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.     

A Sulfated Glycosaminoglycan Linkage Region Is a Novel Type of Human Natural Killer-1 (HNK-1) Epitope Expressed on Aggrecan in Perineuronal Nets

Human natural killer-1 (HNK-1) carbohydrate (HSO₃-3GlcAβ1-3Galβ1-4GlcNAc-R) is highly expressed in the brain and required for learning and neural plasticity. We previously demonstrated that expression of the HNK-1 epitope is mostly abolished in knockout mice for GlcAT-P (B3gat1), a major glucuronyltransferase required for HNK-1 biosynthesis, but remained in specific regions such as perineuronal nets (PNNs) in these mutant mice. Considering PNNs are mainly composed of chondroitin sulfate proteoglycans (CSPGs) and regulate neural plasticity, GlcAT-P-independent expression of HNK-1 in PNNs is suggested to play a role in neural plasticity. However, the function, structure, carrier glycoprotein and biosynthetic pathway for GlcAT-P-irrelevant HNK-1 epitope remain unclear. In this study, we identified a unique HNK-1 structure on aggrecan in PNNs. To determine the biosynthetic pathway for the novel HNK-1, we generated knockout mice for GlcAT-S (B3gat2), the other glucuronyltransferase required for HNK-1 biosynthesis. However, GlcAT-P and GlcAT-S double-knockout mice did not exhibit reduced HNK-1 expression compared with single GlcAT-P-knockout mice, indicating an unusual biosynthetic pathway for the HNK-1 epitope in PNNs. Aggrecan was purified from cultured cells in which GlcAT-P and -S are not expressed and we determined the structure of the novel HNK-1 epitope using liquid chromatography/mass spectrometry (LC/MS) as a sulfated linkage region of glycosaminoglycans (GAGs), HSO₃-GlcA-Gal-Gal-Xyl-R. Taken together, we propose a hypothetical model where GlcAT-I, the sole glucuronyltransferase required for synthesis of the GAG linkage, is also responsible for biosynthesis of the novel HNK-1 on aggrecan. These results could lead to discovery of new roles of the HNK-1 epitope in neural plasticity.     

Differential expression of two glucuronyltransferases synthesizing HNK-1 carbohydrate epitope in the sublineages of the rat myogenic progenitors

HNK-1 epitope is a cell-surface carbohydrate mediating various cell-cell or cell-substrate interactions. We found HNK-1 epitope in longitudinally arrayed fibers in the subpopulation of the epaxial myotome, and hypaxial myoblasts migrating into the limb bud in the rat embryo. We next investigated the expression patterns of genes encoding two glucuronyltransferases (GlcAT-P, GlcAT-D) and sulfotransferase (Sul-T), which are required for biosynthesis of HNK-1 epitope. GlcAT-P gene was expressed in the non-migrating longitudinal fibers, whereas GlcAT-D gene was expressed in the migrating myoblasts in the limb bud. Sul-T gene expression was ubiquitously observed in all these myogenic populations. Thus, differential expression of GlcAT genes may relate to the epaxial/hypaxial or migrating/non-migrating myoblast lineages.     

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.     

Cloning and chromosomal mapping of human glucuronyltransferase involved in biosynthesis of the HNK-1 carbohydrate epitope

The HNK-1 carbohydrate is expressed on various cell adhesion molecules in the nervous system and is suggested to play a role in cell-cell and cell-substrate interactions. Here we describe the isolation of a cDNA encoding human glucuronyltransferase (GlcAT-P), which is a key enzyme in the biosynthesis of the HNK-1 carbohydrate. The primary structure deduced from the cDNA sequence predicted a type II transmembrane protein of 334 amino acids. Human GlcAT-P was 98.2% identical with rat GlcAT-P in amino acid sequence, the exception being the length of the cytoplasmic tail. Northern blot analysis indicated that human GlcAT-P is expressed mainly in the brain. There is a single copy of the human GlcAT-P gene (HGMW-approved symbol B3GAT1), and it was mapped to chromosome 11q25.     

Mice deficient in nervous system-specific carbohydrate epitope HNK-1 exhibit impaired synaptic plasticity and spatial learning

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.     

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.     

Physical and functional association of glucuronyltransferases and sulfotransferase involved in HNK-1 biosynthesis

HNK-1 carbohydrate expressed predominantly in the nervous system is considered to be involved in cell migration, recognition, adhesion, and synaptic plasticity. Human natural killer-1 (HNK-1) carbohydrate has a unique structure consisting of a sulfated trisaccharide (HSO3-3GlcAbeta1-3Galbeta1-4GlcNAc-) and is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST). Considering that almost all the HNK-1 carbohydrate structures so far determined in the nervous system are sulfated, we hypothesized that GlcAT-P or GlcAT-S functionally associates with HNK-1ST, which results in efficient sequential biosynthesis of HNK-1 carbohydrate. In this study, we demonstrated that both GlcAT-P and GlcAT-S were co-immunoprecipitated with HNK-1ST with a transient expression system in Chinese hamster ovary cells. Immunofluorescence staining revealed that these enzymes are mainly co-localized in the Golgi apparatus. To determine which domain is involved in this interaction, we prepared the C-terminal catalytic domains of GlcAT-P, GlcAT-S, and HNK-1ST, and we then performed pulldown assays with the purified enzymes. As a result, we obtained evidence that mutual catalytic domains of GlcAT-P or GlcAT-S and HNK-1ST are important and sufficient for formation of an enzyme complex. With an in vitro assay system, the activity of HNK-1ST increased about 2-fold in the presence of GlcAT-P or GlcAT-S compared with that in its absence. These results suggest that the function of this enzyme complex is relevant to the efficient sequential biosynthesis of the HNK-1 carbohydrate.     

