Environmental and biofilm-dependent changes in a Bacillus cereus secondary cell wall polysaccharide

Bacterial species from the Bacillus genus, including Bacillus cereus and Bacillus anthracis, synthesize secondary cell wall polymers (SCWP) covalently associated to the peptidoglycan through a phospho-diester linkage. Although such components were observed in a wide panel of B. cereus and B. anthracis strains, the effect of culture conditions or of bacterial growth state on their synthesis has never been addressed. Herein we show that B. cereus ATCC 14579 can synthesize not only one, as previously reported, but two structurally unrelated secondary cell wall polymers (SCWP) polysaccharides. The first of these SCWP, →4)[GlcNAc(β1-3)]GlcNAc(β1-6)[Glc(β1-3)][ManNAc(α1-4)]GalNAc(α1-4)ManNAc(β1→, although presenting an original sequence, fits to the already described the canonical sequence motif of SCWP. In contrast, the second polysaccharide was made up by a totally original sequence, →6)Gal(α1-2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(α1-6)GlcNAc(β1→, which no equivalent has ever been identified in the Bacillus genus. In addition, we established that the syntheses of these two polysaccharides were differently regulated. The first one is constantly expressed at the surface of the bacteria, whereas the expression of the second is tightly regulated by culture conditions and growth states, planktonic, or biofilm.     

Characterization of the arabinogalactan protein 31 (AGP31) of Arabidopsis thaliana: new advances on the Hyp-O-glycosylation of the Pro-rich domain

Proteins are important actors in plant cell walls because they contribute to their architecture and their dynamics. Among them, hydroxyproline (Hyp)-rich glycoproteins constitute a complex family of O-glycoproteins with various structures and functions. In this study, we characterized an atypical Hyp-rich glycoprotein, AGP31 (arabinogalactan protein 31), which displays a multidomain organization unique in Arabidopsis thaliana, consisting of a short arabinogalactan protein (AGP) motif, a His stretch, a Pro-rich domain, and a C-terminal PAC (PRP-AGP containing Cys) domain. The use of various mass spectrometry strategies was innovative and powerful: it permitted us to locate Hyp residues, to demonstrate the presence of carbohydrates, and to refine their distribution over the Pro-rich domain. Most Hyp were isolated within repeated motifs such as KAOV, KSOV, K(PO/OP)T, K(PO/OP)V, T(PO/OP)V, and Y(PO/OP)T. A few extensin-like motifs with contiguous Hyp (SOOA and SOOT) were also found. The Pro-rich domain was shown to carry Gal residues on isolated Hyp but also Ara residues. The existence of new type Hyp-O-Gal/Ara-rich motifs not recognized by the β-glucosyl Yariv reagent but interacting with the peanut agglutinin lectin was proposed. In addition, the N-terminal short AGP motif was assumed to be substituted by arabinogalactans. Altogether, AGP31 was found to be highly heterogeneous in cell walls because arabinogalactans could be absent, Hyp-O-Gal/Ara-rich motifs of different sizes were observed, and truncated forms missing the C-terminal PAC domain were found, suggesting degradation in muro and/or partial glycosylation prior to secretion.     

Characterization and scope of S-layer protein O-glycosylation in Tannerella forsythia

Cell surface glycosylation is an important element in defining the life of pathogenic bacteria. Tannerella forsythia is a Gram-negative, anaerobic periodontal pathogen inhabiting the subgingival plaque biofilms. It is completely covered by a two-dimensional crystalline surface layer (S-layer) composed of two glycoproteins. Although the S-layer has previously been shown to delay the bacterium's recognition by the innate immune system, we characterize here the S-layer protein O-glycosylation as a potential virulence factor. The T. forsythia S-layer glycan was elucidated by a combination of electrospray ionization-tandem mass spectrometry and nuclear magnetic resonance spectroscopy as an oligosaccharide with the structure 4-Me-β-ManpNAcCONH(2)-(1→3)-[Pse5Am7Gc-(2→4)-]-β-ManpNAcA-(1→4)-[4-Me-α-Galp-(1→2)-]-α-Fucp-(1→4)-[-α-Xylp-(1→3)-]-β-GlcpA-(1→3)-[-β-Digp-(1→2)-]-α-Galp, which is O-glycosidically linked to distinct serine and threonine residues within the three-amino acid motif (D)(S/T)(A/I/L/M/T/V) on either S-layer protein. This S-layer glycan obviously impacts the life style of T. forsythia because increased biofilm formation of an UDP-N-acetylmannosaminuronic acid dehydrogenase mutant can be correlated with the presence of truncated S-layer glycans. We found that several other proteins of T. forsythia are modified with that specific oligosaccharide. Proteomics identified two of them as being among previously classified antigenic outer membrane proteins that are up-regulated under biofilm conditions, in addition to two predicted antigenic lipoproteins. Theoretical analysis of the S-layer O-glycosylation of T. forsythia indicates the involvement of a 6.8-kb gene locus that is conserved among different bacteria from the Bacteroidetes phylum. Together, these findings reveal the presence of a protein O-glycosylation system in T. forsythia that is essential for creating a rich glycoproteome pinpointing a possible relevance for the virulence of this bacterium.     

