Slow spectral diffusion of the NO stretching mode of [RuCl5(NO)]2- in D2O studied by 2D-IR spectroscopy and molecular dynamics simulations.

The vibrational dynamics of the NO stretching mode of [RuCl5(NO)]2- in D2O were investigated by nonlinear infrared (IR) spectroscopy. We performed IR pump-probe measurements to obtain the vibrational lifetime of this molecule. The lifetime is 31 ps, which is sufficiently long enough to study the vibrational frequency fluctuation on a slower time scale with high precision. By two-dimensional IR spectroscopy, the frequency-frequency time correlation function (FFTCF) of the NO stretching mode was characterized with a delta function plus a double-exponential function. The time constant of the slower component was ∼10 ps. We also found that the time constant does not strongly depend on temperature. In order to investigate the microscopic origin of this component, we performed classical molecular dynamics simulations. It was found that the hydration structure around the NO group was influenced by the negatively charged Cl ligands. To calculate the FFTCF decay, we employed an approximate theoretical model based on the vibrational solvatochromism theory. It was demonstrated that water fluctuations around the Cl ligands projected on the NO group correspond to the 10 ps decay component in the FFTCF. The fluctuation is related to the orientational dynamics of the water molecules attracted by the Cl ligands. By comparing the FFTCF parameters of the present solute with those of previously reported metal complexes and SCN- in D2O, we conclude that the presence of different electrostatic environments around the vibrational probe and the other interaction sites of the solute is important for understanding the slow decay component in the FFTCFs.

ZG16p, an Animal Homologue of Plant ß-Prism Fold Lectins: Purification Methods of Natural and Recombinant ZG16p and Inhibition Assay of Cancer Cell Growth Using ZG16p.

ZG16p is a soluble 16-kDa protein abundantly expressed in the pancreas and gut, and has a ß-prism fold structure similar to that of mannose-binding Jacalin-related lectins (mJRLs) such as BanLec, Heltuba, and Artocarpin. ZG16p binds to mannose via the well-conserved GXXXD loop among mJRLs and sulfated glycosaminoglycans (e.g., heparin and heparan sulfate) via the basic patch of molecular surface. In addition to the above binding activities, ZG16p has inhibitory activity against proliferation of colon cancer cells. This manuscript describes purification of rat pancreatic ZG16p and recombinant ZG16p expressed in Escherichia coli expression system, and cell growth inhibition assay using ZG16p as an inhibitor.

Annexin Lectins: Ca2+-Dependent Heparin-Binding Activity, Phosphatidylserine-Binding Activity, and Anticoagulant Activity.

Among numerous heparin-binding proteins identified in animal tissues and body fluids, annexins are unique because their activies depend on their Ca2+ binding. Annexins are known to have other Ca2+-dependent activities. For example, they bind to phosphatidylserine in the plasma membrane, and some of them exhibit potent anticoagulant activity. This chapter describes three protocols that measure the Ca2+-dependent activities using recombinant annexins: solid-phase heparin-binding assay using bovine serum albumin-conjugated heparin, solid-phase phosphatidylserine-binding assay, and plasma coagulation inhibition assay.

Annexin A4 inhibits sulfatide-induced activation of coagulation factor XII.

BACKGROUND: Factor XII (FXII) is a plasma serine protease that initiates the intrinsic pathway of blood coagulation upon contact with anionic substances, such as the sulfated glycolipid sulfatide. Annexins (ANXs) have been implicated in the regulation of the blood coagulation reaction by binding to anionic surfaces composed of phospholipids and sulfated glycoconjugates, but their physiological importance is only partially understood. OBJECTIVE: To test the hypothesis that ANXs are involved in suppressing the intrinsic pathway initiated by sulfatide, we examined the effect of eight recombinant ANX proteins on the intrinsic coagulation reaction and their sulfatide binding activities. METHODS: Recombinant ANXs were prepared in Escherichia coli expression systems and their anticoagulant effects on the intrinsic pathway initiated by sulfatide were examined using plasma clotting assay and chromogenic assay. ANXA4 active sites were identified by alanine scanning and fold deletion in the core domain. RESULTS AND CONCLUSIONS: We found that ANXA3, ANXA4, and ANXA5 strongly inhibited sulfatide-induced plasma coagulation. Wild-type and mutated ANXA4 were used to clarify the molecular mechanism involved in inhibition. ANXA4 inhibited sulfatide-induced auto-activation of FXII to FXIIa and the conversion of its natural substrate FXI to FXIa but showed no effect on the protease activity of FXIIa or FXIa. Alanine scanning showed that substitution of the Ca2+ -binding amino acid residue in the fourth fold of the core domain of ANXA4 reduced anticoagulant activity, and deletion of the entire fourth fold of the core domain resulted in complete loss of anticoagulant activity.

