3Gal Beta Research Articles

Structural analysist of N-glycans from gull egg white glycoproteins and egg yolk IgG

We previously showed that the expression of (Gal alpha 1-4Gal)-bearing glycoproteins among birds is related to their phylogeny. However, precise structures of (Gal alpha 1-4Gal)-containing N-glycans were only known for pigeon egg white glycoproteins and IgG. To compare structural features of (Gal alpha 1-4Gal)-containing N-glycans from other species, we analyzed N-glycans of gull egg white (GEW)-glycoproteins, ovomucoid, and ovotransferrin, and gull egg yolk IgG by HPLC, mass spectrometry (MS), and MS/MS analyses. GEW-glycoproteins included neutral, monosialyl, and disialyl N-glycans, and some of them contained Gal alpha 1-4Gal sequences. Bi-, tri-, and tetra-antennary oligosaccharides that lacked bisecting GlcNAc were the major core structures, and incomplete alpha-galactosylation and sialylation as well as the presence of diLacNAc on the branches generated microheterogeneity of the N-glycan structures. Moreover, unlike pigeon egg white glycoproteins, the major sialylation in GEW-glycoproteins is alpha2,3-, but not alpha2,6-linked sialic acids (NeuAc). In addition to the complex-type oligosaccharide, hybrid-type oligosaccharides that lack bisecting GlcNAc were also abundant in GEW-glycoproteins. Gull egg yolk IgG also contained Gal alpha 1-4Gal beta 1-4GlcNAc beta 1- sequences, but unlike pigeon IgG, no Gal alpha 1-4Gal beta 1-4Gal beta 1-4GlcNAc beta 1- sequence was detected. Bi- and tri-antennary complex-type oligosaccharides with bisecting GlcNAc and with core fucosylation as well as high-mannose-type oligosaccharides were the major structures in gull IgG. Our data indicated that some N-glycans from both GEW-glycoproteins and gull IgG contain the Gal alpha 1-4Gal beta 1-4GlcNAc beta 1- sequence, but the ratio of alpha-Gal-capped residues to non-alpha-Gal-capped residues in the nonreducing termini of N-glycans is much lower than that in those of pigeon glycoproteins.

Novel Leb-like Helicobacter pylori-binding glycosphingolipid created by the expression of human -1,3/4-fucosyltransferase in FVB/N mouse stomach

The "Le(b) mouse" was established as a model for investigations of the molecular events following Le(b)-mediated adhesion of Helicobacter pylori to the gastric epithelium. By the expression of a human alpha-1,3/4-fucosyltransferase in the gastric pit cell lineage of FVB/N transgenic mice, a production of Le(b) glycoproteins in gastric pit and surface mucous cells was obtained in this "Le(b) mouse," as demonstrated by binding of monoclonal anti-Le(b) antibodies. To explore the effects of the human alpha-1,3/4-fucosyltransferase on glycosphingolipid structures, neutral glycosphingolipids were isolated from stomachs of transgenic alpha-1,3/4-fucosyltransferase-expressing mice. A glycosphingolipid recognized by BabA-expressing H. pylori was isolated and characterized by mass spectrometry and proton NMR as Fuc alpha 2Gal beta 3(Fuc alpha 4)GalNAc beta 4 Gal beta 4 Glc beta 1Cer, i.e., a novel Le(b)-like glycosphingolipid on a ganglio core. In addition, two other novel glycosphingolipids were isolated from the mouse stomach epithelium that were found to be nonbinding with regard to H. pylori. The first was a pentaglycosylceramide, GalNAc beta 3 Gal alpha 3(Fuc alpha 2)Gal beta 4 Glc beta 1Cer, in which the isoglobotetrasaccharide has been combined with Fuc alpha 2 to yield an isoglobotetraosylceramide with an internal blood group B determinant. The second one was an elongated fucosyl-gangliotetraosylceramide, GalNAc beta 3(Fuc alpha 2)Gal beta 3GalNAc beta 4Gal beta 4 Glc beta 1Cer.

