Beta(1,2)-xylose and alpha(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens

Primary structures of the N-glycans of two major pollen allergens (Lol p 11 and Ole e 1) and a major peanut allergen (Ara h 1) were determined. Ole e 1 and Ara h 1 carried high mannose and complex N-glycans, whereas Lol p 11 carried only the complex. The complex structures all had a beta(1,2)-xylose linked to the core mannose. Substitution of the proximal N-acetylglucosamine with an alpha(1, 3)-fucose was observed on Lol p 11 and a minor fraction of Ole e 1 but not on Ara h 1. To elucidate the structural basis for IgE recognition of plant N-glycans, radioallergosorbent test analysis with protease digests of the three allergens and a panel of glycoproteins with known N-glycan structures was performed. It was demonstrated that both alpha(1,3)-fucose and beta(1,2)-xylose are involved in IgE binding. Surprisingly, xylose-specific IgE antibodies that bound to Lol p 11 and bromelain did not recognize closely related xylose-containing structures on horseradish peroxidase, phytohemeagglutinin, Ole e 1, and Ara h 1. On Lol p 11 and bromelain, the core beta-mannose is substituted with just an alpha(1,6)-mannose. On the other xylose-containing N-glycans, an additional alpha(1,3)-mannose is present. These observations indicate that IgE binding to xylose is sterically hampered by the presence of an alpha(1,3)-antenna.     

N-linked oligosaccharides of glycoproteins from Ginkgo biloba pollen, an allergenic pollen.

The pollen of Ginkgo biloba is one of the allergens that cause pollen allergy symptoms. The plant complex type N-glycans bearing beta1-2 xylose and/or alpha1-3 fucose residue(s) linked to glycoallergens have been considered to be critical epitopes in various immune reactions. In this report, the structures of N-glycans of total glycoproteins prepared from Ginkgo biloba pollens were analyzed to confirm whether such plant complex type N-glycans occur in the pollen glycoproteins. The glycoproteins were extracted by SDS-Tris buffer. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine and the resulting pyridylaminated (PA-)N-glycans were purified by a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, IS-MS, and MS/MS. The plant complex type structures (GlcNAc2Man3Xyl1Fuc1GlcNAc2 (31%), GlcNAc2Man3Xyl1GlcNAc2 (5%), Man3Xyl1Fuc1GlcNAc2 (13%), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (8%), and GlcNAc1Man3Xyl1GlcNAc2 (17%)) have been found among the N-glycans of the glycoproteins of Ginkgo biloba pollen, which might be candidates for the epitopes involved in Ginkgo pollen allergy. The remaining 26% of the total pollen N-glycans have the typical high-mannose type structures: Man8GlcNAc2 (11%) and Man6GlcNAc2 (15%).     

Glycoform analysis of Japanese cypress pollen allergen, Cha o 1: a comparison of the glycoforms of cedar and cypress pollen allergens

A Japanese cypress (Chamaecyparis obtusa) pollen allergen, Cha o 1, is one of the major allergens that cause allergic pollinosis in Japan. Although it has been found that Cha o 1 is glycosylated and that the amino acid sequence is highly homologous with that of Japanese cedar pollen allergen (Cry j 1), the structure of N-glycans linked to Cha o 1 remains to be determined. In this study, therefore, we analyzed the structures of the N-glycans of Cha o1. The N-glycans were liberated by hydrazinolysis from purified Cha o 1, and the resulting sugar chains were N-acetylated and pyridylaminated. The structures of pyridylaminated N-glycans were analyzed by a combination of exoglycosidase digestion, two dimensional (2D-) sugar chain mapping, and electrospray ionization mass spectrometry analysis. Structural analysis indicated that the major N-glycan structure of Cha o1 is GlcNAc2Man3Xyl1Fuc1GlcNAc2 (89%), and that high-mannose type structures (Man9GlcNAc2, Man7GlcNAc2) occur as minor components (11%).     