A novel protocol for the preparation of active recombinant human pancreatic lipase from Escherichia coli.

An active recombinant human pancreatic lipase (recHPL) was successfully prepared for the first time from the Escherichia coli expression system using short Strep-tag II (ST II). The recHPL-ST II was solubilized using 8 M urea from E.coli lysate and purified on a Strep-Tactin-Sepharose column. After refolding by stepwise dialyses in the presence of glycerol and Ca2+ for 2 days followed by gel filtration, 1.8-6 mg of active recHPL-ST II was obtained from 1 L of culture. The recHPL was non-glycosylated, but showed almost equal specific activity, pH-dependency and time-dependent stability compared to those of native porcine pancreatic lipase (PPL) at 37°C. However, the recHPL lost its lipolytic activity above 50°C, showing a lower heat-stability than that of native PPL, which retained half its activity at this temperature.

Lectin ZG16p inhibits proliferation of human colorectal cancer cells via its carbohydrate-binding sites.

Zymogen granule protein 16 (ZG16p) is a soluble lectin that binds to both mannose and heparin/heparan sulfate. It is highly expressed in the human digestive tract and is secreted into the mucus. In this study, we investigated the effect of ZG16p on the proliferation of human colorectal cancer cells. Overexpression of ZG16p in Caco-2 cells decreased cell growth. Recombinant ZG16p markedly inhibited proliferation of Caco-2, LS174T, HCT116 and HCT15 cells. Caco-2 cell growth was not inhibited by two mutated ZG16p proteins, D151A and M5 (K36A, R37A, R53A, R55A and R79A) lacking mannose- and heparin-binding activities, respectively. Immunofluorescent cell staining revealed that ZG16p-D151A maintained its binding to the Caco-2 cell surface, whereas ZG16p-M5 failed to bind to the cells. These results suggest that ZG16p interacts with the cell surface via basic amino acids substituted in ZG16p-M5 and inhibits Caco-2 cell proliferation via Asp151. In addition, growth of patient-derived colorectal tumor organoids in a 3D intestinal stem cell system was suppressed by ZG16p but not by ZG16p-M5. Taken together, our findings indicate that ZG16p inhibits the growth of colorectal cancer cells via its carbohydrate-binding sites in vitro and ex vivo. In this study, a novel pathway in cancer cell growth regulation through cell surface carbohydrate chains is suggested.

Defining the Interaction of Human Soluble Lectin ZG16p and Mycobacterial Phosphatidylinositol Mannosides.

ZG16p is a soluble mammalian lectin that interacts with mannose and heparan sulfate. Here we describe detailed analysis of the interaction of human ZG16p with mycobacterial phosphatidylinositol mannosides (PIMs) by glycan microarray and NMR. Pathogen-related glycan microarray analysis identified phosphatidylinositol mono- and di-mannosides (PIM1 and PIM2) as novel ligand candidates of ZG16p. Saturation transfer difference (STD) NMR and transferred NOE experiments with chemically synthesized PIM glycans indicate that PIMs preferentially interact with ZG16p by using the mannose residues. The binding site of PIM was identified by chemical-shift perturbation experiments with uniformly (15)N-labeled ZG16p. NMR results with docking simulations suggest a binding mode of ZG16p and PIM glycan; this will help to elucidate the physiological role of ZG16p.

Solid-phase assay of lectin activity using HRP-conjugated glycoproteins.

Various enzyme-conjugated probes have been widely used for detection of specific interactions between biomolecules. In the case of glycan-protein interaction, horseradish peroxidase (HRP)-conjugated glycoproteins (HRP-GPs) are useful for the detection of carbohydrate-binding activity of plant and animal lectins. In this chapter, a typical solid-phase assay of the carbohydrate-binding activity of Sophora japonica agglutinin I, a Gal/GalNAc-specific lectin, using HRP-conjugated asialofetuin is described. HRP-GPs are versatile tools for probing lectin activities in crude extracts, screening many samples at one time, and applicable not only for solid-phase binding assays but also samples which are dot- or Western-blotted onto the membrane.

Structural basis for multiple sugar recognition of Jacalin-related human ZG16p lectin.

