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Blocking translocation of cell surface molecules from the ER to the cell surface by intracellular antibodies targeted to the ER.

Böldicke T - J. Cell. Mol. Med. (2007 Jan-Feb)

Bottom Line: A particular advantage of the intrabody technology over existing ones is the possibility of inhibiting selectively post-translational modifications of proteins.The main applications of ER intrabodies so far have been (i) inactivation of oncogenic receptors and (ii) functional inhibition of virus envelope proteins and virus-receptor molecules on the surface of host cells.In cancer research, the number of in vivo mouse models for evaluation of the therapeutic potential of intrabodies is increasing.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz Centre for Infection Research, Department of Gene Regulation and Differentiation,Braunschweig, Germany. thomas.boeldicke@helmholtz-hzi.de

ABSTRACT
Intracellular antibodies (intrabodies) constitute a potent tool to neutralize the function of target proteins inside specific cell compartments (cytosol, nucleus, mitochondria and ER). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals and complements or replaces knockdown techniques such as antisense-RNA, RNAi and RNA aptamers. This article focuses on intrabodies targeted to the ER. Intracellular anti-bodies expressed and retained inside the ER (ER intrabodies) are shown to be highly efficient in blocking the translocation of secreted and cell surface molecules from the ER to the cell surface. The advantage of ER intrabodies over cytoplasmic intrabodies is that they are correctly folded and easier to select. A particular advantage of the intrabody technology over existing ones is the possibility of inhibiting selectively post-translational modifications of proteins. The main applications of ER intrabodies so far have been (i) inactivation of oncogenic receptors and (ii) functional inhibition of virus envelope proteins and virus-receptor molecules on the surface of host cells. In cancer research, the number of in vivo mouse models for evaluation of the therapeutic potential of intrabodies is increasing. In the future, endosomal localized receptors involved in bacterial and viral infections, intracellular oncogenic receptors and enzymes involved in glycosylation of tumour antigens might be new targets for ER intrabodies.

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ScFv intrabody targeted to the ER. Shown is the ER intrabody as scFv fragment. VH= variable domain of the heavy chain, VL= variable domain of the light chain. The VH and VL domains are fused by a 15 amino acid flexible linker shown as a black line. The red line at the N-terminus of the VH domain represents the ER signal peptide. The red rectangle at the C-terminus of the VL domain represents the ER retention sequence and the yellow rectangle the c-myc tag. In addition is shown the target protein (cell surface molecule) and the hERD2 receptor that binds to the ER retention sequence of the scFv fragment. The complex consisting of the scFv fragment and the target protein binds to the hERD2 receptor inside the cis-Golgi and is transported through the Golgi apparatus back to the ER where the scFv-target protein complex is released. (CGN: cis-Golgi Network, TGN: trans-Golgi Network).
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fig01: ScFv intrabody targeted to the ER. Shown is the ER intrabody as scFv fragment. VH= variable domain of the heavy chain, VL= variable domain of the light chain. The VH and VL domains are fused by a 15 amino acid flexible linker shown as a black line. The red line at the N-terminus of the VH domain represents the ER signal peptide. The red rectangle at the C-terminus of the VL domain represents the ER retention sequence and the yellow rectangle the c-myc tag. In addition is shown the target protein (cell surface molecule) and the hERD2 receptor that binds to the ER retention sequence of the scFv fragment. The complex consisting of the scFv fragment and the target protein binds to the hERD2 receptor inside the cis-Golgi and is transported through the Golgi apparatus back to the ER where the scFv-target protein complex is released. (CGN: cis-Golgi Network, TGN: trans-Golgi Network).

Mentions: ER intrabodies will give insights into the function of newly detected cell surface molecules and, furthermore, some will have potential application as therapeutic antibodies. A large number of different molecules with specific biological functions are expressed on the cell surface and are involved in cell growth, apoptosis, differentiation, adhesion, bacterial and viral infection and antigen presentation. ER intrabodies are transported to the lumen of the ER and bind to their specific secretory molecule (Fig. 1A). After transport via COPII-coated vesicles, the intrabody-target protein complex binds via the C-terminal retention sequence (KDEL) inside the cis Golgi network to the human ER receptor hERD2 [22]. Proteins retained inside the ER share a common carboxy-terminal tetrapeptide: KDEL [23]. Initially it was shown that fusion of the sequence SEKDEL from the resident luminal ER protein grp78 to lysozyme led to 100% retention [23]. In addition, fusion of the tetrapeptide KDEL to the secreted protein human proneuropeptide Y (pro-NPY) led to retention, too [24]. For the retention of ER intrabodies, the sequence SEKDEL has been used in almost all studies [4].


Blocking translocation of cell surface molecules from the ER to the cell surface by intracellular antibodies targeted to the ER.

