Oligosaccharide Alditols Research Articles

Carbohydrate structure of human pancreatic elastase 1

Human pancreatic elastase 1 (E1) is a glycoprotein containing two potential N-glycosylation sites, one of which carries a carbohydrate moiety [Wendorf, Geyer, Sziegoleit & Linder (1989) FEBS Lett. 249, 275-278]. In order to study its glycosylation, glycoprotein isolated from post-mortem pancreas tissue of 75 donors was digested with trypsin. Oligosaccharides were liberated from resulting glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glycosaminyl)-asparagine amidase F, radiolabelled by reduction with KB3H4 and separated by h.p.l.c. and gel filtration. Major oligosaccharide alditol fractions, representing 67.8 mol% of total glycans, were characterized by methylation analysis and sequential degradation with exoglycosidases. The results revealed that about two-fifths of the partially truncated, mainly biantennary, complex-type glycans found comprised blood group A, B, Lea (or X), difucosyl A or difucosyl B determinants, which could be assigned to lactosamine antennae linked to Man(alpha 1-3)- residues of the sugar chains.

Primary structure of O‐linked carbohydrate chains in the cellulosome of different Clostridium thermocellum strains

The cell-free forms of the multiple cellulase-containing protein complex (cellulosome), isolated from the cellulolytic bacterium Clostridium thermocellum strains YS, ATCC 27405 and LQRI, have a total carbohydrate content of 5-7% (by mass), consisting of O-linked oligosaccharide chains. The carbohydrate chains were liberated by alkaline-borohydride treatment and fractionated as oligosaccharide alditols via gel-permeation chromatography and HPLC. The fractions were investigated by 500-MHz 1H-NMR spectroscopy in combination with monosaccharide and methylation analysis and with fast-atom-bombardment mass spectrometry (FAB-MS). In addition to the previously described major oligosaccharide, (formula; see text) [Gerwig, G. J., de Waard, P., Kamerling, J. P., Vliegenthart, J. F. G., Morgenstern, E., Lamed, R. & Bayer, E. A. (1989) J. Biol. Chem. 264, 1027-1035], the following partial structures of this compound could be established: (formula; see text). Cell-free and cell-associated forms of the cellulosome of C. thermocellum, as determined for strain YS, have the same oligosaccharide pattern. Based on the oligosaccharide structures, a biosynthetic pathway is suggested.

The carbohydrate structures of a mouse monoclonal IgG antibody OKT3

A mouse monoclonal antibody OKT3, of IgG2a isotype, was isolated from hybridoma culture fluid. Sugar analysis showed the presence of sialic acid, galactose, mannose, fucose, and N-acetylglucosamine, i.e. sugars typical for N-glycosidically linked carbohydrate chains. The absence of N-acetylgalactosamine revealed that O-glycosidically linked carbohydrates were not present. The purified antibody was reduced, alkylated, and separated into heavy and light chains, and all carbohydrates were shown to be associated with the heavy chains. The N-linked carbohydrate chains were isolated as alditols using strong alkaline-borohydride degradation and further fractionated on a concanavalin A-Sepharose column and high performance ion exchange chromatography with pulsed amperometric detection. Structural analysis was carried out on the isolated oligosaccharide alditols by chemical analyses, fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. Triantennary and biantenary types of structures were found. The triantennary structures were present as trisialo and tetrasialo forms without fucose; the tetrasialo forms were shown to contain a sequence of Neu5Ac alpha 2-3Gal beta 1-3[Neu5Ac alpha 2-6]GlcNAc beta 1- on one of the branches. The biantennary structures were present as completely sialylated nonfucosylated species and as asialo-, agalacto-, and partially fucosylated structures.

Primary structure of glycans isolated from human leucocyte lactotransferrin. Absence of fucose residues questions the proposed mechanism of hyposideraemia

Lactotransferrin was highly purified from lysates of human neutrophilic leucocytes by immuno-affinity chromatography. A comparative analysis of the molar carbohydrate compositions of human leucocyte lactotransferrin and human milk lactotransferrin reveals that the glycans of leucocyte lactotransferrin differ essentially by the absence of fucose residues. Structural analysis combining methylation-mass spectrometry and 400 MHz 1H-n.m.r. spectrometry of oligosaccharide alditols released from human leucocyte lactotransferrin shows the presence of two disialylated and non-fucosylated biantennary glycans of the N-acetyl-lactosaminic type. These results question a previously proposed mechanism for hyposideraemia in which the leucocyte lactotransferrin was involved and in which the fucose residues played a key role.

