O-linked Sugar Chains Research Articles

Role of Cellulose-Binding Domain of Exocellulase I from White Rot Basidiomycete Irpex lacteus.

The core fragment (designated P-42), devoid of the cellulose-binding domain (CBD) in the C-terminus and prepared from Irpex lacteus exocellulase I (Ex-1), was isolated by limited proteolysis using papain. Both the hydrolytic activity and binding ability of the isolated P-42 toward insoluble cellulose were lower than those of the native Ex-1, whereas Ex-1 and P-42 showed similar hydrolytic activities toward soluble substrates. These results indicate that the CBD of I. lacteus Ex-1 is the important domain which could enhance hydrolytic activity and binding ability of the enzyme toward insoluble cellulose. In addition, the isolated P-42 was different from the native Ex-1 in terms of enzymatic properties such as pH and temperature stabilities. These differences in stability, with regard to pH and temperature, between P-42 and the native Ex-1 are probably due to the O-linked sugar chains existing in the linker region.

Role of cellulose-binding domain of exocellulase I from White rot basidiomycete Irpex lacteus

The core fragment (designated P-42), devoid of the cellulose-binding domain (CBD) in the C-terminus and prepared from Irpex lacteus exocellulase I (Ex-1), was isolated by limited proteolysis using papain. Both the hydrolytic activity and binding ability of the isolated P-42 toward insoluble cellulose were lower than those of the native Ex-1, whereas Ex-1 and P-42 showed similar hydrolytic activities toward soluble substrates. These results indicate that the CBD of I. lacteus Ex-1 is the important domain which could enhance hydrolytic activity and binding ability of the enzyme toward insoluble cellulose. In addition, the isolated P-42 was different from the native Ex-1 in terms of enzymatic properties such as pH and temperature stabilities. These differences in stability, with regard to pH and temperature, between P-42 and the native Ex-1 are probably due to the O-linked sugar chains existing in the linker region.

A journey to the world of glycobiology.

Finding of the deletion phenomenon of certain oligosaccharides in human milk and its correlation to the blood types of the donors opened a way to elucidate the biochemical basis of blood types in man. This success led to the idea of establishing reliable techniques to elucidate the structures and functions of the N-linked sugar chains of glycoproteins. N-Linked sugar chains were first released quantitatively as oligosaccharides by enzymatic and chemical means, and labelled by reduction with NaB3H4. After fractionation, structures of the radioactive oligosaccharides were determined by a series of methods developed for the studies of milk oligosaccharides. By using such techniques, structural rules hidden in the N-linked sugar chains, and organ- and species-specific N-glycosylation of glycoproteins, which afforded a firm basis to the development of glycobiology, were elucidated. Finding of galactose deficiency in the N-linked sugar chains of serum IgG from patients with rheumatoid arthritis, and malignant alteration of N-glycosylation in various tumors opened a new research world called glycopathology. However, recent studies revealed that several structural exceptions occur in the sugar chains of particular glycoproteins. Finding of the occurrence of the Galbeta1-4Fucalpha1- group linked at the C-6 position of the proximal N-acetylglucosamine residue of the hybrid type sugar chains of octopus rhodopsin is one of such examples. This finding indicated that the fucosyl residue of the fucosylated trimannosyl core should no more be considered as a stop signal as has long been believed. Furthermore, recent studies on dystroglycan revealed that the sugar chains, which do not fall into the current classification of N and O-linked sugar chains, are essential for the expression of the functional role of this glycoprotein. It was found that expression of many glycoproteins is altered by aging. Among the alterations of the glycoprotein patterns found in the brain nervous system, the most prominent evidence was found in P0. This protein is produced in non-glycosylated form in the spinal cord of young mammals. However, it starts to be N-glycosylated in the spinal cord of aged animals. These evidences indicate that various unusual sugar chains occur as minor components in mammals, and play important roles in particular tissues.

Unique catalytic and molecular properties of hydrolases from Aspergillus used in Japanese bioindustries.

