Isolation and characterization of sulphated oligosaccharides released from bovine corneal keratan sulphate by the action of endo-beta-galactosidase

A series of oligosaccharides has been isolated from the keratan sulphate peptidoglycan (3 M NaCl fraction) of bovine cornea after digestion with the endo-beta-galactosidase of Bacteroides fragilis. Structural information on the major oligosaccharides was obtained from (a) their susceptibilities to endo-beta-galactosidase before and after desulphation, (b) their elution positions on a column of Bio-Gel P-4 and retention times on a high-performance anion-exchange column and (c) negative-ion fast-atom-bombardment mass spectrometry. More than 75% of the oligosaccharides were sulphated unbranched poly(N-acetyllactosamine) sequences, (-3/4GlcNAc beta 1-3Gal beta 1-)n, and approximately 3% was the neutral disaccharide, GlcNAc beta 1-3Gal. The sulphated disaccharide, GlcNAc-SO-3 beta 1-3Gal, accounted for almost 35% of the oligosaccharide material while 40% consisted of four oligosaccharides, unbranched tetra-, hexa-, octa- and decasaccharides of poly(N-acetyllactosamine) type, having 3, 5, 7 and 9 sulphate residues respectively. Proton nuclear magnetic resonance studies at 500 MHz (Hounsell, E. F., et al. following paper in this journal) have shown that a sulphate residue is attached to the C-6 position of each N-acetylglucosamine and each internal galactose residue of these four oligosaccharides which express to varying degrees the antigenic determinants recognised by three monoclonal antibodies to keratan sulphate (Mehmet, H. et al., paper which follows the next paper in this journal).     

Oligosaccharides obtained from a blood-group-Sd(a+) Tamm-Horsfall glycoprotein. An n.m.r. study.

Treatment of Tamm-Horsfall urinary glycoprotein with Bacteroides fragilis endo-beta-galactosidase over a range of enzyme concentrations, pH and temperature resulted in the release of a small but constant proportion of the terminal sugars, which indicates the presence in the glycoprotein of relatively few enzyme-susceptible -GlcNAc beta 1-3Gal beta 1-4GlcNAc- units. Three oligosaccharides were isolated from the enzyme digest and characterized as Gal beta 1-4GlcNAc beta 1-3Gal, NeuAc alpha 2-3Gal beta 1-4 GlcNAc beta 1-3Gal and GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta 1-4GlcNAc beta 1-3Gal by methylation analysis and exo-glycosidase digestion. The alditols of these oligosaccharides and related structures were examined by 500 MHz 1H-n.m.r. spectroscopy aided by spin-spin decoupling and two-dimensional correlated spectroscopy. An almost complete assignment of proton shifts was possible, and significant differences between the signals of some of the protons in the blood-group-Sda-active oligosaccharide III and literature values for the corresponding signals in the structurally related Cad-blood-group determinant are noted.     

Endo-beta-D-galactosidases of Bacteroides fragilis and Escherichia freundii hydrolyze linear but not branched oligosaccharide domains of glycolipids of the neolacto series

The specificities of the endo-beta-galactosidases of Bacteroides fragilis and Escherichia freundii towards linear and branched oligosaccharides of the lacto-N-glycosyl series were investigated using as substrates glycolipids containing (a) linear neolactotetra - or hexaosyl sequences, (b) branched biantennary neolactooctaosyl sequences, and (c) triantennary neolactononaor dodecaglycosyl sequences. Glycolipid and oligosaccharide hydrolysis products were identified by tlc and/or paper chromatography. The rate of hydrolysis was assessed in time course experiments in which the oligosaccharides released were quantified as 3H-labeled alditols. The salient observations were as follows. (i) With the substrates thus far tested in the present and a previous study ( Scudder , P., Uemura , K., Dolby , J., Fukuda, M.N., and Feizi , T. (1983) Biochem. J. 213, 485-494), the endo-beta-galactosidases from B. fragilis and E. freudii have indistinguishable specificities. (ii) The beta-galactosidic linkage of the branch point sequence (Formula: see text) is completely resistant to hydrolysis by these enzymes, although the unbranched sequence GlcNAc beta 1-3Gal beta 1-4GlcNAc/Glc is readily cleaved. (iii) At an optimal concentration of detergent, the endo-beta-galactosidase susceptibility of the GlcNAc beta 1-3Gal beta 1-4Glc sequence near the ceramide moiety of branched glycolipids is similar to that of the corresponding sequence in linear glycolipids.     

