Structures of the asparagine-linked sugar chain of glucose transporter from human erythrocytes

The asparagine-linked sugar chain of glucose transporter from human erythrocytes was quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. They were converted to radioactive oligosaccharides by NaB3H4 reduction after N-acetylation and fractionated by anion-exchange column chromatography and Bio-Gel P-4 column chromatography after sialidase treatment. Structural study of each oligosaccharide by exo- and endoglycosidase digestion and methylation analysis indicated that the glycoprotein contains a high-mannose-type oligosaccharide, Man9.GlcNAc.GlcNAc, and biantennary complex-type oligosaccharides with Man alpha 1----6(+/- GlcNAc beta 1----4)(Man alpha 1----3) Man beta beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc as their cores and the poly-N-acetyllactosamine composed of about 16 N-acetyllactosaminyl units as their outer chains. These structural features of the sugar moiety of glucose transporter are quite different from those of two major intrinsic glycoproteins of human erythrocytes, glycophorin A and band 3.     

A new Saccharomyces cerevisiae mnn mutant N-linked oligosaccharide structure

We find that the N-linked Man8GlcNAc2- core oligosaccharide of Saccharomyces cerevisiae mnn mutant mannoproteins is enlarged by the addition of the outer chain to the alpha 1----3-linked mannose in the side chain that is attached to the beta 1----4-linked mannose rather than by addition to the terminal alpha 1----6-linked mannose. This conclusion is derived from structural studies on a phosphorylated oligosaccharide fraction and from mass spectral fragment analysis of neutral core oligosaccharides.     

The processing of N-linked glycans in yeast. Mutually exclusive steps in the processing of a Man6 derivative by yeast membrane preparations

When a derivatized oligosaccharide isolated from ovalbumin and containing 6 mannose residues was incubated with yeast membranes and GDP-mannose, two sets of products were obtained, a high molecular weight one containing about 25 mannose residues and a low molecular weight one consisting of compounds with 7, 8, and 9 mannose residues, respectively. When the low molecular weight products were reincubated with the yeast membranes and GDP-mannose, no further mannose incorporation was observed, showing that these compounds must be of the wrong structure as substrates for yeast glycan processing enzymes. The structures were investigated by 1H NMR spectroscopy. The high molecular weight products contained an outer chain of an average length of 18 1----6-linked mannose residues attached to a core structure made up of the original 6 mannose residues with one additional 1----2-linked mannose added. The low molecular weight product with 8 mannose residues was deduced to contain a terminal 1----6-linked mannose (on the 1----6 arm) substituted by mannose at the 2-position, and the ones with 7 and 9 mannose residues were identified as having an additional 1----3-linked mannose on the starting Man6 substrate and on the Man8 product, respectively. The results lend further support to the picture that the processing steps must occur in proper sequence for specific products to form.     

The structure of a neural specific carbohydrate epitope of horseradish peroxidase recognized by anti-horseradish peroxidase antiserum.

Antiserum raised against horseradish peroxidase (HRP) recognizes a neural specific carbohydrate antigen in Drosophila and other insects. The epitopic activity of the carbohydrate moiety of HRP recognized by anti-HRP antiserum was measured by a newly developed enzyme-linked immunosorbent assay, in which HRP glycopeptides conjugated with bovine serum albumin were coated onto the wells and then reacted with goat anti-HRP antiserum. HRP sugar moieties released by almond glycopeptidase A digestion of HRP pepsin digests were subjected to pyridylamination. Pyridylamino oligosaccharides were separated into seven fractions by reverse-phase high performance liquid chromatography. The major fraction, which comprised about 80% of the total sugars, reacted strongly with anti-HRP antiserum. The carbohydrate structure of this fraction was determined by sugar composition analysis and 600-MHz 1H NMR spectroscopy as follows: Man alpha 1----6(Man alpha 1----3)(Xyl beta 1----2)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc. Analyses of reactivity with anti-HRP antiserum of various oligosaccharide derivatives obtained from the major fraction by exoglycosidase digestion and partial acid hydrolysis indicated that alpha 1----6-linked mannose and alpha 1----3-linked fucose are predominantly involved in the epitopic structure.     

