Brain proteolipids in representatives of different vertebrate classes

The Folch-Lees proteolipid complexes of different purity (crude proteolipids and relative pure proteolipids) were isolated from vertebrate brain: mammalia (Macaca irus, Macaca rhesus and white rat), birds (Columbia livia), reptilia (Testudo horsfieldi), amphibia (Rana temporaria) and fishes (Salmo irideus). The proteolipid complexes were isolated by emulsion-centrifugation method. The content of proteolipid protein (mg/g w. w.) correlates with the level of phylogenetic development of the animals studied. It is the highest in monkey brain (10.5 and 8.6 mg/g) and the lowest in fish brain (2.2 mg/g). The yield of proteolipids from the brains of animals studied shows the same pattern. Crude proteolipids of mammalia, birds and reptiles contain 40-50% of protein and 60-50% of lipids. The content of phospholipids is about 40%. Proteolipids of amphibia and fish brain contain less protein--about 30%. In the conditions of mild purification, the protein content in mammalia, birds and reptilia makes up about 70% and lipid content--about 30-35%. The crude and purified proteolipids in all the animals studied (as compared with the original lipid extracts from which they were isolated) are enriched in acid phospholipids: phosphatidyl serine, phosphatidyl inositol and diphosphatidyl glycerol. Acid phospholipids in total lipid extract make up 10-20% of total phospholipids, in crude proteolipids 16-32 and in purified proteolipids--56-75%. There are no marked differences between fatty acid composition of phospholipids in proteolipids and in the same phospholipids isolated from total lipid extract.     

The DM-20 proteolipid is a major protein of the brain. It is synthetized in the fetus earlier than the major myelin proteolipid (PLP)

By studying highly purified CNS proteolipids, we have shown that DM-20 proteolipid, which was considered, until now, to be a minor brain proteolipid is, in fact, almost as abundant as the Major Myelin Proteolipid known also as Proteolipid Protein (PLP). DM-20 proteolipid is even the major brain proteolipid in young foetuses. It is only during myelinisation that the "Proteolipid Protein" increases rapidly and becomes equivalent in weight to DM-20 proteolipid. This study raises the question of the particular function of DM-20 proteolipid.     

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.     

NH2-terminal analysis of Wolfgram and Folch-Lees proteolipid proteins

Abstract— The NH₂-terminal amino acids of Wolfgram and Folch-Lees proteolipids of bovine and human CNS myelin were determined using the cyanate method (Starke & Smyth , 1963) followed by direct amino acid analysis of the products. Glycine predominated in every case and was recovered in amounts similar to the results described by Whikehart & Lees (1973), who used a dansylation technique followed by thin layer chromatography of the DNS-amino acids. In the present study substantial amounts of glutamic acid, serine, alanine and aspartic acid were also recovered, plus traces of other amino acids. Few differences were observed between Wolfgram and Folch-Lees proteolipids. The end group products of purified W1 proteolipid of bovine Wolfgram fraction, of diazometholysed Folch-Lees proteolipid, and of a sample of phosphatidyl serine had essentially the same composition. The similarity of these results, especially for both fractionated and unfractionated Wolfgram proteolipid, may be evidence that the observed products are derived from phosphoglycerides present in proteolipid rather than from the actual NH₂-terminals of the protein.     

A regional study of developing rat brain: The accumulation and distribution of proteolipid proteins

The accumulation and distribution of proteolipid proteins in rat brain and selected brain regions (cerebellum, cerebral cortex, basal ganglia, and hippocampus) were studied during early postnatal development. In whole brain an eightfold increase of proteolipid was observed between ten and 33 days after birth. This was reflected in the separate regions examined where the proteolipid protein content increased six- to ten-fold during the same period. The basal ganglia and cerebral cortex contributed the greatest amount to the total proteolipid present. However, at 28–33 days the greatest concentration (mg/g tissue) was observed in the basal ganglia and hippocampus. When the proteolipid protein preparations were examined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, distinctive, heterogeneous patterns for each brain region were obtained. Proteolipid from basal ganglia (the region richest in white matter) consisted primarily of two major protein bands with apparent molecular weights of approximately 21,500 and 26,000. Both of these bands dramatically increased in quantity during myelination, and the larger protein coelectrophoresed with isolated myelin proteolipid protein. Both bands were also found present in proteolipid preparations from the other brain regions but in varying amounts relative to the total. The data suggest that the increase in proteolipid observed during this developmental period was due in large measure to the accumulation of myelin-specific proteolipids, but also that a significant proportion of the increase was due to the accumulation of nonmyelin components.     

