The glycosyltransferase ST6Gal-I is enriched in cancer stem-like cells in colorectal carcinoma and contributes to their chemo-resistance.

PURPOSE: Presence of cancer stem cells (CSCs) contributes to tumor outgrowth, chemo-resistance and relapse in some cancers including colorectal carcinoma (CRC). The current characterization methods of CSCs in CRC only allows enrichment of CSCs but not their purification. Recent reports showed that ST6 beta-galactoside alpha-2,6-sialyltransferase 1 (ST6Gal-I) plays an essential role in protecting tumor cells against harsh environment like oxidative stress and nutrient deprivation. Therefore, whether ST6Gal-I may be highly expressed in CSCs or whether it may enhance resistance of tumor cells to chemotherapy deserves exploration. METHOD: ST6Gal-I levels were determined in CRC specimens, compared to paired normal colorectal tissue, and examined in CD133+ vs CD133- CRC cells, and CD44+ vs CD44- CRC cells. ST6Gal-I levels and their association with patient survival were examined. In vivo, 2 CRC cell lines Caco-2 and SW48 were transduced with two lentiviruses, one lentivirus carrying a green fluorescent protein reporter and a luciferase reporter under a cytomegalovirus promoter to allow tracing tumor cells by both fluorescence and luciferase activity, and one lentivirus carrying a nuclear red fluorescent protein under the control of ST6Gal-I promoter to allow separation of ST6Gal-I+ vs ST6Gal-I- CRC cells. Tumor sphere formation, resistance to fluorouracil-induced apoptosis, and frequency of tumor formation after serial adoptive transplantation were done on ST6Gal-I+ vs ST6Gal-I- CRC cells. RESULT: ST6Gal-I levels were significantly upregulated in clinically obtained CRC specimens, compared to paired normal colorectal tissue. Poorer patient survival was detected in ST6Gal-I-high CRC, compared to ST6Gal-I-low subjects. Higher levels of ST6Gal-I were detected in CD133+ CRC cells than CD133- CRC cells, and in CD44+ CRC cells than in CD44- CRC cells. Compared to ST6Gal-I- CRC cells, ST6Gal-I+ CRC cells generated significantly more tumor spheres in culture, were more resistant to fluorouracil-induced apoptosis likely through upregulating cell autophagy, and generated tumor more frequently after serial adoptive transplantation. CONCLUSION: ST6Gal-I may be highly expressed in the cancer stem-like cells in CRC and enhances cancer cell resistance to chemotherapy.

Sialic acid expression in the mosquito Aedes aegypti and its possible role in dengue virus-vector interactions.

Dengue fever (DF) is the most prevalent arthropod-borne viral disease which affects humans. DF is caused by the four dengue virus (DENV) serotypes, which are transmitted to the host by the mosquito Aedes aegypti that has key roles in DENV infection, replication, and viral transmission (vector competence). Mosquito saliva also plays an important role during DENV transmission. In this study, we detected the presence of sialic acid (Sia) in Aedes aegypti tissues, which may have an important role during DENV-vector competence. We also identified genome sequences encoding enzymes involved in Sia pathways. The cDNA for Aedes aegypti CMP-Sia synthase (CSAS) was amplified, cloned, and functionally evaluated via the complementation of LEC29.Lec32 CSAS-deficient CHO cells. AedesCSAS-transfected LEC29.Lec32 cells were able to express Sia moieties on the cell surface. Sequences related to α-2,6-sialyltransferase were detected in the Aedes aegypti genome. Likewise, we identified Sia-α-2,6-DENV interactions in different mosquito tissues. In addition, we evaluated the possible role of sialylated molecules in a salivary gland extract during DENV internalization in mammalian cells. The knowledge of early DENV-host interactions could facilitate a better understanding of viral tropism and pathogenesis to allow the development of new strategies for controlling DENV transmission.

Hormonal regulation of pituitary FSH sialylation in male rats.

Sialic acid content in FSH is modulated by GnRH and sexual steroids. Galbeta1,3GlcNAcalpha2,3-sialyltransferase (ST3Gal III) and Galbeta1,4GlcNAcalpha2,6-sialyltransferase (ST6Gal I) incorporate sialic acid residues into FSH oligosaccharides. The aim of the present study was to assess pituitary FSH molecular microheterogeneity and ST3Gal III/ST6Gal I expression during sexual development and after castration in male rats. Preparative isoelectric focusing and lectin chromatography were used to isolate FSH glycosylation variants according to charge and complexity of their oligosaccharides; RT-PCR and immunohistochemistry were employed to analyse sialyltransferase expression. Sexual development was associated with a progressive shift towards more acidic/sialylated FSH glycoforms concomitantly with an increment in ST6Gal I gene and protein expression. After castration, a transient decrease followed by a marked increase in ST6Gal I expression were observed. Less acidic/sialylated FSH glycoforms bearing incomplete oligosaccharides increased after castration, despite high ST6Gal I expression. ST3Gal III expression remained unchanged in all the experimental conditions examined. These results show that the synthesis of FSH isoforms possessing alpha2,6-linked sialic acid is hormonally regulated in male rats.