Reduced neuronal expression of synaptic transmission modulator HNK-1/neural cell adhesion molecule as a potential consequence of amyloid beta-mediated oxidative stress: a proteomic approach

Abstract Oxidative stress imparted by reactive oxygen species (ROS) is implicated in the pathogenesis of Alzheimer's disease (AD). Given that amyloid beta (Abeta) itself generates ROS that can directly damage proteins, elucidating the functional consequences of protein oxidation can enhance our understanding of the process of Abeta-mediated neurodegeneration. In this study, we employed a biocytin hydrazide/streptavidin affinity purification methodology followed by two-dimensional liquid chromatography tandem mass spectrometry coupled with SEQUEST bioinformatics technology, to identify the targets of Abeta-induced oxidative stress in cultured primary cortical mouse neurons. The Golgi-resident enzyme glucuronyltransferase (GlcAT-P) was a carbonylated target that we investigated further owing to its involvement in the biosynthesis of HNK-1, a carbohydrate epitope expressed on cell adhesion molecules and implicated in modulating the effectiveness of synaptic transmission in the brain. We found that increasing amounts of Abeta, added exogenously to the culture media of primary cortical neurons, significantly decreased HNK-1 expression. Moreover, in vivo, HNK-1 immunoreactivity was decreased in brain tissue of a transgenic mouse model of AD. We conclude that a potential consequence of Abeta-mediated oxidation of GlcAT-P is impairment of its enzymatic function, thereby disrupting HNK-1 biosynthesis and possibly adversely affecting synaptic plasticity. Considering that AD is partly characterized by progressive memory impairment and disordered cognitive function, the data from our in vitro studies can be reconciled with results from in vivo studies that have demonstrated that HNK-1 modulates synaptic plasticity and is critically involved in memory consolidation.     

Specific enzyme complex of beta-1,4-galactosyltransferase-II and glucuronyltransferase-P facilitates biosynthesis of N-linked human natural killer-1 (HNK-1) carbohydrate

Human natural killer-1 (HNK-1) carbohydrate is highly expressed in the nervous system and is involved in synaptic plasticity and dendritic spine maturation. This unique carbohydrate, consisting of a sulfated trisaccharide (HSO(3)-3GlcAβ1-3Galβ1-4GlcNAc-), is biosynthesized by the successive actions of β-1,4-galactosyltransferase (β4GalT), glucuronyltransferase (GlcAT-P and GlcAT-S), and sulfotransferase (HNK-1ST). A previous study showed that mice lacking β4GalT-II, one of seven β4GalTs, exhibited a dramatic loss of HNK-1 expression in the brain, although β4GalT-I-deficient mice did not. Here, we investigated the underlying molecular mechanism of the regulation of HNK-1 expression. First, focusing on a major HNK-1 carrier, neural cell adhesion molecule, we found that reduced expression of an N-linked HNK-1 carbohydrate caused by a deficiency of β4GalT-II is not likely due to a general loss of the β1,4-galactose residue as an acceptor for GlcAT-P. Instead, we demonstrated by co-immunoprecipitation and endoplasmic reticulum-retention analyses using Neuro2a (N2a) cells that β4GalT-II physically and specifically associates with GlcAT-P. In addition, we revealed by pulldown assay that Golgi luminal domains of β4GalT-II and GlcAT-P are sufficient for the complex to form. With an in vitro assay system, we produced the evidence that the kinetic efficiency k(cat)/K(m) of GlcAT-P in the presence of β4GalT-II was increased about 2.5-fold compared with that in the absence of β4GalT-II. Finally, we showed that co-expression of β4GalT-II and GlcAT-P increased HNK-1 expression on various glycoproteins in N2a cells, including neural cell adhesion molecule. These results indicate that the specific enzyme complex of β4GalT-II with GlcAT-P plays an important role in the biosynthesis of HNK-1 carbohydrate.     

