A novel class of bifunctional acylpeptidehydrolases – potential role in the antioxidant defensesystems of the Antarctic fish Trematomus bernacchiiMarta Gogliettino1*, Alessia Riccio1*, Marco Balestrieri1, Ennio Cocca1, Angelo Facchiano2,Teresa M. D’Arco1, Clara Tesoro1, Mos�e Rossi1 and Gianna Palmieri1

1 Institute of Protein Biochemistry and Institute of Biosciences and BioResources, National Research Council (CNR-IBP and CNR-IBBR),

Naples, Italy

2 Institute of Food Sciences, National Research Council (CNR-ISA), Avellino, Italy

Keywords

acylpeptide hydrolase; Antarctic fish;

antioxidant defense system; molecular

modeling; protein homeostasis

Correspondence

E. Cocca, IBP and IBBR, Consiglio Nazionale

delle Ricerche, Via Pietro Castellino 111,

80131 Napoli, Italy

Fax: +39 0816132277

Tel: +39 0816132552

E-mail: ennio.cocca@ibbr.cnr.it

*These authors equally contributed to this

work.

(Received 26 July 2013, revised 11 October

2013, accepted 5 November 2013)

doi:10.1111/febs.12610

Oxidative challenge is an important factor affecting the adaptive strategies

of Antarctic fish, but data on antioxidant defenses in these organisms

remain scarce. In this context, a key role could be played by acylpeptide

hydrolase (APEH), which was recently hypothesized to participate in the

degradation of oxidized and cytotoxic proteins, although its physiological

function is still not fully clarified. This study represents the first report on

piscine members of this enzyme family, specifically from the Antarctic tele-

ost Trematomus bernacchii. The cDNAs corresponding to two apeh genes

were isolated, and the respective proteins were functionally and structurally

characterized with the aim of understanding the biological significance of

these proteases in Antarctic fish. Both APEH isoforms (APEH-1Tb and

APEH-2Tb) showed distinct temperature-kinetic behavior, with significant

differences in the Km values. Moreover, beside the typical acylpeptide

hydrolase activity, APEH-2Tb showed remarkable oxidized protein endohy-

drolase activity towards oxidized BSA, suggesting that this isoform could

play a homeostatic role in removing oxidatively damaged proteins, sustain-

ing the antioxidant defense systems. The 3D structures of both APEHs

were predicted, and a possible relationship was found between the

substrate specificity/affinity and the marked changes in the number of

charged residues and hydrophobicity properties surrounding their catalytic

sites. Our results demonstrated the occurrence of two APEH isoforms in

T. bernacchii, belonging to different phylogenetic clusters, identified for the

first time, and showing distinct molecular and temperature–kinetic behav-

iors. In addition, we suggest that the members of the new cluster ‘APEH-2’

could participate in reactive oxygen species detoxification as phase 3

antioxidant enzymes, enhancing the protein degradation machinery.

Introduction

The continent of Antarctica lies almost entirely within

the Antarctic Circle, and is covered by 90% of the

world’s ice. The large populations of fish found in the

nutrient-rich waters of this frozen continent possess

unusual adaptations for surviving the rigors of low

temperatures. In fact, Antarctic fish have evolved a

Abbreviations

Ac-Ala-pNA, acetyl-Ala-p-nitroanilide; Ac-Leu-pNA, acetyl-Leu-p-nitroanilide; Ac-Met-AMC, acetyl-Met-7-amino-4-methylcoumarin; AMC, 7-

amino-4-methylcoumarin; APEH, acylpeptide hydrolase; APEH-1Tb, acylpeptide hydrolase-1 from Trematomus bernacchii; APEH-2Tb,

acylpeptide hydrolase-2 from Trematomus bernacchii; APEHpl, acylpeptide hydrolase from porcine liver; RBC, red blood cell.

FEBS Journal 281 (2014) 401–415 ª 2013 FEBS 401

number of physiological changes, including a low basal

metabolic rate [1,2] and a substantial reduction in the

hematocrit, which reaches its extreme in the complete

loss of both red blood cells (RBCs) and hemoglobin in

the Channichthyidae species [3]. Interestingly, organ-

isms living in the oxygen-rich cold water of Antarctica

have naturally high tissue oxygen concentrations, as

result of their adaptations [4–6]. These high oxidative

conditions, together with the effects that low tempera-

tures have on membrane fluidity [7], have induced the

adoption of alternative strategies, not yet fully under-

stood, such as high efficiency of low molecular mass

scavengers and upregulation of specific gene families

operating in antioxidation, antiapoptosis, protein fold-

ing, lipid metabolism, and other processes [8,9].

Typically, the oxidative stress associated with

increased production of oxidizing species such as free

radicals and other pro-oxidants causes the continuous

formation of damaged proteins within cells [10]. Mam-

malian cells have developed a hierarchy of defense

processes, in which antioxidants provide the first

defensive mechanism, and proteolytic systems act as

secondary defenses [11]. Among these, acylpeptide

hydrolase (APEH), first identified as oxidized protein

hydrolase [12–14], was recently hypothesized to partici-

pate in the degradation of oxidized and cytotoxic pro-

teins [15–17]. For these reasons, APEH may represent

a promising therapeutic target for a wide array of

human diseases caused by the accumulation of dam-

aged proteins [18,19].

APEH (also referred as acylaminoacyl peptidase or

acylamino-acid-releasing enzyme) is a ubiquitous cyto-

solic enzyme belonging to the prolyl oligopeptidase

(POP) family of serine peptidases (clan SC, family S9)

that catalyzes the removal of Na-acetylated amino

acids from blocked peptides, acting as a key regulator

of N-terminally acetylated proteins in human cells [20–22]. To date, APEH has been studied in a number of

eukaryal [22–25] and archaeal [26–28] organisms, and

recently in a psychrophilic bacterium, Sporosarci-

na psychrophila [29,30], but its biological role has not

yet been completely elucidated. The crystal structure

of POPs shows a peptidase domain with a a/b-hydro-lase fold, and an unusual b-propeller, which covers the

catalytic triad [20,31]. This domain makes the mem-

bers of this family able to exclude large, structured

peptides from the active site, protecting them from

proteolysis in the cytosol. In contrast to the other

POPs, only one 3D structure of APEH, that from Ae-

ropyrum pernix K1, has been reported [32]. Unlike its

tetrameric mammalian counterparts, APEH from

A. pernix is a homodimer, with each subunit contain-

ing the typical structural features of the POP family.

We recently reported the isolation and characterization

of two APEHs from the hyperthermophile Sulfolobus

solfataricus that were revealed to be stress-regulated

proteins playing a complementary role in enabling the

survival of this organism under stressful growth

conditions [28].

The purpose of this work was to study the first pis-

cine enzymes of the APEH family in the notothenioid

Trematomus bernacchii, which represents the most

endemic class of fish in Antarctic waters living at tem-

peratures below �1.5 °C [33]. It has been suggested

that notothenioids may have reduced or lost the ability

to increase the levels of secondary antioxidant defenses

in response to environmental stress [34]. Indeed, in the

emerald rockcod T. bernacchii, some common antioxi-

dant enzymes are not naturally enhanced, and an alter-

native biochemical strategy, based on the efficiency of

low molecular mass scavengers, has been hypothesized

to counteract pro-oxidant challenge [9].

