Comparative substrate specificity analysis of recombinant human cathepsin V and cathepsin L

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Archives of Biochemistry and Biophysics 430 (2004) 274–283

ABB

Comparative substrate specificity analysis of recombinanthuman cathepsin V and cathepsin L

Luciano Puzera, Simone S. Cotrina, Marcio F.M. Alvesa,Tobore Egborgec, Mariana S. Araujob, Maria Aparecida Julianoa,

Luiz Julianoa, Dieter Brommec, Adriana K. Carmonaa,*

a Department of Biophysics, Escola Paulista de Medicina, UNIFESP, Rua Tres de Maio, 100 Sao Paulo 04044-020, Brazilb Department of Biochemistry, Escola Paulista de Medicina, UNIFESP, Rua Tres de Maio, 100 Sao Paulo 04044-020, Brazil

c Department of Human Genetics, Mount Sinai School of Medicine, Box 1498, Fifth Avenue at 100th Street, New York 10029, USA

Received 16 June 2004, and in revised form 6 July 2004

Available online 6 August 2004

Abstract

Cathepsins V and L have high identity and few structural differences. In this paper, we reported a comparative study of the

hydrolytic activities of recombinant human cathepsins V and L using fluorescence resonance energy transfer peptides derived from

Abz-KLRSSKQ-EDDnp (Abz = ortho-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine). Five series of

peptides were synthesized to map the S3 to S02 subsites. The cathepsin V subsites S1 and S3 present a broad specificity while

cathepsin L has preference for positively charged residues. The S2 subsites of both enzymes require hydrophobic residues with

preference for Phe and Leu. The S01 and S0

2 subsites of cathepsins V and L are less specific. Based on these data we designed

substrates to explore the electrostatic potential differences of them. Finally, the kininogenase activities of these cathepsins were

compared using synthetic human kininogen fragments. Cathepsin V preferentially released Lys-bradykinin while cathepsin L

released bradykinin. This kininogenase activity by cathepsins V and L was also observed from human high and low molecular

weight kininogens.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Lysosomal proteases; Cysteine proteases; Cathepsin L; Cathepsin V; Fluorogenic substrates; Bradykinin; Kininogen

Mammalian lysosomal cysteine proteases are general-

ly described as enzymes that randomly degrade proteins

in lysosomes. Recently it was demonstrated that some

lysosomal cysteine proteases are also involved in selec-

tive and controlled processes and have specific functionsassociated to their restricted tissue localization [1–4].

This is the case of cathepsin V that is mainly expressed

in thymus, testis, and corneal epithelium [5–7]. The in

vivo role of cathepsin V has been associated with a role

in MHC class II presentation molecule in humans [7,8].

In pathological situations, cathepsin V has been consid-

0003-9861/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.abb.2004.07.006

* Corresponding author. Fax: +55-11-5575-9617.

E-mail address: [email protected] (A.K. Carmona).

ered as a potential diagnostic marker for colon tumors

[5]. In a recent report, cathepsin V was described to be

involved in elastolytic activity of activated macrophages

and together with the lysosomal cathepsins L, K, and S

participates in atherosclerosis [9,10].Despite its selective expression, several pieces of ev-

idence demonstrated a close link between this enzyme

and the ubiquitously expressed cathepsin L. The se-

quences of the two enzymes are quite similar sharing

80% of identity. Cathepsin V was mapped to the chro-

mosomal region, 9q22.2, a site adjacent to the cathep-

sin L locus [7], suggesting the evolution of both

cathepsins from a common ancestor by gene duplica-tion. The similarities in genomic organization suggest

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L. Puzer et al. / Archives of Biochemistry and Biophysics 430 (2004) 274–283 275

that cathepsins V and L diverged late in evolution [7].

The crystal structure of cathepsin V at a resolution of

1.6 A associated with an irreversible vinyl sulfone in-

hibitor was reported and the main differences between

cathepsin V and related enzymes are located at subsites

S2 and S3 [11]. The S3 subsite of cathepsin V is differentfrom those of other cysteine proteases by presenting

Arg at position 70, which may modulate the specificity

features in the enzyme [11]. Differences regarding the

cathepsin V cleavage efficiency were reported compar-

ing with those of cathepsins L and S using few pepti-

dyl-MCA substrates. The study demonstrated that the

kcat/Km values for the hydrolysis of Z-FR-MCA by ca-

thepsin L are approximately 50 times higher than forcathepsin V [7].

In the present work, we show a comparative analysis

of the S3 to S02 subsite specificity of cathepsins V and L

using fluorescence resonance energy transfer (FRET)1

peptides derived from the lead sequence Abz-KLRS

SKQ-EDDnp (Abz = ortho-aminobenzoic acid and

EDDnp = N-(2,4-dinitrophenyl)ethylenediamine). This

peptide was designed based on the results of a solid-phase substrate library for cysteine proteases [12,13].

