Substrate specificities of rat kidney lysosomal and cytosolic alpha-D-mannosidases and effects of swainsonine suggest a role of the cytosolic enzyme in glycoprotein catabolism

Swainsonine is a potent inhibitor of lysosomal alpha-D-mannosidase, causes the production of hybrid glycoproteins, and is reported to produce a phenocopy of hereditary alpha-mannosidosis. We now report that the effects of swainsonine administration in the rat are different in two respects from those found in other animals thus far studied. Swainsonine caused the accumulation of oligosaccharide in kidney and urine but not in liver or brain. The accumulated oligosaccharides were mainly Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc, Man(alpha 1-3)[Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4) GlcNAc, and Man(alpha 1-3)[Man(alpha 1-6)]Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4)GlcNAc. Analogous branched Man4 and Man5 structures are found in pig and sheep tissues, but they are N, N'-diacetylchitobiose derivatives. The substrate specificities of rat kidney lysosomal and cytosolic alpha-D-mannosidases were investigated because in one type of hereditary alpha-mannosidosis, that occurring in man, the major storage products are linear rather than branched oligosaccharides. The lysosomal enzyme showed much greater activity toward linear oligosaccharides than toward the branched oligosaccharides induced in the kidney by swainsonine. On the other hand, cytosolic alpha-D-mannosidase preferred the branched oligosaccharides, a result suggesting that this mannosidase might be inhibitable by swainsonine and that the enzyme might play a normal role in glycoprotein catabolism. Swainsonine was indeed found to inhibit this enzyme at relatively high concentrations (I50 at 100 microM swainsonine), and concentrations of this magnitude were in fact found in the cytosol of kidney of swainsonine-fed rats. The kidney cytosolic alpha-D-mannosidase levels were reduced in these rats and, more important, the accumulated oligosaccharides were present mainly in the cytosol rather than in lysosomes. These results point to possible involvement of cytosolic alpha-D-mannosidase in glycoprotein degradation in the rat.     

Cytosolic glycosidases: do they exist?

The substrate specificity of the alpha-D-mannosidases of rat liver lysosome and cytosol was examined using oligosaccharides of the oligomannosidic type. The hydrolysis products were characterized by 400 MHz 1H-NMR spectroscopy. Both catabolic pathways occur in ordered ways, but are quite different. In fact, the lysosomal pathway is a two-step process: the first step involves a Zn(2+)-independent alpha-1,2-mannosidase activity, whereas the second involves a Zn(2+)-dependent alpha-1,3- and alpha-1,6-mannosidase activity. The final product is the disaccharide Man(beta 1-4)GlcNAc. In contrast, the cytosolic pathway leads, in one step, to a unique hexasaccharide (Man5GlcNAc) which has the same structure as the polyprenolic intermediate synthesized on the cytosolic face of the rough endoplasmic reticulum during the biosynthesis of N-glycosylprotein glycans: Man(alpha 1-2)-Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)] Man(beta 1-4)GlcNAc(beta 1-4)-GlcNAc(alpha)P-P-Dol. In addition, the enzymatic parameters of lysosome, endoplasmic reticulum and cytosol alpha-D-mannosidases are quite different. These results lead to the conclusion that the cytosol contains specific alpha-D-mannosidases which do not originate from lysosomes nor from endoplasmic reticulum. The discovery of cytosolic endo-N-acetyl-beta-D-glucosaminidase active on 'immature complex glycans' (glycopeptides of the oligomannosidic type and of the desialylated N-acetyllactosaminic type) as well as on the glycosyl-dolichol pyrophosphate intermediates allows us to hypothesize that these enzymes belong to a control system of N-glycosylprotein biosynthesis, their role being to destroy unfinished glycans. The fate of the formed oligosaccharide structures is discussed: are they destroyed by cytosolic or lysosomal exoglycosidases, or do they carry an 'oligosaccharin-like activity'?     

