Antigenic sites common to major fish myelin glycoproteins (IP) and to major tetrapod PNS myelin glycoprotein (Po) reside in the amino acid chains

The major myelin glycoproteins in the CNS and PNS of trout (IP) were enzymatically deglycosylated with endoglycosidase F (Endo F) and examined by electro-immunoblotting. Following carbohydrate removal and loss of concanavalin A affinity each of the four IP components underwent a similar reduction in molecular size, corresponding to approximately 3,000 daltons. Immunological cross-reactivities with anti-bovine P_o or anti-trout IP2 antibodies, were however fully retained by the Endo F cleavage products. This strongly implies that the antigenic sites shared by the mammalian P_o protein and the various intermediate glycoproteins of trout CNS and PNS are located in the protein portion. Immunoblot analysis of the PNS myelin proteins from various species of the major vertebrate classes with anti-trout IP2 antiserum revealed striking differences in the immunological properties of the individual P_o components which were not detected when anti-bovine P_o antiserum was used as a probe.     

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.     

Rapid screening of myelin genes in CMT1 patients by SSCP analysis: identification of new mutations and polymorphisms in the P0 gene

Charcot-Marie-Tooth type (CMT1) disease or hereditary motor and sensory neuropathy type I (HMSNI) is an autosomal dominant peripheral neuropathy. In most CMT1 families, the disease cosegregates with a 1.5-Mb duplication on chromosome 17p11.2 (CMT1A). A few patients have been found with mutations in the peripheral myelin protein 22 (PMP-22) gene located in the CMT1A region. In other families mutations have been identified in the major peripheral myelin protein p_o gene localized on chromosome Iq21-q23 (CMT1B). We performed a rapid mutation screening of the PMP-22 and P₀ genes in non-duplicated CMT1 patients by single-strand conformation polymorphism analysis followed by direct polymerase chain reaction sequencing of genomic DNA. Six new single base changes in the P₀ gene were observed: two missense mutations in, respectively, exons 2 and 3, two nonsense mutations in exon 4, and two silent mutations or polymorphisms in, respectively, exons 3 and 6.     

Protease-sensitive thylakoidal import machinery for the Sec-, ΔpH- and signal recognition particle-dependent protein targeting pathways, but not for CFoII integration

Nuclear-encoded proteins are targeted into and across the thylakoid membrane by a surprising variety of mechanisms. Distinct Sec- and ΔpH-dependent mechanisms have been shown to operate for lumenal proteins, and an integral membrane protein, LHCP, has been shown to insert via a signal recognition particle-dependent route. Integration of a further membrane protein, CF_oII, requires neither soluble factors nor energy sources, prompting speculation of a spontaneous insertion mechanism. Although the requirements for soluble factors and energy sources have been determined in some detail, much less is known of the events taking place at the membrane surface. This report examines whether thylakoid proteins are involved in each of these pathways, by testing the effects of trypsin on the capacity of isolated thylakoids to import proteins. Because all of the pathways rely to some extent on the thylakoidal ΔpH, and a light-induced ΔpH is easily destroyed by proteolysis, the conditions under which reverse action of the ATP synthase in the dark generates a high ΔpH even after proteolysis of thylakoids have been established. This system is used to show that protease-sensitive thylakoidal import machinery is crucial for the ΔpH-, Sec- and signal recognition particle-dependent pathways, but not for integration of CF_oII.     

A biochemical comparison of Xenopus laevis and mammalian myelin from the central and peripheral nervous systems

Myelin purified from the central nervous system of Xenopus laevis contained the same major lipid and protein components as human myelin. However, some minor differences in the myelin proteins were noted. The Xenopus basic protein had a higher apparent mol wt. on sodium dodecyl sulfate gels than the corresponding mammalian protein. The absolute specific activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in the Xenopus myelin was considerably higher than in mammals. There were differences in the high mol wt. proteins, and the glycoproteins in Xenopus myelin were more heterogeneous than those in mammals. Peripheral myelin from Xenopus sciatic nerve was compared with that from the rat. The lipids in the two types of myelin were similar. There was a major glycoprotein in the Xenopus myelin corresponding to the P₀ protein and a basic protein of slightly larger mol wt. than the P₁ protein of rat myelin.     