Five series of peptides were generated: Abz-XLRSS

KQ-EDDnp, Abz-KXRSSKQ-EDDnp, Abz-KLXSSK

Q-EDDnp, Abz-KLRXSKQ-EDDnp, and Abz-KLRS

XKQ-EDDnp (X = different amino acids). Based on

these results the peptides Abz-EE-eNH2Cap-ELKLQ-

EDDnp and Abz-KK-eNH2Cap-ELKLQ-EDDnp were

synthesized to explore electrostatic surface differences ofboth enzymes.

The cysteine proteases have been reported to re-

lease kinin from natural kininogen or from synthetic

bradykinin-containing fragment of kininogen. The ki-

ninogenase activity of vegetal cysteine proteases was

reviewed by Prado [14] and those from pathogenic

bacteria were described and related to their invasive-

ness and pathogenicity [15–17]. The cathepsin L-likeproteases from Trypanosoma cruzi (cruzipain) [18]

and from Fasciola hepatica [19], as well as recombi-

nant human cathepsin L itself [20,21], were described

to have kininogenase activities. To further compare

the functions of cathepsins V and L, the kininogenase

activities of both enzymes were examined using syn-

thetic peptides derived from human kininogen as well

as the human high and low molecular weight kinino-gens (HMWK and LMWK).

1 Abbreviations used: Abz, ortho-aminobenzoic acid; DTE, dithio-

erythritol; E64, trans-epoxysuccinyl-LL-leucylamido-(4-guanidino)bu-

tane; EDDnp, N-(2,4-dinitrophenyl)ethylenediamine; HMWK, high

molecular weight kininogen; FRET, fluorescence resonance energy

transfer; LMWK, low molecular weight kininogen; MALDI-TOF,

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

spectroscopy; eNH2Cap, e-amino-caproic acid.

Materials and methods

Enzymes

Recombinant human cathepsins V and L were ex-

pressed in Pichia pastoris as previously described [22].The molar concentrations of these enzymes were deter-

mined by active site titration with E-64 according to

[23]. Stock solutions of the enzymes were prepared in

the presence of 0.01% Tween 20 (v/v) to increase the sta-

bility of the enzyme during the assays [24].

Peptides

The FRET peptides were synthesized by the solid-

phase synthesis method as described elsewhere [25]. Pep-

tide synthesis using the Fmoc-chemistry was performed

on an automated bench-top simultaneous multiple solid-

phase peptide synthesizer (PSSM 8 system, Shimadzu,

Japan). All peptides obtained were purified by semi-pre-

parative HPLC on an Econosil C-18 column. The mo-

lecular weight and purity of synthesized peptides (94%or higher) were checked by amino acid analysis and

MALDI-TOF mass spectrometry, using a TofSpec-E

from Micromass, Manchester, UK. Stock solutions of

the peptides were prepared in DMSO, and the concen-

trations were measured spectrophotometrically using

the molar extinction coefficient of 17,300M�1cm�1 at

365nm.

Enzyme assays

The FRET peptides were assayed in a Hitachi F-

2500 spectrofluorometer, at 37 �C. The assays were per-

formed in 50mM sodium acetate buffer containing

100mM NaCl and 2.5mM EDTA, pH 5.5. The en-

zymes were pre-activated in the presence of 2.5mM

DTE for 5min at 37 �C before the addition of thesubstrates. The fluorescence changes were monitored

continuously at kex = 320nm and kem = 420nm. The

enzyme concentrations for initial rate determinations

were chosen at a level intended to hydrolyze less than

5% of the amount of added substrate. The slope was

converted into micromoles of substrate hydrolyzed

per minute based on a calibration curve obtained from

the complete hydrolysis of each peptide. The inner-fil-ter effect due to intact substrates was corrected using

an empirical equation as previously described [26],

and for 25lM substrate concentration the maximum

correction was less than 15%. The substrate concentra-

tion for kinetic parameter of hydrolysis determination

was between two Km values higher and lower of the

obtained value. The kinetic parameters Km and kcatwere calculated by non-linear regression Grafit pro-gram [27]. The kcat/Km values were calculated as the ra-

tio of these two parameters. The apparent second-order

276 L. Puzer et al. / Archives of Biochemistry and Biophysics 430 (2004) 274–283

rate constant kcat/Km was also determined under pseu-

do first-order conditions, where [S] � Km and per-

formed at two different substrate concentrations for

the substrates with Km values higher than 6lM. For

peptides hydrolyzed at more than one site, the appar-

ent kcat/Km values correspond to the sum of the indi-vidual values of kcat/Km for each cleavage site and

the resulting product from each site corresponds to

the ratio of the individual kcat/Km values [28,29]. In

these cases, the cleavages are mutually exclusive, and

the product of one cleavage is not a substrate for the

cleavage at other site. The errors were less than 5%

for any of the obtained kinetic parameters.

