Growth of Bifidobacterium pseudocatenulatum in medium containing N-acetylsucrosamine: enzyme that induces the growth of this bacterium via degradation of this disaccharide.

Bifidobacterium pseudocatenulatum grows well in the early stages of cultivation in medium containing sucrose (Suc), whereas its growth in medium containing the analogue disaccharide N-acetylsucrosamine (SucNAc) tends to exhibit a considerable delay. To elucidate the cause of this phenomenon, we investigated the proliferation pattern of B. pseudocatenulatum in medium containing D-glucose (Glc) and SucNAc and identified the enzyme that degrades this disaccharide. We found that B. pseudocatenulatum initially proliferates by assimilating Glc, with subsequent growth based on SucNAc assimilation depending on production of the ß-fructofuranosidase, which can hydrolyze SucNAc, after Glc is completely consumed. Thus, B. pseudocatenulatum exhibited a diauxic growth pattern in medium containing Glc and SucNAc. In contrast, when cultured in medium containing Glc and Suc, B. pseudocatenulatum initially grew by degrading Suc via the phosphorolysis activity of Suc phosphorylase, which did not react to SucNAc. These observations indicate that B. pseudocatenulatum proliferates by assimilating Suc and SucNAc via different pathways. The ß-fructofuranosidase of B. pseudocatenulatum exhibited higher hydrolytic activity against several naturally occurring Suc-based tri- or tetrasaccharides than against Suc, suggesting that this enzyme actively catabolizes oligosaccharides other than Suc.

A novel Golgi mannosidase inhibitor: Molecular design, synthesis, enzyme inhibition, and inhibition of spheroid formation.

Effective chemotherapy for solid cancers is challenging due to a limitation in permeation that prevents anticancer drugs from reaching the center of the tumor, therefore unable to limit cancer cell growth. To circumvent this issue, we planned to apply the drugs directly at the center by first collapsing the outer structure. For this, we focused on cell-cell communication (CCC) between N-glycans and proteins at the tumor cell surface. Mature N-glycans establish CCC; however, CCC is hindered when numerous immature N-glycans are present at the cell surface. Inhibition of Golgi mannosidases (GMs) results in the transport of immature N-glycans to the cell surface. This can be employed to disrupt CCC. Here, we describe the molecular design and synthesis of an improved GM inhibitor with a non-sugar mimic scaffold that was screened from a compound library. The synthesized compounds were tested for enzyme inhibition ability and inhibition of spheroid formation using cell-based methods. Most of the compounds designed and synthesized exhibited GM inhibition at the cellular level. Of those, AR524 had higher inhibitory activity than a known GM inhibitor, kifunensine. Moreover, AR524 inhibited spheroid formation of human malignant cells at low concentration (10 µM), based on the disruption of CCC by GM inhibition.

Screening, Synthesis, and Evaluation of Novel Isoflavone Derivatives as Inhibitors of Human Golgi ß-Galactosidase.

The genes GLB1 and GALC encode GLB1 isoform 1 and galactocerebrosidase, respectively, which exhibit ß-galactosidase activity in human lysosomes. GLB1 isoform 1 has been reported to play roles in rare lysosomal storage diseases. Further, its ß-galactosidase activity is the most widely used biomarker of senescent and aging cells; hence, it is called senescence-associated ß-galactosidase. Galactocerebrosidase plays roles in Krabbe disease. We previously reported a novel ß-galactosidase activity in the Golgi apparatus of human cells; however, the protein responsible for this activity could not be identified. Inhibitor-derived chemical probes can serve as powerful tools to identify the responsible protein. In this study, we first constructed a cell-based high-throughput screening (HTS) system for Golgi ß-galactosidase inhibitors, and then screened inhibitors from two compound libraries using the HTS system, in vitro assay, and cytotoxicity assay. An isoflavone derivative was identified among the final Golgi ß-galactosidase inhibitor compound hits. Molecular docking simulations were performed to redesign the isoflavone derivative into a more potent inhibitor, and six designed derivatives were then synthesized. One of the derivatives, ARM07, exhibited potent inhibitory activity against ß-galactosidase, with an IC50 value of 14.8 µM and competitive inhibition with Ki value of 13.3 µM. Furthermore, the in vitro and cellular inhibitory activities of ARM07 exceeded those of deoxygalactonojirimycin. ARM07 may contribute to the development of affinity-based chemical probes to identify the protein responsible for the newly discovered Golgi ß-galactosidase activity. The therapeutic relevance of ARM07 against lysosomal storage diseases and its effect on senescent cells should be evaluated further.