Helicobacter pylori binding to new glycans based on N-acetyllactosamine

Previously we reported binding of Helicobacter pylori to various nonacid and sialylated neolacto carbohydrate structures using a wide range of natural and chemically modified sequences. A novel nonsialylated neolacto-based binding epitope, GlcNAc beta 3Gal beta 4GlcNAc, and analogous structures carrying terminal GalNAc beta 3, GalNAc alpha 3, or Gal alpha 3 showed the binding activity (Miller-Podraza H, Lanne B, Angström J, Teneberg S, Abul-Milh M, Jovall P-A, Karlsson H, Karlsson K-A. 2005. Novel binding epitope for Helicobacter pylori found in neolacto carbohydrate chains. J Biol Chem. 280:19695-19703). The present work reports two other H. pylori-binding nonsialylated neolacto-based structures, GlcA beta 3Gal beta 4GlcNAc beta 3-R and Glc beta 3Gal beta 4GlcNAc beta 3-R, and two amide derivatives (N-methyl and N-ethyl) of GlcA beta 3Gal beta 4GlcNAc beta 3-R which were bound by H. pylori. The latter structures turned out to be more effective as H. pylori binders than the parent saccharide. New reducing-end variants of the neolacto epitope including species containing N-acetyllactosamine linked beta 6 to GlcNAc or Gal with similarity to branched polylactosamines and mucins were prepared and tested. The results extend our previous findings on binding specificities of H. pylori and show that this pathogen is able to interact with an array of N-acetyllactosamine/neolacto structures, which may be of importance for the in vivo interaction of the bacterium with human cells. The information gained in this work may also be of value for rational design of anti-H. pylori drugs.

Enhanced expression of 3-galactosyltransferase 5 activity is sufficient to induce in vivo synthesis of extended type 1 chains on lactosylceramides of selected human colonic carcinoma cell lines

In general, an elevated expression of beta 3-galactosyltransferase (beta 3GalT) activity contributed by beta 3GalT5 correlates well with increased biosynthesis and expression of type 1 chain (Gal beta 1-3GlcNAc beta 1-) derivatives such as Lewis A and sialyl Lewis A, which are mostly recognized as terminal epitopes and not further extended. Most known beta 3-N-acetylglucosaminyltransferases show a higher activity toward extending type 2 chain (Gal beta 1-4GlcNAc beta 1-), and an over-expression of beta 3GalT5 could suppress the formation of the type 2 chain poly-N-acetyllactosaminoglycans. The potential of extending instead the predominant type 1 chain termini synthesized under such circumstances was, however, not investigated, partly due to technical difficulty in unambiguous identification of extended type 1 chains. Using an advanced mass spectrometry-based glycomic mapping and glycan sequencing approach, we show here that type 1 chains carried on the lacto-series glycosphingolipids of colonic carcinoma cells can be extended when the endogenous beta 3GalT activity relative to competing beta 4GalT activity, as defined against a common GlcNAc beta 1-3Gal beta 1-4Glc acceptor, is sufficiently high, as found in Colo205 and SW1116, but not in DLD-1 cells. In support of this positive correlation, the lacto-series glycosphingolipids isolated from stably transfected DLD-1 clones over-expressing beta 3GalT5 were shown to comprise fucosylated dimeric type 1 chains, whereas a mock transfectant and the DLD-1 parent carried only fucosylated dimeric type 2 chains on their lactosylceramides. It suggests that while the natural expression of extended type 1 chain is likely to be determined by many contributing factors including the relative amounts of competing glycosyltransferases and the UDP-Gal level, the enhanced expression of beta 3GalT5 is sufficient to promote in vivo extension of type 1 chains by furnishing a significantly higher amount of type 1 chain precursors relative to competing type 2 chains.

Brain Proton Magnetic Resonance Spectroscopy and Neuromuscular Pathology in a Patient With GM1 Gangliosidosis

The authors report the clinical, neuroradiologic, and neuromuscular pathological findings in a patient with GM1 gangliosidosis. The proton magnetic resonance spectroscopy, previously reported in a single patient with GM1 gangliosidosis, detected a mild reduction of N-acetylaspartate, consistent with relative paucity of axons and neurons and increased levels of myoinositol suggestive of gliotic white matter changes along with the accumulation of an additional compound that could represent either guanidinoacetate or Gal beta 1-6Gal beta 1-4)GlcNAc, an oligosaccharide previously isolated from the urine of GM1 gangliosidosis patients. Although these findings will have to be further confirmed in more patients with GM1 gangliosidosis, they suggest that proton magnetic resonance spectroscopy may provide useful end points to assess the efficacy of novel treatments that could soon become clinically available. Histologically, no significant alterations were found in axons, but there was evidence of redundant and inappropriately folded myelin, which is a feature attributed to disturbed axon-glial interactions.

