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

Functional Proteomics of the Active CysteineProtease Content in Drosophila S2 Cells*Christine Kocks‡��, Rene Maehr§��, Herman S. Overkleeft¶, Evelyn W. Wang**,Lackshmanan K. Iyer‡‡, Ana-Maria Lennon-Dumenil¶¶, Hidde L. Ploegh§,and Benedikt M. Kessler§ §§

The fruit fly genome is characterized by an evolutionaryexpansion of proteases and immunity-related genes. Inorder to characterize the proteases that are active in aphagocytic Drosophila model cell line (S2 cells), we haveapplied a functional proteomics approach that allows si-multaneous detection and identification of multiple prote-ase species. DCG-04, a biotinylated, mechanism-basedprobe that covalently targets mammalian cysteine pro-teases of the papain family was found to detect Drosoph-ila polypeptides in an activity-dependent manner. Chem-ical tagging combined with tandem mass spectrometrypermitted retrieval and identification of these polypep-tides. Among them was thiol-ester motif-containing pro-tein (TEP) 4 which is involved in insect innate immunityand shares structural and functional similarities with themammalian complement system factor C3 and the pan-protease inhibitor alpha2-macroglobulin. We also foundfour cysteine proteases with homologies to lysosomalcathepsin (CTS) L, K, B, and F, which have been impli-cated in mammalian adaptive immunity. The DrosophilaCTS equivalents were most active at a pH of 4.5. Thissuggests that Drosophila CTS are, similar to their mam-malian counterparts, predominantly active in lysosomalcompartments. In support of this concept, we found CTSactivity in phagosomes of Drosophila S2 cells. These re-sults underscore the utility of activity profiling to addressthe functional role of insect proteases in immunity.Molecular & Cellular Proteomics 2:1188–1197, 2003.

Recognition, uptake, and destruction of pathogens byphagocytic cells are fundamental principles of innate immu-nity that are conserved in organisms from insects to humans(1). An important step after the uptake of material into the

endosomal/phagosomal pathway is proteolysis to destroyand clear pathogens (2). Proteolysis along the endocytic path-way has been extensively studied in the mammalian system(3–5). Endocytic proteases, in particular cysteine proteases ofthe cathepsin (CTS)1 family, are actively involved in MHCclass II-restricted antigen presentation and, therefore, play akey role in adaptive immunity (6). Members of the CTS family,such as CTS B (7), CTS S (8, 9), CTS F (10), CTS L (11), and,more recently, CTS Z (12), have been implicated in thisprocess (13, 14).

Little is known about the function of CTS proteases in otherbiological processes that might explain both their evolutionaryconservation and expansion in multicellular organisms. Sev-eral CTS-deficient strains of mice show dramatic phenotypesthat indicate involvement in bone remodeling, development,and apoptosis (15, 16). In addition, dysregulation of CTSactivity is observed in various cancers and correlates withtumor progression (17, 18). Cysteine proteases have beenmainly studied in mice and humans (13, 14); however, func-tional redundancy and significantly overlapping specificities ofCTS make it difficult to explore individual CTS function in miceand humans. Therefore, less complex organisms such as thefruit fly, Drosophila melanogaster, may be helpful in gainingfurther insight into the functional roles of members of thisprotease family.

In the fruit fly, and relative to the genome as a whole,proteases and immunity-related genes are characterized byspecific gene expansions (19–21). Drosophila melanogasterpossesses specialized blood cells, or hemocytes, that phago-cytose microbes in a manner similar to their mammalian coun-terparts (1, 22, 23). In particular, the hemocyte-like Schnei-der’s Drosophila line 2 (S2) has emerged as a model system tostudy phagocytosis and for the identification of phagocyticreceptors that recognize and mediate the engulfment of mi-crobes (24, 25). S2 cells, therefore, are a relevant model forthe application of a functional proteomic approach that per-mits simultaneous detection and identification of multiple pro-tease activities.

Several approaches are now available to assess protease

From the ‡Laboratory of Developmental Immunology, Massachu-setts General Hospital, Department of Pediatrics, Harvard MedicalSchool, Boston, Massachusetts 02114; ¶Leiden Institute of Chemis-try, Gorlaeus Laboratories, Leiden University, 2300 RA Leiden, TheNetherlands; **Surface Logix, Inc., Brighton Massachusetts 02135;‡‡Bauer Center of Genomics, Bauer Laboratory, Cambridge, Massa-chusetts 02138; ¶¶Institut National de la Sante et de la RechercheMedicale, Institut Curie, 75248 Paris, France; and the §Department ofPathology, Harvard Medical School, Boston, Massachusetts 02115

Received, July 8, 2003, and in revised form, September 11, 2003Published, MCP Papers in Press, September 16, 2003, DOI

10.1074/mcp.M300067-MCP200

1 The abbreviations used are: CTS, cathepsin; S2, Schneider’sDrosophila line 2; NCBI, National Center for Biotechnology Informa-tion; PDI, protein disulfide isomerase; TEP, thiol ester-containingprotein.

Research

© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.1188 Molecular & Cellular Proteomics 2.11This paper is available on line at http://www.mcponline.org

activity on a broad scale. Chemistry-based functional pro-teomics is a promising method used to assign putative geneproducts to an enzyme family (26). The rationale behind thedesign of such a functional proteomics approach is to assessthe activity profiles of protease species in complex biologicalsamples rather than merely their presence or absence. Iden-tification and activity measurement can be performed withoutthe need for purification of the individual enzymes understudy. In addition, this approach allows activity measurementof several enzyme species at the same time, a task that wouldbe more cumbersome using classical biochemical ap-proaches. The success of this approach has been demon-strated for serine proteases (27), deubiquitinating enzymes(28), and cysteine proteases (29). In this study, we have usedDCG-04, a chemical probe that specifically and covalentlytargets mammalian cysteine proteases. Our objective was toidentify DCG-04-reactive polypeptides in Drosophila phago-cytes and to characterize and monitor their activities.

