Purification and properties of the endocellular β-glucosidase of Candida cacaoi Buckley and Van Uden CBS 2020

D. DRIDER, P. POMMARES, P. CHEMARDIN, A. ARNAUD AND P. GALZY. 1993. The endocellular enzyme β-glucosidase of Candida cacaoi was purified by ion-exchange chromatography and gel filtration. The molecular weight was 220 ± 10 kDa; its optimum pH was between 4 and 5.5 and its optimum temperature was 60C. This enzyme was active against soluble glucosides tested with β(1–2), β(1–3), β(1–4) and even α(1–4) and α(1–6) and was inhibited by D-glucono-δ-lactone. The enzyme was constitutive but its synthesis was repressed by glucose.     

Chemical and Immunological Characterization of Galactosyl-β1-3-Globoside in Bovine, Human, and Rat Brain

Abstract: Our studies of bovine brain neutral glycosphingolipids (Ngsls) have revealed the presence of several short-chain (containing -CHO 1–4) and previously uncharacterized long-chain (−CHO > 4–5) Ngsls. We reported the structural characterization of brain GgOse₄Cer (GA1) and have now purified another brain Ngsl to homogeneity. The purified Ngsl migrated close to standard GgOse₄Cer and nLcOse₅Cer on a TLC plate employing two different solvent systems. The carbohydrate molar composition indicated the presence of Gal/Glc/GalNAc in a ratio of 2.8:1.0:0.9. Five permethylated alditol acetate peaks were characterized as 2,3,4,6-OMe₄Gal, 2,4,6-OMe₃Gal, 2,3,6-OMe₃Gal, 2,3,6-OMe₃Glc, and 4,6-OMe₂GalNAcMe by gas chromatography-mass spectrometry. The anomeric carbohydrate sequence has been determined by specific exoglycosidase digestion. Six-hundred megahertz ¹H NMR spectroscopy of the oligosaccharide released by ceramide glycanase hydrolysis confirmed the structure of the Ngsl as Galβ1-3GalNAcβ1-3Galα1-4Galβ1-4Glc\1-1Cer or IV³GalGbOse₄Cer. Using the immunooverlay technique with anti-stage-specific embryonic antigen 3 antibody, it was found in bovine, rat, and normal adult human brain and bovine myelin, but not in human or rat myelin.     

Identification of a novel gene involved in pilin glycosylation in Neisseria meningitidis

The pili of Neisseria meningitidis are a key virulence factor, being major adhesins of this capsulate organism that contribute to specificity for the human host. Recently it has been reported that meningococcal pili are post-translationally modified by the addition of an O-linked trisaccharide, Gal (β1–4) Gal (α1–3) 2,4-diacetimido-2,4,6-trideoxyhexose. Using a set of random genomic sequences from N. meningitidis strain MC58, we have identified a novel gene homologous to a family of glycosyltransferases. A plasmid clone containing the gene was isolated from a genomic library of N. meningitidis strain MC58 and its nucleotide sequence determined. The clone contained a complete copy of the gene, here designated pglA (pilin glycosylation). Insertional mutations were constructed in pglA in a range of meningococcal strains with well-defined lipopolysaccharide (LPS) or pilin-linked glycan structures to determine whether pglA had a role in the biosynthesis of these molecules. There was no alteration in the phenotype of LPS from pglA mutant strains as judged by gel migration and the binding of monoclonal antibodies. In contrast, decreased gel migration of the pilin subunit molecules of pglA mutants was observed, which was similar to the migration of pilins of galE mutants of same strains, supporting the notion that pglA is a glycosyltransferase involved in the biosynthesis of the pilin-linked trisaccharide structure. The pglA mutation, like the galE mutation reported previously, had no effect on pilus-mediated adhesion to human epithelial or endothelial cells. Pilin from pglA mutants were unable to bind to monospecific antisera recognizing the Gal (β1–4) Gal structure, suggesting that PglA is a glycosyltransferase involved in the addition of galactose of the trisaccharide substituent of pilin.     