Development of Specific Fluorogenic Substrates for Human ß-N-Acetyl-D-hexosaminidase A for Cell-Based Assays.

Inhibitors of human ß-N-acetyl-D-hexosaminidase (hHEX) A and human O-GlcNAcase (hOGA) reportedly play roles in multiple diseases, suggesting their potential for pharmacological chaperone (PC) therapy of Sandhoff disease (SD) and Tay-Sachs disease (TSD), as lysosomal storage diseases, and Alzheimer's disease and progressive supranuclear palsy, respectively. In particular, hHEXA inhibitors as PCs have been shown to successfully enhance hHEXA levels, leading to the chronic form of SD and TSD. In the diagnosis of enzyme deficiencies in SD and TSD, artificial hHEXA substrates based on 4-methylumbelliferone as a fluorophore are available and generally used; however, they do not have sufficient performance to screen for potential inhibitors for a PC therapy from compound libraries. Further, there are currently few fluorogenic substrates for hHEXA suitable for such requirements and there are no substrates ideal for cell-based inhibitor screening. Here, we clarified the difference in enzyme active site structure between hHEXA and hOGA from their tertiary structures. To develop lysosome-localized hHEXA-specific fluorogenic substrates based on the difference in their active site structures, our developed quinone methide cleavage substrate design platform was applied for the molecular design of substrates. Thereafter, we synthesized via the shortest route and evaluated novel three-color fluorogenic substrates for hHEXA that exhibited excellent specificity and sensitivity in three human cell lines. The designed substrates represent the first-in-a class of new substrates that can be utilized to screen hHEXA inhibitors in adherent human cultured cells.

Chitin Heterodisaccharide, Released from Chitin by Chitinase and Chitin Oligosaccharide Deacetylase, Enhances the Chitin-Metabolizing Ability of Vibrio parahaemolyticus.

Vibrio parahaemolyticus RIMD2210633 secretes both chitinase and chitin oligosaccharide deacetylase and produces ß-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) from chitin. Previously, we reported that GlcNAc-GlcN induces chitinase production by several strains of Vibrio harboring chitin oligosaccharide deacetylase genes (T. Hirano, K. Kadokura, T. Ikegami, Y. Shigeta, et al., Glycobiology 19:1046-1053, 2009). The metabolism of chitin by Vibrio was speculated on the basis of the findings of previous studies, and the role of chitin oligosaccharide produced from chitin has been well studied. However, the role of GlcNAc-GlcN in the Vibrio chitin degradation system, with the exception of the above-mentioned function as an inducer of chitinase production, remains unclear. N,N'-Diacetylchitobiose, a homodisaccharide produced from chitin, is known to induce the expression of genes encoding several proteins involved in chitin metabolism in Vibrio strains (K. L. Meibom, X. B. Li, A. Nielsen, C. Wu, et al., Proc Natl Acad Sci U S A 101:2524-2529, 2004). We therefore hypothesized that GlcNAc-GlcN also affects the expression of enzymes involved in chitin metabolism in the same manner. In this study, we examined the induction of protein expression by several sugars released from chitin using peptide mass fingerprinting and confirmed the expression of genes encoding enzymes involved in chitin metabolism using real-time quantitative PCR analysis. We then confirmed that GlcNAc-GlcN induces the expression of genes encoding many soluble enzymes involved in chitin degradation in Vibrio parahaemolyticus Here, we demonstrate that GlcNAc-GlcN enhances the chitin-metabolizing ability of V. parahaemolyticusIMPORTANCE We demonstrate that ß-N-acetyl-d-glucosaminyl-(1,4)-d-glucosamine (GlcNAc-GlcN) enhances the chitin-metabolizing ability of V. parahaemolyticus Members of the genus Vibrio are chitin-degrading bacteria, and some species of this genus are associated with diseases affecting fish and animals, including humans (F. L. Thompson, T. Iida, and J. Swings, Microbiol Mol Biol Rev 68:403-431, 2004; M. Y. Ina-Salwany, N. Al-Saari, A. Mohamad, F.-A. Mursidi, et al., J Aquat Anim Health 31:3-22, 2019). Studies on Vibrio are considered important, as they may facilitate the development of solutions related to health, food, and aquaculture problems attributed to this genus. This report enhances the current understanding of chitin degradation by Vibrio bacteria.

Design and synthesis of cell-permeable fluorescent nitrilotriacetic acid derivatives.

Fluorescence labeling of the target molecules using a small molecule-based probe is superior than a method using genetically expressed green fluorescence protein (GFP) in terms of convenience in its preparation and functionalization. Fluorophore-nitrilotriacetic acid (NTA) conjugates with several ester protecting groups were synthesized and evaluated for their cell membrane permeability by fluorescence microscopy analysis. One of the derivatives, acetoxymethyl (AM)-protected NTA conjugate is hydrolyzed, resulting in intracellular accumulation, thus providing localized fluorescence intensity in cells. This modification is expected as an effective method for converting a non-cell membrane permeable NTA-BODIPY conjugates to a cell membrane permeable derivatives.

Identification of Small-Molecule Inhibitors of Human Golgi Mannosidase via a Drug Repositioning Screen.

Three Golgi mannosidases (GMs), namely Golgi α-mannosidases IA, IB, and IC, remove mannose residues from N-glycans and regulate the quality control and transportation of nascent proteins. GM inhibitors regulate several biological events such as cell-cell communication, differentiation, and apoptosis in cancer cells. As a result, GM inhibitor-based therapies have gained significant attention for cancer treatment. However, to date, no GM inhibitor has been approved and none is in clinical development for anti-cancer treatment. Meanwhile, drug repositioning plays an important role in identifying potential inhibitors that vary in molecular structure and properties to bypass much of the early cost and time. We performed a drug repositioning screen of a compound library that included approved drugs. The estrogen receptor antagonists tamoxifen and raloxifene inhibited human GMs at the cellular level. Sulindac, a nonsteroidal anti-inflammatory drug, also inhibited GMs. Our results demonstrated the efficacy of this screening strategy and revealed lead compounds for anti-cancer drug development.

In vivo programming of endogenous antibodies via oral administration of adaptor ligands.

Vaccination is a reliable method of prophylaxis and a crucial measure for public health. However, the majority of vaccines cannot be administered orally due to their degradation in the harsh gut environment or inability to cross the GI tract. In this study, we report the first proof-of-concept study of orally producible chemically programmed antibodies via specific conjugation of adaptor ligands to endogenous antibodies, in vivo. Pre-immuniztion with 2,4-dinitrophenyl (DNP), or the reactive hapten, 1,3-diketone (DK), or a novel reactive hapten, vinyl sulfone (VS) in mice, followed by oral administration of adaptor ligands composed of the hapten and biotin to the pre-immunized mice resulted in successful in vivo formation of the biotin-hapten-antibody complexes within 2h. Pharmacokinetic evaluations revealed that apparent serum concentrations of programmed antibodies were up to 144nM and that the serum half-lives reached up to 34.4h. These findings show promise for the future development of orally bioavailable drug-hapten-antibody complexes asa strategy to quickly and easily modulate immune targets for aggressive pathogens as well as cancer.

Chitin oligosaccharide deacetylase from Shewanella baltica ATCC BAA-1091.

Chitin oligosaccharide deacetylase (COD) from bacteria that have been examined so far typically comprise two carbohydrate-binding domains (CBDs) and one polysaccharide deacetylase domain. In contrast, Shewanella baltica ATCC BAA-1091 COD (Sb-COD) has only one CBD, yet exhibits chitin-binding properties and substrate specificities similar to those of other CODs.

