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.     

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.     

Evidence for new beta1-3 galactosyltransferase activity involved in biosynthesis of unusual N-glycan harboring T-antigen in Apis mellifera

In a previous study (Y. Kimura et al., Biosci. Biotechnol. Biochem., 70, 2583-2587, 2006), we found that new complex type N-glycans harboring Thomsen-Friedenreich antigen (Galbeta1-3GalNAc) unit occur on royal jelly glycoproteins, suggesting the involvement of a new beta1-3galactosyltransferase in the synthesis of the unusual complex type N-glycans. So far, such beta1-3galactosyltransferase activity, which can transfer galactosyl residues with the beta1-3 linkage to beta1-4 GalNAc residues in N-glycan, has not been found among any eucaryotic cells. But using GalNAc(2)GlcNAc(2)Man(3)GlcNAc(2)-PA as acceptor N-glycan, we detected the beta1-3 galactosyltransferase activity in membrane fraction prepared from honeybee cephalic portions. This result indicates that honeybee expresses a unique beta1-3 galactosyltransferase involved in biosynthesis of the unusual N-glycan containing a tumor related antigen in the hypopharyngeal gland.     

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.     

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.     

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.     

N-glycan structures of pigeon IgG: a major serum glycoprotein containing Galalpha1-4 Gal termini.

We had shown previously that all major glycoproteins of pigeon egg white contain Galalpha1-4Gal epitopes (Suzuki, N., Khoo, K. H., Chen, H. C., Johnson, J. R., and Lee, Y. C. (2001) J. Biol. Chem. 276, 23221-23229). We now report that Galalpha1-4Gal-bearing glycoproteins are also present in pigeon serum, lymphocytes, and liver, as probed by Western blot with Griffonia simplicifolia-I lectin (specific for terminal alpha-Gal) and anti-P1 (specific for Galalpha1-4Galbeta1-4GlcNAcbeta1-) monoclonal antibody. One of the major glycoproteins from pigeon plasma was identified as IgG (also known as IgY), which has Galalpha1-4Gal in its heavy chains. High pressure liquid chromatography, mass spectrometric (MS), and MS/MS analyses revealed that N-glycans of pigeon serum IgG included (i) high mannose-type (33.3%), (ii) disialylated biantennary complex-type (19.2%), and (iii) alpha-galactosylated complex-type N-glycans (47.5%). Bi- and tri-antennary oligosaccharides with bisecting GlcNAc and alpha1-6 Fuc on the Asn-linked GlcNAc were abundant among N-glycans possessing terminal Galalpha1-4Gal sequences. Moreover, MS/MS analysis identified Galalpha1-4Galbeta1-4Galbeta1-4GlcNAc branch terminals, which are not found in pigeon egg white glycoproteins. An additional interesting aspect is that about two-thirds of high mannose-type N-glycans from pigeon IgG were monoglucosylated. Comparison of the N-glycan structures with chicken and quail IgG indicated that the presence of high mannose-type oligosaccharides may be a characteristic of these avian IgG.     

Trypanosoma brucei glycoproteins contain novel giant poly-N-acetyllactosamine carbohydrate chains

The flagellar pocket of the bloodstream form of the African sleeping sickness parasite Trypanosoma brucei contains material that binds the beta-d-galactose-specific lectin ricin (Brickman, M. J., and Balber, A. E. (1990) J. Protozool. 37, 219-224). Glycoproteins were solubilized from bloodstream form T. brucei cells in 8 M urea and 3% SDS and purified by ricin affinity chromatography. Essentially all binding of ricin to these glycoproteins was abrogated by treatment with peptide N-glycosidase, showing that the ricin ligands are attached to glycoproteins via N-glycosidic linkages to asparagine residues. Glycans released by peptide N-glycosidase were resolved by Bio-Gel P-4 gel filtration into two fractions: a low molecular mass mannose-rich fraction and a high molecular mass galactose and N-acetylglucosamine-rich fraction. The latter fraction was further separated by high pH anion exchange chromatography and analyzed by gas chromatography mass spectrometry, one- and two-dimensional NMR, electrospray mass spectrometry, and methylation linkage analysis. The high molecular mass ricin-binding N-glycans are based on a conventional Manalpha1-3(Manalpha1-6)Manbeta1-4-GlcNAcbeta1-4GlcNAc core structure and contain poly-N-acetyllactosamine chains. A significant proportion of these structures are extremely large and of unusual structure. They contain an average of 54 N-acetyllactosamine (Galbeta1-4GlcNAc) repeats per glycan, linked mostly by -4GlcNAcbeta1-6Galbeta1-interrepeat linkages, with an average of one -4GlcNAcbeta1-3(-4GlcNAcbeta1-6)Galbeta1- branch point in every six repeats. These structures, which also bind tomato lectin, are twice the size reported for the largest mammalian poly-N-acetyllactosamine N-linked glycans and also differ in their preponderance of -4GlcNAcbeta1-6Galbeta1- over -4GlcNacbeta1-3Galbeta1- interrepeat linkages. Molecular modeling suggests that -4GlcNAcbeta1-6Galbeta1- interrepeat linkages produce relatively compact structures that may give these giant N-linked glycans unique physicochemical properties. Fluorescence microscopy using fluorescein isothiocyanatericin indicates that ricin ligands are located mainly in the flagellar pocket and in the endosomal/lysosomal system of the trypanosome.     

