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1.
Plant Signal Behav ; 18(1): 2173146, 2023 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-36723515

RESUMO

The plant extracellular ATP (eATP) receptor, P2K2, binds eATP with strong ligand affinity through its extracellular lectin domain. Ligand binding activates the intracellular kinase domain of P2K2 resulting in a variety of intracellular responses and, ultimately, increased plant immunity to invading fungal and bacterial pathogens. Here, using a computational prediction approach, we developed a tertiary structure model of the P2K2 extracellular lectin domain. In silico target docking of ATP to the P2K2-binding site predicted interaction with several residues through hydrophobic interactions and hydrogen bonding. Our confirmation of the modeling was obtained by showing that H99, R144, and S256 are key residues essential for in vitro binding of ATP by P2K2.


Assuntos
Trifosfato de Adenosina , Receptores Purinérgicos P2 , Ligantes , Sítios de Ligação , Trifosfato de Adenosina/metabolismo , Lectinas/metabolismo
2.
Int J Mol Sci ; 24(2)2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36674948

RESUMO

In Alzheimer's disease (AD), the reduction in acetylcholinesterase (AChE) enzymatic activity is not paralleled with changes in its protein levels, suggesting the presence of a considerable enzymatically inactive pool in the brain. In the present study, we validated previous findings, and, since inactive forms could result from post-translational modifications, we analyzed the glycosylation of AChE by lectin binding in brain samples from sporadic and familial AD (sAD and fAD). Most of the enzymatically active AChE was bound to lectins Canavalia ensiformis (Con A) and Lens culinaris agglutinin (LCA) that recognize terminal mannoses, whereas Western blot assays showed a very low percentage of AChE protein being recognized by the lectin. This indicates that active and inactive forms of AChE vary in their glycosylation pattern, particularly in the presence of terminal mannoses in active ones. Moreover, sAD subjects showed reduced binding to terminal mannoses compared to non-demented controls, while, for fAD patients that carry mutations in the PSEN1 gene, the binding was higher. The role of presenilin-1 (PS1) in modulating AChE glycosylation was then studied in a cellular model that overexpresses PS1 (CHO-PS1). In CHO-PS1 cells, binding to LCA indicates that AChE displays more terminal mannoses in oligosaccharides with a fucosylated core. Immunocytochemical assays also demonstrated increased presence of AChE in the trans-Golgi. Moreover, AChE enzymatic activity was higher in plasmatic membrane of CHO-PS1 cells. Thus, our results indicate that PS1 modulates trafficking and maturation of AChE in Golgi regions favoring the presence of active forms in the membrane.


Assuntos
Acetilcolinesterase , Doença de Alzheimer , Cricetinae , Animais , Humanos , Acetilcolinesterase/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Doença de Alzheimer/metabolismo , Lectinas/metabolismo , Encéfalo/metabolismo , Cricetulus , Presenilina-2/genética , Mutação
3.
BMC Biol ; 21(1): 7, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36635688

RESUMO

BACKGROUND: Innate immunity is the first line of defense against pathogens. In animals, the Toll pathway, the Imd pathway, the complement system, and lectins are well-known mechanisms involved in innate immunity. Although these pathways and systems are well understood in vertebrates and arthropods, they are understudied in other invertebrates. RESULTS: To shed light on immunity in the nemertean Lineus ruber, we performed a transcriptomic survey and identified the main components of the Toll pathway (e.g., myD88, dorsal/dif/NFκB-p65), the Imd pathway (e.g., imd, relish/NFκB-p105/100), the complement system (e.g., C3, cfb), and some lectins (FreD-Cs and C-lectins). In situ hybridization showed that TLRß1, TLRß2, and imd are expressed in the nervous system; the complement gene C3-1 is expressed in the gut; and the lectins are expressed in the nervous system, the blood, and the gut. To reveal their potential role in defense mechanisms, we performed immune challenge experiments, in which Lineus ruber specimens were exposed to the gram-negative bacteria Vibrio diazotrophicus. Our results show the upregulation of specific components of the Toll pathway (TLRα3, TLRß1, and TLRß2), the complement system (C3-1), and lectins (c-lectin2 and fred-c5). CONCLUSIONS: Therefore, similarly to what occurs in other invertebrates, our study shows that components of the Toll pathway, the complement system, and lectins are involved in the immune response in the nemertean Lineus ruber. The presence of these pathways and systems in Lineus ruber, but also in other spiralians; in ecdysozoans; and in deuterostomes suggests that these pathways and systems were involved in the immune response in the stem species of Bilateria.


