Biomineralization of Schlumbergerella floresiana, a significant carbonate-producing benthic foraminifer.

See discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/261288180

Biomineralization of Schlumbergerella
floresiana, a significant carbonate-producing
benthic foraminifer
ARTICLE in GEOBIOLOGY · JULY 2014
Impact Factor: 3.83 · DOI: 10.1111/gbi.12085

CITATION

READS

1

74

10 AUTHORS, INCLUDING:
Anna Sabbatini

Arul Marie

20 PUBLICATIONS 242 CITATIONS

51 PUBLICATIONS 781 CITATIONS

Università Politecnica delle Marche

SEE PROFILE

Muséum National d'Histoire Naturelle

SEE PROFILE

Annachiara Bartolini

Gusti Ngurah Mahardika

69 PUBLICATIONS 1,304 CITATIONS

42 PUBLICATIONS 110 CITATIONS

Muséum National d'Histoire Naturelle

SEE PROFILE

Udayana University

SEE PROFILE

Available from: Gusti Ngurah Mahardika
Retrieved on: 19 October 2015

Geobiology (2014)

DOI: 10.1111/gbi.12085

Biomineralization of Schlumbergerella floresiana, a
significant carbonate-producing benthic foraminifer

OUET,3 A. MARIE,4 A. BARTOLINI,2 L. LANDEMARRE,5
A . S A B B A T I N I , 1 , 2 L . B ED
M. X. WEBER,6,7 I. GUSTI NGURAH KADE MAHARDIKA,8 S. BERLAND,3 F. ZITO9 AND

EC-PEYRE

2
M . - T . V EN
1

Department of Life and Environmental Sciences (Di.S.V.A.), Polytechnic University of Marche, Ancona, Italy
Centre de Recherche sur la Pal
e obiodiversit
e et les Pal
e oenvironnements, UMR 7207 CNRS MNHN UPMC, Mus
e um
National d’Histoire Naturelle, Paris Cedex 05, France
3
Biologie des Organismes et Ecosyste mes Aquatiques, UMR CNRS 7208/IRD 207, Mus
e um National d’Histoire Naturelle,
Paris Cedex 05, France
4
D
e partement R
e gulation D
e veloppement et Diversit
e Mol
e culaire, UMR CNRS 7245, Plateforme de Spectrom
e trie de masse
et de Prot
e omique, Mus
e um National d’Histoire Naturelle, Paris Cedex 05, France
5
GLYcoDiag, UFR Sciences, Orl
e ans Cedex 2, France
6
Department of Biology, Pennsylvania State University, 208 Mueller Lab, University Park, PA 16802, USA
7
Unidad Acad
e mica de Sistemas Arrecifales, Instituto de Ciencias del Mar y Limnologia, Universidad Nacional Aut
onoma

de M
e xico, Puerto Morelos, Q. Roo, M
e xico
8
The Animal Biomedical and Molecular Biology Laboratory, Udayana University, Bali, Indonesia
9
Institut de Biologie Physico-Chimique, CNRS/Universit
e Paris-7 UMR 7099, Paris, France
2

ABSTRACT
Most foraminifera that produce a shell are efficient biomineralizers. We analyzed the calcitic shell of the
large tropical benthic foraminifer Schlumbergerella floresiana. We found a suite of macromolecules containing many charged and polar amino acids and glycine that are also abundant in biomineralization proteins of other phyla. As neither genomic nor transcriptomic data are available for foraminiferal
biomineralization yet, de novo-generated sequences, obtained from organic matrices submitted to MS BLAST
database search, led to the characterization of 156 peptides. Very few homologous proteins were matched
in the proteomic database, implying that the peptides are derived from unknown proteins present in the
foraminiferal organic matrices. The amino acid distribution of these peptides was queried against the UNIPROT database and the mollusk UNIPROT database for comparison. The mollusks compose a well-studied phylum that yield a large variety of biomineralization proteins. These results showed that proteins extracted
from S. floresiana shells contained sequences enriched with glycine, alanine, and proline, making a set of
residues that provided a signature unique to foraminifera. Three of the de novo peptides exhibited
sequence similarities to peptides found in proteins such as pre-collagen-P and a group of P-type ATPases
including a calcium-transporting ATPase. Surprisingly, the peptide that was most similar to the collagen-like
protein was a glycine-rich peptide reported from the test and spine proteome of sea urchin. The molecules,
identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses, included

acid-soluble N-glycoproteins with its sugar moieties represented by high-mannose-type glycans and carbohydrates. Describing the nature of the proteins, and associated molecules in the skeletal structure of living
foraminifera, can elucidate the biomineralization mechanisms of these major carbonate producers in marine
ecosystems. As fossil foraminifera provide important paleoenvironmental and paleoclimatic information, a
better understanding of biomineralization in these organisms will have far-reaching impacts.
Received 12 June 2013; accepted 25 February 2014
Corresponding author:
a.sabbatini@univpm.it

© 2014 John Wiley & Sons Ltd

A.

Sabbatini.

Tel.:

0039 0712204329;

fax:

0039 0712204650;

e-mail:

1

2

A. SABBATINI et al.

INTRODUCTION
Foraminifera are eukaryotic unicellular micro-organisms
that live in almost all marine environments. Most species
construct tests (or shells). During the course of their evolution, they experimented with different materials to build
their tests, namely organic material, calcium carbonate
(CaCO3), opaline silica, and agglutinating sediment grains.
Calcitic foraminifera are among the major calcium
carbonate producers in the oceans (Langer et al., 1997;
Hohenegger, 2006; Langer, 2008; Ziegler & Uthicke,
2011) with intricate and beautiful test morphologies. Their

biomineralization processes are still poorly understood.
However, the elemental and isotopic composition of carbonate tests can be used to trace environmental signals,
including variations in ocean temperature, productivity,
oxygen level, and salinity. Foraminifera constitute an
important tool for paleoenvironmental and paleoclimatic
reconstructions because of their abundance and nearly
continuous fossil record (Gooday, 2003).
Calcification in foraminifera is likely to be a biologically
controlled process similar to hard-part (shell, skeleton) formation observed in many metazoan taxa (e.g., mollusks, corals, sea urchins). In foraminifera, two main models of
calcification have been proposed. The first model concerns
the imperforate high-Mg calcite foraminifera (order Miliolida). In this group, calcification occurs in the intracellular vesicles, where calcite needles are formed and later transported
to the site of formation of the new chamber (Towe & Cifelli,

A

1967). The second model applies to the bilamellar perforate
hyaline calcitic foraminifera (orders Buliminida, Rotaliida,
and Globigerinida). In these groups, the calcification occurs
in situ. An organic matrix (Hemleben et al., 1977; B
e et al.,
1979), also called the ‘primary organic lining’ (Angell, 1979,

