Functional knockdown of VCAM-1 at the posttranslational level with ER retained antibodies.
Vascular cell adhesion molecule 1 (VCAM-1) is involved in the recruitment of leukocytes to inflammatory sites. In this study we present the first functional knockdown of VCAM-1 using an ER retained antibody construct. We generated a knockdown construct encoding the VCAM-1 specific single chain variable fragment scFv6C7.1 fused to the C-terminal ER retention sequence KDEL. HEK-293:VCAM-YFP cells stably expressing a VCAM-YFP fusion protein were transiently transfected with the knockdown construct and showed down-regulation of surface VCAM-1. Knockdown efficiency of the system is time-dependent due to used transient transfection of the intrabody construct. Furthermore, intrabody mediated knockdown of HEK-293:VCAM-YFP cells also impaired cell-cell interaction with Jurkat cells that are endogenously expressing VLA-4, the physiological partner of VCAM-1. Posttranslational knockdown with ER retained antibodies seems to be a promising technique, as shown here for VCAM-1.
Strebe
N
N
Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany.
Guse
A
A
Schüngel
M
M
Schirrmann
T
T
Hafner
M
M
Jostock
T
T
Hust
M
M
Müller
W
W
Dübel
S
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects* / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D051379:Mice; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D019010:Vascular Cell Adhesion Molecule-1