Assuntos
Invertebrados , Vibrioses , Animais , Vibrioses/veterinária , NF-kappa B , Imunidade Inata , Lectinas
4.
Molecules ; 28(2)2023 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-36677706

RESUMO

The glycosylation of proteins is one of the most common post-translational modifications (PTMs) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome, maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of tear film might promote the development of chronic eye diseases, indicating glycoproteins as a valuable source for biomarker discovery or drug target identification. Therefore, the present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and to characterize their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we first accumulated native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA, and UEA I, termed 4L) was applied to glycopeptide enrichment from human tear sample digests, followed by MS-based detection and localization of their specific N-glycosylation sites. As the main result, our study identified a total of 26 N glycosylation sites of 11 N-glycoproteins in the tear sample pools of healthy individuals (n = 3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP), and extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, already associated with the pathogenesis of various chronic eye diseases such as dry eye syndrome (DES). In conclusion, the results of the present study will serve as an important tear film N-glycoprotein catalog for future studies focusing on human tear film and ocular surface-related inflammatory diseases.


Assuntos
Glicoproteínas , Lectinas , Lágrimas , Humanos , Glicopeptídeos/química , Glicoproteínas/química , Glicosilação , Lectinas/química , Espectrometria de Massas/métodos , Processamento de Proteína Pós-Traducional , Lágrimas/química
5.
Int J Mol Sci ; 24(1)2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36614320

RESUMO

A late stage of several retinal disorders is retinal detachment, a complication that results in rapid photoreceptor degeneration and synaptic damages. Experimental retinal detachment in vivo is an invasive and complicated method performed on anesthetized animals. As retinal detachment may result in visual impairment and blindness, research is of fundamental importance for understanding degenerative processes. Both morphological and ethical issues make the porcine retina a favorable organotypic model for studies of the degenerative processes that follow retinal detachment. In the cultured retina, photoreceptor degeneration and synaptic injuries develop rapidly and correlate with resident microglial cells' transition into a reactive phenotype. In this immunohistochemical study, we have begun to analyze the transition of subsets of reactive microglia which are known to localize close to the outer plexiform layer (OPL) in degenerating in vivo and in vitro retina. Biomarkers for reactive microglia included P2Ry12, CD63 and CD68 and the general microglial markers were CD11b, Iba1 and isolectin B4 (IB4). The reactive microglia markers labeled microglia subpopulations, suggesting that protective or harmful reactive microglia may be present simultaneously in the injured retina. Our findings support the usage of porcine retina cultures for studies of photoreceptor injuries related to retinal detachment.


Assuntos
Degeneração Retiniana , Descolamento Retiniano , Animais , Suínos , Microglia , Retina , Lectinas
6.
Int J Biol Sci ; 19(2): 362-376, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36632465

