Inhibition of cytoplasmic antigen, glucose- 6-phosphate dehydrogenase, by VH-CH1, an intracellular Fd fragment antibody derived from a semisynthetic Fd fragment phage display library.
A library of Fd fragment antibody binding proteins was created by random mutation of 15 nucleotides within the CDRIII region of the immunoglobulin heavy chain gene and displayed as Fd coat protein fusion constructs of M13 phage. The library was screened for those VHbinding sites that bound glucose-6-phosphate dehydrogenase (G6PD). One isolate (DH27bp) inhibited G6PD activity by 85 %. The DH27bpgene was re-engineered, placed in a eukaryotic expression vector having an isopropyl-beta-delta-thiogalactopyranoside (IPTG) inducible promoter, and transfected and then expressed in Chinese hamster V79 cells. G6PD activity was completely inhibited. Removal of IPTG reverted the cell to full G6PD activity. The intracellular dynamics of the G6PD/DH27bpcomplex showed that when the proteasomes of cells expressing DH27bpwere inhibited (N -acetyl-Leu-Leu-norleucinal or lactacystin) G6PD activity increased. Metabolic labelling of newly synthesized IPTG-induced proteins during/absence of proteasomal inhibitors showed that both G6PD and DH27bpare signaled for degradation when the intracellular complex is formed. Furthermore, semi-quantitative RT/PCR demonstrated that G6PD mRNA is upregulated over the time course of G6PD inactivation by DH27bpFd binding protein. These effects were not observed in those cells expressing a non-mutated Fd (UMHC) or in IPTG-treated non-transduced V79 cells. Our results demonstrate that an Fd-based intracellular binding protein can find and disable the function of a specific intracellular target and once the Fd expression is repressed the activity of intracellular targeted protein can revert to normal.
Mulligan-Kehoe
M J
MJ
National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. mary.j.mulligan-kehoe@dartmouth.edu
Russo
A
A
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D005954:Glucosephosphate Dehydrogenase / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000276:immunology; D007127:Immunoglobulin Constant Regions / Q000494:pharmacology*; D007143:Immunoglobulin Heavy Chains / Q000494:pharmacology*; D007135:Immunoglobulin Variable Region / Q000494:pharmacology*; D017912:Inovirus; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000378:metabolism; D019151:Peptide Library; D046988:Proteasome Endopeptidase Complex; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D015853:Cysteine Proteinase Inhibitors; D007127:Immunoglobulin Constant Regions; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007976:Leupeptins; D009097:Multienzyme Complexes; D019151:Peptide Library; D011994:Recombinant Proteins; C063261:acetylleucyl-leucyl-norleucinal; C067713:lactacystin; D007544:Isopropyl Thiogalactoside; D005954:Glucosephosphate Dehydrogenase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine