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To investigate the relationship between phylogeny and glycan structures, we analyzed the structure of planarian N-glycans. The planarian Dugesia japonica, a member of the flatworm family, is a lower metazoan. N-glycans were prepared from whole worms by hydrazinolysis, followed by tagging with the fluorophore 2-aminopyridine at their reducing end. The labeled N-glycans were purified, and separated by three HPLC steps. By comparison with standard pyridylaminated N-glycans, it was shown that the N-glycans of planarian include high mannose-type and pauci-mannose-type glycans. However, many of the major N-glycans from planarians have novel structures, as their elution positions did not match those of the standard glycans. The results of mass spectrometry and sugar component analyses indicated that these glycans include methyl mannoses, and that the most probable linkage was 3-O-methylation. Furthermore, the methyl residues on the most abundant glycan may be attached to the non-reducing-end mannose, as the glycans were resistant to α-mannosidase digestion. These results indicate that methylated high-mannose-type glycans are the most abundant structure in planarians.

Herein, I describe pyridylamination for versatile analysis of sugar chains. The reducing ends of the sugar chains are tagged with 2-aminopyridine and the resultant chemically stable fluorescent derivatives are used for structural/functional analysis. Pyridylamination is an effective "operating system" for increasing sensitivity and simplifying the analytical procedures including mass spectrometry and NMR. Excellent separation of isomers is achieved by reversed-phase HPLC. However, separation is further improved by two-dimensional HPLC, which involves a combination of reversed-phase HPLC and size-fractionation HPLC. Moreover, a two-dimensional HPLC map is also useful for structural analysis. I describe a simple procedure for preparing homogeneous pyridylamino sugar chains that is less laborious than existing techniques and can be used for functional analysis (e.g., sugar-protein interaction). This novel approach was applied and some of the results are described: i) a glucosyl-serine type sugar chain found in blood coagulation factors; ii) discovery of endo-beta-mannosidase (EC 3.2.1.152) and a new type plant alpha1,2-L-fucosidase; and iii) novel substrate specificity of a cytosolic alpha-mannosidase. Moreover, using homogeneous sugar chains of a size similar to in vivo substrates we were able to analyze interactions between sugar chains and proteins such as enzymes and lectins in detail. Interestingly, our studies reveal that some enzymes recognize a wider region of the substrate than anticipated.

Summary. Background: Tissue factor pathway inhibitor (TFPI) is a physiological protease inhibitor that inhibits the initial reactions of the extrinsic blood coagulation pathway. Most TFPI in human plasma is associated with lipoproteins; however, the most functionally active form is thought to be the free, full-length form (f-pTFPI). Cell culture derived TFPI and recombinant TFPI (rTFPI) exhibit variations in their respective anticoagulant activity, which may be caused by post-translational modifications, such as the frequent differences in sugar chain structures among recombinant proteins. Sugar chain structures in rTFPI expressed in Chinese hamster ovary (CHO) cells have been reported previously, but those of plasma TFPI have not been. Objectives: To purify f-pTFPI and analyze the sugar chain structures. Results and conclusion: f-pTFPI was purified to homogeneity from blood plasma using a combination of anion-exchange, heparin affinity, immunoaffinity, and reversed-phase chromatographies, resulting in a yield of 76%. f-pTFPI showed a partially phosphorylated glycoprotein comprising a total of 276 amino acids by peptide mapping. The sugar chain structures were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion of the pyridylamino sugar chains and the following results were obtained. (Sialyl) Galβ1-3GalNAc was linked to Thr175, partially to Thr14 and Ser174; sialyl complex-type sugar chains to Asn117 and Asn167, whereas Asn228 was not glycosylated. Neuraminidase-resistant acidic sugar chains including sulfated sugar chains were not observed significantly. The protease inhibitory activities of f-pTFPI towards activated factor (F) X and tissue factor-activated FVII complex were identical to those of full-length rTFPI expressed in CHO cells.