RESUMO

Hepatocellular carcinoma (HCC) is the third-leading cause of cancer deaths globally. Although considerable progress has been made in the treatment, clinical outcomes of HCC patients are still poor. Therefore, it is necessary to find novel prognostic factors upon which prevention and treatment strategies can be formulated. Ficolin-3 (FCN3) protein is a member of the human ficolin family. It activates complement through pathways associated with mannose-binding lectin-associated serine proteases. Herein, we identified that FCN3 was downregulated in HCC tissues and decreased FCN3 expression was closely related to poor prognosis. Overexpression of FCN3 induced apoptosis and inhibited cell proliferation via the p53 signaling pathway. Mechanistically, FCN3 modulated the nuclear translocation of eukaryotic initiation factor 6 (EIF6) by binding ribosome maturation factor (SBDS), which induced ribosomal stress and activation of the p53 pathway. In addition, Y-Box Binding Protein 1 (YBX1) involved in the transcription and translation level regulation of FCN3 to SBDS. Besides, a negative feedback loop in the downstream of FCN3 involving p53, YBX1 and SBDS was identified.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Neoplasias Hepáticas/metabolismo , Proliferação de Células/genética , Linhagem Celular Tumoral , Proteínas/metabolismo , Lectinas/metabolismo
7.
Sci Rep ; 13(1): 525, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36631627

RESUMO

A novel antibacterial immunostimulant using Platinum nanoparticles (PtNPs) and lectin from Metapenaeus dobsoni (Md-Lec) was developed. The Md-Lec and PtNPs (Pt-lec) hybrid formed through non-covalent interaction exhibits antimicrobial activity against fish specific pathogens by affecting membrane integrity and producing excess reactive oxygen species. The therapeutic efficacy of Pt-lec was demonstrated through rescuing Aeromonas hydrophila infected Nile Tilapia. Pt-lec prevents the infection spreading and reduces the bacterial bioburden in less than 12 h, and as a result of this the fish were restored to normalcy. To assess immunostimulation, we studied the expression of three different immune related genes, namely LEC, Myd88 and COX-2 in the gills, liver, spleen and kidney of fish under various experimental conditions. Our results showed that Pt-lec treatment appeared to be better when compared to lectin alone in enhancing the expression of Myd88 and COX-2, but LEC was not as expected. These results suggest that Pt-lec has the ability to protect Nile Tilapia against bacterial infection by restricting bacterial bioburden through their direct effects on the bacterial membrane and indirectly through their effects on host immune-related gene expression. This hybrid could have potential "green" application in fish farming in rescuing infected animals when compared to widely and unregulated antibiotics.


Assuntos
Anti-Infecciosos , Ciclídeos , Doenças dos Peixes , Infecções por Bactérias Gram-Negativas , Nanopartículas Metálicas , Penaeidae , Animais , Ciclídeos/microbiologia , Platina/farmacologia , Lectinas/farmacologia , Ciclo-Oxigenase 2 , Fator 88 de Diferenciação Mieloide , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/veterinária , Antibacterianos/farmacologia , Imunização , Doenças dos Peixes/microbiologia , Aeromonas hydrophila
8.
J Immunother Cancer ; 11(1)2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36653070

RESUMO

BACKGROUND: Cell therapies for solid tumors are thwarted by the hostile tumor microenvironment (TME) and by heterogeneous expression of tumor target antigens. We address both limitations with a novel class of chimeric antigen receptors based on plant lectins, which recognize the aberrant sugar residues that are a 'hallmark' of both malignant and associated stromal cells. We have expressed in T cells a modified lectin from banana, H84T BanLec, attached to a chimeric antigen receptor (H84T-CAR) that recognizes high-mannose (asparagine residue with five to nine mannoses). Here, we tested the efficacy of our novel H84T CAR in models of pancreatic ductal adenocarcinoma (PDAC), intractable tumors with aberrant glycosylation and characterized by desmoplastic stroma largely contributed by pancreatic stellate cells (PSCs). METHODS: We transduced human T cells with a second-generation retroviral construct expressing the H84T BanLec chimeric receptor, measured T-cell expansion, characterized T-cell phenotype, and tested their efficacy against PDAC tumor cells lines by flow cytometry quantification. In three-dimensional (3D) spheroid models, we measured H84T CAR T-cell disruption of PSC architecture, and T-cell infiltration by live imaging. We tested the activity of H84T CAR T cells against tumor xenografts derived from three PDAC cell lines. Antitumor activity was quantified by caliper measurement and bioluminescence signal and used anti-human vimentin to measure residual PSCs. RESULTS: H84T BanLec CAR was successfully transduced and expressed by T cells which had robust expansion and retained central memory phenotype in both CD4 and CD8 compartments. H84T CAR T cells targeted and eliminated PDAC tumor cell lines. They also disrupted PSC architecture in 3D models in vitro and reduced total tumor and stroma cells in mixed co-cultures. H84T CAR T cells exhibited improved T-cell infiltration in multicellular spheroids and had potent antitumor effects in the xenograft models. We observed no adverse effects against normal tissues. CONCLUSIONS: T cells expressing H84T CAR target malignant cells and their stroma in PDAC tumor models. The incorporation of glycan-targeting lectins within CARs thus extends their activity to include both malignant cells and their supporting stromal cells, disrupting the TME that otherwise diminishes the activity of cellular therapies against solid tumors.


