Improvement of the Nuclease Resistance and Immunostimulatory Activity of CpG Oligodeoxynucleotides by Conjugation to Sugar-Immobilized Gold Nanoparticles.

Adjuvants are essential substances for vaccines and immunotherapies that enhance antigen-specific immune responses. Single-stranded oligodeoxynucleotides containing an unmethylated CpG motif (CpG ODNs) are agonistic ligands for toll-like receptor 9 that initiate an innate immune response. They represent promising adjuvants for antiviral and antitumor immunotherapies; however, CpG ODNs have some limitations, such as poor nuclease resistance and low cell membrane permeability. Therefore, an effective formulation is needed to improve the nuclease resistance and immunostimulatory effects of CpG ODNs. Previously, we demonstrated the selective delivery of a small molecule toll-like receptor 7 ligand to immune cells through sugar-binding receptors using sugar-immobilized gold nanoparticles (SGNPs), which significantly enhanced the potency of the ligand. In this study, we examined SGNPs as carriers for partially phosphorothioated A-type CpG ODN (D35) and an entirely phosphorothioated B-type CpG ODN (K3) and evaluated the functionality of the sugar moiety on SGNPs immobilized with CpG ODN. SGNPs immobilized with D35 (D35-SGNPs) exhibited improved nuclease resistance and the in vitro and in vivo potency was significantly higher compared with that of unconjugated D35. Furthermore, the sugar structure on the GNPs was a significant factor in enhancing the cell internalization ability, and enhanced intracellular delivery of D35 resulted in improving the potencies of the A-type CpG ODN, D35. SGNPs immobilized with K3 (K3-SGNPs) exhibited significantly higher induction activities for both humoral and cellular immunity compared with unconjugated K3 and D35-SGNPs. On the other hand, sugar structure on K3-SGNPs did not affect the immunostimulatory effects. These results indicate that the sugar moiety on K3-SGNPs primarily functions as a hydrophilic dispersant for GNPs and the formulation of K3 to SGNPs contributes to improving the immunostimulatory activity of K3. Because our CpG ODN-SGNPs have superior induction activities for antigen-specific T-cell mediated immune responses, they may be effective adjuvants for vaccines and immunotherapies.

Optimized immobilization of single chain variable fragment antibody onto non-toxic fluorescent nanoparticles for efficient preparation of a bioprobe.

Single-chain variable fragment antibody (scFv) is a small molecular weight antibody that can be used for both therapeutic and diagnostic purposes. To visualize the interaction with the target biomolecules, scFv must be labeled with fluorescent molecules. In this study, to achieve the efficient labeling of scFv, we developed scFv-fluorescent nanoparticle conjugates to utilize scFv as bioprobes. As fluorescent carriers, cadmium-free ZnS-AgInS2/ZnS core/shell nanoparticles were used, and scFv was immobilized onto the nanoparticles via the interaction of nickel ions on nitrilotriacetic acid and hexahistidine (His-tag) fused with scFv. UV-Vis, fluorescence spectra, NMR, and dynamic laser scattering were used to characterize the scFv immobilized fluorescent nanoparticles (scFv-FNPs). The amounts of scFv on FNPs were controlled by the concentration of scFv. The scFv-FNPs that were prepared were non-toxic and selectively bound to cancer cells. The scFv-FNPs could be used as bioanalytical tools, and the immobilization method described here is a promising method for labeling biomolecules with the His-tag.

Synthesis of mucin type core 3 and core 5 structures and their interaction analysis with sugar chips.

For the functional analysis of mucin related glycan, we synthesized core 3 and 5 structures of mucin type O-glycan and investigated their binding interaction with lectins using sugar chip technology. The construction of Tn antigen moiety containing α-N-acetylgalactosamine residue was achieved by α-selective glycosylation of 2-azido-6-tert-butyldiphenylsilyl-3,4-di-O-chloroacetyl-2-deoxy-galctopyranosyl imidate and glucose moiety, which acts as a hydrophilic spacer when immobilized on a gold-coated sensor chip. Core 3 and core 5 structures were synthesized by the glycosylation of appropriate N-acetylglucosamine and N-galactosamine donors, respectively, and were converted into sugar-chain ligand conjugates according to the method reported. The interaction analysis of lectins with sugar chips coated with ligand conjugates was performed with a surface plasmon resonance (SPR) biosensor. The specific interaction was observed between the core 3 structure and Jacalin (JAC) and kinetic parameters were estimated as ka = 1.5 × 104, kd = 5.8 × 10-3, and KD = 3.8 × 10-7.

Glyco-Nanoadjuvants: Impact of Linker Length for Conjugating a Synthetic Small-Molecule TLR7 Ligand to Glyco-Nanoparticles on Immunostimulatory Effects.

Immunotherapy has become a powerful clinical strategy for treating infectious diseases and cancer. Synthetic small-molecule toll-like receptor 7 (TLR7) ligands are attractive candidates as immunostimulatory agents for immunotherapy. TLR7 is mainly localized in intracellular endosomal compartments so that the formulation of their small-molecule ligands with macromolecules enhances endocytic uptake of TLR7 ligands and improves the pharmaceutical properties. Previously, we demonstrated that gold nanoparticles co-immobilized with a TLR7 ligand derivative, that is, a conjugate of synthetic small-molecule TLR7 ligand (1V209) and thioctic acid (TA) via 4,7,10-trioxa-1,13-tridecanediamine, and α-mannose (1V209-αMan-GNPs: glyco-nanoadjuvants) significantly enhances immunostimulatory effects. In the present study, we designed a second-generation glyco-nanoadjuvant that possesses a poly(ethylene glycol) (PEG) chain as a spacer between 1V209 and GNPs and investigated the impact of linker length in 1V209 derivatives on the immunostimulatory activities. We used different chain lengths of PEG (n = 3, 5, 11, or 23) as spacers between 1V209 and thioctic acid to prepare four 1V209-αMan-GNPs. In the in vitro study using primary mouse bone-marrow-derived dendritic cells, 1V209-αMan-GNPs that immobilized with longer 1V209 derivatives, especially the 1V209 derivative possessing PEG23 (1V209-PEG23-TA), showed the highest potency toward induction both for interleukin-6 and type I interferon production than those derivatives with shorter PEG chains. Furthermore, 1V209-αMan-GNPs that immobilized with 1V209-PEG23-TA showed significantly higher adjuvant effects for inducing both humoral and cell-mediated immune responses against ovalbumin in the in vivo immunization study. These results indicate that the linker length for immobilizing small-molecule TLR7 ligand on the GNPs significantly affects the adjuvant activity of 1V209-αMan-GNPs and that 1V209-αMan-GNPs immobilized with 1V209-PEG-23-TA could be superior adjuvants for immunotherapies.

Glyco-Nanoadjuvants: Sugar Structures on Carriers of a Small Molecule TLR7 Ligand Affect Their Immunostimulatory Activities.

Toll-like receptors (TLRs) are pattern recognition receptors that activate innate immunity, and their ligands are promising adjuvants for vaccines and immunotherapies. Small molecule TLR7 ligands are ideal vaccine adjuvants as they induce not only proinflammatory cytokines but also type I interferons. However, their application has only been approved for local administration due to severe systemic immune-related adverse events. In a previous study, we prepared the gold nanoparticles coimmobilized with synthetic small molecule TLR7 ligand, 1V209, and α-mannose (1V209-αMan-GNPs). 1V209-αMan-GNPs were selectively delivered via a cell surface sugar-binding protein, mannose receptor, which enabled selective delivery of TLR7 ligands to immune cells. Besides the mannose receptor, immune cells express various sugar-binding proteins such as macrophage galactose binding lectins and sialic acid-binding immunoglobulin-type lectins and recognize distinct sugar structures. Hence, in the present study, we investigated whether sugar structures on GNPs affect the efficiency and selectivity of intracellular delivery and subsequent immunostimulatory potencies. Five neutral sugars and two sialosides were selected and each sugar was coimmobilized with 1V209 onto GNPs (1V209-SGNPs) and their innate immunostimulatory potencies were compared to that of 1V209-αMan-GNPs. The in vitro study using mouse bone marrow derived dendritic cells (BMDCs) demonstrated that α-glucose, α-N-acetylglucosamine, or α-fucose immobilized 1V209-SGNPs increased interleukin-6 and type I interferon release similar to that of 1V209-αMan-GNPs, whereas galacto-type sugar immobilized 1V209-SGNPs predominantly enhanced type I interferon release. In contrast, sialoside immobilized 1V209-SGNPs did not enhance the potency of 1V209. In the in vivo immunization study using ovalbumin as a model antigen, neutral sugar immobilized 1V209-SGNPs induced comparable T helper-1 immune response to that of 1V209-αMan-GNPs and by 10-fold higher than that of sialoside immobilized 1V209-SGNPs. These results indicate that the sugar structures on 1V209-SGNPs affect their immunostimulatory activities, and functionalization of the carrier particles is important to shape immune responses.

