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.

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 SO 4 -3GlcA␤1-3Gal␤1-3(؎Sia␣2-6)Gal␤1-4Xyl. 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.

White- and Yolk-riboflavin binding proteins were isolated from hen eggs, and characterized as to their chemical properties. White- and Yolk-RBPs had almost same amino acid compositions except for glutamic acid, but their carbohydrate compositions were different from each other. The complete amino acid sequence of White-RBP was determined by conventional methods. White-RBP comprised 219 amino acid residues, and the amino-terminus was pyroglutamic acid (pyrrolidonecarboxylic acid). Two amino acids, lysine and asparagine, were found at the fourteenth residue from the amino-terminus. Carbohydrate chains were linked to asparagine residues at positions 36 and 147. Both White- and Yolk-RBPs were phosphorylated. In White-RBP either six or seven of nine serine residues between Ser(185) and Ser(197) were phosphorylated. The amino acid sequences around phosphoserines showed that phosphorylation might occur at a serine residue in one of the following sequences; Ser-X-Glu or Ser-X-Ser(P).

Riboflavin-binding protein of hen egg white (egg-white RBP) comprised 219 amino acid residues and nine disulfide bonds. To identify the locations of these bonds, the native protein was oxidized with cyanogen bromide and digested with trypsin, thermolysin, and Staphylococcus aureus V8 protease. The cystine-containing peptides were isolated by HPLC. Amino acid analyses and amino acid sequence analyses of the reduced pyridylethylated derivatives of the cystine peptides showed that seven of the disulfide bonds were as follows: Cys(24)-Cys(73), Cys(57)-Cys(138), Cys(64)-Cys(110), Cys(99)-Cys(169), Cys(116)-Cys(134), Cys(103)-Cys(152), Cys(167)-Cys(202). The other two disulfide bonds were either Cys(5)-Cys(32) and Cys(33)-Cys(77) or Cys(5)-Cys(33) and Cys(32)-Cys(77).

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]

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

The beta-elimination and nucleophile addition reactions of the substituted serine and threonine residues were studied using several synthesized fluorescence-labeled phosphopeptides and a salmon egg polysialoglycoprotein (PSGP). The reagents used were 1 M CH3SH-0.43 M NaOH, 1 M NaBH4-0.1 M NaOH, 1 M CH3NH2-0.1 M NaOH, and 1 M Na2SO3-0.1 M NaOH. The beta-elimination reaction of a phosphoserine peptide, Gly-Ser(PO4)-Glu-AEAP, was about 20 times faster than that of the corresponding phosphothreonine peptide. The carboxyl-side amino acid of the phosphoamino acids in peptides greatly affected the beta-elimination rate. The beta-elimination reaction rates of O-glycosyl serine and threonine in the polysialoglycoprotein were similar and were about a half of that of the phosphoserine peptide. The rates of addition of the three nucleophiles and hydrogen to alpha-aminoacrylic acid (beta-elimination product of substituted serine) in the peptide decreased in the order of CH3SH, Na2SO3, CH3NH2, and H2(NaBH4), and the addition to alpha-aminocrotonic acid (beta-elimination product of substituted threonine) in the order of Na2SO3, CH3NH2, CH3SH, and H2. These results indicated that sulfite is the most recommended nucleophile because of its high addition rate. If sulfite addition is carried out in the presence of NaBH4, sugar chains can be released as alditols, converting the sugar-attaching amino acids to beta-sulfoamino acids.

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.