[16] Dipeptidyl-peptidase IV from rat liver

[16] Dipeptidyl-peptidase IV from rat liver

[16] DIPEPTIDYL-PEPTIDASE IV 215 EcoRV fragment of pPROII-12 as a probe, only one clone 19H3 (phage 336) hybridized with the probe. Comparison of t...

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[16]

DIPEPTIDYL-PEPTIDASE IV

215

EcoRV fragment of pPROII-12 as a probe, only one clone 19H3 (phage 336) hybridized with the probe. Comparison of the ptrB sequence with the restriction map of Kohara eta/. 27'28 placed the gene at 1981 to 1984 kbp on the E. coli physical map (Fig. 4). The location of the ptrB gene on the EcoRV-EcoRV fragment of 19H3 was also confirmed by the nucleotide sequencing.

[16] D i p e p t i d y l - p e p t i d a s e IV f r o m R a t L i v e r

By Y u m o IKEHARA, SHIGENORI OGATA, and YOSHIO MISUM! Dipeptidyl-peptidase IV, or dipeptidyl aminopeptidase IV (DPPIV) (EC 3.4.14.5), is a serine protease that cleaves N-terminal dipeptides from oligo- and polypeptides with a penultimate prolyl residue. ~-3 DPPIV is a membrane-bound glycoprotein localized on the cell surface (ectoenzyme), in contrast to other DPPs localized in the lysosome (DPPI and DPPII) and in the cytoplasm (DPPIII). In polarized epithelial cells such as those in the liver, small intestine, and kidney proximal tubules, DPPIV is localized in the apical domain of the plasma membrane/-6 Although widely distributed in a variety of tissues, the enzyme has been purified to homogeneity from kidney, 7'8 small intestine, 9 submaxillary gland, ~° and liver.ll'12 The purified enzyme is found to be a dimer, comprising two identical subunits of 110-130 kDa that are variable depending on species and tissues, possibly due to the extent of glycosylation. t V. Hopsu-Havu and G. G. Glenner, Histochemie 7, 197 (1966). 2 j. K. McDonald and C. Schwabe, in "Proteinases in Mammalian Cells and Tissues" (A. J. Barrett, ed.), p. 371. North-Holland Publ., Amsterdam 1976. 3 A. J. Kenny, in "Proteinases in Mammalian Cells and Tissues" (A. J. Barrett, ed.), la. 417. North-Holland Publ., Amsterdam, 1976. 4 K. M. Fukasawa, K. Fukasawa, N, Sahara, M. Harada, Y. Kondo, and I. Nagatsu, J. Histochem. Cytochem. 29, 337 (1981). 5 j. R. Bartles, L. T. Braiterman, and A. Hubbard, J. Biol. Chem. 260, 12792 (1986). 6 S. Hartel, R. Grossrau, C. Hanski, and W. Reutter, Histochem, J. 89, 151 (1988). 7 A. J. Kenny, A. G. Booth, S. G. George, J. Ingrain, D. Kershaw, E. J. Wood, and A. R. Young, Biochem. J. 157, 169 (1976). 8 T. Yoshimoto and R. Walter, Biochim. Biophys. Acta 485, 391 (1977). 9 B. Svensson, M. Danielsen, M. Staun, L. Jeppesen, O. Noren, and H. Sj6str6m, Eur. J. Biochem. 90, 489 (1978). 10 K. Kojima, T. Hama, T. Kato, and T. Nagatsu, J. Chromatogr. 189, 233 (1980). 11 j. Elovson, J. Biol. Chem. 255, 5807 (1980). t2 K. M. Fukasawa, K. Fukasawa, B. Y. Hiraoka, and M. Harada, Biochirn. Biophys. Acta 657, 179 (1981).

METHODS IN ENZYMOLOGY, VOL. 244

Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.

