Functional Proteomics of the Active Cysteine Protease Content in Drosophila S2 Cells

Abstract

The fruit fly genome is characterized by an evolutionary expansion of proteases and immunity-related genes. In order to characterize the proteases that are active in a phagocytic Drosophila model cell line (S2 cells), we have applied a functional proteomics approach that allows simultaneous detection and identification of multiple protease species. DCG-04, a biotinylated, mechanism-based probe that covalently targets mammalian cysteine proteases of the papain family was found to detect Drosophila polypeptides in an activity-dependent manner. Chemical tagging combined with tandem mass spectrometry permitted retrieval and identification of these polypeptides. Among them was thiol-ester motif-containing protein (TEP) 4 which is involved in insect innate immunity and shares structural and functional similarities with the mammalian complement system factor C3 and the pan-protease inhibitor alpha2-macroglobulin. We also found four cysteine proteases with homologies to lysosomal cathepsin (CTS) L, K, B, and F, which have been implicated in mammalian adaptive immunity. The Drosophila CTS equivalents were most active at a pH of 4.5. This suggests that Drosophila CTS are, similar to their mammalian counterparts, predominantly active in lysosomal compartments. In support of this concept, we found CTS activity in phagosomes of Drosophila S2 cells. These results underscore the utility of activity profiling to address the functional role of insect proteases in immunity. The fruit fly genome is characterized by an evolutionary expansion of proteases and immunity-related genes. In order to characterize the proteases that are active in a phagocytic Drosophila model cell line (S2 cells), we have applied a functional proteomics approach that allows simultaneous detection and identification of multiple protease species. DCG-04, a biotinylated, mechanism-based probe that covalently targets mammalian cysteine proteases of the papain family was found to detect Drosophila polypeptides in an activity-dependent manner. Chemical tagging combined with tandem mass spectrometry permitted retrieval and identification of these polypeptides. Among them was thiol-ester motif-containing protein (TEP) 4 which is involved in insect innate immunity and shares structural and functional similarities with the mammalian complement system factor C3 and the pan-protease inhibitor alpha2-macroglobulin. We also found four cysteine proteases with homologies to lysosomal cathepsin (CTS) L, K, B, and F, which have been implicated in mammalian adaptive immunity. The Drosophila CTS equivalents were most active at a pH of 4.5. This suggests that Drosophila CTS are, similar to their mammalian counterparts, predominantly active in lysosomal compartments. In support of this concept, we found CTS activity in phagosomes of Drosophila S2 cells. These results underscore the utility of activity profiling to address the functional role of insect proteases in immunity. Recognition, uptake, and destruction of pathogens by phagocytic cells are fundamental principles of innate immunity that are conserved in organisms from insects to humans (1Hoffmann J.A. Reichhart J.A. Drosophila innate immunity: An evolutionary perspective.Nat. Immunol. 2002; 3: 121-126Google Scholar). An important step after the uptake of material into the endosomal/phagosomal pathway is proteolysis to destroy and clear pathogens (2Bogyo M. Ploegh H.L. Antigen presentation. A protease draws first blood.Nature. 1998; 396: 625-627Google Scholar). Proteolysis along the endocytic pathway has been extensively studied in the mammalian system (3Villadangos J.A. Ploegh H.L. Proteolysis in MHC class II antigen presentation: Who's in charge?.Immunity. 2000; 12: 233-239Google Scholar, 4Riese R.J. Chapman H.A. Cathepsins and compartmentalization in antigen presentation.Curr. Opin. Immunol. 2000; 12: 107-113Google Scholar, 5Watts C. Antigen processing in the endocytic compartment.Curr. Opin. Immunol. 