The action of a bacterial agarase on agarose, porphyran and alkali -treated porphyran

1. A purified extracellular agarase from a Cytophaga species was used to hydrolyse agarose, porphyran and alkali-treated porphyran. 2. The hydrolysate from agarose was separated by gel filtration into the series of neoagarosaccharides, the predominant member of which was the tetrasaccharide. 3. Enzyme action on alkali-treated porphyran gave neoagarosaccharides and other oligosaccharides containing 6-O-methyl-d-galactose units. From the composition of these oligosaccharides it is deduced that action of the enzyme on a d-galactosidic linkage is four to five times faster than on the 6-O-methyl-d-galactosidic linkage. 4. Enzyme action on native porphyran gives a similar series of oligosaccharides but in smaller yield, much of the polysaccharide being either not degraded or only degraded to a series of large, highly sulphated oligosaccharides. 5. For porphyran, it is concluded that 6-O-methyl ether groups are distributed randomly on half the d-galactose units, but that the 6-sulphate groups on l-galactose units tend to occur in blocks.     

The specificity of an agarase from a Cytophaga species

1. The extracellular agarase from a Cytophaga species was shown to have no action on neoagarobiose, neoagarotetraose or their analogues containing 6-O-methyl-d-galactose residues. 2. The action of the enzyme on neoagaro-octaose suggests that scission of the central beta-d-galactosidic linkage, to form two molecules of tetrasaccharide, is the preferred mode of action; however, both exterior d-galactosidic linkages in the octasaccharide and both in neoagarohexaose are hydrolysed at a somewhat lower rate. 3. Sulphated oligosaccharides produced by prolonged enzyme action on porphyran have a minimum degree of polymerization of about 8-10units. 4. For such sulphated oligosaccharides to be further hydrolysed by enzyme action, it is suggested that an unmodified neoagarotetraose residue must be present in the oligosaccharide. 6. A new method for determining the degree of polymerization of these large oligosaccharides is described.     

The galactan sulfate from the edible,red alga Porphyra columbina

A galactan sulfate has been isolated from the seaweed Porphyra columbina, and its structure established by a combination of methylation, methanolysis, treatment with alkali followed by methylation, and ¹³C-n.m.r. spectroscopy. The polysaccharide belongs to the porphyran class, and consists of 3-linked β-d-galactosyl residues and 4-linked α-l-galactosyl residues. 3,6-Anhydro-l-galactose and l-galactose 6-sulfate residues total approximately half of the sugar units, the other half being made up of d-galactose and 6-O-methyl-d-galactose residues. Some evidence is presented that suggests that the galactan sulfate does not have a completely alternating structure.     

Beta-agarases I and II from Pseudomonas atlantica. Substrate specificities

Beta-Agarase I and II were characterised by their action on agar-type polysaccharides and oligosaccharides. Beta-Agarase I, an endo-enzyme, was specific for regions containing a minimum of one unsubstituted neoagarobiose unit [3,6-anhydro-alpha-L-galactopyranosyl-(1 leads to 3)-D-galactose], hydrolysing at the reducing side of this moiety. Yaphe demonstrated that agar was degraded by this enzyme to neoagaro-oligosaccharides limited by the disaccharide but with a predominance of the tetramer [Yaphe, W. (1957) Can. J. Microbiol. 3, 987-993]. Beta-Agarase I slowly degraded neoagarohexaose but not the homologous tetrasaccharide. [1-3H]Neoagarohexaitol was cleaved to neoagarotetraose and [1-3H]neoagarobiitol. The highly substituted agar, porphyran was degraded to methylated, sulphated and unsubstituted neoagaro-oligosaccharides which were invariably terminated at the reducing end by unsubstituted neoagarobiose. The novel enzyme, beta-agarase II, was shown to be an endo-enzyme. Preliminary evidence indicated this enzyme was specific for sequences containing neoagarobiose and/or 6(1)-O-methyl-neoagarobiose. It degraded agar to neoagaro-oligosaccharides of which the disaccharide was limiting and predominant. Beta-Agarase II rapidly degraded isolated neogarotetraose and neoagarohexaose to the disaccharide. With [1-3H]neoagarohexaitol, exo-action was observed, the alditol being cleaved to neoagarobiose and [1-3H]neoagarotetraitol. Neoagarotetraitol was hydrolysed at 4% of the rate observed for the hexaitol. Porphyran was degraded to oligosaccharides, the neutral fraction comprising 24% of the starting carbohydrate. This fraction was almost exclusively disaccharides (22.4%) containing neoagarobiose (7.4%) and 6(1)-O-methyl-neoagarobiose (15%). Beta-Agarase II is probably the 'beta-neoagarotetraose hydrolase' reported by Groleau and Yaphe as an exoenzyme against neoagaro-oligosaccharides [Groleau, D. and Yaphe, W. (1977) Can. J. Microbiol. 23, 672-679].     

