Sianturi J, Manabe Y, Li HS, Chiu LT, Chang TC, et al.
Angewandte Chemie (International ed. in English) •
Cancer treatment with antibodies (Abs) is one of the most successful therapeutic strategies for obtaining high selectivity. In this study, α-gal-Ab conjugates were developed that dramatically increased cellular cytotoxicity by recruiting natural Abs through the interaction between α-gal and anti-gal Abs. The potency of the α-gal-Ab conjugates depended on the amount of α-gal conjugated to the antibody: the larger the amount of α-gal introduced, the higher the level of cytotoxicity observed. The conjugation of antibodies with an α-gal dendrimer allowed the introduction of large amounts of α-gal to the Ab, without loss of affinity for the target cell. The method described here will enable the re-development of Abs to improve their potency.
The oligosaccharides from fission yeast Schizosaccharomyces pombe contain large amounts of D-galactose (Gal) in addition to D-mannose (Man), in contrast to the budding yeast Saccharomyces cerevisiae. Detailed structural analysis has revealed that the Gal residues are attached to the N- and O-linked oligosaccharides via α1,2- or α1,3-linkages. Previously we constructed and characterized a septuple α-galactosyltransferase disruptant (7GalTΔ) anticipating a complete lack of α-Gal residues. However, the 7GalTΔ strain still contained oligosaccharides consisting of α1,3-linked Gal residues, indicating the presence of at least one more additional unidentified α1,3-galactosyltransferase. In this study we searched for unidentified putative glycosyltransferases in the S. pombe genome sequence and identified three novel genes, named otg1(+)-otg3(+) (α one, three-galactosyltransferase), that belong to glycosyltransferase gene family 8 in the Carbohydrate Active EnZymes (CAZY) database. Gal-recognizing lectin blotting and HPLC analyses of pyridylaminated oligosaccharides after deletion of these three additional genes from 7GalTΔ strain demonstrated that the resultant disruptant missing 10 α-galactosyltransferase genes, 10GalTΔ, exhibited a complete loss of galactosylation. In an in vitro galactosylation assay, the otg2(+) gene product had Gal transfer activity toward a pyridylaminated Man(9)GlcNAc(2) oligosaccharide and pyridylaminated Manα1,2-Manα1,2-Man oligosaccharide. In addition, the otg3(+) gene product exhibited Gal transfer activity toward the pyridylaminated Man(9)GlcNAc(2) oligosaccharide. Generation of an α1,3-linkage was confirmed by HPLC analysis, α-galactosidase digestion analysis, (1)H NMR spectroscopy, and LC-MS/MS analysis. These results indicate that Otg2p and Otg3p are involved in α1,3-galactosylation of S. pombe oligosaccharides.
Korekane H, Korekane A, Yamaguchi Y, Kato M, Miyamoto Y, et al.
Glycoconjugate journal •
Extracellular superoxide dismutase (EC-SOD), the major SOD isoenzyme in biological fluids, is known to be N-glycosylated and heterogeneous as was detected in most glycoproteins. However, only one N-glycan structure has been reported in recombinant human EC-SOD produced in Chinese hamster ovary (CHO) cells. Thus, a precise N-glycan profile of the recombinant EC-SOD is not available. In this study, we report profiling of the N-glycan in the recombinant mouse EC-SOD produced in CHO cells using high-resolution techniques, including the liberation of N-glycans by treatment with PNGase F, fluorescence labeling by pyridylamination, characterization by anion-exchange, normal and reversed phase-HPLC separation, and mass spectrometry. We succeeded in identifying 26 different types of N-glycans in the recombinant enzyme. The EC-SOD N-glycans were basically core-fucosylated (98.3% of the total N-glycan content), and were high mannose sugar chain, and mono-, bi-, tri-, and tetra-antennary complex sugar chains exhibiting varying degrees of sialylation. Four of the identified N-glycans were uniquely modified with a sulfate group, a Lewis(x) structure, or an α-Gal epitope. The findings will shed new light on the structure-function relationships of EC-SOD N-glycans.
Fujimura T, Takahagi Y, Shigehisa T, Nagashima H, Miyagawa S, et al.
Molecular reproduction and development •
The objective of the present study was to isolate alpha 1,3-galactosyltransferase (GalGT)-gene double knockout (DKO) cells using a novel simple method of cell selection method. To obtain GalGT-DKO cells, GalGT-gene single knockout (SKO) fetal fibroblast cells were cultured for three to nine passages and GalGT-null cells were separated using a biotin-labeled IB4 lectin attached to streptavidin-coated magnetic beads. After 15-17 days of additional cultivation, seven GalGT-DKO cell colonies were obtained from a total of 2.5 x 10(7) GalGT-SKO cells. A total of 926 somatic nuclear transferred embryos reconstructed with the DKO cells were transferred into eight recipient pigs, producing four farrowed, three liveborns, and six stillborns. Absence of GalGT gene in the cloned pigs was confirmed by PCR and Southern blotting. Flow cytometric analysis revealed that alphaGal antigens were not present in the cells of the cloned DKO pigs.
Miyao Y, Ikenaka K, Kishima H, Tamura M, Nakamura K, et al.
Human gene therapy •
Serum-induced inactivation of retroviruses is the most critical limitation for in vivo gene transfer therapy. To solve this problem, we searched for reagents that protect retroviruses from inactivation. The effects of the protease inhibitors FOY-007 and FOY-305 and of an inhibitor of the complement pathway FUT-175, all of which have been used clinically, were investigated. All of these agents protected against the inactivation of retroviruses by human serum, with 1 microM FUT-175 providing the most effective protection. Thus, the co-administration of FUT-175 with retroviruses may make retrovirus-mediated in vivo gene transfer feasible for the treatment of patients. FUT-175 dose-dependently inhibited the classical pathway of complement in a hemolysis protection assay of sensitized sheep erythrocytes with guinea pig serum or by cell-lysis assay of mouse fibroblasts with human serum. However, increasing the FUT-175 concentration by 10-fold (10 microM) did not produce further protection against retroviral inactivation in most human sera. There was also no correlation between the serum-induced inactivation of retroviruses and either the amount of anti-alpha-galactosyl (anti-alpha-Gal) antibody or the complement activity in human serum. These results suggest that retroviruses are not inactivated by utilizing the same pathway leading to cell lysis by the classical complement system.