Synthesis and immunostimulatory activity of sugar-conjugated TLR7 ligands.

Toll-like receptors (TLRs) are a type of pattern recognition receptors (PRRs), which are activated by recognizing pathogen-associated molecular patterns (PAMPs). The activation of TLRs initiates innate immune responses and subsequently leads to adaptive immune responses. TLR agonists are effective immuomodulators in vaccine adjuvants for infectious diseases and cancer immunotherapy. In exploring hydrophilic small molecules of TLR7 ligands using the cell-targeted property of a vaccine adjuvant, we conjugated 1V209, a small TLR7 ligand molecule, with various low or middle molecular weight sugar molecules that work as carriers. The sugar-conjugated 1V209 derivatives showed increased water solubility and higher immunostimulatory activity in both mouse and human cells compared to unmodified 1V209. The improved immunostimulatory potency of sugar-conjugates was attenuated by an inhibitor of endocytic process, cytochalasin D, suggesting that conjugation of sugar moieties may enhance the uptake of TLR7 ligand into the endosomal compartment. Collectively our results support that sugar-conjugated TLR7 ligands are applicable to novel drugs for cancer and vaccine therapy.

Gold Nanoparticles Coimmobilized with Small Molecule Toll-Like Receptor 7 Ligand and α-Mannose as Adjuvants.

Adjuvants enhance the immune response during vaccination. Among FDA-approved adjuvants, aluminum salts are most commonly used in vaccines. Although aluminum salts enhance humoral immunity, they show a limited effect for cell-mediated immune responses. Thus, further development of adjuvants that induce T-cell-mediated immune response is needed. Toll-like receptors (TLRs) recognizing specific pathogen-associated molecular patterns activate innate immunity, which is crucial to shape adaptive immunity. Using TLR ligands as novel adjuvants in vaccines has therefore attracted substantial attention. Among them a small molecule TLR7 ligand, imiquimod, has been approved for clinical use, but its use is restricted to local administration due to unwanted adverse side effects when used systematically. Since TLR7 is mainly located in the endosomal compartment of immune cells, efficient transport of the ligand into the cells is important for improving the potency of the TLR7 ligand. In this study we examined gold nanoparticles (GNPs) immobilized with α-mannose as carriers for a TLR7 ligand to target immune cells. The small molecule synthetic TLR7 ligand, 2-methoxyethoxy-8-oxo-9-(4-carboxy benzyl)adenine (1V209), and α-mannose were coimmobilized via linker molecules consisting of thioctic acid on the GNP surface (1V209-αMan-GNPs). The in vitro cytokine production activity of 1V209-αMan-GNPs was higher than that of the unconjugated 1V209 derivative in mouse bone marrow-derived dendritic cells and in human peripheral blood mononuclear cells. In the in vivo immunization study, 1V209-αMan-GNPs induced significantly higher titers of IgG2c antibody specific to ovalbumin as an antigen than did unconjugated 1V209, and splenomegaly and weight loss were not observed. These results indicate that 1V209-αMan-GNPs could be useful as safe and effective adjuvants for development of vaccines against infectious diseases and cancer.

Cell Profiling Based on Sugar-Chain-Cell Binding Interaction and Its Application to Typing and Quality Verification of Cells.

Developing methods to determine cell type and cell state has been a significant challenge in the field of cancer diagnosis as well as in typing and quality verification for cultured cells. Herein, we report a cell profiling method based on binding interactions between cell-surface sugar-chain-binding proteins and sugar-chain-immobilized fluorescent nanoparticles (SFNPs), together with a method for cell typing and cell quality verification. Binding profiles of cells against sugar chains were analyzed by performing flow cytometry analysis with SFNPs. Discrimination analysis based on binding profiles could classify cell type and evaluate the quality of cultured cells. By applying our method to differentiated cells originating from conventional cell lines and also to mouse embryotic stem cells, we could detect the cells before and after differentiation. Our method can be utilized not only for the biofunctional analysis of cells but also for diagnosis of cancer cells and quality verification of cultured cells.

Development of sugar chain-binding single-chain variable fragment antibody to adult T-cell leukemia cells using glyco-nanotechnology and phage display method.