Mass-spectrometric analysis of myelin proteolipids reveals new features of this family of palmitoylated membrane proteins

In this study, we have investigated the structure of the native myelin proteolipid protein (PLP), DM-20 protein and several low molecular mass proteolipids by mass spectrometry. The various proteolipid species were isolated from bovine spinal cord by size-exclusion and ion-exchange chromatography in organic solvents. Matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) of PLP and DM-20 revealed molecular masses of 31.6 and 27.2 kDa, respectively, which is consistent with the presence of six and four molecules of thioester-bound fatty acids. Electrospray ionization-MS analysis of the deacylated proteins in organic solvents produced the predicted molecular masses of the apoproteins (29.9 and 26.1 kDa), demonstrating that palmitoylation is the major post-translational modification of PLP, and that the majority of PLP and DM-20 molecules in the CNS are fully acylated. A series of myelin-associated, palmitoylated proteolipids with molecular masses raging between 12 kDa and 18 kDa were also isolated and subjected to amino acid analysis, fatty acid analysis, N- and C-terminal sequencing, tryptic digestion and peptide mapping by MALDI-TOF-MS. The results clearly showed that these polypeptides correspond to the N-terminal region (residues 1-105/112) and C-terminal region (residues 113/131-276) of the major PLP, and they appear to be produced by natural proteolytic cleavage within the 60 amino acid-long cytoplasmic domain. These proteolipids are not postmortem artifacts of PLP and DM-20, and are differentially distributed across the CNS.     

Fatty acid composition of phospholipids of myelin and synaptosomal proteolipid complexes from vertebrate brain.

1. Fatty acid composition of five main phospholipids of vertebrate brain myelin and synaptosomal proteolipids and membranes was studied. 2. Higher content of monoenoic and lower content of saturated and polyenoic fatty acids was found to be characteristic of phospholipids from myelin and myelin proteolipids as compared to phospholipids from synaptosomal proteolipids and membranes of vertebrates (from fishes to mammalians). Fatty acid composition of phospholipids of proteolipid complexes and of the membranes, from which they were isolated, were found to be similar in various species studied. 3. Microviscosity was found to be higher in myelin as compared to synaptosomal membranes of frog Rana temporaria and in rabbit Lepus cuniculus. It appears to be due to the difference in proteolipid content and in lipid composition of myelin and synaptosomal membranes.     

Isolation and identification of proteolipid proteins injimpy mouse brain

Proteolipids were isolated from 20 day old normal andjimpy mouse brain by extraction into chloroform-methanol (21, w/v), delipidated by size-exclusion HPLC, and analyzed by SDS-PAGE, Western blots, amino acid analyses, and N-terminal sequencing. SDS-PAGE showed that a major proteolipid fromjimpy mouse brain had an apparent molecular weight of 23 kDa, intermediate to that of PLP and DM-20 from normal mouse brain. Western blots with 3 different antibodies which recognize residues 200–224, 116–150, and 270–276 respectively recognized immunoreactive material in normal andjimpy PLP. Since antibody reactive with 270–276 did not recognizejimpy PLP, an altered C-terminus of thejimpy protein is suggested. These results demonstrated that a PLP can be partially purified fromjimpy mouse brain. Amino acid analyses failed to show the predicted increase in cysteinyl residues (predicted from cDNA) injimpy PLP. However, whenjimpy brain proteolipids were subjected to N-terminal sequencing, Gly, Leu, Leu, Gly the first four amino acids of PLP were detected. Thus, the partial purification of a proteolipid fromjimpy mouse brain, whose characteristics (apparent molecular weight, immunoreactivity, N-terminal sequence and relative net charge) strongly suggested that PLP of altered size is present injimpy mouse brain.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate toluidine salt - MBP myelin basic protein - NBT -nitro blue tetrazolium chloride - PITC phenylisothiocyanate - PLP myelin proteolipid protein - PVDF polyvinylidene difluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis Special issue dedicated to Dr. Marjorie B. Lees.     