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.

UDP-galacturonic acid, the activated form of galacturonic acid (GalUA), is synthesized both de novo and by salvage pathways. The UDP-GalUA pyrophosphorylase gene involved in the salvage pathway has not been identified. Here we show that UDP-sugar pyrophosphorylase from Pisum sativum with a broad specificity has UDP-GalUA pyrophosphorylase activity. The enzyme catalyzed the formation of UDP-GalUA and pyrophosphate from GalUA 1-phosphate and UTP with an equilibrium constant value of 0.24. The recombinant UDP-sugar pyrophosphorylase had optimal pH of 6.0, and the apparent K(m) values for GalUA 1-phosphate, UTP, UDP-GalUA, and pyrophosphate were 2.27, 1.15, 0.70, and 1.26 mM, respectively. In the presence of inorganic pyrophosphatase, the recombinant enzyme produced UDP-GalUA in an 84% yield (based on the GalUA 1-phosphate substrate) on a preparative scale. Thus, this UDP-sugar pyrophosphorylase is useful for the highly efficient production of UDP-GalUA for studies on pectin biosynthesis.

N-配糖体のコア構造中のManβ1-4GlcNAc結合を加水分解するエンド-β-マンノシダーゼ(EC3.1.2.152) が高等植物より見いだされた。我々は、最近、エンド-β-マンノシダーゼの精製、遺伝子クローニングに成功した。エンド-β-マンノシダーゼのオーソログ遺伝子は植物種のみからしか見いだせず、この酵素は植物に特異的な機能をもっていると考えられる。このエンドグリコシダーゼの基質特異性は、(Man)nManα1-6Manβ1-4GlcNAcβ1-4GlcNAc (n=0～2)のManβ1-4GlcNAc結合を加水分解するというユニークなものである。これらの基質はManα1-3Manβ結合を優先的に加水分解するタチナタマメα-マンノシダーゼ様酵素がハイマンノース型糖鎖に作用することによって生じる。つまり、植物細胞内でエンド-β-マンノシダーゼとタチナタマメα-マンノシダーゼ様酵素が協同的に働いてハイマンノース型糖鎖をN,N'-ジアセチルキトビオースにまで加水分解していると考えられる。また、このエンド-β-マンノシダーゼはトランスグリコシレーション活性を持ち、化学合成時に立体制御が困難なβ-マンノシドを形成することができる。

Some mutants of Caenorhabditis elegans show altered patterns of ectopic binding with wheat germ agglutinin (WGA). Some of these mutants also have defects of morphogenesis and movement during development. To clarify the structures of WGA-ligands in C. elegans that may be involved in developmental events, we have analyzed glycan structures capable of binding WGA. We isolated glycoproteins from wild-type C. elegans by WGA-affinity chromatography, and analyzed their glycan structures by a combination of hydrazine degradation and fluorescent labeling. The glycoproteins had oligomannose-type and complex-type N-glycans that included agalacto-biantenna and agalacto-tetraantenna glycans. Although the complex-type glycans carried beta-GlcNAc residues at their non-reducing ends, they did not bind to the WGA-agarose-resin. Thus, it was suggested that these N-glycans were not responsible for WGA-binding of the isolated glycoproteins. Hydrazinolysis of the glycoproteins also released a considerable amount of GalNAc monosaccharide. It was surmised that N-acetylgalactosamine was derived from mucin-type O-glycans with the Tn-antigen structure (GalNAcalpha1-O-Ser/Thr). WGA-blotting assay of neoglycoproteins revealed that a cluster of Tn-antigens was a good ligand for WGA. These results suggested that the WGA-ligand in C. elegans is a cluster of alpha-GalNAc monosaccharides linked to mucin-like glycoprotein(s). The observations reported in this paper emphasize the possible significance of mucin-type O-glycans in the development of a multicellular organism.

