Juan Perez-Vilar

Mucin granule intraluminal organization in living mucous/goblet cells : Roles of protein post-translational modifications and secretion

Recent studies suggest that the mucin granule lumen consists of a matrix meshwork embedded in a fluid phase. Secretory products can both diffuse, although very slowly, through the meshwork pores and interact noncovalently with the matrix. Using a green fluorescent protein-mucin fusion protein (SHGFP-MUC5AC/CK) as a FRAP (fluorescence recovery after photobleaching) probe, we have assessed in living mucous cells the relative importance of different protein post-translational modifications on the intragranular organization. Long term inhibition of mucin-type O-glycosylation, sialylation, or sulfation altered SHGFP-MUC5AC/CK characteristic diffusion time (t½), whereas all but sulfation diminished its mobile fraction. Reduction of protein disulfide bonds with tris(hydroxypropyl)phosphine resulted in virtually complete immobilization of the SHGFP-MUC5AC/CK intragranular pool. However, when activity of the vacuolar H+-ATPase was also inhibited, disulfide reduction decreased SHGFP-MUC5AC/CK t½ while diminishing its intraluminal concentration. Similar FRAP profiles were observed in granules that remained in the cells after the addition of a mucin secretagogue. Taken together these results suggest that: (a) the relative content of O-glycans and intragranular anionic groups is crucial for protein diffusion through the intragranular meshwork; (b) protein-protein, rather than carbohydrate-mediated, interactions are responsible for binding of SHGFP-MUC5AC/CK to the immobile fraction, although the degree of matrix O-glycosylation and sialylation affects such interactions; (c) intragranular organization does not depend on covalent multimerization of mucins or the presence of native disulfide bonds in the intragranular mucin/proteins, but rather on specific protein-mediated interactions that are important during the early stages of mucin matrix condensation; (d) alterations of the intragranular matrix precede granule discharge, which can be partial and, accordingly, does not necessarily involve the disappearance of the granule.

pH-dependent intraluminal organization of mucin granules in live human mucous/goblet cells

To study the mechanism of gel-forming mucin packaging within mucin granules, we generated human mucous/goblet cells stably expressing a recombinant MUC5AC domain fused to green fluorescent protein (GFP). The fusion protein, named SHGFP-MUC5AC/CK, accumulated in the granules together with native MUC5AC. Inhibition of protein synthesis or disorganization of the Golgi complex did not result in diminished intragranular SHGFP-MUC5AC/CK signals, consistent with long-term storage of the fusion protein. However, SHGFP-MUC5AC/CK was rapidly discharged from the granules upon incubation of the cells with ATP, an established mucin secretagogue. Several criteria indicated that SHGFP-MUC5AC/CK was not covalently linked to endogenous MUC5AC. Analysis of fluorescence recovery after photobleaching suggested that the intragranular SHGFP-MUC5AC/CK mobile fraction and mobility were significantly lower than in the endoplasmic reticulum lumen. Incubation of the cells with bafilomycin A1, a specific inhibitor of the vacuolar H+-ATPase, did not alter the fusion protein mobility, although it significantly increased (∼20%) the intragranular SHGFP-MUC5AC/CK mobile fraction. In addition, the granules in bafilomycin-incubated cells typically exhibited a heterogeneous intraluminal distribution of the fluorescent fusion protein. These results are consistent with a model of mucin granule intraluminal organization with two phases: a mobile phase in which secretory proteins diffuse as in the endoplasmic reticulum lumen but at a lower rate and an immobile phase or matrix in which proteins are immobilized by noncovalent pH-dependent interactions. An intraluminal acidic pH, maintained by the vacuolar H+-ATPase, is one of the critical factors for secretory protein binding to the immobile phase and also for its organization.

Mucin Granules are in Close Contact with Tubular Elements of the Endoplasmic Reticulum

Live cell imaging methods were used to characterize goblet cells expressing a MUC5AC domain fused to enhanced green fluorescent protein that labels the granule lumen. Golgi complex and endosome/lysosome elements largely resided in the periphery of the granular mass. On the contrary, a tubular meshwork of endoplasmic reticulum (ER) was in close contact with the mucin granules. This meshwork could be identified in fixed native human bronchial goblet cells labeled with an anti-calreticulin antibody. The potential biological significance of this ER network for mucin secretion is discussed.

Gastrointestinal Mucus Gel Barrier

A family of glycoproteins, known as gel-forming mucins, endow gastrointestinal mucus with its characteristic viscoelastic and biological properties. In the mucus, these large oligomeric glycoproteins are organized into entangled networks that occasionally can be stabilized by non-covalent interactions as in the stomach lumen. This network is a formidable chemical and physical barrier that not only protects the underlying epithelia but also limits the usefulness of orally administered drugs. In this chapter, I review the molecular and cellular properties of gel-forming mucins and how these macromolecules are organized into a tri-dimensional network to form the gastrointestinal mucus gel barrier.

Formation of mucin granules

In this chapter, the formation of the mucin granule, the characteristic secretory granule found in mucus/goblet cells, is discussed in view of our knowledge on the structure and biosynthesis of gel-forming mucins, granule intraluminal organization and the theoretical framework provided by the main sorting/trafficking modelsthoughtto operate in the secretory pathway. Contrary to most reviews on intracellular trafficking, a soluble protein cargo, rather than membrane components, occupies the central stage of our discussion. Considering the large sizes and properties of mucin precursors, it is very difficult not to envision an active role for these macromolecules during its intracellular trafficking and storage.