Production of N-acetyl-D-glucosamine from alpha-chitin by crude enzymes from Aeromonas hydrophila H-2330

The selective and efficient production of N-acetyl-D-glucosamine (GlcNAc) was achieved from flake type of alpha-chitin by using crude enzymes derived from Aeromonas hydrophila H-2330.     

Long - lasting synchrony of the division of enteric bacteria

Recent finding of α-N-acetylglucosamine(1)phospho(6)mannose diesters in lysosomal enzymes suggested that formation of mannose 6-phosphate residues involves transfer of N-acetylglucosamine 1-phosphate to mannose. Using dephosphorylated β-hexosaminidase as acceptor and [β-³²P]UDP-N-acetylglucosamine as donor for the phosphate group, phosphorylation of β-hexosaminidase by microsomes from rat liver, human placenta and human skin fibroblasts was achieved. The reaction was not affected by tunicamycin. Acid hydrolysis released mannose 6-[³²P]phosphate from the phosphorylated β-hexosaminidase. Our results suggest that lysosomal enzymes are phosphorylated by transfer of N-acetylglucosamine 1-phosphate from UDP-N-acetylglucosamine. The transferase activity was deficient in fibroblasts from patients affected with l-cell disease. This deficiency is proposed to be the primary enzyme defect in l-cell disease.     

Structures of the N-linked carbohydrate of ascorbic acid oxidase from zucchini

N-glycan moiety of ascorbic acid oxidase from zucchini (Cucurbita pepo) has been described to be a core-pentasaccharide with a xylose [D'Andrea et al. (1988) Glycoconjugate J 5:151-7]. Ascorbic acid oxidase is sometimes used to characterize antibodies directed against carbohydrate determinants on plant glycoproteins. To prevent misinterpretations of immunological data, the structure of the N-glycan of ascorbic acid oxidase has been reinvestigated. The oligosaccharides were released by almond N-glycosidase and analysed as their pyridylamino derivatives by 2D-HPLC and exoglycosidase digestions. The main structure resembled the typical complex plant N-glycan consisting of a core-pentasaccharide decorated with xylose and 3-linked fucose. The other abundant species lacked the fucose residue. Small amounts of these glycans carried a GlcNAc residue on the 6-arm. Therefore, ascorbic acid oxidase will not only react with antibodies directed against the xylosylated region but also with those binding to N-glycans with 3-linked fucose.     

Altered Cell surface glycoproteins in phytohemagglutinin-resistant mutants of Chinese hamster ovary cells

Purified membranes from surface-labelled phytohemagglutinin-resistant (Pha^R) and wild-type chinese hamster ovary cells have been analysed by sodium dodecyl sulphate gel electrophoresis. Gel patterns were compared for cells labelled via galactose oxidase and B³H₄ or lactoperoxidase and radioactive iodide. The results suggest that Pha^R cells are altered in the carbohydrate portion of a number of their membrane glycoproteins.     

Separation of glycoprotein-derived oligosaccharides by thin-layer chromatography.

A thin-layer chromatography (tlc) system has been developed for the separation of glycoprotein-derivedoligosaccharides. The method involves chromatography on silica gel using n-propanol/acetic acid/water (3:3:2 v/v) as the solvent. This tlc method was used to separate pathological oligosaccharides isolated from individuals with G_M1 gangliosidosis and with neuraminidase deficiency. The results indicate the potential usefulness of the system in the analysis of complex carbohydrates.     

Isolation and characterization of the potential receptor for wheat germ agglutinin from human neutrophils

Neutrophils participate in host protection and central to this process is the regulation of oxidative mechanisms. We purified by affinity chromatography the receptor for the GlcNAc-specific WGA from CD14+ CD16+ cell lysates (WGAr). The receptor is a 141 kDa glycoprotein constituted by two subunits of 78 and 63 kDa. It is mainly composed of Ser, Asx, and Gly, and, in a minor proportion, His, Cys, and Pro. Its glycan portion contains GlcNAc, Gal, and Man; NeuAc and GalNAc were identified in a minor proportion. The amino acid sequence of the WGA receptor was predicted from tryptic peptides by MALDI-TOF, both subunits showed homology with cytokeratin type II (26 and 29% for the 78 and 63 kDa subunits, respectively); the 78 kDa subunit showed also homology with the human transferrin receptor (24%). Antibodies against WGAr induce higher oxidative burst than WGA, determined by NBT reduction; however, this effect was inhibited (p < 0.05) with GlcNAc suggesting that WGAr participates as mediator in signal transduction in neutrophils.     

