Myelin-associated Glycoprotein Antiserum Research Articles

Distribution of P0 protein and the myelin-associated glycoprotein in peripheral nerves from Trembler mice

The Trembler mouse has a dysymelination of peripheral nerves that includes hypomyelination, failure of myelin compaction, and demyelination/remyelination. We have localized the myelin proteins P0 and myelin associated glycoprotein in Trembler peripheral nerve and correlated their distributions with the ultrastructure of myelin internodes. Immunocytochemically, myelin-associated glycoprotein was localized in Schwann cell periaxonal membranes, Schmidt-Lanterman incisures, paranodal loops, and internal and external mesaxons. P0 staining was located over compact myelin and regions of Schwann cell cytoplasm rich in Golgi membranes. An unusual abundance of small, P0-stained, Golgi-related vesicles was found in some Schwann cells. P0 protein was also detected in multiple spiral wraps of myelin-associated glycoprotein-positive mesaxon membranes. At some sites the periodicity of the myelin membranes was intermediate to that found in mesaxon membranes and compact myelin. The distance between apposing extracellular leaflets was similar to that found in mesaxon membranes, while the cytoplasmic leaflets were fused but twice as thick as normal major dense lines. These intermediate membranes were stained by P0 and myelin-associated glycoprotein antiserum. These studies suggest that altered transport and/or translocation of P0 and myelin-associated glycoprotein results in defective myelin compaction in Trembler peripheral nerve.

Mouse monoclonal and rabbit polyclonal antibodies prepared to human myelin-associated glycoprotein also react with glycosphingolipids of peripheral nerve

A panel of mouse monoclonal antibodies and rabbit polyclonal antisera that were raised to myelin-associated glycoprotein (MAG) were screened for reactivity with acidic glycolipids from brain and peripheral nerve by enzyme-linked immunosorbent assay (ELISA) and/or a thin-layer chromatogram overlay technique. Seven out of 7 mouse monoclonal antibodies that recognize carbohydrate epitopes in human MAG also reacted with acidic glycolipids from human and cat peripheral nerve, while monoclonal antibodies that react with polypeptide epitopes on MAG did not react with these glycolipids. Rabbit anti-human MAG antisera also strongly reacted with the glycolipids from peripheral nerve, while rabbit antisera raised to rat MAG did not. None of the antibodies reacted with similar glycolipid fractions prepared from adult human brain. Overlay of thin-layer chromatograms revealed that all the mouse and rabbit antibodies showing reactivity with peripheral nerve glycolipids were binding to the same two sphingoglycolipids that react with human anti-MAG IgM paraproteins in neuropathy and with HNK-1 (anti-Leu-7), a mouse IgM monoclonal antibody that identifies a subset of human lymphocytes with natural killer function. Thus, the carbohydrate epitope(s) in MAG which is shared with nerve acidic glycolipids appears to be highly immunogenic in mice and rabbits. Further, it is clear that the antibodies that react with the carbohydrate moieties of human MAG cannot be used as specific probes for this glycoprotein.

Effects of glycoprotein synthesis inhibitor on myelination in rat cerebellum

Effects of a glycoprotein synthesis inhibitor on myelination were investigated in rat cerebellum. The glycoprotein synthesis inhibitor, tunicamycin (TM), was injected intracranially into newborn rats. The activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in the cerebellum was significantly reduced in 2-week-old animals and was restored to the normal level by age 3 weeks. When TM was injected into newborn rats every 3-4 days for a total of 6 times, CNPase activity was still low at 3 and 4 weeks. Immunohistochemical stainings for CNPase and myelin-associated glycoprotein (MAG) were performed on paraffin sections of multiple-TM-injected cerebellum at 3 weeks. The intensity of the staining with MAG antiserum in the white matter was clearly decreased in TM-treated cerebellum compared with the control. The myelin in the granule cell layer was poorly stained with CNPase antiserum in TM-treated cerebellum. Subcellular fractionation was carried out and the CNPase activity in each fraction was measured. The CNPase activity in the myelin fraction (P2A) from the TM-treated cerebellum was significantly lower than that in the control. In contrast, the activity in the synaptosomal (P2B) and microsomal (P3) fractions from the multiple-TM-injected cerebellum was higher than in those from the controls. Polyacrylamide gel electrophoretic patterns of the P2A fractions were analyzed. The P2A fraction from TM-treated cerebellum contained less Wolfgram protein than the control. These results suggest that glycoprotein synthesis plays certain roles in myelination in the central nervous system.

Immunocytochemical studies of quaking mice support a role for the myelin-associated glycoprotein in forming and maintaining the periaxonal space and periaxonal cytoplasmic collar of myelinating Schwann cells.

