Purification, Substrate Specificity, and Classification of Tripeptidyl ...

THEJOURNAL OF

Vol.261, No. 5, Issue of February 15, pp. 2409-2417 1986 Printed in Ij.S.A.

BIOLOGICAL CHEMtSTRY

0 1986 by The American Society of Biological Chemists, Inc.

Purification, Substrate Specificity, and Classification of Tripeptidyl Peptidase 11” (Received for publication, April 22, 1985)

Ros-Mari Bhlow, Birgitta Tomkinson, Ulf Ragnarssong, and Orjan Zetterqvist From the Departmentof Medical andPhysiological Chemistry and the $Department of Biochemistry, Universityof Uppsala, Biomedical Center,S-751 23 Uppsala, Sweden

An extralysosomal tripeptide-releasing aminopepti- group had been described when Doebber et al. (2) identified dase was recently discovered in rat liver (Bidow, R.- tripeptidyl peptidase activity in the lysosomal fraction of M., Ragnarsson, U., and Zetterqvist,0. (1983)J. Biol. bovine pituitary glands with an acidic pH optimum and M, Chem. 258, 11622-11628). In the present work this 57,000 for the native protein. Recently, McDonald et al. (3) tripeptidyl peptidase is shown to occur in several rat reported the presence of a similar peptidase, in ovary lysotissues and in human erythrocytes. The erythrocyte somes from pregnant pigs, with an acidic pH optimum and enzyme was purified about 80,000-fold from a hemo- Mr 55,000. These authors have suggested (3) that thelysosolysate while the rat liver enzyme was purified about mal tripeptide-releasing aminopeptidases be tentatively 4,000-fold from a homogenate. Upon polyacrylamide named tripeptidyl peptidase I by analogy with the recomgel electrophoresis in sodium dodecyl sulfate under mended name “dipeptidyl peptidase,” used by the IUB Noreducing conditions more than 90%of the protein was menclature Committee for the dipeptide-releasing aminopeprepresented by a polypeptide of M, 135,000 in both tidases (4).The systematic name would, by analogy, be tricases. In addition, the two enzymes eluted at similar positions in the various chromatographic steps, showed peptidylpeptide hydrolase. McDonald et al. (3) also suggested a pH optimum around that theextralysosomal tripeptidyl aminopeptidase identified similar specific activity, and had by our group (1) be named tripeptidyl peptidase 11. Accord7.5. A tryptic pentadecapeptide from the a-chain of hu- ingly, we will use this nomenclature in what follows. man hemoglobin, Val-Gly-Ala-His-Ala-Gly-Glu-Tyr- In order to make possible the further characterization of Gly-Ala-Glu-Ala-Leu-Glu-Arg, i.e. residues 17-31, tripeptidyl peptidase I1 an extensive purification was atwas found to be sequentially cleaved by the erythrocyte tempted. Rat liver was selected as the source of the enzyme enzyme into five tripeptides, beginning from theNH, since the activity is high, and previous studies of tripeptidyl terminus. Chromogenic tripeptidylamides showed var- peptidase I1 were performed on a partially purified rat liver ious rates of hydrolysis at pH 7.5. With Ala-Ala-Phe- enzyme. However, since human erythrocytes were found to 4-methyl-7-coumarylarnide,K , was 16 HM and V,, 13 contain significant amounts of the enzyme and were available pmol min-l*mg”, comparable to the standard substrate in large amounts and due to the potential interest of having Arg-Arg-Ala-Ser(32P)-Val-Ala values ( K , 13 PM and access to a human tripeptidylpeptidase, the enzyme was also V,, 24 pmol*min-’=mgL1). purified from this source. The two enzyme preparations are The tripeptidyl peptidase of human erythrocytes was compared. The human erythrocyte enzyme was further charclassified as a serine peptidase from its irreversible acterized with respect to substrate specificity and catalytic inhibition by phenylmethanesulfonyl fluoride and dl- type. isopropyl fluorophosphate. The rate of inhibition was decreased by the presence of an efficient competitive EXPERIMENTAL PROCEDURES~ (Ki 1.5 inhibitor, Val-Leu-Arg-Arg-Ala-Ser-Val-Ala PM). [3H]Diisopropylphosphatewas incorporated to the RESULTS AND DISCUSSION extent of 0.7-0.9 mol/mol of M, 135,000 subunit, Tissue Distribution of Rat Tripeptidyl Peptidase II-Triwhich confirms the high purity of the enzyme. peptidyl peptidase I1 was initially detected in the extralysosoma1 fraction of rat liver (1).From Table 111 it is evident that theenzyme, as measured by the standardassay, is present In a recentwork the existence of an extralysosomal tripep- in extracts of a number of rat tissues. The measured activity tidy1 peptidase with a neutral p H optimum and of high mo- was essentially due to tripeptidyl peptidase I1 for the following lecular weight was reported (1).The enzyme was detected in reasons. First, the enzyme activity was measured in thepostrat liver by use of 32P-labeledphosphopeptides representing lysosomal fraction, i.e. 40,000 X g X 25 min supernatants. the phosphorylated site of rat liver pyruvate kinase. Previ- Second, the enzyme activity was measured at pH 6.5 at which ously, only one enzyme with the ability to release tripeptides pH possible interference from lysosomal tripeptidyl peptidase, from the NH, terminus of a substrate with a free a-amino *This workwas supported by the Swedish Medical Research Council (Project 13X-04485), the Swedish Natural Science Research Council (Project K3020), Magnus Bergvalls Stiftelse, 0. E. och Edla Johanssons vetenskapliga stiftelse, and the Medical Faculty, University of Uppsala. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Portions of this work (including “Experimental Procedures,” Figs. 1 and 2, and Tables I and 11) are presented in miniprint at theend of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85M-1337, cite the authors, and include a check or money order for $4.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

