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1.
Mar Drugs ; 22(9)2024 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-39330281

RESUMO

We here report the purification of a novel member of the galectin family, the ß-galactoside-binding lectin hRTL, from the marine sponge Chondrilla australiensis. The hRTL lectin is a tetrameric proto-type galectin with a subunit molecular weight of 15.5 kDa, consisting of 141 amino acids and sharing 92% primary sequence identity with the galectin CCL from the congeneric species C. caribensis. Transcriptome analysis allowed for the identification of additional sequences belonging to the same family, bringing the total number of hRTLs to six. Unlike most other galectins, hRTLs display a 23 amino acid-long signal peptide that, according to Erdman degradation, is post-translationally cleaved, leaving an N-terminal end devoid of acetylated modifications, unlike most other galectins. Moreover, two hRTLs display an internal insertion, which determines the presence of an unusual loop region that may have important functional implications. The characterization of the glycan-binding properties of hRTL revealed that it had high affinity towards TF-antigen, sialyl TF, and type-1 N-acetyl lactosamine with a Galß1-3 structure. When administered to DLD-1 cells, a colorectal carcinoma cell line expressing mucin-associated TF-antigen, hRTL could induce glycan-dependent cytotoxicity.


Assuntos
Antígenos Glicosídicos Associados a Tumores , Neoplasias Colorretais , Galectinas , Animais , Galectinas/farmacologia , Galectinas/metabolismo , Galectinas/isolamento & purificação , Galectinas/genética , Humanos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/tratamento farmacológico , Linhagem Celular Tumoral , Antígenos Glicosídicos Associados a Tumores/metabolismo , Poríferos , Antineoplásicos/farmacologia , Antineoplásicos/química , Sequência de Aminoácidos , Amino Açúcares
2.
J Org Chem ; 89(17): 11875-11890, 2024 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-39178339

RESUMO

N-Acetyllactosamine is a common saccharide motif found in various biologically active glycans. This motif usually works as a backbone for additional modifications and thus significantly influences glycan conformational behavior and biological activity. In this work, we have investigated the type-2 N-acetyllactosamine scaffold using the complete series of its monodeoxyfluorinated analogs. These glycomimetics have been studied by molecular mechanics, quantum mechanics, X-ray crystallography, and various NMR techniques, which have provided a comprehensive and complete insight into the role of individual hydroxyl groups in the conformational behavior and lipophilicity of N-acetyllactosamine.


Assuntos
Amino Açúcares , Amino Açúcares/química , Estrutura Molecular , Cristalografia por Raios X , Halogenação , Modelos Moleculares , Espectroscopia de Ressonância Magnética , Teoria Quântica , Conformação Molecular
3.
Anal Bioanal Chem ; 416(23): 5191-5203, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39095616

RESUMO

The widespread application of enzymes in industrial chemical synthesis requires efficient process control to maintain high yields and purity. Flow injection analysis-electrospray ionization-mass spectrometry (FIA-ESI-MS) offers a promising solution for real-time monitoring of these enzymatic processes, particularly when handling challenging compounds like sugars and glycans, which are difficult to quickly analyze using liquid chromatography-mass spectrometry due to their physical properties or the requirement for a derivatization step beforehand. This study compares the performance of FIA-MS with traditional hydrophilic interaction liquid chromatography (HILIC)-ultra high-performance liquid chromatography (UHPLC)-mass spectrometry (MS) setups for the monitoring of the enzymatic synthesis of N-acetyllactosamine (LacNAc) using beta-1,4-galactosyltransferase. Our results show that FIA-MS, without prior chromatographic separation or derivatization, can quickly generate accurate mass spectrometric data within minutes, contrasting with the lengthy separations required by LC-MS methods. The rapid data acquisition of FIA-MS enables effective real-time monitoring and adjustment of the enzymatic reactions. Furthermore, by eliminating the derivatization step, this method offers the possibility of being directly coupled to a continuously operated reactor, thus providing a rapid on-line methodology for glycan synthesis as well.