216

SERINEPEPTIDASES

[16]

Assay Methods

Direct Photometric Method Principle. The release ofp-nitroaniline from dipeptidyl-p-nitroanilides is photometrically determined at 385 nm after the indicated incubation period. ~3Gly-Pro-p-nitroanilide tosylate is used as a routine substrate, but any Xaa-Pro-p-nitroanilide can be used for the assay. This method is useful for following the progress of purification and for laboratories without a recording spectrophotometer or fluorometer. Reagents Glycine-NaOH buffer: 0.3 M, pH 8.7. Substrate: 3 mM Gly-Pro-p-nitroanilide. Gly-Pro-p-nitroanilide tosylate (Peptide Institute, Inc., Osaka, Japan) is dissolved in 2% (v/v) Triton X-100 (1.4 mg of the substrate/ml), stored at 4° (not frozen), and should be used within 4 days. Standard p-nitroaniline solution: 0.3 mM in 2% (v/v) methanol. Acetate buffer: 1.0 M, pH 4.2. Procedure. A reaction mixture (1.0 ml) contains 0.25 ml of 0.3 M glycine-NaOH buffer (pH 8.7), 0.5 ml of the substrate (1.5/zmol), 0.2 ml of H20, and 0.05 ml of an appropriately diluted enzyme solution. A blank tube contains the same assay mixture without the enzyme solution. The mixtures are incubated at 37° for 30 min. The reaction is stopped by adding 3.0 ml of 1 M acetate buffer (pH 4.2). The mixtures are centrifuged at 3000 g for 5 min at room temperature, if necessary. The absorbance of the sample is read at 385 nm and corrected by subtraction of the blank. The quantity ofp-nitroaniline released is determined from the absorbance of the sample relative to that of a standard solution prepared by substituting 0.3 mMp-nitroaniline (0.15 ~mol) for substrate in the assay mixture. One unit of activity is defined as the amount of enzyme that produces 1/zmol ofp-nitroaniline per minute. The specific activity is expressed in units per milligram of protein.

Colorimetric Method Principle. Diazotization of p-nitroaniline formed and coupling with N-(1-naphthyl)ethylenediamine develop a color that is measured at 548 nm.13 This procedure is about 10-fold more sensitive than the direct photometric method described above. 13T. Nagatsu, M. Hino, H. Fuyamada, T. Hayakawa, S. Sakakibara, Y. Nakagawa, and T. Takemoto, Anal. Biochem. 74, 466 (1976).

[16]

DIPEPTIDYL-PEPTIDASEtV

217

Reagents Glycine-NaOH buffer: 0.3 M, pH 8.7. Substrate: 3 mM Gly-Pro-p-nitroanilide tosylate. Standard p-nitroaniline solution: 0.15 mM in 2% methanol. 5% (v/v) Perchloric acid. 0.2% (w/v) Sodium nitrite. 0.5% (w/v) Ammonium sulfamate. 0.05% (v/v) N-(l-Naphthyl)ethylendiamine in 95% (v/v) ethanol. Procedure. A reaction mixture (0.2 ml) contains 50/~1 of 0.3 M glycine-NaOH buffer (pH 8.7), 100/zl of 3 mM Gly-Pro-p-nitroanilide, 30 /~1 of H20, and 20/zl of an enzyme solution. A blank tube contains the same assay mixture except for the enzyme solution. The sample and blank tubes are incubated at 37° for 30 min, and the reaction is stopped by adding 0.8 ml of 5% perchloric acid. The same volume (20/~1) of the enzyme solution is added to the blank tube. The mixtures are centrifuged at 3000 g for 10 min, and 0.5 ml of each supernatant is removed. A standard solution, which contains I00/xl of 0.15 mM p-nitroaniline (15 nmol), 50 ~1 of the buffer, and 50/zl of H20, is treated as above. To all the tubes, 0.5 ml of 0.2% sodium nitrite is added, and the tubes are kept at 4 ° for 10 min. Freshly prepared 0.5% ammonium sulfamate (0.5 ml) is then added. After 1.0 ml of 0.05% N-(1-naphthyl)ethylenediamine in 95% ethanol is added, all the tubes are incubated at 37° for 30 min in the dark. The absorbance of the sample is read at 548 nm after that of the blank is adjusted to 0. The quantity ofp-nitroaniline enzymatically formed is calculated from the absorbance of the sample relative to that of the standard p-nitroaniline solution (15 nmol). In this case the value thus obtained should be multiplied by a factor of 2, because one-half of the reaction mixture has been used for color development. Other Methods Spectrophotometric Method using Gly-Pro-p-Nitroanilide as Substrate. The assay mixture (3.0 ml) contains 0.75 ml of 0.3 M glycine-NaOH (pH 8.7), 1.5 ml of 3 mM Gly-Pro-p-nitroanilide, and 0.6 ml of H20. ~3The temperature of the assay mixture is equilibrated to 37° , and the reaction is started by adding 0.15 ml of enzyme. The blank mixture is prepared by substituting the enzyme solution with H20. The reaction mixtures in the cuvette with a 1-cm light path are maintained at 37°. The absorbance of the sample is read at 385 nm by an automatic recording spectrophotometer that has been zeroed against the blank. The absorbance of a standard solution containing 0.45 ~mol ofp-nitroaniline is also recorded for calculation of the quantity of the substrate hydrolyzed.