Microscale sequencing of O‐linked oligosaccharides using mild periodate oxidation of alditols, coupling to phospholipid and TLC‐MS analysis of the resulting neoglycolipids

In the course of characterising neoglycolipid products derived from mucin oligosaccharide alditols after periodate oxidation and coupling by reductive amination to the aminolipid dipalmitoylglycerophosphoethanolamine, we have obtained evidence that oxidative cleavage occurs specifically at the C4-C5 bond of core N-acetylgalactosaminitol. Two lipid-linked fragments thus obtained from each oligosaccharide alditol are well resolved on thin-layer chromatography and can be sensitively analysed by liquid-secondary-ion mass spectrometry to assign the sequence and branching patterns of oligosaccharides linked at C6 and C3 to the N-acetylgalactosamitol. These conclusions have been reached from detailed studies of the neoglycolipid derivatives of several oligosaccharides (di- to hexasaccharides) which were isolated from human meconium and characterised previously by MS and NMR studies as the free alditols.

Presence of fucosylated triantennary, tetraantennary and pentaantennary glycans in transferrin synthesized by the human hepatocarcinoma cell line Hep G2

Transferrin synthesized by a human hepatocellular carcinoma cell line Hep G2 (called Hep G2 transferrin) was purified from culture media by immunoaffinity chromatography on a rabbit anti-(human serotransferrin) IgG column. The eluted transferrin was then resolved into five peaks on a cation-exchange column using the fast protein liquid chromatography system. The major fraction, named Hep G2 transferrin fraction C, having a molecular mass of 82.5 kDa was found to be homogeneous in polyacrylamide gel electrophoresis and in concanavalin-A-affinity crossed immunoelectrophoresis. A comparative analysis of the molar carbohydrate composition of normal human serotransferrin and of Hep G2 transferrin fraction C shows an increase in the latter in the number of galactose and N-acetylglucosamine residues and in the presence of fucose, which is absent in normal transferrin. By a combination of methylation analysis and NMR spectroscopy, the primary structure of the oligosaccharide alditols released from Hep G2 transferrin fraction C by reductive alkaline cleavage has been established as triantennary, tetraantennary and pentaantennary N-acetyllactosaminic structures with fucose residues (alpha 1-3)-linked to peripheral N-acetylglucosamine residues. These results indicate that the increase in the number of antennae in transferrin glycans synthesized by the hepatocarcinoma cell line is much more pronounced than in liver diseases such as alcoholic cirrhosis and that, in addition, the malignant transformation of human liver induces the presence of fucose.

Primary structure of twenty three neutral and monosialylated oligosaccharides O-glycosidically linked to the human secretory immunoglobulin A hinge region determined by a combination of permethylation analysis and 400-MHz 1H-NMR spectroscopy.

Sialooligosaccharide and asialooligosaccharide alditols, derived from the human milk secretory immunoglobulin A hinge region, have been purified by HPLC using, successively, an amino-bonded silica column and an octadecyl-bonded silica column. Their primary structures were completely resolved by applying a combination of sugar analysis, methylation mass spectrometry and 400-MHz 1H-NMR spectroscopy. In the present report, twenty three oligosaccharide alditols are described and all possess a type two core consisting of the branched trisaccharide: Gal(beta 1-3)[GlcNAc(beta 1-6)]GalNAc-ol. The elongation of this core arises by chain lengthening only on the GlcNAc(beta 1-6) branch for the sialylated compounds, leading to a dodecasaccharide, and on both branches for the neutral compounds leading to a nonasaccharide. Sixteen of the described oligosaccharide structures are original. Moreover, some of the fucosylated structures were found to support Le(a), Le(b) and X blood group determinants.

Structures of Neutral O-Linked Polylactosaminoglycans on Human Skim Milk Mucins: A novel type of linearly extended poly-N-acetyllactosamine backbones with Galβ(1–4)GlcNAcβ(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.