Toll-like receptors 2 and 4 mediate Abeta(1-42) activation of the innate immune response in a human monocytic cell line

The primary molecules for mediating the innate immune response are the Toll-like family of receptors (TLRs). Recent work has established that amyloid-beta (Aβ) fibrils, the primary components of senile plaques in Alzheimer's disease (AD), can interact with the TLR2/4 accessory protein CD14. Using antibody neutralization assays and tumor necrosis factor alpha release in the human monocytic THP-1 cell line, we determined that both TLR2 and TLR4 mediated an inflammatory response to aggregated Aβ(1–42). This was in contrast to exclusive TLR ligands lipopolysaccharide (LPS) (TLR4) and tripalmitoyl cysteinyl seryl tetralysine (Pam₃CSK₄) (TLR2). Atomic force microscopy imaging showed a fibrillar morphology for the proinflammatory Aβ(1–42) species. Pre-treatment of the cells with 10 μg/mL of a TLR2-specific antibody blocked ∼50% of the cell response to fibrillar Aβ(1–42), completely blocked the Pam₃CSK₄ response, and had no effect on the LPS-induced response. A TLR4-specific antibody (10 μg/mL) blocked ∼35% of the cell response to fibrillar Aβ(1–42), completely blocked the LPS response, and had no effect on the Pam₃CSK₄ response. Polymyxin B abolished the LPS response with no effect on Aβ(1–42) ruling out bacterial contamination of the Aβ samples. Combination antibody pre-treatments indicated that neutralization of TLR2, TLR4, and CD14 together was much more effective at blocking the Aβ(1–42) response than the antibodies used alone. These data demonstrate that fibrillar Aβ(1–42) can trigger the innate immune response and that both TLR2 and TLR4 mediate Aβ-induced tumor necrosis factor alpha production in a human monocytic cell line.     

Salmonella enterica serotype Typhimurium Std fimbriae bind terminal α(1,2)fucose residues in the cecal mucosa

The std operon encodes a fimbrial adhesin of Salmonella enterica serotype Typhimurium that is required for attachment to intestinal epithelial cells and for cecal colonization in the mouse. To study the mechanism by which this virulence factor contributes to colonization we characterized its binding specificity. Std-mediated binding to human colonic epithelial (Caco-2) cells could be abrogated by removing N-linked glycans. Adherence of Std fimbriated S.  Typhimurium to Caco-2 cells could be blocked by co-incubation with H type 2 oligosaccharide (Fucα1-2Galβ1-4GlcNAc) or by pretreatment of cells with α1-2 fucosidase. In contrast, pretreatment of Caco-2 cells with neuraminidase or co-incubation with the type 2 disaccharide precursor (Galβ1-4GlcNAc) did not reduce adherence of Std fimbriated S.  Typhimurium. Binding of purified Std fimbriae to Fucα1-2Galβ1-4GlcNAc in a solid phase binding assay was competitively inhibited by Ulex europaeus agglutinin-I (UEA-I), a lectin specific for Fucα1-2 moieties. Purified Std fimbriae and UEA both bound to a receptor localized in the mucus layer of the murine cecum. These data suggest that the std operon encodes an adhesin that binds an α1-2 fucosylated receptor(s) present in the cecal mucosa.     

Cerebral clearance of human amyloid-beta peptide (1-40) across the blood-brain barrier is reduced by self-aggregation and formation of low-density lipoprotein receptor-related protein-1 ligand complexes

Soluble amyloid-β peptide (Aβ) exists in the form of monomers and oligomers, and as complexes with Aβ-binding molecules, such as low-density lipoprotein receptor-related protein-1 (LRP-1) ligands. The present study investigated the effect of self-aggregation and LRP-1 ligands on the elimination of human Aβ(1–40) [hAβ(1–40)] from the rat brain across the blood–brain barrier. Incubation of [¹²⁵I]hAβ(1–40) monomer resulted in time-dependent and temperature-dependent dimer formation, and the apparent elimination rate of [¹²⁵I]hAβ(1–40) dimer was significantly decreased by 92.7% compared with that of [¹²⁵I]hAβ(1–40) monomer. Pre-incubation with LRP-1 ligands, such as activated α2-macroglobulin (α2M), apolipoprotein E2 (apoE2), apoE3, apoE4, and lactoferrin, reduced the elimination of [¹²⁵I]hAβ(1–40). By contrast, pre-administration of the same concentration of these molecules in the rat brain did not significantly inhibit [¹²⁵I]hAβ(1–40) monomer elimination. Purified [¹²⁵I]hAβ(1–40)/activated α2M complex and [¹²⁵I]activated α2M were not significantly eliminated from the rat brain up to 60 min. MEF-1 cells, which have LRP-1-mediated endocytosis, exhibited uptake of [¹²⁵I]activated α2M, and enhancement of [¹²⁵I]hAβ(1–40) uptake upon pre-incubation with apoE, suggesting that [¹²⁵I]activated α2M and [¹²⁵I]hAβ(1–40)/apoE complex function as LRP-1 ligands. These findings indicate that dimerization and LRP-1-ligand complex formation prevent the elimination of hAβ(1–40) from the brain across the blood–brain barrier.     

