Characterization of the substrate specificity of human carboxypeptidase A4 and implications for a role in extracellular peptide processing

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 24, pp. 18385–18396, June 11, 2010

Characterization of the Substrate Specificity of Human

Carboxypeptidase A4 and Implications for a Role in

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Extracellular Peptide Processing^*

Received for publication, August 27, 2009, and in revised form, April 9, 2010 Published, JBC Papers in Press, April 12, 2010, DOI 10.1074/jbc.M109.060350

Sebastian Tanco^‡, Xin Zhang^§, Cain Morano^§, Francesc Xavier Avile´s^‡, Julia Lorenzo^‡1, and Lloyd D. Fricker^§2

From the ^‡Institut de Biotecnologia i de Biomedicina and Departament de Bioquimica, Universitat Autonoma de Barcelona,

Bellaterra (Barcelona), Spain 08193 and the ^§Department of Molecular Pharmacology, Albert Einstein College of Medicine,

Bronx, New York 10461

CPA4 (carboxypeptidase A4) is a member of the metallocar-

boxypeptidase family. CPA4 was originally found in a screen of

mRNAs up-regulated by sodium butyrate-induced differentia-

tion of cancer cells. Further studies suggested a relation between

CPA4 and prostate cancer aggressiveness. In the present study,

we determined that CPA4 is secreted from cells as a soluble

proenzyme (pro-CPA4) that can be activated by endoproteases,

such as trypsin. Three complementary approaches were used to

study the substrate specificity of CPA4; kinetic analysis was per-

formed using a new series of chromogenic substrates and some

biologically relevant peptides, the cleavage of synthetic peptides

was tested individually, and the cleavage of a mixture of >100

mouse brain peptides was examined using a quantitative pep-

tidomics mass spectrometry-based approach. CPA4 was able to

cleave hydrophobic C-terminal residues with a preference for

Phe, Leu, Ile, Met, Tyr, and Val. However, not all peptides with

C-terminal hydrophobic residues were cleaved, indicating the

importance of additional residues within the peptide. Aliphatic,

aromatic, and basic residues in the P1 position have a positive

influence on the cleavage specificity. In contrast, acidic residues,

Pro, and Gly have a negative influence in the P1 position. Some

of the peptides identified as CPA4 substrates (such as neuroten-

sin, granins, and opioid peptides) have been previously shown to

function in cell proliferation and differentiation, potentially

explaining the link between CPA4 and cancer aggressiveness.

Taken together, these studies suggest that CPA4 functions in

neuropeptide processing and regulation in the extracellular

environment.

a catalytic mechanism in which nucleophilic attack on a peptide

bond is mediated by a Zn^2ϩ-activated water molecule (1). Most

metallo-CPs are classified based on amino acid sequence, struc-

ture, and function into subfamilies within clan MC, family M14

(2). These subfamilies include M14A, also known as the CPA/

CPB subfamily; M14B, also known as the CPN/CPE subfamily;

M14C, which includes ␥-D-glutamyl-(L)-meso-diaminopime-

late peptidase I from Bacillus sphaericus; and a proposed fourth

subfamily, including cytosolic CPs (3, 4). Metallo-CPs have also

been divided into subgroups based on substrate specificity:

A-like enzymes with preference for hydrophobic C-terminal

amino acids; B-like enzymes, which cleave C-terminal basic res-

idues; CPs with preference for glutamate in the C-terminal

position; and CPs with a broad substrate specificity.

CPA4 (carboxypeptidase A4) belongs to the M14A subfamily

of carboxypeptidases. The pancreatic members of this subfam-

ily (CPA1, CPA2, and CPB) act in the degradation of dietary

proteins in the digestive tract. Other members of this subfamily

display a wide spectrum of physiological roles. Mast cell car-

boxypeptidase (recently renamed CPA3) is found in the secre-

tory granules of mast cells and plays a role in defense (5). CPA6

is linked to Duane syndrome, a congenital eye movement dis-

order, and has been proposed to participate in the regulation of

neuropeptides in the extracellular matrix within the olfactory

bulb and other parts of the brain (6). Carboxypeptidase U, also

known as CPB2 and TAFI (thrombin-activatable fibrinolysis

inhibitor), participates in fibrinolysis inhibition and possesses

anti-inflammatory properties (7) and for these reasons is of

interest to the pharmaceutical industry (8).

CPA4 was originally referred to as CPA3 (9) but was renamed

CPA4 to reflect the order in which the CPAs were discovered.

Huang et al. (9) identified CPA4 in a search for mRNAs induced

by sodium butyrate in androgen-independent prostate cancer

cells. The histone deacetylase inhibitor trichostatin A also

induced the expression of CPA4 mRNA in PC-3, DU145, and

BPH1 human prostate cancer cell lines (9). Huang et al. (9) also

reported that CPA4 mRNA expression is associated with hor-

mone-regulated tissues, suggesting that it may have a role in cell

growth and differentiation. The human CPA4 gene is located

Carboxypeptidases (CPs)³hydrolyze a single amino acid

from the C terminus of peptides and proteins. Metallo-CPs use

* This work was supported in part by National Institutes of Health Grants

DK-51271andDA-04494(toL. D. F.). ThisworkwasalsosupportedbyEuro-

pean Commission Grant FP7-Health-2009-241919-LIVIMODE (to F. X. A.),

by Spanish Ministry of Science and Technology Grant BIO2007-68046-C02

(to F. X. A.), and by Xarxa de Refere`ncia en Biotecnologia (Generalitat de

Catalunya).

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The on-line version of this article (available at http://www.jbc.org) contains

supplemental Tables S1–S4 and Fig. S1.

¹To whom correspondence may be addressed. Tel.: 34-935811231; Fax:

34-935812011; E-mail: julia.lorenzo@uab.es.

²To whom correspondence may be addressed: Dept. of Molecular Pharma-

cology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx,

NY 10461. Tel.: 718-430-4225; Fax: 718-430-8922; E-mail: lloyd.fricker@

einstein.yu.edu.

tidase; CPB, carboxypeptidase B; ECM, extracellular matrix; ER, endoplas-

mic reticulum; LC, liquid chromatography; MS, mass spectrometry; MALDI-

TOF, matrix-assisted laser desorption ionization time-of-flight; MS/MS,

tandem mass spectrometry; TMAB, 4-trimethylammoniumbutyrate; HA,

hemagglutinin; FA, 3-(2-furyl)acryloyl.

³The abbreviations used are: CP, carboxypeptidase; CPA, A-like carboxypep-

JUNE 11, 2010•VOLUME 285•NUMBER 24

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Characterization of Human Carboxypeptidase A4

on chromosome 7q32, which is a region in the genome that Eagle’s medium with 4500 mg/liter glucose, GlutaMAX-I, and

might contain genes for prostate cancer aggressiveness (10). In pyruvate supplemented with 10% fetal calf serum (Invitrogen).

addition, the CPA4 gene was found to be maternally imprinted The full-length cDNA sequence of human pro-CPA4 was

in a tissue-specific manner (11, 12). Furthermore, imprinting in cloned into pcDNA3.1 vector (Invitrogen), and the hemagglu-

adult benign hypertrophic prostate tissue suggests that tinin (HA) epitope was introduced on the C terminus of CPA4.

mutations or aberrant imprinting in CPA4 may be related to The plasmid pDsRed2-ER from Clontech was used for endo-

prostate cancer aggressiveness (12). Ross et al. (13) studied plasmic reticulum (ER) localization. Cells were grown in

the association of single-nucleotide polymorphisms on the 35-mm dishes and transfected with Lipofectamine 2000

