From the Department of Anatomy and Neurobiology,
Washington University School of Medicine, St. Louis, Missouri 63110 and the § Department of Veterinary Medical Chemistry,
Swedish University of Agricultural Sciences, Biomedical Center,
Box 575, 751 23 Uppsala, Sweden
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ABSTRACT |
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We describe the characterization, cloning, and genetic analysis of tripeptidyl peptidase II (TPP II) from Drosophila melanogaster. Mammalian TPP II removes N-terminal tripeptides, has wide distribution, and has been identified as the cholecystokinin-degrading peptidase in rat brain. Size exclusion and ion exchange chromatography produced a 70-fold purification of dTPP II activity from Drosophila tissue extracts. The substrate specificity and the inhibitor sensitivity of dTPP II is comparable to that of the human enzyme. In particular, dTPP II is sensitive to butabindide, a specific inhibitor of the rat cholecystokinin-inactivating activity. We isolated a 4309-base pair dTPP II cDNA which predicts a 1354-amino acid protein. The deduced human and Drosophila TPP II proteins display 38% overall identity. The catalytic triad, its spacing, and the sequences that surround it are highly conserved; the C-terminal end of dTPP II contains a 100-amino acid insert not found in the mammalian proteins. Recombinant dTPP II displays the predicted activity following expression in HEK cells. TPP II maps to cytological position 49F4-7; animals deficient for this interval show reduced TPP II activity.
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INTRODUCTION |
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Intercellular communication depends critically on both the generation and termination of biological signals. In the case of neuropeptides and neurohormones, the signaling molecules may be cleaved by extracellular enzymes to produce new active peptides (e.g. by angiotensin-converting enzyme, ACE1 (1) and endothelin-converting enzyme (2)) or cleaved by them to be inactivated (e.g. by enkephalinase (3)). These peptidases are found in the extracellular space or on the cell surface and typically have low specificity (for example substance P, neurokinins, neurotensin, and the endothelins can all be cleaved by enkephalinase, reviewed in Refs. 4 and 5). Although a large number of peptidases have been identified in various tissues, only a handful have been shown definitively to be ectoenzymes with neuropeptide-degrading capability (reviewed in Refs. 4 and 5). These observations have led to the current hypothesis that diverse neuropeptides are inactivated by a relatively small number of enzymes. Recently Rose et al. (6) identified a membrane-associated variant of the enzyme tripeptidyl peptidase II (TPP II) as the peptidase responsible for cleavage and inactivation of the mammalian neuropeptide CCK-8. Intravenous injection of the potent TPP II inhibitor, butabindide, has pro-satiety effects on both behavior and gastric emptying (6). Based on this pharmacology, as well as its substrate specificity, and its correlated expression in CCK-responsive tissues, Rose et al. (6) propose that the neuropeptidase activity of TPP II is intimately associated with CCK signaling and not broadly active on diverse neuropeptides.
TPP II was previously isolated and characterized as an extra-lysosomal peptidase that could release N-terminal tripeptides from a wide range of larger substrates (7, 8). TPP II is a serine protease with a subtilisin-like catalytic domain, but compared with other subtilases, it contains an extended C-terminal region (9). The native form of the enzyme has a remarkably high molecular mass (>1000 kDa) that suggests an oligomeric association of the ~138-kDa subunits (7, 8). The cDNAs encoding the human (10, 11), murine (12), and rat (6) enzymes have been cloned, and the genomic region encoding a putative homologue in Caenorhabditis elegans has been sequenced (GenBankTM accession number U23176). In both human and mouse cDNAs (12), an alternatively spliced exon encoding an additional 13 amino acids has been identified, which is involved in complex formation (13). Rose et al. (6) identified both cytoplasmic and membrane-associated forms of rat TPP II and suggested that an alternatively spliced TPP II mRNA was involved in the membrane association through a glycosylphosphatidylinositol anchor.