Assuntos
Análise de Injeção de Fluxo , Glicosiltransferases , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização por Electrospray/métodos , Análise de Injeção de Fluxo/métodos , Cromatografia Líquida de Alta Pressão/métodos , Glicosiltransferases/metabolismo , Amino Açúcares/análise
4.
Glycobiology ; 34(9)2024 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-39115362

RESUMO

α -Lactalbumin, an abundant protein present in the milk of most mammals, is associated with biological, nutritional and technological functionality. Its sequence presents N-glycosylation motifs, the occupancy of which is species-specific, ranging from no to full occupancy. Here, we investigated the N-glycosylation of bovine α-lactalbumin in colostrum and milk sampled from four individual cows, each at 9 time points starting from the day of calving up to 28.0 d post-partum. Using a glycopeptide-centric mass spectrometry-based glycoproteomics approach, we identified N-glycosylation at both Asn residues found in the canonical Asn-Xxx-Ser/Thr motif, i.e. Asn45 and Asn74 of the secreted protein. We found similar glycan profiles in all four cows, with partial site occupancies, averaging at 35% and 4% for Asn45 and Asn74, respectively. No substantial changes in occupancy occurred over lactation at either site. Fucosylation, sialylation, primarily with N-acetylneuraminic acid (Neu5Ac), and a high ratio of N,N'-diacetyllactosamine (LacdiNAc)/N-acetyllactosamine (LacNAc) motifs were characteristic features of the identified N-glycans. While no substantial changes occurred in site occupancy at either site during lactation, the glycoproteoform (i.e. glycosylated form of the protein) profile revealed dynamic changes; the maturation of the α-lactalbumin glycoproteoform repertoire from colostrum to mature milk was marked by substantial increases in neutral glycans and the number of LacNAc motifs per glycan, at the expense of LacdiNAc motifs. While the implications of α-lactalbumin N-glycosylation on functionality are still unclear, we speculate that N-glycosylation at Asn74 results in a structurally and functionally different protein, due to competition with the formation of its two intra-molecular disulphide bridges.


Assuntos
Colostro , Lactalbumina , Leite , Lactalbumina/metabolismo , Lactalbumina/química , Animais , Glicosilação , Colostro/química , Colostro/metabolismo , Bovinos , Leite/química , Leite/metabolismo , Feminino , Lactação/metabolismo , Amino Açúcares/química , Amino Açúcares/metabolismo , Glicopeptídeos/metabolismo , Glicopeptídeos/química , Glicopeptídeos/análise , Lactose/metabolismo , Lactose/química
5.
Glob Chang Biol ; 30(7): e17427, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-39021313

RESUMO

Atmospheric nitrogen (N) deposition in forests can affect soil microbial growth and turnover directly through increasing N availability and indirectly through altering plant-derived carbon (C) availability for microbes. This impacts microbial residues (i.e., amino sugars), a major component of soil organic carbon (SOC). Previous studies in forests have so far focused on the impact of understory N addition on microbes and microbial residues, but the effect of N deposition through plant canopy, the major pathway of N deposition in nature, has not been explicitly explored. In this study, we investigated whether and how the quantities (25 and 50 kg N ha-1 year-1) and modes (canopy and understory) of N addition affect soil microbial residues in a temperate broadleaf forest under 10-year N additions. Our results showed that N addition enhanced the concentrations of soil amino sugars and microbial residual C (MRC) but not their relative contributions to SOC, and this effect on amino sugars and MRC was closely related to the quantities and modes of N addition. In the topsoil, high-N addition significantly increased the concentrations of amino sugars and MRC, regardless of the N addition mode. In the subsoil, only canopy N addition positively affected amino sugars and MRC, implying that the indirect pathway via plants plays a more important role. Neither canopy nor understory N addition significantly affected soil microbial biomass (as represented by phospholipid fatty acids), community composition and activity, suggesting that enhanced microbial residues under N deposition likely stem from increased microbial turnover. These findings indicate that understory N addition may underestimate the impact of N deposition on microbial residues and SOC, highlighting that the processes of canopy N uptake and plant-derived C availability to microbes should be taken into consideration when predicting the impact of N deposition on the C sequestration in temperate forests.


