A family of novel, acidic N-glycans in Bowes melanoma tissue plasminogen activator have L2/HNK-1-bearing antennae, many with sulfation of the fucosylated chitobiose core.

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.     

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.     

Structural analysis of murine zona pellucida glycans. Evidence for the expression of core 2-type O-glycans and the Sd(a) antigen

Murine sperm initiate fertilization by binding to specific oligosaccharides linked to the zona pellucida, the specialized matrix coating the egg. Biophysical analyses have revealed the presence of both high mannose and complex-type N-glycans in murine zona pellucida. The predominant high mannose-type glycan had the composition Man(5)GlcNAc(2), but larger oligosaccharides of this type were also detected. Biantennary, triantennary, and tetraantennary complex-type N-glycans were found to be terminated with the following antennae: Galbeta1-4GlcNAc, NeuAcalpha2-3Galbeta1-4GlcNAc, NeuGcalpha2-3Galbeta1-4GlcNAc, the Sd(a) antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc, NeuGcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc), and terminal GlcNAc. Polylactosamine-type sequence was also detected on a subset of the antennae. Analysis of the O-glycans indicated that the majority were core 2-type (Galbeta1-4GlcNAcbeta1-6[Galbeta1-3]GalNAc). The beta1-6-linked branches attached to these O-glycans were terminated with the same sequences as the N-glycans, except for terminal GlcNAc. Glycans bearing Galbeta1-4GlcNAcbeta1-6 branches have previously been suggested to mediate initial murine gamete binding. Oligosaccharides terminated with GalNAcbeta1-4Gal have been implicated in the secondary binding interaction that occurs following the acrosome reaction. The significant implications of these observations are discussed.     

Poly-N-acetyllactosamine synthesis in branched N-glycans is controlled by complemental branch specificity of I-extension enzyme and beta1,4-galactosyltransferase I.

Poly-N-acetyllactosamine is a unique carbohydrate that can carry various functional oligosaccharides, such as sialyl Lewis X. It has been shown that the amount of poly-N-acetyllactosamine is increased in N-glycans, when they contain Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->4GlcNAcbeta1 -->2)Manalpha1-->6 branched structure. To determine how this increased synthesis of poly-N-acetyllactosamines takes place, the branched acceptor was incubated with a mixture of i-extension enzyme (iGnT) and beta1, 4galactosyltransferase I (beta4Gal-TI). First, N-acetyllactosamine repeats were more readily added to the branched acceptor than the summation of poly-N-acetyllactosamines formed individually on each unbranched acceptor. Surprisingly, poly-N-acetyllactosamine was more efficiently formed on Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chain than in Galbeta1-->4GlcNAcbeta1-->6Manalpha-->R, due to preferential action of iGnT on Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chain. On the other hand, galactosylation was much more efficient on beta1,6-linked GlcNAc than beta1,2-linked GlcNAc, preferentially forming Galbeta1-->4GlcNAcbeta1-->6(GlcNAcbeta1-->2)Manalph a1-->6Manbeta -->R. Starting with this preformed acceptor, N-acetyllactosamine repeats were added almost equally to Galbeta1-->4GlcNAcbeta1-->6Manalpha-->R and Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chains. Taken together, these results indicate that the complemental branch specificity of iGnT and beta4Gal-TI leads to efficient and equal addition of N-acetyllactosamine repeats on both side chains of GlcNAcbeta1-->6(GlcNAcbeta1-->2)Manalpha1-->6Manbet a-->R structure, which is consistent with the structures found in nature. The results also suggest that the addition of Galbeta1-->4GlcNAcbeta1-->6 side chain on Galbeta1-->4GlcNAcbeta1-->2Man-->R side chain converts the acceptor to one that is much more favorable for iGnT and beta4Gal-TI.     