Porphyran primary structure. An investigation using beta-agarase I from Pseudomonas atlantica and 13C-NMR spectroscopy

Porphyran, a highly substituted agarose from Porphyra umbilicalis was degraded by highly purified beta-agarase I from Pseudomonas atlantica. This enzyme cleaved at the reducing side of units of beta-neoagarobiose (3,6-anhydro-alpha-L-galactopyranosyl-(1 leads to 3)-beta-D-galactopyranose). The oligosaccharides were divided into fractions of low and high molecular weight by dialysis. The permeate (23% of total starting carbohydrate) was separated by ion-exchange into neutral and anionic fractions. Gel filtration of the neutral fraction (19%) resolved two major oligosaccharides. These were shown by 13C-NMR spectroscopy to be 6(3)-O-methyl-neoagarotetraose and 6(3),6(5)-di-O-methyl-neoagarohexaose. Gel filtration of the anionic oligosaccharides (3.3%) revealed two novel monosulphated tetrasaccharides, 6-O-sulphato-alpha-L-galacto-pyranosyl-(1 leads to 3)-beta-D-galactopyranosyl-(1 leads to 4)-3,6-anhydro-alpha-L-galactopyranosyl-(1 leads to 3)-D-galactopyranose and its 6(3)-O-methylated derivative. The 13C-NMR data from the sulphated tetrasaccharides provided a novel reference which was used to characterise higher, partially sulphated fragments in the dialysis permeate. The fraction retained on dialysis (77%) had an average degree of polymerisation of 40 and was homologous with the high-molecular-weight anionic permeate. From 13C-NMR spectroscopy porphyran was found to comprise 49% sulphated disaccharide units and these were calculated to occur in stretches averaging 2.0-2.5 contiguous units.     

Studies on the structure of lipopolysaccharides of Salmonella minnesotta and Salmonella typhimurium R strains

1. The composition of the lipopolysaccharides and the corresponding lipid-free polysaccharides from four R-mutants of Salmonella has been studied. All the lipopolysaccharides, from RI and RII serotypes contained d-glucose, d-galactose, heptose, N-acetylglucosamine and 3-deoxy-2-oxo-octonate. The polysaccharide obtained from the RII lipopolysaccharides also contained all these sugars. The polysaccharides from RI lipopolysaccharides lacked N-acetylglucosamine. 2. From partial hydrolysates of the lipopolysaccharides, a number of oligosaccharides have been isolated and partially characterized. Oligosaccharides containing N-acetylglucosamine or glucosamine were obtained only from RII lipopolysaccharides. Several oligosaccharides composed of glucose and galactose were common to RI and RII preparations. 3. A structural unit, based on the oligosaccharides found, is proposed for the RII lipopolysaccharide. It contains the sequence: alpha-N-acetylglucosaminyl- alpha-glucosyl-alpha-galactosyl-glucosyl.... A second alpha-galactosyl residue is bound to position 6 of the last glucosyl group. The complete unit is believed to to be attached to a polyheptose phosphate backbone in the RII antigen. 4. The RI lipopolysaccharide of Salmonella minnesota contains an analogous structure lacking the terminal N-acetylglucosamine residue. 5. A basal structure common to the lipopolysaccharides of several Salmonella species is proposed.     

