Human Myelin/Oligodendrocyte Glycoprotein: A New Member of the L2/HNK-1 Family

Abstract— Myelin/oligodendrocyte glycoprotein (MOG) is a quantitatively minor component of CMS myelin. In this study, human MOG was found to express the L2/HNK-1 epitope on N-linked oligosaccharide structures. This carbohydrate epitope has been found previously in three other characterized human myelin glycoproteins: the my-elin-associated glycoprotein, P₀, and the oligodendrocyte-myelin glycoprotein. It seems, therefore, that the L2/HNK-1 epitope is expressed frequently in human myelin glycoproteins. Serial lectin affinity chromatography of ¹⁴C-glycopeptides indicated that MOG N -oligosaccharide structures are mainly of the complex type, accounting for 77.8% of total radioactivity. In contrast with myelin-asso-ciated glycoprotein and P₀, which express the L2/HNK-1 epitope on fucosylated structures, in MOG the epitope was detected on all glycopeptide fractions obtained by serial lectin affinity chromatography, although a preferential expression of the L2/HNK-1 epitope was observed on fucosylated structures. Finally, the data indicated that, as for other human myelin glycoproteins, only a subpopulation of MOG molecules expresses the L2/HNK-1 epitope.     

Comparison of the Major Proteins of Shark Myelin with the Proteins of Higher Vertebrates

Abstract: On gel electrophoresis in dodecyl sulphate solutions shark CNS myelin showed four bands close in mobility to the proteolipid protein of bovine CNS myelin. They had apparent molecular weights of 21,000, 26,000, 27,000, and 31,500. Unlike bovine proteolipid protein, all of these shark proteins were shown to be glycosylated by staining gels with the periodate-Schiff reagent. Amino acid analyses of the polypeptides eluted from polyacrylamide gels indicated a high content of apolar amino acids and a composition approximating that of the P_o protein of bovine peripheral nervous system (PNS) myelin, rather than that of the CNS proteolipid protein. The shark poly-peptide of apparent molecular weight 31,500 was obtained by elution from dodecyl sulphate gels and antibodies raised against it in rabbits. By probing of electroblots with this antiserum the four shark CNS bands were shown to share common determinants with each other, with a major shark PNS protein and with sheep and chicken major PNS glycoproteins (P_o). The binding of antibody was unaffected by deglycosylation of the shark CNS polypeptides with anhydrous hydrogen fluoride. Together, these results appeared to establish that shark CNS myelin contains four proteins that are closely related to a major shark PNS protein and to the P_o protein of higher species.     

Developmental Expression of HNK-1-Reactive Antigens in Rat Cerebral Cortex and Molecular Heterogeneity of Sulfoglucuronylneolactotetraosylceramide in CNS Versus PNS

Monoclonal antibody HNK-1 reacts with a carbohydrate epitope present in proteins, proteoglycans, and sulfoglucuronylglycolipids (SGGLs). On high-performance TLC plates, SGGLs of the CNS from several species migrated consistently slower than those from the PNS, a result indicating possible differences in the structures. The structural characteristics of the major SGGL, sulfoglucuronylneolactotetraosylceramide (SGGL-1), from CNS was compared with those of SGGL-1 from PNS. Although the composition, sequence, and linkages of the carbohydrate moiety of the SGGL-1 species were identical, SGGL-1 from CNS contained mainly short-chain fatty acids, 16:0, 18:0, and 18:1, amounting to 85% of the total fatty acids, whereas SGGL-1 from PNS contained large proportions (59%) of long-chain fatty acids (greater than 18:0). These differences in the fatty acid composition accounted for the different migration pattern observed. The developmental expression of SGGLs and HNK-1-reactive proteins was studied in rat cerebral cortex between embryonic day (ED) 15 to adulthood. SGGLs in the rat cortex were maximally expressed around ED 19 and almost completely disappeared by postnatal day (PD) 20. This expression was contrary to their increasing expression in the cerebellum and sciatic nerve with postnatal development. Six to eight protein bands with a molecular mass of greater than 160 kDa were HNK-1 reactive in the rat cerebral cortex at different ages. The major HNK-1 reactivity to the 160-kDa protein band seen in ED 19 to PD 10 cortex decreased and completely disappeared from the adult cortex, whereas several other proteins remained HNK-1 reactive even in the adult. Western blot analyses of the neural cell adhesion molecules (N-CAMs) during development of the rat cortex with a polyclonal anti-N-CAM antibody showed that the major HNK-1-reactive protein bands were not N-CAMs. Between PD 1 and 10, 190-200-kDa N-CAM was the major N-CAM, and between PD 15 to adulthood, 180-kDa N-CAM was the only N-CAM present in the rat cortex.     

