Marj Howard

Assembly of the respiratory mucin MUC5B: a new model for a gel-forming mucin.

Background: Mucin polymer formation is a complex intracellular process. Results: MUC5B N-terminal D3-domains form reversible pH-sensitive calcium mediated cross-links between linear MUC5B polymer chains. Conclusion: Intracellular assembly of MUC5B generates disulfide-bonded polymers which form calcium mediated condensed networks in secretory granules. Significance: This identifies a new model for mucin assembly that may be common to other polymeric mucins. Mucins are essential components in mucus gels that form protective barriers at all epithelial surfaces, but much remains unknown about their assembly, intragranular organization, and post-secretion unfurling to form mucus. MUC5B is a major polymeric mucin expressed by respiratory epithelia, and we investigated the molecular mechanisms involved during its assembly. Studies of intact polymeric MUC5B revealed a single high affinity calcium-binding site, distinct from multiple low affinity sites on each MUC5B monomer. Self-diffusion studies with intact MUC5B showed that calcium binding at the protein site catalyzed reversible cross-links between MUC5B chains to form networks. The site of cross-linking was identified in the MUC5B D3-domain as it was specifically blocked by D3 peptide antibodies. Biophysical analysis and single particle EM of recombinant MUC5B N terminus (D1D2D′D3; NT5B) and subdomains (D1, D1-D2, D2-D′-D3, and D3) generated structural models of monomers and disulfide-linked dimers and suggested that MUC5B multimerizes by disulfide linkage between D3-domains to form linear polymer chains. Moreover, these analyses revealed reversible homotypic interactions of NT5B at low pH and in high calcium, between disulfide-linked NT5B dimers, but not monomers. These results enable a model of MUC5B to be derived, which predicts mechanisms of mucin intracellular assembly and storage, which may be common to the other major gel-forming polymeric mucins.

Proteomic analysis of polymeric salivary mucins: no evidence for MUC19 in human saliva

MUC5B is the predominant polymeric mucin in human saliva [Thornton, Khan, Mehrotra, Howard, Veerman, Packer and Sheehan (1999) Glycobiology 9, 293-302], where it contributes to oral cavity hydration and protection. More recently, the gene for another putative polymeric mucin, MUC19, has been shown to be expressed in human salivary glands [Chen, Zhao, Kalaslavadi, Hamati, Nehrke, Le, Ann and Wu (2004) Am. J. Respir. Cell Mol. Biol. 30, 155-165]. However, to date, the MUC19 mucin has not been isolated from human saliva. Our aim was therefore to purify and characterize the MUC19 glycoprotein from human saliva. Saliva was solubilized in 4 M guanidinium chloride and the high-density mucins were purified by density-gradient centrifugation. The presence of MUC19 was investigated using tandem MS of tryptic peptides derived from this mucin preparation. Using this approach, we found multiple MUC5B-derived tryptic peptides, but were unable to detect any putative MUC19 peptides. These results suggest that MUC19 is not a major component in human saliva. In contrast, using the same experimental approach, we identified Muc19 and Muc5b glycoproteins in horse saliva. Moreover, we also identified Muc19 from pig, cow and rat saliva; the saliva of cow and rat also contained Muc5b; however, due to the lack of pig Muc5b genomic sequence data, we were unable to identify Muc5b in pig saliva. Our results suggest that unlike human saliva, which contains MUC5B, cow, horse and rat saliva are a heterogeneous mixture of Muc5b and Muc19. The functional consequence of these species differences remains to be elucidated.

S20.4 The architecture of respiratory mucin macromolecules

Mucus glycoproteins, as found in mucus, are assembled from subunits via the agency of disulphide bonds. Our goal is to understand this assembly in terms of the organisation and interaction of the distinct glycosylated and naked domains within the subunit. We have studied the fragmentation of whole mucins and subunits by a variety of protein core cleaving reagents. The distribution of fragment size and molecular weight after degradation has been assayed with light scattering, rate zonal-centrifugation, electron microscopy and agarose gel electrophoresis. A novel feature of our data is evidence for a protease sensitive domain, present in macromolecules of Mr>10 × 106 which is involved in a supramolecular assembly resulting in molecules of Mr 2 0 3 0 x 106. Antibodies raised to the intact mucin are primarily targeted at this site which is also susceptible to reduction of disulphide bonds, though it is not disrupted by chaotropic agents such as 6 M guanidinium Chloride or detergents such as 0.1 M SDS. The presence of two other distinct proteinase susceptible domains can also be detected in trypsin digestion experiments. One domain is cleaved over a period of 10 h leaving a fragment Mr 2 . 5 5 x 10 ~ whereas further extremely slow degradation observed over 3 0 4 0 h yields a disperse group of fragments of average Mr 1.3 x 106. A coherent model for the architecture of these mucins, based on these data, will be presented.