Abstract Siglec-7 is a human CD33-like siglec, and is localised predominantly on human natural killer (NK) cells and monocytes. Siglec-7 is considered to function as an immunoreceptor in a sialic acid-dependent manner. However, the underlying mechanisms linking sialic acid-binding and function remain unknown. Here, to gain new insights into the ligand-binding properties of Siglec-7, we carried out in silico analysis and site-directed mutagenesis, and found a new sialic acid-binding region (site 2 containing R67) in addition to the well-known primary ligand-binding region (site 1 containing R124). This was supported by equilibrium dialysis, STD-NMR experiments, and inhibition analysis of GD3-binding toward Siglec-7 using synthetic sialoglycoconjugates and a comprehensive set of ganglioside-based glycoconjugates. Our results suggest that the two ligand-binding sites are potentially controlled by each other due to the flexible conformation of the C-C′ loop of Siglec-7.
A biological system is an interacting network controlled by individual biomolecules such as DNA/RNA, proteins, and glycans. The field of structural biology developed with the aim of understanding the function and detailed modes of interaction of biomolecules from the point of view of 3-D structure. Nuclear magnetic resonance (NMR) spectroscopy has demonstrated its potential with a wide range of applications to studies of the structure and function of biological molecules. The unique potential of NMR lies in its ability to generate high-resolution data not only about the structure but also about the dynamics and binding interactions. This chapter describes the basics of solution NMR and its application to the analysis of the structure and interaction of biomolecules, focusing on glycans (glycoproteins and glycolipids) and glycan-binding proteins.
Background. It is important to develop new immunosuppressive agents without clinical drawbacks. In this article, we reveal the possibility of a chemically synthetic sulfonolipid that acts as a novel immunosuppressive drug. Methods. We evaluated the immunosuppressive effect of 3-O-(6-deoxy-6-sulfono-β-D-glucopyranosyl)-1,2-di-O-acylglycerol (β-SQDG) that contains a saturated C18 fatty acid, which is designated as β-SQDG(18:0) by mixed lymphocyte reaction (MLR) and rat allogeneic skin graft. Then, we investigated the mechanism of immunosuppressive effect of β-SQDG(18:0). Results. β-SQDG(18:0) inhibited human MLR in a dose-dependent manner without overt cytotoxic effect and prolonged rat skin allograft rejection in vivo. β-SQDG(18:0) did not inhibit the direct activation of responder T. This reagent could not affect the expression of either major histocompatibility antigen complex (MHC) class I or class II molecules on the cell surface of the stimulator cells, antigen-presenting cells. In contrast, β-SQDG(18:0) was demonstrated to inhibit the binding among allogeneic lymphocytes. However, the expression of known cell surface accessory and adhesion molecules, such as CD4, CD28, leukocyte function-associated antigen 1, intercellular adhesion molecule 1, and CTLA-4, was not affected by β-SQDG(18:0) treatment. Conclusions. β-SQDG(18:0) might be a new class of the immunosuppressive reagent, and the inhibition of responder T-lymphocyte activation in MLR by β-SQDG(18:0) may be responsible for certain three-dimensional structures of this reagent or its quinovose binding to sulfonic acid.
Recent advances in synthesizing methodologies and purification techniques significantly increase the accessibility of the major glycan structures, which are heterogeneously present on the glycoproteins and glycolipids. In contrast, it is often difficult to obtain sufficient amounts of minor structures consisting of a number of sugar units with reasonable purity sufficient for biological studies. In terms of the accessibility of the desired glycan sequences, it is useful in certain instances to deal with not the full sequence but with the required oligosaccharides, called glycan epitopes. Glycan epitopes have been used for the structural and functional analysis of the proteins that recognize particular glycans. For instance, lectins usually recognize the glycan epitope of a few sugar units, not in a “key-and-keyhole” manner but in a “pattern recognition receptor” mode. Such oligosaccharide epitopes can be prepared through regular chemical synthesis. This report describes recent progress on the NMR analysis of glycan–glycan and protein–glycan interactions using chemically synthesized glycan epitopes.
ZG16p is a soluble mammalian lectin, the first to be described with a Jacalin-related β-prism-fold. ZG16p has been reported to bind both to glycosaminoglycans and mannose. To determine the structural basis of the multiple sugar-binding properties, we conducted glycan microarray analyses of human ZG16p. We observed that ZG16p preferentially binds to α-mannose-terminating short glycans such as Ser/Thr-linked O-mannose, but not to high mannose-type N-glycans. Among sulfated glycosaminoglycan oligomers examined, chondroitin sulfate B and heparin oligosaccharides showed significant binding. Crystallographic studies of human ZG16p lectin in the presence of selected ligands revealed the mechanism of multiple sugar recognition. Manα1-3Man and Glcβ1-3Glc bound in different orientations: the nonreducing end of the former and the reducing end of the latter fitted in the canonical shallow mannose binding pocket. Solution NMR analysis using (15)N-labeled ZG16p defined the heparin-binding region, which is on an adjacent flat surface of the protein. On-array competitive binding assays suggest that it is possible for ZG16p to bind simultaneously to both types of ligands. Recognition of a broad spectrum of ligands by ZG16p may account for the multiple functions of this lectin in the formation of zymogen granules via glycosaminoglycan binding, and in the recognition of pathogens in the digestive system through α-mannose-related recognition.
24dCho, which perfectly retains the cholesterol's membrane properties, was developed to examine cholesterol's interactions and membrane partitions using solid state 2H NMR.
Amphotericin B, a long-used antifungal drug, forms fungicidal ion-permeable channels across cell membranes. Using solid-state nuclear magnetic resonance spectroscopy and molecular dynamics simulations, we experimentally elucidated the three-dimensional structure of the molecular assemblies formed by this drug in membranes in the presence of the fungal sterol, ergosterol. A stable assembly of seven drug molecules was observed to form an ion conductive channel. The structure somewhat resembled the upper half of the barrel-stave model proposed in the 1970s but different substantially in the number of molecules and their arrangement. Based on the structure obtained, the aggregation of the channel assemblies in membranes was investigated and a mechanism was proposed in which complexation with ergosterol stabilizes the drug’s assemblies, leading to their aggregation, and in turn enhancing channel activity. The high-resolution structure is consistent with many previous findings, including structure-activity relationships of the drug, and the channel aggregation provides a more reasonable explanation for the selective toxicity of this drug to fungi.
