The primary structure of bovine brain myelin lipophilin (proteolipid apoprotein)

The amino-acid sequence of bovine myelin lipophilin (proteolipid apoprotein, Folch-protein) has been completed. Lipophilin is a 276 amino acid residues containing, extremely hydrophobic membrane protein with molecular mass 30,000 Da. The sequence determination was based on automated Edman degradation of four tryptophan and four cyanogen bromide fragments and of proteolytic peptides of complete lipophilin as well as the fragments obtained by chemical cleavage. Four additional sequences were determined which led to the completion of the primary structure. Lipophilin is esterified at threonine-198 by long chain fatty acids (palmitic, stearic and oleic acid). The attachment site has been established at the same threonine residue in three different peptides isolated from thermolysinolytic, papainolytic and chymotrypsinolytic hydrolysates. This threonine residue is part of a hydrophilic segment of lipophilin. The covalent fatty acyl bond is being discussed together with important structural and functional properties of this membrane protein which can be derived from sequence information. New separation and purification methods of hydrophobic and hydrophilic polypeptides for this sequence determination (fractional solubilization, silica gel exclusion, high-performance liquid chromatography) had to be elaborated as indispensable tools. They are generally applicable to the structural analysis of hydrophobic membrane proteins. Four long (26, 29, 40 and 36 residues) and one medium long (12 residues) hydrophobic segments are separated by four predominantly positively and one negatively charged hydrophilic segments. On the basis of structural data a model for the membrane integration of lipophilin is proposed.     

Conformation of brain proteolipid apoprotein

The conformation of brain proteolipid apoprotein (PLA) has been investigated using infrared spectroscopy and freeze-fracture electron microscopy. For this purpose, spectroscopic samples consisting of a mixture of liquid paraffin and wet protein have been prepared. These systems have allowed us to record the infrared spectra of PLA at neutral pH. The amide I and III regions reveal the existence of a predominantly -helical structure, as well as the presence of minor -strands and random coil forms. The effect of sonication and a non-denaturing detergent, (n-octyl--d-glucopyranoside), on the structure of the protein have also been investigated. Sonication produces an increase of the and unordered structures at the expense of the -helical conformation. These structural changes are enhanced in the presence of the non-ionic detergent n-octyl--d-glucopyranoside. Lipids protect the native protein structure from the effects of sonication. The aforementioned detergent changes the PLA conformation by increasing the -helical content at the expense of -sheet and random coil forms. Therefore the PLA structure seems to be similar to the structures of other proteins intrinsic to non-neural membranes. The effects investigated also suggest that PLA behaves in a conformationally flexible manner.     

Isolation and terminal sequence determination of the major rat brain myelin proteolipid P7 apoprotein

The major rat brain myelin proteolipid P7 apoprotein has been isolated in pure form by a preparative sodium dodecylsulphate gel electrophoresis system. Automated Edman degradation permitted the establishment of the N-terminal sequence up to the 20th amino acid. The C-terminal sequence was determined by the action of carboxypeptidase A.     

Comparative study of myelin proteolipid apoprotein solvation by multilayer membranes of synthetic DPPC and biological lipid extract from bovine brain. An FT-IR investigation

The interaction between the aqueous form of the myelin proteolipid apoprotein (PLA) and model membranes prepared with either synthetic dipalmitoylphosphatidyl choline (DPPC) or biological lipids extracted from bovine brain (BE) has been investigated by Fourier-Transform IR spectroscopy. IR spectra obtained with lyophilized samples of PLA demonstrated 2 main peaks (amide I and amide II) culminating at 1656 cm-1 and 1545 cm-1, which we assigned to helical conformation. When PLA was solvated in DPPC or BE membranes, both the amide I and amide II features remained located at 1655 cm-1 and 1545 cm-1, although their half-width significantly decreased, demonstrating that the lipid environment favoured alpha helix structures. However differences between both mixtures were detected by measuring the amide I and amide II half-widths as a function of the L:P molar ratio. Moreover, analysis of the 1545/1515 peak intensity ratio brought evidence of different localization and/or molecular arrangement of the protein segments containing tyrosine residues, depending on the lipid composition of the membrane. According to previously published models, these data suggest that recombinants prepared with PLA and BE multilayers better mimic the biological membrane than do DPPC-PLA mixtures.     

