Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A mucus-secreting human colonic cancer cell line. Purification and partial characterization of the secreted mucins.

ABSTRACT: A stably differentiated clonal derivative (Cl.16E) of the human colonic adenocarcinoma cell line HT29 secretes in culture high-Mr glycoproteins that were purified from the serum-free conditioned medium by preparative SDS/polyacrylamide-gel electrophoresis. Analysis of the oligosaccharides released from the [3H]glucosamine-labelled high-Mr glycoproteins by alkaline-borohydride treatment showed that this material consisted of O-linked oligosaccharides (without any detectable N-linked oligosaccharides) that were eluted as three fractions from Bio-Gel P-6 columns. The main oligosaccharide fraction obtained after such treatment and desialylation was eluted together with a six-unit glucose polymer from a Bio-Gel P-4 column. Polyclonal antibodies were raised against the high-Mr glycoproteins, and in immunoblot analysis they reacted specifically with the high-Mr glycoproteins present in the conditioned medium. Furthermore, immunohistochemical staining of sections in paraffin wax revealed that these antibodies labelled normal human gastrointestinal mucins. We conclude that (1) the high-Mr glycoproteins prepared by SDS/polyacrylamide-gel electrophoresis are pure mucus glycoproteins on the basis of sensitivity to alkaline-borohydride treatment, monosaccharide composition and immunochemical and immunohistological findings, and (2) these mucins have antigenic determinants in common with the normal human gastrointestinal mucins.

SUBMITTER: Maoret JJ

PROVIDER: S-EPMC1138434 | biostudies-other | 1989 Mar

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Characterization of pig colonic mucins.

Project description:Pig colonic mucins isolated from the adherent mucus gel in the presence of proteinase inhibitors were solubilized by homogenization and the component mucins fractionated by CsC1 density-gradient centrifugation. Polymeric and reduced pig colonic mucin were both largely excluded on Sepharose CL-2B, papain-digested colonic mucin was included. The M(r) values of polymeric, reduced and digested mucins were 5.5 x 10(6), 2.1 x 10(6) and 0.6 x 10(6) respectively. This suggests that pig colonic mucin is comprised of 2-3 subunits, each subunit containing 3-4 glycosylated regions. The intrinsic viscosities of polymeric, reduced and digested mucin were 240 ml.g-1, 100 ml.g-1 and 20 ml.g-1 respectively. Polymeric pig colonic mucin comprised 16% protein per mg of glycoprotein and was rich in serine, threonine and proline (43% of total amino acids). There were approx. 150 disulphide bridges and 53 free thiol groups per mucin polymer. A seventh of the protein content was lost on reduction. This protein was particularly rich in proline and the hydrophobic amino acids. Papain-digested pig colonic mucin contained 11% protein per mg of glycoprotein and was rich in serine, threonine, glutamate and aspartate. All types of amino acids with the exception of aspartate were lost on digestion. The amino acid analysis of the proteolytically digested regions of pig colonic mucin are markedly different to the tandem repeat regions of the human mucin genes shown to be expressed in the colon.

| S-EPMC1217439 | biostudies-other

Transglutaminase 3 crosslinks the secreted gel-forming mucus component Mucin-2 and stabilizes the colonic mucus layer.

Project description:The colonic mucus layer is organized as a two-layered system providing a physical barrier against pathogens and simultaneously harboring the commensal flora. The factors contributing to the organization of this gel network are not well understood. In this study, the impact of transglutaminase activity on this architecture was analyzed. Here, we show that transglutaminase TGM3 is the major transglutaminase-isoform expressed and synthesized in the colon. Furthermore, intrinsic extracellular transglutaminase activity in the secreted mucus was demonstrated in vitro and ex vivo. Absence of this acyl-transferase activity resulted in faster degradation of the major mucus component the MUC2 mucin and changed the biochemical properties of mucus. Finally, TGM3-deficient mice showed an early increased susceptibility to Dextran Sodium Sulfate-induced colitis. Here, we report that natural isopeptide cross-linking by TGM3 is important for mucus homeostasis and protection of the colon from inflammation, reducing the risk of colitis.

| S-EPMC8752817 | biostudies-literature

Increase in secreted airway mucins and partial Muc5b STAT6/FoxA2 regulation during Pneumocystis primary infection.

