Pluripotency transcription factor Sox2 is strongly adsorbed by heparin but requires a protein transduction domain for cell internalization

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Abstract

The binding of protein transduction domain (PTD)-conjugated proteins to heparan sulfate is an important step in cellular internalization of macromolecules. Here, we studied the pluripotency transcription factor Sox2, with or without the nonaarginine (R9) PTD. Unexpectedly, we observed that Sox2 is strongly adsorbed by heparin and by the fibroblasts without the R9 PTD. However, only the R9Sox2 fusion protein is internalized by the cells. These results collectively show that binding to heparan sulfate is not sufficient for cellular uptake, thereby supporting a recent hypothesis that other proteins play a role in cell internalization of PTD-conjugated proteins.

Highlights

► Both R9Sox2 and Sox2 bind heparin with comparable affinity. ► Both R9Sox2 and Sox2 bind to fibroblasts, but only R9Sox2 is internalized. ► Internalization efficiency of R9Sox2 is 0.3% of the administered protein. ► Heparan sulfate adsorption may be part of a mechanism for managing cell death.

Introduction

Nuclear reprogramming of differentiated cells into pluripotent cells, called induced pluripotent stem cells (iPSCs), holds great promise for patient-specific regenerative medicine [1], [2]. In the early studies, the genes encoding the transcription factors (TFs) that enabled nuclear reprogramming (Sox2, Oct3/4, c-Myc, Klf4, Nanog, and Lin28) were introduced to the differentiated cells using viral vectors. However, viral delivery of these genes is not appropriate for clinical use, since the delivered genes integrate into the host genome and can cause tumors by mutating and destabilizing the host genome [3], [4]. Therefore, our laboratory [5], along with others [6], [7], [8], [9], [10], sought to generate iPSCs by delivering these transcription factors directly to the differentiated cells.

Intracellular delivery of these TFs requires transduction across the plasma membrane of the cell. The first barrier to entry that is encountered by the TFs is the heparan sulfate proteoglycans. Cellular uptake of macromolecules, such as these TFs, can be facilitated by fusing them to a protein transduction domain (PTD), also called a cell-penetrating peptide. PTDs are short polypeptides composed of mostly basic amino acids such as arginine and lysine [11], [12]. They are thought to be internalized via fluid phase endocytosis (specifically, macropinocytosis), which is triggered by electrostatic interactions between the positively-charged PTD and the negatively-charged heparan sulfate proteoglycans on the cell surface [13], [14], [15], [16], [17], [18], [19], [20]. However, more recent studies suggest that other mechanisms may also be involved. For example, Gump and coworkers showed that cellular uptake of a PTD fusion protein still occurs in cells deficient in glycosaminoglycans (GAGs). Internalization of the PTD fusion protein was prevented only when proteins on the cell surface were digested [21]. Naik and coworkers showed that the cellular internalization of both the free polyarginine R16 peptide and the R16-DNA complexes was independent of cell surface GAGs [22]. Therefore, hitherto-unidentified proteins or receptors may also mediate cellular uptake of arginine-rich PTDs.

We studied the heparin binding and the internalization of one of the pluripotency transcription factors, SRY (sex determining region Y)-box 2 (Sox2), with or without the nonaarginine (R9) PTD. Using radiolabeled R9Sox2 and Sox2, we showed that both proteins bind equally well to heparin and to fibroblasts. However, only R9Sox2 is able to enter the fibroblasts. Our results thus support the hypothesis that adsorption of the PTD fusion protein to the cell surface is not sufficient for its cellular uptake.

Section snippets

Materials and methods

The transcription factors R9Sox2 and Sox2 were synthesized, purified and quantified as described [5]. The E. coli S30 cell extract used in the cell-free synthesis of R9Sox2 and Sox2 was prepared as described in reference [23], with modifications reported in reference [24]. The protocols for the heparin and cell binding studies are described below.

Results and discussion

Our initial aim in this project was to evaluate the functionality of the N-terminal nonaarginine (R9) protein transduction domain (PTD) of the purified R9Sox2 transcription factor by first assessing its affinity to the heparan sulfate proteoglycan layer on the surface of the fibroblasts. Heparin-coated Sepharose beads provided a uniform and well-characterized surrogate for the heparan sulfate proteoglycan layer. First, we evaluated the differential affinities of R9Sox2 and Sox2 as a function of

Acknowledgments

This work was supported by a grant from the Wallace H. Coulter Foundation. WCY was supported by NDSEG and NSF graduate fellowships.

References (31)

Cited by (1)

  • Transcription factors: Time to deliver

    2018, Journal of Controlled Release
    Citation Excerpt :

    Thus, great potential is credited to delivery systems having a mechanism of active endosomal escape. The use of different low molecular weight endosomolytic compounds such as chloroquine [206–233] or chemical epigenetic regulators [60,148] can increase the reprogramming efficiency of TFs fused with CPPs, but in vivo translation with this approach remains questionable. The combination of a small molecule transduction agent and hyperosmolality could trigger uptake and intracellular release of proteins [251], but this approach is not suitable for in vivo applications.

1

Present Address: Biogen Idec Inc., 5000 Davis Drive, Research Triangle Park, NC 27709, USA.

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