Assuntos
Carbono , Florestas , Nitrogênio , Microbiologia do Solo , Solo , Nitrogênio/metabolismo , Carbono/metabolismo , Carbono/análise , Solo/química , Amino Açúcares/metabolismo , Amino Açúcares/análise , Árvores/crescimento & desenvolvimento , Árvores/metabolismo
6.
Molecules ; 29(7)2024 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-38611734

RESUMO

Intracellular protein complexes, known as inflammasomes, activate caspase-1 and induce the secretion of pro-inflammatory cytokines, namely interleukin (IL)-1ß and -18. Korean Red Ginseng extract (RGE) is a known immunomodulator and a potential candidate for the regulation of inflammasomes. The saponins, such as ginsenosides, of RGE inhibit inflammasome signaling, while non-saponin substances containing amino sugars promote the priming step, up-regulating inflammasome components (pro-IL-1ß, NLRP3, caspase-1, and Asc). In this study, the amino sugar-enriched fraction (ASEF), which increases only non-saponin components, including amino sugars, without changing the concentration of saponin substances, was used to investigate whether saponin or non-saponin components of RGE would have a greater impact on the priming step. When murine macrophages were treated with ASEF, the gene expression of inflammatory cytokines (IL-1α, TNFα, IL-6, and IL-10) increased. Additionally, ASEF induced the priming step but did not affect the inflammasome activation step, such as the secretion of IL-1ß, cleavage of caspase-1, and formation of Asc pyroptosome. Furthermore, the upregulation of gene expression of inflammasome components by ASEF was blocked by inhibitors of Toll-like receptor 4 signaling. Maltol, the main constituent of ASEF, promoted the priming step but inhibited the activation step of the inflammasome, while arginine, sugars, arginine-fructose-glucose, and fructose-arginine, the other main constituents of ASEF, had no effect on either step. Thus, certain amino sugars in RGE, excluding maltol, are believed to be the components that induce the priming step. The priming step that prepares the NLRP3 inflammasome for activation appears to be induced by amino sugars in RGE, thereby contributing to the immune-boosting effects of RGE.


Assuntos
Ginsenosídeos , Inflamassomos , Animais , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Amino Açúcares , Arginina , Caspase 1 , Frutose , Interleucina-1alfa , Interleucina-1beta , Extratos Vegetais/farmacologia
7.
Zhongguo Zhong Yao Za Zhi ; 49(5): 1225-1239, 2024 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-38621969

RESUMO

Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was employed to investigate the impacts of Pruni Semen processed with different methods(raw and fried) on the liver and spleen metabolism in mice. A total of 24 male mice were randomly assigned to three groups: raw Pruni Semen group, fried Pruni Semen group, and control(deionized water) group. Mice in the three groups were orally administrated with 0.01 g·mL~(-1) Pruni Semen decoction or deionized water for one week. After that, the liver and spleen tissues were collected, and liquid chromatography-mass spectrometry(LC-MS)-based metabolomic analysis was carried out to investigate the impact of Pruni Semen on the liver and spleen metabolism in mice. Compared with thte control group, the raw Pruni Semen group showed up-regulation of 11 metabolites and down-regulation of 57 metabolites in the spleen(P<0.05), as well as up-regulation of 15 metabolites and down-regulation of 58 metabolites in the liver(P<0.05). The fried Pruni Semen group showed up-regulation of 31 metabolites and down-regulation of 10 metabolites in the spleen(P<0.05), along with up-regulation of 26 metabolites and down-regulation of 61 metabolites in the liver(P<0.05). The differential metabolites identified in the raw Pruni Semen group were primarily associated with alanine, aspartate, and glutamate metabolism, purine metabolism, amino sugar and nucleotide sugar metabolism, and D-glutamine and D-glutamate metabolism. The differential metabolites identified in the fried Pruni Semen group predominantly involved riboflavin metabolism, amino sugar and nucleotide sugar metabolism, purine metabolism, alanine, aspartate, and glutamate metabolism, D-glutamine and D-glutamate metabolism, and glutathione metabolism. The findings suggest that both raw and fried Pruni Semen have the potential to modulate the metabolism of the liver and spleen in mice by influencing the glutamine and glutamate metabolism.