Neuropeptides are important signaling molecules in insects (for review see Refs. 14 and 15), and the enzymes involved in neuropeptide regulation appear to be highly conserved (16). Previous studies of neuropeptidases in Drosophila have defined enzyme activities resembling enkephalinase (17, 18), angiotensin-converting enzyme (19, 20), and aminopeptidase activity (21). In each case, the Drosophila activities displayed similar substrate specificities and inhibitor sensitivities to known mammalian enzymes. Furthermore, the gene encoding the Drosophila ACE, AnCE, demonstrates a high degree of sequence similarity to one form of mammalian ACE (20). We propose to use Drosophila genetics to investigate the extent to which TPP II enzyme function has been conserved. In the current study, we show that a TPP II-like activity is present in Drosophila extracts. Furthermore, we partially purify this enzyme activity and characterize it in comparison to the mammalian enzyme. We have cloned and expressed a dTPP II cDNA. Finally, we use genetics to demonstrate that this cloned gene is largely responsible for the observed TPP II activity in tissue extracts.
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EXPERIMENTAL PROCEDURES |
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Materials-- Chromogenic substrates were obtained from Bachem (Bubendorf, Switzerland) and Sigma. N-Ethylmaleimide, phenylmethanesulfonyl fluoride, pepstatin, and p-chloromercuribenzoate were purchased from Sigma; iodoacetamide was obtained from BDH, and bestatin was from Boehringer Mannheim (Bromma, Sweden). Sepharose CL-4B was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden), and DEAE-cellulose (DE52) was from Whatman. A Nucleosil C18 column (10 µm; 4 × 250 mm) for HPLC was packed by Skandinaviska GeneTec AB (Kungsbacka, Sweden). Human TPP II was purified from red blood cells as described previously (8) with modifications subsequently reported (9, 22). The inhibitor butabindide was a generous gift from Drs. Schwartz and Ganellin (6).
Insect Culture--
Standard culture methods were used to obtain
large numbers of adult Drosophila melanogaster (Oregon R
strain) from which eggs were collected on agar-apple juice plates. Eggs
were collected in 24-h intervals, washed in H2O, and stored
at 70 °C until needed. Flies used for enzyme purification were
14-18 days old, quieted, and collected at 4 °C and frozen to
70 °C for storage. The deficiency lines Df(2R)vg 33 (49D;50A), Df(2R)vg 56 (49D;49F), Df(2R)vg B (49D3-4;50A1-2) and
Df(2R)vg C (49B2-3;49E7-F1) were mapped according to
complementation analysis inter se and with stocks bearing
mutations (23, 24). The following 12 ethyl methanesulfonate and one irradiated mutant stocks which potentially represent dTPP II
were analyzed: vr3-2, vr4-57, vr5-5,
vr5-48, vr6-6, vr6-R6 (gamma ray), vr 8-9, vr9-11, vr9,15-43, vr
9-23, vr11-14, vr13-24, and
vr17-32.
TPP II Enzyme Activity Measurements-- TPP II-like activity was measured in a 96-well plate by combining 100 µl of sample mixed with 50 µl of 0.8 mM Ala-Ala-Phe-pNA (AAF-pNA) and 50 µl of a 0.2 M potassium phosphate buffer, pH 7.5, that contained 8 mM DTT. The plate was incubated at 37 °C and the change in absorbance at 405 nm was measured in a Multiscan PLUS reader (Labsystems). Tissue extract and cell lysate assays contained 100 µM bestatin to inhibit aminopeptidases. The enzyme preparations were diluted in a 100 mM potassium phosphate buffer, pH 7.5, that contained 30% glycerol and 1 mM DTT; this solution stabilizes the mammalian enzyme (7).
