Insulin/protein kinase B signalling pathway upregulates metastasis-related phenotypes and molecules in H7721 human hepatocarcinoma cell line.

The effect of insulin on cancer metastatic potential was studied in a human hepatocarcinoma cell line, H7721. Cell adhesion to human umbilical vein endothelial cells (HUVECs) and laminin as well as chemotactic cell migration and invasion were selected as the indices of metastasis-related phenotypes for assessment of metastatic potential ex vivo. The results indicated that insulin enhanced all of these metastasis-related phenotypes. After the cells were treated with specific inhibitor of PI3K (LY294002) or transfected with antisense cDNA of PKB (AS-PKB), all of the above phenotypes were attenuated, and they could not be significantly stimulated by insulin, indicating that the insulin effect on metastatic potential was mediated by PI3K and PKB. Only the monoclonal antibody to the sialyl Lewis X (SLe(x)), but not antibodies to other Lewis antigens, significantly blocked the cell adhesion to HUVECs, cell migration and invasion, suggesting that SLe(x) played a crucial role in the metastatic potential of H7721 cells. The upregulation of cell surface SLe(x) and alpha-1,3-fucosyltransferase-VII (alpha-1,3 Fuc T-VII, enzyme for SLe(x) synthesis) was also mediated by PI3K and PKB, since LY294002 and AS-PKB also reduced the expressions of SLe(x) and alpha-1,3 FucT-VII, and attenuated the response to insulin. Furthermore, the alterations in the expressions of PKB protein and activity were correlated to the changes of metastatic phenotypes and SLe(x) expression. Taken together, the insulin/PKB signalling pathway participated in the enhancement of metastatic potential of H7721 cells, which was mediated by the upregulation of the expression of SLe(x) and alpha-1,3 FucT-VII.

[Regulation by ovarian hormones of alpha 1,3-fucosyltransferase gene (FUT9) expression in human endometrium].

Recently it was found that alpha1,3-fucosyltransferase (FUT9) was an essential enzyme involved in the synthesis of Le(X) oligosaccharide. In this article, the expression of FUT9 gene at different hormonal levels and the regulating mechanism of Le(X) synthesis in human endometrium were investigated by using RT-PCR Immuno histochemistry staining and Western blotting. FUT9 mRNA was detected with human endometrium, and the levels of mRNA in secretory endometrium and in proliferative endometrium from patients who had taken mifepristone before operation, were higher than tha t of proliferative endometrium (P<0.01). Le(X) antigen was mainly located on the surface of epithelium. The floating level of Le(X) antigen expression was similar to that of FUT9 gene. There were three (33 kD, 35 kD and 85 kD) LeX carrying proteins expressed in human endometrium, mifepristone treatment made 35 kD b and disappear. The results indicate that FUT9 gene is obviously expressed in human endometrium and can be upregulated by progesterone, and ovary hormones regulate the expression of Le(X) at the level of transcription.

Soyasaponin I, a potent and specific sialyltransferase inhibitor.

A growing number of reports demonstrate that hypersialylation, which is observed in certain pathological processes, such as oncogenic transformation, tumor metastasis, and invasion, is associated with enhanced sialyltransferase (ST) activity. There is therefore a need for the development of ST inhibitors to modulate ST activity and thus alleviate the disease processes caused by STs. In the present study, soyasaponin I had been discovered to be a potent and specific ST inhibitor by screening strategy from 7500 samples including micribial extracts and natural products. Kinetic analysis shows that it is a CMP-Neu5Ac competitive inhibitor with for ST3Gal I with an inhibition constant (K(i)) of 2.1 microM. In addition, it is only active against ST, but not against the other tested glycosyltransferases and glycosidases. Our study is the first report to discover ST inhibitor by screening method and also to provide the new chemical structure information that should be useful in the development of other novel ST inhibitors.

The ruminant parasite Haemonchus contortus expresses an alpha1,3-fucosyltransferase capable of synthesizing the Lewis x and sialyl Lewis x antigens.