Normal human serum contains high levels of anti‐Galα1‐4GlcNAc antibodies

Natural xenoreactive antibodies (Abs) directed against the Bdi-epitope (Gal alpha 1-3Gal beta) on the cells of non-primate mammals take part in hyperacute rejection of xenotransplanted organs. We found that some Abs, which were one-step affinity purified on Bdi-Sepharose, cross-reacted with the disaccharide Gal alpha 1-4GlcNAc beta. The epitope Gal alpha 1-4GlcNAc has not been identified on mammals or bacterial polysaccharides yet. To isolate the antibodies of the corresponding specificity the disaccharide was immobilized on Sepharose and antibodies were affinity purified from pooled serum of blood group O individuals. These one-step purified Abs cross-reacted with Bdi, but after a prior absorption step on Bdi-Sepharose no cross-reactivity with Bdi was observed any longer. Surprisingly, the quantity of anti-Gal alpha 1-4GlcNAc isolated from the same serum pool, 4-7 microg/ml, was equal to that of anti-Bdi or more. Independently of ABO blood groups all the tested healthy donors had anti-Gal alpha 1-4GlcNAc Abs at a similar level. Monospecific anti-Gal alpha 1-4GlcNAc Abs were not cytotoxic towards porcine cells. 1. The actual concentration of monospecific, xenoreactive Gal alpha 1-3Gal beta Abs in blood may be considerably lower than the value referred to in the literature for 'anti-alpha Gal' or 'anti-Galili' antibodies. 2. Anti-Gal alpha 1-4GlcNAc Abs seem not to be important for xenotransplantation.

Natural protection from zoonosis by alpha‐gal epitopes on virus particles in xenotransmission

Clinical transplantation has become one of the preferred treatments for end-stage organ failure, and one of the novel approaches being pursued to overcome the limited supply of human organs involves the use of organs from other species. The pig appears to be a near ideal animal due to proximity to humans, domestication, and ability to procreate. The presence of Gal-alpha1,3-Gal residues on the surfaces of pig cells is a major immunological obstacle to xenotransplantation. Alpha1,3galactosyltransferase (alpha1,3GT) catalyzes the synthesis of Gal alpha 1-3Gal beta 1-4GlcNAc-R (alpha-gal epitope) on the glycoproteins and glycolipids of non-primate mammals, but this does not occur in humans. Moreover, the alpha-gal epitope causes hyperacute rejection of pig organs in humans, and thus, the elimination of this antigen from pig tissues is highly desirable. Recently, concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of alpha-gal on their viral particles. In this study, transgenic cells expressing alpha1,3GT were selected using 1.25 mg/ml neomycin. The development of HeLa cells expressing alpha1,3GT now allows accurate studies to be conducted on the function of the alpha-gal epitope in xenotransmission. The expressions of alpha-gal epitopes on HeLa/alpha-gal cells were demonstrated by flow cytometry and confocal microscopy using cells stained with IB4-fluorescein isothiocyanate lectin. Vaccinia viruses propagated in HeLa/alpha-gal cells also expressed alpha-gal on their viral envelopes and were more sensitive to inactivation by human sera than vaccinia virus propagated in HeLa cells. Moreover, neutralization of vaccinia virus was inhibited in human serum by 10 mm ethylene glycol bis(beta-aminoethylether)tetraacetic acid (EDTA) treatment. Our data indicated that alpha-gal epitopes are one of the major barriers to zoonosis via xenotransmission.

Lectinochemical studies on the glyco-recognition factors of a Tn (GalNAcα1→Ser/Thr) specific lectin isolated from the seeds of Salvia sclarea

The lectin extracted from the seeds of Salvia sclarea (SSL) recognizes the Tn antigen (GalNAc alpha1-->Ser/Thr) expressed in certain human carcinomas. In previous studies, knowledge of the binding properties of SSL was restricted to GalNAcalpha1--> related oligosaccharides and glycopeptides. Thus, the requirements of functional groups in monosaccharide and high-density polyvalent carbohydrate structural units for SSL binding and an updated affinity profile were further evaluated by enzyme-linked lectinosorbent (ELLSA) and inhibition assays. Among the glycoproteins (gps) tested for interaction, a high density of exposed Tn-containing glycoproteins such as in the armadillo salivary Tn glycoprotein and asialo ovine salivary glycoprotein reacted best with SSL. When the gps were tested for inhibition of SSL binding, which was expressed as 50% nanogram inhibition, the high density polyvalent Tn present in macromolecules was the most potent inhibitor. Among the monosaccharide and carbohydrate structural units studied, which were expressed as nanomole inhibition, GalNAc alpha1-->3GalNAc beta1-->3Gal alpha1-->4Gal beta1-->4Glc (Fp), GalNAc alpha1-->3Gal beta1-->4Glc (A(L)), GalNAc alpha1-->3GalNAc beta1-->Me (F beta), GalNAc alpha1-->3GalNAc alpha1-->Me (F alpha) and GalNAc alpha1--> Ser/Thr (Tn) were the most active ligands, being 2.5-5.0 x 10(3) and 1.25-2.5 times more active than Gal and GalNAc, respectively. From the results, it is suggested that the combining site of SSL is a shallow groove type, recognizing the monosaccharide of GalNAc as the major binding site or Tn up to the Forssman pentasaccharide (Fp). It can be concluded that the three critical factors for SSL binding are the -NH CH(3)CO at carbon-2 in Gal, the configuration of carbon-3 in GalNAc, and the polyvalent Tn (GalNAc alpha1-->Ser/Thr) present in macromolecules. These results should assist in understanding the glyco-recognition factors involved in carbohydrate-lectin interactions in biological processes. The effect of the polyvalent F alpha, F beta and GalNAc beta1-->3Gal alpha1--> (P alpha) glycotopes on binding should be examined. However, this is hampered by the lack of availability of suitable reagents.

