Purification and Characterization of a Mitogenic Lectin from Cephalosporium, a Pathogenic Fungus Causing Mycotic Keratitis

Ophthalmic mycoses caused by infectious fungi are being recognized as a serious concern since they lead to total blindness. Cephalosporium is one amongst several opportunistic fungal species implicated in ophthalmic infections leading to mycotic keratitis. A mitogenic lectin has been purified from the mycelia of fungus Cephalosporium, isolated from the corneal smears of a keratitis patient. Cephalosporium lectin (CSL) is a tetramer with subunit mass of 14 kDa, agglutinates human A, B, and O erythrocytes, and exhibits high affinity for mucin compared to fetuin and asialofetuin but does not bind to simple sugars indicating its complex sugar specificity. CSL showed strong binding to normal human peripheral blood mononuclear cells (PBMCs) to elicit mitogenic activity. The sugar specificity of the lectin and its interaction with PBMCs to exhibit mitogenic effect indicate its possible role in adhesion and infection process of Cephalosporium

Transfection of PC3 and LNCaP C-81 cells with MUC1 cDNA results in production of sLe^a.

<p>Full length MUC1 cDNA composed of tandem repeats and N-terminal FLAG epitope was used to transfect PC3 and LNCaP C-81 cells. After 72 h, cell lysates from both vehicle control and MUC1 cDNA-transfected cells were analyzed by sLe^a (KM231) western blotting. The MUC1 band corresponding to 250 kDa appeared after MUC1 cDNA transfection in both cell lines. The 130 kDa band in frame A probably represents truncated MUC1 in MUC1 cDNA-transfected cells. Antibodies did not detect any sLe^a band in vehicle-treated cells. For equal protein loading, β-actin was used and run on 12% SDS-PAGE.</p

Prostatic Cell-Specific Regulation of the Synthesis of MUC1-Associated Sialyl Lewis a

<div><p>Sialyl Lewis antigens are selectin ligands involved in leukocyte trafficking and cancer metastasis. Biosynthesis of these selectin ligands occurs by the sequential actions of several glycosyltransferases in the Golgi apparatus following synthesis of the protein backbone in the endoplasmic reticulum. In this study, we examine how the synthesis of sialyl Lewis a (sLe^a) is regulated in prostatic cells and identify a mucin that carries this glycotope. We treat human prostatic cells including one normal and three cancerous cells with histone deacetylase inhibitors, valproic acid, tricostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA), and then monitor the expression of sLe^a. We have found that SAHA enhances the production of sLe^a in normal prostatic RWPE-1 cells but not prostatic cancer cells. Employing siRNA technology and co-immunoprecipitation, we show that the sLe^a is associated with MUC1, which is confirmed by confocal immunofluorescence microscopy and proximity ligation assay. The SAHA-induced production of sLe^a in RWPE-1 cells is resulted from upregulation of <i>B3GALT1</i> gene via enhancement of acetylated histone-3 and histone-4. Interestingly, PC3 and LNCaP C-81 cells do not produce detectable amounts of sLe^a despite expressing high levels of B3GALT1. However, the MUC1-associated sLe^a is generated in these cells after introduction of MUC1 cDNA. We conclude that the synthesis of sLe^a is controlled by not only peptide backbone of the glycoprotein but also glycoprotein-specific glycosyltransferases involved in the synthesis of sLe^a. Further, the SAHA induction of this selectin ligand in normal prostatic cells may pose a potentially serious side effect of this drug recently approved by the US Food and Drug Administration.</p> </div

Knockdown of <i>B3GALT1</i> mRNA reduces sLe^a in SAHA-treated RWPE-1 cells.

<p>(A) Anti-sLe^a western blot of the lysates of control cells, SAHA plus scrambled siRNA, or SAHA plus <i>B3GALT1</i> siRNA. (B) Knockdown of <i>B3GALT1</i> gene in RWPE-1 cells was confirmed by 77% reduction of <i>B3GALT1 mRNA</i> measured by quantitative real-time PCR analysis. Relative expression level of <i>B3GALT1</i> gene was determined according to ΔCt method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057416#s2" target="_blank">materials and methods</a>) after normalization with <i>GAPDH</i> ( = 100%) and then expressed as % of <i>B3GALT1</i> mRNA relative to same in the control ( = 100%) (n = 3). (C) Anti-MUC1 western blot of the lysates of cells treated with SAHA, SAHA plus scrambled siRNA, or SAHA plus <i>B3GALT1</i> siRNA. Knockdown of 77% of <i>B3GALT1</i> mRNA reduces sLe^a in SAHA-treated RWPE-1 cells without affecting MUC1 glycoprotein. Experiments were performed (3x) with similar results.</p

Proximity ligation assay of MUC1 and sLe^a in RWPE-1 and PC3 cells as analyzed by confocal microscopy.

