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.     

Oligosaccharide specificities of Phaseolus vulgaris leukoagglutinating and erythroagglutinating phytohemagglutinins. Interactions with N-glycanase-released oligosaccharides

The structural determinants required for interaction of oligosaccharides with leukoagglutinating phytohemagglutinin (L-PHA) and erythroagglutinating phytohemagglutinin (E-PHA) from Phaseolus vulgaris have been studied by immobilized lectin affinity chromatography. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with columns of L- and E-PHA-agarose. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. In virtually all cases, L- and E-PHA yielded identical results, indicating that their specificities for reduced oligosaccharides are similar. Both lectins retarded oligosaccharides bearing alpha 2,3- but not alpha 2,6-linked sialic acid. Desialylated oligosaccharides containing one, two, three, or four peripheral N-acetyllactosamine-type branches were retarded to varying extents by both lectins; however, this interaction was decreased or eliminated by removal of Gal. Desialylated oligosaccharides containing a bisecting GlcNAc residue attached to the beta-linked core Man displayed the greatest interaction with both lectins. Structures containing terminal sulfate or GalNAc did not interact with either lectin. In some instances, the specificities of L- and E-PHA lectins for free, reduced oligosaccharides differed from those established using glycopeptides. Therefore, the structural requirements for interaction with lectins such as L- and E-PHA must be fully and systematically defined using the appropriate authentic standards in order to use lectin affinity chromatography for the fractionation and characterization of free oligosaccharides.     

Sulfated HNK-1 epitope in developing and mature kidney: a new marker for thin ascending loop of Henle and tubular injury in acute tubular necrosis.

The HNK-1 carbohydrate epitope is a 3-sulfo-glucuronyl residue attached to lactosamine structures on glycoproteins, proteoglycans, or glycolipids mostly expressed in the nervous system. Here, using monoclonal antibodies against the sulfated HNK-1 carbohydrate epitope, we first examined its distribution in developing and adult kidneys, then its expression in kidneys with tubular necrosis and renal neoplasms. This HNK-1 epitope was expressed in the human, rabbit, and rat, but not mouse kidney. It was detected within a subset of epithelial cells in the renal vesicle and in comma- and S-shaped bodies during early stages of nephrogenesis. In ureteral bud derivatives, the epitope was present transiently in the area where the collecting duct fused with the nephron. In the adult kidney, expression of the HNK-1 epitope became mainly restricted to the thin ascending loop of Henle where this epitope was carried by heparan- and chondro-proteoglycan. In pathological conditions, HNK-1 epitope expression increased dramatically in proximal epithelial tubule cells in kidneys with acute tubular necrosis. In tumors, the HNK-1 epitope was expressed in the epithelial component of nephroblastomas and in a subgroup of papillary renal cell carcinomas. These data suggest that molecules carrying the sulfated HNK-1 carbohydrate epitope may play an important role in critical stages of renal development and in the physiology of thin ascending loop of Henle.     

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.     

Expression of carbohydrate epitopes L2/HNK-1 and L3 in the larva and imago of Drosophila melanogaster and Calliphora vicina

Summary The carbohydrate epitopes L2/HNK-1 and L3 belong to two overlapping families of adhesion molecules in the vertebrate, and probably the invertebrate nervous systems. To investigate their pattern of expression during the development of insects, cryosections of late third instar larvae and imagoes of Drosophila melanogaster and Calliphora vicina were studied by indirect immunofluorescence using several monoclonal antibodies to the L2/HNK-1 and one monoclonal antibody to the L3 epitope. Each monoclonal antibody to the L2/HNK-1 epitope showed a different immunohistological staining pattern, which differed from that of the L3 monoclonal antibody. In both insect species the immunohistological staining patterns for the two carbohydrate epitopes were similar at the two developmental stages, with immunoreactivity not confined to the nervous system. In larvae, immunoreactivities of the monoclonal antibodies L2.334 and L3.492 were predominantly associated with the extracellular matrix as indicated by co-localization with laminin, particularly in the imaginal discs, while L2.349 revealed a more cell surface-associated distribution. In imagoes, immunoreactivities were detectable in most organs studied.     

