Prolyl Endopeptidase from Flavobacterium Meningosepticum : Cloning and Sequencing of the Enzyme Gene1
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J. Biochem. 110, 873-878 (1991) Prolyl Endopeptidase from Flavobacterium meningosepticum : Cloning and Sequencing of the Enzyme Gene1 Tadashi "Yoshimoto,*,2 Akio Kanatani,* Taiji Shimoda,* Tetsuya Inaoka,** Toshio Kokubo,** and Daisuke Tsuru* *Schoolof PharmaceuticalSciences , Nagasaki University,Nagasaki, Nagasaki 852; and **InternationalResearch Laboratory,Ciba-Geigy (Japan) Limited, Takarazuka,Hyogo 665 Receivedfor publication,July 12, 1991 The prolyl endopeptidase [EC 3.4.21.26] gene of Flavobacterium meningosepticum was cloned in Escherichia coli with the aid of an oligonucleotide probe which was prepared based on the amino acid sequence. The hybrid plasmid, pFPEP1, with a 3.5kbp insert at the HincII site of pUC19 containing the enzyme gene, was subcloned into pUC19 to construct plasmid pFPEP3. The whole nucleotide sequence of an inserted HincII-BamHI fragment of plasmid pFPEP3 was determined by the dideoxy chain-terminating method. The purified prolyl endopeptidase was labeled with tritium DFP, and the sequence surrounding the reactive serine residue was found to be Ala (551)-Leu-Ser-Gly-Arg-*Ser-Asn(557). Ser-556 was identified as a reactive serine residue. The enzyme consists of 705 amino acid residues as deduced from the nucleotide sequence and has a molecular weight of 78,705, which coincides well with the value estimated by ultra centrifugal analysis. The amino acid sequence was 38.2% homologous to that of the porcine brain prolyl endopeptidase [Rennex et al. (1991) Biochemistry 30, 2195-2203] and 24.5% homologous to E. coli protease II, which has substrate specificity for basic amino acids [Kanatani et al. (1991) J. Biochem. 110,315-320]. Prolyl endopeptidases [EC 3.4.21.26] have been purified nucleotide sequencing of the prolyl endopeptidase gene from lamb (1, 2), rat (3- 7), rabbit (8, 9), bovine (10-13), from F. meningosepticum, and a structural comparison with porcine (14, 15), carrot (16), mushroom (17, 18), and related enzymes. Flavobacterium meningosepticum (19, 20). Substrate speci ficities of these enzymes have been studied using several MATERIALS AND METHODS synthetic substrates and natural peptides (1, 7, 21-23), and on the basis of these substrate specificities, potent Materials-Restriction enzymes, BAL31 nuclease, T4- inhibitors, Z-Gly-Pro-CH2C1, Z-Pro-prolinal, and Z-Thio- DNA ligase, kilo and M13 sequencing kits, and primer for pro-thioprolinal, etc., have been synthesized (9, 24-30). sequencing were purchased from Takara Shuzo and Intracellular inhibitors have also been found in porcine Toyobo. [32P]dCTP (110TBq/mmol) and [35S]dCTP (37 pancreas (31) and rat brain (6). These inhibitors act TBq/ mmol) were purchased from ICN Radiochemicals and specifically upon prolyl endopeptidases from several ori Amersham. Sequenase was obtained from Toyobo and gins. The enzymes were characterized as serine proteinases Agarose I from Dojin Chemicals. Lysozyme, RNase A, and because of their sensitivity to DFP. However, the enzymes DNA from salmon were from Sigma. Alkaline phosphatase from animals and plants also showed susceptibility to from calf intestine and Pseudomonas fragi endoproteinase PCMB, while the enzyme from Flavobacterium (19, 20) Asp-N were obtained from Boehringer-Mannheim. Lysyl was insensitive to PCMB. In addition, the isoelectric point endopeptidase from Achromobacter lyticus was from Wako of microbial prolyl endopeptidase was different from those Pure Chemical. of the enzymes from animals and plants. Thus, it would be Bacterial Strains, Plasmid, and Media-E. coli JM83 very interesting to clarify and compare the protein struc (ara, ƒ¢ (lac-proAB), strA, _??_80 dlacZM15), and DH1 (F-, tures of these enzymes from an evolutional point of view. relAl, gyrA96, thi-1, hsdRl7, supEp44, relAl) were used Recently, the amino acid sequence of porcine brain prolyl as hosts. The plasmids, pUC18 and pUC19, were used for endopeptidase was deduced from cDNA analysis of the cloning, and the last one was also used for sequencing. enzyme gene (32). We attempted to clone the enzyme gene Bacteria were grown in LB-broth. from Flavobacterium. This article deals with cloning and Isolation of DNA and Transformation-The chromo somal DNA of F. meningosepticum was prepared by the 1This work was supported in part by a Grant-in-Aid for Scientific method of Saito and Miura (33). Plasmid DNA was isolated Research from the Ministry of Education, Science and Culture of by the alkaline extraction procedure (34) or by CsCl- Japan and a grant from Nagase Science and Technology Foundation ethidium bromide equilibrium density gradient centrifuga of Japan. 