Assessment of glycosaminoglycan-protein linkage tetrasaccharides as acceptors for GalNAc- and GlcNAc-transferases from mouse mastocytoma

Two glycosaminoglycan-protein linkage tetrasaccharide-serine compounds, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser and GlcAβ1-3Gal(4-O-sulfate)β1-3Galβ1-4Xylβ1-O-Ser, were tested as hexosamine acceptors, using UDP-[3H]GlcNAc and UDP-[3H]GalNAc as sugar donors, and solubilized mouse mastocytoma microsomes as enzyme source. The nonsulfated Ser-tetrasaccharide was found to function as an acceptor for a GalNAc residue, whereas the Ser-tetrasaccharide containing a sulfated galactose unit was inactive. Characterization of the radio-labelled product by digestion with α-N-acetylgalactosaminidase and β-N-acetylhexosaminidase revealed that the [3H]GalNAc unit was α-linked, as in the product previously synthesized using serum enzymes, and not β-linked as found in the chondroitin sulfate polymer. Heparan sulfate/heparin biosynthesis could not be primed by either of the two linkage Ser-tetrasaccharides, since no transfer of [3H]GlcNAc from UDP-[3H]GlcNAc could be detected. By contrast, transfer of a [3H]GlcNAc unit to a [GlcAβ1-4GlcNAcα1-4]2-GlcAβ1-4-aMan hexasaccharide acceptor used to assay the GlcNAc transferase involved in chain elongation, was readily detected. These results are in agreement with the recent proposal that two different N-acetylglucosaminyl transferases catalyse the biosynthesis of heparan sulfate. Although the mastocytoma system contains both the heparan sulfate/heparin and chondroitin sulfate biosynthetic enzymes the Ser-tetrasaccharides do not seem to fulfil the requirements to serve as acceptors for the first HexNAc transfer reactions involved in the formation of these polysaccharides. This revised version was published online in November 2006 with corrections to the Cover Date.     

A membrane-associated isozyme of invertase in yeast precursor of the external glycoprotein

An enzymatic test is described which allows the localization of yeast invertase activity directly on sodium dodecyl sulfate gels. When crude membrane fractions are prepared from Saccharomyces cerevisiae cells which are actively synthesizing external invertase, these membranes show an activity band on sodium dodecyl sulfate gels additional to the external and the internal invertase. In the soluble fraction this form was completely absent. It has a molecular weight of approximately 190 000 and was 50 000 smaller than the external invertase. It showed kinetic characteristics of a precursor of the external enzyme. Thus it appeared transiently, when yeast cells were shifted from a condition of non-synthesizing external invertase to one where the enzyme was synthesized. When the increase in the external enzyme slowed down after some time, the membrane-associated form almost completely disappeared.The addition of tunicamycin to yeast cells synthesizing external invertase inhibited further synthesis of the enzyme by 97%; also the formation of the membrane-associated form was strongly inhibited. The amount of it present before the addition of tunicamycin completely disappeared in the presence of the antibiotic. The precursor form, therefore, seems to possess already those carbohydrate parts which contain N-acetylglucosamine and are transferred via dolichyl phosphate-bound intermediates. The membrane-associated precursor amounts to less than 5% of the total invertase activity of a yeast cell.     

Synthesis of N-glycan units for assessment of substrate structural requirements of N-acetylglucosaminyltransferase III

N-Acetylglucosaminyltransferase (GnT) III is a glycosyltransferase which produces bisected N-glycans by transferring GlcNAc to the 4-position of core mannose. Bisected N-glycans are involved in physiological and pathological processes through the functional regulation of their carrier proteins. An understanding of the biological functions of bisected glycans will be greatly accelerated by use of specific inhibitors of GnT-III. Thus far, however, such inhibitors have not been developed and even the substrate-binding mode of GnT-III is not fully understood. To gain insight into structural features required of the substrate, we systematically synthesized four N-glycan units, the branching parts of the bisected and non-bisected N-glycans. The series of syntheses were achieved from a common core trimannose, giving bisected tetra- and hexasaccharides as well as non-bisected tri- and pentasaccharides. A competitive GnT-III inhibition assay using the synthetic substrates revealed a vital role for the Manβ(1–4)GlcNAc moiety. In keeping with previous reports, GlcNAc at the α1,3-branch is also involved in the interaction. The structural requirements of GnT-III elucidated in this study will provide a basis for rational inhibitor design.     