Simple, automated, high resolution mass spectrometry method to determine the disulfide bond and glycosylation patterns of a complex protein: subgroup A avian sarcoma and leukosis virus envelope glycoprotein

Enveloped viruses must fuse the viral and cellular membranes to enter the cell. Understanding how viral fusion proteins mediate entry will provide valuable information for antiviral intervention to combat associated disease. The avian sarcoma and leukosis virus envelope glycoproteins, trimers composed of surface (SU) and transmembrane heterodimers, break the fusion process into several steps. First, interactions between SU and a cell surface receptor at neutral pH trigger an initial conformational change in the viral glycoprotein trimer followed by exposure to low pH enabling additional conformational changes to complete the fusion of the viral and cellular membranes. Here, we describe the structural characterization of the extracellular region of the subgroup A avian sarcoma and leukosis viruses envelope glycoproteins, SUATM129 produced in chicken DF-1 cells. We developed a simple, automated method for acquiring high resolution mass spectrometry data using electron capture dissociation conditions that preferentially cleave the disulfide bond more readily than the peptide backbone amide bonds that enabled the identification of disulfide-linked peptides. Seven of nine disulfide bonds were definitively assigned; the remaining two bonds were assigned to an adjacent pair of cysteine residues. The first cysteine of surface and the last cysteine of the transmembrane form a disulfide bond linking the heterodimer. The surface glycoprotein contains a free cysteine at residue 38 previously reported to be critical for virus entry. Eleven of 13 possible SUATM129 N-linked glycosylation sites were modified with carbohydrate. This study demonstrates the utility of this simple yet powerful method for assigning disulfide bonds in a complex glycoprotein.     

N-glycans of Phaeodactylum tricornutum diatom and functional characterization of its N-acetylglucosaminyltransferase I enzyme

N-glycosylation, a major co- and post-translational event in the synthesis of proteins in eukaryotes, is unknown in aquatic photosynthetic microalgae. In this paper, we describe the N-glycosylation pathway in the diatom Phaeodactylum tricornutum. Bio-informatic analysis of its genome revealed the presence of a complete set of sequences potentially encoding for proteins involved in the synthesis of the lipid-linked Glc(3)Man(9)GlcNAc(2)-PP-dolichol N-glycan, some subunits of the oligosaccharyltransferase complex, as well as endoplasmic reticulum glucosidases and chaperones required for protein quality control and, finally, the α-mannosidase I involved in the trimming of the N-glycan precursor into Man-5 N-glycan. Moreover, one N-acetylglucosaminyltransferase I, a Golgi glycosyltransferase that initiates the synthesis of complex type N-glycans, was predicted in the P. tricornutum genome. We demonstrated that this gene encodes for an active N-acetylglucosaminyltransferase I, which is able to restore complex type N-glycans maturation in the Chinese hamster ovary Lec1 mutant, defective in its endogeneous N-acetylglucosaminyltransferase I. Consistent with these data, the structural analyses of N-linked glycans demonstrated that P. tricornutum proteins carry mainly high mannose type N-glycans ranging from Man-5 to Man-9. Although representing a minor glycan population, paucimannose N-glycans were also detected, suggesting the occurrence of an N-acetylglucosaminyltransferase I-dependent maturation of N-glycans in this diatom.     

Detailed structural and quantitative analysis reveals the spatial organization of the cell walls of in vivo grown Mycobacterium leprae and in vitro grown Mycobacterium tuberculosis

The cell wall of mycobacteria consists of an outer membrane, analogous to that of gram-negative bacteria, attached to the peptidoglycan (PG) via a connecting polysaccharide arabinogalactan (AG). Although the primary structure of these components is fairly well deciphered, issues such as the coverage of the PG layer by covalently attached mycolates in the outer membrane and the spatial details of the mycolic acid attachment to the arabinan have remained unknown. It is also not understood how these components work together to lead to the classical acid-fast staining of mycobacteria. Because the majority of Mycobacterium tuberculosis bacteria in established experimental animal infections are acid-fast negative, clearly cell wall changes are occurring. To address both the spatial properties of mycobacterial cell walls and to begin to study the differences between bacteria grown in animals and cultures, the cell walls of Mycobacterium leprae grown in armadillos was characterized and compared with that of M. tuberculosis grown in culture. Most fundamentally, it was determined that the cell wall of M. leprae contained significantly more mycolic acids attached to PG than that of in vitro grown M. tuberculosis (mycolate:PG ratios of 21:10 versus 16:10, respectively). In keeping with this difference, more arabinogalactan (AG) molecules, linking the mycolic acids to PG, were found. Differences in the structures of the AG were also found; the AG of M. leprae is smaller than that of M. tuberculosis, although the same basic structural motifs are retained.     

Clostridium difficile has an original peptidoglycan structure with a high level of N-acetylglucosamine deacetylation and mainly 3-3 cross-links

The structure of the vegetative cell wall peptidoglycan of Clostridium difficile was determined by analysis of its constituent muropeptides with a combination of reverse-phase high pressure liquid chromatography separation of muropeptides, amino acid analysis, mass spectrometry and tandem mass spectrometry. The structures assigned to 36 muropeptides evidenced several original features in C. difficile vegetative cell peptidoglycan. First, it is characterized by a strikingly high level of N-acetylglucosamine deacetylation. In addition, the majority of dimers (around 75%) contains A(2)pm(3) → A(2)pm(3) (A(2)pm, 2,6-diaminopimelic acid) cross-links and only a minority of the more classical Ala(4) → A(2)pm(3) cross-links. Moreover, a significant amount of muropeptides contains a modified tetrapeptide stem ending in Gly instead of D-Ala(4). Two L,D-transpeptidases homologues encoding genes present in the genome of C. difficile 630 and named ldt(cd1) and ldt(cd2), were inactivated. The inactivation of either ldt(cd1) or ldt(cd2) significantly decreased the abundance of 3-3 cross-links, leading to a marked decrease of peptidoglycan reticulation and demonstrating that both ldt(cd1)-and ldt(cd2)-encoded proteins have a redundant L,D-transpeptidase activity. The contribution of 3-3 cross-links to peptidoglycan synthesis increased in the presence of ampicillin, indicating that this drug does not inhibit the L,D-transpeptidation pathway in C. difficile.     

Crystal Structure of the Minor Pilin FctB Reveals Determinants of Group A Streptococcal Pilus Anchoring

Cell surface pili are polymeric protein assemblies that enable bacteria to adhere to surfaces and to specific host tissues. The pili expressed by Gram-positive bacteria constitute a unique paradigm in which sortase-mediated covalent linkages join successive pilin subunits like beads on a string. These pili are formed from two or three distinct types of pilin subunit, typically encoded in small gene clusters, often with their cognate sortases. In Group A streptococci (GAS), a major pilin forms the polymeric backbone, whereas two minor pilins are located at the tip and the base. Here, we report the 1.9-Å resolution crystal structure of the GAS basal pilin FctB, revealing an immunoglobulin (Ig)-like N-terminal domain with an extended proline-rich tail. Unexpected structural homology between the FctB Ig-like domain and the N-terminal domain of the GAS shaft pilin helps explain the use of the same sortase for polymerization of the shaft and its attachment to FctB. It also enabled the identification, from mass spectral data, of the lysine residue involved in the covalent linkage of FctB to the shaft. The proline-rich tail forms a polyproline-II helix that appears to be a common feature of the basal (cell wall-anchoring) pilins. Together, our results indicate distinct structural elements in the pilin proteins that play a role in selecting for the appropriate sortases and thereby help orchestrate the ordered assembly of the pilus.     