1980), initiates calcification. Precipitation likely occurs in a confined extracellular space, where ion concentration can be regulated behind membranes (Angell, 1979; Hemleben et al., 1986;
Erez, 2003; Weiner & Dove, 2003; Bentov & Erez, 2005, 2006;
Bentov et al., 2009; de Nooijer et al., 2009; Fig. 1).
This bilamellar model likely contributed to the evolutionary success of these foraminifera, which are incredibly
abundant and diverse in past and present oceans. The test
consists of a series of chambers that are constructed
sequentially. The protoplasm of the cell pervades the existing chambers and extends outside the test, functioning in
excretion, food capture, and chamber construction. Aided
by ectoplasmic pseudopods, the first step in building a new
chamber is the delineation of a space that partially isolates
the organism from its environment. In this space, an
organic matrix organized in a thin layer, called the primary
organic membrane (POM; Erez, 2003; Weiner & Dove,
2003), may act as template or mold for the nucleation and
the precipitation of CaCO3 on both its sides, resulting in
inner and outer layers of calcite (Fig. 1). With the formation of every newly secreted chamber, the whole of the
pre-existing test is covered with an outer layer of calcite,
which precipitates on an organic layer called the organic
outer layer (OOL). Another major organic layer associated

B

Fig. 1 Schematic model describing the calcification mechanism in bilamellar perforate hyaline calcitic foraminifera. (A) Lamination scheme modified, from Kunioka et al. (2006); the test consists of a series of chambers that are constructed sequentially, each newly added chamber being composed of two layers of
calcite. With the formation of each newly secreted chamber, the entire pre-existing test is covered with an outer layer of calcite, which precipitates on an
organic layer called the organic outer layer (OOL). Another major organic layer associated with the test, the inner organic layer (IOL), covers the internal surface of the carbonate chamber. (B) Detail of A) describing the building of a new chamber. The cytoplasm of the cell pervades the existing chambers and
extends outside the test, delimiting space that partially isolates the organism from its environment. In this space, an organic matrix organized in a thin layer,
called the primary organic membrane (POM), may act as template or mold for the nucleation and the precipitation of CaCO3 on both its sides, resulting in
inner and outer layers of calcite. This process involves vacuolization of seawater by endocytosis; various pumps and possibly channels operate to increase the
pH, Ca2+, and inorganic carbon pool in these vacuoles. These modified seawater vacuoles are exocytosed into the delimited biomineralization space where
CaCO3 is precipitated over the existing shell. Basic scheme modified from de Nooijer et al. (2009).

© 2014 John Wiley & Sons Ltd

Peptides in shell of S. floresiana
with the test, the inner organic layer (IOL), covers the
internal surface of the carbonate chamber. However, this
inner layer appears to be separate from the protoplasm of
the foraminifer. It is not known whether these organic layers (POM, IOL, OOL) are similar in composition,
although Spero (1988) has described a common formation
process. Despite a comprehensive review by Robbins &

Donachy (1991), they could not determine the composition of the POM, OOL, and IOL. They proposed two
alternative hypotheses: (i) Each layer is composed of different proteins with specific mineralization regulatory functions and (ii) these organic matrices are composed of
similar proteins, which control calcification phases based
on their structural configuration. Organic matrices in other
biocalcifying organisms are composed of proteins, glycoproteins, or polysaccharides (Weiner & Addadi, 1997) that
intimately control the shape and properties of the growing
inorganic-organic composite materials.
Our current knowledge of the macromolecular components (e.g., proteins, sugars, and lipids) in the organic matrix
of foraminiferal shells is very poor (Hedley, 1963; Banner
et al., 1973; Weiner & Erez, 1984). Previous research has
focused mainly on protein components in terms of amino
acid composition (King & Hare, 1972a; Weiner & Erez,
1984; Haugen et al., 1989; Robbins & Donachy, 1991;
Robbins & Healy-Williams, 1991; Stathoplos & Tuross,
1994). Very few studies have tried to identify the amino acid
sequences of matrix shell proteins in foraminifera (Robbins
et al., 1993). In addition, very few taxa and species have
been examined. For example, most studies investigated fossil
planktonic foraminifera (King & Hare, 1972a; Haugen
et al., 1989; Robbins & Brew, 1990; Robbins & Donachy,

1991; Robbins & Healy-Williams, 1991; Stathoplos &
Tuross, 1994). Prior to this study, the only living tropical
benthic foraminifer to be analyzed in this way was Heterostegina depressa d’Orbigny, 1826 (Weiner & Erez, 1984).
In this study, we performed a biochemical and proteomic
approach to investigate the macromolecular components
(amino acids and sugars) of the organic matrix of the tests of
the benthic foraminifer Schlumbergerella floresiana (Schlumberger, 1896), a member of the family Calcarinidae. This species has the same double-layered primary wall as other
canaliculated, perforated foraminifera (i.e., Rotaliidae),
although the shell of species belonging to Calcarinidae is
complex and the mode of construction is not well resolved
(Hottinger & Dreher, 1974; Hottinger & Leutenegger,
1980; Hottinger, 1986). Like the so-called larger, symbiontbearing foraminifera, they are abundant in the western
Indo-Pacific, inhabiting shallow water in high-energy environments. With high population densities, they are important
carbonate producers (up 80%) in coral reef environments
(Hallock, 1981; Langer, 2008). For these reasons, they represent an important model for studying calcification
(Plate 1).

© 2014 John Wiley & Sons Ltd

3

Elucidating the molecular mechanisms of biomineralization will depend on a detailed characterization of shell
matrix proteins. We therefore identified amino acid
sequences from shell matrix proteins in S. floresiana. We
compared the amino acid sequences found in this study
with the amino acid distribution of proteins from all taxa
present in UNIPROT database. We wanted to know whether
proteins involved in foraminiferal calcitic biomineralization
contain sequences that are unique to these unicellular
micro-organisms or whether they share some biochemical
features with biocalcifying metazoan organisms. The foraminifer data were also compared to mollusks, taking advantage of the variety of shell structures and compositions
present in that group, as revealed by comprehensive biochemical, genetic, and proteomic studies.

