Identification of Molecular Intermediates in the Assembly Pathway of the MUC5AC Mucin

Abstract

MUC5AC mucins secreted by HT-29 cells in culture are oligomeric glycoproteins with characteristics similar to the MUC5AC mucins isolated from human airway sputum (Sheehan, J. K., Brazeau, C., Kutay, S., Pigeon, H., Kirkham, S., Howard, M., and Thornton, D. J. (2000) Biochem. J. 347, 37-44). Therefore we have used this cell line as a model system to investigate the biosynthesis of this major airway mucin. Initial experiments showed that the MUC5AC mucins isolated from the cells were liable to depolymerization depending on the conditions used for their solubilization. Prevention against reduction resulted in large oligomers associated with the cells, similar to those secreted into the medium. Using a combination of density gradient centrifugation and agarose gel electrophoresis coupled with probes specific for different forms of the mucin we identified five major intracellular populations of the MUC5AC polypeptide (unglycosylated monomer and dimer, GalNAc-substituted dimer, fully glycosylated dimer, and higher order oligomers). Pulse-chase studies were performed to follow the flow of radioactivity through these various intracellular forms into the mature oligomeric mucin secreted into the medium (a process taking ϳ2-4 h). The results show that the mucin polypeptide undergoes dimerization and then becomes substituted with GalNAc residues prior to glycan elaboration to produce a mature mucin dimer, which then undergoes multimerization. These data indicate that this oligomeric mucin follows a similar assembly to the von Willebrand factor glycoprotein to yield long linear disulfide-linked chains.In vertebrates, epithelia are protected on their luminal side by mucus gels synthesized and secreted by specialized cells in the surface and/or underlying submucosa. These gels act as a physical barrier as well as provide the infrastructure to support an array of protective molecules and mechanisms for sequestering chemical toxins and pathogens. Furthermore, they contribute to the homeostasis and stability of the underlying epithelium. The molecular framework of mucus is provided by large, oligomeric, O-linked glycoproteins termed mucins. These macromolecules are polydisperse in mass (2-40 MDa) and size (0.5-10 m in length) and are assembled from a disulfide linkage of monomers (2-3 MDa) (1-5). Entanglement of the mucin chains is considered to be a major factor for gel formation, and as a consequence their size distribution is a key determinant of the physical properties of the mucus.In humans, four genes encoding oligomeric mucins have been identified: MUC5AC, MUC5B, MUC2, and MUC6, and these are found in a cluster on chromosome 11 (6). Their expression appears to be selectively controlled in a manner favoring tissue-specific expression, but the functional reasons for this are unclear. Sequence similarities of Cys-rich domains in the N and C termini of these mucins with another oligomeric, Olinked glycoprotein, von Willebrand factor (VWF), 1 suggest that mucins are assembled in a manner similar to VWF (7). Indeed, the consens...

Synaptotagmin 2 Couples Mucin Granule Exocytosis to Ca2+ Signaling from Endoplasmic Reticulum

“…4A). The heavy staining of goblet cells for PDI is consistent with the demands of synthesizing disulfidelinked polymeric mucins (31). Ca 2ϩ is heavily buffered in the cytoplasm, so changes in concentration are highly localized.…”

“…To identify rough ER, human lung sections were labeled with antibodies to the immature, nonglycosylated form of MUC5AC (immatMUC5AC) (31). Confocal microscopy showed extensive colocalization of immatMUC5AC and PDI (Fig.…”

Serine Proteases Degrade Airway Mucins in Cystic Fibrosis

“…1A. Due to the heterogeneity of the mucins, multiple bands can appear, as previously described (20,24,40).…”

Phylogenetic and Functional Analysis of Histidine Residues Essential for pH-dependent Multimerization of von Willebrand Factor

“…Like VWF, the homologous gel-forming mucins MUC2, MUC5AC, MUC5B, MUC6, MUC19, and FIMB1 have N-terminal D1D2DЈD3 domains (31). MUC2 (32), MUC5AC (33), and MUC19 (34) have been shown to form interchain disulfide bonds between N-terminal DЈD3 domains. As in the case of VWF, these mucins form dimers in the ER that assemble into disulfide-linked multimers in the Golgi (31), suggesting a conserved role for mucin D1D2DЈD3 domains in the pH-dependent assembly of multimers.…”