The Galα1,3Galβ1,4GlcNAc-R (α-Gal) epitope: A carbohydrate of unique evolution and clinical relevance

In 1985, we reported that a naturally occurring human antibody (anti-Gal), produced as the most abundant antibody (1% of immunoglobulins) throughout the life of all individuals, recognizes a carbohydrate epitope Galα1–3Galβ1–4GlcNAc-R (the α-gal epitope). Since that time, an extensive literature has developed on discoveries related to the α-gal epitope and the anti-Gal antibody, including the barrier they form in xenotransplantation and their reciprocity in mammalian evolution. This review covers these topics and new avenues of clinical importance related to this unique antigen/antibody system (α-gal epitope/anti-Gal) in improving the efficacy of viral vaccines and in immunotherapy against cancer.     

Enzymatic characterization of CMP-NeuAc:Galβ1-4GlcNAc-R α(2-3)-sialyltransferase from human placenta

In this report we present the enzymatic characterization of CMP-NeuAc:Gal1-4GlcNAc-R (2-3)-sialyltransferase from human placenta using placenta membranes as an enzyme preparation. This sialyltransferase is highly sensitive to detergents and prefers type 2 chain (Gal1-4GlcNAc) over type 1 chain (Gal1-3GlcNAc) acceptors. Oligosaccharides and glycopeptides were better acceptor substrates than glycoproteins. Of the branched oligosaccharides, those with a bisectedN-acetylglucosamine (GlcNAc) structure appeared to be poorer substrates, while triantennary structures containing a Gal1-4GlcNAc1-4Man1-3Man branch were preferred. Product characterization, using 400 MHz¹H-NMR spectroscopy, confirmed that sialic acid was introduced into the Gal1-4GlcNAc-R units of the acceptor substrates in an (2-3) linkage, and revealed that this sialytransferase does not prefer either of the two branches of a complex type diantennary glycopeptide acceptor for sialic acid attachment. These properties distinguish this enzyme from all other sialyltransferases characterized to date.Abbreviations NeuAc N-acetylneuraminic acid - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - GP-F2 and GP-F4 diantennary complex type glycopeptides from desialylated fibrinogen - GP-Trf diantennary complex type glycopeptide from desialylated transferrin - LNT Gal1-3GlcNAc1-3Gal1-4Glc (lacto-N-tetraose) - 6-sialytransferase CMP-NeuAc:Gal1-4GlcNAc-R (2-6)-sialytransferase - 3-sialytransferaseO CMP-NeuAc:Gal1-3GalNAc-R (2-3)-sialyltransferase - 3-sialytransferase I CMP-NeuAc:Gal1-3(4)GlcNAc-R (2-3)-sialyltransferase - 3-sialytransferase II CMP-NeuAc:Gal1-4GlcNAc-R (2-3)-sialytransferase     

Presence of natural anti-Galα1-4GalNAcβ1-3Gal (anti-NOR) antibodies in animal sera

Rare polyagglutinable NOR erythrocytes contain unusual globoside extention products terminating with a Galα1-4GalNAcβ1-3Gal- unit. This trisaccharide epitope is recognized by recently characterized antibodies naturally occurring in most human sera (Duk et al., Glycobiology, 15, 109, 2005). These antibodies represent two major types of fine specificity. All these antibodies are most strongly inhibited by Galα1-4GalNAcβ1-3Gal (NOR-tri), and weakly by Galα1-4Gal. However, the type 1 antibodies are strongly inhibited by Galα1-4Galβ1-3Gal-R and weakly by Galα1-4GalNAc, while the type 2 antibodies show the opposite reactivities with these two oligosaccharides. Similar antibodies have now been found in horse, rabbit and pig sera. The antibodies were purified from animal sera by affinity chromatography on Galα1-4GalNAcβ1-3Gal-human serum albumin(HSA)-Sepharose 4B conjugate. The specificity of the antibodies was determined by binding to ELISA plates coated with several α-galactosylated oligosaccharide-polyacrylamide (PAA) or -HSA conjugates and by inhibition with synthetic oligosaccharides. The purified antibodies bound specifically to conjugates containing NOR-tri. The inhibition of binding showed that the animal sera also contain two types of anti-NOR antibodies: type 2 was found in the horse serum, and a mixture of both types was present in rabbit and pig serum. These results indicate that anti-NOR, a new and distinct kind of anti-αGal antibody, are present in animal sera and show similar specificties and diversity as their counterparts found in human sera.     

Synthesis of Galβ1-4GlcNAcβ- and Galβ1-3GalNAcα-O-L-serine Derivatives employing glycosidases

A new approach for the highly specific preparation of L-serine conjugates of lactosamine and Gal1-3GalNAc is described. Thus, the L-serine derivative of lactosamine Gal1-4GlcNAc-O-(N-Z)-Ser-OEt, was obtained from lactose, employing GlcNAc-O-(N-Z)-Ser-OEt as acceptor and a yeast -galactosidase as catalyst Galp 1-3GalNAc-O-(N-Alloc)-Ser-OMe was obtained from lactose, employing GalNAc-O-(N-Alloc)-Ser-OMe as acceptor and -galactosidase from bovine testes as catalyst.     

Monoclonal antibodies that recognize the trisaccharide epitope Galα1-3Galβ1-4GlcNAc present on Ehrlich tumor cell membrane glycoproteins

Monoclonal antibodies were prepared against the trisaccharide Gal1-3Gal1-4GlcNAc, a sequence which occurs on the surface of Ehrlich ascites tumor cells as well as in thyroglobulin, laminin and a variety of other proteins. This was accomplished by immunizing BALB/c mice with the fraction of Ehrlich cell membrane glycoproteins obtained by affinity chromatography on aGriffonia simplicifolia I (GS I) column which selectively binds -d-galactosyl-terminated structures. Detection of Gal1-3Gal1-4GlcNAc-specific antibodies was accomplished by employing glycoproteins containing the trisaccharide sequence; fusion with spleen cells from an immunized mouse was accomplished in the presence of polyethylene glycol (PEG1500). An enzyme-linked immunosorbent assay (ELISA) system was used to identify two clones (2.10G and 6.8E), which recognized the desired trisaccharide conjugate. These clones also recognized a thyroglobulin fraction isolated by GS I affinity chromatography and murine laminin, both of which possess the Gal1-3Gal1-4GlcNAc sequence. Inhibition of antibody-trisaccharide reactivity, examined employing an ELISA assay, revealed that two trisaccharides, Gal1-3Gal1-4GlcNAc/Glc, were the best inhibitory haptens; Gal1-4GlcNAc (LacNAc), Gal1-3Gal and Gal1-4Glc (lactose) were poor inhibitors. Indirect immunofluorescence staining of unfixed Ehrlich cells using the monoclonal antibody at 4° C revealed fluorescence over the entire cell surface. Indirect immunogold labeling of semithin and ultrathin sections of aldehyde fixed and Lowicryl K4M-embedded Ehrlich cells resulted in specific labeling of the cell surface and internal structure. Immunoblot analysis revealed that removal of the -galactosyl residues of laminin by -galactosidase abolished reactivity with the monoclonal antibodies. The availability of this antibody, which belongs to the IgM family of immunoglobulins, now makes possible the detection of this sugar sequence on cells and tissue sections, as well as on glycoproteins in solution.     

The photosynthetic F1-α3β3 and α1β1 catalytic core complexes

Minimal photosynthetic catalytic F₁() core complexes, containing equimolar ratios of the and subunits, were isolated from membrane-bound spinach chloroplast CF₁ and Rhodospirillum rubrum chromatophore RrF₁. A CF₁-₃₃ hexamer and RrF₁-₁₁ dimer, which were purified from the respective F₁() complexes, exhibit lower rates and different properties from their parent F₁-ATPases. Most interesting is their complete resistance to inhibition by the general F₁ inhibitor azide and the specific CF₁ inhibitor tentoxin. These inhibitors were earlier reported to inhibit multisite, but not unisite, catalysis in all sensitive F₁-ATPases and were therefore suggested to block catalytic site cooperativity. The absence of this typical property of all F₁-ATPases in the ₁₁ dimer is consistant with the view that the dimer contains only a single catalytic site. The ₃₃ hexamer contains however all F₁ catalytic sites. Therefore the observation that CF₁-₃₃ can bind tentoxin and is stimulated by it suggests that the F₁ subunit, which is required for obtaining inhibition by tentoxin as well as azide, plays an important role in the cooperative interactions between the F₁-catalytic sites.Abbreviations CF₀F₁ chloroplast F₀F₁ - CF₁ chloroplast F₁ - CF₁ chloroplast F₁ subunit - CF₁ chloroplast F₁ subunit - CF₁() a complex containing equal amounts of the CF₁ and subunits - MF₁ mitochondrial F₁ - RrF₀F₁ Rhodospirillum rubrum F₀F₁ - RrF₁ R. rubrum F₁ - RrF₁ R. rubrum F₁ subunit - RrF₁ R. rubrum F₁ subunit - RrF₁() a complex containing equal amounts of the RrF₁ and subunits - Rubisco Ribulose-1,5-bisphosphate carboxylase - TF₁ thermophilic bacterium PS3 F₁     

