Peter J. Brophy

Protein zero of peripheral nerve myelin: Biosynthesis, membrane insertion, and evidence for homotypic interaction

Protein zero (P0), an integral membrane glycoprotein synthesized by Schwann cells, is the major glycoprotein of peripheral nerve myelin. The predicted disposition of P0 with respect to the membrane bilayer postulates the existence of extracellular and intracellular domains, that mediate compaction of the myelin lamellae. We used in vitro translations programmed with sciatic nerve mRNA and cells transfected with a P0 cDNA construct to study the biosynthesis and topology of P0 in the bilayer. The behavior of P0 at the cell surface, when expressed under physiological conditions, was also examined. We have verified the topological predictions of an earlier model, derived from analysis of a P0 cDNA, and provide evidence that the extracellular domain of P0 mediates homotypically cell-cell interactions in the transfectants.

Periaxin, a novel protein of myelinating schwann cells with a possible role in axonal ensheathment

We report the cloning and subcellular localization of a novel Schwann cell-specific protein of 147 kd that we have named periaxin. Periaxin has a remarkable domain of repetitive pentameric units in the primary sequence. It is expressed in the first uncompacted whorls of membrane that ensheathe the axon, and further synthesis of the protein in the rat sciatic nerve parallels the deposition of myelin. In mature myelin, periaxin colocalizes with the myelin-associated glycoprotein in the cytoplasm-filled periaxonal regions of the sheath but is excluded from compact myelin. We propose that periaxin has a role in axon-glial interactions, possibly by interacting with the cytoplasmic domains of integral membrane proteins such as myelin-associated glycoprotein in the periaxonal regions of the Schwann cell plasma membrane.

The distribution of myelin basic protein mRNAs within myelinating oligodendrocytes

The nervous system contains oligodendrocytes with processes that are greatly extended in space. It is now clear that there are numerous complex, poorly understood mechanisms by which polypeptides are synthesized and delivered to their sites of function in these cells. One mechanism is by the active positioning of mRNAs encoding certain proteins to restricted intracellular subdomains. Perhaps the best studied example of this in the vertebrate CNS is the translocation of myelin basic protein mRNAs to the forming myelin sheath, where the newly synthesized polypeptides, which avidly associate with membranes, can be directly incorporated into the myelin membrane. Evidence for this conclusion is presented here in the context of related work on the general phenomenon of mRNA translocation that is under analysis in other systems.

Spin-label ESR studies on the interaction of bovine spinal cord myelin basic protein with dimyristoylphosphatidylglycerol dispersions.

Electron spin resonance (ESR) spectroscopy and chemical binding assays were used to study the interaction of bovine spinal cord myelin basic protein (MBP) with dimyristoylphosphatidylglycerol (DMPG) membranes. Increasing binding of MBP to DMPG bilayers resulted in an increasing motional restriction of PG spin-labeled at the C-5 atom position in the acyl chain, up to a maximum degree of association of 1 MBP molecule per 36 lipid molecules. ESR spectra of PG spin-labels labeled at other positions in the sn-2 chain showed a similar motional restriction, while still preserving the chain flexibility gradient characteristic of fluid lipid bilayers. In addition, labels at the C-12 and C-14 atom positions gave two-component spectra, suggesting a partial hydrophobic penetration of the MBP into the bilayer. Spectral subtractions were used to quantitate the membrane penetration in terms of the stoichiometry of the lipid-protein complexes. Approximately 50% of the spin-labeled lipid chains were directly affected at saturation protein binding. The salt and pH dependence of the ESR spectra and of the protein binding demonstrated that electrostatic interaction of the basic residues of the MBP with the PG headgroups is necessary for an effective association of the MBP with phospholipid bilayers. Binding of the protein, and concomitant perturbation of the lipid chain mobility, was reduced as the ionic strength increased, until at salt concentrations above 1 M NaCl the protein was no longer bound. The binding and ESR spectral perturbation also decreased as the protein charge was reduced by pH titration to above the pI of the protein at approximately pH 10.(ABSTRACT TRUNCATED AT 250 WORDS)

