Gene expression levels of ß4-galactosyltransferase 5 correlate with the tumorigenic potentials of B16-F10 mouse melanoma cells.

Our previous studies showed that mouse ß4-galactosyltransferase 5 (ß4GalT5) is a lactosylceramide (Lac-Cer) synthase, and that its gene expression increases by 2- to 3-fold upon malignant transformation of cells. In the present study, we examined whether or not the tumorigenic and metastatic potentials of B16-F10 mouse melanoma cells can be suppressed by reducing the expression of the ß4GalT5 gene. We isolated a stable clone named E5 whose ß4GalT5 gene expression level was reduced to 35% that of a control clone C1 by transfection of its antisense cDNA. Thin-layer chromatography analysis of glycosphingolipids showed that the amounts of Lac-Cer and ganglioside GM3 are significantly less in clone E5 than in clone C1. Clone C1 and E5 cells were each transplanted subcutaneously or injected intravenously into C57BL/6 mice, and the sizes of tumors and numbers of colonies formed in the lungs were determined. The average tumor size and average number of colonies formed with clone E5 were decreased to 44 and 49%, respectively, of those formed with clone C1. Furthermore, the numbers and sizes of colonies formed in the soft agarose gels, and the volumes of tumors formed in athymic mice with fibroblasts from wild type, heterozygous and homozygous ß4GalT5-knockout mouse embryos upon transformation with the polyoma virus oncogene correlated with the ß4GalT5 gene dosage. These results strongly indicate that the amounts of Lac-Cer synthesized by ß4GalT5 correlate with the tumorigenic potentials of malignantly transformed cells.

Involvement of murine ß-1,4-galactosyltransferase V in lactosylceramide biosynthesis.

Human ß-1,4-galactosyltransferase (ß-1,4-GalT) V was shown to be involved in the biosynthesis of N-glycans, O-glycans and lactosylceramide (Lac-Cer) by in vitro studies. To determine its substrate specificity, enzymatic activity and its products were analyzed using mouse embryonic fibroblast (MEF) cells from ß-1,4-GalT V (B4galt5)-mutant mice. Analysis of expression levels of the ß-1,4-GalT I-VI genes revealed that the expression of the ß-1,4-GalT V gene in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells are a half and null when compared to that of B4galt5 ( +/+ )-derived MEF cells without altering the expression levels of other ß-1,4-GalT genes. These MEF cells showed no apparent difference in their growth. When ß-1,4-GalT activities were determined towards GlcNAcß-S-pNP, no significant difference in its specific activity was obtained among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. No significant differences were obtained in structures and amounts of N-glycans and lectin bindings to membrane glycoproteins among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. However, when cell homogenates were incubated with glucosylceramide in the presence of UDP-[(3)H]Gal, Lac-Cer synthase activity in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells decreased to 41% and 11% of that of B4galt5 ( +/+ )-derived MEF cells. Consistent with this, amounts of Lac-Cer and its derivative GM3 in B4galt5 ( -/- ) -derived MEF cells decreased remarkably when compared with those of B4galt5 ( +/+ )-derived MEF cells. These results indicate that murine ß-1,4-GalT V is involved in Lac-Cer biosynthesis.

Comparative proteomics analysis of differentially expressed proteins in soybean cell wall during flooding stress.

Flooding is a major problem for soybean crop as it reduces the growth and grain yield. To investigate the function of the soybean cell wall in the response to flooding stress, cell wall proteins were analyzed. Cell wall proteins from roots and hypocotyls of soybeans, which were germinated for 2 days and subjected to 2 days of flooding, were purified, separated by two-dimensional polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. Sixteen out of 204 cell wall proteins showed responses to flooding stress. Of these, two lipoxygenases, four germin-like protein precursors, three stem 28/31 kDa glycoprotein precursors, and one superoxide dismutase [Cu-Zn] were downregulated. A copper amine oxidase was found to have shifted from the basic to acidic zone following flooding stress. Based on these results, it was confirmed by the lignin staining that the lignification was suppressed in the root of soybean under the flooding stress. These results suggest that the roots and hypocotyls of soybean caused the suppression of lignification through decrease of these proteins by downregulation of reactive oxygen species and jasmonate biosynthesis under flooding stress.