2409

Peptidase

2410

Tripeptidyl

11

TABLE I11 Tripeptidyl peptidase activityin the postlysosomal fractionof different tissues from rat Postlysosomal fractions of the different tissues and hemolysate were prepared as described under “Experimental Procedures.” Tripeptidyl peptidase activity was measured by the standard assay. Activity values refer to 1 g of wet tissue. Tissue

n

Enzyme activity &

S.D.

units

Liver Heart Kidney Spleen Skeletal muscle Blood cells

5 5 5 5 4 3

163 1.53 45 f 18 94 k 26 52 f 11 50 C 23 52f 7

that may have leaked out during the preparation procedure, is small (2, 3). Third, in addition to measurements of the enzyme activity with the standard assay, the electrophoretic method (1) was used. The latter discriminates between the phosphopeptides Ala-Ser(P)-Val-Ala andSer(P)-Val-Ala, both of which may be formed from the standard substrate Arg-Arg-Ala-Ser(P)-Val-Ala by crude tissueextractsand bound to theQAE-Sephadex of the standardassay (I).By the electrophoretic method it was found that at least 90% of the peptide-cleaving activity listed in Table I11 was contributed by a peptidase cleaving the Ala-Ser(P) bond. Tripeptidyl Peptidase ZI in Human Blood Cells-Since tripeptidyl peptidase 11 activity was present in rat blood cells and human tripeptidyl peptidase I1 was considered to be of particular interest, the presence of the enzyme in human blood cells was also investigated. The peptidase activity, measured by the standard assay, was found to be about 50 units/ml of packed cells from freshly drawn human blood. The corresponding value for blood used in the heart-lung machine was 10-20 units. The distribution of tripeptidyl peptidase I1 between different blood cell species was studied with the aid of cell separation on Percoll density gradients as described under “Experimental Procedures.” It is apparent from Fig. 3 that essentially all of the tripeptidyl peptidase activity in human blood is derived from erythrocytes. These data suggested that human erythrocytes are a suitable source for preparation of tripeptidyl peptidase 11. Purification and Comparison of Tripeptidyl Peptidase 11 from HumanErythrocytes and Rat Liver-Tripeptidyl peptidase I1 from human erythrocytes and rat liver was prepared as described under “Experimental Procedures.” The recovery of the erythrocyte enzyme was about E%,which allows the conclusion that the purified enzyme represents a main fraction of the tripeptidyl peptidase of the original hemolysate. Whether the lower, i.e. 2%, yield of the rat liver enzyme reflects a greater initial heterogeneity or a greater instability of the liver enzyme remains to be elucidated. At the time of the purification of the ratliver enzyme no protease inhibitors were included in the buffers since the catalytic type of the enzyme was unknown. However, since hemolysis is known to activate a soluble calcium-dependent endopeptidase (19) and pilot experiments showed that EGTA did not inhibit tripeptidy1 peptidase I1 of human erythrocytes, this compound was included in the hemolysate. Although the activity of the enzyme/g of tissue, wet weight, was an order of magnitude lower in human erythrocytes than in rat liver, the procedure for purification of the erythrocyte enzyme gained from the nearly 10-fold higher recovery and the larger amounts of starting material. The human erythrocyteenzyme shows identity with the extralysosomal rat liver enzyme in several re-

