HNK-1 Epitope Research Articles

Molecular and Cellular Insights: A Focus on Glycans and the HNK1 Epitope in Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is a synaptic disorder with a GABA/glutamate imbalance in the perineuronal nets and structural abnormalities such as increased dendritic spines and decreased long distance connections. Specific pregnancy disorders significantly increase the risk for an ASD phenotype such as preeclampsia, preterm birth, hypoxia phenomena, and spontaneous miscarriages. They are associated with defects in the glycosylation-immune placental processes implicated in neurogenesis. Some glycans epitopes expressed in the placenta, and specifically in the extra-villous trophoblast also have predominant functions in dendritic process and synapse function. Among these, the most important are CD57 or HNK1, CD22, CD24, CD33 and CD45. They modulate the innate immune cells at the maternal-fetal interface and they promote foeto-maternal tolerance. There are many glycan-based pathways of immunosuppression. N-glycosylation pathway dysregulation has been found to be associated with autoimmune-like phenotypes and maternal-autoantibody-related (MAR) autism have been found to be associated with central, systemic and peripheric autoimmune processes. Essential molecular pathways associated with the glycan-epitopes expression have been found to be specifically dysregulated in ASD, notably the Slit/Robo, Wnt, and mTOR/RAGE signaling pathways. These modifications have important effects on major transcriptional pathways with important genetic expression consequences. These modifications lead to defects in neuronal progenitors and in the nervous system's implementation specifically, with further molecular defects in the GABA/glutamate system. Glycosylation placental processes are crucial effectors for proper maternofetal immunity and endocrine/paracrine pathways formation. Glycans/ galectins expression regulate immunity and neurulation processes with a direct link with gene expression. These need to be clearly elucidated in ASD pathophysiology.

Discovery Sulfoglycomics and Identification of the Characteristic Fragment Ions for High-Sensitivity Precise Mapping of Adult Zebrafish Brain-Specific Glycotopes.

Mass spectrometry–based high-sensitivity mapping of terminal glycotopes relies on diagnostic MS2 and/or MS3 ions that can differentiate linkage and define the location of substituents including sulfates. Unambiguous identification of adult zebrafish glycotopes is particularly challenging due to the presence of extra β4-galactosylation on the basic building block of Galβ1-4GlcNAc that can be fucosylated and variably sialylated by N-acetyl, N-glycolyl, or deaminated neuraminic acids. Building on previous groundwork that have identified various organ-specific N- and O-glycans of adult zebrafish, we show here that all the major glycotopes of interest can be readily mapped by direct nano-LC-MS/MS analysis of permethylated glycans. Homing in on the brain-, intestine-, and ovary-derived samples, organ-specific glycomic reference maps based on overlaid extracted ion chromatograms of resolved glycan species, and composite charts of summed intensities of diagnostic MS2 ions representing the distribution and relative abundance of each of the glycotopes and sialic acid variants were established. Moreover, switching to negative mode analysis of sample fractions enriched in negatively charged glycans, we show, for the first time, that a full range of sulfated glycotopes is expressed in adult zebrafish. In particular, 3-O-sulfation of terminal Gal was commonly found, whereas terminal sulfated HexNAc as in GalNAcβ1-4GlcNAc (LacdiNAc), and 3-O-sulfated hexuronic acid as in HNK-1 epitope (SO3-3GlcAβ1-3Galβ1-4GlcNAc) were identified only in the brain and not in the intestine or ovaries analyzed in parallel. Other characteristic structural features of sulfated O- and N-glycans along with their diagnostic ions detected in this discovery mode sulfoglycomic work collectively expand our adult zebrafish glycome atlas, which can now allow for a more complete navigation and probing of the underlying sulfotransferases and glycosyltransferases, in search of the functional relevance of zebrafish-specific glycotopes. Of particular importance is the knowledge of glycomic features distinct from those of humans when using adult zebrafish as an alternative vertebrate model, rather than mouse, for brain-related glyco-neurobiology studies.