Novel poly-GalNAcbeta1-4GlcNAc (LacdiNAc) and fucosylated poly-LacdiNAc N-glycans from mammalian cells expressing beta1,4-N-acetylgalactosaminyltransferase and alpha1,3-fucosyltransferase

Glycans containing the GalNAcbeta1-4GlcNAc (LacdiNAc or LDN) motif are expressed by many invertebrates, but this motif also occurs in vertebrates and is found on several mammalian glycoprotein hormones. This motif contrasts with the more commonly occurring Galbeta1-4GlcNAc (LacNAc or LN) motif. To better understand LDN biosynthesis and regulation, we stably expressed the cDNA encoding the Caenorhabditis elegans beta1,4-N-acetylgalactosaminyltransferase (GalNAcT), which generates LDN in vitro, in Chinese hamster ovary (CHO) Lec8 cells, to establish L8-GalNAcT CHO cells. The glycan structures from these cells were determined by mass spectrometry and linkage analysis. The L8-GalNAcT cell line produces complex-type N-glycans quantitatively bearing LDN structures on their antennae. Unexpectedly, most of these complex-type N-glycans contain novel "poly-LDN" structures consisting of repeating LDN motifs (-3GalNAcbeta1-4GlcNAcbeta1-)n. These novel structures are in contrast to the well known poly-LN structures consisting of repeating LN motifs (-3Galbeta1-4GlcNAcbeta1-)n. We also stably expressed human alpha1,3-fucosyltransferase IX in the L8-GalNAcT cells to establish a new cell line, L8-GalNAcT-FucT. These cells produce complex-type N-glycans with alpha1,3-fucosylated LDN (LDNF) GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-R as well as novel "poly-LDNF" structures (-3GalNAcbeta1-4(Fucalpha 1-3)GlcNAcbeta1-)n. The ability of these cell lines to generate glycoprotein hormones with LDN-containing N-glycans was studied by expressing a recombinant form of the common alpha-subunit in L8-GalNAcT cells. The alpha-subunit N-glycans carried LDN structures, which were further modified by co-expression of the human GalNAc 4-sulfotransferase I, which generates SO4-4GalNAcbeta1-4GlcNAc-R. Thus, the generation of these stable mammalian cells will facilitate future studies on the biological activities and properties of LDN-related structures in glycoproteins.     

Structural analysis of murine zona pellucida glycans. Evidence for the expression of core 2-type O-glycans and the Sd(a) antigen

Murine sperm initiate fertilization by binding to specific oligosaccharides linked to the zona pellucida, the specialized matrix coating the egg. Biophysical analyses have revealed the presence of both high mannose and complex-type N-glycans in murine zona pellucida. The predominant high mannose-type glycan had the composition Man(5)GlcNAc(2), but larger oligosaccharides of this type were also detected. Biantennary, triantennary, and tetraantennary complex-type N-glycans were found to be terminated with the following antennae: Galbeta1-4GlcNAc, NeuAcalpha2-3Galbeta1-4GlcNAc, NeuGcalpha2-3Galbeta1-4GlcNAc, the Sd(a) antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc, NeuGcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc), and terminal GlcNAc. Polylactosamine-type sequence was also detected on a subset of the antennae. Analysis of the O-glycans indicated that the majority were core 2-type (Galbeta1-4GlcNAcbeta1-6[Galbeta1-3]GalNAc). The beta1-6-linked branches attached to these O-glycans were terminated with the same sequences as the N-glycans, except for terminal GlcNAc. Glycans bearing Galbeta1-4GlcNAcbeta1-6 branches have previously been suggested to mediate initial murine gamete binding. Oligosaccharides terminated with GalNAcbeta1-4Gal have been implicated in the secondary binding interaction that occurs following the acrosome reaction. The significant implications of these observations are discussed.     

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.     