We defined 1 unit to equal a change in absorbance of 0.001/min, which under these conditions corresponds to the hydrolysis of 0.04 nmol of the substrate, using the molar absorbance of 9.6 mMEnzyme Purification-- Drosophila TPP II enzyme was partially purified from embryos and from adult flies in a two-step purification process. Tissues were homogenized at 1 g/4 ml in 50 mM Tris-HCl, pH 7.5, using a probe tip homogenizer (Ultra-turrax T25) with 2 pulses (10 s each) followed by sonication (MSE Soniprep 150) with 3 pulses (5 s each). Extract samples were prepared by centrifugation at 14,000 × g for 30 min and filtration of the supernatant through glass wool. The filtered extract was loaded onto a Sepharose CL-4B column (Fig. 1A). The active fractions were pooled and further purified by DEAE-cellulose chromatography (Fig. 1C). The column was eluted with a potassium gradient, and fractions were collected and analyzed by the standard assay. Active fractions were pooled for further characterization. The amount of protein in the samples was measured by the modified Bradford method (27, 28) with bovine serum albumin as standard.
Western Blot Analysis--
Samples were mixed with sample buffer
to give final concentrations of 2.3% SDS, 5% -mercaptoethanol, and
10% glycerol. The samples were heated for 5 min at 95 °C before
loading onto an 8% polyacrylamide gel. The SDS-polyacrylamide gel
electrophoresis and Western blot analysis were performed essentially as
described previously (29) using chicken anti-human TPP II antibody.
Silver staining of the polyacrylamide gel was performed according to Morrisey (30).
Characterization of dTPP II Activity-- Assays to determine substrate specificity were conducted according to the standard procedure, but various chromogenic substrates were substituted for the standard AAF-pNA, all at a concentration of 0.2 mM. During inhibitor sensitivity assays, 0.5 mg/ml bovine serum albumin was substituted for DTT in the enzyme dilution buffer to stabilize the enzyme because the presence of 1 mM DTT would hamper the effect of the thiol-reactive compounds. The final concentration of DTT in the incubation was therefore about 50 µM. In control experiments greater than 95% of the activity was retained under these conditions. Inhibitor solutions were prepared according to manufacturers' protocols, and none of the solvents used had any effect on dTPP II activity at the concentrations used. The enzyme was preincubated with the inhibitor for 30 min at 22 °C before addition of the substrate.
For the determination of Km, 2-5 units of enzyme prepared from Drosophila according to Table I or purified recombinant dTPP II were incubated in triplicates with AAF-pNA at concentrations of 1.0, 0.5, 0.2, 0.1, 0.05, and 0.025 mM in 0.1 M potassium phosphate buffer, pH 7.5, that contained 15% glycerol and 2.5 mM DTT and 1% Me2SO. The Km was calculated from Lineweaver-Burk plots to 157 and 138 µM using two different Drosophila enzyme preparations and to 107 and 103 µM for two different preparations of recombinant dTPP II. For the determination of Ki for butabindide, the enzyme was incubated with AAF-pNA at concentrations of 0.5, 0.1, or 0.025 mM in the absence or presence of butabindide (0.025, 0.1, or 0.4 µM) at 37 °C and pH 7.5, as described above. The Ki was determined in two separate experiments to 0.12 and 0.15 µM using two different Drosophila enzyme preparations and 0.11 and 0.14 µM for two different preparations of recombinant dTPP II.HPLC Analysis--
For analysis of cleavage products, 6 units of
enzyme were incubated with the nonapeptide GVLRRASVA (10 nmoles) in 0.1 M potassium phosphate buffer, pH 7.5, that contained 2 mM DTT and 3% glycerol, in a final volume of 100 µl at
37 °C. As a control the enzyme was incubated without the substrate.
The reaction was interrupted by diluting the sample 10-fold with buffer
A, and the sample was stored at 20 °C. An aliquot (200 µl) was
loaded on a Nucleosil C18 column that was equilibrated with
a mixture containing 90% (v/v) buffer A (60 mM sodium
phosphate buffer, pH 2.8, 15 mM sodium pentane sulfonate,
and 17.7 mM triethylamine) and 10% buffer B (18 mM sodium phosphate buffer, pH 2.8, 15 mM
sodium pentane sulfonate, 17.7 mM triethylamine, and 35%
(v/v) ethanol). As per Rosén et al. (31), elution was
performed at 1 ml/min, with a gradient of 10-90% buffer B. The
gradient was started at 10 min and finished at 40 min, and the column
was thereafter eluted with 90% buffer B for 20 min.
