Characterization of Myelin-Associated Glycoprotein (MAG) Proteolysis in the Human Central Nervous System

Purified human central nervous system myelin contains an endogenous cysteine protease which degrades the 100-kDa myelin-associated glycoprotein into a slightly smaller 90-kDa derivative called dMAG, and which has been implicated in demyelinating diseases. The native proteolytic site in human MAG was determined in order to characterize this cysteine protease in humans further. This was accomplished by identifying the carboxy-terminus of purified dMAG. The results of these experiments, in conjunction with peptidolysis assays of myelin, demonstrated that the enzyme which proteolyses MAG is extracellular and has cathepsin L-like specificity. Furthermore, it was shown that this cathepsin L-like activity potentially was regulated by the endogenous extracellular inhibitor cystatin C.     

Myelin instability and oligodendrocyte metabolism in myelin-deficient mutant mice

During the active phase of myelination in myelin-deficient mutant mice (mld), myelin basic protein (MBP) synthesis is defective and the myelin lamellae are uncompacted. In these mutants, we found a fast metabolism of the myelin-associated glycoprotein (MAG) and of sulfatides, and the presence of cholesterol esters and a degradation product of MAG, dMAG, indicating that mld myelin was unstable. The increased synthesis of MAG and Wolfgram protein, two proteins present in uncompacted myelin sheath and paranodal loops, was demonstrated by high levels of messengers. Simultaneously, we found an accumulation of inclusion bodies, vacuoles, and rough endoplasmic reticulum in mld oligodendrocytes. This material was heavily immunostained for MAG. Furthermore, the developmental change between the two molecular forms of MAG (p72MAG/p67MAG) was delayed in mld mice. In 85-d-old mld mice, the MBP content increased and myelin lamellae became better compacted. In these mutants, dMAG was absent and MAG mRNAs were found in normal amounts. Furthermore, the fine structure of mld oligodendrocytes was normal and the MAG immunostaining was similar to age-matched controls. These results support a functional role for MBP in maintaining the metabolic stability and the compact structure of myelin. Furthermore, in the absence of MBP and myelin compaction, the regulation of the synthesis of at least two membrane proteins related to myelin cannot proceed.     

Myelin-Associated Glycoprotein and Related Glycoconjugates in Developing Cat Peripheral Nerve: A Correlative Biochemical and Morphometric Study

The expression and accumulation of the myelin-associated glycoprotein (MAG) and other glycoconjugates have been studied during myelination in the developing cat peripheral nervous system. The glycoconjugates studied have in common a similar carbohydrate determinant which is bound by many antibodies, including the mouse monoclonal antibody HNK-1, and human IgM paraproteins from patients with neuropathy. In addition to MAG, the reactive glycoconjugates include a 60-kilodalton (kD) glycoprotein and a group of 20-26 kD glycoproteins, as well as a group of recently identified acidic glycolipids, the major one of which is sulfate-3-glucuronyl paragloboside (SGPG). The accumulation of these glycoproteins and glycolipids is compared with the established myelin proteins P0, P1, and P2 and with morphometric indices of myelin volume and axonal perimeter. The study demonstrates that MAG appears and accumulates very early during myelination, being present at 15% of the maximum level prior to the appearance of P0, and at 80% of the maximum level when P0 is at 30% of its maximum level. In the adult, the level of MAG falls to 60% maximum. The 60 kD and 20-26 kD glycoproteins accumulate at the same time as or later than P0, suggesting that they are either compact myelin proteins or in membranes closely associated with compact myelin. SGPG accumulates with P0 early in myelination, but falls to 60% of maximum in the adult. By comparing biochemical and morphometric data, we demonstrate that P0 and other compact myelin proteins accumulate synchronously with the increase in myelin area. MAG accumulation, however, is closely related to changes in axonal perimeter, consistent with a predominant localization of MAG to the periaxonal membranes in the peripheral nervous system.     

Susceptibility of the Myelin-Associated Glycoprotein and Basic Protein to a Neutral Protease in Highly Purified Myelin from Human and Rat Brain