218

SERINE PEPTIDASES

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Fluorometric Method Using Gly-Pro-2-Naphthylamide as Substrate. Free 2-naphthylamine is intensely fluorescent whereas dipeptidyl-2-naphthylamides are only slightly fluorescent.TM This fact permits continuous monitoring of the progress of hydrolysis by means of a recording fluorometer or spectrofluorometer. The assay mixture contains 1.0 ml of 0.3 M Tris-HCl (pH 8.0), 1.0 ml of 6.0 mM Gly-Pro-2-naphthylamide, and 0.8 ml of H20. After the assay mixture is warmed to 37°, the reaction is started by adding 0.2 ml of enzyme, followed by recording the rate of change in fluorescence at 410 nm relative to the 2-naphthylamine standard (0.01 raM). The excitation wavelength is 340 nm. Purification Procedure Two forms of DPPIV, papain-cleaved soluble form and Triton X-100solubilized membrane form, are purified from plasma membranes of rat liver. Plasma membranes are prepared by the method of Ray ~5with a slight modification. 16The specific activity of DPPIV in the isolated membranes is increased by about 25-fold as compared with that of homogenates. The plasma membranes are stored at -20 ° until use.

Purification of the Soluble Form Unless otherwise indicated, the following procedures are conducted at a b o u t 4°. z7

Step 1. Papain Treatment. Plasma membranes (506 mg) are suspended in 30 ml of 20 mM Tris-HCl (pH 7.5) containing 5 mM L-cysteine. Papain (31.3 U/mg protein, type III from Sigma, St. Louis, MO) is added to the membrane suspension (final concentration of papain, 1 mg/ml), and the mixture is stirred at 37° for 3 hr. The mixture, to which MgCI2 is added (to 1 mM) to inhibit papain activity, is then centrifuged at 105,000 g for 1 hr. About 90% of total DPPIV activity in the membranes is recovered in the supernatant. Step 2. Ammonium Sulfate Precipitation. Saturated ammonium sulfate solution is added dropwise to the supernatant with continuous mixing to 60% saturation, followed by centrifugation of the mixture at 15,000 g for 20 min. The supernatant is then adjusted to 90% saturation of ammonium sulfate, and the mixture is again centrifuged as above. The precipitates 14R. D. C. Macnair and A. J. Kenny, Biochem. J. 179, 379 (1979). 15T. K. Ray, Biochim. Biophys. Acta 196, 1 (1970). 16 y. Ikehara, K. Takahashi, K. Mansho, S. Eto, and K. Kato, Biochim. Biophys. Acta 4/0, 202 (1977). 17 S. Ogata, Y. Misumi, and Y. Ikehara, J. Biol. Chem. 264, 3596 (1989).