Susceptibility of myelin proteins to a neutral endoproteinase: The degradation of myelin basic protein (MBP) and P2 protein by purified bovine brain multicatalytic proteinase complex (MPC)

Multicatalytic proteinase complex (MPC) was isolated from bovine brain and the susceptibility of myelin basic protein (MBP) and P₂ protein of bovine central and peripheral nervous system was examined. SDS-polyacrylamide electrophoretic analysis of purified MPC revealed protein bands of molecular weight ranging from 22–35 kDa. The enzyme is activated by SDS at a concentration less than 0.01%. Upon incubation with MPC, purified MBP and P₂ proteins were degraded into smaller fragments. There was a 57% and 100% loss of MBP at 2 and 6 hours of incubation. The P₂ protein which is not susceptible to any endogenous non-lysosomal enzyme thus far studied was digested into small peptide fragments only in the presence of SDS (0.01%) and not in its absence. These results indicate that MPC which is active at physiological conditions may have a role in the turnover of myelin proteins and in demyelinating diseases.     

Sulfate metabolism of rat P0 glycoprotein: some observations

It has been known for some time that P₀, the major intrinsic protein in PNS myelin, contains sulfate. The position of sulfate has been described for beef PNS myelin, but rat PNS myelin differs somewhat from that of the beef, therefore an investigation of the location of sulfate in rat P₀ was undertaken. Weanling rat nerves were incubated with [³H] amino acid mixture and [³⁵S]O₄, and purified myelin was prepared, and the proteins separated on polyacrylamide gels. The bulk of the [³⁵S]O₄ was incorporated into P₀, but smaller peaks of sulfate label were found in the higher molecular weight proteins. With tunicamycin in the incubation mixture, sulfate incorporation was inhibited. Incubation of the labeled myelin mixture with endo F or glycanase resulted in total loss of sulfate label on P₀, therefore all of the [³⁵S]O₄ was incorporated into the oligosaccharide chain, with none on the polypeptide. Castanospermine and deoxymannojirimycin inhibited [³⁵S]O₄ incorporation into P₀, but no inhibition was exerted by swainsonine. These results indicate that sulfate resides in the core of the oligosaccharide chain, with none in the terminal region. Such a structure would correlate with the lack of an HNK-1 epitope, absent in the rat, but found in P₀ of many species.Abbreviations Used Endo H endoglycosidase H - Endo F endoglycosidase F - GalNAc N-acetyl galactosamine - GlcNAc N-acetyl glucosamine - MAG myelin-associated glycoprotein - Man mannose Special issue dedicated to Dr. Marjorie B. Lees.     

Lipid and Protein Alterations of Spinal Cord and Cord Myelin of Multiple Sclerosis

Abstract: A comprehensive study was carried out to clarify the chemical compositions of spinal cord, cord myelin, and myelin subfractions of multiple sclerosis (MS). The protein compositions of normal-appearing cerebral white matter and cerebral plaque and periplaque tissues were also analyzed for comparison. MS whole cord samples were found to contain higher amounts of water compared with normal samples. The total lipid contents were below normal. Among the individual lipids, cholesterol content remained unchanged, whereas cholesteryl esters appeared increased in MS cords. The acidic phospholipid concentrations were found to be lower than normal. Glycolipids, such as cerebrosides G_M4, G_M1, and G_D1b, which are abundant in myelin, were all decreased. However, the concentrations of G_M3 and G_D3, which are more characteristic of reactive astrocytes, were highly elevated. The total protein content of MS cord samples was decreased, and the decrease was attributable to the loss of myelin proteins as evidenced by the low recovery of myelin. The concentrations of myelin-specific proteins, such as proteolipid protein and myelin basic protein, were significantly reduced. Other changes in the protein compositions included the accretion of two low molecular weight proteins of approximately 11,000 and 12,000, and the appearance of a periodic acid-Schiff-positive protein with the same electrophoretic mobility as the P₀ protein. Analysis of the isolated myelin indicated that it had a grossly normal protein composition. However, the two low molecular weight proteins and the P₀ protein appeared to be enriched in an upper-phase cord subtraction. We attribute the appearance of the two low molecular weight proteins to the breakdown of proteolipid protein and/or myelin basic protein as a result of demyelination, and the appearance of P₀ to the involvement of PNS myelin. The latter finding provides the first biochemical evidence that in MS cord, remyelination can be achieved in part by invading Schwann cells and/or by the small number of Schwann cells that may be present in the cord.     

