Phosphorylation of myelin basic protein by glycogen phosphorylase kinase

The ability of homogeneous glycogen phosphorylase kinase (Phk) from rabbit skeletal muscle to phosphorylate bovine brain myelin basic protein (MBP) was investigated. Phk could incorporate a maximum of 1.9 mol phosphate/mol MBP. The apparent Km and Vmax for Phk phosphorylation of MBP were 27 microM and 90 nmol/min per mg enzyme, respectively. Properties of MBP phosphorylation by Phk are similar to those of phosphorylase as a substrate. Only serine residues of MBP are phosphorylated by Phk. Phosphorylation sites of MBP by Phk are not identical to those by cAMP-dependent protein kinases.     

Phosphorylation on basic amino acids in myelin basic protein.

Isolated rat brain myelin when incubated with γ³²P labelled ATP yields proteins bearing acid labile, base stable phosphoryl groups. Phosphorylated myelin basic protein can be isolated and degraded with trypsin and pronase to yield principally phosphoarginine and phosphohistidine. Only a very small amount of phosphorerine survives the base treatment used in the isolation procedure.     

Phosphorylation of atrial natriuretic peptides by cyclic AMP-dependent protein kinase

Atrial natriuretic peptides refer to a family of related peptides secreted by atria that appear to have an important role in the control of blood pressure. The structure of these peptides shows the amino acid sequence Arg101-Arg102-Ser103-Ser104, which is a typical recognition sequence (Arg-Arg-X-Ser) for phosphorylation by cyclic AMP-dependent protein kinase. With this background, we tested two synthetic atrial natriuretic peptides (Arg101-Tyr126 and Gly96-Tyr126) as substrates for in vitro phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase. The tested atrial natriuretic peptides were found to be substrates for the reaction. Sequence studies demonstrated that the site of phosphorylation was located, as expected, at Ser104. Kinetic studies demonstrate that both atrial natriuretic peptides are excellent substrates for cyclic AMP-dependent protein kinase. In particular, the longer peptide Gly96-Tyr126 exhibited an apparent Km value of about 0.5 microM, to our knowledge the lowest reported Km for a cyclic AMP-dependent protein kinase substrate. Preliminary studies to measure the biological activity of the in vitro phosphorylated atrial peptides indicate that these compounds are more effective than the corresponding dephospho forms in stimulating Na/K/Cl cotransport in cultured vascular smooth muscle cells.     

Phosphorylation sites of bovine brain myelin basic protein phosphorylated with Ca2+-calmodulin-dependent protein kinase from rat brain

The phosphorylation sites of myelin basic protein from bovine brain were determined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala- and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue.     

Specificity of T lymphocyte lines for peptides of myelin basic protein

T lymphocyte lines specific for myelin basic protein (BP) can mediate experimental autoimmune encephalomyelitis (EAE), or can protect against the active induction of the disease. To investigate the antigenic fine specificity of guinea pig (GP) BP-specific T cell lines raised from different rat strains, and to determine whether functionally different T lymphocyte lines and clones recognized the same or different regions of the BP molecule, the proliferation responses of line cells were assessed after stimulation with purified peptides of GP-BP. Lewis rat T cell lines and clones selected for responses to whole GP-BP responded selectively to the 68-88 amino acid sequence of GP-BP, but not to the 1-37, 43-67, or 89-169 sequences. The region of GP-BP recognized by Lewis T cells was additionally defined to include the 75-80 amino acid sequence, because a T cell clone responded equally to GP and rat BP which differed by only one amino acid at position 79, but did not respond to human or bovine BP, which had a Gly-His insertion in this region. T lymphocyte lines derived from the F344 and PVG (Weizmann) rat strains shared the same selective response to peptide 68-88, but lines from BN rats responded to an epitope(s) outside of the 68-88 sequence. The functional capacity of the various T cell lines to mediate experimental autoimmune encephalomyelitis (EAE) or to induce resistance against EAE was independent of their specificity for the different GP-BP peptides; lines specific for epitope(s) within or excluded from the 68-88 sequence could be encephalitogenic depending on their strain of origin, and various lines specific for the 68-88 peptide could induce both disease and protection, disease only, or neither activity.     

Cell-mediated immunoreactivity of tumor-bearing mice with myelin basic protein and related peptides

Spleen cells (SC) from tumor-bearing mice and mice immunized with porcine myelin basic protein (MBP) reacted in vitro in E-rosette augmentation assays with MBP and certain of its constituent peptides. Peptides 1-115, 43-169, 64-83, 113-121, and 153-161 reacted significantly with both types of SC, while peptide 1-19 reacted only with SC from MBP-immunized mice. The phenomenon of specific inhibition of peptide reactivity by a moderate excess of a related protein was used to identify peptides as accessible epitopes of that protein. Peptide 113-121 was specifically inhibited by excess MBP when reacted with both types of SC, whereas peptide 64-83 was inhibited by excess MBP only when reacted with SC from MBP-immunized mice. These reactions suggest that the immunizing antigen in tumor-bearing mice is related to MBP but differs in epitopes associated with peptides 1-19 and 64-83.     