[16]

DIPEPTIDYL-PEPTIDASEIV

219

thus obtained with ammonium sulfate between 60 and 90% saturation are dissolved in 3 ml of 20 mM Tris-HCl (pH 7.5) containing 0.2 M NaCI and dialyzed overnight against 2 liters of the same solution. Step 3. Gel Filtration. The sample is subjected to gel filtration through a Sephacryl S-300 column (2.5 x 100 cm, Pharmacia, Piscataway, N J) equilibrated with 20 mM Tris-HCl (pH 7.5)/0.2 M NaCI. The column is eluted with the same buffer at a flow rate of 6 ml/hr, and 2-ml fractions are collected. DPPIV appears as a single peak at an elution position with Mr 220,000. Four fractions with DPPIV activity are combined and the sample is adjusted to contain 0.5 M NaC1 in the above buffer.

Step 4. Wheat-Germ Agglutinin (WGA)-Sepharose Chromatography. The sample obtained by gel filtration is applied onto a WGA-Sepharose column (2 × 15 cm, Pharmacia) equilibrated with 20 mM Tris-HC1 (pH 7.5)/0.5 M NaCI. The column is washed with 200 ml of the same solution, and adsorbed proteins including DPPIV are eluted with 0.2 M N-acetylglucosamine (GlcNAc) in the same buffer (Fig. 1). Fractions with DPPIV activity are combined and concentrated to about 1 ml in an ultrafiltration cell with an XM50 membrane (Amicon, Derivers, MA).

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Fraction Number (5 ml/tube) FIG. 1. Chromatography of the soluble form of DPPIV on a WGA-Sepharose column. An enzyme fraction obtained by Sephacryl S-300 chromatography was subjected to chromatography through a WGA-Sepharose column (2 × 15 cm) that had been equilibrated with 20 mM Tris-HC1 (pH 7.5) containing 0.5 M NaCI. After the column was washed with 200 ml of the same solution, adsorbed proteins including DPPIV were e!uted with 0.2 M GIcNAc in the same solution (indicated by an arrow). Protein concentration was measured by absorbance at 280 nm (----) and DPPIV activity was determined with Gly-Pro-p-nitroanilide as substrate (----e--).

220

SERINEPEPTIDASES

[16]

Step 5. Polyacrylamide Gel Electrophoresis. Aliquots (70/zl/gel) of the sample are subjected to electrophoresis on disc gels (7.5% acrylamide, 1 x 13 cm) at pH 8.6 according to Davis. 18Immediately after electrophoresis, the gels are stained for DPPIV activity at 37° for 5 min in 0.2 M TrisHCI (pH 7.8) containing 0.5 mM Gly-Pro-2-naphthylamide and Fast Garnet GBC (1.25 mg/ml).8 Stained areas of the gels are cut out. Segments obtained from 15 gels are packed into a 10-cm syringe and squeezed through a 21-gauge needle. The gel homogenates in 20 ml of 20 mM Tris-HC1 (pH 7.5) are stirred for 2-3 hr and then centrifuged at 30,000 g for 30 min. The supernatant is concentrated to I ml in the ultra_filtration cell as above. The enzyme thus purified is found to be a single protein with 103 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. 17

Purification of the Membrane Form Step 1. Extraction with Triton X-IO0. Plasma membranes (903 rag) are suspended in 60 ml of 20 mM Tris-HCl (pH 7.5) and adjusted to contain 0.5% Triton X-100. The mixture is stirred for 30 min and then centrifuged at 105,000 g for 1 hr. Of DPPIV activity in the mixture, 97% is recovered in the supernatant. ~7 Step 2. Chromatography on an Affi-Gel Blue Column. The sample is adjusted to contain 0.2 M NaCI in the solubilizing buffer and applied to an Affi-Gel Blue column (3 x 30 cm, Bio-Rad, Richmond, CA) which has been equilibrated with 20 mM Tris-HCl (pH 7.5)/0.2 M NaCI/0.5% Triton X-100. The column is subjected to stepwise elutions with 0.2 M NaCI (600 ml), 0.6 M NaC1 (700 ml), 1.0 M NaCI (900 ml), and 1.5 M NaCI (600 ml) in the above buffer. About 85% of the enzyme activity applied is eluted with 1.0 M NaC1, as shown in Fig. 2. Fractions 152 to 190 of the activity peak are pooled and used for the next step. Step 3. Chromatography on WGA-Sepharose Column. The sample is directly applied to the WGA-Sepharose column (2 × 15 cm) equilibrated with 20 mM Tris-HCl (pH 7.5)/0.5 M NaCI/0.5% Triton X-100. The column is washed with 200 ml of the equilibrating buffer and then with 200 ml of 20 mM Tris-HCl/0. I M NaC1/0.1% Triton X-100. DPPIV adsorbed to the column is eluted with 0.2 M GlcNAc in the latter buffer. Fractions of the active peak are pooled and concentrated over the XM50 membrane in the ultrafiltration cell. In this case, concentrating the protein is accompanied by an increase in concentration of Triton X-100, which is, however, 18 B. J. Davis, Ann. N . Y . Acad. Sci. 121, 404 (1964).

