RESUMO
Mucins are major components of the mucus. Besides the highly O-glycosylated tandem repeat domains, mucins contain Cys domains (CysDs). CysDs contain conserved disulfide-forming cysteine residues as well as a WxxW motif. Since this is the consensus sequence for tryptophan C-mannosylation, mucin CysDs have been suggested to be targets for C-mannosyltransferases, but this has never been directly shown. Here, we recombinantly expressed human mucin CysDs in Chinese hamster ovary (CHO) cells and analyzed the C-mannosylation status. Mass spectrometric analysis revealed that the putative C-mannose site is not or only barely C-mannosylated. However, mutation of the adjacent cysteine residues enabled C-mannosylation to occur. In contrast to mucin CysDs, the homologous CysD of human cartilage intermediate layer protein 1 (CILP1) lacks these cysteine residues preceding the WxxW motif. We show that CILP1 CysD is C-mannosylated, but introducing a cysteine at the -2 position causes this modification to be lost. We thus conclude that the presence of cysteine residues prevents the modification of the WxxW motif in CysDs.
Assuntos
Cricetulus , Cisteína , Manose , Cisteína/metabolismo , Cisteína/genética , Cisteína/química , Humanos , Animais , Células CHO , Manose/metabolismo , Manose/química , Glicosilação , Mucinas/metabolismo , Mucinas/química , Mucinas/genética , Domínios Proteicos , Sequência de Aminoácidos , Motivos de Aminoácidos , Sequência Conservada , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/químicaRESUMO
Mucus forms the first defense line of human lungs, and as such hampers the efficient delivery of therapeutics to the underlying epithelium. This holds particularly true for genetic cargo such as CRISPR-based gene editing tools which cannot readily surmount the mucosal barrier. While lipid nanoparticles (LNPs) emerge as versatile non-viral gene delivery systems that can help overcome the delivery challenge, many knowledge gaps remain, especially for diseased states such as cystic fibrosis (CF). This study provides fundamental insights into Cas9 mRNA or ribonucleoprotein-loaded LNP-mucus interactions in healthy and diseased states by assessing the impact of the genetic cargo, mucin sialylation, mucin concentration, ionic strength, pH, and polyethylene glycol (PEG) concentration and nature on LNP diffusivity leveraging experimental approaches and Brownian dynamics (BD) simulations. Taken together, this study identifies key mucus and LNP characteristics that are critical to enabling a rational LNP design for transmucosal delivery.
Assuntos
Fibrose Cística , Muco , Nanopartículas , Polietilenoglicóis , Fibrose Cística/metabolismo , Humanos , Nanopartículas/química , Muco/metabolismo , Polietilenoglicóis/química , Lipídeos/química , Mucinas/metabolismo , Mucinas/química , Técnicas de Transferência de Genes , LipossomosRESUMO
N-acetylgalactosaminyl-transferases (GalNAc-Ts) initiate mucin-type O-glycosylation, an abundant and complex posttranslational modification that regulates host-microbe interactions, tissue development, and metabolism. GalNAc-Ts contain a lectin domain consisting of three homologous repeats (α, ß, and γ), where α and ß can potentially interact with O-GalNAc on substrates to enhance activity toward a nearby acceptor Thr/Ser. The ubiquitous isoenzyme GalNAc-T1 modulates heart development, immunity, and SARS-CoV-2 infectivity, but its substrates are largely unknown. Here, we show that both α and ß in GalNAc-T1 uniquely orchestrate the O-glycosylation of various glycopeptide substrates. The α repeat directs O-glycosylation to acceptor sites carboxyl-terminal to an existing GalNAc, while the ß repeat directs O-glycosylation to amino-terminal sites. In addition, GalNAc-T1 incorporates α and ß into various substrate binding modes to cooperatively increase the specificity toward an acceptor site located between two existing O-glycans. Our studies highlight a unique mechanism by which dual lectin repeats expand substrate specificity and provide crucial information for identifying the biological substrates of GalNAc-T1.
