Sulfoglucuronyl Glycolipids Bind Laminin

Previous studies have shown that HNK-1 antibody reactive glycoconjugates, including the glycolipids 3-sulfoglucuronylneolactotetraosylceramide (SGGL-1) and 3-sulfoglucuronylneolactohexaosylceramide (SGGL-2), are temporally and spatially regulated antigens in the developing mammalian cortex. Extracellular matrix glycoprotein laminin is involved in cell adhesion by interacting with cell surface components and also promotes neurite outgrowth. Laminin has been shown to bind sulfatide. The interaction of sulfated glycolipids SGGL-1 and SGGL-2 with laminin was studied by employing a solid-phase radioimmunoassay and by HPTLC-immunoblotting. Laminin binding was detected with anti-laminin antibodies followed by 125I-labelled Protein A and autoradiography. Laminin binds SGGL-1 and SGGL-2, besides sulfatide, but does not bind significantly gangliosides and neutral glycolipids. The binding of SGGLs to laminin was two to three times less compared to sulfatide when compared on a molar basis. Desulfation of SGGLs and sulfatide by mild acid treatment resulted in abolition of laminin binding. On the other hand, chemical modification of glucuronic acid moiety by either esterification or reduction of the carboxyl group had no effect. This showed that the sulfate group was essential for laminin binding. Of the various glycosaminoglycans tested, only heparin inhibited the binding of laminin to SGGLs and sulfatide in a dose-dependent manner. This indicated that SGGLs and sulfatide bind to the heparin binding site present in the laminin molecule. The availability of HNK-1 reactive glycolipids and glycoproteins such as SGGLs and several neural cell adhesion molecules to bind laminin at critical stages of neural development may serve as important physiological signals.     

Identity of nuclear high-mobility-group protein, HMG-1, and sulfoglucuronyl carbohydrate-binding protein, SBP-1, in brain

High-mobility-group (HMG) proteins are a family of non-histone chromosomal proteins which bind to DNA. They have been implicated in multiple aspects of gene regulation and cellular differentiation. Sulfoglucuronyl carbohydrate binding protein, SBP-1, which is also localized in the neuronal nuclei, was shown to be required for neurite outgrowth and neuronal migration during development of the nervous system. In order to establish relationship between SBP-1 and HMG family proteins, two HMG proteins were isolated and purified from developing rat cerebellum by heparin-sepharose and sulfatide-octyl-sepharose affinity column chromatography and their biochemical and biological properties were compared with those of SBP-1. Characterization by high performance liquid chromatography--mass spectrometry (HPLC-MS), partial peptide sequencing and western blot analysis showed the isolated HMG proteins to be HMG-1 and HMG-2. Isoelectric focusing, HPLC-MS and peptide sequencing data also suggested that HMG-1 and SBP-1 were identical. Similar to SBP-1, both HMG proteins bound specifically to sulfated glycolipids, sulfoglucuronylglycolipids (SGGLs), sulfatide and seminolipid in HPTLC-immuno-overlay and solid-phase binding assays. The HMG proteins promoted neurite outgrowth in dissociated cerebellar cells, which was inhibited by SGGLs, anti-Leu7 hybridoma (HNK-1) and anti-SBP-1 peptide antibodies, similar to SBP-1. The proteins also promoted neurite outgrowth in explant cultures of cerebellum. The results showed that the cerebellar HMG-1 and -2 proteins have similar biochemical and biological properties and HMG-1 is most likely identical to SBP-1.     

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.     

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)     

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.     

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.     

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.     

