Influence of polar residue deletions on lipid-protein interactions with the myelin proteolipid protein. Spin-label ESR studies with DM-20/lipid recombinants

The lipid specificities of two related integral membrane proteins of central nervous system myelin, the proteolipid (PLP) and DM-20 proteins, which differ only by the deletion of a polar stretch of 35 contiguous amino acid residues, were studied with spin-labeled lipids after reconstitution into dimyristoyl-phosphatidylcholine. The selectivity in populating lipid association sites at the protein interface and in modulating the lipid exchange between protein and bulk lipid sites was quantitated by the relative association constants and the off-rate constants for exchange, respectively, for both proteins. The sequence deleted in DM-20 (residues 116-150 of PLP) is found to play a major role in determining the lipid selectivity for the parent PLP protein.     

Synthesis of palmitoyl-thioester T-cell epitopes of myelin proteolipid protein (PLP). Comparison of two thiol protecting groups (StBu and Mmt) for on-resin acylation.

In order to test the effect of thiopalmitoylation on the encephalitogenic properties of two proteolipid protein (PLP) T-cell epitopes, we have studied the on-resin S-palmitoylation of peptides, synthesized using the Fmoc/tBu strategy. The use of two Cys protecting groups was investigated: the tert-butylsulfenyl (StBu) and the methoxytrityl (Mmt). Our studies show that the ease of deprotection of the thiol protected with StBu was sequence dependent. The deprotection of Cys(StBu) was difficult in the case of the two peptides PLP(104-117) and PLP(139-151). Neither of the two Cys(StBu) (Cys108 and Cys140, respectively) could be deprotected with tributylphosphine. Beta-mercaptoethanol was only efficient for the deprotection of Cys(StBu)140 at 85 degrees C and at 135 degrees C for Cys108. The two palmitoylated peptides could be obtained in good yield starting from Cys protected with Mmt. Our conclusion is that the Mmt group is the more versatile protecting group of the thiol for use in the on-resin synthesis of thiopalmitoylated peptides.     

Analysis of myelin proteolipid protein and F0ATPase subunit 9 in normal andjimpy CNS

Membrane fractions and chloroform-methanol (C-M) extracts ofjimpy (jp) and normal CNS at 17–20 days were examined by immunoblot and sequence analysis to determine whether myelin proteolipid protein (PLP) or DM-20 could be detected in jp CNS. No reactivity was detected in jp samples with several PLP antibodies (Abs) except with one Ab to amino acids 109–128 of normal PLP. Proteins in the immunoreactive bands 26 Mr comigrating with PLP were sequenced for the first 10–12 residues. A sequence corresponding to PLP was found in normal CNS, as expected, but not in the band from jp CNS. Our results provide no evidence for an aberrant form of PLP in jp CNS at 17–20 days. This and other studies suggest that the abnormalities in jp brain are not due to toxicity of the mutant jp PLP/DM-20 proteins. Interestingly, a sequence identical to the amino terminus of the mature proton channel subunit 9 of mitochondrial F₀ ATPase was detected in the immunoreactive bands 26 Mr in both normal and jp samples. This identification was supported by reactivity with an Ab to the F₀ subunit and by labeling with dicyclohexylcarbodiimide (DCCD). In contrast to PLP isolated from whole CNS, PLP isolated from myelin was devoid of F₀ subunit 9 based on sequence analysis and lack of reactivity with an Ab to the F₀ subunit, yet still reacted with DCCD. This finding rules out the possibility that contaminating F₀ ATPase gives rise to the DCCD binding exhibited by PLP and confirms the possibility that PLP has proton channel activity, as suggested by Lin and Lees (1,2).Abbreviations used Ab antibody - CM conditioned medium - C M chloroform-methanol - DCCD dicyclohexylcarbodiimide - jp jimpy - Mr mobility (apparent m.w×10^–3) - PLP proteolipid protein - PVDF polyvinylidene difluoride     

Cell-free acylation of rat brain myelin proteolipid protein and DM-20.