ZG16p is a soluble mammalian lectin, the first to be described with a Jacalin-related ß-prism-fold. ZG16p has been reported to bind both to glycosaminoglycans and mannose. To determine the structural basis of the multiple sugar-binding properties, we conducted glycan microarray analyses of human ZG16p. We observed that ZG16p preferentially binds to α-mannose-terminating short glycans such as Ser/Thr-linked O-mannose, but not to high mannose-type N-glycans. Among sulfated glycosaminoglycan oligomers examined, chondroitin sulfate B and heparin oligosaccharides showed significant binding. Crystallographic studies of human ZG16p lectin in the presence of selected ligands revealed the mechanism of multiple sugar recognition. Manα1-3Man and Glcß1-3Glc bound in different orientations: the nonreducing end of the former and the reducing end of the latter fitted in the canonical shallow mannose binding pocket. Solution NMR analysis using (15)N-labeled ZG16p defined the heparin-binding region, which is on an adjacent flat surface of the protein. On-array competitive binding assays suggest that it is possible for ZG16p to bind simultaneously to both types of ligands. Recognition of a broad spectrum of ligands by ZG16p may account for the multiple functions of this lectin in the formation of zymogen granules via glycosaminoglycan binding, and in the recognition of pathogens in the digestive system through α-mannose-related recognition.

HMMC-1, a human monoclonal antibody to fucosylated core 1 O-glycan, suppresses growth of uterine endometrial cancer cells.

HMMC-1 is a human monoclonal antibody that reacts with a fucosylated and extended core 1 O-glycan, Fucα1-2Galß1-4GlcNAcß1-3Galß1-3GalNAc-Ser/Thr, as an epitope. In the present study, we examined the effects of HMMC-1 on cell proliferation of two human uterine endometrial cancer cell lines, HEC8 and HEC9, to investigate the role of glycoproteins bearing the HMMC-1 epitope in cancer progression. HEC9 cells expressed high levels of the HMMC-1 epitope, but HMMC-1 reactivity was hardly detected in HEC8 cells. In a mouse model of lymph node metastasis using orthotopic implantation, HEC8 and HEC9 showed low (10%) and high (80%) metastatic potency, respectively. Growth of HEC9, but not HEC8, was remarkably inhibited by addition of HMMC-1 to the culture medium. Cell cycle analysis and expression analysis showed that HMMC-1 treatment increased the G(1) phase population of HEC9 cells and induced cyclin-dependent kinase inhibitors p16 and p21. Two glycoproteins, 97 and 137 kDa, with a strong reactivity to HMMC-1 were purified, and the 97-kDa glycoprotein was identified as CD166, an immunoglobulin superfamily cell adhesion molecule assumed to be involved in cancer metastasis. CD166 gene-silencing dramatically reduced HMMC-1 epitope expression and growth in HEC9 cells, indicating that CD166 is the primary glycoprotein presenting the HMMC-1 epitope in HEC9 cells. Collectively, HMMC-1 might arrest the cell cycle in the G(1) phase by binding to O-glycans on the CD166 expressed in HEC9 cells, raising the possibility that HMMC-1 extensively inhibits invasive growth of HMMC-1 epitope-positive uterine endometrial cancer cells by targeting the cancer-associated form of CD166.

ZG16p, an animal homolog of ß-prism fold plant lectins, interacts with heparan sulfate proteoglycans in pancreatic zymogen granules.

ZG16p is a soluble 16 kDa pancreatic protein having structural similarities with plant ß-prism fold lectins such as the banana lectin BanLec and the jackfruit lectin jacalin. ZG16p is postulated to be involved in the formation of zymogen granules by interacting with proteoglycans (PGs) localized in pancreatic exocrine granule membranes, but direct evidence was lacking. We characterized the structural properties of rat pancreatic zymogen granule PGs and examined their interaction with ZG16p. Structural analysis of the glycosaminoglycans (GAGs) showed that rat pancreatic zymogen granule PGs have heparan sulfate chains with a unique property, a high degree of sulfation (ΔUA-GlcNAc:ΔUA-GlcNS:ΔUA-GlcNAc6S:ΔUA-GlcNS6S:ΔUA2S-GlcNS:ΔUA2S-GlcNS6S, 27.9:16.6:5.7:22.5:6.2:21.1). After heparin lyase II digestion, the core proteins derived from the PGs were detected at molecular weights of 66,000 and 35,000-40,000. An overlay binding assay revealed that ZG16p binds specifically to heparan sulfate PGs by recognizing their GAG chains. Affinity chromatography demonstrated that ZG16p binds most strongly to heparin among the zymogen granule proteins. Site-directed mutational analysis revealed that the basic amino acid residues located in two putative carbohydrate-binding sites (CBSs) of ZG16p, which were found in association with the crystal structure of BanLec, are responsible for the recognition of heparin. These observations suggest that ZG16p is the primary binding partner of the granule heparan sulfate PGs. ZG16p may cross-link the granule heparan sulfate chains via two CBSs and facilitate the formation of a submembranous matrix, a sorting platform for enzyme proteins on the luminal side of the zymogen granule membrane.