EXPERIMENTAL PROCEDURES

Cell, Culture Conditions, and Reagents—A particularly phagocyticsub-line of S2 cells was obtained from A. Pearson (Department ofPediatrics, Massachusetts General Hospital, Boston, MA) (23, 30). S2cells were grown at 26-27 °C in Schneider’s Insect Medium (Sigma-Aldrich, St. Louis, MO) or Schneider’s Drosophila Medium (InvitrogenLife Technologies, Carlsbad, CA) supplemented with 10% heat-inac-tivated fetal bovine serum (tested to support insect cell growth; In-vitrogen Life Technologies) and maintained at a density of 2 � 105 to8 � 106 cells/ml in 12.5 ml per T75 plastic tissue culture flask toensure exponential growth. Under these conditions, S2 cells dividedevery 16–18 h. Chemicals were obtained from Sigma-Aldrich, unlessindicated otherwise.

Active Site Labeling of Cysteine Proteases in Cell Lysates andDetection by Streptavidin Blotting—JPM-565 and DCG-04 were syn-thesized and purified as previously described (31, 32). Cells wereharvested by centrifugation at 4 °C, washed in Robb’s DrosophilaPBS pH 6.8 (52), and cell pellets corresponding to 5 � 107 cells werefrozen at �80 °C. Cell pellets were thawed on ice and lysed in 100 �llysis buffer, pH 5.0 (50 mM sodium acetate, pH 5.0, 5 mM magnesiumchloride, 0.5% Nonidet-P40), incubated for 30 min and centrifuged for15 min at 13,000 � g to remove nuclei. Protein concentration of thecell extract was measured using the Bio-Rad Protein Assay (Hercules,CA) with BSA as standard (average yield, 1–2 mg protein per 5 � 107

cells). Cell lysate (25 �g protein) was incubated with DCG-04 for 60min at 37 °C. When JPM-565 was used as competitor, cell lysateswere pre-incubated with JPM-565 for 15 min before addition ofDCG-04. The reaction was stopped by the addition of double-con-centrated reducing Laemmli sample buffer and boiling for 10 min.Samples were analyzed by SDS-PAGE and transferred to polyvinyli-dene membrane (Immobilon P; Millipore, Billerica, MA). After blockingovernight in PBS, pH 7.2, containing 10% non-fat dry milk, the mem-brane was incubated with streptavidin-horseradish peroxidase (1:2,500; Amersham Pharmacia Biotech, Uppsala, Sweden) in PBS con-taining 0.2% Tween 20 for 60 min at room temperature followed byextensive washing in PBS-Tween. DCG-04-reactive polypeptideswere detected by enhanced chemiluminescence (Western Lightning;PerkinElmer, Wellesley, MA).

Isolation of DCG-04-Reactive Enzymes for Ms-Based Identifica-tion—Cell lysate from 5.4 � 109 cells was prepared at pH 5.0, asdescribed above. The lysate was divided into five samples, eachcorresponding to approximately 35 mg of protein. The samples were

pre-cleared with 150 �l bead volume streptavidin-agarose (Pierce,Rockford, IL) to decrease nonspecific background activities. Threesamples were incubated with 5 �M DCG-04 for 60 min at 37 °C, onesample was pre-treated with 25 �M JPM-565 before addition ofDCG-04, and one sample received neither DCG-04 nor JPM-565. Tostop the reaction, SDS was added to a final concentration of 0.5%,and the samples were incubated for 5 min at 95 °C (in order todenature the DCG-04-tagged proteins to make the biotin moietyaccessible). Affinity enrichment of DCG-04-reactive polypeptides wasperformed using streptavidin-agarose, as previously described (12,32). Briefly, the buffer was exchanged to pull-down buffer (50 mM Tris,pH 7.4, 150 mM NaCl) using a PD-10 column (Amersham PharmaciaBiotech), and each sample was incubated with 150 �l bed volumestreptavidin-agarose for 60 min at room temperature. After washingwith excess pull-down buffer, bound polypeptides were eluted fromthe beads by addition of 100 �l reducing Laemmli sample buffer andboiling for 10 min. DCG-04-reactive proteins were separated by SDS-PAGE (12.5%). One DCG-04-reacted sample and the control sampleswere stained with silver (see Fig. 3), 5% of each of these samples wasused for streptavidin blotting (not shown), and the two remainingDCG-04-reacted samples were pooled and stained with CoomassieBrilliant Blue G as follows: the gel was fixed in H2O/25% isopropanol/10% acetic acid for 45 min, stained with 10% acetic acid/0.006%Coomassie Brilliant Blue G overnight, and destained with 10% aceticacid for 2h. Visible bands from the Coomassie- and silver-stained gelswere excised and processed for tandem mass spectrometry analysis.

Tryptic Digestion and Analysis by Electrospray Tandem Mass Spec-trometry—In-gel tryptic digestion was performed essentially as de-scribed (33). The samples were subjected to a nanoflow liquid chro-matography system (CapLC; Waters, Medford, MA) equipped with apicofrit column (75-�m ID � 9.8 cm; NewObjective, Woburn, MA), ata flow rate of �150 nl/min using a nanotee (Waters), 16:1 split (initialflow rate 5.5 �l/min). The liquid chromatography system was directlycoupled to a quadrupole time-of-flight micro-tandem mass spectrom-eter (Micromass, Manchester, UK). Analysis was performed in surveyscan mode, and parent ions with intensities greater than 7 weresequenced in MS/MS mode using MassLynx 4.0 software (Micro-mass). MS/MS data were processed and subjected to databasesearches using ProteinLynx Global Server 1.1 software (Micromass)against Swiss-Prot TrEMBL/New (www.expasy.ch), or Mascot (Ma-trixscience, www.matrixscience.com/, (34)) against the National Cen-ter for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov/)non-redundant database (NCBInr). Alternatively, the Drosophila pro-tein database from NCBI was also used for Drosophila-specific pro-tein identification. In all searches, oxidation of methionine and carb-amidomethylation of cysteine residues were considered asmodifications. Matches for proteins were accepted as significant ifscores were more than 75 using Protein Lynx Global Server 1.1, orbased on the Mascot Probability Mowse Score. At least two peptideswere found for each identified protein species. Information about theDrosophila gene products were obtained from the FlyBase database(FlyBase.bio.indiana.edu) and NCBI (www.ncbi.nlm.nih.gov/entrez/query.fcgi)

