Structural studies of two ovalbumin glycopeptides in relation to the endo-beta-N-acetylglucosaminidase specificity.

Heterogeneities of the two ovalbumin glycopeptides, (Man)5(GlcNAc)2Asn and (Man)6(GlcNAc)2Asn, were revealed by borate paper electrophoresis of oligosaccharide alcohols obtained from the glycopeptides by endo-beta-N-acetylglucosaminidase H digestion and NaB3H4 reduction. The structures of the major components of the oligosaccharides were determined by the combination of methylation analysis, acetolysis, and alpha-mannosidase digestion. Based on the results, the whole structures of the major components of (Man)5(GlcNAc)2Asn and (Man)6(GlcNAc)2Asn were elucidated as Manalpha1 leads to 6[Manalpha1 leads to 3]-Manalpha1 leads to 6[Manalpha1 leads to 3[Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc leads to Asn and Manalpha1 leads to 6[Manalpha1 leads to 3]Manalpha1 leads to 6[Manalpha1 leads to 2Manalpha1 leads to 3]Manbeta1 leads to 4GlcNAcbeta1 leads to GlcNAc leads to Asn, respectively. Since endo-beta-N-acetylglucosamini dase D hydrolyzes (Man)5(GlcNAc)2Asn but not (Man)6(GlcNAc)2Asn, the presence of the unsubstituted alpha-mannosyl residue linked at the C-3 position of the terminal mannose of Manbeta1 leads to 4GlcNAcbeta1 leads to 4 GlcNAcAsn core must be essential for the action of the enzyme.     

Studies on the hydrazinolysis of glycoproteins. Core structures of oligosaccharides obtained from Porcine thyroglobulin and pineapple stem bromelain.

Hydrazinolysis of porcine thyroglobulin glycopeptides and of pineapple stem bromelain [EC 3.4.22.4] permitted the isolation of almost intact carbohydrate chains of these glycoproteins. On the basis of permethylation analyses of the released oligosaccharides after reduction with NaBH4, the core structures of Unit A-type and Unit B-type carbohydrate chains of porcine thyroglobulin were deduced to be Manalpha1 leads to 6[Manalpha1 leads to 3]Manbeta1 leads to 4GlcNAcbeta1 leads to 4[Ralpha1 leads to 6]GlcNAc leads to Asn (Unit A-type, R=H; Unit B-type, R=Fuc), and that of bromelain was found to be Manalpha1 leads to 6[R'1 leads to 2]Manbeta1 leads to 4GlcNAcbeta1 leads to 4[R1 leads to 3]GlcNAc leads to Asn (R'=Xylbeta and R=Fucalpha, or R'=Fucalpha and R=Xylbeta). From these results, it appears that the hydrazinolysis method is applicable to wide variety of glycoproteins which have an N-glycosylamine linkage between the carbohydrate and peptide moieties, regardless of the type of linkage to the most proximal N-acetylglucosamine residue which is bound to asparagine.     

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.     

Structural characterization of N-glycans of cauxin by MALDI-TOF mass spectrometry and nano LC-ESI-mass spectrometry

Cauxin is a carboxylesterase-like glycoprotein excreted as a major component of cat urine. Cauxin contains four putative N-glycosylation sites. We characterized the structure of an N-linked oligosaccharide of cauxin using nano liquid chromatography (LC)-electrospray ionization (ESI) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) and MS/MS, and high-performance liquid chromatography (HPLC) with an octadecylsilica (ODS) column. The structure of the N-linked oligosaccharide of cauxin attached to (83)Asn was a bisecting complex type, Galbeta1-4GlcNAcbeta1-2Manalpha1-3(Galbeta1-4GlcNAcbeta1-2Manalpha1-6)(GlcNAcbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc.     

