Bio-Gel P-100 Column Research Articles

DEVELOPMENT OF ENTEROBACTER CLOACAE IP8 MUTANT STRAIN PRODUCING THERMOSTABLE CELLULASE

Mutagenesis of Enterobacter cloacae IP8 for enhanced cellulase production was carried out using ultraviolet (UV) irradiation and ethyl methanesulphonate (EMS) treatment. The mutant strain exhibited cellulolytic activity which was 2.18-fold higher than that of the wildtype strain. The optimal conditions for cellulase production were an incubation period of 28 h, a temperature of 45 oC, and pH 7.0, using CMC and peptone as carbon and nitrogen sources, respectively. The cellulases from both strains were purified by using ammonium sulfate precipitation, CM Sephadex C-50, and Biogel P-100 column chromatography. The specific activity of the purified cellulase from the mutant strain was 29.47 U/mg while that of wildtype cellulase was 21.5 U/mg. Biochemical characterization of the purified enzyme revealed the optimum pH and temperature of 8.0 and 65 oC, respectively, for the cellulase from the mutant strain, and 7.0 and 60 oC for the wild-type cellulase. The mutant cellulase was thermally stable up to 70 oC retaining 86.5% of its original activity after 180 h. Metal ions Na+ and Ca2+ remarkably enhanced the activity of the cellulase from both strains while Al3+ and the chelating agent, EDTA, strongly inhibited the activity. Mutagenesis of E. cloacae IP8 using combined UV and EMS treatment led to the development of mutant strain with enhanced capacity for the production of cellulase exhibiting novel properties such as thermostability, alkalinity, low Km, and high Vmax values. Therefore, the enzyme from the mutant strain of E. cloacae IP8 has the potential for broad industrial applications.

Combinatorial Enzyme Approach to Convert Wheat Insoluble Arabinoxylan to Bioactive Oligosaccharides

Combinatorial enzyme technology was applied for the conversion of wheat insoluble arabinoxylan to oligosaccharide structural variants. The digestive products were fractionated by Bio-Gel P4 column and screened for bioactivity. One fraction pool was observed to exhibit antimicrobial property resulting in the suppression of cell growth of the test organism ATCC 8739 E. coli. It has a MIC value of 1.5% (w/v, 35°C, 20 hr) and could be useful as a new source of prebiotics or preservatives. The present results further confirm the science and useful application of combinatorial enzyme approach.

English

The stomach lysozymes of ruminants play a paramount role in digestion and offer the chance to probe evolutionary changes in complex organisms on a biochemical basis. In this paper, we focus on the characterization of the catalytic properties of Tibetan ruminant (TR) stomach lysozymes and provide a comparative study of TR stomach lysozymes and nonplateau ruminant stomach lysozymes. The stomach lysozymes were purified with a Carboxymethyl (CM) Sepharose Fast Flow (FF) column and a Bio-Gel P-100 column. The purified stomach lysozymes were characterized by sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) and matrix-assisted laser desorption/ionization time-of-flight (MALDITOF) analyses. The antimicrobial activity was evaluated using an agar diffusion method, and the pH, ion strength, temperature, pepsin and trypsin effects on enzymatic activities were evaluated by normal biochemistry methods. The stomach lysozymes of TRs (cattle:TC, yak:TY, and sheep:TS) exhibit maximum activity at pH 5 and ionic strengths below 0.02. TC and TS stomach lysozymes are more resistant to higher temperatures than nonplateau lysozymes. Stomach lysozymes of TRs exhibit greater enzymatic activity than nonplateau lysozymes, in the presence of pepsin and trypsin. Stomach lysozymes from TS retain 86% of their initial enzymatic activity against pepsin inactivation. After trypsin treatment, the stomach lysozymal activities of TRs were significantly affected by trypsin inactivation, with the exception of TS, whose enzymatic activities in the presence of trypsin were 5- and 10-fold higher than those of nonplateau lysozymes. The stomach lysozymes of TRs are more resistant to environmental factors such as pH, temperature, pepsin, and trypsin than nonplateau lysozyme C.

