Illuminating vital surface molecules of symbionts in health and disease.

The immunomodulatory surface molecules of commensal and pathogenic bacteria are critical to microorganisms' survival and the host's response1,2. Recent studies have highlighted the unique and important responses elicited by commensal-derived surface macromolecules3-5. However, the technology available to track these molecules in host cells and tissues remains primitive. We report, here, an interdisciplinary approach that uses metabolic labelling combined with bioorthogonal click chemistry (that is, reactions performed in living organisms)6 to specifically tag up to three prominent surface immunomodulatory macromolecules-peptidoglycan, lipopolysaccharide and capsular polysaccharide-either simultaneously or individually in live anaerobic commensal bacteria. Importantly, the peptidoglycan labelling enables, for the first time, the specific labelling of live endogenous, anaerobic bacteria within the mammalian host. This approach has allowed us to image and track the path of labelled surface molecules from live, luminal bacteria into specific intestinal immune cells in the living murine host during health and disease. The chemical labelling of three specific macromolecules within a live organism offers the potential for in-depth visualization of host-pathogen interactions.

Piperidine-based glycodendrons as protein N-glycan prosthetics.

The generation of homogeneously glycosylated proteins is essential for defining glycoform-specific activity and improving protein-based therapeutics. We present a novel glycodendron prosthetic which can be site-selectively appended to recombinant proteins to create 'N-glycosylated' glycoprotein mimics. Using computational modeling, we designed the dendrimer scaffold and protein attachment point to resemble the native N-glycan architecture. Three piperidine-melamine glycodendrimers were synthesized via a chemoenzymatic route and attached to human growth hormone and the Fc region of human IgG. These products represent a new class of engineered biosimilars bearing novel glycodendrimer structures.

In vivo imaging and tracking of host-microbiota interactions via metabolic labeling of gut anaerobic bacteria.

The intestine is densely populated by anaerobic commensal bacteria. These microorganisms shape immune system development, but understanding of host-commensal interactions is hampered by a lack of tools for studying the anaerobic intestinal environment. We applied metabolic oligosaccharide engineering and bioorthogonal click chemistry to label various commensal anaerobes, including Bacteroides fragilis, a common and immunologically important commensal. We studied the dissemination of B. fragilis after acute peritonitis and characterized the interactions of the intact microbe and its polysaccharide components in myeloid and B cell lineages. We were able to assess the distribution and colonization of labeled B. fragilis along the intestine, as well as niche competition after coadministration of multiple species of the microbiota. We also fluorescently labeled nine additional commensals (eight anaerobic and one microaerophilic) from three phyla common in the gut--Bacteroidetes, Firmicutes and Proteobacteria--as well as one aerobic pathogen (Staphylococcus aureus). This strategy permits visualization of the anaerobic microbial niche by various methods, including intravital two-photon microscopy and non-invasive whole-body imaging, and can be used to study microbial colonization and host-microbe interactions in real time.

The cancer glycocalyx mechanically primes integrin-mediated growth and survival.

Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx. We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function.

Glycocalyx engineering reveals a Siglec-based mechanism for NK cell immunoevasion.

The increase of cell surface sialic acid is a characteristic shared by many tumor types. A correlation between hypersialylation and immunoprotection has been observed, but few hypotheses have provided a mechanistic understanding of this immunosuppressive phenomenon. Here, we show that increasing sialylated glycans on cancer cells inhibits human natural killer (NK) cell activation through the recruitment of sialic acid-binding immunoglobulin-like lectin 7 (Siglec-7). Key to these findings was the use of glycopolymers end-functionalized with phospholipids, which enable the introduction of synthetically defined glycans onto cancer cell surfaces. Remodeling the sialylation status of cancer cells affected the susceptibility to NK cell cytotoxicity via Siglec-7 engagement in a variety of tumor types. These results support a model in which hypersialylation offers a selective advantage to tumor cells under pressure from NK immunosurveillance by increasing Siglec ligands. We also exploited this finding to protect allogeneic and xenogeneic primary cells from NK-mediated killing, suggesting the potential of Siglecs as therapeutic targets in cell transplant therapy.

Glycotherapy: new advances inspire a reemergence of glycans in medicine.

The beginning of the 20(th) century marked the dawn of modern medicine with glycan-based therapies at the forefront. However, glycans quickly became overshadowed as DNA- and protein-focused treatments became readily accessible. The recent development of new tools and techniques to study and produce structurally defined carbohydrates has spurred renewed interest in the therapeutic applications of glycans. This review focuses on advances within the past decade that are bringing glycan-based treatments back to the forefront of medicine and the technologies that are driving these efforts. These include the use of glycans themselves as therapeutic molecules as well as engineering protein and cell surface glycans to suit clinical applications. Glycan therapeutics offer a rich and promising frontier for developments in the academic, biopharmaceutical, and medical fields.

