Substances released from pathogens and damaged cells are specifically captured by various pattern recognition receptors in the host immune system. Of these it is the C-type lectin receptors (CLRs) whose function it is to recognize various types of ligands including glycans, glycolipids, lipids and proteins via small C-type lectin domains and to then transduce signals across the membrane. The released glycolipids are specifically recognized by various CLRs involved in the immune response. In this review, I briefly introduce structural aspects of the glycolipid recognition mechanisms of three CLRs involved in mycobacterial infection. These are Mincle, DCAR and Dectin-2. Mincle binds trehalose dimycolate and glycerol monomycolate which are cell wall components of mycobacteria, but it also binds to various (glyco)lipids such as β-glucosylceramide and cholesterol crystals derived from damaged cells. 3D structural analysis has revealed that Mincle has a sugar binding site that recognizes the trehalose disaccharide unit and a hydrophobic groove that accommodates the acyl chains. DCAR interacts with another mycobacterial glycolipid, phosphatidyl-myo-inositol mannoside. DCAR also accepts acyl chains via a hydrophobic groove, but it is situated in a very different position from that in Mincle. Dectin-2 binds mannose-capped lipoarabinomannan (Man-LAM), a glycolipid of pathogenic mycobacteria, via only the disaccharide unit, Manα1-2Man, and not the lipid moiety.
When diploid cells of the budding yeast Saccharomyces cerevisiae are incubated under starvation conditions, they differentiate into a dormant and stress-resistant form of haploid cells termed spores. Spores have a cell wall (spore wall) which is more complex than that of vegetative cells. The spore wall is composed of the following layers, from inside to outside: mannan, glucan, chitosan, and dityrosine. While the inner two layers contain shared components between the vegetative cell wall and spore wall, chitosan and dityrosine are unique components of the spore wall. The outer two layers are dispensable for the viability of spores. However, the unique features of these layers endow spores with distinct properties, such as stress resistance. Some of these properties allow spores to be used for beneficial purposes. For example, removal of the dityrosine layer by genetic manipulation leads to exposure of the chitosan layer on the spore surface; such mutant spores can be used as chitosan particles. Furthermore, spores can retain soluble secretory proteins in the spore wall because the dityrosine layer works as a diffusion barrier. Thus, if secretory forms of enzymes are expressed in sporulating cells, the enzymes are entrapped in the spore wall. Spores containing enzymes in the spore wall can be used as enzyme capsules.
We recently revealed that the muscular dystrophy-related O-mannosyl glycan of α-dystroglycan contains a tandem structure consisting of ribitol phosphate, a newly identified glycan constituent in mammals. This unique structure is formed by the ribitol phosphate transferases FKTN and FKRP. However, the synthetic mechanisms, in particular, the substrate recognition mechanism and catalytic machinery of these enzymes, are unknown. This review article initially outlines the synthetic mechanisms of O-mannosyl glycan of α-dystroglycan, illustrates the results of our recent structure-function analysis of FKRP, and finally introduces the latest findings regarding the mechanisms by which a ribitol phosphate is transferred to the glycan.
Glycosylation is performed by various glycosidases and glycosyltransferases. Glycan formation and degradation by these enzymes is crucial for various physiological events in cells. Recently, functional analysis of glycan-processing enzymes has actively been performed, and the enzymes are focused on novel drug targets. However, previously reported inhibitors for the glycan-processing enzymes have encountered major problems. Additionally, screening systems for the inhibitors of glycan-processing enzymes are lacking. Accordingly, we are developing cell-based fluorescence imaging systems for glycan-processing glycosidases and screening of their novel inhibitors. In this paper, we introduce the development of a quinone methide cleavage (QMC) fluorescent substrate platform for the glycan-processing glycosidase and the discovery of novel glycosidase inhibitors using these platform-based fluorescent substrates.
As a family of secreted signaling proteins, Wnt contributes to carcinogenesis, stem cell regulation, and various developmental processes in metazoans. Although Wnt has been considered as a morphogen that provides positional information via its concentration gradient, mechanisms by which Wnt proteins disperse in tissues are not fully understood. Heparan sulfate proteoglycans (HSPGs) are involved in distribution and signaling of secreted signaling proteins, including Wnt. Recently we found that HSPGs in Xenopus embryos and HeLa cells form clusters having different degrees of N-sulfation, which we refer to as “HS clusters.” HS clusters are generated from glypicans and modified by N-deacetylase/N-sulfotransferase in Xenopus embryos. They can be classified into N-acetyl-rich and N-sulfo-rich clusters. Importantly, N-sulfo-rich HS clusters may function as scaffolds for endogenous Wnt8, required for both its distribution and signaling. In this review, biological roles of Wnt signaling in embryogenesis and regulation of Wnt distribution and signaling by HSPGs are introduced together with our discovery of HS clusters.
