Assembly of the respiratory mucin MUC5B: a new model for a gel-forming mucin.
Assembly of the respiratory mucin MUC5B: a new model for a gel-forming mucin.
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DOI：
10.1074/jbc.m114.566679
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发表时间：
2014-06-06
期刊：
影响因子：
--
通讯作者：
Thornton DJ
中科院分区：
文献类型：
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作者：
Ridley C;Kouvatsos N;Raynal BD;Howard M;Collins RF;Desseyn JL;Jowitt TA;Baldock C;Davis CW;Hardingham TE;Thornton DJ
背景：粘蛋白聚合物的形成是一个复杂的细胞内过程。结果：MUC5B N-末端D3-结构域在线性MUC5B聚合物链之间形成可逆的pH敏感性钙介导的交联。结论：MUC5B在细胞内组装形成二硫键聚合物，在分泌颗粒中形成钙介导的凝聚网络。意义：这确定了一个新的粘蛋白组装模型，可能是共同的其他聚合粘蛋白。粘蛋白是粘液凝胶中的重要组分，其在所有上皮表面形成保护屏障，但关于其组装、颗粒内组织和分泌后展开以形成粘液仍有许多未知。MUC5B是呼吸道上皮细胞表达的主要多聚粘蛋白，我们研究了其组装过程中涉及的分子机制。完整的多聚MUC5B的研究揭示了一个单一的高亲和力钙结合位点，不同于每个MUC5B单体上的多个低亲和力位点。用完整MUC5B进行的自扩散研究表明，在蛋白质位点处的钙结合催化MUC5B链之间的可逆交联以形成网络。在MUC5B D3结构域中鉴定了交联位点，因为它被D3肽抗体特异性阻断。重组MUC5B N端（D1D2D ′ D3; NT5B）和亚结构域（D1、D1-D2、D2-D ′-D3和D3）的生物物理分析和单颗粒EM生成了单体和二硫键连接的二聚体的结构模型，并表明MUC5B通过D3结构域之间的二硫键多聚化形成线性聚合物链。此外，这些分析揭示了NT5B在低pH值和高钙时，二硫键连接的NT5B二聚体之间，而不是单体之间的可逆同型相互作用。这些结果使得MUC5B的模型能够被导出，该模型预测粘蛋白细胞内组装和储存的机制，这可能是其他主要的凝胶形成聚合粘蛋白所共有的。
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.