An efficient protocol for the preparation of linear arabino-oligosaccharides

In this study, we report the development of an efficient protocol in the preparation of linear arabino-oligosaccharides derived from sugar beet arabinan. By optimizing hydrolytic conditions and separation on tandem Bio-Gel P4 columns, we obtained arabino-oligosaccharides with various degrees of polymerization (DP) from 2 to 15. All of these α-1,5-linked arabino-oligosaccharides are highly pure (>95%) as determined by HPAEC, MALDI-TOF mass spectrometry and 13C NMR spectroscopy. Due to their purity, these oligosaccharides can be used as standards to identify other oligosaccharides and as substrates to characterize new arabinan-specific enzymes, as well as for the development of new functional oligosaccharides.

Production and purification of mannan oligosaccharide with epithelial tight junction enhancing activity.

BackgroundMannanan oligosaccharide (MOS) is well-known as effective supplement food for livestock to increase their nutrients absorption and health status, but the structure and identification of bioactive MOS remain unclear. In this study, MOS production was accomplished, using enzymatic hydrolysis of pretreated coconut meal substrate with recombinant mannanase.MethodsThe mannanase gene was cloned from Bacillus subtilis cAE24, then expressed in BL21. Purified Mannanase exhibit stability over a wide range of pH and temperature from pH 6–8 and 4 °C to 70 °C, respectively. SEM analysis revealed that sonication could change the surface characteristic of copra meal, which gave better MOS yield, compared to untreated substrates. The separation and purification of each MOS were achieved using Biogel-P2 column chromatography. Determination of biological active MOS species was also investigated. T84 cells were cultured and treated with each of the purified MOS species to determine their tight junction enhancing activity.ResultsScanning electron microscope imaging showed that pretreatment using sonication could disrupt the surface of copra meal better than grinding alone, which can improve the production of MOS. Pentamer of MOS (M5) significantly increased tight junction integration of T84 cells measured with TEER (p < 0.0001).

Simple Preparation of Diverse Neoagaro-Oligosaccharides

A simple method for obtaining pure and well-defined oligosaccharides was established by hydrolyzing agar with β-agarase from Vibrio natriegens. The conditions for enzymolysis were optimized as follows: a temperature of 45 °C, a pH of 8.5, a substrate concentration of 0.3%, an enzyme amount of 100 U/g and an enzymolysis time of 20 h. Neoagaro-oligosaccharides with different degrees of polymerization were obtained by hydrolyzing agar with β-agarase for different lengths of time. After removing pigments using activated carbon and salts by dialyzing, the enzyme hydrolysis solution was separated with Bio-Gel P2 column chromatography. Neoagaro-oligosaccharides with different degrees of polymerization were acquired. By comparing with authentic standard substances, along with further confirmation by FTIR, MS and NMR, structures of the purified neoagaro-oligosaccharides were identified as neoagarobiose (NA2), neoagaroteraose (NA4), neoagarohexaose (NA6), neoagarooctaose (NA8), neoagaro-decaose (NA10) and neoagarododecaose (NA12) with purities of more than 97.0%. The present study established a method for the preparation of various neoagaro-oligosaccharides that may be of great significance for further study of their bioactivities.

Structural and Functional Study of d-Glucuronyl C5-epimerase

Heparan sulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. D-glucuronyl C5-epimerase (Glce) is a crucial enzyme in HS synthesis, converting D-glucuronic acid to L-iduronic acid to increase HS flexibility. This modification of HS is important for protein ligand recognition. We have determined the crystal structures of Glce in apo-form (unliganded) and in complex with heparin hexasaccharide (product of Glce following O-sulfation), both in a stable dimer conformation. A Glce dimer contains two catalytic sites, each at a positively charged cleft in C-terminal α-helical domains binding one negatively charged hexasaccharide. Based on the structural and mutagenesis studies, three tyrosine residues, Tyr(468), Tyr(528), and Tyr(546), in the active site were found to be crucial for the enzymatic activity. The complex structure also reveals the mechanism of product inhibition (i.e. 2-O- and 6-O-sulfation of HS keeps the C5 carbon of L-iduronic acid away from the active-site tyrosine residues). Our structural and functional data advance understanding of the key modification in HS biosynthesis.

