Soluble and bound acid protease activity of myelin from bovine cerebral white matter and spinal cord

Isolated myelin of bovine spinal cord was found to degrade exogenous myelin basic protein (MBP) at pH 4.4. Electrophoretic peptide patterns were consistent with limited proteolysis of MBP. Some of the proteolytic activity was soluble at increased ionic strength, some remained bound, withstanding extraction at 37°C for up to 12 hr. While being measurable with exogenous MBP, bound protease degraded neither bound MBP nor any other major intrinsic myelin protein. Both soluble and bound protease activity was completely inhibited by pepstatin A. The patterns of limited proteolysis of MBP they produced were identical. Myelin of cerebral white matter also exhibited soluble and bound acid protease activity which was likewise inhibited by pepstatin A. Protease activity of spinal cord and cerebral myelin is therefore suggested to be due to a cathepsin D-like endopeptidase, present in a loosely and tightly bound form. Both forms increased by 50 to 80% in activity when myelin was isolated from mixtures of white and cortical gray matter. While increased soluble activity of myelin is consistent with binding of cathepsin D of lysosomal origin during the isolation of myelin the tightly bound form might point to a principal mechanism through which exogenous proteins may become attached to the myelin sheath in vivo.     

A novel metalloproteinase associated with brain myelin membranes. Isolation and characterization

Brain myelin membrane preparations contain a metalloproteinase activity which degrades myelin basic protein (MBP). The activity was associated with lentil lectin-binding glycoproteins solubilized from myelin and could be detected in the presence of the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS). The metalloproteinase represented about 5% of this glycoprotein fraction and was isolated from it by chromatography on DEAE-Sephacel, CM-Sepharose, and Superose 6. The proteinase had an apparent relative molecular weight (Mr) of approximately 58,000 both by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr value was unaffected by the presence of reducing agents but was diminished to about 52,000 by treating the proteinase with endoglycosidase F. The purified proteinase cleaved many bonds in MBP but did not generate trichloroacetic acid-soluble products. Two major polypeptides, putatively MBP1-73 and MBP74-170, were prominent in digests of MBP by either the purified enzyme or myelin membranes. The proteinase was active between pH 7 and 9 and was inhibited by phenanthroline and dithiothreitol but not phosphoramidon or inhibitors of serine or cysteine proteinases. Histones, but not azocasein, also served as substrates for the proteinase. From its enzymic and molecular characteristics the myelin-derived metalloproteinase appears distinct from previously described enzymes.     

Susceptibility of myelin proteins to a neutral endoproteinase: The degradation of myelin basic protein (MBP) and P2 protein by purified bovine brain multicatalytic proteinase complex (MPC)

Multicatalytic proteinase complex (MPC) was isolated from bovine brain and the susceptibility of myelin basic protein (MBP) and P₂ protein of bovine central and peripheral nervous system was examined. SDS-polyacrylamide electrophoretic analysis of purified MPC revealed protein bands of molecular weight ranging from 22–35 kDa. The enzyme is activated by SDS at a concentration less than 0.01%. Upon incubation with MPC, purified MBP and P₂ proteins were degraded into smaller fragments. There was a 57% and 100% loss of MBP at 2 and 6 hours of incubation. The P₂ protein which is not susceptible to any endogenous non-lysosomal enzyme thus far studied was digested into small peptide fragments only in the presence of SDS (0.01%) and not in its absence. These results indicate that MPC which is active at physiological conditions may have a role in the turnover of myelin proteins and in demyelinating diseases.     

Identification of water-soluble proteases in myelin preparations.

Sodium chloride extracts obtained from purified bovine brain myelin were found to contain proteolytic activity capable of degrading isolated myelin basic protein as assessed by SDS gel electrophoresis. Using gels copolymerized with gelatin as substrate, two bands at about 54 and 117-125 KDa, respectively, were detected. Activity corresponding to the 54 KDa band was inhibited by zinc. Data presented in this article suggest that proteolytic activity can be released from the myelin sheath in water-soluble form and recognize MBP as substrate.     

