Animal Glycoproteins Research Articles

Identification and comparison of N-glycome profiles from common dietary protein sources

The N-glycomes of bovine whey, egg white, pea, and soy protein isolates are described here. Glycoproteins from four protein isolates were analyzed by HILIC high performance liquid chromatography and quadrupole time-of-flight tandem mass spectrometry (HILIC-FLD-QTOF-MS/MS). A total of 33 N-glycans were identified from bovine whey and egg white, and 10 structures from soy and pea glycoproteins. The type of N-glycans per glycoprotein source were attributable to differences in biosynthetic glycosylation pathways. Animal glycoprotein sources favored a combination of complex and hybrid glycan configurations, while the plant proteins were dominated by oligomannosidic N-glycans. Bovine whey glycoprotein isolate contained the most diverse N-glycans by monosaccharide composition as well as structure, while plant sources such as pea and soy glycoprotein isolates contained an overlap of oligomannosidic N-glycans. The results suggest N-glycan structure and composition is dependent on the host organism which are driven by the differences in N-glycan biosynthetic pathways.

Novel lamprey antibody recognizes terminal sulfated galactose epitopes on mammalian glycoproteins

The terminal galactose residues of N- and O-glycans in animal glycoproteins are often sialylated and/or fucosylated, but sulfation, such as 3-O-sulfated galactose (3-O-SGal), represents an additional, but poorly understood modification. To this end, we have developed a novel sea lamprey variable lymphocyte receptor (VLR) termed O6 to explore 3-O-SGal expression. O6 was engineered as a recombinant murine IgG chimera and its specificity and affinity to the 3-O-SGal epitope was defined using a variety of approaches, including glycan and glycoprotein microarray analyses, isothermal calorimetry, ligand-bound crystal structure, FACS, and immunohistochemistry of human tissue macroarrays. 3-O-SGal is expressed on N-glycans of many plasma and tissue glycoproteins, but recognition by O6 is often masked by sialic acid and thus exposed by treatment with neuraminidase. O6 recognizes many human tissues, consistent with expression of the cognate sulfotransferases (GAL3ST-2 and GAL3ST-3). The availability of O6 for exploring 3-O-SGal expression could lead to new biomarkers for disease and aid in understanding the functional roles of terminal modifications of glycans and relationships between terminal sulfation, sialylation and fucosylation.

Exploiting the Disialyl Galactose Activity of α2,6-Sialyltransferase from Photobacterium damselae To Generate a Highly Sialylated Recombinant α-1-Antitrypsin.

Sialic acids are sugars present in many animal glycoproteins and are of particular interest in biopharmaceuticals, where a lack of sialylation can reduce bioactivity. Here, we describe how α-2,6-sialyltransferase from Photobacterium damselae can be used to markedly increase the level of sialylation of CHO-produced α-1-antitrypsin. Detailed analysis of the sialylation products showed that in addition to the expected α-2,6-sialylation of galactose, a second disialyl galactose motif Neu5Ac-α2,3(Neu5Ac-α2,6)Gal was produced, which, to our knowledge, had never been detected on a mammalian glycoprotein. We exploited this disialyl galactose activity of the P. damselae in a multienzyme reaction to produce a highly sialylated α-1-antitrypsin. The influence of this unique disialylation on the in vitro activity of α-1-antitrypsin was studied, and a toolkit of mass spectrometry methods for identifying this new disialyl galactose motif in complex mixtures was developed.

Unexpected Diversity of Escherichia coli Sialate O-Acetyl Esterase NanS.