Complete 1H- and 13C-n.m.r. assignments for two sulphated oligosaccharide alditols of hen ovomucin

The complete 1H- and 13C-n.m.r. assignments for β- d-Gal p-(1→4)-β- d-Glc pNAc-6-SO 3H-(1→6)-[β- d-Gal p- (1→3)]- d-GalNAcol and α-NeuAc p-(2→3)-β- d-Gal p-(1→3)-[β- d-Gal p-(1→4)-β- d-Glc pNAc-6-SO 3H-(1→6)]- d-GalNAcol were made by a combination of 2-D correlation experiments (Relayed-Cosy; and 13C, 1H Correlation-shift n.m.r. spectroscopy), and 1-D n.m.r. spectroscopy. The results illustrate the ability of these methods to locate sulphate and NeuAc groups in anionic mucinous glycoproteins.

A carbohydrate epitope associated with human squamous lung cancer : Martensson S, Due C, Pahlsson P et al. Department of Clinical Chemistry, University Hospital, S-221 85 Lund. Cancer Res 1988;48:2125-31

A monoclonal antibody, 43-9F, specifically recognizes a tumor-associated antigen expressed both on surface membrane glycoproteins and on secreted soluble mucins of human squamous lung carcinoma (SLC) cells, and the corresponding antigen can be detected as a circulating tumor marker in plasma of SLC patients. Thin-layer chromatography immunostaining of neutral glycolipids extracted from SLC cells reveals a 43-9F-reactive glycolipid whose carbohydrate structure, as determined by fast atom bombardment-mass spectrometry, is identical with that of an Lea-active pentaglycosylceramide described previously: Gal beta 1-3[Fuc alpha 1-4]-GlcNAc beta 1-3Gal beta 1-4Glc-Cer. However, the Lea-active oligosaccharide hapten, lacto-N-fucopentaose II, with the same carbohydrate structure, fails to inhibit binding of 43-9F, and a well-characterized anti-Lea monoclonal antibody blocks only 40% of 43-9F binding sites on SLC cells, suggesting that the major epitope recognized by 43-9F is more complex than the Lea epitope. To search for a higher affinity 43-9F epitope among more complex oligosaccharides, a mixture of tritiated neutral oligosaccharide alditols from pooled human milk was passed through a 43-9F affinity column. A major retarded oligosaccharide was purified by high-performance liquid chromatography and shown by fast atom bombardment-mass spectrometry to have the following structure: Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4[Fuc alpha 1-3] GlcNAc beta 1-3Gal beta 1-4Glc. Oligosaccharides containing this sugar sequence are at least 100-fold more active than lacto-N-fucopentaose II as competitive inhibitors of 43-9F. Thus, antibody 43-9F binds to the above difucosyl Lea-X determinant with high affinity and weakly cross-reacts with the Lea antigen under some conditions such as occurs in thin-layer chromatography and enzyme-linked immunosorbent assay where multiple weak interactions of the decavalent IgM antibody may occur.

Effect of ethanol on the in vitro sulfation of salivary mucin.

In vitro sulfation of mucus glycoprotein by sulfotransferase from rat submandibular salivary gland and the effect of ethanol on this enzyme activity was investigated. Subcellular fractionation studies revealed that the enzyme which catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to submandibular gland mucus glycoprotein is associated with Golgi-rich membrane fraction. The sulfotransferase enzyme exhibited optimum activity at pH 6.8 in the presence of 0.5% Triton X-100, 4 mM MgCl2, and 25 mM NaF. The enzyme was equally capable of sulfation of the desulfated intact as well as proteolytically degraded desulfated glycoprotein preparations, whereas the acceptor capacity of the intact mucus glycoprotein was 70% lower. The submandibular gland sulfotransferase activity was inhibited by ethanol. The rate of inhibition of mucus glycoprotein sulfation was proportional to the concentration of ethanol up to 0.4 M, at which concentration a 39% reduction in the sulfotransferase activity occurred. The apparent Km value of the enzyme for salivary mucus glycoprotein was 11.1 microM, and the Kl in the presence of ethanol was 0.93 M. The synthesized 35S-labeled glycoprotein gave on CsCl equilibrium density gradient centrifugation 35S-labeled peak which coincided with that of the glycoprotein. Alkaline borohydride treatment of this glycoprotein led to the liberation of the label into the acidic oligosaccharide alditol fraction. As the inhibition by ethanol of sulfotransferase enzyme occurred below its isosmotic concentration to plasma, the observed effect could also be detrimental to salivary mucus glycoprotein sulfation in vivo.

