Anti‐histone H1 IgGs from blood serum of systemic lupus erythematosus patients are capable of hydrolyzing histone H1 and myelin basic protein

Novel hydrolytic activity of the anti‐histone H1 antibodies (Ab) toward histone H1 and myelin basic protein (MBP) was shown. Blood serum of ten patients with clinically diagnosed systemic lupus erythematosus (SLE), and nine healthy donors (control) were screened for the anti‐histone H1 antibody‐ and anti‐MBP antibody‐mediated specific proteolytic activity. IgGs were isolated by chromatography on Protein G‐Sepharose, and four of ten SLE patients appeared to possess IgGs that were capable of cleaving both histone H1 and MBP. Such activity was confirmed to be an intrinsic property of the IgG molecule, since it was preserved at gel filtration at alkaline and acidic pH. At the same time, proteolytic activity was absent in the sera‐derived Ab of all healthy donors under control. Anti‐histone IgGs were purified by the affinity chromatography on histone H1‐Sepharose. Their cross‐reactivity toward cationic proteins (histones, lysozyme, and MBP) and their capability of hydrolyzing histone H1 and MBP were detected. However, these IgGs were not cleaving core histones, lysozyme, or albumin. Capability of cleaving histone H1 and MBP was preserved after additional purification of anti‐histone H1 IgGs by the HPLC gel filtration. The protease activity of anti‐histone H1 IgG Ab was inhibited by serine protease inhibitors. Copyright © 2010 John Wiley & Sons, Ltd.     

Chemical exchange between lamellar and non-lamellar lipid phases. A one- and two-dimensional 31P-NMR study.

One- and two-dimensional 31P-exchange NMR has been used to investigate chemical exchange between coexisting lamellar (L alpha) and non-lamellar (hexagonal HII and cubic I2) lipid phases. Samples of DOPE, DOPE/DOPC (9:1 and 7:3), DOPE/cholesterol sulfate (9:1), DOPC/monoolein (MO) (3:7 and 1:1), and DOPC/DOPE/cholesterol (1:1:2) were macroscopically oriented on glass plates and studied at the 0 degree orientation (angle between the bilayer normal and the external magnetic field), where the L alpha, HII, and I2 resonances are resolved. A reversible L alpha to HII transition was observed for all of the samples except for the DOPC/MO mixtures, which displayed a reversible L alpha to I2 transition. Near-equilibrium mixtures of L alpha and either HII or I2 were obtained after prolonged incubation at a given temperature. Two-dimensional exchange experiments were performed on DOPE at 9-14 degrees C for mixing times ranging from 500 ms to 2 s. For all samples, one-dimensional exchange experiments were performed for mixing times ranging from 100 ms to 4 s, at temperatures ranging from 3 degrees C to 73 degrees C. No evidence of lipid exchange between lamellar and non-lamellar phases was observed, indicating that if such a process occurs it is either very slow on the seconds' timescale, or involves an undetectable quantity of lipid. The results place constraints on the stability or kinetic behaviour of proposed transition intermediates (Siegel, D.P. (1986) Biophys. J. 49, 1155-1170).     

N-succinyldioleoylphosphatidylethanolamine: structural preferences in pure and mixed model membranes

The structural preferences of the pH-sensitive phospholipid, N-succinyldioleoylphosphatidylethanolamine (N-succinyl-DOPE), have been examined alone and in mixtures with DOPE by 31P-NMR, fluorescence energy transfer, and freeze-fracture techniques. The basic polymorphic behavior of pure N-succinyl-DOPE and DOPE/N-succinyl-DOPE lipid systems and the influence of calcium and pH were investigated. It is shown that, similar to other negatively charged acidic phospholipids, N-succinyl-DOPE adopts the bilayer organization upon hydration. This structure is maintained at both pH 7.4 and 4.0 in the presence or absence of calcium. In the mixed lipid system, N-succinyl-DOPE can stabilize the non-bilayer lipid, DOPE, into a bilayer structure at both pH 7.4 and 4.0 at more than 10 mol% N-succinyl-DOPE, although a narrow 31P-NMR lineshape is observed at acidic pH values. This corresponds to the presence of smaller vesicles as shown by quasi-elastic light scattering measurements. Addition of equimolar calcium (with respect to N-succinyl-DOPE) to the DOPE/N-succinyl-DOPE systems induces the hexagonal HII phase at both pH values. In unilamellar systems with similar lipid composition the addition of Ca2+ results in membrane fusion as indicated by fluorescence energy-transfer experiments. These findings are discussed with regard to the molecular mechanism of the bilayer to hexagonal HII phase transition and membrane fusion and the utility of N-succinyl-DOPE containing pH-sensitive vesicles as drug-delivery vehicles.     

The association of myelin basic protein with itself and other proteins

Chromatographic studies were performed to measure myelin basic protein (MBP) interactions by covalently binding a number of different proteins to Sepharose and passing radioactive bovine MBP over these columns. Studies at a variety of pH values, ionic strengths and temperatures revealed that the bovine MBP could interact with itself as well as cytochrome c, lysozyme, and ovalbumin. Chromatographic profiles of elution volume vs. pH revealed that the interaction between MBP and these immobilized proteins was biphasic. The self-association of MBP was found to be strongest between pH 7.4 and 8.1 and at an elevated temperature. Titration of the amino acid residues responsible for the association of MBP with other proteins revealed apparent pKs ranging from 6.10 to 6.70. A pH dependence study at an elevated temperature shifted the apparent pK of the MBP interaction to a lower value with all the proteins except ovalbumin. After destroying 60% of the histidine residues in MBP by photooxidation and passing¹²⁵I-labeled photooxidized MBP over Sepharose columns containing immobilized protein, the second phase in binding was decreased significantly with immobilized cytochrome c, lysozyme, and MBP and to a smaller extent with ovalbumin. These results are consistent with the involvement of deprotonated histidine residues in the MBP-protein associations.     

31P nuclear magnetic resonance studies of the association of basic proteins with multilayers of diacyl phosphatidylserine

Lysozyme, cytochrome c, poly(L-lysine), myelin basic protein and ribonuclease were used to form multilayer dispersions containing about 50% protein (by weight) with bovine brain diacyl phosphatidylserine (PS). 31P nuclear magnetic resonance shift anisotropies, spin-spin (T2) and spin-lattice (T1) relaxation times for the lipid headgroup phosphorus were measured at 36.44 MHz. At pH 7.5, lysozyme, cytochrome c, poly(L-lysine) and ribonuclease were shown to increase the chemical shift anisotropy of PS by between 12-20%. Myelin basic protein altered the shape of the phosphate resonance, suggesting the presence of two lipid components, one of which had a modified headgroup conformation. The presence of cytochrome c led to the formation of a narrow spike at the isotropic shift position of the spectrum. Of the various proteins or peptides we have studied, only poly(L-lysine) and cytochrome c had any effect on the T1 of PS (1050 ms). Both caused a 20-30% decrease in T1 of the lamellar-phase phosphate peak. The narrow peak in the presence of cytochrome c had a very short T1 of 156 ms. The possibility is considered that the cytochrome Fe3+ contributes to the phosphate relaxation in this case. The effect of all proteins on the T2 of the phosphorus resonance was to cause an increase from the value for pure PS (1.6 ms) to between 2 and 5 ms. The results obtained with proteins are compared with the effects of small ions and intrinsic membrane proteins on the order and motion of the headgroups of lipids in bilayers.     

Order and dynamics in the lamellar L alpha and in the hexagonal HII phase. Dioleoylphosphatidylethanolamine studied with angle-resolved fluorescence depolarization.