Endo-beta-galactosidase of Escherichia freundii. Purification and endoglycosidic action on keratan sulfates, oligosaccharides, and blood group active glycoprotein.

Endo-beta-galactosidase was purified 4400-fold from a culture filtrate of Escherichia freundii with 45% recovery. The enzyme preparation was practically free of exoglycosidases, sulfatase, and proteases. This enzyme hydrolyzed several keratan sulfates, endoglycosidically releasing oligosaccharides of various molecular sizes. Among the digestion products of the corneal keratan sulfate, the structure of a disaccharride and a tetrasaccharride were shown to be 2-acetamido-2-deoxy-6-O-sulfo-beta-D-glucosyl-(1 leads to 3)-D-galactose and 2-acetamido-2-deoxy-6-O-sulfo-beta-D-glucosyl-(1 leads to 3)-6-O-sulfo-beta-D-galactosyl-(1 leads to 4)-2-acetamido-2-deoxy-6-O-sulfo-beta-D-glucosyl-(1 leads to 3)-D-galactose, respectively. These oligosaccharide structures indicate that this enzyme specifically hydrolyzes the galactosidic bonds in which nonsulfated galactose residues participate. The enzyme could also hydrolyze a small oligosaccharide such as lacto-N-neotetraitol as follows: Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to 3)Gal(beta 1 leads to 4) sorbitol leads to Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to 3)Gal + sorbitol AB active blood group substance could be hydrolyzed by this enzyme only after Smith degradation. After enzymatic digestion small oligosaccharides and resistant macromolecules were produced. These findings indicate that the enzyme should be useful in studying the precise structures of keratan sulfates, related glycoproteins, and oligosaccharides.     

Kinetic parameters of a beta-D-galactoside alpha 2 leads to 6 sialyltransferase from embryonic chicken liver

A beta-D-galactoside alpha 2 leads to 6 sialyltransferase was purified 500-fold in 14% yield from 14-day embryonic chicken liver. Characterization of the product of the sialyltransferase catalysis was accomplished by separation and permethylation of double-labelled ([14C]NeuAc, [3H]Gal) oligosaccharides following their release from the glycoprotein fetuin by hydrazinolysis. The enzyme transfers NeuAc to Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to)R-terminated oligosaccharides; no activity was found towards Gal(beta 1 leads to 3)GalNAc(alpha 1 leads to)R structures. The trisaccharide. NeuAc(alpha 2 leads to 6)Gal(beta 1 leads to 4)Glc, was shown to be a good inhibitor of the sialyltransferase. Kinetic investigations of the enzyme indicate it to have a sequential, random bi-bi mechanism.     

Structures of neutral O-linked polylactosaminoglycans on human skim milk mucins. A novel type of linearly extended poly-N-acetyllactosamine backbones with Gal beta(1-4)GlcNAc beta(1-6) repeating units