Adult T-cell leukemia (ATL) is an intractable blood cancer caused by the infection of human T-cell leukemia virus type-1, and effective medical treatment is required. It is known that the structure and expression levels of cell surface sugar chains vary depending on cell states such as inflammation and cancer. Thus, it is expected that the antibody specific for ATL cell surface sugar chain would be an effective diagnostic tool and a strong candidate for the development of an anti-ATL drug. Here, we developed a stable sugar chain-binding single-chain variable fragment antibody (scFv) that can bind to ATL cells using a fibre-type Sugar Chip and phage display method. The fiber-type Sugar Chips were prepared using O-glycans released from ATL cell lines. The scFv-displaying phages derived from human B cells (diversity: 1.04 × 108) were then screened using the fiber-type Sugar Chips, and an O-glycan-binding scFv was obtained. The flow cytometry analysis revealed that the scFv predominantly bound to ATL cell lines. The sugar chain-binding properties of the scFv was evaluated by array-type Sugar Chip immobilized with a library of synthetic glycosaminoglycan disaccharide structures. Highly sulphated disaccharide structures were found to have high affinity to scFv.

Structure-Activity Relationship Studies of Pyrimido[5,4-b]indoles as Selective Toll-Like Receptor 4 Ligands.

Previous high throughput screening studies led to the discovery of two novel, nonlipid-like chemotypes as Toll-like receptor 4 (TLR4) agonists. One of these chemotypes, the pyrimido[5,4-b]indoles, was explored for structure-activity relationship trends relative to production of TLR4 dependent cytokines/chemokines, resulting in a semioptimized lead (compound 1) that provided a starting point for further optimization studies. In this report, compounds belonging to three areas of structural modification were evaluated for biological activity using murine and human TLR4 reporter cells, primary murine bone marrow derived dendritic cells, and human peripheral blood mononuclear cells. The compounds bearing certain aryl groups at the C8 position, such as phenyl (36) and ß-naphthyl (39), had potencies significantly greater than compound 1. Compound 36 displayed human TLR4 agonist activity at submicromolar concentrations. The computational analysis suggests that the improved potency of these C8-aryl derivatives may be the result of additional binding interactions at the interface of the TLR4/myeloid differentiation protein-2 (MD-2) complex.

Optical Fiber-Type Sugar Chip Using Localized Surface Plasmon Resonance.

Optical fiber-type Sugar Chips were developed using localized surface plasmon resonance (LSPR) of gold (Au) nanoparticles. The endface of an optical fiber was first aminosilylated and then condensed with α-lipoic acid containing a dithiol group. Second, gold nanoparticles were immobilized onto the endface via an Au-S covalent bond. Finally, sugar moieties were attached to the gold nanoparticle using our original sugar chain-ligand conjugates to obtain fiber-type Sugar Chips, by which the sugar moiety-protein interaction was analyzed. The specificity, sensitivity, and quantitative binding potency against carbohydrate-binding protein were found to be identical to that of a conventional SPR sensor. In this analysis, only a small sample volume (approximately 10 µL) was required compared with 100 µL for the conventional SPR sensor, suggesting that the fiber-type Sugar Chip and LSPR are applicable for nonpure small masses of proteins.

Visual Detection of Human Antibodies Using Sugar Chain-Immobilized Fluorescent Nanoparticles: Application as a Point of Care Diagnostic Tool for Guillain-Barré Syndrome.

Sugar chain binding antibodies have gained substantial attention as biomarkers due to their crucial roles in various disorders. In this study, we developed simple and quick detection method of anti-sugar chain antibodies in sera using our previously developed sugar chain-immobilized fluorescent nanoparticles (SFNPs) for the point-of-care diagnostics. Sugar chain structure on SFNPs was modified with the sugar moieties of the GM1 ganglioside via our original linker molecule to detect anti-GM1 antibodies. The structures and densities of the sugar moieties immobilized on the nanoparticles were evaluated in detail using lectins and sera containing anti-GM1 antibodies from patients with Guillain-Barré syndrome, a neurological disorder, as an example of disease involving anti-sugar chain antibodies. When optimized SFNPs were added to sera from patients with Guillain-Barré syndrome, fluorescent aggregates were able to visually detect under UV light in three hours. The sensitivity of the detection method was equivalent to that of the current ELISA method used for the diagnosis of Guillain-Barré syndrome. These results suggest that our method using SFNPs is suitable for the point-of-care diagnostics of diseases involving anti-sugar chain antibodies.

Heparinase I-specific disaccharide unit of heparin is a key structure but insufficient for exerting anti-prion activity in prion-infected cells.

Glycosaminoglycans reportedly play important roles in prion formation, but because of their structural complexity, the chemical structures affecting prion formation have not been fully evaluated. Here, we compared two types of low molecular weight heparins and found that heparinase I-sensitive structures influenced anti-prion activity in prion-infected cells. Surface plasmon resonance analyses showed significant binding of a representative heparinase I substrate disaccharide unit, GlcNS6S-IdoA2S, to recombinant prion protein (PrP) fragments, such as full-length PrP23-231 and N-terminal domain PrP23-89, but not to PrP89-230. This binding was competitively inhibited by heparin or pentosan polysulfate, but not by Cu(2+). These PrP binding profiles of the disaccharide unit are consistent with those previously reported for heparin. However, synthetic compounds comprising disaccharide unit alone or its multimers exhibited no anti-prion activity in prion-infected cells. Consequently, the findings suggest that the heparin disaccharide unit that binds to the N-terminal region of PrP is a key structure, but it is insufficient for exerting anti-prion activity.

Syntheses of chondroitin sulfate tetrasaccharide structures containing 4,6-disulfate patterns and analysis of their interaction with glycosaminoglycan-binding protein.

Chondroitin sulfate tetrasaccharide ligand conjugates, namely GlcA-GalNAc6S-GlcA-GalNAc4S6S (CS-C+E) 1, GlcA2S-GalNAc6S-GlcA2S-GalNAc4S6S (CS-D+T) 2, GlcA-GalNAc4S6S-GlcA-GalNAc4S (CS-E+A) 3, GlcA-GalNAc4S6S-GlcA-GalNAc6S (CS-E+C) 4, and GlcA-GalNAc4S6S-GlcA-GalNAc4S6S (CS-E+E) 5, were systematically synthesized using a disaccharide building block 6. Synthesized CS tetrasaccharide structures were immobilized onto gold-coated chips to prepare array-type sugar chips, and the binding properties of protein were evaluated by surface plasmon resonance imaging biosensor. CS-D+T, CS-E+A, CS-E+C, and CS-E+E showed greater affinity for basic fibroblast growth factor than did other tetrasaccharides (CS-C+D, C+E, D+D).

Synthesis of a chondroitin sulfate disaccharide library and a GAG-binding protein interaction analysis.

Chondroitin sulfate (CS), which belongs to the glycosaminoglycan (GAG) superfamily, is a linear sulfated polysaccharide involved in various biological processes. CS structure is very heterogeneous and contains various sulfation patterns owing to the multiple and random enzymatic modifications that occur during its biosynthesis. The resultant microdomain structure in the CS chain interacts with specific biomolecules to regulate biological functions. Therefore, an analysis of the structure-activity relationship of CS at the molecular level is necessary to clarify their biofunctions. In this study, we designed the common intermediate possessing an orthogonally removable protective group and systematically synthesized all 16 types of CS disaccharide structure generated by sulfation. In addition, we demonstrated the on-time analysis of the binding properties of GAG-binding proteins using 'Sugar Chip' immobilized CS disaccharide structures by surface plasmon resonance (SPR) imaging, indicating that our chip technology is effective for the evaluation of binding properties.

Cadmium-free sugar-chain-immobilized fluorescent nanoparticles containing low-toxicity ZnS-AgInS2 cores for probing lectin and cells.

Sugar chains play a significant role in various biological processes through sugar chain-protein and sugar chain-sugar chain interactions. To date, various tools for analyzing sugar chains biofunctions have been developed. Fluorescent nanoparticles (FNPs) functionalized with carbohydrate, such as quantum dots (QDs), are an attractive imaging tool for analyzing carbohydrate biofunctions in vitro and in vivo. Most FNPs, however, consist of highly toxic elements such as cadmium, tellurium, selenium, and so on, causing problems in long-term bioimaging because of their cytotoxicity. In this study, we developed cadmium-free sugar-chain-immobilized fluorescent nanoparticles (SFNPs) using ZnS-AgInS2 (ZAIS) solid solution nanoparticles (NPs) of low or negligible toxicity as core components, and investigated their bioavailability and cytotoxicity. SFNPs were prepared by mixing our originally developed sugar-chain-ligand conjugates with ZAIS/ZnS core/shell NPs. In binding experiments with lectin, the obtained ZAIS/ZnS SFNPs interacted with an appropriate lectin to give specific aggregates, and their binding interaction was visually and/or spectroscopically detected. In addition, these SFNPs were successfully utilized for cytometry analysis and cellular imaging in which the cell was found to possess different sugar-binding properties. The results of the cytotoxicity assay indicated that SFNPs containing ZAIS/ZnS have much lower toxicity than those containing cadmium. These data strongly suggest that our designed SFNPs can be widely utilized in various biosensing applications involved in carbohydrates.

Characterization of a gene family encoding SEA (sea-urchin sperm protein, enterokinase and agrin)-domain proteins with lectin-like and heme-binding properties from Schistosoma japonicum.

BACKGROUND: We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. METHODOLOGY/PRINCIPAL FINDINGS: Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin)-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10(-6) M) and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. CONCLUSIONS: The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation), and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted.

Stable sugar-chain-immobilized fluorescent nanoparticles for probing lectin and cells.

Sugar chains are important molecules in cellular recognition and signaling, and quantum dots (QDs) are a very powerful tool for in vitro and in vivo imaging. Herein, we report the preparation of stable sugar-chain-immobilized fluorescent nanoparticles (SFNPs) and their application to the analysis of sugar-chain-protein interactions and cellular imaging. SFNPs were easily prepared by mixing CdTe/CdS core/shell QDs with our previously developed sugar-chain-ligand conjugates. The obtained SFNPs were very stable and could be stored for several months. In the binding analysis, ß-galactose- and α-glucose-immobilized SFNPs were specifically interacted with Ricinus communis agglutinin I and concanavalin A, respectively, and made into aggregates. The binding interaction was detected visually, fluorescently, or both. In the experiment for cellular imaging, it was found that SFNPs were predominantly taken up by human hepatocyto carcinoma cells (HepG2), suggesting the possible usage of our designed SFNPs for various biochemical analyses of sugar chains.

Molecular cloning and characterization of a beta-L-Arabinobiosidase in Bifidobacterium longum that belongs to a novel glycoside hydrolase family.

Extensin is a glycoprotein that is rich in hydroxyprolines linked to ß-L-arabinofuranosides. In this study, we cloned a hypBA2 gene that encodes a novel ß-L-arabinobiosidase from Bifidobacterium longum JCM 1217. This enzyme does not have any sequence similarity with other glycoside hydrolase families but has 38-98% identity to hypothetical proteins in Bifidobacterium and Xanthomonas strains. The recombinant enzyme liberated L-arabinofuranose (Araf)-ß1,2-Araf disaccharide from carrot extensin, potato lectin, and Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara(3)-Hyp) but not Araf-α1,3-Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara(4)-Hyp) or Araf-ß1,2-Araf-ß-Hyp (Ara(2)-Hyp), which indicated that it was specific for unmodified Ara(3)-Hyp substrate. The enzyme also transglycosylated 1-alkanols with retention of the anomeric configuration. This is the first report of an enzyme that hydrolyzes Hyp-linked ß-L-arabinofuranosides, which defines a new family of glycoside hydrolases, glycoside hydrolase family 121.

Multiple mechanisms for exogenous heparin modulation of vascular endothelial growth factor activity.

Heparin and heparin-like molecules are known to modulate the cellular responses to vascular endothelial growth factor-A (VEGF-A). In this study, we investigated the likely mechanisms for heparin's influence on the biological activity of VEGF-A. Previous studies have shown that exogenous heparin's effects on the biological activity of VEGF-A are many and varied, in part due to the endogenous cell-surface heparan sulfates. To circumvent this problem, we used mutant endothelial cells lacking cell-surface heparan sulfates. We showed that VEGF-induced cellular responses are dependent in part on the presence of the heparan sulfates, and that exogenous heparin significantly augments VEGF's cellular effects especially when endogenous heparan sulfates are absent. Exogenous heparin was also found to play a cross-bridging role between VEGF-A(165) and putative heparin-binding sites within its cognate receptor, VEGFR2 when they were examined in isolation. The cross-bridging appears to be more dependent on molecular weight than on a specific heparin structure. This was confirmed by surface plasmon resonance binding studies using sugar chips immobilized with defined oligosaccharide structures, which showed that VEGF-A(165) binds to a relatively broad range of sulfated glycosaminoglycan structures. Finally, studies of the far-UV circular dichroism spectra of VEGF-A(165) showed that heparin can also modulate the conformation and secondary structure of the protein.