Evidence for Quantum Chemical Effects in Receptor-Ligand Binding Between Integrin and Collagen Fragments - A Computational Investigation With an Impact on Tissue Repair, Neurooncolgy and Glycobiology.

The collagen-integrin interactions are mediated by the doubly charged Mg2+ cation. In nature this cation seems to have the optimal binding strength to stabilize this complex. It is essential that the binding is not too weak so that the complex becomes unstable, however, it is also of importance that the ligand-receptor binding is still labile enough so that the ligand can separate from the receptor in a suited environment. In the case of crystal growing for experimentally useful integrin-collagen fragment complexes it turned out that Co2+ cations are ideal mediators to form stable complexes for such experiments. Although, one can argue that Co2+ is in this context an artificial cation, however, it is now of special interest to test the impact of this cation in cell-culture experiments focusing on integrin-ligand interactions. In order to examine, in particular, the role cobalt ions we have studied a Co2+ based model system using quantum chemical calculations. Thereby, we have shown that hybrid and long-range corrected functional, which are approximations provide already a sufficient level of accuracy. It is of interest to study a potential impact of cations on the binding of collagen-fragments including collagens from various species because different integrins have numerous biological functions (e.g. Integrin – NCAM (Neural cell adhesion molecule) interactions) and are triggered by intact and degraded collagen fragments. Since integrin–carbohydrate interactions play a key role when bio-medical problems such as tumor cell adhesion and virus-host cell infections have to be addressed on a sub-molecular level it is essential to understand the interactions with heavy-metal ions also at the sub-atomic level. Our findings open new routes, especially, in the fields of tissue repair and neuro-oncology for example for cell-culture experiments with different ions. Since Co2+ ions seem to bind stronger to integrin than Mg2+ ions it should be feasible to exchange these cations in suited tumor tissues although different cations are present in other metalloproteins which are active in such tissues. Various staining methods can be applied to document the interactions of integrins with carbohydrate chains and other target structures. Thereby, it is possible to study a potential impact of these interactions on biological functions. It was therefore necessary to figure out first which histological–glycobiological experimental settings of tumor cells are suited for our purpose. Since the interactions of several metalloproteins (integrin, ADAM12) with polysialic acid and the HNK-1 epitope play a crucial role in tumor tissues selected staining methods are proper tools to obtain essential information about the impact of the metal ions under study.

Expression Patterns of Extracellular Matrix Proteins during Posterior Commissure Development.

Extracellular matrix (ECM) molecules are pivotal for central nervous system (CNS) development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axon guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during CNS development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn toward the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons.

α-Klotho mice demonstrate increased expression of the non-sulfated N-glycan form of the HNK-1 glyco-epitope in kidney tissue.

The α-Klotho mouse is an animal model that prematurely exhibits phenotypes resembling human aging owing to mutation of the α-Klotho gene. Although α-Klotho mice appear normal at birth, they begin showing multiple age-associated disorders after 3-4 weeks. Meanwhile, overexpression of α-Klotho extends lifespan. Therefore, α-Klotho may be involved in the aging process. The α-Klotho protein has homology to β-glucosidase and is proposed to have glycosidase activity. However, it is unclear whether glycan alterations are present in α-Klotho mice. Here we found increased levels of the non-sulfated HNK-1 glyco-epitope in the kidneys of α-Klotho mice. This phenomenon was also observed in normal aged mice. Immunohistochemical analysis demonstrated that increased non-sulfated HNK-1 glyco-epitope appeared predominantly in the outer half of the renal cortex, where α-Klotho protein is highly expressed. To clarify the cause, the expression of glucuronyltransferase S (GlcAT-S) and the activity of β-glucuronidase were also examined. The expressions of GlcAT-S were comparable in α-Klotho mice and wild-type mice, but β-glucuronidase activity was lower in α-Klotho mice than in wild-type. These results suggest that increased non-sulfated HNK-1 epitope levels in α-Klotho mice may be due to decreased β-glucuronidase activity. Taken together, α-Klotho expression was associated with expression of the non-sulfated HNK-1 epitope.