This review covers the unique catalytic and molecular properties of three proteolytic enzymes and a glycosidase from Aspergillus. An aspartic proteinase from A. saitoi, aspergillopepsin I (EC 3.4.23.18), favors hydrophobic amino acids at P1 and P'1 like gastric pepsin. However, aspergillopepsin I accommodates a Lys residue at P1, which leads to activation of trypsinogens like duodenum enteropeptidase. Substitution of Asp76 to Ser or Thr and deletion of Ser78, corresponding to the mammalian aspartic proteinases, cathepsin D and pepsin, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at 1. In addition, the double mutant T77D/G78(S)G79 of porcine pepsin was able to activate bovine trypsinogen to trypsin by the selective cleavage of the K6-I7 bond of trypsinogen. Deuterolysin (EC 3.4.24.39) from A. oryzae, which contains 1g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. It was concluded that His128, His132, and Asp164 provide the Zn2+ ligands of the enzyme according to a 65Zn binding assay. Deuterolysin is a member of a family of metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand. Acid carboxypeptidase (EC 3.4.16.1) from A. saitoi is a glycoprotein that contains both N- and O-linked sugar chains. Site-directed mutagenesis of the cpdS, cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. A. saitoi carboxypeptidase indicated that Ser153, Asp357, and His436 residues were essential for the enzymic catalysis. The N-glycanase released high-mannose type oligosaccharides that were separated on HPLC. Two, which had unique structures of Man10 GlcNAc2 and Man11GlcNAc2, were characterized. An acidic 1,2-alpha-mannosidase (EC 3.2.1.113) was isolated from the culture of A. saitoi. A highly efficient overexpression system of 1,2-alpha-mannosidase fusion gene (f-msdS) in A. oryzae was made. A yeast mutant capable of producing Man5GlcNAc2 human-compatible sugar chains on glycoproteins was constructed. An expression vector for 1,2-alpha-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. The first report of production of human-compatible high mannose-type (Man5GlcNAc2) sugar chains in S. cerevisiae was described.

Study of the Relationship between Sticky Human Serum IgA1 and Its O-Glycan Glycoform

The high-affinity IgA1 toward jacalin was mostly composed of aggregated IgA1 and abundantly contained the asialo disaccharide, Galβ1,3GalNAc, in the O-linked oligosaccharide in the hinge region [Journal of Biochemistry 120, 92–97 (1996)]. Meanwhile, the removal of sialic acid from IgA1 accelerated the aggregation of the IgA1 molecule [J. Am. Soc. Nephrol. 9, 2048–2054 (1998)]. In order to examine the nature of such a sticky IgA1, affinity chromatography using asialo-IgA1 (deSIgA1)-Sepharose was carried out. Seventeen percent of normal human serum IgA1, 27% of asialo-IgA1 (IgA1-S), and 48% of asialo-, agalacto-IgA1 (IgA1-SG) were bound to the column. Removal of the N-acetylgalactosamine residue from IgA1-SG resulted in a decreasing affinity toward deSIgA1-Sepharose. Thus, the binding ability toward the column was the highest for the IgA1-SG among the deglycosylated IgA1s. On the other hand, heat treatment of IgA1 accelerated the aggregation but decreased its binding ability toward the column. Such heat denaturation probably destroys the structure of the binding site. Since the enzymatic removal of the N-glycan sugar chains did not induce the aggregation and exhibited no effect on the binding, the incomplete O-linked sugar chain on the hinge portion should be directly related to the sticky characteristics of the IgA1 molecule. The binding was non-covalent and not strong because the asialo-, agalacto-hinge glycopeptide was eluted slightly slower than the native one from the column and the bound IgA1 was dissociated in the presence of 1 M NaCl.