Determination of the cleavage sites

The fragments resulting from hydrolysis of FRET

peptides were isolated by HPLC on an Ultrasphere

C-18 column (5lm; 4.6mm · 150mm) and the scissile

bonds were identified by MALDI-TOF mass spectrom-

etry (Tof-Spec-E, Micromass).

The effect of ionic strength on catalytic activity

The influence of NaCl on the catalytic activity of

cathepsins V and L was investigated using Abz-ALRS

SKQ-EDDnp, Abz-KLRSSKQ-EDDnp, and Abz-EL

RSSKQ-EDDnp as substrates, at 37 �C, in 50mM sodi-

um acetate buffer and 2.5mM EDTA, pH 5.5, over a

NaCl range of 0–500mM. The enzymes were pre-acti-vated in the presence of 2.5mM DTE for 5min at

37 �C before the addition of the substrates. The enzy-

matic activity was followed as described above.

Kininogenase activity on HMWK and LMWK

Stoichiometric amounts of cathepsins L and V

(200nM of each enzyme) and LMWK or HMWK(200nM), both supplied by Calbiochem, were used

to study the kininogenase activity. The buffer system

used was 0.10M sodium phosphate, pH 6.5, contain-

ing 1mM EDTA. The enzymes were pre-activated

with 2.5mM DTE for 5min, at 37 �C, before the addi-

tion of the kininogens. The reaction mixtures, in a fi-

nal volume of 80lL, were incubated at 37 �C for 1h.

The assays in the presence of 1.0lM E64 were per-formed under the same conditions. Ethanol (3:1, v/v)

was added and the mixture was centrifuged at 1000g

for 15min. The kinin content in the supernatant was

measured by radioimmunoassay as previously de-

scribed [30]. For comparison, human urinary kallik-

reins (200nM) were incubated with HMWK and

LMWK, both at concentration of 200nM. The assays

were performed in the conditions described above ex-cept for the buffer, which was 0.05M Tris–HCl, con-

taining 0.15M NaCl, pH 9.0.

Results and discussion

Despite the sequence identity and structural similarity

between cathepsins V and L, noteworthy differences re-

garding substrate specificity were observed. The peptides

assayed as substrates derived from the sequenceAbz-KLRSSKQ-EDDnp, and, if not otherwise speci-

fied, were all cleaved only at the Arg–Ser bond by cath-

epsins V and L. This peptide is an analogue of

Abz-KLRFSKQ-EDDnp that was designed based on

the results of a solid-phase substrate library screen for

other cysteine peptidases [12,13] and used in the cathep-

sin K specificity studies [31]. The substitution of Phe by

Ser in the P01 position of the reference sequence avoided

the hydrolysis in more than one peptide bond in the ma-

jority of the substrates. Five series of peptides were gen-

erated with variations at P3 to P02 to explore the

specificity of the S3 to S02 subsites (Tables 1–5). For the

substrates cleaved in more than one peptide bond, it

was previously demonstrated that the apparent kcat/Km

values correspond to the sum of the individual kcat/Km

values for each cleaved bond [28,29]. In comparisonwith cathepsin L, described as the most powerful en-

zyme among lysosomal peptidases [32–34], all the as-

sayed substrates were hydrolyzed by cathepsin V with

at least one order of magnitude lower kcat/Km values

due to lower kcat and higher Km values.

Series of Abz-KLXSSKQ-EDDnp for mapping S1 spec-

ificity

Table 1 shows the kinetic parameters for the hydroly-

sis of the peptide series Abz-KLXSSKQ-EDDnp with

different amino acids in the X position. Except for the

substrate Abz-KLFSSKQ-EDDnp hydrolyzed at the

Phe–Ser and Ser–Ser bonds and Abz-KLLSSKQ-ED-

Dnp hydrolyzed at the Leu–Ser and Ser–Ser bonds, all

the other peptides from this series were cleaved by bothenzymes only at the X–Ser bond, indicating that X occu-

pied the P1 position. These double cleavages resulted

from the possibility of the peptides to provide two alter-

native hydrophobic amino acids to the S2 subsites of the

enzymes.

The results revealed that the S1 subsite of cathepsin V

presents a rather broad specificity hydrolyzing sub-

strates with Leu, Gln, Arg, and Lys in P1 position withsimilar catalytic efficiencies, while cathepsin L presents

a clear preference for Arg followed by Lys residues

(Table 1). The preference of cathepsin L for basic amino

acids at the P1 position is in accordance with the previ-

ously reported results obtained with positional scanning

fluorogenic substrate libraries [34].

