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     

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.     

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.     

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.     

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。     

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.     

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.     

COVID-19 Survivor Patients Carrying the Rs35705950 Risk Allele in MUC5B Have Higher Plasma Levels of Mucin 5B

Background: Genetic susceptibility to infectious diseases is partly due to the variation in the human genome, and COVID-19 is not the exception. This study aimed to identify whether risk alleles of known genes linked with emphysema (SERPINA1) and pulmonary fibrosis (MUC5B) are associated with severe COVID-19, and whether plasma mucin 5B differs according to patients’ outcomes. Materials and methods: We included 1258 Mexican subjects diagnosed with COVID-19. We genotyped rs2892474 and rs17580 of the SERPINA1 gene and rs35705950 of MUC5B. Based on the rs35705950 genotypes, mucin 5B plasma protein levels were quantified. Results: Homozygous for the risk alleles of the three polymorphisms were found in less than 5% of the study population, but no statistically significant difference in the genotype or allele association analysis. At the protein level, non-survivors carrying one or two copies of the risk allele rs35705950 in MUC5B (GT + TT) had lower levels of mucin 5B compared to the survivors (0.0 vs. 0.17 ng/mL, p = 0.0013). Conclusion: The polymorphisms rs28929474 and rs17580 of SERPINA1 and rs35705950 of MUC5B are not associated with the risk of severe COVID-19 in the Mexican population. COVID-19 survivor patients bearing one or two copies of the rs35705950 risk allele have higher plasma levels of mucin 5B.     

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.     

Dickkopf相关蛋白1抑制肺炎支原体P1-C诱导小鼠肺上皮细胞过度分泌MUC5AC

肺炎支原体(Mycoplasma pneumoniae)是儿童和成人最常见的呼吸道感染病原体。临床观察肺炎支原体感染会引起呼吸道黏液大量分泌,给患者呼吸造成困难,已有研究表明肺炎支原体感染会引起大量黏蛋白5AC (mucin 5AC,MUC5AC)的分泌。肺炎支原体P1黏附素通过介导病原体与宿主细胞的黏附在肺炎支原体感染的发病机制中发挥重要作用,其中P1的C-末端残基(P1-C)具有免疫原性。本研究探讨了Wnt(Wingless,Wnt)/β-catenin信号通路抑制因子Dickkopf-1(Dickkopf-1, DKK1)在肺炎支原体P1-C诱导的肺上皮细胞分泌黏蛋白MUC5AC的分子机制。利用扫描电镜(scanning electron microscope, SEM)、苏木精-伊红(hematoxylin-eosin, HE)染色观察肺炎支原体P1-C对小鼠肺上皮细胞(mouse airway epithelial cells, MAECs)黏液分泌的影响;利用蛋白芯片技术检测肺炎支原体P1-C对小鼠气道上皮细胞炎症因子分泌及对相关信号通路的富集分析;采用糖原染色(perio...     

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.     

Secretion of MUC5AC mucin from pancreatic cancer cells in response to forskolin and VIP

Bisphosphonates are potent antiresorptive drugs commonly employed in the treatment of metabolic bone diseases. Despite their frequent use, the mechanisms of bisphosphonates on bone cells have largely remained unclear. Receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for osteoclast formation and activation, whereas osteoprotegerin (OPG) neutralizes RANKL. Various osteotropic drugs have been demonstrated to modulate osteoblastic production of RANKL and OPG. In this study, we assessed the effects of the bisphosphonates pamidronate (PAM) and zoledronic acid (ZOL) on OPG mRNA steady-state levels (by semiquantitative RT-PCR) and protein production (by ELISA) in primary human osteoblasts (hOB). PAM increased OPG mRNA levels and protein secretion by hOB by up to 2- to 3-fold in a dose-dependent fashion with a maximum effect at 10(-6) M (P < 0.001) after 72 h. Similarly, ZOL enhanced OPG gene expression and protein secretion by hOB in a dose-dependent fashion with a maximum effect at 10(-8) M after 72 h, consistent with the higher biological potency of ZOL. Time course experiments indicated a stimulatory effect of PAM and ZOL on osteoblastic OPG protein secretion by 6-fold, respectively (P < 0.001). Pretreatment with PAM and ZOL prevented the inhibitory effects of the glucocorticoid dexamethasone on OPG mRNA and protein production. Analysis of cellular markers of osteoblastic differentiation revealed that PAM and ZOL induced type I collagen secretion and alkaline phosphatase activity by 2- and 4-fold, respectively (P < 0.0001 by ANOVA). In conclusion, our data suggest that bisphosphonates modulate OPG production by normal human osteoblasts, which may contribute to the inhibition of osteoclastic bone resorption. Since, OPG production increases with osteoblastic cell maturation, enhancement of OPG by bisphosphonates could be related to their stimulatory effects on osteoblastic differentiation.     

The isolated MUC5AC gene product from human ocular mucin displays intramolecular conformational heterogeneity

Atomic force microscopy (AFM) has been used to show that human ocular mucins contain at least three distinct polymer conformations, separable by isopycnic density gradient centrifugation. In this work we have used affinity purification against the anti(mucin peptide core) monoclonal antibody 45M1 to isolate MUC5AC gene products, a major component of human ocular mucins. AFM images confirm that the affinity-purified polymers adopt distinct conformations that coidentify with two of those observed in the parent population, and further reveal that these two different conformations can be present within the same polymer. AFM images of the complexes formed after incubation of 45M1 with the parent sample reveal different rates of binding to the two MUC5AC polymer types. The variability of gene products within a mucin population was revealed by analyzing the height distributions along the polymer contour and periodicities in distances between occupied antibody binding sites. AFM analysis of mucin polymers at the single molecule level provides new information about the genetic origins of individual polymers and the contributions of glycosylation to the physicochemical properties of mucins, which can be correlated with information obtained from biochemistry, antibody binding assays, and molecular biology techniques.     

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.     

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.     

ITGB4 deficiency induces mucus hypersecretion by upregulating MUC5AC in RSV-infected airway epithelial cells

Respiratory syncytial virus (RSV) infection is the main cause of bronchiolitis in children. Excessive mucus secretion is one of the primary symbols in RSV related lower respiratory tract infections (RSV-related LRTI), which is closely associated with the occurrence and development of asthma in later life. Integrin β4 (ITGB4) is down-regulated in the airway epithelial cells (AECs) of asthma patients which plays a critical role in the pathogenesis of asthma. However, whether ITGB4 is involved in the pathological processes of RSV infection remains unclear. In this study, we found that decreased expression of ITGB4 was negatively correlated with the level of MUC5AC in childhood AECs following RSV infection. Moreover, ITGB4 deficiency led to mucus hypersecretion and MUC5AC overexpression in the small airway of RSV-infected mice. MUC5AC expression was upregulated by ITGB4 in HBE cells through EGFR, ERK and c-Jun pathways. EGFR inhibitors treatment inhibited mucus hypersecretion and MUC5AC overexpression in ITGB4-deficient mice after RSV infection. Together, these results demonstrated that epithelial ITGB4 deficiency induces mucus hypersecretion by upregulating the expression of MUC5AC through EGFR/ERK/c-Jun pathway, which further associated with RSV-related LRTI.