O-Linked oligosaccharides were isolated from human skim milk mucins and from mucin-derived glycopeptides by reductive beta-elimination. The released alditols were fractionated by DEAE-Sephadex chromatography and purified by high performance liquid chromatography on primary amine bonded phase. The structures of the major neutral oligosaccharide alditols could be established by fast atom bombardment and electron impact mass spectrometry, combined with methylation analysis, 500-MHz 1H nuclear magnetic resonance spectroscopy, and endo-beta-galactosidase (from Bacteroides fragilis, EC 3.2.1.103) digestion (where n = 0-3): (formula; see text) Major O-glycosidically linked oligosaccharides on skim milk mucins are of the Gal beta(1-3)[GlcNAc beta(1-6)] GalNAc core type 2 and exhibit linearly extended backbone chains of the poly N-acetyllactosamine type comprizing up to at least four repeating units, which are linked by the hitherto unknown sequence GlcNAc-beta(1-6) Gal rather than GlcNAc beta(1-3)Gal. A considerable portion of neutral alditols is represented by branched isomers of the linear species, which are distinguished by their content of 3,6-disubstituted galactose and their partial resistance to endo-beta-galactosidase digestion.     

Human serum contains a novel beta 1,6-N-acetylglucosaminyltransferase activity that is involved in midchain branching of oligo (N-acetyllactosaminoglycans).

Incubation of UDP-GlcNAc and radiolabeled GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (1) with human serum resulted in the formation of the branched hexasaccharide GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (2) in yields of up to 22.2%. The novel reaction represents midchain branching of the linear acceptor; the previously known branching reactions of oligo-(N-acetyllactosaminoglycans) involve the nonreducing end of the growing saccharide chains. The structure of 2 was established by use of appropriate isotopic isomers of it for degradative experiments. The hexasaccharide 2 was cleaved by an exhaustive treatment with jack bean beta-N-acetylhexosaminidase, liberating two GlcNAc units and the tetrasaccharide Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (3). Endo-beta-galactosidase from Bacteroides fragilis cleaved 2 at one site only, yielding the disaccharide GlcNAc beta 1-3Gal (4) and the branched tetrasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (5). The structure of 5 was established by partial acid hydrolysis and subsequent identification of the disaccharide GlcNAc beta 1-6Gal (6), together with the trisaccharides GlcNAc beta 1-6Gal beta 1-4GlcNAc (7) and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal (8) among the cleavage products. Galactosylation of 2 with bovine milk beta 1,4-galactosyltransferase and UDP-[6-3H]Gal gave the octasaccharide [6-3H]Gal beta 1-4GlcNAc beta 1-3 Gal beta 1-4GlcNAc beta 1-3([6-3H]-Gal beta 1-4GlcNAc beta 1-6)[U-14C] Gal beta 1-4GlcNAc (17), which could be cleaved with endo-beta-galactosidase into the trisaccharide [6-3H]Gal beta 1-4GlcNAc beta 1-3Gal (18) and the branched pentasaccharide GlcNAc beta 1-3-([6-3H]Gal beta 1-4GlcNAc beta 1-6) [U-14C]Gal beta 1-4GlcNAc (19). Partial hydrolysis of 2 with jack-bean beta-N-acetylhexosaminidase gave the linear pentasaccharide 1 and the branched pentasaccharide Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (20). The serum beta 1,6-GlcNAc transferase catalyzed also the formation of GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc (11) from UDP-GlcNAc and GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc (10). The pentasaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (16), too, served as an acceptor for the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and characterization of a new endo-beta-galactosidase from Diplococcus pneumoniae

An endo-beta-galactosidase, which hydrolyzes the internal beta-galactosidic linkages of R----GlcNAc (or GalNAc) beta 1----3Gal beta 1----4GlcNAc (or Glc), was isolated from the culture supernatant of Diplococcus pneumoniae. The enzyme, named endo-beta-galactosidase DII, hydrolyzed linear N-acetyllactosamine repeating structures in glycolipids and glycopeptides to release oligosaccharides. The specificity of endo-beta-galactosidase DII is the same as that of Escherichia freundii endo-beta-galactosidase as far as described above, but the following differences between these two enzymes were found: Branched lactosaminyl glycolipids and H-antigenic glycolipids were resistant to endo-beta-galactosidase DII, even when linear structure was present at the inner part. Throughout the enzymic hydrolysis, endo-beta-galactosidase DII released mostly small oligosaccharides (tetra-, tri-, and disaccharides) from substrates, suggesting that the enzyme split off the oligosaccharides stepwise from the nonreducing terminal. Lactosaminoglycans were partially hydrolyzed by endo-beta-galactosidase DII to produce small oligosaccharides as the major product and residual glycopeptides. The residual glycopeptides were readily hydrolyzed by E. freundii endo-beta-galactosidase to produce various sizes of oligosaccharides. Keratan sulfate was not degraded by endo-beta-galactosidase DII. These properties of endo-beta-galactosidase DII characterize it as a new endo-beta-galactosidase with a unique specificity.     

