MUC5AC,but not MUC2, is a prominent mucin in respiratory secretions

Airway mucus was collected from healthy and chronic bronchitic subjects. The chronic bronchitic sputum was separated into gel and sol phase by centrifugation and mucins were isolated using isopycnic density-gradient centrfugation in CsCl. The presence of the MUC5AC and MUC2 mucins was investigated with antisera raised against synthetic peptides with sequences from the respective apoproteins. The gel and sol phase of chronic bronchitic sputum as well as healthy respiratory secretions were shown to contain MUC5AC whereas the MUC2 mucin could not be detected. Rate-zonal centrifugation showed that the MUC5AC mucin was large, polydisperse in size and that reduction yielded subunits. Ion-exchange HPLC revealed the presence of two subunit populations in all secretions, the MUC5AC subunits always being the more acidic. MUC5AC is thus the first large, subunit-based, gel-forming respiratory mucin identified and this glycoprotein is biochemically distinct from at least one other population of large, gel-forming mucins also composed of subunits but lacking a genetic identity.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate - CF cystic fibrosis - DFP diisopropylphosphofluoridate - DTT dithiothreitol - EDTA ethylenedinitrilotetraacetic acid - NEM N-ethylmaleimide - PAS periodic acid/Schiffs - PMSF phenylmethylsulphonyl fluoride - Tris Tris(hydroxymethyl)aminomethane - VNTR variable number of tandem repeats     

Regulation of MUC5AC mucin secretion by depletion of AQP5 in SPC-A1 cells

Airway mucus is regulated by many inflammatory mediators such as ILs, TNF-alpha, EGF, PGF2alpha, LT, and so on. Recently, the relationship between membrane ion channel and mucus production has been under investigation. The present study aimed to examine whether AQP5 was involved in modulation of mucin expression and secretion in airway submucosal gland cells (SPC-A1). A recombinant plasmid (pShAQP5) containing small hairpin RNA expression cassette targeting AQP5 sequence was constructed. In pShAQP5 transiently transfected cells, ELISA showed MUC5AC synthesis and secretion were increased by 57.9% and 85.3%, respectively, on day 5 after pShAQP5 transfection. While in five stably transfected clones (shAQP5-G1, G2, G3, A2, and A5), the upregulated levels of MUC5AC mRNA were 118%, 165%, 65%, 123%, and 38%, respectively. The elevated levels of MUC5AC synthesis and secretion varied from 59-156% and 33-166%, respectively. This is the first reliable investigation of the regulation of MUC5AC mucin secretion by silencing AQP5. Further study of the regulatory mechanism between AQPs and mucins may provide new strategies for development of novel antihypersecretory drugs in airway diseases.     

呼吸道黏蛋白5AC基因转录表达的顺式调控元件分析

目的：探讨呼吸道黏蛋白（mucin,MUC）5AC基因5'上游序列顺式调控元件在中性粒细胞弹力酶(neutrophil elastase , NE)诱导MUC5AC基因转录表达的调控机制。方法：应用DNA重组技术,构建含萤光素酶报告基因和MUC5AC启动子不同长度片段的嵌合质粒。采用定点突变技术,在嵌合质粒的基础上构建MUC5AC启动子区特殊蛋白（specificity protein）-1和核因子(nuclear factor, NF)-κB结合位点单独突变体,并测定NE刺激的转染细胞荧光素酶相对活性。结果：成功构建了4种含有不同长度MUC5AC基因启动子序列的荧光索酶报告基因质粒。含有启动子序列-1330bp、-689bp、-324bp的嵌合质粒荧光素酶相对光强度较对照组均显著增加,而含有启动子序列-64bp的嵌合质粒荧光素酶相对光强度与对照组相比差异无统计学意义。NE可诱导含有MUC5AC启动子区NF-кB结合位点单独突变体（pGL3E-MUC5AC-NF-кB-MU）荧光素酶相对光强度增加,而NE不能诱导Sp-l结合位点单独突变体（pGL3E-MUC5AC-SP-1-MU）荧光素酶表达增加。结论：MUC5AC 5'上游序列中-324～-64位点存在参与NE诱导MUC5AC基因表达的重要调控元件,位于此区域的顺式作用元件Sp-1位点在NE诱导MUC5AC基因表达机制中起重要作用,该位点可能作为靶向性基因治疗的关键调控元件。     

The expression of human FUT1 in HT-29/M3 colon cancer cells instructs the glycosylation of MUC1 and MUC5AC apomucins

Recently, we have reported that in normal gastric epithelium, the expression of gastric apomucins MUC5AC and MUC6 is associated with the specific expression of type 1 and type 2 Lewis antigens, and FUT2 and FUT1 fucosyltransferases, respectively. Until now, there are no data demonstrating the direct implication of specific glycosyltransferases in the specific patterns of apomucin glycosylation.HT29/M3 colon cancer cell line express MUC1, MUC5AC, type 1 Lewis antigens and FUT2 but not type 2 structures and FUT1, as it occurs in the epithelial cells of the gastric superficial epithelium. These cells were transfected with the cDNA of human FUT1, the -1,2-fucosyltransferase responsible for the synthesis of type 2 Lewis antigens, to assess the implication of FUT1 in the glycosylation of MUC1 and MUC5AC.The M3-FUT1 clones obtained express high levels of type 2 Lewis antigens: H type 2 and Ley antigens. Immunoprecipitation of MUC1 and MUC5AC apomucins gives the direct evidence that FUT1 catalyses the addition of -1,2-fucose to these apomucins, supporting the hypothesis that the pattern of apomucin glycosylation is not only instructed by the mucin primary sequence but also by the set of glycosyltransferases expressed in each specific cell type.     

Cleavage in the GDPH sequence of the C-terminal cysteine-rich part of the human MUC5AC mucin

MUC5AC is the main gel-forming mucin expressed by goblet cells of the airways and stomach where it protects the underlying epithelia. We expressed the C-terminal cysteine-rich part of the human MUC5AC mucin in CHO-K1 cells (Chinese-hamster ovary K1 cells) where it formed disulfide-linked dimers in the ER (endoplasmic reticulum). After reducing the disulfide bonds of these dimers, not only the expected monomers were found, but also two smaller fragments, indicating that the protein was partially cleaved. The site of cleavage was located at an Asp-Pro bond situated in a GDPH (Gly-Asp-Pro-His) sequence found in the vWD4 (von Willebrand D4) domain. This sequence is also found in the human MUC2 mucin, previously shown to be cleaved at the same site by a slow, non-enzymatic process triggered by a pH below 6 [Lidell, Johansson and Hansson (2003) J. Biol. Chem. 278, 13944-13951]. In contrast with this, the cleavage of MUC5AC started already in the neutral ER. However, it continued and was slightly accelerated at a pH below 6.5, a pH found in the later parts of the secretory pathway. The cleavage generated a reactive group in the new C-terminus that could link the protein to a primary amine. No cleavage of MUC5AC has so far been reported. By using an antibody reacting with the C-terminal cleavage fragment, we could verify that the cleavage occurs in wild-type MUC5AC produced by HT-29 cells. The cleavage of MUC5AC and the generation of the reactive new C-terminus could contribute to the adherent and viscous mucus found at chronic lung diseases such as asthma and cystic fibrosis, characterized by mucus hypersecretion and lowered pH of the airways.     

Inhibition of E-Cadherin Dependent Cell-Cell Contact Promotes MUC5AC Mucin Production through the Activation of Epidermal Growth Factor Receptors

Mucin production by epithelial cells is modulated by many soluble factors, including epidermal growth factor (EGF). E-Cadherin promotes EGF receptor (EGFR)-mediated MUC5AC mucin production in airway epithelial cells in dense cultures, suggesting the involvement of E-cadherin in activating EGFRs and mucin production. However, the role of E-cadherin in modulating mucin production is not completely understood. We examined its role in MUC5AC production in a human lung epithelial cell line, NCI-H292. Treatment of low density NCI-H292 cells with an anti-E-cadherin monoclonal antibody (SHE78-7) inhibited cell-cell contact in the dispersed colonies, but promoted MUC5AC production. Furthermore, treatment of the NCI-H292 cells with anti-E-cadherin antibody stimulated phosphorylation of extracellular signal-regulated kinase (ERK). The enhanced production of MUC5AC was inhibited with an EGFR inhibitor and with a MEK inhibitor, but not with a Src family kinase inhibitor. These results suggest that inhibition of E-cadherin activates EGFRs independently of Src and promotes MUC5AC production through the ERK signaling pathway in sparsely cultured NCI-H292 cells.     

