High-performance liquid chromatography of monosaccharides and oligosaccharides in a complex biological matrix.

Analysis of oligosaccharides in complex biological matrices is hampered by the fact that oligosaccharides, closely related in structure, are difficult to separate from each other and that conventional detection procedures (refraction index and uv detection) are not specific enough for carbohydrates. Prepurification of samples by procedures like desalting or gel filtration is often used but can lead to the loss of specific oligosaccharides. We have used pellicular anion chromatography in combination with a postcolumn reaction for reducing carbohydrates based on 4-aminobenzoylhydrazide. This procedure not only detected normal mono- and oligosaccharides but N-acetylhexosamines and reducing N-acetylhexosamine containing oligosaccharides as well. A sensitivity of about 20-25 pmol for non-GlcNAc containing mono- or oligosaccharides and between 30-50 pmol for GlcNAc or oligosaccharides with GlcNAc at the reducing side was reached. The postcolumn detection was compared with pulsed amperometric detection and appeared to be more specific for mono- and oligosaccharides. Except for deproteination to protect the column, no further sample preparation was needed with this system for our application (urines). In this way pellicular anion chromatography in combination with this postcolumn reaction reaction to be a sensitive and specific HPLC procedure for analysis of monosaccharides and oligosaccharides in complex biological matrices.     

Trafficking of oligomannosides released during N-glycosylation: a clearing mechanism of the rough endoplasmic reticulum

The main reaction of N-glycosylation of proteins is the transfer 'en bloc' of the oligosaccharide moieties of lipid intermediates to an asparagine residue of the nascent protein. For the past 15 years, a few laboratories including ours have shown that the process was accompanied by the release of oligosaccharide-phosphates and of neutral oligosaccharides possessing one GlcNAc (OS-Gn(1)) or two GlcNAc (OS-Gn(2)) at the reducing end. The aim of this review is to gather the evidence for the different origins of these soluble oligomannosides, to examine their subcellular location and intracellular trafficking. Furthermore, using Brefeldin A we demonstrated that this released oligomannoside material could be the substrate for the Golgi glycosidases and glycosyltransferases. Indeed, released oligomannoside never reach the Golgi vesicles either because they are directly produced in the cytosol as has been demonstrated for oligosaccharide-phosphates and for neutral oligosaccharides possessing one GlcNAc at the reducing end or because they are actively transported out of the rough endoplasmic reticulum to the cytosol. One of the functions of oligomannoside trafficking between rough endoplasmic reticulum, cytosol and lysosomes could be to prevent these oligosaccharides for competing with glycosylation in the Golgi.     

Oligosaccharide analyses of glycopeptides of horseradish peroxidase by thermal-assisted partial acid hydrolysis and mass spectrometry

Thermal-assisted partial acid hydrolysis of the carbohydrate moieties of N-glycosylated peptides of horseradish peroxidase (HRP) is used to generate oligosaccharide cleavage ladders. These ladders allow direct reading of components of the oligosaccharides by mass spectrometry. Acid hydrolysis performed with 1.4, 3.1, 4.5, or 6.7M trifluoroacetic acid at 37, 65, or 95 degrees C for 30min to 24h hydrolyzed mainly the oligosaccharide units of glycopeptides with least peptide bond or amino acid side chain hydrolysis. Tryptic N-glycosylated peptides from HRP with molecular weights of 2533, 2612, 3355, 3673, and 5647Da were used as test systems in these experiments. Data showed that the most labile group of oligosaccharides is the fucose (Fuc) and the majority of the end cleavage products are peptides with one or no N-acetylglucosamine (GlcNAc) residue linked to Asparagine (Asn). Additionally, the data agree with previous reports that glycopeptides 3355 and 3673Da carry an oligosaccharide (Xyl)Man3(Fuc)GlcNAc2, glycopeptide 5647Da carries two oligosaccharides (Xyl)Man3(Fuc)GlcNAc2, and glycopeptides 2612 and 2533Da carry (Xyl)Man3GlcNAc2 and (Fuc)GlcNAc, respectively. However, the glycosylation site of the 2612Da peptide at Asn286 is partially occupied. This method is particularly useful in identifying glycopeptides and obtaining monosaccharide compositions of glycopeptides.     