Discovery of human Golgi ß-galactosidase with no identified glycosidase using a QMC substrate design platform for exo-glycosidase.

Post-translational modifications (PTMs) of proteins play important roles in the physiology of eukaryotes. In the PTMs, non-reversible glycosylations are classified as N-glycosylations and O-glycosylations, and are catalyzed by various glycosidases and glycosyltransferases. However, ß-glycosidases are not known to play a role in N- and O-glycan processing, although both glycans provide partial structures as substrates for ß-galactosidase and ß-N-acetylglucosaminidase in the Golgi apparatus of human cells. We explored human Golgi ß-galactosidase using fluorescent substrates based on a quinone methide cleavage (QMC) substrate design platform that was previously developed to image exo-type glycosidases in living cells. As a result, we discovered a novel Golgi ß-galactosidase in human cells. It is possible to predict a novel and important function in glycan processing of this ß-galactosidase, because various ß-galactosyl linkages in N- and O-glycans exist in Golgi apparatus. In addition, these results show that the QMC platform is excellent for imaging exo-type glycosidases.

Enzymatic synthesis of novel oligosaccharides from N-acetylsucrosamine and melibiose using Aspergillus niger α-galactosidase, and properties of the products.

Two kinds of oligosaccharides, N-acetylraffinosamine (RafNAc) and N-acetylplanteosamine (PlaNAc), were synthesized from N-acetylsucrosamine and melibiose using the transgalactosylation activity of Aspergillus niger α-galactosidase. RafNAc and PlaNAc are novel trisaccharides in which d-glucopyranose residues in raffinose (Raf) and planteose are replaced with N-acetyl-d-glucosamine. These trisaccharides were more stable in acidic solution than Raf. RafNAc was hydrolyzed more rapidly than Raf by α-galactosidase of green coffee bean. In contrast, RafNAc was not hydrolyzed by Saccharomyces cerevisiae invertase, although Raf was hydrolyzed well by this enzyme. These results indicate that the physicochemical properties and steric structure of RafNAc differ considerably from those of Raf.

Identification of a novel glycan processing enzyme with exo-acting ß-allosidase activity in the Golgi apparatus using a new platform for the synthesis of fluorescent substrates.

The majority of eukaryotic proteins undergo post-translational modifications (PTMs) involving the attachment of complex glycans, predominantly through N-glycosylation and O-glycosylation. PTMs play important roles in virtually all cellular processes, and aberrant regulation of protein glycosylation and glycan processing has been implicated in various diseases. However, glycan processing on proteins in various cellular contexts has not been visualized. We had previously developed a quinone methide cleavage (QMC) platform for enhanced substrate design. This platform was applied here to screen for novel glycan-processing enzymes. We designed and synthesized fluorescent substrates with ß-allopyranoside residues using the QMC platform. When applied in cell-based assays, the fluorescent substrates allowed rapid and clear visualization of ß-allosidase activity in the Golgi apparatus of human cultured cells. The QMC platform will likely find broad applications in visualizing the activities of glycan processing enzymes in living cells and in studying PTMs.

Facile and stabile linkages through tyrosine: bioconjugation strategies with the tyrosine-click reaction.