Assuntos
Ácido Glutâmico , Baço , Camundongos , Masculino , Animais , Sêmen , Glutamina , Ácido Aspártico , Metabolômica/métodos , Fígado/metabolismo , Alanina/metabolismo , Amino Açúcares/metabolismo , Água/metabolismo , Nucleotídeos/metabolismo , Purinas/metabolismo , Açúcares , Cromatografia Líquida de Alta Pressão , Biomarcadores/metabolismo
8.
Cell Rep ; 43(4): 114105, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38619967

RESUMO

Natural killer (NK) cells are primary defenders against cancer precursors, but cancer cells can persist by evading immune surveillance. To investigate the genetic mechanisms underlying this evasion, we perform a genome-wide CRISPR screen using B lymphoblastoid cells. SPPL3, a peptidase that cleaves glycosyltransferases in the Golgi, emerges as a top hit facilitating evasion from NK cytotoxicity. SPPL3-deleted cells accumulate glycosyltransferases and complex N-glycans, disrupting not only binding of ligands to NK receptors but also binding of rituximab, a CD20 antibody approved for treating B cell cancers. Notably, inhibiting N-glycan maturation restores receptor binding and sensitivity to NK cells. A secondary CRISPR screen in SPPL3-deficient cells identifies B3GNT2, a transferase-mediating poly-LacNAc extension, as crucial for resistance. Mass spectrometry confirms enrichment of N-glycans bearing poly-LacNAc upon SPPL3 loss. Collectively, our study shows the essential role of SPPL3 and poly-LacNAc in cancer immune evasion, suggesting a promising target for cancer treatment.


Assuntos
Células Matadoras Naturais , Polissacarídeos , Humanos , Polissacarídeos/metabolismo , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/imunologia , Amino Açúcares/metabolismo , Genômica/métodos , Rituximab/farmacologia , Rituximab/metabolismo , Linhagem Celular Tumoral
9.
Sci Total Environ ; 925: 171752, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38494032

RESUMO

Plant- and microbial-derived organic carbon, two components of the soil organic carbon (SOC) pool in terrestrial ecosystems, are regulated by increased atmospheric nitrogen (N) deposition. However, the spatial patterns and driving factors of the responses of plant- and microbial-derived SOC to N deposition in forests are not clear, which hinders our understanding of SOC sequestration. In this study, we explored the spatial patterns of plant- and microbial-derived SOC, and their responses to N addition and elucidated their underlying mechanisms in forest soils receiving N addition at four sites with various soil and climate conditions. Plant- and microbial-derived SOC were quantified using lignin phenols and amino sugars, respectively. N addition increased the total microbial residues by 20.5% on average ranging from 9.4% to 34.0% in temperate forests but not in tropical forests, and the increase was mainly derived from fungal residues. Lignin phenols increased more in temperate forests (average of 63.8%) than in tropical forests (average of 15.7%) following N addition. The ratio of total amino sugars to lignin phenols was higher in temperate forests than in tropical forests and decreased with N addition in temperate forests. N addition mainly regulated soil microbial residues by affecting pH, SOC, exchangeable Ca2+, gram-negative bacteria biomass, and the C:N ratio, while it mainly had indirect effects on lignin phenols by altering SOC, soil C:N ratio, and gram-negative bacteria biomass. Overall, our findings suggested that N deposition caused a greater increase in plant-derived SOC than in microbial-derived SOC and that plant-derived SOC would have a more important role in sequestering SOC under increasing N deposition in forest ecosystems, particularly in temperate forests.


Assuntos
Ecossistema , Traqueófitas , Carbono , Solo/química , Nitrogênio/análise , Lignina , Florestas , Microbiologia do Solo , Amino Açúcares , Fenóis
10.
Molecules ; 29(4)2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38398516