"Glyco-deglyco" processes during the biosynthesis of glycoproteins

For the past fifteen years, it has appeared increasingly evident that the N-glycosylation process was accompanied by the release of oligomannoside type oligosaccharides. This material is constituted of oligosaccharide-phosphates and of neutral oligosaccharides possessing one GlcNAc (OS-Gn1) or two GlcNAc (OS-Gn2) at the reducing end. It has been demonstrated that oligosaccharide-phosphates originated from the cleavage, by a specific pyrophosphatase, of non-glucosylated cytosolic faced oligosaccharide-PP-Dol and chiefly the Man5GlcNAc2-PP-Dol. The Man5GlcNAc2-P, as the main product, is recovered in the cytosolic compartment and is further degraded to Man5GlcNAc1 by not-yet depicted enzymes. In contrast, OS-Gn2 produced from hydrolysis of oligosaccharide-PP-Dol (presumably as a transfer reaction onto water) when the amount of protein acceptor is limiting, are generated into the lumen of rough endoplasmic reticulum (rough ER). They are further submitted to processing a-glucosidases and rough ER mannosidase and are (mainly as Man8GlcNAc2) exported into the cytosolic compartment. This material is further degraded into a single component, the Man5GlcNAc1, by the sequential action of a cytosolic neutral chitobiase followed by cytosolic mannosidase(s). Furthermore, OS-Gn1 could have a dual origin: in one hand, they originate from OS-Gn2 by the cytosolic degradation pathway indicated above, on the other hand, we will discuss a possible origin from the degradation or remodelling of newly synthesized glycoproteins. Considered first as a minor phenomenon, these observations have lead to the concept of intracellular oligomannoside trafficking, a process which results from more fundamental phenomena such as the control of the dolichol cycle, and the so-called quality-control of glycoprotein. In this review, we would like to describe the evolution of ideas on the origin, intracellular trafficking and putative roles of these oligomannosides released during during the N-glycosylation process. We propose that these early stage "glyco-deglyco" processes represent a way of control of N-glycosylation and of the fate of N-glycoproteins. This review is dedicated to Pr Paul Boulanger who has spent a large part of his career to determine the structure of proteins in order to understand their functions. If it is well established that many biological functions are born by proteins, it appears more and more evident that co- or post translational modifications are of importance in the modulation of these functions. Among them, the glycosylation appears as a major event which intervene in the 3D structure of the protein, which control his biological time-life and which may act in many recognition processes.     

Glycoprotein synthesis in yeast. Identification of Man8GlcNAc2 as an essential intermediate in oligosaccharide processing

Synthesis of the N-linked oligosaccharides of Saccharomyces cerevisiae glycoproteins has been studied in vivo by labeling with [2-3H]mannose and gel filtration analysis of the products released by endoglycosidase H. Both small oligosaccharides, Man8-14GlcNAc, and larger products, Man greater than 20GlcNAc, were labeled. The kinetics of continuous and pulse-chase labeling demonstrated that Glc3Man9GlcNAc2, the initial product transferred to protein, was rapidly (t1/2 congruent to 3 min) trimmed to Man8GlcNAc2 and then more slowly (t1/2 = 10-20 min) elongated to larger oligosaccharides. No oligosaccharides smaller than Man8GlcNAc2 were evident with either labeling procedure. In confirmation of the trimming reaction observed in vivo, 3H-labeled Man9-N-acetylglucosaminitol from bovine thyroglobulin and [14C]Man9GlcNAc2 from yeast oligosaccharide-lipid were converted in vitro by broken yeast cells to 3H-labeled Man8-N-acetylglucosaminitol and [14C]Man8GlcNAc2. Man8GlcNAc and Man9GlcNAc from yeast invertase and from bovine thyroglobulin were purified by gel filtration and examined by high field 1H-NMR analysis. Invertase Man8GlcNAc (B) and Man9GlcNAc (C) were homogeneous compounds, which differed from the Man9GlcNAc (A) of thyroglobulin by the absence of a specific terminal alpha 1,2-linked mannose residue. The Man9GlcNAc of invertase (C) had an additional terminal alpha 1,6-linked mannose and appeared identical in structure with that isolated from yeast containing the mnn1 and mnn2 mutations (Cohen, R. E., Zhang, W.-j., and Ballou, C. E. (1982) J. Biol. Chem. 257, 5730-5737). It is concluded that Man8GlcNAc2, formed by removal of glucose and a single mannose from Glc3Man9GlcNAc2, is the ultimate product of trimming and the minimal precursor for elongation of the oligosaccharides on yeast glycoproteins. The results suggest that removal of a particular terminal alpha 1,2-linked mannose from Man9GlcNAc2 by a highly specific alpha-mannosidase exposes the nascent Man-alpha 1,6-Man backbone for elongation with additional alpha 1,6-linked mannose residues, according to the following scheme: (formula, see text).     

