A novel metallocarboxypeptidase-like enzyme from the
marine annelid Sabellastarte magnifica – a step into the
invertebrate world of proteases
Maday Alonso-del-Rivero
1
, Sebastian A. Trejo
3
,Mo
´
nica Rodrı
´
guez de la Vega
3
, Yamile Gonza
´
lez
1
,
Silvia Bronsoms
3
, Francesc Canals
2
, Julieta Delfı
´
n
1
, Joaquin Diaz
1
, Francesc X. Aviles
3
and Marı

´
ai
Biomedicina (IBB) and Departament de
Bioquı
´
mica i Biologia Molecular, Universitat
Autonoma de Barcelona, 08193 Bellaterra
(Barcelona), Spain
Fax: +34 93 581 2011
Tel: +34 93 581 1231
E-mail:
(Received 16 March 2009, revised 16 June
2009, accepted 30 June 2009)
doi:10.1111/j.1742-4658.2009.07187.x
After screening 25 marine invertebrates, a novel metallocarboxypeptidase
(SmCP) has been identified by activity and MS analytical approaches, and
isolated from the marine annelid Sabellastarte magnifica. The enzyme,
which is a minor component of the molecularly complex animal body, as
shown by 2D gel electrophoresis, has been purified from crude extracts to
homogeneity by affinity chromatography on potato carboxypeptidase inhib-
itor and by ion exchange chromatography. SmCP is a protease of
33792 Da, displaying N-terminal and internal sequence homologies with
M14 metallocarboxypeptidase-like enzymes, as determined by MS and auto-
mated Edman degradation. The enzyme contains one atom of Zn per mole-
cule, is activated by Ca
2+
and is drastically inhibited by the metal chelator
1,10-phenanthroline, as well as by excess Zn
2+
or Cu

plants and bacteria, have been divided into three main
subfamilies based on structural similarity and sequence
homology. The first one, which includes the digestive
enzymes carboxypeptidase (CP) A (CPA) 1, CPA2,
carboxypeptidase B (CPB) 1 and mast cell CPA3, as
well as CPA4, CPA5 CPA6 and carboxypeptidase O
(CPO) (known at the gene level), has been termed sub-
family M14A or A ⁄ B; the second one, including the
bioactive peptide-processing or regulatory enzymes
(e.g. carboxypeptidases N, E, M and D, amongst oth-
ers) has been termed subfamily M14B or N ⁄ E [3]. Very
recently, a novel subfamily composed of enzymes of
larger size and apparently with a predominant cyto-
solic location, termed M14D, Nna-like or CCPs, has
been proposed [4]. Furthermore, three main classes
may be distinguished according to their substrate spec-
ificity: (a) for aromatic ⁄ hydrophobic residues (A-like),
(b) for basic residues (B-like) and (c) for acidic resi-
dues (O-like) [3,5].
MCP enzymes have been isolated from different
sources [3,5,6], mainly from vertebrates, but a few of
them have come from marine invertebrate organisms:
the digestive crayfish carboxypeptidase (CPB) [7], the
carboxypeptidase E-like enzyme from the sea hare
Aplysia californica, with important regulatory func-
tions in this organism [8], two CPs (A and B types)
from the hepatopancreas of the crab Paralithodes cam-
tschatica [9], the CPA-like protease from squid hepato-
pancreas of Illex illecebrosus [10], and CPs (two A and
one B type) isolated from the pyloric ceca of the starf-

The presence of a carboxypeptidase-like enzyme in
Annelida marine invertebrates has not been described
so far.
The present study describes the enzymatic activity
and MS detection of a novel MCP (termed SmCP)
from S. magnifica, and its occurrence as a minor com-
ponent within the animal body extracts by 2D- PAGE.
The enzyme has been isolated and purified, and then
characterized by size, metal content, location, basic
interactions, sequence analysis of different regions of
the enzyme, and by a description of the main parame-
ters related to enzyme kinetics, specificity and inhibi-
tion ranges, as well as other basic molecular features.
From this, it is apparent that SmCP is a novel M14
MCP (belonging to the pancreatic-like subfamily),
showing simultaneous CPA- and CPO-like activities,
which is an unusual feature. The present study com-
prises an attempt to expand the growing field of the
M14 family of proteolytic enzymes, which is now quite
diverse and contains more than 25 different variants
Fig. 1. S. magnifica Phylum Annelida, Class Polychaeta, Subclass
Palpata, Order Canalipalpata, Suborder Sabellida, Family Sabellidae,
Genus Sabellastarte [14] The ‘tentacle crown’ and the ‘body’ parts
of the animal are clearly visible.
A novel metallocarboxypeptidase from S. magnifica M. Alonso-del-Rivero et al.
4876 FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS
[4–6], but for which only very few members from
invertebrates have been characterized until now.
Results
Detection of MCP activities in marine organisms