Myelin protein changes with digestion of whole sciatic nerve in trypsin.

Whole mouse sciatic nerves were split and incubated in phosphate buffered saline (PBS) and in PBS containing various amounts of trypsin. After 24 hours of exposure to PBS alone there were no changes in the gel electrophoresis pattern of myelin proteins. During the same period of time, trypsin digested major amounts ofboth the main myelin protein (P_O) and the two basic proteins of myelin (P₁, P₂). The basic proteins were undetectable after 24 hours of 1% trypsin digestion while the main myelin protein was not completely digested. The amount of digestion of the myelin proteins was related to the concentration of trypsin and the time of digestion. Myelin proteins were demonstrated by staining with Coomassie blue, periodic acid Schiff (PAS) and by special indirect lighting techniques.     

Partial deficiencies in the myelin proteins of developing mice heterozygous for the shiverer mutation

The central nervous system of the shiverer mouse is known to be severely deficient in myelin. Animals heterozygous for this autosomal-recessive mutation were crossed, and the myelin proteins were examined in the brains and spinal cords of shiverers and unaffected littermates among the offspring. In the brains and spinal cords of nine of the 14 unaffected littermates examined, the quantities of the myelin basic and proteolipid proteins were lower than normal. Furthermore, in the brains of heterozygotes 33 to ～ 150 days old, the myelin basic and proteolipid proteins were reduced in amount, compared to wild-type controls; the myelin basic protein was also present in subnormal amounts in the spinal cords from heterozygous animals at the ages of 17 to 150 days. More severe reductions in the quantities of the myelin proteins were observed in central nervous system tissue from homozygous shiverer mice, and the quantity of the myelin proteolipid protein in the central nervous system of the shiverer mouse, expressed as a ratio to the control value at each age, underwent a developmental decline. In heterozygotes, as well as shiverers, the peripheral nerves were also deficient in the P₁ and P_r proteins, which are the same as the basic proteins in rodent central nervous system myelin. The findings regarding heterozygotes suggest that the defective primary gene product in the shiverer mouse could be the myelin basic protein itself or a protein required for a rate-limiting step in the processing of the myelin basic protein.     

Phosphatase activity of extra-radical arbuscular mycorrhizal hyphae: A review

Phosphatase activity of arbuscular mycorrhizal (AM) fungi has attracted attention in three fairly distinct domains: intracellular enzymes with defined metabolic functions that have been studied in intraradical hyphae, histochemical staining of alkaline phosphatase as an indicator of fungal activity measured both intra- and extraradically, and extracellular activity related to mineralization of organic P (P_o) compounds that may enhance mycorrhizal utilization of an important nutrient pool in soil. This review focuses on the latter subjects with emphasis on extraradical mycelium (ERM), while it draws on selected data from the vast material available concerning phosphatases of other organisms. We conclude that histochemical staining of alkaline phosphatase is a sensitive and suitable method for monitoring the effect of adverse conditions encountered by ERM both as a symbiotically functional entity in soil, and in vitro without modifying interference of soil or other solid substrates. Furthermore, the quantitative importance of extracellular enzymes for P nutrition of AM plants is estimated to be insignificant. This is concluded from the low quantitative contribution extracellular hyphae of AM fungi give to the total phosphatase activity in soil, and from estimations of which processes that may be rate limiting in organic P mineralization. Maximum values for the former is in the order of a few percent. As for the latter, solubilization of P_o seems to be far more important than P_o hydrolysis for utilization of P_o by AM fungi and plants, as both endogenous soil phosphatase activity and phosphatases of other soil organisms are ubiquitous and abundant. Our discussion of mycorrhizal phosphatases supports the view that extracellular phosphatases of roots and micro-organisms are to a large extent released incidentally into soil, and that the source has limited benefit from its activity. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nervous system myelin in the electric ray, Torpedo marmorata: Morphological characterization of the membrane and biochemical analysis of its protein components