Glycoproteins from the ruminant helminthic parasite Haemonchus contortus react with Lotus tetragonolobus agglutinin and Wisteria floribunda agglutinin, which are plant lectins that recognize alpha1,3-fucosylated GlcNAc and terminal beta-GalNAc residues, respectively. However, parasite glycoconjugates are not reactive with Ricinus communis agglutinin, which binds to terminal beta-Gal, and the glycoconjugates lack the Lewis x (Le(x)) antigen or other related fucose-containing antigens, such as sialylated Le(x), Le(a), Le(b) Le(y), or H-type 1. Direct assays of parasite extracts demonstrate the presence of an alpha1,3-fucosyltransferase (alpha1,3FT) and beta1,4-N-acetylgalactosaminyltransferase (beta1,4GalNAcT), but not beta1,4-galactosyltransferase. The H. contortus alpha1,3FT can fucosylate GlcNAc residues in both lacto-N-neotetraose (LNnT) Galalpha1-->4GlcNAcbeta1-->3Galbeta1-->4Glc to form lacto-N-fucopentaose III Galbeta1-->4[Fuca1-->3]GlcNAc beta1-->3Galbeta1-4GIc, which contains the Le(x) antigen, and the acceptor lacdiNAc (LDN) GalNAcbeta1-->4GlcNAc to form GalNAc beta1-->4[Fualpha1-->3]GlcNAc. The alpha1,3FT activity towards LNnT is dependent on time, protein, and GDP-Fuc concentration with a Km 50 microM and a Vmax of 10.8 nmol-mg(-1) h(-1). The enzyme is unusually resistant to inhibition by the sulfhydryl-modifying reagent N-ethylmaleimide. The alpha1,3FT acts best with type-2 glycan acceptors (Galbeta1-->4GlcNAcbeta1-R) and can use both sialylated and non-sialylated acceptors. Thus, although in vitro the H. contortus alpha1,3FT can synthesize the Le(x) antigen, in vivo the enzyme may instead participate in synthesis of fucosylated LDN or related structures, as found in other helminths.

The alpha 1-->3 fucosylation at the penultimate GlcNAc catalyzed by fucosyltransferase VII is blocked by internally fucosylated residue in sialosyl long-chain poly-LacNAc: enzymatic basis for expression of physiological E-selectin epitope.

Sialosyl-fucosyl poly-LacNAc without sialosyl-Lex epitope in myeloid cell line HL60 was shown to be the ligand for E-selectin-dependent adhesion, particularly under dynamic flow conditions, in our previous study (Handa K, Stroud MR, Hakomori S, Biochemistry 36, 12412-12420, 1997). HL60 cells express only fucosyl-transferase (FT) IV and VII. X3NeuAcVII3FucnLc10, a representative component showing E-selectin-dependent binding under dynamic flow conditions, is not alpha 1-->3 fucosylated at the penultimate GlcNAc catalyzed by FT-VII, but is alpha 1-->3 fucosylated at the internal GlcNAc catalyzed by FT-IV. VI3NeuAcnLc6 is converted to VI3NeuAcIII3FucnLc6 by FT-IV, but is also converted to VI3NeuAcV3FucnLc6 by FT-VII. Thus, penultimate fucosylation catalyzed by FT-VII is not restricted for nLc6 backbone, but is highly restricted for nLc10 backbone. The cooperative effect of FT-IV and FT-VII for synthesis of poly-LacNAc having sialosyl-Lex with internal fucosylation may be blocked or highly restricted in poly-LacNAc having more than two LacNAc units, because preferential alpha 1-->3 fucosylation by FT-IV takes place at internal GlcNAc, inhibiting penultimate fucosylation by FT-VII.

Elevation of an alpha(1,3)fucosyltransferase activity correlated with apoptosis in the human colon adenocarcinoma cell line, HT-29.