Quantitative MUC5AC and MUC6 mucin estimations in gastric mucus by a least-squares minimization method

We have determined the molar proportions of the MUC5AC and MUC6 mucus glycoproteins (mucins) in mucus from the normal and pathological human gastric antrum using a least-squares minimization analysis applied to amino acid compositions. We noted that the content of MUC5AC mucin in mucus from individuals without gastroduodenal disease was very high, suggesting that the integrity and barrier properties of the adherent gastric mucus layer are normally maintained by building-block structures formed from this mucin alone. We observed that the molar content of MUC6 mucin doubled (without significance) in mucus from patients with duodenal ulcer, and increased five times (with high significance) in mucus from patients with gastric ulcer, when compared with that in mucus from individuals without gastroduodenal disease.     

Distinct localization of MUC5B glycoforms in the human salivary glands

Salivary mucins, encoded by the MUC5B gene, make up a heterogeneous family of molecules, which are secreted by several glands, including the submandibular, sublingual, and palatine glands. Previous studies have shown that heterogeneity in the salivary mucin population is related to its multiglandular origin. In the present study we address the question to what extent the mucin (MUC5B) population from a single human salivary gland is made up of different glycoforms. Using monoclonal antibodies to defined protein and sulfated carbohydrate epitopes specific to MUC5B, we conduct an immunohistochemical study of different salivary gland types, including submandibular, sublingual, and labial glands. In all tissues studied we found a mosaic expression pattern of sulfo-Lewis a antigen, recognized by mAb F2, which in salivary glands is exclusively present on MUC5B. On the other hand, mucous acini were uniformly labeled by mAb EU-MUC5Bb, evoked against a peptide-stretch of the tandem repeat region of MUC5B. Double staining with both antibodies confirmed the presence of MUC5B-positive/sulfo-Lewis a-positive cells, as well as MUC5B-positive/sulfo-Lewis a-negative cells within one glandular unit. These results indicate that one and the same salivary gland synthesizes different MUC5B glycoforms.     

Influence of the amino acid sequence on the MUC5AC motif peptide O-Glycosylation by Human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase(s)

The present work was carried out to study the role of the peptide moiety in the addition of O-linked N-acetylgalactosamine to human apomucin using human crude microsomal homogenates from gastric mucosa (as enzyme source) and a series of peptide acceptors representative of tandem repeat domains deduced from the MUC5AC mucin gene (expressed in the gastric mucosa). Being rich in threonine and serine placed in clusters, these peptides provided several potential sites for O-glycosylation. The glycosylated products were analysed by a combination of electrospray mass spectrometry and capillary electrophoresis in order to isolate the glycopeptides and to determine their sequence by Edman degradation. The O-glycosylation of our MUC5AC motif peptides gave information on the specificity and activity of the gastric microsomal UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase(s). The proline residues and the induced-conformations are of great importance for the recognition of MUC5AC peptides but they are not the only factors for the choice of the O-glycosylation sites. Moreover, for the di-glycosylated peptides, the flanking regions of the proline residues strongly influence the site of the second O-glycosylation.     

Integrin β1 subunit regulates cellular and secreted MUC5AC and MUC5B production in NCI–H292 human lung epithelial cells

The surface of the human respiratory tract is covered with a mucus layer containing mucin 5AC (MUC5AC) and mucin 5B (MUC5B) as the main components. This layer contributes to biological defense by eliminating irritants, but excessive MUC5AC secretion by the airway epithelial cells exacerbates asthma. Therefore, regulating mucin production is important for asthma treatment. In this study, the effects of integrin β1 subunit on MUC5AC and MUC5B production were examined in NCI–H292 human lung cancer epithelial cells. When integrin β1 was overexpressed, cellular and secreted MUC5AC levels were decreased, whereas cellular MUC5B production was increased. Conversely, integrin β1 depletion using siRNA increased cellular and secreted MUC5AC production, but decreased cellular MUC5B production. Further, the activity of extracellular signal-regulated kinase (ERK), which promotes MUC5AC production, was decreased by integrin β1 overexpression and increased by its depletion. These results suggest that integrin β1 suppresses MUC5AC production and promotes MUC5B production by downregulating ERK.     