The ratios of kcat/Km values of cathepsins L and V

presented in Table 1 clearly show the preference of ca-thepsin V compared to cathepsin L for residues contain-

ing aliphatic side chains. These results are in accordance

Table 1

Kinetic parameters for the hydrolysis of the peptide series Abz-KLXSSKQ-EDDnp by recombinant human cathepsins V and L for the

characterization of the S1 subsite specificity

X Cathepsin V Cathepsin L

kcat (s�1) Km (lM) kcat/Km (mM�1 s�1) *L/V kcat (s

�1) Km (lM) kcat/Km (mM�1 s�1)

F *kcat/Km = 1160 (FflS 40%; SflS 60%) 5.3 *kcat/Km = 6176 (FflS 70%; SflS 30%)

A 0.6 2.0 300 3.9 1.1 0.95 1158

V 0.5 0.77 649 2.6 0.5 0.28 1786

L *kcat/Km = 2222 (LflS 60%; SflS 40%) 0.9 *kcat/Km = 2000 (LflS 70%; SflS 30%)

I 0.2 0.54 370 6.4 0.4 0.17 2353

G 0.3 0.78 385 4.0 0.4 0.26 1538

M 0.4 1.3 308 2.8 0.4 0.47 851

P 0.7 8.6 81 0.2 0.1 6.0 17

N 0.4 2.3 174 4.2 1.1 1.5 733

Q 0.8 0.95 842 1.9 0.9 0.56 1607

S 0.2 3.4 59 23 1.5 1.1 1364

T 0.2 1.3 154 15.4 1.3 0.55 2364

D 0.04 6.8 5.9 9.0 0.2 3.8 53

E 0.3 6.3 48 9.8 2.6 5.5 473

K 1.9 2.0 950 19 3.1 0.17 18,235

R 1.3 1.2 1083 28.3 4.6 0.15 30,667

H 0.6 1.0 600 10.3 1.3 0.21 6190

Conditions for hydrolysis are described in Materials and methods. The apparent kcat/Km values for the substrates hydrolyzed in more than one peptide

bond (*kcat/Km) represent the sum of the individual kcat/Km values for each cleavage. *L/V are the ratios of kcat/Km values of cathepsins L and V.

Table 2

Kinetic parameters for the hydrolysis of the peptide series Abz-KXRSSKQ-EDDnp by recombinant human cathepsins V and L for the



kcat (s�1) Km (lM) kcat/Km (mM�1 s�1) kcat (s


F 2.7 2.5 926 13.5 0.26 51,923

Y 1.0 3.4 324 7.8 0.42 18,571

W 0.15 7.2 21 1.9 3.8 500

V 1.4 4.9 286 6.4 0.59 10,847

L 1.3 1.2 1083 4.6 0.15 30,667

I 0.6 1.7 353 2.3 0.17 13,529

G 0.07 7.4 9.5 0.5 9.6 52

M 0.2 7.5 27 4.0 2.8 1429

P 0.1 12.6 7.9 1.2 6.7 179

N 0.05 8.1 6.2 0.4 5.0 80

Q 0.1 9.8 10 1.7 8.5 200

S 0.05 13 3.8 1.9 12 158

T 0.07 9.8 7.1 0.4 4.9 82

D 0.03 15 2.0 0.4 35 11

E 0.08 12 6.7 0.4 9.7 52

R 0.15 7.5 20 3.2 2.6 1231

H 0.1 5.2 19 1.3 3.4 382

Conditions for hydrolysis are described in Materials and methods. All the peptides were cleaved at Arg–Ser bond as determined by HPLC/mass

spectroscopy.


with the recent report of higher elastolytic activity of ca-

thepsin V compared to cathepsin L [9], because elastin is

composed of large hydrophobic domains, which are very

rich in aliphatic residues as Pro, Val, Leu, and Ile [35].

Therefore, this preference of the S1 subsite of cathepsin

V seems to be a feature that distinguishes it from cathep-

sin L.

The differences in the electrostatic potentials on themolecular surfaces of cathepsins V and L may explain

these differences as cathepsin L is negative over extended

regions of the surface including the vicinity of the active

site cleft, while cathepsin V shows only few localized

patches of negative surface potentials and the entire

left-hand region of the visible surface tends to be weakly

positive [7]. Substrates with negatively charged amino

acids (Glu, Asp) and polar non-charged residues (Asn,

Ser, Thr) were poorly hydrolyzed by cathepsins V and

L. The peptide containing Pro was hydrolyzed withthe lowest kcat/Km value in this series by both enzymes.

It is noteworthy that the auto-activation of procathepsin

Table 3

Kinetic parameters for the hydrolysis of the peptide series Abz-XLRSSKQ-EDDnp by recombinant human cathepsins V and L for the





F *kcat/Km = 2250 (RflS 60%; LflR 40%) *kcat/Km = 10,000 (RflS 60%; LflR 40%)

A 4.0 1.6 2500 5.3 0.45 11,778

V 2.6 1.3 2000 2.1 3.4 6176

L *kcat/Km = 2667 (RflS 60%; LflR 40%) *kcat/Km = 5385 (RflS 60%; LflR 40%)

I 1.7 0.81 2125 2.3 0.23 10,000

G 0.1 5.3 19 0.1 2.3 43

N 3.2 1.5 2133 3.5 0.71 4930

Q 3.2 1.4 2286 3.8 0.75 5067

E 3.5 3.3 1061 4.0 2.2 1818

K 1.3 1.2 1083 4.6 0.15 30,667

R 3.0 1.4 2143 3.8 0.16 23,750

H 3.4 1.5 2267 5.9 0.35 16,857

Conditions for hydrolysis are described in Materials and methods. The apparent kcat/Km values for the substrates hydrolyzed in more than one

peptide bond (*kcat/Km) represent the sum of the individual kcat/Km values for each cleavage.

