ABSTRACT
Chemokines provide directional cues for leukocyte migration and activation that are essential for normal leukocytic trafficking and for host responses during processes such as inflammation, infection, and cancer. Recently we reported that matrix metalloproteinases (MMPs) modulate the activity of the CC chemokine monocyte chemoattractant protein-3 by selective proteolysis to release the N-terminal tetrapeptide. Here we report the N-terminal processing, also at position 4-5, of the CXC chemokines stromal cell-derived factor (SDF)-1alpha and beta by MMP-2 (gelatinase A). Robustness of the MMP family for chemokine cleavage was revealed from identical cleavage site specificity of MMPs 1, 3, 9, 13, and 14 (MT1-MMP) toward SDF-1; selectivity was indicated by absence of cleavage by MMPs 7 and 8. Efficient cleavage of SDF-1alpha by MMP-2 is the result of a strong interaction with the MMP hemopexin C domain at an exosite that overlaps the monocyte chemoattractant protein-3 binding site. The association of SDF-1alpha with different glycosaminoglycans did not inhibit cleavage. MMP cleavage of SDF-1alpha resulted in loss of binding to its cognate receptor CXCR-4. This was reflected in a loss of chemoattractant activity for CD34(+) hematopoietic progenitor stem cells and pre-B cells, and unlike full-length SDF-1alpha, the MMP-cleaved chemokine was unable to block CXCR-4-dependent human immunodeficiency virus-1 infection of CD4(+) cells. These data suggest that MMPs may be important regulatory proteases in attenuating SDF-1 function and point to a deep convergence of two important networks, chemokines and MMPs, to regulate leukocytic activity in vivo.
Subject(s)
Chemokines, CXC/antagonists & inhibitors , Matrix Metalloproteinases/metabolism , Amino Acid Sequence , Binding Sites , Cell Line , Chemokine CXCL12 , Chemotaxis , Enzyme-Linked Immunosorbent Assay , Hemopexin/metabolism , Humans , Hydrolysis , Protein Binding , Proteoglycans/metabolismABSTRACT
On the cell surface, the 59-kDa membrane type 1-matrix metalloproteinase (MT1-MMP) activates the 72-kDa progelatinase A (MMP-2) after binding the tissue inhibitor of metalloproteinases (TIMP)-2. A 44-kDa remnant of MT1-MMP, with an N terminus at Gly(285), is also present on the cell after autolytic shedding of the catalytic domain from the hemopexin carboxyl (C) domain, but its role in gelatinase A activation is unknown. We investigated intermolecular interactions in the gelatinase A activation complex using recombinant proteins, domains, and peptides, yeast two-hybrid analysis, solid- and solution-phase assays, cell culture, and immunocytochemistry. A strong interaction between the TIMP-2 C domain (Glu(153)-Pro(221)) and the gelatinase A hemopexin C domain (Gly(446)-Cys(660)) was demonstrated by the yeast two-hybrid system. Epitope masking studies showed that the anionic TIMP-2 C tail lost immunoreactivity after binding, indicating that the tail was buried in the complex. Using recombinant MT1-MMP hemopexin C domain (Gly(285)-Cys(508)), no direct role for the 44-kDa form of MT1-MMP in cell surface activation of progelatinase A was found. Exogenous hemopexin C domain of gelatinase A, but not that of MT1-MMP, blocked the cleavage of the 68-kDa gelatinase A activation intermediate to the fully active 66-kDa enzyme by concanavalin A-stimulated cells. The MT1-MMP hemopexin C domain did not form homodimers nor did it bind the gelatinase A hemopexin C domain, the C tail of TIMP-2, or full-length TIMP-2. Hence, the ectodomain of the remnant 44-kDa form of MT1-MMP appears to play little if any role in the activation of gelatinase A favoring the hypothesis that it accumulates on the cell surface as an inactive, stable degradation product.
Subject(s)
Matrix Metalloproteinase 2/chemistry , Matrix Metalloproteinase 2/metabolism , Metalloendopeptidases/chemistry , Metalloendopeptidases/metabolism , Tissue Inhibitor of Metalloproteinase-2/chemistry , Animals , Cell Membrane/metabolism , Cells, Cultured , Chromatography , Concanavalin A/pharmacology , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Epitopes , Glycine/chemistry , Immunohistochemistry , Kinetics , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/physiology , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , Rats , Receptors, Peptide/chemistry , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Two-Hybrid System TechniquesABSTRACT
Tissue degradation by the matrix metalloproteinase gelatinase A is pivotal to inflammation and metastases. Recognizing the catalytic importance of substrate-binding exosites outside the catalytic domain, we screened for extracellular substrates using the gelatinase A hemopexin domain as bait in the yeast two-hybrid system. Monocyte chemoattractant protein-3 (MCP-3) was identified as a physiological substrate of gelatinase A. Cleaved MCP-3 binds to CC-chemokine receptors-1, -2, and -3, but no longer induces calcium fluxes or promotes chemotaxis, and instead acts as a general chemokine antagonist that dampens inflammation. This suggests that matrix metalloproteinases are both effectors and regulators of the inflammatory response.
Subject(s)
Cytokines , Inflammation/metabolism , Matrix Metalloproteinase 2/metabolism , Monocyte Chemoattractant Proteins/metabolism , Animals , Calcium/metabolism , Catalytic Domain , Cell Line , Chemokine CCL7 , Chemokines/antagonists & inhibitors , Chemokines/metabolism , Chemotaxis, Leukocyte , Collagen/metabolism , Enzyme Activation , Gene Library , Hemopexin/chemistry , Hemopexin/metabolism , Humans , Inflammation/pathology , Mass Spectrometry , Matrix Metalloproteinase 2/chemistry , Mice , Protein Binding , Protein Structure, Tertiary , Receptors, Chemokine/antagonists & inhibitors , Receptors, Chemokine/metabolism , Recombinant Proteins/metabolism , Tissue Inhibitor of Metalloproteinase-2/metabolism , Two-Hybrid System TechniquesABSTRACT
Recombinant yeast nucleosome assembly protein (yNAP-1) facilitates the formation of uniformly spaced nucleosomes from high molecular weight DNA and core histone octamers. No additional factors or metabolites are required. The repeat length of the chromatin produced is about 146 base pairs. To obtain the most distinct nucleosomal ladders, the core histones must preexist as an octamer complex. yNAP-1 forms complexes with core histones as judged by native gel electrophoresis, chemical cross-linking, limited histone proteolysis, and affinity blotting. A discrete complex was observed with a probable ratio of yNAP-1 to histone octamer of 4:1. Chromatin produced by salt dialysis does not contain uniformly spaced nucleosomes, but subsequent incubation with yNAP-1 creates uniform spacing. Trypsin-treated core octamers that lack amino termini, although capable of forming core particles with core-length DNA by salt dialysis, are not assembled by yNAP-1 into uniformly spaced nucleosomes on high molecular weight DNA. Proteolytic removal of the amino termini of the core histones precludes complex formation between a histone octamer and yNAP-1. Affinity blotting also demonstrates that yNAP-1 binds linker histones and high mobility group (HMG)-1/HMG-2 but not HMG-14. Competition experiments with poly-L-arginine, poly-L-lysine, and protamine reveal that yNAP-1 binds to core and linker histones more tightly despite the much higher positive charge densities of the former molecules. Naturally occurring acetylated histone H4 species show no evidence for differential yNAP-1 binding. yNAP-1 is not bound tightly to the resulting chromatin after deposition and thus could act catalytically.