Preparation and purification of raffinose family oligosaccharides from Rehmannia glutinosa Libosch. by fast protein liquid chromatography coupled with refractive index detection

Raffinose family oligosaccharides (RFOs) were extracted and isolated from Rehmannia glutinosa Libosch. by using microwave-assisted extraction and fast protein liquid chromatography coupled with refractive index detection (FPLC-RID). After extraction with 60% ethanol (v/v) and decolorization with MCI gel CHP20P, total oligosaccharides were purified by using FPLC-RID coupled with the Bio-Gel P2 column eluted with water at the flow rate of 0.3mL/min. Three pure oligosaccharides (DP3–DP5) were obtained with the yields of 1.72%, 7.52% and 0.51%, respectively, as well as identified as raffinose, stachyose and verbascose from the results of liquid chromatography coupled with tandem mass spectroscopy (LC–MS/MS) and gas chromatography coupled with mass spectroscopy (GC–MS) analysis. A large-scale of pure RFOs was automatically and efficiently separated from R. glutinosa by using FPLC-RID method, which was beneficial for the qualitative and quantitative analysis of RFOs in R. glutinosa and their products.

Detoxification of cyanide in insects. I. Purification and some properties of rhodanese from the gut of the variegated grasshopper Zonocerus variegatus (Orthoptera: Pyrgomorphidae)

The purification and characterization of rhodanese, an enzyme that catalyses the detoxification of cyanide, from the gut of the variegated grasshopper ( Zonocerus variegatus L.) were carried out to understand the biochemical basis of the survival of this grasshopper living on cyanogenic plants such as cassava. All experiments, including enzyme assay, were carried out at room temperature and all buffers contained 10 m m sodium thiosulphate to stabilize the enzyme. Grasshoppers were caught alive from a cassava farm within the locality and kept frozen until analysis. Each grasshopper was dissected and the gut was removed quickly. Approximately 102 g of the gut were homogenized in three volumes of 0.1 m acetate glycine buffer (pH 7.8) containing ɛ-amino- n -caproic acid. The supernatant was collected by centrifugation at 12,000 rpm, for 30 min at 40 °C. The enzyme was purified to homogeneity by a combination of procedures such as ammonium sulphate precipitation, ion-exchange chromatography (CM-Sephadex and DEAE-Sephadex), gel filtration (Sephadex G-75) and Agarose-Blue affinity chromatography. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE) and non-SDS–PAGE were used to ascertain the purity of the enzyme. The native and subunit molecular weights of the enzyme were determined by gel filtration on a Bio-Gel P-200 column and SDS–PAGE, respectively. Kinetic parameters were determined by using varying concentrations of one of the substrates at a fixed concentration of the other, and vice versa. Furthermore, the effects of temperature, pH and cations on the activity of the enzyme were investigated. The purified enzyme had a specific activity of 51.7 μmol thiocyanate formed/ml/min/mg protein (U/mg protein) with a yield of about 29%. The apparent molecular weight of the enzyme estimated by Sephadex G-75 gel filtration was 35,400 ± 482 Da and its subunit molecular weight determined by SDS–PAGE was 33,000 ± 212 Da. The K m values of KCN and Na 2 S 2 O 3 were found to be 29.63 ± 02.87 and 26 ± 03.04 m m , respectively. An optimum pH and temperature of 7.0 and 35 °C, respectively, were obtained for the enzyme. The results of enzyme inhibition showed that the activity of the enzyme was not affected by NH 4 Cl, MgCl 2 , CoCl 2 , CaCl 2 , MnCl 2 , NiCl 2 and SnCl 2 , but inhibited by ZnCl 2 and BaCl 2 . In conclusion, these results suggest that the survival of Z. variegatus depends on the presence of the enzyme rhodanese, which shows high activity and has suitable kinetic properties in the gut of the grasshopper that feeds mainly on cassava leaves which are cyanogenic.