Identification of mono- and disulfated N-acetyl-lactosaminyl Oligosaccharide structures as epitopes specifically recognized by humanized monoclonal antibody HMOCC-1 raised against ovarian cancer.

A humanized monoclonal antibody raised against human ovarian cancer RMG-I cells and designated as HMOCC-1 (Suzuki, N., Aoki, D., Tamada, Y., Susumu, N., Orikawa, K., Tsukazaki, K., Sakayori, M., Suzuki, A., Fukuchi, T., Mukai, M., Kojima-Aikawa, K., Ishida, I., and Nozawa, S. (2004) Gynecol. Oncol. 95, 290-298) was characterized for its carbohydrate epitope structure. Specifically, a series of co-transfections was performed using mammalian expression vectors encoding specific glycosyltransferases and sulfotransferases. These experiments identified one sulfotransferase, GAL3ST3, and one glycosyltransferase, B3GNT7, as required for HMOCC-1 antigen formation. They also suggested that the sulfotransferase CHST1 regulates the abundance and intensity of HMOCC-1 antigen. When HEK293T cells were co-transfected with GAL3ST3 and B3GNT7 expression vectors, transfected cells weakly expressed HMOCC-1 antigen. When cells were first co-transfected with GAL3ST3 and B3GNT7 and then with CHST1, the resulting cells strongly expressed HMOCC-1 antigen. However, when cells were transfected with a mixture of GAL3ST3 and CHST1 before or after transfection with B3GNT7, the number of antigen-positive cells decreased relative to the number seen with only GAL3ST3 and B3GNT7, suggesting that CHST1 plays a regulatory role in HMOCC-1 antigen formation. Because these results predicted that HMOCC-1 antigens are SO(3) → 3Galß1 → 4GlcNAcß1 → 3(±SO(3) → 6)Galß1 → 4GlcNAc, we chemically synthesized mono- and disulfated and unsulfated oligosaccharides. Immunoassays using these oligosaccharides as inhibitors showed the strongest activity by disulfated tetrasaccharide, weak but positive activity by monosulfated tetrasaccharide at the terminal galactose, and no activity by nonsulfated tetrasaccharides. These results establish the HMOCC-1 epitope, which should serve as a useful reagent to further characterize ovarian cancer.

Caenorhabditis elegans proteins captured by immobilized Galß1-4Fuc disaccharide units: assignment of 3 annexins.

Galß1-4Fuc is a key structural motif in Caenorhabditis elegans glycans and is responsible for interaction with C. elegans galectins. In animals of the clade Protostomia, this unit seems to have important roles in glycan-protein interactions and corresponds to the Galß1-4GlcNAc unit in vertebrates. Therefore, we prepared an affinity adsorbent having immobilized Galß1-4Fuc in order to capture carbohydrate-binding proteins of C. elegans, which interact with this disaccharide unit. Adsorbed C. elegans proteins were eluted with ethylenediaminetetraacetic acid (EDTA) and followed by lactose (Galß1-4Glc), digested with trypsin, and were then subjected to proteomic analysis using LC-MS/MS. Three annexins, namely NEX-1, -2, and -3, were assigned in the EDTA-eluted fraction. Whereas, galectins, namely LEC-1, -2, -4, -6, -9, -10, and DC2.3a, were assigned in the lactose-eluted fraction. The affinity of annexins for Galß1-4Fuc was further confirmed by adsorption of recombinant NEX-1, -2, and -3 proteins to the Galß1-4Fuc column in the presence of Ca(2+). Furthermore, frontal affinity chromatography analysis using an immobilized NEX-1 column showed that NEX-1 has an affinity for Galß1-4Fuc, but no affinity toward Galß1-3Fuc and Galß1-4GlcNAc. We would hypothesize that the recognition of the Galß1-4Fuc disaccharide unit is involved in some biological processes in C. elegans and other species of the Protostomia clade.