In Torpedo, PNS as well as CNS myelines are characterized by clearly separated double intraperiod lines. CNS myelin of Torpedo contains two glycosylated hydrophobic proteins labelled T1 (25,800 Da1) and T2 (29,700 Da1), and two basic proteins BP1 and BP2, migrating like mammalian large basic protein (BP2) and pre-small basic protein (BP1) (Barbarese et al., 1977). PNS myelin of Torpedo carries only BP1 and is characterized by a closely spaced doublet of the glycosylated hydrophobic proteins Con A+ (29,700 Da1) and Con A? (31,000 Da1); the latter does not bind Concanavalin A. These glycosylated proteins (T1, T2, Con A+, Con A?) contain mannose, N-acetylglucosamine and galactose, but lack fucose and sialic acids. They have isoleucine at their amino terminus. They bind anti-rat PNS myelin P₀ antibodies but do not react with anti-rat CNS myelin PLP antibodies. Limited proteolyses of isolated proteins suggest sequence homologies between T1 and T2, and possibly between Con A+ and Con A?. The two basic proteins BP1 and BP2 bind antibodies directed against human myelin basic protein. All Torpedo myelin proteins electrofocus in pH regions characteristic of their mammalian counterparts.     

EFFECT OF PROTEOLYTIC ATTACK ON THE STRUCTURE OF CNS MYELIN MEMBRANE

Rat CNS myelin particles have been incubated with trypin and acetyltrypsin under conditions which ensured a selective and substantial removal of the basic proteins leaving acidic Wolfgram and proteolipid proteins. Some trypsin became associated with the basic protein denuded pellet while no attachment of acetyltrypsin was observed. The removal of basic proteins ‘solubilized’ some myelin and produced a lighter ‘fluff’ layer on top of the myelin pellet, but this amounted to no more than 10 per cent of the total myelin lamellae. Electron microscopy indicated a more dense-straining interperiod line in a small percentage of lamellae which otherwise remained normal. Selective extraction of complex lipids with solvents of increasing polarity, nuclear magnetic resonance spectra and X-ray diffraction patterns showed no significant changes on removing basic proteins from myelin. The results are interpreted as suggesting that the basic proteins are not uniformly distributed in myelin but preferentially located in the outside layers of the myelin sheath and that they play little part in stabilizing the bulk of the myelin membrane structure.     

GANGLIOSIDES OF PERIPHERAL NERVE MYELIN

—Gangliosides have been isolated from myelin obtained from three types of peripheral nerve: bovine spinal roots, bovine sciatic nerve and human sciatic nerve. Yields in most cases were 218–287 μg of lipid-bound sialic acid per g myelin, less than half that previously obtained from CNS myelin. Myelin accounted for approx 60% of total ganglioside present in whole spinal root. The human sample contained only N-acetylneuraminic acid but the two bovine preparations contained that as well as N-glycolylneuraminic acid; N-acetylglucosamine and N-acetylgalactosamine were both present in all three preparations. Sphingosine was the major long-chain base in each preparation while 4-eicosasphingenine (d20:1) comprised about 14% in the two bovine samples and 3% in the human sample. The major fatty acids in all preparations were 16:0, 18:0, 22:0, 24:0 and 24:1. Sialosylgalactosyl ceramide (G₇), a ganglioside characteristic of CNS myelin, was not detected in any of the PNS samples. The majority of gangliosides in bovine spinal root myelin were monosialo species, although the structures differed in some respects from those of CNS myelin. The molar concentration of lipid-bound sialic acid in PNS myelin is roughly equivalent to that of the P₁ basic protein.     

