J. Biochem. 110, 873-878 (1991)

Prolyl from Flavobacterium meningosepticum : Cloning and Sequencing of the 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 [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 [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 . 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 , 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, Ģ (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. 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. Well G contains saline. whole nucleotide sequence of the HincII-BamHI fragment is shown in Fig. 4. Within this sequence, there was an open Since a HincII-hydrolysate was found to hybridize at 3.5 reading frame consisting of 2,115 nucleotides which began kbp, which was a suitable size considering the molecular at an ATG codon. The amino acid sequences determined by weight of the enzyme, while others were smaller or bigger, sequencing of peptides were coincident with that deduced HincII was used for the following treatment (Fig. 1). The from the nucleotide sequence of the gene. A common hybridized HincII fragment was extracted from agarose sequence surrounding the reactive serine residue, Gly-X- gel, ligated at the HincII site of pUC19, and used to Ser-X-Gly, found in several serine enzymes was present at transform E. coli DHl. Five colonies out of approximately amino acid residues 554-558. 3,500 colonies hybridized to the oligonucleotide probe. The The enzyme is composed of 705 amino acid residues and plasmid extracted from the transformants had a 3.5kbp has a calculated molecular weight of 78,705. The amino acid insert in pUC19, named pFPEP1. After the restriction site composition determined by amino acid analysis of the of the fragment was determined, the HincII-BamHI frag purified enzyme (19) and that deduced from the nucleotide ment (2.9kbp) was subcloned into pUC19 to construct sequence of the gene are in fairly good agreement, within plasmid pFPEP3, with which E. coli JM83 was trans- the experimental errors of amino acid analysis. formed. Since the enzyme productivity of the transformant Amino Acid Sequence around the Reactive Serine Resi- was confirmed by the immunoprecipitation method (Fig. due-[3H]DFP-labeled prolyl endopeptidase was cleaved 2), this DNA fragment was used for nucleotide sequencing. by CNBr and the resultant peptide fragments were separat Nucleotide Sequence-The restriction endonuclease ed by HPLC. Figure 5 shows the HPLC profile of peptides mapping and sequencing strategy are shown in Fig. 3. The obtained by CNBr cleavage on a Vydac C4 column. More

Vol. 110, No. 6, 1991 876 T. Yoshimoto et al.

Fig. 5. HPLC profile of CNBr cleavage fragments of [3H]DFP- labeled prolyl endopeptidase. The CNBr-cleaved fragments were applied to a column of Vydac C4. The experimental details are described in the text.

TABLE II. Amino acid composition and sequence of peak 8 on the HPLC profile shown in Fig. 5. A. Amino acid composition

aHSE, homoserine. Fig. 4. Nucleotide sequence of the prolyl endopeptidase gene and deduced amino acid sequence of the enzyme. The nucleotide sequence is numbered from the top of the initiation codon of the tritium. This sequence corresponds to amino acid residues enzyme gene.Numbering of the amino acids starts at the initiation 551-557, and the reactive serine residue is identified as codonof the mature protein of the prolyl endopeptidase.Amino acid Ser-556. sequencesof endoproteinaseAsp-N or lysylendopeptidase fragments Homology Search-The amino acid sequence deduced of the enzyme, determined by Edman degradation,are specified from the nucleotide sequence of the prolyl endopeptidase belowthe amino acid sequenceby arrows. gene of F. meningosepticum was 38.2% homologous to that of the enzyme gene from porcine brain and 24.5% homol ogous to E. coli protease II (Fig. 6). In particular, the amino than 60% of the radioactivity was detected in peak 8. The acid sequence around the reactive serine residue, which was amino acid composition and sequence of this peptide are identified as Ser-554 in the porcine brain enzyme (32) shown in Table II, A and B. Radioactivity (1.4 x 104 dpm) using [3H]DFP, was well conserved among these three was detected at cycle 6 (Table IIB), and the amino acid enzymes. sequence was established to be Ala-Leu-Ser-Gly-Arg- The genes of the other two proline-specific peptidases, *Ser-Asn where the 6th serine residue was labeled with dipeptidyl aminopeptidase IV (39), and aminopeptidase P

