A Proteomic Approach for the Identification of Cell-surface Proteins Shed by Metalloproteases

Proteolytic cleavage (shedding) of extracellular domains of many membrane proteins by metalloproteases is an important regulatory mechanism used by mammalian cells in response to environmental and physiological changes. Here we describe a proteomic system for analyzing cell surface shedding. The method utilized short-term culture supernatants from induced cells as starting material, followed by lectin-affinity purification, deglycosylation, and polyacrylamide gel electrophoresis separation. Relative quantitation of proteins was achieved via isotope dilution. In this study, a number of proteins already known to be shed were identified from activated monocytes and endothelial cells, thereby validating the method. In addition, a group of proteins were newly identified as being shed. The method provides an unbiased means to screen for shed proteins. Proteolytic cleavage (shedding) of extracellular domains of many membrane proteins by metalloproteases is an important regulatory mechanism used by mammalian cells in response to environmental and physiological changes. Here we describe a proteomic system for analyzing cell surface shedding. The method utilized short-term culture supernatants from induced cells as starting material, followed by lectin-affinity purification, deglycosylation, and polyacrylamide gel electrophoresis separation. Relative quantitation of proteins was achieved via isotope dilution. In this study, a number of proteins already known to be shed were identified from activated monocytes and endothelial cells, thereby validating the method. In addition, a group of proteins were newly identified as being shed. The method provides an unbiased means to screen for shed proteins. Proteolysis of cell membrane-bound proteins provides a post-translational means of regulating protein function and has been shown to control the production of many soluble cytokines, receptors, adhesion molecules, and growth factors through the process termed ectodomain shedding (1Schlondorff J. Blobel C.P. Metalloprotease-disintegrins: modular proteins capable of promoting cell-cell interactions and triggering signals by protein-ectodomain shedding.J. Cell Sci. 1999; 112: 3603-3617Google Scholar, 2Mullberg J. Althoff K. Jostock T. Rose-John S. The importance of shedding of membrane proteins for cytokine biology.Eur. Cytokine Netw. 2000; 11: 27-38Google Scholar). Abnormal shedding can contribute to diseases such as rheumatoid arthritis and cancer (3Blobel C.P. Remarkable roles of proteolysis on and beyond the cell surface.Curr. Opin. Cell Biol. 2000; 12: 606-612Google Scholar). A key player in ectodomain shedding is the ADAM (adisintegrin and metalloprotease) family of metalloproteases (1Schlondorff J. Blobel C.P. Metalloprotease-disintegrins: modular proteins capable of promoting cell-cell interactions and triggering signals by protein-ectodomain shedding.J. Cell Sci. 1999; 112: 3603-3617Google Scholar, 2Mullberg J. Althoff K. Jostock T. Rose-John S. The importance of shedding of membrane proteins for cytokine biology.Eur. Cytokine Netw. 2000; 11: 27-38Google Scholar). ADAMs are characterized by a conserved domain structure that consists of an N-terminal signal sequence followed by the pro-domain, the metalloprotease, and disintegrin domains, a cysteine-rich region usually containing an epidermal growth factor repeat, a transmembrane domain, and a cytoplasmic tail (4Black R.A. White J.M. ADAMs: focus on the protease domain.Curr. Opin. Cell Biol. 1998; 10: 654-659Google Scholar). Tumor necrosis factor-α converting enzyme (TACE/ADAM-17) 1The abbreviations used are: TACE, tumor necrosis factor-α-converting enzyme; 1D, one-dimensional; 2D, two-dimensional; DRM, Dexter-ras-myc; HMVEC, human adult dermal microvascular endothelial cells; WGA, wheat germ agglutinin; MS/MS, tandem mass spectrometry; IC-3, Immunex compound-3; PMA, phorbol 12-myristate 13-acetate; TNF-α, tumor necrosis factor-α; IL, interleukin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; LDL, low density lipoprotein; SHPS-1, SH2 domain-containing tyrosine phosphatase substrate 1; h, human; r, receptor. was the first ADAM family protease to be characterized as a sheddase. It was originally identified by its ability to cleave membrane-bound proTNF-α, the precursor form of TNF-α, resulting in the release of soluble TNF-α from cells (5Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. A metalloproteinase disintegrin that releases tumor necrosis factor-α from cells.Nature. 