Báo cáo khoa học: Nautilin-63, a novel acidic glycoprotein from the shell nacre of Nautilus macromphalus doc

Nautilin-63, a novel acidic glycoprotein from the
shell nacre of Nautilus macromphalus
Benjamin Marie
1,2
, Isabelle Zanella-Cle
´
on
3
, Marion Corneillat
4
, Michel Becchi
3
,Ge
´
rard Alcaraz
2,4
,
Laurent Plasseraud
2,5
, Gilles Luquet
1,2
and Fre
´
de
´
ric Marin
1,2
1 UMR 5561 CNRS, Bioge
´
osciences, Dijon, France
2 Universite

´
de Bourgogne, Dijon, France
3 IFR 128 BioSciences, UMR 5086 CNRS, IBCP, Universite
´
de Lyon 1, Lyon, France
4 UPSP PROXISS, De
´
partement Agronomie Environnement, AgroSup, Dijon, France
5 ICMUB, UMR CNRS 5260, Faculte
´
des Sciences Mirande, Dijon, France
Keywords
biomineralization; de novo sequencing;
immunolocalization; mollusc shell nacre;
organic matrix
Correspondence
B. Marie or F. Marin, UMR 5561 CNRS
Bioge
´
osciences, Universite
´
de Bourgogne,
6 Boulevard Gabriel Dijon 21000, France
Fax: +33 3 80 39 63 87
Tel: +33 3 80 39 63 72
E-mail: ;

(Received 29 September 2010, revised 18
March 2011, accepted 11 April 2011)
doi:10.1111/j.1742-4658.2011.08129.x

In molluscs, and more generally in metazoan organisms, the production of
a calcified skeleton is a complex molecular process that is regulated by the
secretion of an extracellular organic matrix. This matrix constitutes a cohe-
sive and functional macromolecular assemblage, containing mainly pro-
teins, glycoproteins and polysaccharides that, together, control the
biomineral formation. These macromolecules interact with the extruded
precursor mineral ions, mainly calcium and bicarbonate, to form complex
organo-mineral composites of well-defined microstructures. For several rea-
sons related to its remarkable mechanical properties and to its high value
in jewelry, nacre is by far the most studied molluscan shell microstructure
and constitutes a key model in biomineralization research. To understand
the molecular mechanism that controls the formation of the shell nacreous
layer, we have investigated the biochemistry of Nautilin-63, one of the
main nacre matrix proteins of the cephalopod Nautilus macromphalus.
After purification of Nautilin-63 by preparative electrophoresis, we demon-
strate that this soluble protein is glycine-aspartate-rich, that it is highly gly-
cosylated, that its sugar moieties are acidic, and that it is able to bind
chitin in vitro. Interestingly, Nautilin-63 strongly interacts with the mor-
phology of CaCO
3
crystals precipitated in vitro but, unexpectedly, it exhib-
its an extremely weak ability to inhibit in vitro the precipitation of CaCO
3
.
The partial resolution of its amino acid sequence by de novo sequencing of
its tryptic peptides indicates that Nautilin-63 exhibits short collagenous-like
domains. Owing to specific polyclonal antibodies raised against the purified
protein, Nautilin-63 was immunolocalized mainly in the intertabular nacre
matrix. In conclusion, Nautilin-63 exhibits ‘hybrid’ biochemical properties
that are found both in the soluble and insoluble proteins, rendering it diffi-

cult to classify according to the standard view on nacre proteins.
Database
The protein sequences of N63 appear on the UniProt Knowledgebase under accession number
P86702.
Abbreviations
AIM, acid-insoluble matrix; ASM, acid-soluble matrix; CBB, Coomassie brillant blue; EST, expressed sequence tag; HPAE-PAD, high
performance anion exchange-pulsed amperometric detection; LSB, Laemmli sample buffer; N63, Nautilin-63; SEM, scanning electron
microscopy; TFMS, trifluoromethanesulfonic acid.
FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS 2117
Introduction
The calcified shells, which protect the mollusc soft tis-
sues, comprise layered structures that are produced
extracellularly by the calcifying epithelium of the man-
tle. The shell layers are composites of calcium carbon-
ate crystals, which are densely packed together with an
array of biomacromolecules that form a 3D frame-
work. Although the organic shell matrix (comprising
mainly proteins, glycoproteins and polysaccharides)
represents only a very small part of the CaCO
3
shell
weight (between 1% and 5% for the nacreous layer), it
is now well known to be essential for the control of
the biomineral formation [1]. In particular, it is
assumed to interact in different ways with the mineral
phase at the nano- to microscale. Indeed, the organic
shell matrix is considered to create a suitable environ-
ment for mineralization to occur [1–3], to promote or
inhibit crystal nucleation [4], to select calcium carbon-
ate polymorph (aragonite and ⁄ or calcite) [5], to allow

crystals to grow in privileged directions [3] and to con-
tribute to the spatial arrangement of crystals to form
well-defined microstructures [2,3]. At the atomic scale,
this matrix slightly modifies the crystal lattice parame-
ters, although this effect is poorly understood [6].
Because of its admirable biomechanical properties [7],
its use in pearl industry and, finally, its potential use in
dentistry and bone surgery [8,9], nacre is by far the most
studied nonhuman organo-mineral biocomposite. It has
a remarkable regular lamellar structure consisting of
uniformly thick layers (approximately 0.5 lm) of tablet-
like aragonite crystals separated by interlamellar layers
of organic matrix. This apparent simple geometry facili-
tates various structural investigations from micro- to
nanoscales [10–12]. Nacre, or its precursor, ‘foliated ara-
gonite’, appeared early in mollusc history, somewhere in
the Cambrian [13]. It constitutes the inner layer of sev-
eral extant mollusc shells, including that of bivalves,
gastropods, cephalopods and monoplacophorans. There
are, however, structural differences between cephalo-
pod, gastropod and bivalve nacres. Although the bivalve
exhibits a characteristic ‘brick-wall’ nacre microstruc-
ture, those of cephalopods and gastropods are a contin-
uous superimposition of tablets forming characteristic
columnar microstructures. Observations of growing
nacre show that each tablet nucleates at a specific loca-
tion on the matrix surface [14]. Today, the general con-
sensus is that nacre tablets grow from their center and
expand laterally until reaching the confluence with
neighboring tablets [10]. Histochemical observations of

Nautilus nacre [15,16] indicate a concentric distribution
of reactive groups, similar to carboxylates or sulfates,
from the center to the periphery of each single tablet.
A recent ultrastructural study has shown that nacre tab-
lets are individually coated by a 5 nm thick layer of
amorphous calcium carbonate [17]. Atomic force
microscopy studies by Rousseau et al. [18] have shown
that each tablet is constituted of nanograins encapsu-
lated in a continuous network of an organic intracrystal-
line phase. Summarizing the different recent advances
on molluscan nacre, Addadi et al. [19] have proposed a
coherent and dynamic model for nacre formation, as
described below.
The organic matrix constitutes the framework in
which nacre tablets form. The major constituents of the
matrix are the polysaccharide b-chitin, together with a
relatively complex assemblage of hydrophobic and
hydrophilic proteins. These macromolecules, which
control the crystal deposition and microstructure self-
assembly, are finally occluded either between the super-
imposed parallel lamellae (‘interlamellar matrix’), at the
boundary of adjacent mature nacre tablets (‘intertabu-
lar matrix’), or within the crystallites (‘intracrystalline
matrix’). First, the interlamellar matrix is assumed to
be predominantly constituted of b-chitin fibrils that are
aligned with the a-axis of the growing aragonitic tablets
[20], suggesting that they can be, directly or indirectly,
implicated in the control of the crystal orientation [21].
On the other hand, the b- and c-axes of the nacre
tablets are oriented in parallel to the growing front in

bivalves, whereas this is not the case in gastropods [22].
These data suggest that chitin is therefore either not
fulfilling this role in nacre formation or that not all
nacres are constructed in the same way, as recently sug-
gested by Jackson et al. [23]. However, structuring the
interface between the formed mineral front and the
secreting mantle tissues, these different matrices are
considered to precede immediately the new minerali-
zation [19]. Second, the hydrophobic matrix, which
contains silk-like proteins [2], is rich in Gly and Ala, or
Gly alone, constituting one of the major protein frac-
tions of the matrix, which can be extracted by decalcifi-
cation of the nacre. These hydrophobic silk-like
proteins are considered to form a hydrogel phase,
supersaturated in calcium ions, between the chitin
sheets. In this gel, the nacre tablets nucleate and grow
[11]. During their growth, they push aside and com-
press the silk-like protein gel. When adjacent tablets
come to confluence, the gel polymerizes and remains
‘sandwiched’, forming the intertabular matrix [24].
Third, acidic hydrophilic proteins containing carboxyl-
ate or sulfate reactive groups [25] are dispersed in the
gel; they are considered to act as nucleating centers for
each tablet; at the same time, they constitute a tenuous
Nautilin-63, a novel shell nacre protein B. Marie et al.
2118 FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS
organic lace inside which mineral nanograins (initially
amorphous) self-organize, orient and crystallize in a
coordinated manner. Once formed, each tablet contains
this intracrystalline matrix.