Molecular Biology-- A partial clone, PGA9, recovered from a 9- to 12-h embryonic cDNA library (K. Zinn, CalTech) showed high sequence homology to the 5' end of the mammalian TPP II.2 The missing 3' portion of the dTPP II gene was cloned using two PCR steps. Nested, gene-specific oligonucleotides were made in the sense direction corresponding to positions +2171 (CCAAGCAATCCTGTCGT) and +2278 (TGCATTGCCAAGTACTG) of the PGA9 clone. In the first round of PCR, oligonucleotide +2171 and the lambda gt11 reverse vector oligonucleotide were used to amplify templates from 5 µl of an amplified stock of the Zinn library. Klentaque LA (Sigma) enzyme was added prior to a 5-min 95 °C phage-denaturing step followed by 30 cycles of 20-s 96 °C denaturing, 30-s 50 °C annealing, and 3-min 68 °C extension. 5% of the unpurified product of the first PCR reaction served as template for a second round of amplification with the nested gene-specific oligonucleotide +2278 and the lambda gt11 reverse vector oligonucleotide. The same cycling conditions, without the initial denaturing step, were used and produced a distinct band. This product was cloned into the T/A vector according to manufacturer's recommendations (Promega, Madison, WI) with the exception that the Klentaque LA enzyme was used. A full-length clone, LD18681, containing EST sequence number AA538993, was obtained from Genome Systems (St. Louis, MO). Genomic clones containing dTPPII sequences were isolated from a Charon 4A library using the PGA9 clone and standard techniques (32). The nucleotide sequence was determined by automated sequencing (Applied Biosystems) using gene- and vector-specific oligonucleotides.
Low stringency Southern blot analysis was performed using a 50% formamide hybridization solution at 37 °C. The filter was rinsed with 2× SSC, 0.1% SDS at room temperature.Expression and Purification of Recombinant dTPP II-- The dTPP II cDNA clone LD18681 was amplified by PCR using the vector oligonucleotide T3 and a gene-specific oligonucleotide that included the stop codon and introduced a NotI restriction site for cloning into the expression vector pCDNA3 (Invitrogen, Carlsbad, CA). Clones were recovered in both the forward and reverse orientation. The mouse TPP II gene was cloned into the EcoRI site of the same vector (13). The constructs were introduced into HEK cells in culture by lipid transfection (TransITTM) according to manufacturer's protocol (Mirus Corp., Madison, WI) and incubated 40-60 h. Cells were harvested, rinsed in PBS, lysed in 50 mM Tris, pH 7.5, with 1% Triton X-100 (100 µl/106 cells), and centrifuged at 14,000 × g for 30 min. The supernatant was diluted 10-fold in 100 mM potassium phosphate buffer that contained 30% glycerol and 1 mM DTT. TPP II activity was assayed according to standard procedure.
For the preparation of stable transformants the constructs were introduced into HEK cells in culture by the calcium phosphate precipitation method, and stable clones were selected after growing the cells in medium containing Geneticin, as described previously (13). Approximately 30 × 106 cells were harvested, lysed (10 µl lysis buffer/106 cells), centrifuged, and diluted as described above. The diluted supernatant (2.6 ml) was loaded onto a Sepharose column for chromatography and analysis as described in Fig. 1A. Pooled fractions were loaded as a 10-ml sample onto a DEAE-cellulose column, and chromatography was performed as described in Fig. 1C. The peak fractions were pooled and used for further characterization.Cytological Location-- Two independent strategies were used to identify the cytological location of the dTPP II gene. In situ hybridization of the genomic phage clones to polytene chromosomes of salivary glands of third instar larvae was carried out using a biotin-avidin detection system (ENZO Detek) and standard techniques (33). As the second strategy, we screened a Drosophila P1 library (34) (Genome Systems) with a 1-kb 3' fragment of the dTPP II cDNA.