Therefore, in this study, we functionally and struc-

turally characterized two new APEHs (APEH-1Tb and

APEH-2Tb) from T. bernacchii, and isolated the

cDNAs of the corresponding coding genes, in order to

better understand their function in the physiology of

Antarctic fish and their involvement in the response to

the strong environmental oxidative stress.

Our investigations showed significant differences in

the catalytic and kinetic behavior of the isolated

enzymes, which were also reflected in a diverse charge

distribution of residues surrounding the active site in

the 3D models. Interestingly, APEH-2Tb was able to

efficiently hydrolyze oxidized BSA, unlike APEH-1Tband the mammalian counterpart, indicating that it

could play a key role in destroying oxidatively dam-

aged proteins and in the antioxidant defense systems.

Moreover, the expression levels of apeh-2Tb appeared

to be higher than those of apeh-1Tb in all tissues ana-

lyzed, reinforcing the hypothesis of a crucial function

associated with APEH-2Tb.

Results

Cloning of apeh-1Tb and apeh-2Tb cDNAs and

phylogenetic analysis

As previously described, APEH is a ubiquitous cytosolic

protein that seems to have an almost universal distribu-

tion in all living organisms [21,22,25,26,28,29], although

much is still unknown about its biological function.

Interestingly, although a unique gene has been found so

far for mammalian members of APEH family, searches

in different protein databases have revealed the exis-

tence of two homologous sequences of APEH in several

402 FEBS Journal 281 (2014) 401–415 ª 2013 FEBS

APEH from the Antarctic fish Trematomus bernacchii M. Gogliettino et al.

fish species. Therefore, to gain new insights into the

molecular structure and evolution of this class of prote-

ases, which are thought to play a key role in protein

turnover and antioxidant defense mechanisms, we

decided, as a first approach, to investigate apeh gene

multiplicity in T. bernacchii, and explore the sequence–structure–function relationship of the gene product(s).

Starting from total RNA from the liver, two partial

cDNAs, corresponding to apeh-1Tb and apeh-2Tb, were

cloned by RT-PCR, predicting two uncomplete APEH

proteins of 717 (APEH-1Tb) and 677 (APEH-2Tb)

amino acids (Fig. S1). The deduced amino acid

sequences were aligned with their homologs from sev-

eral vertebrates and two invertebrates, retrieved from

the Swiss-Prot/TrEMBL database (see Fig. S2 and

Table S1 for species names and accession numbers),

and a phylogenetic tree was constructed (Fig. 1), sup-

ported by high bootstrap in maximum likelihood

analysis. Interestingly, as shown in Fig. 1, two APEH

isoforms were retrieved for all of the teleosts and

also for the bird species Gallus gallus. Moreover, with

the APEH sequences of two invertebrates

(Branchiostoma floridae and Ciona intestinalis) as out-

groups, the analysis revealed two distinct clusters for

APEHs: the first cluster, which we call APEH-1, con-

tains APEH-1Tb, the APEH-1-like proteins from all of

the teleosts, the bird G. gallus, the reptile Anolis caro-

linensis, the amphibian Xenopus tropicalis, and the

entire subgroup of the mammalian APEHs; the second

cluster, which we call APEH-2, contains APEH-2Tb,

the second APEH isoform from all of the teleosts and

that from G. gallus, grouped with the APEH from the

other bird, Taeniopygia guttata.

Isolation, purification and sequence analysis of

APEH-1Tb and APEH-2Tb

As mentioned above, APEH from T. bernacchii

appears to be present as two isoforms, but whether

this multiplicity correlates with distinct biological func-

tions is unclear.

To aid in our understanding of the physiological sig-

nificance of these APEH enzymes, the two isoforms

were purified, and their kinetic and structural features

were studied in comparison with those of the mamma-

lian counterpart.

In mammals, APEH has been found in many differ-

ent types of cells and tissues, such as blood [12–14],brain [35,36], liver [23], and kidney [37]. Therefore,

RBCs were chosen as the starting sample to optimize

a purification procedure, which allowed us to isolate

the most abundant of the two APEH isoforms, corre-

sponding to a 75-kDa band detected on a western blot

(Fig. 2B). The enzyme was purified ~ 350-fold with an

activity recovery of 35% (Table S2A), and protein

homogeneity was proved by SDS/PAGE (Fig. 2A)

and gel filtration chromatography (Fig. S3). The

amount of APEH was ~ 0.1 mg, and the specific

activity was measured as 3.9 9 105 U/mg. Edman

degradation analysis of the isolated protein revealed a

unique N-terminus, VSAHVSEEQL(20–29), which

completely matched the sequence predicted from the

coding gene apeh-2 (Fig. S1B), except for the first 19

amino acids, which were lacking in the purified APEH

(named APEH-2Tb), possibly because of a proteolytic

event.

The second APEH isoform was partially purified

(Table S2B) from the protein liver extract (Fig. 2C),

H. sapiensM.s musculus

B. taurus

S. scrofa

M. domestica

G. gallus 1

G. gallus 2

A. carolinensis

O. niloticus 1O. niloticus 2

T. nigroviridis 1

T. nigroviridis 2

T. rubripes 1

T. rubripes 2

D. rerio 1

D. rerio 2

T. guttata

Fig. 1. Phylogenetic analysis of APEH

proteins in the animal kingdom. The

cladogram includes the sequences

retrieved from 16 organisms, two of which

are invertebrates and were used as sister

groups in the analysis. The branches of

the fish APEH-1 cluster are in light blue,

and those of the fish APEH-2 cluster are

in red. The mammal APEH-1 cluster is

shadowed in blue. Numbers at nodes

represent the confidence limits computed

with the bootstrap procedure (100

replicates).

FEBS Journal 281 (2014) 401–415 ª 2013 FEBS 403

M. Gogliettino et al. APEH from the Antarctic fish Trematomus bernacchii

as its proteolytic activity was scarcely detected in the

hemolysates. The enzyme preparation showed a

unique 80-kDa immunoreactive band (Fig. 2B), and

the N-terminus was identified as XEKQVVTNPE by

Edman degradation analysis performed on the SDS/

PAGE electroblotted protein. This sequence was iden-

tical to that deduced from the coding gene apeh-1Tb,

except for the first amino acid (Fig. S1A), which

remained uncertain even after repeated N-terminal

sequence analysis. This APEH isoform was named

APEH-1Tb.

Molecular properties of APEH-1Tb and APEH-2Tb

The degree of oligomerization of the two APEHs

(APEH-1Tb and APEH-2Tb) was investigated by gel fil-

tration chromatography with two different size-exclu-

sion columns. These analyses indicated that, like the

mammalian APEH from porcine liver (APEHpl), both

T. bernacchii isoforms are tetrameric enzymes with an

apparent molecular mass of ~ 300 kDa, composed of

identical monomers of 80 kDa (APEH-1Tb) and

75 kDa (APEH-2Tb) (Fig. S3).

In evaluating the molecular properties of APEH-1Tband APEH-2Tb in comparison with those of the

mammalian homolog APEHpl, we found that all

enzymes showed a bell-shaped pH–activity profile

with an optimal pH value of 8.0 (Fig. 3A). More-

over, APEH-2Tb showed higher activities at lower

temperatures and activity over a broader temperature

range (Fig. 3B) than APEH-1Tb and APEHpl, which

showed a very similar trend. However, the optimal

temperatures determined for the two T. bernacchii

APEH isoforms remained 25–30 °C above the

optimum growth temperature for this Antarctic spe-

cies, as has also been observed for other proteases

and enzymes from psychrophilic organisms [29,30].