Active Site Labeling of Cysteine Proteases in Phagosomes of LiveCells Using Latex Beads—Phagosomal cysteine protease labeling hasbeen adapted from Lennon-Dumenil et al. (12). S2 cells were platedon 12-well plates (106 cells/well) 1 day before the experiment.Streptavidin-coated latex beads (2-�m diameter; Polysciences,Warrington, PA) were incubated with DCG-04 for 60 min at roomtemperature. Beads were washed three times with PBS and resus-pended in complete culture medium. DCG-04-loaded beads wereadded to the cells and incubated for 30 min at 27 °C (pulse). Phag-ocytosis was halted at 4 °C, cells were collected and pelleted at 2,000rpm (325 � g) for 3 min, resuspended in complete medium, and

Active Cysteine Protease Content in Drosophila S2 Cells

Molecular & Cellular Proteomics 2.11 1189

non-internalized beads were removed by repeated centrifugation af-ter layering on a cushion of heat-inactivated fetal bovine serum (latexbeads remain in interphase). Cells were resuspended in completemedium and incubated in 12-well plates at 27 °C for various periodsof time (chase). At the desired time points, cells were harvested onice, pelleted and lysed in hot SDS-sample buffer containing 100 �M

JPM-565, boiled for 10 min, cooled to room temperature, and passedthrough a 22.5-gauge hypodermic needle to shear DNA before pel-leting the released (phagocytosed) latex beads at 13,000 rpm(13,000 � g) for 5 min. Samples were analyzed by SDS-PAGE andstreptavidin-blotting. Phagocytic uptake of streptavidin beads wascontrolled by light microscopy and uptake of deep-blue dyed latexbeads (0.8-�m diameter; Sigma-Aldrich) by light and electron micros-copy (data not shown).

RESULTS

Protease Activity Profiling in Drosophila S2 Cells Using theMechanism-Based, Epoxide Inhibitor-Derived ChemicalProbe DCG-04—The epoxide inhibitor JPM-565 has beenused as an irreversible inhibitor, with broad reactivity towardcysteine proteases (31, 35). Based on the structure of thiscompound, a derivative was developed to include a biotinaffinity tag (DCG-04; Fig. 1A) (32). This compound permitstargeting of active cysteine proteases present in crude ex-tracts via covalent attachment to the cysteine active siteresidue (Fig. 1B, (29)). In order to test whether DCG-04 reactswith Drosophila proteins, we incubated this probe with crudepH 5.0-level cell extracts prepared from S2 cells. DCG-04reactive polypeptides were separated by SDS-PAGE and vi-sualized by streptavidin blotting. As shown in Fig. 2, weobserved labeling in the molecular mass ranges of 26–29 and33–37 kDa. Preheating of the extract before addition ofDCG-04 abolished labeling (Fig. 2). Competition for labelingwith increasing amounts of non-biotinylated probe (JPM-565) resulted in decreased recovery of these proteins (Fig.2). Thus, DCG-04 appeared to react specifically in a dose-and conformation-dependent manner with several Drosophilapolypeptides.

Identification of DCG-04-Reactive Polypeptides in Dro-sophila S2 Cell Extracts—In order to identify the polypeptides

labeled by DCG-04, we used a strategy based on streptavi-din-agarose affinity purification and tandem mass spectrom-etry as outlined in Fig. 1B. Drosophila S2 cell extracts wereeither left untreated or incubated with DCG-04 in the presenceor absence of non-biotinylated competitor, and DCG-04-re-active polypeptides were purified on streptavidin-agarose.SDS-PAGE of the eluted material, followed by silver staining,revealed several DCG-04-reactive polypeptides whose abun-dance was reduced when a five-fold excess of non-biotiny-lated competitor was included (Fig. 3). Subsequent analysisusing tandem mass spectrometry (liquid chromatography-MS/MS) identified a total of 20 protein species, for which atleast two peptide matches were found. We grouped thesepolypeptides into two categories, based on the absence (Ta-ble I) or presence (Table II) of a catalytically active thiol group.Due to the absence of such a thiol group, the polypeptideslisted in Table I are likely to be nonspecific contaminants,co-purified in our isolation procedure. They appear to bemostly metabolic enzymes but also include cytoskeleton pro-teins and streptavidin. Less frequently observed background

FIG. 2. Specific targeting of DCG-04-reactive proteases in Dro-sophila S2 cells in a concentration, conformation, and activesite-dependent manner. Crude extracts prepared from DrosophilaS2 cells were labeled for 1 hr at 37 °C in the presence or absence of10 �M DCG-04 and varying amounts of non-biotinylated inhibitorJPM-565, preceeded, or not, by preheating for 5 min at 100 °C.Proteins were then separated by SDS-PAGE on 12.5% gels, andlabeled polypeptides were visualized by streptavidin blotting.

FIG. 1. Structure and reaction mechanism of the cysteine protease-reactive compound DCG-04. A, Structure of the epoxide inhibitorDCG-04 composed of a biotin affinity tag, a peptide scaffold, and an epoxide electron withdrawing group (EWG). B, Schematic representationof the reaction mechanism and sample processing for liquid chromatography (LC)-MS/MS analysis.


1190 Molecular & Cellular Proteomics 2.11

proteins included catalase, involved in oxidative stress andaging.

We found six polypeptides that might exert reactivity to-ward DCG-04. All had an N-terminal signal sequence indicat-ing export into a secretory compartment (Table II). One of

these was protein disulfide isomerase (PDI), an enzyme con-taining two thiols as active site residues. PDI catalyzes disul-fide formation in newly synthesized polypeptides in the endo-plasmic reticulum in a two-step reaction, which includes twoalternative intermediate, mixed disulfides between the en-zyme and substrate (36). Therefore, epoxysuccinyl com-pounds such as DCG-04 and JPM-565 may specifically bindto reactive thiol groups of such nature.

Two polypeptides at �120 and 160 kDa were identified asthiol ester-containing protein (TEP) 4 (Fig. 3 and Table II). TEP4 belongs to a group of five thiol ester-containing Drosophilaproteins that show substantial structural and functional simi-larities, including a highly conserved thiol ester motif, to botha central component of mammalian complement system, fac-tor C3, and to a pan-protease inhibitor, alpha2-macroglobulin(37). TEP 4 contains an internal beta-cysteinyl-gamma-glu-tamyl thiol ester (38). It is possible that DCG-04 forms acovalent adduct with the proteolytically activated nascentstate of the thiol ester (39), although the details of this reactionmechanism remain unclear. TEP 4 appears to be expressedconstitutively in Drosophila larvae but is significantly up-reg-ulated after challenge with pathogens (37). In a mosquitohemocyte-like cell line, a related protein, TEP 1, serves as acomplement-like opsonin and promotes phagocytosis ofsome Gram-negative bacteria. This activity is dependent onits internal thiol ester bond (40).