Occurrence of GalNAcbeta1-4GlcNAc unit in N-glycan of royal jelly glycoprotein

Elsewhere, we characterized the structure of twelve N-glycans purified from royal jelly glycoproteins (Kimura, Y. et al., Biosci. Biotechnol. Biochem., 64, 2109-2120 (2000)). Structural analysis showed that the typical high-mannose type structure (Man9-4GlcNAc2) accounts for about 72% of total N-glycans, a biantennary-type structure (GlcNAc2Man3GlcNAc2) about 8%, and a hybrid-type structure (GlcNAc1Man4GlcNAc2) about 3%. During structural analysis of minor N-glycans of royal jelly glycoproteins, we found that one had an N-acetyl-galactosaminyl residue at the non reducing end; most of such residues have been found in N-glycans of mammalian glycoproteins. By exoglycosidase digestion, methylation analysis, ion-spray (IS)-MS analysis, and 1H NMR spectroscopy, we identified the structure of the N-glycan containing GalNAc as; GlcNAc(beta)1-2Man(alpha)1-6(GalNAcbeta1 - 4GIcNAcbeta1 - 2Man(alpha)1 - 3)Manbeta1 - 4GlcNAc(beta)1-4GlcNAc. This result suggested that a beta1-4 GalNAc transferase is present in hypopharyngeal and mandibular glands of honeybees.     

Site-specific glycosylation analysis of the bovine lysosomal alpha-mannosidase

Lysosomal alpha-mannosidase is a broad specificity exoglycosidase involved in the ordered degradation of glycoproteins. The bovine enzyme is used as an important model for understanding the inborn lysosomal storage disorder alpha-mannosidosis. This enzyme of about 1,000 amino acids consists of five peptide chains, namely a- to e-peptides and contains eight N-glycosylation sites. The N(497) glycosylation site of the c-peptide chain is evolutionary conserved among LAMANs and is very important for the maintenance of the lysosomal stability of the enzyme. In this work, relying on an approach based on mass spectrometric techniques in combination with exoglycosidase digestions and chemical derivatizations, we will report the detailed structures of the N-glycans and their distribution within six of the eight N-glycosylation sites of the bovine glycoprotein. The analysis of the PNGase F-released glycans from the bovine LAMAN revealed that the major structures fall into three classes, namely high-mannose-type (Fuc(0-1)Glc(0-1)Man(4-9)GlcNAc(2)), hybrid-type (Gal(0-1)Man(4-5)GlcNAc(4)), and complex-type (Fuc(0-1)Gal(0-2)Man(3)GlcNAc(3-5)) N-glycans, with core fucosylation and bisecting GlcNAc. To investigate the exact structure of the N-glycans at each glycosylation site, the peptide chains of the bovine LAMAN were separated using SDS-PAGE and in-gel deglycosylation. These experiments revealed that the N(497) and N(930) sites, from the c- and e-peptides, contain only high-mannose-type glycans Glc(0-1)Man(5-9)GlcNAc(2), including the evolutionary conserved Glc(1)Man(9)GlcNAc(2) glycan, and Fuc(0-1)Man(3-5)GlcNAc(2), respectively. Therefore, to determine the microheterogeneity within the remaining glycosylation sites, the glycoprotein was reduced, carboxymethylated, and digested with trypsin. The tryptic fragments were then subjected to concanavalin A (Con A) affinity chromatography, and the material bound by Con A-Sepharose was purified using reverse-phase high-performance liquid chromatography (HPLC). The tandem mass spectrometry (ESI-MS/MS) and the MALDI analysis of the PNGase F-digested glycopeptides indicated that (1) N(692) and N(766) sites from the d-peptide chain both bear glycans consisting of high-mannose (Fuc(0-1)Man(3-7)GlcNAc(2)), hybrid (Fuc(0-1) Gal(0-1)Man(4-5)GlcNAc(4)), and complex (Fuc(0-1)Gal(0-2)Man(3)GlcNAc(4-5)) structures; and (2) the N(367) site, from the b-peptide chain, is glycosylated only with high-mannose structures (Fuc(0-1)Man(3-5)GlcNAc(2)). Taking into consideration the data obtained from the analysis of either the in-gel-released glycans from the abc- and c-peptides or the tryptic glycopeptide containing the N(367) site, the N(133) site, from the a-peptide, was shown to be glycosylated with truncated and high-mannose-type (Fuc(0-1)Man(4-5)GlcNAc(2)), complex-type (Fuc(0-1)Gal(0-1)Man(3)GlcNAc(5)), and hybrid-type (Fuc(0-1)Gal(0-1)Man(5)GlcNAc(4)) glycans.     

Structure of ten free N-glycans in ripening tomato fruit. Arabinose is a constituent of a plant N-glycan.