The Interplay of Proton,Electron, and Metabolite Supply for Photosynthetic H2 Production in Chlamydomonas reinhardtii

To obtain a detailed picture of sulfur deprivation-induced H₂ production in microalgae, metabolome analyses were performed during key time points of the anaerobic H₂ production process of Chlamydomonas reinhardtii. Analyses were performed using gas chromatography coupled to mass spectrometry (GC/MS), two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GCxGC-TOFMS), lipid and starch analysis, and enzymatic determination of fermentative products. The studies were designed to provide a detailed metabolite profile of the solar Bio-H₂ production process. This work reports on the differential analysis of metabolic profiles of the high H₂-producing strain Stm6Glc4 and the wild-type cc406 (WT) before and during the H₂ production phase. Using GCxGC-TOFMS analysis the number of detected peaks increased from 128 peaks, previously detected by GC/MS techniques, to ∼1168. More detailed analysis of the anaerobic H₂ production phase revealed remarkable differences between wild-type and mutant cells in a number of metabolic pathways. Under these physiological conditions the WT produced up to 2.6 times more fatty acids, 2.2 times more neutral lipids, and up to 4 times more fermentation products compared with Stm6Glc4. Based on these results, specific metabolic pathways involving the synthesis of fatty acids, neutral lipids, and fermentation products during anaerobiosis in C. reinhardtii have been identified as potential targets for metabolic engineering to further enhance substrate supply for the hydrogenase(s) in the chloroplast.     

Mannose efflux from the cells: a potential source of mannose in blood

All mammals have 50-100 μM mannose in their blood. However, the source of the dynamic pool of mannose in blood is unknown. Most of it is thought to be derived from glucose in the cells. We studied mannose uptake and release by various cell types. Interestingly, our results show that mannose taken up by the cells through transporters is handled differently from the mannose released within the cells due to glycan processing of protein-bound oligosaccharides. Although more than 95% of incoming mannose is catabolized, most of the mannose released by intracellular processing is expelled from the cells as free mannose predominantly via a nocodazole-sensitive sugar transporter. Under physiological conditions, incoming mannose is more accessible to hexokinase, whereas mannose released within the cells is protected from HK and therefore has a different fate. Our data also suggest that generation of free mannose due to the processing of glycoconjugates composed of glucose-derived mannose and its efflux from the cells can account for most of the mannose found in blood and its steady state maintenance.     

Affinity Purification of the Arabidopsis 26 S Proteasome Reveals a Diverse Array of Plant Proteolytic Complexes

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis.     

O-antigen and core carbohydrate of Vibrio fischeri lipopolysaccharide: composition and analysis of their role in Euprymna scolopes light organ colonization

Vibrio fischeri exists in a symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes, where the squid provides a home for the bacteria, and the bacteria in turn provide camouflage that helps protect the squid from night-time predators. Like other gram-negative organisms, V. fischeri expresses lipopolysaccharide (LPS) on its cell surface. The structure of the O-antigen and the core components of the LPS and their possible role in colonization of the squid have not previously been determined. In these studies, an O-antigen ligase mutant, waaL, was utilized to determine the structures of these LPS components and their roles in colonization of the squid. WaaL ligates the O-antigen to the core of the LPS; thus, LPS from waaL mutants lacks O-antigen. Our results show that the V. fischeri waaL mutant has a motility defect, is significantly delayed in colonization, and is unable to compete with the wild-type strain in co-colonization assays. Comparative analyses of the LPS from the wild-type and waaL strains showed that the V. fischeri LPS has a single O-antigen repeat composed of yersiniose, 8-epi-legionaminic acid, and N-acetylfucosamine. In addition, the LPS from the waaL strain showed that the core structure consists of L-glycero-D-manno-heptose, D-glycero-D-manno-heptose, glucose, 3-deoxy-D-manno-octulosonic acid, N-acetylgalactosamine, 8-epi-legionaminic acid, phosphate, and phosphoethanolamine. These studies indicate that the unusual V. fischeri O-antigen sugars play a role in the early phases of bacterial colonization of the squid.     

A Novel 3-Methylhistidine Modification of Yeast Ribosomal Protein Rpl3 Is Dependent upon the YIL110W Methyltransferase

