Table 1. Effect of different linkers and acidic inhibitory domains on ACPP association with Jurkat and HT-1030 cells assayed by flow cytometry.
| Sequence | Uptake before cleavage | Uptake after cleavage (reagent) | Increase caused by cleavage |
|---|---|---|---|
| EEEEEDDDDK*AXRRRRRRRRRXC(Fl) | 0.18 | 2.4 (EK) | 13 |
| EEEEEDDDDK*ARRRRRRRRRXC(Fl) | 0.07 | 1.2 (EK) | 17 |
| EDDDDK*AXRRRRRRRRRXC(Fl) | 0.30 | 2.3 (EK) | 8 |
| EEDDDDK*ARXRRXRRXRRXRRXC(Fl) | 0.015 | 0.11 (EK) | 7 |
| DDDDDDK*ARRRRRRRRRXC(Fl) | 0.05 | 0.77 (EK) | 16 |
| EEDDDDK*AXrrrrrrrrrXC(Fl) | 0.07 | 1.2 (EK) | 17 |
| eeeeeeXPLG*LAGrrrrrrrrrXc(Fl) 10 min | 0.086, 0.034 | 1.3, 1.3 (MMP-2) | 16, 39 |
| eeeeeeXPLG*LAGrrrrrrrrrXc(Fl) 60 min | 0.11 | 2.1 (MMP-2) | 19 |
| UeeeeeeeeXPLG*LAGrrrrrrrrrXk(Fl) | 0.006 | 0.74 (MMP-2) | 123 |
| eeeeeeXPLG*LAGrrrrrrrrrXc(Cy5) | nc | nc (MMP-2) | 36 |
| UeeeeeeXPLG*LAGrrrrrrrrrXc(Cy5) | nc | nc (MMP-2) | 20 |
| UeeeeeeeeXPLG*LAGrrrrrrrrrXk(Cy5.5) | nc | nc (MMP-2) | 17 |
| [11-kDa PEG]XeeeeeeeeeXPLG*LAGrrrrrrrrrXk(Cy5) | 0.012, 0.10 | 0.82, 2.1 (MMP-2) | 68, 21 |
| 0.10 | 1.87 (MMP-9) | 18 | |
| [11-kDa PEG]XeeeeeeeeeXLALGPGrrrrrrrrrXk(Cy5)† | 0.019 | 0.021 (MMP-2) | 1.13 |
| — | 0.020 (MMP-9) | 1.07 | |
| Fl-XrrrrrrrrrXPLG*LAGeeeeeeee-βAla | 0.004 | 0.06 (MMP-2) | 16 |
| Fl-XrrrrrrrrrXSGRS*Aeeeeeeee-βAla | 0.012 | 0.05 (uPA) | 4 |
| eeeeeeXSGRS*AXrrrrrrrrrXc(Cy5) | nc | nc (uPA) | 11 |
| Fl-rrrrrrrrrc-*-ceeeeee‡ | 0.092 | 0.72 (reduction) | 8 |
For sequences, lowercase characters indicate d-amino acid. All peptides were amidated at C terminus. Values represent the results of triplicate experiments performed on the same day. Some entries have two values because the triplicate experiments were repeated on another day.
Cleavage site; U, succinoyl; X, 6-aminohexanoyl; Fl, fluorescein. Uptake before and after cleavage was measured by FACS, normalized to Fl-GGRRRRRRRRRR or rrrrrrrrrk(Cy5), except for some measurements not calibrated (nc) with respect to either reference peptide. EK, enterokinase; uPA, urokinase plasminogen activator.
This scrambled control should be uncleavable, so the rightmost column refers to increase due to enzyme exposure rather than cleavage.
Disulfide-linked. In “ceeeeee,” the N terminus is d-glu and the amidated C terminus is d-cys.