CPA4 gene and its relation to prostate cancer and found that according to the manufacturer’s instructions.

the nonsynonymous coding single-nucleotide polymorphism

(G303C) rs2171492 was associated with an increased risk of transfection, medium was removed, centrifuged, and collected

aggressive disease in younger men. on ice. The dishes were gently washed with phosphate-buffered

Cell Fractionation and Processing—Forty-eight hours after

Despite the potential importance of CPA4 toward prostate saline, and cells were removed by pipetting up and down in a

and other cancers, no previous studies have examined the sub- mild extraction buffer consisting of 50 m^MTris-HCl, pH 8.0.

strate specificity of CPA4 in detail. The three-dimensional The cell suspension was sonicated and centrifuged at 16,000 ϫ

structure of CPA4 has been determined for the zymogen state g for 10 min at 4 °C. The pellet was resuspended in an equivalent

(14) and for the active form in complex with latexin, a brain- amount of 50 m^MTris-HCl, pH 8.0, containing 500 mM NaCl

derived CP inhibitor (15), and in complex with a hexapeptide and 1% Nonidet P-40 and centrifuged as above to produce a

(16). This latter study also compared the cleavage of a handful detergent/high salt extract. Following the removal of cells, the

of synthetic peptides by CPA4, but no kinetic details were pro- plate was washed several times with phosphate-buffered saline,

vided. To gain a better understanding of the enzymatic proper- and the extracellular matrix (ECM) was extracted by adding hot

ties of CPA4, we used a variety of approaches to characterize SDS-PAGE sample buffer directly to the plate. All homogenates

the substrate specificity. A series of dipeptide chromogenic were frozen and stored at Ϫ70 °C until analysis. For trypsin

substrates were synthesized and tested with purified CPA4. A activation assays, 200 ng of trypsin (Sigma) were added to 20 ␮l

number of synthetic peptides were individually tested with of the extracellular medium of transfected HEK 293T cells and

purified CPA4; many of these peptides correspond to biologi- incubated for 30 min at 37 °C.

cally active peptides. A third approach involved incubating

Immunoblot—For immunoblotting, equivalent amounts of

CPA4 with a mixture of over 100 endogenous peptides each lysate or fraction were analyzed by SDS-10% PAGE, and

extracted from mouse brain, using a quantitative peptidomics the separated proteins were electrophoretically transferred

approach to identify substrates and products. The subcellular onto polyvinylidene difluoride filters (Millipore Corp.). Non-

distribution, secretion, and pH optimum of CPA4 were also specific binding sites on the polyvinylidene difluoride filters

examined. Taken together, these studies provide a complemen- were blocked by incubation with 5% skim milk for 12 h. The

tary and thorough analysis of the cleavage specificity of CPA4 polyvinylidene difluoride filters were then incubated with

and suggest that this enzyme functions as a soluble extracellular anti-HA antibody (dilution 1:3,000; Sigma) for 1 h at 25 °C.

peptidase that removes C-terminal hydrophobic/aliphatic res- After washing with phosphate-buffered saline containing 0.05%

idues from secreted peptides.

Tween 20, the filters were incubated with goat anti-mouse IgG-

peroxidase (dilution 1:10,000; Pierce) for 1 h. After three rinses,

immunoreactive bands were visualized with a chemilumines-

EXPERIMENTAL PROCEDURES

Protein Production and Purification—Human pro-CPA4 cence detection kit (ECL; Millipore).

(PCPA4) was produced using the vector pPIC9 and the methy-

Immunofluorescence—HeLa, HEK 293T, PC-3, DU145, or

lotrophic yeast Pichia pastoris as an expression host and puri- LNCaP cells were co-transfected with HA-tagged PCPA4 and

fied as described elsewhere (15). The active enzyme was pDsRed2-ER using Lipofectamine 2000 (Invitrogen) according

obtained through tryptic activation (at a 1:10 w/w ratio) for 60 to the manufacturer’s protocol. After 48 h of incubation, cells

min at room temperature, and the resulting product was sub- were fixed in methanol, permeabilized with 0.1% Triton X-100,

sequently purified by anion-exchange chromatography (TSK- and incubated with anti-HA antibody (clone HA-7 (Sigma);

¨

DEAE 5PW) using an FPLC-Akta system with a linear salt gra- 1:2,000 dilution), followed by conjugated Alexa-488 anti-

dient from 0 to 30% of 0.4 ^Mammonium acetate in 20 mM mouse-IgG (Molecular Probes; 1:500 dilution) and 4Ј,6Ј-dia-

Tris-HCl (pH 10.5). Eluted fractions were analyzed by SDS- midino-2-phenylidole. Coverslips were mounted with Fluo-

PAGE, and the purest samples containing the active enzyme prep mounting medium (Biomerieux) and analyzed with a

were pooled, desalted, and concentrated to 1 mg/ml by Amicon confocal laser-scanning microscope (Leica Microsystems, Ban-

centrifugal filter devices.

nockburn, IL).

Cell Culture and Transfection—HeLa (human cervix adeno-

Substrate Synthesis—Chromogenic substrates were synthe-

carcinoma), PC-3, DU145, and LNCaP (all three cell lines are sized according to Blumberg et al. (17). 3-(2-furyl)acryloyl

human prostate carcinoma), 1BR3.G (human skin fibroblasts), (FA)-Phe-N-hydroxysuccinimide was prepared according to

and HEK 293T (human embryo kidney) cells were purchased the following procedure. A solution of 15 mmol of FA-Phe and

from the American Type Culture Collection (Manassas, VA) 15 mmol of N-hydroxysuccinimide in 60 ml of dioxane-dimeth-

and were cultured in the recommended growth medium: ylformamide (1:1) was cooled to 4 °C, and 15 mmol of dicyclo-

Eagle’s minimum essential medium or Dulbecco’s modified hexylcarbodiimide was added. The solution was stirred at 4 °C

18386 JOURNAL OF BIOLOGICAL CHEMISTRY

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Characterization of Human Carboxypeptidase A4

for 16 h. The dicyclohexyl urea was filtered, and the solution peria, CA) with a linear gradient of 5–35% acetonitrile and fol-

was evaporated in vacuo. Furylacryloyl dipeptides were pre- lowing the absorbance at either 214 or 280 nm. Reactions were

pared by reacting FA-Phe-N-hydroxysuccinimide with the performed at 37 °C for 30 min in 50 m^MTris-HCl, 100 mM

appropriate amino acid according to the following procedure. NaCl, pH 7.5, and stopped by the addition of 0.1% trifluoroace-

The N-hydroxysuccinimide ester (5 mmol) in 25 ml of dioxane tic acid. Substrate concentrations ranged from 0.2 to 5 times the

was added at room temperature to a solution of the amino acid K_mwhenever possible, and the enzyme concentration was cho-

(6 mmol) and sodium bicarbonate (12 mmol) in 25 ml of water. sen so that hydrolysis was less than 10% of product formed. The

The mixture was kept at room temperature for 6 h, the dioxane kinetic parameters, k_catand K_m, were obtained by direct fit to a

evaporated, and the residue acidified with 1 ^NHCl. After the Michaelis-Menten curve using the Grafit program.

mixture was cooled overnight at 4 °C, the precipitate was col-

lected by filtration and dried.

