Structural studies on the O-glycosidically linked carbohydrate chains of glucoamylase G1 from Aspergillus niger

Glucoamylase G1 from Aspergillus niger contains an unusual type of carbohydrate-protein linkage, involving mannose O-glycosidically linked to serine and threonine. The majority of the neutral oligosaccharides of glucoamylase G1 are located in a region of about 70 amino acid residues which carries about 35 oligosaccharide units [(1983) Carlsberg Res. Commun. 48, 517-527]. Structural analysis was performed on the O-linked carbohydrates of a tryptic fragment from glucoamylase G1 comprising the segment characterized by a high degree of glycosylation. The carbohydrate structures released by trifluoroacetolysis were elucidated using sugar analysis, methylation analysis, mass spectrometry, chromium trioxide oxidation, digestion with alpha-mannosidase and 1H-NMR spectroscopy. The following structures could be identified. (formula; see text)     

Bovine testicular beta-galactosidase: purification of enzyme fractions that exhibit high affinity for phosphomannosyl receptors

An improved method is described for the preparation of bovine testicular beta-galactosidase that allows the isolation of enzyme fractions that bind avidly to phosphomannosyl receptors. The procedure permits removal of a contaminating beta-hexosaminidase and yields nearly homogeneous beta-galactosidase. Enzyme eluted from DEAE-Sephacel was arbitrarily divided into pools that exhibited differing ability to bind phosphomannosyl receptors. A high binding fraction was rapidly assimilated by cultured cells and bound to both low and high molecular weight phosphomannosyl receptors. Carbohydrate analysis of the high binding fraction indicates an average content of one complex and one high mannose oligosaccharide chain per molecule and an average mannose 6-phosphate content of two residues per molecule. However, electrofocusing studies indicated that all the fractions were heterogeneous with respect to sialic acid and phosphate content. The purification procedure also provides highly purified beta-galactosidase suitable for removing beta-galactosidase residues from a variety of complex carbohydrates.     

New exopolysaccharides produced by Aureobasidium pullulans grown on glucosamine

The polysaccharides produced by Aureobasidium pullulans, grown using glucosamine as the carbon source, were investigated by means of methylation analysis, affinity chromatography and NMR spectroscopy. The results indicated that, besides a small amount of pullulan, this micro-organism was capable of producing-in low yields-mixtures of at least two different complex polysaccharides containing mainly mannose and galactose. (1)H NMR spectra of two fractions obtained by lectin affinity chromatography indicated that one polymer was constituted exclusively of mannose residues while the other contained both galactofuranosyl and mannopyranosyl residues.     

Methylation analysis of the carbohydrate portion of fibronectin isolated from human plasma

Fibronectin was isolated from pooled human plasma by affinity chromatography on Sepharose-coupled gelatin and subjected to methylation analysis. The results indicate that the carbohydrate chains in fibronectin are very similar to those found in other serum glycoproteins, i.e. a common inner core structure consisting of two N-acetylglucosamine and three mannose residues attached to the polypeptide backbone via a N-acetylglucosamine-asparagine linkage. To this inner core are attached two side chains made up of N-acetylglucosamine, galactose and terminated by sialic acid.     

Immunochemical characterization of Candida albicans cell wall antigens: specific determinant of Candida albicans serotype A mannan

We investigated the chemical structure of the specific determinant in the mannan of Candida albicans M-1012 (serotype A) strain. Acetolysis of the mannan, obtained by alkali extraction and purified as the copper complex, gave mannose and six oligosaccharides (from di- to hexasaccharide) and a small amount of a heptasaccharide. We examined the inhibition by these oligosaccharides up to hexaose of the precipitin reaction between anti-factor 6 serum specific for serotype A and homologous mannan, and found that the mannohexaose was the most effective inhibitor. These, and results obtained by proton magnetic resonance (PMR) spectroscopy, methylation analysis, and other structural studies, suggest that the main component of this hexaose consists of one terminal alpha (1-3) linkage in addition to four alpha (1-2) linkages, and that this alpha (1-3)-containing mannohexaose may be responsible for the specificity of antigenic factor 6. Further results obtained by analyses of polarimetry, PMR spectroscopy, and chromium trioxide oxidation-methylation of C. albicans M-1012 mannan has a beta-linkage in addition to alpha-linkages, and that the mode of the beta-linkage is mainly (1-6) linkage. Further evidence obtained by Smith degradation-methylation analysis and by quantitative precipitin reactions of intact and acid-degraded mannan suggests that the antigenic determinant of antigenic factor 6 may be bound, via the beta (1-6) linkage, to C-6 of mannose residues involved in oligosaccharide side chains of serotype A mannan.     

