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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.

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.

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.

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.

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 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).

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.

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.

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.

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.

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.

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...

An alpha-mannosidase was purified from the magnum section of Japanese quail oviduct by ammonium sulfate precipitation, DEAE-Sephacel chromatography, Sephacryl S-300 chromatography, mannan-Sepharose 4B chromatography, and hydroxyapatite chromatography. The purified alpha-mannosidase (referred to as neutral alpha-mannosidase) showed a single band on polyacrylamide gel with or without sodium dodecyl sulfate. Its molecular weight was found to be 330,000 by gel chromatography. Neutral alpha-mannosidase hydrolyzed p-nitrophenyl alpha-D-mannopyranoside and the pyridylamino derivative of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc (Km value was 3 mM). Mannosyl alpha 1-2 linkages in the pyridylamino derivative of Man alpha 1-2 Man alpha 1-6(Man alpha 1-2Man alpha 1-3)Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc were hardly hydrolyzed. Its optimum pH was found to be 7.0. The activity of the enzyme was activated by CO2+, and was potently inhibited by Cu2+, Hg2+, swainsonine, and 1-deoxymannojirimycin.

The active site of human salivary alpha-amylase is composed of tandem subsites (S3, S2, S1, S1',S2', etc.) geometrically complementary to several glucose residues, and the glycosidic linkage of the substrate is split between S1 and S1'. As a matter of convenience, the subsites to which the non-reducing-end part (glycone) and the reducing-end part (aglycone) of the substrate being hydrolyzed are bound are named the glycone-binding site (S3, S2, S1) and the aglycone-binding site (S1', S2'), respectively. The features of the aglycone-binding site of human salivary alpha-amylase were examined by means of transglycosylation reaction using phenyl alpha-maltoside (GG phi: G-G-phi) and its derivatives (GAG phi: G-AG-phi, GCG phi: G-CG-phi, AGG phi: AG-G-phi, and CGG phi: CG-G-phi) in which one of the glucose residues (G) has been converted to 6-amino-6-deoxy-glucose (AG) or glucuronic acid (CG) residue as the acceptor. A fluorogenic derivative of maltotetraose, p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D -glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG4P, FG-G-G-G-P), was used as the substrate. HSA catalyzed both hydrolysis of FG4P to FG3 (FG-G-G) and p-nitrophenyl alpha-glucoside (G-P) and transfer of the FG3 residue of FG4P to the acceptors. Transfer to GAG phi occurred more effectively than to GG phi. Transfers to GCG phi and CGG phi were less than to GG phi and very little transfer to AGG phi occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Polygalacturonic acid (PGA) synthase successively transfers galacturonic acid to oligogalacturonic acid by an alpha1,4-linkage to synthesize PGA, the backbone of plant pectic homogalacturonan. PGA synthase has not been purified to date due to its instability in vitro. In this study, we found stable conditions in vitro and separated a minimum active component of the enzymes from pea and azuki bean epicotyls. The PGA synthase lost its activity in 500 mM of sodium chloride or potassium chloride, while it was relatively stable at low salt concentrations. Under low salt concentrations, three peaks bearing PGA synthase activity were separated, by gel filtration and sucrose density gradient centrifugation. The molecular masses of these enzymes solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonic acid were estimated to be 21,000, 5,000, and 590 kDa. The two higher molecular mass PGA synthases converted to smaller PGA synthase proteins when treated with high salt concentrations, while retaining their activity, indicating that PGA synthase has a minimum active component for its activity.