Structural investigation on degraded Spondias dulcis gum

Autohydrolysis of an aqueous solution of purified, exudate gum from Spondias dulcis trees yielded a degraded gum containing d-galactose, l-arabinose, and d-galacturonic acid in the mole ratios of 3:3:1. Methylation studies were conducted on the degraded gum and its carboxyl-reduced derivative. Three neutral and three acidic oligosaccharides were obtained on graded hydrolysis of the degraded gum, and these were characterized. Based on the results, a tentative structure was proposed for the repeating unit in the polysaccharide. The results of periodate oxidation supported the structure assigned. The anomeric configurations of the sugar residues were determined by studies of oxidation with chromium trioxide.     

Studies on the glycopeptides isolated from the urinary protein in heavy-chain disease

The urinary protein excreted in heavy-chain disease was separated by ion-exchange chromatography into two broad fractions designated Cra-1 and Cra-2. For a dimeric molecular weight of approx. 51000, Cra-1 contained 3-4 residues of 6-deoxy-l-galactose (l-fucose), 10 of d-mannose, 5-6 of d-galactose, 12 of 2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine) and 4-5 of N-acetylneuraminic acid (sialic acid), whereas the corresponding values for Cra-2 were 2, 10, 7, 12 and 7. Cra-2 contained in addition 1 residue of 2-acetamido-2-deoxy-d-galactose (N-acetyl-d-galactosamine). Cra-1 contained an average of four oligosaccharide units, two of which contained 1 residue of 6-deoxy-l-galactose, 3 of d-mannose, 1 of d-galactose and 3 of 2-acetamido-2-deoxy-d-glucose, whereas the other two units contained the same proportions of 6-deoxy-l-galactose, d-mannose and 2-acetamido-2-deoxy-d-glucose but 2 residues of d-galactose and 2 of N-acetylneuraminic acid. Cra-2 also contained an average of four oligosaccharide units, but the range of glycopeptides was much wider, containing 0-1 residue of 6-deoxy-l-galactose, 2-3 of d-mannose, 2-3 of d-galactose, 2-3 of 2-acetamido-2-deoxy-d-glucose and 1-3 of N-acetylneuraminic acid. Possible reasons for this heterogeneity are discussed. Glycopeptides were also isolated from Cra-2 that contained 1 residue of d-mannose, 2 of d-galactose, 1 of 2-acetamido-2-deoxy-d-galactose and 0-3 of N-acetylneuraminic acid.     

Immunochemical studies on shigella dysenteriae type 9 bacterial polysaccharide

On graded hydrolysis and Smith degradation, the O-somatic polysaccharide isolated from Shigella dysenteriae type 9 bacteria, strain NCTC 7919, yielded five oligosaccharides which were characterized. The positions of the O-acetyl and pyruvic acetal groups in the repeating unit were identified. Immunochemical studies indicated that d-galactose is the immunodominant sugar in the polysaccharide, and one of the oligomers, having the structure Gal-(1→3)-GlcNAc-(1→3)-Gal-(1→4)-Man, showed maximum inhibition of the homologous precipitation.     

Structural features of the mucilage from the stem pith of kiwifruit (actinidia deliciosa): part I,structure of the inner core

The mucilage found in the stem pith of Actinidia deliciosa contains d-glucuronic acid, d-mannose, l-fucose, l-arabinose, and d-galactose in the molar ratios 1.0:1.5:2.0:4.0. The native, carboxyl-reduced, and partially acid-hydrolysed polysaccharides were subjected to methylation analysis. Partial acid hydrolysis of the methylated, carboxyl-reduced glucuronomannan core produced a series of methylated oligosaccharides which, as their alditol derivatives, were isolated by reverse-phase h.p.l.c. and characterised by e.i.- and f.a.b.-m.s. The data suggest that the polysaccharide contains a →4)-β-d-GlcpA-(1→2)-α-d-Manp-(1→ backbone with most of the d-mannosyl and d-glucosyluronic acid residues substituted through positions 3 with oligosaccharides containing l-arabinose, α-l-fucose, and β-d-galactose.     