The sialic acids (N-acylneuraminates) are a group of nine-carbon keto-sugars existing mainly as terminal residues on animal glycoprotein and glycolipid carbohydrate chains. Bacterial commensals and pathogens exploit host sialic acids for nutrition, adhesion, or antirecognition, where N-acetyl- or N-glycolylneuraminic acids are the two predominant chemical forms of sialic acids. Each form may be modified by acetyl esters at carbon position 4, 7, 8, or 9 and by a variety of less-common modifications. Modified sialic acids produce challenges for colonizing bacteria, because the chemical alterations to N-acetylneuraminic acid (Neu5Ac) confer increased resistance to sialidase and aldolase activities essential for the catabolism of host sialic acids. Bacteria with O-acetyl sialate esterase(s) utilize acetylated sialic acids for growth, thereby gaining a presumed metabolic advantage over competitors lacking this activity. Here, we demonstrate the esterase activity of Escherichia coli NanS after purifying it as a C-terminal HaloTag fusion. Using a similar approach, we show that E. coli strain O157:H7 Stx prophage or prophage remnants invariably include paralogs of nanS often located downstream of the Shiga-like toxin genes. These paralogs may include sequences encoding N- or C-terminal domains of unknown function where the NanS domains can act as sialate O-acetyl esterases, as shown by complementation of an E. coli strain K-12 nanS mutant and the unimpaired growth of an E. coli O157 nanS mutant on O-acetylated sialic acid. We further demonstrate that nanS homologs in Streptococcus spp. also encode active esterase, demonstrating an unexpected diversity of bacterial sialate O-acetyl esterase. The sialic acids are a family of over 40 naturally occurring 9-carbon keto-sugars that function in a variety of host-bacterium interactions. These sugars occur primarily as terminal carbohydrate residues on host glycoproteins and glycolipids. Available evidence indicates that diverse bacterial species use host sialic acids for adhesion or as sources of carbon and nitrogen. Our results show that the catabolism of the diacetylated form of host sialic acid requires a specialized esterase, NanS. Our results further show that nanS homologs exist in bacteria other than Escherichia coli, as well as part of toxigenic E. coli prophage. The unexpected diversity of these enzymes suggests new avenues for investigating host-bacterium interactions. Therefore, these original results extend our previous studies of nanS to include mucosal pathogens, prophage, and prophage remnants. This expansion of the nanS superfamily suggests important, although as-yet-unknown, functions in host-microbe interactions.

Structure of the capsular polysaccharides and lipopolysaccharides from Haemophilus parasuis strains ER-6P (serovar 15) and Nagasaki (serovar 5)

Haemophilus parasuis is a Gram-negative bacterium from the family Pasteurellaceae and a swine pathogen. H. parasuis is found in the upper respiratory tract of piglets and produces Glässer’s disease, an invasive disease characterized by polyserositis. H. parasuis contains a short lipopolysaccharide (LPS) or lipooligosaccharide (LOS) reported to play a partial role in interaction with host cells. The presence of capsule has been phenotypically demonstrated in certain H. parasuis strains and its role in virulence has been suggested, but the chemical structure of the surface polysaccharides of this bacterium was unknown. The structure of capsular polysaccharide (CPS) and LOS from virulent strains ER-6P and Nagasaki was studied by NMR spectroscopy, mass spectrometry and chemical methods. CPS from both strains had the same main chain with disaccharide repeating unit, substituted with α-Neu5R-(2-3)-α-GalNAc-(1-P-(strain ER-6P) or α-Neu5R-(2-3)-α-Gal-(1-P-strain Nagasaki) side chains, where R is the N-acetyl or N-glycolyl group. Glycolyl-neuraminic acid is widely found in animal glycoproteins, but it apparently has not been found in bacteria before, and might be important for the biology of this microorganism. Ac and Gc were present in equal amounts in the strain ER-6P but Nagasaki contained only about 20% of Gc substituent. Both strains produced the same LPS of a rough type with a single phosphorylated Kdo linking core and lipid A parts. LOS structure was similar to some strains of H. influenzae and contained a globotetraose terminal sequence.

Preventing Ralstonia solanacearum adhesion with glycans from cashew, cocoa, coffee, pumpkin, and tomato seed extract

Ralstonia solanacearum wilts many plants, causing heavy agricultural losses. Its pathogenic strain ATCC 11696 produces 2 hemagglutinating lectins: RSL and RS-IIL. These lectins may bind to terminal l-fucose-, d-arabinose-, and d-mannose-bearing seedling xylem cell wall glycans, thus enabling pathogen adhesion to them, with devastating infection establishment. Blocking the active sites of these lectins with seed embryo-surrounding oligo- and poly-saccharides hampers binding of the lectins to the embryos. The current study shows that seeds of cashew, cocoa, coffee, pumpkin, and tomato contain low and high molecular mass glycans that block RSL and RS-IIL (like its homologous Pseudomonas aeruginosa PA-IIL lectin). The blocking of the pathogen lectins, which is attributable to the documented composition of the oligo- and poly-saccharides of these seeds, is similar to that observed with animal glycoproteins of avian egg whites (protecting their embryos from infections) and of milk and royal jelly, which likewise protect mammal and bee neonates, respectively. RSL was most strongly inhibited by cashew seed glycans, and RS-IIL by coffee seed glycans. Western blot analyses with these lectins instead of antibodies revealed the hitherto undescribed presence of lectin-binding glycoproteins in the coffee, pumpkin, tomato, and cashew (but not cocoa) seeds. The use of these lectins for unveiling potent embryo-protecting seed glycans might be helpful for seedling-bioprotection projects similar to those planned for animal protection against antibiotic-resistant infections.