Table 4

Kinetic parameters for the hydrolysis of Abz-KLRXSKQ-EDDnp derivatives by recombinant human cathepsins V and L for the characterization of

the S10 subsite




F *kcat/Km = 727 (RflF 70%; SflK 30%) *kcat/Km = 49000 (RflF 60%; SflK 40%)

V 0.9 0.6 1500 5.5 0.23 23,913

L *kcat/Km = 3151 (RflL 55%; SflK 45%) *kcat/Km = 16923 (RflL 60%; SflK 40%)

I 0.8 1.2 667 3.9 0.40 9750

G 0.9 1.5 400 3.4 0.17 20,000

P Resistant Resistant

N 1.9 1.9 1000 6.3 0.20 31,500

Q 1.3 1.6 813 5.8 0.27 21,481

S 1.3 1.2 1083 4.6 0.15 30,667

E 1.1 4.1 268 4.9 1.7 2882

R 0.3 1.1 273 3.7 0.18 20,556

H 1.5 2.2 682 7.3 0.20 36,500



Table 5

Kinetic parameters for the hydrolysis of Abz-KLRSXKQ-EDDnp series by recombinant human cathepsins V and L for the characterization of the

S2 0 subsite specificity

Cathepsin V Cathepsin L

X kcat (s�1) Km (lM) kcat/Km (mM�1 s�1) kcat (s


F *kcat/Km = 2154 (RflS 70%; KflQ 30%) *kcat/Km = 25,000 (RflS 55%;KflQ 45%)

A 1.2 0.8 1500 4.1 0.23 17,826

V — — 2143 3.2 0.13 24,615

L *kcat/Km = 3286 (RflS 60%; KflQ 40%) *kcat/Km = 26,470 (RflS 80%; KflQ 20%)

I 1.5 0.6 2500 2.4 0.13 18,461

G 2.5 2.1 1190 4.8 0.24 20,000

P 2.0 3.3 606 6.0 0.35 17,143

N 1.2 1.9 632 2.3 0.19 12,105

Q 1.7 1.8 944 4.2 0.19 22,105

S 1.3 1.2 1083 4.6 0.15 30,667

E 2.2 3.6 611 4.1 0.72 5694

R 1.5 2.0 750 3.6 0.17 21,176

H 2.0 1.2 1667 3.7 0.14 26,429





V occurs by cleaving the peptide bond Asp(113)–

Leu(114) at pH 4.5 at which pH approximately 50% of

b-carboxyl of Asp is protonated since the pK of this

group is 4.0–4.5 [11]. The peptide Abz-KLDSSKQ-ED-

Dnp was the poorest substrate when the kinetic param-

eters of hydrolysis by cathepsin V were determined atpH 5.5, but its kcat/Km values obtained at pH 4.5 and

4.0 were 58 and 63mM�1 s�1, respectively. These in-

creases in the kcat/Km values confirm that the negative

charge group of Asp at P1 is highly unfavorable and

its protonation allowed the cleavage of the procathepsin

V at Asp carboxyl group during enzyme activation.

Series of Abz-KXRSSKQ-EDDnp for mapping S2 spec-

ificity

Table 2 shows the kinetic parameters for the hydroly-

sis of the peptide series Abz-KXRSSKQ-EDDnp. Cath-

epsins V and L cleaved all the peptides from this series

exclusively at the Arg–Ser bond, indicating that X occu-

pied the P2 position. The preference for hydrophobic al-

iphatic or aromatic residues at the P2 position is welldefined and typical for cysteine peptidases of the papain

superfamily [1–3]. Indeed, data from Table 2 confirmed

that the S2 subsite of cathepsins V presents a binding site

that accommodates very well hydrophobic amino acids

[7]. The peptides with Leu and Phe were hydrolyzed by

cathepsins V and L with the highest kcat/Km values. How-

ever, the S2 subsite of cathepsin V exhibits a slight prefer-

ence for Leu residues while cathepsin L better accepts thepeptide containing Phe in P2 position. This result con-

firmed that cathepsin V has an intermediate specificity

between cathepsins L and S due to its preference for

Leu in P2, as previously suggested using dipeptidyl-

MCA substrates [7]. The peptides containing Tyr, Val,

and Ile were also hydrolyzed by cathepsins V and L but

with lower efficiency. The peptide with Trp was the poor-

est substrate among those containing hydrophobic ami-no acids for both enzymes. Therefore, in cathepsin V,

besides the larger size of its S2 subsite than those of cath-

epsins S or L [11,36], it was not enough to fit the bulkier

indole site chain into the S2 pocket. All other substitu-

tions resulted in very poor substrates for both enzymes.