Glycomics analysis of Schistosoma mansoni egg and cercarial secretions

The parasitic helminth Schistosoma mansoni is a major public health concern in many developing countries. Glycoconjugates, and in particular the carbohydrate component of these products, represent the main immunogenic challenge to the host and could therefore represent one of the crucial determinants for successful parasite establishment. Here we report a comparative glycomics analysis of the N- and O-glycans derived from glycoproteins present in S. mansoni egg (egg-secreted protein) and cercarial (0-3-h released protein) secretions by a combination of mass spectrometric techniques. Our results show that S. mansoni secrete glycoproteins with glycosylation patterns that are complex and stage-specific. Cercarial stage secretions were dominated by N-glycans that were core-xylosylated, whereas N-glycans from egg secretions were predominantly core-difucosylated. O-Glycan core structures from cercarial secretions primarily consisted of the core sequence Galbeta1-->3(Galbeta1-->6)GalNAc, whereas egg-secreted O-glycans carried the mucin-type core 1 (Galbeta1-->3GalNAc) and 2 (Galbeta1-->3(GlcNAcbeta1-->6)GalNAc) structures. Additionally we identified a novel O-glycan core in both secretions in which a Gal residue is linked to the protein. Terminal structures of N- and O-glycans contained high levels of fucose and include stage-specific structures. These glycan structures identified in S. mansoni secretions are potentially antigenic motifs and ligands for carbohydrate-binding proteins of the host immune system.     

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.     

Structure and function of HNK-1 sulfotransferase. Identification of donor and acceptor binding sites by site-directed mutagenesis.

HNK-1 glycan, sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAc-->R, is uniquely enriched in neural cells and natural killer cells and is thought to play important roles in cell-cell interaction. HNK-1 glycan synthesis is dependent on HNK-1 sulfotransferase (HNK-1ST), and cDNAs encoding human and rat HNK-1ST have been recently cloned. HNK-1ST belongs to the sulfotransferase gene family, which shares two homologous sequences in their catalytic domains. In the present study, we have individually mutated amino acid residues in these conserved sequences and determined how such mutations affect the binding to the donor substrate, adenosine 3'-phosphate 5'-phosphosulfate, and an acceptor. Mutations of Lys(128), Arg(189), Asp(190), Pro(191), and Ser(197) to Ala all abolished the enzymatic activity. When Lys(128) and Asp(190) were conservatively mutated to Arg and Glu, respectively, however, the mutated enzymes still maintained residual activity, and both mutant enzymes still bound to adenosine 3',5'-diphosphate-agarose. K128R and D190E mutant enzymes, on the other hand, exhibited reduced affinity to the acceptor as demonstrated by kinetic studies. These findings, together with those on the crystal structure of estrogen sulfotransferase and heparan sulfate N-deacetylase/sulfotransferase, suggest that Lys(128) may be close to the 3-hydroxyl group of beta-glucuronic acid in a HNK-1 acceptor. In contrast, the effect by mutation at Asp(190) may be due to conformational change because this amino acid and Pro(191) reside in a transition of the secondary structure of the enzyme. These results indicate that conserved amino acid residues in HNK-1ST play roles in maintaining a functional conformation and are directly involved in binding to donor and acceptor substrates.     

Functional expression and genomic structure of human N-acetylglucosamine-6-O-sulfotransferase that transfers sulfate to beta-N-acetylglucosamine at the nonreducing end of an N-acetyllactosamine sequence

The cDNA and gene encoding human N-acetylglucosamine-6-O-sulfotransferase (Gn6ST) have been cloned. Comparative analysis of this cDNA with the mouse Gn6ST sequence indicates 96% amino acid identity between the two sequences. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active sulfotransferase, which transferred sulfate to the terminal GlcNAc in GlcNAcbeta1-O-CH(3), GlcNAcbeta1-3Galbeta1-O-CH(3) and GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Gl cNAc but not in GlcNAcalpha1-4GlcAbeta1-3Galbeta1-3Galbeta1-4 Xylbeta1-O-Ser. In addition, neither Galbeta1-4GlcNAcbeta1-O-naphthalenemethanol nor GalNAcbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4X ylbeta1-O-Ser were utilized as acceptors. These findings indicate that a terminal beta-linked GlcNAc residue is necessary for acceptor substrates of Gn6ST. The human Gn6ST gene spans about 7 kb, consists of two exons and exhibits an intron-less coding region.     

