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1.
Methods Mol Biol ; 2370: 281-299, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34611875

RESUMO

In this chapter we describe in detail methods for lectin staining of (1) tissues, and (2) cells to identify and map endogenous glycosylation. We also describe (3) dual antibody and lectin staining of tissues to associate glycosylation with particular proteins or cells in tissues.


Assuntos
Anticorpos , Lectinas , Glicosilação , Histocitoquímica , Lectinas/metabolismo , Coloração e Rotulagem
2.
Cells ; 10(12)2021 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-34944083

RESUMO

Considering the fact that many retinal diseases are yet to be cured, the pathomechanisms of these multifactorial diseases need to be investigated in more detail. Among others, oxidative stress and hypoxia are pathomechanisms that take place in retinal diseases, such as glaucoma, age-related macular degeneration, or diabetic retinopathy. In consideration of these diseases, it is also evidenced that the immune system, including the complement system and its activation, plays an important role. Suitable models to investigate neuroretinal diseases are organ cultures of porcine retina. Based on an established model, the role of the complement system was studied after the induction of oxidative stress or hypoxia. Both stressors led to a loss of retinal ganglion cells (RGCs) accompanied by apoptosis. Hypoxia activated the complement system as noted by higher C3+ and MAC+ cell numbers. In this model, activation of the complement cascade occurred via the classical pathway and the number of C1q+ microglia was increased. In oxidative stressed retinas, the complement system had no consideration, but strong inflammation took place, with elevated TNF, IL6, and IL8 mRNA expression levels. Together, this study shows that hypoxia and oxidative stress induce different mechanisms in the porcine retina inducing either the immune response or an inflammation. Our findings support the thesis that the immune system is involved in the development of retinal diseases. Furthermore, this study is evidence that both approaches seem suitable models to investigate undergoing pathomechanisms of several neuroretinal diseases.


Assuntos
Ativação do Complemento/imunologia , Via Clássica do Complemento/imunologia , Hipóxia/imunologia , Retina/imunologia , Retina/patologia , Células Ganglionares da Retina/patologia , Animais , Apoptose/efeitos dos fármacos , Cobalto/toxicidade , Ativação do Complemento/efeitos dos fármacos , Via Alternativa do Complemento/efeitos dos fármacos , Via Alternativa do Complemento/imunologia , Via Clássica do Complemento/efeitos dos fármacos , Proteínas do Sistema Complemento/metabolismo , Peróxido de Hidrogênio/toxicidade , Lectinas/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Estresse Oxidativo/efeitos dos fármacos , Células Ganglionares da Retina/efeitos dos fármacos , Neurônios Retinianos/efeitos dos fármacos , Neurônios Retinianos/patologia , Estresse Fisiológico/efeitos dos fármacos , Suínos
3.
Curr Protoc ; 1(11): e305, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34826352

RESUMO

All eukaryotic cells are covered with a dense layer of glycoconjugates, and the cell walls of bacteria are made of various polysaccharides, putting glycans in key locations for mediating protein-protein interactions at cell interfaces. Glycan function is therefore mainly defined as binding to other molecules, and lectins are proteins that specifically recognize and interact non-covalently with glycans. UniLectin was designed based on insight into the knowledge of lectins, their classification, and their biological role. This modular platform provides a curated and periodically updated classification of lectins along with a set of comparative and visualization tools, as well as structured results of screening comprehensive sequence datasets. UniLectin can be used to explore lectins, find precise information on glycan-protein interactions, and mine the results of predictive tools based on HMM profiles. This usage is illustrated here with two protocols. The first one highlights the fine-tuned role of the O blood group antigen in distinctive pathogen recognition, while the second compares the various bacterial lectin arsenals that clearly depend on living conditions of species even in the same genus. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Searching for the structural details of lectins binding the O blood group antigen Basic Protocol 2: Comparing the lectomes of related organisms in different environments.