Peptidomics—Peptides were extracted from whole mouse

brain (minus the olfactory bulb and cerebellum) as described

Kinetic Measurements—The rate of hydrolysis of different (19, 20). In brief, four mice (C57B6/J, 2–3 months old) were

substrates was continuously measured at 25 °C in 50 m^MTris, sacrificed by decapitation, and the head was immediately irra-

0.15 ^MNaCl at the optimal pH for each enzyme. The wave- diated in a conventional microwave oven for 8 s to raise the

lengths varied slightly among substrates: 336 nm for FA-Phe- brain temperature to 80 °C and inactivate proteases that can

Phe, FA-Phe-Ile, and FA-Phe-Trp; 338 nm for FA-Phe-Ala, FA- rapidly degrade cellular proteins during postmortem process-

Phe-His, and FA-Phe-Leu; and 340 nm for FA-Phe-Val and ing of the tissue (21). The mouse brain was isolated, the olfac-

FA-Phe-Met. Initial rates determined from the first 5–10% of tory bulb and cerebellum were discarded, and the remainder of

the time trace of each reaction were obtained at substrate con- the brain was frozen in dry ice and stored at Ϫ70 °C until pep-

centrations close to the K_mvalue whenever possible. The tide extraction. Peptides were extracted by sonication of each

kinetic parameters, k_catand K_m, were obtained using six exper- brain in 1.0 ml of ice-cold water followed by incubation at 70 °C

imental points by direct fit to a Michaelis-Menten curve using a for 20 min, cooling in an ice bath, acidification with 120 ␮l of 0.1

nonlinear least-squares regression analysis. For those sub- M HCl to a final concentration of 10 mM HCl, and centrifugation

strates for which the solubility of the substrates limited the at 13,000 ϫ g for 30 min at 4 °C. The supernatants (peptide

measurements to concentrations well below K_m, the k_cat/K_mextracts) from each of the four mice were pooled and mixed,

values were estimated from the slope of the linear portion and 60-␮l aliquots were neutralized to pH 8 by 75 mM borate

of saturation curve using the simplified equation, v ϭ buffer and incubated in the presence of purified CPA4 (20 ng,

(k_cat/K_m)ꢀ[E₀]ꢀ[S].

670 pg, or 22 pg) or in the absence of enzyme for 90 min. The

Measurement of Equilibrium Dissociation Constants (K_i)— reactions were stopped by the addition of 2.5 m^Mbenzylsuc-

The method for reversible tightly binding inhibitors described cinic acid and heat-inactivated at 80 °C for 10 min. Labeling

by Bieth (18) was used for K_idetermination. Recombinant puri- with 4-trimethylammoniumbutyrate (TMAB) isotopic tags

fied CPA4 at a concentration of 8 n^Mwas assayed against containing either no deuterium (D0) or 3 atoms (D3), 6 atoms

increasing concentrations of inhibitor. Initial rates, determined (D6), or 9 atoms (D9) of deuterium was performed as described

from the first 5–10% of the time trace of each reaction, were (19). After labeling, the TMAB reagent was quenched with gly-

obtained for each inhibitor concentration using the N-(4-me- cine, the four samples were combined, and peptides were iso-

thoxyphenylazoformyl)-^L-phenylalanine substrate (Bachem). lated by passage through a Microconꢁ YM-10 unit (Millipore)

K_ivalues were obtained by direct fitting of fraction velocity to remove proteins of Ͼ10 kDa. The samples were treated with

versus inhibitor concentration data to the Morrison equation hydroxylamine to remove TMAB labels from Tyr residues,

using the Grafit program.

Substrate Specificity and Kinetic Measurements Using Pep- concentrated to ϳ20 ␮l in a vacuum centrifuge.

tides—CPA4 substrate specificity was analyzed using synthetic Mass spectrometry was performed as described previously

desalted with a PepClean^TMC-18 spin column (Pierce), and

peptide substrates. Reactions were performed in 50 m^MTris- (19, 22). In brief, the peptide mixture was trapped and washed

HCl, 100 m^MNaCl, pH 7.5, at 37 °C using peptide and enzyme on a PepMap^TMC18 trapping column (5 ␮m, 100 Å, 300-␮m

amounts of 10 ␮mol and 1 nmol, respectively. Peptide cleavage inner diameter ϫ 5 mm, LC Packing (Marlton, NJ)) and sepa-

reactions were stopped at different times (30 min, 1 h, 2 h, and rated on a Grace Vydac MS C18 capillary column (3 ␮m, 100 Å,

16 h) by dropping the pH to 4 using 1% trifluoroacetic acid. The 75-␮m inner diameter ϫ 150 mm; Hesperia, CA) at 4 ␮l/min

reaction products were desalted using a C18 ZipTip (Milli- with an acetonitrile gradient in 0.1% formic acid. The column

pore), following the manufacturer’s protocol. The peptides eluate was analyzed on an API Q-Star Pulsar-i^TMquadrupole

were eluted with 1 ␮l of 70% acetonitrile, 0.1% trifluoroacetic time-of-flight mass spectrometer (Applied Biosystems/MDS

acid; mixed with an equal volume of a saturated solution of Sciex, Foster City, CA) in information-dependent acquisition

␣-hydroxycinnamic acid in the same solvent; and analyzed in a mode, with a collision energy for tandem mass spectrometry

Bruker Daltonics Ultraflex matrix-assisted laser desorption (MS/MS) (20–45 eV) based on the m/z value and charge state

ionization time-of-flight (MALDI-TOF) mass spectrometer.

For the determination of kinetic constants for the hydrolysis

of the ion selected.

Data were analyzed as described (19). In brief, Mascot (avail-

of peptides, rates of substrate hydrolysis were determined by able from the Matrix Science Web site) was used, followed by

reversed-phase high performance liquid chromatography in a manual verification; identifications were rejected unless 80% or

Waters Alliance apparatus using a Grace Vydac 218TP^TMC18 more of the major fragments observed in MS/MS matched pre-

column (3 ␮m, 100 Å, 4.6-mm inner diameter ϫ 250 mm; Hes- dicted b- or y-series fragments (minimum of five matches).

JUNE 11, 2010•VOLUME 285•NUMBER 24

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Characterization of Human Carboxypeptidase A4

Additional criteria included all of the following: a parent mass

within 40 ppm (preferably 20 ppm) of the theoretical mass; the

observed number of TMAB tags on the peptide matching the

predicted number of free amines available (i.e. Lys residue and

N terminus); and the observed charge state(s) of the peptide

consistent with the expected number of positive charges. The

intensities of the TMAB-labeled isotopic peaks were deter-

mined by measuring peak heights of the monoisotopic peak and

the additional peaks containing one and two atoms of ¹³C. The

peak intensities of the samples incubated with enzyme were

compared with the peak intensity of the sample incubated with-

out enzyme; previous studies examining four aliquots of brain

extract with the four different isotopic tags showed average

ratios of 1.00 (19).

RESULTS

The sequence analysis of PCPA4 protein reveals the presence

of a 16-amino acid-long signal peptide consensus sequence, and

therefore the protein is predicted to be translocated to the

lumen of the ER upon synthesis. From the ER, many options are

possible, including retention in the secretory pathway, target-

ing to lysosomes, or secretion. Because CPA4 lacks any trans-

membrane domains and other members of this subfamily are

secreted, PCPA4 is predicted to also be secreted from cells.