Substances released from pathogens and damaged cells are specifically captured by various pattern recognition receptors in the host immune system. Of these it is the C-type lectin receptors (CLRs) whose function it is to recognize various types of ligands including glycans, glycolipids, lipids and proteins via small C-type lectin domains and to then transduce signals across the membrane. The released glycolipids are specifically recognized by various CLRs involved in the immune response. In this review, I briefly introduce structural aspects of the glycolipid recognition mechanisms of three CLRs involved in mycobacterial infection. These are Mincle, DCAR and Dectin-2. Mincle binds trehalose dimycolate and glycerol monomycolate which are cell wall components of mycobacteria, but it also binds to various (glyco)lipids such as β-glucosylceramide and cholesterol crystals derived from damaged cells. 3D structural analysis has revealed that Mincle has a sugar binding site that recognizes the trehalose disaccharide unit and a hydrophobic groove that accommodates the acyl chains. DCAR interacts with another mycobacterial glycolipid, phosphatidyl-myo-inositol mannoside. DCAR also accepts acyl chains via a hydrophobic groove, but it is situated in a very different position from that in Mincle. Dectin-2 binds mannose-capped lipoarabinomannan (Man-LAM), a glycolipid of pathogenic mycobacteria, via only the disaccharide unit, Manα1-2Man, and not the lipid moiety.
When diploid cells of the budding yeast Saccharomyces cerevisiae are incubated under starvation conditions, they differentiate into a dormant and stress-resistant form of haploid cells termed spores. Spores have a cell wall (spore wall) which is more complex than that of vegetative cells. The spore wall is composed of the following layers, from inside to outside: mannan, glucan, chitosan, and dityrosine. While the inner two layers contain shared components between the vegetative cell wall and spore wall, chitosan and dityrosine are unique components of the spore wall. The outer two layers are dispensable for the viability of spores. However, the unique features of these layers endow spores with distinct properties, such as stress resistance. Some of these properties allow spores to be used for beneficial purposes. For example, removal of the dityrosine layer by genetic manipulation leads to exposure of the chitosan layer on the spore surface; such mutant spores can be used as chitosan particles. Furthermore, spores can retain soluble secretory proteins in the spore wall because the dityrosine layer works as a diffusion barrier. Thus, if secretory forms of enzymes are expressed in sporulating cells, the enzymes are entrapped in the spore wall. Spores containing enzymes in the spore wall can be used as enzyme capsules.
We recently revealed that the muscular dystrophy-related O-mannosyl glycan of α-dystroglycan contains a tandem structure consisting of ribitol phosphate, a newly identified glycan constituent in mammals. This unique structure is formed by the ribitol phosphate transferases FKTN and FKRP. However, the synthetic mechanisms, in particular, the substrate recognition mechanism and catalytic machinery of these enzymes, are unknown. This review article initially outlines the synthetic mechanisms of O-mannosyl glycan of α-dystroglycan, illustrates the results of our recent structure-function analysis of FKRP, and finally introduces the latest findings regarding the mechanisms by which a ribitol phosphate is transferred to the glycan.
Glycosylation is performed by various glycosidases and glycosyltransferases. Glycan formation and degradation by these enzymes is crucial for various physiological events in cells. Recently, functional analysis of glycan-processing enzymes has actively been performed, and the enzymes are focused on novel drug targets. However, previously reported inhibitors for the glycan-processing enzymes have encountered major problems. Additionally, screening systems for the inhibitors of glycan-processing enzymes are lacking. Accordingly, we are developing cell-based fluorescence imaging systems for glycan-processing glycosidases and screening of their novel inhibitors. In this paper, we introduce the development of a quinone methide cleavage (QMC) fluorescent substrate platform for the glycan-processing glycosidase and the discovery of novel glycosidase inhibitors using these platform-based fluorescent substrates.
As a family of secreted signaling proteins, Wnt contributes to carcinogenesis, stem cell regulation, and various developmental processes in metazoans. Although Wnt has been considered as a morphogen that provides positional information via its concentration gradient, mechanisms by which Wnt proteins disperse in tissues are not fully understood. Heparan sulfate proteoglycans (HSPGs) are involved in distribution and signaling of secreted signaling proteins, including Wnt. Recently we found that HSPGs in Xenopus embryos and HeLa cells form clusters having different degrees of N-sulfation, which we refer to as “HS clusters.” HS clusters are generated from glypicans and modified by N-deacetylase/N-sulfotransferase in Xenopus embryos. They can be classified into N-acetyl-rich and N-sulfo-rich clusters. Importantly, N-sulfo-rich HS clusters may function as scaffolds for endogenous Wnt8, required for both its distribution and signaling. In this review, biological roles of Wnt signaling in embryogenesis and regulation of Wnt distribution and signaling by HSPGs are introduced together with our discovery of HS clusters.