Biosynthesis of N-glycans varies significantly among tissues and is strictly regulated spatially and temporally within the tissue. The strict molecular mechanisms that are responsible for control of N-glycan synthesis remain largely unknown. We developed complementary deoxyribonucleic acid (cDNA) macroarray system and analyzed gene expression levels of more than 140 glycosyltransferases and glycosidases in the cerebral cortex from developing and adult mice. We also analyzed the relative amounts of major N-glycans present in the cerebral cortex and examined how the synthesis of N-glycans might be regulated through the expression of these genes. We demonstrated that the content of N-linked oligosaccharides dramatically changed during the course of brain development. Some of these changes could not be explained by alterations in the expression of the corresponding genes. For example, the amount of core fucosylated sugar chains in the early embryonic brain and the expression level of fucosyltransferase VIII, the only gene known to be responsible for core fucosylation, did not change proportionately. This result suggests that post-transcriptional regulation of this gene plays an important role in regulating its enzymatic activity. On the other hand, the amount of beta1,3-galactose residue-containing sugar chains increased postnatally following an increase in the level of beta1,3-galactosyltransferase messenger ribonucleic acid (mRNA). Furthermore, the amount of sugar chains with an outer fucose residue, containing LewisX-BA-2, correlated well with the expression of fusocyltransferase IX mRNA. These findings add to our understanding of the molecular mechanisms responsible for the regulation of N-glycan biosynthesis in the cerebral cortex.

To clarify the structure of non-sialic acid anionic residue on N-glycans in the mammalian tissues, we have isolated sialidase-resistant anionic residue on N-glycans from bovine lung. Analyses by partial acid hydrolysis and glycosidase digestions combined with a two-dimensional HPLC mapping method revealed that the major sialidase-resistant anionic N-glycan had a fucosylbianntenary core structure. The anionic residue was identified as a sulfate ester by methanolysis, anion-exchange chromatography, and mass spectrometry. The linkage position of the sulfate ester was the 6-position of the GlcNAc residue on the Manalpha1-6 branch. This conclusion was based on the results of glycosidase digestions followed by two-dimensional HPLC mapping. Furthermore, the disialylated form of this sulfated glycan was dominant, and no asialo form was detected. The structure of the major anionic N-glycan prepared from bovine lung and having a sulfate was proposed to be the pyridylamino derivative of Siaalpha2-3Galphalbeta1-4(HSO(3)-6)GlcNAcbeta1-2Manalpha1-6(Siaalpha2-3Galbeta1-4GlcNAcbeta1-2Manalpha1-3)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc.

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

Oligosaccharides with biantennae and bisecting N-acetyl glucosamine (GlcNAc) residues attached to the mannose in the beta1-4 trimannosyl core (BA2) are enriched in the brain and considered brain-type sugar chains. We investigated the significance of the interplay between galactosyltransferase I (GalTase I) and BA2 formation in a brain-derived cell line, CG4. Increased GalTase expression in different glial- and neuronal-derived cell lines was accompanied by decreased or undetectable levels of BA2, depending on the level of GalTase expression. Forceful expression of GalTase I in CG4 cells expressing high levels of BA2 and low GalTase activity significantly reduced BA2 levels. In addition, a sixfold increase in an abnormal sugar chain A1(6)G1Fo and a moderate increase in A2G2Fo(6)F were evident. The increased levels of A1(6)G1Fo indicate a diversion or abrogation of the N-linked sugar chain biosynthetic pathway from normal. The accumulation of A1(6)G1Fo and increased A2G2Fo(6)F levels were accompanied by decreased levels of the high mannose-type sugar chains, M5A, M6B, M8A, and M9A. Increased GalTase I expression also led to stunted growth and abnormal morphology of CG4 cells, with increased mortality. Even moderate overexpression of GalTase I thus disrupts the normal biosynthetic pathway of N-linked sugar chains, and high overexpression is fatal to CG4 cells.

Human urine collected from healthy individuals was ultrafiltered and the filtrate was gel-filtered. The fraction including disaccharides was pyridylaminated to convert the reducing sugars to fluorescent pyridylamino (PA)-derivatives. A PA-disaccharide consisting of Fuc and Man was purified by gel filtration, reversed-phase HPLC, and size fractionation HPLC. Structural analysis revealed that the disaccharide was Fuc alpha 1-2Man-PA. The disaccharide is considered to be a metabolite of unknown glycoconjugates.

