D. Denise Wood

Increased Citrullination of Histone H3 in Multiple Sclerosis Brain and Animal Models of Demyelination: A Role for Tumor Necrosis Factor-Induced Peptidylarginine Deiminase 4 Translocation

Modification of arginine residues by citrullination is catalyzed by peptidylarginine deiminases (PADs), of which five are known, generating irreversible protein structural modifications. We have shown previously that enhanced citrullination of myelin basic protein contributed to destabilization of the myelin membrane in the CNS of multiple sclerosis (MS) patients. We now report increased citrullination of nucleosomal histones by PAD4 in normal-appearing white matter (NAWM) of MS patients and in animal models of demyelination. Histone citrullination was attributable to increased levels and activity of nuclear PAD4. PAD4 translocation into the nucleus was attributable to elevated tumor necrosis factor-α (TNF-α) protein. The elevated TNF-α in MS NAWM was not associated with CD3+ or CD8+ lymphocytes, nor was it associated with CD68+ microglia/macrophages. GFAP, a measure of astrocytosis, was the only cytological marker that was consistently elevated in the MS NAWM, suggesting that TNF-α may have been derived from astrocytes. In cell cultures of mouse and human oligodendroglial cell lines, PAD4 was predominantly cytosolic but TNF-α treatment induced its nuclear translocation. To address the involvement of TNF-α in targeting PAD4 to the nucleus, we found that transgenic mice overexpressing TNF-α also had increased levels of citrullinated histones and elevated nuclear PAD4 before demyelination. In conclusion, high citrullination of histones consequent to PAD4 nuclear translocation is part of the process that leads to irreversible changes in oligodendrocytes and may contribute to apoptosis of oligodendrocytes in MS.

Peptidyl argininedeiminase 2 CpG island in multiple sclerosis white matter is hypomethylated

In previous studies, we documented increased citrullinated myelin basic protein (MBP) was present in MBP isolated from multiple sclerosis (MS) normal appearing white matter (NAWM). This increase was due to the myelin enzyme peptidyl argininedeiminase 2 (PAD2). In this study, we show that methylation of cytosine of the PAD2 promoter in DNA from MS NAWM was decreased to one‐third of the level of that in DNA from normal white matter. The PAD2 promoter in DNA from thymus obtained from the same MS patients and white matter DNA from Alzheimers, Huntingtons, and Parkinsons was not hypomethylated. DNA demethylase activity in supernatants prepared from NAWM of MS patients was 2‐fold higher than the DNA demethylase from normal, Alzheimers, Huntingtons and Parkinsons disease white matter. The amount of PAD2 enzyme and citrullinated MBP was increased in MS NAWM. The decreased methylation of cytosines in the PAD2 promoter may explain the increased synthesis of PAD2 protein that is responsible for the increased amount of citrullinated MBP, which in turn results in loss of myelin stability in MS brain.

Participation of Acetylpseudouridine in the Synthesis of a Peptide Bond in Vitro

Uracil, uridine, and pseudouridine were acetylated by refluxing in acetic anhydride, and the products of acetylation were incubated with a synthetic peptide(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) that corresponds to the N-terminal 21 amino acid residues of human myelin basic protein. Peptide bond formation, at the N terminus in peptide 1-21, was obtained with acetyluracil and acetylpseudouridine, but not with acetyluridine. Transfer of an acetyl group from acetyluracil and acetylpseudouridine depended on acetylation in the N-heterocycle. X-ray crystallographic analysis definitively established N-1 as the site of acetylation in acetyluracil. Mass spectrometry of the acetylation products showed that one acetyl group was transferred to peptide 1-21, in water, by either acetyluracil or acetylpseudouridine at pH 6. Release of the acetyl group by acylaminopeptidase regenerated peptide 1-21 (mass spectrometry) and automated sequencing (for five cycles) of the regenerated (deacetylated) peptide demonstrated that the N terminus was intact. The findings are discussed in the context of a possible role for pseudouridine in ribosome-catalyzed peptidyltransfer, with particular reference being made to similarities between the possible mechanism of acyl transfer by acetyluracil/pseudouridine and the mechanism of carboxyl transfer by carboxylbiotin in acetyl CoA carboxylase. The possibility that idiosyncratic appearance of a wide range of acyl substituents in myelin basic protein could be related to a peculiar involvement of ribosomal pseudouridine is mentioned.