Comparative study of lysosomal and cytosolic catabolisms of oligomannosidic-type glycans

In order to study the substrate specificities of the enzymes implicated in the catabolism of oligomannosidic-type glycans, the oligosaccharides Man9GlcNAc and Man5GlcNAc were incubated with rat liver lysosomal and cytosolic alpha-D-mannosidases and the hydrolysis products were characterized by 400 MHz 1H-NMR spectroscopy. Although they both occur in an ordered way, the two catabolic pathways are quite different. The lysomal pathway is realized in two stages: the first leads from Man9GlcNAc to Man5GlcNAc by preferential cleavage of the four alpha-1,2-linked mannose residues, and the second, Zn(2+)-dependent, leads from Man5GlcNAc to Man (beta 1-4) GlcN Ac by hydrolysis of alpha-1, 3- and alpha-1,6-linked residues. On the contrary, the cytosolic pattern leads by a pathway quite different to a unique hexasaccharide Man5GlcNAc which has, curiously, the same structure as one of the polyprenolic intermediates occurring in the cytosol during the biosynthesis of N-glycosylprotein glycans: Man (alpha 1-2) Man (alpha 1-2) Man (alpha 1-3) [Man (alpha 1-6)] Man (beta 1-4) GlcN Ac (beta 1-4) GlcNAc alpha 1-P-P-Dol.     

Swainsonine-induced oligosaccharide excretion in sheep. Time-dependent changes in the oligosaccharide profile.

Urinary oligosaccharides isolated from locoweed-intoxicated sheep were separated and quantified by reversed-phase high pressure liquid chromatography of the perbenzoylated alditols. Mannose-containing oligosaccharides were elevated as early as day 3 of feeding, but maximum levels (approx. 1 mumol/ml) were not attained until after 6 weeks of feeding. The relative abundance of individual oligosaccharides changed over the course of the feeding period. Man3GlcNAc2 reached a peak on day 3 and then rapidly declined. Two isomers were shown to be present in this fraction and the relative proportions altered with the duration of locoweed treatment. The major isomer present at early time points (less than 8 days) co-eluted with synthetic Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc, was digested by endo-beta-N-acetyl-glucosaminidase D, and is probably derived from the trimannosyl core of complex glycoproteins synthesized prior to locoweed treatment. Man3GlcNAc2 isolated from day 53 urine was resistant to endo-beta-N-acetylglucosaminidase D digestion but was cleaved by endo-beta-N-acetylglucosaminidase H. This isomer has the probable structure Man(alpha 1-3)Man(alpha 1-6)Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc, indicative of its origin from hybrid or high-mannose glycoproteins. Man5GlcNAc2 reached a peak on day 13 and then slowly declined, whereas Man4GlcNAc2 increased concomitantly. The rapid increase in Man5GlcNAc2 can probably be attributed to the breakdown of hybrid glycans produced as a result of swainsonine inhibition of Golgi alpha-D-mannosidase II. The onset of observable clinical signs on day 38 closely correlated with the time point at which the level of Man4GlcNAc2 exceeded Man5GlcNAc2. After locoweed feeding was discontinued, the amount of urinary oligosaccharides declined rapidly and reached baseline levels within 12 days.     

Sulfated oligosaccharides isolated from the respiratory mucins of a secretor patient suffering from chronic bronchitis

