ABSTRACT
BACKGROUND: 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. 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. RESULTS: IBs 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. CONCLUSION: Data 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.
Subject(s)
Antibodies/metabolism , Neural Cell Adhesion Molecules/metabolism , Sialic Acids/metabolism , Sialyltransferases/metabolism , Animals , Antibodies/genetics , Antibodies/immunology , Base Sequence , CHO Cells , Cell Line, Tumor , Cricetinae , Cricetulus , Endoplasmic Reticulum/metabolism , HEK293 Cells , Humans , Immunoprecipitation , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Neural Cell Adhesion Molecules/immunology , Plasmids/genetics , Plasmids/metabolism , Rhabdomyosarcoma/metabolism , Rhabdomyosarcoma/pathology , Sialyltransferases/genetics , Sialyltransferases/immunology , Transplantation, HeterologousABSTRACT
Toll-like receptor 9 (TLR9) is a component of the innate immune system, which recognizes the DNA of both pathogens and hosts. Thus, it can drive autoimmune diseases. Intracellular antibodies expressed inside the ER block transitory protein functions by inhibiting the translocation of the protein from the ER to its subcellular destination. Here, we describe the construction and characterization of an anti-TLR9 ER intrabody (αT9ib). The respective single-chain Fv comprises the variable domains of the heavy and light chain of a monoclonal antibody (mAb; 5G5) towards human and murine TLR9. Co-expression of αT9ib and mouse TLR9 in HEK293 cells resulted in co-localization of both molecules with the ER marker calnexin. Co-immunoprecipitation of mouse TLR9 with αT9ib indicated that αT9ib interacts with its cognate antigen. The expression of αT9ib inhibited NF-κB-driven reporter gene activation upon CpG DNA challenge but not the activation of TLR3 or TLR4. Consequently, TLR9-driven TNFα production was inhibited in RAW264.7 macrophages upon transfection with the αT9ib expression plasmid. The αT9ib-encoding open reading frame was integrated into an adenoviral cosmid vector to produce the recombinant adenovirus (AdV)-αT9ib. Transduction with AdVαT9ib specifically inhibited TLR9-driven cellular TNFα release. These data strongly indicate that αT9ib is a very promising experimental tool to block TLR9 signaling.
Subject(s)
Antibodies, Monoclonal/immunology , Antigen-Antibody Reactions/immunology , Single-Chain Antibodies/immunology , Toll-Like Receptor 9/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , Antibody Specificity/immunology , Base Sequence , Calnexin/immunology , Calnexin/metabolism , Cell Line , Cloning, Molecular , Endoplasmic Reticulum/immunology , Endoplasmic Reticulum/metabolism , Flow Cytometry , HEK293 Cells , Humans , Immunoblotting , Luciferases/genetics , Luciferases/metabolism , Macrophages/cytology , Macrophages/immunology , Macrophages/metabolism , Mice , Microscopy, Confocal , Molecular Sequence Data , NF-kappa B/metabolism , Protein Binding/immunology , Single-Chain Antibodies/genetics , Single-Chain Antibodies/metabolism , Toll-Like Receptor 9/genetics , Toll-Like Receptor 9/metabolism , Transfection , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Intrabodies are recombinantly expressed intracellular antibody fragments that can be used to specifically bind and inhibit the function of cellular proteins of interest. Intrabodies can be targeted to various cell compartments by attaching an appropriate localization peptide sequence to them. An efficient strategy with a high success rate is to anchor intrabodies in the endoplasmatic reticulum where they can inhibit transitory target proteins by binding and preventing them to reach their site of action. Intrabodies can be assembled from antibody gene fragments from various sources into dedicated expression vectors. Conventionally, antibody cDNA sequences are derived from selected hybridoma cell clones that express antibodies with the desired specificity. Alternatively, appropriate clones can be isolated by affinity selection from an antibody in vitro display library. Here an evaluation of endoplasmatic reticulum targeted intrabodies with respect to other knockdown approaches is given and the characteristics of various intrabody expression vectors are discussed. A step by step protocol is provided that was repeatedly used to construct intrabodies derived from diverse antibody isotypes producing hybridoma cell clones. The inactivation of the cell surface receptor neural cell adhesion molecule (NCAM) by a highly efficacious novel endoplasmatic reticulum-anchored intrabody is demonstrated.
