Probe design and synthesis of Galβ(1→3)[NeuAcα(2→6)]GlcNAcβ(1→2)Man motif of N-glycan

Synthesis and clusterization of Galβ(1→3)[NeuAcα(2→6)]GlcNAcβ(1→2)Man motif of the N-glycan, as the molecular probes for their biological evaluation, are reported. Key step is the quantitative and the completely α-selective sialylation of the C5-azide N-phenyltrifluoroacetimidate with the disaccharide acceptor, Galβ(1→3)GlcNTroc. Clusterization of the 16 molecules of trisaccharide motif was also achieved by the ‘self-activating click reaction’. These probes could efficiently be labeled by biotin and/or other fluorescence- or radioactive reporter groups through either cross metathesis, acylation, Cu(I)-mediated Huisgen [2+3]-cycloaddition, or the azaelectrocyclization to utilize the various biological techniques.     

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.     

Synthesis of Galβ1-3GlcNAc and Galβ1-3GlcNAcβ-SEt by an enzymatic method comprising the sequential use of β-galactosidases from bovine testes andEscherichia coli

Gal1-3GlcNAc (1) and Gal1-3GlcNAc-SEt (2) were synthesized on a 100 mg scale by the transgalactosylation reaction of bovine testes -galactosidase with lactose as donor andN-acetylglucosamine and GlcNAc-SEt as acceptors. In both cases the product mixtures contained unwanted isomers and were treated with -galactosidase fromEscherichia coli which has a different specificity, under conditions favouring hydrolysis, yielding besides the desired products, monosaccharides and traces of trisaccharides. The products were purified to >95% by gel filtration, with a final yield of 12% of 1 and 17% of 2, based on added acceptor. In a separate experiment Gal1-6GlcNAc-SEt (3) was synthesized by the transglycosylation reaction using -galactosidase fromEscherichia coli. No other isomers were detected. Compound 3 was purified by HPLC.     

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...     

UDP-GlcNAc: Galβ3GalNAc-Mucin: (GlcNAc → GalNAc) β6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Galβ3(GlcNAcβ6) GalNAc-Mucin (GlcNAc → Gal)β3-N-Acetylglucosaminyltransferase from Swine Trachea Epithelium

Summary Two specific -N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a 1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3GalNAc-Mucin to yield Gal3(GlcNAc6)GalNAc-Mucin and a 3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3(GlcNAC6)GalNAc-mucin to yield GlcNAc3Gal3 (GlcNAc6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The 1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the 6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal1,3GalNAc chains was 0.53 µM; for UDP-N-acetylglucosamine, 12 µM; and for Gal 1,3GalNAc NO₂ø, 4 mM. The activity of the 6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal3GalNAc chains in Cowper's gland mucin glycoprotein.The best substrate for the partially purified 3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal1,3(GlcNAc6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal1,3GalNAc side chains.The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the 6- and 3-glucosaminyltransferases were shown to be Gal3(GlcNAC6) GalNAc and GlcNAc3 Gal3(GlcNAC6)GalNAc respectively.Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a 6-glucosaminyltransferase converts Gal3GalNAc chains in mucin glycoproteins to Gal3(GlcNAc6)GalNAc chains. This product is the substrate for a second 3-glucosaminyltransferase which converts the Gal3(GlcNAc6)GalNAc chains to GlcNAc3Gal(GlcNAc6)GalNAc chains in the glycoprotein. The 3-glucosaminyltransferase did not utilize Gal3GalNAc chains as a substrate and this results in an ordered sequence of addition of N-acetylglucosamine residues to growing oligosaccharide chains in tracheal mucin glycoproteins.Abbreviations NeuNAc N-acetylneuraminic acid - GalNAcol N-acetylgalactosaminitol - CGMG Cowper's gland mucin glycoprotein - GalNAc-CGMG Cowper's gland mucin glycoprotein containing GalNAc side chains O-glycosidically linked to serine or threonine - Gal3GalNAc-CGMC Cowper's gland mucin glycoprotein containing Gal3GalNAc side chains - MES 2-(N-morpholino) Ethane Sulfonic acid - PBS Phosphate Buffered Saline     

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     

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.     

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     

Construction of linear GlcNAcβ1-6Galβ1-OR type oligosaccharides by partial cleavage of GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-OR sequences with jack bean β-N-acetylhexosaminidase

Radiolabelled GlcNAc beta 1-3(GlcNAc beta 1-6)Gal (1), GlcNAc beta 1-3)GlcNAc beta 1-6)Gal beta 1-OCH3 (4), GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc (7), and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (10) were cleaved partially with jack bean beta-N-acetylhexosaminidase (EC 3.2.1.30), and the digests were analysed chromatographically. All four oligosaccharides were hydrolysed faster at the (1-6) branch, than at the (1-3) branch, but a high branch specificity was observed only with the glycan 4. The saccharides 1 and 7 resembled each other in the kinetics of the enzyme-catalysed release of their two non-reducing N-acetylglucosamine units, but the glycan 10 was rather different. The partial digestions made it possible to obtain radiolabelled GlcNAc beta 1-6Gal, GlcNAc beta 1-6Gal beta 1-OCH3, GlcNAc beta 1-6Gal beta 1-4Glc, and, in particular, GlcNAc beta 1-6Gal beta 1-4GlcNAc.     

