The third helix of the murine Hoxc8 homeodomain facilitates protein transduction in mammalian cells.
Previously, we have demonstrated that purified Hoxc8 homeoprotein has the ability to penetrate the cellular membrane and can be transduced efficiently into COS-7 cells. Moreover, the Hoxc8 protein is able to form a complex with DNA molecules in vitro and helps the DNA be delivered intracellularly, serving as a gene delivery vehicle. Here, we further analyzed the membrane transduction activity of Hoxc8 protein and provide the evidence that the 16 amino acid (a.a.191-206, 2.23 kDa) third helix of murine Hoxc8 protein is an efficient protein transduction domain (PTD). When the 16 amino acid peptide was fused at the carboxyl terminal of enhanced green fluorescence protein (EGFP), the fusion proteins were transduced efficiently into the primary pig fetal fibroblast cells. The transduction efficiency increased in a concentration-dependent manner up to 1 microM, and appeared to plateau above a concentration of 1 microM. When tandem multimers of PTD, EGFP-PTD(2), EGFP-PTD(3), EGFP-PTD(4), and EGFP-PTD(5), were analyzed at 500 nM of concentration, the penetrating efficiency increased in a dose-dependent manner. As the number of PTDs increased, the EGFP signal also increased, although the signal maintained plateau after EGFP-PTD(3). These results indicate that the 16 amino acid third helix is the key element responsible for the membrane transduction activity of Hoxc8 proteins, and further suggest that the small peptide could serve as a therapeutic delivery vehicle for large cargo proteins.
Department of Anatomy, Embryology Laboratory, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Sodaemun-gu, Seoul, Republic of Korea.
Kyoung-Ah
KA
Kong
Jogeswar
J
Gadi
Hyoung Woo
HW
Park
Jinwoong
J
Bok
Myoung Hee
MH
Kim
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014677:Pharmaceutical Vehicles / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000235:genetics; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013552:Swine
D018398:Homeodomain Proteins; C489816:Hoxc8 protein, mouse; D010455:Peptides; D014677:Pharmaceutical Vehicles; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins