Abstract
BackgroundPolysialic acid (polySia) is a carbohydrate modification of the neural cell adhesion molecule (NCAM), which is implicated in neural differentiation and plays an important role in tumor development and metastasis. Polysialylation of NCAM is mediated by two Golgi-resident polysialyltransferases (polyST) ST8SiaII and ST8SiaIV. Intracellular antibodies (intrabodies; IB) expressed inside the ER and retaining proteins passing the ER such as cell surface receptors or secretory proteins provide an efficient means of protein knockdown. To inhibit the function of ST8SiaII and ST8SiaIV specific ER IBs were generated starting from two corresponding hybridoma clones. Both IBs αST8SiaII-IB and αST8SiaIV-IB were constructed in the scFv format and their functions characterized in vitro and in vivo.ResultsIBs directed against the polySTs prevented the translocation of the enzymes from the ER to the Golgi-apparatus. Co-immunoprecipitation of ST8SiaII and ST8SiaIV with the corresponding IBs confirmed the intracellular interaction with their cognate antigens. In CHO cells overexpressing ST8SiaII and ST8SiaIV, respectively, the transfection with αST8SiaII-IB or αST8SiaIV-IB inhibited significantly the cell surface expression of polysialylated NCAM. Furthermore stable expression of ST8SiaII-IB, ST8SiaIV-IB and luciferase in the rhabdomyosarcoma cell line TE671 reduced cell surface expression of polySia and delayed tumor growth if cells were xenografted into C57BL/6 J RAG-2 mice.ConclusionData obtained strongly indicate that αST8SiaII-IB and αST8SiaIV-IB are promising experimental tools to analyze the individual role of the two enzymes during brain development and during migration and proliferation of tumor cells.
Highlights
Polysialic acid is a carbohydrate modification of the neural cell adhesion molecule (NCAM), which is implicated in neural differentiation and plays an important role in tumor development and metastasis
Polysialylation of NCAM is mediated by two Golgi-resident enzymes, the polysialyltransferases ST8SiaII [10, 11] and ST8SiaIV [12, 13], exhibiting overlapping but distinct expression patterns
Endosialidases are large enzymes with restricted penetration into tumor nodules [27] and systemic application of these kinetically stabilized enzymes may be followed by long term complications that based on current research cannot be calculated. It can be stated, that endosialidases, though attractive, are far away from clinical application. To bypass these limitations and to avoid off-target effects we demonstrate in this study that Polysialic acid (polySia) synthesis can be knocked down by means of intrabody technology
Summary
Polysialic acid (polySia) is a carbohydrate modification of the neural cell adhesion molecule (NCAM), which is implicated in neural differentiation and plays an important role in tumor development and metastasis. Polysialylation of NCAM is mediated by two Golgi-resident polysialyltransferases (polyST) ST8SiaII and ST8SiaIV. To inhibit the function of ST8SiaII and ST8SiaIV specific ER IBs were generated starting from two corresponding hybridoma clones. Both IBs αST8SiaII-IB and αST8SiaIV-IB were constructed in the scFv format and their functions characterized in vitro and in vivo. Polysialylation of NCAM is mediated by two Golgi-resident enzymes, the polysialyltransferases (polySTs) ST8SiaII [10, 11] and ST8SiaIV [12, 13], exhibiting overlapping but distinct expression patterns. Selective knockout of only one polyST gene is not sufficient to fully abrogate polySia synthesis in settings where both enzymes are expressed
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
