Abstract
(pro)MMP-9 binds to CLL cells through the PEX9 domain and contributes to CLL progression. To biochemically characterize this interaction and identify potential therapeutic targets, we prepared GST-PEX9 forms containing structural blades B1B2 or B3B4. We recently described a sequence in blade B4 (P3 sequence) that bound α4β1 integrin and partially impaired cell adhesion and migration. We have now studied the possible contribution of the B1B2 region to cell interaction with PEX9. CLL cells bound to GST-B1B2 and CD44 was the primary receptor. GST-B1B2 inhibited CLL cell migration as effectively as GST-B3B4. Overlapping synthetic peptides spanning the B1B2 region identified the sequence FDAIAEIGNQLYLFKDGKYW, present in B1 and contained in peptide P6, as the most effective site. P6 inhibited cell adhesion to PEX9 in a dose-dependent manner and with an IC50 value of 90 μM. P6 also inhibited cell adhesion to hyaluronan but had no effect on adhesion to VCAM-1 (α4β1 integrin ligand), confirming its specific interaction with CD44. Spatial localization analyses mapped P6 to the central cavity of PEX9, in close proximity to the previously identified P3 sequence. Both P6 and P3 equally impaired cell adhesion to (pro)MMP-9. Moreover, P6 synergistically cooperated with P3, resulting in complete inhibition of CLL cell binding to PEX9, chemotaxis, and transendothelial migration. Thus, P6 is a novel sequence in PEX9 involved in cell-PEX9/(pro)MMP-9 binding by interacting with CD44. Targeting both sites, P6 and P3, should efficiently prevent (pro)MMP-9 binding to CLL cells and its pathological consequences.
Highlights
ProMMP-9 binds to Chronic lymphocytic leukemia (CLL) cells through the hemopexin domain (PEX9), contributing to disease progression
The PEX9 B1B2 Region Inhibits CLL Cell Chemotaxis and Transendothelial Migration—ConstitutiveMMP-9 is important for CLL cell migration and invasion [4, 6], and we previously showed that PEX9 or its B3B4 region inhibit this migration [17]
We have studied whether the B1B2 region of PEX9 had a functional role inMMP-9 interaction with CLL cells
Summary
Patients and Cells—Approval was obtained from the Consejo Superior de Investigaciones Científicas Bioethics Review Board for these studies. For binding assays in solution, 1 ϫ 105 cells were incubated in 100 l adhesion medium (for constitutive (pro)MMP-9 expression) or medium containing the various proteins or peptides for 30 min at room tempera-. After washing with ice-cold medium, cells were incubated with anti-MMP-9 rabbit polyclonal antibodies or control Abs (30 min, 4 °C), washed with cold PBS, and incubated (30 min, 4 °C) with Alexa Fluor 488-labeled secondary Abs. Surfacebound proteins were analyzed by flow cytometry. CLL cells, with or without previous incubation with the appropriate proteins or peptides, were added to the HUVEC monolayer, and, after 24 h, migration was quantitated as explained In both types of assays, migrated cells were expressed as the percentage of the total number of cells added, counted by flow cytometry.
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