Assuntos
Glicoconjugados , Lectinas , Proteínas de Transporte , Lectinas/metabolismo , Polissacarídeos
4.
Int J Mol Sci ; 22(19)2021 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-34638920

RESUMO

Glycan-lectin interactions play an essential role in different cellular processes. One of their main functions is involvement in the immune response to pathogens or inflammation. However, cancer cells and viruses have adapted to avail themselves of these interactions. By displaying specific glycosylation structures, they are able to bind to lectins, thus promoting pathogenesis. While glycan-lectin interactions promote tumor progression, metastasis, and/or chemoresistance in cancer, in viral infections they are important for viral entry, release, and/or immune escape. For several years now, a growing number of investigations have been devoted to clarifying the role of glycan-lectin interactions in cancer and viral infections. Various overviews have already summarized and highlighted their findings. In this review, we consider the interactions of the lectins MGL, DC-SIGN, selectins, and galectins in both cancer and viral infections together. A possible transfer of ways to target and disrupt them might lead to new therapeutic approaches in different pathological backgrounds.


Assuntos
Lectinas/metabolismo , Neoplasias/metabolismo , Polissacarídeos/metabolismo , Viroses/metabolismo , Animais , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Galectinas/química , Galectinas/metabolismo , Humanos , Lectinas/química , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Polissacarídeos/química , Ligação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Selectinas/química , Selectinas/metabolismo , Viroses/virologia
5.
Exp Suppl ; 112: 29-72, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34687007

RESUMO

Immunoglobulin (Ig) glycosylation has been shown to dramatically affect its structure and effector functions. Ig glycosylation changes have been associated with different diseases and show a promising biomarker potential for diagnosis and prognosis of disease advancement. On the other hand, therapeutic biomolecules based on structural and functional features of Igs demand stringent quality control during the production process to ensure their safety and efficacy. Liquid chromatography (LC) and lectin-based methods are routinely used in Ig glycosylation analysis complementary to other analytical methods, e.g., mass spectrometry and capillary electrophoresis. This chapter covers analytical approaches based on LC and lectins used in low- and high-throughput N- and O-glycosylation analysis of Igs, with the focus on immunoglobulin G (IgG) applications. General principles and practical examples of the most often used LC methods for Ig purification are described, together with typical workflows for N- and O-glycan analysis on the level of free glycans, glycopeptides, subunits, or intact Igs. Lectin chromatography is a historical approach for the analysis of lectin-carbohydrate interactions and glycoprotein purification but is still being used as a valuable tool in Igs purification and glycan analysis. On the other hand, lectin microarrays have found their application in the rapid screening of glycan profiles on intact proteins.


Assuntos
Imunoglobulina G , Lectinas , Cromatografia Líquida , Glicosilação , Lectinas/metabolismo , Espectrometria de Massas
6.
Zhongguo Dang Dai Er Ke Za Zhi ; 23(8): 828-834, 2021 Aug 15.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-34511173

RESUMO

OBJECTIVES: To study the expression of adipokines in children with primary nephrotic syndrome (PNS) before and after treatment and its correlation with blood lipids, as well as the role of adipokines in PNS children with hyperlipidemia. METHODS: A total of 90 children who were diagnosed with incipient PNS or recurrence of PNS after corticosteroid withdrawal for more than 6 months were enrolled as subjects. Thirty children who underwent physical examination were enrolled as the control group. Venous blood samples were collected from the children in the control group and the children with PNS before corticosteroid therapy (active stage) and after urinary protein clearance following 4 weeks of corticosteroid therapy (remission stage). ELISA was used to measure the levels of adipokines. An automatic biochemical analyzer was used to measure blood lipid levels. RESULTS: Compared with the control group, the children with PNS had a significantly lower level of omentin-1 in both active and remission stages, and their level of omentin-1 in the active stage was significantly lower than that in the remission stage (P<0.001). For the children with PNS, the level of chemerin in the active stage was significantly higher than that in the remission stage, and the children with PNS in the active stage had a significantly higher level of chemerin than the control group (P<0.001). For the children with PNS, atherogenic index of plasma, atherogenic coefficient (AC), castelli risk index-1 (CRI-1), castelli risk index-2 (CRI-2), and non-high-density lipoprotein in the active stage were significantly higher than those in the remission stage (P<0.001), and these indices in the children with PNS in the active stage were significantly higher than those in the control group (P<0.001). The children with PNS in the remission stage had significantly higher atherogenic index of plasma, AC, CRI-1, and non-high-density lipoprotein than the control group (P<0.001). Compared with the control group, the children with PNS in the remission stage had significantly higher serum levels of total cholesterol, triglyceride, high-density lipoprotein, low-density lipoprotein, apolipoprotein B, and apolipoprotein A (P<0.01). In the children with PNS, the ratio of omentin-1 before and after corticosteroid therapy was positively correlated with that of high-density lipoprotein, 24-hour urinary protein excretion, and high-density lipoprotein/apolipoprotein A before and after treatment, and it was negatively correlated with the ratio of AC and CRI-1 before and after treatment (P<0.05). The PNS children with low omentin-1 levels in the active stage had significantly higher levels of CRI-1, CRI-2, AC, and apolipoprotein B/apolipoprotein A ratio than those with high omentin-1 levels (P<0.05). CONCLUSIONS: Omentin-1 may be associated with disease activity, dyslipidemia, and proteinuria in children with PNS. Blood lipid ratios may be more effective than traditional blood lipid parameters in monitoring early cardiovascular risk in children with PNS.