Structural study on the carbohydrate moiety of human placental alkaline phosphatase

Alkaline phosphatase purified from human placenta contains a single asparagine-linked sugar chain in one molecule. The sugar chain was quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction, and separated by paper electrophoresis into one neutral and two acidic fractions. By a combination of sequential exoglycosidase digestion and methylation analysis, the structures of oligosaccharides in the neutral fraction were confirmed to be as follows: Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc. The acidic oligosaccharide fractions were mixtures of mono- and disialyl derivatives of the neutral fraction. All the sialic acid residues of the sugar chains occur as the NeuAc alpha 2----3Gal group. In the case of monosialyl derivatives, the N-acetylneuraminic acid was exclusively linked to the Man alpha 1----3 arm.     

Structures of sugar chains of human kidney gamma-glutamyltranspeptidase

gamma-Glutamyltranspeptidase purified from human kidneys contains 4-5 asparagine-linked sugar chains in each molecule. The sugar chains were released from the polypeptide portion of the enzyme by hydrazinolysis as oligosaccharides and separated by paper electrophoresis into one neutral and two acidic fractions. By sequential exoglycosidase digestion and methylation analysis, the neutral fraction, which comprised 69% of total oligosaccharides, was shown to be a mixture of bisected bi- and triantennary complex-type sugar chains with and without a fucose on the proximal N-acetylglucosamine residue and with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups in their outer chain moieties. The acidic oligosaccharide fractions were mixtures of mono- and disialyl derivatives of bisected triantennary complex-type oligosaccharides with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc group in their outer chain moieties. Some of the outer chains of the acidic oligosaccharides were considered to be sialylated X-antigenic structures.     

The structure of the asparagine-linked sugar chains of bovine brain ribonuclease

The asparagine-linked sugar chains of bovine brain ribonuclease were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. After N-acetylation, they were converted into radioactively-labeled oligosaccharides by NaB3H4 reduction. The radioactive oligosaccharide mixture was fractionated by ion-exchange chromatography, and the acidic oligosaccharides were converted into neutral oligosaccharides by sialidase digestion. The neutral oligosaccharides were then fractionated by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exoglycosidase digestion in combination with methylation analysis revealed that bovine brain ribonuclease showed extensive heterogeneity. It contains bi- and tri-antennary, complex-type oligosaccharides having alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-beta-D- GlcpNAc-(1----4)-[alpha-L-Fucp-(1----6)]-D-GlcNAc as their common core. Four different outside oligosaccharide chains, i.e., beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----, alpha-Neu5Ac-(2----6)-beta-D- Galp-(1----4)-beta-D-GlcpNAc-(1----, alpha-Neu5Ac-(2----3)-beta-D-Galp-(1----4)- beta-D-GlcpNAc-(1----, and alpha-D-Galp-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----, were found. The preferential distribution of the alpha-D-Galp-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc group on the alpha-D-Manp-(1----6) arm is a characteristic feature of the sugar chains of this enzyme.     

Structural study of the sugar chains of human leukocyte cell adhesion molecules CD11/CD18.

Leu-CAMs (CD11/CD18) consisting of LFA-1, Mac-1, and p150/95 are leukocyte cell surface glycoproteins that are involved in various leukocyte functions. The asparagine-linked sugar chains were released as oligosaccharides from Leu-CAMs by hydrazinolysis. About 12 mol of sugar chains was released from 1 mol of Leu-CAMs. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. All of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. The neutral and sialdase-treated acidic oligosaccharides were fractionated by chromatography on lectin columns followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that Leu-CAMs contain mainly high mannose type and high molecular weight complex type sugar chains. The latter sugar chains were of bi-, tri-, and tetraantennary complex types with the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----and/or the Gal beta 1----3GlcNAc beta 1----groups together with the Gal beta 1----4GlcNAc group in their outer-chain moieties. In addition to these sugar chains, a small amount of monoantennary complex type and hybrid type sugar chains was found in Leu-CAMs. Furthermore, analysis of the asparagine-linked sugar chains released from the beta-subunit of Leu-CAMs by a series of lectin chromatography showed that subunit-specific glycosylation is not observed between the alpha- and beta-subunits of Leu-CAMs.     