b-chitin, the silk-like proteins and the acidic proteins
are considered to be the three major components of
the nacre organic matrix. However, several other nacre
proteins have been identified and characterized over
the past 16 years; a review on mollusc shell proteins is
provided by Marin et al. [26]. These proteins, which
exhibit diverse putative functionalities according to
their sequences, are not taken in account in the model.
Furthermore, from the dozen primary structures of
nacre proteins that have been described so far, most of
them correspond to proteins of the pearl oyster or the
abalone models. None of them were described from
the cephalopod nacre.
Although the Nautilus nacre has already been the
focus of several ultrastructural [15,16,27–29] or bio-
chemical investigations of the bulk shell matrix [30–32],
only a few studies have dealt with the detailed charac-
terization of its shell proteins or their amino acid
sequence characterization [33,34]. Because one of the
keys to elucidating the molecular mechanisms of bio-
mineralization depends on a detailed characterization
of matrix proteins, as well as on the understanding of
their functions, we chose to focus on Nautilin-63
(N63), a major protein of the nacre of the cephalopod
Nautilus macromphalus. N63 comprises an acid-soluble
acidic shell matrix glycoprotein, which is specific to the
nacreous layer.
Results
N63 purification by preparative electrophoresis
Because N63 was found to be one of the main proteins

of the nacre acid-soluble matrix (ASM) [34], it was
investigated further. In our previous study, using a 2D
gel, we determined that N63 corresponded to a single
acidic protein, and not a mixture of proteins of the
same molecular weight but with different isoelectric
points: indeed, this protein migrated as a single acidic
spot. The fractionation of the nacre ASM preparative
electrophoresis resulted in the effective one-step purifi-
cation of N63. The purity of the N63 extract was
checked by monodimensional gel electrophoresis with
silver nitrate staining (Fig. 1A).
FTIR
Figure 1B shows the FTIR profile of N63 and of the
nacre ASM. Both samples exhibit characteristic bands
of proteinaceous and ⁄ or glycoproteinaceous compo-
nents [35]: the thick bands around 3270 cm
)1
are
attributed to the -OH and the amide A groups (N-H
bonds), the two small bands at 2915 and 2850 cm
)1
were assigned to the C-H bonds, and the two notewor-
thy bands near 1640 and 1530 cm
)1
were ascribed to
the amide I (C=O bond) and the amide II (C-N bond)
groups, respectively, which are commonly associated
with proteins. Carboxylate (COO
)
) and sulfate (SO

4
2)
)
absorption bands are also present in both samples,
around 1420 and 1235 cm
)1
. We note that N63 exhib-
its a remarkably strong carbohydrate absorption band
(C-O bond) around 1060 cm
)1
. These observations
suggest that N63 is an acidic glycoprotein.
Amino acid composition of N63
The purified N63 was analyzed for its amino acid com-
position and was compared with those of the nacre
ASM, which was obtained previously for the same spe-
cies [34] (Table 1). The six dominant amino acid resi-
dues are Asx (18%), Gly (17%), Thr (11%), Ala (9%),
Glx (8%) and Pro (8%). By comparison with the
Fig. 1. Purification and characterization of
N63. (A) 12% SDS ⁄ PAGE of ASM (and of
N63) after its purification by preparative
electrophoresis. The gel was stained with
silver nitrate. The apparent molecular
weights of the molecular markers (MM) are
indicated on the left. (B) Infrared spectra of
the ASM (gray line) and the purified N63
(black line).
B. Marie et al. Nautilin-63, a novel shell nacre protein
FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS 2119

amino acid composition of the bulk matrix, N63 is
strongly enriched in Thr and Pro residues but depleted
in Gly and Asx residues. A search based on the simi-
larity of amino acid composition (AACompIdent:
did not produce any
significant hits.
Monosaccharide composition of N63
The purified N63 was analyzed for monosaccharide
composition, which was subsequently compared with
that of the whole ASM [34] (Table 2). We note that,
similar to ASM, N63 contains a high amount of mono-
saccharide fraction (total of 275 ngÆlg
)1
). The five
dominant monosaccharides are glucose (22%), galac-
tose (17%), glucosamine (17%), glucuronic acid (15%)
and galactosamine (12%). By comparison with the
monosaccharide composition of the bulk matrix, N63
appears strongly enriched in glucosamine and galactos-
amine but also strongly depleted in glucose. Interest-
ingly, for both samples, we noted an unknown peak on
high performance anion exchange-pulsed amperometric
detection (HPAE-PAD) chromatograms, which eluted
in the ‘acidic monosaccharides’ area, near the expected
galacturonic acid peak [36,37]. The identity of this peak
needs to be investigated further.
Chemical deglycosylation of N63
In a previous study [34], the periodic acid–Schiff and
Alcian blue staining on SDS ⁄ PAGE suggested that
N63 is an acidic glycoprotein. To confirm this finding,

the nacre ASM was chemically deglycosylated with tri-
fluoromethanesulfonic acid (TFMS) at 0 °C. The ASM
and the deglycosylated-ASM were compared on
SDS ⁄ PAGE gels with double Coomassie brillant blue
(CBB) ⁄ silver and Alcian blue staining (Fig. 2A). This
Table 1. Composition of the nacre ASM and purified N63: amino
acid composition. Data are presented as the molar percentage of
total amino acids for each extract. Note that Asx = Asn + Asp and
Glx = Gln + Glu. Cysteine residues were quantified after oxidation.
Tryptophan residues were not detected (ND) as a result of the
hydrolysis conditions.
Amino acid
% of total amino acids
ASM N63
Asx 20.8 18.0
Glx 7.5 8.2
Ser 6.8 8.6
His 2.1 1.3
Gly 21.4 16.8
Thr 4.8 10.6
Ala 7.3 8.7
Arg 3.1 2.2
Tyr 2.9 1.1
Cys 1.1 ND
Val 3.1 4.4
Met 0.7 0.4
Phe 2.7 1.9
Ile 2.3 2.6
Leu 3.2 4.3
Lys 3.9 2.9

