Mucin domains are densely O-glycosylated modular protein domains that are found in a wide variety of cell surface and secreted proteins. Mucin-domain glycoproteins are known to be key players in a host of human diseases, especially cancer, wherein mucin expression and glycosylation patterns are altered. Mucin biology has been difficult to study at the molecular level in part because methods to manipulate and structurally characterize mucin domains are lacking. One major issue is that these domains are resistant to degradation by trypsin, meaning the majority of their sequence space is often left unanalyzed. Selective mucin degradation or enrichment, especially in a sequence- and glycan-specific manner, can facilitate study of these proteins by mass spectrometry.
We first expressed and characterized a bacterial mucinase, StcE, and demonstrated that it selectively cleaves mucins in a glycan- and peptide- specific manner. We went on to use its unique properties to improve sequence coverage, glycosite mapping, and glycoform analysis of recombinant human mucins by mass spectrometry. To expand on this work, we expressed and characterized several other bacterial mucinases to generate a mucin-selective enzymatic toolkit. Their activities were confirmed using a panel of O-glycoproteins by mass spectrometry. We manually validated peptide sequences from MS/MS spectra to elucidate all cleaved peptides present in the mucinase-digested samples but not in the control samples, revealing that each enzyme has a slightly different cleavage motif. Interestingly, all of the enzymes rely on a combination of peptide sequence and glycosylation status. Together with StcE, we have characterized a total of five bacterial mucinases capable of digesting mucins into peptides amenable for mass spectrometric analysis.
Further, given the enzymes’ selectivity for mucin-domain glycoproteins, we reasoned that they could be employed to purify mucins from protein mixtures. Thus, inactivated mucinases were conjugated to aldehyde beads using reductive amidation. Using the enzyme-conjugated beads, we demonstrate that we can selectively enrich for mucin-domain glycoproteins from lysate and crude cancer patient ascites fluid. We are thus attempting to define the “mucinome”, as a comprehensive list of mucin-domain glycoproteins does not exist. Future experiments will be devoted to isolation, digestion, and characterization of mucins from human cancer patient ascites fluid, with the ultimate goal of identifying diagnostic and/or prognostic markers of disease states.