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Fraction Number (lO rnl/tube) FtG. 2. Chromatography of the membrane form of DPPIV on an Affi-Gel Blue column. An enzyme fraction obtained by extraction of plasma membranes with 0.5% Triton X-100 was applied to an Affi-Gel Blue column (3 x 30 cm) that had been equilibrated with 20 mM Tris-HCl (pH 7,5) containing 0.2 M NaCI and 0.1% Triton X-100. Elution was carded out by stepwise increase of NaC1 (0.2, 0.6, and 1.0 M) in the same buffer containing 0.1% Triton X-100. Protein concentration, ----; DPPIV activity, ----0--.

considerably lowered by repeating a dilution of the condensed sample with 20 mM Tris-HC1 (pH 7.5). The sample is finally concentrated to 1.5 ml. Step 4. Polyacrylamide Gel Electrophoresis. Polyacrylamide gel electrophoresis and extraction of DPPIV from gels are carried out in the presence of 0.1% Triton X-100. All the other conditions are the same as those described for the soluble DPPIV. The purified membrane form, when analyzed by SDS-polyacrylamide gel electrophoresis, is found to be a single protein of 109 kDa, slightly larger than the soluble form. J7 The purification procedures of the soluble and membrane forms of DPPIV are summarized in Table I. Properties

Stability DPPIV is a relatively stable enzyme in most respects. 19 The enzyme is known to survive autolysis for up to 24 hr at pH 4 and 37°, with a recovery of more than 50% of the activity .7 The purified enzyme, however, t9 T. Yoshimoto, M. Fischl, R. C. Orlowski, and R. Walter, J. Biol. Chem. 253, 3708 (1978).

222

SERINE PEPTIDASES

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TABLE I PURIFICATION OF SOLUBLE AND MEMBRANEFORMS OF RAT LIVER DPPIV

Step Soluble form Plasma membranes a Papain solubilized Ammonium sulfate Sephacryl S-300 WGA-Sepharose Electrophoresis Membrane form Plasma membranes b Triton X-100 extract Afli-Gel Blue WGA-Sepharose Electrophoresis

Total protein (rag)

Total activity (units)

Specific activity (units/rag)

Purification factor

Yield (%)

506 184 33.8 5.6 2.4 1.5

170.0 156.2 144.7 120.6 112.0 99.4

0.34 0.85 4.28 21.5 46.7 66.3

1 2.5 12.6 63.2 137.4 195.0

100 91.9 85.1 70.9 65.9 58.5

903 275 43.6 6.9 2.6

343 333 254.8 242.2 160.2

0.38 1.21 5.84 35.1 61.6

1 3.2 15.4 92.4 162.1

100 97.1 74.3 70.6 46.7

a Prepared from 60 rat livers (wet weight, 780 g). b Prepared from 110 rat livers (wet weight, 1430 g).

becomes unstable below pH 5.0, but remains stable in neutral and alkaline solutions. No significant loss of the activity is caused by incubation at 45 ° and pH 7.5 for 30 min. It was reported that the purified enzyme was remarkably stable in 8 M urea, but was inactivated by a 4-hr exposure to 12 M urea, 2°

Activators DPPIV requires neither metals nor any other cofactors for its activity. 2

Inhibitors The enzyme is very sensitive to diisopropyl fluorophosphate (DFP) but much less sensitive to other serine enzyme inhibitors such as phenylmethylsulfonyl fluoride (PMSF) and diethyl 4-nitrophenylphosphate (E600). 7 Most of the usual sulfhydryl reagents have little or no effect, nor do chelating agents. 20 A. Barth, H. Schulz, and K. Neubert, Acta Biol. Med. Ger. 32, 157 (1974).