Abstract: Incubation of highly purified human myelin at 25° and pH 8 in ammonium bicarbonate buffer resulted in the conversion of the myelin-associated glycoprotein (MAG) to a smaller derivative (dMAG) with an apparent molecular weight about 10,000 less. dMAG was stable and was not degraded to lower-molecular-weight breakdown products. Incubation of myelin under these conditions also resulted in the degradation of basic protein, but at a much slower rate. Half of the MAG was converted to dMAG in about 30 min, whereas degradation of half of the basic protein required 18 h of incubation. There was no significant loss of proteolipid, the Wolfgram doublet, or other myelin proteins during incubation for up to 18 h under these conditions. The formation of dMAG and the degradation of basic protein appear to be mediated by similar enzymatic activities; both processes exhibited broad pH optima in the neutral range, were prevented by briefly heating the myelin to 70° before incubation, and were stimulated by ammonium bicarbonate and other salts. Incubation of purified rat myelin also resulted in the formation of dMAG and the degradation of basic protein, but the conversion to dMAG occurred much more slowly than in human myelin preparations. In the rat, the percentage decreases in intact MAG and in basic protein were similar to each other and proceeded at rates comparable to the loss of basic protein in human myelin. These studies confirm and extend earlier demonstrations of neutral protease activity in purified myelin, and show that cleavage of MAG is one of the effects of this activity. The proteolytic activity affecting MAG and basic protein was not significantly reduced by further purification of the myelin on sucrose or CsCl gradients, suggesting that the neutral protease may be a myelin-related enzyme. The very high susceptibility of human MAG to this enzyme indicates that the effect of neutral protease on this glycoprotein should be considered in connection with demyelinating diseases.     

Generation and characterization of mouse monoclonal antibodies to the myelin-associated glycoprotein (MAG)

A panel of mouse monoclonal antibodies to rat and human myelin-associated glycoprotein (MAG) was developed. Normal mice were unresponsive to rat MAG, and successful immunization with rat MAG was only achieved in autoimmune NZB mice. By contrast, all strains of mice were responsive to human MAG. The monoclonal antibodies developed differ with respect to immunoglobulin type, their specificity for human and/or rat MAG, and their recognition of protein or carbohydrate epitopes in MAG. In general, the antibodies that react with the protein backbone recognize both rat and human MAG, whereas a large number of the monoclonal antibodies recognize a carbohydrate determinant in human MAG that is not in rat MAG. Immunocytochemical staining of adult human spinal cord with the monoclonal antibodies resulted in periaxonal staining of myelin sheaths, similar to that produced by well-defined, rabbit, polyclonal anti-MAG serum. In addition, the antibodies recognizing, carbohydrate determinants in human MAG strongly stained oligodendrocyte cytoplasm. These monoclonal antibodies will be of value for the further chemical and biological characterization of MAG.     

Effects of Trypsin and Plasmin Treatment of Myelin on the Myelin-Associated Glycoprotein and Basic Protein

Human and rat myelin preparations were incubated with varying concentrations of trypsin and plasmin to determine the effects of these proteolytic enzymes on myelin-associated glycoprotein (MAG), basic protein, and other myelin proteins and to compare the effects with those of the neutral protease that was reported to be endogenous in myelin. Basic protein was most susceptible to degradation by both trypsin and plasmin, whereas MAG was relatively resistant to their actions. Under the assay conditions used, the highest concentrations of trypsin and plasmin degraded greater than 80% of the basic protein but less than 30% of the MAG, and lower concentrations caused significant loss of basic protein without appreciably affecting MAG. Neither trypsin nor plasmin caused a specific cleavage of MAG to a derivative of MAG (dMAG) in a manner analogous to the endogenous neutral protease. Thus the endogenous protease appears unique in converting human MAG to dMAG much more rapidly than it degrades basic protein. MAG is slowly degraded along with other proteins when myelin is treated with trypsin or plasmin, but it is less susceptible to their action than is basic protein.     

Myelin-Associated Glycoprotein and Other Proteins in Trembler Mice

The myelin-associated glycoprotein (MAG) and other myelin proteins were quantitated in homogenates of whole sciatic nerve from adult and 20-day-old Trember mice. In the nerves of adult mice, the concentration of MAG was increased from 1.1 ng/micrograms of total protein in the controls to 1.4 ng/micrograms protein in the Tremblers. By contrast, the concentrations of P0 glycoprotein and myelin basic proteins were reduced to 27% and 20% of control levels, respectively. Immunoblots demonstrated that P2 was also greatly reduced in the Trembler nerves. The specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) was 65% of the control level. Immunoblot analysis showed that MAG had a higher than normal apparent Mr in the sciatic nerves of the Trembler mice, but its apparent Mr was normal in the brains of these mutants. In 20-day-old Tremblers, the P0 and myelin basic protein were reduced slightly less to about 40% of the level in the nerves of age-matched controls. CNP and MAG levels were not significantly different from those in controls, and MAG exhibited a shift toward higher apparent Mr similar to that in the adults. The maintenance of high MAG levels despite the severe deficit of myelin, as reflected by the decrease of the major myelin proteins, is consistent with the immunocytochemical localization of MAG in periaxonal Schwann cell membranes, Schmidt-Lantermann incisures, lateral loops, and the outer mesaxon and its absence from compact myelin. The abnormal form of MAG in the peripheral nervous system (PNS) of the Trembler mice may contribute to the pathology in this mutant.     