Endo-beta-mannosidase is an endoglycosidase that hydrolyzes only the Man beta 1-4GlcNAc linkage of the core region of N-linked sugar chains. Recently, endo-beta-mannosidase was purified to homogeneity from Lilium longiflorum (Lily) flowers, its corresponding gene was cloned and important catalytic amino acid residues were identified [Ishimizu T., Sasaki A., Okutani S., Maeda M., Yamagishi M. & Hase S. (2004) J. Biol. Chem.279, 38555-38562]. In the presence of Man beta 1-4GlcNAc beta 1-4GlcNAc-peptides as a donor substrate and p-nitrophenyl beta-N-acetylglucosaminide as an acceptor substrate, the enzyme transferred mannose to the acceptor substrate by a beta1-4-linkage regio-specifically and stereo-specifically to give Man beta 1-4GlcNAc beta 1-pNP as a transfer product. Further studies indicated that not only p-nitrophenyl beta-N-acetylglucosaminide but also p-nitrophenyl beta-glucoside and p-nitrophenyl beta-mannoside worked as acceptor substrates, however, p-nitrophenyl beta-N-acetylgalactosaminide did not work, indicating that the configuration of the hydroxyl group at the C4 position of an acceptor is important. Besides mannose, oligomannoses were also transferred. In the presence of (Man)(n)Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc-peptides (n = 0-2) and pyridylamino GlcNAc beta 1-4GlcNAc, the enzyme transferred (Man)(n)Man alpha 1-6Man en bloc to the acceptor substrate to produce pyridylamino (Man)(n)Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc (n =0-2). Thus, the lily endo-beta-mannosidase is useful for the enzymatic preparation of oligosaccharides containing the mannosyl beta 1,4-structure, chemical preparations of which have been frequently reported to be difficult.

化学的に均一な糖鎖 (主に蛍光ラベルされた糖鎖) を用いたいくつかの糖鎖関連酵素の基質特異性解析は、酵素の生体内での基質を明らかにする。その解析により、酵素が加水分解部位あるいは糖転移部位のみならず、その部位からかなり離れた基質部分も認識する場合のあることが明らかになってきた。5糖、6糖、場合によっては10糖以上の単位で糖鎖を認識し、ある部分は厳密に、ある部分はあいまいに認識している。また基質特異性が相補的な酵素の組み合わせがあることも明らかになってきた。これらの一例を記述し、糖鎖関連酵素の厳密なそして寛容な広い領域の基質認識の生物学的意義について議論したい。

Endo-β-mannosidase is a novel endoglycosidase that hydrolyzes the Manβ1-4GlcNAc 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-β-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 Manα1-6Manβ1-4Glc-NAcβ1-4GlcNAc into Manα1-6Man and GlcNAcβ1-4Glc-NAc-PA, showing that AtEBM is an endo-β-mannosidase. AtEBM hydrolyzed MannManα1-6Manβ1-4GlcNAcβ1-4GlcNAc-PA (n = 0–2) but not PA-sugar chains containing Manα1-3Manβ or Xyloseβ1-2Manβ as for the lily endo-β-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 β-mannosidases. Indeed, AtEBM-like genes are only found in plants, indicating that endo-β-mannosidase is a plant-specific enzyme. The role of this enzyme in the processing and/or degradation of N-glycan will be discussed.

Solid-phase synthesis of glycopeptide generally requires the protection of both peptide side chains and hydroxyl groups of the carbohydrate portion. However, if the mild coupling conditions are used, the protection of the carbohydrate portion can be omitted. In this paper, we demonstrated it by the synthesis of Fmoc-serine carrying unmasked xylosyl glucose followed by the solid-phase synthesis of epidermal growth factor (EGF)-like domain of factor IX (45-87) using the unit. The product was well characterized by enzymatic digestion, amino acid analysis and mass spectrometry. The secondary structure of the product as well as glucosylated and non-glycosylated EGF-like domain was characterized by circular dichroism (CD) spectroscopy. Published in 2003.

