Intestinal MUC2 Mucin Supramolecular Topology by Packing and Release Resting on D3 Domain Assembly

Abstract

MUC2 is the major gel-forming mucin of the colon forming a protective gel barrier organized into an inner stratified and an outer loose layer. The MUC2 N-terminus (D1-D2-D'D3 domains) has a dual function in building a net-like structure by disulfide-bonded trimerization and packing the MUC2 polymer into an N-terminal concatenated polygonal platform with the C-termini extending perpendicularly by pH- and calcium-dependent interactions. We studied the N-terminal D'D3 domain by producing three recombinant variants, with or without Myctag and GFP, and analyzed these by gel filtration, electron microscopy and single particle image processing. The three variants were all trimers when analyzed upon denaturing conditions, but eluted as hexamers upon gel filtration under native conditions. Studies by electron microscopy and 3D maps revealed cage-like structures with two- and three-fold symmetries. The structure of the MUC2 D3 domain confirms that the MUC2 mucin forms branched net-like structures. This suggests that the MUC2 mucin is stored with two N-terminal concatenated ring platforms turned by 180° against each other implicating that every second unfolded MUC2 net in mature mucus is turned upside-down.

New developments in goblet cell mucus secretion and function

“…1 (22). More recent structural studies of the MUC2 N-terminal D3 domain suggested that every second MUC2 mucin layer is rotated 180° and the N-terminal ring structures meet (29). Upon secretion, the densely packed mucin expands >1,000-fold into large nets (Fig.…”

Assembly Mechanism of Mucin and von Willebrand Factor Polymers

“…To carry out these essential functions, gel-forming mucins are cross-linked into extended disulfide-linked architectures. The respiratory mucins form bundles of linear covalent polymers (Thornton et al, 2018), and it was proposed that the intestinal mucins form a stacked hexagonal network (Ambort et al, 2012;Nilsson et al, 2014). The non-mucin molecule VWF, produced by endothelial cells and platelets to function in hemostasis and thrombosis (Springer, 2014), evolved by exploiting aspects of the mucin disulfide-mediated assembly mechanism despite its distinct physiological context ( Figure 1A).…”

The Properties of the Mucus Barrier, a Unique Gel–How Can Nanoparticles Cross It?

“…The secreted mucus is believed to consist of layers of these nets ( Figure 3D). It is important to note the pore size of these nets should be approximately 1 µm [36,[56][57][58].…”

“…When inhaled particles or microbes reach the lung they become attached to the mucus gel layer and are cleared from the lungs by the action of the cilia moving the mucus layer up and out of the lungs. MUC2 nets Theoretical 1 µM [36,56,57,64] Pig jejunal mucus Particle tracking Ex vivo scraped mucus from the proximal jejunum 500 nm [20] Pig jejunal mucus Atomic force microscopy Jejunal mucus collected with protease inhibitors. Overnight extraction in 4M guanidinium chloride followed by density gradient purification.…”