Some CPs, such as CPA1, remain as soluble proteins in the

extracellular environment (23), whereas others like CPA6 (6) or

FIGURE 1. PCPA4 is a soluble secreted protein. A, PCPA4 transfection in

mammalian cells. HEK 293T cells were transfected with empty vector (Ϫ) or

plasmid expressing HA-tagged PCPA4 (ϩ), and different fractions were

collected and prepared. Equal amounts of each fraction were analyzed by

Western blot using an HA antibody. B, trypsin processing. Extracellular

media was treated with (ϩ) or without (Ϫ) trypsin and analyzed by West-

ern blot. C, subcellular distribution of PCPA4. HEK 293T cells were trans-

fected with HA-tagged PCPA4 and pDsRed-ER cDNAs. After fixation, trans-

fected cells were incubated with mouse anti-HA antibody, followed by

anti-mouse IgG Alexa 488 (green) and 4Ј,6Ј-diamidino-2-phenylidole

(DAPI) to detect cell nuclei.

mouse CPZ (24) are retained within the ECM. In relation to kDa band corresponding to PCPA4 and the appearance of a 35

proprotein processing, some pro-CPs are activated before or kDa band corresponding to the expected molecular weight of

upon secretion by proteases present in the secretory pathway the active enzyme (Fig. 1B). Enzymatic analysis of these samples

(6), whereas others are activated after secretion by extracellular shows no detectable CP activity in the extracellular medium of

proteases (23). To study whether PCPA4 is secreted and acti- PCPA4-transfected cells, but incubation with trypsin resulted

vated, HEK 293T cells were transiently transfected with HA- in the appearance of A-type CP enzymatic activity against

tagged PCPA4 cDNA. After 48 h of transfection, cells, media, the N-(4-methoxyphenylazoformyl)-^L-phenylalanine substrate

and ECM were collected, fractionated, and analyzed by West- (data not shown). The finding that PCPA4 can be activated with

ern blot. A band of an apparent molecular mass of 50 kDa cor- trypsin indicates that it is properly folded and that the absence

responding to the full-length PCPA4 was detected predomi- of detectable zymogen processing in the HEK 293T cells as well

nantly in the cell culture medium (Fig. 1A, lane 5). PCPA4 was as in various other cell lines is due to insufficient levels of the

also detected in two different cell extracts; the zymogen appears activating enzyme and not due to the improper folding of

in a low salt extract but also at low levels in a detergent/high salt PCPA4 in these cell lines.

extract (Fig. 1A, lanes 1 and 3). The absence of any PCPA4 in

The presence of PCPA4 in the cells was further investigated

the ECM shows that the protein remains as a soluble protein in by immunocytochemistry. PCPA4 was found to show some

the extracellular medium. In all cases, PCPA4 is found in the overlap with an ER marker (Fig. 1C), which is consistent with

full-length form, indicating that its activating protease is not the finding that intracellular PCPA4 is present in the secretory

present or active in sufficient levels in the system. To investi- pathway of the cell.

gate if this behavior would be similar in other cell lines, we

Although there are several reports about CPA4 structure (14,

repeated the transfection of PCPA4 in five different cell lines: 15), expression (9), and relation to prostate cancer aggressive-

DU145 and PC-3, which are prostate cancer cells previously ness (11, 12), a detailed analysis of its enzymatic properties has

found to express PCPA4 in relatively high levels (9); LNCaP, a not been reported previously. Recombinant protein obtained in

prostate cancer cell line that does not express PCPA4; HeLa the P. pastoris system as described previously (15) was used for

cells, which arise from a cervical adenocarcinoma; and 1BR.3.G, this purpose. Mature CPA4 was obtained after treatment of

a human skin fibroblast cell line. In all cases, PCPA4 was pre- the proenzyme with trypsin and subsequent purification. As

dominantly found soluble in the extracellular medium in the expected, the active enzyme shows the ability to remove C-ter-

zymogen form (data not shown).

minal residues from both FA-Phe-Phe and FA-Phe-Ala. No

To demonstrate that PCPA4 expressed in HEK 293T cells detectable activity toward substrates with basic (FA-Ala-Arg)

was properly folded and able to produce the enzymatically or acidic (hippuryl-^L-glutamic acid) C-terminal residues was

active form, extracellular medium collected after 48 h of trans- found. This is in accordance with predictions based on its

fection was treated with trypsin. Western blot analysis of these sequence and structure that include CPA4 in the group of

samples shows that trypsin causes the disappearance of the 50 A-type CPs. The Pichia-expressed proenzyme shows 3% of

18388 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 24•JUNE 11, 2010

Characterization of Human Carboxypeptidase A4

residual activity against the N-(4-methoxyphenylazoformyl)-L- 60% inhibition after 5 h of treatment. Recombinant forms of

phenylalanine substrate when compared with the active

enzyme. Although the proenzyme shows residual activity, it

could not be detected in the conditioned medium of PCPA4-

transfected HEK 293T cells because this activity falls below the

limit of detection of the technique.

potato CP inhibitor (27), leech CP inhibitor (28), and tick CP

inhibitor (29) were tested with CPA4 and found to exhibit equi-

librium dissociation constant (K_i) values in the low nanomolar

range (supplemental Table S1 ). The protein inhibitor with the

highest affinity for CPA4 was found to be that from tick Rhipi-

cephalus bursa , for which a constant of 0.8 Ϯ 0.3 n^Mwas

obtained (supplemental Table S1). The K_ivalues for CPA4

determined in the present study are generally comparable with

values previously published for CPA4 with latexin (15) and

Ascaris carboxypeptidase inhibitor (30); these results are

included in supplemental Table S1 for comparison with the

present data.

The influence of pH on CPA4 activity was analyzed using

both FA-Phe-Phe and FA-Phe-Ala. The optimal pH for cleav-

age of either substrate is in the range of 8.5–9 with very low

activity below pH 6 (Fig. 2). This suggests that CPA4 is maxi-

mally active in the extracellular environment after secretion

and has little activity within the acidic environment of the

secretory pathway.

The effect of several different CP inhibitors on CPA4 activity

was assayed. Benzylsuccinic acid (25), a potent synthetic inhib-

itor of the A-type CP subfamily, at a concentration of 100 ␮M

produced 30% inhibition of CPA4 activity and completely

inhibited the enzyme when a 1 m^Mconcentration of the inhib-

itor was used. The Zn^2ϩ-chelating agent 1,10-phenanthroline

(26) showed 70% inhibition of the CP activity after 30 min of

incubation at 1 m^Mconcentration. CPA4 showed resistance

against the action of EDTA, another chelating inhibitor of CPs;

complete inhibition of CPA4 activity was only achieved using a

100 m^Mconcentration of the inhibitor in an overnight incuba-

tion, whereas the same inhibitor concentration caused only

To investigate the substrate specificity of CPA4, a new series

of substrates of the type FA-Phe-Xaa were synthesized. These

substrates incorporated the furylacryloyl moiety as a chro-

mophore, allowing for continuous monitoring of CP activity at

a wavelength that has little interference from proteins or aro-

matic derivatives (31). This new series of substrates provided a

broad range of hydrophobic/aliphatic residues in the C-termi-

nal position so that substrate specificities of A-type CPs could

be evaluated. The different amino acids included in the

C-terminal position were His, Leu, Ile, Phe, Trp, Ala, Val,

and Met. All substrates contain Phe in the penultimate posi-

tion and are of the same length, allowing a direct comparison

of the C-terminal residues of the substrates. Kinetic con-

stants were determined for CPA4 and also for human CPA1

and CPA2 to directly compare these related enzymes with

the same substrates. CPA1 and CPA2 were obtained as

recombinant proteins in the P. pastoris system, purified in

their zymogen form, activated with trypsin, and further puri-

fied as described previously (32, 33). k_cat, K_m, and k_cat/K_m

values are shown in Table 1 and indicate that although all

three enzymes have overlapping specificities, they differ

markedly. Although CPA1 and CPA4 display similar and

broader substrate specificities, CPA2 shows a substrate spec-

ificity that is essentially restricted to large aromatic residues

in the C-terminal position. CPA4 shows a preference for

hydrophobic C-terminal amino acids like Phe, Leu, Met, Val,

and Ile, as judged by k_cat/K_mvalues. Of all of the dipeptide

substrates tested with CPA4, FA-Phe-Trp exhibited the

highest k_cat, but the high K_mresulted in a lower k_cat/K_mfor

this substrate than for FA-Phe-Phe. Overall, the k_catvaried

only 5-fold among the substrates that showed measurable

FIGURE 2. Effect of pH on CPA4 activity. CPA4 activity was determined by

measuring initial rates using 100 ␮M FA-Phe-Phe or FA-Phe-Ala in 50 mM Tris

acetate buffer at the indicated pH at 25 °C. Error bars, S.E. for three independ-

ent determinations.