Functional properties of a manganese-activated exo-polygalacturonase produced by a thermotolerant fungus Aspergillus niveus

A thermotolerant fungus identified as Aspergillus niveus was isolated from decomposing materials and it has produced excellent levels of hydrolytic enzymes that degrade plant cell walls. A. niveus germinated faster at 40°C, presenting protein levels almost twofold higher than at 25°C. The crude extract of the A. niveus culture was purified by diethylaminoethyl (DEAE)-cellulose, followed by Biogel P-100 column. Polygalacturonase (PG) is a glycoprotein with 37.7% carbohydrate, molecular mass of 102.6kDa, and isoelectric point of 5.4. The optimum temperature and pH were 50°C and 4.0-6.5, respectively. The enzyme was stable at pH3.0 to 9.0 for 24h. The DEAE-cellulose derivative was about sixfold more stable at 60°C than the free enzyme. Moreover, the monoaminoethyl-N-aminoethyl-agarose derivative was tenfold more stable than the free enzyme. PG was 232% activated by Mn(2+). The hydrolysis product of sodium polypectate corresponded at monogalacturonic acid, which classifies the enzyme as an exo-PG. The K m, V max, K cat, and K cat/K m values were 6.7mg/ml, 230U/mg, 393.3/s, and 58.7mg/ml/s, respectively. The N-terminal amino acid sequence presented 80% identity with PglB1, PglA2, and PglA3 putative exo-PG of Aspergillus fumigatus and an exo-PG Neosartorya fischeri.

Identical RNA-Protein Interactions in Vivo and in Vitro and a Scheme of Folding the Newly Synthesized Proteins by Ribosomes

A distinct three-dimensional shape of rRNA inside the ribosome is required for the peptidyl transfer activity of its peptidyltransferase center (PTC). In contrast, even the in vitro transcribed PTC RNA interacts with unfolded protein(s) at about five sites to let them attain their native states. We found that the same set of conserved nucleotides in the PTC interact identically with nascent and chemically unfolded proteins in vivo and in vitro, respectively. The time course of this interaction, difficult to follow in vivo, was observed in vitro. It suggested nucleation of folding of cytosolic globular proteins vectorially from hydrophilic N to hydrophobic C termini, consistent with our discovery of a regular arrangement of cumulative hydrophobic indices of the peptide segments of cytosolic proteins from N to C termini. Based on this observation, we propose a model here for the nucleation of folding of the nascent protein chain by the PTC.

Comparative Study of Thiaminase in Headfoot and Hepatopancreas of Limicolaria flammea (Müller, 1774)

Aims: This study aimed at determining the level of thiaminase in the Limicolaria flammea with a view to ascertaining the nutritional and health implications of its consumption. Methodology: Proximate analyses of two tissues, headfoot and hepatopancreas, were investigated to assess their nutritional qualities. The thiaminases in the tissues were purified by chromatographic separations using DEAE-Sephacel ion - exchange and Biogel P-100 columns. Results: The Michealis Menten constant obtained for hepatopancreas thiaminase was 0.83 mM and 0.13 mM for thiamine and aniline respectively; for the headfoot thiaminase, 1.06 mM and 0.16 mM was obtained for thiamine and aniline respectively. An optimum pH of 8.5 was obtained for thiaminase in the two tissues. Temperature optimum of 65°C and 70°C was obtained for the headfoot and hepatopancreas enzymes respectively. The amino acids and glutathione activated the enzyme from hepatopancreas, while the headfoot enzyme was significantly inhibited except proline which showed very high activation. The cations: NH 4 + , Hg

Molecular identification of a polyM-specific alginate lyase from Pseudomonas sp. strain KS-408 for degradation of glycosidic linkages between two mannuronates or mannuronate and guluronate in alginate

