The alpha-1,2 fucosylated tubule system of DU145 prostate cancer cells is derived from a partially fragmented Golgi complex and its formation is actin-dependent.

In previous work, we showed that highly proliferative cells and cancer cells, but not cells with normal growth rate, have tubules rich in alpha-1,2 fucosylated epitopes that extend radially from the nucleus to the cell periphery and form an unusual uptake system. The importance of alpha-1,2 fucosylation in forming tubules was demonstrated by proving that down-regulating the two corresponding fucosyltransferases (FUT1 and FUT2) causes tubule fragmentation. Here, we present evidence that in the prostate cancer cell line DU145, the tubules arise in actively growing cells from vesicles in the medial and trans elements of a partially fragmented Golgi complex, while in not actively growing cells the tubules become completely independent from the Golgi complex. Formation and elongation of the tubules proved to depend on the actin cytoskeleton, since the alpha-1,2 fucosylated protein(s) segregate with the cytoskeleton proteins, and not in the membrane fraction, as do the Golgi markers and other fucosylated proteins, while depolymerization of the actin filaments causes tubule fragmentation and shifting of the alpha-1,2 fucosylated proteins into the membrane fraction.

Formulation of liposomes functionalized with Lotus lectin and effective in targeting highly proliferative cells.

BACKGROUND: Liposomes, used to improve the therapeutic index of new and established drugs, have advanced with the insertion of active targeting. The lectin from Lotus tetragonolobus (LTL), which binds glycans containing alpha-1,2-linked fucose, reveals surface regionalized glycoepitopes in highly proliferative cells not detectable in normally growing cells. In contrast, other lectins localize the corresponding glycoepitopes all over the cell surface. LTL also proved able to penetrate the cells by an unconventional uptake mechanism. METHODS: We used confocal laser microscopy to detect and localize LTL-positive glycoepitopes and lectin uptake in two cancer cell lines. We then constructed doxorubicin-loaded liposomes functionalized with LTL. Intracellular delivery of the drug was determined in vitro and in vivo by confocal and electron microscopy. RESULTS: We confirmed the specific localization of Lotus binding sites and the lectin uptake mechanism in the two cell lines and determined that LTL-functionalized liposomes loaded with doxorubicin greatly increased intracellular delivery of the drug, compared to unmodified doxorubicin-loaded liposomes. The LTL-Dox-L mechanism of entry and drug delivery was different to that of Dox-L and other liposomal preparations. LTL-Dox-L entered the cells one by one in tiny tubules that never fused with lysosomes. LTL-Dox-L injected in mice with melanoma specifically delivered loaded Dox to the cytoplasm of tumor cells. CONCLUSIONS: Liposome functionalization with LTL promises to broaden the therapeutic potential of liposomal doxorubicin treatment, decreasing non-specific toxicity. GENERAL SIGNIFICANCE: Doxorubicin-LTL functionalized liposomes promise to be useful in the development of new cancer chemotherapy protocols.

A fucose-containing O-glycoepitope on bovine and human nucleolin.

In this paper, we demonstrate the existence and localization of fucosyl-containing O-glycoforms of nucleolin in cultured bovine endothelial cells (CVEC) and malignant cultured human A431 cells. The tool for this discovery was an antibody raised against gp273, a glycoprotein ligand for the sperm-egg interaction in the mollusc bivalve Unio elongatulus. The function and immunological properties of gp273 mainly depend on clustered Lewis-like, fucose-containing O-glycans. Here an anti-gp273 antibody was used to evaluate whether glycoepitopes similar to those of gp273 are part of potential ligands of selectins in endothelial cells. We found that anti-gp273 strongly and exclusively interacted with a 110 kDa protein in CVEC and A431 tumor cells. After partial purification, mass spectrometry identified the protein as nucleolin. This was confirmed by comparing anti-gp273 and anti-nucleolin antibody immunoblotting after nucleolin depletion. We confirmed that anti-gp273 binding to nuclear and extranuclear nucleolin was against a fucose-containing O-glycoepitope by immunoblot analysis of the protein after chemically removing O-glycans and by lectin-blot analysis of control and nucleolin-depleted samples. Using anti-gp273 IgG, we detected nucleolin on the plasma membrane and cytoplasm. O-Glycosylation may regulate the plethora of functions in which nucleolin is involved.