was devoid of it.
‘Intensity fading’ MALDI-TOF MS
Once we focused our attention on S. magnifica body
extracts, we found there direct evidence of at least one
MCP enzyme, of approximately 35 kDa by ‘intensity
fading’ MALDI-TOF MS [17]. In the present study,
the added ‘binder’ was the recombinant form of potato
carboxypeptidase inhibitor (rPCI) (4.5 kDa), immobi-
lized on agarose beads, with the aim of both perturb-
ing the MS spectrum and capturing the MCP in the
body extract. The control spectra, as well as the ‘per-
turbed’ one (by rPCI addition, followed by removal of
the captured targets by sedimentation of the beads),
are shown in Fig. 2A,B. It is apparent that some of
the ion signals of the spectra were faded when the
extract was treated with immobilized PCI. Subse-
quently, MS analysis of the protein eluted from the
beads (Fig. 2C) detected a molecular ion of 34 kDa.
This molecular species, which is able to strongly inter-
act with PCI, presumably represents the CP-like
enzyme activity found in S. magnifica body extract.
The experiment indicates not only the occurrence in
the extract of the strong ligand (the enzyme SmCP) for
the added protease inhibitor, but also that this ligand
is probably functional in the very complex extract (i.e.
not in the zymogen state). It is worth noting that the
apparent simplicity of the MALDI-TOF spectrum of
the extract shown in Fig. 2C is most likely caused not
only by the low expansion scale used, but also by
1000

rose addition.
M. Alonso-del-Rivero et al. A novel metallocarboxypeptidase from S. magnifica
FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS 4877
‘signal suppression effects’; such phenomena usually
affect visualization of signals in media crowded in mol-
ecules [17–19], as will be reported and discussed subse-
quently.
Molecular complexity of the S. magnifica body
extract by 2D-PAGE
The molecular complexity of the S. magnifica extracts
(both from the body and from the crown, or mixed)
was demonstrated by 2D-PAGE analysis (Fig. 3). A
great number of visible protein bands [as revealed
either by staining with silver or using difference gel
electrophoresis (DIGE)] appeared in the analysis of
both parts of the animal, with a major presence of
bands in the body (upper part) versus the crown (lower
part). In Fig. 3, we show, in the uncombined
(Fig. 3A,B) or in the combined way (Fig. 3C), the pro-
tein components of both parts of the animal labeled
with fluorescent dyes using the DIGE approach. That
is, the different materials (i.e. crown and body extracts,
purified enzyme) were pre-labeled independently with
DIGE reagents before they were mixed and run simul-
taneously in a single 2D-PAGE separation. The inde-
pendent labeling of the crown and body extracts was
performed not only to allow the differential tracking
of their components, but also to deal with the very
high content of dyes and interfering materials from the
crown, which required a harsh cleaning (and denatur-

