The Primary Structure of Pig Liver Thioltransferase

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 262, No. 14, Issue of May 15, pp by The Amerian Soiety of Biologial Chemists, In. Printed in U.S.A. The Primary Struture of Pig Liver Thioltransferase Zhong-Ru Gan and William W. Wells$ From the Department of Biohemistry, Mihigan State University, East Lansing, Mihigan (Reeived for publiation, Deember 3, 1986) The omplete amino aid sequene of pig liver thioltransferase has been determined. The homogeneous protein was leaved by trypsin, hymotrypsin, Staphyloous aureus V8 protease, and yanogen bromide. The resulting peptides were purified by reversedphase high performane liquid hromatography and ion-exhange fast protein liquid hromatography. Sequening of the fragments was ahieved with either automated Edman degradation or fast atom bombardment-mass spetrometry. Pig liver thioltransferase is a single polypeptide with 15 amino aid residues and an aetylated glutamine N terminus. The protein has 2 ysteine pairs with sequenes of -Cys-Pro-Phe-Cysand -Cys-Ile-Gly-Gly-Cys-, the first pair of whih (CysZ2 and CysZ6) is loated at the potential ative site of the enzyme. The sequene of pig liver thioltransferase displays lose homology (82%) with alf thymus glutaredoxin, suggesting that they belong to the same evolutionary family. Thioltransferase has been found in yeast (l), rat liver (2, 3), bovine liver (4), pig liver (5), and human plaenta (6). There are numerous studies in the literature on its ability to transfer reduing equivalents from GSH to ytosoli protein and nonprotein disulfides and to regulate ytosoli enzyme ativities (7-11). In a reent study, rabbit polymorphonulear leuoytes, stimulated by immune omplex, released inreased thioltransferase into the medium, in vitro (12). In addition, these investigators reported that oxidized inative papain was restored to 85% of original ativity by treatment with both glutathione (GSH) and thioltransferase (12). Furthermore, a thioltransferase-like protein alled soluble protein fator was found to promote the GSH-responsive iodothyronine 5 -deiodinase ativity in rat kidney mirosomes (13-14). However, the omplete physiologial role of thioltransferase remains to be established. Chemial haraterization of thioltransferase from rat (15, mine. The other major ions in Fig. 3 have been assigned to 16), bovine (4), and pig liver (5) has shown that they are small leavage of the peptide bakbone, or amino aid side hains. proteins with moleular weights of approximately 11,. The fast atom bombardment spetrum (not shown) also on- Eah protein ontains 4 ysteine residues, and the enzyme is tained the B, and B2 ions at m/z 171 and 242, respetively, very sensitive to alkylating reagents (5, 16). Carbohydrate onfirming the N-terminal assignments. Aording to the (8.6%) has been found in homogeneous rat liver thioltransferase (15). Further studies on the atalyti mehanism and * This work was supported by Grant AM 3293 from the United States Publi Health Servie and the Department of Biohemistry, Mihigan State University. Mass spetral data were obtained from the Mihigan State University Mass Spetrometry Faility supported by Grant RR-48 from Biotehnology Resoures Branh, Division of Researh Resoures, National Institutes of Health. The osts of publiation of this artile were defrayed in part by the payment of page harges. This artile must therefore be hereby marked advertisement in aordane with 18 U.S.C. Setion 1734 solely to indiate this fat. $ To whom orrespondene should be addressed physiologial funtion of thioltransferase will require knowledge of the primary struture of the protein. In this report, we desribe the amino aid sequene of pig liver thioltransferase. This is the first desription of the sequene of thioltransferase from any soure, and omparison with the sequene of alf thymus glutaredoxin (82% homology) suggests that the two proteins may have idential ellular funtions, although that possibility is not established. EXPERIMENTAL PROCEDURES RESULTS Primary Struture-The amino aid sequene of pig liver thioltransferase together with the positions of peptides used to establish the sequene are shown in Fig. 1. The peptide fragments derived by standard enzymati and hemial leavage were first frationated by reversed-phase HPLC with inreasing aetonitrile in the presene of.1% trifluoroaeti aid. Some peptides were rehromatographed either by reversed-phase HPLC in the presene of25 mm ammonium aetate, ph 7., or by anion-exhange fast protein liquid hromatography. The detailed results of the peptide purifiation and the amino aid ompositions are given in the Miniprint Setion (Figs. 4-7 and Tables 11-V). The amino aid sequene of pig liver thioltransferase is aligned with that of alf thymus glutaredoxin in Fig. 2. The degree of homology between these two proteins is 82%. Trypsin Digestion and Mass Spetrometry-Sine previous work showed that the N terminus of pig liver thioltransferase was bloked (5), the enzyme was diretly subjeted to trypsin digestion. One of the trypti peptides (Tl) was resistant to Edman degradation. When this peptide was examined by fast atom bombardment (2)-mass spetrometry, a protonated moleular ion, (M + H)+ = 96, was observed. The daughter ion spetrum of m/z (Fig. 3) showed fragment ions and the orresponding sequenes (Table I) whih indiated that the N-terminal amino aid of the peptide was aetylated gluta- Portions of this paper (inluding ExperimentalProedures, Figs. 3-7, and Tables I-V) are presented in miniprint at the end of this paper.miniprintiseasilyreadwith the aid of a standard magnifying glass. Full size photoopies are available the from Journal of Biologial Chemistry, 965 Rokville Pike, Bethesda, MD Request Doument No. 86M-4139, ite the authors, and inlude a hek or money order for $5.2 per set of photoopies. Full size photoopies are also inluded in the mirofilm edition of the Journal that is available from Waverly Press. * The abbreviations used are: HPLC, high performane liquid hromatography; SDS, sodium dodeyl sulfate; TLCK, 1-hloro-3-tosyl- amido-7-amino-2-heptanone; TPCK, L-1-tosylamido-2-phenylethyl hloromethyl ketone; PTH, phenylthiohydantoin.

