Microbial glycosphingolipid (GSL)-degrading enzymes with unique specificity are useful tools for GSL research. On the other hand, some microbial glycolipids, not only GSLs but also steryl glucosides, are closely related to pathogenicity, and, thus, the metabolism of microbial glycolipids is attracting attention as a target for antibiotics. This chapter describes the assays and utilization of microbial enzymes useful for glycolipid research and those involved in pathogenicity or host immune reactions.
View Article and Find Full Text PDFSecretogranin III (SgIII) is a member of the chromogranin/secretogranin family of neuroendocrine secretory proteins. Granins are expressed in endocrine and neuroendocrine cells and subsequently processed into bioactive hormones. Although granin-derived peptide expression is correlated with neuroendocrine carcinomas, little is known about SgIII.
View Article and Find Full Text PDFThe significance of glycomic profiling has been highlighted by recent findings that structural changes of glycans are observed in many diseases, including cancer. Therefore, glycomic profiling of the whole body (glycome mapping) under different physiopathological states may contribute to the discovery of reliable biomarkers with disease-specific alterations. To achieve this, standardization of high-throughput and in-depth analysis of tissue glycome mapping is needed.
View Article and Find Full Text PDFThe human placental transfer of maternal IgG is crucial for fetal and newborn immunity. Low-affinity immunoglobulin gamma Fc region receptor IIb2 (FCGR2B2 or FcγRIIb2) is exclusively expressed in an IgG-containing, vesicle-like organelle (the FCGR2B2 compartment) in human placental endothelial cells; thus, we hypothesized that the FCGR2B2 compartment functions as an IgG transporter. In this study, to examine this hypothesis, we performed in vitro bio-imaging analysis of IgG trafficking by FCGR2B2 compartments using human umbilical vein endothelial cells transfected with a plasmid vector containing enhanced GFP-tagged FCGR2B2 (pFCGR2B2-EGFP).
View Article and Find Full Text PDFFrontal affinity chromatography (FAC) is a simple and versatile procedure enabling quantitative determination of diverse biological interactions in terms of dissociation constants (K d), even though these interactions are relatively weak. The method is best applied to glycans and their binding proteins, with the analytical system operating on the basis of highly reproducible isocratic elution by liquid chromatography. Its application to galectins has been successfully developed to characterize their binding specificities in detail.
View Article and Find Full Text PDFHistopathological classification of lung cancer has important implications in the application of clinical practice guidelines and the prediction of patient prognosis. Thus, we focused on discovering glycobiomarker candidates to classify the types of lung cancer tissue. First, we performed lectin microarray analysis of lung cancer tissue specimens and cell lines and identified Aleuria aurantia lectin (AAL), Hippeastrum hybrid lectin (HHL), and Concanavalia ensiformis agglutinin (ConA) as lectin probes specific to non-small cell lung carcinoma (NSCLC).
View Article and Find Full Text PDFAim: The microRNAs (miRNAs) derived from the chromosome 19 miRNA cluster (C19MC) are exclusively expressed in the human placenta, but the origin and functions of C19MC miRNAs are not fully understood. The purpose of this study was to elucidate which cells express C19MC miRNAs in chorionic villi and identify their miRNA targets.
Methods: A combination of laser microdissection (LMD) and real-time polymerase chain reaction (PCR) to examine the localization of five C19MC miRNAs (i.
MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells.
View Article and Find Full Text PDFGalactoseβ1-4Fucose (Galβ1-4Fuc) is a unique disaccharide exclusively found in N-glycans of protostomia, and is recognized by some galectins of Caenorhabditis elegans and Coprinopsis cinerea. In the present study, we investigated whether mammalian galectins also bind such a disaccharide. We examined sugar-binding ability of human galectin-1 (hGal-1) and found that hGal-1 preferentially binds Galβ1-4Fuc compared to Galβ1-4GlcNAc, which is its endogenous recognition unit.
View Article and Find Full Text PDFAminoacyl-tRNA synthetases play a key role in the translation of genetic code into correct protein sequences. These enzymes recognize cognate amino acids and tRNAs from noncognate counterparts, and catalyze the formation of aminoacyl-tRNAs. While Although several tyrosyl-tRNA synthetases (TyrRSs) from various species have been structurally and functionally well characterized, the crenarchaeal TyrRS remains poorly understood.