Processing pathway deduced from the structures of N-glycans in Carica papaya

A processing The processing pathway of N-glycans in Carica papaya was deduced from the structures of N-glycans. The N-glycans were liberated by hydrazinolysis followed by N-acetylation. Their reducing-end sugar residues were tagged with 2-aminopyridine and the pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Eleven PA-sugar chains were found, and their structures were analyzed by two-dimensional sugar mapping combined with partial acid hydrolysis and exoglycosidase digestion. The structures of the N-glycans were of the highmannose types with xylose and fucose; however, among them two new N-glycans, Manalpha1-6(Manalpha1-3)Manalpha1-6(Xylbeta1-2)+ ++Manbeta1-4GlcNAcbeta1- 4(Fucalpha1-3)GlcNAc and Manalpha1-3Manalpha1-6(Xylbeta1-2)Manbeta1-4G lcNAcbeta1-4(Fucalpha1-3 )GlcNAc, were found. Judging from these structures together with Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) (Xylbeta1-2)Manbeta1- 4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc reported previously [Shimazaki, A., Makino, Y., Omichi, K., Odani, S., and Hase, S. (1999) J. Biochem. 125, 560- 565], a processing pathway for N-glycans in C. papaya is inferred in which the activity of Golgi alpha-mannosidase II is incomplete.     

N-Glycan structures of squid rhodopsin.

To determine the glycoforms of squid rhodopsin, N-glycans were released by glycoamidase A digestion, reductively aminated with 2-aminopyridine, and then subjected to 2D HPLC analysis [Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y. & Tomiya, N. (1995) Anal. Biochem.226, 139-146]. The major glycans of squid rhodopsin were shown to possess the alpha1-3 and alpha1-6 difucosylated innermost GlcNAc residue found in glycoproteins produced by insects and helminths. By combined use of 2D HPLC, electrospray ionization-mass spectrometry and permethylation and gas chromatography-electron ionization mass spectrometry analyses, it was revealed that most (85%) of the N-glycans exhibit the novel structure Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4(Galbeta1-4Fucalpha1-6)(Fucalpha1-3)GlcNAc.     

Asparaginyl glycopeptides with a low mannose content are hydrolyzed by endo-beta-N-acetylglucosaminidase H

Substrates susceptible to endo-beta-N-acetylglucosaminidase H were reduced in size through alpha-mannosidase treatment and periodate oxidation to yield the following compounds: (Man)4(GlcNAc)2Asn, [Manalpha 1 leads to 6Manalpha 1 leads to 6(Manalpha 1 leads to 3)Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNACAsn]; (Man)3(GlcNAc)2Asn, [Manalpha 1 leads to 3Man-alpha 1 leads to 6Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn]; (Man)2(GlcNAc)2Asn, [Manalpha 1 leads to 6Manbeta1 leads to 4GlcNAcbeta 1 leads to 4BlcNAcAsm]. Comparison of the relative rates of hydrolysis of these compounds with (Man)5(GlcNAc)2-Asn, the most active substrate to date for the endoglycosidase, revealed (Man)4(GlcNAc)2Asn to be hydrolyzed faster than (Man)5(GlcNAc)2Asn and (Man)3-(GlcNAc)2Asn to be equal to or slightly better than (Man)5(GlcNAc)2Asn as a substrate. (Man)2(GlcNAc)2-Asn was completely hydrolyzed but at a rate that was about 10(4) slower than (Man)5(GlcNAc)2Asn, which is comparable to that for (Man)3(GlcNAc)2Asn(aa)x [Manalpha 1 leads to 6(Manalpha 1 leads to 3)Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn(aa)x], obtained from immunoglobulin M. (Man)1(GlcNAc)2Asn, [Manbeta 1 leads to 4GlcNAcbeta 1 leads to 4GlcNAcAsn] was hydrolyzed at a 100-fold slower rate than the latter glycopeptide. The effective range of endo-beta-N-acetylglucosaminidase H has thus been extended to compounds containing as few as 2 mannosyl residues.     

Poly-N-acetyllactosamine synthesis in branched N-glycans is controlled by complemental branch specificity of I-extension enzyme and beta1,4-galactosyltransferase I.