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.

In order to detect sugar chains specific to a tissue or a disease, we introduced the differential display of glycans using pyridylaminated (PA) sugar chains combined with two-dimensional HPLC sugar mapping. The method is fairly sensitive, and 2 mg of freeze-dried tissue was enough to detect the peaks due to PA- N -linked sugar chains, without any pretreatment of the tissue. The structure of a specific PA-sugar chain identified is determined by using two-dimensional HPLC sugar mapping combined with exoglycosidase digestion. The method was applied to find brain-type and developmentally regulated sugar chains.

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 rice bran 57-kDa protein was isolated by affinity chromatography with fibronectin immobilized on agarose. This fibronectin-binding protein designated as RB-57 had an amino-terminal amino acid sequence identical with that of a putative mature form of rice hydroxyproline-rich glycoprotein. A distinct feature of the amino acid composition of RB-57 was the high contents of hydroxyproline and proline representing about 45% of the total amino acids. The sugar analysis indicated that arabinose represented 46.8% of the total carbohydrates. RB-57 showed cell adhesion activity for murine Lewis lung carcinoma cells. The result suggests that RB-57 may play a role in plant cell adhesion, although cell adhesion-promoting activity for plant cells remains to be tested.

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.

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.

S-RNases encoded by the S-locus of rosaceous and solanaceous plants discriminate between the S-alleles of pollen in gametophytic self-incompatibility reactions, but it is not clear how. We report the structures of N-glycans attached to each of the N-glycosylation sites of seven S-RNases in Pyrus pyrifolia of the Rosaceae. The structures were identified by chromatographic analysis of pyridylaminated sugar chains prepared from S4-RNase and by liquid chromatography/electrospray ionization-mass spectrometric analysis of the protease digests of reduced and S-carboxymethylated S-RNases. S4-RNase carries various types of sugar chains, including plant-specific ones with beta1--2-linked xylose and alpha1--3-linked fucose residues. More than 70% of the total N-glycans of S4-RNase are, however, an N-acetylglucosamine or a chitobiose (GlcNAcbeta1--4GlcNAc), which has not been found naturally. The N-acetylglucosamine and chitobiose are mainly present at the N-glycosylation sites within the putative recognition sites of the S-RNase, suggesting that these sugar chains may interact with pollen S-product(s).

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.

A partially automated technique for the isolation and characterization of N-linked sugar chains from glycoproteins of crude tissue samples is established. The N-linked sugar chains from the acetone-extracted tissues are made free by a process of hydrazinolysis and subsequently N-acetylated by GlycoPrep 1000 (Oxford Glycosystems). These free sugar chains are further converted to pyridylamino derivatives by GlycoTag (Takara). Characterization of these sugar chains is achieved by a combination of HPLC columns using a highly sensitive fluorescence detector at femtomole levels. Tissue sample can be successfully pyridylaminated and analyzed to give highly reproducible results with consistent yield, requiring fewer purification steps, minimum skills, and less time. Moreover, fixed tissues can also be analyzed employing this technique, giving a similar sugar chain pattern compared to normal tissue samples. Using this method we show that the pattern of N-linked sugar chains present in human sera or in one small region of brain is strikingly similar among the different individuals. However, the absence of a highlighted peak in one of the samples suggests this method can be extrapolated to identify changes, if any, associated with disorders such as inflammation or cancer. Furthermore, this two-dimensional display of sugar chains would discover the function-specific molecules as we see in proteins.

O-linked sugar chains with xylose as a reducing end linked to human urinary soluble thrombomodulin were studied. Sugar chains were liberated by hydrazinolysis followed by N-acetylation and tagged with 2-aminopyridine. Two fractions containing pyridylaminated Xyl as a reducing end were collected. Their structures were determined by partial acid hydrolysis, two-dimensional sugar mapping combined with exoglycosidase digestions, methylation analysis, mass spectrometry, and NMR as SO4-3GlcAbeta1-3Galbeta1-3(+/-Siaalpha2-6)Galbeta1+ ++-4Xyl. These sugar chains could bind to an HNK-1 monoclonal antibody. This is believed to be the first example of a proteoglycan linkage tetrasaccharide with glucuronic acid 3-sulfate and sialic acid.

