Figure 1.
CMP is an inhibitor of ST8SiaII in vitro.
A: Activity of human ST8SiaII with increasing concentrations of DMB-DP3 acceptor over 5 hours, as measured by HPLC. B: Lineweaver-Burk plot* generated from A.Km of DMB-DP3 for ST8SiaII (derived from x axis intercept) was calculated as 38.9 µM. Data points are from a single determination representative of two independent experiments. C: HPLC trace extract showing that CMP (250 µM) inhibits polysialylation of DMB-DP3 (10 µM) to DMB-DP4 (ST8SiaII 250 ng; CMP-Neu5Ac 500 µM; 4 h; 25° C). D: Dixon Plot* indicating that CMP is a competitive inhibitor of ST8SiaII, Ki = 10 µM (*Data points are from a single determination representative of two independent experiments).
Figure 2.
Semi-quantitative PCR showing expression of polySTs and NCAM and Western blotting showing expression of polySia-NCAM in cell lines.
A: C6-STX cells express ST8SiaII and NCAM, whereas C6-WT cells express NCAM only. B: SH-SY5Y and IMR-32 cells both express ST8SiaII and NCAM. Expression of ST8SiaIV was not detected in SH-SY5Y cells, and was barely detectable in IMR-32 cells. DLD-1 cells do not express NCAM or ST8SiaII. C: C6-STX, SH-SY5Y and IMR-32 cells all express polySia, whereas C6-WT and DLD-1 cells do not.
Figure 3.
Effect of ST8SiaII inhibition on cell surface polySia expression.
Upper: Flow cytometry analysis of effect of 24h exposure to increasing CMP concentrations on polySia expression in IMR-32 cells (NCAM +; ST8SiaII +; polySia +). Lower: Relative mean fluorescence intensities as a result of CMP treatment. Cells were labelled with EndoNA2-eGFP and analysed by flow cytometry. Fluorescence is expressed as a mean percentage of that observed with the control (untreated cells) ± SD of three independent experiments (* P < 0.01, ** P < 0.001). CMP causes a significant and concentration-dependent reduction in polySia expression in IMR-32 cells.
Figure 4.
Effect of ST8SiaII inhibition on recovery of polySia expression following removal by endoN.
C6-STX cells (left) and SH-SY5Y cells (right) immunolabelled with anti-polySia antibody (mAb 735) followed by incubation with TRITC-conjugated secondary antibody [PolySia orange; nuclei stained blue with DAPI]. (a) Negative control (absence of mAb 735); (b) Positive control (absence of endoN/CMP treatment) (c) Removal of polySia with EndoN (0.3 µg/mL); (d) PolySia recovery following 6 h incubation in absence of CMP; (e) PolySia recovery following 6 h incubation with CMP at 0.5 mM; (f) PolySia recovery following 6 h incubation with CMP at 5 mM. CMP clearly prevents the recovery of polySia on the cell surface following biological removal at 5 mM. Scale bar = 50 µm.
Figure 5.
Effect of ST8SiaII inhibition on tumour cell migration.
Effect of CMP treatment on the migration of C6-STX, C6-WT, SH-SY5Y, and DLD-1 cells as assessed by 2D migration assays. Confluent cell monolayers were incubated with fresh complete medium and re-population of scratched wounds after 24 h was assessed. Migration is expressed as a percentage of that observed with untreated cells for a given cell line (complete re-population). CMP treatment at all concentrations decreased the migration capacity of polySia-expressing SH-SY5Y and C6-STX cells in a concentration dependent manner. However, CMP treatment had no significant effect on the migration of C6-WT and DLD-1 cells even at high concentrations. C6-WT and DLD-1 cells do not express polySTs or polySia. Values shown are means ± S.D based on three independent experiments (* P < 0.01).