2001; 13: 26-31Google Scholar). Endocytic proteases, in particular cysteine proteases of the cathepsin (CTS) 1The abbreviations used are: CTS, cathepsin; S2, Schneider's Drosophila line 2; NCBI, National Center for Biotechnology Information; PDI, protein disulfide isomerase; TEP, thiol ester-containing protein. family, are actively involved in MHC class II-restricted antigen presentation and, therefore, play a key role in adaptive immunity (6Lennon-Dumenil A.M. Bakker A.H. Wolf-Bryant P. Ploegh H.L. Lagaudriere-Gesbert C. A closer look at proteolysis and MHC-class-II-restricted antigen presentation.Curr. Opin. Immunol. 2002; 14: 15-21Google Scholar). Members of the CTS family, such as CTS B (7Deussing J. Roth W. Saftig P. Peters C. Ploegh H.L. Villadangos J.A. Cathepsins B and D are dispensable for major histocompatibility complex class II-mediated antigen presentation.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4516-4521Google Scholar), CTS S (8Driessen C. Bryant R.A. Lennon-Dumenil A.M. Villadangos J.A. Bryant P.W. Shi G.P. Chapman H.A. Ploegh H.L. Cathepsin S controls the trafficking and maturation of MHC class II molecules in dendritic cells.J. Cell. Biol. 1999; 147: 775-790Google Scholar, 9Pluger E.B. Boes M. Alfonso C. Schroter C.J. Kalbacher H. Ploegh H.L. Driessen C. Specific role for cathepsin S in the generation of antigenic peptides in vivo.Eur. J. Immunol. 2002; 32: 467-476Google Scholar), CTS F (10Shi G.P. Bryant R.A. Riese S. Verhelst S. Driessen C. Li Z. Bromme D. Ploegh H.L. Chapman H.A. Role for cathepsin F in invariant chain processing and major histocompatibility complex class II peptide loading by macrophages.J. Exp. Med. 2000; 191: 1177-1186Google Scholar), CTS L (11Nakagawa T. Roth W. Wong P. Nelson A. Farr A. Deussing J. Villadangos J.A. Ploegh H.L. Peters C. Rudensky A.Y. Cathepsin L: Critical role in Ii degradation and CD4 T cell selection in the thymus.Science. 1998; 280: 450-453Google Scholar), and, more recently, CTS Z (12Lennon-Dumenil A.M. Bakker A.H. Maehr R. Fiebiger E. Overkleeft H.S. Rosemblatt M. Ploegh H.L. Lagaudriere-Gesbert C. Analysis of protease activity in live antigen-presenting cells shows regulation of the phagosomal proteolytic contents during dendritic cell activation.J. Exp. Med. 2002; 196: 529-540Google Scholar), have been implicated in this process (13Turk B. Turk D. Turk V. Lysosomal cysteine proteases: more than scavengers.Biochim. Biophys. Acta. 2000; 1477: 98-111Google Scholar, 14Turk V. Turk B. Guncar B. Turk D. Kos. J. Lysosomal cathepsins: Structure, role in antigen processing and presentation, and cancer.Adv. Enzyme. Regul. 2002; 42: 285-303Google Scholar). Little is known about the function of CTS proteases in other biological processes that might explain both their evolutionary conservation and expansion in multicellular organisms. Several CTS-deficient strains of mice show dramatic phenotypes that indicate involvement in bone remodeling, development, and apoptosis (15Turk V. Turk B. Turk D. Lysosomal cysteine proteases: Facts and opportunities.EMBO J. 2001; 20: 4629-4633Google Scholar, 16Felbor U. Kessler B. Mothes W. Goebel H.H. Ploegh H.L. Bronson R.T. Olsen B.R. Neuronal loss and brain atrophy in mice lacking cathepsins B and L.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7883-7888Google Scholar). In addition, dysregulation of CTS activity is observed in various cancers and correlates with tumor progression (17Edwards D.R. Murphy G. Cancer. Proteases—Invasion and more.Nature. 1998; 394: 527-528Google Scholar, 18Kos J. Werle B. Lah T. Brunner N. Cysteine proteinases and their inhibitors in extracellular fluids: Markers for diagnosis and prognosis in cancer.Int. J. Biol. Markers. 2000; 15: 84-89Google Scholar). Cysteine proteases have been mainly studied in mice and humans (13Turk B. Turk D. Turk V. Lysosomal cysteine proteases: more than scavengers.Biochim. Biophys. Acta. 2000; 1477: 98-111Google Scholar, 14Turk V. Turk B. Guncar B. Turk D. Kos. J. Lysosomal cathepsins: Structure, role in antigen processing and presentation, and cancer.Adv. Enzyme. Regul. 