Fluorescence depolarization techniques are used to determine the molecular order and reorientational dynamics of the probe molecule TMA-DPH embedded in the lamellar L alpha and the hexagonal HII phases of lipid/water mixtures. The thermotropically induced L alpha----HII phase transition of the lipid DOPE is used to obtain macroscopically aligned samples in the hexagonal HII phase at 45 degrees C from samples prepared in the lamellar L alpha phase at 7 degrees C. The interpretation of angle-resolved fluorescence depolarization experiments on these phases, within the framework of the rotational diffusion model, yields the order parameters (P2) and (P4), and the diffusion constants for the reorientational motions. The reorientational motion rates of the TMA-DPH molecules in the hexagonal HII phase are comparable with those in the lamellar L alpha phase. Furthermore, the lateral diffusion of the probe molecule on the surface of the lipid/water cylinder in the hexagonal phase is found to be considerably slower than the reorientational motion.     

Specificity of oligoclonal IgG bands against myelin proteins in chronic relapsing EAE in guinea pigs

We showed previously by using imprint electroimmunofixation that the oligoclonal IgG in sera and CSF from chronic relapsing EAE in guinea pigs were specific to spinal cord and Mycobacterium tuberculosis. We now show that most oligoclonal IgG bands are directed predominantly against isolated myelin basic protein (MBP). Activity to the latter could be removed from sera or CSF by absorption with MBP but not with histone or lysozyme. The oligoclonal IgG reacted weakly with isolated proteolipid apoprotein, and lacked reactivity to myelin-associated glycoprotein. When the oligoclonal IgG activity to myelin proteins was removed from the sera by absorption with a preparation of delipidated myelin before imprint electroimmunofixation, a few bands in some sera still reacted with whole spinal cord homogenate. These results indicate that, in some sera, a part of the oligoclonal IgG was directed against non-myelin proteins or lipids. In contrast to chronic relapsing EAE, CSF oligoclonal IgG from patients with multiple sclerosis showed no reactivity against human brain homogenate, whole myelin, delipidated myelin, and MBP in imprint electroimmunofixation.     

Mechanism of the interaction between myelin basic protein and the myelin membrane; the role of arginine methylation

The addition of solutions of bovine myelin basic protein to suspensions of unilamellar vesicles prepared from whole myelin suspensions results in the rapid equilibrium association of the vesicles into dimers, followed by time-dependent aggregation reactions. Other cationic proteins also induce the dimerization of the vesicles and equilibrium constants for dimer formation are obtained for bovine myelin basic protein, lysozyme, polyhistidine and myelin basic protein from carp, which differs from the bovine protein in that it contains no methylarginine residues. The bovine protein is more efficient at inducing dimer formation than the carp protein by approximately 0.93 kcal/mole; the carp protein is approximately as effective as the other cationic proteins examined. Complete methylation of the bovine MBP by AdoMet:MBP methyltransferase increases the interaction between MBP and the membrane by approximately 0.13 kcal/mole, consistent with the suggestion that a large portion of the free energy difference between the carp and bovine proteins arises from favorable interactions involving the methylarginine residues.     

An analysis of the regions of the myelin basic protein that bind to phosphatidylcholine

The interactions of phosphatidylcholine (PC) to regions of the myelin basic protein (MBP) was examined. In solid phase binding assays the nature of the binding of unilamellar vesicles of¹⁴C-labeled phosphatidylcholine to bovine 18.5 kDa MBP, its N- and C-terminal peptide fragments, photooxidized 18.5 kDa MBP and the mouse 14 kDa protein, with an internal deletion of residues 117–157, was studied. The data were analyzed by computer-generated Scatchard plots in which non-specific binding was eliminated. Non-cooperative, low affinity binding of PC vesicles to MBP was observed, and this binding found to be sensitive to pH and ionic changes. At an ionic strength of 0.1 and pH 7.4, the binding of PC to the 14 kDa mouse MBP exhibited a K_d similar to that obtained with both the N-terminal and photooxidized 18.5 kDa bovine MBP. The studies indicated that the sites of PC interaction with MBP are located in the N-terminal region of the protein. The C-terminal region appeared to modulate the strength of the interaction slightly. Under similar conditions, lysozyme did not bind PC liposomes, and histone bound them nonspecifically.     