The diversity of O-linked glycans expressed during Drosophila melanogaster development reflects stage- and tissue-specific requirements for cell signaling

Appropriate glycoprotein O-glycosylation is essential for normal development and tissue function in multicellular organisms. To comprehensively assess the developmental and functional impact of altered O-glycosylation, we have extensively analyzed the non-glycosaminoglycan, O-linked glycans expressed in Drosophila embryos. Through multidimensional mass spectrometric analysis of glycans released from glycoproteins by beta-elimination, we detected novel as well as previously reported O-glycans that exhibit developmentally modulated expression. The core 1 mucin-type disaccharide (Galbeta1-3GalNAc) is the predominant glycan in the total profile. HexNAcitol, hexitol, xylosylated hexitol, and branching extension of core 1 with HexNAc (to generate core 2 glycans) were also evident following release and reduction. After Galbeta1-3GalNAc, the next most prevalent glycans were a mixture of novel, isobaric, linear, and branched forms of a glucuronyl core 1 disaccharide. Other less prevalent structures were also extended with HexA, including an O-fucose glycan. Although the expected disaccharide product of the Fringe glycosyltransferase, (GlcNAcbeta1-3)fucitol, was not detectable in whole embryos, mass spectrometry fragmentation and exoglycosidase sensitivity defined a novel glucuronyl trisaccharide as GlcNAcbeta1-3(GlcAbeta1-4)fucitol. Consistent with the spatial distribution of the Fringe function, the GlcA-extended form of the Fringe product was enriched in the dorsal portion of the wing imaginal disc. Furthermore, loss of Fringe activity reduced the prevalence of the O-Fuc trisaccharide. Therefore, O-Fuc glycans necessary for the modulation of important signaling events in Drosophila are, as in vertebrates, substrates for extension beyond the addition of a single HexNAc.     

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.     

Characteristic structural features of schistosome cercarial N-glycans: expression of Lewis X and core xylosylation

Schistosomal egg N-glycans are the only examples in nature that have been structurally shown to contain beta2-xylosylation, alpha6-fucosylation, and alpha3-fucosylation on the N,N'-diacetyl chitobiose core. We present evidence that core difucosylated and xylosylated N-glycans are characteristics of Schistosoma japonicum eggs but not of the cercariae and adults, for which neither core xylosylation nor alpha3-fucosylation could be readily detected. In contrast, a majority of the N-glycans from Schistosoma mansoni cercariae but not the adults are core xylosylated. Tandem mass spectrometry analysis coupled with chromatographic mapping, sequential exoglycosidase digestion, and methylation analysis were employed to unambiguously define the structures of core beta2-xylosylated, alpha6-fucosylated N-glycans from S. mansoni cercariae. Unexpectedly, a majority of these N-glycans were found to carry Lewis X determinant, Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->, on the nonreducing termini of mono- and biantennary structures. The Lewis X-containing glycoproteins were found to be distinct from those carrying the complex, multifucosylated glycocalyx O-glycans reported previously. The corresponding N-glycans from S. japonicum cercariae are likewise dominated by Lewis X termini but without the core xylosylation. We concluded that the invading cercariae present an important and abundant source of Lewis X antigens, which may contribute to the induced humoral response upon infection. Following transformation and development into the adults, the N-glycans synthesized comprise a significantly larger amount of high mannose and fucosylated pauci-mannose structures in comparison with the cercarial N-glycans. A portion of the mono- and biantennary complex types were identified to carry Lewis X and fucosylated LacdiNAc termini, which could also be detected by mass spectrometry analysis on larger, complex-type structures.     

Core alpha1-->3-fucose is a common modification of N-glycans in parasitic helminths and constitutes an important epitope for IgE from Haemonchus contortus infected sheep