Böldicke T - J. Cell. Mol. Med. (2007 Jan-Feb)

ScFv intrabody targeted to the ER. Shown is the ER intrabody as scFv fragment. VH= variable domain of the heavy chain, VL= variable domain of the light chain. The VH and VL domains are fused by a 15 amino acid flexible linker shown as a black line. The red line at the N-terminus of the VH domain represents the ER signal peptide. The red rectangle at the C-terminus of the VL domain represents the ER retention sequence and the yellow rectangle the c-myc tag. In addition is shown the target protein (cell surface molecule) and the hERD2 receptor that binds to the ER retention sequence of the scFv fragment. The complex consisting of the scFv fragment and the target protein binds to the hERD2 receptor inside the cis-Golgi and is transported through the Golgi apparatus back to the ER where the scFv-target protein complex is released. (CGN: cis-Golgi Network, TGN: trans-Golgi Network).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4401220&req=5

fig01: ScFv intrabody targeted to the ER. Shown is the ER intrabody as scFv fragment. VH= variable domain of the heavy chain, VL= variable domain of the light chain. The VH and VL domains are fused by a 15 amino acid flexible linker shown as a black line. The red line at the N-terminus of the VH domain represents the ER signal peptide. The red rectangle at the C-terminus of the VL domain represents the ER retention sequence and the yellow rectangle the c-myc tag. In addition is shown the target protein (cell surface molecule) and the hERD2 receptor that binds to the ER retention sequence of the scFv fragment. The complex consisting of the scFv fragment and the target protein binds to the hERD2 receptor inside the cis-Golgi and is transported through the Golgi apparatus back to the ER where the scFv-target protein complex is released. (CGN: cis-Golgi Network, TGN: trans-Golgi Network).
Mentions: ER intrabodies will give insights into the function of newly detected cell surface molecules and, furthermore, some will have potential application as therapeutic antibodies. A large number of different molecules with specific biological functions are expressed on the cell surface and are involved in cell growth, apoptosis, differentiation, adhesion, bacterial and viral infection and antigen presentation. ER intrabodies are transported to the lumen of the ER and bind to their specific secretory molecule (Fig. 1A). After transport via COPII-coated vesicles, the intrabody-target protein complex binds via the C-terminal retention sequence (KDEL) inside the cis Golgi network to the human ER receptor hERD2 [22]. Proteins retained inside the ER share a common carboxy-terminal tetrapeptide: KDEL [23]. Initially it was shown that fusion of the sequence SEKDEL from the resident luminal ER protein grp78 to lysozyme led to 100% retention [23]. In addition, fusion of the tetrapeptide KDEL to the secreted protein human proneuropeptide Y (pro-NPY) led to retention, too [24]. For the retention of ER intrabodies, the sequence SEKDEL has been used in almost all studies [4].

Bottom Line: A particular advantage of the intrabody technology over existing ones is the possibility of inhibiting selectively post-translational modifications of proteins.The main applications of ER intrabodies so far have been (i) inactivation of oncogenic receptors and (ii) functional inhibition of virus envelope proteins and virus-receptor molecules on the surface of host cells.In cancer research, the number of in vivo mouse models for evaluation of the therapeutic potential of intrabodies is increasing.

View Article: PubMed Central - PubMed

Affiliation: Helmholtz Centre for Infection Research, Department of Gene Regulation and Differentiation,Braunschweig, Germany. thomas.boeldicke@helmholtz-hzi.de

ABSTRACT
Intracellular antibodies (intrabodies) constitute a potent tool to neutralize the function of target proteins inside specific cell compartments (cytosol, nucleus, mitochondria and ER). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals and complements or replaces knockdown techniques such as antisense-RNA, RNAi and RNA aptamers. This article focuses on intrabodies targeted to the ER. Intracellular anti-bodies expressed and retained inside the ER (ER intrabodies) are shown to be highly efficient in blocking the translocation of secreted and cell surface molecules from the ER to the cell surface. The advantage of ER intrabodies over cytoplasmic intrabodies is that they are correctly folded and easier to select. A particular advantage of the intrabody technology over existing ones is the possibility of inhibiting selectively post-translational modifications of proteins. The main applications of ER intrabodies so far have been (i) inactivation of oncogenic receptors and (ii) functional inhibition of virus envelope proteins and virus-receptor molecules on the surface of host cells. In cancer research, the number of in vivo mouse models for evaluation of the therapeutic potential of intrabodies is increasing. In the future, endosomal localized receptors involved in bacterial and viral infections, intracellular oncogenic receptors and enzymes involved in glycosylation of tumour antigens might be new targets for ER intrabodies.

Show MeSH
Related in: MedlinePlus