Lysosomal cysteine protease, cathepsin B, is targeted to lysosomes by the mannose 6-phosphate-independent pathway in rat hepatocytes: site-specific phosphorylation in oligosaccharides of the proregion

Cathepsin B, a lysosomal cysteine protease, is synthesized as a glycoprotein with two N-linked oligosaccharide chains, one of which is in the propeptide region while the other is in the mature region. When cultured rat hepatocytes were labeled with [(32)P]phosphate, (32)P-labeled cathepsin B was immunoprecipitated only in the proform from cell lysates and medium. Either Endo H or alkaline phosphatase treatment of (32)P-labeled procathepsin B demonstrated the acquisition of a mannose 6-phosphate (Man 6-P) residue on high mannose type oligosaccharides. To identify the site of phosphorylation, immunoisolated (35)S- or (32)P-labeled procathepsin B was incubated with purified lysosomal cathepsin D, since cathepsin D cleaves 48 amino acid residues from the N-terminus of procathepsin B, in which one N-linked oligosaccharide chain was also included [Kawabata, T. et al. (1993) J. Biochem. 113, 389-394]. Treatment of intracellular (35)S-labeled procathepsin B with a molecular mass of 39-kDa with cathepsin D resulted in the production of the 31-kDa intermediate form, but the (32)P-label incorporated into procathepsin B disappeared after treatment with cathepsin D. These results indicate that the phosphorylation of procathepsin B is restricted to an oligosaccharide chain present in the propeptide region. Interestingly, cathepsin B sorting to lysosomes was not inhibited by NH(4)Cl treatment and about 90% of the intracellular procathepsin B initially phosphorylated was secreted into the medium without being dephosphorylated intracellularly, and did not bind significantly to cation-independent-Man 6-P receptor, suggesting the failure of Man 6-P-dependent transport of procathepsin B to lysosomes. Additionally, about 50% of the newly synthesized (35)S-labeled cathepsin B was retained in the cells in mature forms consisting of a 29-kDa single chain form and a 24-kDa two chain form, while part of the procathepsin B was associated with membranes in a Man 6-P-independent manner. Taken together, these results show that in rat hepatocytes, cathepsin B is targeted to lysosomes by an alternative mechanism(s) other than the Man 6-P-dependent pathway.     

Comparative study of the sugar chains of factor VIII purified from human plasma and from the culture media of recombinant baby hamster kidney cells.

The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not. Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1----3Gal group. A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group. Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.     

Structural study of the sugar moieties of monoclonal antibodies secreted by human-mouse hybridoma

Six monoclonal antibodies, three each of human IgG1 and IgG2 subclasses, were obtained from human-mouse hybridomas. Structural study of their asparagine-linked sugar chains was performed to elucidate the regulatory mechanism of secreted monoclonal IgG glycosylation. The sugar moieties were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. They were converted into radioactive oligosaccharides by NaB3H4 reduction after N-acetylation. Structural study of each oligosaccharide by lectin affinity column chromatography, sequential exoglycosidase digestion, and methylation analysis indicated that almost all of them were biantennary complex-type sugar chains containing Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (+/- Fuc alpha 1----6)GlcNAc as core structures. Bisecting N-acetylglucosamine residue, which is present in human IgG but not in mouse IgG, could not be detected at all. The molar ratio of each oligosaccharide from the six IgG samples was different. However, no subclass specificity was detected except that all IgG1 contained neutral, mono-, and disialylated sugar chains, whereas IgG2 did not contain disialylated ones. The molar ratio of N-acetylneuraminic acid to N-glycolylneuraminic acid was also different for each IgG. All six IgGs contained monoantennary complex-type and high mannose-type oligosaccharides which had never been detected in serum IgGs of various mammals so far investigated. These results indicated that the processing of asparagine-linked sugar chains of IgG is less complete in human-mouse hybridoma than in human or mouse B cells, and that the glycosylation machinery of the mouse cells is dominant in the hybrid cells.     