The myelin-associated glycoprotein (MAG) is an integral membrane glycoprotein that is located in the periaxonal membrane of myelin-forming Schwann cells. On the basis of this localization, it has been hypothesized that MAG plays a structural role in (a) forming and maintaining contact between myelinating Schwann cells and the axon (the 12-14-nm periaxonal space) and (b) maintaining the Schwann cell periaxonal cytoplasmic collar of myelinated fibers. To test this hypothesis, we have determined the immunocytochemical localization of MAG in the L4 ventral roots from 11-mo-old quaking mice. These roots display various stages in the association of remyelinating Schwann cells with axons, and abnormalities including loss of the Schwann cell periaxonal cytoplasmic collar and dilation of the periaxonal space of myelinated fibers. Therefore, this mutant provides distinct opportunities to observe the relationships between MAG and (a) the formation of the periaxonal space during remyelination and (b) the maintenance of the periaxonal space and Schwann cell periaxonal cytoplasmic collar in myelinated fibers. During association of remyelinating Schwann cells and axons, MAG was detected in Schwann cell adaxonal membranes that apposed the axolemma by 12-14 nm. Schwann cell plasma membranes separated from the axolemma by distances greater than 12-14 nm did not react with MAG antiserum. MAG was present in adaxonal Schwann cell membranes that apposed the axolemma by 12-14 nm but only partially surrounded the axon and, therefore, may be actively involved in the ensheathment of axons by remyelinating Schwann cells. To test the dual role of MAG in maintaining the periaxonal space and Schwann cell periaxonal cytoplasmic collar of myelinated fibers, we determined the immunocytochemical localization of MAG in myelinated quaking fibers that displayed pathological alterations of these structures. Where Schwann cell periaxonal membranes were not stained by MAG antiserum, the cytoplasmic side of the periaxonal membrane was "fused" with the cytoplasmic side of the inner compact myelin lamella and formed a major dense line. This loss of MAG and the Schwann cell periaxonal cytoplasmic collar usually resulted in enlargement of the 12-14-nm periaxonal space and ruffling of the apposing axolemma. In myelinated fibers, there was a strict correlation between the presence of MAG in the Schwann cell periaxonal membrane and (a) maintenance of the 12-14-nm periaxonal space, and (b) presence of the Schwann cell periaxonal cytoplasmic collar.(ABSTRACT TRUNCATED AT 400 WORDS)

Myelin-associated glycoprotein and myelinating Schwann cell-axon interaction in chronic B,B'-iminodipropionitrile neuropathy.

The myelin-associated glycoprotein (MAG) is a heavily glycosylated integral membrane glycoprotein which is a minor component of isolated rat peripheral nervous system (PNS) myelin. Immunocytochemically MAG has been localized in the periaxonal region of PNS myelin sheaths. The periaxonal localization and biochemical features of MAG are consistent with the hypothesis that MAG plays a role in maintaining the periaxonal space of myelinated fibers. To test this hypothesis, MAG was localized immunocytochemically in 1-micron sections of the L5 ventral root from rats exposed to B,B'-iminodipropionitrile. In chronic states of B,B'-iminodipropionitrile intoxication, Schwann cell periaxonal membranes and the axolemma invaginate into giant axonal swellings and separate a central zone of normally oriented axoplasm from an outer zone of maloriented neurofilaments. Ultrastructurally, the width of the periaxonal space (12-14 nm) in the ingrowths is identical to that found in normally myelinated fibers. These Schwann cell ingrowths which are separated from compact myelin by several micra are stained intensely by MAG antiserum. Antiserum directed against Po protein, the major structural protein of compact PNS myelin, does not stain the ingrowths unless compact myelin is present. These results demonstrate the periaxonal localization of MAG and support a functional role for MAG in maintaining the periaxonal space of PNS myelinated fibers.

Myelin-associated glycoprotein (MAG) distribution in human central nervous tissue studied immunocytochemically with monoclonal antibody

Recent biochemical data show that myelin-associated glycoprotein (MAG) is the antigen for a monoclonal antibody found in sera of patients with IgM paraproteinemia and neuropathy (Braun et al. 1982). Immunoreactivity of this antibody with CNS has not been described. To study this, monoclonal anti-MAG was used in the avidin-biotin-peroxidase complex method (Hsu et al. 1981) to immunostain paraffin and epon sections of human CNS. Well characterized polyclonal MAG antiserum (Quarles et al. 1981) was employed in comparison tests. In paraffin sections of developing CNS, both monoclonal and polyclonal MAG antisera stained oligodendroglia and myelin. In adult CNS, periaxonal regions of myelin sheaths were immunostained in paraffin sections and semithin epon sections treated with monoclonal and polyclonal anti-MAG. In electron-microscopic experiments that included milder pretreatment of epon thin sections and more precise reaction product localization, entire thicknesses of myelin sheaths were immunostained. Thus.

