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Limited Proteolysis of the Bifunctional Thymidylate Synthase-Dihydrofolate Reductase from Leishmania Tropica (Bifunctional Enzyme/Protein Domains/Protozoa) EDWARD P

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Limited Proteolysis of the Bifunctional Thymidylate Synthase-Dihydrofolate Reductase from Leishmania Tropica (Bifunctional Enzyme/Protein Domains/Protozoa) EDWARD P Proc. Nati. Acad. Sci. USA Vol. 82, pp. 7188-7192, November 1985 Biochemistry Limited proteolysis of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania tropica (bifunctional enzyme/protein domains/protozoa) EDWARD P. GARVEY AND DANIEL V. SANTI* Departments of Biochemistry and Biophysics and Pharmaceutical Chemistry, University of California, San Francisco, CA 94143 Communicated by Thomas C. Bruice, June 27, 1985 ABSTRACT The structure and activity of the bifunctional found to be a dimer of apparently identical subunits of Mr thymidylate synthase-dihydrofolate reductase (TS-DHFR) 56,000 (5). To date, there has been no direct evidence for from the protozoan parasite Leishmania tropica were examined either homology or lack of homology between L. tropica by limited proteolysis with five different endopeptidases. Each TS-DHFR and the two individual enzymes from any non- reaction resulted in a rapid, time-dependent loss of TS activity protozoan species. and no effect on DHFR activity. The proteolytic products were Limited proteolysis has been used to examine the structure examined by NaDodSO4/PAGE; each digestion produced a of a number of multifunctional proteins. Results of these fragment of apparent Mr 35,000, and three of the five studies have led to proposals for the arrangement of the digestions generated a fragment of Mr 20,000. Attempts to various functions on the polypeptide (6, 7) and have produced separate the fragments under nondenaturing conditions failed, evidence that many multifunctional proteins exist as a series suggesting that the proteolyzed protein remains a dimer with of separate, independent domains (each domain reflecting a the gross structure ofthe subunits more or less undisturbed. In function) (7-9). We undertook the limited proteolysis of L. contrast, kinetic data indicate that some aspects of higher- tropica TS-DHFR in hopes of separating the two activities as order structure in the native protein are affected by proteolysis. proteolytic fragments, the size of each approximating their The fragments (Mr 36,600 and 20,000) generated by non-protozoan counterparts (i.e., a TS fragment of Mr Staphylococcus aureas V8 protease were subjected to sequence 35,000 and a DHFR fragment of Mr 20,000). We report analysis. Whereas neither the native protein nor the Mr 36,600 here the results of the limited proteolysis of L. tropica fragment yielded an NH2-terminal amino acid, we obtained the TS-DHFR. We propose an arrangement of the enzymatic sequence of the first 28 amino acids of the Mr 20,000 fragment. activities within the TS-DHFR polypeptide and suggest a This sequence bore strong homology with sequences situated structural model of the region that is selectively cleaved by within TS of human, Lactobacillus casei, Escherichia coli, and five different endopeptidases. bacteriophage T4. These and other data indicate that the TS-DHFR polypeotide consists of a DHFR sequence at the EXPERIMENTAL PROCEDURES blocked NH2-terminal and a TS sequence at the COOH- Enzymes, Antisera, and Activity Measurements. The bi- terminal end of the protein. The region that is the target of the functional TS-DHFR from 10-propargyl-5,8-dideazafolate- five proteases corresponds to a highly variable region within the resistant L. tropica (selection to be described elsewhere) was sequences of the other four TSs. We suggest that an insertion purified to homogeneity by methotrexate-Sepharose CL-6B occurs within the TS-DHFR sequence, positioned on the chromatography, as described (5). L-1-Tosylamido-2-phen- surface of the protein and quite vulnerable to the action of ylethyl chloromethyl ketone (TPCK)-treated trypsin, N-p- endopeptidases. tosyl-L-lysine chloromethyl ketone (TLCK)-treated a- chymotrypsin, Staphylococcus aureus V8 protease, Strepto- Thymidylate synthase (5,10-methylenetetrahydrofolate: myces griseus type XIV protease, elastase, and soybean dUMP C-methyltransferase, EC 2.1.1.45) and dihydrofolate trypsin inhibitor were purchased from Sigma. Rabbit antise- reductase (5,6,7,8-tetrahydrofolate:NADP' oxidoreductase, rum to L. tropica TS-DHFR was obtained after subcutaneous EC 1.5.1.3) catalyze consecutive reactions in the de novo injection of 150 ,Ag ofpure TS-DHFR mixed 1:1 (vol/vol) with synthesis of dTMP. In sources as varied as bacteriophage, Freund's complete adjuvant, followed 3 weeks later by a bacteria, and vertebrates, these two enzymes exist as distinct booster injection of 100 k&g of TS-DHFR mixed 1:1 with and readily separable enzymes (for reviews, see refs. 1 and Freund's incomplete adjuvant. Rabbit antiserum raised 2). In contrast, a bifunctional protein, thymidylate synthase- against Escherichia coli TS was provided by F. Maley (New dihydrofolate reductase (TS-DHFR), has been