Characterization of a mannan-like oligosaccharide from Mycobacterium smegmatis

A nitrogen-free neutral mannooligosaccharide, similar in structure to the polysaccharide component of yeast mannoproteins, has been isolated from Mycobacterium smegmatis ATCC-356. It has a molecular weight of 3200 and is terminated at the reducing end by mannose. nuclear magentic resonance spectroscopy, methylation analysis, selective enzymic degradation and acetolysis indicates that the molecule consists of an alpha1 --> 6-linked backbone to which single mannose units are attached in alpha1 --> 2 linkage as sidechains.     

The histochemistry of galactose residues of complex carbohydrates as studied by peroxidase-labeled ricinus communis agglutinin

Summary A peroxidase-labeled Ricinus communis agglutinin diaminobenzidine (PO-RCA-DAB) procedure has been utilized to determine the light microscopic localization of galactose residues of complex carbohydrates in a variety of tissues from different vertebrate species. In the same tissues, the localization of mannose and glucose residues was studied by means of a concanavalin A peroxidase diaminobenzidine (Con A-PO-DAB) procedure for comparison. The distribution pattern of galactose residues was found to be in distinct contrast with that of mannose and glucose residues in connective and epithelial tissues such as those involved in the umbilical cord, cartilage, aorta, comb, thyroid gland, submaxillary gland, duodenum, jejunum, colon and gill of various vertebrates. The results of the present study indicate that the PO-RCA-DAB procedure is useful in providing a means of dividing periodic acid-Schiff reactive neutral complex carbohydrates into subgroups in comparison with the intensity of their Con A-PO-DAB reaction in the histochemistry of complex carbohydrates.A part of this investigation has been presented at the Symposium on The Changing Directions of Carbohydrate Histochemistry in the 5th International Congress of Histochemistry and Cytochemistry held in Bucharest (Romania) in 1976     

两种灵芝孢子粉多糖的分离纯化和结构

本文采用水提醇沉法从灵芝孢子粉中提取其粗多糖,经Sepharose CL-6B凝胶柱层析分离得两种主要成分LBPI和LBPII,经高效液相色谱鉴定,均为高均一性成分,分子量分别为9.17×10 ⁴和1.86×10 ⁴;经酸水解、乙酰化和气相色谱分析,确定LBPI的单糖组成为甘露糖、半乳糖和葡萄糖,LBPII的单糖组成为鼠李糖、甘露糖、半乳糖和葡萄糖;通过高碘酸氧化、甲基化和GC-MS进行结构分析,确定LBPI中葡萄糖残基连接方式为1→、1→4,6和1→3,6连接,半乳糖残基为1→6连接,甘露糖残基为1→3,6连接,LBPII中鼠李糖残基连接方式为1→连接,葡萄糖残基为1→、1→4、1→6、1→4,6和1→3,6连接,半乳糖残基为1→6连接,甘露糖残基为1→2,3,6连接。综上,两种多糖LBPI和LBPII均为多分支的中型杂多糖,但两者的单糖组成和连接方式存在差异,这两种多糖成分均为首次报道,可望为灵芝孢子粉的成分、活性研究和资源开发提供理论依据。     

The identification and characterization of xenoantigenic nonhuman carbohydrate sequences in membrane proteins from porcine kidney

The immunogenic nonhuman carbohydrate sequences in membrane proteins from porcine kidney were identified and characterized using MALDI-TOF MS and ESI-QTOF-MS. The MALDI profile, investigated by incubation with exoglycosidases, showed a series of about 40 carbohydrates that were identified as high mannose glycans (Man(3-9)GlcNAc2) and complex bi-, tri-, and tetra-antennary glycans with and without core fucose. The antennae of many of the complex glycans were terminated with alpha-galactose residues, with the numbers of these residues ranging from one up to the number of antennae. Negative ion ESI-MS/MS spectra confirmed the location of the alpha-galactose residues on the ends of the antennae. This total glycan profile of the membrane proteins from porcine kidney will thus provide important information for the study of molecular interactions between antigenic carbohydrates and proteins in xenotransplantation.     