Assuntos
Citocinas/metabolismo , Hiperlipidemias , Lectinas/metabolismo , Síndrome Nefrótica , Adipocinas , Quimiocinas , Criança , Citocinas/genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Lectinas/genética , Lipídeos , Síndrome Nefrótica/tratamento farmacológico , Proteinúria
7.
Int J Mol Sci ; 22(16)2021 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-34445445

RESUMO

Ascariasis is a global health problem for humans and animals. Adult Ascaris nematodes are long-lived in the host intestine where they interact with host cells as well as members of the microbiota resulting in chronic infections. Nematode interactions with host cells and the microbial environment are prominently mediated by parasite-secreted proteins and peptides possessing immunomodulatory and antimicrobial activities. Previously, we discovered the C-type lectin protein AsCTL-42 in the secreted products of adult Ascaris worms. Here we tested recombinant AsCTL-42 for its ability to interact with bacterial and host cells. We found that AsCTL-42 lacks bactericidal activity but neutralized bacterial cells without killing them. Treatment of bacterial cells with AsCTL-42 reduced invasion of intestinal epithelial cells by Salmonella. Furthermore, AsCTL-42 interacted with host myeloid C-type lectin receptors. Thus, AsCTL-42 is a parasite protein involved in the triad relationship between Ascaris, host cells, and the microbiota.


Assuntos
Ascaris suum/metabolismo , Interações Hospedeiro-Parasita , Mucosa Intestinal/metabolismo , Lectinas Tipo C/metabolismo , Lectinas/metabolismo , Salmonella , Animais , Ascaríase/metabolismo , Ascaríase/microbiologia , Ascaris suum/microbiologia , Ascaris suum/fisiologia , Linhagem Celular , Lectinas/fisiologia , Proteínas Recombinantes , Sus scrofa/microbiologia , Sus scrofa/parasitologia
8.
Sci Rep ; 11(1): 16109, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373510

RESUMO

Scedosporium apiospermum is an emerging opportunistic fungal pathogen responsible for life-threatening infections in humans. Host-pathogen interactions often implicate lectins that have become therapeutic targets for the development of carbohydrate mimics for antiadhesive therapy. Here, we present the first report on the identification and characterization of a lectin from S. apiospermum named SapL1. SapL1 was found using bioinformatics as a homolog to the conidial surface lectin FleA from Aspergillus fumigatus known to play a role in the adhesion to host glycoconjugates present in human lung epithelium. In our strategy to obtain recombinant SapL1, we discovered the importance of osmolytes to achieve its expression in soluble form in bacteria. Analysis of glycan arrays indicates specificity for fucosylated oligosaccharides as expected. Submicromolar affinity was measured for fucose using isothermal titration calorimetry. We solved SapL1 crystal structure in complex with α-methyl-L-fucoside and analyzed its structural basis for fucose binding. We finally demonstrated that SapL1 binds to bronchial epithelial cells in a fucose-dependent manner. The information gathered here will contribute to the design and development of glycodrugs targeting SapL1.