Newly Discovered Neutral Glycosphingolipids in Aureobasidin A-resistant Zygomycetes: IDENTIFICATION OF A NOVEL FAMILY OF GALA-SERIES GLYCOLIPIDS WITH CORE Galα1-6Galβ1-6Galβ SEQUENCES

We found for the first time that Zygomycetes species showed resistance to Aureobasidin A, an antifungal agent. A novel family of neutral glycosphingolipids (GSLs) was found in these fungi and isolated from Mucor hiemalis, which is a typical Zygomycetes species. Their structures were completely determined by compositional sugar, fatty acid, and sphingoid analyses, methylation analysis, matrix-assisted laser desorption ionization time-of-flight/mass spectrometry, and (1)H NMR spectroscopy. They were as follows: Gal beta 1-6Gal beta 1-1Cer (CDS), Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CTS), Gal alpha 1-6Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CTeS), and Gal alpha 1-6Gal alpha 1-6Gal alpha 1-6Gal beta 1-6Gal beta 1-1Cer (CPS). The ceramide moieties of these GSLs consist of 24:0, 25:0, and 26:0 2-hydroxy acids as major fatty acids and 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid. However, the glycosylinositolphosphoceramide families that are the major GSLs components in fungi were not detected in Zygomycetes at all. This seems to be the reason that Aureobasidin A is not effective for Zygomycetes as an antifungal agent. Our results indicate that the biosynthetic pathway for GSLs in Zygomycetes is significantly different from those in other fungi and suggest that any inhibitor of this pathway may be effective for mucormycosis, which is a serious pathogenic disease for humans.

Lewis X-containing glycans are specific and potent competitive inhibitors of the binding of ZP3 to complementary sites on capacitated, acrosome-intact mouse sperm.

Mammalian fertilization requires a cascade of interactions between sperm and the egg's zona pellucida (ZP). O-linked glycans on mouse glycoprotein ZP3 have been implicated in mediating one step of the fertilization process, the firm adhesion of acrosome-intact sperm to the ZP. Experiments to identify structural requirements of a sperm-binding glycan have demonstrated that a Lewis X (Le(x))-containing glycan (Gal beta 4[Fuc alpha 3]GlcNAc-R) was a potent, competitive inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273: 1888-1895). However, those experiments did not define the particular step in the fertilization pathway that was blocked. The experiments described herein test the hypothesis that Le(x)-containing glycans are specific, competitive inhibitors of the binding of Alexa Fluor 568 fluorochrome (Alexa(568))-labeled ZP3 to sperm and, thus, bind the same sperm surface sites as ZP3. Dose-response analyses demonstrated that these glycans are potent inhibitors (IC(50) approximately 180 nM), which at saturation, reduced Alexa(568)-ZP3 binding by approximately 70%. A Lewis A (Le(a))-capped glycan (Gal beta 3[Fuc alpha 4]GlcNAc) was also a potent inhibitor (IC(50) approximately 150-200 nM), but at saturation, it reduced Alexa(568)-ZP3 binding by only 30%. In contrast, nonfucosylated glycans with nonreducing GlcNAc beta 4 or Gal beta 4 residues did not compete; neither did sialyl-Le(x) (Neu5Ac alpha 3Gal beta 4[Fuc alpha 3]GlcNAc-Lewis X) nor sulfo-Le(x) (3'-O-SO(3)-Lewis X). However, at saturation, Gal alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc reduced Alexa(568)-ZP3 binding by approximately 70% but with moderate apparent affinity (IC(50) approximately 3000 nM). Fluorescence microscopy revealed that Alexa(568)-labeled Le(x)-Lac-BSA, Le(a)-Lac-BSA, and ZP3 bound to the same sperm surface domains. However, Le(a)-Lac did not inhibit binding of Alexa(568)-Le(x)-Lac-BSA, and Le(x)-Lac did not inhibit binding of Alexa(568)-Le(a)-Lac-BSA. Finally, Le(x)-Lac and Le(a)-Lac had an additive inhibitory effect on Alexa(568)-ZP3 binding. Thus, Le(x) is a ligand for a major class of ZP3 binding sites on mouse sperm, whereas Le(a) binding defines a different but less-abundant class of sites.