Purification and characterization of minor brain proteolipids: use of fast atom bombardment-mass spectrometry for peptide sequencing

A combination of lipophilic gel permeation chromatography and ion-exchange chromatography in organic solvents was used to purify low molecular weight proteolipids from bovine brain. Cleavage peptides were purified by HPLC and studied mainly by the fast atom bombardment--mass spectrometry technique. A proteolipid of Mr 14 000 contains several peptides from the first 113 amino acids of the major myelin proteolipid (MMPL) plus an extra unknown blocked N-terminal peptide. A proteolipid of Mr 16 000 contains smaller peptides belonging to a C-terminal fragment of MMPL of about 160 residues. These two proteolipids do not seem to be artifacts from MMPL.     

N- and C-terminal amino acids of proteolipids from the rat brain and various other organs

N- and C-terminal amino acids of proteolipid proteins from the whole brain and some other organs were investigated. N-terminal amino acids were identified by the dansylation procedure. C-terminal amino acids were determined after the enzymatic hydrolysis with carboxy peptidases A and B with the following dansylation. Phenyl alanine and lysine were identified as C-terminal amino acids of the proteolipids from the whole brain and only lysine--as the C-terminal amino acid of proteolipids from the heart, liver, kidney (cortical and medullary parts) and skeletal muscle. The corresponding N-terminal amino acids of the proteolipids from the whole brain were aspartic acid and glycine and of proteolipids from the heart, liver, kidney (cortical and medullary parts) and skeletal muscle--only aspartic acid. A comparison of the data obtained with the previous ones has shown that in the brain there exist only two types of proteolipids--one characteristic of myelin, another-- of mitochondria, and in other organs--only one characteristic of mitochondria.     

Purification and immunohistochemical localization of rat brain myelin proteolipid protein.

Abstract— A homogeneous preparation of proteolipid protein (PLP) from rat brain myelin was isolated by preparative gel electrophoresis in sodium dodecyl sulfate and chemically characterized. The results of amino acid and N-terminal amino acid analyses are reported. The same preparation of myelin PLP was used to produce specific precipitating antibodies. Rabbit and goat antisera to myelin PLP each gave a single precipitin line with purified PLP dissolved in Triton X-100. Under identical conditions, no precipitation was observed with antiserum to myelin basic protein or with control serum. Immunofluorescence localization employing antiserum to PLP demonstrated bright specific fluorescence restricted to the myelin sheaths of axons in all anatomical areas of the rat brain examined. Neuronal cell bodies and their dendrites were completely negative with respect to the presence of proteolipid protein. PLP could not be localized in the cell bodies or fibrous processes in any of the glial elements in the adult rat brain. However, myelin PLP was clearly visible in the cytoplasm and processes of actively myelinating oligodendrocytes in the corpus callosum in the brains of 10-day-old rats.     

Amino- and carboxyl-terminal amino acids of proteolipid proteins

The amino- and carboxyl-terminal amino acids of proteolipids from neural and non-neural sources were investigated. Amino-terminal amino acids were identified and quantitated by the dansyiation procedure. Carboxyl-terminal amino acids were determined after hydrazinolysis or enzymatic hydrolysis with carboxypeptidases. Proteolipid from white matter showed two terminal amino acids, regardless of the method of preparation. The major N-terminal amino acid was glycine and the minor one was glutamic acid or glutamine. The corresponding C-terminal amino acids were phenylalanine and glycine. Preparations of white matter proteolipid, therefore, contained more than one protein or protein chain. Proteolipids from brain mitochondria, heart, liver and kidney were characterized by N-terminal aspartic acid or asparagine and C-terminal lysine residues and they exhibited an amino acid composition which differed from white matter proteolipid. Our results suggest the existence of two classes of proteolipids, a myelin type and a non-myelin type. Synaptic membrane and grey matter proteolipids exhibited characteristics of both classes.     

Proteolipids in the developing brains of normal mice and myelin deficient mutants

Abstract— A developmental study of proteolipids from brains of normal mice and two myelin deficient mutants, jimpy and quaking, was performed. The proteolipids were obtained by diethyl ether precipitation of washed total lipid extracts from whole brains and were analysed on polyacrylamide gels containing sodium dodecyl sulphate. The amount of ether precipitable material extractable from normal brains increased almost six-fold between 12 and 21 days posr partum. This increase was not observed with the mutant mice. Polyacrylamide gel electrophoretic analysis of the proteolipid fraction showed it to be heterogeneous, with eight major protein bands. Two of these proteins increased rapidly in quantity in normal mice between 13 and 21 days. These two proteins were present, in severely reduced quantities in the brains of jimpy and quaking mice at all ages examined. One of these proteolipids was the major species present in proteolipid extracts from the brains of normal mature mice. This protein coelectrophoresed with proteolipid isolated from purified myelin and has been tentatively identified as the myelin proteolipid. The other proteolipid which was deficient in jimpy and quaking brains was not characterized, but it appeared to be of extra-myelin origin, and suggests that parts of the brain other than the myelin sheath may be involved in the jimpy and quaking disorders.     