Stable expression of a human-like sialylated recombinant thyrotropin in a Chinese hamster ovary cell line expressing alpha2,6-sialyltransferase.

A CHO cell line, previously genetically modified by the introduction of rat alpha2,6-sialyltransferase cDNA, generated for the first time a human-like sialylated recombinant hTSH (hlsr-hTSH) more similar to the native hormone, with 61% of alpha2,3- and 39% of alpha2,6-linked sialic acid residues. The best clone, when submitted to gene amplification with up to 8 microM methotrexate, presented a secretion level of approximately 2 microg hTSH/10(6)cells/day, useful for product purification and characterization. The relative molecular masses (M(r)) of the heterodimer and of the alpha- and beta-subunits of purified hlsr-hTSH, determined by MALDI-TOF mass spectrometry, and the relative hydrophobicities, determined by RP-HPLC, were not remarkably different from those presented by two r-hTSH preparations secreted by normal CHO cells. Some differences were observed, though, in N-glycan composition, with more tri- and much more tetra-sialylated structures in hlsr-hTSH. When analyzed via an in vivo bioassay based on hTSH-induced T(4) release in mice, hlsr-hTSH was shown to be equipotent (p>0.05) with the commercial preparation of r-hTSH (Thyrogen), and 1.6-fold more potent than native hTSH (p<0.001).

[Role of sialyltransferase and neuraminidase in the epididymis of rats]. / Papel de la sialiltransferasa y neuramidasa en el epidídimo de la rata.

The sialylation and desialylation in the rat epididiymis is the transference or remotion of sialic acid, to or from an adequate acceptor (glyco or sialogycoprotein). The sialylation of the glycoprotein or glycolipids is determinated by sialyltransferase activity; the homogenates from caput region showed 14 times more sialyltransferase activity than homogenates from cauda epididymis. The desialylation of the sialoglycoproteins is quantified by neuraminidase activity, and it can be observed that the homogenate from the caput zone shows 3 times more activity than homogenates from cauda epididymis. When relatione the activities of sialytransferase/neuraminidase of the caput zone and the cauda zone, the obtained values were 0.19 +/- 0.03 and 0.04 +/- 0.005, respectively. The relation between the activities of neuraminidase/sialiltransferase of the caput and the cauda zone showed the following valves: 5 +/- 0.6 and 25 +/- 3.3, respectively. The results have shown that sialylation is more active in the caput than in the cauda. Besides, while valvating the desialylation we observe that it is more effective in the cauda epididymis.

Purification and characterization of an endogenous inhibitor of the sialyltransferase CMP-N-acetylneuraminate: lactosylceramide alpha 2,6-N-acetylneuraminyltransferase (EC 2.4.99.-).

The presence in the 100,000 g supernatant of rat brain homogenate of an inhibitor of the sialyltransferase has been confirmed. It is also present in chicken and bovine brain and in other rat and bovine organs. The inhibitor has been purified, a preparation with a specific activity 130-fold higher than that of the original 100,000 g supernatant of brain being obtained. It runs as a single peak in polyacrylamide-gel electrophoresis; when run in the presence of SDS, two components appeared. The apparent Mr of the components were 14,800 and 22,400. The inhibitor has been characterized as a heat-stable protein of acidic nature. It has effect on the glycolipid and the glycoprotein sialyltransferase activities but has no effect on the galactosaminyltransferase activity.

Direct sialic acid transfer from a protein donor to glycolipids of trypomastigote forms of Trypanosoma cruzi.

Labeled sialoglycolipids were purified from tissue culture-derived trypomastigotes incubated with [3H]fetuin. Thin layer chromatography of [3H]sialoglycolipids showed three components with the same migration as gangliosides extracted from parasites incubated with [3H]palmitic acid. Neuraminidase treatment or mild acid hydrolysis confirmed the presence of [3H]sialyl residues in sialoglycolipids synthesized after [3H]fetuin incubation. Labeling was not observed when parasites were incubated with free [3H]sialic acid (C7 derivative), suggesting that sialyl residues are directly transferred in vivo to gangliosides, by an enzymatic reaction possibly catalysed by a sialyl transferase (transglycosylase). Sonicated extracts of trypomastigotes incubated with [3H]fetuin catalysed the labeling of endogenous glycoconjugates as well as of bovine brain gangliosides. The transglycosylase activity was found associated with the particulate fraction and could be solubilized with Triton X-100. The specific activity of the sialic acid transglycosylase in epimastigotes is 17% of that found in trypomastigotes. Addition of an excess free sialic acid did not inhibit the reaction, suggesting that transfer does not occur via a pool of free sialic acid.