MATERIALS AND METHODS
Sample collection and shell material preparation
Large specimens of S. floresiana (about 2 mm in diameter)
were collected at Amed, on the north coast of Bali, in February 2011. Megalospheric tests were gently scraped off the top
surfaces of reef rubble in five to seven meters of water. Specimens were transported to the laboratory, alive in seawater.
They were maintained alive in a glass Petri dish under a light
microscope for 2 days and observed to verify the presence of
cytoplasm. Living specimens were transferred to new dishes
with filtered seawater, and the outer surface of the shells was
carefully cleaned with a small brush. Sediment and extraneous
material adhering to it was gently scraped off, and the tests were
rinsed clean. Specimens were immersed in 2.5% NaOCl for
10 min and agitated gently to remove all superficial contaminants and traces of cytoplasm from the inner and the outer surface of the test. The shells were then thoroughly rinsed in pure
fresh water and preserved in 70% ethanol until analysis.
To image the shell ultrastructural features and localize the
organic layer(s), scanning electron micrographs of S. floresiana were generated using a ZEISS Supra 55 SEM (Carl
Zeiss, Oberkochen, Germany) at the laboratory Magie of
University UPMC-Paris VI (Plate 1). Some sections of shell
were etched with EDTA 0.5 M for 2 min, to better reveal
the morphological details and the calcitic lamellas in the
walls (Plate 1). Note that after the NaOCl blanching, the
thick IOL is still present and visible in the shell (Plate 1).

EXPERIMENTAL PROCEDURES
Organic matrix extraction procedure
The shells of S. floresiana were crushed to a fine powder
(4 g) using a mortar and pestle; 4 M guanidine thiocyanate
(Sigma, St. Louis, MO, USA) prepared with 100 mM Tris–
HCl (pH 7.4) was used to lyse contaminating cellular

4

A. SABBATINI et al.

Plate 1 Scanning electron micrographs showing the morphology, structure, and microstructure of the Schlumbergerella floresiana’s shell. (1) View of a specimen treated with 2.5% NaOCl. Scale bar: 400 lm. (2) Detail of micrograph 1 showing the surface ornamentation and pillars (pi). Scale bar: 100 lm. (3)
View of a spine. Scale bar: 100 lm. (4) Detail of a complete megalospheric specimen broken in the transversal plane. Scale bar: 400 lm. (5) Detail of another
specimen focusing on the megalospheric proloculus (p) and the chamberlet (ch) arrangement. Scale bar: 100 lm. (6) Detail of a specimen treated with EDTA
0.5 M showing the shell geometrical structure with chamberlet (ch) and the calcitic lamellae (cl). Scale bar: 100 lm. (7) Section made by Schlumberger of the
syntype of Schlumbergerella floresiana helping the taxonomic attribution of analyzed specimens in this study. Collection of Micropaleontology, MNHN, Paris.
(8) Detail of a specimen treated with EDTA 0.5 M showing the inner organic layer (IOL) detached from the inner surface of the calcified chamberlet wall.
Scale bar: 100 lm. (9) Section of another specimen, treated with EDTA 0.5 M, showing details of the alternating pattern of neighboring chamberlets (ch); stolons (st) connecting the chamberlets; IOL forming a continuous osmiophilic sheet covering the inner surface of the calcified chamberlet wall and filling the
pore pits and calcitic lamellae (cl). Scale bar: 50 lm.

material, derived from the foraminifera and xenobiotic
organisms. The solution was shocked for 10 min at room
temperature (1 vortex 10 s/min for 10 min). The sample
was incubated with guanidine thiocyanate until green pigments were eliminated, and several extractions of 10 min
were executed. The supernatant, corresponding to the guanidine thiocyanate-soluble matrix (GT-SM), was then dialyzed against 50 mM ammonium bicarbonate through a
membrane with a cutoff of 500 Da (Spectra/Por) at 4 °C.
The process took 2 days and required several water
changes. Finally, the samples were freeze-dried and
weighed. The guanidine thiocyanate-insoluble fraction was
decalcified in cold 6.5 M acetic acid with intermittent shaking until cessation of CO2 release. The solution was centrifuged at 3000 9 g for 5 min. The resulting pellet
corresponds to the acid-insoluble matrix (A-IM). It was
rinsed twice with milliQ water and centrifuged again at
3000 9 g for 5 min. After the second wash, the pellet was
freeze-dried and weighed. The supernatant, which was
saved after the demineralization of the guanidine thiocya-

nate-insoluble fractions, yielded the acid-soluble matrix (ASM) after centrifugation at 3000 9 g for 5 min. It was
extensively dialyzed against 50 mM ammonium bicarbonate
with a molecular weight cutoff of 500 Da (Spectra/Por) at
4 °C. The dialysis took 2 days and three water changes.
The resulting samples were freeze-dried and weighed. We
obtained a series of progressively cleaner organic matrices.
The GT-SM obtained from the guanidine thiocyanate
probably contained contaminants and some proteins (or
macromolecules) from the foraminiferal shell. The A-SM
fraction would be characterized by more typical shell proteins (or macromolecules). Finally, the AI-SM fraction
likely contained proteins and/or other organic materials
entrapped in shell calcium carbonate.
Neutral sugar assay
The amount of neutral sugars contained in guanidine thiocyanate-soluble (GT-SM) and acid-soluble matrices (A-SM) was
determined using the resorcinol-sulfuric acid micro-method

© 2014 John Wiley & Sons Ltd

Peptides in shell of S. floresiana
with D-glucose as standard (Monsigny et al., 1988). The protein content of acid-soluble (GT-SM, A-SM) and acid-insoluble (A-IM) fractions was derived from the amino acid
composition analysis described in the next paragraph.
Protein and amino acid composition of GT-SM, A-SM and
A-IM
Crude extracts of the GT-SM and A-SM (between 200
and 300 lg) and the A-IM extract (300 lg) were hydrolyzed using 1% phenol 6N HCl for 20 h at 110 °C. We
used a known amount of norleucine as an internal standard. After the HCl evaporated, the protein samples were
analyzed in a L-8800 Hitachi amino acids analyser (postcolumn derivatization with ninhydrin after ion-exchange
chromatography separation). All analyses were performed
at the Institut Pasteur (Paris, France).
GT-SM, A-SM and A-IM analysis on monodimensional
SDS-PAGE
SDS-PAGE analysis of protein extracts was performed on a
mini-Protean II device (BIO-RAD, Hercules, CA, USA;
Pharmacia, New York, NY, USA) according to Laemmli
(1970) using 12% polyacrylamide gels in reducing and
denaturing conditions. Briefly, 100 lg of GT-SM and
10 lg of A-SM extracts were loaded on gels. Soluble proteins from the A-IM extract were recovered after boiling
(5 min) approximately 1 mg of extract in 100 lL of Laemmli buffer. Ten microliters of this extract was used for
electrophoresis. Proteins were stained with silver nitrate
after two fixation steps of 1 h each: the first in 50%
ethanol, 13% acetic acid, and 18% formaldehyde and the
second in 50% ethanol, 13% acetic acid, and 10% glutaraldehyde. Then, gels were washed with 50% ethanol for
30 min and with water for another 1 h. Protein oxidation
was carried out using 32 lM dithiothreitol (DTT) for 1 h
followed by incubation with 0.2% AgNO3 for 1 h. The
color was developed in 3% (w/v) sodium carbonate solution containing 0.05% formaldehyde (Gotliv et al., 2003).
Proteomic analysis of GT-SM, A-SM and A-IM, and data
treatment
Five hundred micrograms of lyophilized soluble extracts
(GT-SM and A-SM) was dissolved in 50 mM ammonium
bicarbonate and reduced at 60 °C for 20 min with 1 mM
dithiothreitol (DTT). After being cooled to room temperature, alkylation was performed for 1 h in the dark with 5 mM
iodoacetamide under stirring. Then, the samples were tenfold diluted in ammonium bicarbonate containing 5% acetonitrile before addition of 5 lg of trypsin (Sigma, Proteomics
Grade, St. Louis, MO, USA). About 34 mg of insoluble (AIM) fraction was suspended in 50 mM ammonium bicarbon-