Anti-myelin associated glycoprotein antibodies recognize HNK-1 epitope on CNS

Antibodies to myelin-associated glycoprotein (MAG) are associated with demyelinating polyneuropathy and are specific for the HNK-1 epitope. To test if anti-MAG IgM recognize HNK-1 on CNS, sera from 20 patients and 238 controls were tested on rat slices by indirect immunofluorescence (IIF). IgM from anti-MAG positive patients, but not from control sera, stained rat brain with perineuronal or neuropil pattern, depending on the CNS region. IIF titers significantly correlated with ELISA anti-MAG titers. The staining of patients’ sera were inhibited by mouse anti-HNK-1 monoclonal antibody. Our results demonstrate that anti-MAG IgM recognizes HNK-1 outside the peripheral nerve myelin carriers.

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-Glc pA-(1→3)-β- d-Gal p-(1→4)-β- d-Glc p-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.

Identification of the glycosylation pattern of meat proteins in tissues from bovine myelin

A variant form of Creutzfeld-Jacob disease in humans is associated with the consumption of food contaminated with the bovine central neural system. This has focused attention on the need for procedures to detect tissues of the neural system in meat and meat products. A method was developed for the identification of myelin glycoproteins in bovine neural tissue. The glycosylated structures of glycoproteins in different protein mixtures from central nervous system (CNS) and peripheral nervous system (PNS) isolated from bovine myelin were identified by the specific lectin-glycoprotein reactions. Digoxigenin-labeled lectins bonded to the terminal glycoconjugate sequences of nine CNS glycoproteins ranging from 15 to 200 kDa and four PNS glycoproteins ranging from 22 to 105 kDa. Datura stramonium (DSA) recognized the epitope Galbeta1-4GlcNAc by two CNS and three PNS glycoproteins. Maackia amurensis (MAA) recognized the epitope NeuAcalpha2-3Gal by four CNS and two PNS glycoproteins. The peanut Arachis hypogaea (PNA) reacted with the high molecular CNS glycoprotein (200 kDa) with the sequence Galbeta1-3GalNAc. Galanthus nivalis (GNA) bonded to mannose subunits linked alpha1-3 and alpha1-6 by six CNS glycoproteins with molecular weights between 17 and 200 kDa. Four of these glycoproteins were recognized from monoclonal antibodies against the "HNK-1 epitope".

Prosencephalic neural folds give rise to neural crest cells in the Australian lungfish, Neoceratodus forsteri

Here we present a fate map of the prosencephalic neural fold (PNF) for the Australian lungfish. The experimental procedures were carried out on lungfish embryos at Kemp's stage 24 using three different approaches. First, either medial PNF (MPNF) or lateral PNF (LPNF) were ablated and the embryos cultured until they reached Kemp's stage 42 and 44. Ablation of the LPNF provided phenotypes with arrested development of the eye, reduction of periocular pigmentation, frontonasal deformity, and a slightly reduced olfactory organ, whereas the MPNF-ablated phenotypes resulted in arrested development of the cornea and frontonasal deformity. Second, we labeled the mid-axial level of the PNF with vital DiI and traced the migration of labeled cells following culture to Kemp's stage 33. Labeled PNF-derived cells populated a basal layer of the olfactory placode, migrated into the frontonasal region, the antero-dorsal periocular quadrant, and also terminated at positions where the forebrain meninges form at later stages. Third, we examined HNK-1 immunoreactivity in the forebrain-related region. We conclude that in the Australian lungfish: (1) LPNF-derived neuroepithelium gives rise to the basal layer and contributes to the apical layer of the olfactory placode; (2) PNF-derived NC cells appear to give rise to meningeal, periocular, and frontonasal ectomesenchyme and likely infiltrate the olfactory placode as developmental precusors of the terminal nerve; (3) HNK-1 epitope is temporarily expressed in cells of the neural tube, NC cells, and neurogenic placodal cells. Our experiments have provided the first evidence for a premandibular NC stream (sensu Kundrát, 2008) in a fish.