Assuntos
Mucinas , N-Acetilgalactosaminiltransferases , Mucinas/química , Mucinas/metabolismo , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/metabolismo , Lectinas , Especificidade por Substrato , Estrutura Terciária de Proteína , Polipeptídeo N-Acetilgalactosaminiltransferase , AçúcaresRESUMO
Muco-obstructive diseases change airway mucus properties, impairing mucociliary transport and increasing the likelihood of infections. To investigate the sorption properties and nanostructures of mucus in health and disease, we investigated mucus samples from patients and cell cultures (cc) from healthy, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) airways. Atomic force microscopy (AFM) revealed mucin monomers with typical barbell structures, where the globule to spacer volume ratio was the highest for CF mucin. Accordingly, synchrotron small-angle X-ray scattering (SAXS) revealed more pronounced scattering from CF mucin globules and suggested shorter carbohydrate side chains in CF mucin and longer side chains in COPD mucin. Quartz crystal microbalance with dissipation (QCM-D) analysis presented water sorption isotherms of the three types of human airway mucus, where, at high relative humidity, COPD mucus had the highest water content compared to cc-CF and healthy airway mucus (HAM). The higher hydration of the COPD mucus is consistent with the observation of longer side chains of the COPD mucins. At low humidity, no dehydration-induced glass transition was observed in healthy and diseased mucus, suggesting mucus remained in a rubbery state. However, in dialyzed cc-HAM, a sorption-desorption hysteresis (typically observed in the glassy state) appeared, suggesting that small molecules present in mucus suppress the glass transition.
Assuntos
Fibrose Cística , Doença Pulmonar Obstrutiva Crônica , Humanos , Água/química , Espalhamento a Baixo Ângulo , Difração de Raios X , Muco/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Mucinas/químicaRESUMO
Background: Changes in protein glycosylation have been reported in various diseases, including cancer; however, the consequences of altered glycosylation in meningiomas remains undefined. We established two benign meningioma cell lines-SUT-MG12 and SUT-MG14, WHO grade I-and demonstrated the glycan and glycosyltransferase profiles of the mucin-type O-linked glycosylation in the primary benign meningioma cells compared with two malignant meningioma cell lines-HKBMM and IOMM-Lee, WHO grade III. Changes in O-linked glycosylation profiles in malignant meningiomas were proposed. Methods: Primary culture technique, morphological analysis, and immunocytochemistry were used to establish and characterize two benign meningioma cell lines. The glycan profiles of the primary benign and malignant meningiomas cell lines were then analyzed using lectin cytochemistry. The gene expression of O-linked glycosyltransferases, mucins, sialyltransferases, and fucosyltransferases were analyzed in benign and malignant meningioma using the GEO database (GEO series GSE16581) and quantitative-PCR (qPCR). Results: Lectin cytochemistry revealed that the terminal galactose (Gal) and N-acetyl galactosamine (GalNAc) were highly expressed in primary benign meningioma cells (WHO grade I) compared to malignant meningioma cell lines (WHO grade III). The expression profile of mucin types O-glycosyltransferases in meningiomas were observed through the GEO database and gene expression experiment in meningioma cell lines. In the GEO database, C1GALT1-specific chaperone (COSMC) and mucin 1 (MUC1) were significantly increased in malignant meningiomas (Grade II and III) compared with benign meningiomas (Grade I). Meanwhile, in the cell lines, Core 2 ß1,6-N-acetylglucosaminyltransferase-2 (C2GNT2) was highly expressed in malignant meningiomas. We then investigated the complex mucin-type O-glycans structures by determination of sialyltransferases and fucosyltransferases. We found ST3 ß-galactoside α-2,3-sialyltransferase 4 (ST3GAL4) was significantly decreased in the GEO database, while ST3GAL1, ST3GAL3, α1,3 fucosyltransferases 1 and 8 (FUT1 and FUT8) were highly expressed in malignant meningioma cell lines-(HKBMM)-compared to primary benign meningioma cells-(SUT-MG12 and SUT-MG14). Conclusion: Our findings are the first to demonstrate the potential glycosylation changes in the O-linked glycans of malignant meningiomas compared with benign meningiomas, which may play an essential role in the progression, tumorigenesis, and malignancy of meningiomas.
