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Endo-beta-mannosidase, which hydrolyses the Manbeta1-4GlcNAc linkage of N-glycans in an endo-manner, was discovered in plants. During the course of the purification of the enzyme from lily flowers, we found a higher molecular mass form of the enzyme (designated as EBM II). EBM II was purified by column chromatography to homogeneity and its molecular composition revealed EBM II to be comprised of endo-beta-mannosidase and an associated protein. The cDNA of this associated protein encodes a protein with slight homology to the fucosidase domain of bifidus AfcA. EBM II has alpha1,2-L-fucosidase activity and acts on a fucosylated xyloglucan nonasaccharide. The amino acid sequence of this associated protein has no similarity to known plant alpha-L-fucosidases. These results show that EBM II is a novel alpha1,2-L-fucosidase and a protein complex containing endo-beta-mannosidase.

We previously reported that zebrafishalpha1-3fucosyltrasferase 1 (zFT1) was expressed in embryos at the segmentation period, and was capable of synthesizing the Lewis x epitope [Gal beta1-4(Fuc alpha1-3)GlcNAc] [Kageyama et.al, J. Biochem., 125, 838-845 (1999)]. In the current study, we attempted to detect the enzyme products of zFT1 in zebrafish embryos. Oligosaccharides were prepared from the zebrafish embryos at 12, 18 and 48 h after fertilization and labelled with a fluorophore, 2-aminopyridine, for highly sensitive detections. Pyridylamino (PA)-oligosaccharides that were alpha1-3/4fucosidase sensitive and time-dependently expressed at 18 h after fertilization were identified as candidates for the in vivo products synthesized by zFT1. Structures of these oligosaccharides were determined by a combination of exoglycosidase digestions and two-dimensional HPLC sugar mapping to be the biantennary complex-type structures with two Lewis x epitopes: (Gal beta1-4)(0,1,2)-{Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-6[Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-3]}Man beta1-4GlcNAc, and (Gal beta1-4)(0,1)-{Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-6[Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-3]} Man beta1-4GlcNAc beta1-4GlcNAc. The presence of Lewis x structure of these oligosaccharides together with their expression time suggests that they are products of zFT1. Remarkably, most of these oligosaccharides were free form. Furthermore, we detected an endo-beta-N-acetylglucosaminidase activity in the 18 h embryo. These results suggest that the oligosaccharides synthesized by zFT1 are present in the embryo at the segmentation period in free form, owing to the liberation from glycoproteins with endo-beta-N-acetylglucosaminidase(s) and/or glycoamidase(s).

A unique O-glycan structure, Xylalpha1-3Xylalpha1-3Glcbeta1-O-Ser is found on the consensus sequence C-X-S-X-P-C (X denotes any amino acid) in epidermal growth factor (EGF)-like domains of plasma proteins such as clotting factor VII and IX. One of the enzymes involved in the biosynthesis of this trisaccharide, UDP-d-xylose:beta-d-glucoside 1,3-d-xylosyltransferase has been identified in HepG2 cells (Omichi, K., Aoki, K., Minamida, S., and Hase, S. Eur. J. Biochem. 245, 143-146 [1997]). Here, we report that this enzyme activity can be detected in bovine liver and that the enzyme has been purified from the microsomal fraction. The enzyme was purified 6200-fold in terms of specific activity and ran as a single band on native-PAGE and isoelectric focusing gel electrophoresis. The best acceptor substrate of those tested was the EGF-like domain of bovine factor IX carrying beta-glucoside at Ser53. The Km value for this substrate was 34 muM. Comparison of initial velocity with various acceptor substrates shows that this xylosyltransferase recognizes not only the glucose moiety to which xylose is transferred but also the tertiary structure of the EGF-like domain. With regard to the donor substrate, the enzyme does not recognize UDP-d-glucose but does recognize UDP-d-xylose.

Pyridylamino sugar chains were converted to the corresponding reducing sugar chains by first converting them to 1-amino-1-deoxy derivatives using the method previously reported [S. Hase, J. Biochem. 112, 266-268 (1992)] and then converting the products to the corresponding reducing sugar chains using the Sommlet reaction. The reaction conditions were optimized so as to obtain the maximal product yield using 1-amino-1-deoxylactose and 1-amino-1-deoxy-N-acetylglucosamine. When the established procedure was successively applied to pyridylamino high-mannose and complex-type sugar chains, the corresponding reducing sugar chains were obtained in yields of 30%.