Enzymic and chemical fragmentation of the apoprotein of the major rat brain myelin proteolipid: Sequence data

Sequence studies of the apoprotein of the major rat brain myelin proteolipid are made difficult by the aggregation of the water-soluble form of the protein and of its split peptides. By digestion with Staphylococcus aureus V8 protease and Lysobacter enzymogenes Lys-C endoproteinase or after partial acid hydrolysis, a new series of fragments has been obtained. Sequence data from these fragments permitted the alignment of tryptic peptides and BNPS-skatole fragments analyzed in previous studies. A nearly complete sequence of the C-terminal moiety of the apoprotein is described; in addition, large segments of the N-terminal part have been characterized.     

Structural studies of the apoprotein of the Folch-Pi bovine brain myelin proteolipid : characterization of the CNBr-fragments and of a long C-terminal sequence.

The action of cyanogen bromide on the quite insoluble bovine proteolipid apoprotein allowed the determination of four peptide fragments: two of them constituted a 19 amino acid long C-terminal sequence of the apoprotein. Our results were in favour of the existence of only one subunit presenting a molecular weight closely related to 25,000 for which a schematic representation is given.     

Preparation of the proteolipid apoprotein from bovine heart, liver and kidney.

Proteolipid apoproteins have been prepared from heart, kidney, and liver by dialysis in chloroform/methanol against chloroform/methanol, acidified chloroform/methanol, and chloroform/methanol in succession. They are free of lipids (less than 0.05% P; less than 0.1% carbohydrate). They show a high content of non-polar amino acids, methionine, and tryptophan and contain little or no half-cystine. The differ from neural proteolipid apoproteins by absence of half-cystine, and of covalently bound fatty acids. As recovered from chloroform/methanol solutions, they are soluble in chloroform/methanol and insoluble in water, but a water-soluble form can be prepared by changing the solvent from chloroform/methanol to water in a stream of nitrogen. The chloroform-methanol-soluble form and the water-soluble form are interconvertible. ORD and CD spectra of all proteolipid apoproteins indicate 60-70% alpha-helix content in chloroform/methanol solution and 20-30% alpha-helix in water solution. Sodium dodecyl sulfate gel electrophoresis resolves proteolipid apoprotein into two major components corresponding to ca. 12 000 and 34 000 daltons. With sodium dodecyl sulfate/urea numerous bands appear, with a major one at 30 000 daltons and 8 to 10, ranging downward to 2500. For comparison, neural proteolipid apoproteins also show numerous bands with a major one at 25 000. The marked chemical and physical similarities among all proteolipid apoproteins studied suggest a common role in membrane structures.     

In vitro insertion of the myelin proteolipid apoprotein into oligodendrocyte plasma membranes

Uncoated vesicles (UCV) loaded with the myelin proteolipid apoprotein covalently tagged with fluorescein (PLP_F) were found to interact with isolated oligodendrocytes from bovine brain at 4°C as well as at 37°C. After 1.5 hours of incubation, the labeled protein was localized in the cell membranes. After 2.5 hours the fluorescence intensity associated with the oligodendrocytes decreased and completely disappeared at t=3.5 hours. Addition of KCl or EDTA in the incubation medium significantly hindered the interaction with cells. In contrast, the elimination of membrane proteins from UCV did not perturb cell labeling. A specific role of PLP was suggested since UCV loaded with a soluble protein (BSA_F) led to a weak cell labeling.Abbreviations IAF 5-iodacetamidofluorescein - BSA bovine serum albumin - BSA BSA labelled with IAF - PLP proteolipid apoprotein - PLP_F aqueous form of PLP tagged with IAF - CV coated vesicles - UCV uncoated vesicles - UCV^*PLP_F UCV loaded with PLP_F - MV model vesicles This work was suported by Cnrs and INSERM.     

Lipid-protein interaction. The incorporation of myelin proteolipid apoprotein into phosphatidylcholine bilayers

Bovine myelin proteolipid apoprotein (PLA), obtained in high yield and purity by a novel ultrafiltration procedure, has been used to study the perturbations produced by this protein on phosphatidylcholine bilayers, using infrared spectroscopy, nuclear magnetic resonance and fluorescence polarisation. PLA interacts with phospholipids in a similar manner to other intrinsic proteins. For bilayers in the fluid state, the fatty-acyl chain static order, as measured by deuterium NMR, is slightly increased in the presence of the protein, except at very high PLA concentrations. Phosphorus NMR reveals some perturbation of the phospholipid polar group by PLA, but to a smaller degree than occurs with other intrinsic proteins. An increase in static order above tc (the onset temperature for gel-to-fluid transition) is also detected by infrared spectroscopy. Studies using steady-state polarisation of diphenylhexatriene fluorescence indicate that the microviscosity of the bilayer increases as a function of the protein mole fraction. From these data an estimation of the average number of lipids perturbed per protein monomer has been made, and a figure of 37 phospholipid molecules determined. The data are compatible with a picture of a hydrophobic polypeptide, perturbing the phospholipids close to it, but allowing rapid (greater than 10(4) s-1) exchange with all the lipid molecules in the system.     