Concanavalin A interactions with asparagine-linked glycopeptides. Bivalency of bisected complex type oligosaccharides

In the preceding paper (Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., and Brewer, C.F. (1987) J. Biol. Chem. 262, 1288-1293), we have demonstrated that certain high mannose and bisected hybrid type glycopeptides are bivalent for concanavalin A (ConA) binding. In the present study, we have investigated the interactions of ConA with a series of synthetic nonbisected and bisected complex type oligosaccharides and related glycopeptides. The modes of binding of the carbohydrates were studied by nuclear magnetic relaxation dispersion techniques, and their affinities were determined by hemagglutination inhibition measurements. We find that certain bisected complex type oligosaccharides are capable of binding and precipitating the lectin. The corresponding nonbisected analogs, however, bind but do not precipitate the protein. The stoichiometries of the precipitin reactions were investigated by quantitative precipitation analyses. The equivalence zones (regions of maximum precipitation) of the precipitin curves indicate that the bisected complex type oligosaccharides are bivalent for lectin binding. Data for the nonbisected analogs are consistent with their being univalent. The nuclear magnetic relaxation dispersion and precipitation data indicate that nonbisected and bisected complex type carbohydrates bind with different mechanisms and conformations. The former class binds by extended site interactions with the protein involving the 2 alpha-mannose residues on the alpha(1-6) and alpha(1-3) arms of the core beta-mannose residue. The latter class binds by only 1 of these 2 mannose residues, which leaves the other mannose residue free to bind to a second ConA molecule. The role of the bisecting GlcNAc residue in affecting the binding properties of complex type carbohydrates to ConA is discussed, and the results are related to the possible structure-function properties of complex type glycopeptides on the surface of cells.     

Structural studies of a cell wall polysaccharide of Trichosporon asahii containing antigen II.

The structure of a cell-wall polysaccharide containing antigen II from Trichosporon asahii was investigated. A purified glucuronoxylomannan (GXM) antigen was found to contain O-acetyl groups that contribute to the serological reactivity. The structure of GXM was analyzed by partial acid hydrolysis, methylation analysis, controlled Smith degradation, NMR studies, and fluorophore-assisted carbohydrate electrophoresis. GXM has an alpha-(1-->3)-D-mannan backbone with a beta-D-glucopyranosyluronic acid residue bound to O-2 of a mannopyranosyl residue and the same number of beta-D-xylopyranosyl residues as mannose. Side chains of beta-D-xylopyranosyl-D-xylopyranose, forming a nonreducing terminus, and beta-D-xylopyranosyl residues were attached to O-2, O-4, and O-6 of the mannose residues.     

Chromatography of methyl ethers of D-glucosamine

Identification of methyl ethers obtained by methylation and subsequent hydrolysis is a powerful technique for determination of linkage positions in structure studies of complex carbohydrates. Although methyl ethers of neutral sugars have been separated by various chromatographic methods, separation of methyl ethers of 2-amino-2-deoxy-d-glucose has been more difficult. Only recently, successful procedures for separation of methyl ethers of d-glucosamine based on thin-layer (1), gas (2), and column (3) chromatography have appeared in literature. We wish to report here a method for separation of d-glucosamine methyl ethers using a combination of partition and ion-exchange chromatography.     

Recombinant carbohydrate variant of human choriogonadotropin beta-subunit (hCG beta) descarboxyl terminus (115-145). Expression and characterization of carboxyl-terminal deletion mutant of hCG beta in the baculovirus system

A recombinant analog of human choriogonadotropin beta-subunit descarboxyl-terminal peptide (115-145 residues, delhCG beta) was obtained by the expression of corresponding beta cDNA in the baculovirus expression system. The efficiency of expression and secretion was high. The recombinant delhCG beta was purified by immunoaffinity using a specific monoclonal antibody against hCG beta and reverse phase high performance liquid chromatography. The hCG beta analog lacked the carboxyl-terminal 31-residue peptide as well as the four O-linked carbohydrates. Also, the N-linked "complex" type carbohydrates in the deletion mutant were modified to the high mannose type. The apparent molecular weights of delhCG beta in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions were found to be 19,000 and 27,500 respectively. delhCG beta on hydrolysis with endo N-acetylglucosaminidase F or H yielded a 17,500 protein band whereas treatment with N-glycanase gave a protein band with a molecular weight of 16,000. The carbohydrate analysis of delhCG beta, calculated on the basis of 4 residues of N-acetylglucosamine, showed 3 or 4 fucose, 0.6 N-acetylgalactosamine, and 11.4 mannose residues, indicating the high mannose type structures of the two N-linked carbohydrate chains. Despite the carbohydrate modification of the N-linked carbohydrates and the carboxyl-terminal deletion, the delhCG beta had about 87% of the immunological activity of the native hCG beta, indicating no significant conformational alteration induced by the mutation. The delhCG beta combined readily with native hCG alpha, and the reconstituted hCG alpha del beta required 0.031 pmol to achieve 50% inhibition of binding of the tracer with rat lutropin/choriogonadotropin receptor compared with 0.039 pmol by native hCG. Like native hCG, hCG alpha del beta also had most comparable ability to stimulate cAMP accumulation and progesterone production in rat Leydig cells. Thus it is clear from the data that the carboxyl-terminal deletion and thereby the deletion of four O-linked carbohydrates had no effect on its in vitro immunological and biological properties.     