Characterization of oligosaccharides from the urine of loco-intoxicated sheep

Two major oligosaccharides were isolated by preparative HPLC from the urine of a locoweed-fed sheep. Analysis by gas-liquid chromatography and mass-spectrometry indicated compositions of (Man)4(GlcNAc)2 and (Man)5(GlcNAc)2, respectively. Structures were determined by digestion with alpha-D-mannosidase and endo-beta-N-acetylglucosaminidases D and H, and comparison of the products by HPLC with synthetic standards, and oligosaccharides isolated from human mannosidosis urine. Incubation with an exo-beta-N-acetylglucosaminidase was without effect.     

常压室温等离子体诱变高通量筛选N-乙酰氨基葡萄糖高产突变株

采用新型常压室温等离子体(ARTP)诱变产N-乙酰氨基葡萄糖工程菌BSGN6,并应用显色法和96孔板培养方法筛选N-乙酰氨基葡萄糖高产突变株.研究表明显色反应时添加四硼酸钾溶液1 μL、PDABA 125 μL、反应时间5 min、反应温度96 ℃时,N-乙酰氨基葡萄糖检测的准确度最高.通过多轮ARTP诱变及高通量筛选最终获得突变株(4A12),3L罐发酵GlcNAc产量达到36.14 g/L,较出发菌株(BSGN6)提高了65.32％.经过50次传代后性状稳定.ARTP诱变技术作为获得产N-乙酰氨基葡萄糖高产菌株的有效途径,与分子生物学手段相比,本方法更加快捷高效.     

Bisecting GlcNAc mediates the binding of annexin V to Hsp47

The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : beta-D-mannoside beta-1,4-N-acetylglucosaminyltansferase III (GnT-III), is responsible for a variety of biological functions. We have previously shown that annexin V, a member of the calcium/phospholipid-binding annexin family of proteins, has binding activity toward the bisecting GlcNAc structure. In this study, we reported on a search for potential target glycoproteins for annexin V in a rat hepatoma cell line, M31. Using a glutathione S-transferase (GST)-annexin V immobilized sepharose 4B affinity column to trap interacting proteins produced by the GnT-III-transfected M31 cells, we isolated a 47 kDa protein. It was identified as Hsp47 by an N-terminal sequence analysis. Immunoprecipitation experiments showed that annexin V interacted with Hsp47. The association of annexin V and Hsp47 was abolished by treatment with N-glycosidase F or preincubation with sugar chains containing bisecting GlcNAc, suggesting that the bisecting GlcNAc plays an important role in the interaction. An oligosaccharide analysis of Hsp47 purified from GnT-III-transfected M31 cells was shown to have the bisecting GlcNAc structure, as detected by erythroagglutinating phytohemagglutinin (E4-PHA) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a Kd of 5.5 microM, whereas no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the colocalization of annexin V, Hsp47, and a bisecting GlcNAc sugar chain around the Golgi apparatus. Collectively, these results suggest that the binding of annexin V to Hsp47 is mediated by a bisecting GlcNAc oligosaccharide structure and that Hsp47 is an intracellular ligand glycoprotein for annexin V.     

Purification by affinity chromatography of the solubilized oligosaccharyltransferase from hen oviducts using a privileged secondary structure adopting peptide

Dolichyldiphosphoryloligosaccharide-protein oligosaccharyltransferase, which transfers the oligosaccharide from Dol PP-(GlcNAc)₂ (Man)₉(Glc)₃ to asparagine residues of glycoproteins was solubilized from hen oviduct membranes by extraction with detergent and then purified by affinity chromatography on a peptide-Sepharose column. The peptide bound to Sepharose was the tetradecapeptide Thr-His-Thr-Asn-Ile-Ser-Glu-Ser-His-Pro-Asn-Ala-Thr-Phe. In an aqueous buffer solution, it underwent a transition from an unordered to an ordered structure when 10% DMSO was added.     

