PH-dependent Intraluminal Organization of Mucin Granules in Live Human Mucous/Goblet Cells

Juan Perez-Vilar,John C Olsen,Michael Chua,Richard C Boucher

doi:10.1074/jbc.m413289200

Juan Perez-Vilar, John C Olsen + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m413289200

Copy DOI

Abstract

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 (approximately 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.

Highlights

The mucous blanket that covers the gastrointestinal, tracheobronchial, urogenital, auditory, and conjunctiva epithelia and the gills of fishes and the epidermis of amphibians lubricates these mucosas and creates a protective barrier against pathogenic and noxious agents [1]
The CK Domains in MUC5AC and MUC5B Form Disulfidelinked Homodimers—To visualize mucin granules in live cells, we employed green fluorescent protein (GFP) technology in HT29 –18N2 cells, a human colon cell line known to differentiate into mucous/goblet cells when grown in a defined serum-free culture medium [37]
A priori, a fusion protein between GFP and a protein domain in the mucin polypeptide had a reasonable chance to interact with endogenous mucin and to be transported eventually into the mucin granule

Summary

Introduction

The mucous blanket that covers the gastrointestinal, tracheobronchial, urogenital, auditory, and conjunctiva epithelia and the gills of fishes and the epidermis of amphibians lubricates these mucosas and creates a protective barrier against pathogenic and noxious agents [1]. 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).

Results

Conclusion