Genetic evidence for the physiological significance of the D-tagatose 6-phosphate pathway of lactose and D-galactose degradation in staphylococcus aureus

Mutants of Staphylococcus aureus were isolated which were unable to utilize d-galactose or lactose, but which were able to utilize all other carbohydrates tested. Growth of the mutants on a peptone-containing medium was inhibited by d-galactose. Of those mutants selected for further study, one (tagI2) was missing d-galactose 6-phosphate isomerase, one (tagK3) was missing d-tagatose 6-phosphate kinase, and one (tagA4) was missing d-tagatose 1, 6-diphosphate aldolase. Each of these mutants accumulated the substrate of the missing enzyme intracellularly. Spontaneous revertants of each of the mutants simultaneously regained their ability to utilize d-galactose and lactose, lost their sensitivity to d-galactose, regained the missing enzymatic activities, and no longer accumulated intermediates of the d-tagatose 6-phosphate pathway. These data support our previous contention that the physiologically significant route for the metabolism of d-galactose and the d-galactosyl moiety of lactose in S. aureus is the d-tagatose 6-phosphate pathway. Furthermore, a mutant constitutive for all three enzymes of this pathway was isolated, indicating that the products of the tagI, tagK, and tagA genes are under common genetic control. This conclusion was supported by the demonstration that d-galactose 6-phosphate isomerase, d-tagatose 6-phosphate kinase, and d-tagatose 1, 6-diphosphate aldolase are coordinately induced in the parental strain.     

Partial characterization of oligosaccharides expressed on midgut microvillar glycoproteins of the mosquito, Anopheles stephensi Liston

Midguts of the malaria-transmitting mosquito, Anopheles stephensi, were homogenized and microvillar membranes prepared by calcium precipitation and differential centrifugation. Oligosaccharides present on the microvillar glycoproteins were identified by lectin blotting before and after in vitro and in situ treatments with endo- and exo-glycosidases. Twenty-eight glycoproteins expressed a structurally restricted range of terminal sugars and oligosaccharide linkages. Twenty-three glycoproteins expressed oligomannose and/or hybrid N-linked oligosaccharides, some with alpha1-6 linked fucose as a core residue. Complex-type N-linked oligosaccharides on eight glycoproteins all possessed terminal N-acetylglucosamine, and alpha- and beta-linked N-acetylgalactosamine. Eight glycoproteins expressed O-linked oligosaccharides all containing N-acetylgalactosamine with or without further substitutions of fucose and/or galactose. Galactosebeta1-3/4/6N-acetylglucosamine-, sialic acidalpha2-3/6galactose-, fucosealpha1-2galactose- and galactosealpha1-3galactose- were not detected. Terminal alpha-linked N-acetylgalactosamine residues on N-linked oligosaccharides are described for the first time in insects. The nature and function of these midgut glycoproteins have yet to be identified, but the oligosaccharide side chains are candidate receptors for ookinete binding and candidate targets for transmission blocking strategies.     

Mammalian-lung galactan

Exopolysaccharides of Agrobacterium tumefaciens and Rhizobium meliloti, containing d-glucose, d-galactose, pyruvic acid, and O-acetyl groups in the approximate proportions 6:1:1:1.5, were analysed by methylation. They were found to contain the following main structural units (all β-glycosidic): chain residues of (1→3)-linked d-glucose (24%), (1→3)-linked d-galactose (15%), (1→4)-linked d-glucose (20%), and (1→6)-linked d-glucose (18%); (1→4,1→6)-linked branching residues of d-glucose (12%), and terminal d-glucose residues substituted at positions 4 and 6 by pyruvate (11%). Uronic acid-containing exopolysaccharides of Rhizobium leguminosarum, R. phaseoli, and R. trifolii contained d-glucose, d-glucuronic acid, d-galactose, pyruvic acid, and O-acetyl groups in the approximate proportions 5:2:1:2:3. Methylation gave identical patterns of methylated sugar components, from which the following structural elements were deduced: chain residues of (1→3)-linked d-glucose substituted at positions 4 and 6 by pyruvate (13%), (1→4)-linked d-glucose (32%), and (1→4)-linked d-glucuronic acid (20%); (1→4,1→6)-linked branching residues of d-galactose and/or d-glucose (13%), and terminal d-glucose and/or d-galactose residues substituted at positions 4 and 6 by pyruvate (13%).     