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RESULTS |
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Partial Purification of Drosophila TPP II-- To investigate the endogenous TPP II-like activity in Drosophila extracts we used the substrate AAF-pNA (8, 13). The activity was measured in the presence of the inhibitor bestatin in order to protect the substrate from sequential aminopeptidase degradation. We found approximately 0.5 units/µg protein in Drosophila extracts (Table I). To achieve a partial purification of the Drosophila TPP II activity, we used the purification procedure that was previously designed for partial purification of TPP II from rat liver (7). Sepharose CL-4B followed by DEAE-cellulose chromatography produced a 70-fold purification, and 48% total recovery, of TPP II-like activity from the extract (Fig. 1 and Table I). In the Sepharose CL-4B chromatography, the enzyme eluted at a Kav of 0.26, which is comparable to the value for the purified mammalian enzyme (7, 8, 13). This indicates that, like the mammalian enzyme, the native Mr is above 106 for the Drosophila enzyme and suggests that oligomeric dTPP II-included complexes are formed (Fig. 1A). Unlike mammalian TPP II, the Drosophila enzyme was not retained by the DEAE-cellulose column. Based upon the dTPP II gene sequence (see below) the deduced isoelectric point is 7.0 for the dTPP II enzyme, compared with 6.2 for the human enzyme. The prediction of a higher isoelectric point for dTPP II is consistent with the observation that it does not bind under these conditions (Fig. 1C). Nevertheless, a large portion of the contaminating proteins did bind the anion exchanger, and a significant purification factor was achieved (Table I) as illustrated by the silver-stained gel (Fig. 2).
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Characterization of dTPP II-- In order to characterize further the partially purified dTPP II, we investigated its pH optimum, substrate specificity, inhibitor sensitivity, and kinetics. The optimal pH for cleavage of the standard substrate AAF-pNA was between 7.5 and 7.8 (data not shown). For comparison, the mammalian enzyme has a pH optimum of 7.5 with the hexapeptide Arg-Arg-Ala-Ser(P)-Val-Ala as the substrate (7, 8). This feature differentiates TPP II from the lysosomal enzyme TPP I that prefers an acidic environment (35, 36).
The sequential cleavage of a larger substrate to form a series of tripeptides is the defining nature of a tripeptidyl peptidase. The tripeptidyl peptidase specificity of the partially purified dTPP II was confirmed by cleavage of the nonapeptide Gly-Val-Leu-Arg-Arg-Ala-Ser-Val-Ala. Fig. 3 shows that the major products corresponded to tripeptides cleaved sequentially from the N terminus of the nonapeptide. No additional cleavage products were detected.
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Cloning of dTPP II-- A 2.5-kb clone fortuitously isolated by Drs. F. Van Leeuwen and R. Nusse from an expression library provided the 5' portion of the Drosophila TPP II cDNA (PGA9). The Berkeley Drosophila Genome Project (BDGP) has described several dTPP II ESTs (clot 474) containing TPP II sequences, among these, the EST produced from cDNA clone LD18681 indicates additional 5'-untranslated region sequences. By use of PCR a complete cDNA was isolated as described under "Experimental Procedures." The complete cDNA sequence consists of 4309 bp that contains a single long open reading frame encoding 1354 aa (Fig. 4). The putative initiator methionine, deduced by alignment with the human protein, is preceded by the sequence GCAGG which does not correspond well with the consensus for Drosophila mRNAs (37). The cDNA has an untranslated 5' end of 126 bp, containing no other AUGs. The 107-bp 3'-untranslated region ends with 15 A bases and displays the consensus polyadenylation signal AATAAA 12 bp upstream.