ChemInform Abstract: Xylose-Containing Carbohydrate Chains Derived from N-Glycoproteins

Xylose-containing N-linked carbohydrate chains are integral parts of certain plant and animal glycoproteins. In all of the known structures, p-D-xylose is 1-2-linked to p-D- mannose of the trimannosyl-N,N'-diacetylchitobiose unit. If a-L-fucose is present at the asparagine-linked N-acetyl-Pglucosamine, then there are differences in the sites of its attachment, namely, al-3- or al-6-linked, depending on the biological origin. The a+ mannose residues can be substituted with additional monosaccharides or 3-0-methylated. The state of the art with respect to structural analysis, organic synthesis, conformational analysis, biosynthesis, lectin binding, and immunological aspects is reviewed.

The Endoplasmic Reticulum Chaperone Cosmc Directly Promotes in Vitro Folding of T-synthase

The T-synthase is the key beta 3-galactosyltransferase essential for biosynthesis of core 1 O-glycans (Gal beta 1-3GalNAc alpha 1-Ser/Thr) in animal cell glycoproteins. Here we describe the novel ability of an endoplasmic reticulum-localized molecular chaperone termed Cosmc to specifically interact with partly denatured T-synthase in vitro to cause partial restoration of activity. By contrast, a mutated form of Cosmc observed in patients with Tn syndrome has reduced chaperone function. The chaperone activity of Cosmc is specific, does not require ATP in vitro, and is effective toward T-synthase but not another beta-galactosyltransferase. Cosmc represents the first ER chaperone identified to be required for folding of a glycosyltransferase.

Lectins: Carbohydrate-specific Reagents and Biological Recognition Molecules

Lectins: Carbohydrate-specific Reagents and Biological Recognition Molecules

Identification of core 1 O-glycan T-synthase from Caenorhabditis elegans

The common O-glycan core structure in animal glycoproteins is the core 1 disaccharide Galbeta1-3GalNAcalpha1-Ser/Thr, which is generated by the addition of Gal to GalNAcalpha1-Ser/Thr by core 1 UDP-alpha-galactose (UDP-Gal):GalNAcalpha1-Ser/Thr beta1,3-galactosyltransferase (core 1 beta3-Gal-T or T-synthase, EC2.4.1.122). Although O-glycans play important roles in vertebrates, much remains to be learned from model organisms such as the free-living nematode Caenorhabditis elegans, which offer many advantages in exploring O-glycan structure/function. Here, we report the cloning and enzymatic characterization of T-synthase from C. elegans (Ce-T-synthase). A putative C. elegans gene for T-synthase, C38H2.2, was identified in GenBank by a BlastP search using the human T-synthase protein sequence. The full-length cDNA for Ce-T-synthase, which was generated by polymerase chain reaction using a C. elegans cDNA library as the template, contains 1170 bp including the stop TAA. The cDNA encodes a protein of 389 amino acids with typical type II membrane topology and a remarkable 42.7% identity to the human T-synthase. Ce-T-synthase has seven Cys residues in the lumenal domain including six conserved Cys residues in all orthologs. The Ce-T-synthase has four potential N-glycosylation sequons, whereas the mammalian orthologs lack N-glycosylation sequons. Only one gene for Ce-T-synthase was identified in the genome-wide search, and it contains eight exons. Promoter analysis of the Ce-T-synthase using green fluorescent protein (GFP) constructs shows that the gene is expressed at all developmental stages and appears to be in all cells. Unexpectedly, only minimal activity was recovered in the recombinant, soluble Ce-T-synthase secreted from a wide variety of mammalian cell lines, whereas robust enzyme activity was recovered in the soluble Ce-T-synthase expressed in Hi-5 insect cells. Vertebrate T-synthase requires the molecular chaperone Cosmc, but our results show that Ce-T-synthase does not require Cosmc and might require invertebrate-specific factors for the formation of the optimally active enzyme. These results show that the Ce-T-synthase is a functional ortholog to the human T-synthase in generating core 1 O-glycans and open new avenues to explore O-glycan function in this model organism.