2002; 42: 285-303Google Scholar); however, functional redundancy and significantly overlapping specificities of CTS make it difficult to explore individual CTS function in mice and humans. Therefore, less complex organisms such as the fruit fly, Drosophila melanogaster, may be helpful in gaining further insight into the functional roles of members of this protease family. In the fruit fly, and relative to the genome as a whole, proteases and immunity-related genes are characterized by specific gene expansions (19Adams M.D. Celniker S.E. Holt R.A. Evans C.A. Gocayne J.D. Amanatides P.G. Scherer S.E. Li P.W. Hoskins R.A. Galle R.F. George R.A. Lewis S.E. Richards S. Ashburner M. Henderson S.N. et al.The genome sequence of Drosophilamelanogaster.Science. 2000; 287: 2185-2195Google Scholar, 20Christophides G.K. Zdobnov E. Barillas-Mury C. Birney E. Blandin S. Blass C. Brey P.T. Collins F.H. Danielli A. Dimopoulos G. Hetru C. Hoa N.T. Hoffmann J.A. Kanzok S.M. Letunic I. et al.Immunity-related genes and gene families in Anopheles gambiae.Science. 2002; 298: 159-165Google Scholar, 21Ross J. Jiang H. Kanost M.R. Wang Y. Serine proteases and their homologs in the Drosophilamelanogaster genome: An initial analysis of sequence conservation and phylogenetic relationships.Gene. 2003; 304: 117-131Google Scholar). Drosophila melanogaster possesses specialized blood cells, or hemocytes, that phagocytose microbes in a manner similar to their mammalian counterparts (1Hoffmann J.A. Reichhart J.A. Drosophila innate immunity: An evolutionary perspective.Nat. Immunol. 2002; 3: 121-126Google Scholar, 22Ramet M. Manfruelli P. Pearson A. Mathey-Prevot B. Ezekowitz R.A. Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli.Nature. 2002; 416: 644-648Google Scholar, 23Pearson A. Baksa K. Ramet M. Protas M. McKee M. Brown D. Ezekowitz R.A. Identification of cytoskeletal regulatory proteins required for efficient phagocytosis in Drosophila.Microbes and Infection. 2003; (In press)Google Scholar). In particular, the hemocyte-like Schneider's Drosophila line 2 (S2) has emerged as a model system to study phagocytosis and for the identification of phagocytic receptors that recognize and mediate the engulfment of microbes (24Ramet M. Pearson A. Manfruelli P. Li X. Koziel H. Gobel V. Chung E. Krieger M. Ezekowitz R.A. Drosophila scavenger receptor CI is a pattern recognition receptor for bacteria.Immunity. 2001; 15: 1027-1038Google Scholar, 25Franc N.C. Phagocytosis of apoptotic cells in mammals, Caenorhabditis elegansDrosophilamelanogaster: Molecular mechanisms and physiological consequences.Front. Biosci. 2002; 7: d1298-d1313Google Scholar). S2 cells, therefore, are a relevant model for the application of a functional proteomic approach that permits simultaneous detection and identification of multiple protease activities. Several approaches are now available to assess protease activity on a broad scale. Chemistry-based functional proteomics is a promising method used to assign putative gene products to an enzyme family (26Kidd D. Liu Y. Cravatt B.F. Profiling serine hydrolase activities in complex proteomes.Biochemistry. 2001; 40: 4005-4015Google Scholar). The rationale behind the design of such a functional proteomics approach is to assess the activity profiles of protease species in complex biological samples rather than merely their presence or absence. Identification and activity measurement can be performed without the need for purification of the individual enzymes under study. In addition, this approach allows activity measurement of several enzyme species at the same time, a task that would be more cumbersome The of this approach has been for serine proteases Y. Cravatt B.F. protein The serine Natl. Acad. Sci. U. S. A. 1999; Scholar), enzymes A. H. N. T. Ploegh H.L. Kessler Chemistry-based functional proteomics members of the enzyme Biol. 2002; Scholar), and cysteine proteases D. A. Z. A. M. Chemical approaches for the Cell. 2002; Scholar). In this we have used DCG-04, a probe that and covalently targets mammalian cysteine was to polypeptides in Drosophila and to characterize and their activities. A phagocytic of S2 cells was from A. Pearson of A. Baksa K. Ramet M. Protas M. McKee M. Brown D. Ezekowitz R.A. Identification of cytoskeletal regulatory proteins required for efficient phagocytosis in Drosophila.Microbes and Infection. 