Scanning mutagenesis of regions in the Gα protein Gpa1 that are predicted to interact with yeast mating pheromone receptors

The mechanism by which receptors activate heterotrimeric G proteins was examined by scanning mutagenesis of the Saccharomyces cerevisiae pheromone-responsive Gα protein (Gpa1). The juxtaposition of high-resolution structures for rhodopsin and its cognate G protein transducin predicted that at least six regions of Gα are in close proximity to the receptor. Mutagenesis was targeted to residues in these domains in Gpa1, which included four loop regions (β2–β3, α2–β4, α3–β5, and α4–β6) as well as the N and C termini. The mutants displayed a range of phenotypes from nonsignaling to constitutive activation of the pheromone pathway. The constitutive activity of some mutants could be explained by decreased production of Gpa1, which permits unregulated signaling by Gβγ. However, the constitutive activity caused by the F344C and E335C mutations in the α2–β4 loop and F378C in the α3–β5 loop was not due to decreased protein levels, and was apparently due to defects in sequestering Gβγ. The strongest loss of the function mutant, which was not detectably induced by a pheromone, was caused by a K314C substitution in the β2–β3 loop. Several other mutations caused weak signaling phenotypes. Altogether, these results suggest that residues in different interface regions of Gα contribute to activation of signaling.     

Inhibition of Inducible Nitric Oxide Synthase Expression by Endothelin in Rat Glial Cells Prepared from the Neonatal Rat Brain

Abstract: In primary cultured rat glial cells, a combination of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) decreased iNOS expression and nitrite accumulation induced by TNF-α/IL-1β. The inhibitory effect of ET on TNF-α/IL-1β-stimulated iNOS expression appears to be mediated by ET_B receptors, because (1) both ET-1 and ET-3 inhibited the effects of TNF-α/IL-1β on iNOS expression and nitrite accumulation, (2) a selective ET_B receptor agonist, Suc-[Glu⁹,Ala^11,15]-ET-1 (8–21) (IRL1620), decreased the effects of TNF-α/IL-1β, and (3) a selective ET_B receptor antagonist, N-cis -2,6-dimethylpiperidinocarbonyl- l -γ-methylleucyl- d -1-methoxycarbonyltryptophanyl- d -norleucine, abolished the inhibitory effects of ETs and IRL1620. Incubation of glial cells with lipopolysaccharide (LPS) caused an increase in iNOS expression. Simultaneous addition of ET-3 decreased the effects of LPS (10 and 100 ng/ml) on iNOS expression. Furthermore, cyclic AMP-elevating agents (dibutyryl cyclic AMP and forskolin) inhibited TNF-α/IL-1β-induced and LPS-induced iNOS expression and nitrite accumulation. These findings suggest that ETs can decrease TNF-α/IL-1β-induced and LPS-induced iNOS expression via ET_B receptors and that cyclic AMP may be involved in this process.     

Histology and carbohydrate histochemistry of the alimentary canal in the rainbow trout Oncorhynchus mykiss

A histological and histochemical analyses were carried out on the entire alimentary canal of the rainbow trout Oncorhynchus mykiss . In particular the oesophageal region showed presence of terminal β‐D‐galactose(1–3)‐N‐acetylgalactosamine and α‐N‐acetylgalactosamine. In the anterior and posterior regions of the stomach, lining epithelium and gastric pits exhibited the presence of β‐gal and α‐GalNAc. In addition sialoglycoconjugates having sialic acid–β–galactose(1–3)‐N‐acetylgalactosamine and sialic acid‐N‐acetylgalactosamine as terminal tri‐ and di‐saccharides, were demonstrated. In proximal and distal intestine goblet cells showed the presence of sialoglyconjugates, having sialic acid‐β‐gal(1–3)‐GalNAc and sialic acid‐GalNAc as terminal sequences, belonging to N‐linked chains. In the enterocytes of the entire intestine, terminal GlcNAc, α‐Gal, α‐fucose were found.     