TABLE 1

Kinetics constants for peptide substrate hydrolysis by CPA1, CPA2, and CPA4

CPA4

_cat/K_m

CPA1

_cat/K_m

CPA2

Substrate

k

_cat/K_mrelative

k

_cat/K_mrelative

k_cat/K_mrelative

k_cat

K_m

k

k_cat

K_m

k

k_cat

K_m

k

_cat/K_m

to FA-Phe-Phe

Ϫ1

Ϫ5

Ϫ1

Ϫ5

Ϫ1

Ϫ5

s

␮M

M

^Ϫ1ꢀs^Ϫ1ꢀ10

s

␮M

M

^Ϫ1ꢀs^Ϫ1ꢀ10

s

␮M

M

^Ϫ1ꢀs^Ϫ1ꢀ10

FA-Phe-Phe 44.3 55.6

FA-Phe-Trp 57.3 615

FA-Phe-Leu 13.4 19.4

7.97

0.93

6.93

5.32

5.81

0.65

3.38

0.27

1.00

0.12

0.87

0.67

0.73

0.082

0.42

0.034

76.6

81.3

34.0

9.77

51.8

78.0

12.3

NM

20.4

166

37.6

4.90

21.7

10.5

4.03

2.58

14.8

1.04

1.00

0.13

0.58

0.28

0.11

0.069

0.39

0.028

121

65.3

18.6

36.4

16.3

393

33.3

40.2

0.47

0.12

0.040

NM

0.030

NM

1.00

1.21

15.7

9.33

128

0.014

0.0035

0.0012

NM

FA-Phe-Ile

12.4 23.3

5.37 460

NM_aNM

FA-Phe-Met 23.9 40.0

FA-Phe-Ala 24.3 372

300

NM

FA-Phe-Val

FA-Phe-His

19.4 57.3

NM NM

8.95

NM

0.0010

NM

^aNM, not measurable; below the limit of detection.

JUNE 11, 2010•VOLUME 285•NUMBER 24

JOURNAL OF BIOLOGICAL CHEMISTRY 18389

Characterization of Human Carboxypeptidase A4

k_catvalues, whereas the K_mvaried 32-fold, and the k_cat/K_m

varied 12-fold for these substrates (Table 1).

To further characterize the catalytic activity of CPA4, its abil-

ity to cleave a series of peptides was studied. Some of the pep-

tides chosen for this analysis are biologically active and are

known to be secreted into the extracellular fluid of tissues

where CPA4 is expressed. Others are not likely to be biological

substrates of CPA4 but provide further information about the

ability of this enzyme to cleave peptides. All of these peptides

were previously tested against CPA6 (6), so they are useful for

comparison purposes. The enzyme, at a concentration of 1 n^M,

was incubated at 37 °C with the peptides in a final concentra-

tion of 10 ␮M. In order to obtain semiquantitative information

about the cleavage, different times of incubation were analyzed

by MALDI-TOF MS. Although some peptides were extensively

cleaved, other peptides were only partially cleaved, and others

were not cleaved under the conditions tested (supplemental

Table S2). CPA4 was found to completely cleave PEN and Met-

enkephalin-Arg-Phe after 30 min of incubation. Big SAAS and

neurotensin were also extensively cleaved after 1–2 h of incu-

bation. Partial cleavage of ϳ50% of angiotensin was found after

30 min of incubation. Des-Asp¹-angiotensin and Leu-enkepha-

lin were partially cleaved in 2 h, and Met-enkephalin and CPA5

C terminus required an overnight incubation for a partial diges-

tion by CPA4. Only a small percentage of cleavage was detected

for human little SAAS. No digestion of the aldolase C-terminal

peptide was detected, although it was expected to be cleaved

because of the C-terminal Tyr. When a 10-fold higher concen-

tration of enzyme (10 n^M) was used, substantial cleavage of the

C-terminal Tyr was observed after 2 h of incubation. Neither

bradykinin nor des-Arg⁹-bradykinin were substrates for CPA4

(supplemental Table S2 ). Although des-Arg⁹-bradykinin has a

C-terminal Phe, the presence of a Pro in the penultimate posi-

tion may explain why CPA4 is not able to cleave this substrate

under the conditions tested.

FIGURE 3. MALDI-TOF MS analysis of neurotensin cleavage by CPA4.

MALDI-TOF MS spectra of 50 ␮M neurotensin incubated for different times at

37 °Cwith20nM CPA4. Numbersabovethemajorpeaksindicatethemonoiso-

topic masses of the MH^ϩion (m/z). The peak with a mass of 1671.96 Da cor-

responds to neurotensin (theoretical monoisotopic mass of neurotensin ϭ

1671.91 Da), and a first cleavage product with a mass of 1558.95 Da formed in

the presence of CPA4 corresponds to the peptide lacking the C-terminal Leu

(theoretical mass ϭ 1558.84 Da), and a second cleavage product with mass of

1445.88 Da corresponds to the peptide lacking both C-terminal Leu and Ile

(theoretical mass ϭ 1445.75 Da).

TABLE 2

Kinetic constants for the hydrolysis of biological peptides by CPA4

Peptide

k_cat

s

K_m

␮M

k_cat/K_m

Ϫ1

␮M^Ϫ1ꢀmin

Neurotensin

0.240 Ϯ 0.017 0.329 Ϯ 0.049

43.8 Ϯ 9.5

9.43 Ϯ 0.86

2.18 Ϯ 0.31

0.85 Ϯ 0.13

0.374 Ϯ 0.053

Met-enkephalin-Arg-Phe

Angiotensin I

1.45 Ϯ 0.03

8.23 Ϯ 0.34

2.33 Ϯ 0.11

3.43 Ϯ 0.16

9.23 Ϯ 0.64

227 Ϯ 25

165 Ϯ 19

550 Ϯ 52

Leu-enkephalin

Met-enkephalin

tial isotopic tagging and MS was used. For this, peptides were

By using higher concentrations of enzyme (20 n^M) and sub- extracted from heat-inactivated mouse brains and purified

strate (50 ␮M), it was possible to examine the processivity of away from proteins by microfiltration (10-kDa filter cut-off)

CPA4 cleavage. The sequential hydrolysis of amino acids from and then incubated with varying amounts of purified CPA4 or

the C terminus of neurotensin was observed (Fig. 3). The prod- in the absence of enzyme, for 1.5 h (Fig. 4). After heat inactiva-

uct of the cleavage of the C-terminal Leu was detected at 10 min tion of CPA4, the various samples were labeled with one of four

of incubation whereas hydrolysis of the penultimate Ile was isotopic tags, combined, and analyzed by liquid chromatogra-

observed at 1 h of incubation (Fig. 3). Further removal of the phy/mass spectrometry (LC/MS). Over 100 peptides were

