Fig 1.
The most common amino acids around the cleavage sites of both meprin α and β.
The size of the one letter code of the amino acid represents the frequency of that amino acid in that particular position. The figure was generated using icelogo, based on peptide cleavage assays described previously [13]. Peptide sequences were aligned on the scissile bond between P1 and P1′ indicated by a black line. Statistically significant amino acid residue occurrences present (P<0.05) were plotted. Those amino acids that were completely absent are shown below in pink.
Fig 2.
Graphical representation of exendin-4 and its derivatives.
The linkers are highlighted in different colours.
Table 1.
Amino acid sequences of the peptide fragments.
Fig 3.
In vitro assessment of the peptides containing cleavable linkers in CHL cells expressing GLP-1R.
(A) IC50 values of natIn labelled peptides. 0.1–0.6 pmol 111In-Ex4NOD40 was used as tracer. (B) Internalisation kinetics of the 111In-labelled probes. 0.2–1 pmol of the respective 111In-labelled peptide was used as tracer. Non-specific binding was determined by incubation with 1 μM of natIn labelled peptide.
Fig 4.
Plasma stability of 111In-labelled PSI-CLNOD1, PSI-CLNOD2 and PSI-CLNOD3 in fresh human blood plasma.
Fig 5.
Digestion of In-labelled probes with recombinant meprin β.
A, C, E, and G show the metabolism of the corresponding 111In-labelled peptides. Curves B, D, F and H show the degradation of the corresponding natIn-labelled peptides and the formation of different fragments over time.
Fig 6.
Digestion of In-labelled probes with recombinant meprin α.
A, C, E, and G show the metabolism of the corresponding 111In-labelled peptides. Curves B, D, F and H show the degradation of the corresponding natIn-labelled peptides and the formation of different fragments over time.
Table 2.
Cleavage sites of In-labelled peptides digested with meprin α and β.
Fig 7.
Immunofluorescence picture of murine kidney sections.
(A) Anti-Meprin β antibody was used to detect the protease, visualised by fluorescence detection at 488 nm. (B) For the control goat igG was used.
Fig 8.
Ex vivo metabolism of the corresponding peptides in CD1 nu/nu kidney homogenates.
Fig 9.
Biodistribution of 111In-labeled peptides in CD1 nu/nu mice with CHL-GLP1R positive tumour-xenografts.
Values are mean percentages injected dose per gram tissue (n = 4. Error bars SD). Blocking was performed by pre-injection of 100 μg excess of unmodified exendin-4. Mice were sacrificed 4 h after injection. The significance was tested by a two-was ANOVA test corrected for multiple comparisons by Tukey’s honest significance test (**p<0.01; ***p<0.001; ****p>0.0001).