Pro 6.4 8.1
Trp ND ND
Table 2. Composition of the nacre ASM and purified N63: mono-
saccharidic composition. The composition of neutral sugars is
obtained by HPAE-PAD. Data are represented as ngÆlg
)1
of the
total matrix and as a percentage of the total identified carbohydrate
compounds. ND, not detected.
Monosaccharide
ngÆlg
)1
of matrix (% of total)
ASM N63
Fucose 15.1 (6) 21.8 (8)
Rhamnose 13.3 (5) 11.6 (4)
Arabinose ND ND
Galactose 43 (17) 46.0 (17)
Glucose 79.4 (31) 60.3 (22)
Mannose 9.2 (4) 10.4 (4)
Xylose 2.0 (1) 2.3 (1)
Galactosamine 20.6 (8) 33.7 (12)
Glucosamine 33.5 (13) 47.5 (17)
Galacturonic acid
a
ND ND
Glucuronic acid 37.0 (15) 41.4 (15)
Total 253.1 (100) 275 (100)
a
An unattributed band was observed around the galacturonic acid

band.
Fig. 2. Glycosylation (A) and chitin-binding (B) characterizations of
N63 by SDS ⁄ PAGE. (A) 12% SDS ⁄ PAGE of ASM and deglycosylat-
ed-ASM (Deg-ASM) stained with silver nitrate + CBB (left) and with
Alcian blue (right). (B) Chitin-binding ability of N63 (top) and BSA
(down, negative control) on 12% SDS ⁄ PAGE stained with silver
nitrate. Lane 1, water wash; lane 2, 0.2
M NaCl wash; lane 3,
extract with LBS. For both proteins, the same volume of solution
was loaded on the gel.
Nautilin-63, a novel shell nacre protein B. Marie et al.
2120 FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS
double staining allowed visualization of most of the
macromolecular compounds of the ASM on the same
gel. N63 exhibits an important shift of approximately
10 kDa, which represents a loss of apparent molecular
weight of 14%. This shift is primarily the result of the
removal of covalently bound polysaccharides. Interest-
ingly, the positive Alcian blue staining, observed for
the N63 glycoprotein, was completely lost after degly-
cosylation. Because we used Alcian blue under low pH
conditions, this result confirms that an important part
of the polyanionic properties of N63 is a result of the
acidic glycosyl moieties [38,39].
Chitin-binding capability of N63
Framework proteins of the organic nacre matrix are
hypothesized to interact with chitin [19,20]. However,
this property has never been tested previously on this
type of matrix. The putative chitin-binding ability of
N63 was examined consequently (Fig. 2B). The nacre

ASM was incubated in solution with powdered chitin,
and the insoluble mixture was successively washed with
distilled water, saline and finally with hot denaturing
Laemmli sample buffer (LSB) [40]. Each washed sam-
ple was analyzed by SDS ⁄ PAGE, stained with silver
nitrate. BSA, used as a negative control, was com-
pletely washed out with the successive water and saline
treatments, with no band being detected in the LSB
wash (Fig. 2B, bottom, lane 3). By contrast, a minor
part of N63 was desorbed after the water and saline
treatments (Fig. 2B, top, lanes 1 and 2) and the drastic
LSB wash was required for complete N63 desorption
from chitin (Fig. 2B, top, lane 3). This clearly suggests
that N63 has a strong affinity for this insoluble poly-
saccharide, and thus possesses a true chitin-binding
ability.
In vitro inhibition of CaCO
3
precipitation with
N63
The effect of nacre ASM and purified N63 on the
kinetics of CaCO
3
precipitation was determined by
monitoring the pH decrease (Fig. 3). In the blank
experiment (without sample), the pH decreased with-
out any time lag (approximately 120 s), corresponding
to the rapid precipitation of calcium carbonate. When
samples were present in the solution, we observed a
slight inhibition of CaCO

3
precipitation. First, the
effect of the nacre matrix started to occur above 1 lg
of the ASM and the delay of the reaction was dose-
dependent. At approximately 50 lg of nacre ASM, a
complete inhibition of the precipitation of calcium car-
bonate was recorded. The observation of the inhibitory
capacity of this matrix is consistent with previous stud-
ies on the organic soluble matrix of nacre from differ-
ent molluscs [24,41,42]. On the other hand, inhibition
experiments performed with N63 demonstrate that it
presents a five-fold lesser inhibition capacity than the
total ASM. At 25 lg, the N63 inhibition curve can be
superimposed to the 5 lg curve obtained with the total
ASM. Taken together, our observations indicate that,
if nacre ASM exhibits a moderate capacity of inhibi-
tion of CaCO
3
precipitation, this effect is not a result
of N63 because the latter presents only a weak inhibi-
tion capacity, despite the fact that it carries sulfated
(i.e. negatively charged) sugars.
Interaction with CaCO
3
crystals precipitated
in vitro
The effect of purified N63 on the precipitation
and morphology of calcium carbonate crystals grown
in vitro was investigated by scanning electron micros-
copy (SEM) (Fig. 4). When no protein is added, crys-

tals exhibit the typical rhombohedral habitus of calcite
with smooth crystal faces (Fig. 4A). In the presence
of an increasing amount of purified N63 (0.1–
50 lgÆmL
)1
), the crystals produced appear mostly as
polycrystalline aggregates with foliation and microsteps
at the corners (Fig. 4B–F). At the highest concentra-
tion (‡ 10 lgÆmL
)1
), the precipitated CaCO
3
crystals
exhibit specific linear grooves at the edges of the poly-
crystals (Fig. 4E,F). FTIR analysis of the calcium
carbonate polymorph confirmed that these crystals
were only made of calcite. Unexpectedly, at the highest
concentrations of N63, no inhibition of crystal forma-
tion occurs.
Fig. 3. In vitro inhibition of CaCO
3
precipitation by nacre ASM and
N63. The effects of different concentrations (1–50 lg) of purified
N63 and whole ASM were monitored on the pH decrease induced
by the in vitro precipitation of CaCO
3
in a CaCl
2
⁄ NaHCO
3

solution
[4]. Blank tests were performed in the absence of protein.
B. Marie et al. Nautilin-63, a novel shell nacre protein
FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS 2121
These results indicate that N63 interacts obviously
with the precipitation of CaCO
3
because it induces
drastic changes in the crystals morphology but does
not (or very slightly) inhibit their formation, with the
combination of these two effects in a shell matrix pro-
tein being rather unusual.
De novo sequencing of N63
Purified N63 was digested with trypsin before analysis
by MS ⁄ MS. Peptide digests were loaded on a nanoLC
column and analyzed by nanoESI-qQ-TOF. Because
no genomic, nor transcriptomic data are available for
Nautilus, the most intensive MS ⁄ MS peaks were manu-
ally interpreted (de novo sequencing) after considering
the complexity of the spectra and the numerous ion
combinations. For N63, the sequence of 27 peptides,
with lengths comprising between eight and 20 amino
acids, was determined by de novo interpretation of
their respective MS ⁄ MS spectra (Table 3). The partial
protein sequences of N63 appear in the UniProt
Knowledgebase under accession number P86702.
Among them, two peptides (GPAAVVGVL ⁄ IGK and
SFDSWL ⁄ ITK) present a sequence similar to two oth-
ers previously obtained by de novo sequencing of the
whole nacre ASM [34].

The MS ⁄ MS deduced sequences were individually
submitted to a blastp search against Swiss-Prot nrdb
using the EXPASY website (http: ⁄⁄expasy.org ⁄ ), and
to a tblastn search against GenBank and the data-
base for expressed sequence tags (EST) (dbEST) using
the NCBI online tool ()
(Table 3, central and right columns, respectively).
Unexpectedly, we did not find any homology with
already known mollusc shell proteins, and most of the
observed hits concern only one unique peptide and are
related to unknown putative proteins or to proteins
that are not expected to be components of the mollusc
shell matrices. These observations should be confirmed
in future works by the use of complementary tech-
niques.
On the other hand, when the peptide GPAAVVGV-
L ⁄ IGK was previously used for blast against an
in-house database of mollusk shell matrix [34], we noted
that it presents partial similarities with the sequence
GPAAVPVAAG of mucoperlin, a shell matrix protein
from the Pinna nobilis nacreous layer [24].
MS
BLAST search
The de novo-generated sequences of N63 were also sub-
mitted to a MS blast database search, which enables
the identification of the proteins or their assignment to
a family of homologous proteins, considering all their
internal peptide sequences together (Table 4). Sequence
similarities were observed for several peptides of N63
A

B
C
FE
D
Fig. 4. SEM micrographs of synthetic calcium carbonate crystals grown in vitro in the presence of N63 at increasing concentrations
(lgÆmL
)1
). (A) Negative control without N63; (B) 0.1 lgÆmL
)1
; (C) 1 lgÆmL
)1
; (D) 5 lgÆmL
)1
; (E) 10 lgÆmL
)1
; (F) 50 lgÆmL
)1
. Scale bars are
60, 20 and 2 lm, on the left, center and right, respectively.
Nautilin-63, a novel shell nacre protein B. Marie et al.
2122 FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS
with vertebrate collagen XI, cuticle collagen from nem-
atoda, and the spidroin-like protein of an arthropod.
This sequence similarity with collagen-like proteins is
partially supported by the fact that some peptides pres-
ent Gxy repeats. Interestingly, we did not find any
homology with already known mollusc shell proteins.
Table 3. MS ⁄ MS derived sequences of N63 trypsic peptides. BLAST search results against Swiss-Prot and mollusc-restricted EST databases
(taxid:6447) are presented in the central and right columns, respectively. The partial protein sequences of N63 appear in the UniProt Knowl-
edgebase under accession number P86702. Alignment results of the

