Assays and Utilization of Enzymes Involved in Glycolipid Metabolism in Bacteria and Fungi.

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.

Glycobiomarker, Fucosylated Short-Form Secretogranin III Levels Are Increased in Serum of Patients with Small Cell Lung Carcinoma.

Secretogranin 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. We previously identified SgIII by a comparative glycoproteomics approach for elucidation of glycobiomarker candidates in lung carcinoma. Here, we examined the expression, secretion, and glycosylation of SgIII to identify novel biomarkers of small cell lung carcinoma (SCLC). In comparative immunohistochemical analysis and secretion profiling, SgIII was observed in all types of lung cancer. However, low-molecular-weight SgIII (short-form SgIII) was specifically found in SCLC culture medium. Glycoproteomics analysis showed that a fucosylated glycan was attached to the first of three potential N-glycosylation sites and an unfucosylated glycan was detected on the second site; however, the third site was not glycosylated. Next, we performed lectin capture with a fucose-binding lectin and detected short-form SgIII specifically in the sera of patients with SCLC. The results suggested an association between the fucosylated glycoform of short-form SgIII and SCLC. Thus, fucosylated short-form SgIII may be a valuable biomarker for SCLC and could be used to monitor development of the disease. All MS data are available via ProteomeXchange and jPOST with identifiers PXD007626 and JPST000313, respectively.

A standardized method for lectin microarray-based tissue glycome mapping.

The 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. However, this is a great challenge due to the lack of analytical methodology for glycans on small amounts of endogenous glycoproteins. Here, we established a standardized method of lectin-assisted tissue glycome mapping. Formalin-fixed, paraffin-embedded tissue sections were prepared from brain, liver, kidney, spleen, and testis of two C57BL/6J mice. In total, 190 size-adjusted fragments with different morphology were serially collected from each tissue by laser microdissection and subjected to lectin microarray analysis. The results and subsequent histochemical analysis with selected lectins were highly consistent with previous reports of mass spectrometry-based N- and/or O-glycome analyses and histochemistry. This is the first report to look at both N- and O-glycome profiles of various regions within tissue sections of five different organs. This simple and reproducible mapping approach is also applicable to various disease model mice to facilitate disease-related biomarker discovery.

Fc gamma receptor IIb participates in maternal IgG trafficking of human placental endothelial cells.

The 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). FCGR2B2-EGFP signals were detected as intracellular vesicular structures similar to FCGR2B2 compartments in vivo. The internalization and transcytosis of IgG was significantly higher in the pFCGR2B2-EGFP-transfected cells than in the mock-transfected cells, and the majority of the internalized IgG was co-localized with the FCGR2B2-EGFP signals. Furthermore, we isolated FCGR2B2 compartments from the human placenta and found that the Rab family of proteins [RAS-related protein Rab family (RABs)] were associated with FCGR2B2 compartments. Among the RABs, RAB3D was expressed predominantly in placental endothelial cells. The downregulation of RAB3D by small interfering RNA (siRNA) resulted in a marked reduction in the FCGR2B2-EGFP signals at the cell periphery. Taken together, these findings suggest that FCGR2B2 compartments participate in the transcytosis of maternal IgG across the human placental endothelium and that RAB3D plays a role in regulating the intracellular dynamics of FCGR2B2 compartments.

Evaluation of galectin binding by frontal affinity chromatography (FAC).

Frontal 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. As a result, their minimal requirements for recognition of disaccharides, i.e., ß-galactosides, as well as characteristic features of individual galectins, have been elucidated. In this chapter, we describe standard procedures to determine the K d's for interactions between a series of standard glycans and various galectins.

Glycoproteomics approach for identifying Glycobiomarker candidate molecules for tissue type classification of non-small cell lung carcinoma.

Histopathological 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). LC-MS-based analysis was performed for the comprehensive identification of glycoproteins and N-linked glycosylation sites using lectin affinity capture of NSCLC-specific glycoforms of glycoproteins. This analysis identified 1092 AAL-bound glycoproteins (316 gene symbols) and 948 HHL/ConA-bound glycoproteins (279 gene symbols). The lectin microarray-assisted verification using 15 lung cancer cell lines revealed the NSCLC-specific expression of fibronectin. The glycosylation profiling of fibronectin indicated that the peanut agglutinin (PNA) signal appeared to differentiate two NSCLC types, adenocarcinoma and large cell carcinoma, whereas the protein expression level was similar between these types. Our glycoproteomics approach together with the concurrent use of an antibody and lectin is applicable to the quantitative and qualitative monitoring of variations in glycosylation of fibronectin specific to certain types of lung cancer tissue.

Placenta-specific miRNA (miR-512-3p) targets PPP3R1 encoding the calcineurin B regulatory subunit in BeWo cells.

