Intracellular Processing of Human Secreted Polymeric Airway Mucins

Abstract

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.

Keywords: MUC5AC; MUC5B; mucin; mucus.

Figure 1.

Schematic diagram of the major airways polymeric mucins, MUC5AC and MUC5B. The multidomain structure of the MUC5B and MUC5AC polypeptides shows the central mucin domains (MDs) that are enriched in serine, threonine, and proline (STP) residues (STP-rich regions) and are the sites of O-glycan attachment. The MD of MUC5B differs in size and sequence to the MD of MUC5AC. The MDs are interrupted and flanked by Cys domains (Cys) that are folded and stabilized by intramolecular disulfide linkages. The N-terminal (D1, D2, D′, D3) and C-terminal (D4, B, C, CK) protein domains share high sequence similarity with related domains in von Willebrand factor (vWF), and in both vWF and mucins these domains are stabilized by intramolecular disulfide bonds and are important for polymer formation. For vWF, a detailed analysis of these domains and their known and predicted disulfide-bond connectivity has been reported by Zhou and colleagues (44). Intermolecular disulfide linkages between CK domains are responsible for dimerization, whereas intermolecular disulfide linkages between D3 domains are responsible for multimerization of the CK-mediated mucin dimers. This assembly process (see Figure 2) produces linear mucin chains.

Figure 2.

Overview of polymeric mucin intracellular assembly and intragranular packaging. Polymeric mucin synthesis is a multistep process. 1) The N- and C-terminal domains and central Cys domains of the mucin polypeptide are folded in the endoplasmic reticulum (ER) via the formation of multiple intramolecular disulfide bonds. 2) The polypeptide then undergoes dimerization through disulfide linkage between C-terminal CK domains (blue squares). 3) After transit to the cis-Golgi network (CGN), O-glycan addition is initiated by the addition of N-acetylgalactosamine (GalNAc) to serine and threonine residues in the central mucin domains via the action of polypeptide-GalNAc transferases. 4) As the polypeptide dimer transits through the Golgi to the trans-Golgi network (TGN), O-glycan chains are elaborated via the action of multiple glycosyltransferases. 5) The O-glycosylated dimers multimerize by intermolecular disulfide linkages between N-terminal D3 domains (red squares). 6) Multimers are then packaged into secretory granules via noncovalent calcium-dependent interactions between N-terminal protein domains. These reversible interactions between mucins are most active at the lower pH of the secretory granule, and are suggested to organize the polymers within the granule. The structure highlighted (in the red dashed-line box) depicts the putative granular form of MUC5B (looped strands emanating from central nodes) observed by electron microscopy (35). Moreover, Kesimer and colleagues (35) localized the N-termini of MUC5B to the central nodes (red circles) of these structures by immuno-electron microscopy. The full mechanistic details of packaging of the large mucin polymers have not yet been elucidated. The charge shielding of the negatively charged mucin glycans by Ca²⁺ ions (37) is not shown.