Mutual separation of hinge-glycopeptide isomers bearing five N-acetylgalactosamine residues from normal human serum immunoglobulin A1 by capillary electrophoresis

Immunoglobulin A1 (IgA1) from normal human serum is known to have O-linked sugar chains, sialylated Galβ1,3GalNAc, in the hinge portion. In order to reduce the microheterogenity of the sugar chain, the hinge glycopeptide prepared from IgA1 was sequentially treated with neuraminidase and β-galactosidase. The asialo-, agalacto-hinge glycopeptide (HGP-SG) composed of a 33-mer peptide (HP33) and N-acetylgalactosamine (GalNAc) residues was obtained. The HGP-SG was separated into three major peaks, A, B and C, by high-performance liquid chromatography (HPLC). Each glycopeptide fraction was further separated by capillary electrophoresis (CE). Peaks A, B and C with HPLC abundantly contained HP33 bearing five and six N-acetylgalactosamine residues (HGP33-5,6GN), HGP33-4,5GN and HGP33-3,4GN, respectively. Among these glycopeptide peaks, only the HGP33-5GN peak was partly split into two peaks based on the CE analysis – HGP33-5GN-α and -β. The glycopeptide, HGP25-5GN shortened by the thermolysin digest of HGP33-SG was also well separated into the α and β forms by CE analysis. No differences in their mass and peptide portion were observed between HGP25-5GN-α and -β. Therefore, the obtained result might indicate that HGP25-5GN-α was an isomer of HGP25-5GN-β differing in its stereospecific structure of the peptide portion and/or the attachment site of the GalNAc residue.

Aggregated human serum immunoglobulin A1 induced by neuraminidase treatment had a lower number of O-linked sugar chains on the hinge portion

A part of human serum immunoglobulin A1(IgA1) was aggregated by treatment with neuraminidase. Aggregated IgA1 was separated from non-aggregated IgA1 by gel permeation chromatography. The prepared asialo-hinge glycopeptide (asialo-HGP) from both IgA1 subfractions was treated with β-galactosidase to determine the number of O-linked sugar chains attached on the hinge region. Removal of the galactose residue from asialo-HGP resulted in the HPLC separation of three major peaks. MALDI-TOFMS analysis of the glycopeptides also indicated the presence of three HGP components with three, four and five N-acetylgalactosamine (GalNAc) residues, respectively. Comparison of their relative content among the glycopeptide components showed a higher content of the HGP component with a lower number of GalNAc residues on aggregated IgA1. Thus, asialo-HGP prepared from aggregated IgA1 induced by neuraminidase treatment had an incomplete core structure of O-linked oligosaccharides. Especially, the result suggested that the reduced number of the attached O-linked oligosaccharides on IgA1 take part in phenomena such as self-aggregation of asialo-IgA1.

Novel Proteoglycan Linkage Tetrasaccharides of Human Urinary Soluble Thrombomodulin, SO4-3GlcAβ1–3Galβ1–3(±Siaα2–6)Galβ1–4Xyl

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 involvement of mnn4 and mnn6 mutations in mannosylphosphorylation of O-linked oligosaccharide in yeast Saccharomyces cerevisiae

The structure of O-linked acidic oligosaccharide from Saccharomyces cerevisiae was analyzed. The chitinase, exclusively O-glycosylated extracelluar protein, was purified from strains mnn1, mnn1 mnn4, mnn1 mnn6 and Δ kre2 and the oligosaccharides were hydrolyzed by O-linked sugar chain specific hydrazinolysis. The mannosylphosphorylated mannotriose (M3-P-M) was detected in strain mnn1, but not in the other three strains ( mnn1 mnn4, mnn1 mnn6 and Δ kre2). α-Mannosidase treatment and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of mannosylphosphorylated mannotriose revealed that mannosylphosphate was attached to a middle mannose of α-1,2-linked mannotriose. This result indicates that the mnn4 and mnn6 mutations affect the mannosylphosphorylation of O-linked oligosaccharide, together with that of N-linked oligosaccharide. The amount of mannosylphosphorylated mannotriose was 7% of total O-linked oligosaccharides (20% of neutral mannotriose) of chitinase in strain mnn1.

Structural study of the O-linked sugar chains of human leukocyte tyrosine phosphatase CD45.