Protein glycoengineering enabled by the versatile synthesis of aminooxy glycans and the genetically encoded aldehyde tag.

Homogeneously glycosylated proteins are important targets for fundamental research and for biopharmaceutical development. The use of unnatural protein-glycan linkages bearing structural similarity to their native counterparts can accelerate the synthesis of glycoengineered proteins. Here we report an approach toward generating homogeneously glycosylated proteins that involves chemical attachment of aminooxy glycans to recombinantly produced proteins via oxime linkages. We employed the recently introduced aldehyde tag method to obtain a recombinant protein with the aldehyde-bearing formylglycine residue at a specific site. Complex aminooxy glycans were synthesized using a new route that features N-pentenoyl hydroxamates as key intermediates that can be readily elaborated chemically and enzymatically. We demonstrated the method by constructing site-specifically glycosylated variants of the human growth hormone.

Active-site-directed chemical tools for profiling mitochondrial Lon protease.

Lon and ClpXP are the only soluble ATP-dependent proteases within the mammalian mitochondria matrix, which function in protein quality control by selectively degrading misfolded, misassembled, or damaged proteins. Chemical tools to study these proteases in biological samples have not been identified, thereby hindering a clear understanding of their respective functions in normal and disease states. In this study, we applied a proteolytic site-directed approach to identify a peptide reporter substrate and a peptide inhibitor that are selective for Lon but not ClpXP. These chemical tools permit quantitative measurements that distinguish Lon-mediated proteolysis from that of ClpXP in biochemical assays with purified proteases, as well as in intact mitochondria and mitochondrial lysates. This chemical biology approach provides needed tools to further our understanding of mitochondrial ATP-dependent proteolysis and contributes to the future development of diagnostic and pharmacological agents for treating diseases associated with defects in mitochondrial protein quality.

Utilization of synthetic peptides to evaluate the importance of substrate interaction at the proteolytic site of Escherichia coli Lon protease.

Lon, also known as protease La, is an ATP-dependent protease functioning to degrade many unstructured proteins. Currently, very little is known about the substrate determinants of Lon at the proteolytic site. Using synthetic peptides constituting different regions of the endogenous protein substrate lambdaN, we demonstrated that the proteolytic site of Escherichia coli Lon exhibits a certain level of localized sequence specificity. Using an alanine positional scanning approach, we discovered a set of discontinuous substrate determinants surrounding the scissile Lon cleavage site in a model peptide substrate, which function to influence the k(cat) of the peptidase activity of Lon. We further investigated the mode of peptide interaction with the proteolytically inactive Lon mutant S679A in the absence and presence of ADP or AMPPNP by 2-dimensional nuclear magnetic resonance spectroscopy, and discovered that the binding interaction between protein and peptide varies with the nucleotide bound to the enzyme. This observation is suggestive of a substrate translocation step, which likely limits the turnover of the proteolytic reaction. The contribution of the identified substrate determinants towards the kinetics of ATP-dependent degradation of lambdaN and truncated lambdaN mutants by Lon was also examined. Our results indicated that Lon likely recognizes numerous discontinuous substrate determinants throughout lambdaN to achieve substrate promiscuity.

Identification of the proteasome inhibitor MG262 as a potent ATP-dependent inhibitor of the Salmonella enterica serovar Typhimurium Lon protease.

Lon is a homo-oligomeric ATP-dependent serine protease which functions in the degradation of damaged and certain regulatory proteins. The importance of Lon activity in bacterial pathogenicity has led to its emergence as a target in the development of novel antibiotics. As no potent inhibitors of Lon activity have been reported to date, we sought to identify an inhibitor which could serve as a lead compound in the development of a potent Lon-specific inhibitor. To determine whether a nucleotide- or peptide-based inhibitor would be more effective, we evaluated the steady-state kinetic parameters associated with both ATP and peptide hydrolysis by human and Salmonella enterica serovar Typhimurium Lon. Although the ATP hydrolysis activities of both homologues are kinetically indistinguishable, they display marked differences in peptide substrate specificity. This suggests that a peptide-based inhibitor could be developed which would target bacterial Lon, thereby decreasing side-effects due to cross-reactivity with human Lon. Using Salmonella enterica serovar Typhimurium Lon as a model, we evaluated the IC50 values of a series of commercially available peptide-based inhibitors. Those inhibitors which behave as transition state analogues were the most useful in inhibiting Lon activity. The peptidyl boronate, MG262, was the most potent inhibitor tested (IC50 = 122 +/- 9 nM) and required binding, but not hydrolysis, of ATP to initiate inhibition. We hope to use MG262 as a lead compound in the development of future Lon-specific inhibitors.