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Expression and Characterization of Recombinant dTPP II-- To test the activity of the putative dTPP II cDNA, we used transient heterologous expression in HEK-293 cells. Transfection with the dTPP II sequence produced a 1.5-11-fold increase of AAF-pNA cleavage activity in cell extracts (Fig. 5). The mouse sequence produced a 2-7-fold increase, under these same conditions. Butabindide produced approximately 80% inhibition of the measured activity which is consistent with the induction of a TPP II-like activity. Transfection with the dTPP II clone in reverse orientation produced no increase in TPP II activity.
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dTPP II Cytological Location and Genetic Analysis-- In situ hybridization of a dTPP II genomic phage clone to polytene chromosomes produced a major hybridizing band at 49F (Fig. 7A) and a minor band at 95F. A dTPP II-specific probe, representing the 3' portion of the gene, produced a strong hybridization signal on the genomic P1 clone array (34) corresponding to the clone DS01087. This P1 clone has been assigned to the Mp20 contig and maps to the chromosomal interval 49F14-59A1. Low stringency Southern blot analysis of D. melanogaster genomic DNA gave a hybridization pattern corresponding to the recovered genomic clone and no additional bands (data not shown). Together these observations indicate the presence of a single dTPP II gene at position 49F of chromosome 2R. We attribute the weak hybridization at 95F to sequences in the genomic phage clone unrelated to dTPP II.
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DISCUSSION |
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This study has assigned a TPP II identity to a Drosophila gene located at cytological position 49F. This assignment derives from results of biochemical, molecular, and genetic experiments. Antibodies against the human enzyme cross-react with a Drosophila protein of expected size that is enriched through the partial purification of activity. The partially purified activity shows similar pH optimum, substrate specificity, and inhibitor sensitivity compared with the mammalian TPP II. The Drosophila and mammalian enzymes display a high percentage of identical and similar amino acids not only around the catalytic triad but also in the extra insert of 200 aa within the catalytic domain that is characteristic of the TPP II. The analysis of deficiency animals correlated the absence of dTPP II gene sequences with a reduction of dTPP II activity. Finally, transfection of HEK-293 cells with the dTPP II sequence results in an induction of TPP II activity, and the recombinant enzyme shows similar biochemical characteristics to enzyme preparations from Drosophila extracts.
Lacking an endogenous substrate, we used AAF-pNA to assay TPP II activity from Drosophila because this chromogenic peptide has been shown to be a good substrate (8). This assay revealed a high level of TPP II-like activity in both adult and embryonic Drosophila extracts, 0.5 and 0.85 units/µg, respectively. For comparison, when using the substrate RRAS(32P)VA, the TPP II activity in rat liver homogenate was 1.2 units/µg (8); this corresponds to about 0.1 units/µg under the present assay conditions (see "Experimental Procedures").
Our two-step chromatographic purification strategy follows that used for the initial characterization of rat TPP II (7). We have used the partially purified material to begin defining the properties of the insect enzyme in comparison to those of mammalian TPP II. The Drosophila enzyme, both partially purified from tissue and recombinant, cleaved the substrate AAF-pNA with a Km of 0.1 mM, whereas the Km for the human enzyme is 0.02 mM when measured under identical conditions.4 This value corresponds to previously reported Km values for cleavage of a similar substrate AAF-7-amido-4-methylcoumarin by mammalian TPP II: 0.025 mM for rat TPP II (6) and 0.016 mM for human TPP II (8). Based on these results, we hypothesize that human TPP II cleaves AAF-pNA more efficiently than does the Drosophila enzyme. A deviating Km value (0.148 mM) has, however, been reported for human TPP II (41).