Functional Implications of HNK-1 Expression on Invasive Behaviour of Melanoma Cells

HNK-1 epitope, also known as CD57, is expressed on a wide range of cells and has been related to cell to cell and cell to extracellular matrix interactions. Expression of the HNK-1 epitope has been considered a prognostic factor in several tumours, as it has been associated with the risk of metastasis. HNK-1 has been found to be expressed on uveal and cutaneous melanoma and proposed as a useful marker of the risk of metastasis. We have analysed the HNK-1 expression on a large panel of melanoma cell lines, the involvement of this epitope in melanoma cell adhesion, as well as its migrative and invasive behaviour. HNK-1 was highly expressed in 12.9% of melanoma cell lines, and in vitro experiments using invasive melanoma cell lines demonstrated that an HNK-1 blockade reduces cell adhesion to extracellular matrix as well as their migrative and invasive ability. These data support the functional relevance of HNK-1 expression in metastatic melanoma.

HNK-1 immunoreactivity during early morphogenesis of the head region in a nonmodel vertebrate, crocodile embryo

The present study examines HNK-1 immunoidentification of a population of the neural crest (NC) during early head morphogenesis in the nonmodel vertebrate, the crocodile (Crocodylus niloticus) embryos. Although HNK-1 is not an exclusive NC marker among vertebrates, temporospatial immunoreactive patterns found in the crocodile are almost consistent with NC patterns derived from gene expression studies known in birds (the closest living relatives of crocodiles) and mammals. In contrast to birds, the HNK-1 epitope is immunoreactive in NC cells at the neural fold level in crocodile embryos and therefore provides sufficient base to assess early migratory events of the cephalic NC. I found that crocodile NC forms three classic migratory pathways in the head: mandibular, hyoid, and branchial. Further, I demonstrate that, besides this classic phenotype, there is also a forebrain-derived migratory population, which consolidates into a premandibular stream in the crocodile. In contrast to the closely related chick model, crocodilian premandibular and mandibular NC cells arise from the open neural tube suggesting that species-specific heterochronic behavior of NC may be involved in the formation of different vertebrate facial phenotypes.

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.

Differential expression of HNK‐1 and p75NTR in adult canine Schwann cells and olfactory ensheathing cells in situ but not in vitro

Olfactory ensheathing cells (OECs) are promising candidates for autologous cell transplantation therapies of nervous system injury and disease. Large animal models are relevant for transferring experimental data into clinical practice. In vivo studies have suggested that adult canine OECs may display similar regenerating capacities as their rodent counterpart. However, data on their molecular phenotype required for generating pure cell preparations are still scarce. In the present study, we comparatively analyzed expression of the carbohydrate HNK-1 epitope and the neurotrophin receptor p75(NTR) in adult canine Schwann cells and olfactory ensheathing cells in situ and in vitro. Myelinating and nonmyelinating Schwann cells in situ exclusively expressed HNK-1 and p75(NTR), respectively, whereas OECs were negative for both markers. In vitro, OECs and Schwann cells shared cell surface expression of p75(NTR) but not of HNK-1, which could be detected transiently in intracellular vesicles. This suggests that Schwann cells and OECs in vitro phagozytose HNK-1+ cellular debris. The cultivation-induced downregulation of HNK-1 expression in Schwann cells and upregulation of p75(NTR) in OECs argues for the possibility that axonal signals control the expression of both markers in situ. Whereas HNK-1 expression in Schwann cells is most likely controlled by signals inducing myelination, e.g., neuregulin, the mechanisms that may suppress p75(NTR) expression in OECs in situ remain to be elucidated. Interestingly, HNK-1 expression in the adult dog was found in both sensory and motor nerve myelinating Schwann cells. This is reminiscent of humans and differs from rodents; it also underscores the importance of large animal models for translational research.

