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The human natural killer (HNK-1) carbohydrate plays important roles during nervous system development, regeneration after trauma and synaptic plasticity. Four proteins have been identified as receptors for HNK-1: the laminin adhesion molecule, high-mobility group box 1 and 2 (also called amphoterin) and cadherin 2 (also called N-cadherin). Because of HNK-1's importance, we asked whether additional receptors for HNK-1 exist and whether the four identified proteins share any similarity in their primary structures. A set of 40,000 sequences homologous to the known HNK-1 receptors was selected and used for large-scale sequence alignments and motif searches. Although there are conserved regions and highly conserved sites within each of these protein families, there was no sequence similarity or conserved sequence motifs found to be shared by all families. Since HNK-1 receptors have not been compared regarding binding constants and since it is not known whether the sulfated or non-sulfated part of HKN-1 represents the structurally crucial ligand, the receptors are more heterogeneous in primary structure than anticipated, possibly involving different receptor or ligand regions. We thus conclude that the primary protein structure may not be the sole determinant for a bona fide HNK-1 receptor, rendering receptor structure more complex than originally assumed.

The human natural killer-1 (HNK-1) epitope is a unique sulfated trisaccharide sequence presented on O - and N -glycans of various glycoproteins and on glycolipids. It is overexpressed in the nervous system and plays crucial roles in nerve regeneration, synaptic plasticity, and neuronal diseases. However, the investigation of functional roles of HNK-1 in a more complex glycan context at the molecular level remains a big challenge due to lack of access to related structurally well-defined complex glycans. Herein, we describe a highly efficient chemoenzymatic approach for the first collective synthesis of HNK-1-bearing O -mannose glycans with different branching patterns, and for their nonsulfated counterparts. The successful strategy relies on both chemical glycosylation of a trisaccharide lactone donor for the introduction of sulfated HNK-1 branch and substrate promiscuities of bacterial glycosyltransferases that can tolerate sulfated substrates for enzymatic diversification. Glycan microarray analysis with the resulting complex synthetic glycans demonstrated their recognition by two HNK-1-specific antibodies including anti-HNK-1/N-CAM (CD57) and Cat-315, which provided further evidence for the recognition epitopes of these antibodies and the essential roles of the sulfate group for HNK-1 glycan-antibody recognition.

Human natural killer-1 (HNK-1) cell antigen is a glycan epitope involved in several neural events, such as neuritogenesis, myelination, synaptic plasticity and regeneration of the nervous system after injury. We have recently identified the small organic compound ursolic acid (UA) as a HNK-1 mimetic with the aim to test its therapeutic potential in the central nervous system. UA, a plant-derived pentacyclic triterpenoid, is well known for its multiple biological functions, including neuroprotective, antioxidant and anti-inflammatory activities. In the present study, we evaluated its functions in a mouse model of spinal cord injury (SCI) and explored the molecular mechanisms underlying its positive effects. Oral administration of UA to mice 1 h after SCI and thereafter once daily for 6 weeks enhanced the regaining of motor functions and axonal regrowth, and decreased astrogliosis. UA administration decreased levels of proinflammatory markers, including interleukin-6 and tumor necrosis factor-α, in the injured spinal cord at the acute phase of inflammation and activated the mitogen-activated protein kinase and phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathways in the injured spinal cord. Taken together, these results suggest that UA may be a candidate for treatment of nervous system injuries., (Copyright © 2017. Published by Elsevier Inc.)

