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1.
PLoS One ; 12(10): e0184378, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29016609

RESUMO

Glycoprotein receptors are influenced by myriad intermolecular interactions at the cell surface. Specific glycan structures may interact with endogenous lectins that enforce or disrupt receptor-receptor interactions. Glycoproteins bound by multivalent lectins may form extended oligomers or lattices, altering the lateral mobility of the receptor and influencing its function through endocytosis or changes in activation. In this study, we have examined the interaction of Galectin-3 (Gal-3), a human lectin, with adhesion receptors. We measured the effect of recombinant Gal-3 added exogenously on the lateral mobility of the α5ß1 integrin on HeLa cells. Using single-particle tracking (SPT) we detected increased lateral mobility of the integrin in the presence of Gal-3, while its truncated C-terminal domain (Gal-3C) showed only minor reductions in lateral mobility. Treatment of cells with Gal-3 increased ß1-integrin mediated migration with no apparent changes in viability. In contrast, Gal-3C decreased both cell migration and viability. Fluorescence microscopy allowed us to confirm that exogenous Gal-3 resulted in reorganization of the integrin into larger clusters. We used a proteomics analysis to confirm that cells expressed endogenous Gal-3, and found that addition of competitive oligosaccharide ligands for the lectin altered the lateral mobility of the integrin. Together, our results are consistent with a Gal-3-integrin lattice model of binding and confirm that the lateral mobility of integrins is natively regulated, in part, by galectins.


Assuntos
Endocitose/genética , Galectina 3/genética , Integrina alfa5beta1/metabolismo , Proteômica , Adesão Celular/genética , Movimento Celular/efeitos dos fármacos , Galectina 3/administração & dosagem , Regulação da Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HeLa , Humanos , Integrina alfa5beta1/genética , Oligossacarídeos/metabolismo , Ligação Proteica
2.
Carbohydr Res ; 417: 109-16, 2015 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-26454791

RESUMO

A variety of applications in glycobiology exploit affinity chromatography through the immobilization of glycans to a solid support. Although several strategies are known, they may provide certain advantages or disadvantages in how the sugar is attached to the affinity matrix. Additionally, the products of some methods may be hard to characterize chemically due to non-specific reactions. The lack of specificity in standard immobilization reactions makes affinity chromatography with expensive oligosaccharides challenging. As a result, methods for specific and efficient immobilization of oligosaccharides remain of interest. Herein, we present a method for the immobilization of saccharides using N'-glycosylsulfonohydrazide (GSH) carbohydrate donors. We have compared GSH immobilization to known strategies, including the use of divinyl sulfone (DVS) and cyanuric chloride (CC), for the generation of affinity matrices. We compared immobilization methods by determining their immobilization efficiency, based on a comparison of the mass of immobilized carbohydrate and the concentration of active binding sites (determined using lectins). Our results indicate that immobilization using GSH donors can provide comparable amounts of carbohydrate epitopes on solid support while consuming almost half of the material required for DVS immobilization. The lectin binding capacity observed for these two methods suggests that GSH immobilization is more efficient. We propose that this method of oligosaccharide immobilization will be an important tool for glycobiologists working with precious glycan samples purified from biological sources.


Assuntos
Cromatografia de Afinidade/instrumentação , Glicômica/instrumentação , Hidrazinas/química , Oligossacarídeos/química , Sefarose/química , Sítios de Ligação , Cromatografia de Afinidade/métodos , Glicômica/métodos , Lectinas de Plantas/química , Sulfonas/química , Triazinas/química
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