Developmental changes of myelin-associated glycoprotein in rat brain: study on experimental hyperphenylalaninemia

We examined developmental changes of myelin-associated glycoprotein (MAG), basic protein (BP), abd proteolipid protein (PLP) in central nervous system myelin isolated from experimental hyperphenylalaninemic rats (PKU rats) and controls. Higher amounts of MAG, including high-molecular-weight MAG in myelin, were found in 12- to 21-day-old control rats than in adult rats. MAG in developing myelin was at a maximum in 18-day-old rats and began to decrease in 21-day-old rats, while PLP and BP in developing myelin increased at these developmental stages. The level of high-molecular-weight MAG decreased in myelin prepared from 21-day-old rats. These results suggest that the decreasing high-molecular-weight MAG is important for compaction of myelin in the early stage of myelination. In myelin from 12- to 18-day-old PKU rats, the ratio of each protein such as MAG, PLP, or BP to that of control was about 0.5 at 12 days, and increased to almost 1.0 at 18 days. The myelination seems to be initially delayed but to be close to that of controls in PKU rats about 18 days old.     

The amino acid sequences of the myelin-associated glycoproteins: homology to the immunoglobulin gene superfamily

The myelin associated glycoproteins (MAG) are integral plasma membrane proteins which are found in oligodendrocytes and Schwann cells and are believed to mediate the axonal-glial interactions of myelination. In this paper we demonstrate the existence in central nervous system myelin of two MAG polypeptides with Mrs of 67,000 and 72,000 that we have designated small MAG (S-MAG) and large MAG (L-MAG), respectively. The complete amino acid sequence of L-MAG and a partial amino acid sequence of S-MAG have been deduced from the nucleotide sequences of corresponding cDNA clones isolated from a lambda gt11 rat brain expression library. Based on their amino acid sequences, we predict that both proteins have an identical membrane spanning segment and a large extracellular domain. The putative extracellular region contains an Arg-Gly-Asp sequence that may be involved in the interaction of these proteins with the axon. The extracellular portion of L-MAG also contains five segments of internal homology that resemble immunoglobulin domains, and are strikingly homologous to similar domains of the neural cell adhesion molecule and other members of the immunoglobulin gene superfamily. In addition, the two MAG proteins differ in the extent of their cytoplasmically disposed segments and appear to be the products of alternatively spliced mRNAs. Of considerable interest is the finding that the cytoplasmic domain of L-MAG, but not of S-MAG, contains an amino acid sequence that resembles the autophosphorylation site of the epidermal growth factor receptor.     

Specific sequence-directed anti-bilitranslocase antibodies as a tool to detect potentially bilirubin-binding proteins in different tissues of the rat.

The hypothesis that the uneven distribution of bilirubin in the organism, which occurs in hyperbilirubinemia, could reflect an uneven distribution of bilirubin-binding proteins was tested by searching for peptides containing the bilirubin-binding motif identified in bilitranslocase (Battiston et al., 1998). In the rat, positive proteins bands were found to be present only in the liver, gastric mucosa and central nervous system. The electrophoretic mobilities of the positive compounds in the liver and stomach were identical to that of purified bilitranslocase (38 kDa). In the brain, on the contrary, two peptides were found with molecular masses of 79 and 34 kDa, respectively. Their distribution pattern in the central nervous system was different for each of them.     

Developmental and pathophysiological aspects of the myelin-associated glycoprotein

1. A glycoslylated sulfate-containing protein known as myelin-associated glycoprotein (MAG) appears to be unique to the central and peripheral nervous systems. This component has been characterized and cDNA clones have been isolated. 2. MAG is a member of the immunoglobulin superfamily. The principal form of MAG synthesized in brain during active myelination has an apparent molecular weight of 100,000. Alternate exon splicing leads to an additional 5000-dalton smaller form with a different C terminus. 3. In patients with multiple sclerosis, MAG is rapidly lost in areas of active disease. It is immunologically reactive in patients with benign monoclonal gammopathy associated with peripheral neuropathy. 4. The role of MAG in the formation of the myelin sheath and its participation in autoimmune neurological disorders are outlined.     

Keratin expression in cultures of adult human epidermal cells.

Keratins are complex fibrous proteins characteristic of epithelial cells. We have developed a procedure that allows us to culture and passage adult human dermal keratinocytes in the absence of mesenchymal substrates. Electron microscopic examination of stratifying cultures showed the presence of numerous filament bundles, desmosomes and electron dense granules. The expression of different classes of keratin was examined by immunofluorescence, SDS-PAGE and immunoblots using monoclonal antibodies. The analysis of water-insoluble proteins revealed the presence of keratins of molecular weights 40 Kda, 50-52 Kda, 56 Kda and 65-67 Kda. Our results indicate that the terminal differentiation of keratinocytes may not require dermal factors.     

Myelin-Associated Glycoprotein Shares an Antigenic Determinant with a Glycoprotein of Human Melanoma Cells

A sulfated 100K-dalton glycoprotein has been shown to be released into the culture medium of melanoma cells. Monoclonal antibodies 10C5 and 11B5, which were raised to human melanoma cells, as well as HNK-1 bind to this glycoprotein. It is shown here that mouse anti-myelin-associated glycoprotein (MAG) carbohydrate antibodies raised to human MAG and a human IgM paraprotein associated with neuropathy also bind to the same 100K molecule. However, anti-MAG antibodies recognizing peptide epitopes do not appear to react with this glycoprotein of melanoma cells, a result suggesting that its similarity to MAG is restricted to shared carbohydrate moieties. The anti-melanoma antibodies (10C5 and 11B5) resemble HNK-1 in binding to MAG and to some 19-28K-dalton glycoproteins and sulfated, glucuronic acid-containing sphingoglycolipids of the peripheral nervous system (PNS). In addition, the anti-melanoma antibodies cross-react with neural cell adhesion molecule (N-CAM), an observation emphasizing the shared antigenicity between MAG and other adhesion molecules. The results demonstrate that the anti-melanoma antibodies fall into a class of monoclonal antibodies (including HNK-1, human IgM paraproteins associated with neuropathy, anti-human MAG antibodies, and L2 antibodies) that are characterized by reactivity against related carbohydrate determinants shared by human MAG, N-CAM, and several protein and lipid glycoconjugates of the PNS.     

Multiple forms of bile salt hydrolase from Lactobacillus sp. strain 100-100.

Four isozymes of bile salt hydrolase (BSH) have been purified from the cytosol of cells of Lactobacillus sp. strain 100-100. The four proteins were designated BSH A, B, C, and D. They eluted from anion-exchange high-pressure liquid chromatography columns at 0.15, 0.18, 0.21, and 0.25 M NaCl, respectively. They are catalytically similar, except that the Vmax of BSH D is about 10-fold lower than those of the other three isozymes. All four proteins consist of one or two polypeptides. The peptides have molecular weights of 42,000 and 38,000 and are designated alpha and beta, respectively. The approximate native molecular weights of BSH A, B, C, and D are 115,000, 105,000, 95,000, and 80,000, respectively. The native proteins are probably trimers; the four isozymes are the array of possible subunit combinations alpha 3, alpha 2 beta 1, alpha 1 beta 2, and beta 3 for A, B, C, and D, respectively. The two subunits are antigenically distinct. Polyclonal antibodies raised against BSH A (all alpha peptide) react in Western blots (immunoblots) only with proteins containing the alpha peptide; such antibodies raised against BSH D (all beta peptide) react only with proteins containing the beta peptide. The amino acid compositions of the two peptides differ. This is the first report of a bacterium that makes four BSH isozymes.     

Analysis of polypeptide molecular weights by electrophoresis in urea

Ten proteins of differing disulfide contents and isoionic points were subjected to disc gel electrophoresis in the presence of 8 urea-0.9 acetic acid to evaluate the use of this technique in determining polypeptide molecular weights. Comparison of the electrophoretic mobilities before and after reduction of the proteins' disulfide bonds demonstrated that only after all disulfide bonds were broken, could their molecular weights be estimated with any degree of accuracy. The expression of the electrophoretic mobilities as a function of the proteins' effective hydrodynamic sizes, thereby taking into account the extent of constraint by disulfide bonds, allowed a comparison of disulfide cross-linked and linear forms of the protein polypeptides. The extent to which intrinsic charge affects a protein's electrophoretic mobility was estimated by comparing alpha-lactalbumin and lysozyme, two proteins of identical size but vastly different isoionic points. They exhibited a 20% difference in mobilities. An apparent slow reduction of disulfide bonds was observed to occur when proteins were exposed to reducing agent at low pH in 8 urea.     