Molecular characterization of myelin protein zero in Xenopus laevis peripheral nerve: Equilibrium between non-covalently associated dimer and monomer

Myelin protein zero (P0), a glycosylated single-pass transmembrane protein, is essential in the formation and maintenance of peripheral nervous system (PNS) compact myelin. P0 in Xenopus (xP0) exists primarily as a dimeric form that remains stable after various physical and chemical treatments. In exploring the nature of the interactions underlying the dimer stability, we found that xP0 dimer dissociated into monomer during continuous elution gel electrophoresis and conventional SDS-PAGE, indicating that the dimer is stabilized by non-covalent interactions. Furthermore, as some of the gel-purified monomer re-associated into dimer on SDS-PAGE gels, there is likely a dynamic equilibrium between xP0 dimer and monomer in vivo. Because the carbohydrate and fatty acyl moieties may be crucial for the adhesion role of P0, we used sensitive mass spectrometry approaches to elucidate the detailed N-glycosylation and S-acylation profiles of xP0. Asn92 was determined to be the single, fully-occupied glycosylation site of xP0, and a total of 12 glycans was detected that exhibited new structural features compared with those observed from P0 in other species: (1) the neutral glycans were composed mainly of high mannose and hybrid types; (2) 5 of 12 were acidic glycans, among which three were sialylated and the other two were sulfated; (3) none of the glycans had core fucosylation; and (4) no glucuronic acid, hence no HNK-1 epitope, was detected. The drastically different carbohydrate structures observed here support the concept of the species-specific variation in N-glycosylation of P0. Cys152 was found to be acylated with stearoyl (C18:0), whereas palmitoyl (C16:0) is the corresponding predominant fatty acyl group on P0 from higher vertebrates. We propose that the unique glycosylation and acylation patterns of Xenopus P0 may underlie its unusual dimerization behavior. Our results should shed light on the understanding of the phylogenetic development of P0's adhesion role in PNS compact myelin.

Isolation and characterization of a novel uronic acid-containing acidic glycosphingolipid from the ascidian Halocynthia roretzi

A novel uronic acid-containing glycosphingolipid (UGL-1) was isolated from the ascidian Halocynthia roretzi. UGL-1 was prepared from chloroform-methanol extracts and purified by the use of successive column chromatography on DEAE-Sephadex, Florisil, and Iatrobeads. Chemical structural analysis was performed using methylation analysis, gas chromatography, gas chromatography-mass spectrometry, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry, and 1H-NMR spectra. The chemical structure of UGL-1 was determined to be a glucuronic acid-containing glycosphingolipid, Galbeta1-4(Fucalpha1-3)GlcAbeta1-1Cer. The ceramide component was composed of C16:0 and C18:0 acids and C16-, C17-, and C18-phytosphingosines as major components.

Phylogenetic and mutational analyses reveal key residues for UDP‐glucuronic acid binding and activity of β1,3‐glucuronosyltransferase I (GlcAT‐I)

The beta1,3-glucuronosyltransferases are responsible for the completion of the protein-glycosaminoglycan linkage region of proteoglycans and of the HNK1 epitope of glycoproteins and glycolipids by transferring glucuronic acid from UDP-alpha-D-glucuronic acid (UDP-GlcA) onto a terminal galactose residue. Here, we develop phylogenetic and mutational approaches to identify critical residues involved in UDP-GlcA binding and enzyme activity of the human beta1,3-glucuronosyltransferase I (GlcAT-I), which plays a key role in glycosaminoglycan biosynthesis. Phylogeny analysis identified 119 related beta1,3-glucuronosyltransferase sequences in vertebrates, invertebrates, and plants that contain eight conserved peptide motifs with 15 highly conserved amino acids. Sequence homology and structural information suggest that Y84, D113, R156, R161, and R310 residues belong to the UDP-GlcA binding site. The importance of these residues is assessed by site-directed mutagenesis, UDP affinity and kinetic analyses. Our data show that uridine binding is primarily governed by stacking interactions with the phenyl group of Y84 and also involves interactions with aspartate 113. Furthermore, we found that R156 is critical for enzyme activity but not for UDP binding, whereas R310 appears less important with regard to both activity and UDP interactions. These results clearly discriminate the function of these two active site residues that were predicted to interact with the pyrophosphate group of UDP-GlcA. Finally, mutation of R161 severely compromises GlcAT-I activity, emphasizing the major contribution of this invariant residue. Altogether, this phylogenetic approach sustained by biochemical analyses affords new insight into the organization of the beta1,3-glucuronosyltransferase family and distinguishes the respective importance of conserved residues in UDP-GlcA binding and activity of GlcAT-I.

