Katsuko Yamashita

The substrate specificities of endo-β-N-acetylglucosaminidases CII and H

Abstract A minor glycopeptide was newly isolated from the exhaustive pronase digest of crystalline ovalbumin by Dowex-50w column chromatography, and its structure was determined as Manα1→3Manα1→6 (Manα1→3) Manβ1→4GlcNAcβ1→4GlcNAc→Asn. This glycopeptide (GP-VI) has the smallest carbohydrate unit among the ovalbumin glycopeptides so far reported, and is also the smallest glycopeptide of all which are susceptible to endo-β-N-acetylglucosaminidases CII and H. This finding, together with the already reported data of the action of both enzymes to glycopeptides of known structures, elucidates that the structural requirement of CII enzyme for its substrate is R→2Manα1→3 (R→6) Manα1→6 (R→2Manα1→3) (R→4) Manβ1→4GlcNAcβ1→4GlcNAc→Asn, in which R represents either hydrogen or sugars, and that of H enzyme is R→2Manα1→3 (R→6) Manα1→6 (R→4) Manβ1→4GlcNAcβ1→4GlcNAc→Asn.

Substrate specificity of diplococcal β-N-acetylhexosaminidase, a useful enzyme for the structural studies of complex type asparagine-linked sugar chains

Summary The substrate specificity of diplococcal β-N-acetylhexosaminidase was studied in detail by using oligosaccharides of known structure. The enzyme cannot cleave G1cNAcβ1→4Man and G1cNAcβ1→6Man linkages although it readily hydrolyzes G1cNAcβ1→2Man, G1cNAcβ1→3Gal and G1cNAcβ1→6Gal linkages. The G1cNAcβ1→2Man linkage in G1cNAcβ1→4(GlcNAcβ1→2)Man group is cleaved by the enzyme but the linkage in G1cNAcβ1→6(G1cNAcβ1→2)Man group is not, probably because of the steric effect of G1cNAcβ1→6Man residue on G1cNAcβ1→2Man linkage. Similar steric effect is also observed in the case of G1cNAcβ1→2Manα1→6(GlcNAcβ1→2Manα1→3) (G1cNAcβ1→4)Manβ1→4 G1cNAc0T. The enzyme can cleave only one of the two G1cNAcβ1→2Man linkages of the heptaitol and produces G1cNAcβ1→2Manα1→6(Manα1→3) (G1cNAcβ1→4)Manβ1→4G1cNAc0T. The substrate specificity of diplococcal β-N-acetylhexosaminidase can be used effectively for the structural studies of asparagine-linked sugar chains.

Structures of the Asparagine-Linked Sugar Chains of Human Chorionic Gonadotropin

The asparagine-linked sugar chains of human chorionic gonadotropin were released from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. More than 90% of the released radioactive oligosaccharides contained N-acetylneuraminic acid residues. After removal of N-acetylneuraminic acid residues by sialidase treatment, two neutral oligosaccharide fractions were obtained by paper chromatography. Sequential exoglycosidase digestion revealed that one of them was a mixture of two neutral oligosaccharides. The complete structures of the three oligosaccharides were elucidated by methylation analysis. It was confirmed that all the N-acetylneuraminic acid residues of the asparagine-linked sugar chains of human chorionic gonadotropin occur as NeuAc alpha 2 leads to 3Gal groupings by comparing the methylation analysis data for the acidic oligosaccharide mixture before and after sialidase treatment. Based on these results, the structures of the asparagine-linked sugar chains of human chorionic gonadotropin were confirmed to be +/- NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(+/- Fuc alpha 1 leads to 6)GlcNAc and Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3 Gal beta 1 leads to 4 GlcNAc beta 1 leads to Man alpha 1 leads to 3)Man beta 1 leads to 4 GlcNAc beta 1 leads to 4GlcNAc.

Asparagine-linked oligosaccharide chains of IgG: a revised structure.

Summary A complete structure of asparagine linked sugar chains of bovine IgG was determined to be as follows. This structure is inconsistent with the results obtained from myeloma proteins in that incomplete galactosylation occurred in the branch with α1→3 linked mannosyl residue.

An α-mannosidase purified from Aspergillus saitoi is specific for α1,2 linkages

Abstract The substrate specificity of an α-mannosidase purified from Aspergillus saitoi was studied in detail. This enzyme hydrolyzes yeast mannan partially but does not act on p-nitrophenyl α-mannopyranoside. Survey of the action of the enzyme on various oligosaccharides liberated from glycoproteins indicated that the enzyme hydrolyzes Manα1→2Man linkage but not Manα1→3Man and Manα1→6 Man linkages at all. All Manα1→2 residues in intact bovine pancreatic ribonuclease B were removed completely by incubation with the α-mannosidase.