© 2014 John Wiley & Sons Ltd

5

ate and reduced under stirring at 60 °C with 1 mM DTT.
After cooling to room temperature, alkylation was performed in the dark with 5 mM iodoacetamide. After centrifugation, the obtained pellet was suspended in 50 mM
ammonium bicarbonate, 5% acetonitrile, and 20 lg trypsin.
Trypsin digestion of soluble and insoluble extracts was carried out for 18 h at 37 °C. The peptide digest from the
organic matrices was separated on a C18 column (150 9
1 mm, Cluzeau, France) at a flow rate of 40 lL min 1 with
0.1% formic acid (solvent A) and acetonitrile (solvent B),
using a gradient that varied from 3 to 70% of B in 60 min.
The eluted peptides were analyzed in an ESI-QqTOF mass
spectrometer (pulsar Applied Biosystems, Ontario, Canada),
using information-dependent acquisition mode. This mode
allows switching between mass spectrometry (MS) and MS/
MS scans. A 2-sec MS scan was followed by 2-sec MS/MS
acquisition using two most intense multiply charged precursor peptide ions (+2 to +4). The fragmented precursor ions
were excluded for 60 min to avoid reanalysis. Minimum ion
intensity for MS/MS experiments was set to 10 counts, and
the collision energy for the peptide ions was determined
automatically by the acquisition software. Data acquisition
and analyses were carried out with Analyst QS software
(version 1.1, Applied Biosystems, Ontario, Canada).
Database search was carried out with an in-house
version of MASCOT (version 2.2; Matrix Science Ltd.,
London, UK) and also PEAKS DB (Peaks studio 5.3;
Bioinformatics Solutions Inc., Ontario, Canada) using
NCBI nr database (downloaded on February 2012).
Parameters used for database search were as follows: Mass
tolerance for MS and MS/MS were 0.5 and 0.6 Da,
respectively; carbamido-methylation as fixed modification
for cysteine and methionine oxidation as variable modification; and the number of missed cleavages was set to 1.
The quality of the fragmentation mass spectra and peptide-spectrum match were manually verified to confirm
the validity of the results. Results from de novo sequencing (Peaks studio) were filtered by setting total local confidence score (TLC) to three, average local confidence to
50, and mass confidence tag to 35%. Finally, only peptides with at least 10 amino acids were considered as
valid sequences. BLAST (http://blast.ncbi.nlm.nih.gov) was
carried out using the de novo-sequenced peptides to find
homologous peptides or proteins present in the database.
Monosaccharide analysis of GT-SM and A-SM
Analyses were performed at GLYcoDIAG, Applied Glycomics
(Universit
e d’Orl
eans, France). Neutral monosaccharide content was determined after hydrolysis with trifluoroacetic acid
(TFA) conducted as follows. Four hundred micrograms of
dry extracts (guanidine thiocyanate-soluble and A-SM)
obtained from S. floresiana was treated with 2 M TFA in
water (Carlo Erba, Val de Reuil, Fr) for 4 h at 100 °C in

6

A. SABBATINI et al.

capped tubes. Reaction mixture was then chilled at 4 °C for
30 min and dried with speedVac evaporator. High-performance anionic exchange chromatographic (HPAEC) analysis
was performed on a Dionex chromatography system
equipped with a PA1 (4 9 250 mm) column and a pulsed
amperometric detector (PAD). Monosaccharide amounts
were calculated by comparison with a range of known monosaccharide standards (fucose, galactosamine, galactose, glucose, glucosamine, and mannose). Samples (100 lg per
100 lL water) were injected and eluted isocratically: 98% eluent A (water) and 2% eluent B (50 mM NaOH, 1.5 mM
sodium acetate; flow rate 1 mL min 1 for 30 min).
N-linked glycan analysis using the matrix-assisted laser
desorption ionization-time of flight mass spectrometry
Analyses of N-glycans from guanidine thiocyanate-soluble
(GT-SM) and acid-soluble matrices (A-SM) of S. floresiana
were performed at GLYcoDIAG, Applied Glycomics
(Universit
e d’Orl
eans, France). Two milligrams of samples
(GT-SM and A-SM) in 200 lL of phosphate buffer pH
7.6 was used. Proteins were denaturated with SDS and
deglycosylated using 20 units Peptide N-Glycosidase F
(PNGase F, Roche 11365 193 001 lot #12724921) at
37 °C, and released N-glycans were permethylated according to Morelle & Michalski (2007).
Mass spectra of permethylated N-glycans resuspended in
4 lL of 50% methanol/water were acquired in the positive
reflector mode (m/z range 1100–4000 Da; voltage 20 kV;
grid 75%; guide wire 0.002%; delay time 175 ns) on a
MALDI-TOF DE pro (AB SCIEX ex Applied BioSystems,
Inc., Framingham, MA, USA) with DHB as matrix (10 mg/
mL, ratio 1:1). External calibration of spectra permitted a
mass accuracy of 20 ppm. Matrix-assisted laser desorption
ionization-post-source decay (MALDI-PSD) fragmentation
was performed on selected ions to confirm structures.

RESULTS
Yields from shell organic matrix extractions
Using guanidine thiocyanate, we isolated a dry extract,
which represented 0.14% (w/w) of the foraminiferal tests
(Table 1). The A-SM represented 0.13% of the tests
(Table 1), while the A-IM represented 3.1% of the tests.
Most of the organic components of the shell were insoluble in guanidine thiocyanate and acetic acid.
Amino acid analysis, after hydrolysis, revealed that the AIM was 21% (w/w) peptides while the acid-soluble matrices
(GT-SM and A-SM) were less protein rich, 2% (w/w) and
8% (w/w), respectively (Tables 1 and 2). The protein content of the soluble matrices (GT-SM and A-SM) was relatively low and constituted approximately 0.003–0.65% by
weight of the foraminiferal shell (Table 1).