Regulation of TRPV1 Ion Channel by Phosphoinositide (4,5)-Bisphosphate: THE ROLE OF MEMBRANE ASYMMETRY*

Membrane asymmetry is essential for generating second messengers that act in the cytosol and for trafficking of membrane proteins and membrane lipids, but the role of asymmetry in regulating membrane protein function remains unclear. Here we show that the signaling lipid phosphoinositide 4,5-bisphosphate (PI(4,5)P₂) has opposite effects on the function of TRPV1 ion channels depending on which leaflet of the cell membrane it resides in. We observed potentiation of capsaicin-activated TRPV1 currents by PI(4,5)P₂ in the intracellular leaflet of the plasma membrane but inhibition of capsaicin-activated currents when PI(4,5)P₂ was in both leaflets of the membrane, although much higher concentrations of PI(4,5)P₂ in the extracellular leaflet were required for inhibition compared with the concentrations of PI(4,5)P₂ in the intracellular leaflet that produced activation. Patch clamp fluorometry using a synthetic PI(4,5)P₂ whose fluorescence reports its concentration in the membrane indicates that PI(4,5)P₂ must incorporate into the extracellular leaflet for its inhibitory effects to be observed. The asymmetry-dependent effect of PI(4,5)P₂ may resolve the long standing controversy about whether PI(4,5)P₂ is an activator or inhibitor of TRPV1. Our results also underscore the importance of membrane asymmetry and the need to consider its influence when studying membrane proteins reconstituted into synthetic bilayers.     

Myelin Gene Expression in Immortalized Schwann Cells: Relationship to Cell Density and Proliferation

Abstract: Myelin gene expression was investigated in the immortalized S16 Schwann cell line grown in the presence and absence of serum and at different densities. Protein expression was monitored by western blotting, and message levels were determined by RNase protection assays. To study cell proliferation rates at different cell densities and serum conditions. [³H]thymidine uptake assays and cell counts were performed. Although serum deprivation decreased cell proliferation as expected, the proliferation of S16 cells was unchanged or slightly increased at high density under the conditions of our experiments in either serum-containing or serum-free medium. This increased cell division at high density appeared to be due to greater release of an autocrine growth factor to the medium by dense cell populations. For both sparse and dense cells, substantially more P₀ glycoprotein (P₀) and myelin-associated glycoprotein (MAG) per milligram of total cellular protein were expressed when the cells were proliferating slowly in defined medium in comparison with more rapidly proliferating cells in serum-containing medium. Furthermore, in both serum-containing and defined media, dense cell populations expressed more MAG and P₀ than sparse ones. P₀ mRNA and MAG mRNA levels generally paralleled protein levels. The level of mRNA for peripheral myelin protein-22 (PMP-22) was also increased at high cell density but did not change much when proliferation was decreased by serum deprivation. PMP-22 protein was not detected under any of the growth conditions. The changes in expression of these genes with growth conditions may be specific for myelin proteins, because the expression of a nonmyelin glycoprotein, L1, remained constant. The level of cyclic AMP in the cells did not change with the different growth conditions tested. The results indicate that the S16 Schwann cell line mimics primary or secondary Schwann cells by down-regulating myelin gene expression when it proliferates more rapidly in the presence of serum. Furthermore, in both the presence and absence of serum, there was greater expression of myelin genes at high cell density that was not associated with a decreased proliferative rate. Because evidence for a role of secretory factors in affecting myelin gene expression was not obtained by treating sparse S16 cells with medium conditioned by dense S16 cells, the results suggest that the higher expression of myelin genes at high density may be mediated by cell-to-cell contact.     

A Region Containing a Proline-Rich Motif Targets sGi2 to the Golgi Apparatus

The central function of heterotrimeric GTP-binding proteins (G proteins) is the transduction of extracellular signals, via membrane receptors, leading to the activation of intracellular effectors. In addition to being associated with the plasma membrane, the α subunits of some of these proteins have also been localized in intracellular compartments. The mRNA of the G-protein inhibitory α subunit 2 (G_αi2) encodes two proteins, G_αi2 and sG_i2, by an alternative splicing mechanism. sG_i2 differs from G_αi2 in the C-terminal region and localizes in the Golgi in contrast to the plasma membrane localization of G_αi2. In this paper we show that the sequence specific to sG_i2 can direct the Golgi localization of other G_αi subunits, but not of the stimulatory subunit G_αs or of a secreted protein. This indicates that, in addition to the sG_i2 C-terminus, sequences located elsewhere in the protein are required to determine the Golgi localization. Inside the sG_i2 C-terminal region we have identified a 14-amino-acid proline-rich motif which specifies the Golgi localization. Finally, we show that the sG_i2 subunit, once activated, leaves the Golgi to be localized in the endoplasmic reticulum.     

