Biochemical and Biophysical Research Communications
Carboxyterminal cleavage of the chemokines MIG and IP-10 by gelatinase B and neutrophil collagenase
Section snippets
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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