RESUMEN
In human serum immunoglobulin G (IgG), a rare modification of biantennary complex N-glycans lead to a ß1,4-galactosylated bisecting GlcNAc branch. We found that the bisecting GlcNAc on a biantennary core-fucosylated N-glycan was enzymatically galactosylated under stringent reaction conditions. Further optimizations led to an efficient enzymatic approach to this particular modification for biantennary substrates. Notably, tri- and tetra-antennary complex N-glycans were not converted by bovine galactosyltransferase. An N-glycan with a galactosylated bisecting GlcNAc was linked to a lanthanide binding tag. The pseudo-contact shifts (PCS) obtained from the corresponding Dy-complex were used to calculate the conformational preferences of the rare N-glycan. Besides two extended conformations only a single folded conformation was found.
Asunto(s)
Acetilglucosamina/metabolismo , Galactosa/metabolismo , Polisacáridos/biosíntesis , Acetilglucosamina/química , Conformación de Carbohidratos , Galactosa/química , Glicosilación , Humanos , Polisacáridos/químicaRESUMEN
The occurrence of α1,6-linked core fucose on the N-glycans of mammalian glycoproteins is involved in tumor progression and reduces the bioactivity of antibodies in antibody-dependent cell-mediated cytotoxicity (ADCC). Since core-fucosylated N-glycans are difficult to isolate from natural sources, only chemical or enzymatic synthesis can provide the desired compounds for biological studies. A general drawback of chemical α-fucosylation is that the chemical assembly of α1,6-linked fucosides is not stereospecific. A robust and general method for the α-selective fucosylation of acceptors with primary hydroxy groups in α/ß ratios exceeding 99:1 was developed. The high selectivities result from the interplay of an optimized protecting group pattern of the fucosyl donors in combination with the activation principle and the reaction conditions. Selective deprotection yielded versatile azides of all mammalian complex-type core-fucosylated N-glycans with 2-4 antennae and optional bisecting GlcNAc.
Asunto(s)
Acetilglucosamina/química , Fucosa/química , Polisacáridos/química , AnimalesRESUMEN
The occurrence of N-glycans with a bisecting GlcNAc modification on glycoproteins has many implications in developmental and immune biology. However, these particular N-glycans are difficult to obtain either from nature or through synthesis. We have developed a flexible and general method for synthesizing bisected N-glycans of the complex type by employing modular TFAc-protected donors for all antennae. The TFAc-protected N-glycans are suitable for the late introduction of a bisecting GlcNAc. This integrated strategy permits for the first time the use of a single approach for multiantennary N-glycans as well as their bisected derivatives via imidates, with unprecedented yields even in a one-pot double glycosylation. With this new method, rare N-glycans of the bisected type can be obtained readily, thereby providing defined tools to decipher the biological roles of bisecting GlcNAc modifications.
RESUMEN
We determined the specificity of BTL, a lectin from the red marine alga Bryothamnion triquetrum, toward fucosylated oligosaccharides. BTL showed a strict specificity for the core α1,6-fucosylation, which is an important marker for cancerogenesis and quality control of therapeutical antibodies. The double fucosylation α1,6 and α1,3 was also recognized, but the binding was totally abolished in the sole presence of the α1,3-fucosylation. A more detailed analysis of the specificity of BTL showed a preference for bi- and tri-antennary nonbisected N-glycans. Sialylation or fucosylation at the nonreducing end of N-glycans did not affect the recognition by the lectin. BTL displayed a strong affinity for a core α1,6-fucosylated octasaccharide with a Kd of 12 µM by titration microcalorimetry. The structural characterization of the interaction between BTL and the octasaccharide was obtained by STD-NMR. It demonstrated an extended epitope for recognition that includes the fucose residue, the distal GlcNAc and one mannose residue. Recombinant rBTL was obtained in Escherichia coli and characterized. Its binding properties for carbohydrates were studied using hemagglutination tests and glycan array analysis. rBTL was able to agglutinate rabbit erythrocytes with strong hemagglutination activity only after treatment with papain and trypsin, indicating that its ligands were not directly accessible at the cell surface. The hemagglutinating properties of rBTL confirm the correct folding and functional state of the protein. The results show BTL as a potent candidate for cancer diagnosis and as a reagent for the preparation and quality control of antibodies lacking core α1,6-fucosylated N-glycans.
Asunto(s)
Proteínas Algáceas/química , Fucosa/química , Lectinas/química , Polisacáridos/química , Rhodophyta/química , Proteínas Algáceas/biosíntesis , Proteínas Algáceas/aislamiento & purificación , Animales , Sitios de Unión , Conformación de Carbohidratos , Secuencia de Carbohidratos , Eritrocitos/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Lectinas/biosíntesis , Lectinas/aislamiento & purificación , Datos de Secuencia Molecular , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Especificidad por SustratoRESUMEN
In the budding yeast Saccharomyces cerevisiae, self-recognition and the thereby promoted aggregation of thousands of cells into protective flocs is mediated by a family of cell-surface adhesins, the flocculins (Flo). Based on this social behavior FLO genes fulfill the definition of "greenbeard" genes, which direct cooperation toward other carriers of the same gene. The process of flocculation plays an eminent role in the food industry for the production of beer and wine. However, the precise mode of flocculin-mediated surface recognition and the exact structure of cognate ligands have remained elusive. Here, we present structures of the adhesion domain of a flocculin complexed to its cognate ligands derived from yeast high-mannose oligosaccharides at resolutions up to 0.95 Å. Besides a PA14-like architecture, the Flo5A domain reveals a previously undescribed lectin fold that utilizes a unique DcisD calcium-binding motif for carbohydrate binding and that is widely spread among pro- and eukaryotes. Given the high abundance of high-mannose oligosaccharides in yeast cell walls, the Flo5A structure suggests a model for recognition, where social non-self- instead of unsocial self-interactions are favored.
Asunto(s)
Lectinas/química , Oligosacáridos/química , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Sitios de Unión/genética , Calcio/metabolismo , Secuencia de Carbohidratos , Cristalografía por Rayos X , Lectinas/genética , Lectinas/metabolismo , Manosa/química , Manosa/metabolismo , Lectina de Unión a Manosa/química , Lectina de Unión a Manosa/metabolismo , Microscopía Fluorescente , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Oligosacáridos/metabolismo , Unión Proteica , Pliegue de Proteína , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
In this study we report on the design, synthesis, and biological evaluation of pyrrole-2-one 2 to be a highly potent VEGF-R2/3 inhibitor with IC 50 of 31/37 nM. The novel 3,4-diaryl-2 H-pyrrole-2-ones were designed on the basis of the modeled binding mode of the corresponding 1 H-pyrrole-2,5-dione (maleimide) VEGF-R2/3 inhibitor 1 indicating two H-bond ligand-protein interactions in the ATP pocket for the amide 2 but not for the isomer 3. Flexible synthetic routes to 3,4-diaryl-2 H-pyrrole-2-ones and structure-activity relationships for the compounds in a panel of 24 therapeutically relevant protein kinases (IC 50 values) are presented. Accordingly to the in vitro data, compounds 1 and 2 were found to possess highly potent antiangiogenic activities in the cellular HLMEC sprouting assay and also slightly induced apoptosis in HDMECs whereas 3 was determined to be significantly less active. Hence, the pyrrole-2-one moiety was dissected from the corresponding maleimide protein kinase inhibitor as a suitable key pharmacophore.