Endogenous phosphorylation of basic protein in myelin of varying degrees of compaction

Fractions containing myelin of varying degrees of compaction were prepared from human white matter. Protein kinase activity in these fractions was measured by using both endogenous and exogenous myelin basic protein (MBP) as substrates. In both cases, less compact myelin fractions possessed higher levels of protein kinase activity than the compact myelin fraction. In addition, the specific activity of phosphorylated basic protein was greater in the loosely compacted fractions than in compact multilamellar myelin. When basic protein in compact myelin or the myelin fractions was phosphorylated by the endogenous kinase, approximately 70% of the [32P]phosphate was incorporated at a single site, identified as Ser-102. The remaining 30% was found in three other minor sites. Electron microscopy of less compact myelin showed it was composed of fewer lamellae which correlated with a relative decrease in the proportion of cationic charge isomers (microheteromers) when MBP was subjected to gel electrophoresis at alkaline pH. The shift in charge microheterogeneity of basic protein to the less cationic isomers in the less compact myelin fractions correlated with an increase in protein kinase activity and a greater specific activity of phosphorylated basic protein.     

Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse

In attempts to elucidate mechanisms of demyelination in the twitcher mouse (Twi), phosphorylation and methylation of myelin basic protein (MBP) were examined in the brainstem and spinal cord of this species. Phosphorylation of MBP in isolated myelin by an endogenous kinase and an exogenous [³²P]ATP was not impaired and protein kinase C activity in the brain cytosol was not reduced. When the methylation of an arginine residue of MBP was examined in slices of the brainstem and spinal cord, using [³H]methionine as a donor of the methyl groups, no difference was found between Twi and the controls. Radioactivity of the [³H] methionine residue of MBP of Twi was also similar to that of the controls. Thus, accumulation of psychosine in Twi does not interfere with the activity of endogenous kinase, methylation of MBP, and the synthesis and transport of MBP into myelin membrane.     

Protein kinase C in rat brain myelin

It has been suggested that phosphorylation of myelin basic protein (MBP) in CNS is catalyzed by protein kinase C (PKC). In order to demonstrate that PKC in the myelin phosphorylates MBP, PKC was partially purified from rat CNS myelin by solubilization with Triton X-100 followed by a DEAE-cellulose column. MBP and histone III-S were phosphorylated in the presence of Ca²⁺ and phospholipid by rat myelin PKC. High voltage electrophoresis revealed that the phosphoamino acids in MBP by this kinase was serine residue, which is known to be the amino acid phosphorylated by PKC. The activity of PKC extracted from myelin was inhibited by the addition of psychosine to the incubation mixture. To confirm the presence of PKC molecule and to identify the isoform of PKC in the myelin, the solubilized myelin fraction was applied on SDS-PAGE, transferred to a nitrocellulose sheet and stained with anti-PKC monoclonal antibodies. Rat CNS myelin contained the PKC of about 80 kDa (intact PKC), and no proteolytic fragments were observed. PKC isozymes in myelin were type II and III. A developmental study from 14 to 42 postnatal days showed that PKC activity in CNS myelin seemed to parallel the deposition of myelin protein.     

Ganglioside-modulated protein phosphorylation in myelin

Gangliosides have profound effects on the phosphorylation of several proteins in myelin. Addition of polysialogangliosides to purified guinea pig brain myelin enhanced the endogenous phosphorylation of a 62-kDa phosphoprotein, but completely inhibited the phosphorylation of myelin basic protein (MBP) (18.5 kDa). The ganglioside-stimulated phosphorylation of the 62-kDa protein was dose-dependent and -specific. Asialo-GM1, ceramide trihexosides, N-acetylneuraminic acid, or colominic acid alone could not mimic this effect, suggesting that the activation process requires both the hydrophobic head group and the anionic character of the gangliosides. Studies on the time course of this reaction revealed that it was a rapid and reversible process and was affected only very slightly by Ca2+. Thus, the stimulatory effect of gangliosides may not involve Ca2+-gangliosides complexes or proteolysis, but may be mediated through an activation of a ganglioside-dependent protein kinase or due to substrate protein-glycolipid interaction. Modulation of the phosphorylation of MBP by gangliosides varies with the states of phosphorylation of this protein. Prior addition of ganglioside to myelin inhibited the phosphorylation of MBP. However, addition of gangliosides to myelin subsequent to maximal phosphorylation of MBP retarded the dephosphorylation of this protein. Phosphorylation of isolated MBP by protein kinase C was stimulated by gangliosides, provided phosphatidylserine was present. In contrast, the glycolipid inhibited the phosphorylation of a unique site catalyzed by cAMP-dependent protein kinase. This site was distinct from those phosphorylated by protein kinase C and was also sensitive to chymotryptic cleavage. Although the exact physiological significance of protein phosphorylation in myelin has yet to be established, gangliosides may play an important role in the modulation of this reversible post-translational modification mechanism.     