Laminin-1 is a novel carrier glycoprotein for the nonsulfated HNK-1 epitope in mouse kidney

The HNK-1 epitope has a unique structure comprising the sulfated trisaccharide (HSO(3)-3GlcAbeta1-3Galbeta1-4GlcNAc), and two glucuronyltransferases (GlcAT-P and GlcAT-S) are key enzymes for its biosynthesis. However, the different functional roles of these enzymes in its biosynthesis remain unclear. Recently, we reported that a nonsulfated form of this epitope, which is biosynthesized by GlcAT-S but not by GlcAT-P, is expressed on two metalloproteases in mouse kidney. In this study, we found that a novel glycoprotein carrying the nonsulfated HNK-1 epitope in mouse kidney was enriched in the nuclear fraction. The protein was affinity-purified and identified as laminin-1, and we also confirmed the N-linked oligosaccharide structure including nonsulfated HNK-1 epitope derived from laminin-1 by mass spectrometry. Curiously, immunofluorescence staining of kidney sections revealed that laminin-1 appeared not to be colocalized with the nonsulfated HNK-1 epitope. However, proteinase treatment strengthened the signals of both laminin-1 and the nonsulfated HNK-1 epitope, resulting in overlapping of them. These results indicate that the nonsulfated HNK-1 epitope on laminin-1 is usually embedded and masked in the robust basement membrane in tight association with other proteins. To clarify the associated proteins and the functional role of the carbohydrate epitope, we investigated the interaction between laminin-1 and alpha-dystroglycan through their glycans in mouse kidney using the overlay assay technique. We obtained evidence that glucuronic acid as well as sialic acid inhibited this interaction, suggesting that the nonsulfated HNK-1 epitope on laminin-1 may regulate its binding and play a role in maintenance of the proper structure in the kidney basal lamina.     

Binding of Erythroagglutinating Phytohemagglutinin Lectin from Phaseolus vulgaris to the Epidermal Growth Factor Receptor Inhibits Receptor Function in the Human Glioma Cell Line, U373 MG

Abstract: Little is known about the role of the N -linked oligosaccharides in the function of the epidermal growth factor (EGF) receptor (EGF-R). In a human glioma cell line, U373 MG, EGF-Rs contain the bisecting N -linked oligosaccharide sequence recognized by erythroagglutinating phytohemagglutinin lectin from Phaseolus vulgaris (E-PHA). Incubation of E-PHA with cultured U373 MG cells results in inhibition of EGF binding to its receptor and consequently inhibition of EGF-induced autophosphorylation of the receptor. Consistent with the inhibitory effects on the EGF-R, phenotypic events that depend on EGF-R signaling, such as cell spreading and proliferation, were also found to be modified. The effect of this lectin seems to be specific because leukoagglutinating phytohemagglutinin lectin from P. vulgaris (L-PHA), an isolectin of E-PHA, had no effect on EGF-R activity or the biological functions of these cells even though L-PHA was able to bind to the EGF-R. These findings suggest the presence of an important bisecting N -linked oligosaccharide structure in close proximity to the EGF binding site on the receptor. Furthermore, these results suggest the possibility that E-PHA lectin binding may provide an additional approach to blocking EGF-dependent glioma cell growth.     

Evaluation of glycoconjugates on the K562 cell surface by means of lectin binding — comparison with human erythrocytes

The binding of five radiolabelled lectins (Vicia graminea, peanut,Phaseolus vulgaris isolectins E-PHA and L-PHA,Evonymus europaeus) to untreated and desialylated K562 cells and human erythrocytes was compared. The number of glycophorin A receptors recognized on the K562 cells by anti-blood group NV. graminea lectin was comparable to that found on the MN or NN erythrocyte surface. However, K562 cells had a severalfold higher number of oligosaccharide chains (presumablyO-glycosidic) which after desialylation became high-affinity receptors for peanut agglutinin, and of complex typeN-glycosidic chains available for the reaction with E-PHA and also with L-PHA (the latter lectin was not bound to erythrocytes). Moreover, K562 cells not treated with neuraminidase had a significant amount of extremely low affinity receptors for peanut agglutinin, whereas binding of this lectin to untreated erythrocytes was undetectable. On the other hand, the untreated K562 cells did not bind anti-blood group B and HE. europaeus lectin, but a small amount of binding by the desialylated cells was observed. Some other differences observed in the mode of lectin binding to K562 cells and erythrocytes are discussed.     

