Metabolic cleavage of cell-penetrating peptides in contact with epithelial models: human calcitonin (hCT)-derived peptides, Tat(47-57) and penetratin(43-58)
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Metabolic cleavage of cell-penetrating peptides in contact with epithelial models : human calcitonin (hCT)-derived peptides, Tat(47-57) and penetratin(43-58). / Tréhin, Rachel; Nielsen, Hanne Mørck; Jahnke, Heinz-Georg; Krauss, Ulrike; Beck-Sickinger, Annette G.; Merkle, Hans P.
In: Biochemical Journal, Vol. 382, No. Pt 3, 2004, p. 945-56.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Metabolic cleavage of cell-penetrating peptides in contact with epithelial models
T2 - human calcitonin (hCT)-derived peptides, Tat(47-57) and penetratin(43-58)
AU - Tréhin, Rachel
AU - Nielsen, Hanne Mørck
AU - Jahnke, Heinz-Georg
AU - Krauss, Ulrike
AU - Beck-Sickinger, Annette G.
AU - Merkle, Hans P
PY - 2004
Y1 - 2004
N2 - We assessed the metabolic degradation kinetics and cleavage patterns of some selected CPP (cell-penetrating peptides) after incubation with confluent epithelial models. Synthesis of N-terminal CF [5(6)-carboxyfluorescein]-labelled CPP, namely hCT (human calcitonin)-derived sequences, Tat(47-57) and penetratin(43-58), was through Fmoc (fluoren-9-ylmethoxycarbonyl) chemistry. Metabolic degradation kinetics of the tested CPP in contact with three cell-cultured epithelial models, MDCK (Madin-Darby canine kidney), Calu-3 and TR146, was evaluated by reversed-phase HPLC. Identification of the resulting metabolites of CF-hCT(9-32) was through reversed-phase HPLC fractionation and peak allocation by MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry) or direct MALDI-TOF-MS of incubates. Levels of proteolytic activity varied highly between the investigated epithelial models and the CPP. The Calu-3 model exhibited the highest proteolytic activity. The patterns of metabolic cleavage of hCT(9-32) were similar in all three models. Initial cleavage of this peptide occurred at the N-terminal domain, possibly by endopeptidase activity yielding both the N- and the C-terminal counterparts. Further metabolic degradation was by aminopeptidase, endopeptidase and/or carboxypeptidase activities. In conclusion, when in contact with epithelial models, the studied CPP were subject to efficient metabolism, a prerequisite of cargo release on the one hand, but with potential for premature cleavage and loss of the cargo as well on the other. The results, particularly on hCT(9-32), may be used as a template to suggest structural modifications towards improved CPP performance.
AB - We assessed the metabolic degradation kinetics and cleavage patterns of some selected CPP (cell-penetrating peptides) after incubation with confluent epithelial models. Synthesis of N-terminal CF [5(6)-carboxyfluorescein]-labelled CPP, namely hCT (human calcitonin)-derived sequences, Tat(47-57) and penetratin(43-58), was through Fmoc (fluoren-9-ylmethoxycarbonyl) chemistry. Metabolic degradation kinetics of the tested CPP in contact with three cell-cultured epithelial models, MDCK (Madin-Darby canine kidney), Calu-3 and TR146, was evaluated by reversed-phase HPLC. Identification of the resulting metabolites of CF-hCT(9-32) was through reversed-phase HPLC fractionation and peak allocation by MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry) or direct MALDI-TOF-MS of incubates. Levels of proteolytic activity varied highly between the investigated epithelial models and the CPP. The Calu-3 model exhibited the highest proteolytic activity. The patterns of metabolic cleavage of hCT(9-32) were similar in all three models. Initial cleavage of this peptide occurred at the N-terminal domain, possibly by endopeptidase activity yielding both the N- and the C-terminal counterparts. Further metabolic degradation was by aminopeptidase, endopeptidase and/or carboxypeptidase activities. In conclusion, when in contact with epithelial models, the studied CPP were subject to efficient metabolism, a prerequisite of cargo release on the one hand, but with potential for premature cleavage and loss of the cargo as well on the other. The results, particularly on hCT(9-32), may be used as a template to suggest structural modifications towards improved CPP performance.
KW - Amino Acid Sequence
KW - Animals
KW - Biotransformation
KW - Calcitonin
KW - Cell Line
KW - Chromatography, High Pressure Liquid
KW - Dogs
KW - Drug Carriers
KW - Epithelial Cells
KW - Gene Products, tat
KW - Homeodomain Proteins
KW - Humans
KW - Kinetics
KW - Molecular Sequence Data
KW - Molecular Weight
KW - Peptide Fragments
KW - Structure-Activity Relationship
KW - tat Gene Products, Human Immunodeficiency Virus
U2 - 10.1042/BJ20040238
DO - 10.1042/BJ20040238
M3 - Journal article
C2 - 15193145
VL - 382
SP - 945
EP - 956
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - Pt 3
ER -
ID: 43890710