<p>RWPE-1 and PC3 cells grown on cover slips were treated with PBS or SAHA and then fixed in paraformaldehyde. These cells were processed for PLA for MUC1 and sLe^a using an Olink Bioscience PLA kit. Briefly, these cells were treated with mouse anti-sLe^a and rabbit anti-MUC1 antibodies, and then with oligonucleotide-conjugated anti-mouse minus and anti-rabbit plus proximity ligation assay secondary probes. The oligonucleotides conjugated with the plus and minus antibodies were used to generate circular DNA, which was then amplified and tagged with a red fluorescence dye. Following DAPI staining of the nuclei, the cells were examined by confocal fluorescence microscopy. The expression as well as association of sLe^a with MUC1, significantly increased only in SAHA treated RWPE-1 cells as indicated by red foci all over the cells. Bar  =  20 µm.</p

SAHA treatment stimulates the expression of <i>B3GALT1</i> gene in RWPE-1 cells.

<p>Quantitative real-time PCR analysis was carried out on RWPE-1, PC3, DU145 and LNCaP C-81 cells treated with PBS or 5 µM SAHA for 72 h. Relative expression levels of <i>B3GALT1</i> and <i>MUC1</i> genes were sorted according to ΔCt (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057416#s2" target="_blank">Materials and Methods</a>) method, normalized with <i>GAPDH</i> in same cell preparation and expressed as fold changes ± SEM determined by calculating the ratio of the expression level of each gene in SAHA-treated vs. that in PBS-treated control cells. Relative amount of each gene versus that of <i>GAPDH</i> (100%) in PBS-treated control cells was given in the parenthesis (n = 3). The numbers shown in parenthesis represent % basal expression level relative to GAPDH ( = 100%).</p

Effects of HDACi treatment on production of sLe^a in one normal and three cancerous prostatic cells.

<p>(A) RWPE-1, PC3, DU145, and LNCaP C-81 cells were treated with 1 mM valproic acid, 20 µM SAHA or 250 nM TSA for 72 h followed by western analysis of the cell lysates for sLe^a using KM231 Abs. SAHA was the only HDACi which induces marked production of sLe^a in RWPE-1 cells. (B) Dose-dependent (0-20 µM) effect of SAHA on the production of sLe^a in RWPE-1 cells. Minimum concentration of SAHA required to induce the production of sLe^a without causing cytotoxicity is 5 µM. β-actin was used as a loading control on 10% gels and probed with anti-β-actin Abs. Experiments were performed (3x) with similar results.</p

Biosynthetic pathway of nucin core 2-associated sialyl Lewis a (sLe^a) antigen.

<p>Biosynthesis of mucin O-glycans is initiated by the addition of GalNAc to ser or thr of the peptide as catalyzed by polypeptide N-acetylgalactosaminyltransferases (GALNTs). This is followed by the addition of Gal in β1-3 linkage to GalNAc as catalyzed by C1GALT1 enzyme and produces core-1. Core 2 is generated by adding GlcNAc in β1-6 linkage to GalNAc by GCNTs. To synthesize sLe^a, Gal is transferred to GlcNAc in β1-3 linkage to form type 1 chain as catalyzed by B3GALTs, followed by the addition of α2-3NeuAc to Gal as catalyzed by ST3GALTs and then α4-fucose (Fuc) to GlcNAc as catalyzed by α3/4FUTs.</p

Confocal and flow cytometric analysis of RWPE-1 cells.

<p>(A) Confocal immunofluorescence images of MUC1 (red) and sLe^a (green) on RWPE-1 cells treated with PBS (control) or SAHA (5 µM) for 72 h. SLe^a induced by SAHA is colocalized with MUC1. (B) SLe^a mean fluorescence intensity in RWPE-1 cells treated with PBS or SAHA as measured by flow cytometry analysis. (p < 0.05) (C) % of sLe^a-positive cells in PBS- or SAHA-treated cells. The data expressed as mean ± SEM were obtained from three independent experiments. (p < 0.05) Bar  =  20 µm.</p