HNK-1 Epitope-carrying Tenascin-C Spliced Variant Regulates the Proliferation of Mouse Embryonic Neural Stem Cells

Neural stem cells (NSCs) possess high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into neuronal and glial cells. NSCs are the source for neurogenesis during central nervous system development from fetal and adult stages. Although the human natural killer-1 (HNK-1) carbohydrate epitope is expressed predominantly in the nervous system and involved in intercellular adhesion, cell migration, and synaptic plasticity, the expression patterns and functional roles of HNK-1-containing glycoconjugates in NSCs have not been fully recognized. We found that HNK-1 was expressed in embryonic mouse NSCs and that this expression was lost during the process of differentiation. Based on proteomics analysis, it was revealed that the HNK-1 epitopes were almost exclusively displayed on an extracellular matrix protein, tenascin-C (TNC), in the mouse embryonic NSCs. Furthermore, the HNK-1 epitope was found to be present only on the largest isoform of the TNC molecules. In addition, the expression of HNK-1 was dependent on expression of the largest TNC variant but not by enzymes involved in the biosynthesis of HNK-1. By knocking down HNK-1 sulfotransferase or TNC by small interfering RNA, we further demonstrated that HNK-1 on TNC was involved in the proliferation of NSCs via modulation of the expression level of the epidermal growth factor receptor. Our finding provides insights into the function of HNK-1 carbohydrate epitopes in NSCs to maintain stemness during neural development.     

Developmentally and spatially regulated expression of HNK-1 carbohydrate antigen on a novel phosphatidylinositol-anchored glycoprotein in rat brain

HNK-1 carbohydrate antigen in an epitope expressed commonly in many cell surface adhesion and recognition molecules in the nervous system. We purified and characterized from rat brain a novel phosphatidylinositol (PI)-anchored 150-kD glycoprotein belonging to the HNK-1 family. The molecule (PI-GP150) was detected by combination of PI-specific phospholipase C treatment of brain membranes and Western blot analysis with mAb HNK-1. HNK-1-positive PI-GP150 was purified from the PI-PLC-released materials with three successive chromatographies (Sephacryl S-300, mAb HNK-1-Sepharose 4B, and Mono Q) and proven to be a novel molecule by immunoblot and structural analyses. Polyclonal antibody was raised against PI-GP150 and used to show that (a) PI-GP150 is expressed on the surface of neuronal cell bodies and their processes in culture, and (b) PI-GP150 appears during embryonic development and is present throughout all postnatal life in all brain regions. However, the expression of the HNK-1 epitope on PI-GP150 is regulated in both developmental stage-specific and region-specific manners. In newborn rats, the HNK-1 epitope is expressed on PI-GP150 throughout the brain. The level of HNK-1 epitope on PI-GP150 decreases after postnatal day 7 in hindbrain and becomes completely absent in adult myelencephalon and metencephalon. In contrast, HNK-1 epitope on PI-GP150 was constitutively expressed in telencephalon. Thus, while the HNK-1 carbohydrate epitope is strongly coupled to PI-GP150, its expression can be regulated independently of that of PI-GP150. The differential expression of the HNK-1 epitope at different rostro-caudal axial levels was observed also in other HNK-1 family molecules in brain membranes. These results suggest that the HNK-1 epitope plays an important role in adding region-specific and developmental stage-specific modifications on the function of the cell surface molecules.     

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.     

Extensive sequence homologies among lectins from leguminous plants

The first twenty five residues of the amino terminal sequence of the β chains from lentil and pea lectins, of soybean and peanut agglutinins, and of the R and L subunits of phytohemagglutinin (PHA) were compared. Extensive homologies were found, ranging from near identity in the case of the β chains of lentil and pea lectins, to 24% identity between soybean agglutinin and L-PHA (assuming two deletions in the latter). Despite different sugar binding specificities, a common ancestry for the genes coding for leguminous lectins appears to be very likely.     