RESUMO

We recently showed that 6-sulfo sialyl N-acetyllactosamine (LacNAc) in O-linked glycans recognized by the CL40 antibody is abundant in the pleural mesothelium under physiological conditions and that these glycans undergo complementary synthesis by GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5) in mice. GlcNAc6ST3 is essential for the synthesis of R-10G-positive keratan sulfate (KS) in the brain. The predicted minimum epitope of the R-10G antibody is a dimeric asialo 6-sulfo LacNAc. Whether R-10G-reactive KS/sulfated LacNAc oligosaccharides are also present in the pleural mesothelium was unknown. The question of which GlcNAc6STs are responsible for R-10G-reactive glycans was an additional issue to be clarified. Here, we show that R-10G-reactive glycans are as abundant in the pulmonary pleura as CL40-reactive glycans and that GlcNAc6ST3 is only partially involved in the synthesis of these pleural R-10G glycans, unlike in the adult brain. Unexpectedly, GlcNAc6ST2 is essential for the synthesis of R-10G-positive KS/sulfated LacNAc oligosaccharides in the lung pleura. The type of GlcNAc6ST and the magnitude of its contribution to KS glycan synthesis varied among tissues in vivo. We show that GlcNAc6ST2 is required and sufficient for R-10G-reactive KS synthesis in the lung pleura. Interestingly, R-10G immunoreactivity in KSGal6ST (encoded by Chst1) and C6ST1 (encoded by Chst3) double-deficient mouse lungs was markedly increased. MUC16, a mucin molecule, was shown to be a candidate carrier protein for pleural R-10G-reactive glycans. These results suggest that R-10G-reactive KS/sulfated LacNAc oligosaccharides may play a role in mesothelial cell proliferation and differentiation. Further elucidation of the functions of sulfated glycans synthesized by GlcNAc6ST2 and GlcNAc6ST3, such as R-10G and CL40 glycans, in pathological conditions may lead to a better understanding of the underlying mechanisms of the physiopathology of the lung mesothelium.


Assuntos
Amino Açúcares , Sulfato de Queratano , Pleura , Animais , Camundongos , Sulfato de Queratano/metabolismo , Pleura/metabolismo , Oligossacarídeos , Polissacarídeos/metabolismo , Epitélio/metabolismo
11.
Environ Int ; 185: 108496, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38359549

RESUMO

Artificial sweeteners (AS) are extensively utilized as sugar substitutes and have been recognized as emerging environmental contaminants. While the effect of AS on aquatic organisms has garnered recent attention, their effects on soil invertebrates and gut microbial communities remain unclear. To address this knowledge gap, we exposed springtails (Folsomia candida) to both single and combined treatments of four typical AS (sucralose [SUC], saccharin [SAC], cyclamate [CYC], and acesulfame [ACE]) at environmentally relevant concentrations of 0.01, 0.1 and 1 mg kg-1 in soil. Following the first-generational exposure, the reproduction of juveniles showed a significant increase under all the AS treatments of 0.1 mg kg-1. The transcriptomic analysis revealed significant enrichment of several Kyoto Encyclopedia of Gene and Genome pathways (e.g., glycolysis/gluconeogenesis, pentose and glucuronate interconversions, amino sugar, and nucleotide sugar metabolism, ribosome, and lysosome) in springtails under all AS treatments. Analysis of gut bacterial microbiota indicated that three AS (SUC, CYC, and ACE) significantly decreased alpha diversity, and all AS treatments increased the abundance of the genus Achromobacter. After the sixth-generational exposure to CYC, weight increased, but reproduction was inhibited. The pathways that changed significantly (e.g., extracellular matrix-receptor interaction, amino sugar and nucleotide sugar metabolism, lysosome) were generally similar to those altered in first-generational exposure, but with opposite regulation directions. Furthermore, the effect on the alpha diversity of gut microbiota was contrary to that after first-generational exposure, and more noticeable disturbances in microbiota composition were observed. These findings underscore the ecological risk of AS in soils and improve our understanding of the toxicity effects of AS on living organisms.


Assuntos
Microbioma Gastrointestinal , Poluentes Químicos da Água , Edulcorantes/toxicidade , Edulcorantes/análise , Edulcorantes/metabolismo , Solo , Poluentes Químicos da Água/análise , Ciclamatos/análise , Amino Açúcares , Nucleotídeos
12.
Glycobiology ; 34(4)2024 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-38349796