Assuntos
Proteínas Fúngicas/metabolismo , Lectinas/metabolismo , Scedosporium/metabolismo , Sequência de Aminoácidos , Aspergillus fumigatus/metabolismo , Sítios de Ligação/fisiologia , Células Cultivadas , Células Epiteliais/metabolismo , Fucose/metabolismo , Glicoconjugados/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo
9.
Phys Chem Chem Phys ; 23(32): 17656-17662, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34373871

RESUMO

In this manuscript the ability of selenium carbohydrates to undergo chalcogen bonding (ChB) interactions with protein residues has been studied at the RI-MP2/def2-TZVP level of theory. An inspection of the Protein Data Bank (PDB) revealed SeA (A = O, C and S) intermolecular contacts involving Se-pyranose ligands and ASP, TYR, SER and MET residues. Theoretical models were built to analyse the strength and directionality of the interaction together with "Atoms in Molecules" (AIM), Natural Bonding Orbital (NBO) and Non Covalent Interactions plot (NCIplot) analyses, which further assisted in the characterization of the ChBs described herein. We expect that the results from this study will be useful to expand the current knowledge regarding biological ChBs as well as to increase the visibility of the interaction among the carbohydrate chemistry community.


Assuntos
Lectinas/metabolismo , Monossacarídeos/metabolismo , Compostos Organosselênicos/metabolismo , Agaricales/química , Aspergillus oryzae/química , Bases de Dados de Proteínas , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Ligação de Hidrogênio , Lectinas/química , Modelos Moleculares , Monossacarídeos/química , Compostos Organosselênicos/química , Ligação Proteica , Selênio/química , Eletricidade Estática , Termodinâmica
10.
Nature ; 598(7880): 342-347, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34464958

RESUMO

SARS-CoV-2 infection-which involves both cell attachment and membrane fusion-relies on the angiotensin-converting enzyme 2 (ACE2) receptor, which is paradoxically found at low levels in the respiratory tract1-3, suggesting that there may be additional mechanisms facilitating infection. Here we show that C-type lectin receptors, DC-SIGN, L-SIGN and the sialic acid-binding immunoglobulin-like lectin 1 (SIGLEC1) function as attachment receptors by enhancing ACE2-mediated infection and modulating the neutralizing activity of different classes of spike-specific antibodies. Antibodies to the amino-terminal domain or to the conserved site at the base of the receptor-binding domain, while poorly neutralizing infection of ACE2-overexpressing cells, effectively block lectin-facilitated infection. Conversely, antibodies to the receptor binding motif, while potently neutralizing infection of ACE2-overexpressing cells, poorly neutralize infection of cells expressing DC-SIGN or L-SIGN and trigger fusogenic rearrangement of the spike, promoting cell-to-cell fusion. Collectively, these findings identify a lectin-dependent pathway that enhances ACE2-dependent infection by SARS-CoV-2 and reveal distinct mechanisms of neutralization by different classes of spike-specific antibodies.


Assuntos
Anticorpos Neutralizantes/imunologia , Lectinas/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Fusão Celular , Linhagem Celular , Cricetinae , Feminino , Humanos , Lectinas/imunologia , Lectinas Tipo C/metabolismo , Fusão de Membrana , Receptores de Superfície Celular/metabolismo , SARS-CoV-2/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
11.
Am J Physiol Heart Circ Physiol ; 321(3): H599-H611, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34415189