position of SmCP enzyme is shown when it was run in an individual 2D-PAGE (and visualized by immunostaining) The spots labeled with
numbers correspond to molecular species affected by affinity capture on the immobilized inhibitors cystatin C (3, 4, 5, 6, 7 and 14) and
soybean trypsin inhibitor (8, 9, 10, 11, 12 and 13), or on both (1 and 2).
A novel metallocarboxypeptidase from S. magnifica M. Alonso-del-Rivero et al.
4878 FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS
identify at least 14 proteins captured differentially for
the first two microcolumns, which are labeled with
numbers in Fig. 3B (1 and 2 by both; 3, 4, 5, 6, 7 and
14 by the cystatin one; and 8, 9, 10, 11, 12 and 13 by
the SBTI one). An initial validation of these assign-
ments as proteolytic enzymes (awating MS ⁄ MS analy-
sis) was made by ‘intensity fading’ MALDI-TOF MS
using the mentioned set of immobilized inhibitors,
employing a strategy similar to the one for PCI
described above.
It is important to note that the band corresponding
to the SmCP enzyme, the target of the present study,
did not appear at around 34 kDa, which is the mass
assigned to it as a potential MCP (see MALDI-TOF
MS analysis and below), when the extracts (either from
the body or body + crown) were analyzed. However,
such a band is clearly visible when the enzyme is puri-
fied, concentrated and subsequently applied to the 2D-
PAGE (Fig. 3, encircled region). We assume that such
a difference is a result of the very low abundance of
SmCP in the animal. Also, it is relevant that the use of
an antibody raised against the sequence around
Asn144-Arg145, preserved in CPs [4], gave rise to a
spot in the same location by immunostaining (not
shown), confirming its assignment.

Table 1. Summary of a typical purification procedure for SmCP
The assays were carried out as described in the Experimental
procedures. Substrate AAFP at 0.1 m
M, pH 7.5, 25 °C.
Step
Protein
(mg)
Enzymatic
activity
(U)
Specific
sctivity
(UÆmg
)1
)
Yield
(%)
Purification
(n-fold)
Extract 404 114 0.28 100 1
Affinity
chromatography
1.12 90 80.3 79 286
Ion exchange
chromatography
0.23 74 322 65 1150
14.2
12
28
34.1

400
600
800
15 000 20 000 25 000 30 000 35 000
m/z
40 000
Intens. (a.u.)
16 928.956
33 792.855.
A
B
C
Fig. 4. Purification of SmCP from the body extract of S. magnifica
and its molecular weight (A) Ion exchange chromatography on a
TSK-DEAE gel (7.5 · 7.5 cm) column Buffer A: 20 m
M Tris–HCl (pH
8.0); buffer B: 1
M Tris–HCl (pH 8.0) (I) Equilibration: 0% B for
45 min; (II) 60% B for 20 min; and (III) gradient 60% to 80% B for
170 min; flow rate: 68 cmÆh
)1
–––, A
280
; ,EnzAct; –––, Conc
NaCl (B) SDS ⁄ PAGE gel (125%) of the purified enzyme Lane 1,
Standard molecular weights [myosin (203 kDa), galactosidase
(120 kDa), bovine serum albumin (90 kDa), ovoalbumin (51 kDa),
carbonic anhydrase (34.1 kDa), soybean trypsin inhibitor (28 kDa)
and lysosyme (14.2 kDa)] Lane 2: Fraction of S. magnifica purified
by PCI-Sepharose and anionic exchange chromatography (C) MS

subfamily [1,4].
Fig. 5. Alignment of the amino terminal and internal sequences of SmCP with the sequences of carboxypeptidases from other organisms
SmCP sequences were derived after trypsin treatment of the purified enzyme followed by LC-MS ⁄ MS (de novo sequencing) and bioinfor-
matics analyses (see Experimental procedures) Similar and identical residues are shown in light and dark grey, respectively ‘Canonical’ resi-
dues of CP (based on bovine CPA1) that are present in the trypsin peptides of SmCP are labeled with an asterisk The sequences are CPA
from Aedes aegypti (yellow fever mosquito) (Q9U9K2 AEDAE); Carboxypeptidase A1 precursor from Mus musculus (CBPA1 MOUSE); car-
boxypeptidase A2 from Paralichthys olivaceus (Japanese flounder) (Q8QAXN5 PAROL); carboxypeptidase A1 precursor from Sus scrofa
(CBPA1 PIG); carboxypeptidase A1 precursor from Bos taurus (CPBPA1 BOVIN); carboxypeptidase homolog from B. jaraca (Q9PUF2 BOT-
JA); CPO from Homo sapiens (CBPO HUMAN); CPB from Astacus fluviatilis (broad-fingered crayfish) (CBPB ASTFL); CPA precursor from
H. armigera (cotton bollworm) (097434_HELAM); carboxypeptidase precursor from H. armigera (cotton bollworm) (Q6H962_HELAM); MCP
from Culicoides sonorensis (Q5QBL3_9DIPT); and carboxypeptidase A2 precursor from H. sapiens (CBPA2_HUMAN).
A novel metallocarboxypeptidase from S. magnifica M. Alonso-del-Rivero et al.
4880 FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS
Kinetic characterization of SmCP
Kinetic analyses for isolated SmCP was performed using
different types of standard synthetic substrates for carb-
oxypeptidases that were clearly cleaved by the enzyme.
The K
m
, k
cat
and k
cat
⁄ K
m
determined for the enzyme
against AAFP, N -benzoyl-Gly-Phe (Hippuryl-Phe) and
N-(3-[2-furyl]acryloyl)-Phe-Phe (FAPP) as substrates
are shown in Table 2. Such kinetic parameters indicate
that SmCP is highly efficient against the three CPA type