Rapid Communication: Human Peripheral Myelin Protein-22 Carries the L2/HNK-1 Carbohydrate Adhesion Epitope

Abstract: Molecular genetic studies have established that mutations in the gene encoding the 22-kDa peripheral myelin protein (PMP-22) are responsible for hereditary peripheral neuropathies in the trembler mouse and in a subset of humans with Charcot-Marie-Tooth disease, type 1a. The function of the PMP-22 protein remains unknown. Several studies on myelin proteins in the PNS have indicated that the L2/HNK-1 epitope, which is believed to be both a ligand for cellular adhesion and a target for autoimmune monoclonal IgM neuritis, may be found on heretofore unidentified proteins with a molecular mass of 19–28 kDa. In this report, we provide immunological evidence that at least one of these proteins is PMP-22.     

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.     

Detection of Multisulphated N-Linked Glycans in the L2/HNK-1 Carbohydrate Epitope Expressing Neural Adhesion Molecule P0

P0, the most abundant glycoprotein of PNS myelin, is a homophilic and heterophilic adhesion molecule. P0 is known to contain a glycoform population that expresses the L2/HNK-1 carbohydrate epitope found on other neural adhesion molecules, and to be functionally implicated centrally in neural cell adhesion and neurite outgrowth. This carbohydrate epitope has been characterized previously from glycolipid structures and contains a sulphated glucuronic acid residue. However, the L2/HNK-1 carbohydrate epitope has not been characterized in glycoproteins. Because P0 possesses only one glycosylation sequon, the number of P0 glycoforms is equal to the heterogeneity of the glycan species. Here we report that the carbohydrate analysis of L2/HNK-1-reactive P0 showed the presence of anionic structures containing sialic acid and sulphate in various combinations. At least one sulphate residue was present in 80% of the monosaccharide sequences, and 20% contained three sulphates. High-resolution P4 gel chromatography of the desialylated and desulphated oligosaccharides showed substantial heterogeneity of monosaccharide sequences. Sequential exoglycosidase digestions indicated that the majority of the structures were of the hybrid class, although the sulphated structures were found to be endoglycosidase H-resistant.     

Expression and biological functions of sulfoglucuronyl glycolipids (SGGLs) in the nervous system—A review

Sulfoglucuronyl carbohydrate linked to neolactotetraose reacts with HNK-1 antibody. The HNK-1 carbohydrate epitope is found in two major glycolipids, several glycoproteins and in some proteoglycans of the nervous system. Most of the HNK-1 reactive glycoproteins so far identified are neural cell adhesion molecules and/or are involved in cell-cell interactions. HNK-1 carbohydrate is highly immunogenic. Several HNK-1-like antibodies, including IgM of some patients with plasma cell abnormalities and having peripheral neuropathy, have been described. This article summarizes published work mainly on sulfoglucuronyl glycolipids, SGGLs and covers: structural requirements of the carbohydrate epitope for binding to HNK-1 and human antibodies, expression of the lipids in various neural areas, stage and region specific developmental expression in CNS and PNS, immunocytochemical localization, loss of expression in Purkinje cell abnormality murine mutations, biosynthetic regulation of expression by a single enzyme N-acetylglucosaminyl transferase, identification of receptor-like carbohydrate binding neural proteins (lectins), and perceived role of the carbohydrate in physiological functions. The latter includes role in: pathogenesis of certain peripheral neuropathies, in migration of neural crest cells, as a ligand in cell-cell adhesion/interaction and as a promoter of neurite outgrowth for motor neurons. Multiple expression of HNK-1 carbohydrate in several molecules and in various neural cell types at specific stages of nervous system development has puzzled investigators as to its specific biological function, but this may also suggest its importance in multiple systems during cell differentiation and migration processes.Special issue dedicated to Dr. Marjorie B. Lees.     

