Trehalose glycolipids are found in many bacteria in the suborder Corynebacterineae, but methyl-branched acyltrehaloses are exclusive to virulent species such as the human pathogen Mycobacterium tuberculosis. In M. tuberculosis, the acyltransferase PapA3 catalyzes the formation of diacyltrehalose (DAT), but the enzymes responsible for downstream reactions leading to the final product, polyacyltrehalose (PAT), have not been identified. The PAT biosynthetic gene locus is similar to that of another trehalose glycolipid, sulfolipid 1. Recently, Chp1 was characterized as the terminal acyltransferase in sulfolipid 1 biosynthesis. Here we provide evidence that the homologue Chp2 (Rv1184c) is essential for the final steps of PAT biosynthesis. Disruption of chp2 led to the loss of PAT and a novel tetraacyltrehalose species, TetraAT, as well as the accumulation of DAT, implicating Chp2 as an acyltransferase downstream of PapA3. Disruption of the putative lipid transporter MmpL10 resulted in a similar phenotype. Chp2 activity thus appears to be regulated by MmpL10 in a relationship similar to that between Chp1 and MmpL8 in sulfolipid 1 biosynthesis. Chp2 is localized to the cell envelope fraction, consistent with its role in DAT modification and possible regulatory interactions with MmpL10. Labeling of purified Chp2 by an activity-based probe was dependent on the presence of the predicted catalytic residue Ser141 and was inhibited by the lipase inhibitor tetrahydrolipstatin (THL). THL treatment of M. tuberculosis resulted in selective inhibition of Chp2 over PapA3, confirming Chp2 as a member of the serine hydrolase superfamily. Efforts to produce in vitro reconstitution of acyltransferase activity using straight-chain analogues were unsuccessful, suggesting that Chp2 has specificity for native methyl-branched substrates.
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Abstract Cancer therapy has been revolutionized by the recent developments of immune-checkpoint inhibitors (ICI) to harness the power of the immune system in fighting cancer. However, most patients fail to have durable responses or become resistant to ICI, highlighting the need to identify new mechanisms of immune evasion in cancer and develop novel therapeutic modalities. Recently, the glyco-immune checkpoint axis (sialoglycan/Siglec pathway) has emerged as a new mechanism of immune regulation involving both innate and adaptive immunity and an important mechanism of cancer immune escape. Upon ligation of sialoglycan to ITIM-containing Siglecs on immune cells, this pathway suppresses multiple facets of anti-cancer immunity, including cancer antigen release, cancer antigen presentation, and priming and activation of anti-cancer T cells. However, a therapeutic intervention of this axis remains a great challenge due to the overlapping and promiscuous receptor-ligand interactions between 15 Siglecs and dense array of various sialoglycans in humans. To overcome this hurdle and block the glyco-immune checkpoint axis, we describe here a new therapeutic modality named EAGLE (Enzyme-Antibody Glyco-Ligand Editing), which is antibody-like, multi-functional, and comprised of a tumor-associated antigen-binding moiety and a sialidase moiety, allowing selectively removing terminal sialic acids, the critical binding carbohydrate of Siglecs, from sialoglycans on tumor cells. We demonstrated that EAGLE decreased sialic acid levels of tumor cells and enhanced anti-tumor immune responses using multiple human system models mimicking immunosuppressive tumor microenvironment and immunocompetent syngeneic mouse tumor models. EAGLE treatment released cancer cell-mediated immunosuppression, restored dendritic cell functions, enhanced CD8+ T-cell proliferation/activation, and induced proinflammatory cytokines IFNγ, IL-17A, IL-2, IL-6, and TNFα in human coculture assays of cancer cells with dendritic cells or PBMC in the presence or absence of primary endothelial cells. Systematic administration of EAGLE increased tumor-infiltrating immune cells and led to significant anti-tumor activities with complete regressions as monotherapy in syngeneic mouse tumor models. Re-challenge experiments in cured mice from the EAGLE treatment resulted in a complete rejection of tumor cells, demonstrating that EAGLE induced anti-tumor immunological memory. We further revealed that the mechanism of action of EAGLE involved both innate and adaptive immunity because depleting macrophages or CD8+ T-cells decreased or abolished its efficacy. Moreover, EAGLE in combination with anti-PD1 mAb treatment achieved ~100% cures in syngeneic EMT6-Her2 models. In summary, EAGLE is a novel and promising immunomodulatory therapeutic modality inhibiting the glyco-immune checkpoints and has the potential to overcome resistance to current immunotherapies. Citation Format: Lizhi Cao, Adam Petrone, Wayne Gatlin, Jenny Che, Abhishek Das, Robert LeBlanc, Zakir Siddiquee, Sujata Nerle, Michal Stanczak, Michele Mayo, Lihui Xu, Karl Normington, Jeff Brown, Wei Yao, Carolyn Bertozzi, James Broderick, Heinz Läubli, Li Peng. A novel therapeutic modality of inhibiting the glyco-immune checkpoint axis to treat cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-109.
Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Therapeutic approaches such as LYTAC
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 architecture, 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.
The sulfur assimilation pathway is a key metabolic system in prokaryotes that is required for production of cysteine and cofactors such as coenzyme A. In the first step of the pathway, APS reductase catalyzes the reduction of adenosine 5'-phosphosulfate (APS) to adenosine 5'-phosphate (AMP) and sulfite with reducing equivalents from the protein cofactor, thioredoxin. The primary sequence of APS reductase is distinguished by a conserved iron−sulfur cluster motif, -CC-X∼80-CXXC-. Of the sequence motifs that are associated with 4Fe-4S centers, the cysteine dyad is atypical and has generated discussion with respect to coordination as well as the cluster's larger functional significance. Herein, we have used biochemical, spectroscopic, and mass spectrometry analysis to investigate the iron−sulfur cluster and its role in the mechanism of Mycobacterium tuberculosis APS reductase. Site-directed mutagenesis of any cysteine residue within the conserved motif led to a loss of cluster with a concomitant loss in catalytic activity, while secondary structure was preserved. Studies of 4Fe-4S cluster stability and cysteine reactivity in the presence and absence of substrates, and in the free enzyme versus the covalent enzyme−intermediate (E-Cys-S-SO3-), suggest a structural rearrangement that occurs during the catalytic cycle. Taken together, these results demonstrate that the active site functionally communicates with the iron−sulfur cluster and also suggest a functional significance for the cysteine dyad in promoting site differentiation within the 4Fe-4S cluster.