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.

Caenorhabditis elegans is an excellent model for morphogenetic research. However, little information is available on the structure of cell-surface glycans in C. elegans, although several lines of evidence have suggested a role for these glycans in cell-cell interactions during development. In this study, we analyzed N-glycan structures. Oligosaccharides liberated by hydrazinolysis from a total membrane fraction were labeled by pyridylamination, and around 90% of the N-glycans were detected as neutral oligosaccharides. The most dominant structure was Man(alpha)1-6(Man(alpha)1-3)Man(beta)1-4GlcNAc(beta)1-4GlcNAc, which is commonly found in insects. Branching structures of major oligomannose-type glycans were the same as those found in mammals. Structures that had a core fucose or non-reducing end N-acetylglucosamine were also identified, but ordinary complex-type glycans with N-acetyllactosamine were not detected as major components.

The sugar chains of human urinary thrombomodulin were studied. N- and O-linked sugar chains were simultaneously liberated by hydrazinolysis followed by N-acetylation and were tagged with 2-aminopyridine. Then the structures of the N- and O-linked pyridylamino (PA-) sugar chains were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion. The major N-linked sugar chains of human urinary thrombomodulin were found to be monosialo- and disialofucosylbiantennary chains, while the major O-linked sugar chain was +/-Siaalpha2-3Galbeta1-3(+/-Siaalpha2-6)GalNAc. Thrombomodulin also contained the reported structure SO4-3GlcAbeta1-3Galbeta1-3(+/-Siaalpha2-6)Galbeta1-4Xyl [H. Wakabayashi, S. Natsuka, T. Mega, N. Otsuki, M. Isaji, M. Naotsuka, S. Koyama, T. Kanamori, K. Sakai, and S. Hase (1999) J. Biol. Chem. 274, 5436-5442]. In addition to these sugar chains, a single Glc was linked to Ser 287.

The structures of N-linked oligosaccharides present in human sera from 12 healthy volunteers and from 14 patients with non-small cell lung cancer (NSCLC) were analyzed by our recently developed partially automated systematic method. Thirty different structures of oligosaccharides were deduced, and these accounted for 84.1% of the total N-linked oligosaccharides present in human sera. All of the quantified oligosaccharide levels in healthy human sera were within twice the standard deviation. The amount of a triantennary trigalactosylated structure with one outer arm fucosylation (A3G3Fo) was found to be markedly increased in NSCLC patients in comparison to that in healthy volunteers (p < 0.01). No significant positive correlation with other clinical data was found. Serum A3G3Fo levels can thus be a novel marker for the diagnosis of NSCLC.

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.

We previously reported two brain-specific agalactobiantennary N-linked sugar chains with bisecting GlcNAc and alpha1-6Fuc residues, (GlcNAcbeta1-2)(0)(or)(1)Manalpha1-3(GlcNAcbeta1-2M analpha1-6)(GlcNA cbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)Glc NAc [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. 114, 334-338]. Here, the reason for the absence of Gal on the sugar chains was analyzed through the detection of other complex type sugar chains. Analysis of N-linked sugar chains revealed the absence of Sia-Gal and Gal on the GlcNAc residues of brain-specific agalactobiantennary N-linked sugar chains. We therefore investigated the substrate specificity of galactosyltransferase activities in brain using pyridylamino derivatives of agalactobiantennary sugar chains with structural variations in the bisecting GlcNAc and alpha1-6Fuc residues as acceptor substrates. While the beta1-4galactosyltransferases in liver and kidney could utilize all four oligosaccharides as substrates, the beta1-4galactosyltransferase(s) in brain could not utilize the agalactobiantennary sugar chain with both bisecting GlcNAc and Fuc residues, but could utilize the other three acceptors. Similar results were obtained using glycopeptides with agalactobiantennary sugar chains and bisecting GlcNAc and alpha1-6Fuc residues as substrates. The beta1-4galactosyltransferase activity of adult mouse brain thus appears to be responsible for producing the brain-specific sugar chains and to be different from beta1-4galactosyltransferase-I. The agalactobiantennary sugar chain with bisecting GlcNAc and alpha1-6Fuc residues acts as an inhibitor against "brain type" beta1-4galactosyltransferase with a K(i) value of 0.29 mM.