Active Cysteine Proteases with Homology to MammalianCathepsins—We identified multiple polypeptide species thatshowed significant homologies to mammalian CTS proteasesL, B, F and K, respectively (Table II and Fig. 3). The CTS F-likeprotease has not been previously described as an activeentity but is found annotated in the D. melanogaster databaseFlyBase as a putative protease. The CTS B- and K-like pro-teases have never been isolated and characterized as proteo-lytic enzymes from Drosophila, although expression of the

FIG. 3. Identification of DCG-04 reactive polypeptides in Dro-sophila S2 cell extracts. Crude extracts were prepared from 5.4 �109 S2 cells, divided into three aliquots (corresponding to 35 mg each)and incubated either without (lane 1) or with DCG-04 in the presence(lane 3) or absence of non-biotinylated inhibitor JPM-565 (lane 2).DCG-04-reactive polypeptides were enriched with streptavidin-agar-ose (see Fig. 1B), separated by SDS-PAGE (12.5% gel), and stainedwith silver (middle lane). Parallel samples omitting DCG-04 (left lane)or adding excess JPM-565 as inhibitor (right lane) were used toassess the extent of nonspecific labeling or adsorption to streptavi-din-agarose. Bands from parallel Coomassie blue-stained and silver-stained gels were excised and analyzed by tandem mass spectrom-etry. Positions of selected polypeptides are indicated.

TABLE ICommon background proteins found in DCG-04-streptavidin pull-down experiments

ProteinAccession

no.Predicted

molecular weightObserved

molecular weightNumber ofpeptides

Sequencecoverage

(%)

Hsc70–3 gene product AE003487 72216 70 9 15Fimbrin NM_078661 71869 52 5 8RH04426p AY084193 71060 70 3 5Intracellular vitamin D-binding protein AAF66593 70880 42 2 460-kDa heat shock protein, mitochondrial precursor gi 12644042 60771 58 10 23Pyruvate kinase NM_079724 57437 52 3 8Catalase NM_080483 57113 52 2 6Enolase (2-phosphoglycerate dehydratase) gi 119351 46534 50 2 6Alpha-1-antiproteinase precursor CAA44840.1 46086 50 4 11Ald gene product AE003755 43007 27 2 5Argk gene product AE003553 39841 40 4 10Glyceraldehyde-3-phosphate dehydrogenase gi 84977 35361 38 2 8Nucleosome remodeling factor (38 kDa) XM_079890 32625 38 2 7Streptavidin precursor P22629 18815 22 4 35Streptavidin precursor P22629 18815 17 3 32



CTS B-like protease has been observed in Drosophila em-bryos (41) and a K-like enzyme in the flesh fly Sarcophagaperegrina (42). A CTS L-like protease has been reported to beexpressed in embryonal and larval midgut (43) and was foundin granules in the hemocyte-like Drosophila cell line mbn-2,suggesting lysosomal localization (44). The latter three en-zymes have been implicated in general digestive processes,yolk degradation, and immunity, and are strongly conservedin other non-vertebrate species (see FlyBase and Refs. 45 and46)).

Sequence alignment and comparison of these CTS-likeproteases and their corresponding mouse homologs revealeda high degree of conservation centering on two regions in themature parts of the molecules, around the active site residuescysteine (C), histidine (H), and asparagine (N) (Fig. 4A). TheN-terminal pro-regions were less well conserved but of similarlength, except in the case of the Drosophila CTS K equivalent(26–29-kDa protease), which contains an insertion of signifi-cant length not found in the pro-region of the mammalianhomolog CTS K. A dendrogram constructed on the basis ofoverall sequence similarities shows that, with the exception ofCTS K, evolutionary diversification of the CTS subgroupsoccurred before the ancestors of mammalian and invertebrateCTS proteases diverged from each other (Fig. 4B). These datasuggest an evolutionary conserved function for these pro-teases and only later recruitment of these molecules foradaptive immunity in vertebrates.

Drosophila Cathepsins Are Most Active at pH 4.5 and CanBe Detected in Phagosomes of Live Cells—We used activityprofiling to determine under which pH conditions these en-zymes are active. Extracts from S2 cells were prepared at apH level ranging from 4.5–7.5. DCG-04 labeling resulted indistinct polypeptide profiles in a highly pH-dependent manner(Fig. 5A, third panel). At a pH of 4.5, two major bands of 26–29and 33–37 kDa were observed, consistent with the experi-ments described above for pH 5.0. This suggests that these

polypeptides correspond to the Drosophila CTS homologsidentified by tandem mass spectrometry. Labeling of thesepolypeptides was specific, because they were not observed inheat-treated samples (Fig. 5A, second panel) and were com-peted by an excess of non-biotinylated compound (Fig. 5A,fourth panel). The marked pH-dependence of these enzymesindicates a function within the endocytic compartment.

In order to address a possible phagosomal localization ofthese Drosophila CTS proteases in live cells, we used anapproach recently pioneered by our laboratory that monitorsthe proteolytic environment in phagosomes of mammalianmacrophages and dendritic cells (12). Streptavidin-latexbeads loaded with DCG-04 are efficiently internalized byphagocytic cells and allow sampling of the proteolytic envi-ronment during phagosome formation and maturation. Dro-sophila S2 cells were incubated for 0.5 h with DCG-04-loadedbeads. The non-internalized beads were removed, and cellswere lysed after 1–6 h of incubation (chase). To preventpost-lysis reactivity of DCG-04, cells were lysed by the addi-tion of boiling SDS sample buffer containing an excess ofnon-biotinylated competitor. Streptavidin blotting revealed la-beling in the 26–29 kDa range, which increased over time (Fig.5B). This labeling profile was dependent on DCG-04-loadedbeads, because no labeling was observed in the absence oflatex beads or in the presence of latex beads that had notbeen loaded with DCG-04. These results suggest that CTSactivity can be detected by activity profiling in phagosomalcompartments of living Drosophila S2 cells.