J. Biochem. Prolyl Endopeptidase from Flavobacterium meningosepticum 877

Fig. 7. Hydropathy patterns of prolyl endopeptidases from F. meningosepticum (A) and porcine brain (B) and of protease II from E. coli (C). The portion abovethe midpointline representsthe hydrophobicregions. Arrowsindicate the reactive serine residue of Fig. 6. Comparison of amino acid sequences of three serine each enzyme. proteases, prolyl endopeptidases from F. meningosepticum (F-PEP) and porcine brain (P-PEP) and protease II from E. coli (E-PII). Conservedamino acids in the three enzymesare indicatedby asterisks, and those in any two enzymes are indicated by dots. cysteine residues is located near the reactive serine residue # indicatethe proposedactive site serine and histidine residues. as in protease K, a -like protease from Tritra chium album Limber (41), as judged from the sensitivity to PCMB. (40), have previously been cloned, and their nucleotide It is of great interest that the amino acid sequences of the sequences have been reported, but no sequence homology two prolyl endopeptidases are highly homologous to that of was observed between these proline-specific aminopep- protease II from E. coli. As shown in Fig. 6, 152 amino acid tidases and the prolyl endopeptidase described here. residues are conserved between prolyl endopeptidase from porcine brain and E. coli protease II, and 161 residues are DISCUSSION also conserved between prolyl endopeptidase from Flavo bacterium and the protease II. Ninety-six amino acid Prolyl endopeptidases are widely distributed in animals, residues were commonly conserved in these three enzymes. plants, and microorganisms, and they have similar en Thus, we propose for these three enzymes the name "prolyl zymatic properties. However, their isoelectric point and endopeptidase family." Most of the homologous sequences sensitivity to PCMB are clearly distinct between procar were found to be located in the rear half of the enzyme, especially around the reactive serine residue. E. coli yotes and eucaryotes. The Flavobacterium enzyme is a basic protein (20) while the animal enzymes are weakly acidic protease II, like prolyl endopeptidase from F. meningo- (13-15); this discrepancy is explicable in terms of the septicum, was not inhibited by PCMB (20, 42), in contrast difference in ionizable amino acid contents. The prolyl to prolyl endopeptidases from animals. These results endopeptidase from Flauobacterium has two cysteine resi- suggest that prolyl endopeptidases and protease II have dues, but no free cysteine residue is detectable by the been derived from a common ancestral gene which codes for Ellman reagent even under denaturing conditions, suggest- a including serine, and from which proteases ing that the enzyme has a disulfide bridge. In contrast, the with distinct substrate specificities were derived. There- enzyme from porcine brain has 16 cysteine residues (32). after, the prolyl endopeptidase gene of eucaryote seems to Though it is not clear how many residues are present as free have evolved further, with the introduction of a cysteine sulfhydryl groups, it seems likely that at least one of the residue near the catalytic site.

Vol. 110, No. 6, 1991 878 T. Yoshimoto et al.