An alginate lyase gene of a newly isolated Pseudomonas sp. strain KS-408 was cloned by using PCR with the specific primers designed from homologous nucleotide sequences. A partial protein sequence of KS-408 alginate lyase was homology-modeled on the basis of the crystal structure of A1-III alginate lyase from Sphingomonas sp. strain A1. The proposed 3-D structure of KS-408 alginate lyase shows that Asn-198, His-199, Arg-246, and Tyr-253 residues are conserved for the catalytic active site. The recombinant KS-408-1F (with signal peptide) and KS-408-2F (without signal peptide) alginate lyases with the (His)(6) tag consist of 393 (44.5 kDa) and 372 (42.4 kDa) amino acids with isoelectric points of 8.64 and 8.46, respectively. The purified recombinant KS-408 alginate lyase was very stable when it was incubated at 40 °C for 30 min. Alginate oligosaccharides produced by the KS-408-2F alginate lyase were purified on a Bio-Gel P2 column and analyzed by thin-layer chromatography, fast-protein liquid chromatography, and electrospray ionization mass spectrometry. (1)H NMR data showed that the KS-408-2F alginate lyase cleaved the glycosidic linkages between two mannuronates (mannuronate-β(1-4)-mannuronate) or mannuronate and guluronate (mannuronate-β(1-4)-guluronate), indicating that the KS-408 alginate lyase is a polyM-specific lyase.

Molecular Identification of β-Citrylglutamate Hydrolase as Glutamate Carboxypeptidase 3

β-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca(2+), the purified enzyme specifically hydrolyzed β-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn(2+). This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of β-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed β-citrylglutamate in the presence of Ca(2+), and acted on both N-acetyl-aspartylglutamate and β-citrylglutamate in the presence of Mn(2+), whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze β-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its β-citrylglutamate hydrolase activity.

Decolorization of the Textile Dyes Using Purified Banana Pulp Polyphenol Oxidase

Polyphenol oxidase (PPO) purified using DEAE-cellulose and Biogel P-100 column chromatography from banana pulp showed 12.72-fold activity and 2.49% yield. The optimum temperature and pH were found to be 30°C and 7.0, respectively for its activity. Catechol was found to be a suitable substrate for banana pulp PPO that showed Vmax, 0.041 mM min−1 and Km, 1.6 mM. The enzyme activity was inhibited by sodium metabisulfite, citric acid, cysteine, and β-mercaptoethanol at 10 mM concentration. The purified enzyme could decolorize (90%) Direct Red 5B (160 μg mL−1) dye within 48 h and Direct Blue GLL (400 μg mL−1) dye up to 85% within 90 h. The GC-MS analysis indicated the presence of 4-hydroxy-benzenesulfonic acid and Naphthalene-1,2,3,6-tetraol in the degradation products of Direct Red 5B, and 5-(4-Diazenyl-naphthalene-1-ylazo)-8-hydroxy-naphthalene-2-sulfonic acid and 2-(4-Diazenyl-naphthalene-1-ylazo)-benzenesulfonic acid in the degradation products of Direct Blue GLL.

Molecular Identification of N-Acetylaspartylglutamate Synthase and β-Citrylglutamate Synthase

The purpose of the present work was to determine the identity of the enzymes that synthesize N-acetylaspartylglutamate (NAAG), the most abundant dipeptide present in vertebrate central nervous system (CNS), and β-citrylglutamate, a structural analogue of NAAG present in testis and immature brain. Previous evidence suggests that NAAG is not synthesized on ribosomes but presumably is synthesized by a ligase. As attempts to detect this ligase in brain extracts failed, we searched the mammalian genomes for putative enzymes that could catalyze this type of reaction. Mammalian genomes were found to encode two putative ligases homologous to Escherichia coli RIMK, which ligates glutamates to the C terminus of ribosomal protein S6. One of them, named RIMKLA, is almost exclusively expressed in the CNS, whereas RIMKLB, which shares 65% sequence identity with RIMKLA, is expressed in CNS and testis. Both proteins were expressed in bacteria or HEK293T cells and purified. RIMKLA catalyzed the ATP-dependent synthesis of N-acetylaspartylglutamate from N-acetylaspartate and l-glutamate. RIMKLB catalyzed this reaction as well as the synthesis of β-citrylglutamate. The nature of the reaction products was confirmed by mass spectrometry and NMR. RIMKLA was shown to produce stoichiometric amounts of NAAG and ADP, in agreement with its belonging to the ATP-grasp family of ligases. The molecular identification of these two enzymes will facilitate progress in the understanding of the function of NAAG and β-citrylglutamate.

