Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies

The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUCSB. ROA indicated that the protein backbone of MUCSB is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUCSB O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUCSB (D1-D2 -D'-D3 domains, NTSB), C-terminal region of MUCSB (D4-B-C-CK domains, CTSB) and the Cys-domain (within the central mucin domain of MUCSB) were found to be dominated by the beta-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUCSB molecule. Interestingly, the molecule was found to contain a large amount of alpha-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin.