Tài liệu Báo cáo khoa học: Identification of a novel matrix protein contained in a protein aggregate

Identification of a novel matrix protein contained in a

protein aggregate associated with collagen in fish otoliths

Hidekazu Tohse1,2, Yasuaki Takagi2 and Hiromichi Nagasawa1

1 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan

2 Division of Marine Biosciences, Graduate School of Fisheries Science, Hokkaido University, Japan

Organisms can design and shape minerals to the desired

conformation and orientation. Such mineral structures

are called biominerals and cannot be formed by any

non-biological environments. Calcium carbonate is one

of the most common biominerals, formed mainly by

invertebrates, and has three crystal phases: calcite, ara￾gonite and vaterite. Although calcite is the most stable

crystal thermodynamically, many organisms can form

metastable aragonite crystals with desired morphologies

under normal environments of pressure and tempera￾ture. It is thought that the morphology and polymor￾phism of biominerals can be controlled by the proteins,

polysaccharides and complexes (organic matrices)

within the biominerals themselves [1,2].

In the past decade, many proteins have been isolated

from various calcium carbonate biominerals, and their

roles in the formation of crystal morphologies have

been discussed. These isolated single proteins have

some activity in changing crystal morphologies; how￾ever, analyses of the single proteins has not led

to insights into how these morphologies and

polymorphisms are formed in the biominerals, as the

Keywords

biomineralization; calcium binding; calcium

carbonate; collagen; otolith matrix

Correspondence

Y. Takagi, Division of Marine Bioscience,

Graduate School of Fisheries Science,

Hokkaido University, 3-1-1 Minato,

Hakodate, Hokkaido 041-8611, Japan

Tel ⁄ Fax: +81 138 40 5550

E-mail: [email protected]

Database

Nucleotide sequence data are available in

the DDBJ ⁄ EMBL ⁄ GenBank databases under

the accession number AB213022

(Received 31 December 2007, revised 10

March 2008, accepted 13 March 2008)

doi:10.1111/j.1742-4658.2008.06400.x

In the biomineralization processes, proteins are thought to control the

polymorphism and morphology of the crystals by forming complexes of

structural and mineral-associated proteins. To identify such proteins, we

have searched for proteins that may form high-molecular-weight (HMW)

aggregates in the matrix of fish otoliths that have aragonite and vaterite as

their crystal polymorphs. By screening a cDNA library of the trout inner

ear using an antiserum raised against whole otolith matrix, a novel protein,

named otolith matrix macromolecule-64 (OMM-64), was identified. The

protein was found to have a molecular mass of 64 kDa, and to contain

two tandem repeats and a Glu-rich region. The structure of the protein

and that of its DNA are similar to those of starmaker, a protein involved

in the polymorphism control in the zebrafish otoliths [So¨llner C, Burgham￾mer M, Busch-Nentwich E, Berger J, Schwarz H, Riekel C & Nicolson T

(2003) Science 302, 282–286]. 45Ca overlay analysis revealed that the

Glu-rich region has calcium-binding activity. Combined analysis by western

blotting and deglycosylation suggested that OMM-64 is present in an

HMW aggregate with heparan sulfate chains. Histological observations

revealed that OMM-64 is expressed specifically in otolith matrix-producing

cells and deposited onto the otolith. Moreover, the HMW aggregate binds

to the inner ear-specific short-chain collagen otolin-1, and the resulting

complex forms ring-like structures in the otolith matrix. Overall, OMM-64,

by forming a calcium-binding aggregate that binds to otolin-1 and forming

matrix protein architectures, may be involved in the control of crystal

morphology during otolith biomineralization.

Abbreviations

GST, glutathione S-transferase; HMW, high molecular weight; IPTG, isopropyl-b-D-thiogalactopyranoside; OMM-64, otolith matrix

macromolecule-64; PVDF, polyvinylidene difluoride; TFMS, trifluoromethanesulfonic acid.

2512 FEBS Journal 275 (2008) 2512–2523 ª 2008 The Authors Journal compilation ª 2008 FEBS