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.     

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.     

1H-NMR studies at 500 MHz of a neutral disaccharide and sulphated di-, tetra-, hexa- and larger oligosaccharides obtained by endo-beta-galactosidase treatment of keratan sulphate

In the preceding paper in this journal, the major oligosaccharides obtained by endo-beta-galactosidase digestion of bovine corneal keratan sulphate were identified as a neutral disaccharide, GlcNAc beta 1-3Gal, and sulphated di-, tetra-, hexa-, octa- and decasaccharides based on the sequence (-3/4GlcNAc beta 1-3Gal beta 1-)n having 1, 3, 5, 7 and 9 sulphate groups, respectively. In the present study, these oligosaccharides have been analysed by 500-MHz 1H-NMR spectroscopy using spin-decoupling and two-dimensional correlated spectroscopy experiments. The NMR data confirm the beta-configuration of all the interglycosidic linkages and are consistent with an alternating sequence of----4GlcNAc and----3Gal, a non-reducing-end N-acetylglucosamine residue and a reducing-end galactose residue. The NMR data have also established that a sulphate group is linked to the C6 position of all sugar residues except the reducing-end galactose as follows: (Formula: see text). The signals of the protons attached to the sulphated carbon atoms show marked downfield shifts (approximately 0.4 ppm from equivalent protons of non-sulphated carbon atoms), while the protons at C5 vicinal to sulphated atoms show a change of 0.1-0.2 ppm and other protons of the sulphated monosaccharides show smaller changes in chemical shift (0.01-0.1 ppm). The proton at C4 of the non-sulphated reducing-end galactose linked at C3 also shows a significant change in chemical shift (0.03 ppm).     

Urinary oligosaccharides of GM1-gangliosidosis. Different excretion patterns of oligosaccharides in the urine of type 1 and type 2 subgroups

Oligosaccharide patterns obtained by gel filtration of the urine of GM1-gangliosidosis Type 1 patients are quite different from those of GM1-gangliosidosis Type 2. By studies of oligosaccharides in the four major peaks obtained from the Type 1 subgroup using sequential exoglycosidase digestion, methylation analysis, and periodate oxidation, the structures of 15 oligosaccharides: Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6Man beta 1 leads to 4GlcNAc, Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[Gal beta 1 leads to 4GlcNAc beta 1 leads to 4(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 6(Gal beta 1 leads to 4Glc NAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 6[Gal beta 1 leads to 4GlcNAc beta 1 leads to 4(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6, and 3(Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 and 6)Man beta 1 leads to 4GlcNAc, (formula see text) were elucidated. The amounts of total oligosaccharides excreted in the urine of the Type 2 subgroup were approximately one-tenth of those of Type 1. Moreover, the last eight oligosaccharides shown above, which have a Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to outer chain, were completely missing in the urine of Type 2.     