Altered expression of MUC2 and MUC5AC in response to Shigella infection,an in vivo study

Infection of mucosal epithelial cells by Shigella species leads to an intense and acute inflammatory bowel disease that is characterized by watery diarrhea and purulent discharge. Mucin production is a common defense mechanism to protect the underlying mucosa against pathogens. The molecular mechanism(s) underlying mucin induction is unknown in Shigellosis. In this study, we have evaluated the relationship between Shigella infection, the expression of MUC2 and MUC5AC and the participation of signaling molecules TNF-α, PKC and ERK1/2. Shigella infection up-regulated MUC2 and MUC5AC expression in 6–8 h, through activation of TNF-α, PKC and ERK1/2. These results confirm that, in response to Shigella infection, the normal expression pattern of MUC-2 and MUC-5AC is altered. This in vivo study brings new insights into the molecular pathogenesis of Shigellosis and new potential therapeutic targets for Shigellosis.     

The MUC5AC glycoprotein is the primary receptor for Helicobacter pylori in the human stomach

Background and objectives. Helicobacter pylori shows a characteristic tropism for the mucus‐producing gastric epithelium. In infected patients, H. pylori colocalizes in situ with the gastric secretory mucin MUC5AC. The carbohydrate blood‐group antigen Lewis B (LeB) was deemed responsible for the adherence of H. pylori to the gastric surface epithelium. We sought to determine if MUC5AC is the carrier of LeB, and thus if MUC5AC is the underlying gene product functioning as the main receptor for H. pylori in the stomach. Methods. We studied three types of human tissue producing MUC5AC: Barrett's esophagus (BE), normal gastric tissue, and gastric metaplasia of the duodenum (GMD). Tissue sections were immuno‐fluorescently stained for MUC5AC or LeB, and subsequently incubated with one of three strains of Texas red‐labeled H. pylori, one of which was unable to bind to LeB. We determined the colocalization of MUC5AC or LeB with adherent H. pylori. Results. The binding patterns for the two LeB‐binding strains to all tissues were similar, whereas the strain unable to bind to LeB did not bind to any of the tissues. In normal gastric tissue, the LeB‐binding strains always bound to MUC5AC‐ and LeB‐positive epithelial cells. In four nonsecretor patients, colocalization of the LeB‐binding strains was found to MUC5AC‐positive gastric epithelial cells. In BE, the LeB‐binding H. pylori strains colocalized very specifically to MUC5AC‐positive cells. MUC5AC‐producing cells in GMD contained LeB. Yet, LeB‐binding H. pylori not only colocalized to MUC5AC or LeB present in GMD, but also bound to the LeB‐positive brush border of normal duodenal epithelium. Conclusions. Mucin MUC5AC is the most important carrier of the LeB carbohydrate structure in normal gastric tissue and forms the major receptor for H. pylori.     

Solid-Phase Synthesis of Core 8 O-Glycan-Linked MUC5AC Glycopeptide

The benzyl-protected disaccharide building blocks of core 8 O-glycan (15a/15b) for glycopeptide were stereoselectively synthesized by two glycosidation reactions with the glycosyl fluoride method. The building blocks were utilized in the solid-phase synthesis of a glycopeptide carrying two O-glycans with the consensus sequence of the tandem-repeat domain of MUC5AC. The synthetic glycopeptide was detached from the resin with reagent K, and subsequent debenzylation under conditions of low-acidity TfOH afforded glycopeptide 2. The synthetic sample will be used as a suitable standard in studies of the physicochemical or immunochemical characterization of mucin glycoforms.     