The most acidic carbohydrate chains released by alkaline borohydride treatment of the bulk of airway mucins secreted by a patient (blood group O, secretor) suffering from a mildly infected chronic bronchitis have been fractionated using high-performance anion-exchange chromatography (HPAEC) according to a protocol already described [Lo-Guidice et al., J. Biol. Chem. 269 (1994) 18794] and were analyzed using 1H-NMR spectroscopy and matrix-assisted laser-adsorption-time-of-flight (MALDI-TOF) spectrometry. Many fractions corresponded to mixtures of oligosaccharides. This confirmed the wide diversity of the post-translational processes involved in the biosynthesis of airway mucins, which had already been observed in bronchial diseases, such as chronic bronchitis and cystic fibrosis (CF). Seven fractions were directly purified by HPAEC, allowing their structural determination. Six of them corresponded to 3-O-sulfated oligosaccharide chains terminated by a sulfated N-acetyllactosamine, a sulfated Lewis X or a sulfated Lewis A determinant, and the last one corresponded to a 6-O-sulfated chain terminated by a sulfated H-2 determinant. Three oligosaccharides had core type 2 and the other four had core type 4: IIIc2-9: Gal(beta1-3)[HSO(3)-3-Gal(beta1-4)GlcNAc(beta1-6)]GalNAc-ol, IIIc2-10: Gal(beta1-3)[Fuc(alpha1-2)Gal(beta1-4)[HSO(3)-6-]GlcNAc(beta1-6)]GalNAc-ol, IIIc2-4: Fuc(alpha1-2)Gal(beta1-3)[HSO(3)-3-Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)]GalNAc-ol, IIIc2-8: Fuc(alpha1-2)Gal(beta1-3)GlcNAc(beta1-3)[HSO(3)-3-Gal(beta1-4)GlcNAc(beta1-6)]GalNAc-ol, IIIc2-7: Fuc(alpha1-2)Gal(beta1-3)GlcNAc(beta1-3)[Gal(beta1-4)[HSO(3)-6-]GlcNAc(beta1-6)]GalNAc-ol, IIIc2-3: Fuc(alpha1-2)Gal(beta1-3)GlcNAc(beta1-3)[HSO(3)-3-Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)]GalNAc-ol, IIIc1-4: Fuc(alpha1-2)Gal(beta1-3)GlcNAc(beta1-3)[HSO(3) -3-Gal(beta1-3)[Fuc(alpha1-4)]GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-6)]GalNAc-ol. Like previous data concerning the airway mucins from another patient (blood group O and non-secretor) suffering from chronic bronchitis [Lo-Guidice et al., Glycoconj. J. 14 (1997) 113], no disialylated oligosaccharide and no sialylated and sulfated oligosaccharide bearing sialyl Lewis X epitope could be isolated. This is in contrast with the data obtained with the airway mucins secreted by the patient severely infected by Pseudomonas aeruginosa and suffering from CF, suggesting that important differences occur in the biosynthesis of airway mucins secreted by patients suffering from different bronchial diseases with or without severe infection.     

Structural characterization of oligosaccharides in the milk of an African elephant (Loxodonta africana africana)

The oligosaccharides present in the milk of an African elephant (Loxodonta africana africana), collected 4 days post partum, were separated by size exclusion-, anion exchange- and high-performance liquid chromatography (HPLC) before characterisation by (1)H NMR spectroscopy. Neutral and acidic oligosaccharides were identified. Neutral oligosaccharides characterised were isoglobotriose, Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc and a novel oligosaccharide that has not been reported in the milk or colostrum of any other mammal: Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc. Acidic oligosaccharides that are also found in the milk of Asian elephant were Neu5Ac(alpha2-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)Glc, Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc and Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3){Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)}Gal(beta1-4)Glc, while Neu5Gc(alpha2-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)[Gal(beta1-4)GlcNAc(beta1-6)]Gal(beta1-4)Glc and Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3){Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)}Gal(beta1-4)Glc have not been found in Asian elephant milk. The oligosaccharides characterised contained both alpha(2-3)- and alpha(2-6)-linked Neu5Ac residues. They also contain only the type II chain, as found in most non-human, eutherian mammals.     

Primary structure of the glycans from mouse serum and milk transferrins.