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.

A new sensitive and convenient method for the structural analysis of oligosaccharides was developed, in which partial acid hydrolysis was combined with two-dimensional sugar mapping of pyridylamino (PA)-oligosaccharides. A PA-oligosaccharide was partially hydrolyzed, the acid hydrolysis conditions being optimized so as to obtain various PA-oligosaccharide fragments with high yields from different types of PA-oligosaccharides. The acid hydrolyzates were then fractionated every 1 glucose unit by size-fractionation HPLC, and each fraction was analyzed by reversed-phase HPLC. The structure of each PA-oligosaccharide fragment was identified on a two-dimensional sugar map prepared with standard PA-sugar chains, after which the original PA-oligosaccharide was reconstructed from the reducing end terminal based on the obtained structures of the PA-oligosaccharide fragments. The method was successfully applied to resolving the structure of an N-linked sugar chain.

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.

HepG2 cells were examined for the presence of UDP-D-xylose: beta-D-glucoside alpha-1,3-xylosyltransferase activity by using 2-[(2-pyridyl)amino]ethyl beta-D-glucopyranoside (Glc beta-R) as a fluorogenic acceptor. UDP-D-xylose and the acceptor were incubated with the HepG2 microsomal fraction, and the enzymatic reaction mixture was analyzed by HPLC. Structural analysis of the fluorogenic product by alpha-D-xylosidase digestion, FAB-MS, and Smith degradation revealed that it was Xyl alpha 1-3Glc beta-R, thus demonstrating the existence of beta-D-glucoside alpha-1,3-xylosyltransferase. A solubilization study of the enzyme from the microsomal fraction indicated that it differed from UDP-D-xylose: alpha-D-xylodie alpha-1,3-xylosyltransferase of the HepG2 microsomal fraction producing Xyl alpha 1-3Xyl alpha 1-3Glc-R' [R', (2-pyridyl)amino-] from UDP-D-xylose and Xyl alpha 1-3Glc-R' [Minamida, S., Aoki, K., Natsuka, S., Omichi, K., Fukase, K., Kusumoto, S. & Hase, S. (1996) J. Biochem. (Tokyo) 120, 1002-1006]. The newly detected enzyme appears to be involved in the biosynthesis of the Xyl alpha 1-3Glc beta-Ser structure of glycoproteins such as human blood coagulation factors VII and IX.

Uridine 5′-diphospho-α- d -xylopyranose, which donates d -xylose during glycoconjugate biosynthesis, was chemically synthesized from α- d -xylose 1-phosphate and uridine 5′-monophosphoimidazolide.

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.

Two-dimensional HPLC mapping of O-linked sugar chains was studied. Twenty-seven pyridylamino (PA-) derivatives of O-linked sugar chains liberated from glycoproteins by hydrazinolysis-N-acetylation were separated by reversed-phase and size-fractionation HPLC. Combining the elution positions obtained by these two HPLC techniques allows the construction of a two-dimensional sugar map which can be used to analyze the structure of PA-O-linked sugar chains. As in the case of PA-N-linked sugar chains, the additivity rule which correlates chemical structures with elution positions was also found in the case of PA-O-linked sugar chains. This separation system was effectively applied to the elucidation of minute amounts of PA-O-linked oligosaccharides.

Sensitive detection and high chromatographic resolution are required for analysing the structures of oligosaccharides available in only limited amounts. Precolumn derivatization is one of the most suitable methods for this purpose as it can compensate for the shortcomings of the intrinsic nature of oligosaccharides. Recently published methods of precolumn derivatization are reviewed and advantages and problems are discussed.

