Core Functional Sequence of C-terminal GAG-binding Domain Directs Cellular Uptake of IGFBP-3-derived Peptides

Abstract

The current study clarifies the role of the Glycosaminoglycan (GAG)-binding domain of insulin-like growth factor binding protein-3 (IGFBP-3) in cell penetration. The cell penetration function of IGFBP-3 has been mapped to an 18-residue GAG-binding domain in the C-terminal region that mobilizes cellular uptake and nuclear localization of unrelated proteins. Uptake of KW-22, a 22-residue peptide that encompasses the 18-residue GAG-binding domain, and another IGFBP-3 peptide carrying a streptavidin protein cargo was investigated in Chinese hamster ovary (CHO) cells defective at several steps of biosynthesis of cell surface GAGs. The severity of GAG truncation was highly correlated to the impairment of uptake ranging from complete abrogation to only a partial reduction, suggesting that GAG-binding is required for uptake. The 18-residue GAG-binding domain consists of an 8-residue KK-8 basic sequence devoid of Arg and an adjacent 10-residue QR-10 sequence rich in Arg. Peptide mapping of uptake and GAG-binding activities within the KW-22 peptide showed that the 8-residue KK-8 basic peptide retained 80% of GAG-binding activity with no uptake activity while the 10-residue QR-10 peptide retained 53% of uptake activity and 18% of GAG-binding activity. This suggests that KK-8 carries out the majority of GAG-binding function while QR-10 carries out the majority of the cell entry function. To our knowledge, this is the first report of physical separation of the uptake and GAG-binding functions within a short cell penetrating peptide and may shed light on the general mechanism of uptake of Arg-rich CPPs and guide new design of Arg-rich CPP-assisted drug/gene delivery systems.

Insulin-Like Growth Factor Binding Protein-3 (IGFBP-3): Unraveling the Role in Mediating IGF-Independent Effects Within the Cell

“…Mutants of GAGs possessing defects in truncation of GAG could affect the cellular uptake of IGFBP-3 ranging from impaired cellular uptake to complete inability to internalize (Liu et al, 2014). In the same study, Liu et al (2014) demonstrated that IGFBP-3 peptides entered as a CPP in a GAG-dependent manner. An 18-mer peptide including the GAG domain of the C-terminus was synthesized and of these, 8-mer peptides retained a stronger binding to GAGs, whereas 10-mer peptides retained most of the cellular uptake functions.…”

“…domain (Lee et al, 2004), heparin binding domain or GAG binding domain (Smith et al, 1994;Fowlkes and Serra, 1996;Liu et al, 2014), metal binding domain (Singh et al, 2004), and ALS binding domain (Firth et al, 1998b), vide supra Figure 1. Therefore, the C-terminal domain of IGFBP-3 plays an important role in metal binding, cellular uptake, as well as nuclear localization of the protein.…”

“…Polycationic molecules initially associate with the cell surface heparan sulfate or GAG, through their heparin or GAG binding domain involving electrostatic forces of interactions followed by internalization through endocytic pathways, either enclosed within vesicles (endocytosis) or without vesicles (direct transport) (Madani et al, 2011). Cells are known to internalize heparan sulfate proteoglycans through endocytosis and along with this they could potentially internalize external ligands that associate with their GAG domains (Fuchs and Raines, 2004;Goda et al, 2008;Liu et al, 2014).…”

“…Mechanism through which IGFBP-3 interacts with the plasma membrane of the cells or ECM include its ability to associate with GAG through the C-terminal domain (Singh et al, 2004;Liu et al, 2014). The amino acid region spanning from 215 to 232 residues in the C-terminal domain was characterized as a GAG-binding domain, which is important for the internalization of IGFBP-3 (Singh et al, 2004).…”

“…A recent study demonstrated that the cellular uptake function of GAG binding domain and the physical GAG-binding functions of IGFBP-3 can be attributed to the distinct regions within the C-terminal domain (Liu et al, 2014). Mutants of GAGs possessing defects in truncation of GAG could affect the cellular uptake of IGFBP-3 ranging from impaired cellular uptake to complete inability to internalize (Liu et al, 2014). In the same study, Liu et al (2014) demonstrated that IGFBP-3 peptides entered as a CPP in a GAG-dependent manner.…”