Assuntos
Carcinoma Ductal Pancreático , Musa , Neoplasias Pancreáticas , Receptores de Antígenos Quiméricos , Humanos , Musa/metabolismo , Lectinas/metabolismo , Linfócitos T , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Microambiente Tumoral
9.
J Am Chem Soc ; 145(3): 1668-1677, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36640106

RESUMO

Multivalent interactions between carbohydrates and proteins enable a broad range of selective chemical processes of critical biological importance. Such interactions can extend from the macromolecular scale (1-10 nm) up to much larger scales across a cell or tissue, placing substantial demands on chemically patterned materials aiming to leverage similar interactions in vitro. Here, we show that diyne amphiphiles with carbohydrate headgroups can be assembled on highly oriented pyrolytic graphite (HOPG) to generate nanometer-resolution carbohydrate patterns, with individual linear carbohydrate assemblies up to nearly 1 µm, and microscale geometric patterns. These are then photopolymerized and covalently transferred to the surfaces of hydrogels. This strategy suspends carbohydrate patterns on a relatively rigid polydiacetylene (persistence length ∼ 16 nm), exposed at the top surface of the hydrogel above the bulk pore structure. Transferred patterns of appropriate carbohydrates (e.g., N-acetyl-d-glucosamine, GlcNAc) enable selective, multivalent interactions (KD ∼ 40 nM) with wheat germ agglutinin (WGA), a model lectin that exhibits multivalent binding with appropriately spaced GlcNAc moieties. WGA binding affinity can be further improved (KD ∼ 10 nM) using diacetylenes that shift the polymer backbone closer to the displayed carbohydrate, suggesting that this strategy can be used to modulate carbohydrate presentation at interfaces. Conversely, GlcNAc-patterned surfaces do not induce specific binding of concanavalin A, and surfaces patterned with glucuronic acid, or with simple carboxylic acid or hydroxyl groups, do not induce WGA binding. More broadly, this approach may have utility in designing synthetic glycan-mimetic interfaces with features from molecular to mesoscopic scales, including soft scaffolds for cells.


Assuntos
Hidrogéis , Lectinas , Lectinas/metabolismo , Carboidratos/química , Aglutininas do Germe de Trigo/química , Concanavalina A
10.
Int J Mol Sci ; 24(2)2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36674590

RESUMO

Receptors on the immune cell surface have a variety of glycans that may account for the immunomodulation induced by lectins, which have a carbohydrate recognition domain (CRD) that binds to monosaccharides or oligosaccharides in a specific manner. ArtinM, a D-mannose-binding lectin obtained from Artocarpus heterophyllus, has affinity for the N-glycans core. Immunomodulation by ArtinM toward the Th1 phenotype occurs via its interaction with TLR2/CD14 N-glycans on antigen-presenting cells, as well as recognition of CD3γ N-glycans on murine CD4+ and CD8+ T cells. ArtinM exerts a cytotoxic effect on Jurkat human leukemic T-cell line and human myeloid leukemia cell line (NB4). The current study evaluated the effects of ArtinM on murine and human B cells derived from non-Hodgkin's lymphoma. We found that murine B cells are recognized by ArtinM via the CRD, and the ArtinM stimulus did not augment the proliferation rate or production of IL-2. However, murine B cell incubation with ArtinM augmented the rate of apoptosis, and this cytotoxic effect of ArtinM was also seen in human B cell-lines sourced from non-Hodgkin's lymphoma Raji cell line. This cytotoxic effect was inhibited by the phosphatase activity of CD45 on Lck, and the protein kinases of the Src family contribute to cell death triggered by ArtinM.