Heterogeneity in the protein cores of mucins isolated from human middle ear effusions: evidence for expression of different mucin gene products

High molecular weight mucins were isolated and purified from human middle ear effusions of children with Otitis Media with Effusion (OME) classified into three groups, (1) thick and (2) thin from anatomically normal children and (3) effusions from cleft palate patients. Amino acid analyses of the purified mucins from the three pools were similar but not identical with characteristic contents of serine threonine and proline (32%, 28%, and 38% for pools (1) (2) and (3) respectively). Proteinase resistant glycopeptide fragments corresponding to the tandem repeat domains of cloned mucin genes showed marked differences both between the three mucin pools and with the composition of the tandem repeat sequences of the cloned mucin genes expressed in the airways. Studies on the antigenic identity of middle ear mucins found an epitope likely to be present on MUC5AC, but only accounting for a maximum of 15% by weight and no reactivity was found with antibodies to MUC2 or MUC1. A polyclonal antibody raised to thick effusion mucins reacted strongly with human salivary mucin suggesting the presence of MUC5B epitopes. These studies suggest that more than one mucin gene product is secreted by the human middle ear mucosa and that there may be further mucin genes expressed by the middle ear that have yet to be cloned.     

Neutrophil elastase induces MUC5AC secretion via protease-activated receptor 2

Mucus hypersecretion is a major manifestation in patients with chronic inflammatory airway diseases, and mucin5AC (MUC5AC) protein is a major component of airway mucus. Previous studies have demonstrated that neutrophil elastase (NE) stimulates the secretion of MUC5AC from airway epithelial cells, however, the mechanism is poorly understood. NE is a known ligand for protein active receptors (PARs), which have been confirmed to participate in releasing MUC5AC in the airways. However, the role of PARs in NE-induced MUC5AC secretion remains unclear. We demonstrated that airway goblet-like Calu-3 cells express PAR1, PAR2, and PAR3 with a predominant level of PAR2. NE can increase PAR2 expression and MUC5AC release. In our study, we showed that NE binding to PAR2 can increase the cytosolic calcium concentration and subsequently activate PKC, leading to MUC5AC secretion. In order to investigate the mechanism of increased cytosolic calcium in Calu-3 cells, thapsigargin was used to exhaust the endoplasmic reticulum (ER) calcium pools, and 2-aminoethoxydiphenyl borate was used to inhibit the function of the store-operated calcium entry (SOCE) channels in the plasma membrane. We found that the NE-induced increase in intracellular calcium concentration is derived from release of the ER calcium pool and its subsequent calcium internal flux from the extracellular space via SOCE channels, which is dependent on sufficient levels of extracellular calcium.     

Somatostatin stimulates colonic MUC2 expression through SSTR5-Notch-Hes1 signaling pathway

Colonic mucus barrier is regarded as the first defense line against bacteria and antigens from directly attaching to the epithelium, which would further lead to intestinal inflammation activation and pathological conditions. As MUC2 mucin is the predominant component of the mucus, understanding the regulatory mechanisms of MUC2 is important for mucus barrier protection. Somatostatin (SST) has been found to play a role in colon protection through various manners. However, whether SST involves in colonic mucus barrier regulation is still unclear. The aim of this study is to investigate the effects and potential mechanisms of SST on colonic MUC2 expression and mucus secretion. In vivo study, exogenous somatostatin (octreotide) administration effectively stimulated mice colonic MUC2 expression and mucus secretion. In human goblet-like cell LS174T cells, SST exposure also significantly stimulated MUC2 expression and mucus secretion. Further studies indicated that SST receptor 5 (SSTR5) was significantly activated by SST, whereas specific SSTR5 siRNA transfection of LS174T cells significantly blocked SST-induced increase in MUC2 expression and mucus secretion. In addition, SSTR5 agonist L817,818 also upregulated MUC2 expression and mucus secretion in LS174T cells. Mechanistic studies further demonstrated that SST/SSTR5-mediated MUC2 upregulation was dependent on Notch-Hes1 pathway suppression by detecting notch intracellular domain (NICD) and Hes1 proteins. Taken together, our findings suggested that SST could participate in colonic mucus barrier regulation through SSTR5-Notch-Hes1-MUC2 signaling pathway. These findings provide a deep insight into the role of SST on colonic mucus regulation under physiological conditions.