Series of Abz-XLRSSKQ-EDDnp for mapping S3 spec-

ificity

The series Abz-XLRSSKQ-EDDnp with different

amino acids in the X position mapped the specificity

of the S3 subsite of cathepsins V and L (Table 3) since

all the peptides were cleaved only at the Arg–Ser bond,

except the peptides Abz-FLRSSKQ-EDDnp and Abz-

LLRSSKQ-EDDnp, which were hydrolyzed by both en-

zymes at Arg–Ser or Leu–Arg bonds.The P3 specificity of cathepsin V has shown to be

broader than that of cathepsin L (Table 3). The reported

structure of cathepsin V shows that its S3 subsite differs

substantially from those of the other cysteine peptidases

[11], which is composed by Gln 61 and Arg 70. These res-

idues in the papain superfamily present little conservation

in the position [37]. The presence of the guanidino group

in the S3 pocket is especially interesting as none of thelysosomal cathepsins have Arg at this position [11]. The

acceptance by cathepsin V of substrates containing a neg-

atively charged Glu residue in P3 position might be ex-

plained by the interaction with the guanidino group of

the Arg70 in the S3 subsite. In contrast, cathepsin L

showed a high preference for basic amino acid residues

such as Lys and Arg as shown by the high kcat/Km values

(Table 3). These results are in accordance with Pauly et al.[36] who proposed that an acidic residue (Glu 64) in the S3subsite of cathepsin L allows the electrostatic interactions

with positive residues in P3. In addition, the presence of an

amino acid with a side chain in the P3 position seems to be

essential for the activities of cathepsins V and L because

the peptide, Abz-GLRSSKQ-EDDnp, was a very poor

substrate for both enzymes. These results do well agree

with the structural view of substrate–cysteine proteaseinteraction (for review, see [1]).

Series of Abz-KLRXSKQ-EDDnp for mapping S01 spec-

ificity

Table 4 shows the kinetic parameters for the hydrolysis

by cathepsins V and L of the Abz-KLRXSKQ-EDDnp

series. All peptides were cleaved by both enzymes onlyat the Arg-X bond, except Abz-KLRFSKQ-EDDnp,

which was hydrolyzed at the Arg–Phe and Ser–Lys bonds

and Abz-KLRLSKQ-EDDnp cleaved at the Arg–Leu

and Ser–Lys bonds. Significant preference for Val, fol-

lowed by Asn and Ser, was observed for the subsite S01

of cathepsin V. The peptide containing Pro was resistant

to hydrolysis.

Cathepsin L hydrolyzed substrates with His, Asn,Phe, and Ser in P0

1 with high efficiency and hydrolyzes

substrates with Arg, Gln, Gly, and Val at lower rates.

Glu in P01 represented the poorest substrate in the series

(with exception of the non-hydrolyzed Pro-containing

peptide). In contrast, cathepsin V hydrolyzed substrates

with Arg or Glu with a low but similar efficacy. These

differences are likely a consequence of the presence of

Glu141 and Glu192 in cathepsin L [7,35] that makes thissubsite more susceptible to interact with positive charg-

es. In contrast, cathepsin V possesses uncharged Ser141

and a Pro192 in these positions.

Series of Abz-KLRSXKQ-EDDnp for mapping S02 spec-

ificity

Table 5 presents the kinetic parameters for the hydro-lysis of the peptides from the series Abz-KLRSXKQ-

EDDnp. All the peptides of this series were cleaved by

Fig. 1. Effect of NaCl on the hydrolysis of the peptides (A)

Abz-ALRSSKQ-EDDnp, (B) Abz-KLRSSKQ-EDDnp, and (C)

Abz-ELRSSKQ-EDDnp by cathepsin V (s) and cathepsin L (�).


cathepsins V and L only at the Arg–Ser bond, except

Abz-KLRSFKQ-EDDnp and Abz-KLRSLKQ-EDDnp

that were hydrolyzed at the Arg–Set and Lys–Gln

bonds. Cathepsins V present a significant preference

for Val and Ile in P02. Leu seems to be also well accepted

but the kinetic parameter evaluation is precluded by thedouble cleavage sites of the Leu-containing peptide. Ca-

thepsin L seems not to have a clear preference for resi-

dues at P02 position, being the Ser- and Glu-containing

peptides hydrolyzed with the higher and lower kcat/Km

values, respectively.