Mannose 6-phosphate receptor-mediated endocytosis of acid hydrolases: internalization of beta-glucuronidase is accompanied by a limited dephosphorylation

Endocytosis of acid hydrolases via the cell surface mannose 6-phosphate (Man 6-P) receptor results in the delivery of the enzymes to lysosomes. To examine the fate of the ligand-associated phosphorylated high mannose oligosaccharides, we have analyzed the asparagine-linked oligosaccharides attached to beta-glucuronidase after uptake and processing by Man 6-P receptor-positive mouse L cells. beta-Glucuronidase, double-labeled with [2-3H]mannose and [35S]methionine, was isolated from the growth medium of mouse P388D1 cells. 80% of the [3H]mannose associated with the secreted enzyme was recovered as high mannose-type oligosaccharides, and 24-37% of these units were phosphorylated. Three species of phosphorylated oligosaccharides were identified; high mannose-type units containing either one or two phosphomonoesters, and hybrid-type units containing one phosphomonoester and one sialic acid residue. After endocytosis by the L cells, the beta-glucuronidase molecules migrated faster on an SDS gel, suggesting that the enzymes had been processed within lysosomes. Examination of the cell-associated beta-glucuronidase molecules indicated that: (a) the percentage of phosphorylated oligosaccharides remained comparable to the input form of the enzyme, even after a 24-h chase period, (b) the presence of a single species of phosphorylated oligosaccharide that contained one phosphomonoester, and (c) the positioning of the phosphate within the intracellular monophosphorylated species was comparable to the positioning of the phosphate within the two phosphomonoester species originally secreted by the P388D1 cells. Therefore, the internalized beta-glucuronidase molecules undergo a limited dephosphorylation; oligosaccharides containing two phosphomonoesters are converted to monophosphorylated species, but the one phosphomonoester forms are conserved. A comparison of the phosphorylated oligosaccharides recovered from ligands internalized by the L cells at 37 degrees and 20 degrees C indicated that: (a) molecules internalized at 20 degrees C retain a higher percentage of phosphorylated structures; and (b) at both temperatures the predominant phosphorylated oligosaccharide contains a single phosphomonoester group. The results indicate that the Man 6-P recognition marker persists after endocytosis and delivery to lysosomes and support the possibility that the limited dephosphorylation of the oligosaccharides may occur en route to these organelles.     

Chemical investigations on the gum exudate from sajna (Moringa oleifera)

The purified, whole-gum exudate from the drum-stick plant (Moringa oleifera) was found to contain l-arabinose, d-galactose, d-glucuronic acid, l-rhamnose, d-mannose, and d-xylose in the molar ratios of ～ 14.5:11.3:3:2:1:1. A homogeneous, degraded-gum polysaccharide consisting of d-galactose, d-glucuronic acid, and d-mannose in the molar ratios of ～ 11.7:3.9:1, was obtained on mild hydrolysis of the whole gum with acid. Permethylation studies were conducted on the whole gum, the degraded gum, and their carboxyl-reduced products, and the results were in good agreement with those obtained from periodate oxidation followed by Smith degradation. Also, isolation and characterization of the oligosaccharides obtained from the mother liquor during preparation of the degraded gum, and by graded hydrolysis of the degraded gum, were achieved. On the basis of the results obtained from these studies, a tentative structure was assigned to the average repeating-unit of the gum.     