Primary structure of human milk nona- and decasaccharides determined by a combination of fast atom bombardment mass spectrometry and1H-/13C-nuclear magnetic resonance spectroscopy. Evidence for a new core structure,iso-lacto-N-octaose

The structure of a nonasaccharide and of two decasaccharides isolated from human milk has been investigated by using methylation, fast atom bombardment mass spectrometry and 1H-/13C-nuclear magnetic resonance spectroscopy. The structures of these oligosaccharides were: trifucosyllacto-N-hexaose; Fuc alpha 1-2Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3[Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6]Gal beta 1-4Glc, difucosyllacto-N-octaoses; Gal beta 1-3(Fuc alpha 1-4)GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6[Gal beta 1-3GlcNAc beta 1-3]Gal beta 1-4Glc and Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6[Fuc alpha 1-3 Gal beta 1-3GlcNAc beta 1-3]Gal beta 1-4Glc. The two decasaccharides possess a new type of core structure proposed to be named iso-lacto-N-octaose.     

Donor specificity in the glycosylation of Tamm-Horsfall glycoprotein: conservation of the Sda determinant in pairs of twins

The content of the Sd(a) determinant in urinary human Tamm-Horsfall glycoprotein (THp) has been reported to be donor-specific. This feature was further addressed by investigating THp from genetically identical individuals. To this end, THp was isolated from the urine of two monozygotic pairs of twins (A and B). The four samples (THp A1, A2, B1, and B2) were subjected to endo-beta-galactosidase from Bacteroides fragilis leading to the liberation of the Neu5Ac(alpha2-3)Gal (beta1-4)GlcNAc(beta1-3)Gal and Neu5Ac(alpha2-3)[GalNAc(beta1-4)] Gal(beta1-4)GlcNAc(beta1-3)Gal (Sd(a) epitope) motifs, both located at the nonreducing termini of complex type N-glycans. The isolated mixtures of oligosaccharides were analyzed for the absolute and relative amounts of the two oligosaccharides. The obtained data clearly indicate that in THp A1 and A2, and in THp B1 and B2, the molar ratios of the tetra- and Sd(a) pentasaccharide are identical for a pair of twins. This conservation of molar ratios points to an identical relative expression of beta-1,4-N-acetylgalactosaminyltransferase activity involved in the biosynthesis of the Sd(a) determinant. Apparently, the degree of conversion of the tetrasaccharidic Sd(a) precursor into the final pentasaccharidic Sd(a) form can be considered to result from a very closely related pattern of glycosylation for genetically homogeneous individuals.     

A new endo-beta-galactosidase acting on the Gal beta 1-3Gal linkage of the proteoglycan linkage region.

A new type of endo-beta-galactosidase acting on the linkage region of peptidochondroitin sulfate was isolated from the mid-gut gland of the mollusk Patinopecten. The purification procedure included ammonium sulfate precipitation, Sephacryl S-200HR gel filtration, DEAE-Sephacel chromatography, and TSKgel Phenyl-5PW RP high performance liquid chromatography. The purified enzyme was free from exoglycosidases, sulfatases, and phosphatases. The specificity of the enzyme was as follows. 1) It acted on the internal galactoside linkage of sugar chains; 2) it specifically hydrolyzed the galactosylgalactose (Gal beta 1-3Gal) linkage, but not the galactosylxylose (Gal beta 1-4Xyl) linkage in the linkage region of peptidoglycans; 3) the enzyme activity was unaffected by the type of glycosaminoglycan, chondroitin sulfate, dermatan sulfate or heparan sulfate used as a substrate; 4) keratan sulfate and some oligosaccharides from glycolipid were not degraded by the enzyme. These properties of the endo-beta-galactosidase characterize it as a new endo-beta-galactosidase with unique specificity.     

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.     

Assignment of the1H- and13C-NMR spectra of eight oligosaccharides of the lacto-N-tetraose and neotetraose series

The assignment of the 13C- and 1H-NMR spectra of eight oligosaccharides of the lacto-N-tetraose and neotetraose series was obtained from homonuclear and heteronuclear correlation spectroscopy. These analyses were performed on the following compounds: 1. Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc; 2. NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc; 3. Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 4. NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 5. NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4Glc; 6. Fuc alpha 1-2Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 7. Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc; 8. NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc.     