BLAST searches are indicated on the peptide sequences: identical and
synonymous amino acid positions are underlined or shown in bold for the
BLAST search against Swiss-Prot and mollusc ESTs, respectively.
Respective scores (similar amino acids ⁄ total amino acids) of both
BLAST searches are indicated after the name of the matching proteins. The
MS ⁄ MS technique does not allowed distinction between L and I residues, which exhibit identical masses.
M+H
+
De novo sequence
Expasy
BLAST against
Swiss-Prot NCBI
TBLASTN against mollusc ESTs [sp.]
858.46 L ⁄ IPDL ⁄ IASSR – –
858.51 STL ⁄ IPVL ⁄ ITK – –
867.50 GPTGL ⁄ IL ⁄ IGPR – –
887.45 GPYGPL ⁄ IQR – –
949.48 FNL ⁄ IEL ⁄ ISAR – –
967.58 GPAAVVGVL ⁄ IGK – –
983.50 SFDSWL ⁄ ITK – –
1048.58 L ⁄ IGL ⁄ IPGPQGR – –
1078.54 PGPPGPGCR – –
1080.54 FAL ⁄ ISNQCL ⁄ IK – –
1177.65 L ⁄ IAVEFAGQSK – –
1227.61 FSSFL ⁄ IANEGKK – –
1260.53 EGPEGEEGPR – –
1262.53 TEFDGAYFAGGK – –
1356.81 FPVVGKPFPQL ⁄ IK – Unknown (Aplysia californica)(10 ⁄ 12)
1384.71 VFHAEPPFPTSR – Ubiquitin-like (Aplysia californica)(9 ⁄ 12)
1441.68 STYGPSGSQPGK – –

1512.88 KGVVTPFKGNQPL ⁄ IK – –
1528.68 FNDFL ⁄ IVESDSR – –
1567.67 CPPDDSSFER – –
1567.76 SPAVSGHSSPATL ⁄ INSR – –
1569.92 MKPAGFPGKGNGAPL ⁄ IK – Methyltransf. (Aristolochia californica)(9 ⁄ 16)
1740.93 NGL ⁄
IASDPLENL ⁄ IKNR – HEAT-containing (Euprymna scolopes)(11 ⁄ 14)
1748.85 L ⁄ IGSCFPDVL ⁄ IDEPPT – SWIRM-like (Euprymna scolopes)(10 ⁄ 14)
1774.85 S
PFFTGPSGYTSDGL ⁄ INK Methylase
a
(11 ⁄ 17) –
1825.92 TPTVSKTL ⁄ IL ⁄ IL ⁄ ITAAGDPGP GAGK – Zona Pellucida (Lottia gigantea)(12 ⁄ 20)
2020.11 VL ⁄ IESSKTDL ⁄ IVAL ⁄ IQGEFQR – Unknown (Lottia gigantea)(13 ⁄ 18)
a
Methylase of Nitrosococcus oceani [Q3JDX6].
Table 4. Results of the MS BLAST search for the identification of N63 using de novo sequenced peptides.
MS
BLAST
Identification
Total
score
Best
score
Peptide
matches
Calculated
mass (kDa) Phylum
Swiss-Prot
number

Collagen a-1 (XI) 179 47 4 59 Vertebrata Q28083
Collagen a-1 (XI) 176 47 4 120 Vertebrata Q61245
Collagen a-2 (XI) 175 47 4 140 Vertebrata P13942
Cuticule collagen 178 60 4 33 Nematoda Q60LV9
Cuticule collagen 160 60 3 33 Nematoda Q23628
Cuticule collagen 102 58 2 28 Nematoda Q18536
Spidroin 2-like 119 50 3 10 Arthropoda Q9BIU5
Chitinase 3-like protein 96 58 2 43 Vertebrata Q8SPQ0
Chitinase 3-like protein 96 58 2 38 Vertebrata Q29411
B. Marie et al. Nautilin-63, a novel shell nacre protein
FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS 2123
Immunolocalization of N63
Polyclonal antibodies raised against purified N63 were
produced in rabbit. We checked the specificity of these
antibodies by western blotting against both the whole
acid-insoluble matrix (AIM) and ASM extracts
(Fig. 5A,B). For these experiments, no immunological
signal was observed for negative controls performed
with pre-immune sera (data not shown). The antibody
raised against the purified N63 showed a specific
immunoreactivity for the 63 kDa band of the nacre
ASM corresponding to N63. This observation demon-
strates that the antibody recognizes exclusively N63
specific epitopes and that N63 protein is exclusively
present in the ASM.
To localize N63 directly in both the prismatic and the
nacreous layers of the shell of N. macromphalus, the im-
munogold technique was applied on shell cross-sections,
followed by observation by SEM, as described previ-
ously [43], using the antibodies raised against the puri-

fied N63 (Fig. 5C–F). Although a low background was
observed for negative control performed with pre-
immune serum (Fig. 5C), the N63 antibodies exhibit a
clear and specific signal on shell nacre (Fig. 5E,F),
whereas very little signal is observed with the prismatic
layer (Fig. 5D), testifying that N63 is specific of the
nacreous layer. Immunolocalization on nacre cross-sec-
tions (Fig. 5E,F) revealed that N63 is largely distrib-
uted inside nacre tablets, and also in the inter-tablet
matrix that separates nacre tablets of the same layer.
Discussion
In a previous study, we characterized the whole acid
soluble matrix extracted from the nacre of the cephalo-
pod N. macromphalus [34]. In particular, we obtained
approximately 40 short sequences of different shell
proteins, both extracted from the acid-soluble and
from the acid-insoluble matrices. In the present study,
we focus on one shell protein, which we named Nauti-
lin-63 (N63), according to its apparent molecular
weight on a 1D electrophoresis gel.
N63 is an acidic shell matrix glycoprotein, which is
unambiguously specific to the nacreous layer of N. mac-
romphalus. N63 belongs to the acetic acid-soluble frac-
tion, and to this fraction exclusively, because no signal
was detected on western blotting (Fig. 6) and none of
its sequenced peptides were found in the acetic acid-
insoluble fraction in our previous study [34]. In vitro,
N63 binds chitin, interacts with the shape of newly-
grown calcite crystals but, apparently, has a very limited
effect on the precipitation of calcium carbonate.

Although its glucose-rich glycosyl moieties exhibits sul-
fated groups, N63 does not bind calcium ions [34].
From the 27 peptidic sequences (of eight to 20 residues
in length) obtained by de novo sequencing, only seven of
them exhibit similarities with other putative molluscan
proteins, which are not related to calcification. One pep-
tide is partly similar to a short domain of mucoperlin, a
bivalve shell protein. At least five obtained peptides
have a collagen signature, characterized by Gxy triplets.
Such a signature has already been found in a short
domain of lustrin-A, a nacre protein of the abalone
Haliotis rufescens [44]. By the immunogold technique,
N63 appears to be particularly concentrated inside
nacre tablets, as well as between them.
The overall composition of all the obtained peptides
of N63, taken together, is enriched in Gly and Pro
AB
C
D
F
E
Fig. 5. Immunodetection of N63 by western blotting (A–B) and the
immunogold technique (C–F). (A) 12% SDS ⁄ PAGE of nacre AIM
and ASM, stained with silver nitrate. (B) The nacre AIM and ASM
were tested by western blotting and incubated with the polyclonal
antibodies raised against purified N63. (C) SEM micrographs for the
immunogold negative control performed on nacre without anti-N63
specific sera. (D–F) SEM micrographs of the immunogold technique
with anti-N63 specific sera on prismatic (D) and nacreous (E,F) shell
layers. Scale bars = 2 lm.

