@article {66018, title = {An Acquired and Endogenous Glycocalyx Forms a Bidirectional {\textquotedblleft}Don{\textquoteright}t Eat{\textquotedblright} and {\textquotedblleft}Don{\textquoteright}t Eat Me{\textquotedblright} Barrier to Phagocytosis}, journal = {Current Biology}, volume = {31}, year = {2021}, month = {Jan-01-2021}, pages = {77 - 89.e5}, abstract = {

Macrophages continuously survey their environment in search of pathogens or apoptotic corpses or debris. Targets intended for clearance expose ligands that initiate their phagocytosis ("eat me" signals), while others avoid phagocytosis by displaying inhibitory ligands ("don{\textquoteright}t eat me" signals). We report that such ligands can be obscured by the glycosaminoglycans and glycoproteins that coat pathogenic as well as malignant phagocytic targets. In addition, a reciprocal barrier of self-synthesized or acquired glycocalyx components on the macrophage surface shrouds phagocytic receptors, curtailing their ability to engage particles. The coating layers of macrophages and their targets hinder phagocytosis by both steric and electrostatic means. Their removal by enzymatic means is shown to markedly enhance phagocytic efficiency. In particular, we show that the removal of mucins, which are overexpressed in cancer cells, facilitates their clearance. These results shed light on the physical barriers that modulate phagocytosis, which have been heretofore underappreciated.

}, issn = {09609822}, doi = {10.1016/j.cub.2020.09.082}, url = {https://www.sciencedirect.com/science/article/pii/S0960982220314950?via\%3Dihub}, author = {Imbert, Paul R.C. and Saric, Amra and Pedram, Kayvon and Bertozzi, Carolyn R. and Grinstein, Sergio and Freeman, Spencer A.} } @article {66017, title = {An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins}, journal = {Proceedings of the National Academy of Sciences}, volume = {117}, year = {2020}, month = {Jan-09-2020}, pages = {21299 - 21307}, abstract = {

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.

}, issn = {0027-8424}, doi = {10.1073/pnas.2012196117}, url = {http://www.pnas.org/lookup/doi/10.1073/pnas.2012196117https://syndication.highwire.org/content/doi/10.1073/pnas.2012196117http://www.pnas.org/syndication/doi/10.1073/pnas.2012196117}, author = {Shon, D. Judy and Malaker, Stacy A. and Pedram, Kayvon and Yang, Emily and Krishnan, Venkatesh and Dorigo, Oliver and Bertozzi, Carolyn R.} } @article {66016, title = {Lysosome-targeting chimaeras for degradation of extracellular proteins}, journal = {Nature}, volume = {584}, year = {2020}, month = {Jan-08-2021}, pages = {291 - 297}, abstract = {

The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein{\textemdash}for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2\ and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5\ and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins{\textemdash}the products of 40\% of all protein-encoding genes7{\textemdash}are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified{\textemdash}but essential{\textemdash}component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein\ E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.

}, issn = {0028-0836}, doi = {10.1038/s41586-020-2545-9}, url = {http://www.nature.com/articles/s41586-020-2545-9http://www.nature.com/articles/s41586-020-2545-9.pdfhttp://www.nature.com/articles/s41586-020-2545-9.pdfhttp://www.nature.com/articles/s41586-020-2545-9}, author = {Banik, Steven M. and Pedram, Kayvon and Wisnovsky, Simon and Ahn, Green and Riley, Nicholas M. and Bertozzi, Carolyn R.} } @article {66012, title = {The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins}, journal = {Proceedings of the National Academy of Sciences}, volume = {116}, year = {2019}, month = {Sep-04-2019}, pages = {7278 - 7287}, abstract = {

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{\textquoteright}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.

}, issn = {0027-8424}, doi = {10.1073/pnas.1813020116}, url = {http://www.pnas.org/lookup/doi/10.1073/pnas.1813020116https://syndication.highwire.org/content/doi/10.1073/pnas.1813020116http://www.pnas.org/syndication/doi/10.1073/pnas.1813020116}, author = {Malaker, Stacy A. and Pedram, Kayvon and Ferracane, Michael J. and Bensing, Barbara A. and Krishnan, Venkatesh and Pett, Christian and Yu, Jin and Woods, Elliot C. and Kramer, Jessica R. and Westerlind, Ulrika and Dorigo, Oliver and Bertozzi, Carolyn R.} } @article {66014, title = {Physical Principles of Membrane Shape Regulation by the Glycocalyx}, journal = {Cell}, volume = {177}, year = {2019}, month = {Jan-06-2019}, pages = {1757 - 1770.e21}, abstract = {

Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface\ glycocalyx.\ Mucinbiopolymers\ and long-chain\ polysaccharides\ within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled\ membrane structures\ and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the\ extracellular matrix.

}, issn = {00928674}, doi = {10.1016/j.cell.2019.04.017}, url = {https://www.sciencedirect.com/science/article/pii/S0092867419304039?via\%3Dihub}, author = {Shurer, Carolyn R. and Kuo, Joe Chin-Hun and Roberts, LaDeidra Mon{\'e}t and Gandhi, Jay G. and Colville, Marshall J. and Enoki, Thais A. and Pan, Hao and Su, Jin and Noble, Jade M. and Hollander, Michael J. and O{\textquoteright}Donnell, John P. and Yin, Rose and Pedram, Kayvon and M{\"o}ckl, Leonhard and Kourkoutis, Lena F. and Moerner, W.E. and Bertozzi, Carolyn R. and Feigenson, Gerald W. and Reesink, Heidi L. and Paszek, Matthew J.} } @article {66013, title = {Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx}, journal = {Developmental Cell}, volume = {50}, year = {2019}, month = {Jan-07-2019}, pages = {57 - 72.e6}, abstract = {

The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and super-resolution localization microscopy to image two constituents of cell-surface glycans,\ N-acetylgalactosamine (GalNAc) and sialic acid, with 10{\textendash}20\ nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale organization on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to-mesenchymal transition and to oncogenic\ KRAS\ activation. In the latter case, we trace increased height to an effector gene,\ GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology.

}, issn = {15345807}, doi = {10.1016/j.devcel.2019.04.035}, url = {https://www.sciencedirect.com/science/article/pii/S1534580719303326?via\%3Dihub}, author = {M{\"o}ckl, Leonhard and Pedram, Kayvon and Roy, Anish R. and Krishnan, Venkatesh and Gustavsson, Anna-Karin and Dorigo, Oliver and Bertozzi, Carolyn R. and Moerner, W.E.} }