Lewis X-containing neoglycoproteins mimic the intrinsic ability of zona pellucida glycoprotein ZP3 to induce the acrosome reaction in capacitated mouse sperm.

The binding of zona pellucida (ZP) glycoprotein ZP3 to mouse sperm surface receptors is mediated by protein-carbohydrate interactions. Subsequently, ZP3 induces sperm to undergo the acrosome reaction, an obligatory step in fertilization. We have previously identified Lewis X (Le(x); Gal beta 4[Fuc alpha 3]GlcNAc) as a potent inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273:1888-1895). This glycan is recognized by approximately 70% of the ZP3 binding sites on capacitated, acrosome-intact mouse sperm, whereas Lewis A (Le(a); Gal beta 3[Fuc alpha 4]GlcNAc) is recognized by most of the remaining sites (Kerr et al. Biol Reprod 2004; 71:770-777). Herein, we test the hypothesis that Le(x)- and Le(a)-containing glycans, when clustered on a neoglycoprotein, bind ZP3 receptors on sperm and induce sperm to undergo the acrosome reaction via the same signaling pathways as ZP3. Results show that a Le(x)-containing neoglycoprotein induced the acrosome reaction in a dose-dependent and capacitation-dependent manner. A Le(a)-containing neoglycoprotein also induced sperm to undergo the acrosome reaction but was less potent than Le(x)-containing neoglycoproteins. In contrast, neoglycoproteins containing beta4-lactosamine (Gal beta 4GlcNAc), Lewis B (Fuc alpha 2Gal beta 3[Fuc alpha 4]GlcNAc), and sialyl-Le(x) glycans were inactive, as were four other neoglycoproteins with different nonfucosylated glycans. Consistent with these results, unconjugated Le(x)- and Le(a)-capped glycans were dose-dependent inhibitors, which at saturation, reduced the ZP-induced acrosome reaction by about 60% and 30%, respectively. Experiments utilizing pharmacological inhibitors suggest that induction of the acrosome reaction by solubilized ZP and Le(x)- and Le(a)-containing neoglycoproteins require the same calcium-dependent pathway. However, only the ZP-induced acrosome reaction requires a functional G(i) protein. Thus, Le(x)-containing neoglycoproteins bind to a major class of ZP3 receptors on capacitated sperm. A Le(a)-containing neoglycoprotein binds a second ZP3 receptor but is a less-potent inducer of the acrosome reaction.

Studies on gangliosides with affinity for Helicobacter pylori: binding to natural and chemically modified structures

Helicobacter pylori, like many other microbes, has the ability to bind to carbohydrate epitopes. Several sugar sequences have been reported as active for the bacterium, including some neutral, sulfated, and sialylated structures. We investigated structural requirements for the sialic acid-dependent binding using a number of natural and chemically modified gangliosides. We have chosen for derivatization studies two kinds of binding-active glycolipids, the simple ganglioside S-3PG (Neu5Ac alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, sialylparagloboside) and branched polyglycosylceramides (PGCs) of human origin. The modifications included oxidation of the sialic acid glycerol chain, reduction of the carboxyl group, amidation of the carboxyl group, and lactonization. Binding experiments confirmed a preference of H. pylori for 3-linked sialic acid and penultimate 4-linked galactose. As expected, neolacto gangliosides (with Gal beta 4GlcNAc in the core structure) were active in our assays, whereas gangliosides with lacto (Gal beta 3GlcNAc) and ganglio (Gal beta 3GalNAc) carbohydrate chains were not. Negative binding results were also obtained for disialylparagloboside (with terminal NeuAc alpha 8NeuAc) and NeuAc alpha 6-containing glycolipids. Chemical studies revealed dependence of the binding on Neu5Ac and its glycerol and carboxyl side chains. Most of the derivatizations performed on these groups abolished the binding; however, some of the amide forms turned out to be active, and one of them (octadecylamide) was found to be an excellent binder. The combined data from molecular dynamics simulations indicate that the binding-active configuration of the terminal disaccharide of S-3PG is with the sialic acid in the anticlinal conformation, whereas in branched PGCs the same structural element most likely assumes the synclinal presentation.