The concentration-dependent stimulatory and inhibitory effect of N-glycans on tomato (Lycopersicon esculentum Mill.) fruit ripening was recently reported (B. Priem and K.C. Gross [1992] Plant Physiol 98: 399-401). We report here the structure of 10 free N-glycans in mature green tomatoes. N-Glycans were purified from fruit pericarp by ethanolic extraction, desalting, concanavalin A-Sepharose chromatography, and amine-bonded silica high performance liquid chromatography. N-Glycan structures were determined using 500 MHz 1H-nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, and glycosyl linkage methylation analysis by gas chromatography-mass spectrometry. A novel arabinosyl-containing N-glycan, Man alpha 1-->6(Ara alpha 1-->2)Man beta 1-->4GlcNAc beta 1-->4(Fuc alpha 1-->3)GlcNAc, was purified from a retarded concanavalin A fraction. The location of the arabinosyl residue was the same as the xylosyl residue in complex N-glycans. GlcNAc[5']Man3(Xyl)GlcNAc(Fuc)GlcNAc and GlcNAc[5']Man2GlcNAc(Fuc)GlcNAc were also purified from the weakly retained fraction. The oligomannosyl N-glycans Man5GlcNAc, Man6GlcNAc, Man7GlcNAc, and Man8GlcNAc were purified from a strongly retained concanavalin A fraction. The finding of free Man5GlcNAc in situ was important physiologically because previously we had described it as a promoter of tomato ripening when added exogenously. Mature green pericarp tissue contained more than 1 microgram of total free N-glycan/g fresh weight. Changes in N-glycan composition were determined during ripening by comparing glycosyl and glycosyl-linkage composition of oligosaccharidic extracts from fruit at different developmental stages. N-Glycans were present in pericarp tissue at all stages of development. However, the amount increased during ripening, as did the relative amount of xylosyl-containing N-glycans.     

Structural features of N-glycans linked to glycoproteins from oil palm pollen, an allergenic pollen*

The pollen of oil palm (Elaeis guineensis Jacq.) is a strong allergen and causes severe pollinosis in Malaysia and Singapore. In the previous study (Biosci. Biotechnol. Biochem., 64, 820-827 (2002)), from the oil palm pollens, we purified an antigenic glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients. In this report, we describe the structural analysis of sugar chains linked to palm pollen glycoproteins to confirm the ubiquitous occurrence of antigenic N-glycans in the allergenic pollen. N-Glycans liberated from the pollen glycoprotein mixture by hydrazinolysis were labeled with 2-aminopyridine followed by purification with a combination of size-fractionation HPLC and reversed-phase HPLC. The structures of the PA-sugar chains were analyzed by a combination of two-dimensional sugar chain mapping, electrospray ionization mass spectrometry (ESI-MS), and tandem MS analysis, as well as exoglycosidase digestions. The antigenic N-glycan bearing alpha1-3 fucose and/or beta1-2 xylose residues accounts for 36.9% of total N-glycans: GlcNAc2Man3Xyl1Fuc1GlcNAc2 (24.6%), GlcNAc2Man3Xyl1GlcNAc2 (4.4%), Man3Xyl1Fuc1-GlcNAc2 (1.1%), GlcNAc1Man3Xyl1Fuc1GlcNAc2 (5.6%), and GlcNAc1Man3Xyl1GlcNAc2 (1.2%). The remaining 63.1% of the total N-glycans belong to the high-mannose type structure: Man9GlcNAc2 (5.8%), Man8GlcNAc2 (32.1%), Man7GlcNAc2 (19.9%), Man6GlcNAc2 (5.3%).     

Purification and characterization of 31-kDa palm pollen glycoprotein (Ela g Bd 31 K), which is recognized by IgE from palm pollinosis patients

A basic glycoprotein, which was recognized by IgE from oil palm pollinosis patients, has been purified from oil palm pollen (Elaeis guineensis Jacq.), which is a strong allergen and causes severe pollinosis in Malaysia and Singapore. Soluble proteins were extracted from defatted palm pollen with both Tris-HCl buffer (pH 7.8) and Na-acetate buffer (pH 4.0). The allergenic glycoprotein was purified from the total extract to homogeneity with 0.4% yield by a combination of DEAE- and CM-cellulose, SP-HPLC, and gel filtration. The purified oil palm pollen glycoprotein with molecular mass of 31 kDa was recognized by the beta1-2 xylose specific antibody, suggesting this basic glycoprotein bears plant complex type N-glycan(s). The palm pollen basic glycoprotein, designated Ela g Bd 31 K, was recognized by IgE of palm pollinosis patients, suggesting Ela g Bd 31 K should be one of the palm pollen allergens. The preliminary structural analysis of N-glycans linked to glycoproteins of palm pollens showed that the antigenic N-glycans having alpha1-3 fucose and alpha1-2 xylose residues (GlcNAc(2 to approximately 0)Man3Xyl1Fuc(1 to approximately 0)GlcNAc2) actually occur on the palm pollen glycoproteins, in addition to the high-mannose type structures (Man(9 to approximately 5)GlcNAc2).     