The amounts and isomeric structures of free oligosaccharides derived from N-linked sugar chains present in the cytosol fraction of perfused mouse liver were analyzed by tagging the reducing end with 2-aminopyridine followed by 2-dimensional HPLC mapping with standard sugar chains. Sixteen pyridylaminated (PA-) oligomannosides terminating with a PA-GlcNAc residue (GN1-type), three glucose-containing oligomannosides, and four oligomannosides terminating with a PA-di-N-acetylchitobiose (GN2-type) were detected. The total contents of the GN1- and GN2-type oligomannosides were 3. 4 and 0.5 nmol, respectively, per gram of wet tissue. Maltooligosaccharides (dimer to pentamer) were also detected, the total content of which was 13 nmol per gram of wet tissue. Besides these oligosaccharides, a PA-disialobiantennary sugar chain-the sole complex-type sugar chain-was also detected. All the oligomannosides identified had partial structures of Glc(3)Man(9)GlNAc(2)-p-p-dolichol, revealing that they were metabolic degradation products. Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)++ +Manbeta1-4GlcNAc (M5B') was the major oligomannoside, suggesting that cytosolic endo-beta-N-acetylglucosaminidase and neutral alpha-mannosidase participate in the degradation, because these enzymes have suitable substrate specificities for the production of M5B'. Degradation by these enzymes seems to be the main pathway by which oligomannosides are degraded in mouse cytosol; however, small amounts of Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4(GlcNAc)1-2 and related oligomannosides together with parts of their structures were also detected, suggesting that there is another minor route by which cytosolic free oligomannosides are produced.

Some alpha(1,3)fucosylated oligosaccharides serve as counter receptors to lectin-like adhesion proteins or are expressed with temporal precision during embryogenesis, and alpha(1, 3)fucosyltransferase is a key enzyme in the production of these oligosaccharides. Two alpha(1,3)-fucosyltransferase genes, designated zFT1 and zFT2, were cloned from zebrafish. Sequence comparisons with other genes indicated that zFT1 and zFT2 share about 30% amino acid sequence identity with human alpha(1, 3)fucosyltransferases. Although the alpha(1,3)fucosyltransferases cloned so far can be classified into three types-myeloid, Lewis, and leukocyte-by virtue of their amino acid sequences, phylogenetic analysis indicated that neither zFT1 nor zFT2 belongs to any of these categories. The expression of zFT1 or zFT2 in mammalian cells induces alpha(1,3)fucosyltransferase activity to synthesize the Lewis x structure from pyridylaminated lacto-N-neotetraose; however, lacto-N-tetraose does not serve as a substrate. Reverse transcriptase-polymerase chain reaction analysis revealed that zFT1 is transcribed during a restricted period before hatching, whereas the mRNA for zFT2 was detected only after hatching.

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.

The presence of free oligomannosides in cytosol has been demonstrated by metabolic radiolabeling, and Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)++ +Manbeta1-4GlcNAc was detected as the main free oligomannoside in Chinese hamster ovary cells [Kmiécik, D., Herman, V., Stroop, C.J.M., Michalski, J.C., Mir, A.M., Labiau, O., Verbert, A., and Cacan, R. (1995) Glycobiology, 5, 483-494]. In the present paper, the isomeric structures and amounts of oligomannosides in the cytosol fraction of hen oviduct were analyzed by pyridylamination and exoglycosidase digestion. Hen oviduct was used since our group has already characterized the cytosolic neutral alpha-mannosidase and endo-beta-N-acetylglucosaminidase obtained from the same source. The amounts of Man2GlcNAc, Man3GlcNAc, Man4GlcNAc, and Man5GlcNAc were 0.6, 0.6, 0.5, and 0.8 nmol/g tissue, respectively, but Man6GlcNAc-Man9GlcNAc were not detected. The isomeric structures of the Man3GlcNAc-Man5GlcNAc found were (Manalpha1-2)0-2Manalpha1-3(Manalpha1-6)Manbeta1 -4GlcNAc, which were compatible with the substrate specificities of cytosolic endo-beta-N-acetylglucosaminidase and neutral alpha-mannosidase, indicating that these enzymes participate in the formation of the oligomannosides present in the cytosol.

