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Pedram Lab / Publications
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14 Publications

Showing 1-10 of 14 results
05/10/24 | Imaging the extracellular matrix in live tissues and organisms with a glycan-binding fluorophore
Fiore A, Yu G, Northey JJ, Patel R, Ravenscroft TA, Ikegami R, Kolkman W, Kumar P, Grimm JB, Dilan TL, Ruetten VM, Ahrens MB, Shroff H, Lavis LD, Wang S, Weaver VM, Pedram K
bioRxiv. 2024 May 10:. doi: 10.1101/2024.05.09.593460

All multicellular systems produce and dynamically regulate extracellular matrices (ECM) that play important roles in both biochemical and mechanical signaling. Though the spatial arrangement of these extracellular assemblies is critical to their biological functions, visualization of ECM structure is challenging, in part because the biomolecules that compose the ECM are difficult to fluorescently label individually and collectively. Here, we present a cell-impermeable small molecule fluorophore, termed Rhobo6, that turns on and red shifts upon reversible binding to glycans. Given that most ECM components are densely glycosylated, the dye enables wash-free visualization of ECM, in systems ranging from in vitro substrates to in vivo mouse mammary tumors. Relative to existing techniques, Rhobo6 provides a broad substrate profile, superior tissue penetration, nonperturbative labeling, and negligible photobleaching. This work establishes a straightforward method for imaging the distribution of ECM in live tissues and organisms, lowering barriers for investigation of extracellular biology.

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04/25/24 | Expansion of in vitro Toxoplasma gondii cysts using enzymatically enhanced ultrastructure expansion microscopy
Kseniia Bondarenko , Floriane Limoge , Kayvon Pedram , Mathieu Gissot , Joanna C. Young
bioRxiv. 2024 Apr 25:. doi: 10.1101/2024.04.24.590991

Expansion microscopy (ExM) is an innovative approach to achieve super-resolution images without using super-resolution microscopes, based on the physical expansion of the sample. The advent of ExM has unlocked super-resolution imaging for a broader scientific circle, lowering the cost and entry skill requirements to the field. One of its branches, ultrastructure ExM (U-ExM), has become popular among research groups studying Apicomplexan parasites, including the acute stage of Toxoplasma gondii infection. The chronic cyst-forming stage of Toxoplasma, however, resists U-ExM expansion, impeding precise protein localisation. Here, we solve the in vitro cyst’s resistance to denaturation required for successful U-ExM of the encapsulated parasites. As the cyst’s main structural protein CST1 contains a mucin domain, we added an enzymatic digestion step using the pan-mucinase StcE prior to the expansion protocol. This allowed full expansion of the cysts in fibroblasts and primary neuronal cell culture without interference with the epitopes of the cyst-wall associated proteins. Using StcE-enhanced U-ExM, we clarified the shape and location of the GRA2 protein important for establishing a normal cyst. Expanded cysts revealed GRA2 granules spanning across the cyst wall, with a notable presence observed outside on both sides of the CST1-positive layer.

Importance Toxoplasma gondii is an intracellular parasite capable of establishing long-term chronic infection in nearly all warm-blooded animals. During the chronic stage, parasites encapsulate into cysts in a wide range of tissues but particularly in neurons of the central nervous system and in skeletal muscle. Current anti-Toxoplasma drugs do not eradicate chronic parasites and leave behind a reservoir of infection. As the cyst is critical for both transmission and pathology of the disease, we need to understand more fully the biology of the cyst and its vulnerabilities.

The advent of a new super-resolution approach called ultrastructure expansion microscopy allowed in-depth studies of the acute stage of Toxoplasma infection but not the cyst-forming stage, which resists protocol-specific denaturation. Here, we show that an additional step of enzymatic digestion using mucinase StcE allows full expansion of the Toxoplasma cysts, offering a new avenue for a comprehensive examination of the chronic stage of infection using an accessible super-resolution technique.