io

20 Volume (ml)

30

FIG. 3. Tripeptidyl peptidase I1 activity in human blood cells separated in a discontinuous gradient of Percoll. Human blood (2 ml) diluted with 2 ml of 0.9% NaCl was layered on the top of a gradient consisting of three layers of 55, 71, and 74% Percoll, respectively, and centrifuged at 2200 X g for 15 min. White and red blood cells were counted, and the activity of tripeptidyl peptidase II was measured as described under “Experimental Procedures.” The data refer to 1ml of gradient and represent the mean of three different experiments.

spects, e.g. elution positions on DEAE-cellulose, Sepharose CL-4B, and hydroxylapatite chromatography. The active material pooled from the last chromatographic step of the purification of both human erythrocyte and rat liver tripeptidyl peptidase displayed one dominating polypeptide with M, 135,000 upon polyacrylamide gel electrophoresis in SDS’ under reducing conditions (Fig. 4, lanes C and D). The two enzymes were similar also with respect to specific activity. In addition, an optimal activity was obtained around pH 7.5 for both enzymes (not shown). The similarities listed are sufficient to ascribe the humanerythrocyte and rat liver enzymes to the same type of tripeptidyl peptidase, i.e. tripeptidyl peptidase 11, which is distinctly different from the lysosomal tripeptidyl peptidase (tripeptidyl peptidase I) described by others (2, 3). For this reason and due to the relative ease by which it can be prepared, the human erythrocyte enzyme is considered to be a suitablesubject for further characterization of tripeptidyl peptidase 11. Cleavage of a Tryptic Peptide of Human Hemoglobin-In a previous work (I) on rat liver tripeptidyl peptidase 11, the substrate specificity was assayed by use of various 32P-labeled phosphopeptides, representing the phosphorylated site of rat liver pyruvate kinase. An investigation of whether tripeptidyl peptidase I1 of human erythrocytes is active toward additional peptides is described in this and the following paragraph. The tryptic fragment from thea-chain of human hemoglobin

Val17-Gly18-Ala’9-His20-Ala21-Gly22-Gl~23-Ty~4-Gly25-A G l ~ ~ ~ - A l a ~ ~ - L e ~ was ~ ~ -isolated G l u ~as~described - A r ~ under “Experimental Procedures” and found to be sequentially degraded by tripeptidyl peptidase I1 (Table IV). This is consistThe abbreviations used are: SDS, sodium dodecyl sulfate; BSA, bovine serum albumin; DAN, diazoacetyl-norleucine methyl ester; DFP, diisopropyl fluorophosphate; DTT, dithiothreitol; E-64, Ltrans-epoxysuccinyl-leucyl-amido(4-guanidino)-butane; HPLC, high performance liquid chromatography; -MCA, 4-methyl-7-coumarylamide; -P-NA, -@-naphthylamide;NEM, N-ethylmaleimide; p C M R , p chloromercuribenzoate; PGD, potassium phosphate (pH 7.5) at the indicated phosphate concentration, 30% (w/v) glycerol, and 1 mM DTT; PMSF, phenylmethanesulfonyl fluoride; TPP, tripeptidyl peptidase; EGTA, [ethylenebis(oxyethelenenitrilo)]tetraacetic acid.