We studied changes in the carbohydrate expression following apoptotic cell death induced by treatment with interferon (IFN)-gamma and anti-Fas antibody using human colon adenocarcinoma HT-29 cells. An apoptotic cell death of HT-29 accompanied with typical DNA fragmentation was observed when the cells, were cultured sequentially with IFN-gamma and anti-Fas antibody. In flow cytometric analyses, the expression of Le(x) and Le(y) antigen was strongly and slightly enhanced, respectively, on the cell surface in accordance with the apoptosis. When the fucosyltransferase (Fuc-T) activities of the lysates from the treated cells were examined relative to those from untreated cells, a 2.5-fold increase of alpha(1,3)-Fuc-T activities and a slight increase of alpha(1,2)-Fuc-T activities were observed, but little or no increase of alpha(1,4)-Fuc-T activity was detected. In Northern blot analyses using probes for Fuc-T III, IV, V, VI and VII genes, strong RNA messages for Fuc-T III, V and/or VI and a weak RNA message for Fuc-T IV were detected in the untreated HT-29 cells. On the other hand, in the treated cells, the messages for Fuc-T III, V and/or VI were found to almost disappear and the 2.3 kb message for Fuc-T IV was observed to elevate 2.8-fold. Therefore, we suggest that the strongly increased expression of Le(x) antigen found on the HT-29 cell surface might be involved in the process of apoptosis, and that the enhancement of the antigen expression seems to result from the increased activity of alpha(1,3)-Fuc-T encoded mainly by the Fuc-T IV gene.

Specificity studies of the GDP-[L]-fucose: 2-acetamido-2-deoxy-beta-[D]-glucoside (Fuc-->Asn-linked GlcNAc) 6-alpha-[L]-fucosyltransferase from rat-liver Golgi membranes.

The specificity of Golgi-membrane glycoprotein 6-alpha-[L]-fucosyltransferase [GDP-[L]-fucose: 2-acetamido-2-deoxy- beta-[D]-glucoside (Fuc-->Asn-linked GlcNAc) 6-alpha-[L]-fucosyltransferase; EC 2.4.1.68] has been assessed with regard to substrate covalent structures and the effect of a protein matrix on the conformational display of those covalent structures. Specificity was studied by direct comparison of the substrate quality of nine 6-biotinamidohexanoylAsn (= R) derivatives of intermediates and products in the pathway from Man5GlcNAc2-R to a fully sialylated biantennary complex-type glycan. The Man5 derivative and the sialic acid-containing glycans were completely inactive as substrates. The other glycans were all fucosylated; the best substrate was GlcNAcMan3GlcNAc2-R. The protein-matrix effect was studied by comparing the substrate quality of the same 6-biotinamidohexanoylAsn derivatives as well as the corresponding biotinylAsn derivatives free in solution and bound to streptavidin. On the basis of a model derived from the known 3D structure of biotin (biocytin)-saturated streptavidin, it was predicted that the fucosylation site in the substrates would be completely masked in the biotin-binding pocket in the biotinyl derivatives (proximal display), and at least partially masked in the 6-biotinamidohexanoyl derivatives (distal display). The activity measurements were in agreement with these predictions; the glycan structures GlcNAcMan5GlcNAc2-, GlcNAcMan3GlcNAc2-, and GlcNAc2-Man3GlcNAc2- were readily fucosylated as derivatives free in solution, but were totally inert in the proximal complex with streptavidin. In the distal complexes the latter two structures were found to be fucosylated very slowly while the former structure was inactive.

Hormonal regulation of glycosylation process in rat small intestine: responsiveness of fucosyl-transferase activity to hydrocortisone during the suckling period, unresponsiveness after weaning.

The aim of this study was to examine the possible role of certain hormones (especially hydrocortisone) in the developmental variations of intestinal fucosyl-transferase activity in rats. Thyroxine and insulin, injected into suckling rats, did not induce significant modifications of the fucosyl-transferase activity, under the conditions used, whereas this enzyme activity was highly enhanced after administration of glucocorticoids (cortisone and hydrocortisone). Hydrocortisone administration to suckling rats induced a precocious and progressive activation of the fucosyl-transferase activity up to adult level as a function of the duration of treatment. The responsiveness of suckling rats to hydrocortisone, as shown by increased fucosyl-transferase activity, disappeared at the end of the third week (corresponding to the weaning time). These physiological periods of responsiveness and unresponsiveness to hydrocortisone could be related to the binding of the hormones to receptors since the antiglucocorticoid RU 38486 counteracted the effect of hydrocortisone in suckling rats but did not prevent the developmental rise of the fucosyl-transferase activity, when administered in the third week of life. These results suggest that the normal developmental rise of the fucosyl-transferase activity is independent of glucocorticoids.