We previously detected a fucosylagalactobiantenna with a bisecting GlcNAc residue (BA-2) and one lacking the GlcNAc residue linked to the Manalpha1-3 residue of BA-2 (BA-1), which were enriched specifically in mouse brain [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. 114, 334-338]. Pyridylamino sugar chains were prepared from mouse brains of various ages, and BA-1 and BA-2 were quantified after separation by HPLC. In cerebrum, BA-1 was scarcely expressed in newborn brain but gradually increased in amount during development, while expression of BA-2 reached a maximum 1 week after birth followed by a rapid decrease; in adult mice, the amount of BA-1 was almost the same as that of BA-2. In cerebellum, expression of BA-1 was lower than that of BA-2 at all stages. Glycoproteins with the BA-1 and BA-2 structures were enriched in the membrane fraction, and the glycoproteins solubilized were purified by lectin-affinity chromatography and gel filtration. The results indicated that BA-1 and BA-2 occurred in glycoproteins of more than 20 kDa in cerebellum, but most BA-1 and BA-2 were found in a 80-200 kDa fraction in cerebrum. These results show that the two brain-specific sugar chains are developmentally regulated and linked to the membrane-associated glycoproteins of subcellular organellas.

An assay method for glycogen debranching enzyme involving fluorogenic dextrins as substrates was developed. Two dextrins were prepared from 6-O-alpha-D-glucosyl-alpha-cyclodextrin and glucose by taking advantage of the action of Bacillus macerans cyclodextrin glucanotransferase, and converted by pyridylamination to fluorogenic derivatives. Structural analysis of the fluorogenic dextrins by FAB-MS, partial acid hydrolysis, and glucoamylase digestion revealed that they were Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glcalpha1- 4Glcalpha1-4Glc-PA (FD6) and Glcalpha1-4Glcalpha1-4(Glcalpha1-6)Glcalpha1- 4Glcalpha1-4Glcalpha1-4G lc-PA (FD7). Using the glycogen debranching enzyme from rabbit muscle, FD6 and FD7 were, respectively, hydrolyzed to PA-maltopentaose and PA-maltohexaose, in addition to glucose, showing that these two fluorogenic dextrins are suitable substrates for assaying the glycogen debranching enzyme. An assay method involving the separation and quantification by HPLC of the characteristic fluorogenic products was successfully applied to determination of the distribution of the enzyme activity in mouse cerebrum.

We previously reported the detection of novel O-linked sugar chains classified as being of the glucosyl-O-serine type [Hase et al. (1988) J. Biochem. 104, 867-868]. The sugar chains are a disaccharide (Xyl alpha 1-3Glc) and a trisaccharide (Xyl alpha 1-3Xyl alpha 1-3 Glc) linked to serine residues in epidermal growth factor-like domains of human and bovine blood coagulation factors. The structures of these sugar chains suggested the presence of an alpha 1-->3xylosyltransferase for their biosynthesis. We report here on the detection of alpha 1-->3xylosyltransferase activity which catalyzes the transfer of xylose to Xyl alpha 1-3Glc in the human hepatoma cell line HepG2. We employed pyridylaminated Xyl alpha 1-3Glc as a fluorescent acceptor and UDP-D-Xyl as a donor. The reaction product was purified by reversed-phase HPLC, and the structure of the transfer product isolated was confirmed to be pyridylaminated Xyl alpha 1-3Xyl alpha 1-3Glc by Smith degradation, mass spectrometry, and alpha- and beta-xylosidase digestions. The apparent K(m) value for pyridylaminated Xyl alpha 1-3Glc was 52 mM and for UDP-D-Xyl 0.28 mM. Optimum pH was 7.2. The enzyme was inactivated by addition of EDTA, and its activity was restored by addition of Mn2+ and Mg2+. These results indicate the presence of a novel enzyme which is able to transfer xylose to Xyl alpha 1-3Glc, forming Xyl alpha 1-3Xyl alpha 1-3Glc in human cells.

An alpha-mannosidase capable of hydrolyzing three Man alpha 1,2-residues from pyridylamine-(PA-) labeled Man9GlcNAc2 was purified from hen oviduct. The purity of the preparation was analyzed by PAGE; its molecular weight was 42,000 by SDS-PAGE or 50,000 by gel filtration. The pH optimum was 6.5. The enzyme was inactivated with EDTA; enzyme activity was restored by the addition of Ca2+. The enzyme activity was inhibited by 1-deoxymannojirimycin, but not by swainsonine. The substrate specificity of the purified enzyme was analyzed using PA-oligomannose-type sugar chains. When Man9GlcNAc2-PA was digested, Man alpha 1-6(Man alpha 1-2Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4Glc-NAc-PA was obtained as an end product, and the enzyme was incapable of hydrolyzing p-nitrophenyl alpha-D-mannoside and Man alpha 1,3- or Man alpha 1,6-residues. Judging from these characteristics, the enzyme was classified as a Man9-mannosidase or Golgi mannosidase I and speculated to participate in the processing or catabolism of glycoproteins.

