Structural and thermodynamic analyses reveal critical features of glycopeptide recognition by the human PILRα immune cell receptor

金沢大学医薬保健研究域薬学系Before entering host cells, herpes simplex virus-1 uses its envelope glycoprotein B to bind paired immunoglobulin-like type 2 receptor α (PILRα) on immune cells. PILRα belongs to the Siglec (sialic acid (SA)-binding immunoglobulin-like lectin)- like family, members of which bind SA. PILRα is the only Siglec member to recognize not only the sialylated O-linked sugar T antigen (sTn) but also its attached peptide region. We previously determined the crystal structure of PILRα complexed with the sTn-linked glycopeptide of glycoprotein B, revealing the simultaneous recognition of sTn and peptide by the receptor. However, the contribution of each glycopeptide component to PILRα binding was largely unclear. Here, we chemically synthesized glycopeptide derivatives and determined the thermodynamic parameters of their interaction with PILRα. We show that glycopeptides with different sugar units linking SA and peptides (i.e. "GlcNAc-Type" and "deoxy- GlcNAc-Type" glycopeptides) have lower affinity and more enthalpy-driven binding than the wild type (i.e. GalNAc-Type glycopeptide). The crystal structures of PILRα complexed with these glycopeptides highlighted the importance of stereochemical positioning of the O4 atom of the sugar moiety. These results provide insights both for understanding the unique O-glycosylated peptide recognition by the PILRα and for the rational design of herpes simplex virus-1 entry inhibitors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc

Dysprosium Absorption of Aluminum Tolerant- and Absorbing-Yeast

Background: Biosorption plays important roles in the recovery of rare earth metals. The absorption of dysprosium (Dy) was tested in yeast. Interestingly, brewing yeast, Saccharomyces cerevisiae, showed Dy absorption, and two strains, Alt-OF2 and Alt-OF5—previously isolated as highly aluminum-tolerant and -absorbing yeast strains—were screened and shown to be superior in terms of their Dy absorption when compared to S. cerevisiae. Here, we analyzed the Dy absorption in these yeast strains. Methods: Dy absorption in yeast strains was measured using an inductively coupled plasma optical emission spectrometer (ICP-OES). Dy concentration and localization in yeast cells and the effect of treated pH on the Dy absorption were assayed. Results: The Dy absorption of Alt-OF2 and Alt-OF5 was more than two times that of S. cerevisiae. The absorption of Dy took place inside of the cells, and a small amount was found in the cell wall fraction. Conclusion: These results suggest that yeast offers a promising solution to the biosorption of rare earth metals and that it is possible to use the highly absorbent strains to breed a yeast strain that can recover even higher concentrations of Dy

Dysprosium Absorption of Aluminum Tolerant- and Absorbing-Yeast

Background: Biosorption plays important roles in the recovery of rare earth metals. The absorption of dysprosium (Dy) was tested in yeast. Interestingly, brewing yeast, Saccharomyces cerevisiae, showed Dy absorption, and two strains, Alt-OF2 and Alt-OF5—previously isolated as highly aluminum-tolerant and -absorbing yeast strains—were screened and shown to be superior in terms of their Dy absorption when compared to S. cerevisiae. Here, we analyzed the Dy absorption in these yeast strains. Methods: Dy absorption in yeast strains was measured using an inductively coupled plasma optical emission spectrometer (ICP-OES). Dy concentration and localization in yeast cells and the effect of treated pH on the Dy absorption were assayed. Results: The Dy absorption of Alt-OF2 and Alt-OF5 was more than two times that of S. cerevisiae. The absorption of Dy took place inside of the cells, and a small amount was found in the cell wall fraction. Conclusion: These results suggest that yeast offers a promising solution to the biosorption of rare earth metals and that it is possible to use the highly absorbent strains to breed a yeast strain that can recover even higher concentrations of Dy

A stereocontrolled construction of 2-azido-2-deoxy-1,2-cis-α-galactosidic linkages utilizing 2-azido-4,6-O-benzylidene-2-deoxygalactopyranosyl diphenyl phosphates: stereoselective synthesis of mucin core 5 and core 7 structures

TMSOTf-promoted glycosidation of 2-azido-4,6-O-benzylidene-2-deoxygalactosyl diphenyl phosphates with fluorenylmethoxycarbonyl (Fmoc)-protected serine and threonine derivatives in THF/Et2O (1:1) gave glycosyl amino acids in high yields and with excellent levels of α-selectivity (α/β=94:6-95:5). The synthetic utility of the present glycosidation method was demonstrated by a stereoselective synthesis of mucin-type glycopeptide core 5 and core 7 building blocks, which are suitable for Fmoc-based solid-phase synthesis of O-glycopeptides

Rapid screening by cell-based fusion assay for identifying novel antivirals of glycoprotein B-mediated herpes simplex virus type 1 infection

金沢大学医薬保健研究域薬学系Herpes simplex virus type 1 (HSV-1) is a causative agent for a variety of diseases. Although antiherpetic drugs such as acyclovir have been developed to inhibit virus replication through interaction with DNA kinases, their continuous administration leads to an increase in the frequency of drug-resistant HSV-1, which is an important clinical issue that requires urgent solution. Recently, we reported that the sialylated O-linked sugar T antigen (sTn) and its attached peptide region (O-glycosylated sTn peptide) derived from the HSV-1 glycoprotein B (gB) protein inhibited HSV-1 infection by specifically targeting paired immunoglobulin-like type 2 receptor alpha (PILRα) in vitro. In this study, to further identify novel inhibitors of gB-mediated HSV-1 infection in vitro, we established a cell-based fusion assay for rapid drug screening. Chinese hamster ovary (CHO) cells were transfected with expression plasmids for HSV-1 gB, gD, gH, and gL, and T7 RNA polymerase, and were designated as the effector cells. The CHO-K1 cells stably expressing PILRα were transfected with the expression plasmid for firefly luciferase under the T7 promoter, and were designated as the target cells. The effector and target cells were co-cultured, and luminescence was measured when both cells were successfully fused. Importantly, we found that cell-to-cell fusion was specifically inhibited by Oglycosylated sTn peptide in a dose dependent manner. Our results suggested that this virus-free cell-based fusion assay system could be a useful and promising approach to identify novel inhibitors of gB-mediated HSV-1 infection, and will aid in the development of antiviral therapeutic strategies for HSV-1-associated diseases. © 2016 The Pharmaceutical Society of Japan

Total Synthesis of Brasilicardins A and C

The first total synthesis of brasilicardins A and C, novel diterpenoid–saccharide–amino acid hybrid metabolites with unique immunosuppressive activity, is described. The key step is a Diels–Alder/reductive angular methylation sequence capitalizing on a <i>trans</i>-fused bicyclic α-cyano-α,β-enone as its precursor to construct the 8,10-dimethyl-<i>trans</i>/<i>syn</i>/<i>trans</i>-perhydrophenanthrene skeleton. Other notable features include an <i>anti</i>-selective aldol reaction, a stereocontrolled glycosylation of a C2 alcohol, and a one-pot, two-step global deprotection sequence that did not damage these sensitive molecules