New bifunctional reagents, 2-amino-6-carboxyethylpyridine and 2-amino-6-cyanoethylpyridine, were designed and synthesized in order to provide a novel procedure for preparation of neoglycoconjugates from fluorescence-labeled and purified sugar chains. Labeling of model sugar chains with these reagents was effected by reductive amination using BH3.Me2NH to give corresponding 6-carboxyethylpyridylaminated (CEPA-) and 6-cyanoethylpyridylaminated (CNEPA-) derivatives, which were readily purified by reversed phase HPLC. The reagent parts of the labeled products possess the functional groups which then serve as linkers for coupling with matrices such as proteins and polymers. A CEPA-derivative of glucose was directly coupled with the e-amino group of a Lys derivative to give a neoglycoprotein model. CNEPA-sugars were hydrogenated to give 6-aminopropylpyridylaminated (APPA-) derivatives, which can then be readily used for the preparation of various types of neoglycoconjugates by use of appropriate spacer...

Oligosaccharides are often converted to fluorogenic pyridylamino-oligosaccharides (PA-oligosaccharides) to be analyzed sensitively. A method for determining the glycosidic linkage position to the PA-reducing-end residue was developed with PA-disaccharides as model compounds. Periodate oxidation of PA-disaccharides was carried out at 0 degrees C for 15 min or at 4 degrees C for 40 h, and the reaction mixtures were reduced with borohydride. The fluorogenic products obtained at 4 degrees C for 40 h were purified by reversed phase HPLC, and the fraction collected were hydrolyzed with acid. The hydrolysates were analyzed by reversed phase HPLC. PA-glyceraldehyde was formed from 2-substituted PA-disaccharides with PA-hexose, PA-threose (or PA-erythrose) from 3-substituted ones, and PA-glycolaldehyde from 4- or 6-substituted ones. HPLC analysis of the products obtained at 0 degrees C for 15 min revealed a difference between 4- and 6-substituted ones. PA-glyceraldehyde was formed from 6-substituted ones, but not from 4-substituted ones. The linkage position, therefore, can be determined by analyzing fluorogenic product(s). As for PA-disaccharides with PA-N-acetylglucosamine, the linkage position can be simply determined by analysis of 40-h oxidation-reduction mixtures. 2-Acetamido-2-deoxy derivatives of PA-threose, PA-xylose, and PA-glyceraldehyde were formed from 3-, 4-, and 6-substituted ones, respectively. The linkage position analysis was successfully applied to determination of the structures of two Fuc-Man-PAs produced through the transglycosylation action of bovine kidney alpha-L-fucosidase.

Human salivary and pancreatic α-amylases (HSA and HPA) are the respective gene products of the AMY1 and AMY2A genes. AMY2B is a newly found human α-amylase gene. The presence of the AMY2B gene product (HXA) in the urine of healthy humans was examined. A mixture of α-amylases that seemed to contain HXA, judging from the substrate specificity, was purified from urine of healthy volunteers by affinity adsorption on starch and then by ion-exchange chromatography. The mixture was reduced and S -alkylated, and the product was digested with trypsin. The digest was separated by reversed-phase HPLC. LVGLLDLALEKDYVR and LVGLLDLALEK, which were found in the digest, are peptids of HXA, but not of HSA and HPA. The detection of these characteristic peptides of HXA demonstrates the presence of HXA in the urine of healthy humans.

