Purification of glycopeptides of human ceruloplasmin and immunoglobulin D by high-pressure liquid chromatography

To facilitate structural studies of glycoproteins, reverse-phase high-pressure liquid chromatography (HPLC) methods have been developed for preparative isolation of glycopeptides and have been applied to human ceruloplasmin as an example of glycopeptides containing glucosamine (GlcN) and to human immunoglobulin D (IgD) for glycopeptides containing galactosamine (GalN). The use of RP-P columns and of trifluoroacetic acid and heptafluorobutyric acid as counterions was investigated. Various elution systems (both isocratic and programmed gradient) were used with n-propanol to assess the relative hydrophilicity of the peptides. The procedure developed for the GlcN glycopeptides of ceruloplasmin enabled purification of nine major chymotryptic peptides (ranging in size from 15 to 29 residues) and also of many minor peaks. These were characterized by amino acid and endgroup analysis, and the complete sequence of five was determined. These represent three different sites of GlcN attachment in the amino-terminal half of the ceruloplasmin chain. The procedures developed have enabled isolation of glycopeptides from ceruloplasmin having a single GlcN oligosaccharide attached; the latter are valuable for study of the structure and function of the carbohydrate groups. Separation of GalN glycopeptides from IgD was more difficult because of the high content of GalN in the hinge. Purification and sequence analysis was aided by partial removal of sugar by treatment with HF and by other methods. Four (or five) GalN oligosaccharides are attached to serine or threonine residues in the IgD hinge region, and all but one are in close proximity in the repeating sequence Ala-Thr-Thr-Ala-Pro-Ala-Thr-Thr.     

Determination of galactosamine impurities in heparin sodium using fluorescent labeling and conventional high-performance liquid chromatography

Heparin is a sulfated glycosaminoglycan (GAG), which contains N-acetylated or N-sulfated glucosamine (GlcN). Heparin, which is generally obtained from the healthy porcine intestines, is widely used as an anticoagulant during dialysis and treatments of thrombosis such as disseminated intravascular coagulation. Dermatan sulfate (DS) and chondroitin sulfate (CS), which are galactosamine (GalN)-containing GAGs, are major process-related impurities of heparin products. The varying DS and CS contents between heparin products can be responsible for the different anticoagulant activities of heparin. Therefore, a test to determine the concentrations of GalN-containing GAG is essential to ensure the quality and safety of heparin products. In this study, we developed a method for determination of relative content of GalN from GalN-containing GAG in heparin active pharmaceutical ingredients (APIs). The method validation and collaborative study with heparin manufacturers and suppliers showed that our method has enough specificity, sensitivity, linearity, repeatability, reproducibility, and recovery as the limiting test for GalN from GalN-containing GAGs. We believe that our method will be useful for ensuring quality, efficacy, and safety of pharmaceutical heparins. On July 30, 2010, the GalN limiting test based on our method was adopted in the heparin sodium monograph in the Japanese Pharmacopoeia.     

Uridine diphospho sugars and related hexose phosphates in the liver of hexosamine-treated rats: identification using 31P-[1H] two-dimensional NMR with HOHAHA relay

The effects of administration of galactosamine (GalN) and glucosamine (GlcN) on the levels of UDP-sugars and hexose monophosphates in rat livers were studied by a variety of 31P NMR methods. The flux of metabolites in the liver was monitored by in vivo NMR and showed elevated levels of UDP-sugars, and even greater increases in resonances at 4.6 ppm for GlcN treatment and at 2.0 ppm for GalN treatment. The individual compounds corresponding to these changes were identified in PCA liver extracts by 31P-[1H] two-dimensional relay spectroscopy with a HOHAHA-type 1H spin-lock. This method of transferring proton magnetization allows for nearly all of the proton chemical shifts to be observed for the hexose moiety of a UDP-sugar present in a complex mixture. The UDP-sugars in the extracts from treated rats were predominantly UDP-hexosamines. Relay spectra were also used to determine that GalN-1-P was the major component (16.0 mumol/g of liver) of the GalN-treated liver, while both alpha and beta anomers of GlcNAc-6-P were readily identified as the major hexose monophosphates in the GlcN experiment. Spectra from the 1H dimension of relay experiments conducted on extracts were nearly superimposable on relay spectra obtained under the same conditions for mixtures of standard compounds of known structure. UDP-GlcN and UDP-GalN were not commercially available, but their presence was established in the extracts after GalN treatment by obtaining relay spectra for a mixture of the compounds produced in situ enzymatically, without purification.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical characterization of lipopolysaccharides from Proteus strains used in Weil-Felix test

