Abstract
Cyclotides or cyclic cystine-knot peptides have emerged as a promising class of pharmacological ligands that modulate protein function. Interestingly, very few cyclotides have been shown to enter into cells. Yet, it remains unknown whether backbone cyclization is required for their cellular internalization. In this report, we studied the cellular behavior of EETI-II, a model acyclic cystine-knot peptide. Even though synthetic methods have been used to generate EETI-II, recombinant methods that allow efficient large scale biosynthesis of EETI-II have been lagging. Here, we describe a novel protocol for recombinant generation of folded EETI-II in high yields and to near homogeneity. We also uncover that EETI-II is efficiently uptaken via an active endocytic pathway to early endosomes in mammalian cells, eventually accumulating in late endosomes and lysosomes. Notably, co-incubation with a cell-penetrating peptide enhanced the cellular uptake and altered the trafficking of EETI-II, leading to its evasion of lysosomes. Our results demonstrate the feasibility of modulating the subcellular distribution and intracellular targeting of cystine-knot peptides, and hence enable future exploration of their utility in drug discovery and delivery.
Highlights
It was shown that the cyclotides MCoTI-I, MCoTI-II and kalata B1 are internalized into cells via overlapping yet distinct mechanisms[15,16,17]
Further work is needed to develop a more comprehensive understanding of the nature of the interaction of the different cyclotides with the cellular lipid membrane and the intrinsic molecular properties governing cellular uptake. Since it is unknown whether the acyclic homologues of cyclotides can enter into cells and if so, via which endocytic routes, we focused our attention on a member of the squash trypsin inhibitor family, Ecballium elaterium trypsin inhibitor II (EETI-II), and explored its route of internalization
Our strategy for the expression of recombinant EETI-II was to produce a version of EETI-II that is N-terminally fused with histidine (6xHis) and glutathione S-transferase (GST) tags in order to utilize nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography for purification and use GST as a carrier protein to drive high expression and improve solubility (Fig. 1b)
Summary
It was shown that the cyclotides MCoTI-I, MCoTI-II and kalata B1 are internalized into cells via overlapping yet distinct mechanisms[15,16,17]. Further work is needed to develop a more comprehensive understanding of the nature of the interaction of the different cyclotides with the cellular lipid membrane and the intrinsic molecular properties governing cellular uptake Since it is unknown whether the acyclic homologues of cyclotides can enter into cells and if so, via which endocytic routes, we focused our attention on a member of the squash trypsin inhibitor family, Ecballium elaterium trypsin inhibitor II (EETI-II), and explored its route of internalization. Several attempts at biosynthesis of recombinant EETI-II in E. coli have been reported[22,23,24] These methods rely on expression of the molecule of interest fused to a carrier protein that routes its secretion to the periplasmic space (oxidizing conditions) or to inclusion bodies (reducing conditions) which requires additional folding steps after purification. Using cell-penetrating peptides, we demonstrate the feasibility of enhancing the cellular uptake of EETI-II and modulating its trafficking in mammalian cells
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