High body temperature increases gut microbiota-dependent host resistance to influenza A virus and SARS-CoV-2 infection.

Fever is a common symptom of influenza and coronavirus disease 2019 (COVID-19), yet its physiological role in host resistance to viral infection remains less clear. Here, we demonstrate that exposure of mice to the high ambient temperature of 36 °C increases host resistance to viral pathogens including influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). High heat-exposed mice increase basal body temperature over 38 °C to enable more bile acids production in a gut microbiota-dependent manner. The gut microbiota-derived deoxycholic acid (DCA) and its plasma membrane-bound receptor Takeda G-protein-coupled receptor 5 (TGR5) signaling increase host resistance to influenza virus infection by suppressing virus replication and neutrophil-dependent tissue damage. Furthermore, the DCA and its nuclear farnesoid X receptor (FXR) agonist protect Syrian hamsters from lethal SARS-CoV-2 infection. Moreover, we demonstrate that certain bile acids are reduced in the plasma of COVID-19 patients who develop moderate I/II disease compared with the minor severity of illness group. These findings implicate a mechanism by which virus-induced high fever increases host resistance to influenza virus and SARS-CoV-2 in a gut microbiota-dependent manner.

Prognostic value of altered N-glycosylation of circulating glycoproteins in patients with unresectable pancreatic cancer treated with gemcitabine.

OBJECTIVES: The objectives of this study were to examine the whole-serum N-glycan profile of patients with unresectable pancreatic cancer and to evaluate the ability of glycans to predict gemcitabine treatment efficacy and patient survival. METHODS: We collected serum from 52 patients with advanced pancreatic cancer before they began gemcitabine monotherapy. The serum glycan profile was measured through comprehensive quantitative high-throughput glycome analysis and compared with the treatment efficacy and patient survival. RESULTS: Of the 61 glycans detected, the serum levels of glycan 4310 (molecular weight [m/z] 1549.566), 6301 (m/z 2032.724), and 9200 (m/z 2010.692) were high in patients with a short time to tumor progression (TTP). Multivariate analysis revealed that a high glycan 9200 concentration was an independent risk factor for shorter TTP (hazard ratio, 2.11; 95% confidence interval, 1.07-4.17) and poor overall survival (hazard ratio, 2.56; 95% confidence interval, 1.08-6.19). The median TTP of patients with up-regulation of 9200 after gemcitabine treatment was shorter than for the remaining patients (91 vs 301 days; P = 0.0005). A similar relationship was observed for overall survival (median, 181 vs 561 days; P = 0.001). CONCLUSIONS: Glycan 9200 is a possible biomarker predicting gemcitabine efficacy survival in patients with unresectable pancreatic cancer.

Clinical utility of high-throughput glycome analysis in patients with pancreatic cancer.

BACKGROUND: Most of the glycan changes reported in cancers were based on the examinations of a small number of patients or particular proteins. The aim of this study was to determine the changes of the serum N-glycan profile comprehensively in a large number of pancreatic cancer patients and investigate its clinical utility. METHODS: Glycan levels in the serum of 92 pancreatic cancer patients and 243 healthy volunteers (HLT) were examined by comprehensive quantitative high-throughput glycome analysis and were compared with clinical parameters. RESULTS: Out of 66 glycans detected, 15 were differentially expressed in pancreatic cancer, and 10 out of the 15 glycans were significantly up-regulated in cases with distant metastasis. There was a clear increase in overall expression of serum glycans, especially highly-branched glycans with fucose moieties, in pancreatic cancer. Among these 15 glycans, a tri-antennary complex type glycan (m/z 3195) showed the highest area under the receiver operating characteristic curve (AUROC = 0.799) for the diagnosis of pancreatic cancer. The ratio of pairs of glycans on the same path of the biosynthesis pathway (m/z 3195/1914) was found to be significantly higher in pancreatic cancer than in HLT (median = 1.11 and 0.41, respectively; p < 0.0001, AUROC = 0.831). For this pair ratio, the hazard ratio for survival (2.60, 95 % CI = 1.44-4.79) was higher than that of any single glycan and 1-year survival of patients with a high and low ratio was 36.9 and 69.2 %, respectively, (p = 0.001). CONCLUSIONS: Comprehensive glycome analysis can be used to know the presence of pancreatic cancer, distant metastasis, and patient prognosis, simultaneously.

Glycoconjugate Data Bank: Structures--an annotated glycan structure database and N-glycan primary structure verification service.

Glycobiology has been brought to public attention as a frontier in the post-genomic era. Structural information about glycans has been accumulating in the Protein Data Bank (PDB) for years. It has been recognized, however, that there are many questionable glycan models in the PDB. A tool for verifying the primary structures of glycan 3D structures is evidently required, yet there have been no such publicly available tools. The Glycoconjugate Data Bank:Structures (GDB:Structures, http://www.glycostructures.jp) is an annotated glycan structure database, which also provides an N-glycan primary structure (or glycoform) verification service. All the glycan 3D structures are detected and annotated by an in-house program named 'getCARBO'. When an N-glycan is detected in a query coordinate by getCARBO, the primary structure of the glycan is compared with the most similar entry in the glycan primary structure database (KEGG GLYCAN), and unmatched substructure(s) are indicated if observed. The results of getCARBO are stored and presented in GDB:Structures.