Assuntos
Linfoma não Hodgkin , Quinases da Família src , Camundongos , Humanos , Animais , Lectinas/farmacologia , Linhagem Celular , Polissacarídeos/metabolismo , Quinase Syk
11.
PLoS One ; 18(1): e0273955, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36602978

RESUMO

Lactococcus lactis strains are used as starter cultures in the production of fermented dairy and vegetable foods, but the species also occurs in other niches such as plant material. Lactococcus lactis subsp. lactis G50 (G50) is a plant-derived strain and potential candidate probiotics. Western blotting of cell-wall proteins using antibodies generated against whole G50 cells detected a 120-kDa protein. MALDI-TOF MS analysis identified it as YwfG, a Leu-Pro-any-Thr-Gly cell-wall-anchor-domain-containing protein. Based on a predicted domain structure, a recombinant YwfG variant covering the N-terminal half (aa 28-511) of YwfG (YwfG28-511) was crystallized and the crystal structure was determined. The structure consisted of an L-type lectin domain, a mucin-binding protein domain, and a mucus-binding protein repeat. Recombinant YwfG variants containing combinations of these domains (YwfG28-270, YwfG28-336, YwfG28-511, MubR4) were prepared and their interactions with monosaccharides were examined by isothermal titration calorimetry; the only interaction observed was between YwfG28-270, which contained the L-type lectin domain, and d-mannose. Among four mannobioses, α-1,2-mannobiose had the highest affinity for YwfG28-270 (dissociation constant = 34 µM). YwfG28-270 also interacted with yeast mannoproteins and yeast mannan. Soaking of the crystals of YwfG28-511 with mannose or α-1,2-mannobiose revealed that both sugars bound to the L-type lectin domain in a similar manner, although the presence of the mucin-binding protein domain and the mucus-binding protein repeat within the recombinant protein inhibited the interaction between the L-type lectin domain and mannose residues. Three of the YwfG variants (except MubR4) induced aggregation of yeast cells. Strain G50 also induced aggregation of yeast cells, which was abolished by deletion of ywfG from G50, suggesting that surface YwfG contributes to the interaction with yeast cells. These findings provide new structural and functional insights into the interaction between L. lactis and its ecological niche via binding of the cell-surface protein YwfG with mannose.


Assuntos
Lactococcus lactis , Manose , Manose/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Proteínas de Membrana/metabolismo , Saccharomyces cerevisiae , Lectinas/metabolismo , Mucinas/metabolismo
12.
Chem Soc Rev ; 52(2): 536-572, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36545903

RESUMO

Human lectins are involved in a wide variety of biological processes, both physiological and pathological, which have attracted the interest of the scientific community working in the glycoscience field. Multivalent glycosystems have been employed as useful tools to understand carbohydrate-lectin binding processes as well as for biomedical applications. The review shows the different scaffolds designed for a multivalent presentation of sugars and their corresponding binding studies to lectins and in some cases, their biological activities. We summarise this research by organizing based on lectin types to highlight the progression in this active field. The paper provides an overall picture of how these contributions have furnished relevant information on this topic to help in understanding and participate in these carbohydrate-lectin interactions.