1997; 385: 729-733Google Scholar, 6Moss M.L. Jin S.L. Milla M.E. Bickett D.M. Burkhart W. Carter H.L. Chen W.J. Clay W.C. Didsbury J.R. Hassler D. Hoffman C.R. Kost T.A. Lambert M.H. Leesnitzer M.A. McCauley P. McGeehan G. Mitchell J. Moyer M. Pahel G. Rocque W. Overton L.K. Schoenen F. Seaton T. Su J.L. Becherer J.D. Cloning of a disintegrin metalloproteinase that processes precursor tumor necrosis factor-α.Nature. 1997; 385: 733-736Google Scholar). Subsequent work, primarily involving TACE knockout mice and cells (7Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. An essential role for ectodomain shedding in mammalian development.Science. 1998; 282: 1281-1284Google Scholar), indicated that the shedding of a number of other proteins is mediated by TACE. These include transforming growth factor-α, l-selectin, p75 TNF recepor, amyloid protein precursor, CD30, IL-6 receptor, Notch 1 receptor, growth hormone-binding protein, and macrophage colony-stimulating factor receptor (7Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. An essential role for ectodomain shedding in mammalian development.Science. 1998; 282: 1281-1284Google Scholar, 8Buxbaum J.D. Liu K.N. Luo Y. Slack J.L. Stocking K.L. Peschon J.J. Johnson R.S. Castner B.J. Cerretti D.P. Black R.A. Evidence that tumor necrosis factor-α-converting enzyme is involved in regulated α-secretase cleavage of the Alzheimer amyloid protein precursor.J. Biol. Chem. 1998; 273: 27765-27767Google Scholar, 9Brou C. Logeat F. Gupta N. Bessia C. LeBail O. Doedens J.R. Cumano A. Roux P. Black R.A. Israel A. A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE.Mol. Cell. 2000; 5: 207-216Google Scholar, 10Hansen H.P. Dietrich S. Kisseleva T. Mokros T. Mentlein R. Lange H.H. Murphy G. Lemke H. CD30 shedding from Karpas 299 lymphoma cells is mediated by TNF-α-converting enzyme.J. Immunol. 2000; 165: 6703-6709Google Scholar, 11Zhang Y. Jiang J. Black R.A. Baumann G. Frank S.J. Tumor necrosis factor-α-converting enzyme (TACE) is a growth hormone-binding protein (GHBP) sheddase: the metalloprotease TACE/ADAM-17 is critical for (PMA-induced) GH receptor proteolysis and GHBP generation.Endocrinology. 2000; 141: 4342-4348Google Scholar, 12Rovida E. Paccagnini A. Del Rosso M. Peschon J. Dello Sbarba P. TNF-α-converting enzyme cleaves the macrophage colony-stimulating factor receptor in macrophages undergoing activation.J. Immunol. 2001; 166: 1583-1589Google Scholar, 13Althoff K. Reddy P. Voltz N. Rose-John S. Mullberg J. Shedding of interleukin-6 receptor and tumor necrosis factor-α. Contribution of the stalk sequence to the cleavage pattern of transmembrane proteins.Eur. J. Biochem. 2000; 267: 2624-2631Google Scholar). In all these studies, the linkage to TACE was made through a hypothesis-driven approach, rather than via a screening process. Protein shedding is a post-translational event that is independent of the expression level of mRNA; hence, screening of protein shedding events requires a proteomic approach. To isolate shed proteins, many of which are glycosylated, from cell supernatants, we first utilized a lectin-affinity purification step to isolate glycoproteins. An N-deglycosylation step was subsequently used to reduce the heterogeneity of the protein, which enhanced the resolution on a one-dimensional SDS-PAGE (1D-PAGE) gel. To quantitatively compare regulated versus constitutive shedding, stable isotope dilution was performed using a novel thiol-alkylating reagent. From mass spectrometric analysis of tryptic fragments, we have identified several metalloprotease-released proteins, including proteins already known to be shed and others that were not. The DRM TACE+/+ and TACE−/− cell lines (7Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. An essential role for ectodomain shedding in mammalian development.Science. 1998; 282: 1281-1284Google Scholar) were cultured as described (12Rovida E. Paccagnini A. Del Rosso M. Peschon J. Dello Sbarba P. TNF-α-converting enzyme cleaves the macrophage colony-stimulating factor receptor in macrophages undergoing activation.J. Immunol. 