RESUMO

Cell surface biomarkers are fundamental for specific characterization of human pluripotent stem cells (hPSCs). Importantly, they can be applied for hPSC enrichment and/or purification but also to remove potentially teratoma-forming hPSCs from differentiated populations before clinical application. Several specific markers for hPSCs are glycoconjugates comprising the glycosphingolipid (GSL)-based glycans SSEA-3 and SSEA-4. We applied an analytical approach based on multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection to quantitatively assess the GSL glycome of human embryonic stem cells and human induced pluripotent stem cells as well as during early stages of differentiation into mesoderm, endoderm, and ectoderm. Thereby, we identified the GSL lacto-N-tetraosylceramide (Lc4-Cer, Galß1-3GlcNAcß1-3Galß1-4Glc-Cer), which comprises a terminal type 1 LacNAc (T1LN) structure (Galß1-3GlcNAc), to be rapidly decreased upon onset of differentiation. Using a specific antibody, we could confirm a decline of T1LN-terminating glycans during the first four days of differentiation by live-cell staining and subsequent flow cytometry. We could further separate T1LN-positive and T1LN-negative cells out of a mixed population of pluripotent and differentiated cells by magnetic activated cell sorting. Notably, not only the T1LN-positive but also the T1LN-negative population was positive for SSEA-3, SSEA-4, and SSEA-5 while expression of nuclear pluripotency markers OCT4 and NANOG was highly reduced in the T1LN-negative population, exclusively. Our findings suggest T1LN as a pluripotent stem cell-specific glycan epitope that is more rapidly down-regulated upon differentiation than SSEA-3, SSEA-4, and SSEA-5.


Assuntos
Amino Açúcares , Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Humanos , Epitopos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Polissacarídeos/metabolismo , Diferenciação Celular
13.
Chemistry ; 30(5): e202302877, 2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-37909475

RESUMO

Poly-N-acetyl lactosamines (polyLacNAc) are common structural motifs of N- and O-linked glycan, glycosphingolipids and human milk oligosaccharides. They can be branched by the addition of ß1,6-linked N-acetyl-glucosamine (GlcNAc) moieties to internal galactoside (Gal) residues by the I-branching enzyme beta-1,6-N-acetylglucosaminyltransferase 2 (GCNT2). I-branching has been implicated in many biological processes and is also associated with various diseases such as cancer progression. Currently, there is a lack of methods that can install, in a regioselective manner, I-branches and allows the preparation of isomeric poly-LacNAc derivatives. Here, we described a chemo-enzymatic strategy that addresses this deficiency and is based on the enzymatic assembly of an oligo-LacNAc chain that at specific positions is modified by a GlcNTFA moiety. Replacement of the trifluoroacetyl (TFA) moiety by tert-butyloxycarbonyl (Boc) gives compounds in which the galactoside at the proximal site is blocked from modification by GCNT2. After elaboration of the antennae, the Boc group can be removed, and the resulting amine acetylated to give natural I-branched structures. It is also shown that fucosides can function as a traceless blocking group that can provide complementary I-branched structures from a single precursor. The methodology made it possible to synthesize a library of polyLacNAc chains having various topologies.


Assuntos
N-Acetilglucosaminiltransferases , Polissacarídeos , Humanos , Polissacarídeos/química , Amino Açúcares/química , Galactosídeos
14.
Environ Sci Technol ; 58(4): 1966-1975, 2024 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-38153028

RESUMO

Polysaccharides in extracellular polymeric substances (EPS) can form a hybrid matrix network with proteins, impeding waste-activated sludge (WAS) fermentation. Amino sugars, such as N-acetyl-d-glucosamine (GlcNAc) polymers and sialic acid, are the non-negligible components in the EPS of aerobic granules or biofilm. However, the occurrence of amino sugars in WAS and their degradation remains unclear. Thus, amino sugars (∼6.0%) in WAS were revealed, and the genera of Lactococcus and Zoogloea were identified for the first time. Chitin was used as the substrate to enrich a chitin-degrading consortium (CDC). The COD balances for methane production ranged from 83.3 and 95.1%. Chitin was gradually converted to oligosaccharides and GlcNAc after dosing with the extracellular enzyme. After doing enriched CDC in WAS, the final methane production markedly increased to 60.4 ± 0.6 mL, reflecting an increase of ∼62%. Four model substrates of amino sugars (GlcNAc and sialic acid) and polysaccharides (cellulose and dextran) could be used by CDC. Treponema (34.3%) was identified as the core bacterium via excreting chitinases (EC 3.2.1.14) and N-acetyl-glucosaminidases (EC 3.2.1.52), especially the genetic abundance of chitinases in CDC was 2.5 times higher than that of WAS. Thus, this study provides an elegant method for the utilization of amino sugar-enriched organics.