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03/16/22 | Small molecule inhibitors of mammalian glycosylation.
Almahayni K, Spiekermann M, Fiore A, Yu G, Pedram K, Möckl L
Matrix Biology Plus. 2022 Mar 16;16:100108. doi: 10.1016/j.mbplus.2022.100108

Glycans are one of the fundamental biopolymers encountered in living systems. Compared to polynucleotide and polypeptide biosynthesis, polysaccharide biosynthesis is a uniquely combinatorial process to which interdependent enzymes with seemingly broad specificities contribute. The resulting intracellular cell surface, and secreted glycans play key roles in health and disease, from embryogenesis to cancer progression. The study and modulation of glycans in cell and organismal biology is aided by small molecule inhibitors of the enzymes involved in glycan biosynthesis. In this review, we survey the arsenal of currently available inhibitors, focusing on agents which have been independently validated in diverse systems. We highlight the utility of these inhibitors and drawbacks to their use, emphasizing the need for innovation for basic research as well as for therapeutic applications.

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01/01/21 | An Acquired and Endogenous Glycocalyx Forms a Bidirectional “Don’t Eat” and “Don’t Eat Me” Barrier to Phagocytosis
Imbert PR, Saric A, Pedram K, Bertozzi CR, Grinstein S, Freeman SA
Current Biology. Jan-01-2021;31(1):77 - 89.e5. doi: 10.1016/j.cub.2020.09.082

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'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.

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08/17/20 | An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins
Shon DJ, Malaker SA, Pedram K, Yang E, Krishnan V, Dorigo O, Bertozzi CR
Proceedings of the National Academy of Sciences. Jan-09-2020;117(35):21299 - 21307. doi: 10.1073/pnas.2012196117

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.


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07/29/20 | Lysosome-targeting chimaeras for degradation of extracellular proteins
Banik SM, Pedram K, Wisnovsky S, Ahn G, Riley NM, Bertozzi CR
Nature. Jan-08-2021;584(7820):291 - 297. doi: 10.1038/s41586-020-2545-9

The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein—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—the products of 40% of all protein-encoding genes7—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—but essential—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.


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01/06/20 | Bump-and-Hole Engineering Identifies Specific Substrates of Glycosyltransferases in Living Cells
Schumann B, Malaker SA, Wisnovsky SP, Debets MF, Agbay AJ, Fernandez D, Wagner LJ, Lin L, Li Z, Choi J, Fox DM, Peh J, Gray MA, Pedram K, Kohler JJ, Mrksich M, Bertozzi CR
Molecular Cell. Jan-06-2020;78(5):824 - 834.e15. doi: 10.1016/j.molcel.2020.03.030

Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. Protein glycosylation contributes to essential biological processes, but correlating glycan structure, underlying protein, and disease-relevant biosynthetic regulation is currently elusive. Here, we engineer living cells to tag glycans with editable chemical functionalities while providing information on biosynthesis, physiological context, and glycan fine structure. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes.


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03/25/19 | The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins
Malaker SA, Pedram K, Ferracane MJ, Bensing BA, Krishnan V, Pett C, Yu J, Woods EC, Kramer JR, Westerlind U, Dorigo O, Bertozzi CR
Proceedings of the National Academy of Sciences. Sep-04-2019;116(15):7278 - 7287. doi: 10.1073/pnas.1813020116

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.


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01/07/19 | Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx
Möckl L, Pedram K, Roy AR, Krishnan V, Gustavsson A, Dorigo O, Bertozzi CR, Moerner W
Developmental Cell. Jan-07-2019;50(1):57 - 72.e6. doi: 10.1016/j.devcel.2019.04.035

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 10–20 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.

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01/06/19 | Physical Principles of Membrane Shape Regulation by the Glycocalyx
Shurer CR, Kuo JC, Roberts LM, Gandhi JG, Colville MJ, Enoki TA, Pan H, Su J, Noble JM, Hollander MJ, O’Donnell JP, Yin R, Pedram K, Möckl L, Kourkoutis LF, Moerner W, Bertozzi CR, Feigenson GW, Reesink HL, Paszek MJ
Cell. Jan-06-2019;177(7):1757 - 1770.e21. doi: 10.1016/j.cell.2019.04.017

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 glycocalyxMucin biopolymers 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.

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