Glycoproteins secreted from suspension-cultured tobacco BY2 cells have distinct glycan structures from intracellular glycoproteins.

Glycan structures of glycoproteins secreted in the spent medium of tobacco BY2 suspension-cultured cells were analyzed. The N-glycans were liberated by hydrazinolysis and the resulting oligosaccharides were labeled with 2-aminopyridine. The pyridylaminated (PA) glycans were purified by reversed-phase and size-fractionation HPLC. The structures of the PA sugar chains were identified by a combination of the two-dimensional PA sugar chain mapping, MS analysis, and exoglycosidase digestion. The ratio (40:60) of the amount of glycans with high-mannose-type structure to that with plant-complex-type structure of extracellular glycoproteins is significantly different from that (ratio 10:90) previously found in intracellular glycoproteins [Palacpac et al., Biosci. Biotechnol. Biochem. 63 (1999) 35-39]. Extracellular glycoproteins have six distinct N-glycans (marked by *) from intracellular glycoproteins, and the high-mannose-type structures account for nearly 40% (Man5GlcNAc2, 28.8%; Man6GlcNAc2*, 6.4%; and Man7GlcNAc2*, 3.8%), while the plant-complex-type structures account for nearly 60% (GlcNAc2Man3Xyl1GlcNAc2*, 32.1%; GlcNAc1Man3Xyl1GlcNAc2 (containing two isomers)*, 6.2%; GlcNAc2Man3GlcNAc2*, 4.9%; Man3Xyl1Fuc1GlcNAc2, 8.3%; and Man3Xyl1GlcNAc2, 3.7%).     

Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans

The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz 1H-NMR spectrometry. The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing beta1-2 xylosyl and/or alpha1-3 fucosyl residue(s) and occurrence of beta1-4GlcNAc residue in the insect glycoproteins. The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man(9 to approximately 4)GlcNAc2) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc2Man3 GlcNAc2) 8.4%, and hybrid type structure (GlcNAc1 Man4GlcNAc2) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc3 Hex3HexNAc2: 3.0%), D2 (HexNAc2Hex5HexNAc2: 4.5%), and D3 (HexNAc3Hex4HexNAc2: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of beta-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a beta1-4 linkage N-acetylglucosaminyl residue.     

Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato.

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.     

First evidence for occurrence of Galbeta1-3GlcNAcbeta1-4Man unit in N-glycans of insect glycoprotein: beta1-3Gal and beta1-4GlcNAc transferases are involved in N-glycan processing of royal jelly glycoproteins

On a way of structural analysis of total N-glycans linked to glycoproteins in royal jelly (Kimura, Y. et al., Biosci. Biotechnol. Biochem., 64, 2109-2120 (2000), Kimura, M. et al., Biosci. Biotechnol. Biochem., 66, 1985-1989 (2002)), we found that some complex type N-glycans containing a beta1-3galactose residue occur on the insect glycoproteins. Up to date, it has been considered that naturally occurring insect glycoproteins do not bear the galactose-containing N-glycans, therefore, in this report we describe the structural analysis of the complex type N-glycans of royal jelly glycoproteins.By a combination of endo- and exo-glycosidase digestions, IS-MS analysis, and 1H-NMR spectroscopy, the structures of the beta1-3 galactose-containing N-glycan were identified as the following; GlcNAcbeta1-2Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc, Manalpha1-3Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc, and Manalpha1-6(Manalpha1-3)Manalpha1-6[GlcNAcbeta1-2(Galbeta1-3GlcNAcbeta1-4)Manalpha1-3]Manbeta1-4GlcNAcbeta1-4GlcNAc. To our knowledge, this is the first report showing that the Galbeta1-3GlcNAcbeta1-4Man unit occurs in N-glycans of insect glycoproteins, indicating a beta1-3 galactosyl transferase and beta1-4GlcNAc transferase (GNT-IV) are expressed in the honeybee cells.     