Purification of hamster melanoma tyrosinases and structural studies of their asparagine-linked sugar chains☆

In cultured melanotic melanoma, a marked decrease of pigmentation has been found to be induced by the addition of tunicamycin [Y. Mishima and G. Imokawa (1983) J. Invest. Dermatol. 81, 106-114]. Since it appears that this impaired pigmentation arises from the loss of asparagine-linked sugar chains serving as a signal for transport of tyrosinase from GERL (Golgi-associated endoplasmic reticulum of lysosomes) to premelanosomes, tyrosinases from the membrane fraction of Greenes hamster melanoma have been purified, and the structures of their sugar chains have been analyzed. Two kinds of tyrosinases were purified by Triton X-100 solubilization; DEAE-cellulose, Sephadex G-200, and DEAE-Sephadex column chromatography; and preparative polyacrylamide gel electrophoresis. The two tyrosinases were separated by polyacrylamide gel electrophoresis, and both corresponded to Mr 69,000. Their asparagine-linked sugar chains were released by hydrazinolysis and analyzed. The sugar chains of the two tyrosinases were identical except for the sialic acid contents. One mole of each tyrosinase contained 1 mol of high-mannose-type sugar chains and 3 mol of complex-type sugar chains. The former chain has Man3 approximately 5 X GlcNAc2 and the latter has Man3 X GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc as their core structures. The complex-type sugar chains are composed of mono-, bi-, tri-, and tetraantennary sugar chains, with +/- Sia alpha 2----3Gal beta 1----4GlcNAc beta 1----as their outer chains.

Oligosaccharides of human milk: Isolation and characterization of three new disialylfucosyl hexasaccharides☆

Abstract A group of nonasaccharides with monosaccharide composition of NANA:Fuc:GlcNAc:Gal:Glc (2:1:2:3:1) was isolated from human milk. By sequential degradation with glycosidases and quantitative methylation analysis, their structures were elucidated as NANA 2 → 3Galβ1 → 3¦NANA2 → 6¦GlcNAcβ1 → 3¦Fucα1 → 3Galβ1 → 4GlcNAcβ1 → 6¦Galβ1 → 4Glc, NANA 2 → 3Galβ1 → 3¦NANA2 → 6¦GlcNAcβ → 3¦Galβ1 → 4(Fucα1 → 3)GlcNAcβ1 → 6¦Galβ1 → 4Glc, and NANA2 → 3(6)Galβ1 → 4(Fucα1 → 3)GlcNAcβ1 → 3[NANA2 → 6(3)Galβ1 → 4GlcNAcβ1 → 6]Galβ1 → 4Glc. The significance of finding the so far unknown Fucα1 → 3Gal grouping is discussed in relation to the blood group antigens.

Oligosaccharides of human milk. V. Isolation and characterization of a new trisaccharide, 6'-galactosyllactose

Abstract Milk from women of “nonsecretor” status lack lactodifucotetraose. As a result, a minor trisaccharide with the same chromatographic properties as lactodifucotetraose was found. The trisaccharide was purified and characterized as having the following structure. Galβ1 → 6Galβ1 → 4Glc Enzymic studies show that the trisaccharide is synthesized by a specific galactosyltransferase from UDP-Gal and lactose and not by the action of β-galactosidase on lactose.

Organ-specific difference in the sugar chains of γ-glutamyltranspeptidase

Sugar chains of gamma-glutamyltranspeptidase purified from neonatal mouse liver and adult mouse kidney were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. A comparative study of the structures of the oligosaccharides has revealed that the GlcNAc beta 1 leads to 4Man beta 1 leads to group is found in the sugar chains of kidney enzyme but not in those of liver enzyme. This is considered as an organ-specific difference common to mammals because the same phenomenon was found in bovine and rat enzymes.

Sialic acid-containing sugar chains of hen ovalbumin and ovomucoid

The acidic oligosaccharide fractions released from hen ovalbumin and ovomucoid by hydrazinolysis contain both sialyloligosaccharides and sulfated oligosaccharides. The sialyloligosaccharides were converted into neutral oligosaccharides by sialidase digestion, and separated from sulfated oligosaccharides by paper electrophoresis. Structural studies of these neutral oligosaccharides showed that the oligosaccharides of ovalbumin have different structural characteristics from those of ovomucoid, i.e., all sialyloligosaccharides from ovomucoid contain a pentasaccharide, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-GlcpNAc- (1----4)-GlcpNAc, as a common core, and the smallest oligomannosyl core of the sugar chains from ovalbumin is alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)-[alpha-D-Manp -(1----3)]-beta-D- Manp-(1----4)-GlcpNAc-(1----4)-GlcpNAc. By methanolysis followed by N-acetylation, sialyl oligosaccharides, free from sulfated oligosaccharides, were recovered quantitatively from the acidic fractions of ovalbumin and ovomucoid. Methylation analysis of these sialyloligosaccharide mixtures, before and after sialidase digestion, showed that all sialic acid of both glycoproteins occurs as a alpha-Sia-(2----3)-D-Galp group.