The scope, chemoselectivity, and utility of the click-like tyrosine labeling reaction with 4-phenyl-3H-1,2,4-triazoline-3,5(4H)-diones (PTADs) is reported. To study the utility and chemoselectivity of PTAD derivatives in peptide and protein chemistry, we synthesized PTAD derivatives possessing azide, alkyne, and ketone groups and studied their reactions with amino acid derivatives and peptides of increasing complexity. With proteins we studied the compatibility of the tyrosine click reaction with cysteine and lysine-targeted labeling approaches and demonstrate that chemoselective trifunctionalization of proteins is readily achieved. In particular cases, we noted that PTAD decomposition resulted in formation of a putative isocyanate byproduct that was promiscuous in labeling. This side reaction product, however, was readily scavenged by the addition of a small amount of 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) to the reaction medium. To study the potential of the tyrosine click reaction to introduce poly(ethylene glycol) chains onto proteins (PEGylation), we demonstrate that this novel reagent provides for the selective PEGylation of chymotrypsinogen, whereas traditional succinimide-based PEGylation targeting lysine residues provided a more diverse range of PEGylated products. Finally, we applied the tyrosine click reaction to create a novel antibody-drug conjugate. For this purpose, we synthesized a PTAD derivative linked to the HIV entry inhibitor aplaviroc. Labeling of the antibody trastuzumab with this reagent provided a labeled antibody conjugate that demonstrated potent HIV-1 neutralization activity demonstrating the potential of this reaction in creating protein conjugates with small molecules. The tyrosine click linkage demonstrated stability to extremes of pH, temperature, and exposure to human blood plasma indicating that this linkage is significantly more robust than maleimide-type linkages that are commonly employed in bioconjugations. These studies support the broad utility of this reaction in the chemoselective modification of small molecules, peptides, and proteins under mild aqueous conditions over a broad pH range using a wide variety of biologically acceptable buffers such as phosphate buffered saline (PBS) and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) buffers as well as others and mixed buffered compositions.

Formylbenzene diazonium hexafluorophosphate reagent for tyrosine-selective modification of proteins and the introduction of a bioorthogonal aldehyde.

4-Formylbenzene diazonium hexafluorophosphate (FBDP) is a novel bench-stable crystalline diazonium salt that reacts selectively with tyrosine to install a bioorthogonal aldehyde functionality. Model studies with N-acyl-tyrosine methylamide allowed us to identify conditions optimal for tyrosine ligation reactions with small peptides and proteins. FBDP-based conjugation was used for the facile introduction of small molecule tags, poly(ethylene glycol) chains (PEGylation), and functional small molecules onto model proteins and to label the surface of living cells.

Virtual ligand screening of α-glucosidase: Identification of a novel potent noncarbohydrate mimetic inhibitor.

5-Thiazoleacetamide derivatives of AR122 and AR125 were screened as α-glucosidase inhibitors by in silico high-throughput screening from commercial drug-like small compound libraries. Inhibition of α-glucosidase with AR122 and AR125 is time dependent: with no preincubation, AR122 and AR125 are relatively moderate inhibitors, but interestingly, after a 120 min incubation, they were 50-fold more potent (AR122: IC(50)=2.47 µM and AR125: IC(50)=27.1 µM). Plots of ln [residual α-glucosidase activity %] versus preincubation time show a pseudo-first order kinetics for both inhibitors. Through dialysis of enzyme-inhibitor complexes, no activity recovery was shown. These results suggest that AR122 and AR125 constitute a new class of noncarbohydrate mimetic inhibitor with an irreversible mechanism.

Involvement of CarA/LitR and CRP/FNR family transcriptional regulators in light-induced carotenoid production in Thermus thermophilus.

Members of the CarA/LitR family are MerR-type transcriptional regulators that contain a C-terminal cobalamin-binding domain. They are thought to be involved in light-induced transcriptional regulation in a wide variety of nonphototrophic bacteria. Based on the distribution of this kind of regulator, the current study examined carotenoid production in Thermus thermophilus, and it was found to occur in a light-induced manner. litR and carotenoid and cobalamin biosynthesis genes were all located on the large plasmid of this organism. litR or cobalamin biosynthesis gene knockout mutants were unable to switch off carotenoid production under dark conditions, while a mutant with a mutation in the downstream gene adjacent to litR (TT_P0055), which encodes a CRP/FNR family transcriptional regulator, was unable to produce carotenoids, irrespective of light conditions. Overall, genetic and biochemical evidence indicates that LitR is bound by cobalamin and associates with the intergenic promoter region between litR and crtB (phytoene synthase gene), repressing the bidirectional transcription of litR and crtB. It is probable that derepression of LitR caused by some photodependent mechanism induces the expression of TT_P0055 protein, which serves as a transcriptional activator for the crtB operon and hence causes the expression of carotenoid biosynthesis and the DNA repair system under light condition.