Peptidase Tripeptidyl - - -. - , - -Origin

” ”

.. - .

”

-.

.

---

200 000 170 000

116 250

97400 66 200

-*

I

-55400 45 000

36 500

-

-

A

B

C

-Front

-

“

D

E

11

241 1

TABLE V Cleavage of chromogenic tripeptidyl substrates by tripeptidyl peptidase I I Incubations were performed as described under“Experimental Procedures.” The experiments were performed twice, with duplicates. Two preparations of the human erythrocyte enzyme were used, the rate of cleavage of Ala-Ala-Phe-MCA being 7.4 pmol .min” .mg” and 7.1 pmol .min”.mg”, respectively. Mean and S.D. were calculated from single rate values normalized to the mean of the rate with AlaAla-Phe-MCA. Chromogenic substrate Relative rate k S.D. His-Leu-His-(3-NA Phe-Pro-Ala-p-NA Pro-Lys-Ala-8-NA Val-Tyr-Ser-8-NA Ala-Ala-Phe-P-NA Ala-Ala-Phe-MCA

0.12 & 0.02 0.06 i 0.01

i-."

X

z

SIC j 1

:

I -

a 3 -

Inhibitor

2 -

1 -

A

-

I

0.1

0.2

FIG. 5. Lineweaver-Burk diagram showingthe cleavage of the phosphohexapeptide Arg-Arg-Ala-Ser(P)-Val-Alai n the presence of the octapeptide Val-Leu-Arg-Arg-Ala-Ser-ValAla or t h e pentapeptide Arg-Ala-Ser-Val-Ala. The enzyme activity was measured at pH 7.5, as described under "Experimental Procedures." A: 0, without inhibitor; 0, 1 p M octapeptide; A, 2 p~ octapeptide; W, 4 p~ octapeptide. B: 0, without inhibitor; 0, 0.5 IM pentapeptide; A, 1p~ pentapeptide; 2 p~ pentapeptide. Single net values of reaction rate were fitted to the Lineweaver-Burk double reciprocal plot by the least squaresmethod. Inset shows relative slope uersw concentration of inhibitor, using the method for determination of K; described by Cleland (30). The experiment was repeated twice using different enzyme preparations, with similar results. Mean and S.D. of K; values are given in the text.

.,

was about 1 mM which should be compared to a Ki value of about 0.02 mM for Arg-Arg-Ala, a product of the rapidly cleaved standard substrate (data not shown). The pentapeptide Arg-Ala-Ser-Val-Alawas as efficient a competitive inhibitor as the octapeptide (Table VI and Fig. 5B), with an apparent Kiof 0.9 & 0.1 p ~ These . data suggest that theoctaand pentapeptide will offer a suitable basis for the design of

None DFP PMSF Soybean trypsin inhibitor pCMB Iodoacetate Iodoacetamide NEM Hg2+ 2,2'-Dithiodipyridine E-64 Pepstatin Cu2++ DAN cu2+ DAN DTT + EDTA EDTA 1,lO-Phenanthroline

Concentration

0.2 mM 0.2 mM 0.1 mg/ml 0.1 mM 1mM 1 mM 1 mM 0.01 mM 0.1 mM 0.1 mM 1 Kdml 1mM each 1 mM 1 mM 2 r n M each 2 mM 1mM

rate

f S.D.

1.0 -c 0.0 €0.1