Shiverer myelin responded like normal myelin to ZnCl2 in acidic saline; however, in alkaline saline shiverer myelin showed broadened X-ray reflections which indicated disordering of the regularity of the membrane arrays, and additional reflections were recorded which indicated lipid phase separation. This breakdown may come about by the binding of Zn2+ to negatively-charged lipids which could be more exposed due to the absence of myelin basic proteins. Electron density profiles were calculated on the assumption that, except for changes in their packing, the myelin membranes were minimally altered in structure. For both normal and shiverer myelins, treatments under acidic conditions resulted in swelling at the extracellular apposition and a slight narrowing of the cytoplasmic space. This swelling is likely due to adsorption of protons and divalent cations. Interaction between Zn2+ and myelin P0 glycoprotein could preserve an ordered arrangement of the apposed membrane surfaces. Alkaline saline containing ZnCl2 produced compaction at the cytoplasmic apposition in both normal and shiverer myelins possibly through interactions with a portion of P0 glycoprotein which extends into the cytoplasmic space between membranes.     

Cellular Mechanism of Myelination in the Central Nervous System

A study of myelination with electron microscopy has been carried out on the spinal cord of young rats and cats. In longitudinal and transverse sections the intimate relationship of the growing axons with the oligodendrocytes was observed. Early naked axons appear to be embedded within the cytoplasm and processes of the oligodendrocytes from which they are limited only by the intimately apposed membranes of both elements (axon-oligocytic membrane). In a transverse section several axons are observed to be in a single oligodendrocyte. The process of myelination consists in the laying down, within the cytoplasm of the oligodendrocyte and around the axon, of concentric membranous myelin layers. The first of these layers is deposited at a certain distance (200 to 600 A or more) from the axon-oligocytic membrane. This and all the other subsequently formed membranes have higher electron density and are apparently formed by the coalescence and fusion of vesicles (of 200 to 800 A) and membranes found in large amounts within the cytoplasm of the oligodendrocytes. At an early stage the myelin layers may be discontinuous and some vesicular material may even be trapped among them or between the myelin proper and the axon-oligocytic membrane. Then, when the 8th to 10th layer is deposited, the complete coalescence and alignment of the lamellae leads to the characteristic orderly multilayered organization of the myelin sheath. Myelination in the central nervous system appears to be a process of membrane synthesis within the cytoplasm of the oligodendrocyte and not a result of the wrapping of the plasma membranes as postulated in Geren's hypothesis for the peripheral nerve fibers. The possible participation of Schwann cell cytoplasm in peripheral myelination is now being investigated.