View Article and Find Full Text PDFBackground: Galectins form a large family of animal lectins, individual members having variously divergent carbohydrate-recognition domains (CRDs) responsible for extensive physiological phenomena. Sugar-binding affinities of galectins were previously investigated by us using frontal affinity chromatography (FAC) with a relatively small set (i.e.
View Article and Find Full Text PDFBiochem Biophys Res Commun
June 2009
Previously, we developed an alpha2-6-sialic acid (Sia)-specific lectin (SRC) starting from an R-type galactose-specific lectin C-terminal domain. However, it showed relatively low affinity because of its monovalency. Here, we engineered a tandem repeat construct (SRC2) showing substantial affinity for alpha2,6-sialylated N-glycans (in the order of 10(-6)M in K(d)), almost comparable to a natural alpha2-6Sia-specific lectin from Sambucus sieboldiana (SSA).
View Article and Find Full Text PDFGalectin-1 (Gal-1), a member of the beta-galactoside-binding animal lectin family, has a wide range of biological activities, which makes it an attractive target for medical applications. Unlike other galectins, Gal-1 is susceptible to oxidation at cysteine residues, which is troublesome for in vitro/vivo studies. To overcome this problem, we prepared a cysteine-less mutant of Gal-1 (CSGal-1) by substituting all cysteine residues with serine residues.
View Article and Find Full Text PDFThe extensive involvement of glycan-binding proteins (GBPs) as regulators in diverse biological phenomena provides a fundamental reason to investigate their glycan-binding specificities. Here, we developed a glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of GBPs. Eighty-nine selected multivalent glycoconjugates comprising natural glycoproteins, neo-glycoproteins, and polyacrylamide (PAA)-conjugated glycan epitopes were immobilized on an epoxy-activated glass slide.
View Article and Find Full Text PDFGalectins, a group of beta-galactoside-binding lectins, are involved in multiple functions through specific binding to their oligosaccharide ligands. No previous work has focused on their interaction with glycosaminoglycans (GAGs). In the present work, affinities of established members of human galectins toward a series of GAGs were investigated, using frontal affinity chromatography.
View Article and Find Full Text PDFTo investigate the recognition sites of histidine tRNA for histidyl-tRNA synthetase from an extreme hyperthermophilic archaeon, Aeropyrum pernix K1, we examined histidylation activities by using overexpressed histidyl-tRNA synthetase and various histidine tRNA transcripts that were prepared by in vitro transcription system. Results indicated that anticodon was not recognized by the histidyl-tRNA synthetase similar to that of Escherichia coli histidine tRNA recognition system. Discriminator base C73 was weekly recognized and an additional G residue was specifically recognized by the enzyme.
View Article and Find Full Text PDFActa Crystallogr Sect F Struct Biol Cryst Commun
November 2005
Hyperthermophilic archaeal tyrosyl-tRNA synthetase from Aeropyrum pernix K1 was cloned and overexpressed in Escherichia coli. The expressed protein was purified by Cibacron Blue affinity chromatography following heat treatment at 363 K. Crystals suitable for X-ray diffraction studies were obtained under optimized crystallization conditions in the presence of 1.
View Article and Find Full Text PDFNucleic Acids Res Suppl
September 2003
Recognition sites of tyrosine tRNA for tyrosyl-tRNA synthetase from Escherichia coli and extreme thermophilic archaeon, Aeropyrum pernix K1 were examined using various in vitro transcripts. With respect to the long variable arm in E. coli tyrosine tRNA, some base pairs in length was required for tyrosylation.
View Article and Find Full Text PDFTo investigate the recognition sites of tRNA(Thr) for threonyl-tRNA synthetase (ThrRS) from an extreme thermophilic and aerobic archaeon, Aeropyrum pernix K1, threonylation experiments using various in vitro mutant transcripts of tRNA(Thr) were examined. The results indicated that A. pernix ThrRS did recognize the first three base pairs of acceptor stem in addition to the second and the third letters of anticodon of tRNA(Thr), in spite of its N-terminal truncated unique structure.
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