Assuntos
Quitinases , Esgotos , Amino Açúcares , Fermentação , Ácido N-Acetilneuramínico , Quitina/química , Quitina/metabolismo , Polissacarídeos , Quitinases/química , Quitinases/metabolismo , Metano
15.
Appl Environ Microbiol ; 89(12): e0167423, 2023 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-37982622

RESUMO

IMPORTANCE: Central metabolism plays a key role in the control of growth and antibiotic production in streptomycetes. Specifically, aminosugars act as signaling molecules that affect development and antibiotic production, via metabolic interference with the global repressor DasR. While aminosugar metabolism directly connects to other major metabolic routes such as glycolysis and cell wall synthesis, several important aspects of their metabolism are yet unresolved. Accumulation of N-acetylglucosamine 6-phosphate or glucosamine 6-phosphate is lethal to many bacteria, a yet unresolved phenomenon referred to as "aminosugar sensitivity." We made use of this concept by selecting for suppressors in genes related to glucosamine toxicity in nagB mutants, which showed that the gene pair of rok-family regulatory gene rokL6 and major facilitator superfamily transporter gene sco1448 forms a cryptic rescue mechanism. Inactivation of rokL6 resulted in the expression of sco1448, which then prevents the toxicity of amino sugar-derived metabolites in Streptomyces. The systems biology of RokL6 and its transcriptional control of sco1448 shed new light on aminosugar metabolism in streptomycetes and on the response of bacteria to aminosugar toxicity.


Assuntos
Streptomyces coelicolor , Streptomyces , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Glucosamina/metabolismo , Streptomyces/genética , Amino Açúcares/metabolismo , Antibacterianos , Genes Reguladores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica
16.
Biochemistry ; 62(20): 2970-2981, 2023 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-37782650

RESUMO

Covalent modification of lipid A with 4-deoxy-4-amino-l-arabinose (Ara4N) mediates resistance to cationic antimicrobial peptides and polymyxin antibiotics in Gram-negative bacteria. The proteins required for Ara4N biosynthesis are encoded in the pmrE and arnBCADTEF loci, with ArnT ultimately transferring the amino sugar from undecaprenyl-phospho-4-deoxy-4-amino-l-arabinose (C55P-Ara4N) to lipid A. However, Ara4N is N-formylated prior to its transfer to undecaprenyl-phosphate by ArnC, requiring a deformylase activity downstream in the pathway to generate the final C55P-Ara4N donor. Here, we show that deletion of the arnD gene in an Escherichia coli mutant that constitutively expresses the arnBCADTEF operon leads to accumulation of the formylated ArnC product undecaprenyl-phospho-4-deoxy-4-formamido-l-arabinose (C55P-Ara4FN), suggesting that ArnD is the downstream deformylase. Purification of Salmonella typhimurium ArnD (stArnD) shows that it is membrane-associated. We present the crystal structure of stArnD revealing a NodB homology domain structure characteristic of the metal-dependent carbohydrate esterase family 4 (CE4). However, ArnD displays several distinct features: a 44 amino acid insertion, a C-terminal extension in the NodB fold, and sequence divergence in the five motifs that define the CE4 family, suggesting that ArnD represents a new family of carbohydrate esterases. The insertion is responsible for membrane association as its deletion results in a soluble ArnD variant. The active site retains a metal coordination H-H-D triad, and in the presence of Co2+ or Mn2+, purified stArnD efficiently deformylates C55P-Ara4FN confirming its role in Ara4N biosynthesis. Mutations D9N and H233Y completely inactivate stArnD implicating these two residues in a metal-assisted acid-base catalytic mechanism.


Assuntos
Lipídeo A , Polimixinas , Polimixinas/farmacologia , Polimixinas/metabolismo , Lipídeo A/metabolismo , Arabinose/metabolismo , Amino Açúcares/química , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Carboidratos , Proteínas de Bactérias/química
17.
Org Lett ; 25(43): 7873-7877, 2023 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-37862141

RESUMO

A synthetic route to 2,4-diamino-2,4,6-trideoxysugar stereoisomers in 6-7 steps and 22-33% overall yield is described. A key step in this pathway is the carbonyl coupling of d- and l-threoninol or d- and l-allo-threoninol to a phthalimido-allene mediated by chiral iridium-H8-BINAP, which allows for installation of two new chiral centers in one, highly diastereoselective (>20:1 dr) step. This approach provides a more concise, diastereoselective, and versatile method to access these deoxy-amino sugars than is currently available.


