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Intracellular single-chain antibody inhibits integrin VLA-4 maturation and function.

ABSTRACT: A single-chain antibody construct was prepared containing the VH and VL regions of anti-(integrin alpha 4) antibody HP1/2, an interchain linker and a KDEL endoplasmic reticulum retention sequence. Intracellular expression of this single-chain antibody caused cell-surface expression of alpha 4 beta 1 integrin to be decreased by 80% on selected RD cells and by 65-100% on selected Jurkat cells, relative to mock transfectants. Immunoprecipitation from single-chain-antibody-transfected cells showed that the single-chain antibody was complexed with the integrin alpha 4 and beta 1 subunits, and the diminished sizes of alpha 4 and beta 1 were consistent with impaired maturation. Furthermore, cell adhesion to alpha 4 beta 1 ligands [VCAM-1 (vascular cell adhesion molecule-1), FN40 (40 kDa chymotryptic fragment of fibronectin) and CS1] was greatly impaired in both RD and Jurkat cells, and cell spreading on immobilized FN40 protein was almost completely eliminated. Thus we conclude that intracellular single-chain antibodies may be used to reduce or eliminate cell-surface expression of a specific integrin, with specific functional consequences. This approach should be generally applicable to other integrin subunits.

SUBMITTER: Yuan Q

PROVIDER: S-EPMC1217661 | biostudies-other | 1996 Sep

REPOSITORIES: biostudies-other

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Project description:We have isolated a recombinant phage antibody (Phab) that binds a distinct epitope of the subclass of the sigma(54)-dependent prokaryotic enhancer-binding proteins that respond directly to aromatic effectors, e.g., those that activate biodegradative operons of Pseudomonas spp. The DNA segments encoding the variable (V) domains of the immunoglobulins expressed by mice immunized with the C-terminal half of TouR (TouRDeltaA) of Pseudomonas stutzeri OX1 were amplified and rearranged in vitro as single-chain Fv (scFv) genes. An scFv library was thereby constructed, expressed in an M13 display system, and subjected to a panning procedure with TouR. One clone (named B7) was selected with high affinity for TouR and XylR (the regulator of the upper TOL operon of the pWW0 plasmid). The epitope recognized by this Phab was mapped to the peptide TPRAQATLLRVL, which seems to be characteristic of the group of enhancer-binding proteins to which TouR and XylR belong and which is located adjacent to the Walker B motif of the proteins. The Phab B7 was instrumental in measuring directly the intracellular levels of XylR expressed from its natural promoter in monocopy gene dosage in Pseudomonas putida under various conditions. Growth stage, the physical form of the protein produced (XylR or XylRDeltaA), and the presence or absence of aromatic inducers in the medium influenced the intracellular pool of these molecules. XylR oscillated from a minimum of approximately 30 molecules (monomers) per cell during exponential phase to approximately140 molecules per cell at stationary phase. Activation of XylR by aromatic inducers decreased the intracellular concentration of the regulator. The levels of the constitutively active variant of XylR named XylRDeltaA were higher, fluctuating between approximately 90 and approximately 570 molecules per cell, depending on the growth stage. These results are compatible with the present model of transcriptional autoregulation of XylR and suggest the existence of mechanisms controlling the stability of XylR protein in vivo.

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Intracellular single-chain antibody inhibits integrin VLA-4 maturation and function.

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