Gas chromatography and gas chromatography/mass spectrometry for the characterization of complex mixtures of large oligosaccharides

AbstractHigh temperature gas chromatography and gas chromatography/mass spectrometry are used in the characterization of complex natural mixtures of permethylated oligosaccharides released from proteins or lipids. The high resolution allows separation of isomeric compounds and the mass range extends to oligosac‐charides around molecular mass 2000 daltons or 10 sugar residues for isomalto‐oligosaccharides. The mass spectra of permethylated oligosaccharide alditols from mucin glycopeptides are very informative and the approach allows a simple and rapid characterization of these complex components.

Sulfation in vitro of mucus glycoprotein by submandibular salivary gland: effects of prostaglandin and acetylsalicylic acid

Enzymatic sulfation of mucus glycoprotein by rat submandibular salivary and the effect of prostaglandin and acetylsalicylic acid on this process were investigated in vitro. The sulfotransferase enzyme which catalyzes the transfer of sulfate ester group from 3′-phosphoadenosine-5′-phosphosulfate to submandibular gland mucus glycoprotein has been located in the detergent extracts of Golgi-rich membrane fraction of the gland. Optimum enzyme activity was obtained at pH 6.8 with 0.5% Triton X-100, 25 mM NaF and 4 mM MgCl 2, using the desulfated glycoprotein. The enzyme was also capable of sulfation of the intact mucus glycoprotein, but the acceptor capacity of such glycoprotein was 68% lower. The apparent K m of the submandibular gland sulfotransferase for salivary mucus glycoprotein was 11.1 μM. The 35S-labeled glycoprotein product of the enzyme reaction gave in CsCl density gradient a 35S-labeled peak which coincided with that of the glycoprotein. This glycoprotein upon reductive β-elimination yielded several acidic 35S-labeled oligosaccharide alditols which accounted for 75% of the 35S-labeled glycoprotein label. Based on the analytical data, the two most abundant oligosaccharides were identified as sulfated tri- and pentasaccharides. The submandibular gland sulfotransferase activity was stimulated by 16,16-dimethyl prostaglandin E 2 and inhibited by acetylsalicylic acid. The rate of enhancement of the glycoprotein sulfation was proportional to the concentration of prostaglandin up to 2 · 10 −5 M at which point a 31% increase in sulfation was attained. The inhibition of the glycoprotein sulfation by acetylsalicylic acid was proportional to the drug concentration up to 2.5 · 10 −4 M at which concentration a 48% reduction in the sulfotransferase activity occurred. The apparent K i value for sulfation of salivary mucus glycoprotein in presence of acetylsalicylic acid was 58.9 μM. The results suggest that prostaglandins may play a role in salivary mucin sulfation and that this process is sensitive to such nonsteroidal anti-inflammatory agents as acetylsalicylic acid.

Structural studies on oncofetal carbohydrate antigens (Ca 19-9, Ca 50, and Ca 125) carried by O-linked sialyl-oligosaccharides on human amniotic mucins

Mucins were extracted from human amniotic fluid in the presence of 45% vol. phenol and separated from the bulk of smaller-sized glycoproteins by exclusion on Sephacryl S400. The mucin-fraction FW, which still contained a minute proportion of mannose, strongly expressed oncofetal antigens recognized by monoclonal antibodies C 50, NS 19-9, OC 125, Leu M1, 49 H 8, and 115 C 2. The structures of the respective mucin-linked saccharides responsible for Ca 50-, Ca 19-9-, and Le a-related antigenic activities were analyzed before or after reductive β-elimination from sialylglycoproteins, and purification of the derived alditols by gel-permeation chromatography on Bio-Gel P-4 or high performance liquid chromatography. Two ubiquitous (FW2, FW3) and three novel oligosaccharide alditols (FW5) were characterized by f.a.b.- and e.i.-m.s., combined with methylation analysis and chromium trioxide oxidation. The OC 125 epitope on mucin-carried O-glycans was destroyed during reductive cleavage of the saccharides, indicating a conformational involvement of the reducing terminal residue and its mode of conjugation to the protein. Exoglycosidase treatment of the mucin-bound antigen revealed that the epitope structure of OC 125 includes terminal β- d-galactosyl groups, and terminal sialyl groups that are almost inaccessible to Vibrio cholerae sialidase digestion.

