Coarse-grained modeling and dynamics tracking of nanoparticles diffusion in human gut mucus.

The gastrointestinal (GI) tract's mucus layer serves as a critical barrier and a mediator in drug nanoparticle delivery. The mucus layer's diverse molecular structures and spatial complexity complicates the mechanistic study of the diffusion dynamics of particulate materials. In response, we developed a bi-component coarse-grained mucus model, specifically tailored for the colorectal cancer environment, that contained the two most abundant glycoproteins in GI mucus: Muc2 and Muc5AC. This model demonstrated the effects of molecular composition and concentration on mucus pore size, a key determinant in the permeability of nanoparticles. Using this computational model, we investigated the diffusion rate of polyethylene glycol (PEG) coated nanoparticles, a widely used muco-penetrating nanoparticle. We validated our model with experimentally characterized mucus pore sizes and the diffusional coefficients of PEG-coated nanoparticles in the mucus collected from cultured human colorectal goblet cells. Machine learning fingerprints were then employed to provide a mechanistic understanding of nanoparticle diffusional behavior. We found that larger nanoparticles tended to be trapped in mucus over longer durations but exhibited more ballistic diffusion over shorter time spans. Through these discoveries, our model provides a promising platform to study pharmacokinetics in the GI mucus layer.

Assembly and organization of the N-terminal region of mucin MUC5AC: Indications for structural and functional distinction from MUC5B.

Elevated levels of MUC5AC, one of the major gel-forming mucins in the lungs, are closely associated with chronic obstructive lung diseases such as chronic bronchitis and asthma. It is not known, however, how the structure and/or gel-making properties of MUC5AC contribute to innate lung defense in health and drive the formation of stagnant mucus in disease. To understand this, here we studied the biophysical properties and macromolecular assembly of MUC5AC compared to MUC5B. To study each native mucin, we used Calu3 monomucin cultures that produced MUC5AC or MUC5B. To understand the macromolecular assembly of MUC5AC through N-terminal oligomerization, we expressed a recombinant whole N-terminal domain (5ACNT). Scanning electron microscopy and atomic force microscopy imaging indicated that the two mucins formed distinct networks on epithelial and experimental surfaces; MUC5B formed linear, infrequently branched multimers, whereas MUC5AC formed tightly organized networks with a high degree of branching. Quartz crystal microbalance-dissipation monitoring experiments indicated that MUC5AC bound significantly more to hydrophobic surfaces and was stiffer and more viscoelastic as compared to MUC5B. Light scattering analysis determined that 5ACNT primarily forms disulfide-linked covalent dimers and higher-order oligomers (i.e., trimers and tetramers). Selective proteolytic digestion of the central glycosylated region of the full-length molecule confirmed that MUC5AC forms dimers and higher-order oligomers through its N terminus. Collectively, the distinct N-terminal organization of MUC5AC may explain the more adhesive and unique viscoelastic properties of branched, highly networked MUC5AC gels. These properties may generate insight into why/how MUC5AC forms a static, "tethered" mucus layer in chronic muco-obstructive lung diseases.

Mechanobiology of conjunctival epithelial cells exposed to wall shear stresses.

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.

Effects of macromolecular crowding on the folding and aggregation of glycosylated MUC5AC.

OBJECTIVE: To investigate the effects of macromolecular crowding on the folding and aggregation of MUC5AC with different levels of glycosylation during refolding. METHODS: Part 1:An in vitro catalytic reaction comprising the ppGalNAc T2 enzyme, uridine-5'-diphospho-N-galactosamine (UDP-GalNAc) and an 11-amino acid peptide substrate, was used to assess the enzyme activity of the ppGalNAc T2 enzyme in macromolecular crowding environment respectively with bovine serum albumin (BSA), polyethylene glycol (PEG2000), Dextran70 and Ficoll70 at different concentration and temperature. Part 2: The recombinant MUC5AC was expressed in HEK293 cells and purified by nickel column chromatography. The purified protein was treated with PNGase F, and the degree of glycosylation was analyzed by SDS-PAGE. Macromolecular crowding was simulated using PEG2000 at the concentrations of 50, 100, and 200 g/L. Deglycosylated-MUC5AC (d-MUC5AC) and glycosylated MUC5AC (g-MUC5AC) were denatured by GdnHCl and renatured by dilution in a refolding buffer. Protein aggregation was monitored continuously by absorbance reading at 488 nm using a UV spectrophotometer at 25 °C. The refolded proteins were centrifuged, the protein concentration of the supernatant was measured, and refolding yield in different refolding buffers was determined. RESULTS: Enzyme activityof ppGalNAc T2 was observed to increase with increasing crowding agent concentration, with highest enzyme activity at 200 g/L. Compared with the group in the absence of crowding reagent, the refolding yield of g-MUC5AC and d-MUC5AC were reduced significantly in the presence of different concentrations of PEG2000 (200, 100, and 50 g/L). Compared with the dilute solution, aggregation increased significantly in the presence of PEG2000, especially at 200 g/L. Moreover, in the crowded reagent with the same concentration, the refolding yield of d-MUC5AC was higher than that of g-MUC5AC, whereas the degree of aggregation of d-MUC5AC was lower than that of g-MUC5AC. CONCLUSION: The crowded intracellular environment reduces the refolding rate of MUC5AC and strongly induces the misfolding and aggregation of glycosylated MUC5AC.

Potential Interaction between Galectin-2 and MUC5AC in Mouse Gastric Mucus.

Galectins are a group of animal lectins characterized by their specificity for ß-galactosides. Of these, galectin-2 (Gal-2) is predominantly expressed in the gastrointestinal tract. In the current study, we used a mouse gastric mucous fraction to investigate whether Gal-2 is secreted from epithelial cells and identify its potential ligands in gastric mucus. Gal-2 was detected in the mouse gastric mucous fraction and could be eluted from it by the addition of lactose. Affinity chromatography using recombinant mouse galectin-2 (mGal-2)-immobilized adsorbent and subsequent LC-MS/MS identified MUC5AC, one of the major gastric mucin glycoproteins, as a potential ligand of mGal-2. Furthermore, MUC5AC was detected in the mouse gastric mucous fraction by Western blotting, and recombinant mGal-2 was adsorbed to this fraction in a carbohydrate-dependent manner. These results suggested that Gal-2 and MUC5AC in mouse gastric mucus interact in a ß-galactoside-dependent manner, resulting in a stronger barrier structure protecting the mucosal surface.

Different Forms of TFF2, A Lectin of the Human Gastric Mucus Barrier: In Vitro Binding Studies.

Trefoil factor family 2 (TFF2) and the mucin MUC6 are co-secreted from human gastric and duodenal glands. TFF2 binds MUC6 as a lectin and is a constituent of the gastric mucus. Herein, we investigated human gastric extracts by FPLC and identified mainly high- but also low-molecular-mass forms of TFF2. From the high-molecular-mass forms, TFF2 can be completely released by boiling in SDS or by harsh denaturing extraction. The low-molecular-mass form representing monomeric TFF2 can be washed out in part from gastric mucosa specimens with buffer. Overlay assays with radioactively labeled TFF2 revealed binding to the mucin MUC6 and not MUC5AC. This binding is modulated by Ca2+ and can be blocked by the lectin GSA-II and the monoclonal antibody HIK1083. TFF2 binding was also inhibited by Me-ß-Gal, but not the α anomer. Thus, both the α1,4GlcNAc as well as the juxtaperipheral ß-galactoside residues of the characteristic GlcNAcα1→4Galß1→R moiety of human MUC6 are essential for TFF2 binding. Furthermore, there are major differences in the TFF2 binding characteristics when human is compared with the porcine system. Taken together, TFF2 appears to fulfill an important role in stabilizing the inner insoluble gastric mucus barrier layer, particularly by its binding to the mucin MUC6.

α-Synuclein Penetrates Mucin Hydrogels Despite Its Mucoadhesive Properties.

Recent research indicates that the progression of Parkinson's disease can start from neurons of the enteric nervous system, which are in close contact with the gastrointestinal epithelium: α-synuclein molecules can be transferred from these epithelial cells in a prion-like fashion to enteric neurons. Thin mucus layers constitute a defense line against the exposure of noninfected cells to potentially harmful α-synuclein species. We show that-despite its mucoadhesive properties-α-synuclein can translocate across mucin hydrogels, and this process is accompanied by structural rearrangements of the mucin molecules within the gel. Penetration experiments with different α-synuclein variants and synthetic peptides suggest that two binding sites on α-synuclein are required to accomplish this rearrangement of the mucin matrix. Our results support the notion that the translocation of α-synuclein across mucus barriers observed here might be a critical step in the infection of the gastrointestinal epithelium and the development of Parkinson's disease.

