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Molecular & Cellular Proteomics 8:1044-1060, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

MMP-9 Sheds the β₂ Integrin Subunit (CD18) from Macrophages^*,S

Tomá Vaisar^,, Sean Y. Kassim, Ivan G. Gomez^¶, Pattie S. Green, Sara Hargarten, Peter J. Gough^¶,||, William C. Parks, Carole L. Wilson^¶, Elaine W. Raines^¶ and Jay W. Heinecke^,**

From the Departments of Medicine and ^¶ Pathology, School of Medicine, University of Washington, Seattle, Washington 98195

Activated macrophages are essential effectors of immunity and a rich source of matrix metalloproteinase-9 (MMP-9; gelatinase B). To search for cellular substrates of the enzyme, we subjected wild-type macrophages and macrophages expressing an autoactivating form of pro-MMP-9 (M9A macrophages) to proteomics analysis. Two-dimensional liquid chromatography together with tandem mass spectrometry identified 467 proteins in medium conditioned by M9A and/or wild-type macrophages. Subtractive proteomics identified 18 candidate MMP-9 substrates. Biochemical studies confirmed that two transmembrane proteins, β₂ integrin subunit (CD18) and amyloid protein precursor (APP), were enriched in the medium of M9A macrophages. To identify potential cleavage sites, we synthesized an overlapping library of peptides that spanned 60 residues of the ectodomain and transmembrane domain of β₂ integrin. Active MMP-9 cleaved a single peptide, ECVKGPNVAAIVGGT, at residues corresponding to Ala⁷⁰⁵ and Ile⁷⁰⁶ of the β₂ integrin. Peptides corresponding to this cleavage site were detected by tandem mass spectrometric analysis only in medium from M9A macrophages, strongly supporting the proposal that β₂ integrin is shed by autoactivating MMP-9. Our observations indicate that subtractive proteomics in concert with peptide substrate mapping is a powerful approach for identifying proteolytic substrates and suggest that MMP-9 plays previously unsuspected roles in the regulation and shedding of β₂ integrin.

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