Carboxyterminal cleavage of the chemokines MIG and IP-10 by gelatinase B and neutrophil collagenase

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Abstract

Proteolytic processing is an important regulatory mechanism for chemokines. Matrix metalloproteinases (MMPs), such as gelatinase A/MMP-2 and gelatinase B/MMP-9, are known to process the aminoterminal end of various chemokines, including interleukin-8 (IL-8/CXCL-8) and monocyte chemotactic protein-3 (MCP-3/CXCL-7). In the present study, two proteases, gelatinase B and neutrophil collagenase/MMP-8, are shown for the first time to process the carboxyterminal end of two chemokines, monokine induced by interferon (IFN)-γ (MIG/CXCL-9) and IFN-inducible protein-10 (IP-10/CXCL-10). Neutrophil collagenase degrades MIG into small fragments and cleaves IP-10 behind positions 71 and 73. Gelatinase B degrades IP-10 and cleaves MIG at three different sites in its extended carboxyterminal region. This results in the formation of MIG(1–94), MIG(1–93), and MIG(1–90). In general, gelatinase B was more efficient than neutrophil collagenase in processing these chemokines. Alignment of the CXC chemokines with the respective cleavage sites by both MMPs identified the ELR motif as a possible determinant for amino terminal cleavage by these MMPs.

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Materials and methods

Chemokines and MMPs. Natural gelatinase B from human neutrophils was purified to homogeneity and activated with 1:100 stromelysin-1 as described [8]. Recombinant human neutrophil collagenase (R&D, Abingdon, UK) was activated during 1 h with 1 mM para-aminophenyl mercuric acetate (APMA) at 37 °C and subsequently dialyzed against assay buffer (100 mM Tris–HCl, pH 7.5, 100 mM NaCl, 10 mM CaCl2, and 0.01% Tween 20).

The chemokines MIG and IP-10 were purchased from Peprotech (Rocky Hill, NJ) and I-TAC from

Cleavage of MIG by gelatinase B and neutrophil collagenase

To determine whether the CXCR3-binding chemokine MIG is cleaved by gelatinase B or neutrophil collagenase, MIG was incubated with both enzymes at a 1:10 enzyme:substrate ratio and analyzed by SDS–PAGE. A clear bandshift is visible after incubation of MIG with each of the two enzymes, showing that both gelatinase B and neutrophil collagenase can process this chemokine (Fig. 1 and data not shown). Stromelysin-1, used to activate progelatinase B, did not cleave MIG at the used concentration.

Discussion

Processing of chemokines by extracellular proteolysis can occur at both the aminoterminus and the carboxyterminus, depending on the chemokine. Aminoterminal cleavage is the most commonly observed, while carboxyterminal cleavage is limited to a few chemokines, including mouse GCP-2/LIX [16] and the CXCR3 ligands MIG, IP-10, and I-TAC. Several proteases have been reported to process the aminoterminus of chemokines, including the dipeptidyl peptidase CD26, thrombin, plasmin, proteinase-3,

Acknowledgements

This study was supported by the “Geconcerteerde OnderzoeksActies 2002–2006,” the Cancer Research Fund of Fortis AB, the Belgian Federation against Cancer, and the National Fund for Scientific Research (F.W.O.-Vlaanderen). P.P. is a postdoctoral fellow of the F.W.O.-Vlaanderen.

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