![]() We have previously employed electron microscopy (EM) to characterize the organization of this structure ( 9) and in particular used colloidal gold to identify the globular NH 2- and COOH-terminal domains ( Fig. Such a structure is many micrometers in length ( Fig. A MUC5B mucin subunit is ∼400–500 nm in length and arranged into a linear polymeric form. These oligosaccharides are so numerous and organized that the glycosylated peptide cannot be proteolytically cleaved even by peptidases as small as trypsin or papain. 1 A) has five such regions, all of which have densely arranged, charged O-linked oligosaccharides. However, gel-forming mucins, although similar in some respects to the VWF, are much larger at their individual subunit level, each containing a number of highly glycosylated domains. ![]() Both mucins and the VWF molecules are generally stored in granules that are unpacked upon release from the cell. This mechanism suggests that the assembling molecules may form extended helical structures around which glycosylated protein regions, linked by the COOH-terminal domains, are organized. A mechanism for the NH 2-terminal assembly process in VWF has recently been described ( 6). However, whereas the mucin granules are always spheroidal in shape ( 2), the VWF granules are more generally cigar shaped ( 20). Both gel-forming mucins and the VWF assemble into their polymeric structures by first dimerizing in the endoplasmic reticulum via their COOH-terminal domains and subsequently multimerizing through protein domains in the NH 2 termini. This molecule provides a vital role in blood coagulation, and genetic defects known to be associated with it relate to a number of diseases ( 7). The basis of the polymeric assembly of these large mucin glycoproteins is thought to mimic that of another related glycoprotein called von Willebrand factor (VWF) protein. The functions of these molecules are understood in general terms, i.e., they form the basic infrastructure of the epithelial protective gels, but the mechanisms by which they are made and organized within cells and thereafter released are not understood. There are four such molecules, MUC2, MUC5AC, MUC5B, and MUC6, and they form the largest glycoproteins in the body ( 10). In humans, these macromolecules come in two major kinds, the epithelial or transmembrane mucins characterized by COOH-terminal domains that attach to cell surface membranes ( 5) and those termed the gel-forming mucins that are secreted as highly assembled polymeric structures ( 14). Mucins are large complex glycoprotein macromolecules that provide the basis of gels protecting the surface epithelia in many multicellular organisms. For the first time, this provides some insight into how the carbohydrate regions might be organized around the NH 2- and COOH-terminal globular protein domains within the granule and also explains how the mucin can expand so rapidly upon its release. The organization of the mucins in this manner is consistent with efficient packing of a number of large heavily glycosylated monomers while still permitting their rapid unfolding and hydration. The appearance indicates that the assembled mucins in a single granular form are organized around a number of nodes, each attached to four to eight subunits. ![]() ![]() In transmission electron microscopy, this compact mucin has maintained a circular structure that is characterized by flexible chains connected around protein-rich nodes as determined by their ability to bind colloidal gold. It has an average mass of ∼150 × 10 6 Da and size Rg of 150 nm in radius of gyration. In this study, using OptiPrep density gradient ultracentrifugation, we have isolated a small amount of a stable form of the recently secreted and expanding MUC5B mucin, which accounts for less than 2% of the total mucin present. Our major interest is in lung mucins, but most particularly in MUC5B, which is the major gel-forming mucin in mucus, and which provides its major protective matrix. In particular, the initial rate of expansion of the mucins after release from their secretory granules is very rapid (seconds), but no clear mechanism for how it is achieved has emerged. The mechanisms by which they are formed and organized within cells and thereafter released to form mucus gels are not understood. They form the matrix of gels protecting all the surface epithelia and are secreted as disulfide-bonded polymeric structures. Gel-forming mucins are the largest complex glycoprotein macromolecules in the body. ![]()
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