Isolation and structures of glycoprotein-derived free sialooligosaccharides from the unfertilized eggs of Tribolodon hakonensis, a dace. Intracellular accumulation of a novel class of biantennary disialooligosaccharides

Novel acidic oligosaccharides were isolated in abnormally large amounts (about 200 ng/egg) from the unfertilized eggs of Tribolodon hakonensis (a dace, "ugui" in Japanese). The free oligosaccharides were found to consist of a mixture of disialylated species most of which end with beta-mannosyl N-acetylglucosamine structure at their reducing termini, i.e. greater than Man beta 1-4GlcNAc. A minute portion of the sialooligosaccharides was found to have the reducing terminal structure, di-N-acetylchitobiose, i.e. greater than Man beta 1-4GlcNAc beta 1-4GlcNAc. From the structural analysis of these free sialooligosaccharides, the following structures are proposed: (sequence; see text) Occurrence of such a symmetrically or dissymmetrically branched form of the biantennary nonreducing periphery as revealed here is novel. Although it is unknown why and how such high amounts of free oligosaccharides are accumulated in unfertilized eggs, these were presumably protein-linked components and must be released at certain stages of oogenesis.     

Site-specific N-glycosylation analysis of human plasma ceruloplasmin using liquid chromatography with electrospray ionization tandem mass spectrometry

Ceruloplasmin has ferroxidase activity and plays an essential role in iron metabolism. In this study, a site-specific glycosylation analysis of human ceruloplasmin (CP) was carried out using reversed-phase high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). A tryptic digest of carboxymethylated CP was subjected to LC-ESI-MS/MS. Product ion spectra acquired data-dependently were used for both distinction of the glycopeptides from the peptides using the carbohydrate B-ions, such as m/z 204 (HexNAc) and m/z 366 (HexHexNAc), and identification of the peptide moiety of the glycopeptide based on the presence of the b- and y-series ions derived from the peptide. Oligosaccharide composition was deduced from the molecular weight calculated from the observed mass of the glycopeptide and theoretical mass of the peptide. Of the seven potential N-glycosylation sites, four (Asn119, Asn339, Asn378, and Asn743) were occupied by a sialylated biantennary or triantennary oligosaccharide with fucose residues (0, 1, or 2). A small amount of sialylated tetraantennary oligosaccharide was detected. Exoglycosidase digestion suggested that fucose residues were linked to reducing end GlcNAc in biantennary oligosaccharides and to reducing end and/or alpha1-3 to outer arms GlcNAc in triantennary oligosaccharides and that roughly one of the antennas in triantennary oligosaccharides was alpha2-3 sialylated and occasionally alpha1-3 fucosylated at GlcNAc.     

Structural analysis of murine zona pellucida glycans. Evidence for the expression of core 2-type O-glycans and the Sd(a) antigen