TPP II displays low substrate specificity in vitro, as evidenced by the release of tripeptides of no apparent similarity. However, the peptidase is not indiscriminate in that different peptide substrates are cleaved at different rates (7-8, 41). The cleavage rate is sensitive to the N-terminal tripeptide sequence and also to the C-terminal sequences and to the phosphorylation state of the substrate (7). However, the tripeptidyl nature of TPP II is absolute. Our results using a nonapeptide substrate (Fig. 3) as well as the chromogenic tripeptide substrates (Table II) demonstrate that the Drosophila enzyme activity adheres to the strict tripeptidyl peptidase characteristic. Furthermore, although the exact rate of cleavage may vary between the mammalian and the Drosophila enzymes, good substrates for one are also cleaved well by the other. Proline residues within the N-terminal tripeptide sequence affect the cleavage rate by the mammalian enzyme (8). Among the chromogenic tripeptide substrates tested, a proline is accepted only in the second position by both the Drosophila and mammalian enzymes (Table II), although a proline in the first position is accepted by the human enzyme in some peptide substrates (41). In conclusion, even though the general pattern of acceptable substrates is conserved between the enzyme from human and Drosophila, there are small differences in the relative rates of cleavage.
The results of the inhibitor sensitivity for dTPP II (Table III) are consistent with those observed previously for human TPP II (8) as well as with our own preliminary comparison to human TPP II. The inhibitor sensitivities define the enzyme activity as that of a serine protease. Furthermore, dTPP II is sensitive to the same thiol-reactive compounds as is the mammalian enzyme (8), suggesting a similarity in tertiary structure and accessibility to essential cysteine residues. This property is quite remarkable since only 3 out of 16 cysteine residues in the predicted Drosophila enzyme are conserved in the human enzyme (aa 207, 599, and 760). Of these three, only Cys-760 is also conserved in the predicted C. elegans sequence (Fig. 4).
The Drosophila EST project has produced 9 ESTs (clot 474) containing TPP II sequences, all of which derive from the dTPP-II gene we have mapped to 49F. The C. elegans genome project has identified 5 cDNAs which correspond to a predicted C. elegans TPP II gene. The longest C. elegans clone, yk15e7, contains sequence that predicts an initiator methionine 46 amino acids upstream from the methionine which begins the region of high homology. There is no evidence for alternate initiation sites for the mammalian or Drosophila enzyme.
The Sepharose column provides an estimation of the very high molecular weight of the native dTPP II enzyme (Fig. 1A). Thus similar to mammalian TPP II, the Drosophila enzyme is active in a large, oligomeric complex. In addition to this abundant active complex, mammalian TPP II can form an even larger complex, eluting at the void volume of the Sepharose column, as judged by Sepharose chromatography of human erythrocytes (8) and expression experiments of alternate splice forms (13). Since an activity shoulder can be seen in this position also for the Drosophila enzyme (Fig. 1A), it is possible that the Drosophila enzyme can form a similar larger complex. Two features of the mammalian sequence have been implicated in the formation of the oligomeric TPP II complex. The first is a KEKE domain (13); such domains are thought to mediate protein-protein interactions (42). There is no apparent KEKE motif in the Drosophila protein. The second domain implicated in oligomer formation is a 13-amino acid sequence (GQSAAKRQGKFKK, separated from the KEKE domain by 20 amino acids in the human sequence) (13). The Drosophila enzyme does not contain a sequence resembling the 13-amino acid domain at a position aligned to the mammalian splice site (aa 985). In addition, the possibility of an alternative exon at this position is precluded because the genomic sequence for this region corresponds directly to that of the cDNA without interruption. However, dTPP II does contain a sequence closer to the C terminus (GESADKQKEDQKK, aa 1175) that shows some similarity to the 13-amino acid domain. When expressed in a human cell line, the recombinant dTPP II, including the similar Drosophila 13-aa domain, did not give rise to the higher molecular weight complex that elutes in the void volume of the Sepharose column. This suggests that either this 13-amino acid region does not serve a similar complex-forming function in the Drosophila enzyme or that the complex requires additional Drosophila proteins not present in the transfected cell line. These differences in the behavior of the dTPP II enzyme indicate a need for further structure-function studies. Such studies will be facilitated by the ability to perform them in vivo through efficient generation of transgenic Drosophila. Furthermore, the hypothesis that TPP II is involved in CCK physiology (6) raises several questions that can best be addressed by genetic experiments.