Project description:Airway mucus responses to subclinical infections may explain variations in progression of chronic lung diseases and differences in clinical expression of respiratory infections across individuals. Pneumocystis associates to more severe Chronic Obstructive Pulmonary Disease (COPD), asthma, respiratory distress of premature newborns, and is a consistent subclinical infection between 2 and 5 months of age when hospitalizations for respiratory cause and infant mortality are higher. This atypical fungus associates to increased mucin 5AC (MUC5AC), a central effector of Th2-type allergic inflammation, in infant lungs. However, mucus progression, expression of MUC5B essential for airway defense, and potential for pharmacologic modulation of mucus during Pneumocystis infection remain unknown. We measured MUC5B and Pneumocystis in infant lungs, and progression of mucin levels and effect of inhibition of the STAT6/FoxA2 mucus pathway using Kaempferol, a JAK/STAT6 inhibitor, in immunocompetent rats during Pneumocystis primary infection. Pneumocystis associated to increased MUC5B in infant lungs. Muc5b increased earlier and more abundantly than Muc5ac during experimental primary infection suggesting an acute defensive response against Pneumocystis as described against bacteria, while increased Muc5ac levels supports an ongoing allergic, Th2 lymphocyte-type response during primary Pneumocystis infection. Kaempferol partly reversed Muc5b stimulation suggesting limited potential for pharmacological modulation via the STAT6-FoxA2 pathway.

| S-EPMC6376022 | biostudies-literature

Contribution of mucus concentration and secreted mucins Muc5ac and Muc5b to the pathogenesis of muco-obstructive lung disease.

Project description:Airway diseases, including cigarette smoke-induced chronic bronchitis, cystic fibrosis, and primary ciliary dyskinesia are associated with decreased mucociliary clearance (MCC). However, it is not known whether a simple reduction in MCC or concentration-dependent mucus adhesion to airway surfaces dominates disease pathogenesis or whether decreasing the concentration of secreted mucins may be therapeutic. To address these questions, Scnn1b-Tg mice, which exhibit airway mucus dehydration/adhesion, were compared and crossed with Muc5b- and Muc5ac-deficient mice. Absence of Muc5b caused a 90% reduction in MCC, whereas Scnn1b-Tg mice exhibited an ∼50% reduction. However, the degree of MCC reduction did not correlate with bronchitic airway pathology, which was observed only in Scnn1b-Tg mice. Ablation of Muc5b significantly reduced the extent of mucus plugging in Scnn1b-Tg mice. However, complete absence of Muc5b in Scnn1b-Tg mice was associated with increased airway inflammation, suggesting that Muc5b is required to maintain immune homeostasis. Loss of Muc5ac had few phenotypic consequences in Scnn1b-Tg mice. These data suggest that: (i) mucus hyperconcentration dominates over MCC reduction alone to produce bronchitic airway pathology; (ii) Muc5b is the dominant contributor to the Scnn1b-Tg phenotype; and (iii) therapies that limit mucin secretion may reduce plugging, but complete Muc5b removal from airway surfaces may be detrimental.

| S-EPMC5250616 | biostudies-literature

Mucins secreted by a transformed cell line derived from human tracheal gland cells.

