Urinary oligosaccharides of fucosidosis. Evidence of the occurrence of X-antigenic determinant in serum-type sugar chains of glycoproteins.

Urine of a fucosidosis patient contained a large amount of fucosyl oligosaccharides and fucose-rich glycopeptides. Six major oligosaccharides were purified by a combination of Bio-Gel P-2 and P-4 column chromatographies and paper chromatography. Structural studies by sequential exoglycosidase digestion and by methylation analysis revealed that their structures were as follows: Fucalpha1 leads to 6GlcNAc, Fucalpha1 leads to 2Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 2Manalpha1 leads to 3Manbeta1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to3)GlcNAcbeta1 leads to 2Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc, and Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 6Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc. In additon, the structure of a minor decasaccharide was found to be Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to [Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to]Manbeta1 leads to 4GlcNAc.     

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.     

Improved secretion of molecular chaperone‐assisted human IgG in silkworm,and no alterations in their N‐linked glycan structures

Human 29IJ6 IgG was expressed in silkworm using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system. The mean amounts of 296IJ6 IgG produced in larval hemolymph and whole pupae were 30.1 μg/larva and 78.0 μg/pupa, respectively. The use of molecular chaperones including calreticulin (CRT), calnexin (CNX), and immunoglobulin heavy chain binding protein (BiP, GRP78) improved the production of 296IJ6 IgG secretion in the larvae fivefold. The total yield of recombinant 29IJ6 IgG was 239 μg/mL when coexpressed with CRT. However, the overexpression of molecular chaperones had negative effects on secretion. The N‐linked glycans of secreted 296IJ6 IgG in silkworm hemolymph were dominated by paucimannose structures. Small amounts of GlcNAc residues linked to the Manα1,3 branch were detected. When molecular chaperones were coexpressed, the compositions of N‐linked glycans in the IgG1 produced were unchanged compared with those produced without them. This suggests that N‐glycosylation is controlled by a regulatory function in the Golgi apparatus even though the post‐translational modification of 296IJ6 IgG was assisted by the coexpression of molecular chaperones. Therefore, if the glycosylation pathways that coexpress N‐acetylglucosaminyltransferase, galactosyltransferase, and sialyltransferase could be improved, silkworm larvae might prove a useful system for producing human antibodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

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.     

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.     

Endo-beta-mannosidase, a plant enzyme acting on N-glycan: purification, molecular cloning, and characterization

Endo-beta-mannosidase is a novel endoglycosidase that hydrolyzes the Manbeta1-4GlcNAc linkage in the trimannosyl core structure of N-glycans. This enzyme was partially purified and characterized in a previous report (Sasaki, A., Yamagishi, M., Mega, T., Norioka, S., Natsuka, S., and Hase, S. (1999) J. Biochem. 125, 363-367). Here we report the purification and molecular cloning of endo-beta-mannosidase. The enzyme purified from lily flowers gave a single band on native-PAGE and three bands on SDS-PAGE with molecular masses of 42, 31, and 28 kDa. Amino acid sequence information from these three polypeptides allowed the cloning of a homologous gene, AtEBM, from Arabidopsis thaliana. AtEBM was engineered for expression in Escherichia coli, and the recombinant protein comprised a single polypeptide chain with a molecular mass of 112 kDa corresponding to the sum of molecular masses of three polypeptides of the lily enzyme. The recombinant protein hydrolyzed pyridylamino derivatives (PA) of Manalpha1-6Manbeta1-4Glc-NAcbeta1-4GlcNAc into Manalpha1-6Man and GlcNAcbeta1-4Glc-NAc-PA, showing that AtEBM is an endo-beta-mannosidase. AtEBM hydrolyzed Man(n)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA (n = 0-2) but not PA-sugar chains containing Manalpha1-3Manbeta or Xylosebeta1-2Manbeta as for the lily endo-beta-mannosidase. AtEBM belonged to the clan GH-A of glycosyl hydrolases. Site-directed mutagenesis experiments revealed that two glutamic acid residues (Glu-464 and Glu-549) conserved in this clan were critical for enzyme activity. The amino acid sequence of AtEBM has distinct differences from those of the bacterial, fungal, and animal exo-type beta-mannosidases. Indeed, AtEBM-like genes are only found in plants, indicating that endo-beta-mannosidase is a plant-specific enzyme. The role of this enzyme in the processing and/or degradation of N-glycan will be discussed.     

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.     

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.     