Murine sperm initiate fertilization by binding to specific oligosaccharides linked to the zona pellucida, the specialized matrix coating the egg. Biophysical analyses have revealed the presence of both high mannose and complex-type N-glycans in murine zona pellucida. The predominant high mannose-type glycan had the composition Man(5)GlcNAc(2), but larger oligosaccharides of this type were also detected. Biantennary, triantennary, and tetraantennary complex-type N-glycans were found to be terminated with the following antennae: Galbeta1-4GlcNAc, NeuAcalpha2-3Galbeta1-4GlcNAc, NeuGcalpha2-3Galbeta1-4GlcNAc, the Sd(a) antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc, NeuGcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc), and terminal GlcNAc. Polylactosamine-type sequence was also detected on a subset of the antennae. Analysis of the O-glycans indicated that the majority were core 2-type (Galbeta1-4GlcNAcbeta1-6[Galbeta1-3]GalNAc). The beta1-6-linked branches attached to these O-glycans were terminated with the same sequences as the N-glycans, except for terminal GlcNAc. Glycans bearing Galbeta1-4GlcNAcbeta1-6 branches have previously been suggested to mediate initial murine gamete binding. Oligosaccharides terminated with GalNAcbeta1-4Gal have been implicated in the secondary binding interaction that occurs following the acrosome reaction. The significant implications of these observations are discussed.     

Chimeric glycosaminoglycan oligosaccharides synthesized by enzymatic reconstruction and their use in substrate specificity determination of Streptococcus hyaluronidase

A method was developed for the reconstruction of glycosaminoglycan (GAG) oligosaccharides using the transglycosylation reaction of an endo-beta-N-acetylhexosaminidase, testicular hyaluronidase, under optimal conditions. Repetition of the transglycosylation using suitable combinations of various GAGs as acceptors and donors made it possible to custom-synthesize GAG oligosaccharides. Thus we prepared a library of chimeric GAG oligosaccharides with hybrid structures composed of disaccharide units such as GlcA-GlcNAc (from hyaluronic acid), GlcA-GalNAc (from chondroitin), GlcA-GalNAc4S (from chondroitin 4-sulfate), GlcA-GalNAc6S (from chondroitin 6-sulfate), IdoA-GalNAc (from desulfated dermatan sulfate), and GlcA-GalNAc4,6-diS (from chondroitin sulfate E). The specificity of the hyaluronidase from Streptococcus dysgalactiae (hyaluronidase SD) was then investigated using these chimeric GAG oligosaccharides as model substrates. The results indicate that the specificity of hyaluronidase SD is determined by the following restrictions at the nonreducing terminal side of the cleavage site: (i) at least one disaccharide unit (GlcA-GlcNAc) is necessary for the enzymatic action of hyaluronidase SD; (ii) cleavage is inhibited by sulfation of the N-acetylgalactosamine; (iii) hyaluronidase SD releases GlcA-GalNAc and IdoA-GalNAc units as well as GlcA-GlcNAc. At the reducing terminal side of the cleavage site, the sulfated residues on the N-acetylgalactosamines in the disaccharide units were found to have no influence on the cleavage. Additionally, we found that hyaluronidase SD can specifically and endolytically cleave the internal unsulfated regions of chondroitin sulfate chains. This demonstration indicates that custom-synthesized GAG oligosaccharides will open a new avenue in GAG glycotechnology.     

Glycoprotein biosynthesis in plants. Formation of lipid-linked oligosaccharides of mannose and N-acetylglucosamine by mung bean seedlings.

Cell-free enzyme particles from mung bean seedlings catalyze the incorporation of mannose from GDP-[¹⁴C]mannose and GlcNAc from UDP-[³H]GlcNAc into glycolipids and into glycoprotein. The most rapidly labeled product from GDP-mannose was characterized as a mannosyl-phosphoryl-polyisoprenol, whereas that from UDP-GlcNAc was a mixture of GlcNAc-(pyro)phosphoryl-polyisoprenol and a disaccharide composed of two N-acetylglucosamine residues attached to the polyisoprenol by a phosphoryl or pyrophosphoryl linkage. Radioactivity from GDP-mannose and UDP-GlcNAc was also incorporated into more polar lipids which have been partially characterized as a series of oligosaccharide-(pyro)phosphoryl-lipids. The mannose-labeled oligosaccharides released from these lipids by mild acid hydrolysis were found to contain GlcNAc at their reducing end indicating that these oligosaccharides contain both GlcNAc and mannose. Both the GlcNAc-labeled and the mannose-labeled oligosaccharides gave multiple radioactive peaks upon paper chromatography indicating that they are composed of a series of different sized oligosaccharides. Finally, radioactivity from GDP-[¹⁴C]mannose and UDP-[³H]GlcNAc is incorporated into an insoluble component. Ten percent of the mannose label and all of the GlcNAc label in this insoluble material could be solubilized by digestion with Pronase. The glycopeptides released by Pronase digestion appeared to be approximately the same size as the oligosaccharides from the lipid-linked oligosaccharides based on gel filtration chromatography on Sephadex G-50. The results are consistent with a mechanism for glycoprotein synthesis involving lipid-linked oligosaccharide intermediates.     