2 67 Primary Struture of Thioltransferase 1 2 A-Gln-Ala-Ala-Phe-Val-Asn-Ser-Lys-Ile-Gln-Pr~ly-Lys-Val-Val-Val-Phe-Ile-Lys-Pro-Thr- I 1-1 "_"_ T1 T2 T3. "_ v1 FIG. 1. The amino aid sequene of pig liver thioltransferase. The peptidesderivedfromleavage of pig liver thioltransferase with trypsin (TI, hymotrypsin (C), S. aureus V8 protease (V), and yanogen bromide (CNBr) are shown. The peptide sequenes obtained by both amino aid omposition and sequene analysis are indiated by ontinuous solid lines, and those proven by only amino aid omposition analysis by dashed lines. A in the N terminus represents an aetyl group. I 3 4 -Cys-Pro-Phe-Cys-~~-Lys-Thr-Gln~lu-L~-Leu-Ser-Gln-L~-Pro-Ph~Lys-Glu-Gly-~u-Leu- -"_ "-, - I """"""""" P 4 5 C6 "-""""""""""""""""- 4 v Glu-Phe-Val-Asp-Ile-Thr-Ala-Thr-Ser-Asp"Asn-Glu-Ile-Gln-Asp-~r-Leu-Gln-Gln-Leu- "_ """""""" """"""""~"~"""""""""""""- v3 CJ T Thr-Gly-Ala-Arg-Thr-Val-Pr~Arg-Val-Phe-Ile-Gly-Lys-Glu-Cys-Ile-Gly-Gly-Cys-Thr-Asp- " T6 R 9 v4 C8 I, v Leu-Glu-ser-llet-H~s-Lys-Ar~-Gly~lu-Leu-~u-~r-~~-Leu~ln-Gln-Ile-Gly-Ala-Leu-Lys FIG. 2. Alignment of amino aid sequenes of pig liver thioltransferase and alf thymus glutaredoxin. Amino aid sequenes of pig liver thioltransferase (T. Tase) and alf thymus glutaredoxin (G. Redoxin) are given in the onventional single letter ode. A represents an aetyl group. The N terminus of alf thymus is a pyroglutamyl residues (19). A deletion is indiated by the dashed line. The residues in the amino aid sequenes whih differ from eah other are boxed exat mass ( measured versus alulated), amino aid analysis, and the fragment ions, the only other alternatives for the N terminus struture are some isomers of C2H4N-Glu. But none of these possibilities make biohemial sense, leaving N-aetylglutamine as the only reasonable hoie. Cyanogen Bromide Treatment-Sine pig liver thioltransferase has 1 methionine, 2 peptides should be obtained from yanogen bromide leavage of the protein. Sequening of the small peptide from the CNBr leavage mixture (Fig. 5) gave 17 amino aid residues of the C-terminal peptide. However, no phenylthiohydantoin derivative was found after 3 yles of Edman degradation of the peptide representing the big peak of the CNBr peptides. SDS-polyarylamide gel eletrophoresis of the CNBr leavage mixture revealed the presene of two bands, one with the same M, as the intat protein and the other with a M, of 1, (data not shown), indiating that inomplete leavage had ourred. Chymotrypsin Digestion-It has been reported that hymotrypsin may leave peptides at the C terminus of a methionine residue (22) in addition to the typial leavage at amino aids with aromati side hains. One suh leavage ourred at Metm of pig liver thioltransferase (Fig. 1). The best overlap information was provided by the sequenes derived from

3 hymotrypti and yanogen bromide leavage. During this study, we needed one more peptide to overlap the two gaps flanking the fragment T6. Sequening one of the hymotrypti peaks with retention time of 28 min (Fig. 6) indiated that it ontained the peptides Thr'j4 to Phe73 and Thrm Phe73. to This result may be aused by the partial leavage of the protein between Arg67 and Thrm by residual trypsin-like ativity in our ommerial TLCK-treated hymotrypsin. We tried to separate these two peptides by Mono Q and Mono S fast protein liquid hromatography, but without suess. V8 Protease Digestion-To establish the unambiguous sequene of pig liver thioltransferase, the protein was digested with Staphyloous aureus V8 protease. Peptide V5 derived from V8 protease leavage ontained the sequene whih overlaps the trypsin peptides T5 to T7 (Fig. 1). However, the yield of the V8 protease digestion was low. The large peak seen at the high aetonitrile onentration region (Fig. 7) had the same retention time as the intat protein, suggesting that inomplete digestion had ourred. Inreasing