2001; 166: 1583-1589Google Scholar). A TACE-encoding retrovirus was generated as described (14Kinsella T.M. Nolan G.P. Episomal vectors rapidly and stably produce high-titer recombinant retrovirus.Hum. Gene Ther. 1996; 7: 1405-1413Google Scholar) and used to reconstitute functional full-length TACE in TACE−/− DRM cells. The control cells were generated by transfecting TACE−/− DRM cells with retrovirus containing an empty vector. The expression of TACE was confirmed by a functional reconstitution assay. Briefly, DRM TACE−/− monocytes were stimulated with lipopolysaccharide (1 μg/ml), and shedding of TNF and TNF receptor were analyzed by enzyme-linked immunosorbent assay (OptEIA™; Pharmingen). HMVECs (BioWhittaker/Clonetics, Walkersville, MD) were grown in EGM2MV media (BioWhittaker/Clonetics) to passage 6. Cultures were fed with fresh media every 2–3 days and passed every 5 days. To pass, 80–90% confluent cultures were gently trypsinized (BioWhittaker/Clonetics), and T175 flasks were seeded at 10,000 cells/cm2 in 35 ml of media. Prior to stimulation, DRM cells were expanded in 1-liter spinner flasks, seeded at 2.5 × 105 cells/ml, and grown to ∼2–3 × 106 cells/ml in 800 ml of growth media. DRM cells were prepared for stimulation by washing twice with cold, serum-free RPMI 1640 (Invitrogen) and once in cold, phenol red-free, serum-free RPMI 1640 (Invitrogen). Washed cells were placed in T175 flasks at 8 × 106 cells/ml in 25 ml of phenol red- and serum-free RPMI 1640. IC-3 (25 μg/ml) and/or PMA (100 ng/ml) (ICN Biomedicals, Inc., Aurora, OH) were added to appropriate flasks. Flasks were incubated for 90 min at 37 °C with 5% CO2. Supernatants from all flasks were harvested, centrifuged for 10 min at 1200 rpm, 4 °C; 0.22-μm filtered (Corning Glass Inc., Corning, NY) and treated with protease inhibitors (175 μg/ml phenylmethylsulfonyl fluoride, 4.75 μg/ml leupeptin, 6.9 μg/ml pepstatin A, and 2.5 μg/ml EDTA). Supernatants were concentrated (Centricon Plus-80, 10-kDa cut-off; Millipore, Bedford, MA; for volumes up to 80 ml) prior to purification. For HMVEC stimulation, passage 6, 90% confluent cells were used. Growth medium was gently replaced with EBM-2 basal media (BioWhittaker/Clonetics,) and cultures were incubated for 14 h. Medium was gently replaced again with phenol red-free EBM basal media (BioWhittaker/Clonetics), and half the flasks were supplemented with an inflammatory cytokine mixture for 4 h. The cytokine mixture is composed of 100 ng/ml hCD40L (Immunex, Seattle, WA), 2 ng/ml hIL-1β (Immunex), 2 ng/ml hTNFα (BIOSOURCE International, Inc., Camarillo, CA), 100 units/ml human interferon-γ (BIOSOURCE International, Inc., Camarillo, CA), 30 ng/ml human fibroblast growth factor-basic (Chemicon International, Inc., Temecula, CA), 100 ng/ml hTWEAK (Chemicon International), and 10 ng/ml human vascular endothelial growth factor (Chemicon International). After 4 h, PMA (100 ng/ml) (ICN Biomedicals, Inc.) was added to the cytokine-containing flasks, which were incubated for an additional hour. Supernatants from all flasks were harvested as above. For cytokine-stimulated cells the total supernatant protein yield per 108 cells was 6.3 mg, whereas unstimulated control cells yielded 3.0 mg. To isolate soluble glycoproteins in the cell supernatant, lectin-affinity chromatography using agarose-bound wheat germ agglutinin (WGA) (Vector Laboratories, Inc., Burlingame, CA) was performed. Briefly, 2–4 mg of concentrated supernatant proteins were incubated with 250 μl of washed WGA-agarose beads in 4 ml of HEPES/NaCl buffer (10 mm HEPES, pH 7.5, containing 0.15 m NaCl) in a capped Micro Bio-Spin chromatography column (Bio-Rad, Hercules, CA). After incubating at 4 °C for 1 h on a rotary shaker, the column was washed three times with 5 ml of the HEPES/NaCl buffer. The lectin-binding proteins were then eluted with 3 ml of 0.5 m N-acetyl-d-glucosamine in HEPES/NaCl buffer. The excess amount of N-acetyl-d-glucosamine was removed from the WGA eluate by 7.5-fold concentration (Centricon YM-10, 10-kDa cut-off; Millipore, Bedford, MA, for volumes up to 2 ml), followed by protein precipitation at room temperature using a method designed for quantitative recovery of protein in dilute solution in the presence of detergents and lipids (15Wessel D. Flugge U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.Anal. Biochem. 1984; 138: 141-143Google Scholar). N-Glycans were removed from glycoproteins using recombinant N-glycosidase F, also referred to as N-glycanase or PNGaseF (Glyko, Inc., Novato, CA). The deglycosylation reaction was carried out as directed by the vendor. 