Assuntos
Carboidratos , Lectinas , Humanos , Lectinas/metabolismo
13.
Methods Mol Biol ; 2557: 691-707, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36512245

RESUMO

The Golgi apparatus is one of the major sites of protein and lipid glycosylation and processing. Protein N-glycosylation plays critical roles in protein folding, transport, stability, and activity. Various glycosyltransferases and glycoside hydrolases are localized at each cisterna in the Golgi apparatus and synthesize a large variety of N-glycan structures. The biosynthetic pathways of N-glycans are complicated, which hiders the rational design of glycan metabolic pathways. In addition, the analysis of glycan structure requires specialized instruments for analyses such as mass spectrometry, high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy, which are not familiar to all laboratories. Here, we introduce relatively simple methods for N-glycan analysis, including disruption of genes encoding glycosyltransferases or glycoside hydrolases, glycan structural analysis using lectins and mass spectrometry, and visualization of glycan metabolic pathways in silico.


Assuntos
Glicosiltransferases , Lectinas , Espectrometria de Massas , Glicosiltransferases/metabolismo , Polissacarídeos/química , Glicosídeo Hidrolases/metabolismo , Coloração e Rotulagem
14.
Biochem Pharmacol ; 207: 115367, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36481348

RESUMO

Often the outer leaflets of living cells bear a coat of glycosylated proteins, which primarily regulates cellular processes. Glycosylation of such proteins occurs as part of their post-translational modification. Within the endoplasmic reticulum, glycosylation enables the attachment of specific oligosaccharide moieties such as, 'glycan' to the transmembrane receptor proteins which confers precise biological information for governing the cell fate. The nature and degree of glycosylation of cell surface receptors are regulated by a bunch of glycosyl transferases and glycosidases which fine-tune attachment or detachment of glycan moieties. In classical death receptors, upregulation of glycosylation by glycosyl transferases is capable of inducing cell death in T cells, tumor cells, etc. Thus, any deregulated alternation at surface glycosylation of these death receptors can result in life-threatening disorder like cancer. In addition, transmembrane glycoproteins and lectin receptors can transduce intracellular signals for cell death execution. Exogenous interaction of lectins with glycan containing death receptors signals for cell death initiation by modulating downstream signalings. Subsequently, endogenous glycan-lectin interplay aids in the customization and implementation of the cell death program. Lastly, the glycan-lectin recognition system dictates the removal of apoptotic cells by sending accurate signals to the extracellular milieu. Since glycosylation has proven to be a biomarker of cellular death and disease progression; glycans serve as specific therapeutic targets of cancers. In this context, we are reviewing the molecular mechanisms of the glycan-lectin regulatory network as an integral part of cell death machinery in cancer to target them for successful therapeutic and clinical approaches.


Assuntos
Lectinas , Neoplasias , Humanos , Glicômica , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Polissacarídeos/metabolismo , Transferases , Receptores de Morte Celular
15.
Talanta ; 253: 123882, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36088845

RESUMO

A CdS/AuNPs/NiO Z-scheme heterojunction was prepared on a fluorine-doped tin oxide (FTO) electrode by hydrothermal synthesis of NiO on FTO, electrodeposition of AuNPs on NiO/FTO electrode and then cast-coating of CdS quantum dots. The CdS/AuNPs/NiO/FTO electrode gave a notably increased photocurrent versus NiO/FTO, CdS/FTO, AuNPs/NiO/FTO, CdS/AuNPs/FTO and CdS/NiO/FTO electrodes. The CdS/AuNPs/NiO/FTO electrode was further cast-coated with chitosan to immobilize d-mannose by Schiff base reaction, and concanavalin A (ConA) and then horseradish peroxidase (HRP) were captured on the electrode surface by lectin-sugar binding. 4-Chloro-1-naphthol (4-CN) was oxidized to form an insoluble precipitate catalyzed by HRP in the presence of H2O2, and the presence of precipitate on the photoelectrode inhibited the photocurrent in the presence of holes scavenger ascorbic acid. The relevant electrodes were characterized by electrochemistry, quartz crystal microbalance (QCM), UV-vis spectrophotometry, scanning electron microscopy/energy dispersive spectroscopy, and transmission electron microscopy. The QCM revealed that the collection efficiency (η) of the 4-CN-electrooxidation precipitate on the electrode can be as high as 91.8%. Under the optimal conditions, the decline of photocurrent responded linearly to the common logarithm of ConA concentration from 50 pM to 500 nM, with a limit of detection of 17 pM (S/N = 3). Satisfactory results were obtained in the detection of real soybean samples.