Endo-beta-mannosidase, which hydrolyses the Manbeta1-4GlcNAc linkage of N-glycans in an endo-manner, was discovered in plants. During the course of the purification of the enzyme from lily flowers, we found a higher molecular mass form of the enzyme (designated as EBM II). EBM II was purified by column chromatography to homogeneity and its molecular composition revealed EBM II to be comprised of endo-beta-mannosidase and an associated protein. The cDNA of this associated protein encodes a protein with slight homology to the fucosidase domain of bifidus AfcA. EBM II has alpha1,2-L-fucosidase activity and acts on a fucosylated xyloglucan nonasaccharide. The amino acid sequence of this associated protein has no similarity to known plant alpha-L-fucosidases. These results show that EBM II is a novel alpha1,2-L-fucosidase and a protein complex containing endo-beta-mannosidase.

A unique O-glycan structure, Xylalpha1-3Xylalpha1-3Glcbeta1-O-Ser is found on the consensus sequence C-X-S-X-P-C (X denotes any amino acid) in epidermal growth factor (EGF)-like domains of plasma proteins such as clotting factor VII and IX. One of the enzymes involved in the biosynthesis of this trisaccharide, UDP-d-xylose:beta-d-glucoside 1,3-d-xylosyltransferase has been identified in HepG2 cells (Omichi, K., Aoki, K., Minamida, S., and Hase, S. Eur. J. Biochem. 245, 143-146 [1997]). Here, we report that this enzyme activity can be detected in bovine liver and that the enzyme has been purified from the microsomal fraction. The enzyme was purified 6200-fold in terms of specific activity and ran as a single band on native-PAGE and isoelectric focusing gel electrophoresis. The best acceptor substrate of those tested was the EGF-like domain of bovine factor IX carrying beta-glucoside at Ser53. The Km value for this substrate was 34 muM. Comparison of initial velocity with various acceptor substrates shows that this xylosyltransferase recognizes not only the glucose moiety to which xylose is transferred but also the tertiary structure of the EGF-like domain. With regard to the donor substrate, the enzyme does not recognize UDP-d-glucose but does recognize UDP-d-xylose.

For comparative biochemical interest, we analyzed the structures of N-glycans in a squid belonging to the Lophotrochozoa, one of the protostome clades. N-Glycans were prepared from squid skin by hydrazinolysis and re-N-acetylation followed by fluorescent tagging with 2-aminopyridine. The labeled N-glycans were purified, and their structures were determined by the two-dimensional HPLC mapping method combined with glycosidase digestions and mass spectrometry. We found that high mannose-type glycans, paucimannose-type glycans and complex-type glycans with a type-1 structure (Galbeta1-3GlcNAc) were dominant in squid skin. The complex-type glycans detected in the squid were similar to those in vertebrates, but have not yet been found in the Ecdysozoa, which is another protostome clade. However, paucimannose-type glycans are commonly found in the Ecdysozoa. Thus, the N-glycan structures of the squid belonging to the Lophotrochozoa have features common to those in vertebrates and the Ecdysozoa including insects and nematodes.

An endo-beta-mannosidase [EC 3.2.1.152, glycoside hydrolase family 2], which hydrolyzes the Manbeta1-4GlcNAc linkage of N-glycans in an endo-manner, has been found in plant tissues [Ishimizu, T., Sasaki, A., Okutani, S., Maeda, M., Yamagishi, M., and Hase, S. (2004) J. Biol. Chem. 279, 38555-38562]. So far, this glycosidase has been purified only from a monocot plant, a lily. Here, an endo-beta-mannosidase was purified from a dicot plant, cabbage (Brassica oleracea), and characterized. The cabbage endo-beta-mannosidase consists of four polypeptides. These four polypeptides are encoded by a single gene, whose nucleotide sequence is homologous to those of the lily and Arabidopsis endo-beta-mannosidase genes. 1H NMR analysis of the stereochemistry of the hydrolysis of pyridylaminated (PA) Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc showed that the cabbage endo-beta-mannosidase is a retaining glycoside hydrolase, as are other glycoside hydrolase family 2 enzymes. The enzymatic characteristics, including substrate specificity, of the cabbage enzyme are very similar to those of the lily enzyme. These endo-beta-mannosidases specifically act on Man(n)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA (n = 0 to 2). These results suggest that the endo-beta-mannosidase is present in at least the angiosperms, and has common roles, such as the degradation of N-glycans.

