Molecular cloning of pigGnT-I and I.2: an application to xenotransplantation

Xenotransplantation is one of the most attractive solutions for the current worldwide shortage of organs. The knocking out of alpha1,3-galactosyltransferase in pigs resulted in a drastic reduction in xenoantigenicity. However, more recent studies indicate that other xeno-antigens, so-called non-Gal antigens, will also need to be downregulated. In this study, pig N-acetylglucosaminyltransferase I (GnT-I), a key enzyme that initiates the biosynthesis of hybrid- and complex-type N-linked sugar chains, was isolated and the pigGnT-I.2 specific for the O-linked sugar chain was also isolated. Point mutants, pigGnT-I(123) and pigGnT-I(320), were subsequently constructed. While pigGnT-I(123) shows an indistinct dominant negative effect for endogenous GnT-I in pig cells, pigGnT-I(320) had a drastic effect. In addition, in the case of pig cell transfectants with pigGnT-I(320), cell surface carbohydrate structures were significantly altered and its antigenicity to human serum was reduced. Consequently, pigGnT-I(320) appears to be potentially useful in xenotransplantation by remodeling the carbohydrate structures on pig cells.     

Concentration measurements of sucrose and sugar surfactants solutions by using the 1H NMR ERETIC method

The ERETIC method has been used to determine precise concentrations of aqueous solutions of sucrose and sugar surfactants, namely octyl glucoside and fatty acid sucrose esters by (1)H NMR spectroscopy. The effects of NMR tuning, acquisition parameters, and spectrum processing on the measurement have been assessed in these particular cases. The linearity upholds over the whole concentration range, with both sucrose and octyl glucoside, whatever the physicochemical phenomena occurring, either an increasing viscosity or the micellization of the surfactant. For sucrose solutions, an accuracy of 2% is measured for concentrations between 0.1 and 200 mmol/L, which is consistent with literature data.     

Facile synthesis toward the construction of an activity probe library for glycosidases

Chemical probes that selectively label the glycoside hydrolase (GH) subfamilies have proven to be a powerful tool in GH-related research. We have previously demonstrated the design and synthesis of an activity probe for beta-glucosidase adopting a cassette-like design in a model study. Herein we report an improved synthetic route using (4-hydroxyphenyl)acetic acid 2-cyanoethyl ester as the precursor for the latent trapping device. Parallel syntheses were performed for the preparation of a library based on the structure of a key intermediate. The recognition head of this library covers a series of six sugars, including alpha- and beta-d-Glc, alpha- and beta-d-Gal, alpha-d-Man, and alpha-l-Fuc. Each member in this versatile intermediate library could serve as the building block in constructing an activity probe for GHs. As demonstrated in this study, three probes that have the 1,2-cis configuration were thus prepared for the first time to target alpha-d-glucosidase, alpha-d-galactosidase, and alpha-l-fucosidase, respectively.     

A role for the L-selectin adhesion system in mediating cytotrophoblast emigration from the placenta

Cytotrophoblast (CTB) aggregates that bridge the gap between the placenta and the uterus are suspended as cell columns in the intervillous space, where they experience significant amounts of shear stress generated by maternal blood flow. The proper formation of these structures is crucial to pregnancy outcome as they play a vital role in anchoring the embryo/fetus to the decidua. At the same time, they provide a route by which CTBs enter the uterine wall. The mechanism by which the integrity of the columns is maintained while allowing cell movement is unknown. Here, we present evidence that the interactions of L-selectin with its carbohydrate ligands, a specialized adhesion system that is activated by shear stress, play an important role. CTBs in cell columns, particularly near the distal ends, stained brightly for L-selectin and with the TRA-1-81 antibody, which recognizes carbohydrate epitopes that support binding of L-selectin chimeras in vitro. Function-perturbing antibodies that inhibited either receptor or ligand activity also inhibited formation of cell columns in vitro. Together, these results suggest an autocrine role for the CTB L-selectin adhesion system in forming and maintaining cell columns during the early stages of placental development, when the architecture of the basal plate region is established. This type of adhesion may also facilitate CTB exit from cell columns, a prerequisite for uterine invasion.     

A label-free continuous total-internal-reflection-fluorescence-based immunosensor

In this study, we continuously monitored, second-by-second, concentration changes of two different carbohydrates (maltose and panose) by using monoclonal antibodies in an optical immunosensor based on total internal reflection fluorescence. Earlier studies have demonstrated that these antibodies increase their intrinsic tryptophan fluorescence upon binding of carbohydrate antigens. Using the four immobilized monoclonal antibodies with low affinities (K(d)>10(-6)M), fast kinetics (k(off)>1s(-1)), and high reversibility gave opportunities for developing a continuous immunosensor without any need for regeneration. Since intrinsic fluorescence was used, no extrinsic labeling was necessary. Sensitivity was in the range of 1-5 microM for panose, and 10-15 microM for maltose and the loss of intensity was as low as 3.5% per hour during measurements. Calculations of DeltaH degrees and DeltaS degrees from the temperature dependence of K(d) indicated an enthalpic driven antigen-antibody binding event that is diminished upon antibody immobilization. We feel certain that weakly interacting antibodies can be used in future applications for continuous monitoring where there is a need to achieve instantaneous information on the concentration of an analyte.     

Extending Tlusty's rate distortion index theorem method to the glycome: Do even ‘low level’ biochemical phenomena require sophisticated cognitive paradigms?

