Scientifc report for the project:

Structural Analysis of O-glycosylated Domains During Mucin Supramolecular Assembly
Deborah Fass
Department of Chemical and Structural Biology
Weizmann Institute of Science, Israel
AbstractGel-forming mucin glycoproteins constitute the mucus that protects exposed epithelia in the body fromphysical and biological hazards. Mucins contain long segments, i.e., thousands of amino acids, rich inproline, threonine, and serine (PTS). The threonine and serine side chains become heavily glycosylated
during mucin bioassembly, and these post-translational modifications are essential for mucin function. Inthe major lung and intestinal mucins, the PTS regions are interrupted by multiple disulfide-rich domains ofabout 100 amino acids, called CysD domains. We aimed to gain insight into the function and organizationof the PTS segments of mucins, as well as their relationship to CysD domains. To study mucin PTS regions,
we took bottom-up and top-down approaches. In the bottom-up approach, we produced recombinant aminoterminal regions of different mucins containing different lengths and compositions of PTS segments andstudied their modes of supramolecular assembly. In this research, we found that inclusion of long PTS
segments (~400 amino acids) makes supramolecular assembly less robust in vitro, and more work is needed
to determine whether it is possible to obtain information from such recombinant constructs. However, by
examining different gel-forming mucins that naturally contain PTS regions of different lengths between
their CysD domains, we are beginning to understand how co-evolution of PTS regions and CysD domains
produces different types of mucin supramolecular assemblies, likely leading to different biophysicalproperties. In the top-down approach, we imaged mucin-containing secretory granules by transmission
electron microscopy (TEM) and scanning transmission electron microscopy after focused ion beam (FIB)
milling of detached colon epithelial cells. In contrast to similar, control experiments done on endothelialcells, which confirmed that we could detect the organization of von Willebrand factor tubules in Weibel
Palade bodies, no underlying order was seen in mucus vesicles in lamellae prepared by FIB milling.
However, we are still working towards imaging large and mature colon goblet-cell granules (~10 µmdiameter) in intact colon epithelium, for which technical challenges in freezing of tissues for subsequent
FIB milling must be overcome.
Objectives
The overall objective of the project was to determine whether there is structural order to glycosylated PTS
regions in mucins and how these regions affect mucin bioassembly in cells prior to secretion. The two
specific aims were 1) to determine whether and how long PTS segments influence the in vitro
supramolecular assembly of the amino-terminal regions of mucins and 2) whether order (on the scale oftens of nanometers) can be detected in mucin-containing vesicles and granules in goblet cells. For the firstaim, we prepared recombinant segments of multiple mucins, beginning at the amino termini and extending
through one or more PTS regions and CysD domains. We then examined the ability of these fragments toundergo supramolecular assembly when placed in the pH and salt conditions representative of the Golgi
apparatus and mucin granules in cells. For the second aim, we isolated epithelial cells from colon tissue,
allowed them to adhere to electron microscopy grids, cryo-preserved the cells, and then performed FIB
milling of the frozen cells to generate thin (200-300 nm) lamellae in regions of mucin-containing vesicles.
Lamellae were then imaged by TEM.

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