Rat liver chitobiase: purification, properties, and role in the lysosomal degradation of Asn-linked glycoproteins

Chitobiase, the lysosomal glycosidase responsible for splitting the GlcNAc beta-D-(1-4)GlcNAc moiety in Asn-linked glycoproteins, was purified over 600-fold from frozen rat livers utilizing an assay with di-N-acetylchitobiose as the substrate. The final preparation showed a major polypeptide of Mr 43,000 (sodium dodecylsulfate-polyacrylamide gel electrophoresis) that was determined to be the chitobiase by an immunological method. The purified chitobiase also hydrolyzed tri- and tetrasaccharides of chitin, which like di-N-acetylchitobiose were not substrates if first reduced by NaBH4. The initial products formed during hydrolysis of the tetrasaccharide were trisaccharide and GlcNAc. These results imply that chitobiase is a "reducing-end exohexosaminidase" which cleaves single GlcNAc units only from the reducing end of oligosaccharides. Fucose, typically found linked to the reducing-end GlcNAc in complex oligosaccharide chains, was found to block this reaction. Additional substrates that were hydrolyzed included GlcNAc beta-D-(1-4)MurNAc, the repeating structure from bacterial cell wall peptidoglycan, and the Man beta-D-(1-4)GlcNAc reducing-end component of glycoproteins. Km and Vm for hydrolysis of these substrates were of similar magnitude as for di-N-acetylchitobiose (6.3 mM and 15 mumol/min/mg protein, respectively). Liver tissues from nin mammalian species were surveyed for the presence of chitobiase activity. The activity was found in rat, mouse, rabbit, and guinea pig liver (Stirling [(1974) FEBS Lett. 39, 171-175] previously observed the enzyme in human liver), but not in dog, sheep, pig, cat, and cow liver. The presence or absence of chitobiase so far observed was found to exactly correlate with the type of oligosaccharide fragments found to accumulate in animals containing genetic or inhibitor-induced lysosomal storage pathologies. The presence of the chitobiase corresponds to the occurrence of one GlcNAc unit at the reducing end of stored oligosaccharides, while the absence of this glycosidase yields fragments with an intact GlcNAc beta-D-(1-4)GlcNAc moiety. These results verify our previous proposal that lysosomal disassembly of glycoproteins to free amino acids and sugars is an ordered, bidirectional pathway in which chitobiase (when present) catalyzes the last step during digestion of the protein-oligosaccharide linkage region.     

Evidence that transient glucosylation of protein-linked Man9GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2 occurs in rat liver and Phaseolus vulgaris cells

Formation of protein-linked Glc1Man9GlcNAc2 , Glc1Man8GlcNAc2 , and Glc1Man7GlcNAc2 was detected in rat liver slices and Phaseolus vulgaris seeds incubated with [U-14C]glucose. Similar compounds were not synthesized in Saccharomyces cerevisiae cells incubated under similar conditions. Rat liver microsomes were incubated with [glucose-U-14C] Glc3Man9GlcNAc2-P-P-dolichol or UDP-[U-14C]Glc as glycosyl donors. Only in the latter condition protein-linked Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2 were formed. Addition of mannooligosaccharides that strongly inhibited alpha 1-2-mannosidases to incubation mixtures containing rat liver microsomes and UDP-[U-14C]Glc did not prevent formation of protein-bound Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2 . Furthermore, the presence of amphomycin in reaction mixtures containing liver membranes and UDP-[U-14C]Glc completely abolished synthesis of glucosylated derivatives of dolichol without affecting formation of protein-linked Glc1Man9GlcNAc2 , Glc1Man8GlcNAc2 , and Glc1Man7GlcNAc2 . The results reported above indicated that under the experimental conditions employed protein-bound Glc1Man9GlcNAc2 , Glc1Man8GlcNAc2 , and Glc1Man7GlcNAc2 were formed by glucosylation of unglucosylated oligosaccharides. Results obtained in pulse-chase experiments performed in vitro also supported this conclusion. UDP-Glc appeared to be the donor of the glucosyl residues. The rough endoplasmic reticulum was found to be the main subcellular site of protein glucosylation. It is tentatively suggested that this process could prevent extensive degradation of oligosaccharides by mannosidases during transit of glycoproteins through the endoplasmic reticulum.     