The O-linked sugar chains of the human leukocyte cell surface glycoprotein CD45 were released as tritium-labeled oligosaccharides by beta-elimination in the presence of NaB3H4. Mono Q column chromatography revealed that they comprise neutral (64%) and acidic (36%) oligosaccharides, the latter of which were converted to neutral ones by Arthrobacter ureafaciens sialidase treatment. Structural studies of each oligosaccharide fractionated on a Bio-Gel P-4 column by sequential exoglycosidase digestion and by methylation analysis revealed that human leukocyte CD45 contains mainly core 1 and core 2 oligosaccharides, 15% of which are modified with poly (N-acetyllactosamine) chains in different extensions. CD45 consists of several isoforms which were isolated after cell surface sialic acid residues were labeled by periodate/NaB3H4 treatment. Bio-Gel P-6 column chromatography of a mixture of the tritium-labeled glycopeptide/oligosaccharides obtained by pronase-digestion followed by mild alkaline borohydride treatment showed that distribution of the sialylated core 2 oligosaccharides is different among CD45 isoforms.

An IgG Monoclonal Antibody against Dictyostelium discoideum Glycoproteins Specifically Recognizes Fucα1,6GlcNAcβ in the Core of N-Linked Glycans: LOCALIZED EXPRESSION OF CORE-FUCOSYLATED GLYCOCONJUGATES IN HUMAN TISSUES

Core fucosylation of N-linked oligosaccharides (GlcNAcbeta1, 4(Fucalpha1,6)GlcNAcbeta1-Asn) is a common modification in animal glycans, but little is known about the distribution of core-fucosylated glycoproteins in mammalian tissues. Two monoclonal antibodies, CAB2 and CAB4, previously raised against carbohydrate epitopes of Dictyostelium discoideum glycoproteins (Crandall, I. E. and Newell, P. C. (1989) Development 107, 87-94), specifically recognize fucose residues in alpha1,6-linkage to the asparagine-bound GlcNAc of N-linked oligosaccharides. These IgG3 antibodies do not cross-react with glycoproteins containing alpha-fucoses in other linkages commonly seen in N- or O-linked sugar chains. CAB4 recognizes core alpha1,6 fucose regardless of terminal sugars, branching pattern, sialic acid linkage, or polylactosamine substitution. This contrasts to lentil and pea lectins that recognize a similar epitope in only a subset of these structures. Additional GlcNAc residues found in the core of N-glycans from dominant Chinese hamster ovary cell mutants LEC14 and LEC18 progressively decrease binding. These antibodies show that many proteins in human tissues are core-fucosylated, but their expression is localized to skin keratinocytes, vascular and visceral smooth muscle cells, epithelia, and some extracellular matrix-like material surrounding subpopulations of lymphocytes. The availability of these antibodies now allows for an extended investigation of core fucose epitope expression in development and malignancy and in genetically manipulated mice.

Glycosylation of Bile-Salt-Stimulated Lipase from Human Milk: Comparison of Native and Recombinant Forms

Bile-salt-stimulated lipase (BSSL) is an enzyme present in human milk. BSSL is important for fat digestion in infants. It contains one site for N-glycosylation and a serine/threonine-rich domain which is highly O-glycosylated. Both N- and O-linked sugar chains were studied on native BSSL from three donors and compared to the glycosylation of recombinant BSSL produced in Chinese hamster ovary or mouse fibroblast (C-127) cell lines. The carbohydrate composition of oligosaccharides was mapped using sugar and methylation analyses, enzyme-linked immunosorbant assay, and different separation techniques. Native BSSL was found to be highly glycosylated (19–26%). It contained a high amount of fucosylated oligosaccharides and expressed both Lewis a and Lewis b blood group antigens. None of the recombinant BSSL forms contained fucose. N-linked structures on native BSSL were identified as mainly mono- and disialylated biantennary complex type structures with or without fucose substitution. High-pH anion-exchange chromatography analysis indicated that the recombinant forms of BSSL contained similar types ofN-glycan structures differing mainly in their content of sialic acid and by the absence of fucose residues. Native BSSL contained predominantly large O-linked oligosaccharides. This was in contrast to the recombinant forms of BSSL which contained mainly short typeO-glycans with a high content of sialic acid. Interestingly, the estimated number ofO-glycans attached to native BSSL was lower than that for the recombinant forms.

