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1.
Nat Microbiol ; 2: 17099, 2017 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-28650431

RESUMO

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.


Assuntos
Bactérias Anaeróbias/química , Bactérias Anaeróbias/ultraestrutura , Lipopolissacarídeos/análise , Peptidoglicano/ultraestrutura , Animais , Bactérias/imunologia , Bactérias/patogenicidade , Bactérias Anaeróbias/imunologia , Bactérias Anaeróbias/metabolismo , Química Click , Fluorescência , Interações Hospedeiro-Patógeno , Intestinos/citologia , Intestinos/imunologia , Intestinos/microbiologia , Intestinos/fisiopatologia , Lipopolissacarídeos/imunologia , Redes e Vias Metabólicas , Camundongos , Peptidoglicano/imunologia , Coloração e Rotulagem , Simbiose
2.
Bioorg Med Chem ; 24(20): 4791-4800, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27283789

RESUMO

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.


Assuntos
Dendrímeros/química , Piperidinas/química , Polissacarídeos/química , Proteínas/química , Dendrímeros/síntese química , Humanos , Estrutura Molecular , Triazinas/química
3.
Nat Med ; 21(9): 1091-100, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26280120

RESUMO

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.


Assuntos
Bacteroides fragilis/isolamento & purificação , Intestinos/microbiologia , Microbiota , Animais , Bactérias Anaeróbias/isolamento & purificação , Feminino , Corantes Fluorescentes , Glicocálix/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peritonite/microbiologia
4.
Nature ; 511(7509): 319-25, 2014 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-25030168

RESUMO

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.


Assuntos
Glicocálix/metabolismo , Glicoproteínas/metabolismo , Integrinas/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Mama/citologia , Mama/metabolismo , Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Fibroblastos , Glicocálix/química , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Integrinas/química , Camundongos , Terapia de Alvo Molecular , Mucina-1/metabolismo , Metástase Neoplásica/patologia , Células Neoplásicas Circulantes , Ligação Proteica , Receptores de Superfície Celular
5.
Nat Chem Biol ; 10(1): 69-75, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24292068

RESUMO

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.


Assuntos
Glicocálix/imunologia , Evasão da Resposta Imune , Células Matadoras Naturais/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/fisiologia , Linhagem Celular Tumoral , Humanos
6.
Chem Biol ; 21(1): 16-37, 2014 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-24269151

RESUMO

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.


Assuntos
Medicina/métodos , Polissacarídeos/uso terapêutico , Humanos , Polissacarídeos/química
8.
J Am Chem Soc ; 133(40): 16127-35, 2011 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-21866965

RESUMO

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.


Assuntos
Amino Açúcares/química , Glicoproteínas/química , Hormônio do Crescimento Humano/química , Aldeídos/química , Amino Açúcares/síntese química , Glicosilação , Hormônio do Crescimento Humano/genética , Humanos , Modelos Moleculares , Oximas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
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