Gene cloning and expression of cadherin in midgut of Helicoverpa armigera and its Cry1A binding region

Cadherins belong to one of the families of animal glycoproteins responsible for calcium-dependent cell-cell adhesion. Recent literatures showed that the cadherin-like in midgut of several insects served as the receptor of Bt toxin Cry1A and the variation of cadherin-like is related to insect's resistance to Cry1A. The full-length cDNA encoding cadherin-like of Helicoverpa armigera is cloned by degenerate PCR and RACE techniques and the gene was designated as BtR-harm, which is 5581 bp in full-length, encoding 1730 amino acid residues (BtR-harm was deposited in GenBank and the accession number is AF519180). Its predicted molecular weight and isoelectric point were 195.39 kDa and 4.23, respectively. The inferred amino acid sequence includes a signal sequence, 11 cadherin repeats, a membrane-proximal region, a transmembrane region and a cytoplasmic region. Sequence analysis indicated that the deduced protein sequence was most similar to the cadherin-like from Heliothis virescens with 84.2% identity and highly similar to three other lepidopteran cadherin from Bombyx mori, Manduca sexta and Pectinophora gossypiella, with the sequence identities of 60.3.6%, 57.5% and 51.0%, respectively. The cDNA encoding cadherin gene was expressed successfully in E. coli and the recombinant proteins can bind with Cry1Ac. Truncation analysis and binding experiment of BtR-harm revealed that the Cry1A binding region was a contiguous 244-amino acid sequence, which located between amino acid 1217 and 1461. Semi-quantitative RT-PCR analysis showed that BtR-harm was highly expressed in midgut of H. armigera, very low expressed in foregut and hindgut and was not expressed in other tissues. After H. armigera producing resistance to Cry1Ac, the expression quantity of BtR-harm significantly decreased in midgut of H. armigera. It is the first confirmation that BtR-harm can function as receptor of Cry1Ac in H. armigera and the binding region was located on a contiguous 244 amino acid sequence, suggesting that the decrease of expression quantity of BtR-harm is one of the main reasons for H. armigera resistance to Cry1Ac.

Xylem Sap Lectin, XSP30, Recognizes GlcNAc Sugar Chains of Glycoproteins in Cucumber Leaves.

The lectin activity of 30-kDa xylem sap protein (XSP30) found in cucumber (Cucumis sativas L.) xylem sap was analyzed by lectin blot coupled with immunological detection of XSP30. XSP30 is homologous to galactose-binding lectin, but it had only weak binding activity against asialofetuin, an animal glycoprotein with a terminal galactose. This reaction was not reduced by galactosidase treatement of asialofetuin. XSP30 bound strongly to soybean agglutinin, whose sugar chains consist solely of mannose and N-acetylglucosamine (GlcNAc), but bound only weakly to soybean peroxidase and γ-globulin, whose GlcNAc is fucosylated. The binding activity was inhibited by tri-N-acetylchitotriose (GlcNAc)3. Leaf parenchyma cells were stained with XSP30 and aniti-XSP30 antibody; this staining was reduced by proteinase treatment of the sections, and XSP30 bound to proteins in leaf particulate fraction. These results suggest that XSP30 transported from root via xylem sap binds to chitobiose, GlcNAc-GlcNAc, in N-linked glycans of leaf glycoproteins.

Involvement of beta 1,4 galactosyltransferase 1 and Gal beta1-->4GlcNAc groups in human hepatocarcinoma cell apoptosis.

Beta 1,4 galactosyltransferase 1 (beta 1,4GT1) synthesizes Gal beta 1-->4GlcNAc 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 beta 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 beta 1-->4GlcNAc groups during apoptosis. Further study showed that the 7721 hepatocarcinoma cells transiently transfected with beta 1,4GT1 were more susceptible to the apoptosis induced by cycloheximide. The increased susceptibility was in accordance to the transfection concentration of beta 1,4GT1, which also led to the increased Gal beta 1-->4GlcNAc groups on the transfected cell surface. All the observations suggested that beta 1,4GT1 and Gal beta 1-->4GlcNAc groups might be associated with the apoptosis of human hepatocarcinoma cells.