2-Acylamido Analogues of N-Acetylglucosamine Prime Formation of Chitin Oligosaccharides by Yeast Chitin Synthase 2

Chitin, a homopolymer of β1,4-linked N-acetylglucosamine (GlcNAc) residues, is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs2, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N′-diacetylchitobiose (GlcNAc₂) was the major reaction product. Formation of both COs and insoluble chitin was also stimulated by GlcNAc₂ and by N-propanoyl-, N-butanoyl-, and N-glycolylglucosamine. MALDI analyses of the COs made in the presence of 2-acylamido analogues of GlcNAc showed they that contained a single GlcNAc analogue and one or more additional GlcNAc residues. These results indicate that Chs2 can use certain 2-acylamido analogues of GlcNAc, and likely free GlcNAc and GlcNAc₂ as well, as GlcNAc acceptors in a UDP-GlcNAc-dependent glycosyltransfer reaction. Further, formation of modified disaccharides indicates that CSs can transfer single GlcNAc residues.     

Structural diversity of cytosolic free oligosaccharides in the human hepatoma cell line, HepG2

It is thought that free oligosaccharides in the cytosol are an outcome of quality control of glycoproteins by endoplasmic reticulum-associated degradation (ERAD). Although considerable amounts of free oligosaccharides accumulate in the cytosol, where they presumably have some function, detailed analyses of their structures have not yet been carried out. We isolated 21 oligosaccharides from the cytosolic fraction of HepG2 cells and analyzed their structures by the two-dimensional high-performance liquid chromatography (HPLC) sugar-mapping method. Sixteen novel oligosaccharides were identified in the cytosol in this study. All had a single N-acetylglucosamine at their reducing-end cores and could be expressed as (Man)n (GlcNAc)1. No free oligosaccharide with N,N'-diacetylchitobiose was detected in the cytosolic fraction of HepG2 cells. This suggested that endo-beta-N-acetylglucosaminidase was a key enzyme in the production of cytosolic free oligosaccharides. The 21 oligosaccharides were classified into three series--series 1: oligosaccharides processed from Manalpha1-2Manalpha1-6 (Manalpha1-2Manalpha1-3)Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3) Manbeta1-4GlcNAc (M9A') and Manalpha1-2Manalpha1-6(Manalpha1-3) Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc (M8A') by digestion with cytosolic alpha-mannosidase; series 2: oligosaccharides processed with Golgi alpha-mannosidases in addition to endoplasmic reticulum (ER) and cytosolic alpha-mannosidases; and series 3: glucosylated oligosaccharides produced from Glc1Man9GlcNAc1 by hydrolysis with cytosolic alpha-mannosidase. The presence of the series "2" oligosaccharides suggests that some of the misfolded glycoproteins had been processed in pre-cis-Golgi vesicles and/or the Golgi apparatus. When the cells were treated with swainsonine to inhibit cytosolic alpha-mannosidase, the amounts of M9A' and M8A' increased remarkably, suggesting that these oligosaccharides were translocated into the cytosol. Four oligosaccharides of series "2" also increased. In contrast, there were obvious reductions in Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc (M5B'), the end product from M9A' by digestion with cytosolic alpha-mannosidase, and Manalpha1-6(Manalpha1- 2Manalpha1-3)Manbeta1-4GlcNAc, derived from series "2" oligosaccharides by digestion with cytosolic alpha-mannosidase. Our data suggest that (1) some of the cytosolic oligosaccharides had been processed with Golgi alpha-mannosidases, (2) the major oligosaccharides translocated from the ER were M9A' and M8A', and (3) M5B' and Glc1M5B' were maintained at relatively high concentrations in the cytosol.     

