Intracellular targeting and functional analysis of single-chain Fv fragments in mammalian cells

Cardinale, Alessio; Filesi, Ilaria; Mattei, Sonia; Biocca, Silvia

doi:10.1016/j.ymeth.2004.04.006

Cited by 25 publications

(13 citation statements)

References 43 publications

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“…Immunofluorescence Staining-Immunofluorescence was carried out as described (35). For surface staining of PrP C , cells were rinsed with ice-cold PBS and incubated for 1 h at 4°C with MAb 7A12 (36) diluted in Opti-MEM (Invitrogen) containing 0.2 mg/ml BSA.…”

Section: Methodsmentioning

confidence: 99%

Trapping Prion Protein in the Endoplasmic Reticulum Impairs PrPC Maturation and Prevents PrPSc Accumulation

Cardinale

Filesi

Vetrugno

et al. 2005

Journal of Biological Chemistry

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The conversion of the normal cellular prion protein (PrP C ) into the abnormal scrapie isoform (PrP Sc ) is a key feature of prion diseases. The pathogenic mechanisms and the subcellular sites of the conversion are complex and not completely understood. In particular, little is known on the role of the early compartment of the secretory pathway in the processing of PrP C and in the pathogenesis of prion diseases. In order to interfere with the intracellular traffic of endogenous PrP C we have generated two anti-prion single chain antibody fragments (scFv) directed against different epitopes, each fragment tagged either with a secretory leader or with the ER retention signal KDEL. The stable expression of these constructs in PC12 cells allowed us to study their specific effects on the synthesis, maturation, and processing of endogenous PrP C and on PrP Sc formation. We found that ER-targeted anti-prion scFvs retain PrP C in the ER and inhibit its translocation to the cell surface. Retention in the ER strongly affects the maturation and glycosylation state of PrP C , with the appearance of a new aberrant endo-H sensitive glycosylated species. Interestingly, ER-trapped PrP C acquires detergent insolubility and proteinase K resistance. Furthermore, we show that ER-targeted anti-prion antibodies prevent PrP Sc accumulation in nerve growth factor-differentiated PC12 cells, providing a new tool to study the molecular pathology of prion diseases.

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Section: Methodsmentioning

confidence: 99%

Trapping Prion Protein in the Endoplasmic Reticulum Impairs PrPC Maturation and Prevents PrPSc Accumulation

Cardinale

Filesi

Vetrugno

et al. 2005

Journal of Biological Chemistry

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“…The En-GFP protein was specifically retained with both 4F11 and SP4F11 and did not bind the resin in the absence of antibodies. It is noteworthy that 4F11 bound En-GFP less efficiently than did SP4F11, possibly because intracellular single-chain antibodies are partially misfolded owing to the reduction of their disulfide bonds in the reductive intracellular milieu (Cardinale et al, 2004).…”

Section: Engineering Tools To Block Extracellular Enmentioning

confidence: 97%

“…This approach has the advantage of blocking En functions without modifying the En sequence. Most importantly, the antibody can be targeted to the extracellular or the intracellular compartment depending on the presence of a signal peptide for secretion (Cardinale et al, 2004;Lesaffre et al, 2007).…”

Section: Engineering Tools To Block Extracellular Enmentioning

confidence: 99%

Engrailed homeoprotein acts as a signaling molecule in the developing fly

et al. 2011

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SUMMARYHomeodomain transcription factors classically exert their morphogenetic activities through the cell-autonomous regulation of developmental programs. In vertebrates, several homeoproteins have also been shown to have direct non-cell-autonomous activities in the developing nervous system. We present the first in vivo evidence for homeoprotein signaling in Drosophila. Focusing on wing development as a model, we first demonstrate that the homeoprotein Engrailed (En) is secreted. Using singlechain anti-En antibodies expressed under the control of a variety of promoters, we delineate the wing territories in which secreted En acts. We show that En is a short-range signaling molecule that participates in anterior crossvein development, interacting with the Dpp signaling pathway. This report thus suggests that direct signaling with homeoproteins is an evolutionarily conserved phenomenon that is not restricted to neural tissues and involves interactions with bona fide signal transduction pathways.

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“…18 Affinity purified anti-rabbit LOX-1 (Santa Cruz), goat anti-calnexin (Santa Cruz), and mouse anti-Golgi 58K (Sigma) protein were used as primary antibodies. Texas Red goat anti-rabbit IgG (Calbiochem), Texas Red goat anti-mouse IgG (Calbiochem), and Rhodamine Red-Xconjugated affinipure rabbit anti-goat IgG (Jackson Immunoreasearch) were used as secondary antibodies.…”

Section: Immunofluorescence Stainingmentioning

confidence: 99%

In Vivo and In Vitro Studies Support That a New Splicing Isoform of OLR1 Gene Is Protective Against Acute Myocardial Infarction

Mango

Biocca

Vecchio

et al. 2005

Circulation Research

115

123

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Abstract-Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), encoded by the OLR1 gene, is a scavenger receptor that plays a fundamental role in the pathogenesis of atherosclerosis. LOX-1 activation is associated with apoptosis of endothelial cells, smooth muscle cells (SMCs), and macrophages. This process is an important underlying mechanism that contributes to plaque instability and subsequent development of acute coronary syndromes. Independent association genetic studies have implicated OLR1 gene variants in myocardial infarction (MI) susceptibility. Because single nucleotide polymorphisms (SNPs) linked to MI are located in intronic sequences of the gene, it remains unclear as to how they determine their biological effects. Using quantitative real-time PCR and minigene approach, we show that intronic SNPs, linked to MI, regulate the expression of a new functional splicing isoform of the OLR1 gene, LOXIN, which lacks exon 5. Macrophages from subjects carrying the "non-risk" disease haplotype at OLR1 gene have an increased expression of LOXIN at mRNA and protein level, which results in a significant reduction of apoptosis in response to oxLDL. Expression of LOXIN in different cell types results in loss of surface staining, indicating that truncation of the C-terminal portion of the protein has a profound effect on its cellular trafficking. Furthermore, the proapoptotic effect of LOX-1 receptor in cell culture is specifically rescued by the coexpression of LOXIN in a dose-dependent manner. The demonstration that increasing levels of LOXIN protect cells from LOX-1 induced apoptosis sets a groundwork for developing therapeutic approaches for prevention of plaque instability.

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Intracellular targeting and functional analysis of single-chain Fv fragments in mammalian cells

Cited by 25 publications

References 43 publications

Trapping Prion Protein in the Endoplasmic Reticulum Impairs PrPC Maturation and Prevents PrPSc Accumulation

Trapping Prion Protein in the Endoplasmic Reticulum Impairs PrPC Maturation and Prevents PrPSc Accumulation

Engrailed homeoprotein acts as a signaling molecule in the developing fly

In Vivo and In Vitro Studies Support That a New Splicing Isoform of OLR1 Gene Is Protective Against Acute Myocardial Infarction

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