the inubation time and enzyme/protein ratio did not appreiably improve the peptide yield from the V8 protease digestion. It is not lear why pig liver thioltransferase is resistant to S. aureus V8 protease. An unusual leavage by V8 protease, whih normally prefers peptides adjaent to glutami aid residues, ourred at the peptide bond between Gly65 and Ala66. To onfirm this kind of leavage both peptides V4 and V5 were sequened, and the results agreed well with the reported leavage (Fig. 1). DISCUSSION The high degree of homology between pig liver thioltransferase and alf thymus glutaredoxin supports the speulation that these two polypeptides, although isolated from different speies, are essentially idential. It has been argued that thioltransferase from rat liver (15) and glutaredoxin from alf thymus (23), although similar in moleular weight, show signifiantly different atalyti properties (11). However, these kineti differenes may also be due to speies variation. We have isolated a homogeneous peptide from rat liver ytosol (16), alf liver ytosol (data not shown), and pig liver ytosol (5) whih we labeled thioltransferase in aordane with the studies of Mannervik and his o-workers (3,8, 15), but whih have properties similar to the enzyme originally desribed by Raker (2) and designated g1utathione:homoystine transhydrogenase. Glutaredoxin was originally disovered (24) in a mutant of Esherihia oli laking thioredoxin (25), but with a fully ative NADPH-dependent ribonuleotide redutase funtion. Interestingly, a more onvenient alternative assay for the measurement of glutaredoxin utilizes 2-hydroxyethyl disulfide, GSH, glutathione redutase, and NADPH instead of the ribonuleotide redutase-oupled assay. The former assay and that using S-sulfoysteine as substrate are also ommonly used in the detetion of thioltransferase ativity (15,16). Therefore, to resolve the question of identity, further studies must be onduted that inlude the assay of pig liver thioltransferase for ribonuleotide redutase ativity and a thorough omparison of the kineti properties of both thioltransferase and glutaredoxin isolated from the same speies. Additional evidene an be obtained by immunohemial ross-reativity studies, but it would be surprising if proteins with 82% homology in primary struture did not reat with antibodies raised against one another. The N-terminal amino aid residue of pig liver thioltransferase is N-aetylated glutamine, whereas glutaredoxin from alf thymus has a pyroglutamyl N terminus as determined by pyroglutamyl aminopeptidase and arboxypeptidase digestion Struture Primary of Thioltransferase 671 (19). Aetylation of protein N-terminal amino aid groups has been found among a variety of strutural proteins (26). Aetylation of a glutamine N terminus is even more unusual as the great majority of proteins in this group possess a serine or an alanine at this position. N-Aetylated thioltransferase is also not similar to other aetylated proteins whih typially have harged residues in the N-terminal regions (26). The strutural or funtional signifiane of N-terminal aetylation is unknown. It appears to have no funtion in synthesis or folding of proteins. One possibility is to protet the protein against degradation by aminopeptidases or athepsin C ativ- ity. This speulation is in aord with the fat that a high proportion of aetylated proteins are strutural, in whih stability towards degradation would seem essential. Thus, most viral apsid proteins, all major musle proteins, some histones, and several fibrous proteins are a-amino aetylated. One interesting analogy is found in the ase of ytohrome of mammalian origin (27), sine this protein and thioltransferase have eletron arrier funtions, albeit with quite different hemial groups, and similar moleular weights. Another apparent differene between thioltransferase and glutaredoxin is that pig liver