Background: The human natural killer-1 (HNK-1) carbohydrate, a unique trisaccharide possessing sulfated glucuronic acid in a non-reducing terminus (HSO 3 -3GlcAß1-3Galß1-4GlcNAc-), is highly expressed in the nervous system and its spatiotemporal expression is strictly regulated. Mice deficient in the gene encoding a key enzyme, GlcAT-P, of the HNK-1 biosynthetic pathway exhibit almost complete disappearance of the HNK-1 epitope in the brain, significant reduction of long-term potentiation, and aberration of spatial learning and memory formation. In addition to its physiological roles in higher brain function, the HNK-1 carbohydrate has attracted considerable attention as an autoantigen associated with peripheral demyelinative neuropathy, which relates to IgM paraproteinemia, because of high immunogenicity. It has been suggested, however, that serum autoantibodies in IgM anti-myelin-associated glycoprotein (MAG) antibody-associated neuropathy patients show heterogeneous reactivity to the HNK-1 epitope., Scope of Review: We have found that structurally distinct HNK-1 epitopes are expressed in specific proteins in the nervous system. Here, we overview the current knowledge of the involvement of these HNK-1 epitopes in the regulation of neural plasticity and discuss the impact of different HNK-1 antigens of anti-MAG neuropathy patients., Major Conclusions: We identified the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor subunit GluA2 and aggrecan as HNK-1 carrier proteins. The HNK-1 epitope on GluA2 and aggrecan regulates neural plasticity in different ways. Furthermore, we found the clinical relationship between reactivity of autoantibodies to the different HNK-1 epitopes and progression of anti-MAG neuropathy., General Significance: The HNK-1 epitope is indispensable for the acquisition of normal neuronal function and can be a good target for the establishment of diagnostic criteria for anti-MAG neuropathy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The design of molecules that mimic biologically relevant glycans is a significant goal for understanding important biological processes and may lead to new therapeutic and diagnostic agents. In this study we focused our attention on the trisaccharide human natural killer cell-1 (HNK-1), considered the antigenic determinant of myelin-associated glycoprotein and the target of clinically relevant auto-antibodies in autoimmune neurological disorders such as IgM monoclonal gammopathy and demyelinating polyneuropathy. We describe a structure-activity relationship study based on surface plasmon resonance binding affinities aimed at the optimization of a peptide that mimics the HNK-1 minimal epitope. We developed a cyclic heptapeptide that shows an affinity of 1.09×10 -7  m for a commercial anti-HNK1 mouse monoclonal antibody. Detailed conformational analysis gave possible explanations for the good affinity displayed by this novel analogue, which was subsequently used as an immunological probe. However, preliminary screening indicates that patients' sera do not specifically recognize this peptide, showing that murine monoclonal antibodies cannot be used as a guide to select immunological probes for the detection of clinically relevant human auto-antibodies., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nerve conduits prefilled with hydrogels are frequently explored in an attempt to promote nerve regeneration. This study examines the interplay in vivo between the porosity of the conduit wall and the level of bioactivity of the hydrogel used to fill the conduit. Nerve regeneration in porous (P) or nonporous (NP) conduits that were filled with either collagen only or collagen enhanced with a covalently attached neurite-promoting peptide mimic of the glycan human natural killer cell antigen-1 (m-HNK) were compared in a 5 mm critical size defect in the mouse femoral nerve repair model. Although collagen is a cell-friendly matrix that does not differentiate between neural and nonneural cells, the m-HNK-enhanced collagen specifically promotes axon growth and appropriate motor neuron targeting. In this study, animals treated with NP conduits filled with collagen grafted with m-HNK (CollagenHNK) had the best overall functional recovery, based on a range of histomorphometric observations and parameters of functional recovery. Our data indicate that under some conditions, the use of generally cell friendly fillers such as collagen may limit nerve regeneration. This finding is significant, considering the frequent use of collagen-based hydrogels as fillers of nerve conduits.

High-resolution electrospray mass spectra in positive and negative ion modes (MS and MS/MS) were measured and described for biotinylated hexaethylene glycol (HEG) connected molecular probes bearing HNK-1 (abbreviation of human natural killer cell-1 epitope) antigenic trisaccharide (1) and its non-sulfated analogue (2). For molecular probe 2, in its CID MS/MS of [M+2Na](2+), unexpected peak at m/z 530.2475 [C22H41N3O8SNa](+) was observed and attributed to the fragmentation of the aglycone at the end of the HEG chain distant from the biotin fragment. No homologous ions having the difference C2H4O smaller than that one were observed. The same cleavage was revealed in negative ion spectra. A similar fragmentation was found for other non-sulfated, biotinylated HEG-spacered molecular probes thus demonstrates this type of fragmentation characteristic for such glycosides., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The human natural killer-1 (HNK-1) carbohydrate comprising a sulfated trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-) is expressed on N-linked and O-mannose-linked glycans in the nervous system and involved in learning and memory functions. Although whole/core glycan structures and carrier glycoproteins for the N-linked HNK-1 epitope have been studied, carrier glycoproteins and the biosynthetic pathway of the O-mannose-linked HNK-1 epitope have not been fully characterized. Here, using mass spectrometric analyses, we identified the major carrier glycoprotein of the O-linked HNK-1 as phosphacan in developing mouse brains and determined the major O-glycan structures having the terminal HNK-1 epitope from partially purified phosphacan. The O-linked HNK-1 epitope on phosphacan almost disappeared due to the knockout of protein O-mannose β1,2-N-acetylglucosaminyltransferase 1, an N-acetylglucosaminyltransferase essential for O-mannose-linked glycan synthesis, indicating that the reducing terminal of the O-linked HNK-1 is mannose. We also showed that glucuronyltransferase-P (GlcAT-P) was involved in the biosynthesis of O-mannose-linked HNK-1 using the gene-deficient mice of GlcAT-P, one of the glucuronyltransferases for HNK-1 synthesis. Consistent with this result, we revealed that GlcAT-P specifically synthesized O-linked HNK-1 onto phosphacan using cultured cells. Furthermore, we characterized the as-yet-unknown epitope of the 6B4 monoclonal antibody (mAb), which was thought to recognize a unique phosphacan glycoform. The reactivity of the 6B4 mAb almost completely disappeared in GlcAT-P-deficient mice, and exogenously expressed phosphacan was selectively recognized by the 6B4 mAb when co-expressed with GlcAT-P, suggesting that the 6B4 mAb preferentially recognizes O-mannose-linked HNK-1 on phosphacan. This is the first study to show that 6B4 mAb-reactive O-mannose-linked HNK-1 in the brain is mainly carried by phosphacan.