Design of a selective substrate for cathepsin V and the

effect of ionic strength on the catalytic activities

In an attempt to improve the cathepsin V selectivity,

the differences at the electrostatic surfaces of cathepsins

V and L were explored. As discussed above, cathepsin L

is negatively charged over extended area of the molecular

surface including the vicinity of the active site cleft, while

in cathepsinV the entire left-hand region of the visible sur-

face is weakly positive [7]. To induce the interaction of thepeptides in the extended regions of the surface, we elon-

gated the substrate Abz-ELKLQ-EDDnp into the non-

primed site by introducing a pair of positively charged

residues, Lys–Lys, or of negatively charged, Glu–Glu,

attached to eNH2Cap group used as spacer. The follow-

ing peptides were synthesized: Abz-EE-eNH2Cap-ELK-

LQ-EDDnp and Abz-KK-eNH2Cap-ELKLQ-EDDnp.

The peptide Abz-KK-eNH2Cap-ELKLQ-EDDnp was abetter substrate for both enzymes than Abz-EE-eNH2-

Cap-ELKLQ-EDDnp (Table 6). However, Abz-EE-

eNH2Cap-ELKLQ-EDDnp was the only substrate

hydrolyzed by cathepsin V with kcat/Km value higher than

that of cathepsin L. Therefore, the electrostatic potential

differences between the two proteases seem to be a signif-

icant feature for cathepsin V.

We investigated the NaCl effect on the kcat/Km param-eter of hydrolysis by both cathepsins of the substrates

Abz-ALRSSKQ-EDDnp, Abz-KLRSSKQ-EDDnp,

and Abz-ELRSSKQ-EDDnp. These peptides with char-

ge differences in the P3 position were chosen because, as

discussed above, S3–P3 interactions depend only on the

P3 side chain. Fig. 1 presents the relative values of kcat/

Km for the hydrolysis of the three peptides by cathepsins

Table 6

Kinetic parameters for the hydrolysis of selective substrates based on the ele

Peptide Cathepsin V

kcat (s)�1 Km (lM) kca

Abz-ELKLQ-EDDnp 0.6 2.0 30

Abz-EE-eNH2Cap-ELKLQ-EDDnp 1.3 1.7 76

Abz-KK-eNH2Cap-ELKLQ-EDDnp 1.7 0.8 212

Conditions for hydrolysis are described in Materials and methods. The cleav

mass spectroscopy.

V and L in the presence of various NaCl concentrations

(0–500mM). Significant reductions of the specificity

constants, kcat/Km, for all three substrates were detected

for both enzymes in the presence of NaCl. However, the

ctrostatic potential at the molecular surface of cathepsins V and L

Cathepsin L

t/Km (mMs)�1 kcat (s)�1 Km (lM) kcat/Km (mMs)�1

0 1.2 1.1 1091

5 0.4 2.5 160

5 1.9 0.6 3167

age site for both enzymes was Lys–Leu bond as determined by HPLC/


hydrolysis of Abz-KLRSSKQ-EDDnp by cathepsin V

was more sensitive to NaCl when compared to the other

two peptides, which were still hydrolyzed with significant

efficiency until a concentration of 500mM. The pattern of

the NaCl effect on the hydrolysis of these peptides by ca-

thepsin L is similar but less pronounced to that observedfor cathepsin V. These results indicated that, for both en-

zymes, the positive charge of the P3 residue seems to have

significant electrostatic interaction with the enzymes,

even with cathepsin V that has less negative charges on

its surface when compared to cathepsin L. NaCl was re-

ported to also reduce the elastolytic activity of cathepsin

V, but to have no effect on the hydrolysis of the short sub-

strate Z-FR-MCA [9].

Hydrolysis of kininogen-related peptide

It has been described that cathepsin L releases kinin

from HMWK [20] and kininogen-derived peptides [21].

To verify the kininogenase activity of cathepsin V

and compare it with that of cathepsin L, the human

kininogen fragment containing bradykinin, Abz-LGMISLMKRPPGWSPFRSSRIW-NH2, was synthesized. In

this peptide, two residues of Trp were introduced in

the position 5 of bradykinin and five residues down-

stream the bradykinin sequence in order to provide fluo-

rescence detection of all potential fragments. Table 7

shows the cleavage sites of this peptide by cathepsins

Table 7

Hydrolysis of fluorescent-labeled kininogen-related peptide (Abz-LG

MISLMKRPPGWSPFRSSRIW-NH2) by cathepsins L and V and

identification of Trp-containing fragments

Enzyme Abz-LGMISLM›K›RPPGWSPFR›SSRIW-NH2

Molecular weight

Calculated Observed

ion (m/z)

Cathepsin L KRPPGWSPFR (30%) 1226.7 1227

RPPGWSPFR (70%) 1098.6 1099

SSRIW-NH2 645.7 647.4

Cathepsin V KRPPGWSPFR (90%) 1226.7 1227

RPPGWSPFR (10%) 1098.6 1099

SSRIW-NH2 645.7 647.4

›Cleavage sites. In bold the Trp-containing kinin collected by HPLC.

