Biomedical subjects

M A Moscarello

Publications and source records attributed to M A Moscarello.

At least 19 recordsLinked to original sources

PubMed2000

Cryoelectron microscopy of protein-lipid complexes of human myelin basic protein charge isomers differing in degree of citrullination.

Myelin basic protein (MBP) is considered to be essential for the maintenance of stability of the myelin sheath. Reduction in cationicity of MBP, especially due to conversion of positively charged arginine residues to uncharged citrulline (Cit), has been found to be associated with multiple sclerosis (MS). Here, the interactions of an anionic phosphatidylserine/monosialoganglioside-G(M1) (4:1, w:w) lipid monolayer with 18.5-kDa MBP preparations from age-matched adult humans without MS (no Cit residues), with chronic MS (6 Cit), and with acute Marburg-type MS (18 Cit) were studied by transmission and ultralow dose scanning transmission electron microscopy under cryogenic conditions. Immunogold labeling and single particle electron crystallography were used to define the nature of the complexes visualized. These electron microscopical analyses showed that the three different MBP charge isomers all formed uniformly sized and regularly shaped protein-lipid complexes with G(M1), probably as hexamers, but exhibited differential association with and organization of the lipid. The least cationic Marburg MBP-Cit(18) formed the most open protein-lipid complex. The data show a disturbance in lipid-MBP interactions at the ultrastructural level that is related to degree of citrullination, and which may be involved in myelin degeneration in multiple sclerosis.

Adult↗

PubMed1999

Highly deiminated isoform of myelin basic protein from multiple sclerosis brain causes fragmentation of lipid vesicles.

Myelin basic protein (MBP) occurs as a number of charge isomers due to phosphorylation, deamidation, and deimination of arginine to citrulline. All of these modifications decrease the net positive charge of the protein and its ability to cause aggregation of negatively charged lipid vesicles. This is used as a model system for the ability of MBP to cause adhesion of the cytosolic surfaces of myelin. Therefore, the effect of two deiminated forms of MBP on lipid vesicles was compared with that of the unmodified, most positively charged isomer, C1, to determine how loss of positively charged arginines would affect the function of MBP. The deiminated forms were the isomer isolated from normal human brains, in which only 6 Arg are deiminated to citrulline (MBP-Cit(6)), and an isomer isolated from the brain of a patient who died with acute, fulminating multiple sclerosis (Marburg type), in which 18 of the 19 Arg were deiminated (MBP-Cit(18)). Whereas C1 caused aggregation of lipid vesicles, resulting in an increase in absorbance due to light scattering, MBP-Cit(18) caused a decrease in absorbance of the lipid vesicles. Size exclusion chromatography and negative staining electron microscopy showed that this was due to fragmentation of the large multilayered vesicles into much smaller vesicles. MBP-Cit(6) caused less aggregation of lipid vesicles than did C1. However, no fragmentation of the vesicles into smaller ones in the presence of C1 and MBP-Cit(6) was detected by size exclusion chromatography or electron microscopy. The membrane fragmentation caused by MBP-Cit(18) is dramatically different from the effects of other forms of MBP from normal brain and may indicate a pathogenic effect of this charge isomer, which may have contributed to the severity of the Marburg type of multiple sclerosis. Alternatively, the deimination may have been a secondary effect resulting from the disease process. Regardless of the role of MBP-Cit(18) in multiple sclerosis, the effect of this modification indicates that, when most of the arginines of MBP are modified to an uncharged amino acid, the protein acquires properties similar to an apolipoprotein; thus, it may take up an amphipathic structure when bound to lipid. A partly amphipathic character may also be related to the role of MBP-Cit(6) in normal immature myelin, where it is the predominant charge isomer.

Animals↗

PubMed1999

The developmental expression and activity of peptidylarginine deiminase in the mouse.