Table 1 Yields of the organic matrix extracts from Schlumbergerella
floresiana
Organic matrix components

GT-SM

A-SM

A-IM

Dry weight (%)*
Protein content (%)†
Neutral sugar content (%)‡
Protein content in shell (%)
Neutral sugar content in shell (%)

0.14
2
33
0.003
0.05

0.13
8
10
0.01
0.013

3.10
21
ND
0.65
ND

GT-SM, guanidine thiocyanate-soluble matrix; A-SM, acid-soluble matrix;
A-IM, acid-insoluble matrix, ND, not determined. *Percentage of organic
matrix components relative to the powdered shell weight. †Percentage of
proteins according to the amino acid composition analysis. ‡Percentage of
neutral sugars measured according to resorcinol/sulfuric acid assay in the
organic matrix components.

Table 2 Amino acid composition of the GT-SM, A-SM and A-IM of
Schlumbergerella floresiana
Amino acids (mol%)

GT-SM*

A-SM†

A-IM‡

Asx
Glx
Ser
Gly
Ala
Thr
Cys
Val
Met
Ile
Leu
Tyr
Phe
b-ala
Lys
His
Arg
Pro
Trp
CysSO3H
CMC
NH3
MetSO2
C/HP

11.3
10.7
8.8
13.0
8.5
5.4
0.8
6.6
0.2
5.2
7.9
3.1
4.4
0.0
5.5
0.6
4.9
4.0
ND
1.0
0.8
/
0.0
0.9
2% (w/w)

14.2
10.5
9.3
10.7
8.0
6.7
0.5
5.9
0.1
4.7
6.1
2.2
3.3
4.1
3.0
0.4
2.5
5.4
ND
1.1
1.3
/
0.0
0.9
8% (w/w)

12.9
11.3
7.3
10.4
7.7
5.5
0.4
6.1
0.1
5.5
7.9
3.2
4.2
0.2
6.2
1.0
4.0
3.9
ND
0.9
0.6
/
0.4
1.0
21% (w/w)

GT-SM, guanidine thiocyanate-soluble matrix; A-SM, acid-soluble matrix;
A-IM, acid-insoluble matrix. Data are presented as molar percentages of
total amino acids for each matrix. Asx (aspartate) = Asp (aspartic
acid) + Asn (asparagine) and Glx (glutamate) = Glu (glutamic acid) + Gln
(glutamine). Cysteine residues were quantified after oxidation. Tryptophan
residues were not detected (ND) due to the hydrolysis conditions. The
C/HP values correspond to the ratio between charged (Asx, Glx, His, Arg,
Lys) and hydrophobic amino acids (Ala, Val, Phe, Pro, Met, Ile, Leu). In this
case, the percentage of proteins among the organic matrices was measured
according phenol/HCL hydrolysis and norleucine assay. *4.650 lg proteins
per 300 lg. †16.184 lg proteins per 200 lg. ‡61.622 lg proteins per
300 lg.

The guanidine thiocyanate-soluble matrix (GT-SM) contained more sugars (33%) than the A-SM (10%). The total
amount of sugars in the soluble matrices (GT-SM and A-

© 2014 John Wiley & Sons Ltd

Peptides in shell of S. floresiana
20

%

GT-SM

A-SM

A-IM

16
12
8
4

H
is
Ar
g
Pr
o

Al
a
Th
r
C
ys
Va
l
M
et
Ile
Le
u
Ty
r
Ph
e
βal
a
Ly
s

r

ly
G

x

Se

G

As

lx

0

Fig. 2 Amino acid composition of the three fractions from Schlumbergerella floresiana. The guanidine thiocyanate-soluble matrix (GT-SM) is represented by filled diamonds, the soluble mineral-associated matrix (A-SM) by
filled squares, and the insoluble matrix (A-IM) by filled triangles. Differences in amino acid composition between the three organic matrices are
minor by comparison, and the one-way ANOVA test carried out to ascertain
these differences is not significant. Asx, aspartate; Glx, glutamate; Ser, serine; Gly, glycine; Ala, alanine; Thr, threonine; Cys, cysteine; Val, valine;
Met, methionine; Ile, isoleucine; Leu, leucine; Tyr, tyrosine; Phe, phenylalanine; b-ala, b-alanine; Lys, lysine; His, histidine; Arg, arginine; Pro, proline.

SM) represented approximately 0.05–0.013% by weight of
the S. floresiana shells (Table 1).
Amino acid composition of the GT-SM, A-SM and A-IM
The amino acid compositions of the three matrices were
recorded in Table 2. Six amino acid residues dominated
all matrices: aspartate (varying from 11 to 14%), glycine

MW
(KDa)

7

(10–13%), glutamate (10–11%), serine (7–9%), alanine (7–
8%), and leucine (6–8%; Fig. 2). The hydrophobic amino
acids (alanine, valine, leucine, isoleucine, phenylalanine, proline, and methionine) comprised 37% of the GT-SM, 34% of
the A-IM, and 33% of the A-SM. Charged and polar amino
acids (aspartate, glutamate, histidine, arginine, and lysine)
and glycine were between ~13% in the GT-SM and ~10% in
the A-SM and A-IM. The C/HP values corresponded to the
ratio between charged and hydrophobic amino acids; they
were around one for all organic matrix components.
Analysis of shell organic matrices on SDS-PAGE
Gel electrophoresis and silver nitrate staining revealed the
basic properties of the polypeptides that comprised the
organic matrices of S. floresiana (Fig. 3). The guanidine
thiocyanate-soluble matrix (GT-SM) was characterized by
one major band of ~ 26 kDa and multiple faint bands of
higher and lower molecular weight producing a ladder-like
pattern. The gel image of the A-IM showed a smear of
macromolecules with molecular weight varying from ~77
to ~48 kDa. The A-SM contained a range of molecular
sizes. Within the smear, two bands were more evident,
migrating at ~101 and ~89 kDa.
Treating the A-IM with DTT during the reduction step
induced the release of one main protein at 56 kDa and several
proteins of lower molecular weight. Boiling the A-IM
in the Laemmli buffer before electrophoresis released pro-

Acid-Soluble Matrix
Guanidine
Acid-Soluble
Thiochyanate

Acid-Insoluble Matrix

DTT
(Room temperature)

Laemmli Buffer
Boiling (100°C)

97
101
89
66

77
68
61

45

48

56
50
45
41

61
53

33
30

26

14

28

18
SDS PAGE, Silver nitrate staining

Fig. 3 Macromolecular composition of the organic matrices isolated from Schlumbergerella floresiana. Electrophoretic analyses of the guanidine thiocyanatesoluble matrix (100 lg dry weight), acid-soluble matrix (4 lg dry weight), and soluble extracts obtained from acid-insoluble matrix with DTT or in Laemmli
buffer boiling analyses were performed using 12% SDS-PAGE under denaturing conditions. Gels were stained with silver nitrate. MW, molecular weight.
Proteins extracted from the shell were separated on 12% SDS-PAGE before silver nitrate staining of proteins.