Studies on the phosphorylation of myelin basic protein by protein kinase C and adenosine 3':5'-monophosphate-dependent protein kinase

The substrate specificity of protein kinase C was studied and compared with that of cyclic AMP-dependent protein kinase (protein kinase A) by using bovine brain myelin basic protein as a model substrate. This basic protein was phosphorylated at multiple sites by both of these protein kinases. In this analysis, the basic protein was thoroughly phosphorylated in vitro with [gamma-32P]ATP and each protein kinase, and then digested with trypsin. The resulting radioactive phosphopeptides were isolated by gel filtration followed by high performance liquid chromatography on a reverse-phase column. Subsequent amino acid analysis and/or sequential Edman degradation of the purified phosphopeptides, together with the known primary sequence of this protein, revealed that Ser-46 and Ser-151 were specifically phosphorylated by protein kinase C, whereas Thr-34 and Ser-115 were phosphorylated preferentially by protein kinase A. Both kinases reacted with Ser-8, Ser-11, Ser-55, Ser-110, Ser-132, and Ser-161 at various reaction velocities. Contrary to protein kinase A, protein kinase C appears to react preferentially with seryl residues that are located at the amino-terminal side close to lysine or arginine. The seryl residues that are phosphorylated commonly by these two protein kinases have basic amino acids at both the amino- and carboxyl-terminal sides. These results provide some clues to understanding the rationale that these kinases may show different but sometimes similar functions depending on the structure of target phosphate acceptor proteins.     

Preparation and properties of monoclonal antibodies to myelin basic protein and its peptides

Techniques are described which have enabled the production and characterisation of monoclonal antibodies to myelin basic protein. These are shown by enzyme immunoassay to react with six different epitopes. Two of these are to peptide 82–91, a region claimed to be present in the spinal fluid of patients with demyelinating disease. One of these, an IgG_2a, is shown to react only with peptides in which the 91–92 phe-phe bond has broken. The other, an IgM, also reacts with whole myelin basic protein. The IgG_2a antibody is shown to have an affinity suitable for use in immunoassay of peptide 82–91. The enzyme immunoassay procedures described help to minimise the work load involved in the preparation and characterisation of monoclonal antibodies to this protein.     

Enzymic O-glycosylation of synthetic peptides from sequences in basic myelin protein.

Nine synthetic peptides containing sequences in the region of a threonine residue at position 98 of bovine basic myelin protein were prepared by the Merrifield solid-phase method and tested for their ability to be glycosylated with [14C]uridinediphospho-N-acetylgalactosamine and a crude detergent-solubilized preparation of uridinediphospho-N-acetylgalactosamine:mucin polypeptide N-acetylgalactosaminyltransferase obtained from porcine submaxillary glands. The tetrapeptide Thr-Pro-Pro-Pro and all larger peptides containing this sequence were glycosylated. The glycosylation was greater for peptides containing residues N-terminal to the Thr-Pro-Pro-Pro. Under the conditions used, the peptide Val-Thr-Pro-Arg-Thr-Pro-Pro-Pro was glycoslyated twice as much as bovine basic myelin protein. Thr-Pro and Thr-Pro-Pro, as well as 10 other synthetic peptides which did not contain the Thr-Pro-Pro-Pro sequence, were not glycosylated. Treatment of the glycopeptide of Phe-Lys-Asn-Leu-Val-Thr-Pro-Arg-Thr-Pro-Pro-Pro-Ser with an alpha-N-acetylgalactosaminidase released N-acetylgalactosamine from the peptide, indicating that the hexosamine was covalently bonded to the peptide in an alpha linkage.     

An idiotype shared by monoclonal antibodies to different peptides of human myelin basic protein.