Structural study of asparagine-linked oligosaccharide moiety of taste-modifying protein, miraculin

The structures of the N-linked oligosaccharides of miraculin, which is a taste modifying glycoprotein isolated from miracle fruits, berries of Richadella dulcifica, are reported. Asparagine-linked oligosaccharides were released from the protein by glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by high performance liquid chromatography (HPLC) on an ODS-silica column. More than five kinds of oligosaccharide fractions were separated by the one chromatographic run. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amidesilica column. Furthermore, high resolution proton nuclear magnetic resonance (1H NMR) measurements were carried out. It was found that 1) five oligosaccharides obtained are a series of compounds with xylose-containing common structural core, Xyl beta 1----2 (Man alpha 1----6) Man beta 1----4-GlcNAc beta 1----4 (Fuca1----3)GlcNAc, 2) a variety of oligosaccharide structures are significant for two glycosylation sites, Asn-42 and Asn-186, and 3) two new oligosaccharides, B and D, with unusual structures containing monoantennary complex-type were characterized. (formula; see text)     

Structures of asparagine-linked oligosaccharides of human placental fibronectin

The asparagine-linked sugar chains of fibronectin purified from human placenta were quantitatively released as oligosaccharides by hydrazinolysis. After N-acetylation, they were converted to radioactive oligosaccharides by NaB3H4 reduction. The radioactive oligosaccharides were fractionated by their charge on an anion-exchange column chromatography. All of the acidic oligosaccharides could be converted to neutral oligosaccharides by sialidase digestion. These oligosaccharides were then fractionated by serial affinity chromatography using immobilized lectin columns. Study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed the following information as to the structures of the sugar chains of human placental fibronectin: 1) nine sugar chains are included in one molecule; 2) all sialic acid residues are exclusively linked at the C-3 position of the galactose residues; 3) bi-, tri-, and tetraantennary complex-type oligosaccharides with the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (+/- Fuc alpha 1----6)-GlcNac as their cores were found; 4) the bisecting N-acetylglucosamine residue and the Gal beta 1----4GlcNAc beta 1----repeating groups are included in some of the sugar chains.     

Carbohydrate structures of nonspecific cross-reacting antigen-2, a glycoprotein purified from meconium as an antigen cross-reacting with anticarcinoembryonic antigen antibody. Occurrence of complex-type sugar chains with the Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains

Nonspecific cross-reacting antigen-2 (NCA-2) is a glycoprotein purified from meconium as a closely correlated entity with carcinoembryonic antigen (CEA). As in the case of CEA, only asparagine-linked sugar chains are included in NCA-2. In order to elucidate the structural characteristics of the sugar chains of NCA-2, they were quantitatively released from the polypeptide backbone by hydrazinolysis and reduced with NaB3H4 after N-acetylation. The radioactive oligosaccharides were fractionated by paper electrophoresis, serial chromatography on immobilized lectin columns, and Bio-Gel P-4 (under 400 mesh) column chromatography. Structures of the oligosaccharides were estimated from the data of the binding specificities of immobilized lectin columns and the effective size of each oligosaccharide determined by passing through a Bio-Gel P-4 column and were then confirmed by endo-beta-galactosidase digestion, sequential digestion with exoglycosidases with different aglycon specificities, and methylation analysis. NCA-2 contains a similar number (27 mol) of sugar chains in one molecule compared with CEA (24-26 mol). However, all sugar chains of NCA-2 were complex-type in contrast to CEA, approximately 8% of the sugar chains of which were high mannose-type (Yamashita, K., Totani, K., Kuroki, M., Matsuoka, Y., Ueda, I., and Kobata, A. (1987) Cancer Res. 47, 3451-3459). About 80% of the oligosaccharides from NCA-2 contain bisecting N-acetylglucosamine residues, and the percent molar ratio of mono-, bi, tri, and tetraantennary oligosaccharides was 2:14:57:27. (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, and GalNAc beta 1----3Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc were found as their outer chain moieties. Approximately 60% of the oligosaccharides from NCA-2 contain the Gal beta 1----4 or 3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----group in their outer chains.     