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‐Cit6), 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‐Cit18). Whereas C1 caused aggregation of lipid vesicles, resulting in an increase in absorbance due to light scattering, MBP‐Cit18 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‐Cit6 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‐Cit6 was detected by size exclusion chromatography or electron microscopy. The membrane fragmentation caused by MBP‐Cit18 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‐Cit18 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‐Cit6 in normal immature myelin, where it is the predominant charge isomer. J. Neurosci. Res. 57:529–535, 1999.

An immunochemical comparison of human myelin basic protein and its modified, citrullinated form, C8

An immunochemical analysis was conducted to compare the C1 isomer of human myelin basic protein (MBP) with the newly described and less cationic, citrullinated isomer of MBP referred to as C8. Ten polyclonal antisera directed at multiple epitopes or restricted regions of MBP were used in radioimmunoassays to examine MBP-C1 and MBP-C8. Antisera reactive with MBP peptide 1-14 clearly distinguished MBP-C1 from MBP-C8. Antisera to human MBP peptides 10-19 and 90-170, but not to MBP peptide 69-89, showed modest differences between MBP-C1 and MBP-C8. The MBP-C8s from multiple sclerosis (MS) and non-MS brain reacted essentially the same. With murine monoclonal antibodies and enzyme-linked immunosorbent assay (ELISA), differences between MBP-C8 and other isomers were shown for anti-MBP 10-19 but not for anti-MBP 1-9 or anti-MBP 80-89. These findings imply differences in sequence or conformation in the structure of MBP-C7 compared to MBP-C1, most notably near the amino terminus.

Expression and properties of the recombinant murine Golli-myelin basic protein isoform J37.

A recombinant form of the murine Golli‐myelin basic protein (MBP) isoform J37 (rmJ37) has been expressed in Escherichia coli and isolated to 95% purity via metal chelation and ion exchange chromatography. The protein did not aggregate lipid vesicles containing acidic phospholipids, unlike the 18.5 kDa isoform of MBP. This result is consistent with J37 having a functional role prior to the assembly of compact myelin. Circular dichroic spectroscopy showed that rmJ37 had a large proportion of random coil in aqueous solution but gained α‐helix and β‐sheet in the presence of monosialoganglioside GM1 and PI(4)P. Thus, like “classic” MBP, J37 is intrinsically unstructured, and its conformation depends on its environment and bound ligands. Analyses of the amino acid sequence of rmJ37 predicted an N‐terminal calmodulin (CaM)‐binding site. It was determined via a gel‐shift assay and fluorescence spectroscopy that rmJ37 and CaM interacted in a 1:1 ratio in a Ca2+‐dependent manner. However, the interaction was weak compared with 18.5 kDa MBP.

Immunological analysis of the amino terminal and the C8 isomer of human myelin basic protein

The citrullination and N-terminus acylation of myelin basic protein (MBP) increases the heterogeneity among the MBP isoforms. The present study was undertaken to further characterize the immune response to the citrullinated form (C8) of MBP as well as to the variably acylated N-terminus of MBP. Six well-characterized murine monoclonal antibodies (mAbs) to human MBP-C8 or MBP peptides (four mAbs to MBP acetyl 1-9, one mAb to MBP 10-19 and one mAb to MBP 80-89), one murine T cell line (PL11) to human MBP peptide acetyl 1-9 and one Lewis rat T cell line (RT-1) to guinea pig (GP) MBP peptide 68-88 were used to assess reactivity with MBP-C1, MBP-C8, and MBP peptides including a series of MBP peptide 1-21 containing 0, 2, 4, 6, 8 or 10 carbon fatty acids. Enzyme-linked immunosorbent assay (ELISA) results revealed that all of the mAbs reacted with human MBP-C1 and MBP-C8 except anti-MBP 10-19 and anti-MBP-C8. The former reacted only with MBP-C1 and the latter only with MBP-C8. The presence and length of acylation of MBP peptide 1-21 modified reactivity. Three mAbs to MBP acetyl 1-9 reacted only with acetyl 1-21, and one mAb anti-MBP actyl 1-9 reacted with all of MBP 1-21 preparations whether acylated or not. mAb anti-MBP-C8 generally reacted better with acylated MBP 1-21 having longer fatty acids. The PL11 T cell line strongly proliferated to human MBP-C1, MBP-C8 and MBP acetyl 1-9, responded, but less well, to MBP 1-21 with longer fatty acids and failed to respond to nonacylated MBP peptide 1-21. The RT-1 cell line responded strongly to GP MBP peptide 68-88, marginally to MBP-C8 and failed to respond to MBP-C1 or any of the other MBP peptides. Specific immune responses to different MBP charge isomers and different N-terminal acylating groups of MBP may play a role in immune-mediated demyelination.