Nautilin-63, a novel shell nacre protein B. Marie et al.
2124 FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS
residues, whereas the overall amino acid composition
of the isolated protein, after its purification, is enriched
in Asx, Gly and Thr residues, which constitutes a typi-
cal shell protein signature. This apparent discrepancy
between de novo sequencing data and the amino acid
composition may be explained in different ways: first,
the de novo sequences represent only approximately
one half of the protein, thus giving a partial picture of
its primary structure. Second, the de novo sequencing
by MS presents technical limitations: on the one hand,
it is ineffective on protein domains, which lack appro-
priate cleavage sites (Arg and Lys in the case of trypsic
digestion); on the other hand, the peptides resulting
from the digestion are not detectable, if not ionized.
Thus, the technique might introduce a bias in the rep-
resentation of the analyzed peptides for the complete
N63 sequence. In the present case, at the very least, it
is likely that some of the nonsequenced peptides con-
stituting the unknown part of N63 are enriched in Asx
and Thr residues.
What might be the role of N63 in the formation of
nacre? Our biochemical data, when combined, give an
unusual mosaic picture, which does not simply fit into
the general structural framework given few years ago
by Nudelman et al. [16] and Addadi et al. [19], and
also recalled in the Introduction of the present study.
First, several of the peptides determined by de novo
sequencing are hydrophobic, and might suggest that

N63 is part of the hydrogel where nascent nacre tablets
grow. By contrast, N63 is a soluble and acidic protein,
and is present not only around nacre tablets, but also
inside the tablets. Second, because N63 contains sul-
fated polysaccharides, it is tempting to assume that
this protein is part of the nacre-nucleating complex
(i.e. the central domain observed for each nacre tab-
lets), whatever it is, either a central spot, as suggested
by Crenshaw and Ristedt [15] or a central ring as
reported by Nudelman et al. [16]. Once again, our
immunogold labeling data do not support this hypoth-
esis because we did not observe spots in the centre of
nacre tablets, but did observe the peripheral distribu-
tion of N63 around the tablets. Finally, we clearly
demonstrate that N63 binds chitin in vitro. This may
suggest that this protein is able to form macromolecu-
lar complexes with chitin, which, in other words,
means that it should be co-localized with chitin at the
interlamellar interface. Obviously, our immunogold
labeling data do not reveal such a location.
The fact that N63 can, at the same time, strongly
interact with the shape of CaCO
3
crystals precipitated
in vitro without (or slightly) inhibiting their formation
is puzzling. Indeed, we observed that, for many mol-
lusc shell proteins, the ability to interact with CaCO
3
crystals and the capacity to inhibit the precipitation of
CaCO

3
were often associated, as observed for P95 or
for caspartin [24,26]. The present case constitutes the
first report indicating that these two properties can be
disconnected in a calcifying matrix protein. This unu-
sual property could be somehow related to the fact
that N63 does not bind calcium ions in solution as
previously noted [34].
Taken together, these findings suggest that N63
exhibits ‘hybrid’ biochemical properties, some of which
are usually found in framework matrix proteins (i.e.
hydrophobicity, intertabular localization, ‘collagen sig-
nature’, absence of calcium-binding, weak ability to
inhibit CaCO
3
precipitation), whereas others are com-
monly met in the soluble matrix components (i.e. solu-
bility, acidity on 2D gels, enrichment in Asx residues,
capacity to interact with CaCO
3
crystals). This clearly
suggests that the models established in recent years for
Nautilus nacre [16,19] must, in some ways, be refined,
by taking in account proteins of ‘intermediate’ bio-
chemical properties, such as N63. In the absence of a
clear correlation between the structure of N63, its bio-
chemical properties and a defined function, we suggest
that N63 is a multifunctional protein that plays a key
role in binding chitin, and thus in participating in the
structuring of the organic framework, at the same time

as finely interacting with the mineral phase. It is possi-
ble that these two functions are displayed sequentially
(chitin-binding, followed by mineral interaction).
We are fully aware that trying to decipher the func-
tion of one single component of the nacre matrix will
not provide an explanation of the whole process of nacre
fabrication. As highlighted in a previous study [16],
‘none of the components of the organic matrix functions
in isolation. The organic matrix is a structural entity in
which the assembly of all components is essential for the
correct regulation of crystal nucleation, growth, mor-
phology, and polymorph type’. However, we consider
that the precise characterization of separate nacre mac-
romolecular constituents will provide the complete bio-
chemical framework required to precisely analyze the
growth of nacre tablets. This framework constitutes the
prerequisite for studying protein–protein and protein–
polysaccharide interactions, and for any attempts
aiming to understand the supramolecular chemistry that
contributes to the emergence of nacre microstructure.
Experimental procedures
Shell preparation and nacre matrix extraction
Fresh shells of the cephalopod N. macromphalus,
150 ⁄ 200 mm in length, were collected on the coast of New
B. Marie et al. Nautilin-63, a novel shell nacre protein
FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS 2125
Caledonia (Pacific). The external prismatic layer was
removed by abrasion under cold water. The shells were
mechanically crushed and fragments of the siphon were
removed. The nacre fragments were immersed in 1% (v ⁄ v)

NaOCl for 24 h to remove superficial contaminants, and
then thoroughly rinsed with water. All of the subsequent
extraction procedure was performed at 4 °C. The nacre
powder (< 200 lm) was decalcified overnight in cold dilute
acetic acid (5%, v ⁄ v) added by an automatic titrimeter
(Titronic Universal; Schott Instruments GmbH, Mainz,
Germany), until pH 4 was obtained. The solution was cen-
trifuged at 3900 g for 30 min. The pellet, corresponding to
the AIM, was rinsed six times with MilliQ water (Millipore
Corp., Billerica, MA, USA) and finally freeze-dried. The
supernatant comprising the ASM was filtered (5 lm) before
being concentrated with an Amicon ultrafiltration system
on a Millipore
Ò
membrane (YM10; 10 kDa cut-off). The
concentrated solution (approximately 5–10 mL) was exten-
sively dialyzed against MilliQ water (3 days, several water
changes) before being freeze-dried and weighed.
SDS
⁄
PAGE and gel staining procedures
The separation of matrix components was performed under
denaturing conditions by monodimensional sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS ⁄ PAGE)
containing 12% polyacrylamide (Mini-protean 3; Bio-Rad,
Hercules, CA, USA). Protein concentration of the ASM
was estimated by using the BCA-200 Protein Assay kit
(Pierce, Rockford, IL, USA). The nacre matrices were
directly suspended in LSB containing b-mercaptoethanol
and heat-denatured [40]. One milligram of AIM was sus-

pended in LBS, heat-denatured (10 min at 100 °C) and then
centrifuged at 3900 g for 30 s. Twenty microlitres of the
supernatant containing the Laemmli-soluble AIM were
loaded onto gel. Fifty micrograms of ASM were loaded in
each well. Because the classical CBB staining is often ineffi-
cient at revealing all the proteins associated with calcified
tissues, we chose to visualize proteins on the gel with both
silver nitrate [45] and CBB R-250. Glycosylation of ASM
macromolecules was studied qualitatively on denaturing
mini-gels by Alcian Blue 8GX staining [39] at pH 1 for the
detection of sulfated sugars [38].
Deglycosylation with TFMS
Chemical deglycosylation of 5 mg of ASM was performed
with 1.5 mL of TFMS ⁄ anisole (2 : 1, v ⁄ v) for 3 h, under a
nitrogen atmosphere, with constant stirring [46]. The tem-
perature was maintained at 0 °C, to preclude peptidic bond
hydrolysis. After neutralization with 2 mL of 50% cold
pyridine, the aqueous phase was extracted twice with
diethyl ether and then extensively dialyzed against water
(5 days) before being lyophilized. Fetuin was treated simi-
larly and used as a positive control. All the deglycosylated
extracts were analyzed on monodimensional SDS ⁄ PAGE
followed by silver nitrate and Alcian blue staining.
Chitin-binding ability
A chitin-binding assay was performed in solution as
described previously [47], with some modifications. One
milligram of nacre ASM and 500 lg of BSA (used as
negative control) were dissolved in 200 lL of water and
incubated with 10 mg of chitin (C9752; Sigma-Aldrich,
St Louis, MO, USA) for 2 h at 25 °C under constant stir-