Incubation of rat brain myelin with [3H]palmitic acid in the presence of ATP, CoA and MgCl2 or [14C]-palmitoyl-CoA in a cell-free system resulted in the selective labelling of 'PLP' [proteolipid protein; Folch & Lees (1951) J. Biol. Chem. 191, 807-817] and 'DM-20' [Agrawal, Burton, Fishman, Mitchell & Prensky (1972) J. Neurochem. 19, 2083-2089] which, after polyacrylamide-gel electrophoresis in SDS, were revealed by fluorography. These results provide evidence of the association of fatty acid-CoA ligase and acyltransferase in isolated myelin. Palmitic acid is covalently bound to PLP and DM-20, because 70 and 92% of the radioactivity was removed from proteolipid proteins after treatment with hydroxylamine and methanolic NaOH respectively. Incubation of myelin with [3H]palmitic acid in the absence of ATP, CoA, MgCl2, or all three, decreased incorporation of fatty acid into PLP to 3, 55, 18 and 2% respectively. The cell-free system exhibits specificity with respect to the chain length of the fatty acids, since myristic acid is incorporated into PLP at a lower rate when compared with palmitic and oleic acids. The acylation of PLP is an enzymic reaction, since (1) maximum incorporation of [3H]palmitic acid into PLP occurred at physiological temperatures and decreased with an increase in the temperature; (2) acylation of PLP with [3H]palmitic acid and [14C]palmitoyl-CoA was severely inhibited by SDS (0.05%); and (3) the incorporation of fatty acid and palmitoyl-CoA into PLP was substantially decreased by the process of freezing-thawing and freeze-drying of myelin. We have provided evidence that all of the enzymes required for acylation of PLP and DM-20 are present in isolated rat brain myelin. Acylation of PLP in a cell-free system with fatty acids and palmitoyl-CoA suggests that a presynthesized pool of non-acylated PLP and DM-20 is available for acylation.     

A method for measurement of free and bound plasma cyst(e)ine

A method for the measurement of free and bound cyst(e)ine and the sum thereof has been developed. In adult human blood, cyst(e)ine is distributed equally between that which is bound to plasma proteins and that which is free. Cyst(e)ine is bound predominantly to albumin, and this binding is not an in vitro artifact. Cysteine bound to plasma proteins may be displaced by homocysteine which competes for the available sulfhydryl groups of plasma proteins. Rats starved for 8 days had a significant decrease in both plasma free cyst(e)ine and bound cysteine. These data suggest that present methods for the determination of plasma cyst(e)ine under-estimate the quantity of cyst(e)ine in the plasma available for cellular metabolism.     

G Run-mediated recognition of proteolipid protein and DM20 5' splice sites by U1 small nuclear RNA is regulated by context and proximity to the splice site

Highly conserved G runs, G1M2 and ISE, regulate the proteolipid protein (PLP)/DM20 ratio. We have investigated recruitment of U1 small nuclear ribonuclear protein (snRNP) by G1M2 and ISE and examined the effect of splice site strength, distance, and context on G run function. G1M2 is necessary for initial recruitment of U1snRNP to the DM20 5' splice site independent of the strength of the splice site. G1M2 regulates E complex formation and supports DM20 splicing when functional U1snRNP is reduced. By contrast, the ISE is not required for the initial recruitment of U1snRNP to the PLP 5' splice site. However, in close proximity to either the DM20 or the PLP 5' splice site, the ISE recruits U1snRNP to both splice sites. The ISE enhances DM20 splicing, whereas close to the PLP 5' splice site, it inhibits PLP splicing. Splicing enhancement and inhibition are mediated by heterogeneous nuclear ribonuclear protein (hnRNP)H/F. The data show that recognition of the DM20 5' splice site depends on G run-mediated recruitment of U1snRNA, whereas a complex interaction between the ISE G runs, context and position determines the functional outcome on splicing. The data suggest that different mechanisms underlie G run-mediated recognition of 5' splice sites and that context and position play a critical role.     