identified in a York State Department of Health, Albany); rabbit antiserum number of genera of protozoa which span a diverse group of against E. coli RT500 DHFR was provided by D. Baccanari the subkingdom (3, 4). This protein ranges in molecular (Burroughs Wellcome, Research Triangle Park, NC). Goat weight from about 110,000 to 140,000, with subunits of anti-rabbit Ig antibody-alkaline phosphatase conjugate was molecular weight 55,000-65,000. As has been noted (3-5), from Boehringer Mannheim. the subunit size of the protozoan TS-DHFR is close to the DHFR activity (10) and TS activity (11) were determined sum of the subunit size of TS (Mr 35,000) and DHFR (Mr spectrophotometrically at 250C. Also, DHFR was quantitat- 20,000) found in most other sources, suggesting that the ed by binding to [3H]methotrexate (1), and TS Was quanti- gene encoding TS-DHFR may have resulted from the fusion tated by binding to 5-fluoro-2'deoxy[3H]uridylate [3H]- of independent TS and DHFR genes. TS-DHFR from a FdUMP and (±)-5,10-methylenetetrahydrofolate (CH2H4- methotrexate-resistant cell line of the protozoan parasite folate) (12), as previously described. Leishmania tropica has been purified to homogeneity and Abbreviations: TS, thymidylate synthase; DHFR, dihydrofolate The publication costs of this article were defrayed in part by page charge reductase; FdUMP, 5-fluoro-2'-deoxyuridylate; CH2H4folate, (+)- payment. This article must therefore be hereby marked "advertisement" 5,10-methylenetetrahydrofolate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 7188 Downloaded by guest on September 25, 2021 Biochemistry: Garvey and Santi Proc. Natl. Acad. Sci. USA 82 (1985) 7189 Table 1. Summary of limited proteolysis of L. tropica TS-DHFR t112, min Loss of enzymatic activity Loss of Mr 56,000 Predominant fragments Protease Specificity* TS DHFR subunitt generated,t M, x i0-3 S. aureus V8 protease Glu, Asp 18 No loss 40 37.3 - 36.6t (225 min) 20 Trypsin Arg, Lys 6 No loss 16 34.5 33.1 -* 31.6 (200 min) 21 -* 19 a-Chymotrypsin Tyr, Trp, Phe, Leu 20 No loss 35 Quartet, -37 (90 min) 19 Elastase Uncharged, nonaromatic 5 No loss 10 35 (135 min) mns§ S. griseus type XIV protease Nonspecific 10 No loss 14 36 (200 min) mns§ *Nature of amino acid residues donating carbonyl portions of susceptible peptide bands. tDetermined by NaDodSO4/PAGE. tArrows denote fragment generated from previous fragment during course of reaction. §Multiple nonspecific bands. Gel Electrophoresis. NaDodSO4/PAGE (10-15% poly- RESULTS acrylamide) was performed essentially as described by The bifunctional TS-DHFR from L. tropica was subjected to Laemmli (13). Nondenaturing PAGE (12.5% polyacrylamide) limited proteolysis by use offive different endopeptidases: S. was performed according to Davis (14). Proteins were trans- aureus V8 protease, trypsin, a-chymotrypsin, elastase, or S. ferred from polyacrylamide gels to nitrocellulose filters (15) griseus type XIV protease (Table 1). Each of the five and probed with antibody (16); the alkaline phosphatase proteolytic reactions was monitored for enzymatic activities; conjugate was assayed as described (17). Peptides were in each digest there was a relatively rapid, time-dependent isolated from polyacrylamide gels for peptide mapping (18) inactivation of TS, which followed apparent first-order ki- and for NH2-terminal sequence analysis (19). netics for at least two half-lives (t1l2 < 20 min), and, under the Limited Proteolysis. TS-DHFR (0.1-0.5 mg/ml) in 50 mM conditions used, no loss ofDHFR activity. Curiously, in each Tes, pH 7.4/2 mM dithiothreitol/1 mM EDTA/5% (vol/vol) of the five digests, the rate of TS inactivation was approxi- glycerol was digested with endopeptidase (1:100 ratio, wt/wt) mately twice the rate of proteolysis (Table 1); when TS was or exopeptidase (5-10:100 ratio) at 25°C. To monitor enzy- completely inactivated, -50% of the Mr 56,000 subunit matic activities, aliquots (2-10 ,ul) were added directly to 1-ml remained intact. The extent ofproteolysis was also measured assay solutions. DEAE-Sepharose chromatography was as by assaying the ability ofTS or DHFR to bind their respective reported (5); the bound, proteolyzed TS-DHFR was eluted stoichiometric inhibitors, [3H]FdUMP or [3H]methotrexate. with a linear 0-0.2 M KCI gradient. Methotrexate-Sepharose V8 protease caused a time-dependent loss in the ability of TS chromatography was as described (5), except for the follow- to bind [3H]FdUMP and CH2H4folate, with the rate of loss ing additional washes of the bound, proteolyzed TS-DHFR occurring approximately twice as slowly as the rate of loss in before elution: 0.1% Triton X-100/10 mM potassium phos- catalytic activity (41/2 35 min vs. 18 min) and at approxi- mately the same rate as proteolysis (t1/2 - 35 min vs. 40 min). phate, pH 7.0, and 0.1% Nonidet P-40/10 mM potassium When TS-DHFR was digested with trypsin, there was no loss phosphate, pH 7.0. Enzyme was eluted with 1 mM 7,8- in the ability of DHFR to bind methotrexate; instead, the dihydrofolate in 50 mM Tes, pH 7.4/2 mM dithiothreitol/l amount of methotrexate bound to DHFR increased 2.3-fold mM EDTA.
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