RESUMO

Sphingosine-1-phosphate (S1P) is a bioactive mediator in inflammation. Dysregulated S1P is demonstrated as a cause of heart failure (HF). However, the time-dependent and integrative role of S1P interaction with receptors in HF is unclear after myocardial infarction (MI). In this study, the sphingolipid mediators were quantified in ischemic human hearts. We also measured the time kinetics of these mediators post-MI in murine spleen and heart as an integrative approach to understand the interaction of S1P and respective S1P receptors in the transition of acute (AHF) to chronic HF (CHF). Risk-free 8-12 wk male C57BL/6 mice were subjected to MI surgery, and MI was confirmed by echocardiography and histology. Mass spectrometry was used to quantify sphingolipids in plasma, infarcted heart, spleen of mice, and ischemic and healthy human heart. The physiological cardiac repair was observed in mice with a notable increase of S1P quantity (pmol/g) in the heart and spleen significantly reduced in patients with ischemic HF. The circulating murine S1P levels were increased during AHF and CHF despite lowered substrate in CHF. The S1PR1 receptor expression was observed to coincide with the respective S1P quantity in mice and human hearts. Furthermore, selective S1P1 agonist limited inflammatory markers CCL2 and TNF-α and accelerated reparative markers ARG-1 and YM-1 in macrophages in the presence of Kdo2-Lipid A (KLA; potent inflammatory stimulant). This report demonstrated the importance of S1P/S1PR1 signaling in physiological inflammation during cardiac repair in mice. Alteration in these axes may serve as the signs of pathological remodeling in patients with ischemia.NEW & NOTEWORTHY Previous studies indicate that sphingosine-1-phosphate (S1P) has some role in cardiovascular disease. This study adds quantitative and integrative systems-based approaches that are necessary for discovery and bedside translation. Here, we quantitated sphinganine, sphingosine, sphingosine-1-phosphate (S1P) in mice and human cardiac pathobiology. Interorgan S1P quantity and respective systems-based receptor activation suggest cardiac repair after myocardial infarction. Thus, S1P serves as a therapeutic target for cardiac protection in clinical translation.


Assuntos
Insuficiência Cardíaca/metabolismo , Lisofosfolipídeos/metabolismo , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Esfingosina/análogos & derivados , Baço/metabolismo , Animais , Arginase/metabolismo , Células Cultivadas , Quimiocina CCL2/metabolismo , Humanos , Lectinas/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/fisiologia , Regeneração , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , Receptores de Esfingosina-1-Fosfato/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo
12.
Molecules ; 26(16)2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34443386

RESUMO

Lectins facilitate cell-cell contact and are critical in many cellular processes. Studying lectins may help us understand the mechanisms underlying tissue regeneration. We investigated the localization of an R-type lectin in a marine annelid (Perinereis sp.) with remarkable tissue regeneration abilities. Perinereis nuntia lectin (PnL), a galactose-binding lectin with repeating Gln-X-Trp motifs, is derived from the ricin B-chain. An antiserum was raised against PnL to specifically detect a 32-kDa lectin in the crude extracts from homogenized lugworms. The antiserum detected PnL in the epidermis, setae, oblique muscle, acicula, nerve cord, and nephridium of the annelid. Some of these tissues and organs also produced Galactose (Gal) or N-acetylgalactosamine (GalNAc), which was detected by fluorescent-labeled plant lectin. These results indicated that the PnL was produced in the tissues originating from the endoderm, mesoderm, and ectoderm. Besides, the localizing pattern of PnL partially merged with the binding pattern of a fluorescent-labeled mushroom lectin that binds to Gal and GalNAc. It suggested that PnL co-localized with galactose-containing glycans in Annelid tissue; this might be the reason PnL needed to be extracted with haptenic sugar, such as d-galactose, in the buffer. Furthermore, we found that a fluorescein isothiocyanate-labeled Gal/GalNAc-binding mushroom lectin binding pattern in the annelid tissue overlapped with the localizing pattern of PnL. These findings suggest that lectin functions by interacting with Gal-containing glycoconjugates in the tissues.


Assuntos
Anelídeos/metabolismo , Organismos Aquáticos/metabolismo , Lectinas/metabolismo , Animais , Misturas Complexas , Ligantes , Polissacarídeos/metabolismo
13.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445134

RESUMO

Coxsackievirus A24 variant (CVA24v) is the primary causative agent of the highly contagious eye infection designated acute hemorrhagic conjunctivitis (AHC). It is solely responsible for two pandemics and several recurring outbreaks of the disease over the last decades, thus affecting millions of individuals throughout the world. To date, no antiviral agents or vaccines are available for combating this disease, and treatment is mainly supportive. CVA24v utilizes Neu5Ac-containing glycans as attachment receptors facilitating entry into host cells. We have previously reported that pentavalent Neu5Ac conjugates based on a glucose-scaffold inhibit CVA24v infection of human corneal epithelial cells. In this study, we report on the design and synthesis of scaffold-replaced pentavalent Neu5Ac conjugates and their effect on CVA24v cell transduction and the use of cryogenic electron microscopy (cryo-EM) to study the binding of these multivalent conjugates to CVA24v. The results presented here provide insights into the development of Neu5Ac-based inhibitors of CVA24v and, most significantly, the first application of cryo-EM to study the binding of a multivalent ligand to a lectin.


