Characterization of chitinase-like proteins (Cg-Clp1 and
Cg-Clp2) involved in immune defence of the mollusc
Crassostrea gigas
Fabien Badariotti, Christophe Lelong, Marie-Pierre Dubos and Pascal Favrel
Universite
´
de Caen Basse-Normandie, IBFA, UMR M100 IFREMER, Physiologie et Ecophysiologie des Mollusques Marins, Laboratoire de
Biologie et Biotechnologies Marines, Caen, France
Glycoside hydrolase family 18 (GH18) is a phylogenet-
ically conserved group of proteins present in eukaryo-
tes, prokaryotes and viruses. The GH18 family is
characterized by a Glyco_18 domain adopting an
(a ⁄ b)
8
triose phosphate isomerase-barrel structure that
consists of a specific arrangement of eight parallel
b-strands, forming the barrel core, surrounded by eight
a-helices [1]. This family classification, based only on
similarities in amino acid sequences, groups together
chitinases and proteins devoid of catalytic activity due
to the substitution of a critical amino acid in the cata-
lytic centre. This latter singular class of proteins, called
chitinase-like proteins (CLPs), has been identified only
recently in plants [2], mammals [3], insects [4] and mol-
luscs [5]. CLPs have been implicated in many biologi-
cal processes, such as control of nodulation [2] and
growth ⁄ differentiation balance during development in
plants [6]. Insect CLPs such as imaginal disc growth
factors represent the first proliferating polypeptides
reported from invertebrates [7]. These mitogenic
growth factors cooperate with insulin to stimulate pro-

molluscan chitinase-like protein, named Crassostrea gigas (Cg)-Clp1, was
shown to control the proliferation and synthesis of extracellular matrix
components of mammalian chondrocytes. However, the precise physiologi-
cal roles of Cg-Clp1 in oysters remain unknown. Here, we report the clo-
ning and the characterization of a new chitinase-like protein (Cg-Clp2)
from the oyster Crassostrea gigas. Gene expression profiles monitored by
quantitative RT-PCR in adult tissues and through development support its
involvement in tissue growth and remodelling. Both Cg-Clp1- and Cg-
Clp2-encoding genes were transcriptionally stimulated in haemocytes in
response to bacterial lipopolysaccharide challenge, strongly suggesting that
these two close paralogous genes play a role in oyster immunity.
Abbreviations
Cg-Clp1 ⁄ 2, Crassostrea gigas chitinase-like protein 1 ⁄ 2; CLP, chitinase-like protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
GH, glycoside hydrolase; HC-gp39, human cartilage glycoprotein-39 (also called YKL-40); LPS, lipopolysaccharide; YKL-40, 40 kDa
mammalian protein with the N-terminus YKL.
3646 FEBS Journal 274 (2007) 3646–3654 ª 2007 The Authors Journal compilation ª 2007 FEBS
from the oyster Crassostrea gigas [5]. Interestingly, this
protein, named C. gigas chitinase-like protein 1 (Cg-
Clp1) was found to be involved in the control of
growth and remodelling processes in a manner similar
to its YKL-40 mammalian counterpart. These findings
argue for an early evolutionary origin and a high con-
servation of this class of proteins at both the structural
and functional levels. Given the multiplicity of CLPs
in humans and insects [14], we hypothesized that
homologues of the previously characterized Cg-Clp1
remain to be found in C. gigas.
In this article, we report the characterization of a
new CLP, named Cg-Clp2, from the oyster C. gigas.
The tissue distribution and temporal pattern of expres-

charide [17] ( (Fig. 1).
Cg-Clp2 sequence identity with other proteins
Optimal alignment of Cg-Clp2 with Cg-Clp1 and other
GH18 family members revealed regions of significant
identity, especially in the Glyco_18 domain. The glu-
tamate residue known to be critical for chitinase activ-
ity [18] is substituted by a glutamine, suggesting that
this protein lacks chitinolytic activity, as was shown
previously for recombinant Cg-Clp1 [5] and other
CLPs [19]. Following the Glyco_18 domain, Cg-Clp2
displays an additional 90 amino acid C-terminal
sequence of unknown function (Fig. 1). Hence, the
overall structure of Cg-Clp2 is similar to that of
Cg-Clp1.
Expression of Cg-Clp2 transcripts during
development and in adult tissues
To gain insights into possible physiological functions
of Cg-Clp2, determination of its tissue distribution and
temporal pattern of expression during development
was performed by real time RT-PCR (Fig. 2A). Cg-
Clp2 transcripts were mainly expressed during larval
metamorphosis, in the mantle edge and the digestive
gland. During the reproductive cycle, expression was
high in gonads during the postspawning period but
not in stage I, when gonial multiplication starts [20].
To investigate which types of cell were responsible for
Cg-Clp2 expression in the mantle edge, in situ hybrid-
ization experiments were performed (Fig. 2B). Tran-
scripts were detected in both epithelial and conjunctive
cells of the mantle.