© 2014 John Wiley & Sons Ltd

8

A. SABBATINI et al.

teins that were close to proteins released during the alkylation
step with DTT. However, the extract was enriched with one
protein of ~ 28 kDa (Fig. 3). In the presence of DTT or mercaptoethanol, the released proteins suggested that disulfide
bridges were present between proteins in the A-IM.
Peptide sequences of the shell organic matrices
Liquid chromatography with tandem mass spectrometric
(LC/MS-MS) analysis of the soluble matrices (GT-SM,
A-SM) and A-IM was performed after hydrolysis with trypsin.
We queried our data against the MASCOT database (http://
www.matrixscience.com/) to identify sequences. The search
identified five proteins for the A-SM and six proteins for the
A-IM. No proteins were identified from the guanidine thiocyanate-soluble matrix (GT-SM; Table 3). In the A-SM,
most of the sequences were related to chlorophyll proteins.
Cytoplasm-localized proteins, including tubulin, actin, and a
type of heat shock protein, were present in the A–IM. P-type
ATPases from A-IM were found in bacteria.
Genomic resources for S. floresiana are limited to the complete small subunit ribosomal DNA gene sequence (http://
Table 3 Results of the

MASCOT

search for the identification of the sequenced peptides from Schlumbergerella floresiana

Peptide sequence
Acid-soluble matrix (A-SM)
LAVNIVPFPR
ALTVPELTQQMFDAK
FPGQLNSDLR
INVYYNEATGGR
TTDVLALFR

NEGADYFNNEVGPQILR

LGANVSSAQGPTGLGK
AAEDPEMETFYTK
DIEGTGNEFVGDFR
Acid-insoluble matrix (A-IM)
ALQDTGEVVGMTGDGVNDAPALK
MPSAVGYQPTLATEMGALQER
INVYYNEATGGR
AILMDLEPGTMDSVR
ALTVPELTQQMFDAK
DLYTNSVLSGGTTMFPGIDVR
TTDVLALFR

NEGADYFNNEVGPQILR

GVVDSEDLPLNISR

forambarcoding.unige.ch/). For other foraminiferal taxa,
data are also available on small and large subunits of rDNA
genes and sequences for a- and b-tubulin and actin genes
(Bowser et al., 2006; Pawlowski et al., 2012). Recently,
Habura et al. (2011) provided the first consistent body of
sequence information about the non-coding regions of the
genome of a giant unilocular agglutinated foraminifer. However, genomic resources are not yet available for S. floresiana
or other calcitic foraminifera. As a result, we interpreted de
novo sequences from the MS/MS spectra. We observed 33
peptide sequences for the guanidine thiocyanate-soluble
matrix (GT-SM). Each contained 10 to 19 amino acids. In
addition, 47 peptide sequences with 10 to 16 amino acid residues were associated with the A-SM. For the A-IM, 76 peptide sequences were identified, and each contained 10–18
amino acids (see Supporting Information Table S1 for the de
novo-sequenced peptides). In total, 156 peptide sequences
were identified from the protein extracts that we isolated from
S. floresiana shells. These observations showed that only a
few peptides with the same mass were present in the three
organic matrices (see Supporting Information Table S1 for
the de novo-sequenced peptides).

Protein name

m/z*

Peptide ion
score (P < 0.05)

Protein score

Phylum

Accession
number

Nuclear beta-tubulin, partial
Nuclear beta-tubulin, partial
Nuclear beta-tubulin, partial
Nuclear beta-tubulin, partial
Ribulose-1,5-bisphosphate
carboxylase/oxygenase
large subunit
Ribulose-1,5-bisphosphate
carboxylase/oxygenase
large subunit
Photosystem II CP43
chlorophyll apoprotein
Photosystem II D2 protein
Fucoxanthin chlorophyll
a/c protein 4

563.4306
570.0440
573.8793
678.9166
518.3765

51
59
38
38
53

187
187
187
187
132

Cryptophyta
Cryptophyta
Cryptophyta
Cryptophyta
Dinoflagellata

gi
gi
gi
gi
gi

968.5769

79

132

Dinoflagellata

gi 66730925

728.9862

79

79

Bacillariophyta

gi 1346847

774.4551
778.4594

79
70

77
70

Bryophyta
Bacillariophyta

gi 314956519
gi 224012385

758.8235
761.4759
678.930
824.5124
846.5621
753.8223
518.3881

73
81
38
59
63
82
48

73
81
159
159
159
82
125

Bacteria (Firmicutes)
Proteobacteria
Ciliophora
Ciliophora
Ciliophora
Foraminifera
Dinoflagellata

gi
gi
gi
gi
gi
gi
gi

646.0761

77

125

Dinoflagellata

gi 66730925

757.488

59

59

Mollusca

gi 205362524

P-type ATPase, translocating
F1FO ATPase beta-subunit
Beta-tubulin
Beta-tubulin
Beta-tubulin
Actin type 1
Ribulose-1,5-bisphosphate
carboxylase/oxygenase
large subunit
Ribulose-1,5-bisphosphate
carboxylase/oxygenase
large subunit
Heat shock protein 90

4105831
4105831
4105831
4105831
66730925

383191697
501149
135498
135498
135498
59859676
66730925

A-SM, acid-soluble matrix; A-IM, acid-insoluble matrix. *Mass/charge (m/z) of the fragmented peptides from which the sequences are matched;
charge = +2.