We have shown that Rpl3, a protein of the large ribosomal subunit from baker''s yeast (Saccharomyces cerevisiae), is stoichiometrically monomethylated at position 243, producing a 3-methylhistidine residue. This conclusion is supported by top-down and bottom-up mass spectrometry of Rpl3, as well as by biochemical analysis of Rpl3 radiolabeled in vivo with S-adenosyl-l-[methyl-³H]methionine. The results show that a +14-Da modification occurs within the GTKKLPRKTHRGLRKVAC sequence of Rpl3. Using high-resolution cation-exchange chromatography and thin layer chromatography, we demonstrate that neither lysine nor arginine residues are methylated and that a 3-methylhistidine residue is present. Analysis of 37 deletion strains of known and putative methyltransferases revealed that only the deletion of the YIL110W gene, encoding a seven β-strand methyltransferase, results in the loss of the +14-Da modification of Rpl3. We suggest that YIL110W encodes a protein histidine methyltransferase responsible for the modification of Rpl3 and potentially other yeast proteins, and now designate it Hpm1 (Histidine protein methyltransferase 1). Deletion of the YIL110W/HPM1 gene results in numerous phenotypes including some that may result from abnormal interactions between Rpl3 and the 25 S ribosomal RNA. This is the first report of a methylated histidine residue in yeast cells, and the first example of a gene required for protein histidine methylation in nature.     

Kre6 protein essential for yeast cell wall beta-1,6-glucan synthesis accumulates at sites of polarized growth

Saccharomyces cerevisiae Kre6 is a type II membrane protein with amino acid sequence homology with glycoside hydrolase and is essential for β-1,6-glucan synthesis as revealed by the mutant phenotype, but its biochemical function is still unknown. The localization of Kre6, determined by epitope tagging, is a matter of debate. We raised anti-Kre6 rabbit antiserum and examined the localization of Kre6 and its tagged protein by immunofluorescence microscopy, subcellular fractionation in sucrose density gradients, and immunoelectron microscopy. Integration of the results indicates that the majority of Kre6 is in the endoplasmic reticulum; however, a small but significant portion is also present in the secretory vesicle-like compartments and plasma membrane. Kre6 in the latter compartments is observed as strong signals that accumulate at the sites of polarized growth by immunofluorescence. The truncated Kre6 without the N-terminal 230-amino acid cytoplasmic region did not show this polarized accumulation and had a severe defect in β-1,6-glucan synthesis. This is the first evidence of a β-1,6-glucan-related protein showing the polarized membrane localization that correlates with its biological function.     

Structure of a galactomannan isolated from the cell wall of the fungus Lineolata rhizophorae

The structure of an alkali-extracted water-soluble polysaccharide isolated from the cell wall of the marine fungus Lineolata rhizophorae has been elucidated by chemical and spectroscopic means. The idealized repeating unit of this novel structure is being m ≈ 41, n ≈ 2, and p ≈ 5.     

N-acetylglucosamine inhibits T-helper 1 (Th1)/T-helper 17 (Th17) cell responses and treats experimental autoimmune encephalomyelitis

Current treatments and emerging oral therapies for multiple sclerosis (MS) are limited by effectiveness, cost, and/or toxicity. Genetic and environmental factors that alter the branching of Asn (N)-linked glycans result in T cell hyperactivity, promote spontaneous inflammatory demyelination and neurodegeneration in mice, and converge to regulate the risk of MS. The sugar N-acetylglucosamine (GlcNAc) enhances N-glycan branching and inhibits T cell activity and adoptive transfer experimental autoimmune encephalomyelitis (EAE). Here, we report that oral GlcNAc inhibits T-helper 1 (Th1) and T-helper 17 (Th17) responses and attenuates the clinical severity of myelin oligodendrocyte glycoprotein (MOG)-induced EAE when administered after disease onset. Oral GlcNAc increased expression of branched N-glycans in T cells in vivo as shown by high pH anion exchange chromatography, MALDI-TOF mass spectroscopy and FACS analysis with the plant lectin l-phytohemagglutinin. Initiating oral GlcNAc treatment on the second day of clinical disease inhibited MOG-induced EAE as well as secretion of interferon-γ, tumor necrosis factor-α, interleukin-17, and interleukin-22. In the more severe 2D2 T cell receptor transgenic EAE model, oral GlcNAc initiated after disease onset also inhibits clinical disease, except for those with rapid lethal progression. These data suggest that oral GlcNAc may provide an inexpensive and nontoxic oral therapeutic agent for MS that directly targets an underlying molecular mechanism causal of disease.     