Porphyran induces apoptosis related signal pathway in AGS gastric cancer cell lines

Porphyrans, the sulfated polysaccharides, are the main components of Porphyra. The potential apoptotic activities of porphyran were evaluated using AGS human gastric cancer cells. Porphyran did not affect the growth of normal cells, but did induce cancer cell death in a dose-dependent manner. The addition of 0.1% porphyran also reduced DNA synthesis after 24 h of exposure, suggesting that porphyran inhibits cancer cell growth by both decreasing cell proliferation and inducing apoptosis. AGS cells treated with porphyran displayed a marked increase in poly(ADP-ribose) polymerase (PARP) cleavage, as well as caspase-3 activation. The ability of porphyran to promote apoptosis may contribute to its usefulness as an agent capable of significantly inhibiting cell growth in AGS human gastric cancer cells. Insulin-like growth factor-I receptor (IGF-IR) phosphorylation was decreased in porphyran-treated AGS cells compared to control cells, which correlated with Akt activation. Thus, porphyran appears to negatively regulate IGF-IR phosphorylation by causing a decrease in the expression levels in AGS gastric cancer cells, and then inducing caspase-3 activation.     

Structural characterization of neutral and acidic oligosaccharides in the milks of strepsirrhine primates: greater galago, aye-aye, Coquerel’s sifaka and mongoose lemur

The structures of milk oligosaccharides were characterized for four strepsirrhine primates to examine the extent to which they resemble milk oligosaccharides in other primates. Neutral and acidic oligosaccharides were isolated from milk of the greater galago (Galagidae: Otolemur crassicaudatus), aye-aye (Daubentoniidae: Daubentonia madagascariensis), Coquerel's sifaka (Indriidae: Propithecus coquereli) and mongoose lemur (Lemuridae: Eulemur mongoz), and their chemical structures were characterized by (1)H-NMR spectroscopy. The oligosaccharide patterns observed among strepsirrhines did not appear to correlate to phylogeny, sociality or pattern of infant care. Both type I and type II neutral oligosaccharides were found in the milk of the aye-aye, but type II predominate over type I. Only type II oligosaccharides were identified in other strepsirrhine milks. α3'-GL (isoglobotriose, Gal(α1-3)Gal(β1-4)Glc) was found in the milks of Coquerel's sifaka and mongoose lemur, which is the first report of this oligosaccharide in the milk of any primate species. 2'-FL (Fuc(α1-2)Gal(β1-4)Glc) was found in the milk of an aye-aye with an ill infant. Oligosaccharides containing the Lewis x epitope were found in aye-aye and mongoose lemur milk. Among acidic oligosaccharides, 3'-N-acetylneuraminyllactose (3'-SL-NAc, Neu5Ac(α2-3)Gal(β1-4)Glc) was found in all studied species, whereas 6'-N-acetylneuraminyllactose (6'-SL-NAc, Neu5Ac(α2-6)Gal(β1-4)Glc) was found in all species except greater galago. Greater galago milk also contained 3'-N-glycolylneuraminyllactose (3'-SL-NGc, Neu5Gc(α2-3)Gal(β1-4)Glc). The finding of a variety of neutral and acidic oligosaccharides in the milks of strepsirrhines, as previously reported for haplorhines, suggests that such constituents are ancient rather than derived features, and are as characteristic of primate lactation is the classic disaccharide, lactose.     

Structural investigations of the galactan of the snail Lymnaea stagnalis

A galactan, isolated from the spawn of the snail Lymnaea stagnalis, contained d-galactose and 0.9% of nitrogen, but neither l-galactose nor phosphate groups. The [α]_D²⁰ values of the galactan and its first Smith-degradation product were +19.5° and +20°, respectively. During each of two consecutive Smith-degradations of the galactan, 1 mol of periodate was consumed and 0.45 mol of formic acid was liberated per mol of “anhydrogalactose” unit. Methylation analyses of the galactan and its first Smith-degradation product yielded equal proportions of 2,3,4,6-tetra-O-methyl- and 2,4-di-O-methyl-galactose. Only small quantities of 2,4,6- (4.9 mol%) and 2,3,4-tri-O-methylgalactose (0.7 mol%) were formed from the galactan, whereas the first Smith-degraded product gave 15.6 and 20.4 mol%, respectively. The product of the second Smith-degradation disintegrated and the following oligosaccharides were identified: β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→3)-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→6)-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→6)-d-Gal-β-d-Gal-(1→3)-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→3)-[β-d-Gal-(1→6)]-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→3)-β-d-Gal-(1→6)-β-d-Gal-(1→1)-l-Gro, and β-d-Gal-(1→3)-β-d-Gal-(1→3)-β-d-Gal-(1→1)-l-Gro. Thus, the galactan is highly branched with the backbone containing sequences of either exclusively (1→6)-linked or of more or less regularly alternating (1→3)- and (1→6)-linked units. The side chains vary in length and in the degree of branching. In immunoprecipitin studies, a high degree of species-specificity was seen when various snail galactans were tested with the antiserum to the Lymnaea stagnalis galactan.     