Gangliosides and Nogo receptors independently mediate myelin-associated glycoprotein inhibition of neurite outgrowth in different nerve cells

In the injured nervous system, myelin-associated glycoprotein (MAG) on residual myelin binds to receptors on axons, inhibits axon outgrowth, and limits functional recovery. Conflicting reports identify gangliosides (GD1a and GT1b) and glycosylphosphatidylinositol-anchored Nogo receptors (NgRs) as exclusive axonal receptors for MAG. We used enzymes and pharmacological agents to distinguish the relative roles of gangliosides and NgRs in MAG-mediated inhibition of neurite outgrowth from three nerve cell types, dorsal root ganglion neurons (DRGNs), cerebellar granule neurons (CGNs), and hippocampal neurons. Primary rat neurons were cultured on control substrata and substrata adsorbed with full-length native MAG extracted from purified myelin. The receptors responsible for MAG inhibition of neurite outgrowth varied with nerve cell type. In DRGNs, most of the MAG inhibition was via NgRs, evidenced by reversal of inhibition by phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves glycosylphosphatidylinositol anchors, or by NEP1-40, a peptide inhibitor of NgR. A smaller percentage of MAG inhibition of DRGN outgrowth was via gangliosides, evidenced by partial reversal by addition of sialidase to cleave GD1a and GT1b or by P4, an inhibitor of ganglioside biosynthesis. Combining either PI-PLC and sialidase or NEP1-40 and P4 was additive. In contrast to DRGNs, in CGNs MAG inhibition was exclusively via gangliosides, whereas inhibition of hippocampal neuron outgrowth was mostly reversed by sialidase or P4 and only modestly reversed by PI-PLC or NEP1-40 in a non-additive fashion. A soluble proteolytic fragment of native MAG, dMAG, also inhibited neurite outgrowth. In DRGNs, dMAG inhibition was exclusively NgR-dependent, whereas in CGNs it was exclusively ganglioside-dependent. An inhibitor of Rho kinase reversed MAG-mediated inhibition in all nerve cells, whereas a peptide inhibitor of the transducer p75(NTR) had cell-specific effects quantitatively similar to NgR blockers. Our data indicate that MAG inhibits axon outgrowth via two independent receptors, gangliosides and NgRs.     

Purification and partial characterization of two basic proteins from human peripheral nerve.

Two basic proteins, denoted P1 and P2 protein, were purified from human sciatic nerve. The isolation was achieved by the following steps: delipidation with chloroform/methanol mixtures, dry acetone and dry ether; acid extraction at pH 2; ion exchange chromatography on QAE-Sephadex A-25 and gel filtration on Sephadex G-100. P1, P2 proteins and the basic protein of the central nervous system have been shown to have different electrophoretic mobility, and each of the two peripheral basic proteins was shown to be homogeneous by disc electrophoresis. The molecular weight of P1 protein is around 14 100 and that of P2 protein is around 12 200, as determined by ultracentrifugal analysis. There was some difference in the amino acid composition of human P1 and P2 protein, and a marked difference between their composition and the composition of central basic protein and bovine peripheral P1 and P2 proteins which were described previously. When injected to animals, P1 protein induced only experimental allergic neuritis while P2 protein induced both mild experimental allergic neuritis and experimental allergic encephalomyelitis. Thus, the human P1 protein is similar to the bovine P1 protein and human P2 protein is similar to bovine P2 protein, concerning their electrophoretic mobilities, molecular weights and biological properties.     

The expression of recombinant large myelin-associated glycoprotein cytoplasmic domain and the purification of native myelin-associated glycoprotein from rat brain and peripheral nerve

The myelin-associated glycoprotein (MAG) is a transmembrane protein of the immunoglobulin superfamily existing as two isoforms (L-MAG and S-MAG) that are differentially expressed by myelinating glial cells of the central and peripheral nervous systems, where MAG represents 1 and 0.1% of the total myelin proteins, respectively. The polypeptide chains of the two isoforms differ only by the carboxy terminus of their respective cytoplasmic domains, which most probably determine the isoform-specific functions. Here, we describe the expression of the L-MAG cytoplasmic domain as a GST fusion protein. The recombinant protein was used to raise polyclonal antibodies against the L-MAG-specific carboxy terminus and against the region of the MAG cytoplasmic domain common to both S-MAG and L-MAG. These antibodies, which function in dot blotting, Western blotting, and immunoprecipitation, were used to immunopurify native MAG from both rat brain and peripheral nerves in quantities and purity sufficient for the realization of most biochemical and functional studies. The antibodies and the recombinant and native MAG proteins provide much needed tools for the study of the common and isoform-specific properties and functions of L-MAG and S-MAG.