[16]

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TABLE II SUBSTRATE SPECIFICITY OF PURIFIED RAT LIVER DPPIV a Substrate

Relative rate (%)

Gly-Pro-pNA Lys-Pro-pNA Arg-Pro-pNA Glu-Pro-pNA AIa-AIa-pNA GIy-AIa-pNA Gly-Hyp-pNA Gly-Leu-pNA Ala-pNA

100 72.4 65.7 56.2 8.5 4.3 11.8 0 0

a Relative rates of hydrolysis were determined photometrically using dipeptide p-nitroanilide (pNA) derivatives at 1.5 mM in 75 mM glycine-NaOH buffer (pH 8.7). Hyp, Hydroxyproline.

Substrate Specificity The substrate specificity of DPPIV has been studied by many investigators using the enzymes purified from various sources in combination with various substrates. 2,7,1°,19-24 Table II shows the results obtained with DPPIV purified from rat liver. The conclusions obtained by these specificity studies are summarized as follows. (I) DPPIV exhibits a strong preference for substrates having a penultimate prolyl residue, which can be replaced only by alanine 2°'22 and hydroxyproline ~° with much lower rates of hydrolysis. (2) When the substrates have the penultimate prolyl residue, the identity of the N-terminal residue is not important for the enzyme activity. (3) No action is detected on NH2-blocked derivatives such as benzyloxycarbonyl and tert-butyloxycarbonyl peptides, 2,22 indicating that the N-terminal residue must have a free amino group. (4) Peptides containing proline 23 or hydroxyproline 24 at the third position (X-Pro-Pro- or X-Pro-Hyp-) cannot serve as a substrates for the enzyme. 21 H. C. Krutzsch and J. J. Pisano, Biochim. Biophys. Acta 576, 280 (1979). 22 V. K. Hopsu-Havu, P. Rintola, and G. G. Glenner, Acta Chem. Scand. 22, 299 (1968). 23 j. K. McDonald, B. B. Zeitman, and S. Ellis, Nature (London) 225, 1048 (1970). 24 H. Oya, M. Harada, and T. Nagatsu, Arch. Oral. Biol. 19, 489 (1974).

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SERINEPEPTIDASES

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Structural Features Primary Structure The entire amino acid sequence of DPPIV for rat, ~7 human, 25,26and mouse 27 has been predicted by molecular cloning and sequencing of the respective cDNAs (Fig. 3). The subunit of rat DPPIV contains 767-amino acid residues with a calculated size of 88,107 Da. The predicted N-terminal sequence with a characteristic sequence for the membrane translocation signal is completely identical to that chemically determined for the purified membrane form of DPPIV. 17In addition, the N-terminal sequence of the papain-solubilized form is identified in the predicted sequence starting at the 35th position from the N terminus (Fig. 3). Thus, it is evident that the signal peptide of DPPIV is not cleaved off during biosynthesis but functions as the membrane-anchoring domain, as demonstrated for other ectoenzymes such as aminopeptidases A and N, sucrase-isomaltase, and y-glutamyl transpeptidase (y-glutamyltransferase). The presence of eight potential N-linked glycosylation sites in the molecule accounts for the difference in molecular mass between the predicted polypeptide (88 kDa) and the purified glycoprotein (109 kDa). The predicted amino acid sequence of rat DPPIV exhibits 84.9 and 91.2% identity to that of the human and mouse enzyme, respectively. The C-terminal sequences of about 250 residues in these DPPIVs are more than 95% identical, and also have a significant similarity to those of other serine peptidases that belong to the prolyl oligopeptidase family. 28,29 Active Sites The active site serine of rat DPPIV has been identified by chemical analysis of [3H]DFP-labeled DPPIV and confirmed by site-directed mutagenesis/expression analysis. 3° The purified enzyme was labeled with the active-site -directed reagent [3H]DFp7 and digested with lysyl endopeptidase. High-performance liquid chromatography of the digested peptides 25 y. Misumi, Y. Hayashi, F. Arakawa, and Y. Ikehara, Biochim. Biophys Acta 1131, 333 (1992). D. Darmoul, M. Lacasa, L. Baricault, D. Marguet, C. Sapin, P. Trotot, A. Barbat, and G. Trugnan, J. Biol. Chem. 267, 4824 (1992). 27 D. Marguet, A. M. Bernard, I. Vivier, D. Darmoul, P. Naquet, and M. Pierres, J. Biol. Chem. 267, 2200 (1992). 28 N. D. Rawlings, L. Polgar, and A. J. Barrett, Biochem. J. 279, 907 (1991). 29 N. D. Rawlings and A. J. Barrett, Biochem. J. 290, 205 (1993). 30 S. Ogata, Y. Misumi, E. Tsuji, N. Takami, K. Oda, and Y. Ikehara, Biochemistry 31, 2582 (1992).