A mAb of the IgG1/kappa isotype was raised against human myelin basic protein (MBP) peptide acetyl 1-9. This mAb, termed F23, reacted with human MBP and human MBP peptides acetyl 1-9, 1-14, and 1-44, but not with MBP peptides 10-19, 80-89, or 45-89. According to the guidelines of the molecular recognition theory, a complementary peptide to human MBP peptide 1-9 was synthesized and used to raise murine mAb with anti-Id activity. Two mAb anti-Id, F25F7 and F25C8, both of the IgM/kappa isotype, were selected for further study. These anti-Id reacted with F23, the mAb for which they were selected, and also reacted with another mAb, which was of the IgG1/kappa isotype and was raised to human MBP peptide 80-89. There was no reaction with another control mAb of the IgG1/kappa isotype or murine myeloma IgG1. By immunoblotting techniques, it was demonstrated that the Id on each of the mAbs to MBP peptides was located on the kappa L chain but also could be recognized in nonreduced IgG. The cross-reactive anti-Id suppressed antibody secretion of Id-producing hybridoma cells in an Id-specific manner, and kinetic studies suggest an intracellular mechanism for the suppression. These cross-reactive Id among antibodies to different MBP peptides imply that the same V region genes of kappa L chains are involved in the selection of antibodies to an autoantigen, like MBP, and may play a role in the modulation of immune responses against MBP in certain inflammatory demyelinating diseases.     

Spontaneous vesicularization of myelin lipids is counteracted by myelin basic protein

Hand-vortexed dispersions of several lipids (cerebrosides, sulfatides, PC, PE, PS and sphingomyelin), mixed in the ratios found for these categories of lipids in myelin, exhibit 31P-NMR spectra which have contributions from both isotropic and lamellar resonances. Investigation of this system by freeze-fracture electron microscopy and X-ray diffraction revealed that this lipid mixture has spontaneously formed small unilamellar vesicles (SUVs) (diam. approximately 400 A) and large highly convoluted unilamellar vesicles (LUVs) (diam. approximately 1000 A), the latter possibly resulting from aggregation and fusion of the SUV structures. This vesicularization of the myelin lipids was reversed by the addition of myelin basic protein: only large multilamellar aggregates were formed in the presence of protein, as shown by all three experimental methods. Although no rigorous physical-chemical explanation for these phenomena is yet available, the possibility is suggested that the high concentration of cerebrosides and/or phosphatidylethanolamine in this particular mixture of myelin lipids play pivotal roles in the formation of these unusual vesicles. Spontaneous vesicularization of myelin lipids is discussed as a potential pathway toward destabilization of the myelin sheath.     

Recognition and degradation of myelin basic protein peptides by serum autoantibodies: novel biomarker for multiple sclerosis

The pathologic role of autoantibodies in autoimmune disease is widely accepted. Recently, we reported that anti-myelin basic protein (MBP) serum Abs from multiple sclerosis (MS) patients exhibit proteolytic activity toward the autoantigen. The aim of this study is to determine MBP epitopes specific for the autoantibodies in MS and compare these data with those from other neuronal disorders (OND), leading to the generation of new diagnostic and prognostic criteria. We constructed a MBP-derived recombinant "epitope library" covering the entire molecule. We used ELISA and PAGE/surface-enhanced laser desorption/ionization mass spectroscopy assays to define the epitope binding/cleaving activities of autoantibodies isolated from the sera of 26 MS patients, 22 OND patients, and 11 healthy individuals. The levels of autoantibodies to MBP fragments 48-70 and 85-170 as well as to whole MBP and myelin oligodendrocyte glycoprotein molecules were significantly higher in the sera of MS patients than in those of healthy donors. In contrast, selective reactivity to the two MBP fragments 43-68 and 146-170 distinguished the OND and MS patients. Patients with MS (77% of progressive and 85% of relapsing-remitting) but only 9% of patients with OND and no healthy donors were positive for catalysis, showing pronounced epitope specificity to the encephalitogenic MBP peptide 81-103. This peptide retained its substrate properties when flanked with two fluorescent proteins, providing a novel fluorescent resonance energy transfer approach for MS studies. Thus, anti-MBP autoantibody-mediated, epitope-specific binding and cleavage may be regarded as a specific characteristic of MS compared with OND and healthy donors and may serve as an additional biomarker of disease progression.     

D-aspartic acid in purified myelin and myelin basic protein

The presence of the biologically uncommon D-isomer of aspartic acid in the white matter of human brains has been reported previously from this laboratory (1). We now report that the level of D-aspartate in human brains is higher in purified myelin than in white matter and is even higher in the myelin basic protein fraction. There also appears to be a difference in the level of D-aspartate found in human brain as compared to bovine brain, possibly a species or age-related difference.     

Phosphorylation of synthetic peptides by (32P)ATP and cyclic GMP-stimulated protein kinase.