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.

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.

The structures of N-glycans of human blood clotting factor IX were studied. N-Glycans liberated by hydrazinolysis were N-acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine. The pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Each PA-sugar chain was analyzed by two-dimensional sugar mapping combined with glycosidase digestion. The major structures of the N-linked sugar chains of human factor IX were found to be sialotetraantennary and sialotriantennary chains with or without fucose residues. These highly sialylated sugar chains are located on the activation peptide of the protein.

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.

An enzyme catalyzing the hydrolysis of the Manbeta1-4GlcNAc linkage of N-linked sugar chains was partially purified and characterized. Endo-beta-mannosidase activity was detected using pyridylaminated (PA-) Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc as the substrate in a homogenate of lily flowers (Lilium longflorum Thumb). The enzyme was partially purified by ammonium sulfate precipitation, and Q-Sepharose, Superdex 200, hydroxyapatite, Poros PE/M, Mono Q, and Superdex 200 column chromatographies. The optimum pH was 5.0 and the estimated molecular weight of the enzyme was 78,000, as determined by gel filtration. The Km value found for Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA was 1.4 mM. The enzymatic activity was not influenced by the addition of 10 mM EDTA or 2 mM Ca2+. Experiments on the hydrolysis of several PA-N-linked sugar chains revealed that the enzyme hydrolyzed MannManalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA (n = 0-2) into a mixture of MannManalpha1-6Man and GlcNAcbeta1-4GlcNAc-PA, indicating that it is an endoglycosidase in nature. However, the enzyme did not hydrolyze beta1-4mannohexaose or p-nitrophenyl beta-mannopyranoside.

Endo-beta-N-acetylglucosaminidase from hen oviduct (Endo-HO) was purified to homogeneity by ammonium sulfate fractionation and then by column chromatographies on DEAE-Sephacel, hydroxyapatite, Octyl-Sepharose CL-4B, Co2+-chelating Sepharose FF, and YMC-Pack Diol-200G. Partial purification of the enzyme was reported previously [Tarentino, A.L. and Maley, F. (1976) J. Biol. Chem. 251, 6537-6543]. The molecular weight was 54,000 by gel filtration and 52,000 by SDS-PAGE in the presence of 2-mercaptoethanol, indicating that Endo-HO is composed of a single polypeptide chain. The optimum pH was 6.5, and the Km value was 25 microM when pyridylaminated Man6GlcNAc2 was used as a substrate. EDTA and metal cations tested, except Hg2+, had no effects on Endo-HO activity. Substrate specificity results using pyridylaminated N-linked sugar chains revealed that Endo-HO hydrolyzed oligomannose-type sugar chains faster than complex- and hybrid-type chains, and that sugar chains containing the Manalpha1-2Manalpha1-3Manbeta1-4GlcNAcbeta1-GlcN Ac structure were good substrates for the enzyme. These findings suggest that in cytosol the enzyme contributes to the production of a free oligosaccharide with one reducing end N-acetylglucosamine residue in cooperation with neutral alpha-mannosidase, an enzyme that specifically hydrolyzes oligosaccharides to Manalpha1-2Manalpha1-2Manalpha1-3(Manalpha1-6)++ +Manbeta1-4GlcNAc.