2003; (In press)Google Scholar, A. A. Krieger M. of a class scavenger receptor from Natl. Acad. Sci. U. S. A. Scholar). S2 cells were at in Schneider's or Schneider's Drosophila with to support insect cell and at a of 2 to in to these S2 cells were from and were and as on the of papain by Med. Scholar, D. A. M. as activity-dependent cysteine protease profiling and Biol. 2000; 7: Scholar). were by at 4 in Drosophila pH J.A. of of Drosophila melanogaster in Cell. Biol. Scholar), and cell to cells were at were on and in pH pH for and for at to of the cell was the with as protein was with for at was used as cell were with for of The was by the of and for were by and to in pH the was with in for at by in polypeptides were by from cells was at pH as The was into to of protein. The samples were with to activities. samples were with for at was with of DCG-04, and the was to a of and the samples were for at order to the proteins to make the of polypeptides was performed as (12Lennon-Dumenil A.M. Bakker A.H. Maehr R. Fiebiger E. Overkleeft H.S. Rosemblatt M. Ploegh H.L. Lagaudriere-Gesbert C. Analysis of protease activity in live antigen-presenting cells shows regulation of the phagosomal proteolytic contents during dendritic cell activation.J. Exp. Med. 2002; 196: 529-540Google Scholar, D. A. M. as activity-dependent cysteine protease profiling and Biol. 2000; 7: Scholar). the was to pH a and was with for at with polypeptides were from the by of and for proteins were by and the samples were with of of these samples was used for and the samples were and with as the was in for with and with for from the and were and for tandem mass spectrometry was performed as M. and Identification Scholar). The samples were to a system with a at a of a The system was to a mass Analysis was performed in and with than were in were and to or protein identification by sequence mass spectrometry 1999; 20: the National Center for Biotechnology the Drosophila protein from was also used for protein In of and of cysteine were as for proteins were as were more than or on the peptides were found for protein species. about the Drosophila gene products were from the and cysteine protease has been from Lennon-Dumenil et (12Lennon-Dumenil A.M. Bakker A.H. Maehr R. Fiebiger E. Overkleeft H.S. Rosemblatt M. Ploegh H.L. Lagaudriere-Gesbert C. Analysis of protease activity in live antigen-presenting cells shows regulation of the phagosomal proteolytic contents during dendritic cell activation.J. Exp. Med. 2002; 196: 529-540Google Scholar). S2 cells were on the were with for at were with and in were to the cells and for at Phagocytosis was at 4 cells were and at for in and were by after on a of in were in and in at for various of the cells were on and in for to and a to the at for were by and uptake of was by and uptake of by and The inhibitor has been used as an with broad cysteine proteases on the of papain by Med. Scholar, D. Bromme D. as mechanism-based cysteine protease Med. Scholar). on the of this a was to a D. A. M. as activity-dependent cysteine protease profiling and Biol. 2000; 7: Scholar). This permits of active cysteine proteases in to the cysteine active D. A. Z. A. M. Chemical approaches for the Cell. 2002; In order to with Drosophila we this probe with pH cell from S2 cells. polypeptides were by and by in we observed in the mass of and of the of for with of probe in of these proteins to in a and manner with several Drosophila of proteases in Drosophila S2 cells in a and active manner. from Drosophila S2 cells were for at in the presence or of and of inhibitor or by for at were by on and polypeptides were by In order to the polypeptides by DCG-04, we used a on purification and tandem mass spectrometry as in Drosophila S2 cell were or with in the presence or of and polypeptides were on of the by several polypeptides was a of was analysis tandem mass spectrometry a of protein for which at peptide were We these polypeptides into on the or presence