We have measured the expression and activity of peptidylarginine deiminase (PAD, EC 3.5.3.15), the enzyme responsible for converting arginyl residues in proteins to citrullines, in normal mouse brain homogenate. PAD transcripts were detected as early as five days and were maximal at one month of age. The enzyme protein was also detected at 5 days in an antibody dependent assay and was maximal at 2 months of age, 1 month later than the maximum expression of transcripts. As expected, enzyme activity had a similar developmental profile to that of the enzyme protein. In isolated mouse brain compact myelin, the activity was highest at 15 days and fell rapidly to 15% of this level by 1-2 months. In the 'loose' myelin fraction (heavy myelin) it remained at the same high level form from 15 days to 8 months. The activity in compact myelin was about 15 times greater than the activity in brain homogenate, suggesting much of the enzyme was localized to myelin.

Animals↗

PubMed1999

Rapid release and unusual stability of immunodominant peptide 45-89 from citrullinated myelin basic protein.

Myelin basic protein (MBP) exists in a population of isoforms and isomers. The 18.5 kDa MBP-C1, the main human adult isoform, has 170 residues and is relatively unmodified, whereas the same isoform can be citrullinated on six arginine residues to create the MBP-C8 (MBP Cit6) isomer. MBP Cit6 dominates in MS brain, accounting for 45% rather than 25% of the population of MBP isomers. In the fulminant form of MS, known as Marburg's Disease, 18 of the 19 arginines in MBP are citrullinated (MBP Cit18). Citrullination of MBP could lead to instability of myelin or limited remyelination. In this investigation, the susceptibilities to degradation by cathepsin D of MBP Cit6 and MBP-C1, both from normal and MS brain tissue, and Marburg MBP Cit18 were compared. The pattern of digestion was similar, and no differences of corresponding isomers in normal and MS brain were noted. However, normal MBP Cit6 was degraded 10-fold more rapidly than MBP-C1, and MBP Cit18 was degraded even more rapidly. MBP peptide 45-89 was preserved regardless of isomer type or source. Its generation was directly related to the citrulline content of the MBP substrate being 4 times faster in normal MBP Cit6 and 35 times faster in Marburg MBP Cit18 than in normal MBP-C1. Peptide 45-89 from a citrullinated MBP exhibited more deamidation, and, regardless of source, showed an alpha-helix structure in a lipid mimetic environment. We postulate that the generation of MBP peptides, including those that are dominant and encephalitogenic, is directly related to deimination of arginine to citrulline in MBP.

Brain↗

PubMed1999

Marburg's variant of multiple sclerosis correlates with a less compact structure of myelin basic protein.

Multiple sclerosis (MS) is an autoimmune disease in which the myelin sheath of the central nervous system is degraded, and the 18.5 kDa isoform of myelin basic protein (MBP) is reduced in cationicity. In a unique case of acute, fulminating MS (Marburg's variant), MBP is considerably less cationic than MBP from both normal, and chronic MS-afflicted individuals. This electron microscopical study has identified that, in vitro, the less cationic Marburg MBP isomer forms a more extended protein-lipid complex than MBP from healthy or chronic MS-afflicted individuals. This correlation implies that chemical modifications to MBP in vivo contribute directly to the structural instability of myelin, and subsequent autoantigenic presentation of this protein, observed in vivo in MS.

Acute Disease↗

PubMed1998

Human proteolipid protein (PLP) mediates winding and adhesion of phospholipid membranes but prevents their fusion.

