Domain structure of MUC2 and N-terminal protein and storage of MUC2 N terminus in goblet cell vesicles. (A) MUC2 monomer consists of multiple domains: VWD [D1 (orange), D2 (yellow), D′D3 (blue) and D4 (light gray)], CysD (red), PTS (green), VWB (gray), VWC (dark gray), and cystine-knot CK (black). The MUC2-N plasmid contains the N-terminal VWD (D1-D2-D′D3) domains, CysD1, Myc-tag (black), and GFP (light green). The α-MUC2-N3 antibody recognizes the VWD′D3 domain. (B) Differentiated LS174TdnTCF4 cells expressing MUC2-N (green) were immunostained with anti-MUC2C2^m1 MAb (red) detecting endogenous MUC2 showing colocalization. (Scale bars, 5 μm.) (C) LS174TdnTCF4 cells expressing the MUC2-N protein (green) were induced to differentiate into goblet-like cells. Increasing accumulation of MUC2-N in larger vesicles was observed in the more differentiated cells. Nucleus, DAPI (blue). (D and E) Induced LS174TdnTCF4 with MUC2-N (green) were immunostained for ER with anti-calnexin (red) (D) or for Golgi with anti-GalNAcT2 (red) (E). No colocalization was apparent. (Scale bars, 5 μm.) (F) Lysate from a radioactive pulse-chase experiment (30 min, 3 h, 20 h) of LS174TdnTCF4 (noninduced) expressing the MUC2-N protein that was immunoprecipitated using anti-myc MAb and analyzed by nonreduced SDS/PAGE. MUC2-N is first detected as a monomer, and during processing when passing the secretory pathway it forms trimers that were stored within the cells for more than 20 h.

Domain organization of MUC2-N six-sided rings. (A) The MUC2-N trimer is made up of three disulfide-linked (white bars) VWD3 (blue) domains in the core structure. At pH 7.4 the VWD1-D2 (orange and yellow) domains extend out and at pH 6.2 with Ca²⁺ flipped back by noncovalent interactions to the VWD3 core (Lower). In VWF the N-terminal VWD′D3 domains form a disulfide-linked (white bars) dimer. At pH 6.2 two VWD1-D2 domains interact with the VWD3 dimer, thus forming a tetrameric complex (Upper). (B) At pH 6.2 with Ca²⁺ in the VWF-N two disulfide-linked VWD3 domains bind on each side one VWD1-D2 peptide thereby forming the repeating unit of a right-handed helix. The VWF-N helix is composed of four repeating units per turn. (C) Analogously, the MUC2-N repeating unit is composed of three disulfide-linked VWD3 domains that bind on each side one VWD1-D2 peptide. Six repeating units make up one ring. (D) EM (negatively stained) showing the gold-labeled α-MUC2-N3 antibodies for the VWD3 domains bound to the corners of six different MUC2-N rings formed at pH 6.2 with Ca²⁺ (each arrow shows one labeled corner). (E) 2D reconstruction of MUC2-N six-sided rings by EM shows a six-folded symmetry, thereby suggesting six repeating units per ring. Each repeating unit is made up of three VWD3 domains and three VWD1-D2 peptides. (Scale bars, 40 nm in D, 7.5 nm in E.)

Packing and unfolding of MUC2. (A and B) Packing of MUC2 at pH 6.2 in presence of Ca²⁺ in secretory granulae of goblet cell. (A) EM of a cross-section of secretory granulae from a human goblet cell showing ring-like structures. (Scale bars, 200 nm at left, 100 nm at top right, 40 nm at bottom right.) (B) MUC2 is organized at one end around the MUC2-N concatenated rings stabilized by noncovalent interactions of N-terminal VWD (orange, yellow, and blue) domains and at the C-terminal end by CK (black) dimers. The extended mucin domains drawn to scale (green) connect neighboring VWD′D3 trimers (blue) of the MUC2-N ring platform (Lower) with the C-terminal ends (Upper). (C and D) Unfolding of MUC2 at high pH and low Ca²⁺ after release from goblet cell secretory granulae. (C) During unpacking the MUC2-N rings fall apart, and the mucin domains begin to stretch out. (D) The released MUC2 is assumed to adopt an outstretched net-like flat sheet composed of trimeric disulfide-linked MUC2-N (in the corners) and dimeric MUC2-C (in the middle of the sides). The expanded net is >1,000-fold larger than the granulae rings. (E and F) The inner mucus layer of the colon show stratified sheets (5) as predicted from the unfolded MUC2 model. (E) EM of the inner mucus layer organized as stratified sheets (s) on top of an enterocyte with its glycocalyx from a colon tissue section. (Scale bar, 2 μm.) (F) Mouse colon tissue section fixed in Carnoy and stained with the anti-MUC2C3 antiserum (green) and FISH (red) as revealed by fluorescence microscopy of the stratified MUC2 inner mucus layer (s) devoid of bacteria and the outer mucus layer (o) with bacteria. (Scale bar, 25 μm.)

MUC2-N forms pH- and calcium-dependent rings. (A) Gel filtration of MUC2-N at pH 7.4, pH 6.2 with EDTA or Ca²⁺, and pH 5 with Ca²⁺. The MUC2-N trimer formed larger aggregates at lower pH that did not enter the gel beads. (B and C) Density gradient centrifugation of MUC2-N protein incubated at pH 8 with EDTA (B) or pH 6.2 with Ca²⁺ (C). As analyzed by reducing SDS/PAGE and silver staining, the MUC2-N trimer was found in fractions 14–18 in high and low pH conditions, whereas larger aggregates were detected in fractions 20–22 only at pH 6.2 with Ca²⁺. (D) EM (negative stain) showing that MUC2-N trimer adopts an outstretched appearance at pH 7.4, with three globular structures extending out of a trefoil core fragment, suggesting that the VWD1-D2 domains are connected via a flexible linker to the disulfide-bonded (indicated by white bars) VWD3 trimeric core (E) (same color code as in Fig. 1). (F) EM showing that MUC2-N forms five-, six-, and seven-sided ring structures at pH 6.2 with Ca²⁺ composed of multiple MUC2-N trimers. (G) Glutaraldehyde cross-linking at pH 6.2 with Ca²⁺ before density gradient centrifugation (fraction 22 from C) allowed for isolating confluent 2D sheets of concatenated MUC2-N rings as seen by EM (negative stain). (Scale bars, 50 nm in D, 100 nm in F and G.)

Dissolution of MUC2-N aggregates by Hepes-EDTA or NaHCO₃-treatment. (A) MUC2-N aggregates formed at pH 5 with 50 mM CaCl₂ were analyzed by gel filtration with or without addition of 100 mM sodium bicarbonate (pH 8.3). Addition of sodium bicarbonate partially dissolved the MUC2-N aggregates. (B and C) MUC2-N aggregates formed at pH 6.2 with Ca²⁺ were dialyzed against 20 mM Hepes (pH 8), 10 mM EDTA (B) or 50 mM NaHCO₃ (pH 8.3) (C) and then analyzed by density gradient centrifugation and reducing SDS/PAGE and silver staining. Higher pH conditions or sodium bicarbonate treatment both partially dissolved the MUC2-N aggregates that were formed at low pH with Ca²⁺ in fractions 20–22.