[16]

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248 250

498 497

S . V N D . G . . . . . . .

.

98 100

148 150

D V . Y . . . T .

I LPPHFDKSKKY . . . . . . . . .

.

I TEEKI PN . . . . R . . .

.

. . . .

rat human

P

.

FGD D E . . H

548 547

R . ,.

598 597

.

.

647 . .

.

.

69B . 697 . .

.

74B . 747

767 766

FiG. 3. Primary structures of rat and human DPPIV predicted by the c D N A sequences. Amino acid residues of human DPPIV that are identical with those of the rat enzyme are indicated by a dot. Dashes indicate gaps introduced into the sequences so that they could be aligned. The boxed N-terminal sequence represents a transmembrane domain that also functions as an uncleavable signal peptide during the biosynthesis of DPPIV. An arrow indicates the papain cleavage site for release of the soluble form. Asparagine residues with stars indicate potential N-glycosylation sites. The boxed sequence G-W-S-Y-G corresponds to the consensus active site sequence G-X-S-X-G proposed for serine proteinases. The catalytic triad residues serine, aspartic acid, and histidine are indicated by arrowheads.

226

SERINE PEPTIDASES

[16]

yielded a single 3H-labeled peptide, which was analyzed for amino acid sequence and radioactivity distribution. A comparison of the determined sequence with the predicted primary structure of DPPIV revealed that the [3H]DFP was bound to Ser-631 within the sequence Gly-Trp-Ser-TyrGly (positions 629-633), which corresponds to the consensus Gly-X-SerX-GIy motif proposed for serine proteases. 31The DPPIV cDNA was modified by site-directed mutagenesis, followed by expression of the mutagenized cDNAs in COS-1 cells. The complete loss of the enzyme activity was caused by any single substitution of Gly-629, Ser-631, or Gly-633, indicating that the sequence Gly-X-Ser-X-Gly (positions 629-633) is essential for the expression of the DPPIV activity (Fig. 3). A comparison of the DPPIV sequence (C-terminal 250 residues) with those of other serine peptidases of the prolyl oligopeptidase family suggested candidate residues, aspartic acid and histidine, which may form part of a catalytic triad, za Site-directed mutagenesis and expression of the cDNA in COS-1 cells have demonstrated that Asp-709 and His-741 are the other essential residues, possibly required for the catalytic triad of rat DPPIV together with Ser-631 (Fig. 3). This was also confirmed by the same techniques for the human (Asp-708 and His-740) 25and mouse DPPIV (Asp-702 and His-734)? 2 It is of interest to note that the sequential order Ser-631-Asp-709-His-741 of the putative catalytic triad of DPPIV is distinct from that of the classical serine proteases, including the chymotrypsin family (His-Asp-Ser) and the subtilisin family (Asp-His-Ser), Mutation