The structures of the sugar chains of hen yolk riboflavin-binding protein (RBP) were established. Asparagine-linked sugar chains of yolk-RBP were liberated by hydrazinolysis. Free amino groups of the sugar chains were acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine. Fluorescent pyridylamino (PA-) derivatives of the sugar chains were purified by gel-filtration and reversed-phase HPLC. Seven PA-sugar chains were isolated, and the structure of each was determined by composition analysis, sequential exoglycosidase digestion, methylation analysis, and 500-mHz 1H-NMR spectroscopy. These analyses showed that the main sugar chains had sialylbiantenna and sialyltriantenna structures. PA-sugar chains of plasma-RBP were also isolated, and the structures of the PA-sugar chains of yolk- and plasma-RBPs were compared as to their elution patterns on anion-exchange chromatography and reversed-phase HPLC. The plasma RBP had almost the same sugar chains as the yolk RBP did, indicating that sugar chains are not modified during incorporation into the oocyte.

The di- and trisaccharide sequences and their conjugates with serine recently identified in the first epidermal growth factor-like domain of bovine and human blood-clotting factors VII, IX, and protein Z were synthesized in order to elucidate their biological function. The disaccharide α-D-Xylp(1→3)-D-Glcp was prepared from 2,3,4-tri-O-benzyl-α-D-xylopyranosyl fluoride and allyl 2,4,6-tri-O-acetyl-α-D-grucopyranoside (9a) using tin(II) chloride and silver perchlorate as glycosylating reagents. The trisaccharide α-D-Xylp(1→3)-α-D-Xylp(1→3)-D-Glcp was prepared from O-(2,3,4-tri-O-benzyl-α-D-xylopyranosyl)-(1→3)-2,4-di-O-benzyl-α,β-D-xylopyranosyl fluoride and 9a with the same reagents. Their conjugates with serine, α-D-Xylp(1→3)-β-D-Glcp(1→3)-L-Ser and α-D-Xylp(1→3)-α-D-Xylp(1→3)-β-D-Glcp(1→3)-L-Ser, were synthesized by the glycosylation of N-benzyloxycarbonyl-L-serine benzyl ester with the corresponding glycosyl trichloroacetimidates of the above saccharides, followed by removal of the protecting groups. T...

Comparative analyses of the N-glycosylation pattern of hemolymph glycoproteins from Biomphalaria glabrata strains Puerto Rico (BgPR) and Salvador (BgBS-90), differing in their susceptibility towards Schistosoma mansoni infection, were performed by Western blotting, enzyme-linked immunosorbent assays, two-dimensional high-performance liquid chromatography and mass spectrometry. Obtained data demonstrated an enhanced expression of serologically cross-reacting, fucosylated carbohydrate epitopes by the highly susceptible BgPR-strain in comparison to the resistant BgBS-90-strain. In particular, glycoproteins of BgPR snails exhibited larger amounts of glycans with (β1-2)-linked xylose or terminal Fuc(α1-3)GalNAc(β1-4)[±Fuc(α1-3)]GlcNAc(β1-)-units which are known to mediate cross-reactivity with schistosomal glycoconjugates. This finding could be corroborated by immunohistochemical studies showing again an enhanced expression of such carbohydrate epitopes in BgPR tissue. Hence, our results provide evidence for a correlation of B. glabrata susceptibility towards S. mansoni infection and the expression of carbohydrate determinants shared by the parasite and its intermediate host.

A method for preparation of pyridylamino (PA-) derivatives of O-linked sugar chains from glycoproteins was developed. A glycopeptide containing O-linked Gal beta 1-3GalNAc was prepared from fetuin and treated with anhydrous hydrazine followed by N-acetylation of free amino groups. Sugar chains released were pyridylaminated with improved reaction conditions and excess reagents were removed by gel filtration. Gal beta 1-3GalNAc-PA obtained together with PA-Gal as a by-product was quantified by HPLC. Conditions for the hydrazine treatment were investigated and the treatment at 40 degrees C for 350 h gave the best results for releasing O-linked sugar chains. The total yield of Gal beta 1-3GalNAc-PA from the glycopeptide was 53% under the established conditions and that of PA-Gal was 18%. The present method was applied to a glycoprotein, and the expected PA-O-linked sugar chains were obtained. Under these conditions, N-linked sugar chains were also released.