Alteration of brain type N-glycans in neurological mutant mouse brain

We have previously detected two brain-specific and development-dependent N-glycans [H. Shimizu, K. Ochiai, K. Ikenaka, K. Mikoshiba, and S. Hase (1993) J. Biochem. 114, 334-338]. In the present study we attempted to analyze specific N-glycans detected in neurological mutant mice. N-glycans in cerebrum and cerebellum obtained from 3-week-old neurological mutant mice (jimpy, staggerer, and shiverer) were compared with those obtained from normal mice. N-glycans liberated from the cerebrum and cerebellum by hydrazinolysis-N-acetylation were pyridylaminated, and pyridylamino derivatives of N-glycans thus obtained were separated into neutral and five acidic fractions by anion exchange chromatography. PA-N-glycans in each fraction were compared with those obtained from normal mice by reversed-phase HPLC, and the following results were obtained. The ratio of the two brain-type N-glycans, Manalpha1-3(GlcNAcbeta1-2Manalpha1-6)(GlcNAcbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc (BA-1) to GlcNAcbetaManalpha1-3(GlcNAcbeta1-2Manalpha1-6)(GlcNAcbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fuca1-6)GlcNAc (BA-2), was higher in staggerer mice than other mutant mice and normal mice. Sia-Gal-BA-2, triantennary N-glycans, and bisected biantennary N-glycans were found in the cerebellum of shiverer and staggerer mice but not in normal or jimpy mice. High-mannose type N-glycans were not altered in mutant mice brains. The amounts of disialylbiantennary N-glycans and disialylfucosylbiantennary N-glycans were lower in jimpy mouse cerebellum than in normal mouse cerebellum, but were higher in shiverer mouse. Some alterations of N-glycans specific to mutations were successfully identified, suggesting that expression of component(s) of the N-glycan biosynthetic pathway was specifically affected in neurological mutations.     

Molecular cloning and characterization of the Caenorhabditis elegans alpha1,3-fucosyltransferase family

The genome of Caenorhabditis elegans encodes five genes with homology to known alpha1,3 fucosyltransferases (alpha1,3FTs), but their expression and functions are poorly understood. Here we report the molecular cloning and characterization of these C. elegans alpha1,3FTs (CEFT-1 through -5). The open-reading frame for each enzyme predicts a type II transmembrane protein and multiple potential N-glycosylation sites. We prepared recombinant epitope-tagged forms of each CEFT and found that they had unusual acceptor specificity, cation requirements, and temperature sensitivity. CEFT-1 acted on the N-glycan pentasaccharide core acceptor to generate Manalpha1-3(Manalpha1-6)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-Asn. In contrast, CEFT-2 did not act on the pentasaccharide acceptor, but instead utilized a LacdiNAc acceptor to generate GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc, which is a novel activity. CEFT-3 utilized a LacNAc acceptor to generate Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc without requiring cations. CEFT-4 was similar to CEFT-3, but its activity was enhanced by some divalent cations. Recombinant CEFT-5 was well expressed, but did not act on available acceptors. Each CEFT was optimally active at room temperature and rapidly lost activity at 37 degrees C. Promoter analysis showed that CEFT-1 is expressed in C. elegans eggs and adults, but its expression was restricted to a few neuronal cells at the head and tail. We prepared deletion mutants for each enzyme for phenotypic analysis. While loss of CEFT-1 correlated with loss of pentasaccharide core activity and core alpha1,3-fucosylated glycans in worms, loss of other enzymes did not correlate with any phenotypic changes. These results suggest that each of the alpha1,3FTs in C. elegans has unique specificity and expression patterns.     

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.     

Structural characteristics of the N-glycans of two isoforms of prostate-specific antigens purified from human seminal fluid

Prostate-specific antigen (PSA) is a glycosylated chymotrypsin-like serine protease and is found mainly in prostatic tissue and seminal fluid. We purified two forms of PSA (PSA-A and PSA-B) from human seminal fluid with pI values of approx. 7.2 and approx. 6.9, respectively. To characterize the N-glycans of the two isoforms, the sugar chains were liberated by hydrazinolysis followed by N-acetylation, and derivatized with 2-aminobenzamide. Both PSA-A and PSA-B contained mono- and disialylated sugar chains, although PSA-B had a much higher content of the latter. After removal of sialic acid residues by sialidase digestion, mono- and biantennary N-glycans and three outer chain moieties (Galbeta1-4GlcNAcbeta1-, GlcNAcbeta1-, GalNAcbeta1-4GlcNAcbeta1-) were found in both samples. However, the ratios of each N-glycan were different. These results indicate that PSA-A and PSA-B differ not only in their sialic acid contents, but also in their outer chain features.     

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.     

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.     

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.     

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.     

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.