The lipopolysaccharides (LPS) extracted from Proteus strains OX2, OX19, and OXK used as antigens in the Weil-Felix test, were characterized by chemical analysis and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). To separate the O-polysaccharide, core-oligosaccharide, and lipid A moieties, each LPS was treated with 2% acetic acid, centrifuged, and applied to Sephadex G-50 column. The core-oligosaccharides contained L-glycero-D-mannoheptose, D-glycero-D-mannoheptose, glucose (Glc), galactose, 3-deoxy-D-mannooctulosonic acid, uronic acid, phosphate, glucosamine (GlcN), and galactosamine (GalN). The lipid A preparations contained GlcN, GlcN-phosphate, and three fatty acids (myristic, plamitic, and beta-hydroxymyristic acids). However, the O-polysaccharides of OX2- and OXK-LPS had different chemical compositions which consisted of Glc, GlcN, and quinovosamine, and Glc, uronic acid, and GalN, respectively, while OX19-LPS seemed to lack O-polysaccharide.     

Involvement of oxidative DNA damage and apoptosis in antitumor actions of aminosugars.

We investigated the mechanisms of apoptosis and DNA damage induced by aminosugars in relation to their antitumor actions. The order of cytotoxic effects of aminosugars was D-mannosamine (ManN) > D-galactosamine (GalN) > D-glucosamine (GlcN). A comparison of the frequency of apoptotic cells showed the same order. DNA ladders were formed by only ManN and the formation of DNA ladders was inhibited by a caspase inhibitor. Pulsed-field gel electrophoresis showed that ManN caused cellular DNA cleavage at a lower concentration than those causing apoptosis. Cellular DNA cleavage was inhibited by catalase and enhanced by a catalase inhibitor. Flow cytometry showed that ManN enhanced the production of intracellular peroxides. These results suggest that ManN-induced apoptosis is preceded by H2O2-mediated DNA damage. The order of the extent of damage to 32P-labeled DNA fragments by aminosugars plus Cu(II) was ManN > GalN > GlcN. The DNA damage was inhibited by catalase and bathocuproine, suggesting that H2O2 reacts with Cu(I) to form the metal-peroxide complex capable of causing DNA damage. Two mechanisms of H2O2 generation from aminosugars were proposed: one is the major pathway to form a dioxo compound and NH4+; the other is the minor pathway to form a pyrazine derivative through the condensation of two molecules of an aminosugar. The order of reactivity to generate these products was ManN > GalN > GlcN. On the basis of these results, it is concluded that aminosugars, especially ManN, produce H2O2 to cause DNA damage, which mediates apoptosis resulting in tumor growth inhibition.     

Quantitative capillary electrophoresis determination of oversulfated chondroitin sulfate as a contaminant in heparin preparations

A simple, accurate, and robust quantitative capillary electrophoresis (CE) method for the determination of oversulfated chondroitin sulfate (OSCS) as a contaminant in heparin (Hep) preparations is described. After degradation of the polysaccharides by acidic hydrolysis, the hexosamines produced (i.e., GlcN from Hep and GalN from OSCS) were derivatized with anthranilic acid (AA) and separated by means of CE in approximately 10 min with high sensitivity detection at 214 nm (limit of detection [LOD] of ∼200 pg). Furthermore, AA-derivatized GlcN and GalN showed quite similar molar absorptivity, allowing direct and simple quantification of OSCS in Hep samples. Moreover, a preliminary step of specific enzymatic treatment by using chondroitin ABC lyase may be applied for the specific elimination of interference in the analysis due to the possible presence in Hep samples of natural chondroitin sulfate and dermatan sulfate impurities, making this analytical approach highly specific for OSCS contamination given that chondroitin ABC lyase is unable to act on this semisynthetic polymer. The CE method was validated for specificity, linearity, accuracy, precision, LOD, and limit of quantification (LOQ). Due to the very high sensitivity of CE, as little as 1% OSCS contaminant in Hep sample could be detected and quantified. Finally, a contaminated raw Hep sample was found to contain 38.9% OSCS, whereas a formulated contaminated Hep was calculated to have 39.7% OSCS.     