Cyclic GMP-stimulated protein kinase from pig lung has been shown to phosphorylate synthetic peptides. The rate of phosphorylation was about one order of magnitude higher than that for mixed histones at a comparable concentration, $\text{i.e.}$ 0.1 mM. The peptides represented sites, phosphorylatable by cyclic AMP-stimulated protein kinase, in pyruvate kinase type L from rat liver, calf thymus histone H2B and the α-subunit of rabbit muscle phosphorylase b kinase. The shortest pyruvate kinase peptide that could be phosphorylated at a significant rate by cyclic GMP-stimulated protein kinase was Arg-Arg-Ala-Ser-Val-Ala, which is one amino acid residue longer than the minimal substrate of cyclic AMP-stimulated protein kinase. The apparent K_m was 0.3 mM which is about 10 times higher than that with cyclic AMP-stimulated protein kinase. The K_m was only slightly decreased upon successive extension of the peptide in the N-terminal direction to Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala. Modification of the sequence showed the importance of two adjacent arginyl residues, and substitution of arginine for the C-terminal alanine abolished the measurable activity. Thus, it has been demonstrated that there are both differences and similarities in substrate specificity of the two protein kinases.     

Hydrolysis of myelin basic protein in human myelin by terminal complement complexes

The participation of terminal complement complexes (TCC) in demyelination has been shown in rodent cerebellar cultures. Since TCC modulates activities of various membrane-associated enzymes and increases the level of cellular Ca2+ we investigated whether TCC could activate Ca2+-dependent neutral proteases in myelin that would lead to hydrolysis of myelin basic protein (BP). Addition of antibody and C7-deficient serum plus C7 to sealed myelin vesicles of two to six bilayers caused significant BP hydrolysis compared to the hydrolysis caused by antibody and C7-deficient serum. Significant hydrolysis occurred at the stage of C5b6,7 assembly, which increased in magnitude at the C5b6-8 stage. C5b6-9 formation did not enhance the effect of C5b6-8. BP hydrolysis by C5b6,7 did not require Ca2+ whereas the effect of C5b6-8/C5b6-9 was, in part, Ca2+-dependent. We postulated that TCC formation in myelin membranes causes activation of myelin-associated neutral proteases with subsequent hydrolysis of BP as a consequence of complement peptide insertion and channel formation. Such processes may alter the structure of myelin and augment the action of other inflammatory cells and their products in demyelinating diseases that could ultimately lead to the loss of myelin.     

Ganglioside-modulated protein phosphorylation. Partial purification and characterization of a ganglioside-stimulated protein kinase in brain

Gangliosides have profound modulatory effects on protein phosphorylation in brain. A protein kinase activated directly by gangliosides has been partially purified from the particulate fractions of guinea pig brain through extraction with nonionic detergent, ion-exchange chromatography, hydrophobic chromatography, and gel filtration. This novel ganglioside-stimulated protein kinase is distinct from cAMP-dependent, Ca2+/calmodulin-dependent, and Ca2+/phospholipid-dependent protein kinases. The partially purified kinase preparation could undergo ganglioside-stimulated autophosphorylation of a major phosphoprotein with Mr corresponding to 68,000. It also could phosphorylate exogenous substrates such as the synthetic peptide Leu-Arg-Arg-Ala Ser-Leu-Gly. The requirement of gangliosides for the activation of kinase activity is dose-dependent and specific. Among the various gangliosides tested, GT1b and GD1a were found to be the most potent activators, whereas GD1b and GM1 were slightly less effective. The activation process is rapid and does not require the presence of Ca2+, suggesting that the stimulatory effect of gangliosides is not mediated through limited proteolysis or Ca2+-glycolipid complexes. Although the exact physiological significance of the ganglioside-stimulated protein kinase is not known at present, it is possible that certain functions related to gangliosides in the nervous system are mediated through the activation of this novel enzyme.     

Phosphorylation of caldesmon by protein kinase C

Protein kinase C catalyzes phosphorylation of caldesmon, an F-actin binding protein of smooth muscle, in the presence of Ca2+ and phospholipid. Protein kinase C incorporates about 8 mol of phosphate/mol of chicken gizzard caldesmon. When calmodulin was added in the medium, there was an inhibition of phosphorylation. The fully phosphorylated, but not unphosphorylated, caldesmon inhibited myosin light chain kinase activity. The possibility that protein kinase C plays some role in smooth muscle contractile system through caldesmon, warrants further attention.     

Phosphorylation and functional modification of calmodulin-dependent protein kinase IV by cAMP-dependent protein kinase

Calmodulin-dependent protein kinase IV (CaM-kinase IV), a neuronal calmodulin-dependent multifunctional protein kinase, undergoes autophosphorylation in response to Ca2+ and calmodulin, resulting in activation of the enzyme (Frangakis et al. (1991) J. Biol. Chem. 266, 11309-11316). In contrast, the enzyme was phosphorylated by cAMP-dependent protein kinase, leading to a decrease in the enzyme activity. Thus, the results suggest differential regulation of CaM-kinase IV by two representative second messengers, Ca2+ and cAMP.