Human natural killer-1 (HNK-1) carbohydrate, comprising a unique trisaccharide HSO(3)-3GlcAβ1-3Galβ1-4GlcNAc, shows well-regulated expression and unique functions in the nervous system. Recent studies have revealed sophisticated and complicated expression mechanisms for HNK-1 glycan. Activities of biosynthetic enzymes are controlled through the formation of enzyme-complexes and regulation of subcellular localization. Functional aspects of HNK-1 carbohydrate were examined by overexpression, knockdown, and knockout studies of these enzymes. HNK-1 is involved in several neural functions such as synaptic plasticity, learning and memory, and the underlying molecular mechanisms have been illustrated upon identification of the target carrier glycoproteins of HNK-1 such as the glutamate receptor subunit GluA2 or tenascin-R. In this review, we describe recent findings about HNK-1 carbohydrate that provide further insights into the mechanism of its expression and function in the nervous system.

Pseudomonas aeruginosa (PA) can cause infections in compromised hosts by interacting with the glycocalyx of host epithelial cells. It binds to glycostructures on mucosal surfaces via two lectins, which are carbohydrate-binding proteins, named PA-IL and PA-IIL, and blocking this interaction is, thus, an attractive anti-adhesive strategy. The aim of this study was to determine by ciliary beat frequency (CBF) analysis whether monosaccharides or peptides mimicking glycostructures represent blockers of PA lectin binding to human airway cilia. The treatment with monosaccharides and peptides alone did not change the CBF compared to controls and the tested compounds did not influence the cell morphology or survival, with the exception of peptide pOM3. PA-IL caused a decrease of the CBF within 24 h. D-galactose as well as the peptides mimicking HNK-1, polysialic acid and fucose compensated the CBF-modulating effect of PA-IL with different affinities. PA-IIL also bound to the human airway cilia in cell culture and resulted in a decrease of the CBF within 24 h. L(-)-fucose and pHNK-1 blocked the CBF-decreasing effect of PA-IIL. The HNK-1-specific glycomimetic peptide had a high affinity for binding to both PA-IL and PA-IIL, and inhibited the ciliotoxic effect of both lectins, thus, making it a strong candidate for a therapeutic anti-adhesive drug.

The human natural killer cell carbohydrate, HNK-1, plays function-conducive roles in peripheral nerve regeneration and synaptic plasticity. It is also the target of autoantibodies in polyneuropathies. It is thus important to synthesize structurally related HNK-1 carbohydrates for optimizing its function-conducive roles, and for diagnosis and neutralization of autoantibodies in the fatal Guillain-Barré syndrome. As a first step toward these goals, we have synthesized several HNK-1 carbohydrate derivatives to assess the specificity of monoclonal HNK-1 antibodies from rodents: 2-aminoethyl glycosides of selectively O-sulfated trisaccharide corresponding to the HNK-1 antigen, its nonsulfated analogue, and modified structures containing 3-O-fucosyl or 6-O-sulfo substituents in the N-acetylglucosamine residues. These were converted, together with several related oligosaccharides, into biotin-tagged probes to analyze the precise carbohydrate specificity of two anti-HNK-1 antibodies by surface plasmon resonance that revealed a crucial role of the glucuronic acid in antibody binding. The contribution of the different oligosaccharide moieties in the interaction was shown by saturation transfer difference (STD) NMR of the complex consisting of the HNK-1 pentasaccharide and the HNK-1 412 antibody., (© 2011 American Chemical Society)

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., (2010 Elsevier Ltd. All rights reserved.)

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.

Glycosylation is a major post-translational protein modification, especially for cell surface proteins, which play important roles in a variety of cellular functions, including recognition and adhesion. Among them, we have been interested in HNK-1 (human natural killer-1) carbohydrate, which is characteristically expressed on a series of cell adhesion molecules in the nervous system. The HNK-1 carbohydrate has a unique structural feature, i.e. a sulfated glucuronic acid is attached to the non-reducing terminal of an N-acetyllactosamine residue (HSO(3)-3GlcAbeta1-3Galbeta1-4GlcNAc-). We have cloned and characterized the biosynthetic enzymes (two glucuronyltransferases and a sulfotransferase), and also obtained evidence that the HNK-1 carbohydrate is involved in synaptic plasticity and memory formation. In this review, we describe recent findings regarding the expression mechanism and functional roles of this carbohydrate.

