Wietske E.C.M. Schiphorst

LacdiNAc-glycans constitute a parasite pattern for galectin-3-mediated immune recognition

Although Galβ1–4GlcNAc (LacNAc) moieties are the most common constituents of N-linked glycans on vertebrate proteins, GalNAcβ1–4GlcNAc (LacdiNAc, LDN)-containing glycans are widespread in invertebrates, such as helminths. We postulated that LDN might be a molecular pattern for recognition of helminth parasites by the immune system. Using LDN-based affinity chromatography and mass spectrometry, we have identified galectin-3 as the major LDN-binding protein in macrophages. By contrast, LDN binding was not observed with galectin-1. Surface plasmon resonance (SPR) analysis and a solid phase binding assay demonstrated that galectin-3 binds directly to neoglycoconjugates carrying LDN glycans. In addition, galectin-3 bound to Schistosoma mansoni soluble egg Ags and a mAb against the LDN glycan inhibited this binding, suggesting that LDN glycans within S. mansoni soluble egg Ags contribute to galectin-3 binding. Immunocytochemistry demonstrated high levels of galectin-3 in liver granulomas of S. mansoni-infected hamsters, and a colocalization of galectin-3 and LDN glycans was observed on the parasite eggshells. Finally, we demonstrate that galectin-3 can mediate recognition and phagocytosis of LDN-coated particles by macrophages. These findings provide evidence that LDN-glycans constitute a parasite pattern for galectin-3-mediated immune recognition.

The acceptor substrate specificity of human β4-galactosyltransferase V indicates its potential function in O-glycosylation

In order to assess the function of the different human UDP‐Gal:GlcNAc β4‐galactosyltransferases, the cDNAs of two of them, β4‐GalT I and β4‐GalT V, were expressed in the baculovirus/insect cell expression system. The soluble recombinant enzymes produced were purified from the medium and used to determine their in vitro substrate specificities. The specific activity of the recombinant β4‐GalT V was more than 15 times lower than that of β4‐GalT I, using GlcNAcβ‐S‐pNP as an acceptor. Whereas β4‐GalT I efficiently acts on all substrates having a terminal β‐linked GlcNAc, β4‐GalT V appeared to be far more restricted in acceptor usage. β4‐GalT V acts with high preference on acceptors that contain the GlcNAcβ1→6GalNAc structural element, as found in O‐linked core 2‐, 4‐ and 6‐based glycans, but not on substrates related to N‐linked or blood group I‐active oligosaccharides. These results suggest that β4‐GalT V may function in the synthesis of lacNAc units on O‐linked chains, particularly in tissues which do not express β4‐GalT I, such as brain.

Specificity of Sialyltransferase: Structure of α1‐Acid Glycoprotein Sialylated in vitro

The terminal galactosyl units of desialylated alpha1-acid glycoprotein were selectively labeled with tritium by a galactose oxidase/NaB3H4 procedure. The 3H-labeled glycoprotein was effective as an acceptor in sialytransferase reactions catalyzed by rat liver microsomes in vitro with unlabeled CMP-N-acetyl-neuramininic acid as sialic acid donor. Permethylation/hydrolysis of glycopeptides derived from the resialylated 3H-labeled glycoprotein yielded radioactive 2,3,4-trimethylgalactose indicating that rat liver microsomes are capable of transferring sialic acid to position C-6 of the terminal galactosyl units of desialylated alpha1-acid glycoprotein. No indication was obtained for transfer of sialic acid to other positions. This result is discussed in view of the multiplicity of positions of attachment of sialic acid to galactosyl residues in native alpha1-acid glycoprotein.