N-acetyl-D-galactosamine/N-acetyl-D-glucosamine--recognizing lectin from the snail Cepaea hortensis: purification, chemical characterization, cloning and expression in E. coli

From the albumin gland of the snail Cepaea hortensis we isolated and characterized a new N-acetyl-D-galactosamine/N-acetyl-D-glucosamine (GalNAc/GlcNAc) specific lectin (CHA-II) which was purified by a combination of affinity chromatography on GalNAc-agarose and gel filtration. The purified native lectin was found to be a multimeric protein, as revealed by SDS-PAGE and MALDI-TOF analysis. In SDS-PAGE the denatured and reduced lectin showed two bands of molecular masses with 17 and 15.5 kDa which reacted equally with anti-CHA-II rabbit antiserum. The lectin was O- and N-glycosylated with [(Gal)2-Man]2-Man-GlcNAc-GlcNAc-Asn as a probable structure for the oligosaccharide. Isoelectric focusing revealed a heterogeneous protein of at least four bands around pH 8.7. Tryptic peptides of CHA-II were N-terminally sequenced and highly degenerated gene specific oligonucleotide primers (GSPs) had been constructed. Using total RNA isolated from albumin glands, cDNAs were produced by the running race technique. Specific PCR fragments were obtained by PCR using GSPs, the universal primer and 5'- or 3'-RACE-cDNAs. The amplified fragments were cloned into the vector pDrive and were sequenced. The resulting total cDNA sequence consisted of 496 base pairs including an open reading frame of 360 base pairs which encoded a protein of 120 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to comprise 99 amino acid residues with a calculated molecular weight of 11,239 Da. The PCR fragment encoding the mature protein was cloned into the vector pQE30 and expressed in E. coli. Recombinant CHA-II lectin was produced as inclusion bodies and extracted by 6 M guanidine hydrochloride. After refolding, the recombinant CHA-II agglutinated specifically human red blood cells of groups A and AB. In immunodiffusion experiments using rabbit antiserum raised against the native lectin, the protein showed a precipitation line of identity with the native lectin.     

Biophysical characterization and structural determination of the potent cytotoxic Psathyrella asperospora lectin

A lectin with strong cytotoxic effect on human colon cancer HT29 and monkey kidney VERO cells was recently identified from the Australian indigenous mushroom Psathyrella asperospora and named PAL. We herein present its biochemical and structural analysis using a multidisciplinary approach. Glycan arrays revealed binding preference towards N‐acetylglucosamine (GlcNAc) and, to a lesser extent, towards sialic acid (Neu5Ac). Submicromolar and millimolar affinity was measured by surface plasmon resonance for GlcNAc and NeuAc, respectively. The structure of PAL was resolved by X‐ray crystallography, elucidating both the protein's amino acid sequence as well as the molecular basis rationalizing its binding specificity. Proteins 2017; 85:969–975. © 2016 Wiley Periodicals, Inc.     

一种来源于兼性嗜碱菌Bacillus pseudofirmus 703的β-N-乙酰氨基葡萄糖苷酶

N-乙酰氨基葡萄糖苷酶作用于肽聚糖或几丁质,从其非还原末端水解产生β-D-N-乙酰氨基葡萄糖单体,该酶在细胞壁代谢过程中起重要作用,在医药和生物技术领域也有广泛的应用。【目的】克隆表达来源于兼性嗜碱菌Bacillus pseudofirmus 703的β-N-乙酰葡糖胺糖苷酶NagZ703,为获得乙酰氨基葡萄糖单体奠定基础。【方法】以B.pseudofirmus703基因组DNA为模板,克隆得到了β-N-乙酰氨基葡萄糖苷酶基因NagZ703,通过构建pET28a-nagZ703表达载体,在大肠杆菌BL21(DE3)中诱导表达NagZ703,利用镍柱纯化得到NagZ703纯蛋白,并对其酶学和生化性质进行分析。【结果】NagZ703与其同源蛋白多序列比对分析结果表明,NagZ703属于糖苷水解酶3家族(GH3),由2个结构域构成,催化活性中心由位于N端结构域的Arg232-His234-Arg318组成,和研究最多的Bacillussubtilis168来源的BsNagZ氨基酸的序列相似性为37%。酶学性质分析表明,以对硝基酚-β-乙酰氨基葡萄糖苷(pNP-β-GlcNAc)为底物,NagZ703的最适反应温度和pH分别为60°C和pH 6.5,比酶活为10.79 U/mg,其Km和Vmax分别为0.276 mmol/L和0.612 mmol/(mg·min)。该酶具有较好的稳定性,在50°C处理30 min,或在pH 6.0–10.5条件下,4°C保存12 h后,仍保留80%以上的酶活力。EDTA不影响该酶的活性,推测其为非金属依赖酶,且Hg2+可完全抑制酶活性。【结论】本研究将兼性嗜碱菌Bacillus pseudofirmus 703来源的β-N-乙酰葡糖胺糖苷酶NagZ703在大肠杆菌中成功表达和纯化,并分析了其酶学性质;NagZ703的最适pH为6.5,没有表现出耐盐嗜碱的特征;NagZ703能水解胶体几丁质产生GlcNAc,为酶解生产GlcNAc提供了一条可行的思路。     