Synthesis of parasite specific IgE plays a critical role in the defence against helminth infections. We report here that IgE from serum from Schistosoma mansoni infected mice and Haemonchus contortus infected sheep recognizes complex-type N-glycans from Arabidopsis thaliana, which contain R-GlcNAcbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-Asn (core alpha1-->3-Fuc) and Xylbeta1-->2Manbeta1-->4GlcNAcbeta1-R (core beta1-->2-Xyl) modifications, and honeybee phospholipase A2, which carries N-glycans that contain the core alpha1-->3-Fuc epitope. Evidence is presented that core alpha1-->3-fucosylated N-glycans bind a substantial part of the parasite specific IgE in serum of H. contortus infected sheep. These results suggest that the core alpha1-->3-Fuc antigen may contribute to induction of a Th2 response leading to the production of IgE. In addition we show here that N-glycans carrying core alpha1-->3-Fuc and beta1-->2-Xyl antigens are synthesized by many parasitic helminths and also by the free living nematode Caenorhabditis elegans. Since N-glycans containing the core alpha1-->3-Fuc have also been implicated in honeybee and plant induced allergies, this conserved glycan might represent an important common IgE epitope.     

Identification and characterization of a novel UDP-GalNAc:GlcAbeta-R alpha1,4-N-acetylgalactosaminyltransferase from a human sarcoma cell line.

We recently discovered a novel alpha-N-acetylgalactosaminyltransferase in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190-22195, 1995) and also in mouse mast cytoma cells (Lidholt et al., Glycoconjugate J., 14, 737-742, 1997), which catalyzed the transfer of an alpha-GalNAc residue to the linkage tetrasaccharide-serine, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, derived from proteoglycans. In this study, we characterized this enzyme using a preparation obtained from the serum-free culture medium of a human sarcoma (malignant fibrous histiocytoma) cell line by phenyl-Sepharose chromatography. Structural characterization by1H NMR spectroscopy of the reaction product using the linkage tetrasaccharide-serine, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, as a substrate demonstrated that the enzyme was a UDP-GalNAc:GlcAbeta1-R alpha1,4-N -acetylgalactosaminyltransferase. This is the first identification of an alpha1,4-N-acetylgalactosaminyltransferase. Using N -acetylchondrosine GlcAbeta1-3GalNAc as an alternative substrate, the enzyme required divalent cations for the transferase reaction, with maximal activity at 20 mM Mn2+and exhibited a dual optimum at pH 6.5 and pH 7.4 depending upon the buffers used, with the highest activity in a 50 mM 2-( N -morpholino)ethanesulfonic acid buffer at pH 6.5. The apparent Km values obtained for N -acetylchondrosine, the linkage tetrasaccharide-serine, and UDP-GalNAc were 1060 microM, 188 microM, and 27 microM, respectively. This suggested that the linkage tetrasaccharide-serine was a good acceptor substrate for the enzyme. In addition, the enzyme utilized glucuronylneolactotetraosylceramide GlcAbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4G lcbeta1-1Cer but not sulfoglucuronylneolactotetraosylceramide GlcA(3-O -sulfate)beta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Gl cbeta1-1Cer as acceptor substrates. The possibility of involvement of this enzyme in the biosynthesis of glycosaminoglycan as well as other GlcA-containing glycoconjugates is discussed.     

Characterization of the oligosaccharide component of microsomal beta-glucuronidase from rat liver

The oligosaccharides of microsomal beta-glucuronidase were analysed by gel permeation and weak anion exchange chromatography following hydrazine release. N-linked glycans, constituted 80% of the total glycan pool and were mainly of the tri- and biantennary complex type with or without core and arm fucose. The major oligosaccharide, that comprised 30.6% of all the species analysed, was structurally identified by reagent array analysis method and found to be a triantennary complex structure, Galbeta1,4GlcNAcbeta1,2Manalpha1,6(3)(Galbeta1,4GlcNAcbeta1,4(Galbeta1,4GlcNAcbeta1,2) Manalpha1,3(6))Manbeta1,4GlcNAcbeta1,4 GlcNAc. O-Linked glycans comprised 20% of the total glycan pool, the major species being Galbeta1,3GalNAc. All of the N- and O-linked glycans were charged. Most of the negative charge was due to sialic acid (85.0%) with the remainder being phosphate present as phosphomonoesters (7.3%) and phosphodiesters (5%). This is the first report of O-linked carbohydrate chains in microsomal beta-glucuronidase. The presence of O-linked glycans and branched N-linked glycans in a microsomal enzyme, in relation to the current view of glycosyltransferase compartmentalization in the Golgi is discussed.