Tyr was not detected, even upon prolonged incubation, pre- detected in the LC/MS analysis, including many of the brain

sumably because of the negative influence of the Pro in the P1 peptides tested in supplemental Table S2 (neurotensin, PEN,

position of this peptide (Fig. 3). The action of CPA4 (20 n^M) big SAAS, Met-enkephalin-Arg-Phe (i.e. the proenkephalin

with angiotensin (200 ␮M) also showed processivity; the heptapeptide), little SAAS, Leu-enkephalin, Met-enkephalin,

removal of C-terminal Leu, penultimate His, and antepenulti- and big LEN). Angiotensin and bradykinin were not detected in

mate Phe is sequentially observed at 10 min, 30 min, and 4 h of the brain peptidome, reflecting the low abundance of these pep-

incubation, respectively (data not shown).

tides in the brain. Quantification of the relative peptide levels in

Five of the peptides identified as CPA4 substrates were fur- the enzyme-treated and control incubations was performed by

ther investigated by determining the kinetic constants for determining peak intensity; representative data are indicated in

CPA4 catalyzed hydrolysis. The data are summarized in Table 2 Fig. 4. Many of the observed peptides showed roughly equal

and show a broad variation in K_mand k_catvalues for the differ- peak intensities; these are neither substrates nor products of

ent peptides. CPA4 cleaves neurotensin and Met-enkephalin- CPA4 under the incubation conditions employed. Some pep-

Ϫ1

Arg-Phe with high efficiency (43.8 and 9.43 ␮M

min

,

tides showed a decrease in peak intensity only for the sample

incubated with the highest amount of enzyme; these are weak

respectively) and low K_m(0.329 and 9.23 ␮M, respectively).

To evaluate the ability of CPA4 to cleave a larger number of substrates of CPA4. Other peptides showed a large decrease in

peptides, a quantitative peptidomics approach using differen- peak intensity for both the highest and the medium amount of

18390 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 24•JUNE 11, 2010

Characterization of Human Carboxypeptidase A4

showed four equal peaks for all of

the peptides observed in the present

study (19). Approximately half of

the detected peptides could be iden-

tified by MS/MS sequence analysis.

In addition to these sequenced pep-

tides, several other peptides were

tentatively identified based on

matches to known or predicted pep-

tides; the criteria for these matches

included an observed monoisotopic

mass that was within 0.004% of the

theoretical mass, a correct charge

(equal to the number of basic res-

idues plus the N terminus), and a

correct number of isotopic tags

incorporated (based on the num-

ber of free amines in the peptide).

These tentatively identified pep-

tides are included in Tables 3–5 and

supplemental Table S3 with the

sequence surrounded by parenthe-

ses; all other sequences in these

tables were confirmed by MS/MS

sequencing (in addition to fitting

the above criteria).

Analysis of the C-terminal resi-

due (i.e. the P1Ј position) of the 16

best substrates of CPA4 identified

from the brain peptidome showed a

predominance of Leu and Phe, with

14 of the 16 peptides containing one

of these two amino acids (Table 3

and Fig. 5). The other two peptides

in this group had C termini of Tyr or

Val. Similar analysis of the 24 weak

substrates of CPA4 showed these 4

amino acids to be present in the

C-terminal position, in addition to

Ile, Met, Ala, Thr, and His (Table 4

and Fig. 5). Analysis of the products

of CPA4 focused on the down-

FIGURE 4. Quantitative peptidomics scheme and representative results. Peptides were extracted from

microwave-irradiated mouse brain and separated from proteins by microfiltration with a 10-kDa filter. Aliquots

were incubated with purified CPA4 (20 ng or 30-fold dilutions) or in the absence of CPA4. After incubation, the

samples were labeled with one of four stable isotopic TMAB tags that differ in the number of hydrogens and

deuteriums. Samples were then pooled and analyzed by LC/MS. Representative data are shown. Peptides that

showed less than a 25% decrease in peak intensity for the sample incubated with the highest concentration of

enzyme (the D3 TMAB tag) were considered non-substrates, as shown for the proenkephalin fragment SPQLE-

DEAKELQ. Peptides that showed a major decrease with the highest concentration of enzyme (Ͼ50% decrease)

but less than a 25% decrease with the medium concentration of enzyme were considered weak substrates. An

exampleofaweaksubstrateistheneuropeptidedynorphinA8. Peptidesthatshowedasubstantialdecreasein

peak intensity for the highest concentration of enzyme and at least a 25% decrease for the medium concen-

tration of enzyme were considered good substrates. An example is little SAAS, an 18-residue peptide with a

C-terminal Leu. Peptides that showed an increase in peak intensity for the samples incubated with enzyme

wereconsideredproductsofCPA4. AnexampleislittleSAAS1–17, whichlackstheC-terminalLeuoflittleSAAS.

stream residue that was removed; of

the 10 products that were identified,

six required removal of a Leu, and

the other four required removal of

an Ile, Val, Tyr, or Ala (Table 5).

Analysis was also performed on the

peptides that were neither sub-

strates nor products of CPA4 (sup-

plemental Table S3 and Fig. 5). Of

the 50 peptides in this category,

only a small number had C-termi-

enzyme; these are good substrates of CPA4. Finally, some pep- nal Leu or Phe residues; the majority of these peptides con-

tides showed a large increase in peak intensity when incubated tained C-terminal residues that were basic (Lys and Arg),

with enzyme; these are products of CPA4. Control studies in acidic (Asp and Glu), polar (Ser, Asn, and Gln), or otherwise

which aliquots of mouse brain peptides were isotopically tagged not represented in the best substrate group (Ala, His, and

and analyzed by LC/MS, without incubation with enzyme, Pro).

JUNE 11, 2010•VOLUME 285•NUMBER 24

JOURNAL OF BIOLOGICAL CHEMISTRY 18391

Characterization of Human Carboxypeptidase A4

TABLE 3

Good substrates of CPA4

Good substrates are defined as those peptides affected (Ͼ25% decrease) by the medium concentration of enzyme and with the high concentration of enzyme greatly

reducing the level of peptide (Ͼ80% decrease). Sequences were determined by MS/MS analysis, as described under in “Experimental Procedures.” Mox, oxidized methio-

nine; pE, pyroGlu; z, charge; T, number of isotopic tags incorporated into peptide; Obs M, observed monoisotopic mass; Theor M, theoretical monoisotopic mass; ppm,

difference between Obs M and Theor M in parts per million; Ratio CPA4/no enzyme, the ratio in peak intensity between the sample incubated with enzyme and the sample

incubated without enzyme. When the peak intensity was below the background, the ratio is expressed as Ͻ0.10 or Ͻ0.20, depending on the signal strength and background

levels.

Gly in the P1 position. In contrast, only two of the non-cleaved

peptides had P1 aliphatic, aromatic, or basic residues (Fig. 6).

Furthermore, many of these non-cleaved peptides had acidic or

␣-helix-breaking residues in the P1 position. Thus, it appears

that although the C-terminal residue plays a critical role in

determining specificity of CPA4, the P1 residue also contrib-

utes to the cleavage specificity.