ring. Samples were centrifuged (13 000 g for 5 min) and the
supernatants were taken away and preserved. The residues
were then rinsed three times with 500 lL of distilled water,
before washing with 300 lL of 0.2 m NaCl and centrifuga-
tion. The precipitates were boiled with LSB for 10 min at
99 °C. Each supernatant and washing solution was ana-
lyzed on SDS ⁄ PAGE under denaturing conditions. After
electrophoresis, the gels were stained with silver nitrate [45].
Purification of N63 by preparative SDS
⁄
PAGE
The nacre ASM was fractionated on a preparative 12%
polyacrylamide gel under denaturing conditions as described
previously [48]. Eighty fractions (5 mL each) were eluted
from the preparative gel. Aliquots of the fractions were
tested by SDS ⁄ PAGE with silver nitrate staining. Fractions
containing the N63 protein were pooled, concentrated, thor-
oughly dialyzed against MilliQ water and freeze-dried.
Infrared analysis of N63
Infrared spectra were directly recorded on lyophilized sam-
ples of nacre ASM and of purified N63 at a 2 cm
)1
resolution
on a FTIR spectrometer (Vector 22; Bruker, Ettlingen, Ger-
many) equipped with a Specac Golden GateÔ ATR device in
the wave number range 4000–500 cm
)1
. For each extract, we
obtained several spectra with a high reproducibility.
Amino acid composition of N63

The amino acid composition of the purified N63 was
determined by Eurosequence (Groningen, The Nether-
lands). Freeze-dried samples were hydrolyzed with 5.7 m
HCl in the gas phase for 1.5 h at 150 °C. The resulting
hydrolysate was analyzed on an HP 1090 Aminoquant
(Hewlett-Packard, Palo Alto, CA, USA) [49] by an auto-
mated two-step precolumn derivatization with O-phthalal-
dehyde for primary and N-(9-fluorenyl)methoxycarbonyl
for secondary amino acids. Cysteine residues were quanti-
fied after oxidation. The hydrolysis procedure does not
allow the quantification of tryptophan residues. Experi-
mentally determined amino acid values may deviate up to
approximately 10%. For comparison, the amino acid
Nautilin-63, a novel shell nacre protein B. Marie et al.
2126 FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS
composition of the nacre ASM was previously determined
by Marie et al. [34].
Monosaccharide composition of N63
Lyophilized sample N63 (100 lg) was hydrolyzed in 100 lL
of 2 m trifluoroacetic acid at 105 °C for 4 h, and then evapo-
rated to dryness before being resuspended with 100 lLof
50 mm NaOH. The acidic, neutral and amino sugar contents
of the hydrolysates were determined by HPAE-PAD on an
ICS 3000 instrument (Dionex Corp., Sunnyvale, CA, USA)
with a CarboPac PA100 column (Dionex P ⁄ N043055) in
accordance with the manufacturer’s instructions. Nonhydro-
lyzed samples were analyzed, similarly, to detect free mono-
saccharides that could have contaminated the sample during
dialysis. For comparison, the monosaccharide composition
of the nacre ASM was that described previously [34].

In vitro inhibition of CaCO
3
precipitation in the
presence of purified N63
Purified N63 was subsequently assayed for in vitro inhibition
of calcium carbonate precipitation [4]. Three millilitres of
40 mm CaCl
2
were rapidly added to 3 mL of 40 mm
NaHCO
3
containing variable amounts of protein samples
(1–50 lg). For each experiment, the pH was constantly
recorded with a combined glass electrode coupled with a pH-
meter (model GLP21; Crison, Barcelona, Spain) connected to
a personal computer. Each concentration was tested in trip-
licate. Blank tests were performed in the absence of protein.
Growth of calcite crystals in the presence of
purified N63
CaCO
3
precipitation was performed in vitro by slow diffusion
of ammonium carbonate vapor in a calcium chloride solution
[50]. The adapted test comprised: 500 lLof10mm CaCl
2
,
containing different amounts of purified N63 (0.1–
20 lgÆmL
)1
) were introduced in a eight-well culture slide (BD

Falcon; Becton-Dickinson Biosciences, Franklin Lakes, NJ,
USA). The culture slide was closed with a plastic cover,
which had been pierced with 1 mm holes. It was incubated
for 48 h at 4 °C in a closed vessel containing crystals of
ammonium bicarbonate. After incubation, the slides were
gently dried by capillarity and then by rapid incubation at
50 °C. Blank controls were performed without any sample.
Samples were subsequently carbon-sputtered and observed at
15 keV by SEM (JEOL 6400; JEOL Ltd., Tokyo, Japan).
Production of polyclonal antibodies raised
against purified N63
Polyclonal antibodies raised against purified N63 were
obtained by immunizing subcutaneously (100 lg of N63 per
injection) a female New Zealand white rabbit according to
a standard procedure (Covalab, Lyon, France), at days 1,
14 and 28. The blood was collected at days 1 (preserum,
4–5 mL), 39 (first bleeding, 12–15 mL) and 56 (final bleed-
ing, 40–50 mL). The serum containing the polyclonal anti-
bodies was used for further immunodetection tests.
Immunodetection of N63 by western blotting
The specificity of the antibodies was assayed by western
blotting with soluble and insoluble matrices extracted from
nacreous, as described previously [51]. After electrophoretic
fractionation by SDS ⁄ PAGE, the different matrices were
blotted onto poly(vinylidene difluoride) Immobilon-P
membrane (Millipore Corp.) with Mini-Trans Blot module
(Bio-Rad) for 90 min at 120 mA. The polyclonal antibodies
raised against purified N63 (dilution 1 : 500) were used at a
dilution of 1 : 500. Goat anti-rabbit, peroxidase-conjugated
serum (A3164; Sigma) was used as a secondary antibody.

The membrane was subsequently incubated in luminol
buffer for 5 min and exposed to X-Omat film in the dark
(Eastman Kodak Co., Rochester, NY, USA).
Immunolocalization of N63 on shell nacre
Immunogold labeling was performed on shell fragments as
described previously [43], using the antibody raised against
purified N63, diluted 1 : 500, and a secondary antibody
(goat anti-rabbit, dilution 1 : 400], coupled with 5 nm gold
particles (catalogue number EM.GAR5; British Biocell
International, Cardiff, UK). The size of the gold particles
was increased further by incubating the shell fragments in a
silver-enhancing solution. The samples were then dried,
and carbon sputtered before observation by SEM (Philips
XL-30 LaB6) under back-scattering electron mode. Blank
experiments were performed similarly without the first anti-
body step or with pre-immune serum.
Protein cleavage
In-gel trypsin digestion was performed on N63. After
electrophoresis under denaturing conditions in 15% bis-
acrylamide SDS ⁄ PAGE and staining with CBB (Biosafe;
Bio-Rad), the gel piece containing the N63 was destained
by successive washes with 20 mm NH
4
HCO
3
buffer and
H
2
O ⁄ CH
3

CN (50 : 50) mixture. Reduction was performed
with 50 lLof10mm dithiothreitol in 50 mm NH
4
HCO
3
for 15 min at 50 °C. Alkylation was performed with 50 lL
of 100 mm iodoacetamide for 30 min at room temperature
in the dark. The gel pieces were successively dried, rehy-
drated and dried again using 300 lLofCH
3
CN, 200 lLof
50 mm NH
4
HCO
3
and 300 lLofCH
3
CN, respectively.
The gel pieces were treated with 0.4 lg of trypsin (Sequence
grade; Promega, Madison, WI, USA) in 20 lLof50mm
B. Marie et al. Nautilin-63, a novel shell nacre protein
FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS 2127
NH
4
HCO
3
for 45 min at 50 °C under agitation at
800 r.p.m. The supernatant was removed and retained. The
gel pieces were extracted with 30 l LofH
2