Exocellular glycopeptide from a Penicillium charlesii mutant incapable of growth on D-galactose.

The compositions of exocellular saccharide-containing polymers from six mutants of Penicillium charlesii incapable of growing on galactose were investigated. The polymers from the mutants contain a much smaller percentage of galactose than that reported for the peptidophosphogalactomannan (PPGM) from the wild-type organism (Gander et al. 1974). A polymer containing only one galactosyl residue per 49 mannosyl residues was investigated in detail. This polymer is a glycopeptide (peptidomannan) with an amino acid composition similar to that of peptidophospogalactomannan and a mass of about 23,000 daltons. Treatment of peptidomannan with 0.4 N NaOH releases mannan, mannopentaose, mannotetraose, mannotriose, mannobiose, and mannose residues, which are attached to the peptide by O-glycosidic linkage to seryl and threonyl groups. The quantity of glycerol and threitol, derived from mannosyl and internal galactofuranosyl residues, respectively, following Smith degradation, showed that peptidomannan contains 2 mol of internal galactofuranosyl residues per mol of polymer. The polymer contains only 3 mol of (1 yields 5)-linked galactofuranosyl residues per mol of polymer, as described by analysis of the methylation products. Methylation analysis also indicates that the polysaccharide contains primarily (1 yields 2)-linked (67.5%) and (1 yields 6)-linked (20.2%) mannopyranosyl residues. However, acetolysis of the polymer suggests that 37% of the residues are (1 yields 6)-linked. Mannopentaose, mannotetraose, mannotriose, mannobiose, and mannose in a molar ratio of 0.30:0.11:0.15:0.39:0.06, respectively, are released by acetolysis. This result is similar to that obtained with peptidophosphogalactomannan. We conclude that the occurrence of large numbers of galactofuranosyl residues in the major extracellular glycopeptide is not an obligatory requirement for glycopeptide formation.     

Structure of the complex oligosaccharides of fetuin.

The complete structure of the complex oligosaccharides of fetuin has been established. The three fractions of complex oligosaccharide which were isolated by ion exchange chromatography following pronase digestion (F-I, F-II, and F-III) had identical molar ratios of sialic acid (Sia), galactose, mannose, and N-acetylglucosamine of 3:3:3:5. A combination of methylation analyses, Smith periodate degradations, and endoglycosidase and exoglycosidase digestions were utilized to establish the structure which is proposed to be: (see article of journal). Features of this structure not previously established include the presence of 2 residues of alpha2,3- and 1 residue of alpha2,6-linked sialic acid and their location relative to the mannose branch points. Also unusual is the presence of an alpha-linked branch mannose with substituents at positions 2 and 4 which is in turn linked to position 6 of the beta-linked, branch mannose. These features result in unexpected resistance to specific exoglycosidases.     

Carbohydrate composition of central nervous system synapses: Analysis of isolated synaptic junctional complexes and postsynaptic densities

The composition of specialized structures present at synapses within the central nervous system was elucidated by biochemical analysis of fractions enriched in synaptic junctional complexes and postsynaptic densities. The results indicate that the synaptic junctional complex is primarily protein together with some glycoproteins. The synaptic junctional complex proteins are similar in amino acid composition to synaptic membrane proteins; they are not especially rich in basic residues, as previously suggested. The major carbohydrates present in the synaptic junctional complex and postsynaptic density glycoproteins are mannose, galactose, and glucosamine, with lesser amounts of fucose, $\text{N-}\text{acetylneuraminic}$ acid, and galactosamine. Comparison with the synaptic membrane fraction indicates that galactose is more concentrated in the synaptic junctional complex and mannose in the postsynaptic density. Glucose is dramatically enriched in both these fractions. Sucrose binding during isolation may partially account for the glucose enrichment.     