The synthesis and biological evaluation of some carbocyclic analogues of PUGNAc

The synthesis of some analogues of O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenylcarbamate, PUGNAc, an inhibitor of β-N-acetylglucosaminidases, is described. The analogues were tested against a range of β-N-acetylglucosaminidases to establish any biological activity. As well, the analogues were tested as inhibitors of a uridine diphosphate-N-acetyl-d-glucosamine: polypeptidyl transferase, OGT, a critical protein involved in the post-translational modification of nuclear and cytosolic proteins by N-acetyl-d-glucosamine.     

Glycosylation of endogenous proteins through dolichol derivatives in reticulocyte plasma membranes

It has been found that rabbit reticulocyte plasma membranes contain the enzymatic activities responsible for the synthesis of all the polyprenol-bound sugars that are intermediates in the glycosylation of proteins. The final reaction in which an oligosaccharide is transferred from a dolichol pyrophosphate derivative to an endogenous protein was detected in reticulocyte but not in erythrocyte plasma membranes.     

The chitin catabolic cascade in the marine bacterium Vibrio cholerae: characterization of a unique chitin oligosaccharide deacetylase

Chitin, one of the most abundant organic substances in nature, is consumed by marine bacteria, such as Vibrio cholerae, via a multitude of tightly regulated genes (Li and Roseman 2004, Proc Natl Acad Sci USA. 101:627-631). One such gene, cod, is reported here. It encodes a chitin oligosaccharide deacetylase (COD), when cells are induced by chitobiose, (GlcNH(2))(2), or crude crab shells. COD was molecularly cloned (COD-6His), overproduced, and purified to apparent homogeneity. COD is secreted at all stages of growth by induced V. cholerae. The gene sequence predicts a 26 N-terminal amino acid signal peptide not found in the isolated protein. COD is very active with chitin oligosaccharides, is virtually inactive with GlcNAc, and slightly active with colloidal ([(3)H]-N-acetyl)-chitin. The oligosaccharides are converted almost quantitatively to products lacking one acetyl group. The latter were characterized by mass spectrometry (ESI-MS), and treatment with nitrous acid. COD catalyzes the following reactions (n = 2-6): (GlcNAc)(n)--> GlcNAc-GlcNH(2)-(GlcNAc)(n-2) + Ac(-). That is, COD hydrolyzes the N-acetyl groups attached to the penultimate GlcNAc residue. The gene bank sequence data show that cod is highly conserved in Vibrios and Photobacteria. One such gene encodes a deacetylase isolated from V. alginolytics (Ohishi et al. 1997, Biosci Biotech Biochem. 61:1113-1117; Ohishi et al. 2000, J Biosci Bioeng. 90:561-563), that is specific for (GlcNAc)(2), but inactive with higher oligosaccharides. The COD enzymatic products, GlcNAc-GlcNH(2)-(GlcNAc)(n), closely resemble those obtained by hydrolysis of the chitooligosaccharides with Nod B: GlcNH(2)-(GlcNAc)(3-4). The latter are key intermediates in the biosynthesis of Nod factors, critically important in communications between the symbiotic nitrogen fixing bacteria and plants. Conceivably, the COD products play equally important roles in cellular communications that remain to be defined.