Sulfated HNK-1 Epitope in Developing and Mature Kidney: A New Marker for Thin Ascending Loop of Henle and Tubular Injury in Acute Tubular Necrosis

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

Collagen type IX and HNK-1 epitope in tears of patients with pseudoexfoliation syndrome

Pseudoexfoliation syndrome (PEX) is an age-related condition, which may cause open-angle glaucoma and has increasing interest since it seems to affect additional human tissues, i.e., cardiovascular tissue, skin, and still lacks elucidated pathogenesis. Collagen type IX and HNK-1 epitope have been considered characteristic constituents of the aqueous humour of PEX patients, since their amounts were increased in PEX aqueous humour compared to normal eyes. Since it has been proposed that the initial manifestations of PEX syndrome occur in conjunctiva, the present study was undertaken to investigate the presence of the same antigens in tears of PEX patients and their possible use as the biochemical markers for early diagnosis. Tears of PEX patients and healthy individuals were subjected to western blotting analysis for various basement membrane components identified in aqueous humour. It was found that collagen type IX and HNK-1 epitope were present in tears, the amount of the former being increased 2.7 times compared to normal (P<0.05), surprisingly high as compared with total protein or lysozyme activity in tears, which were found to be increased in PEX patients about 25% with no statistical differences (P approximately 0.4). The results suggest the possible use of tears' collagen type IX for the diagnosis of PEX syndrome.

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.

Changes in HNK-1 epitope and collagen type IX in the aqueous humour of patients with pseudoexfoliation syndrome

Purpose. To investigate alterations in the proteoglycan (PG) and glycosaminoglycan (GAG) content of the aqueous humour in patients with pseudoexfoliation syndrome (PEX). Materials and methods. Aqueous humor samples were obtained during cataract surgery from nineteen patients bearing PEX features and twenty-three age-matched normal controls. Protein and IgG were quantified densitometrically after their electrophoretic separation. Collagen type IX, 3-sulphoglucuronic acid (HNK-1 epitope), biglycan and heparan sulphate proteoglycans were detected in Western and dot blots by using specific monoclonal antibodies (MAbs). The immunochemical analysis was performed in native aqueous humour or after degradation of the glycosaminoglycans with chondroitinases. Results. Degradation of the samples with chondroitinases ABC, AC and B revealed that, in the aqueous humour from PEX eyes, collagen type IX and biglycan had a more dermatan sulphate than did normal eyes. In addition, more HNK-1 epitope was observed in PEX eyes, which after similar enzymatic treatment was found to be located mainly in dermatan sulphate sequences. 3-sulphoglucuronic acid was a constituent of the GAG chains of the collagen type IX. We found that the electrophoretic mobility of the bands of collagen type IX and HNK-1 epitope was exactly the same in the aqueous humour of normal and PEX samples; both migrated as four bands at 120, 113, 92.6 and 56 kDa. The PGs bearing heparan sulphate were found only in normal samples. Other PGs were not detected. Conclusion. Because no significant difference was observed in the concentration of albumin and IgG in PEX and normal samples, the blood-aqueous barrier was probably not significantly compromised in PEX patients with cataract but without open-angle glaucoma. The results support the hypothesis that the pathogenesis of PEX can be linked to disturbed metabolism of GAGs and PGs.