AIM: 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.e. miR-512-3p, miR-518b, miR-520a, miR-524 and miR-1323) in the human placenta was performed. Furthermore, to identify miR-512-3p-target genes, we analyzed gene expression profiles of the trophoblast cell line BeWo using a DNA microarray. Predicted target genes were validated by real-time PCR, western blotting, and 3'-untranslated region reporter assay. RESULTS: By LMD and subsequent PCR analysis, five C19MC miRNAs examined in this study were predominantly expressed in villous trophoblast cells; little expression, if any, was observed in villous stroma cells or fetal endothelial cells. Microarray data showed that 334 genes were downregulated in BeWo cells treated with Pre-miR-512-3p (mature miR-512-3p mimic). We found six candidate target genes of miR-512-3p using DNA microarray data and target prediction software. Furthermore, we revealed that protein phosphatase 3, regulatory subunit B, alpha (PPP3R1), one of the six genes, was a miR-512-3p target using an in vitro experimental validation system. CONCLUSION: These data suggest that miR-512-3p participates in human trophoblast function[s] by targeting PPP3R1, encoding a regulatory subunit of calcineurin.

MiR-376c down-regulation accelerates EGF-dependent migration by targeting GRB2 in the HuCCT1 human intrahepatic cholangiocarcinoma cell line.

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. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3'-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.

Mammalian galectins bind galactoseß1-4fucose disaccharide, a unique structural component of protostomial N-type glycoproteins.

Galactoseß1-4Fucose (Galß1-4Fuc) 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 Galß1-4Fuc compared to Galß1-4GlcNAc, which is its endogenous recognition unit. We also tested other human and mouse galectins, i.e., hGal-3, and -9 and mGal-1, 2, 3, 4, 8, and 9. All of them also showed substantial affinity to Galß1-4Fuc disaccharide. Further, we assessed the inhibitory effect of Galß1-4Fuc, Galß1-4Glc, and Gal on the interaction between hGal-1 and its model ligand glycan, and found that Galß1-4Fuc is the most effective. Although the biological significance of galectin-Galß1-4Fuc interaction is obscure, it might be possible that Galß1-4Fuc disaccharide is recognized as a non-self-glycan antigen. Furthermore, Galß1-4Fuc could be a promising seed compound for the synthesis of novel galectin inhibitors.

Studies on crenarchaeal tyrosylation accuracy with mutational analyses of tyrosyl-tRNA synthetase and tyrosine tRNA from Aeropyrum pernix.

Aminoacyl-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. In this study, we performed mutational analyses on tyrosine tRNA (tRNA(Tyr)) and TyrRS from the crenarchaeon, Aeropyrum pernix, to investigate the molecular recognition mechanism. Kinetics for tyrosylation using in vitro transcript indicated that the discriminator base A73 and adjacent G72 in the acceptor stem are identity elements of tRNA(Tyr), whereas the C1 base and anticodon had modest roles as identity determinants. Intriguingly, in contrast to the identity element of eukaryotic/euryarchaeal TyrRSs, the first base-pair (C1-G72) of the acceptor stem was not essential in crenarchaeal TyrRS as a pair. Furthermore, A. pernix TyrRS mutants were constructed at positions Tyr39 and Asp172, which could form hydrogen bonds with the 4-hydroxyl group of l-tyrosine. The tyrosylation activities with the mutants resulted that Asp172 mutants completely abolished tyrosylation activity, whereas Tyr39 mutants had no effect on activity. Thus, crenarchaeal TyrRS appears to adopt different molecular recognition mechanism from other TyrRSs.

The Galß-(syn)-gauche configuration is required for galectin-recognition disaccharides.

BACKGROUND: 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., 41) of oligosaccharides. However, total understanding of a consensus rule for galectin-recognition saccharides is still hampered by the lack of fundamental knowledge about their sugar-binding specificity toward a much larger panel of oligosaccharides in terms of dissociation constant (K(d)). METHODS: In the present study, we extended a FAC analysis from a more systematic viewpoint by using 142 fluorescent-labeled oligosaccharides, initially with focus on functional human galectins-1-9. Binding characteristics were further validated with 11 non-human galectins and 13 non-galectin Gal/GalNAc-binding lectins belonging to different families. RESULTS: An empirical [Galß-equatorial] rule for galectin-recognition disaccharides was first derived by our present research and previous works by others. However, this rule was not valid for a recently reported nematode disaccharide, "Galß1-4-L-Fuc" [Butschi et al. PLoS Pathog, 2010; 6(1):e1000717], because this glycosidic linkage was directed to 'axial' 4-OH of L-Fuc. After careful reconsideration of the structural data, we reached an ultimate rule of galectin-recognition disaccharides, which all of the galectins so far identified fulfilled, i.e., under the re-defined configuration "Galß-(syn)-gauche". The rule also worked perfectly for differentiation of galectins from other types of lectins. GENERAL SIGNIFICANCE: The present attempt should provide a basis to solve the riddle of the glyco-code as well as to develop therapeutic inhibitors mimicking galectin ligands.