Poly-N-acetyllactosamine is a unique carbohydrate that can carry various functional oligosaccharides, such as sialyl Lewis X. It has been shown that the amount of poly-N-acetyllactosamine is increased in N-glycans, when they contain Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->4GlcNAcbeta1 -->2)Manalpha1-->6 branched structure. To determine how this increased synthesis of poly-N-acetyllactosamines takes place, the branched acceptor was incubated with a mixture of i-extension enzyme (iGnT) and beta1, 4galactosyltransferase I (beta4Gal-TI). First, N-acetyllactosamine repeats were more readily added to the branched acceptor than the summation of poly-N-acetyllactosamines formed individually on each unbranched acceptor. Surprisingly, poly-N-acetyllactosamine was more efficiently formed on Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chain than in Galbeta1-->4GlcNAcbeta1-->6Manalpha-->R, due to preferential action of iGnT on Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chain. On the other hand, galactosylation was much more efficient on beta1,6-linked GlcNAc than beta1,2-linked GlcNAc, preferentially forming Galbeta1-->4GlcNAcbeta1-->6(GlcNAcbeta1-->2)Manalph a1-->6Manbeta -->R. Starting with this preformed acceptor, N-acetyllactosamine repeats were added almost equally to Galbeta1-->4GlcNAcbeta1-->6Manalpha-->R and Galbeta1-->4GlcNAcbeta1-->2Manalpha-->R side chains. Taken together, these results indicate that the complemental branch specificity of iGnT and beta4Gal-TI leads to efficient and equal addition of N-acetyllactosamine repeats on both side chains of GlcNAcbeta1-->6(GlcNAcbeta1-->2)Manalpha1-->6Manbet a-->R structure, which is consistent with the structures found in nature. The results also suggest that the addition of Galbeta1-->4GlcNAcbeta1-->6 side chain on Galbeta1-->4GlcNAcbeta1-->2Man-->R side chain converts the acceptor to one that is much more favorable for iGnT and beta4Gal-TI.     

Deletion of the glucosidase II gene in Trypanosoma brucei reveals novel N-glycosylation mechanisms in the biosynthesis of variant surface glycoprotein

The trypanosomatids are generally aberrant in their protein N-glycosylation pathways. However, protein N-glycosylation in the African trypanosome Trypanosoma brucei, etiological agent of human African sleeping sickness, is not well understood. Here, we describe the creation of a bloodstream-form T. brucei mutant that is deficient in the endoplasmic reticulum enzyme glucosidase II. Characterization of the variant surface glycoprotein, the main glycoprotein synthesized by the parasite with two N-glycosylation sites, revealed unexpected changes in the N-glycosylation of this molecule. Structural characterization by mass spectrometry, nuclear magnetic resonance spectroscopy, and chemical and enzymatic treatments revealed that one of the two glycosylation sites was occupied by conventional oligomannose structures, whereas the other accumulated unusual structures in the form of Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, and Glcalpha1-3Manalpha1-2Manalpha1-2Manalpha1-3(Galbeta1-4GlcNAcbeta1-2Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc. The possibility that these structures might arise from Glc1Man9GlcNAc2 by unusually rapid alpha-mannosidase processing was ruled out using a mixture of alpha-mannosidase inhibitors. The results suggest that bloodstream-form T. brucei can transfer both Man9GlcNAc2 and Man5GlcNAc2 to the variant surface glycoprotein in a site-specific manner and that, unlike organisms that transfer exclusively Glc3Man9GlcNAc2, the T. brucei UDP-Glc: glycoprotein glucosyltransferase and glucosidase II enzymes can use Man5GlcNAc2 and Glc1Man5GlcNAc2, respectively, as their substrates. The ability to transfer Man5GlcNAc2 structures to N-glycosylation sites destined to become Man(4-3)GlcNAc2 or complex structures may have evolved as a mechanism to conserve dolichol-phosphate-mannose donors for glycosylphosphatidylinositol anchor biosynthesis and points to fundamental differences in the specificities of host and parasite glycosyltransferases that initiate the synthesis of complex N-glycans.     

Human IgG1 expression in silkworm larval hemolymph using BmNPV bacmids and its N-linked glycan structure

A Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expressing heavy and light chains of human 29IJ6 IgG was constructed and used to secrete recombinant antibody into silkworm larval hemolymph. Fifth instar silkworm larvae were reared and injected into the dorsum of the larvae with recombinant cysteine protease- and chitinase-deficient BmNPV (BmNPV-CP(-)-Chi(-)) bacmid/29IJ6 IgG and harvested after approximately 6 days. The total yield of recombinant 29IJ6 IgG was 36 microg/larvae, which is equivalent to 8 mg/kg of larvae. The recombinant antibody was purified to homogeneity using a HiTrap rProtein A FF column with a purification yield of 83.1%. The purified protein was identified by Western blot and ELISA experiments. The N-linked glycan structure of the purified protein was determined by the HPLC mapping method. The N-glycans of the 29IJ6 IgG glycoprotein produced in, and secreted by the silkworm larvae were composed exclusively of two kinds of paucimannose-type oligosaccharides, Manalpha1-6Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc and Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc.     