Generation and Regulation of β-Amyloid Peptide Variants by Neurons

Abstract: Studies of processing of the Alzheimer β-amyloid precursor protein (βAPP) have been performed to date mostly in continuous cell lines and indicate the existence of two principal metabolic pathways: the "β-secretase" pathway, which generates β-amyloid (Aβ_1–40/42; ∼4 kDa), and the "α-secretase" pathway, which generates a smaller fragment, the "p3" peptide (Aβ_17–40/42; ∼3 kDa). To determine whether similar processing events underlie βAPP metabolism in neurons, media were examined following conditioning by primary neuronal cultures derived from embryonic day 17 rats. Immunoprecipitates of conditioned media derived from [³⁵S]methionine pulse-labeled primary neuronal cultures contained 4- and 3-kDa Aβ-related species. Radiosequencing analysis revealed that the 4-kDa band corresponded to conventional Aβ beginning at position Aβ(Asp¹), whereas both radio-sequencing and immunoprecipitation-mass spectrometry analyses indicated that the 3-kDa species in these conditioned media began with Aβ(Glu¹¹) at the N terminus, rather than Aβ(Leu¹⁷) as does the conventional p3 peptide. Either activation of protein kinase C or inhibition of protein phosphatase 1/2A increased soluble βAPP_α release and decreased generation of both the 4-kDa Aβ and the 3-kDa N-truncated Aβ. Unlike results obtained with continuously cultured cells, protein phosphatase 1/2A inhibitors were more potent at reducing Aβ secretion by neurons than were protein kinase C activators. These data indicate that rodent neurons generate abundant Aβ variant peptides and emphasize the role of protein phosphatases in modulating neuronal Aβ generation.     

Polyclonal Antibodies Directed Against an Epitope Specific for the α4-Subunit of GABAA Receptors Identify a 67-kDa Protein in Rat Brain Membranes

Abstract: Polyclonal antibodies were raised to the C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α4-subunit. These anti-peptide α4 (517–523) antibodies specifically identified a protein with apparent molecular mass 67 kDa in rat brain membranes. This protein was enriched by immunoaffinity chromatography of brain membrane extracts on Affigel 10 coupled to the anti-peptide α4 (517–523) antibodies and could then be identified by the anti-α4-antibodies as well as by the GABA_A receptor subunit-specific monoclonal antibody bd-28. This appears to indicate that the 67-kDa protein is the α4-subunit of GABA_A receptors. Intact GABA_A receptors appeared to be retained by the immunoaffinity column because other GABA_A receptor subunit proteins like the β2/β3-subunits and the γ2-subunit were detected in the immunoaffinity column eluate. Furthermore, in addition to the 67-kDa protein, a 51-kDa protein could be detected by the antibody bd-28 and the anti-peptide α4 (517–523) antibody in the immunoaffinity column eluate. A protein with similar apparent molecular mass was identified by the α1-subunit-specific anti-peptide α1 (1–9) antibody. In contrast to the α1-subunit, the 51-kDa protein identified by the anti-α4 antibody could not be deglycosylated by N -Glycanase. The identity of the 51-kDa protein identified by the anti-α4-antibodies thus must be further investigated.     

Multiple Determinants of Dihydro-β-Erythroidine Sensitivity on Rat Neuronal Nicotinic Receptor α Subunits

Abstract: Neuronal nicotinic acetylcholine receptors are differentially sensitive to blockade by the competitive antagonist dihydro-β-erythroidine. Both α and β subunits participate in determining sensitivity to this antagonist. The α subunit contribution to dihydro-β-erythroidine sensitivity is illustrated by comparing the α4β4 receptor and the α3β4 receptor, which differ in sensitivity to dihydro-β-erythroidine by ∼120-fold. IC₅₀ values for blocking α4β4 and α3β4, responding to EC₂₀ concentrations of acetylcholine, were 0.19 ± 0.06 and 23.1 ± 10.2 µ M , respectively. To map the sequence segments responsible for this difference, we constructed a series of chimeric α subunits containing portions of the α4 and α3 subunits. These chimeras were coexpressed with β4, allowing pharmacological characterization. We found determinants of dihydro-β-erythroidine sensitivity to be distributed throughout the N-terminal extracellular domain of the α subunit. These determinants were localized to sequence segments 1–94, 94–152, and 195–215. Loss of determinants within segment 1–94 had the largest effect, decreasing dihydro-β-erythroidine sensitivity by 4.3-fold.     