ProcessingO-glycan core 1, Galβ1-3GalNAcα-R. Specificities of core 2, UDP-GlcNAc: Galβ1-3GalNAc-R(GlcNAc to GalNAc) β6-N-acetylglucosaminyltransferase and CMP-sialic acid:Galβ1-3GalNAc-R α3-sialyltransferase

To elucidate control mechanisms ofO-glycan biosynthesis in leukemia and to develop biosynthetic inhibitors we have characterized core 2 UDP-GlcNAc:Gal1-3GalNAc-R(GlcNAc to GalNAc) 6-N-acetylglucosaminyl-transferase (EC 2.4.1.102; core 2 6-GlcNAc-T) and CMP-sialic acid: Gal1-3GalNAc-R 3-sialyltransferase (EC 2.4.99.4; 3-SA-T), two enzymes that are significantly increased in patients with chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML). We observed distinct tissue-specific kinetic differences for the core 2 6-GlcNAc-T activity; core 2 6-GlcNAc-T from mucin secreting tissue (named core 2 6-GlcNAc-T M) is accompanied by activities that synthesize core 4 [GlcNAc1-6(GlcNAc1-3)GalNAc-R] and blood group I [GlcNAc1-6(GlcNAc1-3)Gal-R] branches; core 2 6-GlcNAc-T in leukemic cells (named core 2 -GlcNAc-T L) is not accompanied by these two activities and has a more restricted specificity. Core 2 6-GlcNAc-T M and L both have an absolute requirement for the 4- and 6-hydroxyls ofN-acetylgalactosamine and the 6-hydroxyl of galactose of the Gal1-3GalNAc-benzyl substrate but the recognition of other substituents of the sugar rings varies, depending on the tissue. 3-sialytransferase from human placenta and from AML cells also showed distinct specificity differences, although the enzymes from both tissues have an absolute requirement for the 3-hydroxyl of the galactose residue of Gal1-3GalNAc-Bn. Gal1-3(6-deoxy)GalNAc-Bn and 3-deoxy-Gal1-3GalNAc-Bn competitively inhibited core 2 6-GlcNAc-T and 3-sialyltransferase activities, respectively.Abbreviations AFGP antifreeze glycoprotein - AML acute myeloid leukemia - Bn benzyl - CML chronic myelogenous leukemia - Fuc l-fucose - Gal, G d-galactose - GalNAc, GA N-acetyl-d-galactosamine - GlcNAc, Gn N-acetyl-d-glucosamine - HC human colonic homogenate - HO hen oviduct microsomes - HPLC high performance liquid chromatography - mco 8-methoxycarbonyl-octy - Me methyl - MES 2-(N-morpholino)ethanesulfonate - MK mouse kidney homogenate - onp o-nitrophenyl - PG pig gastric mucosal microsomes - pnp p-nitrophenyl - RC rat colonic mucosal microsomes - SA sialic acid - T transferase Enzymes: UDP-GlcNAc:Gal1-3GalNAc-R (GlcNAc to GalNAc) 6-N-acetylglucosaminyltransferase,O-glycan core 2 6-GlcNAc-transferase, EC 2.4.1.102; CMP-sialic acid: Gal1-3GalNAc-R 3-sialyltransferase,O-glycan 3-sialic acid-transferase, EC 2.4.99.4.     

Crystal structure of α-1,3-galactosyltransferase (α3GT) in a complex with p-nitrophenyl-β-galactoside (pNPβGal)

The specificities of glycosyltransferases make them useful for the synthesis of biologically active oligosaccharides, but also restrict their range of products. In substrate engineering, substrate promiscuity is enhanced by attaching removable interactive groups to weak substrates. Thus, the attachment of β p-nitrophenyl converts galactose from a poor into a good substrate of α-1,3-galactosyltransferase. The crystallographic structure of a complex of α3GT containing p-nitrophenyl-β-galactoside shows that the p-nitrophenyl binds similarly to the N-acetylglucosamine of the substrate, N-acetyllactosamine, interacting with the indole of Trp249. p-Nitrophenyl, unlike N-acetylglucosamine, makes no H-bonds but has more non-polar interactions, making it an effective monosaccharide mimetic.     