Structures of the sugar chains of recombinant macrophage colony-stimulating factor produced in Chinese hamster ovary cells.

The structures of the N- and O-linked sugar chains of recombinant human macrophage colony-stimulating factor (rhM-CSF) from Chinese hamster ovary (CHO) cells were studied. rhM-CSF is a homodimeric glycoprotein. Sugar composition analysis revealed that rhM-CSF contained 4.1 mol N-acetylgalactosamine, 10.3 mol N-acetylglucosamine, 5.0 mol mannose, 10.0 mol galactose, 1.4 mol fucose, and 11.8 mol sialic acid per mol of the monomer. The N- and O-linked sugar chains 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. The structures of the PA-sugar chains were analyzed by a combination of reversed-phase and size-fractionation HPLC, and exoglycosidase digestions, from which the structures of the rhM-CSF sugar chains were estimated to be as follows: monosialo biantennary sugar chain (9 mol%), monosialo fucosylbiantennary sugar chain (10 mol%), disialo biantennary sugar chain (30 mol%), disialo fucosylbiantennary sugar chain (28 mol%), disialo triantennary sugar chain (7 mol%), trisialo triantennary sugar chain (11 mol%), and trisialo fucosyltriantennary sugar chain (5 mol%) for the N-linked sugar chains, and asialo (27 mol%), monosialo (51 mol%), and disialo (22 mol%) Galbeta1-3GalNAc for the O-linked sugar chains. Sialic acid residues were linked to the N-linked sugar chains through an alpha2-3 linkage.

Production and characterization of extracellular uronic acid-containing glycoproteins from Fusarium oxysporum

Fusarium and Giberella species were found to secrete acidic polysaccharides when grown in Czapex-Dox medium. Fusarium oxysporum showed the highest rate of secretion of acidic sugars into the growth medium. The acidic sugar-containing glycoprotein from F. oxysporum was purified from the culture filtrate to apparent homogeneity on ultracentrifugation analysis, by ammonium sulfate precipitation, Toyopearl HW-55 gel filtration and charcoal chromatography. The purified acidic sugar-containing glycoprotein has a 10% protein content and a content of 40% acidic sugars as glucuronic acid, and the neutral sugars present in this glycoprotein are mainly mannose, galactose, and glucose. The protein moiety of the glycoprotein contains a high proportion of serine and threonine residues. Treatment of the glycoprotein with alkaline solution resulted in an increase in absorbance at 241 nm, and alkaline borohydride treatment resulted in a marked decrease in the number of serine and threonine residues. We conclude that the glycoprotein has O-linked sugar chains which are mainly attached to serine and threonine residues of the protein moiety. The primary structure of the acidic polysaccharide from F. oxysporum was analyzed mainly by NMR spectrometry. The main parts of this polysaccharide have structures similar to those of uronic acid-containing polysaccharide from Fusarium sp. M7-1.

Purification and characterization of novel whey glycoprotein WGP-88 which binds to a monoclonal antibody to PAS-4 glycoprotein in the bovine milk fat globule membrane.

A monoclonal antibody to the PAS-4 glycoprotein (78 kDa) of the bovine milk fat globule membrane (MFGM) specifically recognized PAS-4, and was named KAS4. A component recognized by KAS4 was found in whey protein, this being a glycoprotein of 88 kDa by SDS-PAGE and named WGP-88. WGP-88 was purified and characterized in comparison with PAS-4. WGP-88 had apparent pI values of 6.45 and 6.39, while those of PAS-4 were 7.39 and 7.35. Neuraminidase digestion shifted the pI values of WGP-88 to 6.57 and of PAS-4 to 7.52. WGP-88 was rich in polar amino residues (44.9 mol%), while PAS-4 was abundant in nonpolar amino acid residues (48.7 mol%). WGP-88 contained 17.1% of carbohydrate and PAS-4 had 7.2%. The results of reductive hydrolysis, N-glycanase digestion, and a lectin blot analysis suggested that N- and O-linked sugar chains were contained in both glycoproteins. WGP-88 and PAS-4 had a different N-terminal amino acid sequence. WGP-88 and PAS-4 respectively inhibited competitively the binding of KAS4 to PAS-4 and WGP-88. Our studies revealed WGP-88 recognized by KAS4 mAb to be a novel whey protein and to have different biochemical properties from those of PAS-4.