2 To whom correspondence should be addressed . tion. E. coli DH1 and E. coli JM83 were transformed with Abbreviations: DFP, diisopropyl phosphorofluoridate; HPLC, high- hybrid plasmids by Hanahan's method (35). performance liquid chromatography; TFA, trifluoroacetic acid. Purification and Sequence Analysis of Prolyl Endopep- Vol. 110, No. 6, 1991 873 874 T. Yoshimoto et al. tidase-The enzyme was purified from the cell-free extract mg) was incubated with 1 mg of CNBr in 1ml of 70% formic of F. meningosepticum as described previously (20), except acid under nitrogen gas in the dark for 24 h at room for an additional final purification step by HPLC. To temperature, diluted with water, and then lyophilized. The remove salt in the preparation, the enzyme was applied to resultant peptide fragments were separated and purified by a TSKgel octadecyl-NPR column (4.6 x 35mm). The puri HPLC as described above, except that a Vydac C4 column fied enzyme (0.5 mg) in 1ml of 10mM ammonium bicar was used instead of C18. bonate buffer, pH 8.0, containing 4 M urea was hydrolyzed by 1ƒÊg of endoproteinase Asp-N at 37•Ž for 24 h. Peptide RESULTS fragments were isolated from the digestion mixture by reverse-phase HPLC; the peptide mixture was applied to a Partial Sequence Determination of Prolyl Endopeptidase Vydac C18 column (4.6 x 250 mm) equilibrated with from F. meningosepticum-The purified enzyme was 0.075% trifluoroacetic acid (TFA) at 25•Ž and eluted with digested with endoproteinase Asp-N and lysyl endopep an increasing gradient of the solvent system of acetonitrile/ tidase, and the digestion mixtures were separated by 2-propanol (3:1) containing 0.06% TFA at a flow rate of 1.0 HPLC. The amino acid sequences of 24 peptides isolated ml/min. The elution of peptides was monitored by measur were determined by Edman degradation (Table I). These ing absorbance at 214 nm. The enzyme was also digested amino acid sequences were well coincident with those with lysyl endopeptidase in 20mM Tris-HC1 buffer, pH deduced from nucleotide sequencing of the enzyme gene, as 9.0, containing 4 M urea in a manner similar to that used for shown later (see Fig. 4). endoproteinase Asp-N digestion and the resultant peptide Construction of Gene Library and Screening of Prolyl fragments were purified by HPLC as above. Amino acid Endopeptidase Gene-Chromosomal DNA of F. meningo sequences of peptides were determined by manual Edman septicum was digested with each of SacI, PvuII, PstI, degradation (36), and their amino acid compositions were HindIII, HincII, EcoRV, EcoRI, and BgIII. The hydrol analyzed by the PTC (phenyl thiocarbamyl)-amino acid ysates were subjected to agarose gel electrophoresis, and method (37), after hydrolysis with 6 N HO containing a the products were transferred to nitrocellulose filters and trace amount of phenol at 150•Ž for 1 h. hybridized with a 32P-labeled synthetic oligonucleotide. Immunological Studies-Antiserum for prolyl endopep tidase from F. meningosepticum was that prepared previ TABLEI. Amino acid sequences of the proteolytic fragments ously (20). Immunodiffusion was performed overnight in of prolyl endopeptidase obtained by digestion with endopro 1.2% agarose gel in 70mM phosphate buffer, pH 7.5, teinase Asp-N or lysyl endopeptidase. D and K indicatefragments containing 0.9% NaCl. obtainedby digestionwith endopeptidaseAsp-N and lysyl endopep DNA ProbesOligonucleotides were synthesized with an tidase, respectively.C, Edmandegradation cycle; AA, amino acid; Y, Applied Biosystems Model 381A DNA synthesizer. After yield (pmol). removal of the dimethoxytrityl group at the 5•L end of the oligonucleotide, the product was cleaved from the support. The probe used for this experiment was synthesized as mixed and inosine-replaced nucleotides as follows. Subcloning and Nucleotide Sequencing-Restriction endonuclease fragments of the chromosomal DNA were subcloned into pUC18 and pUC19 following the reported protocols (34). The HincII and 3•L end BamHI fragment was further digested by BAL31 nuclease and kilo-sequencing method, and then subcloned into pUC19. The recombinant plasmids were isolated by alkaline lysis. After polyethy lene glycol precipitation, nucleotide sequencing was carried out by the method of Hattori and Sakaki (38), except that Klenow fragment was replaced by Sequenase. Active Site Labeling of Prolyl Endopeptidase-The enzyme (1mg) was incubated with a 100-fold molar excess of [3H]DFP in 1ml of 20mM Tris-HC1 buffer, pH 7.0, at 37•Ž. After 1 h, unlabeled DFP was added to obtain a final concentration of 1mM, and the mixture was incubated for an additional 1 h before extensive dialysis against distilled water and lyophilization. The tritium-labeled enzyme (1 J. Biochem. Prolyl Endopeptidase from Flavobacterium meningosepticum 875 Fig. 1. Electrophoresis of restriction enzyme digests of chromosomal DNA from F. meningo septicum (A) and their Southern plot analyses (B) with a probe. Fig. 2. Double immunodiffusion profile of cell-free extracts of Fig. 3. Restriction map and sequencing strategy of the prolyl transformants against antiserum for prolyl endopeptidase from endopeptidase gene. The black box represents the enzyme coding F. meningosepticum. Well A contains antiserum against the en region. The arrows indicate the sequencingdirection and extent of zyme. Well B contains the enzyme preparation of the wild strain. DNAfragments digested by restriction enzymesor exonucleaseIII. Wells C, D, E, and F contain cell-free extracts of E. coli JM83/ pFEP3, JM83/pFPE2, DHl/pFPEl, and JM83.