Watanabe et al. 33 reported the defect of DPPIV in a substrain of Fischer-344 rats. The molecular basis for the enzyme deficiency was examined in the subsequent studies. 34'35 Cloning and sequencing of DPPIV cDNAs revealed a point mutation (G ~ A at nucleotide 1897) in the cDNA from the enzyme-defective rat, which leads to substitution of Gly-633 Arg in the active site sequence Gly-Trp-Ser-Tyr-Gly (Fig. 3). Pulse-chase experiments with primary-cultured rat hepatocytes showed that the mutant DPPIV, although being synthesized as a precursor with the same molecular mass (103 kDa) as the wild type, was rapidly degraded within the endoplas31S. Brenner, Nature (London) 334, 528 (1988). 32F. David, A.-M. Bernard, M. Peirres, and D. Margue,J. Biol. Chem. 268, 17247(1993). 33y. Watanabe, T. Kojima, and Y. Fujimoto, Experientia 43, 400 (1987). 34E. Tsuji, Y. Misumi, T. Fujiwara, N. Takami, S. Ogata, and Y. Ikehara, Biochemistry 31, 11921(1992). 35T. Fujiwara, E. Tsuji, Y. Misumi, N, Takarni, and Y. Ikehara, Biochem. Biophys. Res. Commun. 185, 776 (1992).

[17]

ACYLAMINOACYL-PEPTIDASE

227

mic reticulum without being processed into the mature form (109 kDa), resulting in no expression of DPPIV on the cell surface. Site-directed mutagenesis and expression of the cDNA confirmed that the rapid degradation of DPPIV in the endoplasmic reticulum is caused by a single substitution not only of Gly-633 but also of Gly-629 to any other residue. 34'35 Thus, it is evident that the consensus active site motif is essential for the expression of DPPIV on the cell surface as well as for its catalytic activity.

[ 17] A c y l a m i n o a c y l - p e p t i d a s e

By WANDA M. JONES, ANDREA SCALONI, and JAMES M. MANNING Acylaminoacyl-peptidase (EC 3.4.19.1) catalyzes the removal of a blocked amino acid from a blocked peptide as described in the following equation: X-aa I - aa a • • • aa, ~ X-aa I + aa2 • • • aan The enzyme is also referred to by the names acylpeptide hydrolase, ~-3 acylamino acid-releasing enzyme 4,s and acylaminoacyl-peptide hydrolase. 6 The products of the reaction are an acyl amino acid and a peptide with a free N terminus shortened by one amino acid. The enzyme acts on a variety of substrates, including peptides with different N-terminal acyl groups, i.e., acetyl, chloroacetyl, formyl, and carbamyl groups. 7 The optimum length of the blocked peptide substrate is 2-3 amino acids, but larger peptide substrates are also cleaved at slower rates) For instance, the blocked 13-residue peptide, a-melanocyte-stimulating hormone (aMSH), is a substrate. 7 On the other hand, N-terminally blocked proteins are not substrates for the enzyme. 9 Acylaminoacyl-peptidase could conI W. Gade and J. L. Brown, J. Biol, Chem. 253, 5012 (1978). 2 W. M. Jones and J. M. Manning, Biochem. Biophys. Res. Commun, 126, 933 (1985). 3 K. Kobayashi, L.-W. Lin, J. E. Yeadon, L. B, Klickstein, and J. A. Smith, J. Biol. Chem. 264, 8892 (1989). 4 S. Tsunasawa, K. Narita, and K. Ogata, J, Biochem. (Tokyo) 77, 89 (1975). 5 M. Mitta, K. Asada, Y. Uchimura, F. Kimizuka, I. Kato, F. Sakiyama, and S. Tsunasawa, J. Biochem. (Tokyo) 106, 548 (1989). 6 G. Radhakrishna and F. Wold, J. Biol. Chem. 264, 11076 (1989). 7 W. M, Jones, L. R. Manning, and J. M. Manning, Biochem. Biophys. Res, Commun~ 139, 244 (1986). 8 W. M. Jones, A. Scaloni, F. Bossa, A. M. Popowicz, O. Schneewind, and J. M. Manning, Proc. Natl. Acad. Sci. U.S.A. 88, 2194 (1991). 9 T. C. Farries, A. Harris, A. D. Auffret, and A. Aitken, Eur. J, Biochem. 196, 679 (199l).

METHODS 1N ENZYMOLOGY, VOL. 244

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