Assuntos
Neoplasias Meníngeas , Meningioma , Humanos , Glicosilação , Sialiltransferases/genética , Mucinas/química , Glicosiltransferases/metabolismo , Polissacarídeos/química , Fucosiltransferases/metabolismo , Lectinas/metabolismoRESUMO
PURPOSE OF REVIEW: Gut microbiota-mucosa-epithelial cells co-exist in an intricate three-way relationship that underpins gut homeostasis, and ultimately influences health and disease conditions. The O-glycans of mucin glycoproteins have been uncovered as a centrepiece of this system, although understanding the phenomena at play at the molecular level has been challenging and subject to significant traction over the last years. The purpose of this review is to discuss the recent advances in the phenomena that mediate microbiota and mucus multidirectional interactions in the human gut. RECENT FINDINGS: The mucus biosynthesis and degradation by both commensal and pathogenic bacteria is under tight regulation and involves hundreds of carbohydrate-active enzymes (CAZy) and transporters. The fucosylation of O-glycans from mucin-2 seems to dictate binding by pathogenic species and to influence their virulence. Less clear is the influence of O-glycans in quorum sensing and biofilm formation. We have reviewed the advances in the in vitro models available to recreate the phenomena that capture the physiological context of the intestinal environment, emphasising models that include mucus and other aspects relevant to the physiological context. SUMMARY: The recent findings highlight the importance of merging advances in analytical (glycans analysis) and omics techniques along with original robust in vitro models that enable to deconstruct part of the high complexity of the living gut and expand our understanding of the microbes-mucosa relationships and their significance in health and disease.
Assuntos
Mucosa Intestinal , Mucinas , Humanos , Mucosa Intestinal/metabolismo , Mucinas/química , Mucinas/metabolismo , Células Epiteliais/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , BactériasRESUMO
Mucin glycans are an important component of the mucus barrier and a vital defence against physical and chemical damage as well as pathogens. There are 20 mucins in the human body, which can be classified into secreted mucins and transmembrane mucins according to their distributions. The major difference between them is that secreted mucins do not have transmembrane structural domains, and the expression of each mucin is organ and cell-specific. Under physiological conditions, mucin glycans are involved in the composition of the mucus barrier and thus protect the body from infection and injury. However, abnormal expression of mucin glycans can lead to the occurrence of diseases, especially cancer, through various mechanisms. Therefore, targeting mucin glycans for the diagnosis and treatment of cancer has always been a promising research direction. Here, we first summarize the main types of glycosylation (O-GalNAc glycosylation and N-glycosylation) on mucins and the mechanisms by which abnormal mucin glycans occur. Next, how abnormal mucin glycans contribute to cancer development is described. Finally, we summarize MUC1-based antibodies, vaccines, radio-pharmaceuticals, and CAR-T therapies using the best characterized MUC1 as an example. In this section, we specifically elaborate on the recent new cancer therapy CAR-M, which may bring new hope to cancer patients.
Assuntos
Mucinas , Neoplasias , Humanos , Mucinas/química , Mucina-1/metabolismo , Polissacarídeos/química , Glicosilação , Neoplasias/terapiaRESUMO
Mucin-domain glycoproteins are highly O-glycosylated cell surface and secreted proteins that serve as both biochemical and biophysical modulators. Aberrant expression and glycosylation of mucins are known hallmarks in numerous malignancies, yet mucin-domain glycoproteins remain enigmatic in the broad landscape of cancer glycobiology. Here we review the multifaceted roles of mucins in cancer through the lens of the analytical and biochemical methods used to study them. We also describe a collection of emerging tools that are specifically equipped to characterize mucin-domain glycoproteins in complex biological backgrounds. These approaches are poised to further elucidate how mucin biology can be understood and subsequently targeted for the next generation of cancer therapeutics.
