Intracellular single-chain antibody inhibits integrin VLA-4 maturation and function.
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.
Yuan
Q
Q
Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, U.S.A.
Strauch
K L
KL
Lobb
R R
RR
Hemler
M E
ME
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000494:pharmacology*; D002448:Cell Adhesion; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000494:pharmacology; D039041:Integrin alpha4beta1; D016023:Integrins / Q000096:biosynthesis / Q000276:immunology / Q000502:physiology*; D019169:Jurkat Cells; D051379:Mice; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D016201:Receptors, Lymphocyte Homing / Q000096:biosynthesis / Q000276:immunology / Q000502:physiology*; D018000:Receptors, Peptide / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D014162:Transfection
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D039041:Integrin alpha4beta1; D016023:Integrins; C064459:KDEL receptor; D016201:Receptors, Lymphocyte Homing; D018000:Receptors, Peptide; D011994:Recombinant Proteins