Proteolipid protein (PLP or lipophilin) is a highly conserved, strongly hydrophobic, integral membrane protein, and is the major protein component of central nervous system myelin. Although PLP has been implicated in many functions, its in vivo role is still uncertain. Here, we report the investigation of PLP's putative adhesive function using purified PLP and reconstituted phospholipid vesicles made of either 100% phosphatidylcholine (PC), or a mixture of 92% PC and 8% phosphatidylserine (PS), by weight. PLP-induced changes in the phospholipid bilayer surfaces were directly examined by transmission electron microscopy. We found that upon the introduction of PLP, larger lipid vesicles became smaller and unilamellar. At the PLP:lipid molar ratio of 1:20, vesicle membranes rolled onto themselves forming 'croissant'-like structures that subsequently adhered to each other. The phenomena of PLP-induced bilayer rolling and adhesion were dependent on the concentration of PLP and the period of incubation, but were independent of the presence of calcium and types of phospholipids (PC or PC:PS). Furthermore, the presence of PLP in the lipid bilayers prevented the fusion of membranes. These findings show that PLP can induce membrane 'winding' while preventing the fusion of adjacent lipid bilayers. Hence, our data provide direct evidence for PLP's suspected function of membrane adhesion, and also suggest that PLP could potentially play a role in the formation of the myelin sheath.

Apoproteins↗

PubMed1998

A novel microtubule independent effect of paclitaxel: the inhibition of peptidylarginine deiminase from bovine brain.

Although the principal effect of paclitaxel (taxol) is in preventing depolymerization of microtubules, other effects have been described recently. In the present manuscript, we demonstrate an inhibitory effect on the enzyme peptidylarginine deiminase (PAD) which converts peptidyl bound arginine to citrulline. To study the mechanism of action of the drug on PAD, a number of studies were carried out with purified enzyme. With the synthetic substrate benzoyl-arginine ethyl ester (BAEE), almost total inhibition of activity was observed at 12. 5 mM. With myelin basic protein (MBP) as a substrate, deimination of arginyl residues was prevented by 0.5 mM paclitaxel. The velocity-substrate curve was unusual since substrate enhancement was observed at 5 mM BAEE. These data suggested the presence of two binding sites on the enzyme. Inhibition of activity by paclitaxel was non-competitive for both sites.

Animals↗

PubMed1998

Mechanism of demyelination in DM20 transgenic mice involves increased fatty acylation.

Transgenic mice (ND4) containing 70 copies of the transgene encoding DM20 were clinically normal up to 3 months of age, spontaneously demyelinated there-after and died in 8-10 months. Whereas the myelin fraction from normal mice increased in amount from 1-4 months as expected, the corresponding fraction in ND4 mice remained constant over this period. In order to study the mechanism by which decreased myelin synthesis was manifest in the ND4 mouse, we investigated the amounts of proteolipids at various ages. The amount of proteolipid protein (PLP) was greatly decreased after 1-2 months in the ND4 mice. Although the message for DM20 was increased (transgene mRNA), very little DM20 was found in myelin at 1 month. It subsequently increased so that at 3-4 months the amount of DM20 in myelin isolated from transgenic animals was much higher than in normal mice. Characterization of the DM20 and PLP at 1 month of age showed that the amount of fatty acid (stearate and palmitate) was increased and the N-terminal glycine was methylated. These data suggested that high copy numbers of the cDNA for DM20 affected post translational events which we postulate affected the proper insertion of both DM20 and PLP in the myelin bilayer.

Acylation↗

PubMed1997

Three-dimensional structure of myelin basic protein. I. Reconstruction via angular reconstitution of randomly oriented single particles.

Myelin basic protein (MBP) plays an integral role in the structure and function of the myelin sheath. In humans and cattle, an 18.5-kDa isoform of MBP predominates and exists as a multitude of charge isomers resulting from extensive and varied post-translational modifications. We have purified the least modified isomer (named C1) of the 18.5-kDa isoform of MBP from fresh bovine brain and imaged this protein as negatively stained single particles adsorbed to a lipid monolayer. Under these conditions, MBP/C1 presented diverse projections whose relative orientations were determined using an iterative quaternion-assisted angular reconstitution scheme. In different buffers, one with a low salt and the other with a high salt concentration, the conformation of the protein was slightly different. In low salt buffer, the three-dimensional reconstruction, solved to a resolution of 4 nm, had an overall "C" shape of outer radius 5.5 nm, inner radius 3 nm, overall circumference 15 nm, and height 4.7 nm. The three-dimensional reconstruction of the protein in high salt buffer, solved to a resolution of 2.8 nm, was essentially the same in terms of overall dimensions but had a somewhat more compact architecture. These results are the first structures achieved directly for this unusual macromolecule, which plays a key role in the development of multiple sclerosis.