Influence of D-galactosamine on the synthesis of sugar nucleotides and glycoconjugates in rat hepatocytes

Rat hepatocytes were incubated in the presence of a high concentrationof the hepatopathogenic agent D-galactosamine (GalN), and theeffect on the cellular concentrations of pyrimidine nucleotidesand nucleotide sugars was determined. The UTP pool became depleted.The pools of UMP and CMP in RNA decreased to 72%, indicativefor an inhibition of RNA synthesis. UDP-HexNAc (where HexNAcis GlcNAc + GalNAc) and UDP-HexN (where HexN is GlcN + GalN)levels increased, and those of UDP-hexose and UDP-GlcA (whereGlcA is glucuronic acid) decreased. The cellular concentrationof CTP did not change, whereas that of CMP-NeuAc (where NeuAcis N-acetylneuraminic add) showed a 2-fold increase. Labellingwith [¹⁴C]orotic acid and [³H]cytidine showed that the metabolicflow via the de novo pathway was not changed. The depletionof the so-called overflow pool of UTP [Pels Rijcken et al, Biochem.J., 293, 207–213, 1993] caused a release of the feedbackinhibition by UTP and thus an increased flow through the salvagepathway. Finally, it appeared that GalN, when added to hepatocytes,gives rise to a pool of UDP-GlcNAc (where GlcNAc is N-acetylglueosamine)that is separate from the pool of UDP-GlcNAc that is derivedfrom GlcN. D-galactosamine glycosylation sugar nucleotide biosynthesis     

Anatomically determined polydispersity of proteoglycans of immature articular cartilage

Proteoglycans of the articulating and growing zones of minimum- and maximum-contact areas of calf articular cartilage were studied. Material was extracted sequentially (0.15 m sodium acetate, 2 m CaCl₂, and 4 m guanidinium chloride) in the presence of protease inhibitors. The very small proportion of material extracted by 0.15 m sodium acetate was poor in carbohydrates, but rich in serine, glycine, and glutamic acid, and had a K_av of 0.42 on Sepharose 2B. Proteoglycan extracted from the articulating zone was of smaller average hydrodynamic size (K_av of monomer, 0.42) than that from the growing zone (K_av of monomer, 0.32), but the attached chondroitin sulfate chains were of similar size. Proteoglycan prepared from the articulating area of minimum contact was chondroitin sulfate enriched (molar ratio of GalN:GlcN, 27) in comparison to that prepared from other regions of the articular cartilage (GalN:GlcN, 9–12). It is suggested that age-related maturation may be modified by physiological load or stress.     

Composition of soluble mineralizing matrices in zooxanthellate and non-zooxanthellate scleractinian corals: Biochemical assessment of photosynthetic metabolism through the study of a skeletal feature

Summary Soluble organic matrices extracted from aragonitic skeletons produced by recent zooxanthellate and non-zooxanthellate scleractinian corals were studied after suitable hydrolyses, by HPLC chromatographies allowing characterization of their amino acid and monasaccharide compositions. Clear compositional differences can be correlated with the symbiotic or non-symbiotic character in both proteic (via Asp, Glu, Ala and Ser) and glucidic phases of soluble matrices (via GalN, GlcN and Gal), providing new criteria to assess the impact of photosynthetic metabolism on skeletal features of scleractinian corals.     

Quantitative microbial indices in biogas and raw cattle slurries

Biogas slurry, the secondary product of the anaerobic digestion process, is increasingly being used as fertilizer. Information is available on its chemical and physical properties and their effects on plant growth. However, there is a demand to characterize the microbial quality of slurries, which may control further mineralization processes after application to soil. In this study, biogas and raw slurries obtained from six farms were analyzed for their ergosterol and amino sugar concentrations as indices for microbial biomass. A reliable, precise method for determining ergosterol in slurries is presented. Biogas slurries contained significantly less ergosterol (?34%), muramic acid (MurN; ?42%), galactosamine (GalN; ?32%), and fungal glucosamine (GlcN; ?40%) than raw slurries. The mean fungal GlcN to ergosterol ratio (50) and also the mean fungal carbon (C) to bacterial C ratio (0.29) did not significantly differ between the slurry types. The mean microbial C concentration in the biogas slurries was significantly lower than in the raw slurries. Consequently, the contribution of microbial C to slurry organic C was 3.6% in the biogas slurries and 5.7% in the raw slurries. Microbial C revealed significant nonlinear relationships with the fiber and ash concentration, pH, as well as the C/N ratio of the slurries.     

