Calcium-dependent protein interactions in MUC5B provide reversible cross-links in salivary mucus
Calcium-dependent protein interactions in MUC5B provide reversible cross-links in salivary mucus
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DOI：
10.1074/jbc.m304632200
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发表时间：
2003-08-01
影响因子：
4.8
通讯作者：
Thornton, DJ
中科院分区：
文献类型：
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作者：
Raynal, BDE;Hardingham, TE;Thornton, DJ
唾液内的大分子组织进行了研究，通过示踪剂扩散测量的荧光聚苯乙烯微球的荧光恢复光漂白后，使用共聚焦显微镜（共聚焦-FRAP）。微球扩散存在浓度依赖性降低;这在钙（10 mM）存在下大得多，并且通过加入EGTA（10 mM）降低。这些对示踪剂扩散的影响表明，天然唾液中含有一个大分子组织，对游离钙浓度敏感。这得到了唾液中高分子量粘蛋白级分的重均分子量（10-62 × 10（6））和唾液特性粘度（733 - 1203 ml/g）增加的支持，两者均由钙引起。对示踪剂在唾液中扩散的变化的分析表明，表观孔径增加了20倍（从10 mM CaCl 2中的130 nm增加到生理浓度下10 mM EGTA中的2600 nm）。该效应对钙具有特异性，不受高达2 M NaCl的影响。钙结合活性包含在从Sepharose CL-2B排除的唾液的高浮力密度部分中。钙离子与该组分结合的Kd值约为7 × 10（-6）M，用6 M氯化胍处理和轻度还原后，这种结合被不可逆地破坏，表明它与蛋白质位点结合。通过正离子电喷雾电离-串联质谱分析，该唾液部分显示含有MUC 5 B作为单一主要蛋白质种类。结果表明，唾液中的寡聚MUC 5 B通过钙介导的蛋白质交联组装成更大的线性或分支组装体。
The macromolecular organization within saliva was investigated by tracer diffusion measurements of fluorescent polystyrene microspheres by fluorescence recovery after photobleaching using a confocal microscope (confocal-FRAP). There was a concentration-dependent reduction in microsphere diffusion; this was much greater in the presence of calcium (10 mM) and was reduced by the addition of EGTA (10 mM). These effects on tracer diffusion showed that native saliva contained a macromolecular organization that was sensitive to free calcium concentrations. This was supported by a major increase in the weight average molecular weight of the high molecular weight mucin fraction in saliva (10-62 x 10(6)) and an increase in intrinsic viscosity of saliva (733 to 1203 ml/g) both caused by calcium. Analysis of the change in tracer diffusion in saliva showed a 20-fold increase in the apparent pore size (from 130 nm in 10 mM CaCl2 to 2600 nm in 10 mM EGTA at physiological concentration). The effect was specific for calcium and was unaffected by up to 2 M NaCl. The calcium binding activity was contained in a high buoyant density fraction of saliva excluded from Sepharose CL-2B. Calcium binding to this fraction gave an approximate K-d of 7 x 10(-6) M, and the binding was irreversibly destroyed by treatment with 6 M guanidinium chloride and by mild reduction, suggesting it to be to a protein site. This fraction of saliva was shown to contain MUC5B as the single major protein species by positive ion electrospray ionization-tandem mass spectrometry analysis. The results suggested that oligomeric MUC5B in saliva is assembled into much larger linear or branched assemblies through calcium-mediated protein cross-links.