Proteolytic processing and inactivation of CCL2/MCP-1 by meprins

Abstract

Monocyte chemotactic protein 1 (CCL2/MCP-1) is a small chemokine involved in the recruitment and trafficking of mononuclear immune cells to inflammation sites. Our studies demonstrate that the metalloendopeptidases meprin A (purified from kidney cortex), recombinant meprin α, and recombinant meprin β can all process CCL2/MCP-1. The cleavage sites were determined by amino acid sequencing and mass spectrometry analysis of the generated products, and the biological activity of the products was evaluated by chemotactic migration assay using THP-1 cells. The cleavage sites generated by the meprin isoforms revealed that meprin A and meprin α cleaved the N-terminal domain of mouse CCL2/MCP-1 at the Asn⁶ and Ala⁷ bond, resulting in significant reduction in the chemotactic activity of the cleaved CCL2/MCP-1. Meprin β was unable to cleave the N-terminus of mouse CCL2/MCP-1 but cleaved the C-terminal region between Ser⁷⁴ and Glu⁷⁵. Human CCL2/MCP-1 that lacks the murine C-terminal region was also cleaved by meprin α at the N-terminus resulting in significant loss of CCL2/MCP-1 biological activity, whereas meprin β did not affect the biological activity. These studies suggest that meprin α and meprin β may play important roles in regulating the CCL2/MCP-1 chemokine activity during inflammation.

Keywords: Chemokine; Inflammation; Meprin A; Meprin α; Meprin β; Metalloproteinase; Monocyte chemotactic protein 1.

Fig. 1

Cleavage of mouse CCL2/MCP-1 by meprins. Activated recombinant rat meprin α, recombinant human meprinβΔ, and meprin A from rat kidney membranes (100 ng) were incubated with full-length mature mouse CCL2/MCP-1 (100 ng) at 37 °C for 90 min. Samples (50 ng) were separated by 10% SDS-PAGE and products detected on western blots with an anti-CCL2/MCP-1 rabbit polyclonal antibody (1:500 dilution). Untreated CCL2/MCP-1 (10 ng) was used as a reference (lane 5). Kaleidoscope Marker (Bio-Rad), Marker 1 (not visible, lane 4) was used to monitor samples during gel run. Sizes of Magic Mark XP (Invitrogen) Marker 2 protein standards (separate lane) are indicated at right. Separate lane as shown was obtained from the same western blot.

Fig. 2

Sites of enzyme cleavage by meprins in the full-length mature murine CCL2/MCP-1 sequence. Sequence of full-length mature mouse CCL2/MCP-1 showing the sites of enzyme cleavage for recombinant human meprin βΔ, recombinant rat meprin α, and meprin A purified from rat kidney. Dashed arrows indicate the putative C-terminal amino acid of the fragment produced with meprin α and meprin A. The N-termini were determined by Edman degradation, the C-terminus produced by meprin α was determined by LC/MS/MS. Sequence of truncated mature mouse CCL2/MCP-1 is underlined.

Fig. 3

Time course of CCL2/MCP-1 cleavage by meprins. Panel A. Recombinant mature mouse CCL2/MCP-1 (1.0 µM) was digested with activated human promeprin α (0.10 µM) or human promeprin β (0.10 µM) respectively at 37 °C for 2, 30, and 90 min. Actinonin (50 µM) was added to block meprin activity. Aliquots (100 ng) were separated on 15% SDS-PAGE, blotted on PVDF membranes and blots probed with rabbit polyclonal antibody against CCL2/MCP-1. Incubation of CCL2/MCP-1 with trypsin/SBTI was used to demonstrate complete deactivation of trypsin by SBTI used in the activation of promeprins. Panel B. Recombinant mature mouse CCL2/MCP-1 (1.0 µM) was digested with activated human promeprin α (0.10 µM) or human promeprin β (0.10 µM) respectively at 37 °C for 2, 30, 90, and 180 min and subjected to SDS-PAGE as described for Panel A.

Fig. 4

Biological activity assay of cleaved CCL2/MCP-1 fragments. Panel A. Western blot of cleaved CCL2/MCP-1 fragments used in CCL2/MCP-1 migration assay. Recombinant mature mouse CCL2/MCP-1 was digested with activated human promeprin α and activated promeprin β at 37 °C for 90 min. Aliquots of the individual digests were removed and used for the migration assay and aliquots (50 ng) of the remaining samples analyzed by western blot as described in Materials and Methods. A representative western blot is shown. Panel B. The chemotactic migration assays were performed in triplicate in 8.0 µm transwell plates. THP-1 cells were seeded at 3×10⁶ cells/mL in RPMI medium containing 0.2% BSA in the upper chamber. Digested or undigested mouse or human CCL2/MCP-1 (5.0 nM) in RPMI medium/0.2% BSA was placed in the bottom chamber. Cells were allowed to migrate at 37 °C in a humidified CO₂-incubator for 4 h. Cell counting kit-8 reagent was added to the bottom chamber and plates incubated for 4 h at 37 °C in the CO₂-incubator. Cell migration was monitored by reading absorbance at 450 nm in a SpectramaxM5 microplate reader. Cell migration obtained with digested CCL2/MCP-1 was normalized to migration obtained with untreated CCL2/MCP-1. The values displayed represent averages ± SEM of four independent experiments (separate cleavage reactions). Results of Student's T-test are shown with **P<0.01 and ***P<0.001.