Regarding thermal stability (Fig. 3C–F), both piscine

APEHs retained ~ 80% of enzymatic activity after

7 h of incubation at 10 °C and 20 °C, whereas the

residual activity of APEH-1Tb was halved after 5 h

of incubation at 37 °C, which caused complete inacti-

vation of APEH-2Tb. In contrast, no decrease in

activity was observed for mammalian APEH under

the same experimental conditions, whereas all of the

enzymes showed a half-life of a few minutes at

50 °C. These results underline the fact that APEH-

2Tb has the typical features of cold-adapted enzymes:

both the optimal temperature for catalytic activity

and the temperature limit for retaining thermostabil-

ity were lower than the values obtained for the meso-

philic counterparts. However, APEH-1Tb seems to

show properties intermediate between those of

APEHpl and APEH-2Tb.

Kinetic and thermodynamic parameters of

APEH-1Tb and APEH-2Tb

The thermal dependence of APEH kinetic parameters

for the enzymes from T. bernacchii was studied in

comparison with those of the mammalian counterpart

APEHpl. As reported in the literature, mammalian

APEHs usually cleave the peptides after small and

large neutral amino acids, whereas archaeal APEHs

prefer large, hydrophobic residues. Therefore, to exam-

ine the exopeptidase activity of T. bernacchii APEHs,

we decided to use two substrates differing in the size

of the neutral amino acid.

APEH -1Tb80 kDa

APEH -2Tb75 kDa

kDa212.0

158.0

116.097.2

66.4

55.6

kDa

55.6

97.2116.0

158.0212.0

66.4

1 2 31 2 3

1 2 3

80 kDa75 kDa

A C

B

Fig. 2. SDS/PAGE and immunoblotting

analyses of APEH isoforms from

T. bernacchii. (A) SDS/PAGE analysis of

APEH-2Tb from blood. Lane 1: molecular

mass markers (myosin, 212 kDa; MBP-

b-galactosidase, 158 kDa; b-galactosidase,

116 kDa; phosphorylase-b, 97.2 kDa;

serum albumin, 66.4 kDa; glutamine

dehydrogenase, 55.6 kDa). Lane 2:

hemolysate. Lane 3: purified APEH-2Tb. (B)

Immunoblotting analysis of APEH-1Tb and

APEH-2Tb. Lane 1: APEH-1Tb from liver.

Lane 2: APEH-2Tb from blood. Lane 3:

APEHpl used as control. (C) SDS/PAGE

analysis of APEH-1Tb from liver. Lane 1:

partially purified APEH-1Tb. Lane 2: protein

liver extract. Lane 3: molecular mass

markers.

404 FEBS Journal 281 (2014) 401–415 ª 2013 FEBS

APEH from the Antarctic fish Trematomus bernacchii M. Gogliettino et al.

Notably, the Michaelis–Menten constants of APEH-1Tband APEH-2Tb were affected differently by increased

temperatures, with the most common substrates of

eukaryal APEHs, acetyl-Ala-p-nitroanilide (Ac-

Ala-pNA) and acetyl-Met-7-amino-4-methylcoumarin

(Ac-Met-AMC). No remarkable variations were

observed in the binding affinities (Km) of APEH-1Tband APEH-2Tb in the temperature range 15–40 °Cversus Ac-Ala-pNA, as also revealed for APEHpl,

although the Km values of APEH-1Tb were, signifi-

cantly, the lowest measured at all temperatures

(Fig. 4A). The difference in the thermodynamic

parameters was even more marked at higher tempera-

tures. Similar results were obtained (Fig. 4B) for both

the cold-adapted and the mammalian APEHs with

Ac-Met-AMC as the substrate. These data confirm

that the two ‘APEH’ clusters revealed by the phylo-

genetic analysis (Fig. 1) include functionally distinct

homologs, as also shown with acetyl-Leu-p-nitroani-

lide (Ac-Leu-pNA) as substrate, which was not

hydrolyzed by APEH-2Tb, in contrast to APEH-1Tband APEHpl (Table 1).

Regarding the kinetic constants, APEH-2Tb and

APEHpl showed increased kcat values with increas-

ing temperature when either Ac-Ala-pNA or Ac-

Met-AMC was used to detect the enzyme activities,

although the values for APEH-2Tb were signifi-

cantly lower than those determined for the mam-

malian counterpart at all temperatures tested

(Fig. 4C–E), suggesting that the adaptation could

be incomplete, as illustrated in most reported cases

[38].

Therefore, the effect of temperature on the catalytic

efficiency (kcat/Km) was determined (Fig. 4D–F). Twoaspects are worth mentioning: the efficiencies of

APEH-2Tb and APEHpl were maximal when the tem-

peratures were close to their respective thermal envi-

ronmental conditions, and sharply decreased outside

this range. This behavior, which was clearly shown

with Ac-Ala-pNA as the substrate, illustrates the typi-

cal property of the cold-adapted enzymes, which show

optimal catalytic performance at low temperatures.

Contrasting results were obtained with Ac-Met-AMC

as the substrate (Fig. 4E,F).

In a previous study [28], we found that hyper-

thermophilic APEHs showed both endopeptidase and

exopeptidase activities, highlighting the fact that

these archaeal enzymes were less specialized [26] than

the mammalian ‘true’ exopeptidase APEHs. How-

ever, when the small peptide substrates (see Experi-

mental procedures), differing in the size and nature

of amino acids, were tested with the piscine APEHs,

no peptide fragments were detected in the reaction

mixtures, suggesting that the typical endopeptidase

020406080

100120

0 200 400

0

20

40

60

80

100

0 20 40 60 80

T (°C)0

20

40

60

80

100

0 5 10

Res

dual

act

ivity

(%)

pH

A B

37 °C

0

20

40

60

80

100

0 40 80 120

Res

idua

l act

ivity

(%)

50 °C

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D

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0 200 400

10 °C

C

20 °C

E

Time (min) Time (min)60 180 300

0

20

40

60

80

100

0 20 40 60

Fig. 3. Molecular properties of

T. bernacchii APEH isoforms as compared

with APEHpl. (A) pH–activity profile. (B)

Influence of temperature on enzyme

activities. (C–F) Thermoresistance of

APEHs at: (C) 10 °C, (D) 20 °C, (E) 37 °C,

and (F) 50 °C. All experiments were

performed in triplicate on three different

protein preparations, with Ac-Ala-pNA as

the substrate. APEH-2Tb, APEH-1Tb and

APEHpl are indicated by squares, circles,

and triangles, respectively.

FEBS Journal 281 (2014) 401–415 ª 2013 FEBS 405

M. Gogliettino et al. APEH from the Antarctic fish Trematomus bernacchii

activity of the serine proteases was lacking in these

isoforms.

It is important to emphasize that these studies are

based only on the performance of isolated proteins at

temperatures that are often much higher than those

tolerated by the whole organism. Only in vivo kinetic

measures, which take into account a large number of

physiological and biochemical factors, could ade-

quately reveal the cold-adapted catalytic features of

these enzymes.

Oxidized protein endohydrolase activity of APEH

isoforms from T. bernacchii

Primary defense systems including antioxidants and

antioxidative enzymes, which prevent the generation

of free radicals or break radical chain reactions, are

known to protect living cells from oxidative damage

[10]. Secondary antioxidant defense systems, includ-

ing proteases that preferentially degrade oxidatively

damaged proteins, are also known to exist within

Table 1. Optimal kinetic parameters of T. bernacchii APEHs in comparison with those of APEHpl.