A polysaccharide from Streptococcus sanguis 34 that inhibits coaggregation of S. sanguis 34 with Actinomyces viscosus T14V.

Coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34 depends on interaction of a lectin on A. viscosus T14V with a cell surface carbohydrate on S. sanguis 34. This carbohydrate was isolated, and its chemical makeup was established. The carbohydrate remained attached to S. sanguis 34 cells through extraction with Triton X-100 and treatment with pronase. It was cleaved from the cell residue by autoclaving and purified by differential centrifugation and column chromatography on DEAE-Sephacel and Sephadex G-75. The polysaccharide contained phosphate which was neither inorganic nor monoester. Treatment with NaOH-NaBH4, followed by Escherichia coli alkaline phosphatase, or with 48% HF at 4 degrees C, followed by NaBH4, yielded inorganic phosphate and oligosaccharide alditols. Therefore, the polysaccharide is composed of oligosaccharide units joined together by phosphodiester bridges. The structure and stereochemistry of the main oligosaccharide alditol was established previously (F. C. McIntire, C. A. Bush, S.-S. Wu, S.-C. Li, Y.-T. Li, M. McNeil, S. Tjoa, and P. V. Fennessey, Carbohydr. Res. 166:133-143). Permethylation analysis, 1H and 31P nuclear magnetic resonance studies on the whole polysaccharide revealed the position of the phosphodiester linkages. The polysaccharide is mainly a polymer of (6) GalNAc(alpha 1-3)Rha(beta 1-4)Glc(beta 1-6)Galf(beta 1-6)GalNAc(beta 1- 3)Gal(alpha 1)-OPO3. It reacted as a single antigen with antiserum to S. sanguis 34 cells and was a potent inhibitor of coaggregation between A. viscosus T14V and S. sanguis 34. Quantitative inhibition of precipitation assays with oligosaccharides, O-allyl N-acetylgalactosaminides, and simple sugars indicated that specific antibodies were directed to the GalNAc end of the hexasaccharide unit. In contrast, coaggregation was inhibited much more effectively by saccharides containing betaGalNAc. Thus, the specificity of the A. viscosus T14V lectin is strikingly different from that of antibodies directed against the S. sanguis 34 polysaccharide.

Chemical structure of neutral sugar chains isolated from human mature milk κ-casein

The carbohydrate chains linked to human κ-casein from mature milk were released by alkaline borohydride treatment as reduced oligosaccharides. The neutral oligosaccharides of lower molecular weight were fractionated and purified by gel filtration and preparative thin layer chromatographies. Seven neutral oligosaccharides (a di- (0.5%), two tetra- (30.5%), two penta- (5.4%) and teo hexasaccharide alditols (10.9%)) were obtained in homogeneity, and followed by methylation analysis with gas-liquid chromatography-mass spectrometry and by anomer analysis with 13C nuclear magnetic resonance. Their chemical structures were identified to be Gal β1 → 3GalNAc-ol (I), Gal β1 → 3[Gal β1 → 4GlcNAc β1 → 6]GalNAc-ol (II), Gal β1 → 3 [Fuc α1 → 4GlcNAc β1 → 6]GalNAc-ol (III), GlcNAc β1 → 3 / 6Gal β1 → 3[Gal β1 → 4GlcNAc β1 → 6] GalNAc-ol (IV), GlcNAc β1 → 3 / 6Gal β1 → 3[Fuc α1 → 4GlcNAc β1 → 6]GalNAc-ol (V), Fuc α1 → 4GlcNAc β1 → 3 / 6Gal β1 → 3[Gal β1 → 4GlcNAc β1 → 6]GalNAc-ol (VI) and Fuc α1 → 4GlcNAc β1 → 3 / 6Gal β1 → 3[Fuc α1 → 4GlcNAc β1 → 6]GalNAc-ol (VII). Five oligosaccharide alditols (III–VII) were the novel carbohydrate chains of κ-casein from mammalian milk.

