Cell penetration properties of maurocalcine, a natural venom peptide active on the intracellular ryanodine receptor.
Maurocalcine (MCa) is a 33-amino acid residue peptide toxin initially isolated from the scorpion Scorpio maurus maurus. Its structural and functional features make it resembling many Cell Penetrating Peptides. In particular, MCa exhibits a characteristic positively charged face that may interact with membrane lipids. External application of MCa is known to produce Ca2+-release from intracellular stores within seconds. MCa binds directly to the skeletal muscle isoform of the ryanodine receptor, an intracellular channel target of the endoplasmic reticulum, and induces long-lasting channel openings in a mode of smaller conductance. The binding sites for MCa have been mapped within the cytoplasmic domain of the ryanodine receptor. In this manuscript, we further investigated how MCa proceeds to cross biological membranes in order to reach its target. A biotinylated derivative of MCa (MCab) was chemically synthesized, coupled to a fluorescent streptavidin indicator (Cy3 or Cy5) and the cell penetration of the entire complex followed by confocal microscopy and FACS analysis. The data provide evidence that MCa allows the penetration of the macro proteic complex and therefore may be used as a vector for the delivery of proteins in the cytoplasm as well as in the nucleus. Using both FACS and confocal analysis, we show that the cell penetration of the fluorescent complex is observed at concentrations as low as 10 nM, is sensitive to membrane potential and is partly inhibited by heparin. We also show that MCa interacts with the disialoganglioside GD3, the most abundant charged lipid in natural membranes. Despite its action on ryanodine receptor, MCa showed no sign of cell toxicity on HEK293 cells suggesting that it may have a wider application range. These data indicate that MCa may cross the plasma membrane directly by cell translocation and has a promising future as a carrier of various drugs and agents of therapeutic, diagnostic and technological value.
Inserm U607, Canaux Calciques, Fonctions et Pathologies, 17 rue des Martyrs, 38054 Grenoble Cedex 09, France.
Sylvie
S
Boisseau
Kamel
K
Mabrouk
Narendra
N
Ram
Nicolas
N
Garmy
Véronique
V
Collin
Abir
A
Tadmouri
Mohamad
M
Mikati
Jean-Marc
JM
Sabatier
Michel
M
Ronjat
Jacques
J
Fantini
Michel
M
De Waard
D000595:Amino Acid Sequence; D002232:Carbocyanines / Q000032:analysis / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004337:Drug Carriers / Q000737:chemistry / Q000378:metabolism*; D004705:Endocytosis; D005434:Flow Cytometry; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D015688:Oncogene Protein pp60(v-src) / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D021381:Protein Transport; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012604:Scorpion Venoms / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity
D002232:Carbocyanines; D004337:Drug Carriers; D008563:Membrane Lipids; D010446:Peptide Fragments; D010455:Peptides; D019837:Ryanodine Receptor Calcium Release Channel; D012604:Scorpion Venoms; C089187:cyanine dye 3; C085321:cyanine dye 5; C406406:maurocalcine; C098708:peptide A; D015688:Oncogene Protein pp60(v-src)