Substrate Enzyme Km (mM) kcat (min�1) kcat/Km (min�1�mM�1)

Ac-Ala-pNA APEH-2Tb (1.2 9 10�1) � (1.0 9 10�2)a (1.0 9 102) � 7.0b (2.8 9 102) � 3.0c

APEH-1Tb (1.4 9 10�2) � (5.0 9 10�3)d ND ND

APEHpl (0.9 9 10�1) � (2.0 9 10�2)a (2.1 9 103) � (1.3 9 102)b (6.2 9 104) � (3.0 9 103)b

Ac-Met-AMC APEH-2Tb (1.1 9 10�1) � (1.0 9 10�2)d (4.3 9 108) � (3.0 9 107)b (2.6 9 109) � (1.0 9 108)b

APEH-1Tb (0.3 9 10�1) � (1.0 9 10�2)d ND ND

APEHpl (5.5 9 10�2) � (7.0 9 10�3)d (1.7 9 109) � (1.0 9 108)b (1.9 9 1010) � (1.2 9 109)c

Ac-Leu-pNA APEH-2Tb NA NA NA

APEH-1Tb (0.9 9 10�1) � (2.0 9 10�2)a ND ND

APEHpl (2.3 9 10�1) � (3.0 9 10�2)e (5.1 9 103) � (2.0 9 102)f (7.7 9 104) � (1.0 9 103)f

Kinetic parameters measured with Ac-Met-AMC as the substrate are expressed in arbitrary units. NA, no activity; ND, not determined.a Measured at 15 °C. bMeasured at 50 °C. cMeasured at 37 °C. dMeasured at 25 °C. eMeasured at 40 °C. fMeasured at 60 °C.

0

0.2

0.4

0.6

0.8

10 20 30 40 500

0.1

0.2

0.3

10 20 30 40 50

0.1

0.2

6 7 8 9K

m A

c-A

la-p

NA

(mM

)

pH

Km

Ac-

Met

-AM

C (m

M)

1.0 x 10

1.0 x 102

1.0 x 103

1.0 x 104

1.0 x 10

1.0 x 102

1.0 x 103

1.0 x 104

10 20 30 40 50 10 20 30 40 50

Log

10 [k

cat A

c-A

la-p

NA

(min

–1)]

Log

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cat/K

m A

c-A

la-p

NA

(min

–1 m

M–1

)]

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1.0 x 108

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1.0 x 1010

10 20 30 40 50 10 20 30 40 50Log

10 [k

cat A

c-M

et-A

MC

(min

–1)]

Log

10 [k

cat/K

m A

c-M

et-A

MC

(min

–1 m

M–1

)]

T (°C) T (°C)

A B

C D

E F

Fig. 4. Effects of temperature on kinetic

parameters of APEH-1Tb and APEH-2Tb. (A,

B) Temperature–Km profiles of

T. bernacchii and porcine APEHs. (B,

insert) pH–Km profiles of T. bernacchii

APEH isoforms with Ac-Met-AMC as the

substrate. (C, D) Effect of temperature on

the reaction velocity (C) and the catalytic

efficiency (D) of APEH-2Tb and APEHpl

with Ac-Ala-pNA as the substrate. (E, F)

Effect of temperature on the reaction

velocity (E) and the catalytic efficiency (F)

of APEH-2Tb and APEHpl with Ac-Met-

AMC as the substrate. All experiments

were performed in triplicate on three

different protein preparations. Data are

expressed as means � standard

deviations. Standard deviation values

lower than 5% are not shown. APEH-2Tb,

APEH-1Tb and APEHpl are indicated by

squares, circles, and triangles,

respectively.

406 FEBS Journal 281 (2014) 401–415 ª 2013 FEBS

APEH from the Antarctic fish Trematomus bernacchii M. Gogliettino et al.

cells such as reticulocytes and RBCs. Recently, an

oxidized protein hydrolase was discovered in the

human RBC cytosol [13], and was found to be iden-

tical to APEH. The oxidized protein hydrolase pref-

erentially degraded oxidatively damaged proteins in

living cells, and it was shown to play a preventive

role against oxidative stress, enhancing stress toler-

ance [14,17]. In this study, to clarify the physiologi-

cal role of APEH-1Tb and APEH-2Tb, we examined

whether these enzymes were able to degrade oxidized

proteins, with BSA as a substrate model and

APEHpl as a control.

Unoxidized or oxidized BSA was incubated with

APEHs for up to 48 h, and each reaction mixture

was subjected to SDS/PAGE analysis. The intensity

of the oxidized BSA band decreased remarkably fol-

lowing treatment with APEH-2Tb (Fig. 5C), with the

concomitant detection of lower molecular mass frag-

ments (Fig. 5B). In contrast, only 15% of substrate

reduction was found with APEHpl (Fig. 5A,C) or

APEH-1Tb (Fig. 5C), although fluctuations in the

substrate intensity values were obtained following

APEH-1Tb incubation. Interestingly, when unoxidized

BSA was used as the substrate with all of the APE-

Hs, no BSA fragments were detected, indicating an

‘atypical’ endopeptidase activity associated with the

eukaryal members of the APEH family. Indeed, it

has been suggested that the increased susceptibility

of BSA to degradation by APEH upon oxidation

could be attributable to a conformational change

that brings the cleavage sites to the surface of the

molecule, where they become easily accessible to the

enzyme [39].

Expression analysis of apeh-1Tb and apeh-2Tb

The transcriptional levels of apeh-1Tb and apeh-2Tbwere analyzed in several cells and tissues of T. bernac-

chii by quantitative real-time PCR. Total RNAs from

RBCs, heart, brain, head kidney, spleen, gills, ovary,

testis and liver of adult specimens were retrotran-

scribed, and dilutions of the resulting cDNAs were

used to determine the expression levels of apeh-1Tb and

apeh-2Tb in comparison with those of the b-actin gene,

which were used for normalization. All data shown are

expressed as fold mean expression from triplicate

experiments, and were calculated with the Pfaffl equa-

tion [40]. The expression patterns are shown in Fig. 6:

apeh-1Tb transcriptional levels were higher, in order of

size, in ovary, liver, and RBCs (Fig. 6A), and apeh-2Tbwas more highly expressed in liver, heart, and ovary

(Fig. 6B); moreover, the transcriptional levels of apeh-

2Tb were higher than those of apeh-1Tb in all of the

samples, with the apeh-2Tb/apeh-1Tb ratio ranging from

17 in ovary to 173 in liver (Fig. 6C).