Suppression of beta 1,3galactosyltransferase beta 3Gal-T5 in cancer cells reduces sialyl-Lewis a and enhances poly N-acetyllactosamines and sialyl-Lewis x on O-glycans.

We investigated the role of beta 3 Gal-T5, a member of the beta 1,3galactosyltransferase (beta 1,3Gal-T) family, in cancer-associated glycosylation, focusing on the expression of sialyl-Lewis a (sLea, the epitope of CA19.9 antigen), poly N-acetyllactosamines, and sialyl-Lewis x (sLex) antigen. A clone permanently expressing an antisense fragment of beta 3Gal-T5 was obtained from the human pancreas adenocarcinoma cell line BxPC3 and characterized. Both beta 1,3Gal-T activity and sLea expression are dramatically impaired in the clone. Analysis of the oligosaccharides synthesized in cells metabolically labelled with tritiated galactose shows that a relevant amount of radioactivity is associated to large O-glycans. Endo-beta-galactosidase mostly releases NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal and NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal from such O-glycans of BxPC3 membranes, but GlcNAc beta 1-3Gal and type 2 chain oligosaccharides, including NeuAc alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-3Gal, from those of the antisense clone. Furthermore, BxPC3 cells secrete sLea in the culture media but not sLex, while antisense clone secretes mostly sLex, and accumulation of both antigens is prevented by benzyl-alpha-GalNAc. These data indicate that beta 3Gal-T5 suppression turns synthesis of type 1 chain O-glycans to poly N-acetyllactosamine elongation and termination by sLex. In other cell lines and clones, beta 3Gal-T5 transcript, beta 1,3Gal-T activity, and sLea antigen are also correlated, but quantitatively the relative expression ratios are very different from cell type to cell type. We suggest that beta 3Gal-T5 plays a relevant role in gastrointestinal and pancreatic tissues counteracting the glycosylation pattern associated to malignancy, and is necessary for the synthesis and secretion of CA19.9 antigen, whose expression still depends on multiple interacting factors.

Molecular Cloning and Expression of a Second Chondroitin N-Acetylgalactosaminyltransferase Involved in the Initiation and Elongation of Chondroitin/Dermatan Sulfate

We identified a novel human chondroitin N-acetylgalactosaminyltransferase, designated chondroitin GalNAcT-2 after a BLAST analysis of the GenBank(TM) data base using the sequence of a previously described human chondroitin N-acetylgalactosaminyltransferase (chondroitin GalNAcT-1) as a probe. The new cDNA sequence contained an open reading frame encoding a protein of 542 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 60% identity to that of human chondroitin GalNAcT-1. Like chondroitin GalNAcT-1, the expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which not only transferred beta1,4-N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc to a polymer chondroitin representing growing chondroitin chains (beta-GalNAc transferase II activity) but also to GlcUA beta 1-3Gal beta 1-O-C(2)H(4)NHCbz, a synthetic substrate for beta-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein-linkage region of chondroitin sulfate. In contrast, the tetrasaccharide serine (GlcUA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser) derived from the linkage region, which is an inert acceptor substrate for chondroitin GalNAcT-1, served as an acceptor substrate. The coding region of this enzyme was divided into seven discrete exons, which is similar to the genomic organization of the chondroitin GalNAcT-1 gene, and was localized to chromosome 10q11.22. Northern blot analysis revealed that the chondroitin GalNAcT-2 gene exhibited a ubiquitous but differing expression in human tissues, and the expression pattern differed from that of chondroitin GalNAcT-1. Thus, we demonstrated redundancy in the chondroitin GalNAc transferases involved in the biosynthetic initiation and elongation of chondroitin sulfate, which is important for understanding the biosynthetic mechanisms leading to the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate and heparin/heparan sulfate chains.

