The cyclic cystine knot miniprotein MCoTI-II is internalized into cells by macropinocytosis.
The cyclotides are macrocyclic knotted proteins characterized by a compact topology and exceptional stability. Accordingly it has been hypothesized that they may be useful as protein engineering frameworks for the stabilization and delivery of bioactive peptide sequences. This study examined the internalization of cyclotides into mammalian cells, a vital step for the delivery of bioactive peptide sequences to intracellular targets. Although the entry of various linear peptides into cells has been reported previously, this is the first report of internalization of a macrocyclic peptide. Cell uptake was examined for representatives of two cyclotide subfamilies; the first was MCoTI-II, a member of the trypsin inhibitor subfamily, which was internalized by a macrophage and breast cancer cell line and the second, the prototypic cyclotide kalata B1 from the Möbius subfamily, which remained extracellular. Biotin labeled MCoTI-II entered macrophages by macropinocytosis, resulting in vesicular encapsulation without trafficking to lysosomes for degradation. The ready uptake, coupled with low cytotoxicity, indicates that MCoTI-II has the potential to transport grafted bioactivities to intracellular targets, making it a potentially valuable framework in drug design applications.
Greenwood
Kathryn P
KP
Institute for Molecular Bioscience and Australian Research Council Special Research Centre for Functional and Applied Genomics, The University of Queensland, Brisbane, Queensland 4072, Australia.
Daly
Norelle L
NL
Brown
Darren L
DL
Stow
Jennifer L
JL
Craik
David J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D047169:Cyclotides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D003911:Dextrans / Q000378:metabolism; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D051907:Lysosomal Membrane Proteins / Q000032:analysis; D008247:Lysosomes / Q000737:chemistry / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000032:analysis; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D029775:Momordica / Q000737:chemistry; D010873:Pinocytosis / Q000502:physiology*; D012235:Rhodamines / Q000378:metabolism; D012639:Seeds / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D033921:Vesicular Transport Proteins / Q000032:analysis; D052118:Lysosomal-Associated Membrane Protein 1
D047169:Cyclotides; D003911:Dextrans; D051907:Lysosomal Membrane Proteins; D008565:Membrane Proteins; D012235:Rhodamines; D033921:Vesicular Transport Proteins; C067661:Fluoro-Ruby; C052255:Lamp1 protein, mouse; D052118:Lysosomal-Associated Membrane Protein 1; C093846:early endosome antigen 1; C091930:kalata B1; C409923:trypsin inhibitor MCoTI-II