Membrane protease proteomics: Isotope-coded affinity tag MS identification of undescribed MT1-matrix metalloproteinase substrates

Abstract

By proteolytic modification of low abundant signaling proteins and membrane receptors, proteases exert potent posttranslational control over cell behavior at the postsecretion level. Hence, substrate discovery is indispensable for understanding the biological role of proteases in vivo. Indeed, matrix metalloproteinases (MMPs), long associated with extracellular matrix degradation, are increasingly recognized as important processing enzymes of bioactive molecules. MS is now the primary proteomic technique for detecting, identifying, and quantitating proteins in cells or tissues. Here we used isotopecoded affinity tag labeling and multidimensional liquid chromatography inline with tandem MS to identify MDA-MB-231 breast carcinoma cell proteins shed from the cell surface or the pericellular matrix and extracellular proteins that were degraded or processed after transfection with human membrane type 1-MMP (MT1-MMP). Potential substrates were identified as those having altered protein levels compared with the E240A inactive MT1-MMP mutant or vector transfectants. New substrates were biochemically confirmed by matrix-assisted laser desorption ionization-time-of-flight MS and Edman sequencing of cleavage fragments after incubation with recombinant soluble MT1-MMP in vitro. We report many previously uncharacterized substrates of MT1-MMP, including the neutrophil chemokine IL-8, secretory leukocyte protease inhibitor, pro-tumor necrosis factor alpha, death receptor-6, and connective tissue growth factor, indicating that MT1-MMP is an important signaling protease in addition to its traditionally ascribed roles in pericellular matrix remodeling. Moreover, the high-throughput and quantitative nature of isotope-coded affinity tag labeling combined with tandem MS sequencing is a previously undescribed degradomic screen for protease substrate discovery that should be generally adaptable to other classes of protease for exploring proteolytic function in complex and dynamic biological contexts.

Fig. 1.

Characterization of cells expressing MT1-MMP and inactive mutant E240A MT1-MMP. (A) Cell lysates from MT1-MMP, E240A, and vector-transfected MDA-MB-231 cells were analyzed by SDS/PAGE (10%) and Western blotting. Full-length MT1-MMP (55 kDa) and the 44-kDa form are indicated. (B) Cells labeled with either α-FLAG (Sigma) or α-human MT1-MMP antibody (AB815, Chemicon) (dark line) or no primary antibody (light line) were analyzed by fluorescent flow cytometry on a FACScan. (C) Media from MT1-MMP and vector-transfected cells, with or without exogenous proMMP-2 (72 kDa) added, were analyzed by gelatin zymography. Fully activated (62-kDa) and activation intermediate (68-kDa) MMP-2 are indicated.

Fig. 2.

MT1-MMP processing of IL-8 and SLPI. (A–C) CXC chemokines or SLPI were incubated with sMT1-MMP for 18 h at 37°C. Samples were then separated on 15% Tris-tricine gels under nonreducing conditions. (A) MT1-MMP cleavage of IL-8 and sequence of the cleavage site. (B) MT1-MMP did not cleave GRO-α or -γ as analyzed by SDS/PAGE. MALDI-TOF MS revealed identical masses for both chemokines in the presence or absence of MT1-MMP. (C) SLPI was detected by Western blotting by using α-SLPI antibody. SLPI cleavage was blocked by TIMP-2 added with sMT1-MMP or after preincubation (not shown). N-terminal sequences are shown.

Fig. 3.

MT1-MMP shedding of fibronectin and death receptor-6. (A) MT1-MMP and vector-transfected MDA-MB-231 cells were incubated in serum-free DMEM with or without inhibitors (TIMP-1 and -2, 100 nM). Shed fibronectin was then purified from the 48-h media by using a gelatin-Sepharose column and eluted with 8 M urea in PBS before analysis by SDS/PAGE (5%) and Western blotting with α-fibronectin monoclonal antibody. (B) Fibronectin was incubated with sMT1-MMP (+, ++) in vitro for18hat37°C. (C) DR6/Fc fusion protein was treated with increasing amounts of sMT1-MMP and separated by SDS/PAGE (10%). Cleavage products are indicated by arrows. (D) DR6/Fc was cleaved with Factor Xa (5 h, 37°C) before digestion with sMT1-MMP (18 h, 37°C). Samples were then electrophoresed and Western blotted by using α-DR6 antibody. Factor Xa (1) and MT1-MMP (2) cleaved-DR6/Fc products are shown.

Fig. 4.

MT1-MMP processing of proTNFα and CTGF. (A) ProTNFα/GST fusion protein was incubated with sMT1-MMP (18 h, 37°C) with or without BB2116. Samples were separated by SDS/PAGE (5–15%). MT1-MMP cleavage sites within the TNFα proregion were determined by N-terminal sequencing as schematically shown. Mature TNFα commences at VRSSSRT. (B) CTGF was treated with sMT1-MMP (+, ++)for 18 h at 37°C and analyzed by SDS/PAGE (15%) and MALDI-TOF MS. MT1-MMP cleavage sites were located by N-terminal sequencing to be in the CTGF linker. The IGF-binding protein module (IGFBP), von Willebrand factor type 1C module (vWFC-1), thrombospondin 1 module (TSP-1), and the cysteine-knot-containing C-terminal module (CYS) of CTGF are indicated. Insufficient protein was present to sequence the 18.8-kDa fragment. No sequence was attainable for intact CTGF, indicative of a modified N terminus.