Project description:High-molecular-mass glycoconjugates are secreted by the continuous cell line MM-39, which has been obtained from cultured human tracheal gland cells transformed by simian virus 40. They were purified on Sepharose(R) CL-4B and then by two steps of density-gradient centrifugation. High-molecular-mass glycoproteins resistant to digestion by hyaluronidase, chondroitin ABC lyase and heparitinase were obtained, in addition to hyaluronic acid and proteoglycans. They were susceptible to beta-elimination. They contained polylactosaminoglycan chains as well as carbohydrate chains with a terminal sialic acid in the NeuAc alpha2-3 sequence. Most of them have a buoyant density of 1.45 g/ml in CsCl-density-gradient centrifugation, except for MUC1. The MM-39 cells were also characterized by a high expression of MUC1 and MUC4 genes, but they did not express MUC2, MUC3, MUC5B and MUC5AC. Therefore the MM-39 cells synthesized mucin-like glycoproteins as well as lysozyme and mucous proteinase inhibitor [Merten, Kammouni, Renaud, Birg, Mattéi and Figarella (1996) Am. J. Respir. Cell. Mol. Biol. 15, 520-528]; they should be considered as having a mixed, both serous and mucous, phenotype.

| S-EPMC1218688 | biostudies-other

Intracellular Processing of Human Secreted Polymeric Airway Mucins.

Project description:Mucociliary clearance is a crucial component of innate defense of the lung. In respiratory diseases, such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis, mucus with abnormal properties contributes to obstruction of the airways. The failure in function of mucus in airway clearance and pathogen protection leads to chronic infection and risk of death. Polymeric mucins (MUC5AC and MUC5B) provide the structural framework of the airway mucus gel. The intracellular synthesis and assembly of these enormous, polymeric O-linked glycoproteins is a complex, multistage process involving intra- and intermolecular disulfide bond formation and extensive addition of O-glycan chains. The fully formed polymers are packaged in a highly organized and condensed form within secretory granules inside specialized secretory cells, and after the appropriate stimulus, mucins are released and expand to form mucus. This short article brings together the current knowledge on the different steps in the production of mucin polymers and the molecular mechanisms that condense them into a packaged form in secretory granules. It is by unraveling the molecular mechanisms that control intracellular mucin supramolecular structure that we might gain new insight into what determines mucus gel properties in health and disease.

| S-EPMC6276985 | biostudies-literature

Purification and characterization of a plasminogen activator secreted by a pig kidney cell line.

Project description:The plasminogen activator secreted by calcitonin-treated pig kidney cells was purified, characterized and compared with human urinary urokinase. The purification procedure was based on the following steps: sulphopropyl-Sephadex chromatography, p-aminobenzamidine-Sepharose chromatography, preparative sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectrofocusing. The purified enzyme was obtained from the conditioned medium with a yield of 13% and a purification factor of 390-fold. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under non-reducing conditions showed one closely spaced doublet with an Mr of 50 000; in the presence of reducing agents, two additional bands of Mr 30 000 and 20 000 appeared. The purified enzyme resembles the 53 000-Mr components of human urinary urokinase in amino acid composition and two-dimensional tryptic peptide maps and in its catalytic properties, and the two enzymes cross-react immunologically with rabbit antibodies raised against either. The enzyme appears to be different from tissue plasminogen activator secreted by HeLa cells.

| S-EPMC1153570 | biostudies-other

Purification and Partial Characterization of ?-Glucosidase in Chayote (Sechium edule).

Project description:?-Glucosidase (EC 3.2.1.21) is a prominent member of the GH1 family of glycoside hydrolases. The properties of this ?-glucosidase appear to include resistance to temperature, urea, and iodoacetamide, and it is activated by 2-ME, similar to other members. ?-Glucosidase from chayote (Sechium edule) was purified by ionic-interchange chromatography and molecular exclusion chromatography. Peptides detected by LC-ESI-MS/MS were compared with other ?-glucosidases using the BLAST program. This enzyme is a 116 kDa protein composed of two sub-units of 58 kDa and shows homology with Cucumis sativus ?-glucosidase (NCBI reference sequence XP_004154617.1), in which seven peptides were found with relative masses ranging from 874.3643 to 1587.8297. The stability of ?-glucosidase depends on an initial concentration of 0.2 mg/mL of protein at pH 5.0 which decreases by 33% in a period of 30 h, and then stabilizes and is active for the next 5 days (pH 4.0 gives similar results). One hundred ?g/mL ?-D-glucose inhibited ?-glucosidase activity by more than 50%. The enzyme had a Km of 4.88 mM with p-NPG and a Kcat of 10,000 min(-1). The optimal conditions for the enzyme require a pH of 4.0 and a temperature of 50 °C.