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.     

The thiol groups of the Folch-Pi protein from bovine white matter. Exposure, reactivity and significance.

The number and the reactivity of accessible thiol groups of the Folch-Pi apoprotein and proteolipid (50% of myelin proteins) were studied, by using a specific thiol-disulphide interchange reaction, in connection with the known solubility of this protein in organic and aqueous solvents. The high reactivity of 2,2'-dipyridyl disulphide towards thiol groups leads to the titration of 4.8 mol of SH groups/mol of protein (Mr 30000) in alkaline and acidic chloroform/methanol (2:1, v/v). Unlike previous findings, this value was consistently found from batch to batch and remained stable with time. In the proteolipid 1 mol of SH groups/mol was not accessible as compared with the apoprotein. In aqueous solvents, a similar number of 4.4 mol of SH groups/mol was also found. For the first time, kinetic studies carried out in chloroform/methanol discriminated between two classes of thiol groups. The reaction of 2 mol of SH groups/mol was characterized by apparent second-order rate constants whose values were 5-10-fold higher than those of the other class. Kinetic studies and cyanylation experiments in aqueous solvents also indicated the high reactivity of these thiol groups with Ellman's reagent. Together with kinetic results, studies on the stoichiometry of the interchange reaction of equimolar solutions of protein and disulphide indicate that these highly reactive thiol groups are near to each other in the amino acid sequence. The location of the thiol groups at the boundary between hydrophilic and hydrophobic domains of the Folch-Pi protein is suggested in connection with their possible structural and biological significance.     

Cysteine-108 is an acylation site in myelin proteolipid protein.

Myelin proteolipid protein (PLP) contains covalently bound long-chain fatty acids. A large proportion of these acyl moieties are bound in thioester linkages, as demonstrated by alkylation of newly formed SH groups upon deacylation. To identify the Cys residue(s) involved in the thioester linkage(s), reduced and carboxyamidomethylated proteolipid protein was labeled with [14C]iodoacetamide upon deacylation with neutral hydroxylamine. The labeled protein was digested with trypsin or pepsin, and peptides analyzed by RP-HPLC. Identification of the isolated radioactive peptides by amino acid analysis, peptide sequencing and/or fast-atom bombardment-mass spectrometry revealed that Cys108 in the bovine PLP sequence is an acylated site. The sequence surrounding the palmitoylation site in the myelin PLP is strikingly similar to that found in rhodopsin. Furthermore, as in rhodopsin and other members of the G protein-coupled receptor family, this Cys residue is located within a hydrophilic, basic, and possibly cytoplasmic, domain.     

Exogenous cysteine and cystine promote cell proliferation in CaCo-2 cells

Abstract. Previous studies have shown that intracellular glutathione, a ubiquitous intracellular thiol, is related to cell proliferation and that cysteine or its disulphide form, cystine, also induces cell proliferation. Cysteine is a thiol containing amino acid and a rate-limiting precursor of glutathione. Therefore, it is still unresolved as to whether the proliferative effect of cysteine or cystine is entirely mediated by a change in the intracellular glutathione status. The objective of this study was to delineate the relationship among cysteine/cystine (thereafter referred to as cyst(e)ine), intracellular glutathione and cell proliferation in the human colon cancer CaCo-2 cell line. CaCo-2 cells were cultured in cyst(e)ine-free Dulbecco's Modified Eagle Medium without serum, and treated with 200 µ m cysteine and/or 200–400 µ m cystine for 24 h. In the presence of DL-buthionine-[S, R]-sulfoximine (BSO), a glutathione synthesis inhibitor, exogenously administered cyst(e)ine did not change the intracellular glutathione content, but increased the intracellular cysteine as well as cystine level. Addition of exogenous cyst(e)ine following 5 m m BSO treatment significantly increased cell proliferation as measured by ³H-thymidine incorporation and protein content. Cell cycle analyses revealed that cyst(e)ine promoted cell progression from the G₁ phase to the S phase. Correspondingly, cyst(e)ine treatment induced expression of cyclin D1 and phosphorylation of retinoblastoma protein (Rb). In conclusion, these data indicate that both cysteine and cystine have proliferative effects in CaCo-2 cells independent of an increase in intracellular glutathione. Induction of cyclin D1, phosphorylation of Rb, and subsequent facilitation of G₁-to-S phase transition were involved in the proliferative effect of exogenous cyst(e)ine.     