© 2014 John Wiley & Sons Ltd

Peptides in shell of S. floresiana
MS BLAST

search

The de novo-generated sequences from the foraminiferal
organic matrices were submitted to the MS BLAST database to
identify proteins by sequence similarity searches using peptide
sequences produced by the interpretation of tandem mass
spectra (Shevchenko et al., 2001). We identified three peptides from the A-IM with similar amino acid sequences. The
first was related to the pre-collagen-P from the mollusk, Mytilus galloprovincialis. This peptide was a glycine-rich peptide containing Gly-X-Y repeats. X and Y represented proline
and hydroxyproline, respectively. The second peptide
blasted to sequences associated with chloroplast genes that
coded for Photosystem II cytochrome c550 of Bacillariophyta. These hits can likely be attributed to the diatom
symbiont associated with S. floresiana (Table 4). The third
peptide was related to several ATPase families: calciumtransporting ATPase in an arthropod (Simulium nigrimanum), the plasma membrane hydrogen-transporting ATPase
from Streptophyta (Upland Cotton), and a P-type ATPase
from a rare enteric gram-negative bacterium (Rahnella
aquatilis).
Monosaccharide composition of the GT-SM and A-SM
Neutral sugar analysis indicated that the guanidine thiocyanate-soluble matrix contained 29% (w/w) neutral sugars.
The GT-SM matrix contained traces of galactose (0.9%)
and mannose (1.5%). The A-SM was represented by 9%
(w/w) of neutral monosaccharide. Both matrices were rich in
D-glucose; they contained 98% and 82% total sugars, respectively. They also contained galactose (9%), mannose (5%),
and glucosamine (3%) as minor components (Table 5).
GT-SM and A-SM characterization of the N-glycans
We identified N-linked glycans after digestion of the
GT-SM and A-SM with PNGase F. Upon permethylation,
the N-glycan profiles determined by matrix-assisted laser
desorption ionization-time of flight mass spectrometry
(MALDI-TOF-MS) analyses identified oligomannoses containing 5 to 9 mannoses in both extracts (Table 6; see
Supporting Information Fig. S1 for MALDI-TOF-MS
spectra analysis). To confirm the presence of particular
Table 4 Results of the

MS BLAST

9

N-glycans (Man5 GlcNAc2 and Man9 GlcNAc2) in the
guanidine thiocyanate-soluble matrix (GT-SM), the ions at
m/z 1579 and 2396 were fragmented by MALDI-PSD
(see Supporting Information Fig. S2 for MALDI-PSD spectrum analysis). Fragmentation indicated that the core of the
freed N-glycans was composed of N-acetyl-chitobiose, a
dimer of b-1,4-linked glucosamine units. This represented
the canonical signature of N-glycans. The fragmentation
analysis allowed us to propose a chain structure for the two
N-glycans (Fig. 4). After digestion of the extracts with the
highly specific enzyme PNGase F, release of glycans indicated the presence of N-glycoproteins in the shell organic
matrices of S. floresiana.
GT-SM and A-SM characterization of hexose polymers
In the mass spectra of permethylated N-glycans obtained
from the MALDI-TOF-MS analyses, we also noticed the
presence of hexose polymers in the acid-soluble matrices
(GT-SM and A-SM; Table 7; see Supporting Information
Figs S1 and S2 for MALDI-TOF-MS spectra analysis).
These polysaccharide chains contained 6–14 hexoses. They
were likely composed of glucose according to the neutral
sugar composition of both extracts.

DISCUSSION
The organic matrix plays an important role in biomineral
formation. However, the components of these matrices in
foraminifera remain unknown. Until now, only a few foraminiferal matrix components have been identified and
characterized at the protein level (Weiner & Erez, 1984;
Robbins & Donachy, 1991; Robbins & Healy-Williams,
1991). However, these characterizations will become more
common as transcriptomes and other genomic resources
are developed for foraminifera (Burki et al., 2006; Habura
et al., 2011; Pawlowski et al., 2012).
Does organic matrix extraction correspond to
morphological compartments?
We collected three organic matrix extracts containing
proteins (GT-SM, A-SM, and A-IM) by employing our

search for the identification of de novo-sequenced peptides from Schlumbergerella floresiana

de novo-sequenced peptide

MS BLAST

identification

Acid-insoluble matrix (A-IM)
GAGAPGLGGFGGFGAGSR (1)
LGLDPEALSLATPR (2)
TGDGVNDAPALK (3)
TGDGVNDAPALK (3)

Pre-collagen-P
Photosystem II cytochrome c550 (chloroplast)
Ca2+ transporting ATPase
H+ transporting ATPase

m/z*

E-value

Phylum

Accession number

962.1813
940.124
758.8235
758.8235

0.13
0.0008
0.009
0.006

Mollusca
Bacillariophyta
Arthropoda
Streptophyta

Q8MW54
YP_005089680.1
ACZ28168.1
AAC04385

A-IM, acid-insoluble matrix. Numbers in brackets indicate the three de novo-sequenced peptides highlighted in Table S1 that show matches during
search. *Mass/charge (m/z) of the fragmented peptides from which the sequences are obtained; charge = +2.

© 2014 John Wiley & Sons Ltd

MS BLAST

10

A. SABBATINI et al.

Table 5 Monosaccharide composition of the acid-soluble matrices of
Schlumbergerella floresiana

Monosaccharides
Fucose
Galactosamine
Galactose
Glucosamine
Glucose
Mannose
Total

GT-SM*
(lg per
400 lg of
matrix)
ND
ND
1.047  0.00
ND
111.5  1.43
1.716  0.01
114.26
29% (w/w)

GT-SM
(% of
the total)
ND
ND
0.9
ND
97.6
1.5

A-SM†
(lg per
400 lg
of matrix)
0.06
0.098  0.03
3.15  0.07
1.073  0.27
28.9  5.88
1.79  0.31
35.14
9% (w/w)

A-SM
(% of
the total)
0.2
0.3
9.0
3.1
82.4
5.1

GT-SM, guanidine thiocyanate-soluble matrix; A-SM, acid-soluble matrix.
Neutral sugar compositions were obtained by high-performance anion
exchange-pulsed amperometric detection (HPAE-PAD) after acid hydrolysis.
Data are presented in lg per 400 lg of the total matrix and in percentage
of the total identified carbohydrate compounds. Average  standard deviation (SD) is reported. ND = not detected, the signal is under the detection
limit (0.01 ppm or 4 ng per 400 lg of sample). *114.26 lg neutral sugars
per 400 lg. †35.14 lg neutral sugars per 400 lg.

