title: Acidic domain in dentin phosphophoryn facilitates cellular uptake: implications in targeted protein delivery.
abstract: Dentin phosphophoryn is nature's most acidic protein found predominantly in the dentin extracellular matrix. Its unique amino acid composition containing Asp-Ser (DS)-rich repeats makes it highly anionic. It has a low isoelectric point (pI 1.1) and, therefore, tends to be negatively charged at physiological pH. Phosphophoryn is normally associated with matrix mineralization as it can bind avidly to Ca(2+). It is well known that several macromolecules present in the extracellular matrix can be internalized and localized to specific intracellular compartments. In this study we demonstrate that dentin phosphophoryn (DPP) is internalized by several cell types via a non-conventional endocytic process. Utilizing a DSS polypeptide derived from DPP, we demonstrate the repetitive DSS-rich domain facilitates that endocytosis. As a proof-of-concept, we further demonstrate the use of this polypeptide as a protein delivery vehicle by delivering the osteoblast transcription factor Runx2 to the nucleus of mesenchymal cells. The functionality of the endocytosed Runx2 protein was demonstrated by performing gene expression analysis of Runx2 target genes. Nuclear localization was also demonstrated with the fusion protein DSS-Runx2 conjugated to quantum dots in two- and three-dimensional culture models in vitro and in vivo. Overall, we demonstrate that the DSS domain of DPP functions as a novel cell-penetrating peptide, and these findings demonstrate new opportunities for intracellular delivery of therapeutic proteins and cell tracking in vivo.
authors.affiliation: Brodie Tooth Development Genetics and Regenerative Medicine Research Laboratory Department of Oral Biology, University of Illinois, Chicago, Illinois 60612, USA.
authors.forename: Sriram
authors.initials: S
authors.lastname: Ravindran
authors.forename: Preston T
authors.initials: PT
authors.lastname: Snee
authors.forename: Amsaveni
authors.initials: A
authors.lastname: Ramachandran
authors.forename: Anne
authors.initials: A
authors.lastname: George
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D000818:Animals; D002460:Cell Line; D057846:Cell-Penetrating Peptides / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D050659:Core Binding Factor Alpha 1 Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D000099311:Dentin Sialophosphoprotein
chemical_list: D057846:Cell-Penetrating Peptides; D050659:Core Binding Factor Alpha 1 Subunit; D016326:Extracellular Matrix Proteins; D010750:Phosphoproteins; D012795:Sialoglycoproteins; D000099311:Dentin Sialophosphoprotein; C493709:RUNX2 protein, human; C493716:Runx2 protein, mouse
keywords: Dentin Phosphophoryn; Drug Delivery System; Endocytosis; Extracellular Matrix Proteins; Nanotechnology; Nuclear Translocation; Quantum Dots