DISCUSSION

Chemical targeting of cysteine proteases by means of amechanism-based probe combined with tandem mass spec-trometry has allowed rapid identification of multiple activeprotease species in a Drosophila cell line with phagocyticproperties. Four of 12 total CTS-like proteases encoded in theDrosophila genome were identified (Tables II and III). Exami-

TABLE IIDCG-04-reactive polypeptides in S2 cell extracts (pH 5) identified by tandem mass spectrometry

TrEMBLaccession

no.

Gene name/symbol/full name(FlyBase)

Molecular function(FlyBase anotation)b

Predictedlength

(aa)

PredictedMW(Da)

ObservedMW(kDa)

No. ofpeptides

Sequencecoverage

(%)

Q9NFV5 CG10363/TEP4/Thiolestercontaining protein 4

Antibacterial; alpha-2 macroglobulin 1496 168491 160 3 2

120 9 5P54399a CG6988/Pdil Protein disulfide

isomeraseProtein disulfide isomerase 496 55781 55 4 10

Q95029a CG6692/Cp1/Cysteineproteinase-1

Cathepsin L 341 37970 33–37 3 9

27–29 10 30Q9VN92 CG12163 Cathepsin F 475 53545 33–37 2 6

27–29 4 12Q9V3U6 CG8947/26–29kD proteinase Cathepsin K 549 62129 27–29 3 7Q9VY87 CG10992 Cathepsin B 340 37424 27–29 2 8

a Swiss-Prot.b Inferred by sequence similarity (flybase.bio.indiana.edu).



nation of S2 cell-specific expressed sequence tags availablein the FlyBase database revealed that only six of these 12CTS-like genes appear to be expressed in S2 cells, including

the four proteases identified by tandem mass spectrometry(Table III and Fig. 4C). Inspection of the sequence of the othertwo genes revealed that the active site residue cysteine was

FIG. 4. Structure-based amino acid sequence alignment of Drosophila and mouse CTS proteases. A, Sequence alignments wereperformed using ClustalW (www.ch.embnet.org/software/ClustalW.html) and Vector NTI (Informax, Frederick, MD) software. Conservedresidues are indicated under the sequence by: *, conserved; :, strongly homologous residues; �, homologous residues. The catalytic cysteineand histidine residues are indicated by dots above the sequence, and the conserved region around the catalytic cysteine is boxed. Thebeginning of the mature enzymes, i.e. beginning and end of single chains or heavy and light chains (by similarity to human enzymes), isindicated above the sequence by angles. (Human CTS K and F are single-chain enzymes, whereas CTS L and B consist of disulfide-bondedheavy and light chains). CTS-derived peptides detected by tandem mass spectrometry are indicated above the respective sequences by lines.B, Dendrogram showing the evolutionary relationships of Drosophila and mouse CTS proteases. Overall sequence identities betweenhomologous mouse and Drosophila pairs are indicated. A and B, The following accession numbers were used: mouse CTS F: Q9R013;Drosophila CTS F: Q9VN92; mouse CTS L: P06797; Drosophila CTS L: Q95029; mouse CTS K: P55097; Drosophila CTS K: Q9V3U6; mouseCTS B: P10605; Drosophila CTS B: Q9VY87. C, sequences around the catalytic residue C for CTS-like proteases in Drosophila genome andexpressed sequence tags.



not conserved, precluding reaction with DCG-04 (Fig. 4C).The Drosophila genome contains a CTS H-like protease that isnot expressed in S2 cells and lacks the catalytic cysteineresidue (Table III and Fig. 4C). This is in contrast to themammalian system in which CTS H was shown to react withDCG-04 (12).

We isolated not only cysteine proteases but also otherenzymes that catalyze thiol-based reactions, such as PDI andTEP 4 (Table II). Although it is possible that DCG-04 directlyreacted with these polypeptides (see “Results”), PDI and TEP4 may have been isolated as a consequence of their associ-ation with cysteine proteases that were targeted by DCG-04.The observation that DCG-04 can be used to isolate insectTEP will be useful in monitoring their activity. Insect TEP haveattracted considerable interest lately because they play a rolein innate immunity in the mosquito vector Anopheles by lim-iting the multiplication of malaria parasites inside the vectororganism (40).3

We observed two forms of Drosophila CTS L and F withmolecular masses of 26–29 and 33–37 kDa that reacted withDCG-04 (Fig. 3 and Table II). Glycosylation has been de-scribed for mammalian CTS proteases (47) and could accountfor this difference in molecular mass. However, it seems morelikely that 33–37-kDa forms correspond to pro-forms of these

enzymes that have retained all (CTS L) or part (CTS F) of theirpro-peptides, as suggested for human and mouse CTS L (47).In line with this possibility, we found that the 33–37-kDa formof Drosophila CTS L contained the tryptic peptideAADESFKGVTFISPAHVTLPK106–126, which is located N-ter-minally of the predicted cleavage site for the mature enzyme(Fig. 4A and see Ref. 13). Labeling of CTS pro-forms byepoxide-based probes such as DCG-04 has been observedpreviously in mammalian cells (48). This phenomenon can beattributed to the small molecular size of the chemical probeand to its covalent reaction mechanism. Although pro-peptideefficiently prevents the access of large polypeptide substratesto the active site, it may not be bound tightly enough toprevent the small-sized DCG-04 molecule from entering theactive site and from irreversibly attaching to the proenzyme.This interpretation may also explain the results obtained byactivity profiling in S2 cell phagosomes (Fig. 5B). In this sub-cellular compartment, DCG-04-reactive polypeptides wereexclusively found in the 26–29 kDa range, suggesting thatonly fully processed, mature CTS proteases are exposed tophagocytosed material.

Whereas the Drosophila CTS L-, B-, and F-like enzymespecies shared significant overall sequence identities withtheir mouse counterparts (Fig. 4, A and B), CTS K (26–29 kDaprotease) appears to have unique properties. This enzymeshowed only 28% overall sequence identity to mouse CTS K,3 E. A. Levashina and F. C. Kafatos, personal communication.