Neutral alpha-mannosidase was purified to homogeneity from hen oviduct. The molecular mass of the enzyme was 480 kDa on gel filtration, and the 110-kDa band on SDS-PAGE in the presence of 2-mercaptoethanol indicated that it is composed of four subunits. The activated enzyme hydrolyzed both p-nitrophenyl alpha-D-mannoside and high mannose-type sugar chains. This substrate specificity is almost the same as that reported for the neutral a-mannosidase from Japanese quail oviduct [Oku and Hase (1991) J. Biochem. 110, 982-989]. Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4GlcNAc (Km =0.44 mM) was hydrolyzed four times faster than Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4GIcNAcbeta1-4GlcNAc, and Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)++ +Manbeta1-4GlcNAc was obtained as the end product from Man9GlcNAc on digestion with the activated alpha-mannosidase. The enzyme was activated 24-fold on preincubation with Co2+. The activation with other metal ions, like Mn2+, Ca2+, Fe2+, Fe3+, and Sr2+, was less than 5-fold, and Zn2+, Cu2+, and Hg2+ inhibited the enzyme activity. The optimum pHs for both the enzyme activity and activation with Co2+ were around 7. The cobalt ion contents of the purified, EDTA-treated, and Co2+-activated enzymes were 1.5, 0.0, and 3.9, respectively, per molecule. Since the Co2+-activated enzyme gradually lost its activity on incubation with EDTA and the activity was restored promptly on the addition of Co2+, the binding of Co2+ to the enzyme seems to be essential for its activation. The results obtained with protease inhibitors together with those of the SDS-PAGE before and after activation, showed that the proteolytic cleavage reported for the activation of monkey brain alpha-mannosidase seems not to be involved.

A cytosolic neutral alpha-mannosidase was purified from bovine liver. Its molecular weight was found to be 500,000 on gel filtration. The activity of the enzyme toward Man alpha 1-6-(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc-PA was increased 26-fold by preincubation with 1 mM Co2+. Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc was hydrolyzed by the enzyme to Man alpha 1-3Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc, which was further hydrolyzed to Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc. The rate of hydrolysis was 15-fold greater than that of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. This substrate specificity suggested that the enzyme could be involved in the degradation of oligomannose-type sugar chains with one GlcNAc residue released from glycoproteins by endo-beta-N-acetylglucosaminidase, and supported a pathway for glycoprotein catabolism via oligomannosyl glycans with one GlcNAc residue proposed on the basis of an earlier study on a cytosolic neutral alpha-mannosidase from Japanese quail oviduct [Oku, H. and Hase, S. (1991) J. Biochem. 110, 982-989].

The existence of alpha-amylase (HXA) encoded by alpha-amylase gene AMY2B in healthy humans was examined using a fluorogenic substrate, FG5P (FG-G-G-G-G-P: FG, 6-deoxy-6-[(2-pyridyl)amino]-D-glucose residue; G, glucose residue; P, p-nitrophenyl residue; -, alpha-1,4-glycosidic bond). Chromatofocusing of urine from a healthy human was carried out. FG5P was digested with the fractions exhibiting alpha-amylase activity and each digest at an early stage was analyzed by HPLC. FG5P was hydrolyzed to FG3 (FG-G-G) and p-nitrophenyl alpha-maltoside (G-G-P), and to FG4 (FG-G-G-G) and p-nitrophenyl alpha-glucoside (G-P). The molar ratios of FG4 to FG3 (FG4/FG3) in the digests with basic fractions were larger than those in the digests of human pancreatic alpha-amylase (HPA, 1.11) and human salivary alpha-amylase (HSA, 0.51). Considering that the value for the AMY2B gene product with yeast (yHXA) is 1.88, a value of more than 1.11 implies that HXA exists. The amount of HXA was determined after removal of HSA on an anti-human salivary alpha-amylase antibody bound column. The FG4/FG3 values for six urine samples free from HSA were 1.23-1.26. Assuming that the FG4/FG3 value for HXA is the same as that for yHXA, the ratios of HXA and HPA were estimated to be 1:5.4-4.1. The results obtained showed that the AMY2B gene is usually expressed as HXA in healthy humans.

Pyridylamino (PA) derivatives of sugar chains were converted to 1-amino-1-deoxy derivatives. PA-lactose as a model compound was reduced with hydrogen, then treated with hydrazine. The product obtained was identified as 1-amino-1-deoxylactitol by mass spectrometry and chromatography with 1-amino-1-deoxylactitol as standard. PA-N-acetylglucosamine was converted to 1-amino-1-deoxy-N-acetylglucosaminitol under the same conditions. As an application, Man alpha 1-6(Man alpha 1-3)Man alpha 1- 6(Man alpha 1-2Man alpha 1-3)-Man beta 1-4GlcNAc beta 1-4GlcNAc-PA was converted to the 1-amino-1-deoxy derivative, which was further derivatized with fluorescein isothiocyanate or biotin sulfo-N-hydroxy-succinimide ester. Binding of these derivatives to concanavalin A dot-blotted on a nitrocellulose membrane was confirmed by fluorescence and by streptavidin-peroxidase conjugate. This conversion allowed replacement of the PA-group in PA-sugar chains which can be easily purified from glycoconjugates.