DISCUSSION

Summary of Cleavage Specificity of CPA4—The major find-

ing of the present study is the cleavage specificity of CPA4,

which has not been previously examined. Because of the poten-

tial link between CPA4 and some types of cancer (described in

the Introduction), it is important to determine the substrate

specificity of this enzyme so that predictions can be made

regarding its biological role(s). Based on the present results,

CPA4 is clearly a CPA-like enzyme with specificity for C-termi-

FIGURE 5. Analysis of the C-terminal residue (P1ꢂ) of peptides detected

using the quantitative peptidomics technique. The number of times each

amino acid was present in the C-terminal position was determined for good

substrates, weak substrates, and non-substrates, as defined in the legend to

nal aliphatic and aromatic residues, consistent with the sub-

strate binding pocket predicted from analysis of the crystal

Fig. 4.

structure of CPA4 (14–16). The three types of substrates used

in the present study (small chromogenic substrates, selected

synthetic peptides, and a mixture of brain peptides) provide

complementary information. Although the small chromogenic

substrates are ideal for kinetic analysis of k_catand K_mvalues,

these are not natural substrates, and the chromogenic group

can potentially affect the cleavage specificity. Synthetic pep-

tides are useful to study precise substrate/product relationships

but are limited by the expense and the time to analyze each

peptide. The quantitative peptidomics approach provides a

great deal of information regarding which peptides are sub-

strates, which are products, and which are not cleaved by CPA4.

However, this approach does not provide kinetic information,

The finding that some but not all of the peptides with C-ter-

minal aliphatic and aromatic residues could be cleaved by

CPA4 under the conditions tested suggested that additional

residues contributed to the preference of this enzyme for par-

ticular peptides. To analyze this, we examined the P1 residue in

all peptides with a C-terminal hydrophobic residue. Altogether,

there were 62 peptides with a C-terminal Leu, Ile, Val, Met, Ala,

Phe, Tyr, His, or Thr; of these, 40 were found to be substrates,

and 22 were not cleaved under the conditions tested. The

majority of the substrates contained an aliphatic residue (Leu,

Ile, Val, Met, or Ala), aromatic (Phe or Tyr), or basic (Arg or

Lys) residue in the P1 position (Fig. 6). Furthermore, none of the and it is not possible to follow individual reactions, as can be

substrates contained an acidic residue (Asp or Glu), a Pro, or a done with the synthetic peptides. Therefore, these three

18392 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 24•JUNE 11, 2010

Characterization of Human Carboxypeptidase A4

TABLE 4

Weak substrates of CPA4

Weak substrates are defined as those peptides that were not substantially affected by the medium concentration of enzyme (less than 25% change) but showed a decrease

with the high concentration of enzyme. Abbreviations are as defined in Table 3. Peptide sequences in parentheses are tentative identifications, based on similarities to known

peptides, without MS/MS sequence information. These similarities include the monoisotopic mass, the charge (based on the number of basic residues in the peptide), and

the number of isotopic tags incorporated into the peptide (based on the number of free amines). See “Experimental Procedures” for further details.

TABLE 5

Products of CPA4

These peptides are defined as those that increased Ͼ100% with one or more of the concentrations of CPA4. Abbreviations are as defined in Table 3. nd, not detected. For

those peptides not detected in the “no enzyme” control, the ratio is expressed using the greater than sign; relative levels between the different concentrations of enzyme are

reflected in the different lower limits for the increase. The amino acid cleaved by CPA4 to generate the observed peptide is indicated in column 4.

approaches have overlapping strengths and collectively provide substrates, allowing measurements of CPA2 toward substrates

a more complete picture than a single approach.

with small hydrophobic C-terminal amino acids, which have

Taken together, the optimal C-terminal residue for CPA4 not previously been reported. In most cases, the improvement

cleavage is Leu, Ile, Val, Met, Tyr, or Phe (Fig. 7). Other accept- of catalytic efficiency for CPA2 toward FA-Phe-Xaa substrates

able amino acids in this position include Ala, His, Thr, or Trp. versus N-benzyloxycarbonyl-Gly_n-Xaa substrates is due to

CPA4 does not cleave C-terminal Pro, basic residues (Arg and lower K_mvalues, but in some cases an increase in k_catvalues also

Lys), acidic residues (Asp and Glu), or polar residues, such as contributes to the difference (33). This effect may be explained

Ser, Asn, or Gln. Although Thr can be considered a polar resi- by the different amino acids in the penultimate position of these

due, it is less polar than Ser. No information on Gly or Cys in the substrates (Phe versus Gly); if CPA2 is similar to CPA4 as

C-terminal position is available. The P1 position also has an described above from the peptidomics analysis, Phe is much

influence on cleavage specificity, with aliphatic, aromatic, and preferred over Gly in the P1 position. In addition, the FA-block-

basic residues favorable in this position (Fig. 7). Certain amino ing group is known to enhance the CP activity (31). The kinetic

acids have a negative influence in the P1 position, such as acidic data for CPA2 fully agree with the data reported by Reverter et

residues, Pro, and Gly (Fig. 7). No information on Trp or Cys is al. (33), showing a preference for FA-Phe-Trp and FA-Phe-Phe.

available for the P1 position.

Compared with FA-Phe-Trp, the k_cat/K_mvalues are 85- and

Comparison among CPAs—The FA-Phe-Xaa series of sub- 335-fold lower for FA-Phe-Leu and FA-Phe-Ile, respectively. In

strates offer higher k_cat/K_mvalues than other small synthetic contrast with these previous reports, we were able to estimate

JUNE 11, 2010•VOLUME 285•NUMBER 24

JOURNAL OF BIOLOGICAL CHEMISTRY 18393

Characterization of Human Carboxypeptidase A4

k

_cat/K_mvalues for FA-Phe-Phe are approximately the same for

CPA1 and CPA2 and 4 times lower for CPA4.

A comparison between CPA4 and CPA6 activities toward the

same group of peptides shows several differences. CPA6 cleaves

both Leu and His from the C terminus of angiotensin I and

des-Asp¹angiotensin I, whereas CPA4 only removes the Leu

and not the His under the conditions described in sup-

plemental Table S2. However, CPA4 can cleave both the Leu

and His in angiotensin when higher enzyme and peptide con-

centrations are used (data not shown). Furthermore, FA-Phe-

His is cleaved by CPA4 at a low rate, and in the peptidomics

study, two peptides that contain C-terminal His residues were

found to be weak substrates of CPA4. Therefore, CPA4 is able

to cleave His from the C terminus of some substrates. In addi-

tion to CPA4 and CPA6, previously it was found that CPA1 (34)

and CPA3 (35) can cleave C-terminal His residues. Although

His is commonly considered to be a basic residue, at neutral pH

values, it will be mainly unprotonated and resemble an aromat-

ic/aliphatic residue. Thus, it is not surprising that this residue is

cleaved by CPA-like enzymes.

FIGURE 6. Analysis of the P1 residue of peptide containing hydrophobic

C-terminal residues. For this analysis, only peptides that were substrates or

potential substrates were considered, based on the analysis shown in Fig. 5.

Those peptides with acidic, basic, or polar C-terminal residues were excluded.

This analysis included 40 substrates (good and weak substrates were com-

bined) and 22 non-substrates with similar C-terminal residues as the sub-

strates (i.e. Leu, Ile, Val, Met, Ala, Phe, Tyr, and His). The frequency of the

various amino acids in the P1 position of each group were determined and

combined into groups based on amino acid type: aliphatic (Leu, Ile, Val, Met,

and Ala), aromatic (Phe and Tyr), basic (Arg and Lys), acidic (Asp and Glu),

␣-helix breakers (Pro and Gly), and polar (Ser, Thr, Gln, and Asn). His was

placed into its own group, and there were no data for Cys or Trp because

these amino acids were not present in the P1 position in any of the peptides

analyzed.

The substrate specificity of CPA4 determined in the present

study fits very well with predictions made from structural anal-

ysis of this enzyme in complex with a hexapeptide (16) and with

results from structural analysis of many CPAs and CPBs (1).