Uptake and incorporation of 14C-labelled carbohydrates in the tissues of the reproductive system of the female fowl

The in vivo incorporation of radioactivity from [14C]GlcN, [14C]GalN, [14C]Glc and [14C]Gal, for different time intervals between 1 and 240 hr into whole tissues, acetone extracted tissues and MPS-P of the different parts of the reproductive system of the female fowl was studied. The incorporation of radioactivity was much more extensive when [14C]GlcN was injected than when [14C]GalN was injected. The incorporation of radioactivity was much more extensive when [14C]HexN was injected than when the corresponding [14C]Hex was injected. This difference of incorporation was greater in the MPS-P than in the fresh or acetone extracted tissues. A comparison was undertaken in the extent that radioactivity was incorporated among the different parts of the reproductive system of the fowl when [14C]HexN and 14C[Hex] were administered.     

Capillary electrophoretic analysis of advanced glycation endproducts formed from the reaction of reducing sugars with the amino group of glucosamine

Glucosamine (GlcN) is an amino sugar sold over-the-counter and is widely used as a dietary supplement to relieve symptoms of osteoarthritis. It is not known whether it is the GlcN alone or one of its many possible nonenzymatic glycation products that is responsible for this effect. The current study demonstrates that reducing sugars form advanced glycation endproducts (AGEs) with GlcN and, as a result, decrease GlcN autocondensation by reducing the availability of the GlcN amino group. Capillary electrophoresis (CE) was used to analyze the in vitro Maillard reaction of GlcN with glyceraldehyde (GA), glucose (Glc), and fructose (Fru) as well as their inhibition of GlcN autocondensation under physiological conditions. Formation of AGEs was monitored by UV and fluorescence spectroscopy. Major components were separated by CE using a bare capillary and UV detection at 214 nm. AGE species were separated by HPLC and were complementary to the CE results. The effects of sugar concentration and incubation time on the AGE profile are also reported for each of the GlcN reducing sugar model systems. A simple and rapid CE method was developed to analyze the AGE formation in this initial report of the reaction of reducing sugars with the amino group of GlcN.     

Characterization of oligosaccharides of highly purified glycoprotein gC of herpes simplex virus type 1 (HSV-1)

The envelope membrane glycoprotein gC of HSV-1 was purified from Triton X-100 extracts of virus-infected BHK-21 or HEp-2 cells by a single step immuno-affinity column using monoclonal anti-gC antibody. The analysis of the purified [³H]G1cN labeled glycoprotein gC (by gel filtration on Bio-Gel P4) before and after digestion with endo-β-N-acetylglucosaminidase (endo D) indicated that gC contains Asn-linked “complex type” oligosaccharides. No “high mannose” type oligosaccharides were detected. Fractionation of radio-labeled glycopeptides of gC on a column of concanavalin A-sepharose suggested that glycopeptides have “diantennary” and “triantennary” and/or “tetra antennary” structures. Tunicamycin inhibited the incorporation of [¹⁴C]GalN or [³H]GlcN into gC in HSV-1 infected BHK-21 or HEp-2 cells. Gel filtration analysis of [³H]GlcN labeled gC following β-elimination reaction failed to indicate O-glycosidically linked oligosaccharides.     

