Mapping the Protein Domain Structures of the Respiratory Mucins: A Mucin Proteome Coverage Study

Mucin genes encode a family of the largest expressed
proteins in the human genome. The proteins are highly substituted
with O-linked oligosaccharides that greatly restrict
access to the peptide backbones. The genomic organization of the N-terminal, O-glycosylated, and C-terminal regions of most of the mucins
has been established and is available in the sequence databases. However,
much less is known about the fate of their exposed protein regions
after translation and secretion, and to date, detailed proteomic studies
complementary to the genomic studies are rather limited. Using mucins
isolated from cultured human airway epithelial cell secretions, trypsin
digestion, and mass spectrometry, we investigated the proteome coverage
of the mucins responsible for the maintenance and protection of the
airway epithelia. Excluding the heavily glycosylated mucin domains,
up to 85% coverage of the N-terminal region of the gel-forming mucins
MUC5B and MUC5AC was achieved, and up to 60% of the C-terminal regions
were covered, suggesting that more N- and sparsely O-glycosylated regions as well as possible other modifications
are available at the C-terminus. All possible peptides from the cysteine-rich
regions that interrupt the heavily glycosylated mucin domains were
identified. Interestingly, 43 cleavage sites from 10 different domains
of MUC5B and MUC5AC were identified, which possessed a non-tryptic
cleavage site on the N-terminal end of the peptide,
indicating potential exposure to proteolytic and/or “spontaneous
cleavages”. Some of these non-tryptic cleavages may be important
for proper maturation of the molecule, before and/or after secretion.
Most of the peptides identified from MUC16 were from the SEA region.
Surprisingly, three peptides were clearly identified from its heavily
glycosylated regions. Up to 25% coverage of MUC4 was achieved covering
seven different domains of the molecule. All peptides from the MUC1
cytoplasmic domain were detected along with the three non-tryptic
cleavages in the region. Only one peptide was identified from MUC20,
which led us to successful antisera raised against the molecule. Taken
together, this report represents our current efforts to dissect the
complexities of mucin macromolecules. Identification of regions accessible
to proteolysis can help in the design of effective antibodies and
points to regions that might be available for mucin–protein
interactions and identification of cleavage sites will enable understanding
of their pre- and post-secretory processing in normal and disease
environments.