Assuntos
Antivirais/farmacologia , Infecções por Coxsackievirus/dietoterapia , Enterovirus Humano C/efeitos dos fármacos , Ácido N-Acetilneuramínico/farmacologia , Conjuntivite Hemorrágica Aguda/tratamento farmacológico , Conjuntivite Hemorrágica Aguda/metabolismo , Conjuntivite Hemorrágica Aguda/virologia , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/virologia , Glucose/metabolismo , Humanos , Lectinas/metabolismo , Ligantes , Polissacarídeos/metabolismo , Receptores Virais/metabolismo
14.
Anal Chem ; 93(31): 10925-10933, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34319080

RESUMO

Glycan arrays are indispensable for learning about the specificities of glycan-binding proteins. Despite the abundance of available data, the current analysis methods do not have the ability to interpret and use the variety of data types and to integrate information across datasets. Here, we evaluated whether a novel, automated algorithm for glycan-array analysis could meet that need. We developed a regression-tree algorithm with simultaneous motif optimization and packaged it in software called MotifFinder. We applied the software to analyze data from eight different glycan-array platforms with widely divergent characteristics and observed an accurate analysis of each dataset. We then evaluated the feasibility and value of the combined analyses of multiple datasets. In an integrated analysis of datasets covering multiple lectin concentrations, the software determined approximate binding constants for distinct motifs and identified major differences between the motifs that were not apparent from single-concentration analyses. Furthermore, an integrated analysis of data sources with complementary sets of glycans produced broader views of lectin specificity than produced by the analysis of just one data source. MotifFinder, therefore, enables the optimal use of the expanding resource of the glycan-array data and promises to advance the studies of protein-glycan interactions.


Assuntos
Lectinas , Polissacarídeos , Algoritmos , Proteínas de Transporte , Lectinas/metabolismo , Software
15.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199928

RESUMO

Pancreatic cancer (PC) is an aggressive cancer with a high mortality rate, necessitating the development of effective diagnostic, prognostic and predictive biomarkers for disease management. Aberrantly fucosylated proteins in PC are considered a valuable resource of clinically useful biomarkers. The main objective of the present study was to identify novel plasma glycobiomarkers of PC using the iTRAQ quantitative proteomics approach coupled with Aleuria aurantia lectin (AAL)-based glycopeptide enrichment and isotope-coded glycosylation site-specific tagging, with a view to analyzing the glycoproteome profiles of plasma samples from patients with non-metastatic and metastatic PC and gallstones (GS). As a result, 22 glycopeptides with significantly elevated levels in plasma samples of PC were identified. Fucosylated SERPINA1 (fuco-SERPINA1) was selected for further validation in 121 plasma samples (50 GS and 71 PC) using an AAL-based reverse lectin ELISA technique developed in-house. Our analyses revealed significantly higher plasma levels of fuco-SERPINA1 in PC than GS subjects (310.7 ng/mL v.s. 153.6 ng/mL, p = 0.0114). Elevated fuco-SERPINA1 levels were associated with higher TNM stage (p = 0.024) and poorer prognosis for overall survival (log-rank test, p = 0.0083). The increased plasma fuco-SERPINA1 levels support the utility of this protein as a novel prognosticator for PC.