The Folch-Lees proteolipid induces phase coexistence and transverse reorganization of lateral domains in myelin monolayers

Solvent solubilized myelin membranes spread as monomolecular layers at the air-water interface show a heterogeneous pattern at all surface pressures. In order to asses the role of myelin protein and lipid components in the surface structuring we compared the topography, as seen by Brewster angle microscopy (BAM) and epifluorescence microscopy, of monolayers made from mixtures containing all myelin lipids (except gangliosides) and variable proportions of Folch-Lees proteolipid protein (PLP, the major protein component of myelin). The presence of the single PLP, in the absence of the other myelin proteins, can reproduce the surface pattern of the whole myelin extract films in a concentration-dependant manner. Moreover, a threshold mole fraction of PLP is necessary to induce the lipid-protein component reorganization leading to the appearance of a rigid (gray) phase, acting as a surface skeleton, at low surface pressures and of fractal clusters at high surface pressures. The average size of those clusters is also dependent on the PLP content in the monolayer and on the time elapsed from the moment of film spreading, as they apparently result from an irreversible lateral aggregation process. The transverse rearrangement of the monolayer occurring under compression was different in films with the highest and lowest PLP mole fractions tested.     

Isolation and characterization of a cDNA encoding the zebra finch myelin proteolipid protein

A cDNA was isolated from a zebra finch telencephalon cDNA library that encodes the myelin proteolipid protein. The clone was 2874 nucleotides long containing an open reading frame of 831 nucleotides that encoded a 277 amino acid myelin proteolipid protein. The 5-and 3 untranslated regions were 112 and 1931 nucleotides, respectively. In Northern blots the clone hybridized to 3 bands of 3.5, 2.4 and 1.5 Kb in mouse brain RNA, but to only a single band of 3.0 kb in zebra finch brain RNA, suggesting the lack of alternative polyadenylation sites within the 3 untranslated region of the zebra finch PLP mRNAs. There was a small degree of homology between the zebra finch and chicken PLP 5 untranslated regions, but relatively little homology of the 5 untranslated regions of the zebra finch PLP cDNA clone with the homologous regions of PLP cDNAs of many mammalian species. Except for a small stretch of considerable homology, there was little overall homology with the 3 untranslated regions of mammalian PLP mRNAs. Approximately 10% (i.e. 28) of the amino acids in the zebra finch PLP differed from mammalian PLP, with most of these changes located within exon 3. There were 16 amino acid changes between zebra finch and chicken, suggesting that greater sequence variation in PLP structure is tolerated among avian species than among mammalian species.Abbreviations DM20 25 kDa proteolipid protein in myelin - PLP classic 30 kDa myelin proteolipid protein Special issue dedicated to Dr. Marjorie B. Lees.     