Assuntos
Lectinas , Nanopartículas Metálicas , Concanavalina A , Ouro , Peróxido de Hidrogênio , Açúcares
16.
J Exp Med ; 220(1)2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36413219

RESUMO

Intelectin-1 (ITLN1) is a lectin secreted by intestinal epithelial cells (IECs) and upregulated in human ulcerative colitis (UC). We investigated how ITLN1 production is regulated in IECs and the biological effects of ITLN1 at the host-microbiota interface using mouse models. Our data show that ITLN1 upregulation in IECs from UC patients is a consequence of activating the unfolded protein response. Analysis of microbes coated by ITLN1 in vivo revealed a restricted subset of microorganisms, including the mucolytic bacterium Akkermansia muciniphila. Mice overexpressing intestinal ITLN1 exhibited decreased inner colonic mucus layer thickness and closer apposition of A. muciniphila to the epithelial cell surface, similar to alterations reported in UC. The changes in the inner mucus layer were microbiota and A. muciniphila dependent and associated with enhanced sensitivity to chemically induced and T cell-mediated colitis. We conclude that by determining the localization of a select group of bacteria to the mucus layer, ITLN1 modifies this critical barrier. Together, these findings may explain the impact of ITLN1 dysregulation on UC pathogenesis.


Assuntos
Colite Ulcerativa , Verrucomicrobia , Humanos , Camundongos , Animais , Verrucomicrobia/metabolismo , Muco/metabolismo , Lectinas , Colite Ulcerativa/metabolismo , Colite Ulcerativa/microbiologia , Colite Ulcerativa/patologia
17.
ACS Chem Biol ; 18(1): 70-80, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36525666

RESUMO

Glycan-binding proteins (GBPs) are widely used reagents for basic research and clinical applications. These reagents allow for the identification and manipulation of glycan determinants without specialized equipment or time-consuming experimental methods. Existing GBPs, mainly antibodies and lectins, are limited, and discovery or creation of reagents with novel specificities is time consuming and difficult. Here, we detail the generation of GBPs from a small, hyper-thermostable DNA-binding protein by directed evolution. Yeast surface display of a variable library of rcSso7d proteins was screened to find variants with selectivity toward the cancer-associated glycan Galß1-3GalNAcα or Thomsen-Friedenreich antigen and various relevant disaccharides. Characterization of these proteins shows them to have specificities and affinities on par with currently available lectins. The proteins can be readily functionalized with fluorophores or biotin using sortase-mediated ligation to create reagents that prove useful for glycoprotein blotting and cell staining applications. The presented methods for the development of GBPs toward specific saccharides of interest will have great impact on both biomedical and glycobiological research.


Assuntos
Proteínas de Transporte , Dissacarídeos , Antígenos Glicosídicos Associados a Tumores , Lectinas/metabolismo
18.
Int J Biol Macromol ; 229: 432-442, 2023 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-36581040

RESUMO

Aphid (Aphis gossypii Glover) attack frequently results in a significant loss of output and deterioration of fruit quality in cucumber (Cucumis sativus L.). Phloem protein 2 (PP2) is conserved as a phloem lectin in plants, and few studies have been conducted on the regulatory mechanism of PP2. Based on our previous study of CsPP2-A1 in cucumber, to further investigate the biological function of CsPP2-A1, we compared the changes of selectivity, non-selectivity, colonization, reproductions of aphids, and the phenotype in wild type (WT), CsPP2-A1 overexpressing (CsPP2-A1-OE), and CsPP2-A1 interfering (CsPP2-A1-RNAi) cucumber plants after inoculation with aphids. We found that CsPP2-A1-OE cucumber plants generated resistance to aphids. The aphid colonization rate and number of reproductions of CsPP2-A1-OE cucumber plants were significantly lower than that of WT and CsPP2-A1-RNAi cucumber plants. Through Pearson's correlation and principal component analysis (PCA), it was found that CsPP2-A1 played a crucial role in the balance of reactive oxygen species (ROS) in plants. Overexpression of the CsPP2-A1 resulted in increased levels of antioxidant enzyme, eliminating ROS and preventing the damage by ROS in cucumber. Furthermore, nutritional imbalance for aphids and content of secondary metabolites were increased in overexpressed CsPP2-A1 cucumber plants, and thus preventing aphid attack. These together may improve cucumber resistance against aphids and the mechanism of CsPP2-A1 defense against aphids was preliminarily explored.