FATTY-ACID PH TITRATION AND THE SELECTIVITY OF INTERACTION WITH EXTRINSIC PROTEINS IN DIMYRISTOYLPHOSPHATIDYLGLYCEROL DISPERSIONS - SPIN LABEL ESR STUDIES

The effects of pH titration on the ESR spectra of 5-(4,4-dimethyloxazolidine-N-oxyl) stearic acid in bilayers of dimyristoyl phosphatidylglycerol complexed with either myelin basic protein, apocytochrome c, cytochrome c or lysozyme have been studied. Binding of the protein increases the outer hyperfine splitting of the stearic acid spin label to an extent which depends on the protonation state of the fatty acid carboxyl group for all proteins studied. The hyperfine splittings have been analysed to determine the relative selectivities of the protonated and unprotonated forms of the fatty acid by assuming that the spectra correspond to fast exchange on the ESR timescale. For myelin basic protein and apocytochrome c, the relative association constants are approximately 80% and 30% greater, respectively for the charged state than for the protonated state of the fatty acid. The pKa of the stearic acid spin label has been determined from the pH titration behaviour of the outer hyperfine splitting in the ESR spectra. The pKa at the lipid/water interface in the absence of protein is 8.0, and at the lipid/peripheral protein interface is 9.6, 9.2, 8.5 and 8.5 for the case of bilayers complexed with the myelin basic protein, apocytochrome c, cytochrome c and lysozyme, respectively. The large upward shifts in pKa are interpreted in terms of a strongly reduced polarity at the lipid/protein interface, suggesting that the dimyristoyl phosphatidylglycerol headgroups become largely dehydrated on binding of either myelin basic protein or apocytochrome c. The extent of this dehydraton is considerably less for cytochrome c and lysozyme.

Temperature sensitivity of potassium flux into red blood cells in the familial pseudohyperkalaemia syndrome.

The temperature dependence of potassium flux into the red cells of normal and pseudohyperkalaemic individuals over the range 4-40 degrees C was measured using 86RbCl as tracer. Flux through the pump was measured as the ouabain-sensitive component (0.2 mM ouabain) and flux via Na+,K+-cotransport was measured as the decrease in the rate of K+ influx in the presence of 1 mM furosemide. The residual passive permeability of the red cell plasma membranes to K+ was that influx which was unaffected by either inhibitor. When Na+ influxes were measured, the ratio of Na+ to K+ transported via the furosemide-sensitive component was 1 over the full temperature range studied. The temperature sensitivity of K+ influx via the pump was normal as was the enzymic activity of the Na+,K+-ATPase. In contrast, the activity of the Na+,K+-cotransport system in pseudohyperkalaemics was more temperature sensitive than that of controls and affected individuals also showed a greater passive permeability to K+ at low temperatures. Red cell membranes from affected individuals have significantly increased amounts of phosphatidylcholine which are balanced, to a degree, by a decreased content of phosphatidylethanolamiane. It is proposed that in this example of familial pseudohyperkalaemia there is an alteration in the structure of the red cell plasma membrane which influences the temperature sensitivity of both its cotransport and passive permeability properties.

Distribution of Myelin Basic Protein and P2 mRNAs in Rabbit Spinal Cord Oligodendrocytes

Abstract: Myelin basic protein (MBP) and P2 protein are small positively charged proteins found in oligodendrocytes of rabbit spinal cord. Both proteins become incorporated into compact myelin. We have begun investigations into the mechanisms by which MBP and P2 become incorporated into the myelin membrane. We find that P2, like the MBPs, is synthesized on free polysomes in rabbit spinal cord. Cell fractionation experiments reveal that rabbit MBP mRNAs are preferentially segregated to the peripheral myelinating regions whereas P2 mRNAs are predominantly localized within the perikaryon of the cell. In vitro synthesized rabbit MBP readily associates with membranes added to translation mixtures, whereas P2 protein does not. It is possible that P2 requires a “receptor” molecule, perhaps a membrane‐anchored protein, for association with the cytoplasmic face of the myelin membrane.