Assuntos
Mucinas , Neoplasias , Humanos , Mucinas/química , Mucinas/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , GlicosilaçãoRESUMO
Acetylgalactosamine (GalNAc)-type O-glycosylation is an essential posttranslational modification (PTM) that plays fundamental roles in biology. Malfunction of this PTM is exemplified by the presence of truncated O-glycans in cancer. For instance, the glycoprotein MUC1 is overexpressed in many tumor tissues and tends to carry simple oligosaccharides that allow for the presentation of different tumor-associated antigens, such as the Tn or sTn antigens (GalNAc-α-1-O-Thr/Ser and Neu5Acα2-6GalNAcα1-O-Ser/Thr, respectively). In other cases, such as tumoral calcinosis associated with O-glycosylation of the fibroblast growth factor 23, O-glycans are absent or less abundant. Significant progress has been made in determining the three-dimensional structures of biomolecules that recognize GalNAc, such as antibodies, lectins, mucinases, GalNAc-transferases, and other glycosyltransferases. Analysis of the complexes between these entities and GalNAc-containing glycopeptides, in most cases derived from crystallographic or NMR analysis, provides an understanding of the key structural elements that control molecular recognition of these glycopeptides. Here, we describe and compare the binding sites of these proteins in detail, focusing on how the GalNAc moieties interact selectively with them. We also summarize the differences and similarities in GalNAc recognition. In general, the recognition of GalNAc-containing glycopeptides is determined by hydrogen bonds between hydroxyl groups and the N-acetyl group of GalNAc with proteins, as well as CH-π contacts in which the hydrophobic α-face of the sugar and the methyl group of NHAc can be involved. The latter interaction usually provides the basis for selectivity. It is worth noting that binding of these glycopeptides depends primarily on recognition of the sugar moiety, with some exceptions such as a few anti-MUC1 antibodies that primarily recognize the peptide backbone and use the sugar to facilitate shape complementarity or to establish a limited number of interactions with the protein. Focusing specifically on the GalNAc moiety, we can observe that there is some degeneracy of interactions within the same protein families, likely due to substrate flexibility. However, when all studied proteins are considered together, despite the commonalities within each protein family, no pattern can be discerned between the different families, apart from the presence of common residues such as Tyr, His, or Asp, which are responsible for hydrogen bonds. The lack of a pattern can be anticipated, given the diverse functions of mucinases, glycosyltransferases, antibodies, and lectins. Finally, it is important to point out that the conformational differences observed in solution in glycopeptides bearing GalNAc-α-1-O-Ser or GalNAc-α-1-O-Thr also can be found in the bound state. This unique characteristic is exploited, for instance, by the enzyme C1GalT1 to broadly glycosylate both acceptor substrates. The findings summarized in this review may contribute to the rational structure-guided development of therapeutic vaccines, novel diagnostic tools for early cancer detection, and new cancer treatments for cancer with tailored anti-Tn or anti-STn antibodies or new drugs to inhibit GalNAc-T isoenzymes.
Assuntos
Glicopeptídeos , Mucinas , Mucinas/química , Mucinas/metabolismo , Glicosilação , Glicopeptídeos/química , Lectinas/química , Carboidratos , Polissacarídeos , Glicosiltransferases , AçúcaresRESUMO
The gut microbiota interacts with the host through the mucus that covers and protects the gastrointestinal epithelium. The main component of the mucus are mucins, glycoproteins decorated with hundreds of different O-glycans. Some microbiota members can utilize mucin O-glycans as carbons source. To degrade these host glycans the bacteria express multiple carbohydrate-active enzymes (CAZymes) such as glycoside hydrolases, sulfatases and esterases which are active on specific linkages. The studies of these enzymes in an in vivo context have started to reveal their importance in mucin utilization and gut colonization. It is now clear that bacteria evolved multiple specific CAZymes to overcome the diversity of linkages found in O-glycans. Additionally, changes in mucin degradation by gut microbiota have been associated with diseases like obesity, diabetes, irritable bowel disease and colorectal cancer. Thereby understanding how CAZymes from different bacteria work to degrade mucins is of critical importance to develop new treatments and diagnostics for these increasingly prevalent health problems. This mini-review covers the recent advances in biochemical characterization of mucin O-glycan-degrading CAZymes and how they are connected to human health.
Assuntos
Microbioma Gastrointestinal , Mucinas , Humanos , Mucinas/química , Mucinas/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Bactérias/metabolismoRESUMO
Mucin glycoproteins are the major component of mucus and coat epithelial cell surfaces forming the glycocalyx. The glycocalyx and mucus are involved in the transport of nutrients, drugs, gases, and pathogens toward the cell surface. Mucins are also involved in diverse diseases such as cystic fibrosis and cancer. Due to inherent heterogeneity in native mucin structure, many synthetic materials have been designed to probe mucin chemistry, biology, and physics. Such materials include various glycopolymers, low molecular weight glycopeptides, glycopolypeptides, polysaccharides, and polysaccharide-protein conjugates. This review highlights advances in the area of design and synthesis of mucin mimic materials, and their biomedical applications in glycan binding, epithelial models of infection, therapeutic delivery, vaccine formulation, and beyond.
