Selection and characterization of KDEL-specific VHH antibody fragments and their application in the study of ER resident protein expression.
Several diseases are caused by defects in the protein secretory pathway of the cell, particularly in the endoplasmic reticulum (ER). These defects are manifested by the activation of the unfolded protein response (UPR) that involves the transcriptional up-regulation of several ER resident proteins, the down-regulation of protein translation and up-regulation of ER associated degradation (ERAD). Although this transcriptional up-regulation of ER resident proteins during ER stress has been well described, data on differential protein expression of these same proteins are hardly available. Tools that would enable the simultaneous analysis of this set of proteins would be of high importance. Since the C-terminal KDEL sequence is a conserved epitope present in a large set of ER resident proteins, an antibody directed against this sequence would be such a tool. Using a carefully designed selection strategy, VHH antibody fragments from a non-immune phage display library were isolated that recognize the KDEL sequence at the C-terminus of proteins, irrespective of the protein context. In an accepted in vitro model for ER stress, this antibody was shown to be an excellent tool to study differences in ER resident protein expression. Furthermore, the application of this antibody showed differences in ER resident protein levels during replicative senescence of human umbilical vein endothelial cells (HUVECs), underlining its significance in biological research. The selection strategy used to obtain these KDEL-specific antibodies opens up ways to select antibodies to other conserved epitopes, such as the nuclear localization signal (NLS) or the peroxisomal targeting sequence, permitting the simultaneous analysis of specific groups of proteins.
Klooster
Rinse
R
Department of Cellular Architecture and Dynamics, Institute of Biomembranes, Department of Biology, Science Faculty, Utrecht University, Utrecht, The Netherlands.
Eman
Michael R
MR
le Duc
Quint
Q
Verheesen
Peter
P
Verrips
C Theo
CT
Roovers
Rob C
RC
Post
Jan A
JA
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D015536:Down-Regulation; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D019151:Peptide Library; D017510:Protein Folding; D018000:Receptors, Peptide / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015854:Up-Regulation
D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; C064459:KDEL receptor; D019151:Peptide Library; D018000:Receptors, Peptide; D011994:Recombinant Proteins