Changes in N-linked oligosaccharides during seed development of Ginkgo biloba

Structural changes in N-linked oligosaccharides of glycoproteins during seed development of Ginkgo biloba have been explored to discover possible endogenous substrate(s) for the Ginko endo-beta-N-acetylglucosaminidase (endo-GB; Kimura, Y., et al. (1998) Biosci. Biotechnol. Biochem., 62, 253-261), which should be involved in the production of high-mannose type free N-glycans. The structural analysis of the pyridylaminated oligosaccharides with a 2D sugar chain map, by ESI-MS/MS spectroscopy, showed that all N-glycans expressed on glycoproteins through the developmental stage of the Ginkgo seeds have the xylose-containing type (GlcNAc2 approximately 0Man3Xyl1Fuc1 approximately 0GlcNAc2) but no high-mannose type structure. Man3Xyl1Fuc1GlcNAc2, a typical plant complex type structure especially found in vacuolar glycoproteins, was a dominant structure through the seed development, while the amount of expression of GlcNAc2Man3Xyl1Fuc1GlcNAc2 and GlcNAc1Man3Xyl1Fuc1GlcNAc2 decreased as the seeds developed. The dominantly occurrence of xylose-containing type structures and the absence of the high-mannose type structures on Ginkgo glycoproteins were also shown by lectin-blotting and immunoblotting of SDS-soluble glycoproteins extracted from the developing seeds at various developmental stages. Concerning the endogenous substrates for plant endo-beta-N-acetylglucosaminidase, these results suggested that the endogenous substrates might be the dolicol-oligosaccharide intermediates or some glycopeptides with the high-mannose type N-glycan(s) derived from misfolded glycoproteins in the quality control system for newly synthesized glycoproteins.     

Biosynthesis of human-type N-glycans in heterologous systems

Insects, yeasts and plants generate widely different N-glycans, the structures of which differ significantly from those produced by mammals. The processing of the initial Glc2Man9GlcNAc2 oligosaccharide to Man8GlcNAc2 in the endoplasmic reticulum shows significant similarities among these species and with mammals, whereas very different processing events occur in the Golgi compartments. For example, yeasts can add 50 or even more Man residues to Man(8-9)GlcNAc2, whereas insect cells typically remove most or all Man residues to generate paucimannosidic Man(3-1)GlcNAc2N-glycans. Plant cells also remove Man residues to yield Man(4-5)GlcNAc2, with occasional complex GlcNAc or Gal modifications, but often add potentially allergenic beta(1,2)-linked Xyl and, together with insect cells, core alpha(1,3)-linked Fuc residues. However, genomic efforts, such as expression of exogenous glycosyltransferases, have revealed more complex processing capabilities in these hosts that are not usually observed in native cell lines. In addition, metabolic engineering efforts undertaken to modify insect, yeast and plant N-glycan processing pathways have yielded sialylated complex-type N-glycans in insect cells, and galactosylated N-glycans in yeasts and plants, indicating that cell lines can be engineered to produce mammalian-like glycoproteins of potential therapeutic value.     

Primary structure of the glycan chains of normal C 1 esterase inhibitor (C 1-INH) after NMR analysis at 400 MHz

The primary structural analysis of O- and N-linked carbohydrate chains of the C-1-esterase inhibitor purified from normal serum was carried out by 400-MHz 1H-NMR spectroscopy. C-1-esterase inhibitor protein of a molecular weight of 116,000 daltons contains 24 O-glycans: NeuAc (alpha 2-3) Gal (beta 1-3) GalNAc, 4 N-glycans: NeuAc (alpha 2-6) Gal (beta 1-4) (GlcNAc (beta 1-2) Man (alpha 1-3) [NeuAc (alpha 2-6) Gal (beta 1-4) GlcNAc (beta 1-2) Man (alpha 1-6)] Man (beta 1-4) GlcNAc (beta 1-4) GlcNAc and 2 N-glycans: NeuAc (alpha 2-3) Gal (beta 1-4) GlcNAc (beta 1-2) Man (alpha 1-3) [NeuAc (alpha 2-3) Gal (beta 1-4) GlcNAc (beta 1-2) Man (alpha 1-6)] Man (beta 1-4) GlcNAc (beta 1-4) GlcNAc. 30% of the N-glycans are fucosylated.     