is marked with a thick black line above the sequence alignment. Arrowheads indicate the positions of residues (D and E) required for catalytic activity in bacterial chitinases [18]. The black
dotted line delimits the Glyco_18 domain. The species abbreviations used are: Dm, Drosophila melanogaster; and Hs, Homo sapiens. GeneBank accession numbers: Cg-Clp1, AJ971241;
Dm IDGF4, NP511101; Hs YKL-40, NP001267; Dm Cht9, NP611543.
Oyster chitinase-like proteins F. Badariotti et al.
3648 FEBS Journal 274 (2007) 3646–3654 ª 2007 The Authors Journal compilation ª 2007 FEBS
haemocytes as compared to freshly harvested circula-
ting cells.
Discussion
In the present study, we identified a second oyster
CLP named Cg-Clp2. Comparative sequence analyses
with other GH18 family members show that Cg-Clp2
displays the same protein organization as the previ-
ously identified Cg-Clp1, with a Glyco_18 domain (in
a catalytically inactive form [5]) followed by an addi-
tional C-terminal sequence of about 90 amino acids of
unknown function. The high degree of identity of the
Cg-Clp1 and Cg-Clp2 Glyco_18 domains (84% iden-
tity) argues for a conservation of the tertiary structure
and associated biochemical properties (such as chitin
binding). Evidence for a high level of conservation of
the tertiary structure of CLPs during evolution is also
supported by the observation that both Cg-Clp1 and
its closest mammalian homologue YKL-40 present
A
B
Fig. 2. Expression of Cg-Clp2 mRNAs in adult tissues and during development measured by real-time quantitative RT-PCR. (A) Each value is
the mean + SE of three pools of four animals (tissues) or the mean of one pool of embryos or larva from one spawn. Expression levels are
related to 100 copies of GAPDH. (B) Localization of Cg-Clp2 mRNA expression in the mantle edge investigated by in situ hybridization.
Arrows indicate hybridization signals.
F. Badariotti et al. Oyster chitinase-like proteins

Statistical analysis of the results was per-
formed with Student’s t-test (*P < 0.05;
**P < 0.02).
Oyster chitinase-like proteins F. Badariotti et al.
3650 FEBS Journal 274 (2007) 3646–3654 ª 2007 The Authors Journal compilation ª 2007 FEBS
during metamorphosis, in the mantle edge and post-
spawning gonads. Metamorphosis represents the ulti-
mate stage of oyster development, and is characterized
by the degeneration of larval tissues, such as the velum
and the foot, and the remodelling of larval tissues to
produce adult tissues (i.e. the development of the gills
and the production of an adult shell), which is accom-
panied by significant growth of the soft body parts
[21]. The mantle edge governs shell formation and
body growth by the secretion of shell organic matrix
and by cell proliferation. As Cg-Clp2 appears to be
expressed in both epithelial and conjunctive cell types
of the mantle edge, this protein could orchestrate the
synthesis of extracellular components and ⁄ or the pro-
liferation of mantle cells, as was proposed for Cg-Clp1
[5]. The postspawning gonad is characterized by the
resorption of gonadic tubules and the rebuilding of
storage tissues [22]. The expression of Cg-Clp2 during
this particular period is somewhat reminiscent of the
finding that certain mammalian CLPs such as CLP-1
and MGP40 are specifically expressed during mam-
mary gland involution [23,24]. Considering Cg-Clp2
patterns of expression, this protein could be involved
in tissue growth and remodelling, as was formerly
postulated for Cg-Clp1 [5].

ponents of mammalian chondrocytes [5] endorses the
possibility that Cg-Clp1 promotes cell (haemocyte)
proliferation and ⁄ or tissue repair, both processes
occurring during immune responses [26,27]. Such a
role was also suggested for insect imaginal disc growth
factors [8,9,28]. As was observed for its murine
homologue (YM-1), which behaves as a chemotactic
cytokine that recruits cells to sites of inflammation and
promotes eosinophilia around larvae of nematode
parasites [10], mediation of immune cell (haemocytes)
migration or aggregation might also represent a
potential function for Cg-Clp1. Because Cg-Clp1 and
Cg-Clp2 are two close paralogues sharing a very sim-
ilar structure, the several roles predicted for Cg-Clp1
in immunity may also be relevant for Cg-Clp2.
Interestingly, haemocyte adhesion to the culture plastic
dish induces on its own a strong increase in Cg-Clp2
transcript expression, whereas no effect was detected
for Cg-Clp1. Such a surprising result was previously
observed for the oyster chitinase Cg-Chit [15]. This
in vitro assay somehow mimics haemocyte conversion
from circulating cells to cells that interact with and
adhere to each other or to a foreign target surface, as
is observed for encapsulation [29]. These ‘activated
haemocytes’ may become immunologically competent
cells capable of producing acute phase immune effec-
tors, as was recently reported for Manduca sexta plas-
matocytes, which express only the specific lectin
‘lacunin’ upon adhering to a foreign surface [30]. This
would explain why stimulation of Cg