O ⁄ CH
3
CN ⁄ H-
COOH (68 : 30 : 2; v ⁄ v ⁄ v) mixture for 30 min at 30 °C
under agitation at 800 r.p.m. Finally, both supernatant
extracts were pooled, dried in a vacuum concentrator and
re-suspended in 13 lL of 0.1% trifluoroacetic acid. Then
5 lL of sample was injected into the nanoLC-nanoESI-
MS ⁄ MS system for analysis.
MS analysis
The samples were loaded onto a trap column (PepMap100
C
18
;5lm; 100 A
˚
; 300 lm · 5 mm; Dionex Corp.), washed
for 3 min at 25 lLÆmin
)1
with 0.05% trifluoroacetic
acid ⁄ 2% acetonitrile, then eluted onto a C
18
reverse phase
column (PepMap100 C
18
;3lm; 100 A
˚
;75lm · 150 mm;
Dionex Corp.). Peptides were separated at a flow rate of
0.300 lLÆmin
)1

with a linear gradient of 5% to 40% aceto-
nitrile in 0.1 m formic acid over 60 min. MS was performed
using a Q-Star XL ESI Quadrupole ⁄ TOF tandem mass
spectrometer, nanoESI-qQ-TOF-MS ⁄ MS (Applied Biosys-
tems, Courtaboeuf, France), coupled to an online nanoLC
system (Ultimate Famos Switchos; Dionex Benelux BV,
Amsterdam, The Netherlands). MS data were acquired
automatically using analyst qs, version 1.1 (Applied Bio-
systems). A 1 s TOF-MS survey scan was acquired over m ⁄ z
400–1600, followed by three 3 s production scans over m ⁄ z
65–2000. The three most intense peptides with charge state
of two to four above a 20 count threshold were selected for
fragmentation and dynamically excluded for 30 s with
± 50 mmu mass tolerance. The collision energy was set by
the software according to the charge and mass of the precur-
sor ion. The MS and MS ⁄ MS data were recalibrated using
internal reference ions from a trypsin autolysis peptide at
m ⁄ z 842.51 [M + H]
+
and m ⁄ z 421.76 [M + 2H]
2+
.
Data treatment
Because of the lack of genome sequence information for
N. macromphalus, sequence identification was performed
manually by de novo sequencing of MS ⁄ MS spectra. All pep-
tides were manually interpreted by assigning the different
peaks to uninterrupted y-orb-ion series and immonium
presence was also confirmed. Then, the de novo interpreta-
tions were compared to those proposed by analyst qs, ver-

sion 1.1, before validation. Only new sequences that were
undoubtedly determined were further considered. It is impor-
tant to note that this technique does not allow a distinction
between isoleucine (I) and leucine (L) residues, which exhibit
identical masses and are indicated in the sequences as L ⁄ I.
The partial protein sequences of N63 appear in the UniProt
Knowledgebase under accession number P86702. The
homology searching was restricted to the sequences contain-
ing at least eight amino acids. MS blast searches [52] were
performed against nonredundant Swiss-Prot database
(nrdb95) under default settings (l-heidel-
berg.de/Blast2/msblast.html) for the merged sequences of
each band into a single query string. When there were several
significant hits, the three or four top hits with the highest
score were considered. The individual peptide sequences were
compared with known protein sequences, according to two
strategies. On the one hand, blast searches were performed
on the complete Swiss-Prot protein database by using
EXPASY–blast online tools () with
default settings. On the other hand, tblastn searches were
performed on the approximately 800 000 mollusc-restricted
ESTs (taxid:6447) that are available online using the NCBI
online tool (). The expect
threshold was set at 100 or 1000 as suggested by the software
developer for short sequence inquiry.
Acknowledgements
The work of B. Marie, G. Luquet and F. Marin is
financially supported by an ANR (ACCRO-EARTH,
ref. BLAN06-2_159971, coordinator G. Ramstein,
LSCE) during the period 2007–2010. The ‘Conseil

Re
´
gional de Bourgogne’ (Dijon, France) provided
additional support for the acquisition of new equip-
ment in the Biogeosciences research unit (UMR CNRS
5561). Complementary financial support was provided
by INSU (Action INTERVIE 2010). B.M. thanks
‘L’aquarium des lagons de Noume
´
a (Nouvelle-Cale
´
do-
nie)’ for providing the fresh shells of N. macromphalus,
Aline Bonnotte (Centre de Microscopie Applique
´
ea
`
la
Biologie, Universite
´
de Bourgogne) for helping with
the SEM, and Danielle Ballivet-Tkatchenko and Lau-
rent Plasseraud (UMR 5188, LSEO, Universite
´
de
Bourgogne) for infrared measurements.
References
1 Mann S (1988) Molecular recognition in biomineraliza-
tion. Nature 332 , 119–124.
2 Sudo S, Fujikawa T, Nagakura T, Ohkubo T, Sakagu-

chi K, Tanaka M, Nakashima K & Takahashi T (1997)
Structure of mollusc shell framework proteins. Nature
387, 563–564.
3 Suzuki M, Saruwatari K, Kogure T, Yamamoto Y,
Nishimura T, Kato T & Nagasawa H (2009) An acidic
matrix protein, Pif, is a key macromolecules for nacre
formation. Science 325, 1388–1390.
4 Wheeler AP, George JW & Evans CA (1981) Control
of CaCO
3
nucleation and crystal growth by soluble
matrix of oyster shell. Science 212, 1397–1398.
5 Falini G, Albeck S, Weiner S & Addadi L (1996) Con-
trol of aragonite and calcite polymorphism by mollusk
shell macromolecules. Science 271, 67–69.
Nautilin-63, a novel shell nacre protein B. Marie et al.
2128 FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS
6 Pokroy B, Fitch AN, Marin F, Kapon N, Adir N &
Zolotoyabko E (2006) Anisotropic lattice distorsion in
biogenic calcite induced by intra-crystalline organic
molecules. J Struct Biol 155, 96–103.
7 Jackson AP, Vincent JV & Turner RM (1988) The
mechanical design of nacre. Proc R Soc Lond 234,
415–440.
8 Atlan G, Balmain N, Berland S, Vidal B & Lopez E
(1997) Reconstruction of human maxillary defects with
nacre powder: histological evidence for bone regenera-
tion. C R Acad Sci Paris 320, 253–258.
9 Westbroek P & Marin F (1998) A marriage of bone
and nacre. Nature 392, 861–862.

10 Lin A & Meyers MA (2005) Growth and structure in
abalone shell. Mater Sci Eng 390, 27–41.
11 Checa AG, Cartwright JH & Willinger MG (2009) The
key role of the surface membrane in why gastropod nacre
grows in towers. Proc Natl Acad Sci USA 106, 38–43.
12 Nudelman F, Shimoni E, Keiln E, Rousseau M,
Bourrat X, Lopez E, Addadi L & Weiner S (2008)
Forming nacreous layer of the shells of the bivalves
Atrina rigida and Pinctada margaritifera: an environ-
mental- and cryo-scanning electron microscopy study.
J Struct Biol 162, 290–300.
13 Vendrasco MJ, Porter SM, Kouchinsky A, Li G &
Fernandez CZ (2010) New data on molluscs and their
shell microstructures from the Middle Cambrian
Gowers Formation, Australia. Palaeontology 53, 97–135.
14 Nakahara H (1991) Nacre formation in bivalve and
gastropod molluscs. In Mechanisms and Phylogeny of
Mineralization in Biological Systems (Suga S & Nakahara
H eds), pp 343–350. Springer-Verlag, New York.
15 Crenshaw M & Ristedt H (1976) The histochemical
localization of reactive groups in septal nacre from
Nautilus pompilius.InThe Mechanisms of Mineralization
in the Invertebrates and Plants (Watabe N & Wilbur
KM eds), pp 355–367. University of South Carolina
Press, Colombia.
16 Nudelman F, Gotliv B, Addadi L & Weiner S (2006)
Mollusk shell formation: mapping the distribution of
organic matrix components underlying a single arago-
nitic tablet in nacre. J Struct Biol 153, 176–187.
17 Nassif N, Pinna N, Gehrke N, Antonietti M, Jaeger C