The Arabidopsis thaliana putative sialyltransferase resides in the Golgi apparatus but lacks the ability to transfer sialic acid

A common feature of the animal sialyltransferases (STs) is the presence of four conserved motifs, namely large (L), small (S), very small (VS) and motif III. Although sialic acid (SA) has not been detected in plants, three orthologues containing sequences similar to the ST motifs have been identified in the Arabidopsis thaliana L. database. In this study, we report that the At3g48820 gene (Gene ID: 824043) codes for a Golgi resident protein lacking the ability to transfer SA to asialofetuin or Galβ1,3GalNAc and Galβ1,4GlcNAc oligosaccharide acceptors. Restoration of deteriorated motifs S, VS and motif III by constructing chimeric proteins consisting of the 28–308 amino acid region of the A. thaliana At3g48820 ST-like protein and the 264–393 amino acid region of the Oryza sativa L. AK107493 ST-like protein, or of the 28–240 amino acid region of the At3g48820 protein and the 204–350 amino acid region of the Homo sapiens L. α2,3-ST ( NP_008858 ) was not able to recover sialyltransferase activity. Altering the appropriate amino acid regions of the A. thaliana At3g48820 ST-like protein to those typical for the mammalian motif III (HHYWE) and VS motif (HDADFE) also did not have any effect. Our data, together with previous results, indicate that A. thaliana in particular, and plants in general, do not have transferases for SA. Substrates for the plant ST-like proteins might be compounds involved in secondary metabolism.     

ON THE STRUCTURE OF TWO NEW GANGLIOSIDES FROM BEEF BRAIN

Abstract— Two new gangliosides were isolated in pure form from beef brain. They were provisionally named ganglioside G5a and G5b. Ganglioside GSa is a monosialoganglioside containing fucose. Its basic neutral glycosphingolipid core is the gangliotetraose ceramide: Gal (1 —> 3) GalNac (1—> 4) Gal (1 —> 4) Glc (1—>) ceramide, most likely with β-linkages. Fucose is linked to the 2-position of external galactose, N -acetylneuraminic acid to the 3- position of internal galactose. Ganglioside G5b is a mixture of at least two isomeric disialogangliosides containing N -acetylneuraminic acid and N -glycolylneura-minic acid. The major isomer has the following structure: NeuNac (α,2—>3) Gal (β,1—>3) GalNac (β, 1 —> 4) (NeuNglα, 2 —> 3) gal (β,1—>4) Glc (β,1 —>)-ceramide. The minor isomer contains N -acetylneur-aminic acid and N -glycolylneuraminic acid in an inverted linkage position.     

The role of inositol lipids in the activation of monocytes by interleukin-1 and bacterial endotoxin

Abstract The effect of interleukin-1 (IL-1) and bacterial endotoxin (lipopolysaccharide, LPS) on the activation of phosphoinositidase C (PIC) and on prostaglandin E₂ release was studied in monocytes (Mø). Both IL-1α and IL-1β increased the release of PGE₂ in a concentration-dependent manner, with EC₅₀s of 0.48 nM and 0.12 nM, respectively. Intact Mø were prelabelled with [³H]inositol and the formation of inositol phosphates (IPs) was estimated by ion exchange chromatography. PIC activity was estimated directly by measuring the conversion of [³H]phosphatidylinositol-4,5,-bisphosphate to aqueous soluble radioactivity by Mø homogenates. IL-1α (5.8 nM) increased the accumulation of IPs within 1–4 minutes and increases in IP₃ and IP₄ occured before the increase in IP₁₊₂ whereas LPS only increased the IPs level after at least 30 min. IL-1α increased PIC activity in Mø homogenates within 15 min with an EC₅₀ of 0.58 nM and IL-1β (0.1 nM) also increased activity. Neither IL-1α nor IL-1β affected the PIC activity of membrane or cytosolic fractions. LPS decreased activity in all fractions. These data indicate that IL-1, but not LPS, can directly lead to an increased activity of PIC which may be involved in eicosanoid formation in Mø.     