Another advantage of using Drosophila is the ability to create and isolate mutants. Toward this goal we have identified three deficiency lines that display reduced TPP II activity due to loss of the gene. Embryos homozygous for the deficiencies Df(2R)vg 56, Df(2R)vg 33, or Df(2R)vg B display abnormal mitosis soon after fertilization (43), and development proceeds until the germ band retraction stage when the embryos die, presumably due to loss of multiple gene functions. Therefore, accurate analysis of a dTPP II null phenotype is not possible with these large deficiencies. These lines define a cytogenetic region that contains 11 known lethal complementation groups (23, 44)5 suggesting the presence of at least 11 vital gene functions. Two of these have been identified as follows: one, vr10, is the dDP transcription factor involved in cell cycle regulation (44), and the other, vr 14, is Su(z)2, a DNA-binding protein (45, 46). We assayed the AAF-pNA cleavage activity for stocks representing one or more alleles from each of the (as yet) unidentified complementation groups. None of the lines showed significant reduction of TPP II-like activity, relative to the AFP-pNA cleavage activity. From this we conclude that the dTPP II gene is not mutated in any of these defined lethal stocks. Further analysis is required to determine if dTPP II is necessary for survival.
The genetic analysis initiated in this study provides the basis for future studies of TPP II function. TPP II is a single copy gene in Drosophila, and deficiencies of the dTPP II locus significantly reduce the enzyme activity. Our results now provide the means to identify single gene mutations of dTPP II and analyze its loss-of-function phenotypes. By using germ line transformation, it will also be possible to test partial restoration and gain-of-function TPP II phenotypes to address the hypothesis that this enzyme is involved in neuropeptide metabolism.
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ACKNOWLEDGEMENTS |
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We are indebted to Drs. F. Van Leeuwen and R. Nusse for providing the clone PGA9 containing the 5' end of the dTPP II cDNA; Dr. K. Zinn for providing the embryonic cDNA library; Dr. Sarah Elgin for providing flies and embryos for enzyme purification; and Dr. C. T. Wu for providing the deficiency and ethyl methanesulfonate Drosophila stocks. We thank the Berkeley Drosophila Genome Project for sequence information and the Bloomington Stock Center for stocks. Butabindide was a generous gift from Dr. J.-C. Schwartz and R. Ganellin. The peptide substrate and references were a generous gift from Dr. Ö. Zetterquist. We thank Dr. K. O'Malley and M. Moffat for assistance on expression experiments and Dr. J. Gordon for the use of enzyme-linked immunosorbent assay reader.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant NS-21749 (to P. T.), the Swedish Medical Research Council Project 09914 (to B. T.), and Magn. Bergvalls Stiftelse.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF035351 for Drosophila, M73047 for human, and U23176 for C. elegans.
¶ To whom correspondence should be addressed: Dept. of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110. Tel.: 314-362-3641; Fax: 314-362-3446; E-mail: Taghertp{at}thalamus.wustl.edu.
1 The abbreviations used are: ACE, angiotensin-converting enzyme; aa, amino acid(s); bp, base pairs; CCK, cholecystokinin; DTT, dithiothreitol; EST, expressed sequence tag; HEK, human embryonic kidney cells; HPLC, high performance liquid chromatography; kb, kilobase pairs; pNA, para-nitroanilide; PCR, polymerase chain reaction; suc, succinyl; TPP II, tripeptidyl peptidase II.
2 R. Nusse and F. Van Leeuwen, personal communication.
3 S. C. P. Renn, and P. H. Taghert, unpublished observations.
4 B. Tomkinson, unpublished data.
5 C. T. Wu, personal communication.
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REFERENCES |
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