A 'serotransferrin-like' protein was purified from mouse milk. This serotransferrin cross-reacts immunologically with the serotransferrin isolated from mouse plasma and not with the mouse lactotransferrin (lactoferrin). Sugar analysis of the three transferrins, i.e. serotransferrin, milk 'serotransferrin-like' protein and lactotransferrin, revealed that the major difference between the glycan primary structure of mouse serotransferrin and those of mouse milk 'serotransferrin-like' protein and lactotransferrin concerns essentially the presence of one fucose residue in the last two proteins. For structural determination, the N-glycosidically linked glycans were released from the protein by a reductive cleavage of the oligosaccharide-protein linkage under strong alkaline conditions. The primary structure of the released oligosaccharide alditols was determined by methylation analysis and 400 MHz 1H-n.m.r. spectroscopy. The oligosaccharide alditols released from milk 'serotransferrin-like' protein and lactotransferrin were identical and were identified as disialylated biantennary glycans of the N-acetyl-lactosamine type with a fucose residue alpha-1,6-linked to the N-acetylglucosamine residue conjugated to the peptide chain and having the following primary structure: NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)GlcNAc(beta 1-4)[Fuc(alpha 1-6)]GlcNAc(beta 1-N)Asn. The serotransferrin glycan has the same primary structure but is only partially fucosylated (10-15%).     

Structural analysis of the major urinary oligosaccharides in feline alpha-mannosidosis.

Two homologous series of urinary oligosaccharides were identified by h.p.l.c. and fast-atom-bombardment mass spectrometry in feline alpha-mannosidosis. The predominant series has the composition Man2-8GlcNAc2 and a minor series the composition Man2-9GlcNAc. The structure of the most abundant oligosaccharide, which accounts for over 80% of the urinary oligosaccharide, was shown to be alpha-D-Manp(1----3)[alpha-D-Manp-(1----6)]beta-D-Manp -(1----4)-beta-D-GlcpNA c-(1----4)-D-GlcNAc by gas chromatography and mass spectrometry. Such a structure is consistent with the incomplete catabolism of complex N-linked glycans due to a deficiency of alpha-D-mannosidase in tissue lacking an endohexosaminidase activity.     

Structural basis of the heterogeneity of asparagine-linked oligosaccharides of a Waldenstrom's macroglobulinemia immunoglobulin M

The extent of glycans heterogeneity in a pathological human immunoglobulin M ZAJ has been studied on oligosaccharides released by hydrazinolysis from the purified glycoprotein. After reduction with NaB3H4, asparagine-linked carbohydrate chains were separated by affinity chromatography on concanavalin A-Sepharose into oligomannosidic and N-acetyllactosaminic types. Glycans of the oligomannosidic type were further fractionated by HPLC and those of the N-acetyllactosamine type by preparative high-voltage electrophoresis. The primary structure of the main oligosaccharides was investigated on the basis of micro-methylation analysis, mass spectrometry and sequential exo-glycosidase digestion. Glycans of the oligomannosidic type varied in size from Man5GlcNAc2 to Man9GlcNAc2. N-Acetyllactosaminic glycans were found of the biantennary, bisected-biantennary and triantennary types. They presented a higher degree of heterogeneity due to the presence of a variable number of NeuAc and fucose residues. The new structures we report here were in addition to the major biantennary one we previously described on the basis of methylation analysis and 500 MHz 1H-NMR spectroscopy (Cahour, A., Debeire, P., Hartmann, L., Montreuil, J., Van Halbeek, H. and Vliegenthart, J.F.G. (1984) FEBS Lett. 170, 343-349): NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[Gal(beta 1-4)Glc-NAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)]Glc-NAc(beta 1-4) [Fuc(alpha 1-6)]GlcNAc.     