Publisher Summary This chapter describes the pre- and post-column detection-oriented derivatization techniques in the high-performance liquid chromatography (HPLC) of carbohydrates. Difficulties arise in analyzing carbohydrates by chromatography in the absence of effective chromophores to allow sensitive detection. Colorimetric analysis (post-column labeling) had long been used; however, the detection method is not suitable for derivatization purposes. Monosaccharides and oligosaccharides are detected without any derivatization by measuring their refractive indices or absorption in an ultraviolet (UV) region at 190–210 nm. Although these procedures are reasonably sensitive and suitable for chromatography, problems are often encountered with interfering peaks that arise from strongly UV-absorbing contaminating materials. Thus, their sensitivities are restricted to the nanomole order. Although a pulsed amperometric detector (PAD) combined with HPLC allows the sensitive detection of carbohydrates, PAD needs eluents with relatively high pH values, which imposes several limitations on the HPLC system and causes some modification of reducing-end sugars.

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.

Astrocytes are implicated in many aspects of brain function; however, it remains unclear whether astrocytes arise from a single cell lineage. It is therefore important to obtain new markers for the astrocyte cell lineage. We show that exogenously added UDP-galactose (UDP-Gal) can be used to metabolically label a subset of glial fibrillary acidic protein-positive (GFAP+) cells. UDP-Gal was incorporated into the cultured embryonic mouse brain slices in a time-dependent manner. Surprisingly, the transferred sugar moiety was no longer Gal but was mainly glucose. Most of the radioactivity was transferred to a polymer of glucose, most likely to be glycogen, and also to glucosyl ceramide. In the slice culture, the reaction products were distributed densely in the ventricular zone and also on process-like structures extending to the pial surface. In dissociation culture, UDP-Gal labeled some of the GFAP+ cells and some of the vimentin+ cells. Because radial glial cells (RGCs) contain glycogen and change from vimentin+ to GFAP+, it is strongly suggested that UDP-Gal labeled RGCs and their descendants. Only 27% of the GFAP+ cells were labeled with UDP-Gal, which suggests that only a subset of astrocytes are derived from RGCs and that there is a discrete group of GFAP+ cells that is not generated from RGCs.

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.

A method for preparing neoglycolipids possessing a sugar chain with a definite structure using two N-linked sugar chains as model compounds was developed. Pyridylaminated sugar chains obtained from glycoproteins were purified by reversed-phase HPLC and a pyridylaminated sugar chain thus obtained was converted to a 1-amino-1-deoxy derivative. The product was conjugated by reductive amination with phosphatidylglycolaldehyde prepared by periodate oxidation of dipalmitoyl phosphatidylglycerol. Neoglycolipids formed were purified by gel filtration from the reaction mixture. The structures of the purified neoglycolipids were confirmed by composition analysis for sugar, fatty acid, phosphorus, and 2-acetamido- 1,2-dideoxy-1-[(hydroxyethyl)amino]- D -glucitol, the link- age region of the sugar chain, and lipid moieties. The method provides a convenient means of preparing neoglycolipids having a definite structure.

Sugar chain types were classified on the basis of reducing end di- and trisaccharide structures. Sugar chains liberated from glycoproteins by the hydrazinolysis-N-acetylation method were pyridylaminated, and pyridylamino (PA-) sugar chains were purified by HPLC. The PA-sugar chains thus purified were partially hydrolyzed with 1 M trifluoroacetic acid. The acid hydrolysis conditions were investigated with the object of obtaining PA-di- and trisaccharides with high yields for different types of PA-sugar chains. The acid hydrolysates were separated by size-fractionation HPLC into PA-mono-, PA-di-, and PA-trisaccharides, and each fraction was analyzed by reversed-phase HPLC. The structures were then identified by comparing the HPLC elution positions with those of authentic PA-oligosaccharides derived from N-linked sugar chains and 12 types of O-linked sugar chains.

Oligomers made of Asp-52-esterified lysozyme or native one showed agglutination for human, mouse, rat and chicken erythrocytes, and also for Hep G2 cells (liver carcinoma-derived cell line) [1] not weaker than wheat germ agglutinin(WGA). Oligomers of Asp-52-esterified lysozyme have about 4-fold stronger agglutinating activity than those of the native one. These results indicate that some enzymes can be converted to lectin-like proteins (neolectin) with binding characteristics similar to their substrats specificity.