A macrophage protein, Ym1, transiently expressed during inflammation is a novel mammalian lectin

Oral infections of mice with Trichinella spiralis induce activation of peritoneal exudate cells to transiently express and secrete a crystallizable protein Ym1. Purification of Ym1 to homogeneity was achieved. It is a single chain polypeptide (45 kDa) with a strong tendency to crystallize at its isoelectric point (pI 5.7). Co-expression of Ym1 with Mac-1 and scavenger receptor pinpoints macrophages as its main producer. Protein microsequencing data provide information required for full-length cDNA cloning from libraries constructed from activated peritoneal exudate cells. A single open reading frame of 398 amino acids with a leader peptide (21 residues) typical of secretory protein was deduced and later deposited in GenBank (accession number M94584) in 1992. By means of surface plasmon resonance analyses, Ym1 has been shown to exhibit binding specificity to saccharides with a free amine group, such as GlcN, GalN, or GlcN polymers, but it failed to bind to other saccharides. The interaction is pH-dependent but Ca2+ and Mg2+ ion-independent. The binding avidity of Ym1 to GlcN oligosaccharides was enhanced by more than 1000-fold due to the clustering effect. Specific binding of Ym1 to heparin suggests that heparin/heparan sulfate may be its physiological ligand in vivo during inflammation and/or tissue remodeling. Although it shares approximately 30% homology with microbial chitinases, no chitinase activity was found associated with Ym1. Genomic Southern blot analyses suggest that Ym1 may represent a member of a novel lectin gene family.     

Biosynthesis of a novel polysaccharide by Acetobacter xylinum

An Acetobacter xylinum adapted to a medium containing N-acetylglucosamine (GlcNAc) has been used to prepare a novel polysaccharide containing residual GlcNAc in cellulose. The maximum amount of incorporation was found to be 4 mol% in cellulose, when a mixed medium containing 1.4% glucose (Glc) and 0.6% GlcNAc was used for the culture of A. xylinum. The resulting polysaccharide was lysozyme-susceptible. The aminosugar residue incorporated into bacterial cellulose was found to be only GlcNAc, even if galactosamine (GalN) and glucosamine (GlcN) were applied, whereas there was little effect by mannosamine (ManN). As the major component of the resulting polysaccharide was Glc residues, even if the only carbon source in the culture medium was GlcNAc, it was suggested that there must be several enzyme systems to convert GlcNAc into Glc in the bacteria. Several ammonium salts were also found to be effective for the incorporation of GlcNAc residues when the incubation system was converted to rotatory and aerobic incubation from static incubation. The amount of residual GlcNAc was remarkably increased by the addition of lysozyme-susceptible phosphoryl-chitin (P-chitin) and increased slightly with addition of P-chitin that was less lysozyme-susceptible. However, little effect was found on addition of highly substituted P-chitin.     

Enzymatic synthesis of UDP-GlcN by a two step hollow fiber enzyme reactor system

UDP-GlcN was synthesized from GlcN and UTP by a two step hollow fiber enzyme reactor method. In step 1, GlcN was converted to GlcN 6-P and then to GlcN 1-P by hexokinase and phosphoglucomutase, respectively, and UTP was used as the phosphate donor. In step 2, GlcN 1-P was converted to UDP-GlcN by UDP glucose pyrophosphorylase. All the enzymes required for the synthesis of UDP-GlcN were enclosed in hollow fiber bundles which allow for the free diffusion of substrates and products across the membranes to and from the enzymes, allow for the reutilization of the enzymes, and simplify the isolation of the product, UDP-GlcN. We show that both UTP and GlcN 6-P are inhibitors of the yeast UDPG pyrophosphorylase and therefore their concentrations must be regulated to obtain maximum yields of UDP-GlcN. The UDP-GlcN produced can be N-acetylated with [14C]acetic anhydride to produce UDP-[14C]GlcNAc. This method can also be used to synthesize [32P]UDP-GlcN and [32P]UDP-GlcNAc from [alpha-32P]UTP and GlcN 1-P.     

Monosaccharide compositions of Danaus plexippus (monarch butterfly) and Trichoplusia ni (cabbage looper) egg glycoproteins

Monosaccharide compositions of eggs from Danaus plexippus (monarch butterfly) and Trichoplusia ni (cabbage looper) were analyzed. Analyses were performed mainly with high performance anion exchange chromatography (HPAEC) using crude extracts of eggs or SDS-PAGE separated and PVDF-blotted protein bands. Man and GlcN were the major components in all cases, but low levels of Gal and Fuc were possibly present in some samples. Some T. ni egg glycoproteins even contained GalN. Although a peak comigrating with Neu5Ac could be detected with HPAEC-PAD or RP-HPLC (fluorometry) after derivatization with 1,2-diamino-4,5-methylenedioxy-benzene, the quantities were too small to be significant as an integral part of the analyzed glycoproteins. These data suggests that most of glycans on the glycoproteins are pauci-Man type N-glycans, but a small portion of N-glycan may be either hybrid type or complex type.     