a well-known organic inhibitor of A-type carboxypep-
tidases, fully cancelled the enzyme activity, at 1 mm.
Furthermore, the addition of the protein inhibitor of
carboxypeptidases PCI (in fact rPCI, a recombinant
form, reactive towards CPA and CPB type enzyme) at
0.4 lm produced a 70% inhibition of SmCP activity.
The apparent K
i
value for this inhibitor towards SmCP
was 7.37 · 10
)8
m; however, the adjusted value
considering the substrate-induced dissociation was
2.45 · 10
)8
m. Another protein inhibitor from leech
(rLCI, also recombinant) at 13.5 lm produced a 70%
inhibition of SmCP activity. The estimated K
i
value
for rLCI was 2.95 · 10
)8
m, and its adjusted value
considering the substrate induced dissociation was
1.45 · 10
)8
m (Table 4). Preincubation of the inhibi-
tors with the enzymes for various periods of time did
not affect its inhibitory activity, suggesting that rLCI
and rPCI are fast tight binding inhibitors.

m
M
)1
Æs
)1
K
m
(mM) k
cat
s
)1
k
cat
⁄ K
m
M
)1
Æs
)1
SmCP 0.05 ± 0.01 42.5 79 · 10
5
0.36 ± 0.03 145 3.8 · 10
5
0.14 ± 0.01 15 1.7 · 10
5
bCPA 0.11 ± 0.01
a
44.0 41 · 10
5
0.88 ± 0.05

Trypsin-chymotrysin
inhibitor (soybean)
3mM 100 9.1 · 10
5
M
1,10-Phenanthroline 1 mM 21 3.03 · 10
5
M
Benzylsuccinic acid 1 mM < 1 3.03 · 10
5
M
EDTA 10 mM 117 3.03 · 10
5
M
PCI 0.4 lM 28.5 1.21 · 10
2
M
LCI 13.5 lM 30 4.1 · 10
2
M
Aprotinin 3 mM 100 9.1 · 10
5
M
Trypsin inhibitor
(soybean)
2mM 100 6.0 · 10
5
M
Table 4. K
i

,Mn
2+
or Mg
2+
enhanced the enzyme
activity of apoSmCP above 100% of the control activ-
ity, whereas the addition of Cd
2+
at 1 mm or Co
2+
at
1mm or 10 mm did not affect the enzymatic activity
of the control (Fig. 6). However, Cu at 1 mm and
10 mm reduced the apoenzyme activity to 11% and
15% of its residual activity. Noteworthy, under our
conditions, the addition of Zn
2+
at 1 mm or 10 mm
brought the activity to 100% (full rescue) and to 70%,
respectively, with the latter assignable to inhibition by
this metal.
Specificity of cleavage
Two different long peptides were used as substrate
models to analyze the ability of SmCP to cleave differ-
ent kinds of residues at the C-terminus, in comparison
Fig. 6. Effect of divalent metals on SmCP
activity The concentrations used in the
assays were 329 n
M for the enzyme SmCP
and 0.1 m

