Tài liệu Báo cáo Y học: Soluble silk-like organic matrix in the nacreous layer of the bivalve

Soluble silk-like organic matrix in the nacreous layer of the bivalve

Pinctada maxima

A new insight in the biomineralization field

Lucilia Pereira-Mourie` s

1

, Maria-Jose´ Almeida1,3, Cristina Ribeiro3

, Jean Peduzzi2

, Michel Barthe´ lemy2

,

Christian Milet1 and Evelyne Lopez1

1

Laboratoire de Physiologie Ge´ne´rale et Compare´e, UMR CNRS 8572, Muse´um National d’Histoire Naturelle, Paris, France; 2

Laboratoire de Chimie des Substances Naturelles, ESA CNRS 8041, Muse´um National d’Histoire Naturelle, Paris, France; 3

INEB – Instituto de Engenharia Biome´dica, Rua do Campo Alegre, Porto, Portugal

Nacre organic matrix has been conventionally classified as

both
water-soluble and
water-insoluble, based on its

solubility in aqueous solutions after decalcification with acid

or EDTA. Some characteristics (aspartic acid-rich, silk￾fibroin-like content) were specifically attributed to either one

or the other. The comparative study on the technique of

extraction (extraction with water alone vs. demineralization

with EDTA) presented here, seems to reveal that this gen￾erally accepted classification may need to be reconsidered.

Actually, the nondecalcified soluble organic matrix, extrac￾ted in ultra-pure water, displays many of the characteristics

of what until now has been called
insoluble matrix. We

present the results obtained on this extract and on a

conventional EDTA-soluble matrix, with various charac￾terization methods: fractionation by size-exclusion and

anion-exchange HPLC, amino acid analysis, glycosami￾noglycan and calcium quantification, SDS/PAGE and

FTIR spectroscopy. We propose that the model for the

interlamellar matrix sheets of nacre given by Nakahara [In:

Biomineralization and Biological Metal Accumulation,

Westbroek, P. & deJong, E.W., eds, (1983) pp. 225–230.

Reidel, Dordrecht, Holland] and Weiner and Traub

[Phil. Trans. R. Soc. Lond. B (1984) 304, 425–434] may no

longer be valid. The most recent model, proposed by

Levi-Kalisman et al. [J. Struct. Biol. (2001) 135, 8–17],

seemed to be more in accordance with our findings.

Keywords: nacre; undecalcified soluble matrix; EDTA￾soluble matrix; hydrophobicity; silk-fibroin-like-proteins.

In the biomineralization field, the mollusk shell is one of

the best studied of all calcium carbonate biominerals.

Particular attention has been given to the organic matrix

[1–5]. The latter is thought to promote the nucleation of

the mineral component, to direct the crystal growth and to

act as glue, preventing fracture of the shell [6–9]. The main

biopolymers present in the organic matrix are essentially

proteins, either glycosylated or not, acidic polysaccharides

and chitin. In nacre, they represent 1–5% (w/w) of the

structure.

From the earliest experiments, it was believed that the

biochemical properties of matrix constituents depend of

the use of a decalcification procedure for removing the

mineral component, which is strongly associated with the

organic matrix [1,3]. Therefore, all investigations up until

now used either EDTA, acetic acid or hydrochloric acid

for this demineralization step and, subsequently, two

fractions of the organic matrix were separated, based on

their solubility in aqueous solutions. Accordingly, a

designation of matrix into two classes, the soluble matrix

and the insoluble matrix, has evolved from this extraction

[10–14].

This paper presents for the first time the results of a

comparative study on the organic matrix extracted from

the nacreous layer of the shell from the pearl oyster

Pinctada maxima by two very different methods. The first

is a nondecalcifying technique obtained by an extraction in

ultra-pure water. This unconventional approach arises

from previous in vivo and in vitro experiments where we

showed that biochemical signals from nacre chips were able

to diffuse in the surrounding media and to induce new

bone formation [15–22]. In an attempt to identify these

signal molecules, we have previously perfected this original

method of extraction of the organic matrix, without any

acid treatment or demineralization, in order to minimize

any possible alteration of the activity of the macromole￾cules [20,23]. The second method is one of the widely used

extraction techniques which involves a demineralization

with EDTA followed by intensive dialysis against distilled

water. The content of the respective soluble matrix extracts

were very different and seemed to raise important

questions about the actual conventional classification of

the soluble (known as acidic and aspartic acid-rich) and

insoluble (said to be hydrophobic and glycine, alanine-rich)

matrices and on the current model of nacre organic matrix

organization.

Correspondence to E. Lopez, Laboratoire de Physiologie Ge´ne´rale

et Compare´e, UMR CNRS 8572, Muse´um National d’Histoire

Naturelle, 7 rue Cuvier, 75231, Paris Cedex 05, France.

Fax: +33 1 40795620, Tel: +33 1 40793622,

E-mail: [email protected]

Abbreviations: EDT A-IM, EDT A-insoluble matrix; EDT A-SM,

EDTA-soluble matrix; GAG, glycosaminoglycan; PG, proteoglycan;

WIM, water-insoluble matrix; WSM, water-soluble matrix.

(Received 22 April 2002, revised 16 August 2002,

accepted 23 August 2002)

Eur. J. Biochem. 269, 4994–5003 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03203.x