Branched RNA nanostructures for RNA interference.

Branched RNAs with three- or four-way junctions were designed by assembling single-stranded RNA for RNA interference. Human Dicer transformed branched RNAs into about 20 base pairs of double-stranded RNA, which is a standard siRNA species. Our tetramer design provides a potent silencing effect over a period of 5 days.

Crystal structures of human secretory proteins ZG16p and ZG16b reveal a Jacalin-related ß-prism fold.

ZG16p is a secretory protein that mediates condensation-sorting of pancreatic enzymes to the zymogen granule membrane in pancreatic acinar cells. ZG16p interacts with glycosaminoglycans and the binding is considered to be important for condensation-sorting of pancreatic enzymes. ZG16b/PAUF, a paralog of ZG16p, has recently been found to play a role in gene regulation and cancer metastasis. However, the detailed functions of ZG16p and ZG16b remain to be clarified. Here, in order to obtain insights into structure-function relationships, we conducted crystallographic studies of human ZG16p lectin as well as its paralog, ZG16b, and determined their crystal structures at 1.65 and 2.75 Å resolution, respectively. ZG16p has a Jacalin-related ß-prism fold, the first to be reported among mammalian lectins. The putative sugar-binding site of ZG16p is occupied by a glycerol molecule, mimicking the mannose bound to Jacalin-related mannose-binding-type plant lectins such as Banlec. ZG16b also has a ß-prism fold, but some amino acid residues of the putative sugar-binding site differ from those of the mannose-type binding site suggesting altered preference. A positively charged patch, which may bind sulfated groups of the glycosaminoglycans, is located around the putative sugar-binding site of ZG16p and ZG16b. Taken together, we suggest that the sugar-binding site and the adjacent basic patch of ZG16p and ZG16b cooperatively form a functional glycosaminoglycan-binding site.

DNA templated nucleophilic aromatic substitution reactions for fluorogenic sensing of oligonucleotides.

We have developed an S(N)Ar reaction-triggered fluorescence probe using a new fluorogenic compound derivatized from 7-aminocoumarin for oligonucleotides detection.

Rapid DNA chemical ligation for amplification of RNA and DNA signal.

Enzymatic ligation methods are useful in the diagnostic detection of DNA sequences. Here, we describe the investigation of nonenzymatic phosphorothioate--iodoacetyl DNA chemical ligation as a method for the detection and identification of RNA and DNA. The specificity of ligation on the DNA target is shown to allow the discrimination of a single point mutation with a drop in the ligation yield of up to 16.1-fold. Although enzymatic ligation has very low activity for RNA targets, this reaction is very efficient for RNA targets. The speed of the chemical ligation with an RNA target achieves a 70% yield in 5 s, which is equal to or better than that of ligase-enzyme-mediated ligation with a DNA target. The reaction also exhibits a significant level of signal amplification under thermal cycling in periods as short as 100-120 min, with the RNA or DNA target acting in a catalytic way to ligate multiple pairs of probes.

Multiple chemical ligation under thermal cycle.

Enzymatic ligation methods are useful in diagnostic detection of DNA sequence. Here we describe the investigation of nonezymatic phosphorothioate-iodoacetyl DNA chemical ligation as a method for detection and identification of RNA and DNA. Specificity of ligation on DNA target is shown to yield discrimination of single point mutation as a drop in two magnitude. Although enzymatic ligation shows very low activity for RNA target, this reaction is found to be very efficient on RNA target. This chemical ligation with RNA target completes 70% within a few seconds, which equal or overcome ligase enzyme-mediated ligation with DNA target. The reaction is also shown to exhibit a significant level of signal amplification under thermal cycle for short time. Further, we found recently that ligation fidelity changed in function of chemical reactivity of probe. This trend was systematically investigated.

Pentamer is the minimum structure for oligomannosylpeptoids to bind to concanavalin A.

Enzyme-linked lectin assay (ELLA) was performed for oligomannosylpeptoids, which were immobilized on microtiter plates through a streptavidin-biotin interaction. The other immobilization methods, a hydrophobic adsorption and a covalent attachment, were found inapplicable to the oligomannosylpeptoids. Penta- and hexamannosylpeptoids with a shorter or longer spacer were found to be significantly recognized by concanavalinA (ConA), while the smaller peptoids showed no bindings. A proportional relationship between the amount of bound ConA and the peptoid density on the microtiter plate was observed, indicating the absence of both cluster and overdense effects that would assist or inhibit the binding increasingly with the ligand density.