Abstract
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, beta(2) 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 beta(2) integrin. Active MMP-9 cleaved a single peptide, ECVKGPNVAAIVGGT, at residues corresponding to Ala(705) and Ile(706) of the beta(2) 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 beta(2) 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 beta(2) integrin.
Highlights
Activated macrophages are essential effectors of immunity and a rich source of matrix metalloproteinase-9 (MMP-9; gelatinase B)
Our observations indicate that subtractive proteomics in concert with peptide substrate mapping is a robust, high throughput technique for identifying cellular substrates that are proteolytically shed from macrophages
Macrophage Matrix metalloproteinases (MMPs)-9 Sheds 2 Integrin Molecular & Cellular Proteomics 8.5 1051 a Annotation based on UniProt Knowledgebase. b ϩ or Ϫ, passes or fails (Bolshev’s sign test at ␣ ϭ 0.05), respectively. c Glycosylphosphatidylinositol-membrane anchored. d No peptide identified in WT cells. e Annotation based on Gene Ontology. f Computational prediction (Phobius and Sig-Pred)
Summary
Cell Culture—The mouse macrophage cell line RAW264.7 was transduced with high titer retrovirus. Conditioned medium from the cells was collected, supplemented with 100 M butylated hydroxytoluene (to inhibit oxidation) and 10 mM EDTA (to inhibit MMP-9 and other metal ion-dependent proteases), and centrifuged for 15 min at 1200 ϫ g. They were separated on a C18 reverse-phase column (Hypersil BioBasic-C18, 5 m, 100 ϫ 0.3 mm; Thermo Electron Corp.) with a linear gradient of 7–35% acetonitrile in 0.1% formic acid over 90 min. A single analysis identified an average of 329 proteins (Ն2 unique peptides and a ProteinProphet probability of Ն0.96) in medium from M9A macrophages. The rank order of protein abundance (as assessed by the number of unique peptides detected) in conditioned M9A and WT medium was compared with that in lysates of macrophages expressing autoactivating MMP-9.
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