Human Neuroectoderm-Derived Cell Line Secretes Fibronectin that Shares the HNK-1/10C5 Carbohydrate Epitope with Neural Cell Adhesion Molecules

A human malignant melanoma cell line, Melur, secretes several glycoproteins that contain a unique carbohydrate epitope shared by neural cell adhesion molecules and recognized by the monoclonal antibodies HNK-1, L2, and 10C5. In this report, we present evidence that one of the major melanoma glycoproteins containing the HNK-1/10C5 epitope is the cell adhesion molecule, fibronectin, or a fibronectin-like molecule. Melanoma-derived fibronectin was isolated from serum-free conditioned medium by gelatin-Sepharose affinity adsorption and shown to react with monoclonal antibodies HNK-1 and 10C5 in Western blot analysis. HNK-1-containing fibronectin was purified on a gelatin-Sepharose column followed by an affinity column using a monoclonal antibody against the HNK-1 carbohydrate. The purified HNK-1-fibronectin then could be incorporated into the extracellular matrix of hamster fibroblasts in vitro, and such a matrix was detectable using the HNK-1 monoclonal antibody in an immunofluorescence assay. Of the seven neuroectoderm-derived tumor cell lines tested, only the Melur melanoma cell secreted fibronectin containing the HNK-1 carbohydrate. Identification of human neuroectoderm-derived fibronectin as a potential carrier of the HNK-1 carbohydrate suggests a new role for fibronectin in neural development and regeneration, and represents a new model for studying the function of this carbohydrate domain in neural cell adhesion.     

Effect of different fixatives on immunocytochemical localization of HNK-1-reactive antigens in cerebellum: a method for differentiating the localization of the same carbohydrate epitope on proteins vs lipids.

Monoclonal antibody (MAb) HNK-1 recognizes a carbohydrate epitope present in certain glycolipids, glycoproteins, and proteoglycans. Five different fixation methods, together with biochemical analyses of the antigens, were evaluated to study immunocytochemical localization of this epitope in layers of adult rat cerebellum; 4% paraformaldehyde/0.5% cetylpyridinium chloride was found to be optimal for overall immunoreactivity, and the antigens were apparent in all cerebellar layers. To differentially localize HNK-1-reactive carbohydrate epitope on proteins vs lipids in cerebellar layers, we tested the effect of 0.2%, 2%, or 4% glutaraldehyde combined with 2% paraformaldehyde (GT/PF) on HNK-1 and other MAb-reactive protein and lipid antigens; 2% or 4% GT/PF significantly reduced or abolished immunoreactivity of MAb HNK-1 and 5F9 (reacting with microtubule-associated protein 2) with cerebellar proteins analyzed on Western blots, but did not decrease HNK-1 reactivity to lipid antigens on HPTLC blots. In cerebellar tissue sections, HNK-1 and 5F9 immunoreactivity was reduced after 2% or 4% GT/PF fixation. However, significant amounts of HNK-1 immunoreactivity remained in molecular layer and deep cerebellar nuclei. GT/PF fixation did not cause significant changes in immunoreactivity patterns of other carbohydrate lipid antigens, such as those that react with MAb A2B5, 7A, and WCC4. Therefore, carbohydrate epitope on lipids, as opposed to that on proteins, may be preferentially detectable by immunocytochemistry after fixation with 2% or 4% GT/PF. The selective localization of HNK-1-reactive carbohydrate in the molecular layer and deep cerebellar nuclei with 2% or 4% GT/PF fixation correlates well with the observed presence of HNK-1-reactive lipids in these areas but not in the granular layer and white matter, as determined by microdissection of the individual layers and biochemical analysis. The application of 2% or 4% GT/PF fixation as a general method for differentiating the same carbohydrate epitope on proteins vs lipids in immunocytochemistry for other tissues and other antibodies remains to be further evaluated.     

Detection of the L2/HNK-1 Carbohydrate Epitope on Glycoproteins and Acidic Glycolipids of the Insect Calliphora vicina