Molecular modeling and protein structural

properties of APEH-1Tb and APEH-2Tb

The structural models of APEH-1Tb and APEH-2Tb,

obtained with a comparative modeling strategy as

described in Experimental procedures, are shown in

Fig. 7A,B. Both proteins are characterized by a two-

domain architecture. The N-terminal domain has the

typical b-propeller structure, formed by seven antipar-

allel four-stranded b-sheet motifs, and the C-terminal

domain is folded as an a/b-domain, with a b-sheet

A

B

0 48 48 24 24 0 hAPEHpl

BSA

APEH-2TbBSA

C

25

50

75

100

0 15 30 45

Rel

ativ

e in

tens

ity (%

)

Time (h)

Fig. 5. SDS/PAGE profiles of oxidized BSA treated with APEH-1Tb,

APEH-2Tb and APEHpl. (A) Unoxidized BSA (left panel) and oxidized

BSA (right panel) were treated with APEHpl at pH 8.0 and 37 °C

for the indicated period. The reaction mixture was subjected to

SDS/PAGE. (B) Unoxidized BSA (left panel) and oxidized BSA (right

panel) were treated with APEH-2Tb at pH 8.0 and 37 °C for the

indicated period. (C) SDS/PAGE data are expressed as percentage

density of BSA at the indicated incubation times versus time 0,

and were obtained by densitometric analysis with CHEMIDOC XRS and

QUANTITY ONE software. Oxidized BSA levels after incubation with

APEH-2Tb, APEH-1Tb and APEHpl are indicated by squares, circles,

and triangles, respectively. Unoxidized control BSA treated with all

of the APEHs is indicated by diamonds. The experiments were

performed in duplicate on two different protein preparations, and

the average of the relative intensities of measurements, performed

in triplicate, are expressed as means � standard deviations.

FEBS Journal 281 (2014) 401–415 ª 2013 FEBS 407

M. Gogliettino et al. APEH from the Antarctic fish Trematomus bernacchii

formed by seven parallel b-strands and one additional

antiparallel b-strand [20].

The multiple alignments of members of the APEH

family, including the two isoforms from T. bernacchii,

together with information available for the other mem-

bers of the family investigated in the past, suggested to

us the position of the amino acids involved in the cata-

lytic site (Fig. S2).

They are Ser587, Asp675 and His707 in APEH-1Tb,

and Ser555, Asp643 and His675 in APEH-2Tb. To

investigate possible differences at the level of specific-

ity, we observed in detail the amino acids in the space

surrounding the catalytic triad, with increasing dis-

tances (Table S3). Most of the side chains in proximity

to the catalytic triad are identical; that is, Val141,

Gly586, His588, Val678 and Ser708 in APEH-1Tb

correspond to Val132, Gly554, His556, Val646 and

Ser676 in APEH-2Tb. At a distance of 2.5��A from the

three amino acids, in APEH-1Tb we found one nega-

tively (Asp674) and one positively (Lys676) charged

residue, whereas in APEH-2Tb we found two positively

(Lys642 and Arg644) charged residues. Another differ-

ence in the region of the catalytic triad is the presence

of Asn706 in APEH-1Tb and Gly674 in the corre-

sponding position in APEH-2Tb. These changes may

affect the substrate specificity/affinity of the two iso-

forms (see Discussion), as supported by the pH–Km

profiles of APEH-1Tb and APEH-2Tb (Fig. 4B, inset).

Indeed, the overall variations in Km with pH were not

significantly altered for APEH-1Tb in the pH range

6.5–9.0, whereas APEH-2Tb strongly differed in its

pH–kinetic response profile. Specifically, the substrate

affinity constant of APEH-2Tb increased above and

below 7.5, assuming a convex shape and highlighting a

specific kinetic pH window, presumably because of the

presence of a different electrostatic environment

around the active site, which progressively changes

with pH variation.

Discussion

Antarctic fish have adapted to extreme environmental

conditions characterized by low seawater temperature

and elevated solubility of oxygen [6]. Under such con-

ditions, oxidative challenge is an important factor in

the adaptive strategies of these organisms. This impor-

tant physiological aspect, together with the prolonged

half-life of oxy-radicals, requires efficient long-term

antioxidant protection.

Generally, eukaryotic cytoplasm contains different

mechanisms for defense against oxidative stress,

which include the direct scavenging and detoxifica-

tion of reactive chemicals (phase 1), and the restora-

tion of reversibly denatured proteins (phase 2) [25].

It has been recently suggested that the antioxidant

apparatus could be improved by a further step

(phase 3) [25], targeted at the elimination of irrevers-

ibly denatured proteins and assisted by a coordinated

action of APEH and the proteasome via biochemical

mechanisms [16,18]. The proteasome is known to

function in the selective destruction of damaged or

oxidized proteins, as one of the ultimate mechanisms

that cells use to ensure the quality of intracellular

proteins [11]. A protective role against oxidative

stress has also been proposed for APEH, as it has

been shown that this enzyme is able to remove dam-

aged protein aggregates in RBCs [13,14], although

the biological function of this enzyme is not yet

understood.

0

50

100

150

0

0.01

0.02

0.03

0.04

0

0.0002

0.0004

0.0006

0.0008

Fold

exp

ress

ion

Fold

exp

ress

ion

Fold

exp

ress

ion

apeh-2Tb/apeh-1TbC

A

B

apeh-2Tb

apeh-1Tb

RBCHea

rt

Brain

Head K

idney

Spleen

Gills

Ovary

Testis

Liver

RBCHea

rt

Brain

Head K

idney

Spleen

Gills

Ovary

Testis

Liver

RBCHea

rt

Brain

Head K

idney

Spleen

Gills

Ovary

Testis

Liver

Fig. 6. Expression analysis of apeh-1Tb and apeh-2Tb. (A, B) The

transcriptional levels of apeh-1Tb (A) and apeh-2Tb (B) in several

cells and tissues of T. bernacchii, normalized with respect to

b-actin expression. (C) The apeh-2Tb /apeh-1Tb fold expression ratio.

All data shown are expressed as mean fold expression from

triplicates.

408 FEBS Journal 281 (2014) 401–415 ª 2013 FEBS

APEH from the Antarctic fish Trematomus bernacchii M. Gogliettino et al.

Indeed, in the Antarctic notothenioids, with respect

to temperate/tropical species, a transcriptomic analysis

of tissues has revealed increased levels of ubiquitin-

conjugated proteins targeted for proteasomal degrada-

tion [41], which are needed to maintain protein

homeostasis in the extreme Antarctic environment.

However, neither the APEH family nor the involve-

ment of a possible APEH–proteasome pathway in pro-

tein turnover and antioxidant mechanisms have so far

been investigated in these Antarctic marine organisms.

In such a context, the purpose of this study was to

explore the first ‘piscine APEH’ from the Antarctic fish

T. bernacchii, investigating the physiological role of

this class of enzymes in supporting the life of organ-

isms in their cold and oxygen-rich environment.

Antarctic teleosts such as T. bernacchii show

reduced amounts of antioxidant enzymes as compared

with the temperate species; thus, the phase 2 defense

machinery could not directly compensate for the

extreme oxygen levels, suggesting a greater dependence

on reactive oxygen species scavengers, whose content

increases, expanding phase 1 as a strategy against oxi-

dative damage [9].

Our results demonstrated the presence of two active

APEH isoforms (APEH-1Tb and APEH-2Tb) in

T. bernacchii, belonging to two different APEH sub-

families, identified for the first time in this study, and

showing distinct molecular and temperature–kineticbehaviors. Specifically, APEH-1Tb was found to be a

member of the APEH-1 cluster, like all of the mam-

malian counterparts, and showed the typical func-

tional properties of APEHs. Remarkably, APEH-2Tb,

in parallel with the exopeptidase activity, showed effi-

cient hydrolytic activity towards oxidized BSA, in

contrast to APEH-1Tb and APEHpl, indicating that

this isoform could play a homeostatic role in sustain-

ing the antioxidative system as an oxidized protein

endohydrolase. Therefore, APEH-2Tb can be supposed

to be the founder of a new cluster, named ‘APEH-2’,

whose members could participate in reactive oxygen

species detoxification as phase 3 antioxidant enzymes,

enhancing proteasome-mediated proteolysis [18,25].