Urinary endo-beta-galactosidase capable of depolymerizing polylactosaminoglycans

Human urine was found to contain an endo-beta-galactosidase capable of depolymerizing sulfated and non-sulfated polylactosaminoglycans. Using 0.05 M sodium phosphate buffer, pH 7.0, this enzyme was not retained by DEAE-Sephadex A-50 or concanavalin A-Sepharose. The urinary endo-beta-galactosidase liberated a disaccharide with chromatographic mobility identical to 6-O-sulfo-GlcNAc beta 1----3Gal as one of the major products from keratan sulfates isolated from whale nasal cartilage, bovine cornea, and human costal cartilage. It also liberated GlcNAc beta 1----3 Gal as one of the major oligosaccharides from erythroglycan. The oligosaccharide profiles produced from various keratan sulfates and erythroglycan by the action of urinary endo-beta-galactosidase are quite similar to those produced by Escherichia freundii endo-beta-galactosidase (Nakagawa, H., Yamada, T., Chien, J.-L., Gardas, A., Kitamikado, M., Li, S.-C., and Li, Y.-T. (1980) J. Biol. Chem. 255, 5955-5959). The presence of urinary endo-beta-galactosidase indicates the existence of a new catabolic pathway for polylactosaminoglycans. This pathway involves the cleavage of internal beta-galactosyl linkages of the glycan chain.     

Chemical characterisation of the oligosaccharides in hooded seal (Cystophora cristata) and Australian fur seal (Arctocephalus pusillus doriferus) milk

Carbohydrates were extracted from hooded seal milk, Crystophora cristata (family Phocidae). Free oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and preparative thin layer or paper chromatography and their structures determined by 1H-NMR. The hooded seal milk was found to contain inositol and at least nine oligosaccharides, most of which had lacto-N-neotetraose or lacto-N-neohexaose as core units, similar to those in milk of other species of Carnivora such as bears (Ursidae). Their structures were as follows: Gal(beta1-4)Glc (lactose); Fuc(alpha1-2)Gal(beta1-4)Glc (2'-fucosyllactose); Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (lacto-N-neotetraose); Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (lacto-N-fucopentaose IV); Gal(beta1-4)GlcNAc(beta1-3)[Gal(beta1-4)GlcNAc(beta1-6)]Gal(1-4)Glc (lacto-N-neohexaose); Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-3)[Gal(beta1-4)GlcNAc(beta1-6)]Gal(beta1-4)Glc (monofucosyl lacto-N-neohexaose a); Gal(beta1-4)GlcNAc(beta1-3)[Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-6)]Gal(beta1-4)Glc (monofucosyl lacto-N-neohexaose b); Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-3)[Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-6)]Gal(beta1-4)Glc (difucosyl lacto-N-neohexaose); Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (para lacto-N-neohexaose); Fuc(alpha1-2)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (monofucosyl para lacto-N-neohexaose). Milk of the Australian fur seal, Arctophalus pusillus doriferus (family Otariidae) contained inositol but no lactose or free oligosaccharides. These results, therefore, support the hypothesis that the milk of otariids, unlike that of phocids, contains no free reducing saccharides.     

Chemical characterisation of the oligosaccharides in a sample of milk of a white-nosed coati, Nasua narica (Procyonidae: Carnivora).

Carbohydrates were extracted from a sample of coati milk and the component oligosaccharides were separated and partially purified by gel filtration and preparative thin layer chromatography. Their structures were determined by 1H-NMR. Fuc alpha 1-->2Gal beta 1-->4Glc Gal alpha 1-->3Gal beta 1-->4Glc Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc Fuc alpha 1-->2Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc Gal alpha 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc The two pentasaccharides are novel sugars. In addition, higher oligosaccharides, whose core units were lacto-N-neohexaose, were found in coati milk. Free lactose constituted only about one-third of the total free milk saccharides. The results are discussed in terms of comparisons with the milk sugars of bears and other species.