The same or a very similar carbohydrate determinant, as represented by some sulfated, glucuronic acid-containing glycosphingolipids of human peripheral nerve, occurs on several adhesion molecules in the mammalian nervous system. In the present study, the occurrence of this epitope on glycoproteins and glycolipids of the fly, Calliphora vicina, was investigated by Western blot analysis and thin-layer chromatogram immunostaining. Several monoclonal antibodies recognizing an epitope on various neural cell adhesion molecules, designated L2 (334, 336, 349, and 412); the monoclonal antibody HNK-1 (recognizing an epitope on human natural killer cells); and a human IgM M-protein were found to react by Western blot analysis with various glycoproteins from larval and adult brains, although the intensity of staining of bands recognized by each antibody varied. Acidic glycolipids from pupae were also recognized, but only by the L2 antibody 334 and IgM M-protein. After desulfation of the acidic glycolipid fraction, the immunostaining pattern remained the same, an observation suggesting that the L2/HNK-1 epitope on insect acidic glycolipids contains a nonsulfated, glucuronic acid moiety. These observations indicate that the L2/HNK-1 carbohydrate structure occurs not only in vertebrates but also in insects on both glycoproteins and glycolipids, a finding suggesting a high degree of phylogenetic stability of this functionally important carbohydrate.     

Antigenic sites common to major fish myelin glycoproteins (IP) and to major tetrapod PNS myelin glycoprotein (Po) reside in the amino acid chains

The major myelin glycoproteins in the CNS and PNS of trout (IP) were enzymatically deglycosylated with endoglycosidase F (Endo F) and examined by electro-immunoblotting. Following carbohydrate removal and loss of concanavalin A affinity each of the four IP components underwent a similar reduction in molecular size, corresponding to approximately 3,000 daltons. Immunological cross-reactivities with anti-bovine P_o or anti-trout IP2 antibodies, were however fully retained by the Endo F cleavage products. This strongly implies that the antigenic sites shared by the mammalian P_o protein and the various intermediate glycoproteins of trout CNS and PNS are located in the protein portion. Immunoblot analysis of the PNS myelin proteins from various species of the major vertebrate classes with anti-trout IP2 antiserum revealed striking differences in the immunological properties of the individual P_o components which were not detected when anti-bovine P_o antiserum was used as a probe.     

Peripheral Nervous System Myelin and Schwann Cell Glycoproteins: Identification by Lectin Binding and Partial Purification of a Peripheral Nervous System Myelin-Specific 170,000 Molecular Weight Glycoprotein

Radioiodinated lectins were used to detect glycoproteins of peripheral nervous system (PNS) myelin (rat, human, bovine) and cultured rat Schwann cells. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and transferred to nitrocellulose filters. The filters were overlaid with radioiodinated lectins of known saccharide affinities. These included concanavalin A, Helix pomatia, Limulus polyphemus, Maclura pomifera, peanut, soybean, Ulex europaeus, and wheat germ agglutinins. Inclusion of the appropriate monosaccharide in the overlay solution (0.2 M) inhibited lectin binding to the nitrocellulose-fixed proteins. Fluorography permitted identification of 26 myelin glycoproteins and many more in Schwann cells. All lectins labeled a band present in myelin, but not Schwann cells, corresponding to the major PNS myelin protein, P0. Our attention focused on a high-molecular-weight myelin glycoprotein [apparent molecular weight (Mr) 170,000], which appeared abundant by Coomassie Blue staining and which was heavily labeled by all lectins except concanavalin A. A protein with approximately this Mr and lectin-binding pattern was present in human and bovine PNS myelin as well, but not detected in rat Schwann cells, CNS myelin, liver and fibroblast homogenates, or cultured bovine oligodendroglia. Hence this 170,000 Mr glycoprotein is apparently unique to PNS myelin.     

Expression of a P0-like glycoprotein in central nervous system myelin of amphibians (Ambystoma mexicanus,xenopus laevis and Rana catesbeiana)

• 1.1. The myelin protein profiles in the CNS and PNS of three species of amphibians were analyzed by biochemical and immunohistochemical methods.
• 2.2. The CNS myelin of the African clawed frog (Xenopus) and the Mexican salamander (axolotl) contained, in addition to proteolipid protein, a unique protein zero (P0)-like protein, whereas the adult bullfrog did not.
• 3.3. A strong expression of the P0-like protein in the bullfrog CNS myelin was found transiently at ontogenetically early phases including at the time of metamorphosis.
• 4.4. The CNS P0-like protein and the PNS P0 protein showed a difference in reactivity with lectins and anti-L2/HNK-1 antibodies, suggesting that the two proteins differ in some aspects of their carbohydrate structures.