Unlike the universal genetic code and ordered protein folding, direct application of Tlusty's method to the glycome produces a reducto ad absurdum: From the beginning a complicated system of chemical cognition is needed so that external information constrains and tunes what would otherwise be a monstrously large 'glycan code error network'. Further, the glycan manufacture machinery itself must be regulated by yet other levels of chemical cognition to ensure that what is produced matches what was chosen for production. Application of a rate distortion index theorem/operator method at this second stage appears possible, permitting analytic characterization of the complicated 'glycan spectra' associated with cellular interactions and their dynamics. The regulation of 'low level' biochemical processes, ranging from gene expression and protein folding through the production of flexible glycan surface signalling fronds, appears to require systems of chemical cognition whose sophistication may rival that of high order neural process.     

Molecular recognition of Candida albicans (1->2)-β-mannan oligosaccharides by a protective monoclonal antibody reveals the immunodominance of internal saccharide residues

A self-consistent model of β-mannan oligosaccharides bound to a monoclonal antibody, C3.1, that protects mice against Candida albicans has been developed through chemical mapping, NMR spectroscopic, and computational studies. This antibody optimally binds di- and trisaccharide epitopes, whereas larger oligomers bind with affinities that markedly decrease with increasing chain length. The (1→2)-β-linked di-, tri-, and tetramannosides bind in helical conformations similar to the solution global minimum. Antibody recognition of the di- and trisaccharide is primarily dependent on the mannose unit at the reducing end, with the hydrophobic face of this sugar being tightly bound. Recognition of a tetrasaccharide involves a frameshift in the ligand interaction, shown by strong binding of the sugar adjacent to the reducing end. We show that frameshifting may also be deliberately induced by chemical modifications. Molecular recognition patterns similar to that of mAb C3.1, determined by saturation transfer difference-NMR, were also observed in polyclonal sera from rabbits immunized with a trisaccharide glycoconjugate. The latter observation points to the importance of internal residues as immunodominant epitopes in (1→2)-β-mannans and to the viability of a glycoconjugate vaccine composed of a minimal length oligosaccharide hapten.     

Drosophila heparan sulfate, a novel design

Heparan sulfate (HS) proteoglycans play critical roles in a wide variety of biological processes such as growth factor signaling, cell adhesion, wound healing, and tumor metastasis. Functionally important interactions between HS and a variety of proteins depend on specific structural features within the HS chains. The fruit fly (Drosophila melanogaster) is frequently applied as a model organism to study HS function in development. Previous structural studies of Drosophila HS have been restricted to disaccharide composition, without regard to the arrangement of saccharide domains typically found in vertebrate HS. Here, we biochemically characterized Drosophila HS by selective depolymerization with nitrous acid. Analysis of the generated saccharide products revealed a novel HS design, involving a peripheral, extended, presumably single, N-sulfated domain linked to an N-acetylated sequence contiguous with the linkage to core protein. The N-sulfated domain may be envisaged as a heparin structure of unusually low O-sulfate content.     

Characterization of a novel Agrobacterium tumefaciens galactarolactone cycloisomerase enzyme for direct conversion of D-galactarolactone to 3-deoxy-2-keto-L-threo-hexarate

Microorganisms use different pathways for D-galacturonate catabolism. In the known microbial oxidative pathway, D-galacturonate is oxidized to D-galactarolactone, the lactone hydrolyzed to galactarate, which is further converted to 3-deoxy-2-keto-hexarate and α-ketoglutarate. We have shown recently that Agrobacterium tumefaciens strain C58 contains an uronate dehydrogenase (At Udh) that oxidizes D-galacturonic acid to D-galactarolactone. Here we report identification of a novel enzyme from the same A. tumefaciens strain, which we named Galactarolactone cycloisomerase (At Gci) (E.C. 5.5.1.-), for the direct conversion of the D-galactarolactone to 3-deoxy-2-keto-hexarate. The At Gci enzyme is 378 amino acids long and belongs to the mandelate racemase subgroup in the enolase superfamily. At Gci was heterologously expressed in Escherichia coli, and the purified enzyme was found to exist as an octameric form. It is active both on D-galactarolactone and D-glucarolactone, but does not work on the corresponding linear hexaric acid forms. The details of the reaction mechanism were further studied by NMR and optical rotation demonstrating that the reaction product of At Gci from D-galactaro-1,4-lactone and D-glucaro-1,4-lactone conversion is in both cases the L-threo form of 3-deoxy-2-keto-hexarate.     

Targeting of Several Glycolytic Enzymes Using RNA Interference Reveals Aldolase Affects Cancer Cell Proliferation through a Non-glycolytic Mechanism

In cancer, glucose uptake and glycolysis are increased regardless of the oxygen concentration in the cell, a phenomenon known as the Warburg effect. Several (but not all) glycolytic enzymes have been investigated as potential therapeutic targets for cancer treatment using RNAi. Here, four previously untargeted glycolytic enzymes, aldolase A, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase, and enolase 1, are targeted using RNAi in Ras-transformed NIH-3T3 cells. Of these enzymes, knockdown of aldolase causes the greatest effect, inhibiting cell proliferation by 90%. This defect is rescued by expression of exogenous aldolase. However, aldolase knockdown does not affect glycolytic flux or intracellular ATP concentration, indicating a non-metabolic cause for the cell proliferation defect. Furthermore, this defect could be rescued with an enzymatically dead aldolase variant that retains the known F-actin binding ability of aldolase. One possible model for how aldolase knockdown may inhibit transformed cell proliferation is through its disruption of actin-cytoskeleton dynamics in cell division. Consistent with this hypothesis, aldolase knockdown cells show increased multinucleation. These results are compared with other studies targeting glycolytic enzymes with RNAi in the context of cancer cell proliferation and suggest that aldolase may be a useful target in the treatment of cancer.