Effect of α(2–6)-linked sialic acid and α(1–3)-linked fucose on the interaction ofN-linked glycopeptides and related oligosaccharides with immobilizedPhaseolus vulgaris leukoagglutinating lectin (L-PHA)

The effects of branching and substitution of branches by sialic acid and fucose on the interaction ofN-linked glycopeptides and related oligosaccharides with immobilizedPhaseolus vulgaris leukoagglutinating lectin (L-PHA) were examined. Asialo bi-, tri-and tetra-antennary glycans were all retarded but to different extents on a long column of L-PHA-agarose. Asialo tri- and tetra-antennary glycans containing the pentasaccharide unit Galβ1-4GlcNAcβ1-2[Galβ1-4GlcNAcβ1-6]Man were strongly retarded, whereas asialo bi- and tri-antennary glycans lacking the Galβ1-4GlcNAcβ1-6 branch were only weakly retarded. In all instances the interaction with the lectin was completely abolished when either α(2–6)-linkedN-acetylneuraminic acid or α(1–3)-linked fucose was present at the galactose orN-acetylglucosamine residue of the Galβ1-4GlcNAcβ1-6Manα1-6 branch, respectively. The same substitutions on the Galβ1-4GlcNAcβ1-6Manα1-6 branch decreased but did not abolish the affinity of the lectin for the glycans. The presence of NeuAcα2-6 and Fucα1-3 on the other two branches did not interfere with the binding of the glycans to L-PHA. Furthermore, it appeared that the presence of the Manβ1-4GlcNAc unit is requried for interaction with the lectin. In order to obtain reliable information on the relative occurrence of tri- and tetra-antennary glycopeptides, this study shows that it is essential to desialylate and to defucosylate the glycans prior to application to L-PHA-agarose.

Characterization of a core α1→3‐fucosyltransferase from the snail Lymnaea stagnalis that is involved in the synthesis of complex‐type N‐glycans

We have identified a core α1→3‐fucosyltransferase activity in the albumin and prostate glands of the snail Lymnaea stagnalis. Incubation of albumin gland extracts with GDP‐[14C]Fuc and asialo/agalacto‐glycopeptides from human fibrinogen resulted in a labeled product in 50% yield. Analysis of the product by 400 MHz 1H‐NMR spectroscopy showed the presence of a Fuc residue α1→3‐linked to the Asn‐linked GlcNAc. Therefore, the enzyme can be identified as a GDP‐Fuc:GlcNAc (Asn‐linked) α1→3‐fucosyltransferase. The enzyme acts efficiently on asialo/agalacto‐glycopeptides from both human fibrinogen and core α1→6‐fucosylated human IgG, whereas bisected asialo/agalacto‐glycopeptide could not serve as an acceptor. We propose that the enzyme functions in the synthesis of core α1→3‐fucosylated complex‐type glycans in L. stagnalis. Core α1→3‐fucosylation of the asparagine‐linked GlcNAc of plant‐ and insect‐derived glycoproteins is often associated with the allergenicity of such glycoproteins. Since allergic reactions have been reported after consumption of snails, the demonstration of core α1→3‐fucosylation in L. stagnalis may be clinically relevant.

Branching and elongation with lactosaminoglycan chains of N-linked oligosaccharides result in a shift toward termination with α2→3-linked rather than with α2→6-linked sialic acid residues

The activity of bovine colostrum CMP‐NeuAc: Galβ1→4GlcNAcβ‐R α2→6‐sialyltransferase (α6‐NeuAcT) toward oligosaccharides that form part of complex‐type, N‐linked glycans appears significantly reduced when a bisecting GlcNAc residue or additional branches are present, or when core GlcNAc residues are absent. By contrast human placenta CMP‐NeuAc:Galβ1→4GlcNAcβ‐Rα2→3‐sialyltransferase (α3‐NeuAcT) is much less sensitive to structural variations in these acceptors. Furthermore the α3‐NeuAcT shows a much higher activity than the α6‐NeuAcT with oligosaccharides that form part of linear and branched lactosaminoglycan extensions. These results indicate that, in tissues that express both enzymes, branching and lactosaminoglycan formation of N‐linked glycans will cause a shift from termination with α2→6‐linked sialic acid to termination with α2→3‐linked sialic acid residues. These findings provide an enzymatic basis for the sialic acid linkage‐type patterns found on the oligosaccharide chains of N‐glycoproteins.