     

Preparation and Characterization of Antibodies Against a Sulfated Glucuronic Acid-Containing Glycosphingolipid

In some patients with demyelinating neuropathy there are immunoglobulin M paraproteins that react with carbohydrate determinants shared by myelin-associated glycoprotein (MAG) and two peripheral nerve acidic glycolipids, termed sulfoglucuronosylglycosphingolipids (SGGLs). To study the antigenicity of these glycolipids, we immunized three New Zealand white rabbits with sulfoglucuronosylparagloboside (SGPG), a major SGGL in peripheral nerve, emulsified in Freund's complete adjuvant and keyhole limpet hemocyanin. All three rabbits inoculated with SGPG showed weight loss and mild weakness, predominantly in their hind feet, 2-5 weeks postinoculation (PI). Two of the three rabbits again showed moderate weakness 3 and 8 months PI, respectively. Electrophysiological studies demonstrated a slowed nerve conduction velocity in the sciatic nerve. Anti-SGPG antibody titers in sera were detected at dilutions of 1:1,000 to 1:2,500 by an enzyme-linked immunosorbent assay. Although all three rabbit sera reacted with SGGLs, two reacted with a desulfated form of SGPG and the other did not, suggesting a fine heterogeneity in antigenic specificity. As with sera from patients with demyelinative paraproteinemia, all rabbit sera reacted with MAG in human CNS and PNS myelin. They also reacted with MAG from bovine CNS myelin as well as several low-molecular-weight glycoproteins in bovine peripheral nerve myelin. Thus, we demonstrated that the rabbit antisera generated against SGPG have the same or similar antigenic specificity as those of the anti-MAG M-proteins from patients with neuropathy. The results suggest that an autoimmune response against the sulfoglucuronosyl residue may participate in the immunopathogenesis of this type of neuropathy.     

ASTROGLIAL UPTAKE IS MODULATED BY EXTRACELLULAR K+

Abstract— Developmental changes of myelin proteins in chick sciatic nerve were studied at the stage of myelination by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The myelin of adult hen peripheral nervous system (PNS) contained two glycoproteins (BR-P0 and PASII), both of which are unique to PNS myelin, in addition to the basic encephalitogenic protein, BP, which is common to CNS and PNS myelin. The other basic protein (BF-P2) found in the PNS of other species was not definitely detectable in hen PNS. At the early stages of myelination (from 14 to 18 embryonic days) the amounts of myelin proteins increased rapidly in parallel with the increase in number of layers of the myelin sheath of the PNS. At 14 embryonic days high molecular weight proteins were dominant, while myelin specific proteins were barely detectable in the PNS myelin fraction. At 18 embryonic days, however, BR-PO, BP and PASII proteins became the main protein components of the PNS myelin, whereas high molecular weight proteins decreased in quantitative importance during development. At the early stage of myelination other glycoproteins were also detectable in the PNS myelin. Radioactive fucose was actively incorporated into the two glycoproteins, BR-P0 and PASII, at the early stage of myelination in vivo. These results suggested that myelin proteins especially glycoproteins, may play an important role in PNS myelin formation.     

Appearance of Myelin proteins during vertebrate evolution

Myelin, defined as an arrangement of spirally fused unit membranes, is an acquisition of vertebrates and first appeared during evolution in Gnathostomata. In all species studied PNS and CNS myelins contain the myelin-associated glycoprotein (MAG) and the myelin basic protein (MBP). Throughout phylogeny PNS myelin is characterized by the major P₀ glycoprotein which is called IP in fishes. The PNS myelin proteins did not evolve further except for the addition of P₂ protein from reptiles onward. In Elasmobranchii and Chondrostei, PNS and CNS myelin proteins are similar. CNS myelin of actinopterygian fishes possesses a 36,000 Da protein (36K) in addition to P₀-like IP glycoproteins. In tetrapod CNS myelin, P₀ is replaced by the proteolipid protein (PLP) and the Wolfgram protein (WP). Of particular interest in a transitional phylogenetic sense are the lungfish Protopterus, carrying glycosylated PLP (g-PLP) but no P₀, 36K or WP, and the bichir Polypterus, showing simultaneous presence of P₀, 36K and PLP.

These results indicate that myelin proteins could be valuable molecular markers in establishing vertebrate phylogenetic relationships and in reconstructing the fish-tetrapod transition.     

