Active Site of Tripeptidyl Peptidase II from Human Erythrocytes Is of The

Proc. Natl. Acad. Sci. USA Vol. 84, pp. 7508-7512, November 1987 Biochemistry Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type (serine peptidase/cysteine peptidase/thennitase/thiol dependency) BIRGITTA ToMKINSON*, CHRISTER WERNSTEDTt, ULF HELLMANt, AND ORJAN ZETTERQVIST* *Department of Medical and Physiological Chemistry, University of Uppsala, Biomedical Center, Box 575, S-751 23 Uppsala, Sweden; and tLudwig Institute for Cancer Research, Uppsala Branch, Box 595, S-751 23 Uppsala, Sweden Communicated by Bruce Merrifield, July 20, 1987 ABSTRACT The present report presents evidence that the EXPERIMENTAL PROCEDURES site of amino acid sequence around the serine of the active Materials. [1,3-3H]iPr2P-F was purchased from Amersham human tripeptidyl peptidase II is of the subtilisin type. The International. Emulsifier scintillator 299 came from Packard. enzyme from human erythrocytes was covalently labeled at its Dithiothreitol and bovine trypsin were from Boehringer active site with [3H]diisopropyl fluorophosphate, and the Mannheim, and L-1-tosylamido-2-phenylethyl chloromethyl protein was subsequently reduced, alkylated, and digested with ketone-treated trypsin was from Worthington. Unlabeled trypsin. The labeled tryptic peptides were purified by gel iPr2P-F was obtained from Fluka, and Tween-20 was from filtration and repeated reversed-phase HPLC, and their amino- Atlas Chemie. Guanidinium chloride, acetonitrile (Lich- terminal sequences were determined. Residue 9 contained the rosolv), trifluoroacetic acid (Uvasol), and iodoacetic acid radioactive label and was, therefore, considered to be the active (recrystallized in petroleum ether before use) were bought serine residue. The primary structure of the part of the active from Merck. Sephadex G-50, fine, was purchased from site (residues 1-10) containing this residue was concluded to be Pharmacia. The Nucleosil C18 column (10 ,um; 4 x 250 mm) Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid for HPLC was packed by Skandinaviska GeneTec AB sequence is homologous to the sequence surrounding the active (Kungsbacka, Sweden), and the Bondapak C18/Corasil pack- serine of the microbial peptidases subtilisin and thermitase. ing material was obtained from Millipore-Waters. These data demonstrate that human tripeptidyl peptidase II Preparation of Reduced and ALkylated, 3H-Labeled TPP-II. represents a potentially distinct class of human peptidases and TPP-II was prepared from human erythrocytes as described raise the question of an evolutionary relationship between the (2) with a few modifications. Outdated erythrocyte concen- active site of a mammalian peptidase and that of the subtilisin trate, obtained from the blood bank at the University Hos- family of serine peptidases. pital of Uppsala, was used. The erythrocytes were washed once with 4 vol of 0.9% (wt/vol) NaCl before lysis with 8 vol of 10 mM potassium phosphate, pH 7.5/2 mM EGTA/1 mM The discovery of an extralysosomal tripeptide-releasing ami- dithiothreitol. The centrifugation used for removing erythro- no-peptidase in rat liver has been reported (1). Its substrate cyte membranes was omitted, and the ion-exchanger was specificity, as studied by use ofa series ofpeptide substrates, added directly to the hemolysate after the addition ofglycerol is complex (1, 2). The peptidase releases tripeptides with little (2). The specific activity of the enzyme preparation used was apparent similarity, although the rate of cleavage varies 4000 units/mg, as measured by the standard assay (1). TPP-II considerably. The enzyme, currently named tripeptidyl pep- (1.0 mg, i.e., 7.7 nmol of subunit) was incubated with 0.2 mM tidase II (TPP-II) (2, 3), has a high native molecular weight [3H]iPr2P-F (1.0 Ci/mmol; 1 Ci = 37 GBq) in a total volume (>106) (1), a subunit molecular weight of 135,000, and a of 750 ,lI for 1.5 hr at 23°C to label the serine residue at the neutral pH optimum (2). The presence ofTPP-II in numerous active site of the enzyme. After this treatment, the enzyme tissues of the rat (2) and in erythrocytes and liver of several had <15% of the original activity. Guanidinium chloride (8 other species (4) has been demonstrated. The human eryth- M), Tween-20 [1.6% (vol/vol)], and dithiothreitol (64 mM) rocyte enzyme has been classified as a serine peptidase due were added to the incubation mixture to give the final to its complete inhibition by diisopropyl fluorophosphate concentrations of 6 M, 0.05%, and 2 mM, respectively. The (iPr2P-F) and phenylmethylsulfonyl fluoride (2). However, pH was adjusted to 8.0 with NaOH. Nitrogen gas was passed the enzyme was also inhibited by some of the thiol reagents through the solution, which was then incubated at 37°C. After tested, e.g., N-ethylmaleimide, p-chloromercuribenzoate, 