Table 8

Catalytic efficiency for hydrolysis by human cathepsins V and L of fluorescenc

and Arg389–Ser390 bonds of human kininogen sequence

Substrates Cathepsin V

Cleavage site kcat/Km (

Abz-MISLMKRPQ-EDDnp M-K (90%) K-R (10%) 404

Abz-GFSPFRSSRQ-EDDnp R-S 2154

Conditions for hydrolysis are described in Materials and methods. The is

percentage of each cleavage was quantified by the area of the peak in the UV

kcat/Km values for each cleavage.

L and V, which fragments were isolated by HPLC and

characterized by MALDI-TOF mass spectroscopy.

Cathepsin L cleaved this peptide at Leu–Met, Met–

Lys, and Arg–Ser bonds generating 30% of Lys-bradyki-

nin (KRPPGFSPFR) and 70% of bradykinin (RPPGFS

PFR). Cathepsin V cleaved preferentially the Met–Lysand Arg–Ser bonds generating 90% of Lys-bradykinin

and 10% of bradykinin. These activities of cathepsins

V and L were also examined by determining the kinetic

parameters for the hydrolysis of the peptides Abz-MIS-

LMKRPQ-EDDnp and Abz-GFSPFRSSRQ-EDDnp

that correspond to the sequences flanking the N- and

C-terminal ends of bradykinin in human kininogen.

Met–Lys and Lys–Arg bonds in the peptide Abz-MISLMKRPQ-EDDnp were cleaved by cathepsins L and

V with the same proportion described above for the

hydrolysis of Abz-LGMISLMKRPPGWSPFRSSRIW-

NH2. Table 8 shows the kcat/Km values for hydrolysis

of these FRET peptides. The Arg–Ser bond is hydro-

lyzed with the highest kcat/Km values, which is in accor-

dance with the observed accumulation of Abz-LGMISL

MKRPPGWSPFR-OH on the hydrolysis of Abz-LGMISLMKRPPGWSPFRSSRIW-NH2 by both peptidases.

As described above, S1 and S01 subsites of cathepsin L

interact very well with positive residues. This fact can

explain the preference of cathepsin L to cleave the

Lys–Arg bond in the kinin-related peptides placing a

Met residue in the subsite S2. This result shows a clear

competition of the subsites S2, S1, and S01 for the best

residue and indicated a high plasticity of cathepsin Lsubsites. The preferential cleavage of Met–Lys bond

Table 9

Radioimmunoassay of the generation of kinin from HMWK and

LMWK by cathepsins L and V and human tissue kallikrein (hK1)

Sample Kinin released (pg)

HMWK (1.9lg) LMWK (1.1lg)

Null 29 53

Cathepsin L 1700 2000

Cathepsin V 975 900

Cathepsin L + E64 50 50

Cathepsin V + E64 40 44

hK1 3500 4000

Experimental conditions are described under Materials and methods.

e resonance energy transfer peptides flanking the scissile Met379–Lys380

Cathepsin L

mM�1 s�1) Cleavage site kcat/Km (mM�1 s�1)

M-K (30%) K-R (70%) 1015

R-S 2410

olated HPLC fragments were identified by mass spectroscopy. The

detector of the HPLC. The value represented the sum of the individual


by cathepsin V seems to be due to its difficulty to accept

positive residues at S01 subsite and for its preference for

amino acids with aliphatic side chain at P2, as described

above.

The releasing of kinin from HMWK and LMWK by

cathepsins L and V was observed using stoichiometricamounts of enzymes and kininogen. The kinins released

were quantified by radioimmunoassay. This activity was

abolished by the addition of the irreversible cysteine

protease inhibitor E64, demonstrating the specificity of

assay. As shown in Table 9 the kininogenase activity of

cathepsin V on HMWK and LMWK kininogens in the

same conditions is approximately 20% of tissue kallikrein

and cathepsin L presented higher kininogenase activitythan cathepsin V. It is not possible to relate a physiologi-

cal role for this kininogenase activity in vitro of cathepsins

V and L using a substrate that contain cystatin domains

and are potent inhibitors of cysteine proteases. However,

we have observed in rat a dose–response decrease of blood

pressure related to kinin release after intravenous admin-

istration of cathepsin L (data not published). In addition,

the kininogenase activities of cathepsins L and V can bepart of their functions in atherosclerosis that is an inflam-

matory disease characterized by extensive modifications

of arterial wall architecture [10].

In conclusion,we described in detail the substrate spec-

ificity of human recombinant cathepsin V compared to

that of cathepsin L. The no striking differences pointed

in our study for the substrate specificity between

cathepsin L and V reflect the high structural similaritybetween the cysteine proteases and the physiological role

of cathepsin V remains a question for further investiga-

tion.

Acknowledgments

This work was supported by Fundacao de AmparoPesquisa do Estado de Sao Paulo (FAPESP), Conselho

Nacional de Desenvolvimento Cientıfico e Tecnologico

(CNPq), Human Frontiers for Science Progress (RG

00043/2000-M), and National Institutes of Health

Grants AR 46182 and AR 48669 (D.B.).

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Documents

Comparative substrate specificity analysis of recombinant human cathepsin V and cathepsin L