Comparison of the N-Linked Oligosaccharide Structures of the Two Major Human Myelin Glycoproteins MAG and P0: Assessment of the Structures Bearing the Epitope for HNK-1 and Human Monoclonal Immunoglobulin M Found in Demyelinating Neuropathy

The epitope for HNK-1 and patient's monoclonal autoantibodies in demyelinating polyneuropathy associated with immunoglobulin M gammopathy is borne by different types of N-linked oligosaccharide structures in human P0 and myelin-associated glycoprotein (MAG). Fourteen glycopeptide fractions bearing different oligosaccharide structures were obtained from either MAG or P0 glycopeptides by serial lectin affinity chromatography on concanavalin A-Sepharose, Phaseolus vulgaris erythrophytohemagglutinin-agarose, Pisum sativum agglutinin-agarose, and Phaseolus vulgaris leucophytohemagglutinin-agarose. As shown by dot-TLC plate immunostaining, the same MAG and P0 glycopeptide fractions were recognized by HNK-1 and patient's immunoglobulin M, confirming that these antibodies display similar specificities. The antigenic carbohydrate was present in glycopeptide fractions that either interact with Pisum sativum agglutinin-agarose or were bound by Aleuria aurantia agglutinin-digoxigenin, indicating that these structures contained alpha(1-6)fucose residues. This study demonstrates that the L2/HNK-1 epitope is borne mainly or even exclusively by N-linked oligosaccharide structures alpha(1-6)fucosylated in the core.     

Molecular cloning and expression of a second glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope

A cDNA encoding a novel glucuronyltransferase was cloned from a rat brain cDNA library. The cDNA sequence contained an open reading frame encoding 324 amino acids, with type II transmembrane topology. The amino acid sequence revealed 49% homology to rat GlcAT-P, a glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope of glycoproteins, [Terayama et al. (1997) Proc. Natl. Acad. Sci. USA 94, 6093-6098] and the highest sequence homology was found in the catalytic region. Northern blot analysis indicated that this newly cloned glucuronyltransferase is expressed in the nervous system, consistent with the selective localization of the HNK-1 carbohydrate epitope in the nervous system. Transfection of this cDNA into COS-1 cells induced the expression of the HNK-1 carbohydrate epitope on cell surfaces, and induced the morphological changes in these cells. These results indicated that this newly cloned cDNA is a second glucuronyltransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope.     

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.     

Sulfoglycolipids bind to adhesive protein amphoterin (P30) in the nervous system.

HNK-1 antibody reactive carbohydrate epitope carried by glycolipids and glycoproteins has been shown to be involved in cell to cell interactions. It has been proposed that the HNK-1 reactive 3-sulfoglucuronyl carbohydrate epitope in glycolipids may interact with a cell surface receptor to promote the biological response in the developing nervous system. The possible occurrence of such a receptor was examined in rat nervous system. A specific binding of sulfoglycolipids to a 30 kD protein from adult rat cerebellum is described. Little binding was found with neutral glycolipids and gangliosides. The 30 kD protein from cerebellum was partially purified on a sulfatide-octyl-Sepharose affinity column. Binding of sulfoglucuronyl glycolipids to a similar 30 kD protein from forebrain previously identified as amphoterin is also shown. Amphoterin is developmentally regulated and is involved in neural cell adhesion and neurite extension.     

Developmental Expression of HNK-1-Reactive Antigens in the Rat Cerebellum and Localization of Sulfoglucuronyl Glycolipids in Molecular Layer and Deep Cerebellar Nuclei