Assuntos
Amino Álcoois , Amino Açúcares , Butileno Glicóis , Estereoisomerismo
18.
Ying Yong Sheng Tai Xue Bao ; 34(10): 2601-2609, 2023 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-37897266

RESUMO

We sieved soils from a Pinus massoniana plantation in the Three Gorges Reservoir area into four aggregate sizes, including aggregates of 2000-8000 µm (large macroaggregates), 1000-2000 µm (coarse aggregates), 250-1000 µm (small macroaggregates), and <250 µm (microaggregates). We analyzed the differences in the acidolyzable organic N components and net N mineralization of the aggregates under different N addition levels (30, 60, and 90 kg N·hm-2·a-1, representing by N30, N60 and N90, respectively). The results showed that net nitrification rate of the aggregates ranged from 0.30-3.42 mg N·kg-1 and accounted for more than 80% of net nitrogen mineralization. Compared with the control, addition of 30, 60, and 90 kg N·hm-2·a-1 increased total N by 24.1%-45.5%, 6.4%-34.3%, and 7.9%-42.4% in the large aggregates, coarse aggregate, small macroaggregates, and microaggregates, increased net N mineralization rate by 1.3-7.2, 1.4-6.6, and 1.8-12.9 times, but decreased the contents of available phosphorus by 9.3%-36.9%, 12.2%-56.7%, and 19.2%-61.9%, respectively. The contents of total acidolyzable N, soil organic matter, and rates of net ammonification, net nitrification, and net N mineralization increased as the aggregate size decreased, while available phosphorus contents showed an opposite trend. The levels of acid-hydrolyzable N components were ranked as acidolyzable amino acid N > acidolyzable ammonia N > acidolyzable unknown N> acidolyzable amino sugar N. Total N was the dominant contributor to the increases in acid-hydrolyzable N components. Results of stepwise multiple regression analyses showed that acidoly-zable amino acid N and acidolyzable amino sugar N were predictors of net ammonification rate. Acidolyzable amino sugar N, acidolyzable amino acid N, and acidolyzable ammonia N were predictors of net nitrification, net nitrogen mineralization rate, and net nitrogen mineralization accumulation. The physical structure of aggregates was associa-ted with soil net N mineralization. Addition of N increased the contents and bioavailability of acidolyzable organic N, a large amount of which contributed to soil organic matter levels and the decrease in available phosphorus.


Assuntos
Nitrogênio , Pinus , Nitrogênio/análise , Amônia/análise , Solo/química , Fósforo/análise , China , Aminoácidos , Amino Açúcares , Carbono/análise
19.
Org Lett ; 25(34): 6413-6418, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37603587

RESUMO

Herein we report the first total synthesis of a densely functionalized tetrasaccharide repeating unit of Vibrio cholerae O43, which contains rare deoxy amino sugars d-quinovosamine and d-viosamine attached with the rare amino acid N-acetyl-l-allothreonine. Synthesis of orthogonally protected rare sugars and unnatural amino acid building blocks, stereoselective construction of three consecutive 1,2-cis glycosidic linkages, amide coupling, and the presence of five nitrogen atoms dispersed over four sugar units as well as the carboxylic acid functionality make the total synthesis a formidable task.


Assuntos
Oligossacarídeos , Vibrio cholerae , Amidas , Aminoácidos , Amino Açúcares
20.
Molecules ; 28(12)2023 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-37375279

RESUMO

Amino sugars are a kind of carbohydrates with one or more hydroxyl groups replaced by an amino group. They play crucial roles in a broad range of biological activities. Over the past few decades, there have been continuing efforts on the stereoselective glycosylation of amino sugars. However, the introduction of glycoside bearing basic nitrogen is challenging using conventional Lewis acid-promoted pathways owing to competitive coordination of the amine to the Lewis acid promoter. Additionally, diastereomeric mixtures of O-glycoside are often produced if aminoglycoside lack a C2 substituent. This review focuses on the updated overview of the way to stereoselective synthesis of 1,2-cis-aminoglycoside. The scope, mechanism, and the applications in the synthesis of complex glycoconjugates for the representative methodologies were also included.


Assuntos
Amino Açúcares , Glicosídeos Cardíacos , Ácidos de Lewis , Carboidratos , Glicoconjugados , Aminoglicosídeos , Estereoisomerismo
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