Proteolysis of peripheral nerve myelin in acute experimental allergic neuritis

Proteolysis of peripheral nerve myelin was studied in rats with experimental allergic neuritis (EAN). In vitro measurements using rat sciatic nerve homogenate and denatured bovine myelin as a substrate showed two myelin specific enzyme activities at pH 3.8 (inhibited by pepstatin) and pH 5.8 (inhibited by PMSF) in the normal rat and newly appearing activities at pH 2.8 (inhibited by pepstatin) and pH 5.0 (not characterized) in the EAN rat. In EAN the proteolytic activity was not restricted to myelin substrate but degraded total sciatic nerve protein as well. Endogenous sciatic nerve protease at pH 5.8 did not significantly change in activity during the course of disease. On the contrary, activity of acid protease at pH 2.8 corresponded well to the disease. Myelin degradation in EAN, therefore, appears to be mainly due to exogenous non-tissue protease.Abbreviations EAN experimental allergic neuritis - EDTA ethylenediaminetetraacetic acid - HBM hydroxymercuro benzoate - PLP proteolipid protein - PMSF phenylmethylsulfonyl fluoride - PNS peripheral nervous system - SDS sodium dodecylsulfate - TCA trichloroacetic acid This work is part of the M.D. thesis of R. B.     

Identification of the ATP.Mg-dependent protein phosphatase activator (FA) as a myelin basic protein kinase in the brain

The activating factor FA of the ATP.Mg-dependent protein phosphatase FcM was purified to near homogeneity from pig brain by a procedure involving chromatography on phosphocellulose, phosvitin-Sepharose 4B, and Blue Sepharose CL-6B. A specific myelin basic protein (MBP) kinase was found to co-purify with FA in a constant ratio throughout purification. It also proved impossible to separate the two activities on nondenaturing gel electrophoresis and 5-20% sucrose density gradient ultracentrifugation. Kinetic study indicated that MBP, presumably a substrate for FA, could compete with FcM for FA and thereby prevent the FA-mediated activation of the FcM activity. All the results taken together demonstrate that MBP kinase and FA are localized on the same protein. This, together with the data that FA, by activating the ATP.Mg-dependent phosphatase, promotes the dephosphorylation of [32P]MBP, phosphorylated by FA itself, suggests the evidence for a protein bearing two opposing activities involved in the regulation of brain functions. Moreover, since FA is tightly associated with the purified brain myelin membrane, the results further support the notion that FA may well be an endogenous protein kinase responsible for the cyclic phosphorylation-dephosphorylation of the central nervous system myelin.     

Serum degradation of myelin basic protein with loss of encephalitogenic activity: evidence for an enzymatic process.

Fibrin deposition in parallel with loss of myelin basic protein (MBP), an antigenic constituent of central nervous system (CNS) myelin, within the lesions of animals with experimental allergic encephalomyelitis (EAE) suggested that degradation of MBP by proteolytic activity associated with blood clotting might be an important immunopathologic event in this prototypic autoimmune disease. Following incubation in normal rat serum at 37 °C for more than 4 hr, but not to any comparable degree in plasma, MBP had little or no encephalitogenic activity when bioassayed in guinea pigs or rats. Fragments of increasingly lower molecular weight were demonstrable by polyacrylamide gel electrophoresis after addition of MBP to rat serum; no fragments appeared after incubating the protein in rat plasma. Little or no loss of encephalitogenic activity was observed when MBP was incubated in serum containing protease inhibitors. These findings indicate that the serum-mediated degradation of MBP and concomitant loss of encephalitogenic activity is due to an enzymatic process associated with the coagulation cascade or/and the complement, kallikrein or fibrinolytic pathways. Implications of these findings concerning EAE and the multiple sclerosis process in man are discussed.     

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.     