Synthesis of Le aLe x oligosaccharide fragments and efficient one-step deprotection

We describe here the synthesis of two oligosaccharide fragments of the tumor associated carbohydrate antigen Le aLe x. While the linear lacto- N-triose I: β- d-Gal p-(1→4)-β- d-GlcNAc p-(1→3)-β- d-Gal p-OMe is a known compound, this is the first reported preparation of the branched tetrasaccharide β- d-GlcNAc p-(1→3)-β- d-Gal p-(1→4)-[α- l-Fuc p-(1→3)]-β- d-GlcNAc p-OMe. Our synthetic schemes involved using an N-trichloroacetylated trichloroacetimidate glucosaminyl donor activated with excess TMSOTf at 0 °C for glycosylation at O-3 of galactosyl residues and that of trichloroacetimidate galactosyl donors activated with excess BF 3·OEt 2 to glycosylate either O-3 or O-4 of glucosamine residues. The fucosylation at O-3 of the glucosamine acceptor was accomplished using a thiofucoside donor activated with copper(II) bromide and tetrabutylammonium bromide. Thus, syntheses of the protected tri- and tetrasaccharides were achieved easily and efficiently using known building blocks. Of particular interest, we also report that these protected oligosaccharides were submitted to dissolving metal conditions (Na–NH 3) to provide in one single step the corresponding deprotected compounds. Under these conditions all protecting groups ( O-acyl, benzylidene, benzyl, and N-trichloroacetyl) were efficiently cleaved. The work-up procedure for such reactions usually involves quenching with excess methanol and then neutralization with acetic acid. In our work the neutralization was carried out using acetic anhydride rather than acetic acid to ensure N-acetylation of the glucosamine residue. Both fully deprotected compounds were then simply purified and desalted by gel permeation chromatography on a Biogel P2 column eluted with water.

Activity, Splice Variants, Conserved Peptide Motifs, and Phylogeny of Two New α1,3-Fucosyltransferase Families (FUT10 and FUT11)

We report the cloning of three splice variants of the FUT10 gene, encoding for active alpha-l-fucosyltransferase-isoforms of 391, 419, and 479 amino acids, and two splice variants of the FUT11 gene, encoding for two related alpha-l-fucosyltransferases of 476 and 492 amino acids. The FUT10 and FUT11 appeared 830 million years ago, whereas the other alpha1,3-fucosyltransferases emerged 450 million years ago. FUT10-391 and FUT10-419 were expressed in human embryos, whereas FUT10-479 was cloned from adult brain and was not found in embryos. Recombinant FUT10-419 and FUT10-479 have a type II trans-membrane topology and are retained in the endoplasmic reticulum (ER) by a membrane retention signal at their NH(2) termini. The FUT10-479 has, in addition, a COOH-ER membrane retention signal. The FUT10-391 is a soluble protein without a trans-membrane domain or ER retention signal that transiently localizes to the Golgi and then is routed to the lysosome. After transfection in COS7 cells, the three FUT10s and at least one FUT11, link alpha-l-fucose onto conalbumin glycopeptides and biantennary N-glycan acceptors but not onto short lactosaminyl acceptor substrates as do classical monoexonic alpha1,3-fucosyltransferases. Modifications of the innermost core GlcNAc of the N-glycan, by substitution with ManNAc or with an opened GlcNAc ring or by the addition of an alpha1,6-fucose, suggest that the FUT10 transfer is performed on the innermost GlcNAc of the core chitobiose. We can exclude alpha1,3-fucosylation of the two peripheral GlcNAcs linked to the trimannosyl core of the acceptor, because the FUT10 fucosylated biantennary N-glycan product loses both terminal GlcNAc residues after digestion with human placenta alpha-N-acetylglucosaminidase.