Assuntos
Fibrose Cística , Mucinas , Humanos , Mucinas/química , Mucinas/metabolismo , Muco/metabolismo , Fibrose Cística/metabolismo , Polissacarídeos/química , GlicopeptídeosRESUMO
High consumption of polyphenol-rich green tea, coffee, fruits, and vegetables is associated with a low risk of human chronic diseases. Recent studies highlight the relevance of polyphenol-mediated gut microbiota modulation and its impact on mucus barrier. Herein, we study the direct interaction of epicatechin (EC), epigallocatechin gallate (EGCG), and tannic acid (TA) with intestinal mucin by isothermal titration calorimetry and multiple particle tracking and the impact on mucus barrier using ex vivo mucus and Caco-2/HT29-MTX cocultures. Results show that pyrogallol-containing polyphenols EGCG and TA exhibit strong binding to intestinal mucin and reinforce mucus barrier, whereas EC does not. ECGG and TA also mitigate gliadin-mediated cytotoxicity and inflammation. The chemical binding of EGCG and TA to the nucleophilic thiol groups of mucins shows their roles as cross-linkers of mucin networks. These results bring a novel understanding of the health benefits of polyphenols and provide support for the consumption of pyrogallol-containing beverages like green tea as a potential dietary therapy for gluten-related disorders.
Assuntos
Catequina , Polifenóis , Células CACO-2 , Catequina/química , Humanos , Mucinas/química , Muco/metabolismo , Polifenóis/química , Polifenóis/farmacologia , Pirogalol , Chá/químicaRESUMO
Mucin 21(Muc21)/epiglycanin is expressed on apical surfaces of squamous epithelia and has potentially protective roles, which are thought to be associated with its unique glycoforms, whereas its aberrant glycosylation is implicated in the malignant behaviors of some carcinomas. Despite the importance of glycoforms, we lack tools to detect specific glycoforms of mouse Muc21. In this study, we generated two monoclonal antibodies (mAbs) that recognize different glycoforms of Muc21. We used membrane lysates of Muc21-expressing TA3-Ha cells or Chinese hamster ovary (CHO)-K1 cells transfected with Muc21 as antigens. Specificity testing, utilizing Muc21 glycosylation variant cells, showed that mAb 1A4-1 recognized Muc21 carrying glycans terminated with galactose residues, whereas mAb 18A11 recognized Muc21 carrying sialylated glycans. mAb 1A4-1 stained a majority of mouse mammary carcinoma TA3-Ha cells in vitro and in engrafted tumors in mice, whereas mAb 18A11 recognized only a subpopulation of these. mAb 1A4-1 was useful in immunohistochemically detecting Muc21 in normal squamous epithelia. In conclusion, these mAbs recognize distinct Muc21 epitopes formed by combinations of peptide portions and O-glycans.
Assuntos
Antineoplásicos Imunológicos , Carcinoma de Células Escamosas , Animais , Anticorpos Monoclonais , Células CHO , Cricetinae , Cricetulus , Camundongos , Mucina-1/química , Mucinas/química , Polissacarídeos/químicaRESUMO
Investigation into Heliobacter pylori binding to Lewis b (Leb) antigens through the blood group antigen binding adhesion protein (BabA) requires structurally well-defined tools. A Leb hexasaccharide thioglycoside donor was chemically prepared through a linear approach starting from D-lactose. This donor can be used to attach reducing end linkers providing a range of options for conjugation techniques or to further extend the oligosaccharide structure. To evaluate its efficiency as a donor, it was coupled to a 6-OH GalNAc acceptor, producing an extended Leb-containing Tn mucin core structure in 84% yield, and to L-serine in 72% yield. The latter compound was subsequently functionalized with a photolabile diazirine linker and biotin, creating a Leb hexasaccharide structure-function tool suitable for lectin tagging interaction studies. This donor opens a wide range of possibilities for conjugation of Leb structures to produce a variety of chemical biology tools to assist in the study of these interactions.
Assuntos
Mucinas , Tioglicosídeos , Antígenos do Grupo Sanguíneo de Lewis , Mucinas/química , Mucinas/metabolismo , Oligossacarídeos/química , SerinaRESUMO
The secreted mucus layer that lines and protects epithelial cells is conserved across diverse species. While the exact composition of this protective layer varies between organisms, certain elements are conserved, including proteins that are heavily decorated with N-acetylgalactosamine-based sugars linked to serines or threonines (O-linked glycosylation). These heavily O-glycosylated proteins, known as mucins, exist in many forms and are able to form hydrated gel-like structures that coat epithelial surfaces. In vivo studies in diverse organisms have highlighted the importance of both the mucin proteins as well as their constituent O-glycans in the protection and health of internal epithelia. Here, we summarize in vivo approaches that have shed light on the synthesis and function of these essential components of mucus.