Structural determination of the oligosaccharides in the milk of an Asian elephant (Elephas maximus)

Milk of an Asian elephant (Elephas maximus), collected at 11 days post partum, contained 91 g/L of hexose and 3 g/L of sialic acid. The dominant saccharide in this milk sample was lactose, but it also contained isoglobotriose (Glc(alpha1-3)Gal(beta1-4)Glc) as well as a variety of sialyl oligosaccharides. The sialyl oligosaccharides were separated from neutral saccharides by anion exchange chromatography on DEAE-Sephadex A-50 and successive gel chromatography on Bio Gel P-2. They were purified by high performance liquid chromatography (HPLC) using an Amide-80 column and characterized by 1H-NMR spectroscopy. Their structures were determined to be those of 3'-sialyllactose, 6'-sialyllactose, monofucosyl monosialyl lactose (Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc), sialyl lacto-N-neotetraose c (LST c), galactosyl monosialyl lacto-N-neohexaose, galactosyl monofucosyl monosialyl lacto-N-neohexaose and three novel oligosaccharides as follows: Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc, and Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc. The higher oligosaccharides contained only the type II chain (Gal(beta1-4)GlcNAc); this finding differed from previously published data on Asian elephant milk oligosaccharides.     

Site-specific N-glycosylation of human chorionic gonadotrophin--structural analysis of glycopeptides by one- and two-dimensional 1H NMR spectroscopy.

Glycopeptides representing individual N-glycosylation sites of the heterodimeric glycoprotein hormone human chorionic gonadotrophin (hCG) were obtained from subunits hCG alpha (N-glycosylated at Asn-52 and Asn-78) and hCG beta (N-glycosylated at Asn-13 and Asn-30) by digestion with trypsin and chymotrypsin, respectively. Following purification by reverse-phase HPLC and identification by amino acid sequencing, the glycopeptides were analysed by one- and two-dimensional 1H NMR spectroscopy. The results are summarized as follows: (i) oligosaccharides attached to Asn-52 of hCG alpha comprised monosialylated 'monoantenary' NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-4'), disialylated diantennary NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[NeuAc alpha 2-3-Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N2), and the monosialylated hybrid-type structures NeuAc alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-3Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-A) and NeuAc alpha 2-3Gal-beta 1-4GlcNAc beta 1-2Man alpha 1-3[Man alpha 1-3(Man alpha 1-6)Man alpha 1-6]Man beta 1-4GlcNAc beta 1-4GlcNAc (N1-AB) in a ratio approaching 5:2:2:1; (ii) Asn-78 of hCG alpha carried N2 and N1-4' almost exclusively (ratio approximately 3:2); (iii) both N-glycosylation sites of hCG beta contained predominantly component N2, partially (approximately 25%) and completely alpha 1-6-fucosylated at the N-acetylglucosamine linked to Asn-13 and Asn-30, respectively. The distinct site-specific distribution of the oligosaccharide structures among individual N-glycosylation sites of hCG appears to reflect primarily the influence of the surrounding protein structure on the substrate accessibility of the Golgi processing enzymes alpha-mannosidase II, GlcNAc transferase II and alpha 1,6-fucosyltransferase.     

Structural variability of the neutral carbohydrate moiety of cow colostrum kappa-casein as a function of time after parturition. Identification of a tetrasaccharide with blood group I specificity

New neutral oligosaccharides from cow colostrum kappa-casein were identified and characterized by 500-MHz 1H-NMR spectroscopy. Their structures are Gal beta(1----3)GalNAc-ol, Gal beta(1----3)[GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Fuc alpha(1----3)[Gal beta(1----4)]GlcNAc beta(1----6)]GalNAc-ol. The tetrasaccharide and the cow colostrum kappa-caseinoglycopeptide which contains this oligosaccharide inhibit the hemagglutination of blood group I human erythrocytes. In cow mature milk only the disaccharide is characterized. The variability of these neutral oligosaccharides in cow kappa-casein as a function of time after calving is studied.     

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.     