F
F
F
2188
2317
2466
1427
1529
1541
1563
1587
1619
1693
1716
1748
ACTH control 60 min
bCPA vs ACTH
2466
2317
ACTH control 60 min
bCPA vs V15E
1793
1716
1748
V15E control 60 min
V15E control 60 min
SmCP + PCI 60 min
15 min
30 min
60 min

with bovine pancreatic CPA (a reference enzyme in the
field). After 15 min of incubation of SmCP with the
adrenocorticotropic hormone (ACTH) fragment used
as substrate (residues 18–39, 2466 Da), the enzyme was
able to release phenylalanine (ACTHdes-F, 2317 Da)
and glutamic acid (ACTHdes-EF, 2188 Da) residues
from the substrate C-terminus (Fig. 7A). No further
amino acids were released after a 30-min incubation
period. Under the same conditions, bovine pancreatic
CPA was only able to hydrolyze the C-terminal phen-
ylalanine residue from ACTH to obtain the ACTHdes-
F (2317 Da). The addition of the protein inhibitor
rPCI prevented cleavage in all cases.
To confirm the capability of SmCP to hydrolyze
acidic residues from the C-terminus of peptides, the
specificity of SmCP against synthetic substrate
[VKKKARKAAGC(Amc)AWE] (V15E peptide) (resi-
due 15, 1716 Da) was evaluated (Fig. 7B). After
15 min of incubation, the release of glutamic acid from
the peptide was observed and, after 60 min, the new
C-terminus residues formed and tryptophan and ala-
nine were further released, as shown by the trimming
scale: 1716, 1587 and 1329 Da. However, bovine pan-
creatic CPA was unable to hydrolyze the first of such
C-terminal residues, glutamic acid, even after 60 min
of incubation. Again, the addition of rPCI prevented
any kind of hydrolysis by the enzyme. The release of a
glutamic acid residue from the C-terminus of peptides
is a very unusual capability of a CPA-like enzyme and
is reminiscent of the so-called CPO forms [3,5].

we named SmCP.
Different fractionation methods have been per-
formed to purify SmCP from the body extract of
S. magnifica. In initial attempts, using anion exchange
and gel filtration chromatographies, we found a frac-
tion with clear carboxypeptidase activity, which, inter-
estingly, conveyed two additional activities against
typical substrates for trypsin-like (benzoyl arginyl ethyl
ester; BAEE) and chymotrypsin-like (benzoyl tyrosine
ethyl ester; BTEE) serine proteases (data not shown).
This suggests that, in the fractionation, SmCP could
co-elute with serine proteases, perhaps establishing bin-
ary or ternary complexes with such enzymes, as shown
in other organisms [21,22]. Nevertheless, the substitu-
tive use of affinity chromatography on rPCI-agarose,
in subsequent experiments, allowed the selective cap-
ture of SmCP and contributed to its separation from
the other enzymes. Potentially, rPCI could promote
the dissociation of SmCP from ‘complexes with serine
proteases’ that it might establish in the crude extracts.
This is an issue that merits further research.
The 2D-PAGE analysis of the crude extracts indi-
cates that they are very complex in protein species,
and that a stainable band at around 35 kDa, attribut-
able to SmCP, is not directly visible with such
approach unless high sensitivity approaches (i.e immu-
nostaining) are employed. This is probably a result of
the low representation of this enzyme in the animal
extracts, in agreement with its subsequent analysis and
visualization in the purified form.

was technically solved, the feasibility and data genera-
tion capability of both the 2D-PAGE and ‘intensity
fading’ MALDI-TOF MS of this annelid indicated
that such proteomic-like approaches (and probably
related ones) are very promising for the analysis of
proteolytic enzymes in marine invertebrates.
A central question in the analysis of novel MCPs
from biological sources is whether they occur in their
precursor or mature forms [2–5]. In the present study,
using direct extracts from S. magnifica, we found only
a monomeric and activated form of SmCP, as shown
by its enzymatic activity, molecular mass, derived
N-terminal sequence and homology analysis. Procarb-
oxypeptidases are usually activated by proteolytic
removal of their activation segment by serine prote-
ases, mostly trypsin. Studies on procarboxypeptidases
from several species have indicated that its activation
is dependent of the environmental ionic conditions
and, sometimes, the influence of quaternary structure
[2,5]. Under our experimental conditions, quick activa-
tion of SmCP by autologous serine-like proteases,
which appeared to be present in large quantities in the
extract, could be favored. On the other hand, the
coincidence between the N-terminal sequences of
SmCP and those from several other MCPs included in
alignments (Fig. 5) also suggests that SmCP has been
purified in the active mature form. In addition, we
found that the sequences of a number of SmCP inter-
nal peptides included important residues that belong to
catalytic site and domain of this enzyme family,