Conversion of brain-specific complex type sugar chains by N-acetyl-beta-D-hexosaminidase B

The N-linked sugar chains, GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(Manalpha1++ +-3)Manbeta1-4GlcNAcb eta1-4(Fucalpha1-6)GlcNAc (BA-1) and GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-4)(GlcNAcbeta1 -2Manalpha1-3)Manb eta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc (BA-2), were recently found to be linked to membrane proteins of mouse brain in a development-dependent manner [S. Nakakita, S. Natsuka, K. Ikenaka, and S. Hase, J. Biochem. 123, 1164-1168 (1998)]. The GlcNAc residue linked to the Manalpha1-3 branch of BA-2 is lacking in BA-1 and the removal of this GlcNAc residue is not part of the usual biosynthetic pathway for N-linked sugar chains, suggesting the existence of an N-acetyl-beta-D-hexosaminidase. Using pyridylaminated BA-2 (BA-2-PA) as a substrate the activity of this enzyme was found in all four subcellular fractions obtained. The activity was much greater in the cerebrum than in the cerebellum. To further identify the N-acetyl-beta-D-hexosaminidase, BA-1 and BA-2 in brain tissues of Hex gene-disrupted mutant mice were detected and quantified. PA-sugar chains were liberated from the cerebrum and cerebellum of the mutant mice by hydrazinolysis-N-acetylation followed by pyridylamination. PA-sugar chains were separated by anion-exchange HPLC, size-fractionation, and reversed-phase HPLC. Each peak was quantified by measuring the peaks at the elution positions of authentic BA-1-PA and BA-2-PA. BA-2-PA was detected in all the PA-sugar chain fractions prepared from Hexa, Hexb, and both Hexa and Hexb (double knockout) gene-disrupted mice, but BA-1 was not found in the fractions from Hexb gene-disrupted and double knockout mice. These results indicate that N-acetyl-beta-D-hexosaminidase B encoded by the Hexb gene hydrolyzed BA-2 to BA-1.     

Fuc-TIX: a versatile alpha1,3-fucosyltransferase with a distinct acceptor- and site-specificity profile

alpha1,3-Fucosyltransferases (Fuc-Ts) convert N-acetyllactosamine (LN, Galbeta1-4GlcNAc) to Galbeta1-4(Fucalpha1-3)GlcNAc, the Lewis x (CD15, SSEA-1) epitope, which is involved in various recognition phenomena. We describe details of the acceptor specificity of alpha1,3-fucosyltransferase IX (Fuc-TIX). The unconjugated N- and O-glycan analogs LNbeta1-2Man, LNbeta1-6Manalpha1-OMe, LNbeta1-2Manalpha1-3(LNbeta1-2Manalpha1-6)Manbeta1-4GlcNAc, and Galbeta1-3(LNbeta1-6)GalNAc reacted well in vitro with Fuc-TIX present in lysates of appropriately transfected Namalwa cells. Fuc-TIX reacted well with the reducing end LN of GlcNAcbeta1-3'LN (underscored site reacted) and GlcNAcbeta1-3'LNbeta1-3'LN (both LNs reacted), but very poorly with the reducing end LN of LNbeta1-3'LN. However, Fuc-TIX reacted significantly better with the non-reducing end LN as compared to the other LN units in the glycans LNbeta1-3'LN and LNbeta1-3'LNbeta1-3'LNbeta1-3'LN, confirming our previous data on LNbeta1-3'LNbeta1-OR. In contrast, the sialylated glycan Neu5Acalpha2-3'LNbeta1-3'LNbeta1-3'LNbeta1-3'LN was fucosylated preferentially at the two most reducing end LN units. We conclude that Fuc-TIX is a versatile alpha1,3-Fuc-T, that (1) generates distal Lewis x epitopes from many different acceptors, (2) possesses inherent ability for the biosynthesis of internal Lewis x epitopes on growing polylactosamine backbones, and (3) fucosylates the remote internal LN units of alpha2,3-sialylated i-type polylactosamines.     