Hypersialylated type-I lactosamine-containing N-glycans found in Artiodactyla sera are potential xenoantigens

There is increasing interest in biologics, i.e. human-originated biological pharmaceutics. Most of the protein drugs developed so far, such as immunoglobulins and erythropoietin, are secreted glycoproteins; as a result, any non-human-type glycans, such as αGal and NeuGc, derived from animal cells and sera must be removed to circumvent undesirable immunogenic reactions. In this study, we made an extensive search for potential xenoantigenic glycans among a panel of mammalian sera. As a result, sera belonging to the order Artiodactyla, i.e. bovine, lamb and goat sera, were found to contain substantial amounts of hypersialylated biantennary glycans closely associated with a type-I lactosamine structure containing a unique tetrasaccharide, Siaα2-3Galβ1-3(Siaα2-6)GlcNAc. In all three Artiodactyla sera, the most abundant structure was Siaα2-3Galβ1-3(Siaα2-6)GlcNAcβ1-2Manα1-3[Siaα2-6Galβ1-4GlcNAcβ1-2Manα1-6]Manβ1-4GlcNAcβ1-4GlcNAc. A dually hypersialylated biantennary structure, Siaα2-3Galβ1-3(Siaα2-6)GlcNAcβ1-2Manα1-3[Siaα2-3Galβ1-3(Siaα2-6)GlcNAcβ1-2Manα1-6]Manβ1-4GlcNAcβ1-4GlcNAc, was also abundant (10%) in bovine serum. The amount of hypersialylated glycans among total sialylated glycans was 46, 26 and 23% in bovine, lamb and goat sera, respectively. On the other hand, such structures could not be detected in the sera of other animals including human. The biological functions and the immunogenicity of the hypersialylated glycans in these animals remain to be elucidated; however, it is worth noting that glycoproteins biosynthesized from Artiodactyla cells and those contaminated with bovine serum might enhance undesirable antigenicity in human patients.     

Engineering the yeast Yarrowia lipolytica for the production of therapeutic proteins homogeneously glycosylated with Man8GlcNAc2 and Man5GlcNAc2

ABSTRACT: BACKGROUND: Protein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective. Yarrowia lipolytica is a suitable host that is generally regarded as safe (GRAS). Yeasts, however, modify their glycoproteins with heterogeneous glycans containing mainly mannoses, which complicates downstream processing and often interferes with protein function in man. Our aim was to glyco-engineer Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation. RESULTS: We engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man8GlcNAc2 or Man5GlcNAc2. First, we inactivated the yeast-specific Golgi alpha-1,6-mannosyltransferases YlOch1p and YlMnn9p; the former inactivation yielded a strain producing homogeneous Man8GlcNAc2 glycoproteins. We tested this strain by expressing glucocerebrosidase and found that the hypermannosylation-related heterogeneity was eliminated. Furthermore, detailed analysis of N-glycans showed that YlOch1p and YlMnn9p, despite some initial uncertainty about their function, are most likely the alpha-1,6-mannosyltransferases responsible for the addition of the first and second mannose residue, respectively, to the glycan backbone. Second, introduction of an ER-retained alpha-1,2-mannosidase yielded a strain producing proteins homogeneously glycosylated with Man5GlcNAc2. The use of the endogenous LIP2pre signal sequence and codon optimization greatly improved the efficiency of this enzyme. CONCLUSIONS: We generated a Y. lipolytica expression platform for the production of heterologous glycoproteins that are homogenously glycosylated with either Man8GlcNAc2 or Man5GlcNAc2 N-glycans. This platform expands the utility of Y. lipolytica as a heterologous expression host and makes it possible to produce glycoproteins with homogeneously glycosylated N-glycans of the human high-mannose-type, which greatly broadens the application scope of these glycoproteins.     

The oligosaccharides of the glycoprotein pheromone of Volvox carteri f. nagariensis Iyengar (Chlorophyceae)