m
and
k
cat
⁄ K
m
for certain substrates [23,24]. In addition,
SmCP has a maximum activity at pH 7.5, in agreement
with the optimum pH activity of almost all M14A CP-
like forms, including marine enzymes [7–13], which lie
in the neutral range (pH 6.5–8.5), and is consistent
with the pH at their sites of biological action [1,2].
As previously shown for mammalian CPs [25–27],
potato and leech proteinaceous inhibitors efficiently
inhibit SmCP, displaying similar K
i
values. In addition,
two smaller organic molecules (benzylsuccinic acid and
1,10-phenantroline) known to act on MCPs are also
able to inhibit the enzyme. By contrast, EDTA, which
chelates metal ions, at 10 mm, failed to inhibit SmCP
activity significantly after 10 min of preincubation,
which is in agreement with the reported properties of
other invertebrate MCPs isolated from the gut of Tion-
ela bisselliella [28] and from Helicoverpa armigera larvae
[29] for which EDTA effects are also time dependent.
The capability of divalent metal ions to substitute
the essential active site Zn
2+
of MCPs [30,31], or bind

its wide specificity on both synthetic and long pep-
tide substrates (Fig. 7), particularly the hydrophobic
ones characteristic of a CPA-like specificity, although
it is unable to hydrolyze those assignable to a CPB-
like specificity (i.e. with Arg or Lys at the C-termi-
nus). By contrast, SmCP is also able to hydrolyze
acidic C-terminal residues, such as glutamic acid.
The latter type of specificity, now termed CPO [5],
was recently described for a CP isolated from the
insect H. armiguera [36], which is unable to hydro-
lyze either CPA or CPB substrates. The strict speci-
ficity of CPO has been proposed to be a result of
the presence of a basic residue at the substrate rec-
ognition pocket [5,37], which is different than those
for the other two general types (A and B). SmCP is
the first marine invertebrate CP to be described with
this specificity.
In conclusion, SmCP shares many similarities with
the M14A MCPs isolated from other sources, such as
molecular mass, N-terminal sequence, the presence of
key catalytic residues, optimum pH, the effect of some
metal ions and salts and the inhibition pattern. On the
other hand, it shows a broad capability for releasing
C-terminus substrate residues, being able to hydrolyze
both CPA and CPO substrates, comprising a mixed
specificity not previously described for CP-like
enzymes. It may be a digestive requirement of the ani-
mal (i.e. the S. magnifica annelid). Before an ample
characterization of other proteolytic enzymes present
in this invertebrate is achieved (several other proteases,

The marine organisms belonging to the kingdom Methazoa
(Phyla: Annelida, Urochordata, Echinodermata, Cnidaria,
Mollusca, Artropoda) were collected in the north coast of
Havana and classified by Cuban specialists at the National
Institute of Oceanology (Havana, Cuba). The organisms
were homogenized in their own sea water liquid (1 : 2,
w ⁄ v). The homogenates were centrifuged at 10 000 g for
30 min at 4 °C. In the case of the marine invertebrate
S. magnifica, belonging to the Phylum Annelida, the ani-
mals were separated into two parts, tentacle crowns and
bodies, which were homogeneized as described above.
Carboxypeptidase assays
The general assay for CPA-like activity was carried out
using AAFP as substrate [23]. It was prepared at 10 mm in
dimethylsulfoxide. From this solution, 10 lL of substrate
was added to 50 lL of extract or enzyme samples in
940 lLof50mm Tris–HCl, 0.5 m NaCl (pH 7.5) for a final
concentration of 0.1 mm in the assay. The hydrolysis of the
chromogenic substrate caused a decrease in A
350
, which was
followed at 15-s intervals for 10 min at 25 °C in a kinetic
spectrophotometer Pharmacia-Biotech (Uppsala, Sweden).
The amount of residual substrate was determined using an
absorption coefficient of 19 · 10
)6
lm
)1
Æcm
)1