A di-N-acetylchitobiase activity is involved in the lysosomal catabolism of asparagine-linked glycoproteins in rat liver

A perfused rat liver was used to study the effects of 5-diazo-4-oxo-L-norvaline on lysosomal glycoprotein catabolism. Addition of this compound (1.0 mM) to the perfusate reduced activity of beta-aspartyl-N-acetylglucosylamine amidohydrolase by 99% in 1 h. Treated livers were unable to completely degrade endocytosed N-acetyl[14C]glucosamine-labeled asialo-alpha 1-acid glycoprotein as evidenced by a 50% reduction in radiolabeled serum glycoprotein secretion compared to controls. This decreased degradation was matched by a lysosomal accumulation of glycopeptides with the structure: GlcNAc beta(1-4)GlcNAc-Asn. The result suggested the presence of an unrecognized glycosidase in rat liver lysosomes, since this remnant was extended by one more GlcNAc residue than would have been expected after specific inactivation of the amidohydrolase. Such a novel enzyme would therefore catalyze cleavage of the N-acetylglucosamine residue at the reducing end of alpha 1-acid glycoprotein oligosaccharides only following removal of the linking Asn. The activity was then detected in lysosomal extracts by using intact asialo-biantennary oligosaccharides labeled with [3H] galactose or N-acetyl[14C]glucosamine residues as a substrate. To prevent simultaneous digestion of the material from its nonreducing end, beta-D-galactosidase in the enzyme extract was first inactivated with the irreversible active site-directed inhibitor, beta-D-galactopyranosylmethyl-p-nitrophenyltriazene. The observed di-N-acetylchitobiose cleaving activity worked optimally at pH 3.4 and was uniquely associated with the lysosomal fraction of the liver homogenate. The enzyme also cleaved triantennary chains and di-N-acetylchitobiose, but failed to hydrolyze substrates that had been reduced with NaBH4. The new glycosidase was well separated from N-acetyl-beta-D-glucosaminidase (assayed with p-nitrophenyl-beta-D-glucosaminide) by gel filtration chromatography and had an apparent molecular weight of 37,000. A similar enzyme that hydrolyzes di-N-acetylchitobiose had previously been found in extracts of human liver (Stirling, J. L. (1974) FEBS Lett. 39, 171-175).     

Immunochemical characterization of antisera reactivities toN-acetyl-d-glucosamine oligosaccharides with theβ(1–4)-glycosidic linkage

The (1–4)-linked oligosaccharides ofN-acetyl-d-glucosamine (GlcNAc) isolated from chitin were used to prepare synthetic immunogens and antigens by reductive amination of (GicNAc)_n to bovine serum albumin (BSA). The rabbit antisera produced to the (GlcNAc)_n-BSA conjugates were characterized using an enzyme-linked immunosorbent assay (ELISA) system under conditions that, only the antibodies with carbohydrate specificity were reactive with the solid-phase adsorbed (GlcNAc)_n-BSA antigens. Inhibition assays using the (GlcNAc)_n-BSA, (GlcNAc)_n oligosaccharides, and the reduced oligosaccharides showed a relative specificity of the antisera for the chain length of the (GlcNAc)_n sequences. For example, the anti-(GlcNAc)₅-and anti-(GlcNAc)₄-sera were inhibited best by the longer chain (GlcNAc)_n ologosaccharides with the antibody combining sites directed mainly to the cyclic GlcNAc residues of the (GlcNAc)_n-BSA conjugates. The antibody combining sites were in part directed to the acyclic moiety of the reducing end of the oligosaccharides as shown by the increased inhibitory activities of the reduced (GlcNAc)_n oligosaccharides particularly, with the anti-(GlcNAc)₂-and anti-(GlcNAc)₃-sera. The best hapten inhibitors for the anti-(GlcNAc)₂-BSA and anti-(GlcNAc)₁-BSA sera were theN-butylamine derivatives of (GlcNAc)₂ and (GlcNAc)₁, respectively, indicating that the antibodies were also reactive with the secondary amine formed between the reducing end of the oligosaccharides and the -amino groups of lysine.Abbreviations ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - GicNAc N-acetyl-d-glucosamine - (GlcNAc)_n Oligosaccharides containing GlcNAc in 1–4 linkages - (GlcNAc)₂ DGlcNAc(1–4)-d-GlcNAc - (GlcNAc)₃ (GlcNAc)₄ and (GlcNAc)₅, the homologous oligosaccharides of (GlcNAc)₂ - PBS phosphate buffered saline (0.01 M sodium phosphate, pH73 containing 0.15%M (NaCl) - PBSA PBS containing 1% BSA and 0.1% Tween-20 - ONPG o-nitrophenyl--d-galactopyranoside     