Animals↗

PubMed1997

Three-dimensional structure of myelin basic protein. II. Molecular modeling and considerations of predicted structures in multiple sclerosis.

A computational model of myelin basic protein (MBP) has been constructed based on the premise of a phylogenetically conserved beta-sheet backbone and on electron microscopical three-dimensional reconstructions. Many residues subject to post-translational modification (phosphorylation, methylation, or conversion of arginines to citrullines) were located in loop regions and thus accessible to modifying enzymes. The triproline segment (residues 99-101) is fully exposed on the back surface of the protein in a long crossover connection between two parallel beta-strands. The proximity of this region to the underlying beta-sheet suggests that post-translational modifications here might have potential synergistic effects on the entire structure. Post-translational modifications that lead to a reduced surface charge could result first in a weakened attachment to the myelin membrane rather than in a gross conformational change of the protein itself. Such mechanisms could be operative in demyelinating diseases such as multiple sclerosis.

Humans↗

PubMed1997

Characterization of myelin basic protein charge microheterogeneity in developing mouse brain and in the transgenic shiverer mutant.

Myelin basic protein (MBP) is a highly heterogeneous family of membrane proteins consisting of several isoforms resulting from alternative splicing and charge isomers arising from posttranslational modifications. Although well characterized in the bovine and human species, those in the mouse are not. With the availability of a number of transgenic and knockout mice, the need to understand the chemical nature of the MBPs has become very important. To isolate and characterize the MBP species in murine brain, two methods were adapted for use with the small amounts of MBP available from mice. The first was a scaled-down version of the preparative CM-52 chromatographic system commonly used to isolate MBP charge isomers; the second was an alkaline-urea slab gel technique that required five times less material than the conventional tube gel system and, from these gels, western blots were readily obtained. Murine MBP was resolved into two populations of charge isomers: the 18.5- and 14-kDa isoforms. Isolation and characterization of these charge isomers or components permitted us to assign possible posttranslational modifications to some of them. Component 1 (C-1), the most cationic isomer, had a molecular weight of 14,140.38 +/- 0.79. C-2 consisted of two 14-kDa species, 14,136.37 +/- 0.74 and 14,204.45 +/- 0.70. Two variants, 14,215.57 +/- 0.94 and 18,413.57 +/- 0.76, constituted C-3. C-4, C-5, and C-8 (the least cationic isomer) each consisted of both 14- and 18.5-kDa isoforms. During myelinogenesis, the 18.5-kDa isoform appeared first (day 4); the 14-kDa isoform appeared at day 16 and subsequently became the dominant isoform. The transgenic shiverer mutant synthesized mainly the 18.5-kDa isoform, but none of the 14-kDa isoform, similar to the 4-day-old mouse. We concluded that the transgenic shiverer was able to initiate myelinogenesis with the 18.5-kDa isoform, but was unable to complete myelinogenesis because of the absence of the 14-kDa isoform.

Animals↗

PubMed1997

Increased expression of CD44 on astrocytoma cells induced by binding myelin basic protein.