Identification of Mycobacterium tuberculosis Clinical Isolates with Altered Phagocytosis by Human Macrophages Due to a Truncated Lipoarabinomannan

Phenotypically distinct clinical isolates of Mycobacterium tuberculosis are capable of altering the balance that exists between the pathogen and human host and ultimately the outcome of infection. This study has identified two M. tuberculosis strains (i.e. HN885 and HN1554) among a bank of clinical isolates with a striking defect in phagocytosis by primary human macrophages when compared with strain Erdman, a commonly used laboratory strain for studies of pathogenesis. Mass spectrometry in conjunction with NMR studies unequivocally confirmed that both HN885 and HN1554 contain truncated and more branched forms of mannose-capped lipoarabinomannan (ManLAM) with a marked reduction of their linear arabinan (corresponding mainly to the inner Araf-alpha(1-->5)-Araf unit) and mannan (with fewer 6-Manp residues and more substitutions in the linear Manp-alpha(1-->6)-Manp unit) domains. The truncation in the ManLAM molecules produced by strains HN885 and HN1554 led to a significant reduction in their surface availability. In addition, there was a marked reduction of higher order phosphatidyl-myo-inositol mannosides and the presence of dimycocerosates, triglycerides, and phenolic glycolipid in their cell envelope. Less exposed ManLAM and reduced higher order phosphatidyl-myo-inositol mannosides in strains HN885 and HN1554 resulted in their low association with the macrophage mannose receptor. Despite reduced phagocytosis, ingested bacilli replicated at a fast rate following serum opsonization. Our results provide evidence that the clinical spectrum of tuberculosis may be dictated not only by the host but also by the amounts and ratios of surface exposed mycobacterial adherence factors defined by strain genotype.

Acceptor Specificity of the Pasteurella Hyaluronan and Chondroitin Synthases and Production of Chimeric Glycosaminoglycans

The hyaluronan (HA) synthase, PmHAS, and the chondroitin synthase, PmCS, from the Gram-negative bacterium Pasteurella multocida polymerize the glycosaminoglycan (GAG) sugar chains HA or chondroitin, respectively. The recombinant Escherichia coli-derived enzymes were shown previously to elongate exogenously supplied oligosaccharides of their cognate GAG (e.g. HA elongated by PmHAS). Here we show that oligosaccharides and polysaccharides of certain noncognate GAGs (including sulfated and iduronic acid-containing forms) are elongated by PmHAS (e.g. chondroitin elongated by PmHAS) or PmCS. Various acceptors were tested in assays where the synthase extended the molecule with either a single monosaccharide or a long chain (approximately 10(2-4) sugars). Certain GAGs were very poor acceptors in comparison to the cognate molecules, but elongated products were detected nonetheless. Overall, these findings suggest that for the interaction between the acceptor and the enzyme (a) the orientation of the hydroxyl at the C-4 position of the hexosamine is not critical, (b) the conformation of C-5 of the hexuronic acid (glucuronic versus iduronic) is not crucial, and (c) additional negative sulfate groups are well tolerated in certain cases, such as on C-6 of the hexosamine, but others, including C-4 sulfates, were not or were poorly tolerated. In vivo, the bacterial enzymes only process unsulfated polymers; thus it is not expected that the PmCS and PmHAS catalysts would exhibit such relative relaxed sugar specificity by acting on a variety of animal-derived sulfated or epimerized GAGs. However, this feature allows the chemoenzymatic synthesis of a variety of chimeric GAG polymers, including mimics of proteoglycan complexes.