were followed to remove divalent metal ions from water
and buffers, which were used in all stages of the analysis.
MALDI-TOF MS identification and interaction
with inhibitors
Enzyme identification in the S. magnifica crude extracts and
interactomic experiments with protein inhibitors of CPs
were carried out using the ‘intensity fading’ MALDI-
TOF MS approach, as previously reported [17–19]. In the
experiment, 1 lLofS. magnifica body extract was mixed
with 2 lL of rPCI immobilized on agarose microbeads and
incubated for 3 min at room temperature. To eliminate the
unbound proteins, the rPCI-agarose matrix was washed
with 10 mm Tris–HCl buffer (pH 7.5) three times. The elu-
tion from rPCI-agarose microbeads was carried out mixing
the matrix with 2 lL of 0.1% formic acid. After 3 min of
incubation, 0.5 lL of the drop was pipetted to be analyzed
by MALDI-TOF MS, as described below.
Affinity capture by immobilized protease
inhibitors
Microcolumns based on agarose matrices (0.1–1 mL) with
immobilized cystatin C (generously provided by M. Abra-
hamson, Division of Clinical Chemistry and Pharmacology,
Lund University, Sweden), SBTI (reference T0637 and pep-
statin A (reference P2032), both from Sigma, were used to
capture proteases from S. magnifica extracts. Extracts were
loaded in 100 m m ammonium bicarbonate (pH 8.5) in the
first two cases, and in 100 mm sodium acetate (pH 5.5) in
the last case. The captured proteins were released by trifluo-
roacetic acid 0.2% (pH 2) in the former cases, and with 1 m
NaCl (pH 5.5) in the latter case, and precipitated by addition

in lysis buffer. For the 2D-DIGE approach, the samples
were labeled with two different CyDye DIGE fluorofors
(Cy2 for body extract and Cy5 for tentacle crown extract)
before performing the 2D-PAGE. Each sample was labeled
with 200 pmol (1 lL) of CyDye per 30 lg of protein, incu-
bated on ice for 30 min in the dark and quenched with
1 lLof10mm lysine and then incubated on ice for 10 min
in the dark, according to the manufacturer’s instructions.
2D-PAGE with immobilized pH gradient was carried out
according to Go
¨
rg et al. [46]. Samples were loaded in the first
dimension IEF, using the cup-loading method, onto previ-
ously rehydrated 11 cm IPG drystrips (GE Healthcare,
Milwaukee, WI, USA) that contain an immobilized linear
gradient in the range pH 3–10. Approximately 30 lgof
tentacles crown and body extracts, after prelabeling, were
loaded and run either independently or jointly in this first
dimension; in the latter case, after a previous mix and load of
equal amounts of extracts from the two parts of the animal.
IEF was performed at 300 V for 1 h, followed by three gradi-
ent steps (1000 V for 30 min; 5000 V for 80 min and 8000 V
A novel metallocarboxypeptidase from S. magnifica M. Alonso-del-Rivero et al.
4886 FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS
for 30 min) and, finally, 8000 V for 2 h. After focusing, the
strips were equilibrated and proteins separated on 15% poly-
acrylamide gels. Electrophoresis was carried out at 4 °C until
the front of fast migrating ions reached the bottom of the gel.
2D-PAGE gels were stained with silver nitrate [47]. The
stained gels were immediately scanned using a Umax Astra

adjust the eluates to pH 8.0. Fractions with CP activity
were concentrated and dialyzed against 20 mm Tris–HCl
buffer (pH 8.0) and applied to an ion exchange column of
TSK-DEAE (0.75 · 7.5 cm), previously equilibrated with
20 mm Tris–HCl buffer (pH 8.0), at a flow rate of
68 cmÆh
)1
. After extensive washing with the equilibration
buffer (45 min, six column volumes), the column was
washed with 60% of buffer B (1 m Tris–HCl, pH 8.0) for
20 min (three column volumes). Bound enzyme was eluted
with a linear gradient from 60% to 80% of buffer B over
170 min (25 column volumes) at the same flow rate.
MS, N-terminal sequence analysis and proteolytic
cleavage
A MALDI-TOF spectrometer was used to analyze the
molecular mass of peptides and proteins (Ultraflex MS;
Bruker, Ettlingen, Germany). Ionization was accomplished
with a 337 nm pulsed nitrogen laser and spectra were
acquired in the linear positive ion mode, using 25 kV acceler-
ation voltage. The analysis of proteins or peptide fragments
was carried out using 3,5-dimethoxy-4-hydroxycinnamic acid
(sinapinic acid) and a-cyano-4-hydroxicinnamic acid as
matrices. Samples were prepared by mixing them with equal
volumes of a saturated solution of the matrices. From this
mixture, 1 lL was spotted on the sample slide and allowed to
evaporate to dryness.
N-terminal amino acid sequence analysis was performed
by automated Edman degradation on an Applied Biosystems
(Applied Biosystems, Foster City, CA, USA) protein

