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

doi:10.1016/j.ymeth.2004.04.006

Methods

Volume 34, Issue 2, October 2004, Pages 171-178

https://doi.org/10.1016/j.ymeth.2004.04.006 Get rights and content

Abstract

In the past decade, intracellular antibodies have proven to be a useful tool in obtaining the phenotypic knock-out of selected gene function in different animal and plant systems. This strategy is based on the ectopic expression of recombinant forms of antibodies targeted towards different intracellular compartments, exploiting specific targeting signals to confer the new intracellular location. The functional basis of this technology is closely linked to the ability of intracellular antibodies to interact with their target antigens in vivo. This interaction allows either a direct neutralising effect or the dislodgement of the target protein from its normal intracellular location and, by this mechanism, the inactivation of its function. By using this approach, the function of several antigens has been inhibited in the cytoplasm, the nucleus, and the secretory compartments. In this article, we shall describe all the steps required for expressing single-chain Fv fragments in different subcellular compartments of mammalian cells and their subsequent use in knock-out experiments, starting from a cloned single-chain Fv fragment. This will include the analysis of the solubility properties of the new scFv fragment in transfected mammalian cells, the intracellular distribution of the antigen–antibody complex, and the resulting phenotype.

Introduction

Intracellular antibodies are a relatively new technology based on the concept that antibody chains or domains, if equipped with suitable localisation signals, can be targeted towards new ectopic intracellular sites to interfere with endogenous antigens [1], [2], [3], [4]. This can be obtained by exploiting the property of dominant and autonomous targeting sequences that can be grafted onto other reporter proteins such as antibodies to confer them a new intracellular localisation. Recombinant antibody domains, in particular single-chain Fv (scFv)¹ fragments, have been successfully expressed inside cells to inhibit the function of several antigens in the cytoplasm [5], [6], the nucleus [7], [8], and the secretory pathway of mammalian cells [9]. This type of strategy has a great potential for a variety of applications, including gene therapy [10], plant biotechnology [11] and, more recently, functional genomics [4], [12], [26].

Crucial to the efficacy of this approach is the capacity of the intracellular antibody to interact with the endogenous antigen within any compartment of a mammalian cell. This interaction may result in a direct inhibition of the target antigen [6], [13], [14], may restore a mutant deficient activity [15] or may interfere with the protein folding of pathological mutant proteins [16]. In a different mode of action, an intracellular antibody can also act by diverting the intracellular traffic of the antigen. This is the case, for example, of membrane receptors, whose appearance at the surface can be inhibited by the interaction with intracellular antibodies retained in the endoplasmic reticulum by retention signals (intracellular anchors) [17], [18].

As it was clear from the very beginning of work on this technology, the folding properties of the antibodies and antibody domains vary according to individual scFv fragments and to the intracellular compartment where they are located. A systematic comparison of scFvs targeted to the same compartment showed that antibody fragments expressed from identical expression vectors have very distinct properties of solubility and stability [19], [20]. Although some scFvs are soluble in the cell cytoplasm, overexpression of these molecules may lead to the formation of intracellular aggregates. For this reason, there is great interest in engineering frameworks suitable for intracellular expression and onto which other specificities could be grafted. Different approaches have been developed to solve this problem, including modification of the sequence of VH and VL domains utilising random mutations to stabilise scFvs with intrinsic stability [21] or, alternatively, genetic selection approaches to derive functional scFvs, which can tolerate the reducing cellular environments of the cell cytoplasm [22], [23], [24].

Notwithstanding the phenomenon of aggregation, cytosolic scFv fragments maintain the capacity to bind the antigen and, by sequestering it in intracellular aggregates, to divert it from its intracellular location and block its function. A number of examples indicate that the antibody-mediated co-aggregation of the antigen represents a mode of action for intracellular antibodies targeted to the cytoplasm and to the nucleus [25], [26], [27], [28].

The purpose of this paper was to discuss certain aspects of the intracellular antibody technology that are worth considering in order to start a project with this powerful approach. In addition, we shall describe methods for the analysis of: (i) the expressed scFv fragments, (ii) in vivo interaction with the endogenous antigen, and (iii) the assessment of the resulting biological activity. Since, during the past decade, we have focussed our studies on the expression of scFv fragments in the cytoplasm and in the nucleus of mammalian cells, we shall provide examples and protocols for the analysis of antibody domains in these compartments. Reagents and methodological considerations related to each issue are described below in the context of our work on p21Ras and heterochromatin proteins 1 (HP1).

Section snippets

Intracellular targeting of scFvs

Starting from a cloned scFv fragment, a set of general vectors have been described for the expression of these fragments in different cellular compartments of mammalian cells [29]. All the plasmids contain an N- or C-terminal localisation signal that allows the correct retargeting of the scFv fragment. Table 1 shows the amino-acid sequences of some of the sorting peptides that have been successfully used so far for targeting antibodies or antibody domains (in particular scFv fragments) to

References (43)

S. Biocca et al.
Trends Cell Biol.
(1995)
J.H. Richardson et al.
Trends Biotechnol.
(1995)
S. Biocca et al.
Biochem. Biophys. Res. Commun.
(1993)
A. Cattaneo et al.
Trends Biotechnol.
(1999)
S. Jung et al.
J. Mol. Biol.
(1999)
M. Visintin et al.
J. Mol. Biol.
(2002)
E. Tse et al.
J. Mol. Biol.
(2002)
A. Cardinale et al.
FEBS Lett.
(1998)
L. Persic et al.
Gene
(1997)
J.D. Chesnut et al.
J. Immunol. Methods
(1996)

M.A. Hink et al.

J. Biol. Chem.

(2000)

G. Fenteany et al.

J. Biol. Chem.

(1998)

S. Biocca et al.

EMBO J.

(1990)

A. Cattaneo et al.

Intracellular antibodies: development and application

(1997)

S. Biocca et al.

Bio/Technology

(1994)

L. Duan et al.

Proc. Natl. Acad. Sci. USA

(1994)

A.M. Mhashilkar et al.

EMBO J.

(1995)

W.A. Marasco et al.

Proc. Natl. Acad. Sci. USA

(1993)

I.J. Rondon et al.

Annu. Rev. Microbiol.

(1997)

P. Tavladoraki et al.

Nature

(1993)

M. Visintin et al.

Proc. Natl. Acad. Sci. USA

(1999)

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

Abstract

Introduction

Section snippets

Intracellular targeting of scFvs

Trends Cell Biol.

Trends Biotechnol.

Biochem. Biophys. Res. Commun.

Trends Biotechnol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

FEBS Lett.

Gene

J. Immunol. Methods

J. Biol. Chem.

J. Biol. Chem.

EMBO J.

Intracellular antibodies: development and application

Bio/Technology

Proc. Natl. Acad. Sci. USA

EMBO J.

Proc. Natl. Acad. Sci. USA

Annu. Rev. Microbiol.

Nature

Proc. Natl. Acad. Sci. USA