Isolation and Characterization of a Proteolipid in Defatted Soybean Meals

A proteolipid was isolated from the chloroform–methanol (2:1, by vol.) extract of defatted soybean meals by a modified Folch method. The proteolipid gave a yield of 0.05% of the defatted meals, and the ratio of protein and lipid was neary 3:4. The complex gave a single band containing both protein and lipid on polyacrylamide gel electrophoresis. TLC analysis of the lipid moiety showed that the major components were glycolipids and phospholipids. The protein moiety contained more hydrophobic amino acids and less acidic amino acids in comparison with the amino acid composition of soybean globulin. The protein moiety contained two kinds of protein component (I and II) which have molecular weights of 13,000 (I) and 15,000 (II) on SDS-urea polyacrylamide gel electrophoresis, and N-terminal amino acids of alanine (I) and glutamic acid (II). The apoprotein is a new protein and different from the whey proteins or globulins of soybean.     

Presence of the Plasma Membrane Proteolipid (Plasmolipin) in Myelin

Plasma membrane proteolipid (plasmolipin), which was originally isolated from kidney membranes, has also been shown to be present in brain. In this study, we examined the distribution of plasmolipin in brain regions, myelin, and oligodendroglial membranes. Immunoblot analysis of different brain regions revealed that plasmolipin levels were higher in regions rich in white matter. Plasmolipin was also detected in myelin, myelin subfractions, and oligodendroglial membranes. Immunocytochemical analysis of the cerebellum revealed that plasmolipin was localized in the myelinated tracts. Plasmolipin levels in myelin were enriched during five successive cycles of myelin purification, similar to the enrichment of myelin proteolipid apoprotein (PLP) and myelin basic protein (MBP). In contrast, levels of Na+,K(+)-ATPase and a 70-kDa protein were decreased. When myelin or white matter was extracted with chloroform/methanol, it contained, in addition to PLP, a significant amount of plasmolipin. Quantitative immunoblot analysis suggested that plasmolipin constitutes in the range of 2.2-4.8% of total myelin protein. Plasmolipin, purified from kidney membranes, was detected by silver stain on gels at 18 kDa and did not show immunological cross-reactivity with either PLP or MBP. Thus, it is concluded that plasmolipin is present in myelin, possibly as a component of the oligodendroglial plasma membrane, but is structurally and immunologically different from the previously characterized myelin proteolipids.     

Selectivity of lipid-protein interaction with myelin proteolipids PLP and DM-20. A fluorescence anisotropy study

The two main myelin proteolipids, PLP (30 kDa) and DM-20 (25 kDa), differ by an internal deletion in DM-20. The deleted fragment, of 35 amino acids (116-150), corresponds to the major hydrophilic domain of PLP. Fluorescence anisotropy experiments using diphenylhexatriene as a fluorescent probe were performed to detect the phase separation induced by these two proteolipids in multilamellar vesicles of binary composition. We found that in vesicles composed of 30% L-alpha-PS and 70% DPPC, the PLP boundary layer contained about 18 motionally restricted phospholipids, almost exclusively L-alpha-PS. On the contrary, the DM-20 boundary layer contained only 14 to 15 phospholipids, with a composition no different from that of the bulk vesicle. In mixtures of DMPG and DPPC, the selectivity of PLP for the acidic phospholipid DMPG was maintained, but was lower than that observed for L-alpha-PS. We assume that this selectivity of PLP stems mainly from electrostatic interactions between the charged residues of the 116-150 fragment, deleted in DM-20, and the acidic phospholipids. These results suggest that fragment 116-150 may play a specific role in the interaction of PLP with the lipid bilayer of the myelin membrane.     

