Summary The translocation of proteins across the bacterial cell membrane is carried out by highly conserved components of the Sec system. Most bacterial species have a single copy of the genes encoding SecA and SecY, which are essential for viability. However, Streptococcus gordonii strain M99 encodes SecA and SecY homologues that are not required for viability or for the translocation of most exported proteins. The genes (secA2 and secY2) reside in a region of the chromosome required for the export of GspB, a 286 kDa cell wall‐anchored protein. Loss of GspB surface expression is associated with a significant reduction in the binding of M99 to human platelets, suggesting that it may be an adhesin. Genetic analyses indicate that M99 has a second, canonical SecA homologue that is essential for viability. At least two other Gram‐positive species, Streptococcus pneumoniae and Staphylococcus aureus, encode two sets of SecA and SecY homologues. One set is more similar to SecA and SecY of Escherichia coli, whereas the other set is more similar to SecA2 and SecY2 of strain M99. The conserved organization of genes in the secY2–secA2 loci suggests that, in each of these Gram‐positive species, SecA2 and SecY2 may constitute a specialized system for the transport of a very large serine‐rich repeat protein.

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. Here, we demonstrate that secreted protease of C1 esterase inhibitor (StcE), a bacterial protease from Escherichia coli, cleaves mucin domains by recognizing a discrete peptide-and glycan-based motif. We exploited StcE's unique properties to improve sequence coverage, glycosite mapping, and glycoform analysis of recombinant human mucins by mass spectrometry. We also found that StcE digests cancer-associated mucins from cultured cells and from ascites fluid derived from patients with ovarian cancer. Finally, using StcE, we discovered that sialic acid-binding Ig-type lectin-7 (Siglec-7), a glycoimmune checkpoint receptor, selectively binds sialomucins as biological ligands, whereas the related receptor Siglec-9 does not. Mucin-selective proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of mucin domain structure and function.O-glycosylation | mucin | protease | glycoproteomics | Siglec

Highlights d Human glycosyltransferases (170 GTf genes) organized in glycosylation pathway maps d The human glycome displayed in a natural context on the cell surface d Sustainable cell-based array resource to dissect biological functions of glycans d Microbial adhesins may bind to clustered patches of Oglycans