Modulation by glycosphingolipids of membrane-membrane interactions induced by myelin basic protein and melittin.

The effect of glycosphingolipids (GSLs) with oligosaccharide chains of different length and charge on membrane-membrane interactions induced by myelin basic protein (MBP) or melittin (Mel) was comparatively investigated with small unilamellar vesicles. MBP induces a fast vesicle aggregation and close membrane apposition. Merging of lipid bilayers and vesicle fusion induced by MBP are slower and less extensive processes compared to membrane apposition. The changes of membrane permeability concomitant to these phenomena are small. The Trp region of MBP remains in a rather polar environment when interacting with vesicles; its accessibility to NO3- or acrylamide quenching depends on the type of GSLs in the membrane. The Trp region of Mel is inserted more deeply into the lipid bilayer and its accessibility to the aqueous quenchers is less dependent on variations of the oligosaccharide chain of the GSLs. Mel induces a faster and more extensive membrane apposition and bilayer merging than does MBP. Extensive vesicle disruption occurs in the presence of Mel. Negatively charged GSLs facilitate membrane proximity and vesicle aggregation but an increase of the oligosaccharide chain length of either neutral or acidic GSLs decreases the interaction among vesicles that are induced by either protein. This effect is independent of the different mode of insertion of MBP and Mel into the membrane. Our results suggest that the modulation by the oligosaccharide chain on the protein-induced interactions between bilayers containing GSLs is probably exerted beyond the level of local molecular interactions between the basic proteins and the lipids.     

Implications of a non-lamellar lipid phase for the tight junction stability. Part I: Influence of basic amino acids, pH and protamine on the bilayer-hexagonal II phase behaviour of PS-containing PE membranes.

Inverted lipid micelles have been proposed, among other biological functions, to constitute the structural basis of the so-called tight junctions, a special cell cell contact found in epithelia and endothelial, which act as a barrier for the paracellular solute passage. As a model system for the opening and closing of this gate, we investigated the formation of the inverted hexagonal phase (HII phase) in lipid bilayer systems consisting of egg phosphatidylethanolamine (egg PE) and mixed egg PE/bovine brain phosphatidylserine (BBPS) membranes. The formation of the HII phase was modulated by Ca2+ ions, pH, basic amino acids and protamine. The lamellar-HII phase transition temperature TH of pure egg PE membranes at pH 7.0 was lowered with increasing Ca2+ concentration. This effect was attenuated by the presence of 50 mM lysine methyl ester. In the mixed lipid system, this effect was also observed, but even more pronounced. However this effect could be compensated for by raising the Ca2+ concentration from 2 to 10 mM. This was not observed in the pure PE system. In the absence of Ca2+, lysine methyl ester and protamine lowered TH in both monocomponent and mixed lipid systems, whereas lysine caused the opposite effect. The pH-dependence of mixed lipid systems, which were investigated up to a BBPS content of 20 mol%, clearly shows that increasing PS content stabilizes the lamellar phase even at low pH. The results obtained with model membranes are discussed with respect to biological implications of the lamellar-HII phase transition for the modulation of tight junction stability.     

Basic Protein in Brain Myelin Is Phosphorylated by Endogenous Phospholipid-Sensitive Ca2+-Dependent Protein Kinase

Abstract: Phosphorylation of myelin basic protein (MBP) in rat or rabbit brain myelin was markedly stimulated by Ca²⁺, and this reaction was not essentially augmented by exogenous phosphatidylserine or calmodulin or both. Solubilization of myelin with 0.4% Triton X-100 plus 4 m M EGTA, with or without further fractionation, showed that Ca²⁺-dependent phosphorylation of MBP required phosphatidylserine, but not calmodulin. DEAE-cellulose chromatography of solubilized myelin revealed a pronounced peak of protein kinase activity stimulated by a combination of Ca²⁺ and phosphatidylserine; a protein kinase stimulated by Ca²⁺ plus calmodulin was not detected. These findings clearly indicate an involvement of phospholipid-sensitive Ca²⁺-dependent protein kinase in phosphorylation of brain MBP, although a possible role for the calmodulin-sensitive species of Ca²⁺-dependent protein kinase in this reaction could not be excluded or established. Phosphorylation of MBP in solubilized rat myelin catalyzed by the phospholipid-sensitive enzyme was inhibited by adriamycin, palmitoylcarnitine, trifluoperazine, melittin, polymyxin B, and N -(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W–7).     

Micron-scale phase segregation in lipid monolayers induced by myelin basic protein in the presence of a cholesterol analog

It was previously shown that myelin basic protein (MBP) can induce phase segregation in whole myelin monolayers and myelin lipid films, which leads to the accumulation of proteins into a separate phase, segregated from a cholesterol-enriched lipid phase. In this work we investigated some factors regulating the phase segregation induced by MBP using fluorescent microscopy of monolayers formed with binary and ternary lipid mixtures of dihydrocholesterol (a less-oxidable cholesterol analog) and phospholipids. The influence of the addition of salts to the subphase and of varying the lipid composition was analyzed. Our results show that MBP can induce a dihydrocholesterol-dependent segregation of phases that can be further regulated by the electrolyte concentration in the subphase and the composition (type and proportion) of non-sterol lipids. In this way, changes of the lipid composition of the film or the ionic strength in the aqueous media modify the local surface density of MBP and the properties (phase state and composition) of the protein environment.     

A thermodynamic and structural study of myelin basic protein in lipid membrane models

Myelin basic protein (MBP) is a major protein of the myelin membrane in the central nervous system. It is believed to play a relevant role in the structure and function of the myelin sheath and is a candidate autoantigen in demyelinating processes such as multiple sclerosis. MBP has many features typical of soluble proteins but is capable of strongly interacting with lipids, probably via a conformation change. Its structure in the lipid membrane as well as the details of its interaction with the lipid membrane are still to be resolved. In this article we study the interaction of MBP with Langmuir films of anionic and neutral phospholipids, used as experimental models of the lipid membrane. By analyzing the equilibrium surface pressure/area isotherms of these films, we measured the protein partition coefficient between the aqueous solution and the lipid membrane, the mixing ratio between protein and lipid, and the area of the protein molecules inserted in the lipid film. The penetration depth of MBP in the lipid monolayer was evaluated by x-ray reflectivity measurements. The mixing ratio and the MBP molecular area decrease as the surface pressure increases, and at high surface pressure the protein is preferentially located at the lipid/water interface for both anionic and neutral lipids. The morphology of MBP adsorbed on lipid films was studied by atomic force microscopy. MBP forms bean-like structures and induces a lateral compaction of the lipid surface. Scattered MBP particles have also been observed. These particles, which are 2.35-nm high, 4.7-nm wide, and 13.3-nm long, could be formed by protein-lipid complexes. On the basis of their size, they could also be either single MBP molecules or pairs of c-shaped interpenetrating molecules.     

Observation of inverted cubic phase in hydrated dioleoylphosphatidylethanolamine membranes

We report the observation of an inverted cubic phase in aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) by small-angle X-ray diffraction. DOPE is a paradigm in the study of nonlamellar phases in biological systems: it exhibits a well-known phase transition from the lamellar (L alpha) to the inverted hexagonal phase (HII) as the temperature is raised. The transition is observed to occur rapidly when a DOPE dispersion is heated from 2 degrees C, where the L alpha phase is stable, to 15 degrees C, where the HII phase is stable. We report on the induction of a crystallographically well-defined cubic lattice that is slowly formed when the lipid dispersion is rapidly cycled between -5 and 15 degrees C hundreds of times. Once formed, the cubic lattice is stable at 4 degrees C for several weeks and exhibits the same remarkable metastability that characterizes other cubic phases in lipid-water systems. X-ray diffraction indicates that the cubic lattice is most consistent with either the Pn3m or Pn3 space group. Tests of lipid purity after induction of the cubic indicate the lipid is at least 98% pure. The cubic lattice can be destroyed and the system reset by cycling the specimen several times between -30 and 2 degrees C. The kinetics of the formation of the cubic are dependent on the thermal history of the sample, overall water concentration, and the extreme temperatures of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of myelin protein genes in the developing brain