Carbohydrate-binding specificity of Con A was characterized by competitive binding studies of pyridylamino (PA) sugar chains. PA-derivatives of 17 oligomannose-type sugar chains, Man1-9GlcNAc2-PA, and those of three complex-type sugar chains were used as ligands. The ratios of bound and free sugar concentrations, [LS]/[S], were determined by means of microequilibrium dialysis followed by high performance liquid chromatography as already reported [Mega, T. & Hase, S. (1991) J. Biochem. 109, 600-603]. The association constant, Ka, was calculated from [LS]/[S] of a sugar chain and that of a standard sugar chain by using the equation Ka = Ka0 x ([S0]/[LS0]) x ([LS]/[S]), where Ka0, [S0], and [LS0] are the association constant, and the free and bound ligand concentrations of the standard sugar chain, respectively. This calculation was effective for the determination of Ka of ligands with similar affinities to the standard sugar chain. The carbohydrate structures with highest affinity for Con A among those tested were found to be: [formula: see text]

The di- and trisaccharide sequences and their conjugates with serine recently identified in the first epidermal growth factor-like domain of bovine and human blood-clotting factors VII, IX, and protein Z were synthesized in order to elucidate their biological function. The disaccharide α-D-Xylp(1→3)-D-Glcp was prepared from 2,3,4-tri-O-benzyl-α-D-xylopyranosyl fluoride and allyl 2,4,6-tri-O-acetyl-α-D-grucopyranoside (9a) using tin(II) chloride and silver perchlorate as glycosylating reagents. The trisaccharide α-D-Xylp(1→3)-α-D-Xylp(1→3)-D-Glcp was prepared from O-(2,3,4-tri-O-benzyl-α-D-xylopyranosyl)-(1→3)-2,4-di-O-benzyl-α,β-D-xylopyranosyl fluoride and 9a with the same reagents. Their conjugates with serine, α-D-Xylp(1→3)-β-D-Glcp(1→3)-L-Ser and α-D-Xylp(1→3)-α-D-Xylp(1→3)-β-D-Glcp(1→3)-L-Ser, were synthesized by the glycosylation of N-benzyloxycarbonyl-L-serine benzyl ester with the corresponding glycosyl trichloroacetimidates of the above saccharides, followed by removal of the protecting groups. T...

The substrate specificity of neutral alpha-mannosidase purified from Japanese quail oviduct [Oku, H., Hase, S., & Ikenaka, T. (1991) J. Biochem. 110, 29-34] was analyzed by using 21 oligomannose-type sugar chains. The enzyme activated with Co2+ hydrolyzed the Man alpha 1-3 and Man alpha 1-6 bonds from the non-reducing termini of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc (M5A), but hardly hydrolyzed the Man alpha 1-2 bonds of Man9GlcNAc2. The hydrolysis rate decreased as the reducing end of substrates became more bulky: the hydrolysis rate for the pyridylamino (PA) derivative of M5A as to that of M5A was 0.8; the values for M5A-Asn and Taka-amylase A having a M5A sugar chain being 0.5 and 0.04, respectively. The end product was Man beta 1-4GlcNAc2. For the substrates with the GlcNAc structure at their reducing ends (Man5GlcNAc, Man6GlcNAc and Man9GlcNAc), the hydrolysis rate was remarkably increased: Man5GlcNAc was hydrolyzed 16 times faster than M5A, and Man2GlcNAc 40 times faster than Man9GlcNAc2. The enzyme did not hydrolyze Man alpha 1-2 residue(s) linked to Man alpha 1-3Man beta 1-4GlcNAc. The end products were as follows: [formula; see text] These results suggest that oligomannose-type sugar chains with the GlcNAc structure at their reducing ends seem to be native substrates for neutral alpha-mannosidase and the enzyme seems to hydrolyze endo-beta-N-acetylgucosaminidase digests of oligomannose-type sugar chains in the cytosol.