Publisher Summary This chapter discusses the high-performance liquid chromatography of pyridylaminated (PA) saccharides. Pyridylamination involves reductive amination and removal of the pyridyl group. These reactions have been used mostly for asparagine-linked saccharides. A sample from glycoconjugates obtained by hydrazinolysis- N -acetylation or by an enzyme reaction is pyridylaminated without purification of saccharides. Most excess reagents are removed first by evaporation or by extraction with chloroform. The small excess that remains is removed by gel filtration. Instead of evaporation, most of the excess reagents can be removed by extraction with chloroform before gel filtration. On a preparative scale, the amount of reagents is increased in proportion to the amount of saccharides or glycoconjugates. The large amounts of the saccharides or glycoconjugates should be employed together with a known PA saccharide as a standard for chromatography while using the pyridylamination technique. The peaks obtained are not always from saccharides, although most peaks are from PA saccharides. This can be confirmed by exoglycosidase digestion or component sugar analysis.

The structures of N-linked oligosaccharides, especially the distribution of sialic acid species, present on porcine plasma vitronectin were elucidated. Oligosaccharides were released from the vitronectin by N-glycosidase F digestion and tagged with 2-aminopyridine, and the pyridylamino-oligosaccharides were fractionated by anion-exchange and reverse-phase HPLC. Nine major pyridyl-amino-oligosaccharides were isolated. The linkages and locations of sialic acids were determined by a novel approach involving desialylation with Salmonella sialidase in combination with acid desialylation. After desialylation, the asialo-forms were analyzed by two-dimensional sugar mapping, component sugar analysis and 400-MHz 1H-NMR spectroscopy. The major oligosaccharides of porcine vitronectin were of the fucosylated biantennary type, with a small amount of the triantennary N-acetyllactosamine type, to which 1-3 mol sialic acids was linked. Sialic acids were linked predominantly through alpha 2-6 linkages, although alpha 2-3 linkages were also present, and fucose was linked to the innermost N-acetylglucosamine through an alpha 1-6 linkage. It was found that every pyridylamino-oligosaccharide population contained N-glycolylneuraminic acid and N-acetylneuraminic acid in a molar ratio of 1:2-9, and that N-glycolylneuraminic acids were located predominantly on the Man alpha 1-6 arm.

Biologically active molecules are often present in minute quantity. Much effort and many samples are required to isolate such molecules and to analyze their structures. When sugar chains are analyzed as molecules with biological activity, a method is needed to isolate and analyze sugar chains with a high sensitivity. Therefore, we developed the pyridylamination method (Hase et al., 1978) for sugar chains and improved this method to apply it to sugar chains from glycoproteins (Hase et al., 1984).

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.

The concentrations of free fucose and other sugars in urine of cancer patients and healthy subjects were analyzed by high-performance liquid chromatography. After the urine samples were dried, we coupled the sugars in the residue with 2-aminopyridine to be detected as fluorescent derivatives. We then analyzed the pyridylamino derivatives of the sugars with an anion-exchange column and borate buffer. The difference between cancer patients and healthy subjects for the mean concentrations of fucose corrected for creatinine was significant (P less than 0.001). We checked the relationship between the concentrations of other sugars and the presence of cancer. This method is highly sensitive, and neither a cleanup procedure before labeling nor purification before injection into the column is needed. Not only fucose but also other sugars can be detected simultaneously, so this method should be useful for studying any changes in sugars in urine in various diseases.

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.

Ten oligomannose-type sugar chains (ManGlcNAc2-Man5GlcNAc2) were prepared from various glycoproteins and fluorescence labeled with 2-aminopyridine. The fluorescent pyridylamino (PA)-sugar chains were first separated into five fractions according to their molecular sizes by HPLC on a TSK gel Amide-80 column. Each fraction was then separated into the component PA-sugar chains by reversed-phase HPLC on a Capcell Pak C18 column according to their chemical structures. The method is useful for studying the substrate specificities of alpha-mannosidases with Man5GlcNAc2-PA as a substrate.