MMP-9 sheds the beta2 integrin subunit (CD18) from macrophages.
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.
Vaisar
Tomás
T
Department of Medicine, School of Medicine, University of Washington, Seattle, Washington 98195, USA.
Kassim
Sean Y
SY
Gomez
Ivan G
IG
Green
Pattie S
PS
Hargarten
Sara
S
Gough
Peter J
PJ
Parks
William C
WC
Wilson
Carole L
CL
Raines
Elaine W
EW
Heinecke
Jay W
JW
D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D018821:CD18 Antigens / Q000737:chemistry / Q000378:metabolism*; D002457:Cell Extracts; D002460:Cell Line; D002853:Chromatography, Liquid; D017077:Culture Media, Conditioned; D004789:Enzyme Activation; D008264:Macrophages / Q000201:enzymology* / Q000378:metabolism; D013058:Mass Spectrometry; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics; D015203:Reproducibility of Results; D013379:Substrate Specificity
D016564:Amyloid beta-Protein Precursor; D018821:CD18 Antigens; D002457:Cell Extracts; D017077:Culture Media, Conditioned; D008565:Membrane Proteins; D010455:Peptides; D021122:Protein Subunits; D020780:Matrix Metalloproteinase 9