An astrocytoma cell line (HTB-14), expressing high amounts of a CD44 variant compared to other astrocytoma lines was shown to bind myelin basic protein to a greater extent than low expressing lines in a concentration-dependent manner. The CD44 variant expressed by HTB-14 cells was determined to migrate in sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular mass of 100 kDa compared to that from white matter which had a molecular mass of 80 kDa. The most cationic component of myelin basic protein (MBP), (component 1) bound more avidly than the least cationic isomer (component 8). Internalization of MBP was demonstrated by immunogold electron microscopy and was localized to the perinuclear area with some gold particles in the cytoplasm but not near the plasma membrane. Colocalization with glial fibrillary acid protein suggested an interaction between these two molecules. Binding and internalization of MBP was accompanied by an increase in CD44 as determined by quantitation of gold particles and the measurement of CD44 by sandwich enzyme-linked immunosorbent assay. The implication of these studies for the mechanism of demyelination is discussed.

Astrocytoma↗

PubMed1996

Loss of myelin basic protein cationicity in DM20 transgenic mice is dosage dependent.

Demyelination in the transgenic mice depended on the dosage of the cDNA for DM20, in which low copy numbers (two to four and 17 copies of the minigene) showed no signs of demyelination. However when transgenic mice with 17 copies were made homozygous with 34 copies of the DM20 minigene (ND3A hm.) demyelination was observed at around 12 to 16 months compared with ND4 mice having 70 copies of the transgene which had an earlier onset of demyelinating symptoms at 3 months, demonstrating a transgene dosage effect. The process by which demyelination was initiated was associated with changes in myelin basic protein. An increased abundance of less cationic MBP (C-8) isomers occurred prior to demyelination. This increase was also associated with increased activity of peptidylarginine deiminase, the enzyme which converts arginine to citrulline in proteins, thereby providing a mechanism for generating less cationic forms of MBP. These data support a dosage effect of the DM20 transgene.

Aging↗

PubMed1996

Myelin basic protein in experimental allergic encephalomyelitis is not affected at the posttranslational level: implications for demyelinating disease.

The microheterogeneity of myelin basic protein, expressed as the ratio between the least cationic (C-8) charge isomer and the most cationic (C-1), was examined in experimental allergic encephalomyelitis (EAE) cases. These included acute EAE of 2 months' duration induced with bovine proteolipid protein in complete Freund's adjuvant (CFA), chronic EAE induced with mouse spinal cord homogenate in varying doses from 0.5 to 2.0 mg in CFA, and chronic relapsing EAE of 12 months' duration induced with synthetic peptide 139-151 of the proteolipid protein sequence. The C-8/C-1 ratio was within the normal range for all groups of animals. However, the C-8/C-1 ratio was six- to sevenfold increased in a spontaneously demyelinating transgenic model, ND4, which contains 70 copies of the cDNA for DM20 (Mastronardi et al.: 1996). Since an increase in the C-8/C-1 ratio was also observed in victims of multiple sclerosis but not other neurological diseases, the ND4 model may address primary changes prior to demyelination, while the EAE model addresses the autoimmune aspects of the disease.

Animals↗

PubMed1996

Modifications of myelin basic protein in DM20 transgenic mice are similar to those in myelin basic protein from multiple sclerosis.

Transgenic mice containing different numbers of transgenes (2-70) of the myelin proteolipid protein DM20 were phenotypically normal up to 3 mo of age, after which the mice containing 70 copies of the transgene spontaneously demyelinated and died at 10-12 mo. Since we demonstrated that demyelination in multiple sclerosis involved specific chemical changes in myelin basic protein (MBP), we investigated the MBP in our transgenic line for similar changes. Both the total amount of MBP in brain and the MBP mRNA levels were unaffected at the different ages. All the isoforms (14-21 kD) of MBP were present, but the microheterogeneity (a posttranslational event) was changed resulting in a higher proportion of the less cationic components reminiscent of the changes in MBP found in multiple sclerosis. An increased amount of the citrullinated form of MBP was found by Western blot analysis. Immunogold labeling of cryosections of brain revealed a greater density of particles with the anticitrulline antibody at 10 mo and that the levels of peptidylarginine deiminase (which deiminates protein-bound arginine to citrulline) were increased. This stable transgenic line represents a useful animal model for the human disease multiple sclerosis.