Characterization of the human mucin 5AC promoter and its regulation by the histone acetyltransferase P300.

Histone acetylation is important in the modification of gene transcription in asthma and is regulated by histone acetyltransferases (HATs). P300 (P300 HAT) is an enzyme that is able to acetylate a wide variety of proteins. The modification of core histones can further regulate gene transcription, cell proliferation and other cell processes. Airway mucus hypersecretion is one of the most serious pathophysiological symptoms of chronic airway inflammatory diseases, and the human mucin 5AC (MUC5AC) gene has been reported to be a major component of respiratory secretions related to asthma and chronic obstructive pulmonary disease. In the present study, the 5' sequence of the human MUC5AC gene with a 1,348­bp DNA sequence was amplified from human A549 cells genomic DNA by polymerase chain reaction (PCR), and the product of the PCR was sequenced. By promoter deletion analysis, five promoter segments with different lengths were amplified by PCR. The products were identified by DNA sequencing and the six promoter segments were inserted into pGL3­enhancer vectors. The core promoter area was identified with a series of 5' deletion promoter plasmids using luciferase reporter assays. MUC5AC promoter activity, and the mRNA and protein expression levels of MUC5AC were observed in P300 wild­type, P300 mutant, P300 small interfering RNA and P300 control groups. The results showed that the core promoter area of MUC5AC was located within the ­935/+48 region and that P300 reduced the expression of MUC5AC in A549 cells.

Generation of 13C-Labeled MUC5AC Mucin Oligosaccharides for Stable Isotope Probing of Host-Associated Microbial Communities.

Stable isotope probing (SIP) has emerged as a powerful tool to address key questions about microbiota structure and function. To date, diverse isotopically labeled substrates have been used to characterize in situ growth activity of specific bacterial taxa and have revealed the flux of bioavailable substrates through microbial communities associated with health and disease. A major limitation to the growth of the field is the dearth of biologically relevant "heavy" labeled substrates. Mucin glycoproteins, for example, comprise an abundant source of carbon in the gut, oral cavity, respiratory tract, and other mucosal surfaces but are not commercially available. Here, we describe a method to incorporate a 13C-labeled monosaccharide into MUC5AC, a predominant mucin in both gastrointestinal and airway environments. Using the lung adenocarcinoma cell line, Calu-3, polarized cell cultures grown in 13C-labeled d-glucose resulted in liberal mucin production on the apical surface. Mucins were isolated by size-exclusion chromatography, and O-linked glycans were released by ß-elimination, permethylated, and analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) techniques. We demonstrate a 98.7% incorporation of 13C in the heterogeneous O-linked oligosaccharides that make up >80% of mucin dry weight. These "heavy" labeled glycoproteins represent a valuable tool for probing in vivo activity of host-associated bacterial communities and their interactions with the mucosal barrier. The continued expansion of labeled substrates for use in SIP will eventually allow bacterial taxa that degrade host compounds to be identified, with long-term potential for improved health and disease management.

Covalently mucoadhesive amphiphilic prodrug of 5-fluorouracil for enhanced permeation and improved oral absorption.

5-Fluorouracil (5-FU) is one of the important antitumor drugs and is widely used to treat various types of cancers. However, its administration is limited to intravenous route due to poor oral bioavailability. Herein, we hypothesized that the maleimide group-containing 5-FU prodrug (EMC-5-FU) could improve the intestinal mucoadhesion because the maleimide end group can covalently target thiol residues of mucin glycoprotein covering the intestinal enterocytes. In vitro bioadhesion results showed that EMC-5-FU exhibited good affinity to the cysteine-rich subdomains of mucin and NMR studies successfully verified the covalent attachment of EMC-5-FU to mucin. The intestinal perfusion study indicated that the intestinal bioadhesion and membrane permeability are greatly enhanced for EMC-5-FU, in comparison with 5-FU. Mucoadhesion investigations on rat intestine intuitively confirmed increased intestinal retention of 5-FU through maleimide-mediated mucoadhesion. Moreover, AUC0-24h of the total 5-FU level for EMC-5-FU solution was 2.65-fold higher compared with 5-FU solution. Our study further suggested that the amphiphilic prodrug EMC-5-FU with good mucoadhesion is a promising delivery strategy form to overcome multiple barriers of oral absorption.