Carbohydrate structures of human interleukin 5 expressed in Chinese hamster ovary cells.

The asparagine-linked sugar chains of recombinant human interleukin 5 produced by Chinese hamster ovary cells were released quantitatively as oligosaccharides by hydrazinolysis. After N-acetylation followed by NaB3H4 reduction, each oligosaccharide was isolated by paper electrophoresis and serial lectin column chromatography. Study of their structures by sequential exoglycosidase digestion in combination with methylation analysis, revealed that they are bi-, tri-, and tetraantennary complex-type with fucosylated and non-fucosylated trimannosyl cores and high mannose type sugar chains. More than 80% of the sugar chains occur as biantennary complex-type sugar chains. Although acidic oligosaccharides amount to only 14% of the total oligosaccharides, their sialic acid residues occur exclusively as the Sia alpha 2----3Gal group. Removal of the sugar moiety from intact recombinant human interleukin 5 produced a 2.5-fold increase of its activity to induce IgM secretion.     

Structures of the sugar chains of mouse immunoglobulin G

The asparagine-linked sugar chains of mouse immunoglobulin G (IgG) were quantitatively liberated as radioactive oligosaccharides from the polypeptide portions by hydrazinolysis followed by N-acetylation, and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin (RCA120) affinity high-performance liquid chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Mouse IgG was shown to contain the biantennary complex type sugar chains. Eight neutral oligosaccharide structures, viz, +/- Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(+/- Gal beta 1---- 4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc, were found after the sialidase treatment. The molar ratio of the sugar chains with 2,1, and 0 galactose residues was 2:5:3. The galactose residue in the monogalactosylated sugar chains was distributed on Man alpha 1----3 and Man alpha 1----6 sides in the ratio of 1:3. The oligosaccharides were almost wholly fucosylated and contained no bisecting N-acetylglucosamine which is present in human, rabbit, and bovine IgGs.     

Diversity of oligosaccharide structures linked to asparagines of the scrapie prion protein

Prion proteins from humans and rodents contain two consensus sites for asparagine-linked glycosylation near their C-termini. The asparagine-linked oligosaccharides of the scrapie isoform of the hamster prion protein (PrP 27-30) were released quantitatively from the purified molecule by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The radioactive oligosaccharides were fractionated into one neutral and three acidic oligosaccharide fractions by anion-exchange column chromatography. All oligosaccharides in the acidic fractions could be converted to neutral oligosaccharides by sialidase digestion. Structural studies on these oligosaccharides including sequential exoglycosidase digestion in combination with methylation analysis revealed that PrP 27-30 contains a mixture of bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6(GlcNAc beta 1----4)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4-(Fuc alpha 1----6)GlcNAc as their core. Variation is produced by the different combination of the oligosaccharides Gal beta 1----4GlcNAc beta 1----, Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----, GlcNAc beta 1----, Sia alpha 2----3Gal beta 1----4GlcNAc beta 1----, and Sia alpha 2----6Gal beta 1----4GlcNAc beta 1---- in their outer chain moieties. When both asparagine-linked consensus sites are glycosylated, the diversity of oligosaccharide structures yields over 400 different forms of the scrapie prion protein. Whether these diverse asparagine-linked oligosaccharides participate in scrapie prion infectivity or modify the function of the cellular prion protein remains to be established.     