The sexuality-inducing glycoprotein of Volvox carteri f. nagariensis was purified from supernatants of disintegrated sperm packets of the male strain IPS-22 and separated by reverse-phase HPLC into several isoforms which differ in the degree of O-glycosylation. Total chemical deglycosylation with trifluoromethanesulphonic acid yields the biologically inactive core protein of 22.5 kDa. This core protein possesses three putative binding sites for N-glycans which are clustered in the middle of the polypeptide chain. The N-glycosidically bound oligosaccharides were obtained by glycopeptidase F digestion and were shown by a combination of exoglycosidase digestion, gaschromatographic sugar analysis and two-dimensional HPLC separation to possess the following definite structures: (A) Man beta 1-4GlcNAc beta 1-4GlcNAc; (B) (Man alpha)3 Man beta 1-4GlcNAc beta 1-4GlcNAc Xyl beta; (C) (Man alpha)2 Man beta 1-4GlcNAc beta 1-4GlcNAc; (D) (Man)2Xyl(GlcNAc)2. Xyl beta Two of the three N-glycosidic binding sites carry one B and one D glycan. The A and C glycans are shared by the third N-glycosylation site. The O-glycosidic sugars, which make up 50% of the total carbohydrate, are short (up to three sugar residues) chains composed of Ara, Gal and Xyl and are exclusively bound to Thr residues.     

Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing

Synthesis of the N-linked oligosaccharides of Saccharomyces cerevisiae glycoproteins has been studied in vivo by labeling with [2-3H]mannose and gel filtration analysis of the products released by endoglycosidase H. Both small oligosaccharides, Man8-14GlcNAc, and larger products, Man greater than 20GlcNAc, were labeled. The kinetics of continuous and pulse-chase labeling demonstrated that Glc3Man9GlcNAc2, the initial product transferred to protein, was rapidly (t1/2 congruent to 3 min) trimmed to Man8GlcNAc2 and then more slowly (t1/2 = 10-20 min) elongated to larger oligosaccharides. No oligosaccharides smaller than Man8GlcNAc2 were evident with either labeling procedure. In confirmation of the trimming reaction observed in vivo, 3H-labeled Man9-N-acetylglucosaminitol from bovine thyroglobulin and [14C]Man9GlcNAc2 from yeast oligosaccharide-lipid were converted in vitro by broken yeast cells to 3H-labeled Man8-N-acetylglucosaminitol and [14C]Man8GlcNAc2. Man8GlcNAc and Man9GlcNAc from yeast invertase and from bovine thyroglobulin were purified by gel filtration and examined by high field 1H-NMR analysis. Invertase Man8GlcNAc (B) and Man9GlcNAc (C) were homogeneous compounds, which differed from the Man9GlcNAc (A) of thyroglobulin by the absence of a specific terminal alpha 1,2-linked mannose residue. The Man9GlcNAc of invertase (C) had an additional terminal alpha 1,6-linked mannose and appeared identical in structure with that isolated from yeast containing the mnn1 and mnn2 mutations (Cohen, R. E., Zhang, W.-j., and Ballou, C. E. (1982) J. Biol. Chem. 257, 5730-5737). It is concluded that Man8GlcNAc2, formed by removal of glucose and a single mannose from Glc3Man9GlcNAc2, is the ultimate product of trimming and the minimal precursor for elongation of the oligosaccharides on yeast glycoproteins. The results suggest that removal of a particular terminal alpha 1,2-linked mannose from Man9GlcNAc2 by a highly specific alpha-mannosidase exposes the nascent Man-alpha 1,6-Man backbone for elongation with additional alpha 1,6-linked mannose residues, according to the following scheme: (formula, see text).     

Glycoform analysis of Japanese cedar pollen allergen, Cry j 1

In our previous study (Y. Kimura et al., Biosci. Biotechnol. Biochem., 69, 137-144 (2005)), we found that plant complex type N-glycans harboring Lewis a epitope are linked to the mountain cedar pollen allergen Jun a 1. Jun a 1 is a glycoprotein highly homologous with Japanese cedar pollen glycoallergen, Cry j 1. Although it has been found that some plant complex type N-glycans are linked to Cry j 1, the occurrence of Lewis a epitope in the N-glycan moiety has not been proved yet. Hence, we reinvestigated the glycoform of the pollen allergen to find whether the Lewis a epitope(s) occur in the N-glycan moiety of Cry j 1. From the cedar pollen glycoallergen, the N-glycans were liberated by hydrazinolysis and the resulting sugar chains were N-acetylated and then coupled with 2-aminopyridine. Three pyridylaminated sugar chains were purified by reversed-phase HPLC and size-fractionation HPLC. The structures were analyzed by a combination of exo- and endo-glycosidase digestions, sugar chain mapping, and electrospray ionization mass spectrometry (ESI-MS). Structural analysis clearly indicated that Lewis a epitope (Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-), instead of the Galbeta1-4(Fucalpha1-6)GlcNAc, occurs in the N-glycans of Cry j 1.