O-Acetylation of peptidoglycan is required for proper cell separation and S-layer anchoring in Bacillus anthracis

O-Acetylation of the MurNAc moiety of peptidoglycan is typically associated with bacterial resistance to lysozyme, a muramidase that serves as a central component of innate immunity. Here, we report that the peptidoglycan of Bacillus anthracis, the etiological agent of anthrax, is O-acetylated and that, unusually, this modification is produced by two unrelated families of O-acetyltransferases. Also, in contrast to other bacteria, O-acetylation of B. anthracis peptidoglycan is combined with N-deacetylation to confer resistance of cells to lysozyme. Activity of the Pat O-acetyltransferases is required for the separation of the daughter cells following bacterial division and for anchoring of one of the major S-layer proteins. Our results indicate that peptidoglycan O-acetylation modulates endogenous muramidase activity affecting the cell-surface properties and morphology of this important pathogen.     

Cross-linking Chemistry of Squid Beak

In stark contrast to most aggressive predators, Dosidicus gigas (jumbo squids) do not use minerals in their powerful mouthparts known as beaks. Their beaks instead consist of a highly sclerotized chitinous composite with incremental hydration from the tip to the base. We previously reported l-3,4-dihydroxyphenylalanine (dopa)-histidine (dopa-His) as an important covalent cross-link providing mechanical strengthening to the beak material. Here, we present a more complete characterization of the sclerotization chemistry and describe additional cross-links from D. gigas beak. All cross-links presented in this report share common building blocks, a family of di-, tri-, and tetra-histidine-catecholic adducts, that were separated by affinity chromatography and high performance liquid chromatography (HPLC) and identified by tandem mass spectroscopy and proton nuclear magnetic resonance (¹H NMR). The data provide additional insights into the unusually high cross-link density found in mature beaks. Furthermore, we propose both a low molecular weight catechol, and peptidyl-dopa, to be sclerotization agents of squid beak. This appears to represent a new strategy for forming hard tissue in animals. The interplay between covalent cross-linking and dehydration on the graded properties of the beaks is discussed.     

Structural and Enzymatic Analysis of TarM Glycosyltransferase from Staphylococcus aureus Reveals an Oligomeric Protein Specific for the Glycosylation of Wall Teichoic Acid

Anionic glycopolymers known as wall teichoic acids (WTAs) functionalize the peptidoglycan layers of many Gram-positive bacteria. WTAs play central roles in many fundamental aspects of bacterial physiology, and they are important determinants of pathogenesis and antibiotic resistance. A number of enzymes that glycosylate WTA in Staphylococcus aureus have recently been identified. Among these is the glycosyltransferase TarM, a component of the WTA de novo biosynthesis pathway. TarM performs the synthesis of α-O-N-acetylglycosylated poly-5′-phosphoribitol in the WTA structure. We have solved the crystal structure of TarM at 2.4 Å resolution, and we have also determined a structure of the enzyme in complex with its substrate UDP-GlcNAc at 2.8 Å resolution. The protein assembles into a propeller-like homotrimer in which each blade contains a GT-B-type glycosyltransferase domain with a typical Rossmann fold. The enzymatic reaction retains the stereochemistry of the anomeric center of the transferred GlcNAc-moiety on the polyribitol backbone. TarM assembles into a trimer using a novel trimerization domain, here termed the HUB domain. Structure-guided mutagenesis experiments of TarM identify residues critical for enzyme activity, assign a putative role for the HUB in TarM function, and allow us to propose a likely reaction mechanism.     

海洋红酵母多糖提取及其对日本蟳血清中部分免疫活性因子的影响

以海洋红酵母为材料, 通过化学抽提法得到多糖, 用经典的Sevag 法进行脱蛋白处理, 经多级沉淀得到纯糖并采用硫酸-蒽酮法测得其中葡萄糖含量; 考马斯亮蓝法分析蛋白质含量。以定量海洋红酵母多糖人工注射日本蟳, 注射等量生理盐水为对照, 定时测定其血清中部分免疫活性因子的活性; 实验表明: 提取多糖为蛋白多糖, 其中葡萄糖含量为3.6%, 蛋白质含量1.9%, 含有多种氨基酸, 其中天冬氨酸含量最多; 注射后12 h 日本蟳血清中总超氧化物歧化酶(T-SOD) 活力达到最高, 酸性磷酸酶(ACP) 活力在注射后24 h 达到最高, 碱性磷酸酶(AKP) 48 h 达到最高, 过氧化氢酶(CAT) 48 h 达到最高, 溶菌酶(LZM) 12 h 即达到最高, 最高点分别高于对照组24%、43%、25%、35%、95%; 72 h 后都恢复至对照组水平。结论: 海洋红酵母多糖注射48 h 内日本蟳体内免疫活性因子均有不同程度的提高, 对日本蟳有较强免疫刺激作用。