Modified maltooligosaccharides, IG-G-G-G-AG-G (IG: 6-deoxy-6-iodo-D-glucopyranose residue, G: D-glucopyranose residue, AG: 6-amino-6-deoxy-D-glucopyranose residue, -: alpha-1,4-glycosidic linkage), IG-G-G-G-AG-M (M: methyl), and IG-G-G-G-AG-phi (phi: phenyl) were prepared by the use of cyclodextrin glucanotransferase in order to examine the third subsite (S3') of the aglycone-binding site of human salivary and pancreatic alpha-amylases. Human alpha-amylases hydrolyzed the modified maltooligosaccharides to IG-G-G and G-AG-G, IG-G-G and G-AG-M, and IG-G-G and G-AG-phi. This implied that G, M, and phi fit into S3'. There was no difference in the rate parameters between the two enzymes. The Km values for the hydrolysis of IG-G-G-G-AG-G by both enzymes were the same as those for IG-G-G-G-AG-M, and twice those for IG-G-G-G-AG-phi. The results showed that S3' of the two enzymes has no affinity for the glucose residue and is not a subsite but a hydrophobic environment.

We used high performance liquid chromatography to determine the concentrations of free ligands after equilibrium dialysis to examine lectin-sugar interactions. The binding of p-nitrophenyl 1-thio-alpha-mannoside, p-nitrophenyl N-acetyl-1-thio-beta-glucosaminide, and the pyridylamino derivative of Man6GlcNAc2 to concanavalin A and Triticum vulgaris lectin was examined. The binding constant, Ka, and the concentration of total binding sites, [L]t, were calculated from the trace amounts of sugars and lectins using the equation, [S]/[LS] = 1/Ka[L]t + [S]/[L]t, where [S] is the concentration of free ligand and [LS] the concentration of bound ligand.

Potential aldehyde groups of several monosaccharides and oligosaccharides were coupled with 2-aminopyridine by reductive amination with sodium cyanoborohydride. The product was isolated by adsorption on a Dowex 50 (H+) column, followed by washing, elution with aqueous ammonia and evaporation. The specimen was analyzed by paper electrophoresis at pH 5.0. Each sugar derivative gave a single spot in addition to one corresponding to excess 2-amino-pyridine when the paper was scanned under a UV lamp. The migration rates of these fluorescent sugars were related to their molecular weights and were independent of their linkage points and anomeric configurations. The reducing end sugar units of oligosaccharide derivatives could be identified by gas-liquid chromatography after hydrolysis of the 2-aminopyridine derivatives of the oligosaccharides.

Crystalline Taka-amylase A, prepared from Takadiastase, was fractionated into four fractions by DEAE-Sephacel and Concanavalin A-Sepharose column chromatography. The relative weight ratio of the fractions was 90 : 4 : 4 : 2. They had similar molecular weights (51,000), amino acid compositions, and hydrolytic activity against soluble starch, but different phenyl maltosidase activities and electrophoretic mobilities on polyacrylamide gel electrophoresis. Three of the fractions mainly had the high mannose type sugar chain with the sugar composition of Man5-GlcNAc2, but the other fraction had only one N-acetylglucosamine residue as the sugar chain. These results suggested that Taka-amylase A was heterogeneous both in the sugar portions and in the polypeptide portions.

Interferon-gamma produced by the human myelomonocyte cell line HBL-38 contained galactose, mannose, fucose, N-acetylglucosamine, and N-acetylneuraminic acid as sugar components. Sugar chains were liberated from interferon-gamma by hydrazinolysis. Free amino groups of the sugar chains were acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine under new reaction conditions in which no sialic acid residue was hydrolyzed. The pyridylamino (PA-) derivatives of the sugar chains thus obtained were purified by gel filtration and reversed-phase HPLC. Seven major PA-sugar chains were isolated and the structure of each purified PA-sugar chain was identified by stepwise exoglycosidase digestion and 500-mHz 1H-NMR spectroscopy. The results indicated that the structures of the major PA-sugar chains were of the biantennary type, to which 0 to 2 mol of fucose and 1 to 2 mol of N-acetylneuraminic acid were linked as shown below. (formula; see text)