2013). To avoid contamination by cellular debris, the
organic film present on the outer surface (i.e., bacterial
biofilm) and symbionts, the first extraction step was performed using denaturing guanidine thiocyanate HCl. This
step was developed to wash away all non-mineral-associated soluble biomolecules from the remaining mineralized
tissue in vertebrates (Termine et al., 1980). The same
approach was used by Stathoplos & Tuross (1994) in
three planktonic foraminiferal species. They verified that
the resulting matrix appeared to be free of ectoplasm and
adherent organic debris as well. The first extraction was
considered an effective cleaning step capable of solubilizing the rest of the cytoplasm, foreign organic film and the
diatom symbionts, because the resulting solution was a
green/brownish color, corresponding to the guanidine
thiocyanate-soluble matrix (GT-SM). However, it is possible that some of the proteins present in the different
organic layers might be removed through the GT-SM
washing process. That fraction might have contained con-

Man 9

Table 6 N-glycan characterization of the acid-soluble matrices of Schlumbergerella floresiana
[M+Na]+ Permeth (mass m/z)

Relative%

Guanidine thiocyanate-soluble matrix (GT-SM)
1375.7
10.4
1579.8
20.8
1783.9
18.0
1988.0
18.7
2192.1
13.9
2396.2
18.1
Acid-soluble matrix (A-SM)
1375.7
3.7
1579.8
13.1
1783.9
17.3
1988.0
26.6
2192.1
21.4
2396.2
17.9

Interpretation

Man4
Man5
Man6
Man7
Man8
Man9

GlcNAc2
GlcNAc2
GlcNAc2
GlcNAc2
GlcNAc2
GlcNAc2

Man4
Man5
Man6
Man7
Man8
Man9

Man4
Man5
Man6
Man7
Man8
Man9

GlcNAc2
GlcNAc2
GlcNAc2
GlcNAc2
GlcNAc2
GlcNAc2

Man4
Man5
Man6
Man7
Man8
Man9

Man 5

Mannose
N-acetylglucosamine

Fig. 4 Proposed structures of the N-glycans of Schlumbergerella floresiana.

GT-SM, guanidine thiocyanate-soluble matrix; A-SM, acid-soluble matrix.

extraction protocol (Table 1). All three had hydrolyzable
amino acids, and they all showed staining upon gel electrophoresis (Table 2; Figs 2 and 3).
Scanning electron micrographs of complete shells
showed that all ectoplasm was likely removed by first
washing with 2.5% NaOCl (Plate 1). Inside the shell, most
of the IOL and diatom symbionts were still visible. Therefore, we cannot exclude the alternative that some residual
cytoplasm (endoplasm) or digested remains of food organisms were still present (Langer & Bell, 1995). We used
this washing protocol because it has been tested on other
living benthic foraminifera (Borrelli et al., 2011) and
NaOCl is often used in the preparation of mollusk shells
(Marie et al., 2007, 2011, 2013; Ramos-Silva et al.,

Table 7 Hexose polymer characterization of the acid-soluble matrices (GTSM and A-SM) of Schlumbergerella floresiana
Hexose polymers
m/z

Interpretation

1293
1497
1701
1906
2110
2314
2518
2723
2927

Hex6
Hex7
Hex8
Hex9
Hex10
Hex11
Hex12
Hex13
Hex14

Hexose polymers present in GT-SM and A-SM were identified after
MALDI-TOF analysis of two samples.

© 2014 John Wiley & Sons Ltd

Peptides in shell of S. floresiana
taminants and also proteins (or macromolecules) associated
with the foraminiferal shell. During the demineralization
with cold 6.5 M acetic acid, the calcite test powder dissolved and exposed the organic layer(s) that were previously protected by the mineral (King & Hare, 1972a;
Weiner & Erez, 1984; Haugen et al., 1989; Robbins &
Healy-Williams, 1991; Stathoplos & Tuross, 1994). Here,
the acid-soluble proteins were extracted (A-SM), and the
A-IM contained the remaining insoluble components. The
fractions (GT-SM, A-SM, and A-IS) were the result of a
progressive cleaning to separate contaminants from shell
proteins potentially involved in the foraminiferal biomineralization mechanism. The amino acid composition of all
matrices of S. floresiana (GT-SM, A-SM, and A-IM) was
similar (Table 2; Figs 2 and 5a). This might indicate that
the shell organic layers (i.e., POM, OOL, IOL) are composed of proteins with comparable physicochemical properties. Alternatively, the similarity in protein composition
could be explained by the presence of only one kind of
layer within the organic matrix. Despite numerous scanning electron microscopic observations of S. floresiana
shell fragments, no POM was clearly observed; however, a
thick IOL was always distinguishable (Plate 1).
These results differ from those of Stathoplos & Tuross
(1994). They reported that the mineral-associated organic
matrix in the planktonic foraminifer Globigerinoides ruber
was significantly richer in aspartate (~30%) than the other
fractions (guanidine-soluble and guanidine-insoluble residues) (~15%). They suggested that different proteins were
concentrated in the A-SM. The soluble fraction of the
organic matrix in a larger tropical benthic foraminifer,
H. depressa, was rich in aspartate (~26%), glycine (~12%),
serine (~12%), glutamate (~11%), and alanine (~9%) residues, while the insoluble fraction was dominated by aspartate (~26%) and double the glycine (~24%; Weiner &
Erez, 1984). At the same time, the hydrophobic amino
acids represented ~17% of the insoluble fractions, and
the values were doubled in the soluble fraction (~34%).
Our results (Table 1) differed quantitatively from those
described by Weiner & Erez (1984). For example in
H. depressa, the soluble fraction contained almost all the
proteins present in the shell; conversely, for S. floresiana,
most of the proteins were concentrated in the insoluble
fraction. These three foraminiferal species may have
evolved to partition protein composition differently. Alternatively, discrepancies between the different datasets could
be related to different extraction methodologies and/or
could be related to a species-specific feature (King & Hare,
1972b).
Aspartate was an abundant amino acid in all matrices
extracted from S. floresiana (11–14% and Fig. 2). This evidence suggested that proteins capable of binding calcium
ions were present and probably involved in the crystal
formation. The high proportion of glycine and alanine in

© 2014 John Wiley & Sons Ltd

A
V 16%
Y

R
N
D

8%

T

C

4%
0%

S

GT-SM

A

12%

W

11

Q

P

E

A-SM
F

A-IM

G
M

H
K

UNIPROT

V 16%

B
Y

A
R
N

12%

W

D

8%

T

C

4%
0%

S

GT-SM

L

Q

P

E

A-SM
F

G

A-IM

M

H
K

Mollusk UNIPROT

V 16%

C
Y

L
A
R
N

12%

W

D

8%

T

C

4%
0%

S

Q

P

UNIPROT
Mollusk UNIPROT

E
F

G
M

H
K

Total foraminiferal organic matrices

L

Fig. 5 Spider chart distribution of amino acid composition of the de novosequenced peptides from the organic matrices (GT-SM, A-SM, and A-IM) of
Schlumbergerella floresiana in relation to UNIPROT database (http://www.ebi.
ac.uk/uniprot/TrEMBLstats/). (A) Proteins retri