We have previously reported the large-scale synthesis of neolactotetraose (Galbeta-4GlcNAcbeta-3Galbeta-4Glc) from lactose in engineered Escherichia coli cells (Priem B, Gilbert M, Wakarchuk WW, Heyraud A and Samain E. 2002. A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria. Glycobiology. 12:235-240). In the present study we analyzed the adaptation of this system to glucuronylated oligosaccharides. The catalytic domain of mouse glucuronyl transferase GlcAT-P was cloned and expressed in an engineered strain which performed the in vivo synthesis of neolactotetraose. Under these conditions, efficient glucuronylation of neolactotetraose was achieved, but some residual neolactotetraose was still present. Although E. coli K-12 has an indigenous UDP-glucose dehydrogenase, the yield of glucuronylated oligosaccharides was greatly improved by the additional expression of the orthologous gene kfiD from E. coli K5. Glucuronylation of neolactohexaose and lactose was also observed. The final glucuronylated oligosaccharides are precursors of the brain carbohydrate motif HNK-1, involved in neural cell adhesion.

The human beta1,3-glucuronosyltransferases galactose-beta1,3-glucuronosyltransferase I (GlcAT-I) and galactose-beta1,3-glucuronosyltransferase P (GlcAT-P) are key enzymes involved in proteoglycan and HNK-1 carbohydrate epitope synthesis, respectively. Analysis of their acceptor specificity revealed that GlcAT-I was selective toward Galbeta1,3Gal (referred to as Gal2-Gal1), whereas GlcAT-P presented a broader profile. To understand the molecular basis of acceptor substrate recognition, we constructed mutants and chimeric enzymes based on multiple sequence alignment and structural information. The drastic effect of mutations of Glu227, Arg247, Asp252, and Glu281 on GlcAT-I activity indicated a key role for the hydrogen bond network formed by these four conserved residues in dictating Gal2 binding. Investigation of GlcAT-I determinants governing Gal1 recognition showed that Trp243 could not be replaced by its counterpart Phe in GlcAT-P. This result combined with molecular modeling provided evidence for the importance of stacking interactions with Trp at position 243 in the selectivity of GlcAT-I toward Galbeta1,3Gal. Mutation of Gln318 predicted to be hydrogen-bonded to 6-hydroxyl of Gal1 had little effect on GlcAT-I activity, reinforcing the role of Trp243 in Gal1 binding. Substitution of Phe245 in GlcAT-P by Ala selectively abolished Galbeta1,3Gal activity, also highlighting the importance of an aromatic residue at this position in defining the specificity of GlcAT-P. Finally, substituting Phe245, Val320, or Asn321 in GlcAT-P predicted to interact with N-acetylglucosamine (GlcNAc), by their counterpart in GlcAT-I, moderately affected the activity toward the reference substrate of GlcAT-P, N-acetyllactosamine, indicating that its active site tolerates amino acid substitutions, an observation that parallels its promiscuous substrate profile. Taken together, the data clearly define key residues governing the specificity of beta1,3-glucuronosyltransferases.

Perineuronal nets (PNs) are lattice-like condensations of the extracellular matrix (ECM) that envelop synapses and decorate the surface of subsets of neurons in the CNS. Previous work has suggested that, despite the fact that PNs themselves are not visualized until later in development, some PN component molecules are expressed in the rodent CNS even before synaptogenesis. In the adult mammalian brain, monoclonal antibody Cat-315 recognizes a glycoform of aggrecan, a major component of PNs. In primary cortical cultures, a Cat-315-reactive chondroitin sulfate proteoglycan (CSPG) is also expressed on neuronal surfaces and is secreted into culture media as early as 24 h after plating. In this study, we show that in primary cortical cultures, the Cat-315 CSPG detected in early neural development is expressed in extrasynaptic sites prior to synapse formation. This suggests that ECM components in the CNS, as in the neuromuscular junction (NMJ), may prepattern neuronal surfaces prior to innervation. We further show that while the Cat-315-reactive carbohydrate decorates aggrecan in the adult, it decorates a different CSPG in the developing CNS. Using receptor protein tyrosine phosphatase beta (RPTPbeta/protein tyrosine phosphatase zeta) knock-out mice and immunoprecipitation techniques, we demonstrate here that in the developing rodent brain Cat-315 recognizes RPTPbeta isoforms. Our further examination of the Cat-315 epitope suggests that it is an O-mannose linked epitope in the HNK-1 family. The presence of the Cat-315 reactive carbohydrate on different PN components--RPTPbeta and aggrecan--at different stages of synapse development suggests a potential role for this neuron-specific carbohydrate motif in synaptogenesis.

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.

