ADP-ribosyltransferase activity in myelin membranes isolated from human brain.

An ADP-ribosyltransferase has been identified in compact myelin and in several white matter fractions which contain less compact myelin, fractionated on the basis of increasing protein/lipid ratios. One fraction the P3A contained the greatest activity although the activity in compact myelin was only slightly less. The ADP-ribosyltransferase activity of solubilized myelin was stimulated by increasing amounts of GTP gamma S and was specific for the beta-isomer of NAD. Although ADP-ribosylation was demonstrated with the heterotrimeric G proteins in the 40-50 kDa range, the substrate for the ADP-ribosyltransferase in the 20 kDa range was identified as MBP. ADP-ribosyltransferase; myelin basic protein; signal transduction.

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.

ADP-ribosylation of human myelin basic protein.

When isolated myelin membranes were ADP-ribosylated by [32P]NAD+ either in the absence of toxin (by the membrane ADP-ribosyltransferase) or in the presence of cholera toxin, the same proteins were ADP-ribosylated in both cases and myelin basic protein (MBP) was the major radioactive product. Therefore, cholera toxin was considered a good model for ADP-ribosylation of myelin proteins. Although purified human MBP migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 20 kDa, the microheterogeneity that is masked under these conditions can be clearly demonstrated on alkaline-urea gels at pH 10.6. At this pH, MBP is resolved into several components that differ one from the other by a single charge (charge isomers). These charge isomers can be resolved on CM52 columns at pH 10.6, and several can be ADP-ribosylated. Component 1 (C-1), the most cationic charge isomer, incorporated 1.79 mol of ADP-ribose/mol of protein. C-2 and C-3 (which differ from C-1 by the loss of one and two positive charges, respectively) incorporated slightly less at 1.67 and 1.63 mol of ADP-ribose/mol of protein, respectively, whereas C-8, the least cationic, incorporated less than 0.11 mol/mol of protein. In the presence of neutral hydroxylamine, the ADP-ribosyl bond was shown to have a half-life of about 80 min, suggesting an N-glycosidic linkage between ADP-ribose and an arginyl residue of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Myelin in multiple sclerosis is developmentally immature.

The etiology of multiple sclerosis (MS) is considered to involve genetic, environmental, infective, and immunological factors which affect the integrity of a normally assembled myelin sheath, either directly or indirectly resulting in demyelination. In a correlative study involving protein chemical, mass spectrometric, and electron microscopic techniques we have determined that myelin obtained from victims of MS is arrested at the level of the first growth spurt (within the first 6 yr of life) and is therefore developmentally immature. The data supporting this conclusion include (a) the pattern of microheterogeneity of myelin basic protein (MBP); (b) the NH2-terminal acylation of the least cationic component of MBP ("C-8"); (c) the phase transition temperature (Tc) of myelin isolated from victims of MS correlated with the increased proportion of the least cationic component of MBP; and (d) immunogold electron microscopy using an antibody specific for "C-8" showed that the distribution of gold particles in a 2-yr-old infant was similar to the distribution found in a victim of MS. We postulate that this developmentally immature myelin is more susceptible to degradation by one or a combination of factors mentioned above, providing the initial antigenic material to the immune system.

Acylation of myelin basic protein peptide 1-21 with alkyl carboxylates 2-10 carbons long affects secondary structure and posttranslational modification.

A peptide consisting of the first 21 residues of human myelin basic protein (MBP) was synthesized. The N-terminal alanine of portions was blocked in separate experiments with alkyl carboxylates varying in size from 2 to 10 carbon atoms. The effects of these different alkyl carboxylates at the N-terminus on the secondary structure was studied by circular dichroism (250-190 nm). In water, the spectra of the unblocked peptide suggested unordered structure with large negative ellipticities at 198 nm. Addition of an acetyl group altered the magnitude of [theta]198 from -21856 +/- 2319 to -11095 +/- 1000 deg cm2 dmol-1, suggesting a significant increase in ordered structure. When peptides with longer alkyl carboxylates, acylated at the N-termini, were studied, the magnitude of theta 198 approached that of the unblocked peptide but greater negative ellipticities were observed for the C8 and C10 alkyl carboxylates. The theta 222 values were generally low (-1803 +/- 463) but increased with increasing length of the alkyl carboxylate to about -3200 deg cm2 dmol-1, suggesting that little alpha-helical structure was present. The spectra were also taken in lipid-mimetic solvents, including 2-propanol, methanol, and lysophosphatidylglycerol (lysoPG). In general the theta 198 and theta 222 values were suggestive of increased structure in these environments compared to water. In 90% 2-propanol the theta 198 of the unblocked peptide did not change when an acetyl group was added to the N-terminus (9088 compared to 8477 deg cm2 dmol-1). Addition of longer alkyl carboxylates correlated with larger, negative ellipticities.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of basic proteins from goldfish myelin.

Myelin basic protein (MBP) from common goldfish (Carassius auratus) myelin was extracted with dilute mineral acid. Immunological cross-reactivity of the goldfish MBP, with polyclonal antisera raised against bovine MBP, suggested that the goldfish protein has epitopes for these antibodies. It also reacted with a monoclonal antibody specific for a seven amino acid epitope (130-137) conserved in the MBP of most mammalian species. To characterize the charge heterogeneity of this protein, we iodinated the protein with 125I and chromatographed it on a carboxymethyl cellulose-52 column together with a nonlabeled acid soluble fraction prepared from human white matter as a carrier protein. All of the goldfish protein was recovered in the unbound fraction, demonstrating that it was less cationic than the carrier protein (human MBP). We have also examined the urea alkaline gel profile of the goldfish MBP together with the human C-1, C-2, C-3, C-4, and C-8 components. The results from these experiments indicated that this MBP extracted from goldfish brain myelin lacked the microheterogeneity that is associated with MBPs from higher vertebrates. The MBPs from goldfish myelin were separated into their isoforms by reversed-phase HPLC. Amino acid compositions were determined for both the 17- and 14-kDa goldfish proteins. Amino acid analysis revealed similarities with the compositions of other MBPs; however, the serine content in both the 17- and 14-kDa proteins was higher than that of the human C-1, the mouse C-1 protein, and the shark proteins. The HPLC-purified 14-kDa goldfish protein was chemically cleaved with CNBr for partial sequence analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

ADP-ribosylation of myelin basic proteins isolated from normal and mutant mouse brains.

Myelin basic proteins (MBPs) have been shown to be ADP-ribosylated in-vitro by cholera toxin in the presence of NAD. Since acid-soluble extracts of brain contain other proteins in the 14-32 kD range (such as histones) in addition to MBP's, the identification of the ADP-ribosylated proteins was uncertain. To determine that only the MBP's were ADP-ribosylated, the acid-soluble fractions from several murine mutants were prepared. Thus, in the Shiverer mutants, none of the proteins in the 14-32 kD range were ADP-ribosylated; in the Myelin-deficient mutant, some of ADP-ribosylation was observed but none in the Jimpy mutant, consistent with our demonstration that the least cationic isomer cannot be ADP-ribosylated.

Guanine nucleotides stimulate hydrolysis of phosphatidyl inositol bis phosphate in human myelin membranes.

Phosphodiesterase activity was stimulated in myelin membranes in the presence of guanine nucleotide analogues. This activity was reduced in myelin membranes which had been adenosine diphosphate ribosylated in the presence of cholera toxin which ADP-ribosylated three proteins of Mr 46,000, 43,000 and 18,500. Aluminum fluoride treatment of myelin had the same stimulatory effects on phosphodiesterase activity as did the guanine nucleotides.