Insulin-sensitive myelin basic protein phosphorylation on tyrosine residues.

Rat brain plasma membranes were solubilized in detergent and a glycoprotein-enriched fraction was obtained by lectin affinity chromatography. This glycoprotein fraction contained insulin receptors, as well as protein kinases capable of phosphorylating some exogenously added substrates such as MAP2 (microtubule associated protein 2) and MBP (myelin basic protein), but not ribosomal protein S6. Phosphoamino acid analysis of MAP2 and MBP showed that phosphotyrosine residues, as well as phosphoserine/phosphotheronine residues, were present in both proteins under basal conditions. Whereas the addition of insulin to the rat brain membrane glycoprotein fraction in vitro had no effect on MAP2 phosphorylation, MBP phosphorylation was stimulated 2.7-fold in response to insulin. This phenomenon was dose-dependent, with half-maximal stimulation of MBP phosphorylation observed with 2 nM insulin. Phosphoamino acid analysis of MBP indicated that insulin stimulated the phosphorylation of tyrosine residues nearly three-fold, whereas the phosphorylation of serine or threonine residues was not increased. These results identify MBP as a substrate for the rat brain insulin receptor tyrosine-specific protein kinase in vitro.     

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 GTPS and was specific for the -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.Abbreviations ADP-ribose adenosine diphosphate ribose - APAD 3-acetylpyridine adenine dinucleotide - ATP adenosine triphosphate - C-1, 2, 3 etc MBP components isolated by CM52 chromatography - EDTA ethylenediaminetetraacetic acid - GTP guanosine triphosphate - GTPS guanosine 5-(3-0-thio)triphosphate - INH isonicotinic acid hydrazide - MBP myelin basic protein - NAD nicotinamide adenine dinucleotide - PMSF phenylmethylsulfonyl fluoride - PLP proteolipid protein Special issue dedicated to Dr. Leon S. Wolfe.     

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.     

Degradation of myelin basic protein by a membrane-associated metalloprotease: Neural distribution of the enzyme

A metalloprotease activity associated with myelin membrane preparations degrades myelin basic protein (MBP), generating a characteristic fragment designated peptide C (MBP 74-170). Using an immunoblotting assay, peptide C-generating activity was detected in mammalian, avian, reptilian, and amphibian brains. The activity was present in rat brain as early as postnatal day 1 and also in adult rat peripheral nerve. Immunohistochemistry with a monoclonal antibody to the purified enzyme revealed that the metalloprotease was present in oligodendrocytes of optic nerve, of both white and grey matter of spinal cord, and also in the cytoplasm of both myelinating and non-myelinating Schwann cells of peripheral nerve.Special issue dedicated to Dr. Alan N. Davison     

Calcium-Stimulated Proteolysis in Myelin: Evidence for a Ca2+-Activated Neutral Proteinase Associated with Purified Myelin of Rat CNS

Incubation of myelin purified from rat spinal cord with CaCl2 (1-5 mM) in 10-50 mM Tris-HCl buffer at pH 7.6 containing 2 mM dithiothreitol resulted in the loss of both the large and small myelin basic proteins (MBPs), whereas incubation of myelin with Triton X-100 (0.25-0.5%) and 5 mM EGTA in the absence of calcium produced preferential extensive loss of proteolipid protein (PLP) relative to MBP. Inclusion of CaCl2 but not EGTA in the medium containing Triton X-100 enhanced degradation of both PLP and MBPs. The Ca2+-activated neutral proteinase (CANP) activity is inhibited by EGTA (5 mM) and partially inhibited by leupeptin and/or E-64c. CANP is active at pH 5.5-9.0, with the optimum at 7-8. The threshold of Ca2+ activation is approximately 100 microM. The 150K neurofilament protein (NFP) was progressively degraded when incubated with purified myelin in the presence of Ca2+. These results indicate that purified myelin is associated with and/or contains a CANP whose substrates include MBP, PLP, and 150K NFP. The degradation of PLP (trypsin-resistant) in the presence of detergent suggests either release of enzyme from membrane and/or structural alteration in the protein molecule rendering it accessible to proteolysis. The myelin-associated CANP may be important not only in the turnover of myelin proteins but also in myelin breakdown in brain diseases.     