A unique molecular chaperone Cosmc required for activity of the mammalian core 1 beta 3-galactosyltransferase.

Human core 1 beta3-galactosyltransferase (C1beta3Gal-T) generates the core 1 O-glycan Galbeta1-3GalNAcalpha1-SerThr (T antigen), which is a precursor for many extended O-glycans in animal glycoproteins. We report here that C1beta3Gal-T activity requires expression of a molecular chaperone designated Cosmc (core 1 beta3-Gal-T-specific molecular chaperone). The human Cosmc gene is X-linked (Xq23), and its cDNA predicts a 318-aa transmembrane protein ( approximately 36.4 kDa) with type II membrane topology. The human lymphoblastoid T cell line Jurkat, which lacks C1beta3Gal-T activity and expresses the Tn antigen GalNAcalpha1-SerThr, contains a normal gene and mRNA encoding C1beta3Gal-T, but contains a mutated Cosmc with a deletion introducing a premature stop codon. Expression of Cosmc cDNA in Jurkat cells restored C1beta3Gal-T activity and T antigen expression. Without Cosmc, the C1beta3Gal-T is targeted to proteasomes. Expression of active C1beta3Gal-T in Hi-5 insect cells requires coexpression of Cosmc. Overexpression of active C1beta3Gal-T in mammalian cell lines also requires coexpression of Cosmc, indicating that endogenous Cosmc may be limiting. A small portion of C1beta3Gal-T copurifies with Cosmc from cell extracts, demonstrating physical association of the proteins. These results indicate that Cosmc acts as a specific molecular chaperone in assisting the foldingstability of C1beta3Gal-T. The identification of Cosmc, a uniquely specific molecular chaperone required for a glycosyltransferase expression in mammalian cells, may shed light on the molecular basis of acquired human diseases involving altered O-glycosylation, such as IgA nephropathy, Tn syndrome, Henoch-Schönlein purpura, and malignant transformation, all of which are associated with a deficiency of C1beta3Gal-T activity.

How proteins bind carbohydrates: lessons from legume lectins.

The pioneering studies of Irvin Liener on soybean agglutinin (SBA) in the early 1950s served as the starting point of our involvement in lectin research during the past four decades. Initially we characterized SBA extensively as a glycoprotein and showed that its covalently linked glycan is an oligomannoside commonly present in animal glycoproteins. We have also introduced the use of the lectin to the study of normal and malignant cells and to the purging of bone marrow for transplantation. Our recent work focuses on the combining site of Erythrina corallodendron lectin, closely related to SBA. In this legume lectin, as in essentially all other members of the same protein family, irrespective of their sugar specificity, interactions with a constellation of three invariant residues (aspartic acid, asparagine, and an aromatic residue) are essential for ligand binding. Lectins from other families, whether of plants or animals, also combine with carbohydrates by H-bonds and hydrophobic interactions, but the amino acids involved may differ even if the specificity of the lectins is the same. Therefore, nature finds diverse solutions for the design of binding sites for structurally similar ligands, such as mono- or oligosaccharides. This diversity strongly suggests that lectins are products of convergent evolution.

Molecular Cloning and Enzymatic Characterization of a UDP-GalNAc:GlcNAcβ-R β1,4-N-Acetylgalactosaminyltransferase fromCaenorhabditis elegans