The expression analysis also revealed a different dis-

tribution of apeh-1Tb and apeh-2Tb transcripts. The

apeh-1Tb mRNA was particularly abundant in ovary,

which is mostly active in terms of protein turnover

during the late spring/early summer in Antarctic

waters, when the fish population is enriched with

females ready to spawn. In contrast, apeh-2Tb tran-

scripts were more abundant in liver, which is one of

A B

C D

Fig. 7. Molecular models and catalytic site

details. (A, B) The whole structures of

APEH-1Tb and APEH-2Tb, respectively. The

backbone ribbon is enriched with

secondary structure elements, shown as

red cylinders (helices) and yellow arrows

(b-strands). The catalytic triad is indicated

by a space-filling representation of the

three amino acids. (C, D) Details of the

catalytic sites in APEH-1Tb and APEH-2Tb,

respectively. The amino acids of the

catalytic triad are drawn in ball-and-stick

representation, with standard atom colors

(C, green; O, red; N, blue). The charged

amino acids within 2.5��A of the triad are

drawn in ball-and-stick representation.

Red: negatively charged amino acids. Blue:

positively charged amino acids.

FEBS Journal 281 (2014) 401–415 ª 2013 FEBS 409

M. Gogliettino et al. APEH from the Antarctic fish Trematomus bernacchii

the most active tissues in terms of protein breakdown.

The different distribution may be related to the pecu-

liar functionality of the corresponding APEH prod-

ucts, APEH-2Tb being exceptionally able to hydrolyze

oxidized proteins. In addition, the apeh-2Tb mRNA

levels were higher than those of apeh-1Tb in all of the

tissues analyzed, especially in those (liver and heart)

presumed to contain greater amounts of oxidized pro-

teins. Surprisingly, the transcriptional level ratios of

the two apeh genes were not in agreement with the rel-

ative protein amounts in liver, where APEH-1Tb was

the most abundant isoform. Indeed, the high RNA/

protein ratio for APEH-2Tb may represent a possible

approach to maintain the capability for an immediate

response to the oxidative environment when the levels

of damaged proteins become too high. Recently, it has

been reported that the Antarctic invertebrates maintain

elevated RNA/protein ratios, through increased RNA

levels to counterbalance the low RNA translational

efficiency at low temperatures; a different strategy has

been seen for the Antarctic eelpout (Pachycara brachy-

cephalum), which shows improved RNA translational

capacity [42]. However, further studies will be required

to determine whether the strategies adopted by Antarc-

tic fish to counteract the lower protein synthesis rates

involve an increase in RNA concentration, an

enhancement of translational capacity, or a combina-

tion of both mechanisms.

The different functional properties of the two APEH

isoforms were also supported by an investigation on

the 3D models and the respective active sites. The

geometry and chemical nature of the residues sur-

rounding the catalytic site are important for defining

the specificity of the enzyme and promoting efficient

substrate binding in order to lower the energy of its

transition state. It is therefore reasonable to hypothe-

size that the long-range interactions could play an

important role in the ‘molecular recognition’. In this

context, the charge distribution resulting from the dif-

ferent polar residues surrounding the catalytic sites of

the two enzymes could explain their distinctive kinetic

and thermodynamic properties, such as the ability of

APEH-2Tb to hydrolyze oxidized BSA. Specifically, we

note that the catalytic site in APEH-2Tb is more posi-

tively charged, and a possible attractive effect could be

exerted on the negatively charged methionine sulfox-

ide, a common modification observed in oxidized

BSA. Furthermore, the APEH-2Tb active site shows an

increase in its hydrophobic nature resulting from the

presence of Gly674 instead of Asn706 at the corre-

sponding position in the APEH-1Tb active site. These

structural features could be responsible for the ampli-

fied susceptibility of oxidized BSA [39], which under-

goes a conformational change that brings the cleavage

sites to the surface of the molecule, where they are eas-

ily accessible by APEH-2Tb. Therefore, the hydropho-

bicity may represent a signal for the degradation of

oxidized proteins by this subfamily of APEHs.

The data described in this work establish a valuable

starting point for studying the phase 3 antioxidant

enzyme systems in the Antarctic notothenioids, which

offer an excellent model with which to examine the

APEH–proteasome proteolytic pathway in cold-

adapted fish.

Experimental procedures

Materials

T. bernacchii specimens were collected in the vicinity of

Mario Zucchelli Station, along the coast of Terra Nova

Bay (74′42°S, 164′07°E), Antarctica, during the Italian

XXV expedition (2009–2010). They were held in running

seawater at �2 °C to +1 °C until tissue sampling. The ani-

mals were killed by truncation of the spinal cord. Tissues

were dissected from adult specimens, and frozen immedi-

ately in liquid nitrogen. Blood was drawn from the caudal

vein with heparinized syringes. RBCs were collected by cen-

trifugation at 3000 g for 5 min, washed in 1.7% NaCl, and

then frozen in liquid nitrogen. Tissues and cells were stored

at �80 °C until use.

Cloning and sequence analysis

Total RNA was isolated from liver of T. bernacchii accord-

ing to the NucleoSpin RNA II kit (Macherey-Nagel) proto-

col, with an on-column DNase I step. RNA concentrations

were determined with a Qubit Fluorometer (Invitrogen).

RNAs were then reverse transcribed with the SuperScript

VILO MasterMix (Invitrogen). The apeh cDNAs were

amplified by PCR with oligonucleotides designed on the

basis of conserved regions of various piscine apeh gene

sequences available in GenBank and BLAST analysis of a not-

othenioid SRA library (SRS255209). The sequences used for

primer design had the following accession numbers:

XM_003963278.1 and XM_003963026.1 (Takifugu rubripes),

XM_003448291.1 and XM_003444841.1 (Oreochromis niloti-

cus), XR_177409.1 and XM_004070616.1 (Oryzias latipes),

and NM_198869.1 and NM_001040347.1 (Danio rerio).

The primers used were as follows: APEH1Tbfor, 5′-ATGAA

CTCACAGGTGGTGACC-3′; APEH1Tbrev, 5′-TCACTTC

CACAAGTGTTGAATTATCCA-3′; APEH2Tbfor, 5′-AT

GGAGCCCAGCCTGGT-3′; and APEH2Tbrev, 5′-CTGTG

TTGAGGAAGCAGTCGG-3′.

The amplification was performed as follows: 94 °C for

2 min, 40 cycles of 94 °C (30 s), 58–60 °C (30 s), and

72 °C (2–2.5 min), and a final extension at 72 °C for

410 FEBS Journal 281 (2014) 401–415 ª 2013 FEBS

APEH from the Antarctic fish Trematomus bernacchii M. Gogliettino et al.

10 min. The PCR products were then analyzed on 1% aga-

rose gel, purified with the StrataPrep DNA Gel Extraction

Kit (Stratagene), and cloned into the StrataClone PCR

Cloning kit (Stratagene). The sequence of positive clones

was determined with an ABI PRISM 3100 automated

sequencer at PRIMM (Milan, Italy).

The sequences were edited and analyzed with the CLC

MAIN WORKBENCH 6.8 program (CLC bio, 2013) and depos-

ited in the GenBank database under the accession numbers

KC626077 and KC626078.