Phospholipid composition of myelin and synaptosomal proteolipid from vertebrate brain

1. The phospholipid composition of the main proteolipid complexes of the nervous system was studied in myelin and synaptosomal membranes from brains of representatives of various vertebrate classes. 2. The relative content of acid phospholipids was much higher in proteolipid complexes from myelin and synaptosomal membranes of all vertebrates studied as compared to their content in the initial lipid extract (28-80% and 11-20% of total phospholipid content, respectively). 3. The relative content of acid phospholipids in proteolipid complexes of myelin membranes was much lower in brain of fishes and amphibia as compared to higher vertebrates. 4. The main acid phospholipids of proteolipid complexes was phosphatidylserine, phosphatidic acid being characteristic for myelin proteolipids and diphosphatidyl glycerol for synaptosomal proteolipids of all vertebrates studied.     

Purification and properties of a glycerophosphocholine phosphodiesterase from bovine brain myelin

An enzyme releasing phosphocholine from glycerophosphocholine was purified to apparent homogeneity based upon SDS-PAGE. The enzyme was liberated from lyophilized bovine myelin by differential detergent extraction and final purification was accomplished with Q-Sepharose Fast Flow chromatography yielding an apparently homogenous protein. The molecular mass based upon PAGE was approximately 14 kDa. The enzyme was also capable of releasing p-nitrophenol from p-nitrophenyl-phosphocholine. Maximal activity was obtained with 0.2 mM ZnCl₂ or 1 mM CoCl₂. p-Nitrophenylphosphocholine and phosphocholine were competitive inhibitors of glycerophosphocholine hydrolysis with Ki's of 0.028 mM and 0.03 mM respectively. Glycerophosphocholine and phosphocholine were competitive inhibitors of p-nitrophenylphosphocholine hydrolysis with Ki's of 0.5 mM and 1.75 mM respectively.Abbreviations SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - GPC glycerophosphocholine - pNPPC p-nitrophenylphosphocholine - OG octyl--glucoside - PMSF phenylmethylsulfonylfluoride - CNPase 23-cyclic nucleotide 3-phosphodiesterase     

Characterization of proteolipid protein fatty acylesterase from rat brain myelin.

A protein fatty acylesterase activity that catalyzes the removal of fatty acid from exogenous proteolipid protein (PLP) has been demonstrated in isolated rat brain myelin. Optimum enzyme activity for the deacylation of PLP was obtained in 0.5% Triton X-100, 1 mM dithiothreitol at pH 7.0 and at 37 degrees C. Other detergents (octyl beta-D-glucoside, Nonidet P-40, and Tween 20) have little or no effect, whereas deacylation was completely abolished by 0.1% sodium dodecyl sulfate or boiling the membrane fraction for 5 min prior to incubation. Under optimal conditions, the rate of deacylation was linear up to 20 min, and the apparent Km for bovine [3H]palmitoyl-PLP was 18 microM. The myelin-associated PLP fatty acylesterase has no apparent requirements for divalent cations (Ca2+, Mg2+, Mn2+), and chelators such as EDTA, [ethylenebis(oxyethylenenitrilo)] tetraacetic acid, and 1,10-phenantroline have little or no effect on enzyme activity. Sulfhydryl and histidine residues are needed for full enzyme activity, whereas the "active serine"-directed inhibitor phenylmethylsulfonyl fluoride has no effect. The myelin-associated protein fatty acylesterase was present throughout brain development and in all myelin subfractions, in agreement with the dynamic metabolism of PLP-bound fatty acids. Enzyme activity was also present in sciatic nerve, brain cortex, and heart whereas liver was devoid of activity. Several esterases, including phospholipase A2, glyoxalase II, and acetylcholinesterase, did not remove fatty acid from PLP. Myelin basic protein, palmitoyl-CoA hydrolase, and myelin-associated nonspecific esterase were also ruled out as the PLP fatty acylesterase. Thus, all data seem to indicate that this enzyme is different from esterases of the lipid metabolism. Finally, stimulation of protein phosphorylation with Ca2+, but not with cyclic-AMP, inhibited PLP deacylation, suggesting that the myelin-associated protein fatty acylesterase activity is regulated by endogenous Ca(2+)-dependent protein kinases.     