Structure determination of five sialylated trisaccharides with core types 1, 3 or 5 isolated from bovine submaxillary mucin

In this study we have investigated the structures of five sialylated trisaccharides released from bovine submaxillary mucin by alkaline borohydride treatment and isolated by high-performance liquid chromatography. Three of the trisaccharides contained NeuAc while two contained NeuGc. One oligosaccharide contained core-type 1, two contained core-type 3 and two contained core-type 5. The structures, determined by a combination of one- and two-dimensional 1H-NMR spectroscopy at 270 MHz and methylation analysis involving gas-liquid chromatography/mass spectrometry, were as follows: A4b, GalNAc alpha(1----3) [NeuAc alpha(2----6)]GalNAcol; A4c, GlcNAc beta(1----3)[NeuAc alpha(2----6)]GalNAcol; A4d, Gal beta(1----3)[NeuAc alpha(2----6)]GalNAcol; A4e, GalNAc alpha(1----3)-[NeuGc alpha(2----6)]GalNAcol; A4f, GlcNAc beta(1----3)[NeuGc alpha (2----6)]GalNAcol. The oligosaccharides occurred in the approximate molar ratios 1.0:12.0:0.3:0.2:2.0. This is the first report of oligosaccharides containing core-type 5 and of the occurrence of oligosaccharides A4b, A4e, and A4f in bovine submaxillary mucin. 1H-NMR data for structure A4e, which is a novel structure, are presented for the first time.     

Chemical characterization of milk oligosaccharides of the polar bear, Ursus maritimus

Two trisaccharides, three tetrasaccharides, two pentasaccharides, one hexasaccharide, one heptasaccharide, one octasaccharide and one decasaccharide were isolated from polar bear milk samples by chloroform/methanol extraction, gel filtration, ion exchange chromatography and preparative thin-layer chromatography. The oligosaccharides were characterized by 1H-NMR as follows: the saccharides from one animal: Gal(alpha1-3)Gal(beta1-4)Glc (alpha3'-galactosyllactose), Fuc(alpha1-2)Gal(beta1-4)Glc (2'-fucosyllactose), Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc (B-tetrasaccharide), GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc (A-tetrasaccharide), Gal(alpha1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc, Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Gl c, Gal(alpha1-3)Gal(beta1-4)GlcNAc(beta1-3)[Gal(alpha1-3)Gal(beta1-4)Glc NAc(beta1-6)]Gal(beta1-4)Glc; the saccharides from another animal: alpha3'-galactosyllactose, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, A-tetrasaccharide, GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)[Fuc(alpha1-3)]Glc (A-pentasaccharide), Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Gl c, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[F uc(alpha1-3)]Glc (difucosylheptasaccharide) and Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)?Gal(alpha1-3) Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)?Gal(beta1-4)Glc (difucosyldecasaccharide). Lactose was present only in small amounts. Some of the milk oligosaccharides of the polar bear had alpha-Gal epitopes similar to some oligosaccharides in milk from the Ezo brown bear and the Japanese black bear. Some milk oligosaccharides had human blood group A antigens as well as B antigens; these were different from the oligosaccharides in Ezo brown and Japanese black bears.     

Complete structure of the carbohydrate moiety of stem bromelain. An application of the almond glycopeptidase for structural studies of glycopeptides.

Asparagine-linked oligosaccharides of stem bromelain glycopeptides were quantitatively released by digestion with the almond glycopeptidase which cleaves beta-aspartylglycosylamine linkage in glycopeptides with oligopeptide moieties. The primary structures of the two oligosaccharide components, (Man)3(Xyl)1(Fuc)1(GlcNAc)2 and (Man)2-(Xyl)1(Fuc)1(GlcNAc)2 were elucidated as Man alpha 1 leads to 6Man alpha 1 leads to 6[Xyl beta 1 leads to 2]Man beta 1 leads to 4GlcNAc beta 1 leads 4[Fuc alpha 1 leads to 3]GlcNAc and Man alpha 1 leads to 6[Xyl beta 1 leads to 2]Man beta 1 leads to 4 GlcNAc beta 1 leads to 4[Fuc alpha 1 leads to 3] GlcNAc, respectively.     