Carbohydrate-protein interactions between HNK-1-reactive sulfoglucuronyl glycolipids and the proteoglycan lectin domain mediate neuronal cell adhesion and neurite outgrowth

Lecticans, a family of chondroitin sulfate proteoglycans, represent the largest group of proteoglycans expressed in the nervous system. We previously showed that the C-type lectin domains of lecticans bind two classes of sulfated cell surface glycolipids, sulfatides and HNK-1-reactive sulfoglucuronylglycolipids (SGGLs). In this paper, we demonstrate that the interaction between the lectin domain of brevican, a nervous system-specific lectican, and cell surface SGGLs acts as a novel cell recognition system that promotes neuronal adhesion and neurite outgrowth. The Ig chimera of the brevican lectin domain bind to the surface of SGGL-expressing rat hippocampal neurons. The substrate of the brevican chimera promotes adhesion and neurite outgrowth of hippocampal neurons. The authentic, full-length brevican also promotes neuronal cell adhesion and neurite outgrowth. These activities of brevican substrates are neutralized by preincubation of cells with HNK-1 monoclonal antibodies and by pretreatment of the brevican substrates with purified SGGLs. Brevican and HNK-1 carbohydrates are coexpressed in specific layers of the developing hippocampus where axons from entorhinal neurons elongate. Our observations suggest that cell surface SGGLs and extracellular lecticans comprise a novel cell-substrate recognition system operating in the developing nervous system.     

L1/HNK-1 Carbohydrate- and β1 Integrin-Dependent Neural Cell Adhesion to Laminin-1

Abstract: We have shown recently that mouse small cerebellar neurons adhere to a short amino acid sequence of the G2 domain of the laminin α1 chain via the cell surface-expressed HNK-1 carbohydrate. Therefore, we were interested in identifying glycoproteins carrying the HNK-1 carbohydrate at the cell surface of these neurons. Adhesion of small cerebellar neurons to laminin is partially dependent on Ca²⁺, Mn²⁺, and Mg²⁺, indicating the involvement of integrins, which were identified as β1, α3, and α6. They could be shown to bind to laminin by a β1-dependent adhesion mechanism. None of these subunits was found to carry the HNK-1 carbohydrate. HNK-1-immunoreactive glycoproteins were immunoprecipitated and shown to consist of predominantly one molecular species, which was identified as the neural cell recognition molecule L1. L1 was demonstrated to bind in a concentration-dependent and saturating manner to laminin. The binding could be partially inhibited by Fab fragments of monoclonal antibodies against the HNK-1 carbohydrate and against the Ig-like domains of L1. Furthermore, antibodies to the Ig-like domains of L1 and β1 integrin inhibited partially cell adhesion to laminin. Determination of the association of L1, β1 integrin, and the HNK-1 carbohydrate on the cell surface of live cerebellar neurons by antibody-induced patching and copatching revealed HNK-1 to be linked to L1, but less so to β1 integrin. However, only negligible association was found between L1 and β1 integrin. Furthermore, it could be shown that adhesion to laminin is mediated by L1/HNK-1- and β1 integrin-dependent mechanisms that act at least partially independent of each other.     

Loss of Sulfoglucuronyl and Other Neolactoglycolipids in Purkinje Cell Abnormality Murine Mutants

A significant reduction in the content of two members of the sulfoglucuronyl-neolacto series of glycolipids (SGGLs), 3-sulfoglucuronyl-lacto-N-neotetraosylceramide (SGGL-1) and 3-sulfoglucuronyl lacto-N-norhexaosylceramide (SGGL-2), in the cerebellum of the Purkinje cell abnormality mutants, Purkinje cell degeneration (pcd/pcd), lurcher (Lc/+), and staggerer (sg/sg), was also confirmed in the mildly affected nervous (nr/nr) mutant. The expression of SGGLs was studied during development of the pcd/pcd mutant cerebellum, and it was shown that the rate of decline in the level of SGGLs practically coincided with the loss of Purkinje cell perikarya. This indicated that SGGLs are primarily localized in Purkinje cells and that initially, at least, there is no genetic defect in the biosynthesis of SGGLs in the mutant. The precursors of SGGLs, viz., lacto-N-neotetraosylceramide (paragloboside) and lacto-N-norhexaosylceramide, as well as other glycolipids derived from these precursors, such as X-determinant fucoglycolipids and disialosyllacto-N-neotetraosylceramide, were also present in normal cerebellum. Levels of paragloboside and its other derivatives, similar to SGGLs, were also significantly reduced in the Purkinje cell abnormality mutants pcd/pcd, sg/sg, Lc/+, and nr/nr but were normal in other cerebellar mutants, such as quaking (qk/qk), weaver (wv/wv), and reeler (rl/rl), where Purkinje cells are not involved. Thus, the entire paragloboside family of glycolipids is primarily associated with Purkinje cells in the cerebellum. Although levels of monoclonal antibody HNK-1-reactive glycolipids were reduced in the Purkinje cell abnormality mutants, HNK-1-reactive glycoproteins were not affected in these mutants.     