The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules. Its structure is characterized by a terminal sulfated glucuronyl acid. The glucuronyltransferases, GlcAT-P and GlcAT-S, are involved in the biosynthesis of the HNK-1 epitope, GlcAT-P as the major enzyme. We overexpressed and purified the recombinant human GlcAT-P from Escherichia coli. Analysis of its enzymatic activity showed that it catalyzed the transfer reaction for N-acetyllactosamine (Galbeta1-4GlcNAc) but not lacto-N-biose (Galbeta1-3GlcNAc) as an acceptor substrate. Subsequently, we determined the first x-ray crystal structures of human GlcAT-P, in the absence and presence of a donor substrate product UDP, catalytic Mn(2+), and an acceptor substrate analogue N-acetyllactosamine (Galbeta1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide. The asymmetric unit contains two independent molecules. Each molecule is an alpha/beta protein with two regions that constitute the donor and acceptor substrate binding sites. The UDP moiety of donor nucleotide sugar is recognized by conserved amino acid residues including a DXD motif (Asp(195)-Asp(196)-Asp(197)). Other conserved amino acid residues interact with the terminal galactose moiety of the acceptor substrate. In addition, Val(320) and Asn(321), which are located on the C-terminal long loop from a neighboring molecule, and Phe(245) contribute to the interaction with GlcNAc moiety. These three residues play a key role in establishing the acceptor substrate specificity.

Polysialylated neural cell adhesion molecule (NCAM) was immunoaffinity-purified from the brains of newborn calves. A degree of polymerization of up to 40 was chromatographically determined for released polysialic acid (PSA) chains. For characterization of N-glycan structures and attachment sites, PSA-NCAM was digested with trypsin, and the generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specific for PSA or the HNK1 epitope, i.e., HSO(3)-3GlcA(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-, yielding PSA-glycopeptides, HNK-glycopeptides and non-PSA/HNK1-(glyco) peptides. Using a combination of enzymatic deglycosylation, peptide fractionation, mass spectrometry and Edman degradation, HNK1-N-glycans could be assigned to glycosylation sites 2, 4, 5 and 6. Non-PSA/HNK1-glycans were assigned to glycosylation site 2, whereas PSA-N-glycans of bovine NCAM had been already previously shown to be restricted to glycosylation sites 5 and 6 (Glycobiology 12 (2002) 47). Respective oligosaccharides were enzymatically released, labeled with 2-aminopyridine and characterized by linkage analysis and mass spectrometry. Carbohydrate chains bearing PSA or the HNK1 epitope comprised mainly fucosylated, partially sulfated diantennary, triantennary or tetraantennary glycans without bisecting GlcNAc or fucosylated diantennary and triantennary species carrying, in part, bisecting GlcNAc residues, respectively. Some N-glycans simultaneously contained both the HNK1-epitope and PSA. Non-PSA/HNK1-glycans exhibited a heterogeneous pattern of partially truncated, mostly diantennary structures with one to three fucose residues, bisecting GlcNAc and/or sulfate residues. In addition, they were demonstrated to carry, to some extent, the Lewis X epitope. When compared with previous data on murine NCAM glycosylation, our results indicate a conservation of structural features and attachment sites for the different types of NCAM N-glycans.

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.

The HNK-1 carbohydrate epitope, a sulfated glucuronic acid at the non-reducing terminus of glycans, is expressed characteristically on a series of cell adhesion molecules and is synthesized through a key enzyme, glucuronyltransferase (GlcAT-P). We generated mice with a targeted deletion of the GlcAT-P gene. The GlcAT-P -/- mice exhibited normal development of gross anatomical features, but the adult mutant mice exhibited reduced long term potentiation at the Schaffer collateral-CA1 synapses and a defect in spatial memory formation. This is the first evidence that the loss of a single non-reducing terminal carbohydrate residue attenuates brain higher functions.

Interleukin 6 (IL-6) is endowed with a lectin activity for oligosaccharide ligands possessing the HNK-1 epitope (3-sulfated glucuronic acid) found on some mammalian glycoprotein N-glycans (Cebo, C., Dambrouck, T., Maes, E., Laden, C., Strecker, G., Michalski, J. C., and Zanetta, J. P. (2001) J. Biol. Chem. 276, 5685-5691). Using high affinity oligosaccharide ligands, it is demonstrated that this lectin activity is responsible for the early dephosphorylation of tyrosine residues found on specific proteins induced by interleukin 6 in human resting lymphocytes. The gp130 glycoprotein, the signal-transducing molecule of the IL-6 pathway, is itself a molecule possessing the HNK-1 epitope. This indicates that IL-6 is a bi-functional molecule able to extracellularly associate its alpha-receptor with the gp130 surface complex. Computational modeling indicates that the lower energy conformers of the high affinity ligands of IL-6 have a common structure. Docking experiments of these conformers suggest that the carbohydrate recognition domain of IL-6 is localized in the domain previously identified as site 3 of IL-6 (Somers, W., Stahl, M., and Seehra, J. S. (1997) EMBO J. 16, 989-997), already known to be involved in interactions with gp130.