Concanavalin A-stimulated expression of gangliosides with GalNAcβ1-4(NeuAcα2-3)Galβ structure in murine thymocytes

We analysed the glycolipids of mouse thymocytes before and after Concanavalin A (Con A) or recombinant interleukin-2 (rIL-2) stimulation by TLC-immunostaining with carbohydrate-specific antiglycolipid antibodies. The thymocytes were cultured in serum-free medium in the presence of 500 ng ml^–1 Con A, 10 U ml^–1 rIL-2 or Con A plus rIL-2 for 6, 12, 24, 48, and 72 h, and were found to start proliferating 24 h after cultivation in the presence of Con A or Con A plus rIL-2, the maximum levels being reached at 72 h and 48 h, respectively, in a thymidine uptake experiment. The concentrations of II³Neu-Gg₄Cer, Gg₄Cer and IV³GalNAc-Gb₄Cer after 48 h Con A stimulation were found to be at almost the original levels. Conversely, II³Neu-Gg₃Cer, which was not detected in the thymocytes at the start, began to appear after 48 h stimulation with Con A and Con A plus rIL-2, and IV³Neu-Gg₅Cer in the cells 48 h after stimulation with Con A and Con A plus rIL-2 has increased to 41 and 44 times higher than in the original cells, respectively, as judged on TLC-immunostaining with monoclonal antibody YHD-06, which detects the GalNAc1-4(NeuAc or NeuGc2-3)Gal-structure. These results indicate that the increased synthesis of both gangliosides, in other words, the activation ofN-acetylgalactosaminyltransferase, is associated with the mitogen-induced proliferation.N-Acetylneuraminic acid was the sole sialic acid in II³Neu-Gg₃Cer which newly appeared in the cells on stimulation, whereas the sialic acid of IV³Neu-Gg₅Cer was a mixture ofN-acetyl- andN-glycoloylneuraminic acids. This result may suggest that the substrates for the two differentN-acetylgalactosaminyltransferases must be different. This GalNAc1-4(NeuAc or NeuGc2-3)Gal-structure was also detected on the surface of the Con A or Con A plus rIL-2 stimulated mouse thymocytes on flow cytometric analysis of cells indirectly stained with monoclonal antibody YHD-06. Abbreviations: carbohydrate and glycolipid nomenclature and abbreviations follow the IUPAC-IUB recommendations or the nomenclature system of Svennerholm L. (1963)J Neurochem 10:613–23.     

GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate) Is the Preferred Substrate for Chondroitin N-Acetylgalactosaminyltransferase-1

A deficiency in chondroitin N-acetylgalactosaminyltransferase-1 (ChGn-1) was previously shown to reduce the number of chondroitin sulfate (CS) chains, leading to skeletal dysplasias in mice, suggesting that ChGn-1 regulates the number of CS chains for normal cartilage development. Recently, we demonstrated that 2-phosphoxylose phosphatase (XYLP) regulates the number of CS chains by dephosphorylating the Xyl residue in the glycosaminoglycan-protein linkage region of proteoglycans. However, the relationship between ChGn-1 and XYLP in controlling the number of CS chains is not clear. In this study, we for the first time detected a phosphorylated tetrasaccharide linkage structure, GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate), in ChGn-1^−/− growth plate cartilage but not in ChGn-2^−/− or wild-type growth plate cartilage. In contrast, the truncated linkage tetrasaccharide GlcUAβ1–3Galβ1–3Galβ1–4Xyl was detected in wild-type, ChGn-1^−/−, and ChGn-2^−/− growth plate cartilage. Consistent with the findings, ChGn-1 preferentially transferred N-acetylgalactosamine to the phosphorylated tetrasaccharide linkage in vitro. Moreover, ChGn-1 and XYLP interacted with each other, and ChGn-1-mediated addition of N-acetylgalactosamine was accompanied by rapid XYLP-dependent dephosphorylation during formation of the CS linkage region. Taken together, we conclude that the phosphorylated tetrasaccharide linkage is the preferred substrate for ChGn-1 and that ChGn-1 and XYLP cooperatively regulate the number of CS chains in growth plate cartilage.     