of a active thiol to the of such a thiol the polypeptides in are to be in to be enzymes also proteins and observed proteins involved in and proteins found in of gene gene gene factor in a polypeptides in S2 cell by tandem mass function by sequence of protein disulfide disulfide by sequence in a We found polypeptides that might an sequence into a of these was protein disulfide an enzyme as active disulfide in polypeptides in the in a which the enzyme and the of protein disulfide of disulfide in a model Scholar). Therefore, such as and may to thiol of such polypeptides at and were as thiol ester-containing protein (TEP) 4 and 4 to a of thiol ester-containing Drosophila proteins that show structural and functional a conserved thiol to both a of mammalian complement factor and to a pan-protease M. E. M. Hoffmann J.A. of a protein in and of Natl. Acad. Sci. U. S. A. 2000; Scholar). 4 an thiol G. I. of thiol ester-containing A. 1998; Scholar). is that a with the of the thiol and of complex Biol. Scholar), the of this 4 to be in Drosophila is significantly after with pathogens M. E. M. Hoffmann J.A. of a protein in and of Natl. Acad. Sci. U. S. A. 2000; Scholar). In a hemocyte-like cell a as a and phagocytosis of This activity is on thiol Blandin S. G. M. role of a protein in phagocytosis by in cells of the Anopheles 2001; Scholar). We multiple species that homologies to mammalian CTS proteases L, B, F and K, II and The CTS protease has been as an active is found in the D. melanogaster as a putative The CTS and proteases have been and characterized as proteolytic enzymes from of the CTS protease has been observed in Drosophila M. P. Drosophila cathepsin A role in Biophys. and a enzyme in the fly Y. A. S. S. of the insect are from a 1999; Scholar). A CTS protease has been to be in and I. H. K. S. Y. A putative cysteine from Drosophilamelanogaster is predominantly in the and J. and was found in in the hemocyte-like Drosophila cell line lysosomal Y. D. Cysteine a cathepsin enzyme in the Drosophilamelanogaster cell line Biol. Scholar). The enzymes have been implicated in and and are conserved in other species and M. Molecular of that cysteine proteinases in the of the and A.M. A.H. of a cathepsin protease and the of the protease in of Biol. 2001; and of these proteases and their homologs a of conservation on in the of the the active cysteine and The were less conserved of similar in the of the Drosophila CTS which an of found in the of the mammalian CTS K. A on the of sequence similarities shows with the of CTS K, evolutionary of the CTS the of mammalian and CTS proteases from other These an evolutionary conserved function for these proteases and of these molecules for adaptive immunity in We used activity profiling to under which pH these enzymes are from S2 cells were at a pH from in profiles in a manner a pH of major of and were with the for pH This suggests that these polypeptides to the Drosophila CTS homologs by tandem mass of these polypeptides was were observed in samples and were by an of The of these enzymes a function the endocytic In order to address a phagosomal of these Drosophila CTS proteases in live cells, we used an approach by that the proteolytic in phagosomes of mammalian and dendritic cells (12Lennon-Dumenil A.M. Bakker A.H. Maehr R. Fiebiger E. Overkleeft H.S. Rosemblatt M. Ploegh H.L. Lagaudriere-Gesbert C. Analysis of protease activity in live antigen-presenting cells shows regulation of the phagosomal proteolytic contents during dendritic cell activation.J. Exp. Med. 2002; 196: 529-540Google Scholar). with are by phagocytic cells and of the proteolytic during and Drosophila S2 cells were for with The were and cells were after of of DCG-04, cells were by the of an of in the which This was on was observed in the of or in the presence of that been with These results that CTS activity can be by activity profiling in phagosomal of Drosophila S2 cells. Chemical of cysteine proteases by of a mechanism-based probe combined with tandem mass spectrometry has identification of multiple active protease species in a Drosophila cell line with phagocytic of