Assuntos
Biomarcadores Tumorais/sangue , Fucose/química , Glicoproteínas/sangue , Lectinas/química , Neoplasias Pancreáticas/diagnóstico , Proteoma/metabolismo , alfa 1-Antitripsina/sangue , Estudos de Casos e Controles , Cromatografia de Afinidade , Feminino , Fucose/metabolismo , Humanos , Lectinas/metabolismo , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Neoplasias Pancreáticas/sangue , Proteoma/análise , Taxa de Sobrevida , alfa 1-Antitripsina/química
16.
Front Immunol ; 12: 683572, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34267752

RESUMO

Background: Immunotherapy is an effective therapeutic approach for multiple human cancer types. However, the correlations between EVA1C and patients' prognosis as well as immune infiltration remain obscure. Herein, we employed transcriptomic and clinical data extracted from two independent databases to systematically investigate the role of EVA1C in the oncological context. Methods: The differential expression of EVA1C was analyzed via TCGA and Oncomine databases. We evaluated the influence of EVA1C on clinical prognosis using Kaplan-Meier plotter. We then used the expression profiler to calculate stromal score, immune score, and ESTIMATE score based on the ESTIMATE algorithm. The abundance of infiltrating immune cells was calculated via TIMER. The correlations between EVA1C expression and immune infiltration levels were analyzed in two independent cohorts. Results: In patients with World Health Organization (WHO) grade II/III glioma, high EVA1C expression was associated with malignant clinicopathological features and poor overall survival in both cohorts. EVA1C expression was positively associated with immune infiltration levels of B cell, CD4+ T cell, neutrophil, macrophage, and dendritic cells (DCs). Besides, EVA1C expression strongly correlated with diverse immune marker sets. And the predictive power of EVA1C was better than that of other indicators in predicting high immune infiltration levels in glioma. Conclusions: For the first time, we identified the overexpression of EVA1C in glioma, which was tightly correlated with the high infiltration levels of multiple immune cells as well as poor prognosis. Meanwhile, EVA1C might be a potential biomarker for predicting high immune infiltration in WHO grade II/III gliomas.


Assuntos
Biomarcadores Tumorais , Neoplasias Encefálicas/etiologia , Neoplasias Encefálicas/patologia , Glioma/etiologia , Glioma/patologia , Lectinas/genética , Proteínas de Membrana/genética , Microambiente Tumoral , Adulto , Feminino , Expressão Gênica , Humanos , Lectinas/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Curva ROC , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/patologia , Adulto Jovem
17.
Artigo em Inglês | MEDLINE | ID: mdl-34274643

RESUMO

Separations based on combinations of 2.1 mm I.D. high-performance affinity microcolumns and capillary electrophoresis were developed and used to characterize the glycoforms of an intact glycoprotein. Human alpha1-acid glycoprotein (AGP) was used as a model analyte due to its heterogeneous glycosylation resulting from variations in its degree of branching, fucosylation, and number of sialic acids. Three separation formats were examined based on microcolumns that contained the lectins concanavalin A (Con A) or Aleuria aurantia lectin (AAL). These microcolumns were used with one another or in combination with capillary electrophoresis. N-Glycan analysis of the non-retained and retained AGP fractions was carried out by using PNGase F digestion and nanoflow electrospray ionization mass spectrometry. Con A microcolumns were found to selectively enrich AGP that contained bi-antennary N-glycans, while AAL microcolumns retained AGP with fucose-containing N-glycans. Results from these separation methods indicated that fucosylation of the N-linked glycans was more abundant when a high degree of branching was present in AGP. Sialic acid residues were more abundant when higher degrees of branching and more fucose residues were present in AGP. The separation and analysis methods that were developed could be used with relatively small amounts of AGP and can be adapted for use with other intact glycoproteins.


Assuntos
Cromatografia de Afinidade/métodos , Eletroforese Capilar/métodos , Lectinas/metabolismo , Orosomucoide , Glicoproteínas/análise , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Lectinas/química , Ácido N-Acetilneuramínico/química , Orosomucoide/análise , Orosomucoide/química , Orosomucoide/isolamento & purificação , Polissacarídeos/química
18.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206350

RESUMO

The monolayer character of two-dimensional materials predestines them for application as active layers of sensors. However, their inherent high sensitivity is always accompanied by a low selectivity. Chemical functionalization of two-dimensional materials has emerged as a promising way to overcome the selectivity issues. Here, we demonstrate efficient graphene functionalization with carbohydrate ligands-chitooligomers, which bind proteins of the lectin family with high selectivity. Successful grafting of a chitooligomer library was thoroughly characterized, and glycan binding to wheat germ agglutinin was studied by a series of methods. The results demonstrate that the protein quaternary structure remains intact after binding to the functionalized graphene, and that the lectin can be liberated from the surface by the addition of a binding competitor. The chemoenzymatic assay with a horseradish peroxidase conjugate also confirmed the intact catalytic properties of the enzyme. The present approach thus paves the way towards graphene-based sensors for carbohydrate-lectin binding.