Myelin Proteolipid Protein Contains Thioester-Linked Fatty Acids

Myelin proteolipid protein (PLP) is known to contain long-chain, covalently bound fatty acids. Previous studies, including our own, have suggested the occurrence of an oxyester type of linkage between fatty acids and PLP. However, we found that protein-SH groups are required in the acylation reaction, suggesting the possible presence of thioesters. In the present study, we have examined the nature of the acyl-PLP linkages by determining whether free thiol groups are generated on removal of fatty acids. Incubation of reduced and carboxyamidomethylated proteolipid apoprotein (RCM-APL) with 0.2 M hydroxylamine and [14C]iodoacetamide at pH 7.5 and 37 degrees C resulted in the release of fatty acids and the concomitant labeling of newly formed thiol groups. Incubation with Tris or methylamine at pH 7.5 failed to remove fatty acids and generate free -SH groups. The possibility that on treatment buried thiol groups became exposed was essentially excluded because (1) similar results were obtained in 2-chloroethanol, a solvent in which acylated and deacylated PLP have the same conformation, and (2) small PLP peptides were labeled only in the presence of hydroxylamine. On incubation with [14C]methylamine at pH 9.0, RCM-APL was not labeled, thus excluding the occurrence of intramolecular thiol esters. On the other hand, fatty acids were released as radioactive N-methyl fatty acylamide, indicating the presence of intermolecular thioesters between fatty acids and protein. These results demonstrate that a large proportion of fatty acids covalently bound to PLP are liked to -SH groups.     

Interactions between aspartate animotransferase protomers observed during holoenzyme reconstitution..

Aspartate aminotransferase from pig heart cytosol consists of 2 identical protomers. Car?ymethylation of the easily accessible cysteines 45 and 82 of the holoenzyme produces a modified protein which however is still fully active. If the apoenzyme obtained from the car?ymethylated holoenzyme is subjected to the thiol specific reagent DTNB, another group per protomer can be titrated. There is some evidence that the group modified is the thiol 390 which is titrated in “syncatalytic” conditions, i.e. 2 mM ketoglutarate and 70 mM glutamate. The full reactivation of the car?ymethylated apoenzyme is obtained on binding of one PLP at each active site of the dimer. During the reactivation the number of thiols 390 accessible to DTNB decreases from 2 to 0 when PLP is added. The variation of the number of reactive thiol residues is not linear with coenzyme binding. Apo-holoenzyme car?ymethylated hybrid does not react with DTNB. Thus, a strong interaction between the 2 protomers is demonstrated.     

The Folch-Lees proteolipid induces phase coexistence and transverse reorganization of lateral domains in myelin monolayers

Solvent solubilized myelin membranes spread as monomolecular layers at the air-water interface show a heterogeneous pattern at all surface pressures. In order to asses the role of myelin protein and lipid components in the surface structuring we compared the topography, as seen by Brewster angle microscopy (BAM) and epifluorescence microscopy, of monolayers made from mixtures containing all myelin lipids (except gangliosides) and variable proportions of Folch-Lees proteolipid protein (PLP, the major protein component of myelin). The presence of the single PLP, in the absence of the other myelin proteins, can reproduce the surface pattern of the whole myelin extract films in a concentration-dependant manner. Moreover, a threshold mole fraction of PLP is necessary to induce the lipid-protein component reorganization leading to the appearance of a rigid (gray) phase, acting as a surface skeleton, at low surface pressures and of fractal clusters at high surface pressures. The average size of those clusters is also dependent on the PLP content in the monolayer and on the time elapsed from the moment of film spreading, as they apparently result from an irreversible lateral aggregation process. The transverse rearrangement of the monolayer occurring under compression was different in films with the highest and lowest PLP mole fractions tested.     

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.     

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.     

Molecular cloning and nucleotide sequence of a cDNA clone coding for rat brain myelin proteolipid

A cDNA library from rat brain was constructed in pBR322 and screened with a 14-mer mixed oligonucleotide probe based on residues 231-235 of bovine proteolipid (PLP). A positive clone was isolated: it contained a 1334-base-pair cDNA insert and was subjected to DNA sequence analysis. The cDNA encoded information for the 276 amino acids of rat PLP. Comparison with bovine PLP sequence showed a complete amino acid sequence homology except for 4 amino acid residues.     

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.