1D-PAGE was performed under reducing using CA). The first of the was carried out using pH from CA). The proteins were with buffer m CHAPS, mm DTT, pH and was performed using the system from The from were used for the Protein were by with (Invitrogen). Protein were from the 1D-PAGE by washing with a mixture of mm were with DTT, with or under and with as described A. M. O. M. spectrometric of proteins polyacrylamide Chem. 1996; Scholar). or was from or and were concentrated by mass spectrometric spectrometric analysis of tryptic was performed on a 1 were by mass analysis using an CA) The was using an CA) at 5 and the was the such that the through the column was The of the column was directed an containing a and a to a The mass was in a and proteins were identified by a protein sequence using the D.M. protein by sequence using mass 1999; Scholar). A was performed for to for shedding events are induced by phorbol such as PMA and by such as IC-3 protein J. 1997; Scholar, Fitzner J.N. Cerretti D.P. M. C. T. K. M. Gerhart M. Kozlosky C.J. March C.J. Black R.A. a of by an of tumor necrosis factor Scholar). To isolate shed proteins, cell supernatants were from cells (7Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. An essential role for ectodomain shedding in mammalian development.Science. 1998; 282: 1281-1284Google Scholar) that were stimulated with PMA in the presence or of From an of mg of supernatant proteins were from cells in the presence of from an mg per cells was in the of were in the total amount of protein in the was that shed proteins composed a of the total and that the of the supernatant proteins were from cell and was confirmed the supernatant proteins were with and analyzed by These that the proteins in the cell supernatant were of proteins, and with this we were to in the pattern on and from of cell supernatants and and is used and is as a for to the proteins in a Y. Y. R. of gel analysis Sci. S. A. 2000; Scholar, the 2000; Scholar). was that additional protein be to quantitative proteins in these proteins or proteins from cell are to be WGA, which a group of can containing or that are to many mammalian and membrane glycoproteins. WGA-agarose was for the purification of glycoproteins from the cell After the lectin-affinity the glycoproteins were to N-deglycosylation by with recombinant N-glycosidase F, which the of reducing heterogeneity and the protein on SDS-PAGE (1D-PAGE) The proteins were analyzed by and 1D-PAGE and with the from cultures containing IC-3 a were to be or of in the supernatants from cells treated with IC-3 were stimulated with PMA in These were prior to WGA of glycoproteins which was of low in the cell containing these were and protein was identified by tandem mass with for and the proteins that were identified from the are 1 transmembrane proteins that the lectin-affinity step was in cytoplasmic proteins. of the tryptic identified were from the extracellular domains of the membrane proteins, as for proteins by identified by tandem mass of Protein were from m is to of the N-glycosidase in a a Protein were from m is to of the N-glycosidase N-deglycosylation protein of in and/or mass resulting from and other proteins as on and many of the than protein protein quantitation via gel and To this we a protein quantitation method that 1D-PAGE with stable isotope dilution. are first by 1D-PAGE of protein with the mass and were then from the gel. were with DTT, and were with or and with The tryptic were and analyzed by mass that were identified from the 1D-PAGE gel all the proteins that were identified in the 1 and In addition, for proteins from which be for quantitation was by the of the and of these used for quantitation are shown of the versus to 1 for from protein, and N-glycosidase A of 1 was for the N-glycosidase F, an amount of N-glycosidase was added to the deglycosylation and protein were the proteins in the cell supernatant purification and shed and proteins, In several membrane proteins, including amyloid protein, SHPS-1, and were to be in in the IC-3 that these proteins were shed via a metalloprotease that can be by was several and the 1D-PAGE gel were with the of a mass protein receptor In the pattern indicated that was shed in the of IC-3 in the presence of the metalloprotease In a the shedding of was The for the of in this gel is be of in gel or that this protein To the shedding events with TACE TACE−/− DRM cells (7Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. An essential role for ectodomain shedding in mammalian development.Science. 