FIG. 5. Drosophila CTS proteases show a narrow activity optimum under acidic conditions and can be detected in the phagosome.Proteins were separated by SDS-PAGE on 12.5% gels, and labeled bands were visualized by streptavidin blotting. A, Cytoplasmic extractsfrom Drosophila S2 cells were prepared at the indicated pH level. Samples were incubated in the presence or absence of DCG-04 and a 10-foldexcess of non-biotinylated competitor JPM-565, followed, or not, by preheating for 5 min at 100 °C. Labeled polypeptides were separated bySDS-PAGE (12.5%) and visualized by streptavidin blotting. B, Labeling of phagosomal cysteine proteases by DCG-04, immobilized onstreptavidin beads, and phagocytosed by live Drosophila S2 cells. Streptavidin latex beads (0.2 �m) were loaded with 10 �M DCG-04. S2 cellswere incubated with DCG-04-loaded or unloaded latex beads for the indicated times (pulse). Non-phagocytosed beads were removed, andcells were incubated for the indicated times (chase).



and 27% to mouse CTS L (Fig. 4). From a single precursor, itis processed to two separate 26 and 29 kDa polypeptides,which then dimerize via disulfide bonds, as shown for itsclose homologue in the flesh fly (42). The 29-kDa subunitcorresponds to a single chain CTS such as mammalian CTSF or K (Swiss-Prot TrEMBL annotation, www.expasy.ch),whereas the 26-kDa subunit has no homolog in mammals,suggesting a specific role of the 26–29-kDa protease ininvertebrates.

DCG-04 activity profiling at different pH levels was used toshow that Drosophila CTS proteases are most active underacidic conditions, suggesting that these enzymes exert theirbiological function in late endocytic or lysosomal cell com-partments (Fig. 5). In contrast, some mammalian CTS pro-teases, such as CTS L, B, and K are also active at a neutral pHlevel (49), which may reflect a role in a wider range of biolog-ical functions in higher vertebrates. However, the predomi-nant presence of CTS in phagosomes of both classes oforganisms (insects and mammals), as well as the phylogeneti-cally conserved branching into CTS subgroups L, F, and B(Fig. 4B), is consistent with a general role in lysosomal prote-

olysis, which precedes their function in antigen processingand presentation in mammalian cells. Drosophila, which lacksan adaptive immune system, is an appropriate organism toinvestigate whether CTS proteases have a function in innateimmunity during a challenge with bacterial or fungal patho-gens. However, examination of published genome-wide mi-croarray data indicated no alteration of Drosophila CTS L, K,B, and F mRNA expression levels in response to immunestimuli (50, 51). In addition, preliminary experiments withcrude extracts from S2 cells exposed to immune stimulatorsrevealed no significant differences in DCG-04-labeling profiles(data not shown). It will therefore be interesting to assessDrosophila CTS activity profiles in a more refined way, forexample, by immunoprecipitation of specific DCG-04-reac-tive CTS proteases from phagosomes of living cells exposedto such stimuli.

Taken together, our results demonstrate the usefulness ofthis functional proteomics approach and provide a basis forthe simultaneous monitoring of multiple protease activities ininsect models at different developmental stages or during animmune challenge.

TABLE IIICathepsin-like genes of the papain cysteine protease family in the genome of Drosophila melanogaster (Flybase 05/23/2003)

Mosthomologous

to

Gene/symbol

ChromosomeCytogenetic

mapName/Information

Found inthis study

Expressedsequence tagsfrom S2 cells

Catalytic Cactive siteconsensus

Cathepsin L CG6692 2R 50C18–20 Cathepsin L; cysteine proteinase-1;Cp1

33–37 kDa;27–29 kDa

Yes, SD15384 Conserved

CG6357 2R 50C6 Sequence similarity withMGDa:Ctla2a, Cytotoxic Tlymphocyte-associated protein-2-alpha; MGI:88554 and cathepsin L

Not found Yes, SD11271 Not conserved

CG6347 2R 50C6 NOT Cathepsin L activity Not found No 1 aa insertionCG1075 3R 83D2 Cathepsin L activity inferred from

sequence similarity with Swiss-Prot Q28944

Not found No Deleted

CG11459 3R 83D2 NOT Cathepsin L activity Not found No 1 aa insertionCG5367 2L 31D10 Cathepsin L activity inferred from

sequence similarity with NCBI_gi:4503153

Not found No Conserved

Cathepsin F CG12163 3R 82F7 Cathepsin F activity inferred fromsequence similarity with EMBL:AJ131851

33–37 kDa;27–29 kDa

Yes, SD14754 Conserved

Cathepsin K CG8947 3L 70C10 26–29 kD-proteinase 27–29 kDa Yes, SD03727 ConservedCG4847 2R 54C1 Cathepsin K activity inferred from

sequence similarity with EMBL:AJ006033

Not found No Conserved

Cathepsin B CG10992 X 12C2 Cathepsin B activity inferred fromsequence similarity withMGD:Ctsb; MGI:88561

27–29 kDa Yes, SD11720 Conserved

CG3074 2R 58C7 Cathespin B activity inferred fromsequence similarity withMGD:Ctsb; MGI:88561

Not found Yes, SD09749 Not conserved;C 3 S � 1aa insertion

Cathepsin H CG6337 2R 50C6 Cathepsin H activity inferred fromsequence similarity withMGD:CtsH; MGI:MGI:107285

Not found No Not conserved;C 3 S � 1aa insertion

a MGD, Mouse Genome Database; MGI, Mouse Genome Informatics.



Acknowledgments—We thank Alan Pearson (Massachusetts Gen-eral Hospital, Boston) for providing S2 cells, R. Alan B. Ezekowitz formanifold support, LeAnn Williams for expert preparation of samplesfor MS/MS analysis, and Thilo Stehle, Edda Fiebiger, and Alan Pear-son for helpful comments on the manuscript.

* This work was supported by a National Institutes of Health Grant,1ROI 62502, a Harvard Center for Neurodegeneration and RepairGrant; an Alexander and Margaret Stewart Trust Foundation Grant (toH. L. P. and B. M. K.), a Multiple Myeloma Research Foundation Sen-ior Research Award (to B. M. K.), and by a supplement to NIH Grant5PO1 AI44220–5 (to R. Alan B. Ezekowitz and C. K.). R. M is arecipient of a Boehringer Ingelheim Fonds fellowship.The costs ofpublication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “adver-tisement” in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.

� C. K. and R. M. contributed equally to this work.§§ To whom correspondence should be addressed: Department of

Pathology, Harvard Medical School, 200 Longwood Avenue, Boston,Massachusetts 02115. Tel.: 617-432-4789; Fax: 617-432-4775;E-mail: [email protected].