| S-EPMC6332095 | biostudies-literature

Searching the Evolutionary Origin of Epithelial Mucus Protein Components-Mucins and FCGBP.

Project description:The gel-forming mucins are large glycosylated proteins that are essential components of the mucus layers covering epithelial cells. Using novel methods of identifying mucins based on profile hidden Markov models, we have found a large number of such proteins in Metazoa, aiding in their classification and allowing evolutionary studies. Most vertebrates have 5-6 gel-forming mucin genes and the genomic arrangement of these genes is well conserved throughout vertebrates. An exception is the frog Xenopus tropicalis with an expanded repertoire of at least 26 mucins of this type. Furthermore, we found that the ovomucin protein, originally identified in chicken, is characteristic of reptiles, birds, and amphibians. Muc6 is absent in teleost fish, but we now show that it is present in animals such as ghost sharks, demonstrating an early origin in vertebrate evolution. Public RNA-Seq data were analyzed with respect to mucins in zebrafish, frog, and chicken, thus allowing comparison in regard of tissue and developmental specificity. Analyses of invertebrate proteins reveal that gel-forming-mucin type of proteins is widely distributed also in this group. Their presence in Cnidaria, Porifera, and in Ctenophora (comb jellies) shows that these proteins were present early in metazoan evolution. Finally, we examined the evolution of the FCGBP protein, abundant in mucus and related to gel-forming mucins in terms of structure and localization. We demonstrate that FCGBP, ubiquitous in vertebrates, has a conserved N-terminal domain. Interestingly, this domain is also present as an N-terminal sequence in a number of bacterial proteins.

| S-EPMC4948705 | biostudies-literature

Partial purification and characterization of phytase from Aspergillus foetidus MTCC 11682.

Project description:Phytase is a phosphatase enzyme widely used as feed additive to release inorganic phosphorus from plant phytate and enhance its uptake in monogastric animals. Although engineered fungal phytases are used most, a natural enzyme gives opportunity to understand novel properties, if any. In the current study, a novel fungal strain, Aspergillus foetidus MTCC 11682 was immobilized on poly urethane cubes and used for phytase production, purification and molecular characterization. Phytase produced by this method was partially purified by ammonium sulphate precipitation and Sephacryl S-200HR gel filtration to 23.4-fold (compared to crude extract) with recovery of 13% protein. Electrophoresis analysis revealed that phytase has molecular weight of 90.5 kDa on non-reducing and 129.6 kDa on reducing SDS-PAGE. The purified phytase exhibited a wider pH and temperature stability. Analysis of the cloned sequence showed that the gene has 1176 bp that encodes for a peptide of 391 amino acids of the core catalytic region. It was also found that phytase from A. foetidus has a sequence identity of 99% with the phytase gene of other Aspergillus species at nucleotide level and 100% at protein level in A. niger, A. awamori, A. oryzae. In silico analysis of sequence identified the presence of two consecutive and one non-consecutive intra chain disulfide bonds in the phytase. This probably contributed to the differential migration of phytase on reducing and non-reducing SDS-PAGE. There are predicted 11 O-glycosylation sites and 8 N-glycosylation sites, possibly contributed to an enhanced stability of enzyme produced by this organism. This study opened up a new horizon for exploring the novel properties of phytase for other applications.

| S-EPMC6320329 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data