Silkworm expression and sugar profiling of human immune cell surface receptor, KIR2DL1

Immune cell surface receptors are directly involved in human diseases, and thus represent major drug targets. However, it is generally difficult to obtain sufficient amounts of these receptors for biochemical and structural studies because they often require posttranslational modifications, especially sugar modification. Recently, we have established a bacmid expression system for the baculovirus BmNPV, which directly infects silkworms, an attractive host for the large-scale production of recombinant sugar-modified proteins. Here we produced the human immune cell surface receptor, killer cell Ig-like receptor 2DL1 (KIR2DL1), by using the BmNPV bacmid expression system, in silkworms. By the direct injection of the bacmid DNA, the recombinant KIR2DL1 protein was efficiently expressed, secreted into body fluids, and purified by Ni²⁺ affinity column chromatography. We further optimized the expression conditions, and the final yield was 0.2 mg/larva. The sugar profiling revealed that the N-linked sugars of the purified protein comprised very few components, two paucimannose-type oligosaccharides, Manα1-6Manβ1-4GlcNAcβ1-4GlcNAc and Manα1-6Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc. This revealed that the protein product was much more homogeneous than the complex-sugar type product obtained by mammalian cell expression. The surface plasmon resonance analysis demonstrated that the purified KIR2DL1 protein exhibited specific binding to the HLA-Cw4 ligand. Moreover, the CD spectrum showed the proper secondary structure. These results clearly suggested that the silkworm expression system is quite useful for the expression of cell surface receptors that require posttranslational modifications, as well as for their structural and binding studies, due to the relatively homogeneous N-linked sugar modifications.     

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.     

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.     

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.     

Substrate specificity and molecular cloning of the lily endo-beta-mannosidase acting on N-glycan

Endo-beta-mannosidase, which hydrolyzes the Manbeta1-4GlcNAc linkage in the trimannosyl core structure of N-glycans, was recently purified to homogeneity from lily (Lilium longiflorum) flowers as a heterotrimer [Ishimizu, T., Sasaki, A., Okutani, S., Maeda, M., Yamagishi, M., and Hase, S. (2004) J. Biol. Chem. 279, 38555-38562]. Here, we describe the substrate specificity of the enzyme and cloning of its cDNA. The purified enzyme hydrolyzed pyridylaminated (PA-) Man(n)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc (n = 0-2) to Man(n)Manalpha1-6Man and GlcNAcbeta1-4GlcNAc-PA. It did not hydrolyze PA-sugar chains containing Manalpha1-3Manbeta and/or Xylbeta1-2Manbeta. The best substrate among the PA-sugar chains tested was Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA with a K(m) value of 1.2 mM. However, the enzyme displayed a marked preference for the corresponding glycopeptide, Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-peptide (K(m) value 75 microM). These results indicate that the substrate recognition by the enzyme involves the peptide portion attached to the N-glycan. Sequence information on the purified enzyme was used to clone the corresponding cDNA. The monocotyledonous lily enzyme (952 amino acids) displays 68% identity to its dicotyledonous (Arabidopsis thaliana) homologue. Our results show that the heterotrimeric enzyme is encoded by a single gene that gives rise to three polypeptides following posttranslational proteolysis. The enzyme is ubiquitously expressed, suggesting that it has a general function such as processing or degrading N-glycans.     

Co(II)-regulated substrate specificity of cytosolic alpha-mannosidase

Cytosolic neutral alpha-mannosidase is a putative catabolic enzyme that produces cytosolic free oligomannosides. Activation of the enzyme by Co(II) treatment has been reported using pyridylamino derivatives of Man(5)GlcNAc and Man(5)GlcNAc2, and p-nitrophenyl alpha-mannoside as substrates, with the Co(II)-treated enzyme releasing four alpha-mannose residues from Man(9)GlcNAc to give Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc as an end product. When Man(9)GlcNAc, which is considered to be the actual substrate in the cytosol, was used as a substrate, we found that even before treatment with Co(II) the enzyme was able to cleave a single Manalpha1-2 residue from Man(9)GlcNAc to give Manalpha1-6(Manalpha1-2Manalpha1-3)Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc as the end product. The K(m) value of the Co(II)-treated enzyme for Man(9)GlcNAc was found to be 37 microM, which is one-twelfth that of the non-treated enzyme, while the values were V(max) values were almost the same, indicating that the affinity of the substrate is higher with Co(II). These results indicate that Co(II) regulates the substrate specificity of the enzyme.     

Silkworm, Bombyx mori larvae expressed the spider silk protein through a novel Bac-to-Bac/BmNPV baculovirus

Abstract:  The silkworm has become an ideal multicellular eukaryotic model system for basic research. The major advantages of expressing foreign genes in silkworm larvae are the low cost of feeding, the extremely high levels of expression achievable compared with expression in cell lines and increased safety because the baculovirus is noninfectious to vertebrates. In this study, we used a recently developed Bombyx mori Nucleopolyhedrovirus (BmNPV) bacmid to express the spider flagelliform silk gene in silkworm larvae. The recombinant bacmid baculoviruses (rBacmid/BmNPV/Flag) were introduced into the first-day larvae of the fifth instar by subcutaneous injection. The worms presented symptoms typical of NPV infection from 72 h after injection compared with control. The haemolymph was collected from the infected larvae 120 h post-infection and the recombinant 6× His-tagged Flag protein was purified by the Ni-NTA spin kit under denaturing conditions with 8  m urea. A 37.0-kDa protein was visualized both in rBacmid/BmNPV/Flag-infected haemolymph and eluting fraction. The results showed that the Bac-to-Bac/BmNPV baculovirus expression system is an efficient tool to express the target gene in silkworm larvae, which takes only 7–10 days for generating recombinant baculovirus, compared with the traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses.     