1 hr, a solution of50 mM iodoacetate was added to give a final and Hg2+. TPP-II could be protected against iPr2P-F, concentration of4.4 mM. Nitrogen was again passed through phenylmethylsulfonyl fluoride, or N-ethylmaleimide by the the reaction mixture, and the alkylation was allowed to use of a competitive inhibitor (2), suggesting that not only a proceed in the dark at 37°C for 1 hr. The reaction was serine, but also a cysteine residue is of importance for the terminated by the addition of 2-mercaptoethanol to a final activity of the enzyme. To obtain an unambiguous classifi- concentration of 1%, and the sample was then dialyzed at cation of TPP-II as a serine peptidase, we considered it 23°C for 23 hr against three 2.5-liter changes of 50 mM necessary to isolate and determine the amino acid sequence ammonium bicarbonate, pH 8.3/0.05% Tween-20. The addi- of the part of the protein that contains the active serine tion of Tween-20 was necessary to prevent losses due to residue. Information on the primary structure of the active unspecific adsorption of the labeled protein. site would also be ofinterest for the investigation ofstructural To determine the apparent specific radioactivity of relationships between TPP-II and other peptidases. [3H]iPr2P-F, by a procedure that makes allowance for pos- sible elimination of3H-labeled isopropyl groups (5), 0.1 mg of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: iPr2P-F, diisopropyl fluorophosphate; >PhNCS, in accordance with 18 U.S.C. §1734 solely to indicate this fact. phenylthiohydantoin; TPP-II, tripeptidyl peptidase II. 7508 Downloaded by guest on October 1, 2021 Biochemistry: Tomkinson et al. Proc. Natl. Acad. Sci. USA 84 (1987) 7509 bovine trypsin was labeled, reduced, alkylated, and dialyzed as described above for TPP-II. After the dialysis, the amount of protein was determined by the modified Bradford method (6, 7), using trypsin as the standard. The radioactivity was measured in a Philips PW4700 liquid scintillator counter after mixing the samples with 5 ml of emulsifier scintillator 299. The apparent specific radioactivity of [3H]iPr2P-F was cal- culated to be 26 x 104 cpm/nmol, and this value was used thereafter for determination of the amounts of labeled peptides. 40C Preparation of Tryptic Peptides. After dialysis, 6.5 nmol of 0 reduced and alkylated 3H-labeled TPP-II was incubated with 0co ._ 4._ L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated 0 trypsin for 3 hr at 370C. The molar ratio of trypsin/TPP-II 04) subunit was 1:28. The digestion was stopped by the addition Q aO of 1 M HCl to give a final pH of2. The digest was then loaded onto a Sephadex G-50 column and chromatographed as 0 described in Fig. 1. As a control, the same amount of x L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated E trypsin (0.2 nmol) was dissolved in 50 mM ammonium Q bicarbonate, pH 8.3/0.05% Tween-20 and chromatographed C._ on the same Sephadex G-50 column. Fractions from the two Sephadex G-50 chromatographies were pooled, as indicated 0 by the bar in Fig. 1, and lyophilized. Each lyophilized 0 Sephadex G-50 pool was dissolved in 470 ,u ofthe ammonium ._0 bicarbonate/Tween buffer. The inclusion of Tween-20 in the 0 buffer allowed a good recovery of the labeled peptides, generally 75-100%, as compared to only 10-30o when Tween-20 was excluded. The dissolved material was purified by reversed-phase HPLC on a Nucleosil C18 column as described in Fig. 2. Fractions from three identical preparations were pooled as indicated. After lyophilization, the Elution time (min) pooled material was dissolved in 320 Al of the ammonium FIG. 2. Reversed-phase chromatography of tryptic peptides of bicarbonate/Tween buffer and rechromatographed as de- 3H-labeled TPP-II after Sephadex G-50 chromatography. An aliquot scribed in Fig. 3. Fractions from two identical preparations (150 pl) of the dissolved Sephadex G-50 pool was loaded on a were pooled, as indicated, and lyophilized. Pools 1 and 2 Nucleosil C18 column equilibrated with 0.1% trifluoroacetic acid and were to acid sequence analysis as described 6% (vol/vol) acetonitrile, pH 2.1, and eluted with a linear acetonitrile subjected amino gradient as indicated. The trifluoroacetic acid concentration was kept below. Pool 3 was dissolved in 160 Al of the ammonium at 0.1% throughout the gradient. The flow rate was 1.0 ml/min, and fractions of 0.5 ml were collected. Radioactivity was determined in 5 ,l ofeach fraction as described. The vertical bars correspond to an absorbance of0.02. The horizontal bar indicates the fractions pooled c for further purification. A, control; B, tryptic peptides of 3H-labeled 0 25 a b c TPP-II; C, enlargement from B, showing all radioactivity above 0~~~~~~ background level (histogram).

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