Glucosamine causes overproduction of reactive oxygen species, leading to repression of hypocotyl elongation through a hexokinase-mediated mechanism in Arabidopsis

Glucosamine (GlcN) is a naturally occurring amino-sugar that is synthesized by amidation of fructose-6-phosphate. Although a number of reports have examined the biological effects of GlcN on insulin resistance in mammalian systems, little is known about its effects on plant growth. In this study, we have shown that exogenous GlcN inhibits hypocotyl elongation in Arabidopsis, whereas glucose and its analogs alleviate this inhibitory effect. The hexokinase (HXK)-specific inhibitor mannoheptulose also restored hypocotyl elongation. The gin2-1 mutants with an alteration in AtHXK1 exhibited higher tolerance to GlcN. We also found that GlcN induces a significant increase in the production of reactive oxygen species (ROS). In addition, the GlcN-mediated inhibition of hypocotyl elongation was relieved by reducing agents such as ascorbic acid and glutathione. GlcN treatment resulted in significant induction of expression of GST1, GST2 and GST6, which are marker genes for ROS production. The gin2 mutation also represses the ROS production and the GST2 induction by GlcN treatment. Taken together, these results provide evidence that GlcN induces HXK-mediated induction of oxidative stress, leading to growth repression in Arabidopsis thaliana.     

Action pattern of Bacillus sp. no. 7-M chitosanase on partially N-acetylated chitosan.

The hydrolyzate of partially N-acetylated chitosan by Bacillus sp. No. 7-M chitosanase was separated by gel filtration on Bio-Gel P-2. Sugar compositions and sequences of the oligosaccharides were identified by exo-splitting with beta-GlcNase, fast atom bombardment mass spectroscopy, and proton NMR spectroscopy. In addition to chitooligosaccharides, (GlcN)2, (GlcN)3, and (GlcN)4, hetero-chitooligosaccharides such as (GlcN)2.GlcNAc.(GlcN)2, GlcN.GlcNAc.(GlcN)3, (GlcN)2.GlcNAc.(GlcN)3, and GlcN.GlcNAc.(GlcN)4 were detected. These results indicate that Bacillus sp. No. 7-M chitosanase is absolutely specific toward the GlcN.GlcN bonds in partially N-acetylated chitosan and at least three GlcN residues were necessary to the hydrolysis of chitosan by chitosanase.     

Characterization of a novel glucosamine-6-phosphate deaminase from a hyperthermophilic archaeon

A key step in amino sugar metabolism is the interconversion between fructose-6-phosphate (Fru6P) and glucosamine-6-phosphate (GlcN6P). This conversion is catalyzed in the catabolic and anabolic directions by GlcN6P deaminase and GlcN6P synthase, respectively, two enzymes that show no relationship with one another in terms of primary structure. In this study, we examined the catalytic properties and regulatory features of the glmD gene product (GlmD(Tk)) present within a chitin degradation gene cluster in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Although the protein GlmD(Tk) was predicted as a probable sugar isomerase related to the C-terminal sugar isomerase domain of GlcN6P synthase, the recombinant GlmD(Tk) clearly exhibited GlcN6P deaminase activity, generating Fru6P and ammonia from GlcN6P. This enzyme also catalyzed the reverse reaction, the ammonia-dependent amination/isomerization of Fru6P to GlcN6P, whereas no GlcN6P synthase activity dependent on glutamine was observed. Kinetic analyses clarified the preference of this enzyme for the deaminase reaction rather than the reverse one, consistent with the catabolic function of GlmD(Tk). In T. kodakaraensis cells, glmD(Tk) was polycistronically transcribed together with upstream genes encoding an ABC transporter and a downstream exo-beta-glucosaminidase gene (glmA(Tk)) within the gene cluster, and their expression was induced by the chitin degradation intermediate, diacetylchitobiose. The results presented here indicate that GlmD(Tk) is actually a GlcN6P deaminase functioning in the entry of chitin-derived monosaccharides to glycolysis in this hyperthermophile. This enzyme is the first example of an archaeal GlcN6P deaminase and is a structurally novel type distinct from any previously known GlcN6P deaminase.