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The linkage position of a glycosidic bond to the reducing-end residue of a pyridylamino (PA-) sugar can be determined sensitively by Smith degradation and HPLC [K. Omichi and S. Hase, (1994) J. Biochem. 115, 429-434]. With the aim of enhancing the sensitivity of this method of linkage position analysis to the fmol-level, use of the 7-(1,3-disulfonaphtyl)amino (DSNA-) group instead of the PA-group as a fluorescent tag was examined. Smith degradation of DSNA-disaccharides with a DSNA-hexose, DSNA-N-acetylglucosamine, or DSNA-N-acetylgalactosamine reducing-end residue was carried out. HPLC and FAB-MS of the fluorogenic Smith degradation products showed that the DSNA.group was stable under the Smith degradation reaction conditions, and that the reaction proceeded in a manner similar to that using PA-disaccharides to give the predicted products. Fluorogenic Smith degradation products specific to the glycosidic linkage position were well separated by reversed-phase HPLC, and were easily assignable by comparing the HPLC elution positions with those of standard compounds. The method was successfully applied to analyzing the structure of an N-linked sugar chain. [ABSTRACT FROM AUTHOR]

Glycogen debranching enzyme was partially purified from bovine brain using a substrate for measuring the amylo-l,6-glucosidase activity. Bovine cerebrum was homogenized, followed by cell-fractionation of the resulting homogenate. The enzyme activity was found mainly in the cytosolic fraction. The enzyme was purified 5,000-fold by ammonium sulfate precipitation, anion-exchange chromatography, gel-filtration, anion-ex-change HPLC, and gel-permeation HPLC. The enzyme preparation had no α-glucosidase or α-amylase activities and degraded phosphorylase limit dextrin of glycogen with phos-phorylase. The molecular weight of the enzyme was 190,000 and the optimal pH was 6.0. The brain enzyme differed from glycogen debranching enzyme of liver or muscle in its mode of action on dextrins with an α-l,6-glucosyl branch, indicating an amino acid sequence different from those of the latter two enzymes. It is likely that the enzyme is involved in the breakdown of brain glycogen in concert with phosphorylase as in the cases of liver and muscle, but that this proceeds in a somewhat different manner. The enzyme activity decreased in the presence of ATP, suggesting that the degradation of brain glycogen is controlled by the modification of the debranching enzyme activity as well as the phosphorylase. [ABSTRACT FROM AUTHOR]

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 highmsnnose types with xylose and fucose; however, among them two new N-glycans, Manα1-6(Manα1-3)Manα1-6(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc and Manα1-3Manα1-6(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAc, were found. Judging from these structures together with Manα1-6(Manα1-3)Manα1-6(Manα1-3)(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)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 α-mannosidase II is incomplete. [ABSTRACT FROM AUTHOR]

Glycogen storage disease type IIIa (GSD IIIa) is an autosomal recessive disorder caused by deficiency of the glycogen-debranching enzyme (AGL). Recent studies of the AGL gene have revealed the prevalent mutations in North African Jewish and Caucasian populations, but whether these common mutations are present in other ethnic groups remains unclear. We have investigated eight Japanese GSD IIIa patients from seven families and identified seven mutations, including one splicing mutation (IVS14+1G→T) previously reported by us, together with six novel ones: a nonsense mutation (L124X), a splice site mutation (IVS29–1G→C), a 1-bp deletion (587delC), a 2-bp deletion (4216–4217delAG), a 1-bp insertion (2072–2073insA), and a 3-bp insertion (4735–4736insTAT). The last mutation results in insertion of a tyrosine residue at a putative glycogen-binding site, and the rest are predicted to cause synthesis of truncated proteins lacking the glycogen-binding site at the carboxyl terminal. Thirteen novel polymorphisms have also been revealed in this study: three amino acid substitutions (R387Q, G1115R, and E1343 K), one silent point mutation (L298L), one nucleotide change in the 5'-noncoding region, and eight nucleotide changes in introns. Haplotype analysis with combinations of these polymorphic markers showed L124X, IVS14+1G→T, and 4216–4217delAG to be on different haplotypes. These results demonstrate the importance of the integrity of the carboxy terminal domain in the AGL protein and the molecular heterogeneity of GSD IIIa in Japan. [ABSTRACT FROM AUTHOR]

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 Manα1–6(Manα1–3)Manα1–6(Manα1–3)(Xylβ1–2) Manβ1–4GlcNAcβ1–4(Fucα1–3)GlcNAc. This sugar chain represents a new plant-type sugar chain with five mannose residues. [ABSTRACT FROM PUBLISHER]

HepG2 cells were examined for the presence of UDP-D-xylose:β-D-glucoside α-1,3-xylosyltransferase activity by using 2-[(2-pyridyl)amino]ethyl β-D-glucopyranoside (Glcβ-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 α-D-xylosidase digestion, FAB-MS, and Smith degradation revealed that it was Xylα1-3Glcβ-R, thus demonstrating the existence of β-D-glucoside α-1,3-xylosyltransferase. A solubilization study of the enzyme from the microsomal fraction indicated that it differed from UDP-D-xylose:α-D-xyloside α-l,3-xylosyltransferase of the HepG2 microsomal fraction producing Xylα1-3Xylα1-3Glc-R′ [R′, (2-pyridyl)amino-] from UDP-D-xylose and Xylαl-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 Xylαl-3Glcβ-Ser structure of glycoproteins such as human blood coagulation factors VII and IX. [ABSTRACT FROM AUTHOR]

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 (Xylα1-3Glc) and a trisaccharide (Xylα1-3Xylα1-3Glc) 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 a1→xylosyltransferase for their biosynthesis. We report here on the detection of a1→3xylosyltransferase activity which catalyzes the transfer of xylose to Xylα1-3Glc in the human hepatoma cell line HepG2. We employed pyridylaminated Xylα1-3Glc 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 Xylαl-3Xylα1-3Glc by Smith degradation, mass spectrometry, and α- and β-xylosidase digestions. The apparent Km value for pyridylaminated Xylα1-3Glc 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 Xylα1-3Glc, forming Xylα1-3Xylα1-3Glc in human cells. [ABSTRACT FROM AUTHOR]