proteases in the Drosophila genome were II and of S2 sequence available in the that of these genes to be in S2 cells, the four proteases by tandem mass spectrometry and of the sequence of the other genes that the active cysteine was with The Drosophila genome a CTS protease that is in S2 cells and the cysteine and This is in to the mammalian system in which CTS was to with (12Lennon-Dumenil A.M. Bakker A.H. Maehr R. Fiebiger E. Overkleeft H.S. Rosemblatt M. Ploegh H.L. Lagaudriere-Gesbert C. Analysis of protease activity in live antigen-presenting cells shows regulation of the phagosomal proteolytic contents during dendritic cell activation.J. Exp. Med. 2002; 196: 529-540Google genes of the papain cysteine protease family in the genome of Drosophila melanogaster in this sequence from S2 active cysteine with T and cathepsin Cathepsin L L activity from sequence with Cathepsin L L activity from sequence with F activity from sequence with activity from sequence with B activity from sequence with B activity from sequence with S activity from sequence with S in a We cysteine proteases also other enzymes that such as and 4 it is that with these polypeptides and 4 may have been as a of their with cysteine proteases that were by The that can be used to insect be in their have play a role in innate immunity in the Anopheles by the of the Blandin S. G. M. role of a protein in phagocytosis by in cells of the Anopheles 2001; Scholar). A. and C. We observed of Drosophila CTS L and F with of and that with and has been for mammalian CTS proteases S.M. S.E. S. of L for species and is required for proteolytic J. and for this in it more that to of these enzymes that have or of their as for and CTS L S.M. S.E. S. of L for species and is required for proteolytic J. Scholar). In line with this we found that the of Drosophila CTS L the peptide which is of the for the enzyme and B. Turk D. Turk V. Lysosomal cysteine proteases: more than scavengers.Biochim. Biophys. Acta. 2000; 1477: 98-111Google Scholar). of CTS by such as has been observed in mammalian cells U. Bryant R.A. Ploegh H.L. Olsen B.R. Mothes W. cathepsin L from J. 2000; Scholar). This can be to the of the probe and to the of to the active it may be to the from the active and from to the This may also explain the results by activity profiling in S2 cell phagosomes In this polypeptides were found in the that CTS proteases are to the Drosophila CTS and enzyme species sequence with their counterparts A and CTS to have This enzyme sequence to CTS K, and to CTS L a it is to and which disulfide as for in the fly Y. A. S. S. of the insect are from a 1999; Scholar). The to a chain CTS such as mammalian CTS F or the has in mammals, a specific role of the protease in activity profiling at pH was used to show that Drosophila CTS proteases are most active under that these enzymes their biological function in endocytic or lysosomal cell In mammalian CTS proteases, such as CTS L, B, and are also active at a pH J.A. Bryant R.A. Deussing J. Driessen C. Lennon-Dumenil A.M. Riese R.J. Roth W. Saftig P. Shi G.P. Chapman H.A. Peters C. Ploegh H.L. involved in MHC class II antigen 1999; Scholar), which may a role in a of biological in the presence of CTS in phagosomes of both of organisms and as as the conserved into CTS L, F, and B is with a role in lysosomal which their function in antigen processing and presentation in mammalian cells. which an adaptive is an to CTS proteases have a function in innate immunity during a with or of of Drosophila CTS L, K, B, and F in to E. P.T. B. analysis of the Drosophila by Natl. Acad. Sci. U. S. A. 2001; Scholar, P. M. C. Reichhart Hoffmann J.A. Hetru C. A analysis of in Natl. Acad. Sci. U. S. A. 2001; Scholar). In addition, with from S2 cells to in profiles be to assess Drosophila CTS activity profiles in a more for by of specific CTS proteases from phagosomes of cells to such results the of this functional proteomics approach and a for the simultaneous of multiple protease activities in insect at or during an We Pearson for S2 cells, R. B. Ezekowitz for for of samples for and and Pearson for helpful on the