Phylogenetic analysis

Multiple amino acid sequence alignment of APEH chains

was obtained by use of the MUSCLE algorithms contained in

the CLC MAIN WORKBENCH 6.8 program. The alignment was

then submitted to the PROT TEST 3.2 program [43] to find

the best-fitting protein evolution model. Maximum likeli-

hood analysis was performed with the ‘A la Carte’ Mode

pipeline available on the Phylogeny.fr website (http://

www.phylogeny.fr/version2_cgi/index.cgi) [44], with the

parameters indicated by the PROT TEST analysis and the fol-

lowing program choice: MUSCLE for alignment; GBLOCKS for

alignment curation; and PHYML for phylogeny (substitution

model, WAG; gamma shape parameter, 1.38; proportion of

invariant, 0.07; bootstrapped data sets, 100). Finally, the

web-based tool PHYLOWIDGET (http://www.phylowidget.org/)

was used to draw the best bootstrap consensus tree [45].

Molecular modeling and structural analysis

The amino acid sequences of APEH-1Tb and APEH-2Tbwere used to create molecular models based on the tem-

plate structure of APEH from Aeropyrum pernix (Protein

Data Bank code: 1VE6). The modeling procedure has been

adapted from a well-assessed strategy used in our labora-

tory [46,47], taking into account specific features of these

sequences. The strategy combines bioinformatics tools to

apply the comparative modeling approach and perform,

just like a workflow, the search for templates, the align-

ment of the sequences, the modeling step, the validation of

the models, and, if needed, an iteration of all steps to

obtain satisfactory results. In detail, the search for similar-

ity with proteins of known 3D structures was performed

with BLAST. The refinement of the alignment of sequences

started from the BLAST result, and took into account sec-

ondary structure position and known properties, so that it

could suggest more than one alignment to be tested. In

addition, owing to the shorter extension of the sequence of

the template protein, different alignments were generated

that differed in the N-terminal half of the APEHs. For

each alignment, 10 models were generated with MODEL-

LER v9.10 by the Sali laboratory (http://www.salilab.org)

and analyzed for their structural properties and quality

with PROCHECK, PROSA, and visual inspection with molecular

graphics tools. For each protein, the best-quality model

was selected for further investigations.

Expression analysis

Total RNAs from several tissues of T. bernacchii were iso-

lated and reverse transcribed under the conditions

described in ‘Cloning and sequence analysis’. A total of

100 ng of cDNA and its dilution series to calculate the effi-

cacy of primers were amplified by quantitative real-time

PCR on an iCycler iQTM (Bio-Rad) with 300 nM gene-spe-

cific primers, MaximaH SYBR Green/Fluorescein qPCR

Master Mix (Thermo Scientific), and the following PCR

conditions: one cycle of 95 °C for 10 min, and 40 cycles of

95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The

expression level of the b-actin gene was used as an internal

control for normalization. Raw cycle threshold values (Ct

values) obtained for apeh-1Tb and apeh-2Tb (target genes)

were compared with the Ct value obtained for b-actin tran-

script levels (reference gene). The final graphical data

were derived from the following equation: R = (Etarget)ΔCt_target (control - sample)/(Eref)

ΔCt_ref (control - sample) [40]. The

Universal Probe Library Assay Design Center (http://

www.roche-applied-science.com/shop/CategoryDisplay?cata

logId=10001&tab=Assay+Design+Center&identifierUni

versal+Probe+Library&langId=-1#tab-3) was used for

the design of primers. The primer pairs used were as follows:

b-actin, 5′-ATGTTCGAGACCTTCAACACC-3′ and 5′-C

GACCAGAGGCGTACAGG-3′; APEH1Tb, 5′-TCTTCC

AGAGTGAGTCACCAAA-3′ and 5′-CTCCCTTCTCAC

GGTCTC-CT-3′; and APEH2Tb, 5′-AGTCTGCAGCAG

TTGGAC-CT-3′ and 5′-GGTCTGTTGACGACCTCCA

G-3′.

Enzyme preparation

APEH from T. bernacchii liver

We conducted partial purification of the target enzyme. Liv-

ers were dissected from different individuals (at least six) of

T. bernacchii. Tissue samples were homogenized in four vol-

umes of ice-cold homogenization buffer (10 mM Tris/HCl,

pH 7.5, containing 150 mM NaCl) with an Ultra-Turrax T25

homogenizer (IKA Works, Staufen, Germany). The homog-

enate was centrifuged at 288 000 g for 1.5 h at 4 °C. Thesupernatant was dialyzed against 25 mM Tris/HCl and 1 M

ammonium sulfate (pH 7.5) (buffer A), and then applied to

a Phenyl Sepharose column (1.6 9 2.5 cm) (Amersham Bio-

sciences) connected to an AKTAFPLC system (Amersham

Biosciences) equilibrated with the same buffer. Bound pro-

teins were eluted with a linear gradient (0–100%) of 25 mM

Tris/HCl (pH 7.5) (buffer B) at a flow rate of 1 mL�min�1.

The active fractions were pooled, dialyzed against 25 mM

FEBS Journal 281 (2014) 401–415 ª 2013 FEBS 411

M. Gogliettino et al. APEH from the Antarctic fish Trematomus bernacchii

Tris/HCl (pH 7.5), applied to a Superose 12 HR 10/30 col-

umn (Amersham Biosciences) connected to an AKTAFPLC

system, and then eluted with 25 mM Tris/HCl (pH 7.5) con-

taining 50 mM NaCl at a flow rate of 0.5 mL�min�1. Active

fractions were pooled, extensively dialyzed against 25 mM

Tris/HCl (pH 7.5) (buffer A), and loaded onto a Q Sepha-

rose Fast Flow ion exchange column (0.86 9 30 cm) (Amer-

sham Biosciences) pre-equilibrated in buffer A with an

AKTAFPLC system. After extensive washing of the column

with the same buffer, bound proteins were eluted with a lin-

ear ionic gradient (0–100%) of 1 M NaCl in buffer A at a

flow rate of 1 mL�min�1. The eluted active fractions were

pooled, and stored in 25 mM Tris/HCl (pH 7.5) containing

5% glycerol. The enzyme purification was followed with Ac-

Met-AMC as the substrate.

APEH from T. bernacchii RBCs

Samples of freshly drawn blood were obtained from the cau-

dal vein with heparinized syringes, and washed three times

with 0.15 M NaCl. Hemolysates were prepared from the

RBCs, separated from the blood plasma by centrifugation

(1067 g, 5 min), and washed twice with cold isotonic solution

(10 mM Tris/HCl, pH 7.6, 1.7% NaCl). Lysis of RBCs was

carried out by incubation in hypotonic solution (25 mM Tris/

HCl, pH 7.5) for 30 min on ice. The ‘soluble fraction’ was

obtained by centrifugation of the lysate at 9.200 g for 40 min

at 4 °C, and applied to a Sephadex-G25 column (GE Health-

care Bio-Sciences) equilibrated in 25 mM Tris/HCl (pH 7.5).

Active fractions were pooled, and loaded onto a DEAE

Sepharose Fast Flow column, previously equilibrated in

25 mM Tris/HCl (pH 7.5) (buffer A). Bound proteins were

eluted with an ionic strength gradient from 10 mM to 1 M

NaCl in buffer A. Active fractions were pooled (’hemoglo-

bin-free cytosolic fraction’), dialyzed extensively against

buffer A and then applied to a DEAE Sepharose Fast Flow

column, pre-equilibrated in buffer A (25 mM Tris/HCl,

pH 7.5, containing 175 mM NaCl). Bound proteins were

eluted with an ionic strength gradient from 175 mM to

350 mM NaCl in buffer A. The active fractions recovered

from the DEAE column were pooled, dialyzed against 1 M

ammonium sulfate in buffer A, and loaded onto a Phenyl Su-

perose PC 1.6/5 (Amersham) column, connected to a

SMART System (Pharmacia), equilibrated in the same buf-

fer. Bound proteins were eluted with a linear gradient of

25 mM Tris/HCl (pH 7.5) (0–100%). Active fractions were

pooled, and the purified protease was stored in 25 mM Tris/

HCl (pH 7.5) containing 5% glycerol. The enzyme purifica-

tion was followed with Ac-Met-AMC as the substrate.