Oligosaccharide specificity of the fucolectin from the bark of laburnum Laburnum anagyroides

A comparative study of thin carbohydrate specificity of the lectin from the bark of laburnum Laburnum anagyroides (LABA) and fucolectin from asparagus pea Tetragonolobus purpureus (TPA) was performed using inhibition of agglutination of the complex formed by H-active neoglycoprotein and nanoparticles of colloidal gold. Both lectins bound most strongly the H type 2 oligosaccharides comprising O-glycanes; however, TPA was almost unable to discriminate between them. LABA bound more weakly the H type 6 trisaccharide (Fuc alpha 1-2Gal beta 1-4Glc) and difucosyllactose (Fuc alpha 1-2Gal beta 1-4[Fuc alpha 1-3]Glc), a glucoanalogue of the Le(y) antigen, and, even more weakly, the Le(a) pentasaccharide lacto-N-fucopentaose II (Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4Glc). However, LABA did not bind the antigens Le(b), Le(c), and Le(d), very poorly interacted with the terminal Le(x), and somewhat more strongly bound the internal Le(x). The lectin also had a hydrophobic binding site. Both lectins exhibited a cluster effect with polymeric ligands (neoglycoproteins).

Elimination of anti-Gal B cells by alpha-Gal ricin1.

A major barrier in pig to human organ transplantation is the binding of human anti-Gal to alpha-gal epitopes (Gal alpha 1-3Gal beta 1-4GlcNAc-R) on pig cells, resulting in hyperacute and acute vascular rejection of pig xenografts. Moreover, the immune system in xenograft recipients is activated by these epitopes to produce high affinity anti-Gal, which is also detrimental to xenografts. Production of anti-Gal can be prevented by specific elimination of anti-Gal B cells. This was achieved with the toxin ricin A, coupled to human alpha1-acid glycoprotein modified to carry alpha-gal epitopes. This complex, designated alpha-gal ricin, is targeted in vivo to anti-Gal B cells by interaction with the immunoglobulin molecules (i.e., B cell receptors) on these cells. Carbohydrate chains on alpha 1-acid glycoprotein were converted to carry alpha-gal epitopes by enzymatic treatment with recombinant alpha 1,3 galactosyltransferase (alpha 1,3GT). This molecule and ricin A were biotinylated and coupled by avidin to generate alpha-gal ricin. The efficacy of alpha-gal ricin in eliminating anti-Gal B cells was studied in the experimental model of alpha 1,3GT knockout (KO) mice. These mice produce large amounts of anti-Gal immunoglobulin G when immunized with pig kidney membranes, as measured by ELISA with alpha-gal epitopes linked to bovine serum albumin (BSA). In the absence of anti-Gal B cells, these mice lack the ability to produce anti-Gal. Repeated administration of alpha-gal ricin into alpha1,3GT KO mice resulted in elimination of anti-Gal B cells, thereby preventing production of anti-Gal immunoglobulin G after immunization with pig kidney membranes. This prevention of anti-Gal production occurred with doses of alpha-gal ricin that were not toxic to the mice and did not affect production of antibodies with other specificities. Administration of alpha-gal ricin results in specific elimination of anti-Gal B cells in alpha 1,3GT KO mice. The elimination of these B cells may prove to be helpful in attempts to achieve immune tolerance to alpha-gal epitopes in primates.

A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria.

When fed to a beta-galactosidase-negative (lacZ(-)) Escherichia coli strain that was grown on an alternative carbon source (such as glycerol), lactose accumulated intracellularly on induction of the lactose permease. We showed that intracellular lactose was efficiently glycosylated when genes of glycosyltransferase that use lactose as acceptor were expressed. High-cell-density cultivation of lacZ(-) strains that overexpressed the beta 1,3 N acetyl glucosaminyltransferase lgtA gene of Neisseria meningitidis resulted in the synthesis of 6 g x L(-1) of the expected trisaccharide (GlcNAc beta 1-3Gal beta 1-4Glc). When the beta 1,4 galactosyltransferase lgtB gene of N. meningitidis was coexpressed with lgtA, the trisaccharide was further converted to lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) and lacto-N-neoheaxose with a yield higher than 5 g x L(-1). In a similar way, the nanA(-) E. coli strain that was devoid of NeuAc aldolase activity accumulated NeuAc on induction of the NanT permease and the lacZ(-) nanA(-) strain that overexpressed the N. meningitidis genes of the alpha2,3 sialyltransferase and of the CMP-NeuAc synthase efficiently produced sialyllactose (NeuAc alpha 2-3Gal beta 1-4Glc) from exogenous NeuAc and lactose.