Assuntos
Afídeos , Cucumis sativus , Animais , Cucumis sativus/genética , Espécies Reativas de Oxigênio , Lectinas
19.
Biochem Biophys Res Commun ; 642: 162-166, 2023 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-36580827

RESUMO

Cutaneous melanoma is one of the most aggressive and deadly types of skin cancer and rates of disease are continuing to increase worldwide. Currently, no serum biomarkers exist for the early detection of cutaneous melanoma. Normal human cells cannot make the sialic acid sugar, Neu5Gc, yet human tumor cells express Neu5Gc and Neu5Gc-containing glycoconjugates have been proposed as tumor biomarkers. We engineered a Neu5Gc-specific lectin based on the pentameric B-subunit of the Shiga toxigenic Escherichia coli subtilase cytotoxin, termed SubB2M. We have detected elevated Neu5Gc-containing biomarkers in the sera of ovarian and breast cancer patients in a highly sensitive surface plasmon resonance (SPR)-based assay using our SubB2M lectin. Here, we used the SubB2M-SPR assay to investigate Neu5Gc-containing glycoconjugates in the serum of cutaneous melanoma patients. We found elevated total serum Neu5Gc levels in primary (n = 24) and metastatic (n = 38) patients compared to cancer-free controls (n = 34). Serum Neu5Gc levels detected with SubB2M can distinguish cutaneous melanoma patients from cancer-free controls with high sensitivity and specificity as determined by ROC curve analysis. These data indicate that serum Neu5Gc-containing glycoconjugates are a novel class of biomarkers for cutaneous melanoma, particularly for primary melanoma, and have the potential to contribute to the early diagnosis of this disease.


Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Melanoma/diagnóstico , Neoplasias Cutâneas/diagnóstico , Ácidos Neuramínicos , Lectinas , Biomarcadores Tumorais , Glicoconjugados
20.
Insect Sci ; 29(3): 717-729, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34473412

RESUMO

In mammals, plant lectinshave been shown to possess immunomodulatory properties, acting in both the innate and adaptive immune system to modulate the production of mediators of the immune response, ultimately improving host defences. At present, knowledge of immunomodulatory effects of plant lectins in insects is scarce. Treatment of insect cells with the Orysa sativa lectin, Orysata, was previously reported to induce cell aggregation, mimicking the immune process of encapsulation. In this project we investigated the potential immunomodulatory effects of this mannose-binding lectin using Drosophila melanogaster S2 cells. Identification of the Orysata binding partners on the surface of S2 cells through a pull-down assay and proteomic analysis revealed 221 putative interactors, several of which were immunity-related proteins. Subsequent qPCR analysis revealed the upregulation of Toll- and immune deficiency (IMD)-regulated antimicrobial peptides (Drs, Mtk, AttA, and Dpt) and signal transducers (Rel and Hid) belonging to the IMD pathway. In addition, the iron-binding protein Transferrin 3 was identified as a putative interactor for Orysata, and treatment of S2 cells with Orysata was shown to reduce the intracellular iron concentration. All together, we believe these results offer a new perspective on the effects by which plant lectins influence insect cells and contribute to the study of their immunomodulatory properties.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Imunidade , Imunidade Inata , Lectinas/farmacologia , Mamíferos/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Lectinas de Plantas/farmacologia , Proteômica
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