Design and synthesis of an ER-specific fluorescent probe based on carboxylesterase activity with quinone methide cleavage process.

CEs are important enzymes that catalyze the hydrolysis of prodrugs. In this Letter, we present a new mechanistic ER-specific fluorescent probe 1 based on CE activity. Permeation of 1 into cells and subsequent hydrolytic activation by CEs causes spontaneously quinone methide cleavage, resulting in bright red fluorescence in ER with high specificity. Probe 1 was developed for CE activity imaging and inhibitor screening at the cellular level.

Expression, purification, physicochemical characterization and structural analysis of cytochrome c554 from Vibrio parahaemolyticus strain RIMD2210633.

The function of cytochrome c(554) of Vibrio parahaemolyticus has not yet been determined. We have determined the physicochemical properties and crystal structure of cytochrome c(554) at 1.8 A in order to help elucidate its function. The physicochemical properties and the tertiary structure of cytochrome c(554) resemble those of dimeric cytochrome c(552) from Pseudomonas nautica, but the Vibrio genus contains no gene for nitrite reductase, cytochrome cd(1), in its genome DNA. These results raise the possibility that both cytochromes denote an electron to an electron carrier and accept an electron from same electron carrier.

Utilization of sucrose and analog disaccharides by human intestinal bifidobacteria and lactobacilli: Search of the bifidobacteria enzymes involved in the degradation of these disaccharides.

The majority of oligosaccharides used as prebiotics typically consist of a combination of 3 kinds of neutral monosaccharides, d-glucose, d-galactose, and d-fructose. In this context, we aimed to generate new types of prebiotic oligosaccharides containing other monosaccharides, and to date have synthesized various oligosaccharides containing an amino sugar, uronic acid, and their derivatives. In this study, we investigated the effects of 4 kinds of sucrose (Suc) analog disaccharides containing d-glucosamine, N-acetyl-d-glucosamine, d-glucuronic acid, or d-glucuronamide as constituent monosaccharides, on the growth of 8 species of bifidobacteria and 3 species of lactobacilli isolated from the human intestine. The results of these experiments were compared with those obtained from identical experiments using Suc. We confirmed that all bacterial strains could utilize Suc as a nutrient source for growth; in contrast, only specific species of bifidobacteria showed growth with Suc analog disaccharides. When oligosaccharides are utilized as a nutrient source by bacteria, they are often broken down into monosaccharides or their derivatives by cellular enzymes before entering the intracellular glycolytic pathway. Therefore, to clarify the above phenomenon involved in the growth of bifidobacteria using Suc analog disaccharides, we investigated the cellular glycosidases of 3 strains of bifidobacteria shown to be capable or incapable of growth in the presence of these disaccharides. As the result, it was confirmed that the strains capable of growth using Suc analog disaccharides show greater productivity of glycosidases that degrade these disaccharides than strains not capable of growth; however, we have not identified the enzymes here.

Enzymatic Synthesis and Structural Confirmation of Novel Oligosaccharide, D-Fructofuranose-linked Chitin Oligosaccharide.

Utilizing transglycosylation reaction catalyzed by ß- N -acetylhexosaminidase of Stenotrophomonas maltophilia , ß-D-fructofuranosyl-(2↔1)-α- N , N ´diacetylchitobioside (GlcNAc 2 -Fru) was synthesized from N -acetylsucrosamine and N , N ´-diacetylchitobiose (GlcNAc 2 ), and ß-D-fructofuranosyl-(2↔1)-α- N , N ´, N ´´-triacetylchitotrioside (GlcNAc 3 -Fru) was synthesized from GlcNAc 2 -Fru and GlcNAc 2 . Through purification by charcoal column chromatography, pure GlcNAc 2 -Fru and GlcNAc 3 -Fru were obtained in molar yields of 33.0 % and 11.7 % from GlcNAc 2 , respectively. The structures of these oligosaccharides were confirmed by comparing instrumental analysis data of fragments obtained by enzymatic hydrolysis and acid hydrolysis of them with known data of these fragments.