& Coelfen H (2005) Amorphous layer around aragonite
platelets in nacre. Proc Natl Acad Sci USA, 102, 12653–
13655.
18 Rousseau M, Lopez E, Stempfle
´
P, Brendle
´
M, Franke
L, Guette A, Naslain R & Bourrat X (2005) Multiscale
structure of sheet nacre. Biomaterials 26, 6254–6262.
19 Addadi L, Joester D, Nudelman F & Weiner S (2006)
Mollusk shell formation: a source of new concepts for
understanding biomineralization processes. Chem Eur J
12, 980–987.
20 Levi-Kalisman Y, Falini G, Addadi L & Weiner S
(2001) Structure of the nacreous organic matrix of a
bivalve mollusk shell examined in the hydrated state
using cryo-TEM. J Struct Biol 135, 8–17.
21 Weiner S & Traub W (1984) Macromolecules in mollusc
shells and their functions in biomineralization. Phil
Trans R Soc Lond 304, 425–434.
22 Hedegaard C & Wenk H (1998) Microstructure and
texture patterns of mollusc shells.
J Molluscan Stud 64,
133–136.
23 Jackson DJ, McDougall C, Woodcroft B, Moase P,
Rose RA, Kube M, Reinhardt R, Rokshar DS,
Montagnani C, Joubert C et al. (2010) Parallel
evolution of nacre building gene sets in molluscs. Mol
Biol Evol 27, 591–608.

24 Marin F, Corstjens B, Gaulejac E, De Vring-de Jong E
& Westbroek P (2000) Mucins and molluscan calcifica-
tion – molecular characterization of mucoperlin, a novel
mucin-like protein from the nacreous shell layer of the
fan mussel Pinna nobilis (Bivalvia, Pteriomorphia).
J Biol Chem 275, 20667–20675.
25 Crenshaw M (1972) The soluble matrix of Mercenaria
mercenaria shell. Biomineral Res Rep 6, 6–11.
26 Marin F, Luquet G, Marie B & Medakovic D (2008)
Molluscan shell proteins: primary structure, origin and
evolution. Curr Top Dev Biol 80, 209–276.
27 Meenakshi VR, Martin AW & Wilbur KM (1974)
Shell repair in Nautilus macromphalus. Mar Biol 27,
27–35.
28 Gre
´
goire C (1987) Ultrastructure of the Nautilus shell.
In Nautilus, The Biology and Paleonthology of a
Living Fossil (Saunders WB & Landman NH eds),
pp. 463–486. Topics in Geology Vol.6, Plenum press,
New York.
29 Velazquez-Castillo RR, Reyes-Gasga J, Garcia-
Gutierrez DI & Jose-Yacaman M (2006) Crystal
structure characterization of nautilus shell at different
length scales. Biomaterials 27, 4508–4517.
30 Weiner S, Lowenstam HA & Hood L (1977) Discrete
molecular weight components of the organic matrices of
mollusc shells. J Exp Mar Biol Ecol 30, 45–51.
31 Keith J, Stockwell S, Ball D, Remillard K, Kaplan D,
Thannhauser T & Sherwood R (1993) Comparative

analysis of macromolecules in mollusc shells. Comp
Biochem Physiol B 105, 487–496.
32 Dauphin Y (2006) Structure and composition of the
septal nacreous layer of Nautilus macromphalus L.
(Mollusca, Cephalopoda). Zoology 109, 85–95.
33 Zhao H, Samata T, Takakura D, Hashimoto R,
Miyazaki Y, Nozawa T & Hikita Y (2003) Organic
matrix proteins preserved in fossil molluscan shells. In
Biomineralization (BIOM2001): Formation, Diversity,
Evolution and Application (Kobayashi I & Ozawa H
eds), pp. 108–111. Tokai University Press, Kanagawa.
34 Marie B, Marin F, Marie A, Be
´
douet L, Dubost L,
Alcaraz G, Milet C & Luquet G (2009) Evolution of
nacre: biochemistry and proteomics of the shell organic
B. Marie et al. Nautilin-63, a novel shell nacre protein
FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS 2129
matrix of the cephalopod Nautilus macromphalus.
ChemBioChem 10, 1495–1506.
35 Worms D & Weiner S (1986) Mollusk shell organic
matrix: Fourier transform infrared study of the acidic
macromolecules. J Exp Zool 237, 11–20.
36 Ferro DR & Provasoli A (1990) Conformer populations
of L-iduronic acid residues in glycosaminoglycan
sequences. Carbohydr Res 195, 157–167.
37 Arias JL & Fernanbez MS (2008) Polysaccharides and
proteoglycans in calcium carbonate-based biomineral-
ization. Chem Rev 108, 4475–4482.
38 Lev R & Spicer S (1964) Specific staining of sulfate

groups with Alcian blue at low pH. J Histochem
Cytochem 12, 309.
39 Wall RS & Gyi TJ (1988) Alcian blue staining of prote-
oglycans in polyacrylamide gels using the ‘critical elec-
trolyte concentration’ approach. Anal Biochem 175,
298–299.
40 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
41 Weiss IM, Go
¨
hring W, Fritz M & Mann K (2001) Per-
lustrin, a Haliotis laevigata (abalone) nacre protein, is
homologous to the insulin-like growth factor binding
protein N-terminal module of vertebrates. Biochem
Biophys Res Commun 285, 244–249.
42 Marie B, Luquet G, Pais de Barros J-P, Guichard N,
Morel S, Alcaraz G, Bollache L & Marin F (2007) The
shell matrix of the freshwater mussel Unio pictorum
(Paleoheterodonta, Unionoida). Involvement of acidic
polysaccharides from glycoproteins in nacre mineraliza-
tion. FEBS J 274, 2933–2945.
43 Marin F, Pokroy B, Luquet G, Layrolle P & De Groot
K (2007) Protein mapping of calcium carbonate biomi-
nerals by immunogold. Biomaterials 28, 2368–2377.
44 Shen X, Belcher AM, Hansma PK, Stucky GD &
Morse DE (1997) Molecular cloning and characteriza-
tion of lustrin-A, a matrix protein from shell and pearl
nacre of Haliotis rufescens. J Biol Chem 272, 32472–
32481.

45 Morrissey JH (1981) Silver stains for proteins in poly-
acrylamide gels: a modified procedure with enhanced
uniform sensitivity. Anal Biochem 117, 307–310.
46 Edge AS, Faltynek CR, Hof L, Reichert LE & Weber P
(1981) Deglycosylation of glycoproteins by trifluorome-
thanesulfonic acid. Anal Biochem 118, 131–137.
47 Inoue H, Ozaki N & Nagasawa H (2001) Purification
and structural determination of a phosphorylated pep-
tide with anti-calcification and chitin-binding activities
in the exoskeleton of the crayfish, Procambarus clarkii.
Biosci Biotechnol Biochem 65, 1840–1848.
48 Marin F, Pereira L & Westbroek P (2001) Large-scale
fractionation of molluscan shell matrix. Prot Expres
Purif 23, 175–179.
49 Schuster J (1988) Determination of amino acids in
biological, pharmaceutical, plant and food samples by
automated precolumn derivatization and high-perfor-
mance liquid chromatography. J Chromatogr 431,
271–284.
50 Addadi L & Weiner S (1985) Interactions between
acidic proteins and crystals: stereochemical requirements
in biomineralization. Proc Natl Acad Sci USA 82,
4110–4114.
51 Marie B, Luquet G, Be
´
douet L, Milet C, Guichard N,
Medakovic D & Marin F (2008) Nacre calcification in
the freshwater mussel Unio pictorum: carbonic anhydr-
ase activity and purification of a 95 kDa calcium-bind-
ing glycoprotein. ChemBioChem 9, 2515–2523.

52 Shevchenko A, Sunyaev S, Loboda A, Shevchenko A,
Bork P, Ens W & Standin KG (2001) Charting the
proteomes of organisms with unsequenced genomes by
MALDI-quadrupole time-of-flight mass spectrometry
and BLAST homology searching. Anal Chem 73, 1917–
1926.
Nautilin-63, a novel shell nacre protein B. Marie et al.
2130 FEBS Journal 278 (2011) 2117–2130 ª 2011 The Authors Journal compilation ª 2011 FEBS