Identification and characterization of a 38 kDa glycoprotein functionally associated with mating activity of Paramecium primaurelia.

In Paramecium primaurelia mating interactions take place immediately after mixing mating-competent cells of opposite mating types. The cells clump in clusters (mating reaction) and then separate in pairs. Previous results have shown that sialic acid-containing glycoconjugates are present on the cell surface and are involved in mating-cell pairing. In order to identify the sialic acid-containing glycoprotein(s), we first metabolically radiolabelled non-mating-competent cells with D-[6-(3)H]galactose, and then analyzed the radiolabelled proteins by anion exchange chromatography. We characterized a 38 kDa (gp38) sialic acid-containing glycoprotein and raised the corresponding polyclonal antibody by means of which we localized the antigen at the level of the oral region of non-mating-competent cells and on the ciliary surface of mating-competent cells. Immunoblot analysis of the ciliary protein fraction showed that the anti-gp38 serum interacted with a 38 kDa protein in both mating types I and II cells. We also demonstrated the functional activity of gp38 in the mating reaction by means of anti-gp38 antibody competition assays.

Abnormal N-glycosylation pattern for brain nucleotide pyrophosphatase-5 (NPP-5) in Mecp2-mutant murine models of Rett syndrome.

Neurological disorders can be associated with protein glycosylation abnormalities. Rett syndrome is a devastating genetic brain disorder, mainly caused by de novo loss-of-function mutations in the methyl-CpG binding protein 2 (MECP2) gene. Although its pathogenesis appears to be closely associated with a redox imbalance, no information on glycosylation is available. Glycoprotein detection strategies (i.e., lectin-blotting) were applied to identify target glycosylation changes in the whole brain of Mecp2 mutant murine models of the disease. Remarkable glycosylation pattern changes for a peculiar 50kDa protein, i.e., the N-linked brain nucleotide pyrophosphatase-5 were evidenced, with decreased N-glycosylation in the presymptomatic and symptomatic mutant mice. Glycosylation changes were rescued by selected brain Mecp2 reactivation. Our findings indicate that there is a causal link between the amount of Mecp2 and the N-glycosylation of NPP-5.

Envelope glycosylation determined by lectins in microscopy sections of Acinetobacter venetianus induced by diesel fuel.

It was suggested in a previous study that cells of Acinetobacter venetianus VE-C3 adhere to diesel fuel by synthesizing a capsular polysaccharide containing glucose and/or mannose. To study the fine structure of cells and localization of bacterial polysaccharide in the presence of diesel fuel, two lectins were used: ConA, an agglutinin from Canavalia ensiformis specific for mannose and/or glucose residues, and PNA, an agglutinin from Arachis hypogaea, for terminal galactose residues. The lectins were conjugated with electron dense ferritin for transmission electron microscopy (TEM) and with fluorescein isothiocyanate (FITC) for scanning confocal laser microscopy (SCLM). Samples were prepared by freeze substitution, which allows glycosylation to be determined in situ in thin sections of specimens. The distribution of glycosylation was imaged with and without treatment of specimens with their specific hapten (glucose and galactose). The glycosylation activity produced a polysaccharide capsule. Emulsified diesel fuel nanodroplets were observed at the cell envelope perimeter. Fine structure of vesicles consisted of polysaccharide and diesel fuel nanodroplets. Lectin blotting analysis showed ConA-positive glycoprotein with an apparent molecular mass of 22 kDa in the outer membrane. Its production was induced by diesel fuel. This glycoprotein was probably responsible for bioemulsifying activity at the cell envelope. Several other glycoproteins were positive for PNA lectin, the main constituent migrating with an apparent molecular weight of 17.8 kDa. However, they were all constitutive and probably involved in cell biofilm formation at the oil surface.