TY - JOUR T1 - Molecular structure, packing and release of MUC2 with relevance to cystic fibrosis JF - European Respiratory Journal JO - Eur Respir J VL - 38 IS - Suppl 55 SP - p4551 AU - Harriet Nilsson AU - Daniel Ambort AU - Gunnar Hansson AU - Hans Hebert Y1 - 2011/09/01 UR - http://erj.ersjournals.com/content/38/Suppl_55/p4551.abstract N2 - In Cystic Fibrosis extremely viscous fluid is built up likely connected to impaired release and expansion of mucins. MUC2 is mainly expressed in small and large intestine, but also in inflamed airways, and it is stored as a multimer in secretory granules of goblet cells at high [Ca2+] and low pH. The extracellular milieu have to trigger the unpacking of MUC2 controled by its N-terminus, a not yet fully understood process. The aim is to elucidate organization and structure of MUC2 when it is packed and secreted.The N-terminal part of MUC2 was expressed in CHO cells. The secreted trimerized recombinant mucin was purified from culture medium by anion exchange chromatography. Crosslinked samples were purified by density ultracentrifugation. Analysis was performed by transmission electron microscopy (TEM). The pH in the buffers was varied in the range from 5.2 to 8 to mimic conditions of secretory pathway and extracellular environment by adding HAc (pH 5.2), MES (pH 6.2) or Tris (pH 7.4 and pH 8) with or without calcium. Samples were adsorbed onto carbon coated EM grids and negative stained. Processing of micrographs was performed using EMAN1 software.When pH was low and or calcium present, rings with an outer and inner diameter of 25-30 and 20-25 nm respectively were observed. Without calcium rings were assembled at pH 5.2 and 6.2, but vanished with increasing pH. 2D refinements of the projections showed rotational 5- or 6- folded symmetry. Assemblies of laterally concatenated rings were obtained in the high density fraction of MUC2 N-terminus. The formations of these rings are probably vital for proper packing and release of full length MUC2.Harriet N. and Daniel A. have contributed equally ER -