1H-NMR analysis of 2-pyridylaminated O-linked sugar chains from fetuin in fetal calf serum.

The standard sugars (GalNAc, Galβ1-3GalNAc) in the core region of O-linked oligo-saccharides were labeled with a fluorescent reagent, 2-aminopyridine, by reductive amination. These sugars were then analyzed by 1H-NMR spectroscopy. The chemical shifts and spectral pattern of the structural-reporter groups of the pyridylaminated sugars agreed with that of the structural-reporter groups of the corresponding O-linked sugar-alditols, except for the H-2 and the N-acetyl protons of N-acetylgalactosamine. It was proven that the structural-reporter groups of the O-linked sugar-alditols are applicable to a structural analysis of the corresponding pyridylaminated sugars. The structures of GalNAc-PA and Galβl-3GalNAc-PA, prepared from fetuin in fetal calf serum, were analyzed using this method.

Structures of Sialylated O-Linked Oligosaccharides of Bovine Peripheral Nerve α-Dystroglycan: THE ROLE OF A NOVEL O-MANNOSYL-TYPE OLIGOSACCHARIDE IN THE BINDING OF α-DYSTROGLYCAN WITH LAMININ

alpha-Dystroglycan is a heavily glycosylated protein, which is localized on the Schwann cell membrane as well as the sarcolemma, and links the transmembrane protein beta-dystroglycan to laminin in the extracellular matrix. We have shown previously that sialidase treatment, but not N-glycanase treatment, of bovine peripheral nerve alpha-dystroglycan greatly reduces its binding activity to laminin, suggesting that the sialic acid of O-glycosidically-linked oligosaccharides may be essential for this binding. In this report, we analyzed the structures of the sialylated O-linked oligosaccharides of bovine peripheral nerve alpha-dystroglycan by two methods. O-Glycosidically-linked oligosaccharides were liberated by alkaline-borotritide treatment or by mild hydrazinolysis followed by 2-aminobenzamide-derivatization. Acidic fractions obtained by anion exchange column chromatography that eluted at a position corresponding to monosialylated oligosaccharides were converted to neutral oligosaccharides by exhaustive sialidase digestion. The sialidases from Arthrobacter ureafaciens and from Newcastle disease virus resulted in the same degree of hydrolysis. The neutral oligosaccharide fraction, thus obtained, gave a major peak with a mobility of 3.8-3.9 glucose units upon gel filtration, and its reducing terminus was identified as a mannose derivative. Based on the results of sequential exoglycosidase digestion, lectin column chromatography, and reversed-phase high-performance liquid chromatography, we concluded that the major sialylated O-glycosidically-linked oligosaccharide of the alpha-dystroglycan was a novel O-mannosyl-type oligosaccharide, the structure of which was Siaalpha2-3Galbeta1-4GlcNAcbeta1-2Man-Ser/Thr (where Sia is sialic acid). This oligosaccharide constituted at least 66% of the sialylated O-linked sugar chains. Furthermore, a laminin binding inhibition study suggested that the sialyl N-acetyllactosamine moiety of this sugar chain was involved in the interaction of the alpha-dystroglycan with laminin.

Detection of UDP-D-xylose: alpha-D-xyloside alpha 1-->3xylosyltransferase activity in human hepatoma cell line HepG2.

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.

Classification of Sugar Chains of Glycoproteins by Analyzing Reducing End Oligosaccharides Obtained by Partial Acid Hydrolysis

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 1Mtrifluoroacetic 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.

Preparation of Pyridylaminated O-Linked Sugar Chains from Glycoproteins Blotted on a Polyvinylidene Difluoride Membrane and Application to Human Granulocyte Colony-Stimulating Factor

Preparation of Pyridylaminated O-Linked Sugar Chains from Glycoproteins Blotted on a Polyvinylidene Difluoride Membrane and Application to Human Granulocyte Colony-Stimulating Factor