Reduction of streptavidin RYDS-mediated renal adhesion by site-directed mutagenesis.
Naturally occurring core-Streptavidin (c-Strep) would serve as a more useful agent in vivo if not for its high kidney retention. This retention is mediated by an integrin-binding motif-RYDS-that shares homology to the more common RGDS. We generated a c-Strep molecule constituting amino acids 13-139 of streptavidin and by site-directed mutagenesis altered the RYDS motif to RYES. RYDS-c-Streptavidin and RYES-c-Streptavidin were expressed in E. coli and purified on a 2-imminobiotin matrix. Each demonstrated an affinity for biotin similar to that of native post-secretory streptavidin while maintaining their ability to form dimers and tetramers. The mutant RYES-c-Streptavidin was no longer able to mediate normal rat kidney cell attachment in an in vitro assay. RYDS-c-Streptavidin-mediated kidney cell attachment was inhibited by competition with c-Streptavidin, RYDS-c-Streptavidin and RGDS-containing peptides but not with an irrelevant peptide or RYES-c-Streptavidin. Therefore, the point mutation D49E generates a molecule, which may not display the in vivo kidney retention observed for RYDS-c-Streptavidin, potentially finding more widespread clinical application.
Murray
Samuel
S
Unit of Dermatology, Department of Medicine, ICSTM, Hammersmith Campus, London W12 0HS, UK. smbhsam@hotmail.com
Maraveyas
Anthony
A
Dougan
Tammy
T
Chu
Anthony C
AC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D002448:Cell Adhesion; D002478:Cells, Cultured; D016023:Integrins / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D051381:Rats; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity
D016023:Integrins; D001710:Biotin; D019809:Streptavidin