farm (Saint Vaast La Hougue, France). The embryonic and
larval stages were produced in the IFREMER shellfish
laboratory of Argenton (France).
RNA purification, reverse transcription, cloning
and sequencing
Total RNA was isolated from the oyster mantle edge using
Tri-Reagent (Sigma-Aldrich, St Louis, MO, USA) accord-
ing to the manufacturer’s instructions. mRNAs were isola-
ted using oligodT coupled to magnetic beads as described
by the manufacturer (Dynal, Invitrogen, Carlsbad, CA,
USA). Reverse transcription was carried out using oli-
go(dT)
17
as primer, 1 lg of mantle edge mRNA, and 200 U
of Moloney murine leukaemia virus reverse transcriptase
(Promega, Madison, WI, USA). cDNAs were used as tem-
plates for PCR amplifications using two degenerated prim-
ers designed to anneal to conserved consensus regions of
GH18 family members (chitinases and CLPs) from different
bilaterian species. The sense primer corresponding to the
LK(I ⁄ M)L(F ⁄ L)(S ⁄ T ⁄ R ⁄ C ⁄ W)VGG amino acid seque-
nce was 5¢-CTN AAR ATN CTN YTN WSN GTN GGN
GG-3¢, whereas the antisense primer corresponding to the
FDGLDLA amino acid sequence was 5¢-GGC NAG RTC
NAG NCC RTC RAA-3¢ (Y ¼ CorT,R¼ AorG,S¼
CorG,W¼ AorT,N¼ A or C or G or T). PCR was
performed in a total volume of 50 lL with 10 ng of mantle
edge cDNA in 10 mm Tris ⁄ HCl (pH 9.0), containing
50 mm KCl, 0.1% Triton X-100, 0.2 m m each dNTP, 1 lm
each primer, 2.5 mm MgCl

Green supermix PCR kit (Biorad) was used for real-time
monitoring of amplification (5 ng of cDNA template, 40
cycles: 95 °C for 15 s, 60 °C for 15 s) with the following
primers: QsCgClp1 (5¢-CTTCCTCCGCTTCCATGA-3¢)
and QaCgClp1 (5¢-CCATGAAGTCCGCGAATC-3¢); and
QsCgClp2 (5¢-GCATAGCGATGTGGACGA-3¢) and
QaCgClp2 (5¢-GAGGACCGAGACCGTGAA-3¢). The
abbreviations ‘Qs’ and ‘Qa’ refer, respectively, to sense and
antisense primers. Accurate amplification of the target
amplicon was checked by obtaining a melting curve.
Using QsGAPDH (5¢-TTCTCTTGCCCCTCTTGC-3¢) and
QaGAPDH (5¢-CGCCCAATCCTTGTTGCTT-3¢), a paral-
lel amplification of oyster glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (CGI548886) reference tran-
scripts was carried out to normalize the expression data of
Cg-Clp1 and Cg-Clp2 transcripts. The relative level of
expression of each target gene was calculated for 100 copies
of GAPDH transcript by using the following formula:
N ¼ 100 · 2
(Ct GAPDH ) cycle threshold transcript of interest)
.
In situ hybridization
A 1283 bp fragment corresponding to the most 3¢-end of
Cg-Clp2 was subcloned in pGEMT easy. This recombinant
plasmid was used as a template for the synthesis of biotin-
labelled sense and antisense cRNA probes according to the
manufacturer’s instructions (NEN Life Sciences, PE, Wal-
tham, MA, USA). Dissected C. gigas mantle edges were
fixed, dehydrated in an increasing alcohol series and xylene,
and embedded in paraplast. Seven-micrometre sections were

In vitro challenge
Primary haemocyte culture was performed as previously
described, with some modifications [33]. Haemolymph was
recovered from the pericardic region of 90 oysters using a
45-gauge needle, and then subsequently transferred to a
sterile tube and simultaneously diluted 1 : 3 in cooled sterile
anticoagulant modified Alsever’s solution (115 mm glucose;
27 mm sodium citrate; 11.5 mm EDTA; 382 m m NaCl).
Haemocytes were rapidly plated at 4 · 10
6
cells per 9.5 cm
2
well, to which three volumes of sterile artificial sea water
were added to allow cell attachment. Cultures were main-
tained at 15 °C in a humidified incubator (CO
2
-free). After
60 min of incubation, cells were washed with Hanks-199
medium modified by the addition of 250 mm NaCl, 10 mm
KCl, 25 mm MgSO
4
, 2.5 m m CaCl
2
, and 10 mm Hepes; the
final pH was 7.4, and the osmolarity was 1100 mOsmolÆ L
)1
.
Cells were then covered with fresh medium supplemented
with l-glutamine (2 mm), concanavalin A (2 mm), strepto-
mycin sulfate (76.1 IUÆmL

Re
´
gional de Basse-Normandie’, the ‘Agence de l’eau
Seine-Normandie’ and FEDER Presage No. 4474
grant (program PROMESSE). The authors are
indebted to all staff of the Argenton IFREMER
experimental hatchery for the production of oyster
embryos and larvae. The authors thank Christophe
Fleury and Emeline Furon (University of Caen) for
technical assistance.
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