Immunoblot identification of phosphorylated basic proteins of rat and rabbit CNS and PNS myelin: Evidence for four phosphorylated basic proteins and P2 in rat PNS myelin

The immunoblot technique permits the visualization of proteins following their separation on acrylamide gels, transfer to cellulose nitrate sheets and subsequent staining with antiserum. We have utilized this technique to demonstrate the presence of four basic proteins in rat PNS myelin with molecular weights of 21K, 18K, 17K, and 14K. Similarly, we demonstrated the presence of two basic proteins in rabbit PNS myelin (molecular weights of 21K and 18K). Exposure of the immunostained cellulose nitrate strips to X-ray film revealed the phosphorylation of four and two basic proteins in rat and rabbit PNS myelin, respectively. These basic proteins were present in the CNS myelin of the two species and were also phosphorylated. Because of the sensitivity of the immunoblot technique, it was also possible for us to visualize the P₂ protein in both rat and rabbit PNS myelin.     

THE PROTEIN COMPOSITION OF ISOLATED MYELIN1

—Three fractions, each containing markedly different proteins, was obtained from myelin: (1) The first fraction was obtained as an insoluble residue when myelin was extracted with neutral chloroform-methanol (CM, 2:1, v/v). It was digestible with trypsin and had an amino acid composition similar to that of the acidic proteolipid protein of Wolfgkam (1966). (2) The second fraction was obtained as a precipitate by the addition of various electrolytes (KCl, NaCl, CaCl₂, MgCl₂ or HCl) to the CM (2:1 v/v) extract. This fraction consisted mainly of a basic protein which exhibited an electrophoretic mobility and amino acid composition indistinguishable from those of the basic protein obtained from white matter (Martensson and LeBaron, 1966). This procedure provided for a simple and rapid isolation of the basic protein from myelin. Depending on the conditions of precipitation, this fraction was either free of lipid or contained tri- and diphosphoinositide. The effects of different ions at differing concentrations and the yield and nature of the precipitate have been studied. (3) A third fraction remained in solution in CM (2:1, v/v) after the addition of the electrolyte. It comprised the bulk of the myelin lipids and a protein fraction which was resistant to digestion with trypsin and had an amino acid composition similar to the classical proteolipid protein of Folch-Pi and Lees (1951). The possibility of a salt-type bonding between the basic protein and the polyphosphoinositides is discussed, and values for tri- and diphosphoinositide in bovine myelin are given.     

Binding of 2′,3′-Cyclic Nucleotide 3′-Phosphodiesterase to Myelin: An In Vitro Study

Abstract: The binding of 2′,3′-cyclic nucleotide 3′-phosphodiesterase isoform 1 (CNP1) to myelin and its association with cytoskeletal elements of the sheath have been characterized with in vitro synthesized polypeptides and purified myelin. We have previously shown that the cysteine residue present in the carboxy-terminal CXXX box of CNP1 is isoprenylated, and that both C₁₅ farnesyl and C₂₀ geranylgeranyl isoprenoids can serve as substrates for the modification. Here, we have mutated the CXXX box to obtain selectively farnesylated CNP1 or geranyl-geranylated CNP1 and found that these two modified forms of CNP1 behave identically in all of the assays performed. Isoprenylation is essential but not sufficient for the binding of in vitro synthesized CNP1 to purified myelin, because a control nonmyelin protein is isoprenylated, yet unable to bind to myelin. In our assay, membrane-bound CNP1 partitions quantitatively into the non-ionic detergent-insoluble phase of myelin, suggesting that CNP1 binds to cytoskeletal elements within myelin. However, isoprenylated CNP1 fails to bind to the cytoskeletal matrix isolated from myelin by detergent treatment, implying that both detergent-soluble and insoluble myelin components are involved in the binding of CNP1. A model for the interactions between CNP1 and myelin is presented, consistent with models proposed for other isoprenylated proteins.