Endogenous Basic Protein Phosphatases in the Brain Myelin

Direct treatment of brain myelin with freezing/thawing in 0.2 M 2-mercaptoethanol stimulated the endogenous myelin phosphatase activity manyfold when 32P-labeled phosphorylase a was used as a substrate, a result indicating that an endogenous myelin phosphatase is a latent protein phosphatase. When myelin was treated with Triton X-100, this endogenous latent phosphatase activity was further stimulated 2.5-fold. Diethylaminoethyl-cellulose and Sephadex G-200 chromatography of solubilized myelin revealed a pronounced peak of protein phosphatase activity stimulated by freezing/thawing in 0.2 M 2-mercaptoethanol and with a molecular weight of 350,000, which is characteristic of latent phosphatase 2, as previously reported. Moreover, endogenous phosphorylation of myelin basic protein (MBP) in brain myelin was completely reversed by a homogeneous preparation of exogenous latent phosphatase 2. By contrast, under the same conditions, endogenous phosphorylation of brain myelin was entirely unaffected by ATP X Mg-dependent phosphatase and latent phosphatase 1, although both enzymes are potent MBP phosphatases. Together, these findings clearly indicate that a high-molecular-weight latent phosphatase, termed latent phosphatase 2, is the most predominant phosphatase responsible for dephosphorylation of brain myelin.     

Cleavage of Myelin Basic Protein by Neutral Protease Activity of Human White Matter and Myelin

Polypeptides arising from neutral in vitro proteolysis of myelin basic protein (MBP) of human brain were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At pH 7 a marked breakdown of MBP resulted in the formation of 8-12 polypeptides ranging from 6 to 17 kd in molecular weight. As neutral proteolytic activity was not eliminated by either gel filtration or cation-exchange chromatography acid-soluble protease(s) involved probably have a size and electric charge similar to that of MBP. The enzymatic nature of neutral proteolysis was ascertained by heat inactivation and inhibition by alpha 2-macroglobulin. Incomplete inhibition of proteolysis and the failure of small peptides (less than 6 kd) to show up on electrophoresis seem to suggest that MBP was degraded by exopeptic proteases as well. Acid extracts of purified myelin yielded polypeptides similar to those of MBP of delipidated white matter. The results are consistent with a sequential limited proteolysis of MBP by neutral proteases probably associated with myelin and possibly related to the in situ catabolism of MBP in man.     

Characterization of fertilization-modulated myelin basic protein kinases from sea star: regulation of Mapk.

The myelin basic protein (MBP)-phosphorylating enzymes present during maturation and early embryogenesis of the sea star (Pisaster ochraceus) were investigated. The major maturation-activated MBP kinase (p45 Mapk) was molecularly cloned based on tryptic sequence information obtained with the purified enzyme and shown to be highly related to human Erk1 with 76% amino acid identity. Kinase assays and immunoblotting studies revealed that Mapk remained highly active until 12 h post-fertilization (PF), after which it declined. By 4 days PF, Mapk protein was no longer detectable. At 3 h PF, about half of the detectable MBP phosphotransferase activity could be attributed to a 75 kDa protein kinase that was distinct from Mapk. Like Mapk, this protein phosphorylated MBP mostly on threonine residues, but it failed to phosphorylate a peptide (APRTPGGRR) based upon the Thr-97 MAP kinase phosphorylation site in MBP. Rather, it phosphorylated a peptide (AAQKRPSQRTKYLA) patterned after the N-terminus of MBP. Our studies also showed a dramatic increase in MBP phosphotransferase activity occurred by 4 days PF that arose from a third kinase that phosphorylated MBP solely on serine residues. This kinase exhibited the following substrate substrate preference: AAQKRPSQRTKYLA, peptide substrate for S6 kinases (AKRRRLSSLRASTSKSESSQK) > MBP > histone H1 > prota-mine > casein > APRTPGGRR. This kinase was not appreciably affected by addition of phosphatidylserine/diacylglycerol, or the staurosporine analogue Roche Compound 3, but it was partly inhibited by a protein kinase C pseudosubstrate peptide. Gel filtration analysis revealed an apparent molecular mass of 41 kDa for the enzyme. Therefore, at least two novel MBP-phosphorylating enzymes distinct from Mapk are preferentially activated following fertilization and early embryogenesis of the sea star.