A family of about 20 novel acidic bi- and tri-antennary N-glycans, amounting to almost half those expressed on Bowes melanoma tissue-plasminogen activator (t-PA) were found to possess Galbeta1-->4GlcNAcbeta1-->, sulfated and sialylated GalNAcbeta1-->4GlcNAcbeta1--> or sulfated GlcAbeta1--> 3Galbeta1-->4GlcNAcbeta1--> antennae, of which those containing sulfated GlcA, depicting the L2/HNK-1 carbohydrate epitope, were preferentially located on the 6 arm. A proportion of the glycans were highly charged, because of multiple and variously distributed sulfation, some of which was located on the fucosylated chitobiose core. Multiple expression of the L2/HNK-1 epitope on a single glycan was observed. The most abundant compound was a biantennary glycan carrying sulfated GlcA on the 6-branched antenna and an alpha2-->6 sialylated GalNAc on the other. The N-glycosylation sequon containing Asn448, which is known to express all of the sulfate-carrying N-glycans contains, unusually, an arginine residue. An electrostatic interaction between this cationic amino acid and the core-sulfate group of the N-glycan is proposed to reduce mobility of the carbohydrate in the region of the t-PA active site. Because of the 'brain-type' nature of the N-glycans described in this neuro-ectodermal cell line, the possibility of neural t-PA interacting with the L2/HNK-1-recognizing molecule, laminin, of the central nervous system extracellular matrix is discussed.

The N-glycosylation pattern of the neural cell adhesion molecule (NCAM), isolated from brains of newborn mice, has been analyzed. Following digestion with trypsin, generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specifically recognizing polysialic acid (PSA) units or the HNK1-carbohydrate epitope. Subsequent analyses of the resulting (glyco)peptides by Edman degradation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed polysialylated glycans to be exclusively linked to glycosylation sites 5 (Asn(431)) and 6 (Asn(460)), whereas glycans carrying the HNK1-epitope could be assigned to sites 2 (Asn(297)), 5, 6, and, to a lesser extent, site 3 (Asn(329)). PSA-, HNK1-, and non-PSA/HNK1-glycan fractions were characterized by carbohydrate constituent and methylation analyses as well as MALDI-TOF-MS in conjunction with chromatographic fractionation techniques. The results revealed that the core structures of PSA-glycans represented predominantly fucosylated, partially sulfated 2,6-branched isomers of triantennary as well as tetraantennary complex-type glycans, whereas carbohydrate chains bearing the HNK1-epitope were dominated by diantennary species carrying in part bisecting GlcNAc residues. Non-PSA/HNK1-glycans exhibited a highly heterogeneous pattern of partially truncated, mostly diantennary structures being characterized by the presence of additional fucose, bisecting GlcNAc and/or sulfate residues. In conclusion, our results revealed that the glycosylation pattern of murine NCAM displays high structural and regional selectivity, which might play an important role in controlling the biological activities of this molecule.

The HNK-1 carbohydrate epitope is part of many cell membrane and extracellular matrix molecules. It has been implicated in cell to cell and cell to extracellular matrix adhesion, and antibodies to the HNK-1 epitope are emerging as a versatile tool in eye research. They have been used to identify a novel cell type in the human eye, the subepithelial matrix cells that reside in the inner connective tissue layer (ICTL) of the ciliary body. Although these cells resemble fibroblasts in ultrastructure, they form a distinct cell population that differs in its antigenic profile from fibroblasts of other tissues. These cells are associated with the elastic fiber system of the ICTL. Other structures in the human eye that harbor the HNK-1 epitope in a nonrandom pattern are the ciliary and iris epithelia, the zonular lamella, the lens capsule, the retina, glial cells of the optic and ciliary nerves, and scleral fibroblasts. The HNK-1 epitope in the eye appears early during embryonic development and is phylogenetically conserved, but many interspecies differences exist in its distribution. The role of the HNK-1 epitope may be to structurally stabilize the ciliary body and the retina, and to participate in zonular attachments. The HNK-1 epitope has been linked with many common eye diseases. The subepithelial matrix cells seem to be susceptible to undergo irreversible damage as a result of glaucoma, thermal injury, and tissue compression. This epitope has proved to be useful in identifying intraocular deposits of exfoliation syndrome. It can explain the adhesiveness of exfoliation material. Intraocular exfoliation material differs in HNK-1 immunoreactivity from the extraocular fibrillopathy of exfoliation syndrome and its presence in fellow eyes also argues against the concept of unilateral exfoliation syndrome. The HNK-1 epitope is found in the extracellular matrix of secondary cataract and anterior subcapsular cataract, and it may contribute to their pathogenesis. Finally, the HNK-1 epitope can be used to trace neuroepithelial derivatives of the optic vesicle in developmental anomalies and in tumors of the eye. Eventual identification of molecules that bear the HNK-1 epitope in the eye will likely shed light on many aspects of ocular physiology and pathobiology