the experimental data to the rectangular hyperbola curve,
using origin software (OriginLab, Northampton, MA,
USA).
pH optimum of SmCP activity
The optimum pH was determined using the AAFP substrate
by measuring the activity of SmCP (1.95 nm in assay) at var-
ious pH values using the buffers: 20 mm sodium phosphate
buffer (pH 11.0 and 12.0); 20 mm Hepes buffer (pH 7.0, 7.5,
8.0 and 8.5); 20 mm carbonate-bicarbonate buffer (pH 9.0,
9.5, 10.0 and 10.5); and 20 mm Tris–HCl buffer (pH 7.0,
7.5, 8.0, 8.5 and 9.0). All other experimental conditions were
as described for the CP assay using AAFP as substrate [23].
Effect of inhibitors and metal cations
Inhibition studies of SmCP by proteinaceous inhibitors was
evaluated against pepstatin A, rPCI, rLCI, aprotinin,
M. Alonso-del-Rivero et al. A novel metallocarboxypeptidase from S. magnifica
FEBS Journal 276 (2009) 4875–4890 ª 2009 The Authors Journal compilation ª 2009 FEBS 4887
soybean trypsin inhibitor, soybean trypsin–chymotrypsin
inhibitor and by synthetic inhibitors, such as E-64, EDTA,
benzylsuccinic acid, 1,10-phenanthroline and Pefabloc. The
effect of divalent metals such as Ca, Mn, Cd, Cu, Mg, Co
and Zn was analyzed using Milli-Q water that had been
passed through Chelax 100 columns according to establish
procedures (Bio-Rad). The enzyme was dialysed against
10 mm EDTA, in 20 mm Tris–HCl (pH 7.5) (metal-free),
overnight at 4 °C. EDTA was removed using a PD10 col-
umn and metal-free buffer. The apoenzyme was then incu-
bated for 10 min with divalent metals before activity was
assayed. The final activity was reported as the percentage
of apoenzyme activity.

sents the enzymatic activity in the absence of the inhibitor.
The experimental points were adjusted to the equation
described for tight binding inhibition [48] by employing
nonlinear fitting using statistica software (StatSoft, Tulsa,
OK, USA), as described previously [49].
Specificity against peptides substrates
The specificity of SmCP was studied using a fragment
derived from ACTH (18–39) as substrate for CPA-like
activity and V15E peptide as substrate for CPO-like activ-
ity. The reaction mixture contained 2.19 nm of SmCP and
1 lm of the peptides in 10 lLof10mm Tris–HCl buffer
(pH 8.0). The assays were monitored by MALDI-TOF MS
at 37 °C for 15, 30 and 60 min. Inhibition assays were per-
formed using PCI (5.6 lm) in the mixture. Similar experi-
ments were performed in parallel with bovine pancreatic
CPA (1 n m).
Acknowledgements
This work was supported by the International Founda-
tion of Science, Sweden (Grants F3342-1 and F3276-1),
by grant BIO2007-6846 (Ministerio de Educacio
´
ny
Ciencia-CICYT ⁄ MCINN, Spain) and by Xarxa de
Refere
`
ncia en Biotecnologia (XeRBa, Generalitat de
Catalunya). M.A.C. acknowledges a Visitor Grant from
AGAUR (Generalitat de Catalunya). Professor Magnus
Abrahamson and colleagues (Lund, Sweden) are
acknowledged for kindly providing immobilized cysta-

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