Identification of a GDP-Fuc:Galβ1–3GalNAc-R (Fuc to Gal)α1–2 fucosyltransferase and a GDP-Fuc:Galβ1–4GlcNAc (Fuc to GlcNAc)α1–3 fucosyltransferase in connective tissue of the snailLymnaea stagnalis

Connective tissue of the freshwater pulmonateLymnaea stagnalis was shown to contain fucosyltransferase activity capable of transferring fucose from GDP-Fuc in 1–2 linkage to terminal Gal of type 3 (Gal1–3GalNAc) acceptors, and in 1–3 linkage to GlcNAc of type 2 (Gal1–4GlcNAc) acceptors. The 1–2 fucosyltransferase was active with Gal1–3GalNAc1-OCH₂CH=CH₂ (K _m=12 mM,V _max=1.3 mU ml^–1) and Gal1–3GalNAc (K _m=20 mM,V _max=2.1 mU ml^–1), whereas the 1–3 fucosyltransferase was active with Gal1–4GlcNAc (K _m=23 mM,V _max=1.1 mU ml^–1). The products formed from Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4GlcNAc were purified by high performance liquid chromatography, and identified by 500 MHz¹H-NMR spectroscopy and methylation analysis to be Fuc1–2Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4(Fuc1–3)GlcNAc, respectively. Competition experiments suggest that the two fucosyltransferase activities are due to two distinct enzymes.Abbreviations 2Fuc-T 1–2 fucosyltransferase - 3Fuc-T 1–3 fucosyltransferase - MeO-3Man 3-O-methyl-D-mannose - MeO-3Gal 3-O-methyl-D-galactose     

Adenovirus-mediated expression of pig α(1,3) galactosyltransferase reconstructs Gal α(1,3) Gal epitope on the surface of human tumor cells

INTRODUCTIONGal a(1, 3) Gal (gal epitope) is a carbohydrate epitope, which is produced in large amounton the cells of pigs, mice and New World monkey(monkey of South America) by the glycosylationenzyme G alal 1 ) 4G IcNAc3- a- D- galactosyltransferase[or(1, 3)GT; EC2.4.1.511111. This enzyme is active in the Golgi appaxatus of cells and transfers galactose from the sugandonor uridine diphoSphate galactose (UDP-galactose) to the acceptor Nacetyllactosamine residue (Galaal-4GlcNAc-R…     

Oligo-N-acetyllactosaminoglycans bearing Galβ1-4(Fucα1-3)GlcNAc sequences reveal lower affinities than their nonfucosylated,or α(1-2) fucosylated counterparts for immobilized wheat germ agglutinin

Relative affinities of several fucosylated and nonfucosylated oligo-N-acetyllactosaminoglycans for immobilized wheat germ agglutinin (WGA) were studied using a chromatographic technique. (1-3) Fucosylation of theN-acetylglucosamine unit(s) in mono- and biantennary saccharides of the Gal1-4GlcNAc-R type strongly reduced the WGA-affinity. In contrast, (1-2) fucosylation of the nonreducing galactose unit(s) of the saccharides did not reduce the affinity.     

Negative Correlation between Natural Human Antibodies Directed to Glycotopes Galβ1-3GlcNAc and Galβ1-4GlcNAc

Russian Journal of Bioorganic Chemistry - In a cohort of 106 donors, we analyzed correlations in the binding of natural antibodies to human glycans in a composition of the glycan array. Along with...     

Synthesis of Gal-β-(1→4)-GlcNAc-β-(1→6)-[Gal-β- (1→3)]-GalNAc-α-OBn oligosaccharides bearing O-methyl or O-sulfo groups at C-3 of the Gal residue: specific acceptors for Gal: 3-O-sulfotransferases

Our recent studies have revealed the existence of two distinct Gal: 3-O-sulfotransferases capable of acting on the C-3 position of galactose in a Core 2 branched structure, e.g., Gal14GlcNAc16(Gal13)GalNac1OBenzyl as acceptor to give 3-O-sulfoGal14GlcNAc13(Gal13)GalNAc1OB 20 and Gal14GlcNAc16(3-O-sulfoGal13)GalNAc1OB 23. We herein report the synthesis of these two compounds and also that of other modified analogs that are highly specific acceptors for the two sulfotransferases. Appropriately protected 1-thio-glycosides 7, 8, and 10 were employed as glycosyl donors for the synthesis of our target compounds.     