Mass spectrometry of hexosamine containing oligosaccharides as permethylated N-trifluoroacetyl derivatives.

Permethylated N-trifluoroacetyl hexosamine containing oligosaccharide alditols are favorable for analysis by mass spectrometry. Electron impact and fast atom bombardment mass spectra of these derivatives are characterized by strong primary and secondary fragments in which a positive charge is localized on the N-trifluoroacetyl hexosamine residue. From the mass spectra the monosaccharide sequence and the position(s) of substitution of a N-trifluoroacetyl hexosamine can be determined.

Continuous-flow fast atom bombardment-mass spectrometry of permethylated oligosaccharides: A comparative study of direct mixture analysis with packed capillary column liquid chromatography-fast atom bombardment-mass spectrometry

Conventional positive fast atom bombardment (FAB) and continuous-flow FAB analysis were carried out with permethylated lacto- N-tetraose. This latter method, a new approach, has been used to analyze a mixture of permethylated oligosaccharides by liquid chromatography—mass spectrometry (LC-MS) with a packed capillary fused-silica column and the continuous-flow probe as interface. Under these conditions, we found the continuous-flow probe to be superior to the conventional probe because its low matrix level increased the signal-to-noise ratio. The analysis of the mixture of permethylated oligosaccharide alditols obtained from hen ovomucoid by LC-MS using the continous-flow probe as interface is described.

Structural determination of the oligosaccharide side chains from a glycoprotein isolated from the mucus of the coral Acropora formosa.

An extracellular mucous glycoprotein has been isolated from the hard coral Acropora formosa. The glycoprotein contains sulfated oligosaccharide side chains attached through O-glycosidic linkages to serine and threonine, the principal amino acids (77%) in the polypeptide. The oligosaccharide side chains consist of D-arabinose, D-mannose, and N-acetyl-D-glucosamine with smaller amounts of D-galactose, L-fucose, and N-acetyl-D-galactosamine, but no sialic or uronic acids. Alkaline borohydride reductive cleavage resulted in a mixture of oligosaccharide alditols. Six oligosaccharides were purified by high performance liquid chromatography. The structures of these oligosaccharides, which do not resemble those of any other glycoprotein so far examined, were determined by a combination of gas chromatography/mass spectrometry analysis of methylation products and NMR spectroscopy. All oligosaccharides contain a reducing terminal mannitol residue with N-acetylglucosamine linked to carbon 2, 4, or 6 of the mannitol. There is no evidence for linkage of N-acetylglucosamine to any other glycoses in the glycoprotein. Galactose was detected in two oligosaccharides linked to the 4-position of mannitol. Arabinose (Ara) was found in only one oligosaccharide. This was probably due to hydrolysis of the labile arabino-furanoside linkages. Evidence is presented which indicates the arabinose occurs primarily at the terminal position of oligosaccharide side chains. The structures of the oligosaccharides isolated from the glycoprotein were: (Formula: see text).

Characterisation of oligosaccharides released from human-blood-group O erythrocyte glycopeptides by the endo-beta-galactosidase of Bacteroides fragilis. A study of the enzyme susceptibility of branched poly(N-acetyllactosamine) structures.

Desialylated human blood group O erythrocyte glycopeptides were digested with the endo-beta-galactosidase of Bacteroides fragilis and the enzyme-released products reduced with NaBH4 and purified by Bio-Gel P-4 chromatography. Three linear and six branched oligosaccharides of poly(N-acetylllactosamine) type, which together accounted for 90% of the oligosaccharide alditols, were characterised by fast-atom-bombardment mass spectrometry and gas-liquid chromatography/mass spectrometry. Linkage and composition data were obtained for the remaining material. The salient findings were (a) the branched oligosaccharide alditols each contained the sequence: (Formula: see text) and (b) there was no evidence for the terminal branch-point sequence: (Formula: see text). Together these observations indicate that, as with erythrocyte glycolipids described previously [Scudder, P., Hanfland, P., Uemura, K. & Feizi, T. (1984) J. Biol. Chem. 259, 6586-6592], the endo-beta-galactosidase of Bacteroides fragilis cannot hydrolyse branch-point beta-galactosidic linkages on erythrocyte membrane glycopeptides.