α2,3-Sialylation of Terminal GalNAcβ1–3Gal Determinants by ST3Gal II Reveals the Multifunctionality of the Enzyme: THE RESULTING Neu5Acα2–3GalNAc LINKAGE IS RESISTANT TO SIALIDASES FROM NEWCASTLE DISEASE VIRUS AND STREPTOCOCCUS PNEUMONIAE

Enzymatic alpha 2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing alpha 2,3-sialylated GalNAc residues have been reported. In the present experiments, recombinant soluble alpha 2,3-sialyltransferase ST3Gal II efficiently sialylated the X(2) pentasaccharide GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, globo-N-tetraose GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and the disaccharide GalNAc beta 1-3Gal in vitro. The purified products were identified as Neu5Ac alpha 2-3GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, Neu5Ac alpha 2-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and Neu5Ac alpha 2-3GalNAc beta 1-3Gal, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enzymatic degradations, and one- and two-dimensional NMR-spectroscopy. In particular, the presence of the Neu5Ac alpha 2-3GalNAc linkage was firmly established in all three products by a long range correlation between Neu5Ac C2 and GalNAc H3 in heteronuclear multiple bond correlation spectra. Collectively, the data describe the first successful sialyltransfer reactions to the 3-position of GalNAc in any acceptor. Previously, ST3Gal II has been shown to transfer to the Gal beta 1-3GalNAc determinant. Consequently, the present data show that the enzyme is multifunctional, and could be renamed ST3Gal(NAc) II. In contrast to ST3Gal II, ST3Gal III did not transfer to the X(2) pentasaccharide. The Neu5Ac alpha 2-3GalNAc linkage of sialyl X(2) was cleaved by sialidases from Arthrobacter ureafaciens and Clostridium perfringens, but resisted the action of sialidases from Newcastle disease virus and Streptococcus pneumoniae. Therefore, the latter two enzymes cannot be used to differentiate between Neu5Ac alpha 2-3GalNAc and Neu5Ac alpha 2-6GalNAc linkages, as has been assumed previously.

Developmental Profile of Neural Cell Adhesion Molecule Glycoforms with a Varying Degree of Polymerization of Polysialic Acid Chains

More precise information on the degree of polymerization (DP) of polysialic acid (polySia) chains expressed on neural cell adhesion molecule (NCAM) and its developmental stage-dependent variation are considered important in understanding the mechanism of regulated polysialylation and fine-tuning of NCAM-mediated cell adhesion by polySia. In this paper, first we performed a kinetic study of acid-catalyzed hydrolysis of polySia and report our findings that (a) in (-->8Neu5Ac alpha 2-->)(n)-->8Neu5Ac alpha 2-->3Gal beta 1-->R, the proximal Neu5Ac residue alpha 2-->3 linked to Gal is cleaved about 2.5-4 times faster than the alpha 2-->8 linkages and (b) in contrary to general belief that alpha 2-->8 linkages in polySia are extremely labile, the kinetic consideration showed that they are not so unstable, and every ketosidic bond is hydrolyzed at the same rate. These findings are the basis of our strategy for DP analysis of polySia on NCAM. Second, using the recently developed method that provides base-line resolution of oligo/polySia from DP 2 to >80 with detection thresholds of 1.4 fmol per resolved peak, we have determined the DP of polySia chains expressed in embryonic chicken brains at different developmental stages. Our results support the presence of numerous NCAM glycoforms differing in DPs of oligo/polySia chains and a delicate change in their distribution during development.

Preparation of autologous leukemia and lymphoma vaccines expressing alpha-gal epitopes.

This study describes a novel method for increasing the immunogenicity of autologous tumor vaccines in leukemia and lymphoma patients by exploiting the natural anti-Gal antibody for in situ targeting of the vaccinating cells to antigen-presenting cells (APCs). Incubation of leukemia or lymphoma cells with neuraminidase and recombinant alpha 1,3-galactosyltransferase results in the synthesis of many alpha-gal epitopes (Gal alpha 1-3Gal beta 1-4GlcNAc-R) on their cell membranes. Vaccination with such processed tumor cells results in the binding of the natural anti-Gal immunoglobulin G (IgG) antibody to these epitopes and opsonization of these cells for effective phagocytosis by APCs, such as dendritic cells and macrophages. These APCs may transport the vaccine to adjacent draining lymph nodes for subsequent effective processing and presentation of tumor-associated antigens (TAA) peptides to activate TAA-specific helper and cytotoxic T cells. Once the TAA-specific cytotoxic T cells are activated, they can leave the lymph node, circulate in the body, and seek metastatic cells expressing TAA to destroy them. Alternatively, activated helper T cells may provide the help required for B cells to produce antibodies to TAA on the leukemia or lymphoma cells. Because every patient receives his or her own TAA within the vaccinating cells, such vaccines are customized for the patient. These autologous tumor vaccines may be used as an adjuvant treatment that complements currently used treatment regimens by providing the immune system with an additional opportunity to be exposed effectively to autologous TAA.