There are three histidine residues, His-230, His-639, 13. Yoshimoto, T., Nishimura, T., Kits, T., & Tsuru, D. (1983) J. and His-675, which are conserved among these three Biochem. 94, 1179-1190 enzymes (Fig. 6), and adjacent residues are also fairly well 14. Moriyama, A. & Sasaki, M. (1983) J. Biochem. 94,1387-1397 conserved. Thus, one of these histidine residues could be a 15. Soeda, S., Ohyama, M., & Nagamatsu, A. (1984) Chem. Pharm. Bull. 32, 1510-1516 plausible candidate for one of the catalytic triad. 16. Yoshimoto, T., Sattar, A.K.M.A., Hirose, W., & Tsuru, D. Codon usage of the prolyl endopeptidase gegeenefrom F. (1987) Biochim. Biophys. Acta 916, 29-37 meningosepticum was quite different from that in the gene 17. Sattar, A.K.M.A., Yoshimoto, T., & Tsuru, D. (1990) J. Bio for the enzyme degrading nylon oligomers from Flavobac chem. 107, 256-261 terium sp. K172 (43). It is reasonable that we could not get 18. Yoshimoto, T., Sattar, A.K.M.A., Hirose, W., & Tsuru, D. any DNA fragment which hybridizes with oligonucleotide (1988) J. Biochem. 104, 622-627 19. Yoshimoto, T., Ando, M., Ohota, K., Kawahara, K., & Tsuru, D. probes based on the codon usage of the latter enzyme gene. (1982) Agric. Biol. Chem. 46, 2157-2158 As shown in Fig. 7, hydropathy profiles of the two prolyl 20. Yoshimoto, T., Walter, R., & Tsuru, D. (1980) J. Biol. Chem. 255, endopeptidases are similar but that of protease II is rather 4786-4792 different from those of both prolyl endopeptidases. How- 21. Hartrodt, B., Neubert, K., Fischer, G., Demuth, U., Yoshimoto, ever, domain analyses of these enzymes by the average T., & Barth, A. (1982) Pharmazie 37, 72-73 distance map method (44) showed similar distributions of 22. Nomura, K. (1986) FEBS Lett. 209, 235-237 23. Walter, R. & Yoshimoto, T. (1978) Biochemistry 17, 4139-4144 domain structures. These results suggest that the backbone 24. Nishikata, M., Yokosawa, H., & Ishii, S. (1986) Chem. Pharm. structures of these enzymes resemble each other. Bull. 34, 2931-2936 Dipeptidyl aminopeptidase IV is also a proline-specific 25. Tsuru, D., Yoshimoto, T., Koriyama, N., & Furukawa, S. (1988) , and the nucleotide sequence of the gene J. Biochem. 104, 580-586 was determined (39). No homology was observed between 26. Yoshimoto, T., Tsuru, D., Yamamoto, N., Ikezawa, R., & this exopeptidase and the prolyl endopeptidase family, Furukawa, S. (1991) Agric. Biol. Chem. 55, 37-43 except for a common sequence around the reactive serine 27. Yoshimoto, T., Kado, K., Matsubara, F., Koriyama, N., Kaneto, H., & Tsuru, D. (1987) J. Pharmacobiodyn. 10, 730-735 residue. Though the reason is not clear, it seems that 28. Yoshimoto, T., Kawahara, K., Matsubara, F., Kado, K., & Tsuru, ancestor proteins of exo- and endo-peptidases may have D.(1985) J. Biochem. 98, 975-979 been distinct. 29. Yoshimoto, T., Orlowski, R.C., & Walter, R. (1977) Biochemistry To clarify the evolutional change of prolyl endopeptidase, 16,2942-2948 we are planning phylogenetic studies. 30. Yokozawa, H., Miyata, M., Sawada, H., & Ishii, S. (1983) J. Biochem. 94, 1067-1076 We thank Miss A. Shikazefor her technicalassistance. 31. Yoshimoto, T., Tsukumo, K., Takatsuka, N., & Tsuru, D. (1982) J. Pharmacobiodyn. 5, 734-740 32. Rennex, D., Hemmings, B.A., Hofsteenge, J., & Stone, S.R. REFERENCES (1991) Biochemistry 30, 2195-2203 1. Yoshimoto,T., Fischl,M., Orlowski,R.C., &Walter, R. (1978)J. 33. Saito, H. & Miura, K. (1963) Biochim. Biophys. Acta 72, 619- Biol. Chem.253, 3708-3716 629 2. Yoshimoto,T., Simmons,W.H., Kits, D., &Tsuru, D. (1981).J. 34. Sambrook, J., Fritsch, F.E., & Maniatis, T. (1989) Molecular Biochem.90, 325-334 Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3. Andrews,P.C., Minth, C. D., &Dixon, J.E. (1982) J. Biochem. Cold Spring Harbor, NY 257,5861-5865 35. Hanahan, D. (1983) J. Mol. Biol. 166, 557-580 4. Green, G.D.J. & Shaw,E. (1983) Arch. Biochem.Biophys. 225, 36. Kobayashi, R. & Tarr, G.E. (1986) Protein Nucleic Acid Enzyme 331-337 31,991-1002 5. Kato, T., Nakano,T., Kojima,K., Nagatsu,T., &Sakakibara, S. 37. Tarr, G.E. (1986) in Method of Protein Microcharacterization (J.E. Shively, ed.) pp. 155-194, Humana Press, NJ (1980) J. Neurochem.35, 527-535 6. Soeda, S., Yamakawa,N., Ohyama,M., Shimeno,H., & Naga- 38. Hattori, M. & Sakaki, Y. (1986) Anal. Biochem. 152, 232-238 matsu, A. (1985) Chem.Pharm. Bull. 33, 2445-2451 39. Ogata, S., Misumi, Y., & Ikehara, Y. (1989) J. Biol. Chem. 264, 7. Yoshimoto,T., Ogita, K., Walter, R., Koida, M., & Tsuru, D. 3596-3601 40. Yoshimoto, T., Tone, H., Honda, T., Osatomi, K., Kobayashi, R., (1979) Biochim.Biophys. Acta 569,184-192 & Tsuru, D. (1989) J. Biochem. 105, 412-416 8. Orlowski, M., Wilk, E., Pearce, S., & Wilk, S. (1979) J. Neurochem.33, 461-469 41. Betzel, C., Pal, G.P., Struck, M., Jany, K.D., & Saenger, W. (1986) FEBS Lett. 197, 105-110 9. Wilk, S. & Orlowski,M. (1983) J. Neurochem.41, 69-75 42. Kanatani, A., Masuda, T., Shimoda, T., Xu, S.L., Yoshimoto, T., 10. Blumberg, S., Teichberg, V.I., Charli, J.L., Hersh, L.B., & & Tsuru, D. (1991) J. Biochem. 110, 315-320 McKelvy,J.F. (1980) Brain Res. 192, 477-486 43. Okada, H., Negoro, S., Kimura, H., & Nakamura, S. (1983) 11. Knisatschek,H. &Bauer, K. (1979) J. Biol. Chem.254, 10936- Nature 306, 203-206 10943 44. Kikuchi, T., Nemethy, G., & Scheraga, H.A. (1988) J. Protein 12. Yoshimoto,T., Oyama,H., Koriyama,N., & Tsuru, D. (1988) Chem. 7, 427-471 Chem.Pharm. Bull. 36, 1456-1462

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