Expression of Kv1.2 in microglia and its putative roles in modulating production of proinflammatory cytokines and reactive oxygen species

Microglial cells are endowed with different potassium ion channels but their expression and specific functions have remained to be fully clarified. This study has shown Kv1.2 expression in the amoeboid microglia in the rat brain between 1 (P1) and 10 (P10) days of age. Kv1.2 expression was localized in the ramified microglia at P14 and was hardly detected at P21. In postnatal rats exposed to hypoxia, Kv1.2 immunoreactivity in microglia was markedly enhanced. Quantitative RT-PCR analysis confirmed Kv1.2 mRNA expression in microglial cells in vitro . It was further shown that Kv1.2 and protein expression coupled with that of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) was significantly increased when the cells were subjected to hypoxia. The same increase was observed in cells exposed to adenosine 5'-triphosphate (ATP) and lipopolysaccharide (LPS). Concomitantly, the intracellular potassium concentration decreased significantly. Blockade of Kv1.2 channel with rTityustoxin-Kα (TsTx) resulted in partial recovery of intracellular potassium concentration accompanied by a reduced expression of IL-1β and TNF-α mRNA and protein expression and intracellular reactive oxygen species (ROS) production. We conclude that Kv1.2 in microglia modulates IL-1β and TNF-α expression and ROS production probably by regulating the intracellular potassium concentration.     

Construction and characterization of stably transfected BHK-21 cells with human-type sialylation characteristic

The human Golgi enzyme CMP-NeuAc:Gal(β1–4)GlcNAc-R α2,6-sialyltransferase (ST6N) was stably coexpressed with human erythropoietin (EPO) from a BHK-21A cell line. The cell line was characterized with respect to the expression and in vitro activity of the ST6N and the endogenous α2,3-sialyltransferase. Detailed structural analysis of the N-linked carbohydrates of the rhuEPO expressed from the new cell line was performed by HPAE-PAD-mapping, MALDI/TOF-MS and methylation analysis after purification of the recombinant protein by immunoaffinity chromatography. This is the first report describing that the human α2,6-sialyltransferase is capable of sialylating, apart from Gal(β1–4)GlcNAc-R, also GalNAc(β1–4)GlcNAc-R motifs in vivo, which is not the case for the endogenous BHK-cell α2,3-sialyltransferase. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purification and properties of a BT-glucosidase of Hanseniaspora vineae Van der Walt and Tscheuschner with the view to its utilization in fruit aroma liberation

The β-glucosidase of Hanseniaspora vineae was purified by ion-exchange chromatography and gel filtration. Its molecular weight was 295000 PT 15000, its optimum pH was between 6 and 6–5, and its optimum temperature was 55°C. The enzyme was active against different soluble glucosides with β(1–2), β(1–3), β(1–4), β(1–6) and even aP(1–4) configurations. A glucosyltransferase activity appeared in the presence of ethanol. The enzyme was constitutive but its synthesis was repressed by glucose.     

Alpha-Galactosyl trisaccharide epitope: Modification of the 6-primary positions and recognition by human anti-αGal antibody

Galactose oxidase (EC 1.1.3.9, GAO) was used to convert the C-6′ OH of Galβ(1 → 4)Glcβ–OBn (5) to the corresponding hydrated aldehyde (7). Chemical modification, through dehydratative coupling and reductive amination, gave rise to a small library of Galβ(1 → 4)Glcβ–OBn analogues (9a–f, 10, 11). UDP-[6-³H]Gal studies indicated that α1,3-galactosyltransferase recognized the C-6′ modified Galβ(1 → 4)Glcβ–OBn analogues (9a–f, 10, 11). Preparative scale reactions ensued, utilizing a single enzyme UDP-Gal conversion as well as a dual enzymatic system (GalE and α1,3GalT), taking full advantage of the more economical UDP-Glc, giving rise to compounds 6, 15–22. Galα(1 → 3)Galβ(1 → 4)Glcβ–OBn trisaccharide (6) was produced on a large scale (2 g) and subjected to the same chemoenzymatic modification as stated above to produce C-6″ modified derivatives (23–30). An ELISA bioassay was performed utilizing human anti-αGal antibodies to study the binding affinity of the derivatized epitopes (6, 15–30). Modifications made at the C-6′ position did not alter the IgG antibody's ability to recognize the unnatural epitopes. Modifications made at the C-6″ position resulted in significant or complete abrogation of recognition. The results indicate that the C-6′ OH of the αGal trisaccharide epitope is not mandatory for antibody recognition. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.