Enzymic glycosphingolipid synthesis on polymer supports. III. Synthesis of GM3, its analog [NeuNAcα(2-3)Galβ(1-4)Glcβ(1-3)Cer] and their lyso-derivatives

Two water-soluble polymers, carrying 0.24 meq g–1 of lactosyl-(1-1)-sphingosine (7) and 0.13 meq g–1 of lactosyl-(1-3)-sphingosine (8) were prepared. The polymers served as acceptors in the -(2-3)-sialyltransferase reaction (up to 55.3 and 38.5% transfer yields, respectively). Subsequent photolysis, released compounds 11 (lyso-GM3) and 12 (lyso-GM3 analog), respectively; acylation and chromatography afforded (5-acetamido-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonic acid)-(2-3)--D-galactopyranosyl-(1-4)--D-glucopyranosyl-(1-1)-(2S, 3R, 4E)-2-octadecanoylamino-4-octadecene-1,3-diol (13, GM3) and (5-acetamido-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonic acid)-(2-3)--D-galactopyranosyl-(1-4)--D-glucopyranosyl-(1-3)-(2S, 3R, 4E)-2-octadecanoylamino-4-octadecene-1,3-diol (14, GM3 analogue), respectively, thus presenting a route to glycosphingolipids possessing the unusual glycosyl-(1-3)-spingosine linkage.     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

The linear tetrasaccharide,Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAc,isolated from radiolabeled teratocarcinoma poly-N-acetyllactosaminoglycan resists the action ofE. freundii endo-β-galactosidase

A novel linear tetrasaccharide, Gal1-4GlcNAc1-6Gal1-4GlcNAc, was isolated from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine teratocarcinoma cells. It was characterized by exo-glycosidase sequencing and by mild acid hydrolysis followed by identification of all partial cleavage products. The tetrasaccharide, and likewise labelled GlcNAc1-6Gal1-4GlcNAc, resisted the action of endo--galactosidase (EC 3.2.1.103) fromE. freundii at a concentration of 125 mU/ml, while the isomeric, radioactive teratocarcinoma saccharides Gal1-4GlcNAc1-3Gal1-4GlcNAc and GlcNAc1-3Gal1-4GlcNAc were cleaved in the expected manner.Abbreviations WGA wheat germ agglutinin - BSA bovine serum albumin - [³H]GlcNAc1-4-GlcNAc1-4GlcNAcOL N,N,NN'-triacetylchitotriose reduced with NaB³H₄     

Differential Binding to Glycotopes Among the Layers of Three Mammalian Retinal Neurons by Man-Containing N-linked Glycan, Tα (Galβ1–3GalNAcα1-), Tn (GalNAcα1-Ser/Thr) and Iβ/IIβ (Galβ1–3/4GlcNAcβ-) Reactive Lectins

Carbohydrate structures between retinal neurons and retinal pigment epithelium (RPE) play an important role in maintaining the integrity of retinal adhesion to underlying RPE, and in retinal detachment pathogenesis. Since relevant knowledge is still in the primary stage, glycotopes on the adult retina of mongrel canines (dog), micropigs and Sprague-Dawley rats were examined by lectino-histochemistry, using a panel of 16 different lectins. Paraffin sections of eyes were stained with biotinylated lectins, and visualized by streptavidin-peroxidase and diaminobenzidine staining. Mapping the affinity profiles, it is concluded that: (i) all sections of the retina reacted well with Morniga M, suggesting that N-linked glycans are present in all layers of the retina; (ii) no detectable human blood group ABH active glycotopes were found among retinal layers; (iii) outer and inner segments contained glycoconjugates rich in ligands reacting with T _α (Galβ1–3GalNAcα1-Ser/Thr) and Tn (GalNAcα1-Ser/Thr) specific lectins; (iv) cone cells of retina specifically bound peanut agglutinin (PNA), which recognizes T _α residues and could be used as a specific marker for these photoreceptors; (v) the retinas of rat, dog and pig, had a similar binding profile but with different intensity; (vi) each retinal layer had its own binding characteristic. This information may provide useful background knowledge for normal retinal physiology and miscellaneous retinal diseases, including retinal detachment (RD) and age-related macular degeneration (ARMD).