Engineering a versatile tandem repeat-type alpha2-6sialic acid-binding lectin.

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). Notably, its binding to branched N-glycans was found to be more selective than SSA. Nevertheless, SRC2 showed no apparent hemagglutinating activity, while it exerted strong erythrocyte-binding activity. This unique feature will help flow cytometry analysis, where usual lectins including SSA agglutinate cells. Some other biochemical properties investigated for SRC2, e.g., high productivity in bacteria and easy release of captured glycoproteins with lactose have demonstrated versatility of this mutant protein as a powerful tool for sialoglycomics.

Functional and structural bases of a cysteine-less mutant as a long-lasting substitute for galectin-1.

Galectin-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. In the case of wild-type Gal-1, the formation of covalent dimers/oligomers was evident after 10 days of storage in the absence of a reducing agent with a concomitant decrease in hemagglutination activity, while CSGal-1 did not form multimers and retained full hemagglutination activity after 400 days of storage. Frontal affinity chromatography showed that the sugar-binding specificity and affinity of Gal-1 for model glycans were barely affected by the mutagenesis. Gal-1 is known to induce cell signaling leading to an increase in the intracytoplasmic calcium concentration and to cell death. CSGal-1 is also capable of inducing calcium flux and growth inhibition in Jurkat cells, which are comparable to or more potent than those induced by Gal-1. The X-ray structure of the CSGal-1/lactose complex has been determined. The structure of CSGal-1 is almost identical to that of wild-type human Gal-1, showing that the amino acid substitutions do not affect the overall structure or carbohydrate-binding site structure of the protein. These results indicate that CSGal-1 can serve as a stable substitute for Gal-1.

Glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of glycan-binding proteins.

The 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. The GBP binding was monitored by an evanescent-field fluorescence-assisted scanner at equilibrium without washing steps. The detection principle also allows direct application of unpurified GBPs with the aid of specific antibodies. Model experiments using plant lectins (RCA120, ConA, and SNA), galectins (3 and 8), a C-type lectin (DC-SIGN) and a siglec (CD22) provided data consistent with previous work within 4 h using less than 40 ng of GBPs per analysis. As an application, serum profiling of antiglycan antibodies (IgG and IgM) was performed with Cy3-labeled secondary antibodies. Moreover, novel carbohydrate-binding ability was demonstrated for a human IL-18 binding protein. Thus, the developed glycan array is useful for investigation of various types of GBPs, with the added advantage of wash-free analysis.

Desulfated galactosaminoglycans are potential ligands for galectins: evidence from frontal affinity chromatography.

Galectins, 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. Structurally-defined keratan sulfate (KS) oligosaccharides showed significant affinity to a wide range of galectins if Gal residue(s) remained unsulfated, while GlcNAc sulfation had relatively little effect. Consistently, galectins showed much higher affinity to corneal type I than cartilageous type II KS. Unexpectedly, galectin-3, -7, and -9 also exerted significant affinity to desulfated, GalNAc-containing GAGs, i.e., chondroitin and dermatan, but not at all to hyaluronan and N-acetylheparosan. These observations revealed that the integrity of 6-OH of betaGalNAc is important for galectin recognition of these galactosaminoglycans, which were shown, for the first time, to be implicated as potential ligands of galectins.

Molecular recognition of histidine tRNA by histidyl-tRNA synthetase from hyperthermophilic archaeon, Aeropyrum pernix K1.

To 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.

Overexpression, purification and crystallization of tyrosyl-tRNA synthetase from the hyperthermophilic archaeon Aeropyrum pernix K1.

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 1.5 M ammonium sulfate using the hanging-drop vapour-diffusion method. The crystals belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 66.1, c = 196.2 A, and diffracted to beyond 2.15 A resolution at 100 K.

Molecular recognition of threonine tRNA by threonyl-tRNA synthetase from an extreme thermophilic archaeon, Aeropyrum pernix K1.

To 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. Discriminator base was not involved in recognition by A. pernix ThRS. These determinants were confirmed by the identity switching experiments from the in vitro mutants of A. pernix tRNA(Pro) and tRNA(Asn).

Differences in tyrosine tRNA identity between Escherichia coli and archaeon, Aeropyrum pernix K1.

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. None of the recognition sites were found in the acceptor stem, except the discriminator base A73 in E. coli tyrosine tRNA. In case of A. pernix tyrosine tRNA, C1-G72 base pair and discriminator base A73 in the acceptor region as well as anticodon were base specifically involved in tyrosylation by A. pernix tyrosyl-tRNA synthetase.