Myelin-Associated Glycoprotein Is the Antigen for a Monoclonal IgM in Polyneuropathy

Recent studies show that IgM monoclonal antibody from patients with IgM paraproteinemia and peripheral neuropathy reacts with a protein component of human PNS myelin and an analogous component or components of human CNS myelin. We have now demonstrated that the antigen for this antibody is a specific glycoprotein component of myelin, referred to as myelin-associated glycoprotein (MAG). Human PNS and CNS myelin proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on pore-gradient slabs, and MAG was identified by the immuno-electroblot procedure with rabbit anti-MAG (rat). The identical band(s) were stained by an analogous procedure with patient serum as the first antibody. Human PNS MAG had an apparent molecular weight of 107,000. Human CNS MAG appeared as three bands: 113,000, 107,000, and 92,000. Passage of myelin proteins through a concanavalin A-Sepharose column removed the staining component. Purified patient IgM, added to a lithium diiodosalicylate extract of myelin, immunoprecipitated MAG. This antibody also cross-reacted with MAG from bovine CNS, but not from rabbit, rat, or mouse.     

Myelin Structure and Composition in Zebrafish

To establish a standard for genotype/phenotype studies on the myelin of zebrafish (Danio rerio), an organism increasingly popular as a model system for vertebrates, we have initiated a detailed characterization of the structure and biochemical composition of its myelinated central and peripheral nervous system (CNS; PNS) tissues. Myelin periods, determined by X-ray diffraction from whole, unfixed optic and lateral line nerves, were approximately 153 and approximately 162 Angstrom, respectively. In contrast with the lability of PNS myelin in higher vertebrates, zebrafish lateral line nerve myelin exhibited structural stability when exposed to substantial changes in pH and ionic strength. Neither optic nor lateral line nerves showed swelling at the cytoplasmic apposition in CaCl(2)-containing Ringer's solution, in contrast with nerves from other teleost and elasmobranch fishes. Zebrafish optic nerve showed greater stability against changes in NaCl and CaCl(2) than lateral line nerve. The nerves from zebrafish having mutations in the gene for myelin basic protein (mbpAla2Thr and mbpAsp25Val) showed similar myelin periods as the wildtype (WT), but gave approximately 20% less compact myelin. Analysis of proteins by SDS-PAGE and Western blotting identified in both CNS and PNS of WT zebrafish two orthologues of myelin P0 glycoprotein that have been characterized extensively in trout--intermediate protein 1 (24 kDa) and intermediate protein 2 (28 kDa). Treatment with endoglycosidase-F demonstrated a carbohydrate moiety of approximately 7 kDa, which is nearly threefold larger than for higher vertebrates. Thin-layer chromatography for lipids revealed a similar composition as for other teleosts. Taken together, these data will serve as a baseline for detecting changes in the structure and/or amount of myelin resulting from mutations in myelin-related genes or from exogenous, potentially cytotoxic compounds that could affect myelin formation or stability.     

Immunocytological localization of the HNK-1 carbohydrate in murine cerebellum,hippocampus and spinal cord using monoclonal antibodies with different epitope specificities

The HNK-1 carbohydrate, an unusual 3′-sulfated glucuronic acid epitope characteristic of many neural recognition molecules, serves as a ligand in neural cell interactions and is differentially expressed in the quadriceps and saphenous branches of the femoral nerve in the PNS of adult mice. Based on these observations, we investigated the possibility that the HNK-1 carbohydrate may be differentially distributed in neurons and fiber tracts also in the CNS thereby contributing to different targeting and guidance mechanisms. We have used antibodies with different HNK-1 epitope specificities to probe for subtle differences in expression patterns. In the adult mouse cerebellum the HNK-1 carbohydrate is detectable in stripe-like compartments in the molecular and Purkinje cell layers, whereas N-CAM and its associated α2,8 polysialic acid does not show this compartmentation. In the adult hippocampus, the HNK-1 carbohydrate localizes to perineuronal nets of inhibitory interneurons and marks the inner third of the molecular layer of the dentate gyrus. In the adult spinal cord, HNK-1 labeling is most pronounced in gray matter areas. White matter enriched regions show differential labeling with regard to fiber tracts and antibody specificity. Whereas the different antibodies do not show differences in staining in the cerebellum and the hippocampus, they show differences in staining pattern of fiber tracts and motoneurons in the spinal cord. The HNK-1 expression pattern also differed in the adult spinal cord from that observed at embryonic day 14 and postnatal day 14. Our observations suggest a functional role in the specification of functionally discrete compartments in different areas of the CNS and during development.