Conversion of GalNAcβ(1–4)GlcNAcβ-OMe into GalNAcβ(1–4)-[Fucα(1–3)]GlcNAcβ-OMe using human milk α3/4-fucosyltransferase synthesis of a novel terminal element in glycoprotein glycans

Incubation of GalNAcβ(1–4)GlcNAcβ‐OMe with GDP‐Fuc in the presence of human milk α3/4‐fucosyltransferase resulted in the formation of GalNAcβ(1–4)[Fucα(1–3)]GlcNAcβ‐OMe. Under conditions that led to complete α3‐fucosylation of Galβ(1–4)GlcNAcβ‐OEt, GalNAcβ(1–4)GlcNAcβ‐OMe was fucosylated for more than 85%. For the identification of the isolated fucosylated products one‐ and two‐ dimensional 1H‐NMR spectroscopy was applied. In vacuo molecular dynamics simulations of Galβ(1–4)[Fucα(1–3)]GlcNAcβ‐OEt and GalNAcβ(1–4)[Fucα(1–3)]GlcNAcβ‐OMe using the CHARMm based force field CHEAT, demonstrated only small differences between the conformations of these compounds. This illustrates the minor conformational influence of the substituent at C‐2′, i.e. a hydroxyl function versus a N‐acetyl group.

Specific detection of N-acetylglucosamine-containing oligosaccharide chains on ovine submaxillary asialomucin.

Human milk beta-N-acetylglucosaminide beta 1 leads to 4-galactosyltransferase (EC 2.4.1.38) was used to galactosylate ovine submaxillary asialomucin to saturation. The major [14C]galactosylated product chain was obtained as a reduced oligosaccharide by beta-elimination under reducing conditions. Analysis by Bio-Gel filtration and gas-liquid chromatography indicated that this compound was a tetrasaccharide composed of galactose, N-acetylglucosamine and reduced N-acetylgalactosamine in a molar ratio of 2:0.9:0.8. Periodate oxidation studies before and after mild acid hydrolysis in addition to thin-layer chromatography revealed that the most probable structure of the tetrasaccharide is Gal beta 1 leads to 3([14C]Gal beta 1 leads to 4GlcNAc beta 1 leads to 6)GalNAcol. Thus it appears that Gal beta 1 leads to 3(GlcNAc beta 1 leads to 6)GalNAc units occur as minor chains on the asialomucin. The potential interference of these chains in the assay of alpha-N-acetylgalactosaminylprotein beta 1 leads to 3-galactosyltransferase activity using ovine submaxillary asialomucin as an acceptor can be counteracted by the addition of N-acetylglucosamine.

Human liver and human placenta both contain CMP-NeuAc:Galβ1→4GlcNAc-R α2→3- as well as α2→6-sialyltransferase activity

A high pH anion exchange chromatographic (HPAEC) system for the separation of isomeric sialo‐oligosaccharide products was developed. Employing this system, using Galβ1→4GlcNAcβ1→2Manα1→6Manβ1→4GlcNAc as a substrate, a Galβ1→4GlcNAc‐R α2→3‐sialyltransferase activity was detected for the first time in human liver. This activity is expressed together with the prevalent α2→6‐sialyltransferase. Furthermore, in addition to the major α2→3‐sialyltransferase, a low but distinct activity of α2→6‐sialyltransferase was detected in human placenta. This activity could not be found by methods based on methylation analysis or high resolution NMR spectroscopy. It is concluded that HPAEC, in combination with the use of the pentasaccharide as an acceptor substrate, is suited for the specific detection of minor, Galβ1→4GlcNAc‐specific sialyltransferase activities.

Bovine heart lectin stimulates β-D-galactoside α2→6 sialyltransferase of bovine colostrum

Preparations of the β-galactoside-binding lectin of bovine heart have been shown to stimulate in vitro the sialylation of the oligosaccharide Ga1β1→4G1cNAc and asialo-α1-acid glycoprotein by bovine colostrum β-D-galactoside α2→6 sialyltransferase. Kinetic data revealed that in the presence of lectin the Km values for Ga1β1→4G1cNAc and CMP-NeuAc were reduced from 25.0 to 11.6 mM and from 0.42 to 0.19 mM respectively, but the Km for asialo-α1-acid glycoprotein and the Vmax values for all three substrates were little affected. Stimulation by the lectin was partially inhibited by Fucα1→2Ga1β1→4G1cNAc. This, together with the effects of certain plant lectins, suggests that the stimulation of sialytransferase may be mediated through the carbohydrate-binding properties of the lectin.