ATP-Evoked Arachidonic Acid Mobilization in Astrocytes Is via a P2Y-Purinergic Receptor

The mouse monoclonal antibody HNK-1 and the human monoclonal IgM antibody present in patients with polyneuropathy both recognize carbohydrate epitope(s) on human myelin-associated glycoprotein and P0. In the present study, the oligosaccharide structures that bear the antibody epitope(s) were investigated. The extracellular derivative of myelin-associated glycoprotein (dMAG) was purified by immunoaffinity chromatography. P0 was electroeluted from gel slices. Western blot analysis of whole glycoproteins demonstrated that the epitopes for HNK-1 and the human monoclonal IgM antibody were different. The glycopeptides obtained by proteolysis of purified dMAG and P0 were separated and characterized by affinity chromatography on concanavalin A-Sepharose. Both dMAG and P0 displayed heterogeneity in their oligosaccharide structures, i.e., they both contained mainly tri- and tetraantennary oligosaccharides (approximately 80%), although biantennary (10%) and high-mannose and/or hybrid (10%) oligosaccharides were present. The human monoclonal IgM antibody epitope was present on all types of isolated oligosaccharide structures from either dMAG and P0. The HNK-1 epitope was present on all types of oligosaccharide structures of dMAG, whereas it was present only on tri- and tetraantennary structures of P0.     

HNK-1 carbohydrate synthesis in retinoic acid-induced P19 cells

Sulfoglucuronyl carbohydrate (SGC), reactive with HNK-1 antibody, is expressed in several glycolipids, glycoproteins and proteoglycans of the nervous system. The interaction of SGC with SGC-binding protein, SBP-1 has been implicated in cell-cell recognition, neurite outgrowth and neuronal migration during development. In sulfotransferase (ST) null mutant mice, which lack SGC, synaptic transmission in pyramidal cells of the hippocampus was increased and long-term potentiation was reduced. However, ST null mice are viable, fertile and have wild type anatomy of all major brain areas and many non-neural organs. Failure to observe severe phenotype in the ST null mice prompted us to determine the compensatory molecular replacement of SGC by analyzing the carbohydrate of glycolipids and glycoprotefins of the mutant nervous system. In the ST null mice, SGC containing molecules were absent and they were replaced by the precursor glucuronyl carbohydrate (GC) containing molecules. Other relevant glycolipids and proteins were not affected. The GC molecules in the mutant were localized at the same anatomical sites as the SGC molecules in the wild type. In vitro binding studies showed that similar to sulfoglucuronyl glycolipids, glucuronyl glycolipids interacted with SBP-1, but with a lower binding capacity. In vitro studies with explant cultures of cerebellum indicated that neurite outgrowth and cell migration were not significantly affected, possibly due to interaction of SBP-1 with the GC molecules. The results indicated that in vivo SBP-1–GC interaction was sufficient enough for normal neurite outgrowth and cell migration in the mutant and thus having a minimal abnormal phenotype.     