The structure of sugar chains of Japanese quail ovomucoid. The occurrence of oligosaccharides not expected from the classical biosynthetic pathway for N-glycans; a method for the assessment of the structure of glycans present in picomolar amounts

While the structure of the major oligosaccharide of Japanese quail ovomucoid was reported earlier (Hase, S. et al. (1982) J. Biochem. 91, 735-737), the structures of the minor oligosaccharide units were investigated for the first time in the present studies. For this purpose, the glycans of the protein were liberated from the polypeptide chain by hydrazinolysis. After N-acetylation, the reducing ends of the oligosaccharides obtained were coupled with 2-aminopyridine, and then the resulting fluorescent derivatives were purified by Bio-Gel P-2 column chromatography and reversed-phase HPLC. The chemical structures of two minor oligosaccharide units were determined with the aid of exoglycosidases, and by methylation analysis and Smith degradation. The results demonstrated that the ovomucoid contains the following two monoantennary glycans: Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc and Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. The latter structure was not predicted by the classical metabolic pathway for the N-glycans to be formed. The structures of three additional minor heterosaccharides were deduced from their elution positions on HPLC together with the results of determination of their molecular sizes and the HPLC elution positions of their enzymatic degradation products. It is noteworthy that for the latter procedure for the estimation of the structures of oligosaccharides only minute quantities of glycans (several hundreds pmol) are required.     

Rat epididymal alpha-D-mannosidase: purification, carbohydrate composition, substrate specificity, and antibody production

Two alpha-D-mannosidases have previously been identified in rat epididymis. This communication reports the purification and characterization of the "acid" alpha-D-mannosidase. The enzyme was purified over 1000-fold to near homogeneity by acetone and (NH4)2SO4 precipitation followed by ion-exchange and hydroxylapatite chromatography. The molecular weight of the enzyme was estimated to be 220,000 by gel filtration. Polyacrylamide gel electrophoresis of the native enzyme under two conditions of buffer and pH showed a single band when stained for protein while electrophoresis under denaturing conditions resulted in bands of apparent Mr 60,000 and 31,000. The enzyme is a glycoprotein containing about 5.6% hexose. In addition to mannose (3.1%) and glucosamine (2.0%), the enzyme also contained small amounts of glucose, fucose, and galactose. Chemical analysis indicated the absence of sialic acid. The substrate specificity of the purified enzyme was investigated using linear and branched mannose-containing oligosaccharides. The enzyme cleaved linear oligosaccharides [Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc and Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc] very efficiently. However, little or no activity was observed toward high mannose oligosaccharides (Man9GlcNAc through Man5GlcNAc) or the branched trimannosyl derivative Man3GlcNAc. This specificity is very similar to that observed with rat kidney lysosomal alpha-D-mannosidase. Additional evidence that the epididymal enzyme is essentially a lysosomal alpha-D-mannosidase is the fact that polyclonal antibody prepared against the purified epididymal enzyme cross-reacted with lysosomal alpha-D-mannosidase from several rat tissues and with acidic alpha-D-mannosidase of a human cell line, results suggesting that the antibody will be useful in studying the biosynthesis and turnover of lysosomal alpha-D-mannosidases in at least two species.     

Chemical characterization of milk oligosaccharides of the Japanese black bear, Ursus thibetanus japonicus