Comparative analysis of oligosaccharide specificities of fucose-specific lectins from Aspergillus oryzae and Aleuria aurantia using frontal affinity chromatography

Aleuria aurantia lectin (AAL) is widely used to estimate the extent of α1,6-fucosylated oligosaccharides and to fractionate glycoproteins for the detection of specific biomarkers for developmental antigens. Our previous studies have shown that Aspergillus oryzae lectin (AOL) reflects the extent of α1,6-fucosylation more clearly than AAL. However, the subtle specificities of these lectins to fucose linked to oligosaccharides through the 2-, 3-, 4-, or 6-position remain unclear, because large amounts of oligosaccharides are required for the systematic comparative analysis using surface plasmon resonance. Here we show a direct comparison of the dissociation constants (K_d) of AOL and AAL using 113 pyridylaminated oligosaccharides with frontal affinity chromatography. As a result, AOL showed a similar specificity as AAL in terms of the high affinity for α1,6-fucosylated oligosaccharides, for smaller fucosylated oligosaccharides, and for oligosaccharides fucosylated at the reducing terminal core GlcNAc. On the other hand, AOL showed 2.9-6.2 times higher affinity constants (K_a) for α1,6-fucosylated oligosaccharides than AAL and only AAL additionally recognized oligosaccharides which were α1,3-fucosylated at the reducing terminal GlcNAc. These results explain why AOL reflects the extent of α1,6-fucosylation on glycoproteins more clearly than AAL. This systematic comparative analysis made from a quantitative viewpoint enabled a clear physical interpretation of these fucose-specific lectins with multivalent fucose-binding sites.     

Purification and characterization of a chitinase from Trichoderma viride

Usukizyme, a commercial enzyme preparation from Trichoderma viride, showed multiple chitin- degrading activities. One of these was purified to homogeneity by sequential DEAE Sepharose CL-6B, Q-Sepharose FF, and Sephacryl S-100 HR column chromatographies. The purified enzyme showed optimum activity at pH 3.5 and 50 degrees -55 degrees C and was stable in the pH range of 3.5-6.0 and up to 45 degrees C. It showed higher activity toward chitosan-7B, a 62% deacetylated chitosan, as opposed to highly deacetylated chitosan substrates. Products of degradation of a 1% (w/v) solution of partially deacetylated chitin (PC-100) were purified on CM-Sephadex C-25 and analyzed by HPLC, exo-glycosidase digestion, and nitrous acid deamination. The enzyme was unable to split the GlcN-GlcN linkages in the substrate. It produced mainly (GlcNAc)(2) and (GlcNAc)(3) along with mixed oligosaccharides. When subjected to nitrous acid degradation, some of the mixed oligosaccharides produced mainly 2-deoxyglucitol, implying the presence of GlcN at the reducing end of the oligosaccharides.     

红花菜豆凝集素的糖结合专一性

经肼解、Ｂｉｏ－Ｇｅｌ Ｐ－２柱层析、ＮａＢ＾３Ｈ４和ＮａＢＨ４还原,制备各种来源的、氚标记在还原末端的、还原末端为Ｎ－乙酰氨基葡萄糖醇的混合寡糖,经Ｂｉｏ－Ｇｅｌ Ｐ－４凝胶柱分离,以及用糖苷酶酶解,制备了各种不同类型的氚标记的寡糖。这些寡糖在固定化的ＰＣＬ－Ｓｅｐｈａｒｏｓｅ柱上亲和层析,根据各种类型寡糖在ＰＣＬ－Ｓｅｐｈａｒｏｓｅ柱上的层析行为,确定红花菜豆（矮生红花变种）凝集素（ＰＣＬ）的