Different acceptor specificities of two glucuronyltransferases involved in the biosynthesis of HNK-1 carbohydrate

The biosynthesis of HNK-1 carbohydrate is mainly regulated by two glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase (HNK-1 ST). To determine how the two glucuronyltransferases are involved in the biosynthesis of the HNK-1 carbohydrate, we prepared soluble forms of GlcAT-P and GlcAT-S fused with the IgG-binding domain of protein A and then compared the enzymatic properties of the two enzymes. Both GlcAT-P and GlcAT-S transferred glucuronic acid (GlcA) not only to a glycoprotein acceptor, asialoorosomucoid (ASOR), but also to a glycolipid acceptor, paragloboside. The activity of GlcAT-P toward ASOR was enhanced fivefold in the presence of sphingomyelin, but there were no effects on that of GlcAT-S. The activities of the two enzymes toward paragloboside were only detected in the presence of phospholipids such as phosphatidylinositol. Kinetic analysis revealed that the K(m) value of GlcAT-P for ASOR was 10 times lower than that for paragloboside. Furthermore, acceptor specificity analysis involving various oligosaccarides revealed that GlcAT-P specifically recognized N-acetyllactosamine (Galbeta1-4GlcNAc) at the nonreducing terminals of acceptor substrates. In contrast, GlcAT-S recognized not only the terminal Galbeta1-4GlcNAc structure but also the Galbeta1-3GlcNAc structure and showed the highest activity toward triantennary N-linked oligosaccharides. GlcAT-P transferred GlcA to NCAM about twice as much as to ASOR, whereas GlcAT-S did not show any activity toward NCAM. These lines of evidence indicate that these two enzymes have significantly different acceptor specificities, suggesting that they may synthesize functionally and structurally different HNK-1 carbohydrates in the nervous system.     

Crystal structure of GlcAT-S, a human glucuronyltransferase, involved in the biosynthesis of the HNK-1 carbohydrate epitope

The HNK-1 carbohydrate epitope is found in various neural cell adhesion molecules. Two glucuronyltransferases (GlcAT-P and GlcAT-S) are involved in the biosynthesis of HNK-1 carbohydrate. Our previous study on the crystal structure of GlcAT-P revealed the reaction and substrate recognition mechanisms of this enzyme. Comparative analyses of the enzymatic activities of GlcAT-S and GlcAT-P showed that there are notable differences in the acceptor substrate specificities of these enzymes. To elucidate differences between their specificities, we now solved the crystal structure of GlcAT-S. Residues interacting with UDP molecule, which is a part of the donor substrate, are highly conserved between GlcAT-P and GlcAT-S. On the other hand, there are some differences between these proteins in the manner they recognize their respective acceptor substrates. Phe245, one of the most important GlcAT-P residues for the recognition of acceptors, is a tryptophan in GlcAT-S. In addition, Val320, which is located on the C-terminal long loop of the neighboring molecule in the dimer and critical in the recognition of the acceptor sugar molecule by the GlcAT-P dimer, is an alanine in GlcAT-S. These differences play key roles in establishing the distinct specificity for the acceptor substrate by GlcAT-S, which is further supported by site-directed mutagenesis of GlcAT-S and a computer-aided model building of GlcAT-S/substrate complexes.     

The role of human natural killer-1 (HNK-1) carbohydrate in neuronal plasticity and disease

BackgroundThe human natural killer-1 (HNK-1) carbohydrate, a unique trisaccharide possessing sulfated glucuronic acid in a non-reducing terminus (HSO₃-3GlcAß1-3Galß1-4GlcNAc-), 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 reviewWe 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 conclusionsWe 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 significanceThe 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.     

Structural basis for acceptor substrate recognition of a human glucuronyltransferase, GlcAT-P, an enzyme critical in the biosynthesis of the carbohydrate epitope HNK-1

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.     

Biosynthesis of HNK-1 glycans on O-linked oligosaccharides attached to the neural cell adhesion molecule (NCAM): the requirement for core 2 beta 1,6-N-acetylglucosaminyltransferase and the muscle-specific domain in NCAM

The HNK-1 glycan, sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->R, is highly expressed in neuronal cells and apparently plays critical roles in neuronal cell migration and axonal extension. The HNK-1 glycan synthesis is initiated by the addition of beta1,3-linked GlcA to N-acetyllactosamine followed by sulfation of the C-3 position of GlcA. The cDNAs encoding beta1,3-glucuronyltransferase (GlcAT-P) and HNK-1 sulfotransferase (HNK-1ST) have been recently cloned. Among various adhesion molecules, the neural cell adhesion molecule (NCAM) was shown to contain HNK-1 glycan on N-glycans. In the present study, we first demonstrated that NCAM also bears HNK-1 glycan attached to O-glycans when NCAM contains the O-glycan attachment scaffold, muscle-specific domain, and is synthesized in the presence of core 2 beta1,6-N-acetylglucosaminyltransferase, GlcAT-P, and HNK-1ST. Structural analysis of the HNK-1 glycan revealed that the HNK-1 glycan is attached on core 2 branched O-glycans, sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNAc. Using synthetic oligosaccharides as acceptors, we found that GlcAT-P and HNK-1ST almost equally act on oligosaccharides, mimicking N- and O-glycans. By contrast, HNK-1 glycan was much more efficiently added to N-glycans than O-glycans when NCAM was used as an acceptor. These results are consistent with our results showing that HNK-1 glycan is minimally attached to O-glycans of NCAM in fetal brain, heart, and the myoblast cell line, C2C12. These results combined together indicate that HNK-1 glycan can be synthesized on core 2 branched O-glycans but that the HNK-1 glycan is preferentially added on N-glycans over O-glycans of NCAM, probably because N-glycans are extended further than O-glycans attached to NCAM containing the muscle-specific domain.     

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.