The specificity of CPA4 for C-terminal hydrophobic residues,

such as Leu, Ile, Val, Met, Tyr, and Phe, and weaker hydrolysis

of Trp match more closely the specificity of CPA1 rather than

CPA2. The S1Ј specificity pocket is delimited by the side chains

in positions 194, 203, 207, 243, 247, 250, 253–255, and 268

(using the numbering system of bovine CPA1). The residue in

position 255 (Ile for CPA4) is considered to be the major deter-

minant of substrate specificity for carboxypeptidases of the

CPA/CPB subfamily (36). In CPA4, the residues in positions

203, 247, and 250 (numbering system of CPA1), which are Met,

Val, and Ala, respectively, also contribute to the hydrophobicity

of the S1Ј binding pocket. A comparison of the amino acids that

shape the S1Ј pocket between CPA1, CPA2, and CPA4 explains

FIGURE7. SummaryofcleavagespecificityofCPA4, basedonresultsfrom

synthetic chromogenic substrates, synthetic peptides, and endogenous

mouse brain peptides. Those amino acids considered most favorable for the strong preference of hCPA2 for bulkier hydrophobic amino

cleavage by CPA4 are listed in the top row and include Leu, Ile, Val, Met, Tyr,

andPheintheP1Ј positionandthesesameresiduesplusArgandLysintheP1

position. Residues that are acceptable in these positions are listed in the mid-

dle row, and those residues that appear to be inhibitory for cleavage are listed

acids in comparison with CPA1 and CPA4 (supplemen-

tal Table S4 and Fig. S1). The presence of Ala in position 268 in

CPA2, in contrast to the Thr present in CPA1 and CPA4, makes

in the bottom row.

the specificity pocket of CPA2 larger and more hydrophobic,

explaining the specificity of CPA2 for large amino acids like Trp

k

_cat/K_mvalues for FA-Phe-Met and FA-Phe-Val that are ϳ1000 (37, 38). The amino acid in position 253 may also be responsible

times lower than the value for FA-Phe-Trp.

for this difference. The preference of CPA1 for C-terminal Phe

Although CPA1 has been well studied over the past 50 years, is more marked than that of CPA4; this preference can be

particularly bovine CPA1 (originally referred to as CPA in the explained by the presence of a more hydrophobic environment

literature), the kinetic constants for human CPA1 have not in the S1Ј pocket of CPA1 provided by Ile²⁴³and Ile²⁴⁷, com-

been previously reported. The preference of CPA1 for Phe in pared with the Thr and Val in similar positions in CPA4.

the C-terminal position is more marked than that of CPA4

In the present study, the S1 subsite was also found to contrib-

when the k_cat/K_mvalues for different substrates are compared. ute to the specificity of CPA4 (Fig. 7); critical residues in this

The k_cat/K_mratio of CPA1 for FA-Phe-Phe is ϳ2-fold greater subsite are the Tyr in position 198 and Ser in position 199 (16).

than that of FA-Phe-Leu and FA-Phe-Val, 4 times higher than Other residues known to contribute to the shape of the S1 sub-

that of FA-Phe-Ile, and 10-fold higher than the corresponding site of CPAs include Val²⁴⁷, Tyr²⁴⁸, Phe²⁷⁹, and Glu²⁷⁰, which

values for FA-Phe-Trp, FA-Phe-Met, and FA-Phe-Ala. When form a mainly hydrophobic pocket that may explain the prefer-

k

_cat/K_mvalues are compared among the three enzymes, CPA1 ence of CPA4 for substrates with hydrophobic amino acids in

and CPA2 appear to be more efficient than CPA4 in the the P1 position. The preference of CPA4 for positively charged

removal of C-terminal residues from peptides. For example, amino acids over negatively charged amino acids in the P1 posi-

18394 JOURNAL OF BIOLOGICAL CHEMISTRY

VOLUME 285•NUMBER 24•JUNE 11, 2010

Characterization of Human Carboxypeptidase A4

tion could be explained by interactions between the side chain Additional roles for some of these products include the stimu-

of the P1 residue with the polar portion of Tyr¹⁹⁸and Ser¹⁹⁹in lation of proliferation in tumor cells (46) and invasive potential

the S1 binding site (1).

of prostate cancer cells partially through enhancement of cell

Potential Function of CPA4—The finding that PCPA4 is mobility (47). Similarly, neurotensin is also a well studied neu-

secreted from cells in a soluble form and, once activated, has a ropeptide that has recently been proposed to contribute to the

neutral pH optimum suggests a function in the extracellular progression of several types of cancer, including prostate,

environment rather than within the secretory pathway. breast, and pancreatic cancer (48–50). Because the enkepha-

Although trypsin was used to activate PCPA4 in the present lins, secretogranins, and neurotensin are secreted from some of

study, this is not likely to represent the physiological activating the same tissues where CPA4 is also secreted, it is likely that this

enzyme because trypsin is pancreatic, whereas CPA4 has a enzyme plays a role in the termination of the action of these

broader distribution. There are a number of trypsin-like peptides as well as many others. A role for CPA4 in the inacti-

enzymes expressed in the brain and other tissues where PCPA4 vation of peptides that function in cell proliferation would

is found (39). Based on modeling and structural considerations, potentially provide a mechanism to account for the previous

the conversion of PCPA4 into active CPA4 requires cleavage at studies linking CPA4 with aggressiveness of some forms of can-

a single Arg in the sequence EGQER-SS. It is possible that the cer. Additional research efforts are needed to characterize and

activation of PCPA4 may be an important control point that understand the biological effects of this enzyme and its single-

regulates the active form of the enzyme.

nucleotide polymorphism variant(s) in the tumoral process.

The distribution of CPA4 provides important clues as to the

function of this enzyme. Huang et al. (9) found CPA4 mRNA to

be expressed at high levels in different prostate cancer cell lines

and at extremely low levels in some normal human tissues,

including prostate and pancreas. CPA4 mRNA was induced in

prostate cancer cell lines upon treatment with 10 m^Msodium

butyrate, indicating that its expression is associated with in

vitro differentiation. It was also reported that CPA4 expression

is associated with hormone-regulated tissues. Through an

imprinting analysis, Kayashima et al. (12) found that CPA4

mRNA was also highly expressed in fetal tissues that are grow-

ing or differentiating with the transcriptionally active state of

chromatin. Using RT-PCR, Bentley et al. (11) detected CPA4

expression in a wide variety of tissues, including placenta, testis,

uterus, heart, brain, intestine, kidney, and muscle. In mouse

brain, in situ hybridization showed relatively high expression of

CPA4 mRNA in the olfactory bulb, specifically in the granular

and mitral layers (40), in an expression pattern similar to that of

CPA6 mRNA (41). A United States patent using TaqMan anal-

ysis of human tissues reported expression of CPA4 in kidney,

spinal cord, and brain, with moderate levels in cortex and con-

siderably higher levels in hypothalamus, especially in the arcu-

ate and paraventricular nuclei (42). Taken together, it is clear

that CPA4 mRNA is present in many tissues.

Acknowledgments—Mass spectrometry was performed in the Labora-

tory for Macromolecular Analysis and Proteomics of the Albert Ein-

stein College of Medicine, directed by Dr. Ruth Angeletti. We acknowl-

edge the technical assistance of the Cell Culture Facility of the Institut

de Biotecnologia i de Biomedicina and the Microscopy Facility, both

at the Universitat Auto`noma de Barcelona. We thank Dr. Peter Lyons

for helpful comments.

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