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     

Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains

GlcNAc1-2Man and GlcNAc1-6Man were synthesized using the reverse hydrolysis activity of -N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal1-4GlcNAc1-2Man and Gal1-4GlcNAc1-6Man were synthesized regioselectively using the transglycosylation activity of -galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies. Abbreviations: pNp--GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside; pNp--Gal, p-nitrophenyl -D-galacto-pyranoside     

Structural Basis Underlying the Binding Preference of Human Galectins-1, -3 and -7 for Galβ1-3/4GlcNAc

Galectins represent β-galactoside-binding proteins and are known to bind Galβ1-3/4GlcNAc disaccharides (abbreviated as LN1 and LN2, respectively). Despite high sequence and structural homology shared by the carbohydrate recognition domain (CRD) of all galectin members, how each galectin displays different sugar-binding specificity still remains ambiguous. Herein we provided the first structural evidence of human galectins-1, 3-CRD and 7 in complex with LN1. Galectins-1 and 3 were shown to have higher affinity for LN2 than for LN1, while galectin-7 displayed the reversed specificity. In comparison with the previous LN2-complexed structures, the results indicated that the average glycosidic torsion angle of galectin-bound LN1 (ψ^LN1 ≈ 135°) was significantly differed from that of galectin-bound LN2 (ψ^LN2 ≈ -108°), i.e. the GlcNAc moiety adopted a different orientation to maintain essential interactions. Furthermore, we also identified an Arg-Asp/Glu-Glu-Arg salt-bridge network and the corresponding loop (to position the second Asp/Glu residue) critical for the LN1/2-binding preference.     

Involvement of β 1,4 galactosyltransferase 1 and Galβ 1→4GlcNAc groups in human hepatocarcinoma cell apoptosis

1,4 galactosyltransferase 1 ( 1,4GT1) synthesizes Gal 14GlcNAc groups in N-linked sugar chains of animal glycoproteins, which have been demonstrated to play an important role in many biological events, including sperm-egg interaction, cell migration and mammalian embryonic development. In this study, the mRNA level of 1,4GT1 was found to increase greatly during the 7721 hepatocarcinoma cells apoptosis induced by cycloheximide. Ricinus Communis Agglutinin-I staining indicated generous increase of Gal 14GlcNAc groups during apoptosis. Further study showed that the 7721 hepatocarcinoma cells transiently transfected with 1,4GT1 were more susceptible to the apoptosis induced by cycloheximide. The increased susceptibility was in accordance to the transfection concentration of 1,4GT1, which also led to the increased Gal 14GlcNAc groups on the transfected cell surface. All the observations suggested that 1,4GT1 and Gal 14GlcNAc groups might be associated with the apoptosis of human hepatocarcinoma cells.     

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.     

One-pot enzymatic synthesis of deoxy-thymidine-diphosphate (TDP)-2-deoxy-α-d-glucose using phosphomannomutase

Production of deoxy-thymidine-diphosphate (TDP)-sugars as substrates of glycosyltransferases, has been one of main hurdles for combinatorial antibiotic biosynthesis, which combines sugar moiety with aglycon of various antibiotics. Here, we report the one-pot enzymatic synthesis of TDP-2-deoxy-glucose employing high efficient TMP kinase (TMK; E.C. 2.7.2.12), acetate kinase (ACK; E.C. 2.7.1.21), and TDP-glucose synthase (TGS; E.C. 2.7.7.24) with phosphomannomutase (PMM; E.C. 5.4.2.8). In this study, replacing phosphoglucomutase (PGM; E.C. 5.4.2) by PMM from Escherichia coli gave four times higher specific activity on 2-deoxy-6-phosphate glucose, suggesting that the activity on 2-deoxy-glucose-6-phosphate was mainly affected by PMM activity, not PGM activity. Using an in vitro system starting from TMP and 2-deoxy-glucose-6-phosphate glucose, TDP-2-deoxy-glucose (63% yield) was successfully synthesized. Considering low productivity of NDP-sugars from cheap starting materials, this paper showed how production of NDP-sugars could be enhanced by controlling mutase activity.     

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Glycosidase-catalysed synthesis of glycosylated amino acids: synthesis of GalNAcα-Ser and GlcNAcβ-Ser derivatives

A glycosidase from Aspergillus oryzae catalysed the stereospecific formation of various derivatives of the Tn antigen, D-GalNAca1O-L-Ser, employing D-GalNAc-OPhNO - p as glycosyl donor and different N- and C-protected L-Ser derivatives as acceptors. The same glycosidase preparation was also useful for stereospecific preparation of D-GalNAca1O-L-Thr and D-GlcNAc1O-L-Ser derivatives. Yields were in the range 10-50% depending on the type of acceptor. Lipase from porcine pancreas was used for specific hydrolysis, generating a Tn antigen derivative with a free carboxyl group. This facilitates the use of the derivatives in e.g. solid phase synthesis of glycopeptides.     

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.     

The α-Gal epitope (Galα1-3Galβ1-4GlcNAc-R) in xenotransplantation

Many patients with failing organs (e.g., heart, liver or kidneys), do not receive the needed organ because of an insufficient number of organ donors. Pig xenografts have been considered as an alternative source of organs for transplantation. The major obstacle currently known to prevent pig to human xenotransplantation is the interaction between the human natural anti-Gal antibody and the alpha-gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R), abundantly expressed on pig cells. This short review describes the characteristics of anti-Gal and of the alpha-gal epitope, their role in inducing xenograft rejection and some experimental approaches for preventing this rejection.     

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.