The 2-aminoethyl glycoside of pentasaccharide 3-O-sulfo-GlcA(beta-1-->3)Gal(beta-1-->4)GlcNAc(beta-1-->3)Gal(beta-1--> 4)Glc(beta (1) and its conjugates with biotin and biotinylated polyacrylic acid were synthesized as molecular probes to investigate the recognition of the HNK-1 epitope containing carbohydrates by proteins. Key steps in the first of two investigated schemes for the preparation of the target compound 1 were (a) assembling of the pentasaccharide backbone (compound 10) by glycosylation of selectively substituted allyl glycoside of the trisaccharide GlcNAc(beta-1-->3)Gal(beta-1-->4)Glc(beta with glucuronyl-galactose glycosyl donor, (b) transformation of the allyl aglycon in 10 into 2-azidoethyl one (to give 11), (c) selective deprotection of the OH group at C-3 of the GlcA residue in 11 via saponification, intramolecular formation of 6,3-lacton (13) and its methanolysis, and (d) subsequent O-sulfation. The alternative scheme with the use of 2-azido-ethyl glycoside of the trisaccharide GlcNAc(beta-1-->3)Gal(beta-1-->4)Glc(beta instead of the allyl glycoside 6 was less effective due to smaller yield at the step of pentasaccharide synthesis. Additionally to 1 the 2-aminoethyl glycosides of the oligosaccharides GlcA(beta-1-->3)Gal(beta-1-->4)GlcNAc(beta-1-->3)Gal(beta-1-->4)Glc(beta, 3-O-sulfo-GlcA(beta-1-->3)Gal(beta, and GlcA(beta-1-->3)Gal(beta were also synthesized.

The acceptor specificity of a rat brain glucuronyltransferase, GlcAT-P, associated with biosynthesis of the HNK-1 epitope on glycoproteins, was investigated using asialoorosomucoid as a model acceptor substrate. Structural analysis of N-linked oligosaccharides, to which glucuronic acid was transferred by GlcAT-P, by means of two-dimensional mapping of pyridylamino-oligosaccharides and MS spectrometry, demonstrated that the enzyme transferred glucuronic acid to bi-, tri-, and tetra-antennary complex type sugar chains, with almost equal efficiency, indicating that the enzyme has no preference as to the number of acceptor sugar branches. Next, we studied the branch specificity of this enzyme by means of the selective branch scission method involving two step exoglycosidase digestion using authentic pyridylamino-oligosaccharides. The GlcAT-P is highly specific for the terminal N-acetyllactosamine structure and no glucuronic acid was incorporated into a Galbeta1-3GlcNAc moiety. The GlcAT-P transferred glucuronic acid to the galactose residues in the N-acetyllactosamine branches of bi-, tri-, and tetra-antennary oligosaccharide chains, with different efficiencies and most preferentially to those in the Galbeta1-4GlcNAcbeta1-4Manalpha1-3 branch.

All members of a glucuronyltransferase (GlcAT) gene family cloned to date contain four conserved regions (modules I-IV), which are widely located in the catalytic domain. In order to understand the biological significance of these modules, we investigated the structure-function relationship of GlcAT-P by means of the combination of site-directed mutagenesis and computer aided three-dimensional modeling. The wild-type and mutant GlcAT-Ps were expressed in Escherichia coli as glutathione-S-transferase (GST)-fused soluble proteins. Most of the mutants in which a polar amino acid within the modules was replaced with alanine lost their transferase activity almost completely, while all of the mutants in which the replacement was outside these modules retained the original catalytic activity. A three-dimensional (3-D) model of GlcAT-P was constructed by computer simulation with the three-dimensional structure of adenylate kinase (1AKE) as a template. This model predicted that the large catalytic domain of GlcAT-P forms a globular shape with a Rossmann-fold motif consisting of five alpha-helix and beta-sheet repeats. The putative catalytic pocket consisting mainly of modules I-III is surrounded by a cluster of polar amino acids, which are essential for the transferase activity and also for the binding to the acceptor substrate (essential amino acids), asialo-orosomucoid. There is the second cluster of essential amino acids almost on the opposite surface of the molecule, in which an aspartic acid repeat (DDD) is located. The biological significance of the second cluster is currently not clear but it may be associated with the interaction of the enzyme with modulation molecules, manganese and membrane phospholipids.

