Tài liệu Báo cáo khóa học: The lysozyme of the starfishAsterias rubens A paradigmatic typei lysozyme

The lysozyme of the starfish Asterias rubens

A paradigmatic type i lysozyme

Sana Bachali1

, Xavier Bailly2

, Jacqueline Jolle` s

3

, Pierre Jolle` s

3,* and Jean S. Deutsch1

1

E´

quipe De´veloppement et E´

volution, UMR 7622 ‘Biologie du de´veloppement’, CNRS et Universite´ P& M Curie, Paris, France; 2

Station Biologique, Roscoff, France; 3

Laboratoire des Prote´ines, Universite´ Paris V, France

On the basis of a partial N-terminal sequence, Jolle`s

and Jolle`s [Jolle`s, J., & Jolle`s, P. (1975) Eur. J. Biochem. 54,

19–23] previously proposed that the lysozyme from the

starfish Asterias rubens represents a new form of lysozyme,

called type i (invertebrate) lysozyme. Indeed, it differed from

both the types c (chicken) and g (goose) known in other

animals, as well as from plant and phage lysozymes.

Recently, several proteins belonging to the same family have

been isolated from protostomes. Here we report the com￾plete mature protein sequence and cDNA sequence of the

lysozyme from Asterias. These sequences vindicate the

previously proposed homology between the starfish, a

deuterostome, and protostome lysozymes. In addition, we

present a structural analysis that allows us to postulate upon

the function of several conserved residues.

Keywords: cDNA; invertebrates; lysozyme; starfish; struc￾ture.

During recent years, interest in a new type of lysozyme, the

invertebrate-type (i-type), has been growing. In 1996 Jolle`s

et al. [1] published the N-terminal sequences of lysozymes

from two coastal bivalves belonging to the genus Mytilus

and of four deep-sea bivalves belonging to the genera

Bathymodiolus and Calyptogena. This lysozyme represented

a model for the digestion of bacteria by the deep-sea

bivalves [2]. A similar lysozyme was then described in other

bivalves, Tapes japonica [3], and Chlamys islandica [4,5].

These authors noticed the striking similarity between the

bivalve lysozyme and another protein, the so-called desta￾bilase identified in the medicinal leech Hirudo medicinalis

[6,7]. It was then determined that the leech destabilase also

has lysozyme activity [8,9].

In a previous work [10], we reported the cDNA

sequence of several bivalve lysozymes. We showed that,

in addition to bivalve lysozymes, homologous sequences

can be found in the genome of the nematode Caenorhab￾ditis elegans and that of the fly Drosophila melanogaster, as

well as expressed sequences tags from penaeid shrimps,

indicating that these species possess putative proteins akin

to the lysozyme i type. We performed a phylogenetic

analysis of all of these sequences together with those of the

more conventional lysozyme c type; the results suggested

that these two lysozymes originate from a common gene

ancestor, at least in the central exon coding for the active

lysozyme domain [10].

In fact, the existence of a new type of lysozyme, lysozyme

i, was proposed as early as 1975, on the basis of the N￾terminal sequence of a lysozyme extracted from the starfish

Asterias rubens [11]. All recently described type-i lysozymes,

including putative proteins derived from nucleic acid

sequences, belong to protostome species. Thus, it seems

worthwhile to revisit the lysozyme i from the deuterostome

invertebrate in which it has been described for the first time.

In the present work, we present the protein sequence and the

complete cDNA sequence of the lysozyme i from A. rubens.

In addition, we present putative models of its secondary and

tertiary structure.

Material and methods

Biological material

The starfish A. rubens was collected near Roscoff (Brittany,

France). For RNA extraction, samples were preserved in

RNAlaterTM solution (Ambion) to inactivate RNAases.

Protein sequencing

The A. rubens lysozyme was prepared according to Jolle`s

and Jolle`s [11]. The lysozyme was reduced according to

Jolle`s et al. [12], using iodoacetamide for alkylation. Diges￾tion by trypsin or carboxypeptidase (Worthington, Lake￾1wood, NJ, USA) or by Staphylococcus aureus V8 proteinase

(Miles) was performed for 18 h at 37
C in 0.1 M ammo￾nium bicarbonate with an enzyme/substrate ratio of 1 : 50.

Cyanogen bromide (Merck) cleavage was performed in

Correspondence to J. S. Deutsch, E´ quipe De´veloppement et E´ volution,

UMR 7622 ‘Biologie du de´veloppement’, CNRS et Universite´ P&M

Curie, 9 quai St-Bernard, case 241, 75252 Paris cedex 05, France.

Fax: +33 14427 3253, Tel.: +33 14427 2576,

E-mail: [email protected]

Note: The nucleotide sequence of the Asterias lysozyme i cDNA is

available in the GenBank database under accession number

AY390770.

*Present address: MNHN, Paris and Mine´ralogie Cristallographie

(LMCP) UMR 7590, Universite´ P & M Curie, Paris (France),

pl. Jussieu, case 115, 75252 Paris cedex 05, France.

(Received 22 September 2003, revised 4 November 2003,

accepted 11 November 2003)

Eur. J. Biochem. 271, 237–242 (2004) FEBS 2003 doi:10.1046/j.1432-1033.2003.03915.x