Molecular mass determination

Protein homogeneity was estimated by SDS/PAGE analysis

[48] with 8% (w/v) acrylamide resolving gel. Standard pro-

teins (Broad Range) were from New England BioLabs.

Molecular masses of the native enzymes were established

by gel filtration chromatography on both Super-

dex 200 PC 3.2/30 and Superose 12 3.2/30 columns (Phar-

macia Biotech), connected to a SMART System and

calibrated with BioRad gel filtration standards (code 151-

1901). The protein concentration was determined with the

Bradford assay method [49].

Enzyme assays

The aminopeptidase activities of APEHs from T. bernacchii

and porcine liver were measured by the use of Ac-Leu-

pNA (Sigma), Ac-Ala-pNA (Bachem), acetyl-Phe-p-nitro-

anilide (Sigma), and Ac-Met-AMC (Bachem). The release

of p-nitroanilide (e410 nm = 8800 M�1�cm�1) was monitored

at 410 nm with a Cary 100 SCAN (Varian) UV–visible

spectrophotometer, equipped with a thermostated cuvette

compartment. The release of fluorescent product (7-amino-

4-methylcoumarin) was monitored in a Perkin-

Elmer LS 50B fluorimeter. The excitation and emission

wavelengths were 380 nm and 460 nm, respectively. All

experiments were carried out in triplicate on three different

protein preparations. Data were fitted to the Michaelis–

Menten equation by nonlinear regression with SIGMA PLOT.

The reaction mixture (1 mL), containing the appropriate

amount of each enzyme in 50 mM Tris/HCl (pH 7.5) and

1% (v/v) Triton X-100, was preincubated at the assay tem-

perature for 2 min. Then, substrate was added and the

release of product was measured. Calculated activities were

based on the initial linear phase of release. One unit of

APEH activity (U) was defined as the amount of enzyme

releasing 1 lmole of substrate per minute under the assay

conditions. All of the enzymatic activities, measured with

Ac-Met-AMC as the substrate, were expressed in arbitrary

units. The endopeptidase activity of APEHs was measured

spectrophotometrically by using the chromogenic substrates

succinyl-glycine-glycine-Phe-p-nitroanilide, carboxybenzyl-Gly-

Gly-Leu-p-nitroanilide, succinyl-Ala-Ala-Ala-p-nitroanilide,

succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and succinyl-Ala-

Ala-Val-Ala-p-nitroanilide, all from Bachem, following the

standard assay procedure described above.

Temperature and pH influence on APEH activity

Determinations of temperature and pH optima of APEHs

were performed with Ac-Ala-pNA as the substrate. The

effect of pH was determined between pH 4.6 and pH 8.9

under the assay conditions described above. Citrate/phos-

phate buffer (50 mM) was used for pH 4.6, and was

replaced by Tris/HCl (50 mM) for pH 6.7–8.9. Relative

activity was expressed as percentage of the maximum of

the enzyme activity under the standard assay conditions.

Optimum temperatures for the enzymes were determined

412 FEBS Journal 281 (2014) 401–415 ª 2013 FEBS

APEH from the Antarctic fish Trematomus bernacchii M. Gogliettino et al.

under the standard assay conditions in the temperature

range 15–70 °C. The thermal stabilities were determined by

measuring residual activities on Ac-Ala-pNA substrate after

incubation of the enzymes at various temperatures (10–

50 °C). Temperatures below 10 °C were not explored,

owing to their dramatic effects on substrate solubility.

Western blot analysis

Aliquots of protein samples were subjected to SDS/PAGE

(8%), and then electroblotted onto poly(vinylidene difluo-

ride) membranes (Immobilon; Millipore). Membranes were

next incubated with the primary antibody (APEH N-18 goat

IgG; 1 : 5000; Santa Cruz Biotechnology) and then with the

horseradish peroxidase-conjugated secondary antibody

(1 : 5000; 1 h at 37 °C; Santa Cruz Biotechnology). The

immune complexes formed were visualized by enhanced

chemiluminescence and autoradiography, according to the

manufacturer’s protocol (Amersham Biosciences) and mea-

sured by densitometry analysis with CHEMIDOC XRS (Bio-

Rad). Protein expression data were quantified with QUAN-

TITY ONE software (Bio-Rad).

N-terminal amino acid sequence analysis

Automated N-terminal sequence analysis of the two APEH

isoforms (corresponding to 80-kDa and 75-kDa immunore-

active bands) electroblotted onto poly(vinylidene difluoride)

membranes (Bio-Rad) was performed with a Perkin-Elmer

Applied Biosystems 477A pulsed-liquid protein sequencer.

The NCBI nonredundant protein dataset was scanned with

the PSI-BLAST program [50], with the experimentally

obtained N-terminus as the query sequence.

Degradation of oxidized BSA by APEHs as

analyzed by SDS/PAGE

A solution of unoxidized or oxidized BSA (1.7 lg), pre-

pared as described by Fujino et al. [14], was treated with

the APEHs (0.7 lg) from T. bernacchii in 30 lL of 25 mM

Tris/HCl (pH 8.0) at 37 °C for 0, 24 and 48 h. Thirty mi-

croliters of the reaction mixtures was subjected to SDS/

PAGE analysis. The same experiment was performed with

APEHpl as a control.

Acknowledgements

The authors wish to thank V. Carratore for expert

assistance with protein sequencing, and G. Monti for

technical support with the biological sample prepara-

tions. This study was conducted in the framework of

the project (PNRA) 2010/A1.08 ‘Role of oxygen in evo-

lution – genes and proteins of polar marine organisms’.

We gratefully acknowledge the logistical support pro-

vided to our research team at Mario Zucchelli Station.

A. Facchiano acknowledges the partial support of the

FLAGSHIP ‘InterOmics’ project (PB.P05), funded by

the Italian MIUR and CNR organizations.

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Supporting information

Additional supporting information may be found in

the online version of this article at the publisher’s web

site:Table S1. APEH chains utilized for phylogenetic

analysis.

Table S2A. Purification of APEH-2Tb from RBCs of

the notothenioid fish Trematomus bernacchii.

Table S2B. Purification of APEH-1Tb from liver of the

notothenioid fish T. bernacchii.

Table S3. List of charged residues found within an

increasing distance from the three amino acids of the

catalytic triad.

Fig. S1. cDNA sequence of apeh-1Tb and apeh-2Tbfrom T. bernacchii with the deduced amino acid

sequences.

Fig. S2. Muscle alignment of APEH-1Tb and APEH-

2Tb sequences with those of APEHs from different

sources.

Fig. S3. Gel filtration chromatography on a Super-

dex 200 column of APEH-2Tb from T. bernacchii.

FEBS Journal 281 (2014) 401–415 ª 2013 FEBS 415

M. Gogliettino et al. APEH from the Antarctic fish Trematomus bernacchii