Transient expression of sialylated glycans during glycoprotein processing by embryonal carcinomas

Embryonal carcinoma and early embryonic cells express unusually large and complex carbohydrates on their surfaces that are lost during differentiation. These carbohydrates are composed of alternating galactose and N-acetylglucosamine residues and have either linear or branched architectures. Compared to the glycans expressed by many differentiated cells these glycans are poorly sialylated. However, metabolic studies reveal that there is a transient expression of sialylated glycans during the processing of glycoproteins by embryonal carcinomas. After a short pulse with mannose the major complex-type glycan is a biantennary glycan with two sialic acids. During subsequent chase periods this glycan species is replaced by unsialylated glycans that have elongated branches composed of alternating galactose and N-acetylglucosamine residues.     

Characterization of the oligosaccharide structures on recombinant human prorenin expressed in Chinese hamster ovary cells.

Prorenin was isolated by immunoprecipitation from the culture medium of Chinese hamster ovary cells transfected with a human prorenin cDNA. The N-linked oligosaccharide structures on the in vivo [3H]mannose-labeled, purified protein were characterized using a combination of serial lectin affinity chromatography, high-pressure liquid chromatography, ion-exchange chromatography, and size-exclusion chromatography and treatment with specific glycosidases and methylation analysis. Approximately 61% of the oligosaccharides on the molecule are complex type, in the form of tetraantennary (2%), 2,6-branched triantennary (13%), 2,4-branched triantennary (3%), and biantennary (43%) structures. The majority of all complex type structures are core-fucosylated. Sialic acids are linked at the C-3 position of terminal galactose, and the degree of sialylation of the bi- and triantennary structures varies between nonsialylated and fully sialylated; no tetraatennary structure contains more than three sialic acid residues. Recombinant prorenin contains 4% hybrid-type structures, all of which carry a terminal sialic acid residue. The remaining 35% of the structures on the molecule are high mannose type, composed of 5, 6, or 7 mannose residues. Approximately 6% of the high mannose type structures and 10% of the hybrid structures are phosphorylated, as judged by their susceptibility to treatment with alkaline phosphatase. Compositional analysis of an unlabeled preparation of the protein suggested the presence of approximately 1.4 oligosaccharide units per molecule.     

The MPB83 antigen from Mycobacterium bovis contains O-linked mannose and (1-->3)-mannobiose moieties

Mycobacterium tuberculosis and Mycobacterium bovis, the causative agents of human and bovine tuberculosis, have been reported to express a range of surface and secreted glycoproteins, although only one of these has been subjected to detailed structural analysis. We describe the use of a genetic system, in conjunction with lectin binding, to characterize the points of attachment of carbohydrate moieties to the polypeptide backbone of a second mycobacterial glycoprotein, antigen MPB83 from M. bovis. Biochemical and structural analysis of the native MPB83 protein and derived peptides demonstrated the presence of 3 mannose units attached to two threonine residues. Mannose residues were joined by a (1 --> 3) linkage, in contrast to the (1 --> 2) linkage previously observed in antigen MPT32 from M. tuberculosis and the (1 --> 2) and (1 --> 6) linkages in other mycobacterial glycolipids and polysaccharides. The identification of glycosylated antigens within the M. tuberculosis complex raises the possibility that the carbohydrate moiety of these glycoproteins might be involved in pathogenesis, either by interaction with mannose receptors on host cells, or as targets or modulators of the cell-mediated immune response. Given such a possibility characterization of mycobacterial glycoproteins is a step toward understanding their functional role and elucidating the mechanisms of mycobacterial glycosylation.     

Structure of a major oligosaccharide of Taka-amylase A.

The structures of a major oligosaccharide of Taka-amylase A, shown below, is proposed based on the results of chemical (methylation and acetolysis) and enzymatic (digestions with exo and endo-glycosidases) analyses. This structure is an amendment of that proposed by Yamaguchi et al. (1971) (J. Biochem. 70, 587-594), in which one more mannose residue is attached (Formula: see text) through an alpha 1,2 linkage to the mannose residue which is alpha 1,3-linked to the intermost mannose residue.