Phosphatidylinositol transfer activity in rat cerebral hemispheres during development.

PHOSPHOLIPID biosynthesis is primarily localized in the endoplasmic reticulum of eukaryotic cells (JELSEMA & MORRE, 1978). It is therefore evident that some mechanism must exist by which phospholipids are transported to the other cellular membranes. Owing to the low solubility of phospholipids in water it has been suggested that phospholipid-exchange proteins might fulfil this intracellular transport function (WIRTZ, 1974). These proteins catalyze the transfer of phospholipids between membranes in uitro and they have been detected in a variety of eukaryotic cells (WIRTZ, 1974). It has recently been shown that proteins present in the soluble fractions of both bovine brain (CAREY & FOSTER, 1977) and rat brain (BRAMMER, 1978) catalyze the transfer of phospholipids to the myelin membrane in uitro. The rapid onset of myelogenesis in the rat brain 12-16days after birth (DAVISON & PETERS, 1970) provides an excellent opportunity for determining whether changes in the activity of phospholipid exchange during this period reflect the vastly increased rate of membrane biosynthesis. Phosphatidylinositol is a component of the myelin membrane and in this study the activity of phosphatidylinositol exchange in the cerebral hemispheres of developing rats has been invesitagted. There is an increase in the activity of phosphatidylinositol transfer which occurs at the same time as the onset of myelogenesis. This suggests that phospholipid-exchange proteins may indeed have a role in the assembly of the myelin membrane.

Biosynthesis of the Myelin 2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterases

We have investigated the site of synthesis of the 2′,3′‐cyclic nucleotide 3′‐phosphodiesterases (CNPs I and II) in rat brain. Rapid kinetics of incorporation of CNPs into oligodendrocyte plasma membrane in the intact brain are consistent with their synthesis on free polysomes. This hypothesis was confirmed by the translation in vitro of RNA isolated from free and bound polysomes, respectively. Unlike myelin basic protein (MBP) mRNAs, CNP mRNAs are not enriched in a myelin‐associated pool of RNA. MBPs, but not CNPs, were found to readily associate in vitro with membrane vesicles derived from rough endoplasmic reticulum. The avidity of MBPs in binding to membranes is probably related to the previously observed spatial segregation of MBP mRNAs into actively myelinating cellular processes of the oligodendrocyte. Such a segregation would ensure that newly synthesized MBPs are immediately incorporated into myelin. In contrast, the CNPs probably associate with the cytoplasmic surface of the oligodendrocyte plasma membrane through interaction with a membrane‐bound receptor.

Lipid composition and physical properties of membranes from C-6 glial cells with altered phospholipid polar headgroups.

Growth of C-6 glial cells in media enriched in the polar headgroup precursors N,N-dimethylethanolamine, N-monomethylethanolamine or ethanolamine for 24 h resulted in the accumulation of the corresponding phospholipids to about 30% of total membrane phospholipid. Under these conditions the cholesterol to phospholipid ratios were unaffected. With the exception of arachidonic acid, which was significantly reduced in the lipids from cells grown in the presence of N-monomethylethanolamine, the fatty acid composition of cells grown under the various conditions was identical. The physical properties of membranes prepared from these cells were compared by electron spin resonance spectroscopy using spin-labelled stearic acid. Modifications in cellular phospholipid composition did not affect either the order parameter or the correlation time of fatty acid spin labels. Since there are no significant effects on the other membrane lipids and since the physical properties of the membranes are maintained, these modifications in phospholipid composition provide a valuable means for studying the role of phospholipid polar headgroups in the function of membrane-bound enzymes and hormone receptors in C-6 cells.