A common terminal structure in glycans from animal glycoproteins and glycolipids is the lactosamine sequence Gal(beta)4GlcNAc-R (LacNAc or LN). An alternative sequence that occurs in vertebrate as well as in invertebrate glycoconjugates is GalNAc(beta)4GlcNAc-R (LacdiNAc or LDN). Whereas genes encoding beta4GalTs responsible for LN synthesis have been reported, the beta4GalNAcT(s) responsible for LDN synthesis has not been identified. Here we report the identification of a gene from Caenorhabditis elegans encoding a UDP-GalNAc:GlcNAc(beta)-R beta1,4-N-acetylgalactosaminyltransferase (Ce(beta)4GalNAcT) that synthesizes the LDN structure. Ce(beta)4GalNAcT is a member of the beta4GalT family, and its cDNA is predicted to encode a 383-amino acid type 2 membrane glycoprotein. A soluble, epitope-tagged recombinant form of Ce(beta)4GalNAcT expressed in CHO-Lec8 cells was active using UDP-GalNAc, but not UDP-Gal, as a donor toward a variety of acceptor substrates containing terminal beta-linked GlcNAc in both N- and O-glycan type structures. The LDN structure of the product was verified by co-chromatography with authentic standards and (1)H NMR spectroscopy. Moreover, Chinese hamster ovary CHO-Lec8 and CHO-Lec2 cells expressing Ce(beta)4GalNAcT acquired LDN determinants on endogenous glycoprotein N-glycans, demonstrating that the enzyme is active in mammalian cells as an authentic beta4GalNAcT. The identification and availability of this novel enzyme should enhance our understanding of the structure and function of LDN-containing glycoconjugates.

Structural characterisation of the native fetuin-binding protein Scilla campanulata agglutinin: a novel two-domain lectin

The three-dimensional structure of a 244-residue, multivalent, fetuin-binding lectin, SCAfet, isolated from bluebell ( Scilla campanulata) bulbs, has been solved at 3.3 Å resolution by molecular replacement using the coordinates of the 119-residue, mannose-binding lectin, SCAman, also from bluebell bulbs. Unlike most monocot mannose-binding lectins, such as Galanthus nivalis agglutinin from snowdrop bulbs, which fold into a single domain, SCAfet contains two domains with approximately 55% sequence identity, joined by a linker peptide. Both domains are made up of a 12-stranded β-prism II fold, with three putative carbohydrate-binding sites, one on each subdomain. SCAfet binds to the complex saccharides of various animal glycoproteins but not to simple sugars.

Diversity in sialic and polysialic acid residues and related enzymes

The occurrence of extensive diversity in the family of sialic acids is now well recognized but biological significance of this diversity has been clarified only partially. In 1986 we showed natural occurrence of deaminated form of neuraminic acid, 2-keto-3-deoxy-D- glycero-D-galacto-nononic acid (KDN) as a capping residue of polysialyl chain of rainbow trout egg polysialoglycoprotein. Subsequent studies of our own and by others showed ubiquitous occurrence of KDN from bacteria to human although expression of this residue in large amounts was limited to certain species of animal. KDN was different from other 'families' of sialic acids usually denoted as modified N-acylneuraminic acids of which O-acetyl substitution is most frequently found. The definition of sialic acid has to be changed by the finding of KDN. Prior to the finding of poly N-acetylneuraminic acid group in mammals, in 1978 we discovered poly N-glycolylneuraminic acid in rainbow trout eggs. This was the first report of the occurrence of polysialic acid structure in animal glycoproteins. We also found the occurrence of polyKDN chains. All these findings made significant contribution to show that diversity of sialic acid family and especially diversity in polysialic acid structure are much more extensive and more ubiquitous than generally believed before. We showed that diversity of polysialic acid is not only originated from the diversity in building block but also the type of interresidue linkage. The diversity of sialic acid and polysialic acid is reflected in diversities in molecules that recognize these structures. We studied some of these molecules that specifically recognize different types of sialic acids and polysialic acids, and biosynthetic mechanism of polysialic acids.

Core alpha1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts.

Carbohydrates have been suggested to account for some IgE cross-reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti-horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose linked to the core mannose of many plant and gastropod N-linked oligosaccharides.

Two-mode Analysis by High-performance Liquid Chromatography ofρ-Aminobenzoic Ethyl Ester-derivatized Monosaccharides

ρ-Aminobenzoic ethyl ester (ABEE)-derivatized monosaccharides were separated by HPLC with a trifluoroacetic acid (TFA) solution or borate buffer as the eluent. In the case of the TFA solution, ABEE-derivatized monosaccharides of the neutral and amino sugars found in animal glycoproteins were separated in a simultaneous analysis. In the case of the borate buffer, ABEE-derivatized monosaccharides of identical molecular weights such as ABEE-Gal, -Glc, and -Man were separated as stereoisomers. Glucuronic acid and galacturonic acid were detected and separated within 8 min. The relationship between the peak areas and the amounts of ABEE-derivatized monosaccharides was linear in the range of 1 to 1000 pmol.