Two trisaccharides, two tetrasaccharides, one penta-, one hexa-, two hepta-, one deca- and two undeca-saccharides were isolated from several Japanese black bear milk samples by chloroform/methanol extraction, gel filtration and preparative thin-layer chromatography. The oligosaccharides were characterized by 1H-NMR as follows: Gal(alpha 1-3)Gal(beta 1-4)Glc (alpha 3'-galactosyllactose), Fuc(alpha 1-2)Gal(beta 1-4)Glc (2'-fucosyllactose), Gal(alpha 1-3)(Fuc(alpha 1-2))Gal(beta 1-4)Glc (B-tetrasaccharide), Gal(alpha 1-3)Gal(beta 1-4)(Fuc(alpha 1-3))Glc, Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]Glc (B-pentasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4)Glc (monofucosylhexasaccharide), Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4)Glc (difucosylheptasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]Glc (difucosylheptasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)[Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-6)]Gal(beta 1-4)Glc (difucosyldecasaccharide), Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)[Gal(alpha 1-3) Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-6)]Gal(beta 1-4)Glc (trifucosylundecasaccharide), Gal(alpha 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)[Gal(alpha 1-3)[Fuc(alpha 1-2)]Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-6)]Gal(beta 1-4)Glc (trifucosylundecasaccharide). Lactose was present only in trace amounts. B-pentasaccharide was a dominant saccharide in early lactation milk, while alpha 3'-galactosyllactose was dominant in milk, later. The milk oligosaccharides of the Japanese black bear were compared with those of the Ezo brown bear.     

Control of glycoprotein synthesis. Detection and characterization of a novel branching enzyme from hen oviduct, UDP-N-acetylglucosamine:GlcNAc beta 1-6 (GlcNAc beta 1-2)Man alpha-R (GlcNAc to Man) beta-4-N-acetylglucosaminyltransferase VI

Hen oviduct membranes were shown to contain high activity of a novel enzyme, UDP-GlcNac:GlcNAc beta 1-6(GlcNAc beta 1-2) Man alpha-R (GlcNAc to Man) beta 4-GlcNAc-transferase VI. The enzyme was shown to transfer GlcNAc in beta 1-4 linkage to the D-mannose residue of GlcNAc beta 1-6 (GlcNAc beta 1-2) Man alpha-R where R is either 1-6Man beta-(CH2)8COOCH3 or methyl. Radioactive enzyme products were purified by several chromatographic steps, including high performance liquid chromatography, and structures were determined by proton nmr, fast atom bombardment-mass spectrometry, and methylation analysis to be GlcNAc beta 1-6 ([14C]GlcNAc beta 1-4) (GlcNAc beta 1-2) Man alpha-R. The enzyme is stimulated by Triton X-100 and has optimum activity at a relatively high MnCl2 concentration of about 100 mM; Co2+, Mg2+, and Ca2+ could partially substitute for Mn2+. A tissue survey demonstrated high GlcNAc-transferase VI activity in hen oviduct and lower activity in chicken liver and colon, duck colon, and turkey intestine. No activity was found in mammalian tissues. Hen oviduct membranes cannot act on GlcNAc beta 1-6Man alpha-R but have a beta 4-GlcNAc-transferase activity that converts GlcNAc beta 1-2Man alpha-R to GlcNAc beta 1-4(GlcNAc beta 1-2) Man alpha-R where R is either 1-6Man beta-(CH2)8COOCH3 or 1-6Man beta methyl. The latter activity is probably due to GlcNAc-transferase IV which preferentially adds GlcNAc in beta 1-4 linkage to the Man alpha 1-3 arm of the GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn core structure of asparagine-linked glycans. The minimum structural requirement for a substrate of beta 4-GlcNAc-transferase VI is therefore the trisaccharide GlcNAc beta 1-6(GlcNAc beta 1-2) Man alpha-; this trisaccharide is found on the Man alpha 6 arm of many branched complex asparagine-linked oligosaccharides. The data suggest that GlcNAc-transferase VI acts after the synthesis of the GlcNAc beta 1-2Man alpha 1-3-, GlcNAc beta 1-2Man alpha 1-6-, and GlcNAc beta 1-6 Man alpha 1-6-branches by GlcNAc-transferases I, II, and V, respectively, and is responsible for the synthesis of branched oligosaccharides containing the GlcNAc beta 1-6(GlcNAc beta 1-4)(GlcNAc beta 1-2)Man alpha 1-6Man beta moiety.