A derivative of allyl 3"-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-beta-lactoside with a free OH group at C-4GlcNAc was glycosylated with trichloroacetimidate of selectively protected GlcA(beta 1-->3)Gal alpha disaccharide in dichloromethane in the presence of trimethylsilyl triflate resulting in a pentasaccharide product with an 82% yield. This product was converted to monohydroxy derivative with a free OH group at C-3GlcA via the formation and the subsequent opening of the 6,3-lactone ring in the glucuronic acid residue. The 3"'-O-sulfation of the monohydroxy derivative, the removal of the protective groups, and the reduction of the allyl aglycon yielded the pentasaccharide propyl glycoside NaSO3-3GlcA(beta 1-->3)Gal(beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc beta-Opr comprising the oligosaccharide chain of the SGGL-1 glycolipid, which is recognized by HNK-1 antibodies. NaSO3-3GlcA(beta 1--> 3)Gal beta OAll, GlcA(beta 1-->3)Gal(beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc beta-OPr and GlcA(beta 1-->3)Gal beta OAll were synthesized in a similar way.

Polysialic acid and HNK-1 carbohydrate are expressed on Gal beta 1-->4GlcNAc outer chains of N-linked sugar chain of neural cell recognition molecules at certain developmental stages and involved in neural tissue formation. Targeted inactivation of the mouse beta-1,4-galactosyltransferase (beta-1,4-GalT) gene resulted in short life of the mice which supposedly do not have such carbohydrate antigens but have no defects in neural tissue formation. Analysis of the mutant mouse brain glycoproteins revealed that polysialic acid and HNK-1 carbohydrate are normally expressed in an age-dependent manner. In support of this, protein bands reacted with Ricinus communis agglutinin-I, which interacts with oligosaccharides terminated with the Gal beta 1-->4GlcNAc group, and beta-1,4-GalT activity toward GlcNAc beta-S-pNP were detected in the mutant mouse brain, indicating that brain contains another functional beta-1,4-GalT important for the expression of the carbohydrate antigens.

We have shown recently that mouse small cerebellar neurons adhere to a short amino acid sequence of the G2 domain of the laminin alpha 1 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 beta 1, alpha 3, and alpha 6. They could be shown to bind to laminin by a beta 1-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 beta 1 integrin inhibited partially cell adhesion to laminin. Determination of the association of L1, beta 1 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 beta 1 integrin. However, only negligible association was found between L1 and beta 1 integrin. Furthermore, it could be shown that adhesion to laminin is mediated by L1/HNK-1- and beta 1 integrin-dependent mechanisms that act at least partially independent of each other.

The HNK-1 epitope has been associated with the metastatic behaviour of uveal melanomas. We characterized HNK-1 antigens on four human uveal (primary and metastatic) and two primary cutaneous melanoma cell lines by immunocytochemistry and Western blot analysis. We also determined the involvement of the HNK-1 epitope in cell-cell interactions on a matrigel layer. Three uveal melanoma cell lines (one primary and two metastatic) and one cutaneous melanoma cell line showed HNK-1 expression by immunocytochemistry. On matrigel, only the HNK-1-positive cutaneous melanoma cell line Bowes grew in a honeycomb-like structure which disappeared after adding HNK-1 antibodies to the culture medium. Immunoblot analysis of the primary uveal melanoma cell line EOM-3 revealed five HNK-1-positive protein bands with apparent molecular weights of 200, 160, 115, 95 and 75 kDa. The cutaneous melanoma cell line Bowes showed three HNK-1-positive protein bands with apparent molecular weights of 150, 135 and 90 kDa. This study shows that two uveal (primary and metastatic) and one primary cutaneous melanoma cell lines express HNK-1 antigens on immunoblot. Only in the HNK-1-positive cutaneous melanoma cell line Bowes did the HNK-1 epitope have a function in intercellular adhesion. Although the primary uveal melanoma cell line EOM-3 showed a similar HNK-1 immunoreactivity, we could not demonstrate HNK-1-mediated cell adhesion. On immunoblot, the two cell lines displayed different HNK-1 antigens, which may explain the difference in cell adhesion.

The HNK-1 carbohydrate epitope, expressed by many neural recognition molecules, is involved in cell interactions that control cell type-specific neurite outgrowth and regeneration. It is also the target for autoimmune IgM antibodies in demyelinating neuropathies of the peripheral nervous system in humans. Despite its acknowledged importance in cell interactions, the HNK-1 carbohydrate structure, when expressed on glycoproteins, is still unknown. Here, we describe the structure of one of the predominant HNK-1-bearing glycans of bovine P0. The epitope consists of the sulfated trisaccharide SO4-3GlcAbeta1-3Galbeta1-4GlcNAc, attached to the alpha1-6 arm of a diantennary core with a bisecting N-acetylglucosamine. It is the first example of a terminal 3-sulfated glucuronic acid on an asparagine-linked carbohydrate. Because the similarity between the glycoprotein-derived structure and the glycosphingolipids carrying HNK-1 is restricted to the terminal sulfated trisaccharide, we conclude that this element is sufficient for HNK-1 immunoreactivity. Knowledge of the HNK-1 structure on proteins is an important prerequisite for the elucidation of its functional role in development and disease.