Developmental Study of Proteolipids in Bovine Brain: A Novel Proteolipid and DM-20 Appear Before Proteolipid Protein (PLP) During Myelination

In a developmental study, we have shown that DM-20 is present before proteolipid protein (PLP) in the fetal bovine cerebral hemispheres. When the white matter appears (27-30 weeks of gestation), the amount of DM-20 drastically increases. DM-20 remains the major proteolipid until birth. PLP is detected only 2-4 weeks after the appearance of white matter, that is, more than 4 weeks after the appearance of DM-20. The early appearance of DM-20 at the beginning of myelination raises the question of its particular function. In the adult bovine cerebral hemispheres, PLP is the major proteolipid but DM-20 remains quantitatively important because the PLP/DM-20 ratio ranges from 1.5 to 1.7. In the same developmental study we have, in the fetal cerebral hemispheres, isolated and characterized a novel proteolipid (apparent Mr 20,000), which appears even before DM-20 and is not detected in the adult brain. It is structurally related to PLP and DM-20 because the first 31 N-terminal amino acid residues are the same. However, in immunoblot, it did not react either with the antitridecapeptide 117-129 antiserum of PLP or with the anti-C-terminal hexapeptide antiserum of PLP.     

Dispersal of proteolipid macroaggregates with trifluoroacetic acid and analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis

The propensity of highly purified proteolipids to form macroaggregates in aqueous solutions, especially when heated with sodium dodecyl sulfate (SDS), with or without thiol reagents, has made qualitative and quantitative analyses of individual species by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) difficult and unreliable. Comparisons of proteolipid profiles from liver, brain, and cultured human keratinocytes demonstrate that 40-72% of the total proteolipid in SDS-PAGE sample buffer is in the form of macroaggregates. Treatment of proteolipids with neat trifluoroacetic acid (TFA) followed by removal of the TFA and incubation in cold SDS-PAGE sample buffer causes complete dispersal of the macroaggregates and allows recovery of virtually all of the proteolipid applied to gels (increasing yields by as much as 3.6 times, depending on tissue type). Gels of TFA-treated samples display differences not only in the relative amounts of individual species but also in novel species not found in untreated samples. Eluted macroaggregates treated with TFA display the same SDS-PAGE banding profiles as TFA-treated whole proteolipids. Hence, routine TFA treatment of proteolipids prior to SDS-PAGE increases total proteolipid yields, allows reliable quantitation of individual apoprotein species, and reveals species previously obscured by the formation of macroaggregates.     

Cyst(e)ine residues of bovine white-matter proteolipid proteins. Role of disulphides in proteolipid conformation.

Cyst(e)ine residues of bovine white-matter proteolipid proteins were characterized in a highly purified preparation. From a total of 10.6 cyst(e)ine residues/molecule of protein, as determined by performic acid oxidation, 2.5-3 thiol groups were freely accessible to iodoacetamide, iodoacetic acid and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), when the proteins were solubilized in chloroform/methanol (C/M) (2:1, v/v). The presence of lipids had no effect on thiol-group exposure. One thiol group available to DTNB in C/M could not be detected when proteolipids were solubilized in the more polar solvent n-butanol. In a C/M solution of purified proteolipid proteins, SDS did not increase the number of reactive thiol groups, but the cleavage of one disulphide bridge made it possible to alkylate six more groups. C.d. and fluorescence studies showed that rupture of this disulphide bond changed the protein conformation, which was reflected in partial loss of helical structure and in a greater exposure to the solvent of at least one tryptophan residue. Cyst(e)ine residues were also characterized in the different components [PLP (principal proteolipid protein), DM20 and LMW (low-Mr proteins)] of the proteolipid preparation. Although the numbers of cyst(e)ine residues in PLP and DM20 were similar, in LMW fewer residues were alkylated under four different experimental conditions. The differences, however, are not simply related to differences in Mr.     

In vitro synthesis of myelin proteolipid protein from rat brain using a total homogenate system

In vitro synthesis of myelin proteolipid protein (PLP) was explored at different ages using rat brain total homogenates, incubated for 30 min with [³H]glycine. Total proteolipids, extracted from the incubated samples, were separated by SDSPAGE and the radioactivity was measured in the band corresponding to myelin PLP. The incorporation into PLP in relation to the incorporation into brain total proteins increased from 0.04% at 10 days of age to 0.63% at 20 days, and declined slowly thereafter. Time course experiments were carried out using brain homogenates obtained from rats of 20 days of age (i.e. at the period of maximal synthesis of PLP). Labeled PLP molecules were already found at 2.5 min of incubation and the incorporation of the label into this protein, relative to the incorporation into total proteins, did not vary throughout the entire incubation time (30 min). Pulsechase experiments using a similar system and adding cycloheximide at different incubation times showed that the appearance of label into mature PLP was immediately blocked by the inhibitor of protein synthesis. These data suggest that PLP is synthesized as such and not as a pre-protein which is subsequently processed to render mature PLP.