Carolyn Bertozzi, Doctoral Dissertation Advisor (AC)
Structure-guided mutagenesis of a mucin-selective metalloprotease from Akkermansia muciniphila alters substrate preferences.
The Journal of biological chemistry
Akkermansia muciniphila, a mucin-degrading microbe found in the human gut, is often associated with positive health outcomes. The abundance of Akkermansia muciniphila is modulated by the presence and accessibility of nutrients, which can be derived from diet or host glycoproteins. In particular, the ability to degrade host mucins, a class of proteins carrying densely O-glycosylated domains, provides a competitive advantage in the sustained colonization of niche mucosal environments. Although Akkermansia muciniphila is known to rely on mucins as a carbon and nitrogen source, the enzymatic machinery used by this microbe to process mucins in the gut is not yet fully characterized. Here, we focus on the mucin-selective metalloprotease, Amuc_0627 (AM0627), which is known to cleave between adjacent residues carrying truncated core 1 O-glycans. We showed that this enzyme is capable of degrading purified mucin 2 (MUC2), the major protein component of mucus in the gut. An X-ray crystal structure of AM0627 (1.9 A resolution) revealed O-glycan binding residues that are conserved between structurally characterized enzymes from the same family. We further rationalized the substrate cleavage motif using molecular modeling to identify nonconserved glycan-interacting residues. We conclude that mutagenesis of these residues resulted in altered substrate preferences down to the glycan level, providing insight into the structural determinants of O-glycan recognition.
View details for DOI 10.1016/j.jbc.2022.101917
View details for PubMedID 35405095
Revealing the human mucinome
OXFORD UNIV PRESS INC. 2021: 1729
View details for Web of Science ID 000754737200119
Classification, structural biology, and applications of mucin domain-targeting proteases.
The Biochemical journal
2021; 478 (8): 1585–1603
Epithelial surfaces throughout the body are coated by mucins, a class of proteins carrying domains characterized by a high density of O-glycosylated serine and threonine residues. The resulting mucosal layers form crucial host-microbe interfaces that prevent the translocation of microbes while also selecting for distinct bacteria via the presented glycan repertoire. The intricate interplay between mucus production and breakdown thus determines the composition of the microbiota maintained within these mucosal environments, which can have a large influence on the host during both homeostasis and disease. Most research to date on mucus breakdown has focused on glycosidases that trim glycan structures to release monosaccharides as a source of nutrients. More recent work has uncovered the existence of mucin-type O-glycosylation-dependent proteases that are secreted by pathogens, commensals, and mutualists to facilitate mucosal colonization and penetration. Additionally, immunoglobulin A (IgA) proteases promote bacterial colonization in the presence of neutralizing secretory IgA through selective cleavage of the heavily O-glycosylated hinge region. In this review, we summarize families of O-glycoproteases and IgA proteases, discuss known structural features, and review applications of these enzymes to glycobiology.
View details for DOI 10.1042/BCJ20200607
View details for PubMedID 33909028
An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins.
Proceedings of the National Academy of Sciences of the United States of America
Densely O-glycosylated mucin domains are found in a broad range of cell surface and secreted proteins, where they play key physiological roles. In addition, alterations in mucin expression and glycosylation are common in a variety of human diseases, such as cancer, cystic fibrosis, and inflammatory bowel diseases. These correlations have been challenging to uncover and establish because tools that specifically probe mucin domains are lacking. Here, we present a panel of bacterial proteases that cleave mucin domains via distinct peptide- and glycan-based motifs, generating a diverse enzymatic toolkit for mucin-selective proteolysis. By mutating catalytic residues of two such enzymes, we engineered mucin-selective binding agents with retained glycoform preferences. StcEE447D is a pan-mucin stain derived from enterohemorrhagic Escherichia coli that is tolerant to a wide range of glycoforms. BT4244E575A derived from Bacteroides thetaiotaomicron is selective for truncated, asialylated core 1 structures commonly associated with malignant and premalignant tissues. We demonstrated that these catalytically inactive point mutants enable robust detection and visualization of mucin-domain glycoproteins by flow cytometry, Western blot, and immunohistochemistry. Application of our enzymatic toolkit to ascites fluid and tissue slices from patients with ovarian cancer facilitated characterization of patients based on differences in mucin cleavage and expression patterns.
View details for DOI 10.1073/pnas.2012196117
View details for PubMedID 32817557
Enzyme toolkit for selective enrichment and analysis of mucin-domain glycoproteins
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. 2019: S42
View details for Web of Science ID 000516535000056