Assuntos
Mucinas , Muco , Células Epiteliais/metabolismo , Glicosilação , Humanos , Mucinas/química , Mucinas/metabolismo , Muco/metabolismo , PolissacarídeosRESUMO
Mucin-type O-glycosylation of serine or threonine residue in proteins is known to be one of the major post-translational modifications. In this study, two novel alkyl glycosides, Nα-lauryl-O-(2-acetamido-2-deoxy-α-d-galactopyranosyl)-l-serineamide (GalNAc-Ser-C12) and Nα-lauryl-O-(2-acetamido-2-deoxy-α-d-galactopyranosyl)-l-threonineamide (GalNAc-Thr-C12) were synthesized as saccharide primers to prime mucin-type O-glycan biosynthesis in cells. Upon incubating human gastric cancer MKN45 cells with the saccharide primers, 22 glycosylated products were obtained, and their structures were analyzed using liquid chromatography-mass spectrometry and enzyme digestion. The amounts of glycosylated products were dependent on the amino acid residues in the saccharide primers. For example, in vitro synthesis of T antigen (Galß1-3GalNAc), fucosyl-T (Fucα1-2Galß1-3GalNAc), and sialyl-T (NeuAcα2-3Galß1-3GalNAc) preferred a serine residue, whereas sialyl-Tn (NeuAcα2-6GalNAc) preferred a threonine residue. Furthermore, the glycosylated products derived from GalNAc-Ser/Thr-C12 and Gal-GalNAc-Ser/Thr-C12 using cell-free synthesis showed the same amino acid selectivity as those in the cell experiments. These results indicate that glycosyltransferases involved in the biosynthesis of mucin-type O-glycans distinguish amino acid residues conjugated to GalNAc. The saccharide primers developed in this study might be useful for comparing mucin-type oligosaccharides in cells and constructing oligosaccharide libraries to study cell function.
Assuntos
Mucinas , Treonina , Glicosilação , Humanos , Mucinas/química , Oligossacarídeos/química , Polissacarídeos/química , Treonina/químicaRESUMO
Mucins are the major proteins that distributed on the intestinal mucosa layer and protect the intestine from pathogens infection. The composition of intestinal mucin O-glycans can affect the health of the gastrointestinal tract in pigs. Porcine intestinal mucosa is widely used as the main raw material of heparin extraction. The heparin extraction residues rich in mucins were usually wasted. The structure of mucin derived O-glycans in porcine intestinal mucosa are currently unknown. In this study, we isolated the mucins from the heparin extraction residues and profiled the O-glycans. After heparin extraction, mucin was digested with trypsin, and separated by strong anion exchange chromatography. The mucin derived O-glycans were release by alkaline ß elimination, and analyzed by ultra high performance liquid chromatography-porous graphitized carbon-Fourier transform mass spectrometry (UPLC-PGC-FTMS/MS). Thirty five kinds of O-glycans were identified, most of which were Core 3-derived glycans. In particular, the O-glycans containing sialic acid Neu5Ac accounted for 71.93% of the total O-glycans, which were different from that of other species, including mouse intestine, fish intestine, and porcine colon. The high content sialylated mucin may explain its effect in biological processes. Furthermore, the immunological activity results indicated that the porcine intestinal mucin could promote phagocytosis and proliferation without any cytotoxic effects, which may aid in the development of immunomodulators.
Assuntos
Heparina/química , Mucosa Intestinal/química , Mucosa Intestinal/metabolismo , Macrófagos/efeitos dos fármacos , Mucinas/química , Mucinas/farmacologia , Animais , Heparina/metabolismo , Macrófagos/imunologia , Camundongos , Células RAW 264.7 , SuínosRESUMO
Mucin-domain glycoproteins comprise a class of proteins whose densely O-glycosylated mucin domains adopt a secondary structure with unique biophysical and biochemical properties. The canonical family of mucins is well-known to be involved in various diseases, especially cancer. Despite this, very little is known about the site-specific molecular structures and biological activities of mucins, in part because they are extremely challenging to study by mass spectrometry (MS). Here, we summarize recent advancements toward this goal, with a particular focus on mucin-domain glycoproteins as opposed to general O-glycoproteins. We summarize proteolytic digestion techniques, enrichment strategies, MS fragmentation, and intact analysis, as well as new bioinformatic platforms. In particular, we highlight mucin directed technologies such as mucin-selective proteases, tunable mucin platforms, and a mucinomics strategy to enrich mucin-domain glycoproteins from complex samples. Finally, we provide examples of targeted mucin-domain glycoproteomics that combine these techniques in comprehensive site-specific analyses of proteins. Overall, this Review summarizes the methods, challenges, and new opportunities associated with studying enigmatic mucin domains.
Assuntos
Mucinas/química , Animais , Biologia Computacional , Glicosídeo Hidrolases/química , Humanos , Espectrometria de Massas/métodos , Peptídeo Hidrolases/química , Domínios Proteicos , Proteólise , Proteoma/química , Proteômica/métodosRESUMO
The human conjunctival epithelial cells (HCEC) line the inner sides of the eyelids and the anterior part of the sclera. They include goblet cells that secret mucus into the tear film that protects the ocular surface. The conjunctival epithelium is subjected to mechano-physical stimuli due to eyelid movement during blinking, during wiping and rubbing the eyes, and when exposed to wind and air currents. We cultured primary HCEC under air-liquid interface (ALI) conditions in custom-designed wells that can be disassembled for installation of the in vitro model in a flow chamber. We exposed the HCEC after ALI culture of 8-10 days to steady and oscillatory airflows. The in vitro model of HCEC was exposed to steady wall shear stresses (sWSS) of 0.5 and 1.0 dyne/cm2 for lengths of 30 and 60 min and to oscillatory wall shear stresses (oWSS) of 0.5 and 0.77 dyne/cm2 amplitudes for a length of 10 min. Cytoskeletal alterations and MUC5AC mucin secretion in response to WSS were investigated using immunohistochemically fluorescent staining and enzyme-linked lectin assay (ELLA), respectively. The results revealed that both exposure times and sWSS values increased the polymerization of F-actin filaments while mucin secretion decreased. However, after a recovery of 24 h in the incubator we observed a decrease of F-actin fibers and mucin secretion only for exposure of 30 min. The length of exposure was more influential on cytoskeletal alterations than the level of sWSS. The very small effect of sWSS on mucin secretion is most likely related to the much smaller amount of goblet cell than in other mucus-secreting tissue. The results for both oWSS amplitudes revealed similar trends regarding F-actin and mucin secretion. Immediately post-exposure we observed an increase in polymerization of F-actin filaments while mucin secretion decreased. However, after 24-h recovery we observed that both F-actin and mucin secretion returned to the same values as for unexposed cultures. The results of this study suggest that WSS should be considered while exploring the physiological characteristics of HCEC.
Assuntos
Túnica Conjuntiva/patologia , Células Epiteliais/patologia , Citoesqueleto de Actina , Actinas/metabolismo , Actinas/fisiologia , Células Cultivadas , Citoesqueleto/metabolismo , Epitélio , Movimentos Oculares , Pálpebras , Células Caliciformes/citologia , Humanos , Técnicas In Vitro , Lectinas/química , Mucina-5AC/química , Mucinas/química , Oscilometria , Resistência ao Cisalhamento , Estresse MecânicoRESUMO
In this paper, two amphiphilic graft copolymers were synthesized by grafting polylactic acid (PLA) as hydrophobic chain and poly(2-methyl-2-oxazoline) (PMeOx) or poly(2-methyl-2-oxazine) (PMeOzi) as hydrophilic chain, respectively, to a backbone of α,ß-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA). These original graft copolymers were used to prepare nanoparticles delivering Zileuton in inhalation therapy. Among various tested methods, direct nanoprecipitation proved to be the best technique to prepare nanoparticles with the smallest dimensions, the narrowest dimensional distribution and a spherical shape. To overcome the size limitations for administration by inhalation, the nano-into-micro strategy was applied, encapsulating the nanoparticles in water-soluble mannitol-based microparticles by spray-drying. This process has allowed to produce spherical microparticles with the proper size for optimal lung deposition, and, once in contact with fluids mimicking the lung district, able to dissolve and release non-aggregated nanoparticles, potentially able to spread through the mucus, releasing about 70% of the drug payload in 24 h.