Assuntos
Grafite/química , Lectinas/metabolismo , Polissacarídeos/química , Peroxidase do Rábano Silvestre , Lectinas/análise , Polissacarídeos/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína
19.
Cancer Sci ; 112(9): 3722-3731, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34115906

RESUMO

The rBC2LCN lectin, known as a stem cell marker probe that binds to an H type 3 fucosylated trisaccharide motif, was recently revealed to also bind to pancreatic ductal adenocarcinoma (PDAC) cells. A lectin-drug conjugate was generated by fusing rBC2LCN with a cytocidal toxin, and it showed a strong anticancer effect in in vitro and in vivo PDAC models. However, it is unclear which molecules are carrier proteins of rBC2LCN on PDAC cells. In this study, we identified a rBC2LCN-positive glycoprotein expressed in PDAC. Tumor lysates of PDAC patient-derived xenografts (PDXs) were coprecipitated with rBC2LCN lectin and analyzed by liquid chromatography-mass spectrometry. A total of 343 proteins were initially identified. We used a web-based database to select five glycoproteins and independently evaluated their expression in PDAC by immunohistochemistry (IHC). Among them, we focused on carcinoembryonic antigen 5 (CEA) as the most cancer-specific carrier protein in PDAC, as it showed the most prominent difference in expression rate between PDAC cells (74%) and normal pancreatic duct cells (0%, P > .0001). rBC2LCN lectin and CEA colocalization in PDAC samples was confirmed by double-staining analysis. Furthermore, rBC2LCN-precipitated fractions were blotted with an anti-CEA polyclonal antibody (pAb), and CEA pAb-precipitated fractions were blotted with rBC2LCN lectin. The results demonstrate that CEA is in fact a ligand of rBC2LCN lectin.


Assuntos
Antígeno Carcinoembrionário/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Proteínas de Transporte/metabolismo , Lectinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Anticorpos/imunologia , Antígeno Carcinoembrionário/imunologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Feminino , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Xenoenxertos , Humanos , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Ligantes , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Transplante de Neoplasias , Neoplasias Pancreáticas/patologia
20.
Artigo em Inglês | MEDLINE | ID: mdl-34126205

RESUMO

The increasing availability of sequenced genomes has enabled a deeper understanding of the complexity of fish lectin repertoires involved in early development and immune recognition. The teleost fucose-type lectin (FTL) family includes proteins that preferentially bind fucose and display tandemly arrayed carbohydrate-recognition domains (CRDs) or are found in mosaic combinations with other domains. They function as opsonins, promoting phagocytosis and the clearance of microbial pathogens. The Antarctic fish Trematomus bernacchii is a Perciforme living at extremely low temperatures (-1.68 °C) which is considered a model for studying adaptability to the variability of environmental waters. Here, we isolated a Ca++-independent fucose-binding protein from the serum of T. bernacchii by affinity chromatography with apparent molecular weights of 32 and 30 kDa under reducing and non-reducing conditions, respectively. We have characterized its carbohydrate binding properties, thermal stability and potential ability to recognize bacterial pathogens. In western blot analysis, the protein showed intense cross-reactivity with antibodies specific for a sea bass (Dicentrarchus labrax) fucose-binding lectin. In addition, its molecular and structural aspects, showing that it contains two CRD-FTLs confirmed that T. bernacchii FTL (TbFTL) is a bona fide member of the FTL family, with binding activity at low temperatures and the ability to agglutinate bacteria, thereby suggesting it participates in host-pathogen interactions in low temperature environments.


Assuntos
Bactérias/metabolismo , Fucose/metabolismo , Lectinas/sangue , Lectinas/fisiologia , Perciformes/fisiologia , Sequência de Aminoácidos , Animais , Regiões Antárticas , Sequência de Bases , Lectinas/isolamento & purificação , Lectinas/metabolismo , Filogenia
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