Animals↗

PubMed1996

Acute multiple sclerosis (Marburg type) is associated with developmentally immature myelin basic protein.

We have studied a case of acute, fulminating multiple sclerosis (MS) (Marburg type) at the pathological and biochemical levels. Postmortem examination of the brain revealed extensive areas of gross rarefaction in the hemispheric white matter. Histologically, well-demarcated areas of demyelination with a large influx of macrophages and a subtle perivascular infiltration of lymphocytes were seen with relative preservation of the axis cylinders. Myelin basic protein (MBP) was isolated and purified [correction of purifed] from noninvolved white matter. It was slightly larger in molecular weight than MBP from normal brain or from chronic MS brain. The increase in mass was accounted for, in part, by the deimination of 18 of 19 arginyl residues to citrulline, making the patient's MBP much less cationic than MBP from normal white matter. When expressed as the ratio of least cationic form of MBP to the most cationic (C-8/C-1), the normal ratio was 0.82, chronic MS 2.5, and the patient in this study 6.7. Because the ratio of 6.7 was similar to 7.5 found for a 15-month-old infant, MBP was considered to be of the immature form. The data are consistent with a genetic factor influencing the charge microheterogeneity of MBP. The resulting less cationic MBP cannot carry out its normal function of compacting multilayers.

Adult↗

PubMed1996

Identification of a mitogen-activated protein kinase site in human myelin basic protein in situ.

Ultrastructural localization of a specific phosphorylated isomer of myelin basic protein (MBP) has been achieved with a monoclonal antibody specific for human MBP sequence, 89-105, in which Thr98 was phosphorylated. Cryosections of human brain white matter revealed that gold particles were found localized almost exclusively to the major dense line demonstrating that threonine 98 in the sequence Thr-Pro-Arg-Thr-Pro-Pro-Pro, a mitogen-activated protein kinase-specific site, was phosphorylated in vivo. In two cases of multiple sclerosis, the density of gold particles in myelin was reduced by about 30%, in one case by 42%, and by 80% in a fourth case. However, gold labelling was seen in areas of demyelination suggesting that the phosphorylated threonyl peptide was protected from degradation.

Amino Acid Sequence↗

PubMed1995

The isolation and characterization of four myelin basic proteins from the unbound fraction during CM52 chromatography.

The unbound fraction from CM52 columns was used as the source of at least four additional myelin basic protein (MBP) molecules. From this fraction we routinely obtained two major fractions called C8-A and C8-B. The C8-A and C8-B fractions were further purified on HPLC. Each contained two proteins in the 17- to 18-kDa range which we called C8-A(H) (higher M(r)), C8-A(L) (lower M(r)), C8-B(H), and C8-B(L). The citrulline values (calculated as citrulline plus ornithine) were high in three of the four proteins, which was accompanied by a compensatory decrease in the arginine values. The compositions clearly identified these four proteins with the citrullinated form of MBP. Western blot analysis showed that both H and L forms reacted with anti MBP antibodies. Partial sequence analysis after cyanogen bromide cleavage, showed that the sequences of both proteins in the C8-B fraction (C8-B(H) and C8-B(L)) were identical to the 18.5-kDa isoform of MBP. Mass spectrometry by electrospray ionization of the C8-B(H) and C8-B(L) provided us with accurate masses of 18,558.08 +/- 8.13 and 17,266.63 +/- 2.24, respectively. We concluded that the H and L proteins from the C8-B fractions were MBPs. Although similar detailed analyses of the C8-A(H) and C8-A(L) have not been done they are also considered to be MBP on the basis of the immunoreactivity with anti MBP antibodies. The origins of these proteins is not known at this time and their functional significance is obscure. The possibility that they are found in early forms of myelin, as components of transitional membranes between oligodendrocytes and myelin or are involved in remyelination, cannot be discounted.

Amino Acid Sequence↗