A non-sulfated form of the HNK-1 carbohydrate is expressed in mouse kidney

The HNK-1 carbohydrate, which is recognized by anti-HNK-1 antibody, is well known to be expressed predominantly in the nervous system. The characteristic structural feature of the HNK-1 carbohydrate is 3-sulfo-glucuronyl residues attached to lactosamine structures (Gal beta1-4GlcNAc) on glycoproteins and glycolipids. The biosynthesis of the HNK-1 carbohydrate is regulated mainly by two glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase. In this study, we found that GlcAT-S mRNA was expressed at higher levels in the kidney than in the brain, but that both GlcAT-P and HNK-1 sulfotransferase mRNAs, which were expressed at high levels in the brain, were not detected in the kidney. These results suggested that the HNK-1 carbohydrate without sulfate (non-sulfated HNK-1 carbohydrate) is expressed in the kidney. We substantiated this hypothesis using two different monoclonal antibodies: one (anti-HNK-1 antibody) requires sulfate on glucuronyl residues for its binding, and the other (antibody M6749) does not. Western blot analyses of mouse kidney revealed that two major bands (80 and 140 kDa) were detected with antibody M6749, but not with anti-HNK-1 antibody. The 80- and 140-kDa band materials were identified as meprin alpha and CD13/aminopeptidase N, respectively. We also confirmed the presence of the non-sulfated HNK-1 carbohydrate on N-linked oligosaccharides by multistage tandem mass spectrometry. Immunofluorescence staining with antibody M6749 revealed that the non-sulfated HNK-1 carbohydrate was expressed predominantly on the apical membranes of the proximal tubules in the cortex and was also detected in the thin ascending limb in the inner medulla. This is the first study indicating the presence of the non-sulfated HNK-1 carbohydrate being synthesized by GlcAT-S in the kidney. The results presented here constitute novel knowledge concerning the function of the HNK-1 carbohydrate.     

Two immunologically and developmentally distinct chondroitin sulfate proteolglycans in embryonic chick brain.

Two different chondroitin sulfate proteoglycans (CSPG) in embryonic chick brain were distinguished by immunoreactivity either with S103L, a rat monoclonal antibody which reacts specifically with an 11-amino-acid region in the chondroitin sulfate domain of the core protein of chick cartilage CSPG (Krueger, R. C., Jr., Fields, T. A., Mensch, J. R., and Schwartz, N. B. (1990) J. Biol. Chem. 265, 12088-12097), or with HNK-1, a mouse monoclonal antibody which reacts with a 3-sulfoglucuronic acid residue on neural glycolipids and glycoproteins (Chou, D. K. H., Ilyas, A., Evans, J. E. Costello, C., Quarles, R. H., and Jungawala, F. B. (1986) J. Biol. Chem. 261, 11717-11725) but not with both antibodies. This specific immunoreactivity was used to separate the two CSPGs for further characterization. The S103L reactive brain proteoglycan had a core protein of similar size to cartilage CSPG (370 kDa) but exhibited a smaller hydrodynamic size (K(av) of 0.308). It was substituted predominantly with chondroitin sulfate chains and virtually no keratan sulfate chains. The HNK-1 reactive CSPG had a smaller core protein (340 kDa), an even smaller hydrodynamic size (K(av) of 0.564), and was substituted with both chondroitin sulfate and keratan sulfate chains. Glycosidase digestion patterns with endo-beta-galactosidase, N-glycosidase F, neuraminidase, and O-glycosidase, and reactivity with an antibody to the hyaluronate binding region also showed significant differences between the two brain CSPGs. Expression of the S103L reactive brain CSPG was developmentally regulated from embryonic day 7 through 19 with a peak in core protein on day 13, and in mRNA expression at day 10. In contrast the HNK-1 reactive brain CSPG was constitutively present from day 7 through hatching. These data suggest that these two distinct core proteins are immunologically and biochemically unique translation products of two different CSPG genes.     