The major myelin proteins fall into two classes, the basic proteins and the proteolipid proteins. In mice, five forms of the myelin basic protein (MBP) have been identified with apparent molecular masses of 21.5 kD, 18.5 kD, 17 kD and 14 kD. The 17 kD MBP variant consists of two molecular forms with similar molecular masses but different amino acid sequences. Cell-free translation studies and analyses of MBP cDNAs have shown that each of the MBP variants is encoded by a separate mRNA of approximately 2 000 bp. The five mouse MBP mRNAs appear to be derived by alternative splicing of exons 2, 5, and 6 of the MBP gene. cDNAs encoding four forms of MBP have been isolated from a human fetal spinal cord library. The mRNAs corresponding to these cDNAs are probably derived by alternative splicing of exons 2 and 5 of the human MBP gene. Proteolipid protein (PLP) cDNAs have been isolated from several species and used to establish that the size of the major PLP mRNA is approximately 3 kb. Multiple size classes of the PLP mRNAs exist in mice and rats whereas the 3 kb mRNA is the predominant form in the developing human spinal cord. In normal mice, maximal expression of the PLP gene lags behind that of the MBP gene by several days. Studies on dysmyelinating mutants have determined some of the molecular defects with respect to these two classes of myelin proteins. For example, there is a deletion of a portion of the MBP gene in the shiverer mutant. In the quaking mutant, the expression of both classes of myelin proteins is significantly reduced prior to 3 weeks. However, after 3 weeks, MBP expression approaches normal levels but the newly synthesized protein fails to be incorporated into myelin. In the jimpy mutant, although the expression of both classes of proteins is reduced, PLP expression is most severely affected.     

Interactions of free and immobilized myelin basic protein with anionic detergents

The interaction of free and immobilized myelin basic protein (MBP) with sodium deoxycholate (DOC) and sodium dodecyl sulfate (NaDodSO4) was studied under a variety of conditions. Free MBP formed insoluble complexes with both detergents. Analysis of the insoluble complexes revealed that the molar ratio of detergent/MBP in the precipitate increased in a systematic fashion with increasing detergent concentration until the complex became soluble. At pH 4.8, equilibrium dialysis studies indicated that approximately 15 mol of NaDodSO4 could bind to the protein without precipitation occurring. Regardless of the surfactant, however, minimum protein solubility occurred when the net charge on the protein-detergent complex was between +18 and -9. Complete equilibrium binding isotherms of both detergents to the protein were obtained by using MBP immobilized on agarose. The bulk of the binding of both detergents was highly cooperative and occurred at or above the critical micelle concentration. At I = 0.1, saturation levels of 2.09 +/- 0.15 g of NaDodSO4/g of protein and 1.03 /+- 0.40 g of DOC/g of protein were obtained. Below pH 7.0 the NaDodSO4 binding isotherms revealed an additional cooperative transition corresponding to the binding of 15-20 mol of NaDodSO4/mol of protein. Affinity chromatography studies indicated that, in the presence of NaDodSO4 (but not in its absence), [125I]MBP interacted with agarose-immobilized histone, lysozyme, and MBP but did not interact with ovalbumin-agarose. These data support a model in which the detergent cross-links and causes precipitation of MBP-anionic detergent complexes. Cross-linking may occur through hydrophobic interaction between detergents electrostatically bound to different MBP molecules.     

髓鞘碱性蛋白对不同头部基团的髓磷脂质单层膜影响的热力学特性

髓鞘碱性蛋白(myelin basic protein,MBP)是中枢神经系统(central nervous system,CNS)髓鞘成熟期的主要蛋白质之一.研究资料表明,MBP与变态反应性脑脊髓炎(allergic encephalomyelitis,EAE)、多发性硬化等多种神经疾病有关,是反映中枢神经系统有无...