1998; 282: 1281-1284Google Scholar) were with full-length TACE. of the protein shedding of the cell with that from TACE−/− cells with an empty by 1D-PAGE analysis of from the 1D-PAGE gel in for several proteins, including receptor amyloid and These proteins are shed by TACE. To this can be used to proteins shed by other cell we carried out a with HMVECs were treated with a mixture of inflammatory followed by PMA to shedding, and the cell supernatant was with supernatant from unstimulated After lectin-affinity purification and the supernatant proteins were analyzed by 1D-PAGE the protein were and of the be to the added as of the cell stimulation the proteins, 1 and endothelial cell protein receptor, were identified from protein that to be of in the Protein using the method that these proteins were in the stimulated cell supernatant we the of IC-3 on are transmembrane proteins and to be by shedding. In endothelial cell protein receptor was identified as a protein in endothelial cells J. D. C.T. release of endothelial cell protein Biol. Chem. 2000; Scholar), validating the method as to proteins already known or as proteins were identified in this using cell These include amyloid protein, l-selectin, and endothelial cell protein receptor (7Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. An essential role for ectodomain shedding in mammalian development.Science. 1998; 282: 1281-1284Google Scholar, 8Buxbaum J.D. Liu K.N. Luo Y. Slack J.L. Stocking K.L. Peschon J.J. Johnson R.S. Castner B.J. Cerretti D.P. Black R.A. Evidence that tumor necrosis factor-α-converting enzyme is involved in regulated α-secretase cleavage of the Alzheimer amyloid protein precursor.J. Biol. Chem. 1998; 273: 27765-27767Google Scholar, 12Rovida E. Paccagnini A. Del Rosso M. Peschon J. Dello Sbarba P. TNF-α-converting enzyme cleaves the macrophage colony-stimulating factor receptor in macrophages undergoing activation.J. Immunol. 2001; 166: 1583-1589Google Scholar, 13Althoff K. Reddy P. Voltz N. Rose-John S. Mullberg J. Shedding of interleukin-6 receptor and tumor necrosis factor-α. Contribution of the stalk sequence to the cleavage pattern of transmembrane proteins.Eur. J. Biochem. 2000; 267: 2624-2631Google Scholar, J. D. C.T. release of endothelial cell protein Biol. Chem. 2000; Scholar, W. J. shedding, and of are stimulated by its Cell Sci. 2000; Scholar, J.L. Shedding as a mechanism of of on stimulated human Immunol. Scholar, D. R. Murphy G. Shedding of is regulated by a receptor and the receptor and is mediated by a Cell Sci. 2001; Scholar, P. Slack J.L. Davis R. Cerretti D.P. Kozlosky C.J. R.A. D. Peschon J.J. Black R.A. analysis of the domain structure of tumor necrosis factor-α-converting enzyme.J. Biol. Chem. 2000; Scholar, P. M. C. The receptor tyrosine cell by Cell. Biol. Scholar). this proteomic was as a method that can be in of protein shedding. In addition, this a number of additional proteins as being shed by including SHPS-1, and TACE was shown to be the protease in the of the and of the identified shed proteins and receptor for which the been is known as a receptor that to LDL, the in and cells by J.L. A for Scholar). family proteins, including a shed protein to be by have been to in a of J. The receptor signal in the 2001; Scholar). The transmembrane is a physiological substrate for phosphatase and to an receptor is in macrophages and and has been in regulating events of receptor and and cell K. T. T. T. K. T. T. S. M. and cell J. 2000; Scholar). 1 is a for the receptor Notch 1 through Notch 1 has been shown to a role in The functional of shedding of SHPS-1, and 1 to be on be to the and stable proteins Y. Y. R. of gel analysis Sci. S. A. 2000; Scholar, the 2000; Scholar), protein and prior to yield a group of low proteins, of which as proteins, can be on a the starting cell supernatants in this is and the electrophoresis is by a lectin-affinity and a low and method for and proteins, was to be with these appropriate isotope and mass we have a method for the of proteins in and for Fitzner for of and for critical of the

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