Sulfation of glucuronic acid in the linkage tetrasaccharide by HNK-1 sulfotransferase is an inhibitory signal for the expression of a chondroitin sulfate chain on thrombomodulin

HNK-1 (human natural killer-1) carbohydrate epitope (HSO₃-3GlcAβ1-3Galβ1-4GlcNAc-) recognized by a HNK-1 monoclonal antibody is highly expressed in the nervous system and biosynthesized by a glucuronyltransferase (GlcAT-P or GlcAT-S), and sulfotransferase (HNK-1ST). A similar oligosaccharide (HSO₃-3GlcAβ1-3Galβ1-3Galβ1-4Xyl) also recognized by the HNK-1 antibody had been found in a glycosaminoglycan (GAG)-protein linkage region of α-thrombomodulin (TM) from human urine. However, which sulfotransferase is involved in sulfation of the terminal GlcA in the GAG-protein linkage region remains unclear. In this study, using CHO-K1 cells in which neither GlcAT-P nor GlcAT-S is endogenously expressed, we found that HNK-1ST has the ability to produce HNK-1 immunoreactivity on α-TM. We also demonstrated that HNK-1ST caused the suppression of chondroitin sulfate (CS) synthesis on TM and a reduction of its anti-coagulant activity. Moreover, using an in vitro enzyme assay system, the HNK-1-positive TM was found not to be utilized as a substrate for CS-polymerizing enzymes (chondroitin synthase (ChSy) and chondroitin polymerizing factor (ChPF)). These results suggest that HNK-1ST is involved in 3-O-sulfation of the terminal GlcA of the linkage tetrasaccharide which acts as an inhibitory signal for the initiation of CS biosynthesis on TM.     

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.     

Identification of a peptide mimic of the L2/HNK-1 carbohydrate epitope

The L2/HNK-1 carbohydrate is carried by many neural recognition molecules and is involved in neural cell interactions during development, regeneration in the peripheral nervous system, synaptic plasticity, and autoimmune-based neuropathies. Its key structure consists of a sulfated glucuronic acid linked to lactosaminyl residues. Because of its biological importance but limited availability, the phage display method was used to isolate a collection of peptide mimics that bind specifically to an L2/HNK-1 antibody. The phages isolated from a 15-mer peptide library by adsorption to this antibody share a consensus sequence of amino acids. The peptide mimicked several important functions of the L2/HNK-1 carbohydrate, such as binding to motor neurons in vitro, and preferential promotion of in vitro neurite outgrowth from motor axons compared with sensory neurons. A scrambled version of the peptide had no activity. The combined observations indicate that we have isolated a mimic of the L2/HNK-1 carbohydrate that is able to act as its functional substitute.     

Occurrence of the HNK-1 epitope (3-sulfoglucuronic acid) in PC12 pheochromocytoma cells, chromaffin granule membranes, and chondroitin sulfate proteoglycans

After biosynthetic labeling of sulfated glycoproteins in rat and goldfish brain and PC12 pheochromocytoma cells with sodium [35S]sulfate, it was observed that all of the bands reactive with the HNK-1 antibody on immunoblots of sodium dodecyl sulfate-polyacrylamide gels corresponded with sulfate-labeled proteins detected by fluorography. These results support data from other studies, which indicate that the HNK-1 epitope is a 3-sulfo-glucuronic acid residue. In addition to its presence in a wide range of nervous tissue glycoproteins, the HNK-1 epitope was also detected in chromaffin granule membranes, chondroitinase ABC, and in chondroitin sulfate proteoglycans of brain, cartilage, and chondrosarcoma. However, it is not present in the heparan sulfate proteoglycan of brain, or in either of two chondroitin sulfate/dermatan sulfate proteoglycans in the chromaffin granule matrix.     

Chemo-bacterial synthesis and immunoreactivity of a brain HNK-1 analogue

This work reports the synthesis and the biological validation of a trisaccharide analogue of the HNK-1 epitope. The 3-O-sulfo-β-d-GlcpA-(1→3)-β-d-Galp-(1→4)-β-d-Glcp-allyl has been prepared by enzymatic glucuronylation of allyl lactoside by an engineered recombinant Escherichia coli strain followed by a chemoselective sulfation. Subsequent covalent attachment of the ozone-oxidised trisaccharide to bovine serum albumin provided a neo-glycoconjugate, which has been interrogated with antibodies specific to the human natural killer carbohydrate epitope HNK-1. ELISA assays confirmed the absolute requirement of the sulfate group for protein recognition and the potential application of this synthetic oligosaccharide as HNK-1 surrogate.     

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.