thioltransferase has an extra trypti peptide between positions 67 to 72. This tetrapeptide (-Thr-Val-Pro-Arg-) was not found in alf thymus glutaredoxin (19). Both proteins have 2 ysteine pairs. The ysteine 22 and 25 pair was proposed as the ative enter in alf thymus glutaredoxin (19). Pig liver thioltransferase has a similar sequene around this ysteine pair, exept that the alf thymus glutaredoxin Tyr24 replaed is by a phenylalanine in pig liver thioltransferase. In a ompanion paper (28), we demonstrate that ysteines 22 and 25 of pig liver thioltransferase represent the ative site of the enzyme and that the ysteine 22 thiol has an unusually aidi reativity (pk, = 2.5). In addition, thioltransferase from rat liver ytoplasm has been reported to ontain 8.6% arbohydrate (8), an unusual finding for a ytoplasmi protein. We have also observed the presene of periodi aid-shiff-positive material within the band orresponding to homogeneous rat liver thioltransferase on SDS-polyarylamide gel eletrophoresis (16). On the other hand, whether glutaredoxin from mammalian soures ontains arbohydrate remains to be eluidated. In summary, those investigators working with glutaredoxin have foused their studies primarily on its ability to funtion in the ribonuleotide redutase system, whereas those investigating thioltransferase have onentrated on a more general role in atalyzing ellular thiol-disulfide transhydrogenation reations. Further studies are required to larify the strutural relationships between thioltransferase and glutaredoxin as well as the relative ontribution of eah, if they are not idential, to ellular funtions involving the thiol/disulfide redox status. Aknowledgments-We wish to thank Dr. Young Moo Lee, diretor of the Maromoleular Struture Faility, Department of Biohemistry, and his staff, Doris Bauer and Melanie Markel, for their invaluable assistane in the amino aid omposition and sequene analyses. We are grateful to Dr. John T. Stults for performing the fast atom bombardment-mass spetrometry analysis and interpretation of the trypti N-terminal peptide. REFERENCES 1. Nagai, S., and Blak, S. 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4 672 Struture Primary 6. Larson, K., Eriksson, V., and Mannervik, B. (1985) Methods Enzymol. 113, States, B., and Segal, S. (1973) Biohem. J. 132, Mannervik, B., and Axelsson, K. (198) Biohem. J. 19, Axelsson, K., and Mannervik, B. (1983) FEBS Lett. 152, Mannervik, B., Axelsson, K., Sundewall, A.-C., and Holmgren, A. (1983) Biohem. J. 213, Ziegler, D. (1985) Annu. Reu. Biohem. 54, Hatakeyama, M., Lee, C., Chon, C., Hayashi, M., and Mizoguhi, T. (1985) Biohem. Biophys. Res. Comrnun. 127, Goswami, A., and Rosenberg, I. N. (1985) J. Biol. Chem. 26, Sawada, K., Hummel, B. C. W., and Walfish, P. (1986) Biohern. J. 234, Axelsson, K., Eriksson, S., and Mannervik, B. (1978) Biohernistry 17, Gan, Z., and Wells, W. W. (1986) J. Biol. Chem. 261, Gray, J. H. (1977) Methods Enzymol. 47, of Thioltransferase 18. Cohen, S. A., Bidlingmeyer, B.A., and Tarvin, T. L. (1986) Nature 32, Klintrot, L.-M., Hoog, J.-O., Jornvall, H., Holmgren, A., and Luthman, M. (1984) Eur. J. Biohem. 144, Biemann, K. (1986) Anal. Chem. 58, 1288A-13A 21. Roepstorff, P., and Fohlman, J. (1984) Biorned. Mass Spetrom. 11, Keil, B. (1982) in Methods in Protein Sequene Analysis (Elzinga, M., ed) pp , Humana Press, Clifton, NJ 23. Luthman, M., and Holmgren, A. (1982) J. Biol. Chem. 257, Holmgren, A. (1976) Pro. Natl. Aad. Si. U. S. A. 73, Holmgren, A., Ohlsson, I., and Grankvist, M.-L. (1978) J. Biol. Chem. 253, Jornvall, H. (1975) J. Theor. Biol. 55,l Fasman, G. D. (ed) (1976) in Handbook of Biohemistry and Moleular Biology, pp , CRC Press, Cleveland, OH 28. Gan, Z.-R., and Wells, W. W. (1987) J. Biol. Chem. 261, I.o E.8 9 N.6 75 if.4 5.E - = s f.2 a * Time (mid..

5 Primary Struture of Thioltransferase 673 E s N.6.4 a9 m