Myelin-associated glycoprotein: electron microscopic immunocytochemical localization in compact developing and adult central nervous system myelin.

Light microscopic immunocytochemical studies have shown that myelin-associated glycoprotein (MAG) is localized in myelin of the developing CNS; but in the adult, MAG appears to be restricted to periaxonal regions of myelinated fibers. To extend these observations, we embedded optic nerves of 15-day-old rats, adult rats, and an adult human in epon after aldehyde and osmium tetroxide fixation. After 5% H2O2 pretreatment, thin sections were immunostained with 1:250-1:5,000 rabbit antiserum to rat CNS MAG according to the avidin-biotin-peroxidase complex (ABC) method. Dense deposits of reaction product covered compact myelin in both developing and adult optic nerves. When we used 1:500, 1:1,000, and 1:2,000 anti-MAG, less intense immunostaining of myelin was found. We also obtained the same localization in compact myelin with the peroxidase-antiperoxidase (PAP) method. With 1:250 anti-MAG, dense deposits of reaction product were not observed on axolemmal membranes or on oligodendroglial membranes located periaxonally and paranodally. In thin sections of adult human optic nerve, anti-MAG also stained compact myelin intensely. When thin sections of rat and human optic nerves were treated with preimmune or absorbed serum, no immunostaining was observed. Immunoblot tests showed that our MAG antisera did not react with any non-MAG myelin proteins. In contrast with earlier light microscopic data, this study shows that MAG localization does not change during CNS development; both developing and adult compact myelin sheaths contain MAG. As many biochemical studies also show that MAG is present in compact myelin, we suggest that this 100,000 dalton glycoprotein now be called myelin glycoprotein (MGP) instead of MAG.

Presence of the myelin-associated glycoprotein correlates with alterations in the periodicity of peripheral myelin.

The myelin-associated glycoprotein (MAG) is an integral membrane protein (congruent to 100,000 mol wt) which is a minor component of purified peripheral nervus system (PNS) myelin. In the present study, MAG was localized immunocytochemically in 1-micrometer thick Epon sections of 7-d and adult rat peripheral nerves, and its localization was compared to that of the major structural protein (Po) of PNS myelin. To determine more precisely the localization of MAG, immunostained areas in 1 micrometer sections were traced on electron micrographs of identical areas from adjacently cut thin sections.l MAG was localized in periaxonal membranes. Schmidt-Lantermann incisures, paranodal membranes, and the outer mesaxon of PNS myelin sheaths. Compact regions of PNS myelin did not react with MAG antiserum. The results demonstrate MAG's presence in "'semi-compact" Schwann cell or myelin membranes that have a gap of 12-14 nm between extracellular leaflets and a spacing of 5 nm or more between cytoplasmic leaflets. In compact regions of the myelin sheath which do not contain MAG, the cytoplasmic leaflets are "fused" and form the major dense line, whereas the extracellular leaflets are separated by a 2.0 nm gap appearing as paired minor dense lines. Thus, it is proposed that MAG plays a role in maintaining the periaxonal space, Schmidt-Lantermann incisures, paranodal myelin loops, and outer mesaxon by preventing "complete" compaction of Schwann cell and myelin membranes. The presence of MAG in these locations also suggests that MAG may serve a function in regulating myelination in the PNS.

Immunocytochemical study of P0 glycoprotein, P1 and P2 basic proteins, and myelin-associated glycoprotein (MAG) in lesions of idiopathic polyneuritis.

To investigate the mechanism of myelin breakdown in idiopathic polyneuritis, paraffin and Epon sections of lesions were immunostained with antisera to four proteins in myelin sheaths. Three of these (P0, P2, and BP) are constituents of compact myelin whereas myelin-associated glycoprotein (MAG) is restricted to membranes near Schwann cell cytoplasm in periaxonal and paranodal regions and in Schmidt-Lanterman clefts. In early lesions, there were focal abnormalities in P2, P0, and BP immunostaining of paranodal and internodal myelin. No single protein was affected selectively and lesions occurred in fibres of all sizes, not just in larger fibres selectively stained by P2 antiserum. Early changes in MAG immunostaining occurred only in regions where myelin immunostaining also was abnormal. More severe, late changes in the distribution of P0, P2, and BP and MAG were consistent with the sequence of myelinated fibre alterations seen in segmental demyelination and Wallerian degeneration. In regenerating fibres, MAG antiserum stained periaxonal regions intensely; thin regenerating myelin sheaths were stained by P0, and BP antisera, but not by P2 antiserum. The results show that myelin sheath changes are identified more easily in immunostained sections than in conventional histological preparations. Our data also suggest that in idiopathic neuritis, myelin sheaths are the primary target and that the breakdown of myelin and its proteins is not secondary to Schwann cell damage.