Lipid-protein and protein-protein interactions in double recombinants of myelin proteolipid apoprotein and myelin basic protein with dimyristoylphosphatidylglycerol.

The integral proteolipid apoprotein (PLP) from bovine spinal cord has been reconstituted in dimyristoylphosphatidylglycerol (DMPG) bilayers, and the mutual interactions on binding the peripheral myelin basic protein (MBP) have been studied. Quantitation of protein and lipid contents in the MBP-PLP-DMPG double recombinants at different PLP:DMPG ratios led to the conclusion that MBP binds only to the DMPG lipid headgroups and is hindered from interaction with the first shell of lipids surrounding the PLP. No specific PLP-MBP association could be detected. Electron spin resonance (ESR) spectra of phosphatidylglycerol spin-labeled at position n = 5 in the sn-2 chain showed that complexation of MBP with the PLP-DMPG recombinants leads to a decrease in lipid chain mobility to an extent which correlates with the degree of MBP binding. At low DMPG:PLP ratios, the perturbations of lipid mobility by both proteins are mutually enhanced. In single recombinants of PLP with DMPG, the ESR spectra of phosphatidylglycerol spin-labeled at position n = 14 in the sn-2 chain indicated that approximately 10 lipids/protein are motionally restricted by direct contact with the intramembranous surface of the protein. This number is in agreement with earlier results for reconstitutions of PLP in dimyristoylphosphatidylcholine (DMPC) [Brophy, P. J., Horváth, L. I., & Marsh, D. (1984) Biochemistry 23, 860-865] and is consistent with a hexameric arrangement of the PLP molecules in DMPG bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino-acid sequence of human and bovine brain myelin proteolipid protein (lipophilin) is completely conserved

Proteolipid protein (PLP) was isolated from white matter of human brain by chloroform/methanol extraction and further purified by chromatography. Performic acid oxidation yielded a product homogeneous in NaDodSO4-polyacrylamide electrophoresis with a molecular mass of 30 kDa. The carboxymethylated PLP was chemically cleaved with cyanogen bromide into four fragments: CNBr I 22-24 kDa, CNBr II 5 kDa, CNBr III 1.4 kDa and CNBr IV 0.7 kDa. HBr/dimethylsulfoxide cleavage at tryptophan residues released four fragments: Trp I 14-16 kDa, Trp II 2.0 kDa, Trp III 5 kDa and Trp IV 7 kDa. Hydrophilic fragments were enriched in 50% formic acid (CNBr II, III, IV and Trp II and III), whereas hydrophobic peptides precipitated from this solvent were CNBr I, Trp I and IV. The fragments were separated by gel filtration with 90% formic acid as solvent and finally purified by gel permeation HPLC (Si 60 and Si 100) for automated liquid and solid-phase Edman degradation. Large fragments were further cleaved with different proteinases (trypsin, V8-proteinase, endoproteinase Lys-C and thermolysin). We used an improved strategy in the sequencing of the human proteolipid protein compared with our approach to the structural elucidation of bovine brain PLP. The amino-acid sequence of human PLP contains 276 residues, the same as found in bovine proteolipid protein. The two sequences proved to be identical. The possible importance of the conservative structure of this integral membrane protein is discussed.     

Purification of myelin basic protein from bovine brain

Myelin basic protein (MBP) is a commonly used substrate for in vitro determination of numerous protein kinase activities. Herein we describe a rapid method for isolating relatively large amounts of MBP from bovine brain with a purity greater than that currently available from commercial sources. Lipids were first extracted from the CNS tissue by homogenization in sec-butanol. Washes under neutral and mildly basic conditions were employed to remove neutral and acidic proteins from the defatted residue. MBP was subsequently extracted under acidic conditions and further purified by chromatography on CM Sephadex C-25. Potential contaminating enzyme activities were destroyed by heart treatment. This method typically yields a recovery of 1.0-1.5 mg MBP per gram of starting material with a purity of greater than 95%. The MBP prepared in this manner was suitable for determination of kinase activities by both solution and the "in gel" kinase assay systems.