350-kDa royal jelly glycoprotein (apisin), which stimulates proliferation of human monocytes, bears the beta1-3galactosylated N-glycan: analysis of the N-glycosylation site

While doing a structural analysis of minor component N-glycans linked to 350-kDa royal jelly glycoprotein (RJGP), which stimulates the proliferation of human monocytes, we found that a Galbeta1-3GlcNAcbeta1-4Man unit occurs on the insect glycoprotein. The structure of the fluorescence-labeled N-glycan was analyzed by sugar component analysis, IS-MS, and (1)H-NMR. The structural analysis showed that the 350-kDa RJGP bears Galbeta1-3GlcNAcbeta1-4(GlcNAcbeta1-2)Manalpha1-3 (Manalpha1-3Manalpha1-6)Manbeta1-4GlcNAcbeta1-4GlcNAc, suggesting this insect glycoprotein is one of the substrates for both beta1-3 galactosyl and beta1-4 N-acetylglucosamininyl transferases. To our knowledge, this is the first report that succeeded in identifying an insect glycoprotein bearing the beta1-3 galactosylated N-glycan.     

Isolation and characterization of linear polylactosamines containing one and two site-specifically positioned Lewis x determinants: WGA agarose chromatography in fractionation of mixtures generated by random, partial enzymatic alpha3-fucosylation of pure polylactosamines

We report that isomeric monofucosylhexasaccharides, Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4(Fucalpha1-3) GlcNAc, Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-3Galbeta1-4 GlcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 GlcNAc, and bifucosylhexasaccharides Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAc, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 (Fucalpha1-3)GlcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4( Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4GlcNAc can be isolated in pure form from reaction mixtures of the linear hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc with GDP-fucose and alpha1,3-fucosyltransferases of human milk. The pure isomers were characterized in several ways;1H-NMR spectroscopy, for instance, revealed distinct resonances associated with the Lewis x group [Galbeta1-4(Fucalpha1-3)GlcNAc] located at the proximal, middle, and distal positions of the polylactosamine chain. Chromatography on immobilized wheat germ agglutinin was crucial in the separation process used; the isomers carrying the fucose at the reducing end GlcNAc possessed particularly low affinities for the lectin. Isomeric monofucosyl derivatives of the pentasaccharides GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1- 4Gl cNAc and Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4G lcN Ac and the tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc were also obtained in pure form, implying that the methods used are widely applicable. The isomeric Lewis x glycans proved to be recognized in highly variable binding modes by polylactosamine-metabolizing enzymes, e.g., the midchain beta1,6-GlcNAc transferase (Lepp?nen et al., Biochemistry, 36, 13729-13735, 1997).     

Structure of a major oligosaccharide of PASII/PMP22 glycoprotein in bovine peripheral nerve myelin

The amino acid sequence of the glycopeptide obtained from bovine PASII/PMP22 protein in the PNS myelin was determined to be Gln-Asn-Cys-Ser-Thr, where the asparagine was glycosylated. To eliminate all the contaminated P(o) glycopeptides from the PASII/PMP22 glycopeptide preparation, we used a fluorescent probe, N-[2-(2-pyridylamino)ethyl]maleimide, which reacts with the cysteine of the PASII/PMP22 glycopeptides. The labeled PASII/PMP22 glycopeptides were isolated by HPLC and were digested further with glycopeptidase A. The resultant oligosaccharides were conjugated with 2-aminopyridine (PA) as a fluorescent tag. One major PA-oligosaccharide, OPPE1, was purified by HPLC. The structure of OPPE1 was elucidated by fast atom bombardment mass spectrometry and (1)H-NMR studies and comparing the derivatives of PA-OPPE1 and PA-oligosaccharides of gamma-globulin on HPLC. The structure, SO(4)-3GlcAbeta1-3Galbeta1-4GlcNAcbeta1-2Manalpha1+ ++-6(GlcNAcbeta1-4) (GlcNAcbeta1-2Manalpha1-3)Manbeta1-4GlcNAcbeta1- 4(Fucalpha1-6)GlcNAc- PA, was identical to the pyridylaminated form of the major oligosaccharide D8 of bovine P(o) previously reported.     