Biochemical and functional characterization of the Tn-specific lectin from Salvia sclarea seeds.

SSL, the lectin isolated from Salvia sclarea seeds, recognizes the Tn antigen (GalNAcalpha-O-Ser/Thr), a specific marker of many human carcinomas. Two-dimensional electrophoresis, amino-acid and amino-sugar analysis, and MALDI-TOF MS showed that SSL is an acidic (pI 5.5), 60-61-kDa dimeric glycoprotein composed of apparently identical subunits linked by a single disulfide bond. The apparent molecular mass of SSL in solution determined by equilibrium sedimentation analytical ultracentrifugation was 59 +/- 9 kDa. This value did not change in the pH range 2.5-8.5, indicating that SSL does not associate into higher order structures. Tandem mass spectrometry and methylation analysis of N-glycans released from SSL by hydrazinolysis indicated that SSL possesses 2-3 glycosylation sites occupied with the typical plant glycans Manalpha1-6[(Manalpha1-3)(Xylbeta1-2)]Manbeta1-4 -GlcNAcbeta1-4(Fucalp ha1-3)GlcNAc and [(Manalpha1-3/6)(Xylbeta1-2)]Manbeta1-4-GlcNAcbeta1 -4(Fucalpha1-3)Glc NAc. The influence of adjacent Tn structures on the binding of two Tn-specific lectins (SSL and the isolectin B4 from Vicia villosa) and an anti-Tn monoclonal antibody (mAb 83D4) was evaluated using synthetic Tn glycopeptides. The binding of both lectins to the synthetic Tn glycopeptides was independent of the density of Tn structures. On the other hand, mAb 83D4 only reacted with glycopeptides displaying two or three consecutive Tn structures.     

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.     

Structural diversity of cytosolic free oligosaccharides in the human hepatoma cell line, HepG2

It is thought that free oligosaccharides in the cytosol are an outcome of quality control of glycoproteins by endoplasmic reticulum-associated degradation (ERAD). Although considerable amounts of free oligosaccharides accumulate in the cytosol, where they presumably have some function, detailed analyses of their structures have not yet been carried out. We isolated 21 oligosaccharides from the cytosolic fraction of HepG2 cells and analyzed their structures by the two-dimensional high-performance liquid chromatography (HPLC) sugar-mapping method. Sixteen novel oligosaccharides were identified in the cytosol in this study. All had a single N-acetylglucosamine at their reducing-end cores and could be expressed as (Man)n (GlcNAc)1. No free oligosaccharide with N,N'-diacetylchitobiose was detected in the cytosolic fraction of HepG2 cells. This suggested that endo-beta-N-acetylglucosaminidase was a key enzyme in the production of cytosolic free oligosaccharides. The 21 oligosaccharides were classified into three series--series 1: oligosaccharides processed from Manalpha1-2Manalpha1-6 (Manalpha1-2Manalpha1-3)Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3) Manbeta1-4GlcNAc (M9A') and Manalpha1-2Manalpha1-6(Manalpha1-3) Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc (M8A') by digestion with cytosolic alpha-mannosidase; series 2: oligosaccharides processed with Golgi alpha-mannosidases in addition to endoplasmic reticulum (ER) and cytosolic alpha-mannosidases; and series 3: glucosylated oligosaccharides produced from Glc1Man9GlcNAc1 by hydrolysis with cytosolic alpha-mannosidase. The presence of the series "2" oligosaccharides suggests that some of the misfolded glycoproteins had been processed in pre-cis-Golgi vesicles and/or the Golgi apparatus. When the cells were treated with swainsonine to inhibit cytosolic alpha-mannosidase, the amounts of M9A' and M8A' increased remarkably, suggesting that these oligosaccharides were translocated into the cytosol. Four oligosaccharides of series "2" also increased. In contrast, there were obvious reductions in Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc (M5B'), the end product from M9A' by digestion with cytosolic alpha-mannosidase, and Manalpha1-6(Manalpha1- 2Manalpha1-3)Manbeta1-4GlcNAc, derived from series "2" oligosaccharides by digestion with cytosolic alpha-mannosidase. Our data suggest that (1) some of the cytosolic oligosaccharides had been processed with Golgi alpha-mannosidases, (2) the major oligosaccharides translocated from the ER were M9A' and M8A', and (3) M5B' and Glc1M5B' were maintained at relatively high concentrations in the cytosol.     

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.