Monoclonal antibody HNK-1-reactive carbohydrate epitope is expressed on proteins, proteoglycans, and sulfoglucuronyl glycolipids (SGGLs). The developmental expression of these HNK-1-reactive antigens was studied in rat cerebellum. The expression of sulfoglucuronyl lacto-N-neotetraosylceramide (SGGL-1) was biphasic with an initial maximum at postnatal day one (PD 1), followed by a second rise in the level at PD 20. The level of sulfoglucuronyl lacto-N-norhexaosyl ceramide (SGGL-2) in cerebellum was low until PD 15 and then increased to a plateau at PD 20. The levels of SGGLs increased during postnatal development of the cerebellum, contrary to their diminishing expression in the cerebral cortex. The expression of HNK-1-reactive glycoproteins decreased with development of the rat cerebellum from PD 1. Several HNK-1-reactive glycoproteins with apparent molecular masses between 150 and 325 kDa were visualized between PD 1 and PD 10. However, beyond PD 10, only two HNK-1-reactive bands at 160 and 180 kDa remained. The latter appeared to be neural cell adhesion molecule, N-CAM-180. A diffuse HNK-1-reactive band seen at the top of polyacrylamide electrophoretic gels was due mostly to proteoglycans. This band increased in its reactivity to HNK-1 between PD 15 and PD 25 and then decreased in the adult cerebellum. The lipid antigens were shown by two complementary methodologies to be localized primarily in the molecular layer and deep cerebellar nuclei as opposed to the granular layer and white matter. A fixation procedure which eliminates HNK-1-reactive epitope on glycoproteins and proteoglycans, but does not affect glycolipids, allowed selective immunoreactivity in the molecular layer and deep cerebellar nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of recombinant human glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans.

We characterized the recombinant glucuronyltransferase I (GlcAT-I) involved in the glycosaminoglycan-protein linkage region biosynthesis. The enzyme showed strict specificity for Galbeta1-3Galbeta1-4Xyl, exhibiting negligible incorporation into other galactoside substrates including Galbeta1-3Galbeta1-O-benzyl, Galbeta1-4GlcNAc and Galbeta1-4Glc. A comparison of the GlcAT-I with another beta1,3-glucuronyltransferase involved in the HNK-1 epitope biosynthesis revealed that the two beta1,3-glucuronyltransferases exhibited distinct and no overlapping acceptor substrate specificities in vitro. Nevertheless, the transfection of the GlcAT-I cDNA into COS-1 cells induced the significant expression of the HNK-1 epitope. These results suggested that the high expression of the GlcAT-I gene rendered the cells capable of synthesizing the HNK-1 epitope.     

Expression of the HNK-1/NC-1 epitope in early vertebrate neurogenesis

Summary A family of glycoconjugates has recently been shown to share a common carbohydrate epitope recognized by the mouse monoclonal antibody HNK-1. The specificity of HNK-1 was found to be similar to that of another monoclonal antibody, NC-1. These two IgM monoclonal antibodies were raised after immunization of mice with a human T-cell line and avian neural crest-derived ganglia, respectively. The antigens recognized by these antibodies include the myelin-associated glycoprotein, MAG, a glycolipid of defined structure, and a set of molecules involved in cell adhesion. The timing and pattern of appearance of these antigens are distinct. Moreover, the epitope may be absent on an antigen at a given stage or in a given tissue. Therefore, although the molecules able to carry the NC-1/ HNK-1 epitope are numerous and expressed in various tissues, the use of the monoclonal antibodies on tissue sections has proven adequate for following the migration of avian neural crest cells, the major cell lineage recognized by NC-1 and HNK-1 during early embryogenesis. Analogies in several other species have been found on the basis of HNK-1 reactivity. In this study we show that NC-1 and HNK-1 can be used successfully to label migrating neural crest cells in dog, pig and human. On the other hand, the NC-l/HNK-1 epitope was not present on migrating crest cells in amphibians or mice and was found only transiently on the neural crest of rats.     

High Expression of the HNK-1/L2 Carbohydrate Epitope in the Major Glycoproteins of Shark Myelin

The major 24- and 28-kDa glycoproteins in shark PNS and CNS myelin express high levels of the adhesion-associated HNK-1/L2 carbohydrate epitope. The 28-kDa protein, but not the 24-kDa protein, cross-reacts strongly with one of two anti-bovine P0 antisera not previously tested against fish myelin proteins. Shark PNS and CNS myelin also contains smaller amounts of high-molecular-weight HNK-1-positive proteins, including a prominent broad band in the 65-85-kDa range. Although myelin-associated glycoprotein (MAG) is well known to react with HNK-1 in some mammals, monoclonal and polyclonal anti-MAG antibodies did not react with the high-molecular-weight HNK-1-positive material in shark myelin, a result suggesting that it is not a MAG-like protein. The high expression of the HNK-1/L2 epitope in glycoproteins of shark myelin, including the major P0-related ones, suggests that this adhesion-related carbohydrate structure may have had an important role in the molecular evolution of the myelinating process.