Tumor antigen occurs in N-glycan of royal jelly glycoproteins: honeybee cells synthesize T-antigen unit in N-glycan moiety

In our previous paper (Kimura, Y., et al., Biosci. Biotechnol. Biochem., 67, 1852-1856, 2003), we found that a complex type N-glycans containing beta1-3 galactose residue occurs on royal jelly glycoproteins. During structural analysis of minor components of royal jelly N-glycans, we found complex type N-glycans bearing both galactose and N-acetylgalactosamine residues. Detailed structural analysis of pyridylaminated oligosaccharide revealed that the newly found N-glycan had a complex type structure harboring a tumor marker (T-antigen) unit: Galbeta1-3GalNAcbeta1-4GlcNAcbeta1-2Manalpha1-6 (Galbeta1-3GalNAcbeta1-4GlcNAcbeta1-2Manalpha1-3) Manbeta1-4GlcNAcbeta1-4GlcNAc. To our knowledge, this may be the first report of the presence of the T-antigen unit in the N-glycan moiety of eucaryotic glycoproteins.     

A new sugar chain of the proteinase inhibitor from latex of Carica papaya

The structure of a sugar chain of the proteinase inhibitor from the latex of Carica papaya was studied. Sugar chains liberated on hydrazinolysis were N-acetylated, and their reducing-end residues were tagged with 2-aminopyridine. One major sugar chain was detected on size-fractionation and reversed-phase HPLC analyses. The structure of the PA-sugar chain was determined by two-dimensional sugar mapping combined with sequential exoglycosidase digestion and partial acid hydrolysis, and by 750 MHz 1H-NMR spectroscopy. The structure found was Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) (Xylbeta1-2)Manbeta1- 4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc. This sugar chain represents a new plant-type sugar chain with five mannose residues.     

Determination by electrospray mass spectrometry and 1H-NMR spectroscopy of primary structures of variously fucosylated neutral oligosaccharides based on the iso-lacto-N-octaose core.

We have isolated a nonfucosylated and three variously fucosylated neutral oligosaccharides from human milk that are based on the iso-lacto-N-octaose core. Their structures were characterized by the combined use of electrospray mass spectrometry (ES-MS) and NMR spectroscopy. The branching pattern and blood group-related Lewis determinants, together with partial sequences and linkages of these oligosaccharides, were initially elucidated by high-sensitivity ES-MS/MS analysis, and then their full structure assignment was completed by methylation analysis and 1H-NMR. Three new structures were identified. The nonfucosylated iso-lacto-N-octaose, Galbeta1-3GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-6[Galbeta1-3GlcNAcbeta1-3]Galbeta1-4Glc, has not previously been reported as an individual oligosaccharide. The monofucosylated and trifucosylated iso-lacto-N-octaose, Galbeta1-3GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-6[Galbeta1-3GlcNAcbeta1-3]Galbeta1-4Glc and Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6[Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3]Galbeta1-4Glc, both containing an internal Lex epitope, are also novel structures.     

Characterization of the oligosaccharide component of microsomal beta-glucuronidase from rat liver

The oligosaccharides of microsomal beta-glucuronidase were analysed by gel permeation and weak anion exchange chromatography following hydrazine release. N-linked glycans, constituted 80% of the total glycan pool and were mainly of the tri- and biantennary complex type with or without core and arm fucose. The major oligosaccharide, that comprised 30.6% of all the species analysed, was structurally identified by reagent array analysis method and found to be a triantennary complex structure, Galbeta1,4GlcNAcbeta1,2Manalpha1,6(3)(Galbeta1,4GlcNAcbeta1,4(Galbeta1,4GlcNAcbeta1,2) Manalpha1,3(6))Manbeta1,4GlcNAcbeta1,4 GlcNAc. O-Linked glycans comprised 20% of the total glycan pool, the major species being Galbeta1,3GalNAc. All of the N- and O-linked glycans were charged. Most of the negative charge was due to sialic acid (85.0%) with the remainder being phosphate present as phosphomonoesters (7.3%) and phosphodiesters (5%). This is the first report of O-linked carbohydrate chains in microsomal beta-glucuronidase. The presence of O-linked glycans and branched N-linked glycans in a microsomal enzyme, in relation to the current view of glycosyltransferase compartmentalization in the Golgi is discussed.