The solution structure of horseshoe crab antimicrobial peptide tachystatin B with an inhibitory cystine-knot motif.

Tachystatin B is an antimicrobial and a chitin-binding peptide isolated from the Japanese horseshoe crab (Tachypleus tridentatus) consisting of two isopeptides called tachystatin B1 and B2. We have determined their solution structures using NMR experiments and distance geometry calculations. The 20 best converged structures of tachystatin B1 and B2 exhibited root mean square deviations of 0.46 and 0.49 A, respectively, for the backbone atoms in Cys(4)-Arg(40). Both structures have identical conformations, and they contain a short antiparallel beta-sheet with an inhibitory cystine-knot (ICK) motif that is distributed widely in the antagonists for voltage-gated ion channels, although tachystatin B does not have neurotoxic activity. The structural homology search provided several peptides with structures similar to that of tachystatin B. However, most of them have the advanced functions such as insecticidal activity, suggesting that tachystatin B may be a kind of ancestor of antimicrobial peptide in the molecular evolutionary history. Tachystatin B also displays a significant structural similarity to tachystatin A, which is member of the tachystatin family. The structural comparison of both tachystatins indicated that Tyr(14) and Arg(17) in the long loop between the first and second strands might be the essential residues for binding to chitin.

Computational design and experimental evaluation of glycosyltransferase mutants: engineering of a blood type B galactosyltransferase with enhanced glucosyltransferase activity.

Glycosyltransferases are an enormous and diverse class of enzyme encompassing 1% of all sequenced genomes. They catalyze the transfer of a monosaccharide from an activated donor such as a sugar-nucleotide to an acceptor molecule. Though the primary sequences of glycosyltransferases have little homology, X-ray structural studies on glycosyltransferases have revealed that there are two main folds and that the orientation of the sugar donors with respect to the folds is highly conserved. It seems that glycosyltransferases have evolved diversified specificities toward donor sugars by changing the amino acids around the monosaccharide moiety without altering the orientation of the nucleotide moiety. In this study, we designed new glycosyltransferases with altered donor specificities by use of a novel empirical model called the Epimer Propensity Index (EPI). The EPI was constructed using 221 carbohydrate-protein complex structures in the Protein Data Bank with either galactose or glucose in the complex. The blood type B synthesizing glycosyltransferase GTB, a galactosyltransferase was our target enzyme. Two GTB mutants designed to exhibit enhanced glucosyltransferase activity were cloned, expressed and characterized experimentally. The predicted GTB mutants, Ser185Asn and Ser185Cys, exhibited 4.3- and 4.8-fold elevations in k(cat)/K(m) for UDP-Glc relative to that of wild-type enzyme.

Alternative splicing in human transcriptome: functional and structural influence on proteins.

Alternative splicing is a molecular mechanism that produces multiple proteins from a single gene, and is thought to produce variety in proteins translated from a limited number of genes. Here we analyzed how alternative splicing produced variety in protein structure and function, by using human full-length cDNAs on the assumption that all of the alternatively spliced mRNAs were translated to proteins. We found that the length of alternatively spliced amino acid sequences, in most cases, fell into a size shorter than that of average protein domain. We evaluated comprehensively the presumptive three-dimensional structures of the alternatively spliced products to assess the impact of alternative splicing on gene function. We found that more than half of the products encoded proteins which were involved in signal transduction, transcription and translation, and more than half of alternatively spliced regions comprised interaction sites between proteins and their binding partners, including substrates, DNA/RNA, and other proteins. Intriguingly, 67% of the alternatively spliced isoforms showed significant alterations to regions of the protein structural core, which likely resulted in large conformational change. Based on those findings, we speculate that there are a large number of cases that alternative splicing modulates protein networks through significant alteration in protein conformation.

Glycoinsulins: dendritic sialyloligosaccharide-displaying insulins showing a prolonged blood-sugar-lowering activity.

Mono-, di-, and trisialyloligosaccharides were introduced to mutant insulins through enzymatic reactions. Sugar chains were sialylated by alpha2,6-sialyltransferase (alpha2,6-SiaT) via an accessible glutamine residue at the N-terminus of the B-chain attached by transglutaminase (TGase). Sia2,6-di-LacNAc-Ins(B-F1Q) and Sia2,6-tri-LacNAc-Ins(B-F1Q), displaying two and three sialyl-N-acetyllactosamines, respectively, were administered to hyperglycemic mice. Both branched glycoinsulins showed prolonged glucose-lowering effects compared to native or lactose-carrying insulins, showing that sialic acid is important in obtaining a prolonged effect. Sia2,6-tri-LacNAc-Ins(B-F1Q), in particular, induced a significant delay in the recovery of glucose levels.