A Rheological Study of the Association and Dynamics of MUC5AC Gels.

The details of how a mucus hydrogel forms from its primary structural component, mucin polymers, remain incompletely resolved. To explore this, we use a combination of macrorheology and single-particle tracking to investigate the bulk and microscopic mechanical properties of reconstituted MUC5AC mucin gels. We find that analyses of thermal fluctuations on the length scale of the micrometer-sized particles are not predictive of the linear viscoelastic response of the mucin gels, and that taken together, the results from both techniques help to provide complementary insight into the structure of the network. In particular, we show that macroscopic stiffening of MUC5AC gels can be brought about in different ways by targeting specific associations within the network using environmental triggers such as modifications to the pH, surfactant, and salt concentration. Our work may be important for understanding how environmental factors, including pathogens and therapeutic agents, alter the mechanical properties of fully constituted mucus.

Mucin-Inspired Lubrication on Hydrophobic Surfaces.

In the human body, high-molecular-weight glycoproteins called mucins play a key role in protecting epithelial surfaces against pathogenic attack, controlling the passage of molecules toward the tissue and enabling boundary lubrication with very low friction coefficients. However, neither the molecular mechanisms nor the chemical motifs of those biomacromolecules involved in these fundamental processes are fully understood. Thus, identifying the key features that render biomacromolecules such as mucins outstanding boundary lubricants could set the stage for creating versatile artificial superlubricants. We here demonstrate the importance of the hydrophobic terminal peptide domains of porcine gastric mucin (MUC5AC) and human salivary mucin (MUC5B) in the processes of adsorbing to and lubricating a hydrophobic PDMS surface. Tryptic digestion of those mucins results in removal of those terminal domains, which is accompanied by a loss of lubricity as well as surface adsorption. We show that this loss can in part be compensated by attaching hydrophobic phenyl groups to the glycosylated central part of the mucin macromolecule. Furthermore, we demonstrate that the simple biopolysaccharide dextran can be functionalized with hydrophobic groups which confers efficient surface adsorption and good lubricity on PDMS to the polysaccharide.

Structural Diversity of Human Gastric Mucin Glycans.

The mucin O-glycosylation of 10 individuals with and without gastric disease was examined in depth in order to generate a structural map of human gastric glycosylation. In the stomach, these mucins and their O-glycosylation protect the epithelial surface from the acidic gastric juice and provide the first point of interaction for pathogens such as Helicobacter pylori, reported to cause gastritis, gastric and duodenal ulcers and gastric cancer. The rational of the present study was to map the O-glycosylation that the pathogen may come in contact with. An enormous diversity in glycosylation was found, which varied both between individuals and within mucins from a single individual: mucin glycan chain length ranged from 2-13 residues, each individual carried 34-103 O-glycan structures and in total over 258 structures were identified. The majority of gastric O-glycans were neutral and fucosylated. Blood group I antigens, as well as terminal α1,4-GlcNAc-like and GalNAcß1-4GlcNAc-like (LacdiNAc-like), were common modifications of human gastric O-glycans. Furthemore, each individual carried 1-14 glycan structures that were unique for that individual. The diversity and alterations in gastric O-glycosylation broaden our understanding of the human gastric O-glycome and its implications for gastric cancer research and emphasize that the high individual variation makes it difficult to identify gastric cancer specific structures. However, despite the low number of individuals, we could verify a higher level of sialylation and sulfation on gastric O-glycans from cancerous tissue than from healthy stomachs.

One-Pot Ligation-Oxidative Deselenization at Selenocysteine and Selenocystine.

The use of native chemical ligation at selenocysteine (Sec) residues with peptide thioesters and additive-free selenocystine ligation with peptides bearing phenyl selenoesters, in concert with one-pot oxidative deselenization chemistry, is described. These approaches provide a simple and rapid method for accessing native peptides with serine in place of Sec at the ligation junction. The efficiency of both variants of the one-pot ligation-oxidative deselenization chemistry is probed through the synthesis of a MUC5AC-derived glycopeptide.

