Pharmacological Inhibition of polysialyltransferase ST8SiaII Modulates Tumour Cell Migration

Polysialic acid (polySia), an α-2,8-glycosidically linked polymer of sialic acid, is a developmentally regulated post-translational modification predominantly found on NCAM (neuronal cell adhesion molecule). Whilst high levels are expressed during development, peripheral adult organs do not express polySia-NCAM. However, tumours of neural crest-origin re-express polySia-NCAM: its occurrence correlates with aggressive and invasive disease and poor clinical prognosis in different cancer types, notably including small cell lung cancer (SCLC), pancreatic cancer and neuroblastoma. In neuronal development, polySia-NCAM biosynthesis is catalysed by two polysialyltransferases, ST8SiaII and ST8SiaIV, but it is ST8SiaII that is the prominent enzyme in tumours. The aim of this study was to determine the effect of ST8SiaII inhibition by a small molecule on tumour cell migration, utilising cytidine monophosphate (CMP) as a tool compound. Using immunoblotting we showed that CMP reduced ST8iaII-mediated polysialylation of NCAM. Utilizing a novel HPLC-based assay to quantify polysialylation of a fluorescent acceptor (DMB-DP3), we demonstrated that CMP is a competitive inhibitor of ST8SiaII (K _i = 10 µM). Importantly, we have shown that CMP causes a concentration-dependent reduction in tumour cell-surface polySia expression, with an absence of toxicity. When ST8SiaII-expressing tumour cells (SH-SY5Y and C6-STX) were evaluated in 2D cell migration assays, ST8SiaII inhibition led to significant reductions in migration, while CMP had no effect on cells not expressing ST8SiaII (DLD-1 and C6-WT). The study demonstrates for the first time that a polysialyltransferase inhibitor can modulate migration in ST8SiaII-expressing tumour cells. We conclude that ST8SiaII can be considered a druggable target with the potential for interfering with a critical mechanism in tumour cell dissemination in metastatic cancers.     

Glycomic strategy for efficient linkage analysis of di-, oligo- and polysialic acids

Sialic acid polymers of glycoproteins and glycolipids are characterized by a high diversity in nature and are involved in distinct biological processes depending inter alia on the glycosidic linkages between the present sialic acid residues. Though suitable protocols are available for chain length and sialic acid determination, sensitive methods for linkage analysis of di-, oligo-, and polysialic acids (di/oligo/polySia) are still pending. In this study, we have established a highly sensitive glycomic strategy for this purpose which is based on permethylation of di/oligo/polySia after tagging their reducing ends with the fluorescent dye 1,2-diamino-4,5-methylenedioxybenzene (DMB). Using DMB-labeled sialic acid di/oligo/polymers glycosidic linkages could be efficiently determined and, optionally, the established working procedure can be combined with HPLC for in depth characterization of distinct di/oligo/polySia chains. Moreover, the outlined approach can be directly applied to mammalian tissue samples and linkage analysis of sialic acid polymers present in biopsy samples of neuroblastoma tissue demonstrating the usefulness of the outlined work flow to screen, for example, cancer tissue for the presence of distinct variants of di/oligo/polySia as potentially novel biomarkers. Hence, the described strategy offers a highly sensitive and efficient strategy for identification of glycosidic linkages in sialic acid di/oligo/polymers of glycoproteins and glycolipids.     

Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.