Processing of MOPC 315 immunoglobulin A oligosaccharides: evidence for endoplasmic reticulum and trans Golgi alpha 1,2-mannosidase activity

The processing of asparagine-linked oligosaccharides on the alpha- chains of an immunoglobulin A (IgA) has been investigated using MOPC 315 murine plasmacytoma cells. These cells secrete IgA containing complex-type oligosaccharides that were not sensitive to endo-beta-N- acetylglucosaminidase H. In contrast, oligosaccharides present on the intracellular alpha-chain precursor were of the high mannose-type, remaining sensitive to endo-beta-N-acetylglucosaminidase H despite a long intracellular half-life of 2-3 h. The major [3H]mannose-labeled alpha-chain oligosaccharides identified after a 20-min pulse were Man8GlcNAc2 and Man9GlcNAc2. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man6GlcNAc2, which was shown to contain a single alpha 1,2-linked mannose residue. Conversion of Man6GlcNAc2 to complex-type oligosaccharides occurred at the time of secretion since appreciable amounts of Man5GlcNAc2 or further processed structures could not be detected intracellularly. The subcellular locations of the alpha 1,2-mannosidase activities were studied using carbonyl cyanide m-chlorophenylhydrazone and monensin. Despite inhibiting the secretion of IgA, these inhibitors of protein migration did not effect the initial processing of Man9GlcNAc2 to Man6GlcNAc2. Furthermore, no large accumulation of Man5GlcNAc2 occurred, indicating the presence of two subcellular locations of alpha 1,2-mannosidase activity involved in oligosaccharide processing in MOPC 315 cells. Thus, the first three alpha 1,2-linked mannose residues were removed shortly after the alpha-chain was glycosylated, most likely in rough endoplasmic reticulum, since this processing occurred in the presence of carbonyl cyanide m-chlorophenylhydrazone. However, the removal of the final alpha 1,2-linked mannose residue as well as subsequent carbohydrate processing occurred just before IgA secretion, most likely in the trans Golgi complex since processing of Man6GlcNAc2 to Man5GlcNAc2 was greatly inhibited in the presence of monensin.     

Structural studies of an acidic polysaccharide from the mucin secreted by Drosera capensis

The polysaccharide of the mucin secreted by the leaves of Drosera capensis is composed of l-arabinose, d-xylose, d-galactose, d-mannose, and d-glucuronic acid in the molar ratio of 3.6:1.0:4.9:8.4:8.2. For structural elucidation, methylation analysis using g.l.c. and g.l.c.-m.s. was performed on the native, the carboxyl-reduced, and the degraded polysaccharides. Partial hydrolysis, periodate oxidation, chromium trioxide oxidation, and uronic acid degradation were also performed on the native and carboxyl-reduced polysaccharides. Partial hydrolysis of the native and carboxyl-reduced polysaccharides gave various oligosaccharides that were characterized and suggest a structure containing a d-glucurono-d-mannan backbone having a repeating unit → 4)-β-d-GlcpA-(1 → 2)-α-d-Manp-(1 →. l-Arabinose and d-xylose are present as nonreducing furanosyl and pyranosyl end-groups, respectively, both attached to O-3 of d-glucuronic acid residues of the backbone. d-Galactose is present as non-reducing pyranosyl end-group linked to O-3 of d-mannose residues.