Cathepsins B and H from porcine spleen. Purification, polypeptide chain arrangements, and carbohydrate content

Procedures for the purification of cathepsins B and H from porcine spleens have been described. The purified porcine cathepsin B (Mr = 27,000) is predominantly a two-chain enzyme with a heavy chain (Mr = 22,000) and a light chain (Mr = 5,000). It also contains two minor forms of cathepsin B with different chain structures. Porcine cathepsin H is a single-chain enzyme with a molecular weight of 25,000. The carbohydrate analyses showed that these enzymes were glycoproteins. A glycopeptide containing 3 amino acids, 2 glucosamines, and 6 mannoses was isolated from cathepsin H. Proton NMR studies revealed that it contained a mixture of 4 high mannose-type of oligosaccharides characteristic of those found on lysosomal enzymes. The carbohydrate of cathepsin B consisted of a single residue of glucosamine and trace mannose. This sugar content is in agreement with the finding that about 80% of the porcine spleen cathepsin B contained a single N-acetylglucosamine while 20% of the enzyme contained a 5-sugar oligosaccharide (Takahashi, T., Schmidt, P. G. and Tang, J. (1984) J. Biol. Chem. 259, 6059-6062). Thus, the studies on carbohydrate contents also indicated the good purity of the enzymes.     

A novel saccharide structure, Xyl 1----3 Gal 1----(SO3-)3,4 Fuc----, is present in acrosome reaction-inducing substance of the starfish, Asterias amurensis.

The jelly coat of echinoderm eggs contains a glycoconjugate, acrosome reaction-inducing substance (ARIS), that is essential for triggering the acrosome reaction in homologous spermatozoa. In the starfish, Asterias amurensis, ARIS is a sulfated glycoprotein of an apparent molecular size of greater than 10(7). Since its biological activity is dependent mostly on its sugar moiety, oligosaccharides liberated by hydrolysis with 10 mM H2SO4 for 60 min at 100 degrees C from pronase digests of ARIS (P-ARIS) were chemically analyzed. The main oligosaccharide purified by high-performance anion-exchange chromatography was determined to be Xyl1----3Gal1----(SO3-)3,4Fuc by compositional analysis and FAB mass spectrometry. This structure indicates that ARIS possesses a novel saccharide chain having sulfated fucose as an internal residue.     

Structural studies on the minor teichoic acid of Bacillus coagulans AHU 1631

The minor teichoic acid linked to glycopeptide was isolated from lysozyme digests of Bacillus coagulans AHU 1631 cell walls, and the structure of the teichoic acid moiety and its junction with the peptidoglycan were studied. Hydrolysis of the teichoic-acid--glycopeptide complex with hydrogen fluoride gave a nonreducing oligosaccharide composed of glucose, galactose and glycerol in a molar ratio of 3:1:1 which was presumed to be dephosphorylated repeating units of the polymer chain. From the results of structural analysis involving NaIO4 oxidation, methylation and acetolysis, the above fragment was characterized as glucosyl(beta 1----3)glucosyl(beta 1----6)galactosyl(beta 1----6)glucosyl(alpha 1----1/3)glycerol. In addition, the Smith degradation of the complex yielded a phosphorus-containing fragment identified as glycerol-P-6-glucosyl(beta 1----1/3)glycerol. These results led to the most likely structure for the repeating units of the teichoic acid, -6[glucosyl(beta 1----3)]glucosyl(beta 1----6)galactosyl(beta 1----6)glucosyl(alpha 1----1/3)glycerol-P-. The minor teichoic acid, just like the major teichoic acid bound to the linkage unit, was released by heating the cell walls at pH 2.5. The mild alkaline hydrolysis of the minor teichoic acid after reduction with NaB3H4 gave labeled saccharides characterized as glucosyl(beta 1----6)galactitol and glucosyl(beta 1----3)glucosyl(beta 1----6)galactitol, together with a large amount of the unlabeled repeating units of the teichoic acid chain. Thus, the minor teichoic acid chain is believed to be directly linked to peptidoglycan at the galactose residue of the terminal repeating unit without a special linkage sugar unit.