A Gastric Glycoform of MUC5AC Is a Biomarker of Mucinous Cysts of the Pancreas.

Molecular indicators to specify the risk posed by a pancreatic cyst would benefit patients. Previously we showed that most cancer-precursor cysts, termed mucinous cysts, produce abnormal glycoforms of the proteins MUC5AC and endorepellin. Here we sought to validate the glycoforms as a biomarker of mucinous cysts and to specify the oligosaccharide linkages that characterize MUC5AC. We hypothesized that mucinous cysts secrete MUC5AC displaying terminal N-acetylglucosamine (GlcNAc) in either alpha or beta linkage. We used antibody-lectin sandwich assays to detect glycoforms of MUC5AC and endorepellin in cyst fluid samples from three independent cohorts of 49, 32, and 66 patients, and we used monoclonal antibodies to test for terminal, alpha-linked GlcNAc and the enzyme that produces it. A biomarker panel comprising the previously-identified glycoforms of MUC5AC and endorepellin gave 96%, 96%, and 87% accuracy for identifying mucinous cysts in the three cohorts with an average sensitivity of 92% and an average specificity of 94%. Glycan analysis showed that MUC5AC produced by a subset of mucinous cysts displays terminal alpha-GlcNAc, a motif expressed in stomach glands. The alpha-linked glycoform of MUC5AC was unique to intraductal papillary mucinous neoplasms (IPMN), whereas terminal beta-linked GlcNAc was increased in both IPMNs and mucinous cystic neoplasms (MCN). The enzyme that synthesizes alpha-GlcNAc, A4GNT, was expressed in the epithelia of mucinous cysts that expressed alpha-GlcNAc, especially in regions with high-grade dysplasia. Thus IPMNs secrete a gastric glycoform of MUC5AC that displays terminal alpha-GlcNAc, and the combined alpha-GlcNAc and beta-GlcNAc glycoforms form an accurate biomarker of mucinous cysts.

Oxidative Deselenization of Selenocysteine: Applications for Programmed Ligation at Serine.

Despite the unique chemical properties of selenocysteine (Sec), ligation at Sec is an under-utilized methodology for protein synthesis. We describe herein an unprecedented protocol for the conversion of Sec to serine (Ser) in a single, high-yielding step. When coupled with ligation at Sec, this transformation provides a new approach to programmed ligations at Ser residues. This new reaction is compatible with a wide range of functionality, including the presence of unprotected amino acid side chains and appended glycans. The utility of the methodology is demonstrated in the rapid synthesis of complex glycopeptide fragments of the epithelial glycoproteins MUC5AC and MUC4 and through the total synthesis of the structured, cysteine (Cys)-free protein eglin C.

Identification of PAM4 (clivatuzumab)-reactive epitope on MUC5AC: a promising biomarker and therapeutic target for pancreatic cancer.

PAM4 is a monoclonal antibody showing high specificity for pancreatic ductal adenocarcinoma (PDAC). Humanized PAM4 labeled with 90Y in combination with low-dose gemcitabine has shown promising therapeutic activity, and is being evaluated in a phase III clinical trial. Prior efforts have suggested that PAM4 potentially reacts with MUC5AC, a secretory mucin expressed de novo in early pancreatic neoplasia and retained throughout disease progression. In present study, we provide further evidence validating MUC5AC as the PAM4 antigen, and locate PAM4-reactive epitope within the N-terminal cysteine-rich subdomain 2 (Cys2), thus differentiating PAM4 from most anti-MUC5AC antibodies known to-date. Specifically, we show (i) PAM4-antigen and MUC5AC were co-localized in multiple human cancer cell lines, including Capan-1, BxPC-3, and CFPAC-1; (ii) MUC5AC-specific siRNA prominently reduced the expression of both MUC5AC and PAM4-antigen in CFPAC-1 cells; (iii) PAM4 preferentially binds to the void-volume fractions from Sepharose-CL2B chromatography of Capan-1 culture supernatants, which were revealed by Western blot to display the ladder pattern characteristic of oligomeric MUC5AC; and (iv) the N-terminal Cys2 within several recombinant MUC5AC fragments is essential for binding to PAM4. These findings shed light on the mechanism of PAM4-based diagnosis and treatment for pancreatic cancer, and guide further exploration of its clinical utility.