REFERENCES

1. Hoffmann, J. A., and Reichhart, J. A. (2002) Drosophila innate immunity: Anevolutionary perspective. Nat. Immunol. 3, 121–126

2. Bogyo, M., and Ploegh. H. L. (1998) Antigen presentation. A protease drawsfirst blood. Nature 396, 625–627

3. Villadangos, J. A., and Ploegh, H. L. (2000) Proteolysis in MHC class IIantigen presentation: Who’s in charge? Immunity 12, 233–239

4. Riese, R. J., and Chapman, H. A. (2000) Cathepsins and compartmental-ization in antigen presentation. Curr. Opin. Immunol. 12, 107–113

5. Watts, C. (2001) Antigen processing in the endocytic compartment. Curr.Opin. Immunol. 13, 26–31

6. Lennon-Dumenil, A. M., Bakker, A. H., Wolf-Bryant, P., Ploegh, H. L., andLagaudriere-Gesbert, C. (2002) A closer look at proteolysis and MHC-class-II-restricted antigen presentation. Curr. Opin. Immunol. 14, 15–21

7. Deussing, J., Roth, W., Saftig, P., Peters, C., Ploegh, H. L., and Villadangos,J. A. (1998) Cathepsins B and D are dispensable for major histocompat-ibility complex class II-mediated antigen presentation. Proc. Natl. Acad.Sci. U. S. A. 95, 4516–4521

8. Driessen, C., Bryant, R. A., Lennon-Dumenil, A. M., Villadangos, J. A.,Bryant, P. W., Shi, G. P., Chapman, H. A., and Ploegh, H. L. (1999)Cathepsin S controls the trafficking and maturation of MHC class IImolecules in dendritic cells. J. Cell. Biol. 147, 775–790

9. Pluger, E. B., Boes, M., Alfonso, C., Schroter, C. J., Kalbacher, H., Ploegh,H. L., and Driessen, C. (2002) Specific role for cathepsin S in the gen-eration of antigenic peptides in vivo. Eur. J. Immunol. 32, 467–476

10. Shi, G. P., Bryant, R. A., Riese, S., Verhelst, S., Driessen, C., Li, Z., Bromme,D., Ploegh, H. L., and Chapman, H. A. (2000) Role for cathepsin F ininvariant chain processing and major histocompatibility complex class IIpeptide loading by macrophages. J. Exp. Med. 191, 1177–1186

11. Nakagawa, T., Roth, W., Wong, P., Nelson, A., Farr, A., Deussing, J.,Villadangos, J. A., Ploegh, H. L., Peters, C., and Rudensky, A. Y. (1998)Cathepsin L: Critical role in Ii degradation and CD4 T cell selection in thethymus. Science 280, 450–453

12. Lennon-Dumenil, A. M., Bakker, A. H., Maehr, R., Fiebiger, E., Overkleeft,H. S., Rosemblatt, M., Ploegh, H. L., and Lagaudriere-Gesbert, C. (2002)Analysis of protease activity in live antigen-presenting cells shows reg-ulation of the phagosomal proteolytic contents during dendritic cellactivation. J. Exp. Med. 196, 529–540

13. Turk, B., Turk, D., and Turk, V. (2000) Lysosomal cysteine proteases: morethan scavengers. Biochim. Biophys. Acta 1477, 98–111

14. Turk, V., Turk, B., Guncar, B., Turk, D., and Kos., J. (2002) Lysosomalcathepsins: Structure, role in antigen processing and presentation, andcancer. Adv. Enzyme. Regul. 42, 285–303

15. Turk, V., Turk, B., and Turk, D. (2001) Lysosomal cysteine proteases: Factsand opportunities. EMBO J. 20, 4629–4633

16. Felbor, U., Kessler, B., Mothes, W., Goebel, H. H., Ploegh, H. L., Bronson,

R. T., and Olsen, B. R. (2002) Neuronal loss and brain atrophy in micelacking cathepsins B and L. Proc. Natl. Acad. Sci. U. S. A. 99, 7883–7888

17. Edwards, D. R., and Murphy, G. (1998) Cancer. Proteases—Invasion andmore. Nature 394, 527–528

18. Kos, J., Werle, B., Lah, T., and Brunner, N. (2000) Cysteine proteinases andtheir inhibitors in extracellular fluids: Markers for diagnosis and prognosisin cancer. Int. J. Biol. Markers 15, 84–89

19. Adams, 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. (2000) The genome sequence of Drosophila melanogaster.Science 287, 2185–2195

20. Christophides, 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. (2002)Immunity-related genes and gene families in Anopheles gambiae. Sci-ence 298, 159–165

21. Ross, J., Jiang, H., Kanost, M. R., and Wang, Y. (2003) Serine proteasesand their homologs in the Drosophila melanogaster genome: An initialanalysis of sequence conservation and phylogenetic relationships. Gene.304, 117–131

22. Ramet, M., Manfruelli, P., Pearson, A., Mathey-Prevot, B., and Ezekowitz,R. A. (2002) Functional genomic analysis of phagocytosis and identifica-tion of a Drosophila receptor for E. coli. Nature 416, 644–648

23. Pearson, A., Baksa, K., Ramet, M., Protas, M., McKee, M., Brown, D., andEzekowitz, R. A. (2003) Identification of cytoskeletal regulatory proteinsrequired for efficient phagocytosis in Drosophila. Microbes and Infection(In press)

24. Ramet, M., Pearson, A., Manfruelli, P., Li, X., Koziel, H., Gobel, V., Chung,E., Krieger, M., and Ezekowitz, R. A. (2001) Drosophila scavenger recep-tor CI is a pattern recognition receptor for bacteria. Immunity 15,1027–1038

25. Franc, N. C. (2002) Phagocytosis of apoptotic cells in mammals, Caenorh-abditis elegans and Drosophila melanogaster: Molecular mechanismsand physiological consequences. Front. Biosci. 7, d1298–1313

26. Kidd, D.,Liu, Y., and Cravatt, B. F. (2001) Profiling serine hydrolase activitiesin complex proteomes. Biochemistry 40, 4005–4015

27. Liu, Y., Patricelli, M. P., and Cravatt, B. F. (1999) Activity-based proteinprofiling: The serine hydrolases. Proc. Natl. Acad. Sci. U. S. A. 96,14694–14699

28. Borodovsky, A., Ovaa, H., Kolli, N., Gan-Erdene, T., Wilkinson, K. D.,Ploegh, H. L., and Kessler, B. M. (2002) Chemistry-based functionalproteomics reveals novel members of the deubiquitinating enzyme fam-ily. Chem. Biol. 9, 1149–1159

29. Greenbaum, D., Baruch, A., Hayrapetian, L., Darula, Z., Burlingame, A.,Medzihradszky, K. F., and Bogyo, M. (2002) Chemical approaches forfunctionally probing the proteome. Mol. Cell. Proteomics 1, 60–68

30. Pearson, A., Lux, A., and Krieger, M. (1995) Expression cloning of dSR-CI,a class C macrophage-specific scavenger receptor from Drosophilamelanogaster. Proc. Natl. Acad. Sci. U. S. A. 92, 4056–4060

31. Meara, J. P., and Rich, D. H. (1996) Mechanistic studies on the inactivationof papain by epoxysuccinyl inhibitors. J. Med. Chem. 39, 3357–3366

32. Greenbaum, D., Medzihradszky, K. F., Burlingame, A., and Bogyo, M.(2000) Epoxide electrophiles as activity-dependent cysteine proteaseprofiling and discovery tools. Chem. Biol. 7, 569–581

33. Kinter, M., and Sherman, N. E. (2000) Protein Sequencing and IdentificationUsing Tandem Mass Spectrometry. Wiley-Interscience, New York

34. Perkins, D. N., Pappin, D. J., Creasy, D. M., and Cottrell, J. S. (1999)Probability-based protein identification by searching sequence data-bases using mass spectrometry data. Electrophoresis 20, 3551–3567

35. Palmer, J. T., Rasnick, D., Klaus, J. L., and Bromme, D. (1995) Vinylsulfones as mechanism-based cysteine protease inhibitors. J. Med.Chem. 38, 3193–3196

36. Darby, N. J., Freedman, R. B., and Creighton, T. E. (1994) Dissecting themechanism of protein disulfide isomerase: catalysis of disulfide bondformation in a model peptide. Biochemistry 33, 7937–7947

37. Lagueux, M., Perrodou, E., Levashina, E. A., Capovilla, M., and Hoffmann,J. A. (2000) Constitutive expression of a complement-like protein in tolland JAK gain-of-function mutants of Drosophila. Proc. Natl. Acad. Sci.U. S. A. 97, 11427–11432

38. Houen, G., and Svendsen, I. (1998) Affinity chromatography of thiol ester-



containing proteins. J. Chromatogr. A 799, 139–14839. Sottrup-Jensen, L. (1989) Alpha-macroglobulins: structure, shape, and

mechanism of proteinase complex formation. J. Biol. Chem. 264,11539–11542

40. Levashina, E. A., Moita, L. F., Blandin, S., Vriend, G., Lagueux, M., andKafatos, F. C. (2001) Conserved role of a complement-like protein inphagocytosis revealed by dsRNA knockout in cultured cells of the mos-quito, Anopheles gambiae. Cell 104, 709–718

41. Medina, M., Leon, P., and Vallejo, C. G. (1988) Drosophila cathepsin B-likeproteinase: A suggested role in yolk degradation. Arch. Biochem. Bio-phys. 263, 355–363

42. Fujimoto, Y., Kobayashi, A., Kurata, S., and Natori, S. (1999) Two subunitsof the insect 26/29-kDa proteinase are probably derived from a commonprecursor protein. J. Biochem. (Tokyo) 125, 566–573

43. Matsumoto, I., Watanabe, H., Abe, K., Arai, S., and Emori, Y. (1995) Aputative digestive cysteine proteinase from Drosophila melanogaster ispredominantly expressed in the embryonic and larval midgut. Eur. J. Bio-chem. 227, 582–587

44. Tryselius, Y., and Hultmark, D. (1997) Cysteine proteinase 1 (CP1), a ca-thepsin L-like enzyme expressed in the Drosophila melanogaster hae-mocyte cell line mbn-2. Insect. Mol. Biol. 6, 173–181

45. Laycock, M. V., MacKay, R. M., Di Fruscio, M., and Gallant, J. M. (1991)Molecular cloning of three cDNAs that encode cysteine proteinases inthe digestive gland of the American lobster (Homarus americanus). FEBSLett. 292, 115–120

46. Butler, A. M., Aiton, A. L., and Warner, A. H. (2001) Characterization of anovel heterodimeric cathepsin L-like protease and cDNA encoding thecatalytic subunit of the protease in embryos of Artemia franciscana.Biochem. Cell Biol. 79, 43–56

47. Smith, S. M., Kane, S. E., Gal, S., Mason, R. W., and Gottesman, M. M.(1989) Glycosylation of procathepsin L does not account for speciesmolecular-mass differences and is not required for proteolytic activity.Biochem. J. 262, 931–938

48. Felbor, U., Dreier, L., Bryant, R. A., Ploegh, H. L., Olsen, B. R., and Mothes,W. (2000) Secreted cathepsin L generates endostatin from collagen XVIII.EMBO J. 19, 1187–1194

49. Villadangos, J. A., Bryant, R. A., Deussing, J., Driessen, C., Lennon-Dume-nil, A. M., Riese, R. J., Roth, W., Saftig, P., Shi, G. P., Chapman, H. A.,Peters, C., and Ploegh, H. L. (1999) Proteases involved in MHC class IIantigen presentation. Immunol. Rev. 172, 109–120

50. De Gregorio, E., Spellman, P. T., Rubin, G. M., and Lemaitre, B. (2001)Genome-wide analysis of the Drosophila immune response by usingoligonucleotide microarrays. Proc. Natl. Acad. Sci. U. S. A. 98,12590–12595

51. Irving, P., Troxler, L., Heuer, T. S., Belvin, M., Kopczynski, C., Reichhart,J. M., Hoffmann, J. A., and Hetru, C. (2001) A genome-wide analysis ofimmune responses in Drosophila. Proc. Natl. Acad. Sci. U. S. A. 98,15119–15124

52. Robb, J. A. (1969) Maintenance of imaginal discs of Drosophila melano-gaster in chemically defined media. J. Cell. Biol. 41, 876–885



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

Documents

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