Gli regulates MUC5AC transcription in human gastrointestinal cells.

MUC5AC is a well-known gastric differentiation marker, which has been frequently used for the classification of stomach cancer. Immunohistochemistry revealed that expression of MUC5AC decreases accompanied with increased malignant property of gastric mucosa, which further suggests the importance of MUC5AC gene regulation. Alignment of the 5'-flanking regions of MUC5AC gene of 13 mammal species denoted high homology within 200 bp upstream of the coding region. Luciferase activities of the deletion constructs containing upstream 451 bp or shorter fragments demonstrated that 15 bp region between -111 and -125 bp plays a critical role on MUC5AC promoter activity in gastrointestinal cells. We found a putative Gli-binding site in this 15 bp sequence, and named this region a highly conserved region containing a Gli-binding site (HCR-Gli). Overexpression of Gli homologs (Gli1, Gli2, and Gli3) clearly enhanced MUC5AC promoter activity. Exogenous modulation of Gli1 and Gli2 also affected the endogenous MUC5AC gene expression in gastrointestinal cells. Chromatin immunoprecipitation assays demonstrated that Gli1 directly binds to HCR-Gli: Gli regulates MUC5AC transcription via direct protein-DNA interaction. Conversely, in the 30 human cancer cell lines and various normal tissues, expression patterns of MUC5AC and Gli did not coincide wholly: MUC5AC showed cell line-specific or tissue-specific expression whereas Gli mostly revealed ubiquitous expression. Luciferase promoter assays suggested that the far distal MUC5AC promoter region containing upstream 4010 bp seems to have several enhancer elements for gene transcription. In addition, treatments with DNA demethylation reagent and/or histone deacetylase inhibitor induced MUC5AC expression in several cell lines that were deficient in MUC5AC expression. These results indicated that Gli is necessary but not sufficient for MUC5AC expression: namely, the multiple regulatory mechanisms should work in the distal promoter region of MUC5AC gene.

A combined small-angle X-ray and neutron scattering study of the structure of purified soluble gastrointestinal mucins.

The structures of purified soluble porcine gastric (Muc5ac) and duodenal (Muc2) mucin solutions at neutral and acidic pH were examined using small-angle X-ray scattering and small-angle neutron scattering experiments. We provide evidence for the morphology of the network above the semidilute overlap concentration and above the entanglement concentration. Furthermore, we investigated the gelation of both types of mucin solutions in response to a reduction in pH, where we observed the formation of large-scale heterogeneities within the polymer solutions, typical of microphase-separated gels. The concentration dependence of the inhomogeneity length scale (Ξ) and the amplitude of the excess scattering intensity [I(ex) (0)] are consistent with previously studied gelled synthetic polymeric systems. The persistence lengths of the chains were found to be similar for both Muc5ac and Muc2 from Kratky plots of the neutron data (8 ± 2 nm).

Quantitative glycoproteomics analysis reveals changes in N-glycosylation level associated with pancreatic ductal adenocarcinoma.

Glycosylation plays an important role in epithelial cancers, including pancreatic ductal adenocarcinoma. However, little is known about the glycoproteome of the human pancreas or its alterations associated with pancreatic tumorigenesis. Using quantitative glycoproteomics approach, we investigated protein N-glycosylation in pancreatic tumor tissue in comparison with normal pancreas and chronic pancreatitis tissue. The study lead to the discovery of a roster of glycoproteins with aberrant N-glycosylation level associated with pancreatic cancer, including mucin-5AC (MUC5AC), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), insulin-like growth factor binding protein (IGFBP3), and galectin-3-binding protein (LGALS3BP). Pathway analysis of cancer-associated aberrant glycoproteins revealed an emerging phenomenon that increased activity of N-glycosylation was implicated in several pancreatic cancer pathways, including TGF-ß, TNF, NF-kappa-B, and TFEB-related lysosomal changes. In addition, the study provided evidence that specific N-glycosylation sites within certain individual proteins can have significantly altered glycosylation occupancy in pancreatic cancer, reflecting the complexity of the molecular mechanisms underlying cancer-associated glycosylation events.